Estrogen decreases bone formation by decreasing remodeling, but formation is decreased less than resorption and

Data and modified from Raisz, L. G. (1999). Physiology and pathophysiology of bone remodeling. Clinical chemistry, 45

PTH is a stimulator of bone resorption and 1,25-Dihydroxy vitamin D has its greatest effect on intestinal calcium and phosphate absorption, but it may also have direct effects on bone and other tissues. It is probably critical for the differentiation of both osteoblasts and osteoclasts and can stimulate bone resorption and formation under some experimental conditions. A third hormone, calcitonin (Table 1), in contrast to PTH and 1,25(OH)2 D3, both of which increase calcium release from the mineralized matrix, calcitonin is an inhibitor of osteoclast activity. It is a potent inhibitor of bone resorption and is used clinically in the treatment of bone diseases. Other systemic hormones are keys in regulating bone remodeling, such as: Growth hormone

**Bone Formation (osteoblast activity)**

**4. Regulation signals into the control of bone remodeling**

parathyroid hormone (PTH) and 1,25-dihydroxy vitamin D (Table 1) [50].

**4.1. Systemic regulation of bone remodeling**

\* PTH and vitamin D decrease collagen synthesis in high doses.

**Table 1.** Local and systemic regulation of bone remodeling.

? = Effects are not Known

8 Topics in Osteoporosis

bone mass increases.

(8B): 1353-1358.

The parathyroid hormone (PTH) increases bone formation in bone diseases. The anabolic effects of PTH on bone formation are mediated through the PTH/PTH-related peptide (PTHrP) receptor-dependent mechanisms that generate multiple G protein-dependent signals (Table 1). PTH mediated cyclic AMP/protein kinase phosphorylates the osteoblast transcription factor Runx2, which in turn upregulates the expression of osteoblast genes. Intermittent PTH also activates ERK1/2-mitogen-activated protein kinase (MAPK) Erk1/2 and phosphatidylinositol phosphate (PI3K) signaling, resulting in increased osteoblastogenesis and osteoblast survival (Figure 3) [53]. PTH induces the synthesis of IGF-I that works with PTH in osteoblasts to stimulate osteoblast proliferation and differentiation as well as indirectly regulates osteoclast activity [54,55]. Also, PTH was inferred to interact with various local signaling molecules, including insulin-like growth factors and Wnt antagonist sclerostin (SOST) [55-57]. It was recently shown that, in addition to reducing SOST, PTH reduces Dkk1 expression and thereby increases Wnt signaling, which contributes to the anabolic effect of PTH in bone [58]. This does not exclude the possibility that PTH receptor signaling may increase bone mass and bone remodeling by affecting Wnt signaling in other cell types. Recent data indicate that the activation of the PTH receptor in T lymphocytes plays a role in PTH-induced bone formation and bone mass by promoting the production of Wnt10b by these cells [59]. These observations and the finding that PTH signaling also acts by phosphorylating the Wnt coreceptor LRP6 and β-catenin indicate that direct and indirect crosstalks between PTH and Wnt signaling are important mechanisms regulating bone formation.

### **4.3. Wnt and Wnt antagonists**

Genetic studies in human and animal models suggest that the canonical Wnt/β-catenin pathway (Table 2), together with BMP signaling and key transcription factor RUNX2(CBFA1/ AML3), has an key role in skeletal development, osteoblast differentiation and bone formation [60,61]. Wnt/β-catenin signaling plays a significant role in promoting mesenchymal commit‐ ment to the osteoblastic lineage during the embryonic bone development. The canonical Wnt/ β-catenin signaling activity is promoted in the forming osteoblast, and this activity promotes osteoblast differentiation during endochondral bone formation, and the skeletal development is affected and the osteoblast differentiation is reduced when Wnt/β-catenin signaling is interrupted in the mesenchyme (Figure 3) [62]. The *in vivo* stimulation of the Wnt10b signaling cascade in the FABP4 promoter-Wnt10b transgenic mice led to a significantly higher bone mass because of the stimulation of osteoblastogenesis and the inhibition of adipogenesis. In addition, the Wnt10b−/−mice had decreased trabecular bone and serum osteocalcin [63]. Recent advan‐ ces have been made in our understanding of the role of Wnt proteins in bone cell biology. It was found that, in addition to Wnt10b [63], several other Wnt proteins (Wnt6a, Wn10a) influence the differentiation of mesenchymal precursors into osteoblasts or adipocytes, and thereby control bone mass [64]. The Wnt signal is modulated by various antagonists, including secreted factors, transmembrane modulators, and intracellular signals. Dickkopf family members (Dkk1 and Dkk2) and secreted frizzled related proteins (Sfrps) are families of extracellular proteins that negatively modulate canonical Wnt signalling [60].

osteoblast function and bone remodeling (Table 2) [67]. Notably, BMPR1A in osteoblasts negatively regulates bone mass and Wnt/β-catenin signaling through upregulation of the Wnt inhibitors Sost and Dkk1 in mice [68]. Also, BMPs promote osteoblastogenesis through the Smad and MAPK pathways, which upregulates the expression of *Runx2* and *Osx*, and thus stimulate the bone formation (Figure 3). BMP signaling is modulated by multiple agonists and antagonists acting at the extracellular level, which are also important for bone remodeling and may be potential therapeutic targets [69]. It was found that the Wnt-induced secreted protein 1 (WISP-1/CCN4) enhances BMP2-induced signaling (Smad-1/5/8 phosphorylation and

activation), resulting in increased osteogenic differentiation and bone mass in mice.

PTH PTH/PTHrP

TGFB TGF-B type II

Brain derived serotonin (BDS)

**4.6. Eph and Ephrin interactions**

**Ligand Receptors Activated pathways Target Cells**

Wnt3a LRP5/LRP6/Frizzled Wnt, PI3K/Akt Osteoblasts

BMP BMPR1A Wnt Osteoblasts/osteoclasts

Ephrins Eph c-Fos-NFATc1 Osteoblasts/osteoclasts

FGF2 FGFR1/2 Erk1/2, PKCa, Wnt Osteoblasts

Wnt5a Ror2 JNK Osteoblasts/osteoclasts

Semaphorin 4D Plexin-B1 RhoA/IGF1 Osteoblasts/osteoclasts

The interactions between Eph and Ephrin play important roles in bone cell differentiation and patterning by exerting effects on osteoblast and osteoclast differentiation, resulting in the

IGF-1/IGFBP2 IGFR Akt, Wnt Osteoblats

EGFR ERBB1-4 Ras-Raf-Map-Kinase

**Table 2.** Signaling pathways affecting bone cells and bone remodeling.

cAMP/PCA, PKC, PI3K/Akt, Wnt

cAMP/PCA, PKC, PI3K/Akt, Wnt

Htr2c Wnt Osteoblasts

Osteoblasts

Molecular Aspects of Bone Remodeling http://dx.doi.org/10.5772/54905 11

Osteoblats/osteoclasts

### **4.4. Transforming growth factor-β**

The transforming growth factor-β (TGF-β) signaling pathway, is known to control bone remodeling and maintenance. However, TGF-β exerts both positive and negative effects on bone cells, causing bone loss or bone gain in mice. There are three isoforms of TGF-β, namely, TGF-β1, TGF-β2, and TGF-β3. TGF-β1, known as the most abundant TGF-β isoform in the bone tissue, has been intensively studied during bone remodeling [65]. A study on the mechanism of TGF-β for osteoblast regulation has indicated that TGF-β1 stimulates bone matrix apposition and osteoblast proliferation in vitro. Additional research revealed that although TGF-β1 stimulates the early differentiation of osteoblast cells, this factor suppresses the late stage of osteoblast differentiation. These signals are transduced together by the activation of R-smads and Cosmads as well as through the mitogen-activated protein kinase (MAPK) pathway (Table 2). A cross talk exists between the TGF-β signal and the parathyroid hormone (PTH) in the regulation of osteoblastogenesis [66]. PTH stimulates the production of TGF-β1 and TGF-β2 in the osteoblast. In addition to regulating the osteoblastic bone formation, TGF-β1 has a key role in regulating bone remodeling by connecting bone formation and bone resorption (Figure 3). TGF-β proteins are present in their latent form in the bone matrix, and osteoclasts can release, as well as activate, TGF-β from the bone matrix via osteoclastic bone resorption. The released TGF-β may in turn stimulate the osteoblastic bone formation [45].

### **4.5. Bone morphogenetic proteins**

Bone morphogenetic proteins (BMPs), they are so named for their osteoinductive properties, and regulate differentiation of mesenchymal cells into components of bone, cartilage or adipose tissue. TGF-β/BMP ligand signal is mediated by serine/threonine protein kinases (receptor types 1 and 2) and a family of receptor substrates (the Smad proteins) that move into the nucleus. BMP signaling is important for skeletal development and maintenance of bone mass through activation of BMP type 1A (BMPR1A) and type 1B receptors that control osteoblast function and bone remodeling (Table 2) [67]. Notably, BMPR1A in osteoblasts negatively regulates bone mass and Wnt/β-catenin signaling through upregulation of the Wnt inhibitors Sost and Dkk1 in mice [68]. Also, BMPs promote osteoblastogenesis through the Smad and MAPK pathways, which upregulates the expression of *Runx2* and *Osx*, and thus stimulate the bone formation (Figure 3). BMP signaling is modulated by multiple agonists and antagonists acting at the extracellular level, which are also important for bone remodeling and may be potential therapeutic targets [69]. It was found that the Wnt-induced secreted protein 1 (WISP-1/CCN4) enhances BMP2-induced signaling (Smad-1/5/8 phosphorylation and activation), resulting in increased osteogenic differentiation and bone mass in mice.


**Table 2.** Signaling pathways affecting bone cells and bone remodeling.

### **4.6. Eph and Ephrin interactions**

ment to the osteoblastic lineage during the embryonic bone development. The canonical Wnt/ β-catenin signaling activity is promoted in the forming osteoblast, and this activity promotes osteoblast differentiation during endochondral bone formation, and the skeletal development is affected and the osteoblast differentiation is reduced when Wnt/β-catenin signaling is interrupted in the mesenchyme (Figure 3) [62]. The *in vivo* stimulation of the Wnt10b signaling cascade in the FABP4 promoter-Wnt10b transgenic mice led to a significantly higher bone mass because of the stimulation of osteoblastogenesis and the inhibition of adipogenesis. In addition, the Wnt10b−/−mice had decreased trabecular bone and serum osteocalcin [63]. Recent advan‐ ces have been made in our understanding of the role of Wnt proteins in bone cell biology. It was found that, in addition to Wnt10b [63], several other Wnt proteins (Wnt6a, Wn10a) influence the differentiation of mesenchymal precursors into osteoblasts or adipocytes, and thereby control bone mass [64]. The Wnt signal is modulated by various antagonists, including secreted factors, transmembrane modulators, and intracellular signals. Dickkopf family members (Dkk1 and Dkk2) and secreted frizzled related proteins (Sfrps) are families of

extracellular proteins that negatively modulate canonical Wnt signalling [60].

released TGF-β may in turn stimulate the osteoblastic bone formation [45].

Bone morphogenetic proteins (BMPs), they are so named for their osteoinductive properties, and regulate differentiation of mesenchymal cells into components of bone, cartilage or adipose tissue. TGF-β/BMP ligand signal is mediated by serine/threonine protein kinases (receptor types 1 and 2) and a family of receptor substrates (the Smad proteins) that move into the nucleus. BMP signaling is important for skeletal development and maintenance of bone mass through activation of BMP type 1A (BMPR1A) and type 1B receptors that control

The transforming growth factor-β (TGF-β) signaling pathway, is known to control bone remodeling and maintenance. However, TGF-β exerts both positive and negative effects on bone cells, causing bone loss or bone gain in mice. There are three isoforms of TGF-β, namely, TGF-β1, TGF-β2, and TGF-β3. TGF-β1, known as the most abundant TGF-β isoform in the bone tissue, has been intensively studied during bone remodeling [65]. A study on the mechanism of TGF-β for osteoblast regulation has indicated that TGF-β1 stimulates bone matrix apposition and osteoblast proliferation in vitro. Additional research revealed that although TGF-β1 stimulates the early differentiation of osteoblast cells, this factor suppresses the late stage of osteoblast differentiation. These signals are transduced together by the activation of R-smads and Cosmads as well as through the mitogen-activated protein kinase (MAPK) pathway (Table 2). A cross talk exists between the TGF-β signal and the parathyroid hormone (PTH) in the regulation of osteoblastogenesis [66]. PTH stimulates the production of TGF-β1 and TGF-β2 in the osteoblast. In addition to regulating the osteoblastic bone formation, TGF-β1 has a key role in regulating bone remodeling by connecting bone formation and bone resorption (Figure 3). TGF-β proteins are present in their latent form in the bone matrix, and osteoclasts can release, as well as activate, TGF-β from the bone matrix via osteoclastic bone resorption. The

**4.4. Transforming growth factor-β**

10 Topics in Osteoporosis

**4.5. Bone morphogenetic proteins**

The interactions between Eph and Ephrin play important roles in bone cell differentiation and patterning by exerting effects on osteoblast and osteoclast differentiation, resulting in the

**4.7. Epidermal growth factor receptor (EGFR)**

increased bone resorption [48].

**4.8. Fibroblast Growth Factors (FGFs)**

The epidermal growth factor receptor (EGFR) is a glycoprotein on the cell surface of a variety of cell types and is characterized by its ligand-dependent tyrosine kinase activity. After ligand binding to the extracellular domain, the EGFRs are activated by homo- or heterodimerization with auto- and transphosphorylation on tyrosine residues at the intracellular domain, and then a variety of signaling pathways, such as Ras-Raf-MAP-kinase and PI-3- kinase-Akt, are activated to influence cell behaviors, such as proliferation, differentiation, apoptosis, and migration (Table 2) [73]. In recent years, several experiments indicate that the epidermal growth factor receptor (EGFR) system plays important roles in skeletal biology and pathology. This network, including a family of seven growth factors – the EGFR ligands – and the related tyrosine kinase receptors EGFR (ERBB1), ERBB2, ERBB3 and ERBB4, regulates aspects such as proliferation and differentiation of osteoblasts, chondrocytes and osteoclasts, parathyroid hormone-mediated bone formation and cancer metastases in bone (Figure 3) [74]. In addition, EGFR signaling affects osteoclasts, albeit this could be an indirect effect mediated by inhibition of OPG expression and increased RANKL expression by osteoblasts [74]. It was recently found that decreasing EGFR expression in pre-osteoblasts and osteoblasts in mice results in decreased trabecular and cortical bone mass as a consequence of reduced osteoblastogenesis and

Molecular Aspects of Bone Remodeling http://dx.doi.org/10.5772/54905 13

Signaling induced by Fibroblast Growth Factors (FGFs) regulate osteoblastogenesis and bone formation. Multiple signaling pathways activated by FGF receptors 1 and 2 control osteoblast proliferation, differentiation, and survival (Table 2). FGFs bind to high affinity FGF receptors (FGFR), leading to FGFR dimerization, phosphorylation of intrinsic tyrosine residues and activation of several signal transduction pathways [75]. Recent studies provided some insights into specific signaling pathways induced by FGF/FGFR signaling that control osteoblasts. Activation of ERK1/2 signaling by FGF was found to be essential for promoting cell prolifer‐ ation in osteoblast precursor cells [76]. In addition, activation of ERK1/2 is involved in FGFR2 mediated osteoblast differentiation. Activation of ERK-MAP kinase by activating FGFR2 mutations results in increased transcriptional activity of Runx2, an essential transcription factor involved in osteoblastogenesis, and increased osteogenic marker gene expression (Figure 3) [77]. Recent data indicate that FGF2 stimulates osteoblast differentiation and bone formation in part by activating Wnt signaling suggesting that Wnt signaling may mediate, at least in part, the positive effect of FGF/FGFR signaling on bone formation in mice [78]. Besides Wnt signaling, FGF/FGFR signaling interacts with other pathways. One interaction involves a negative regulation of the BMP antagonist Noggin by FGF2 during skull development [79]. Another interaction involves the upregulation of the BMP2 gene by endogenous FGF/FGFR signaling in calvarial osteoblasts. In vivo, FGF2 treatment of developing bone fronts promotes BMP2 gene expression through the modulation of Runx2 expression [80]. These studies

support a positive role of FGF and BMP signaling crosstalks on bone formation.

**Figure 3.** Key signaling pathways for regulating osteoblastogenesis in bone remodeling. BMPs/TGF-β, Wnt, intermit‐ tent PTH and Wnt5a-Ror2 stimulate osteoblast differentiation. Eph–Ephrin and RANKL-RANK signal mediate osteo‐ blast–osteoclast interaction. TGF-β1 secretion mediated by osteoclastic bone resorption induces BMSC migration and bone formation. Leptin–brainstem-derived serotonin-sympathetic nervous system and Sema4D pathway suppresses osteoblast proliferation, whereas gut-derived serotonin inhibits osteoblast proliferation.

coupling of bone resorption and bone formation. Eph receptors are tyrosine kinase receptors activated by ligands called ephrins (Eph receptor interacting proteins). Both Ephs and ephrins are divided into two A and B groups [70]. To date, ephrinB2, a transmembrane protein expressed on osteoclasts, and its engagement with its receptor, EphB4, on osteoblasts, lead to bi-directional signaling between these cells; this is one of the cell-cell contact mechanisms that mediate crosstalk between these cells. EphrinB2 (as reverse signaling), located on the surface of osteoclast precursors, suppresses osteoclast precursor differentiation by inhibiting the osteoclastogenic c-Fos-NFATc1 cascade (Table 2) [71]. In addition, the signaling mediated by EphB4 (as forward signaling) located on the surface of osteoblast enhances the osteogenic differentiation. Ephrin B1 induces osteoblast differentiation by transactivating the nuclear location of transcriptional coactivator with PDZ-binding motif (TAZ), a co-activating protein of Runx2. TAZ, together with Runx2, induces osteoblast-related gene expression [72]. The functional role of the EphrinA2–EphA2 complex differs significantly in its interactions compared with the EphrinB2– EphB4 complex. Both the reversed signaling EphrinA2 and forward signaling EphA2 stimulate osteoclast differentiation, but EphA2 has a negative role in bone formation by inhibiting osteoblast differentiation through the regulation of RhoA activity (Figure 3) [71].

### **4.7. Epidermal growth factor receptor (EGFR)**

The epidermal growth factor receptor (EGFR) is a glycoprotein on the cell surface of a variety of cell types and is characterized by its ligand-dependent tyrosine kinase activity. After ligand binding to the extracellular domain, the EGFRs are activated by homo- or heterodimerization with auto- and transphosphorylation on tyrosine residues at the intracellular domain, and then a variety of signaling pathways, such as Ras-Raf-MAP-kinase and PI-3- kinase-Akt, are activated to influence cell behaviors, such as proliferation, differentiation, apoptosis, and migration (Table 2) [73]. In recent years, several experiments indicate that the epidermal growth factor receptor (EGFR) system plays important roles in skeletal biology and pathology. This network, including a family of seven growth factors – the EGFR ligands – and the related tyrosine kinase receptors EGFR (ERBB1), ERBB2, ERBB3 and ERBB4, regulates aspects such as proliferation and differentiation of osteoblasts, chondrocytes and osteoclasts, parathyroid hormone-mediated bone formation and cancer metastases in bone (Figure 3) [74]. In addition, EGFR signaling affects osteoclasts, albeit this could be an indirect effect mediated by inhibition of OPG expression and increased RANKL expression by osteoblasts [74]. It was recently found that decreasing EGFR expression in pre-osteoblasts and osteoblasts in mice results in decreased trabecular and cortical bone mass as a consequence of reduced osteoblastogenesis and increased bone resorption [48].

### **4.8. Fibroblast Growth Factors (FGFs)**

coupling of bone resorption and bone formation. Eph receptors are tyrosine kinase receptors activated by ligands called ephrins (Eph receptor interacting proteins). Both Ephs and ephrins are divided into two A and B groups [70]. To date, ephrinB2, a transmembrane protein expressed on osteoclasts, and its engagement with its receptor, EphB4, on osteoblasts, lead to bi-directional signaling between these cells; this is one of the cell-cell contact mechanisms that mediate crosstalk between these cells. EphrinB2 (as reverse signaling), located on the surface of osteoclast precursors, suppresses osteoclast precursor differentiation by inhibiting the osteoclastogenic c-Fos-NFATc1 cascade (Table 2) [71]. In addition, the signaling mediated by EphB4 (as forward signaling) located on the surface of osteoblast enhances the osteogenic differentiation. Ephrin B1 induces osteoblast differentiation by transactivating the nuclear location of transcriptional coactivator with PDZ-binding motif (TAZ), a co-activating protein of Runx2. TAZ, together with Runx2, induces osteoblast-related gene expression [72]. The functional role of the EphrinA2–EphA2 complex differs significantly in its interactions compared with the EphrinB2– EphB4 complex. Both the reversed signaling EphrinA2 and forward signaling EphA2 stimulate osteoclast differentiation, but EphA2 has a negative role in bone formation by inhibiting osteoblast differentiation through the regulation of RhoA

osteoblast proliferation, whereas gut-derived serotonin inhibits osteoblast proliferation.

**Figure 3.** Key signaling pathways for regulating osteoblastogenesis in bone remodeling. BMPs/TGF-β, Wnt, intermit‐ tent PTH and Wnt5a-Ror2 stimulate osteoblast differentiation. Eph–Ephrin and RANKL-RANK signal mediate osteo‐ blast–osteoclast interaction. TGF-β1 secretion mediated by osteoclastic bone resorption induces BMSC migration and bone formation. Leptin–brainstem-derived serotonin-sympathetic nervous system and Sema4D pathway suppresses

activity (Figure 3) [71].

12 Topics in Osteoporosis

Signaling induced by Fibroblast Growth Factors (FGFs) regulate osteoblastogenesis and bone formation. Multiple signaling pathways activated by FGF receptors 1 and 2 control osteoblast proliferation, differentiation, and survival (Table 2). FGFs bind to high affinity FGF receptors (FGFR), leading to FGFR dimerization, phosphorylation of intrinsic tyrosine residues and activation of several signal transduction pathways [75]. Recent studies provided some insights into specific signaling pathways induced by FGF/FGFR signaling that control osteoblasts. Activation of ERK1/2 signaling by FGF was found to be essential for promoting cell prolifer‐ ation in osteoblast precursor cells [76]. In addition, activation of ERK1/2 is involved in FGFR2 mediated osteoblast differentiation. Activation of ERK-MAP kinase by activating FGFR2 mutations results in increased transcriptional activity of Runx2, an essential transcription factor involved in osteoblastogenesis, and increased osteogenic marker gene expression (Figure 3) [77]. Recent data indicate that FGF2 stimulates osteoblast differentiation and bone formation in part by activating Wnt signaling suggesting that Wnt signaling may mediate, at least in part, the positive effect of FGF/FGFR signaling on bone formation in mice [78]. Besides Wnt signaling, FGF/FGFR signaling interacts with other pathways. One interaction involves a negative regulation of the BMP antagonist Noggin by FGF2 during skull development [79]. Another interaction involves the upregulation of the BMP2 gene by endogenous FGF/FGFR signaling in calvarial osteoblasts. In vivo, FGF2 treatment of developing bone fronts promotes BMP2 gene expression through the modulation of Runx2 expression [80]. These studies support a positive role of FGF and BMP signaling crosstalks on bone formation.

### **4.9. Insulin-like growth factor-I**

The Insulin-like growth factor-I (IGF-I) signaling through its type 1 receptor generates a complex signaling pathway that stimulates cell proliferation, function, and survival in osteoblasts (Table 2) [81]. Accordingly, mice lacking functional IGF-I exhibit severe deficiency in bone formation and a 60% deficit in peak bone mineral density (BMD) [82]. IGF-I can act in an endocrine, paracrine or autocrine manner and is regulated by a family of six IGF binding proteins (IGFBPs). The IGFBPs, have received considerable attention as regulators of IGF actions. The IGFBPs have been reported to have stimulatory or inhibitory actions on the IGFs in bone, and recent experiments have provided evidence that some of IGFBPs function independently of IGF to increase parameters of bone formation. The IGFBPs are often found bound to IGF-I in the circulation or complexed with IGF-I in osteoblasts. IGFBP-3 and -5 are known stimulators of IGF-I actions, whereas IGFBP-1, -2, -4 and -6 are known inhibitors of IGF-I action in bone. Once IGF-I binds to its receptor (type 1 IGF receptor) it initiates a complex signaling pathway including the phosphoinositol 3-kinase (PI3-K)/3-PI-dependent kinase (PDK)-1/Akt pathway and the Ras/Raf/mitogen-activated protein (MAP) kinase pathway which stimulate cell function and/or survival (Figure 3) [83]. Recent findings indicate that many of the IGFBPs and specific proteins in the IGF-I signaling pathways are also potent anabolic factors in regulating osteoblast function and may serve as potential targets to stimulate osteoblast function and bone formation locally.

did not support the Lrp5-GDS-osteoblast model because they found that there was no

Molecular Aspects of Bone Remodeling http://dx.doi.org/10.5772/54905 15

More recently, other signaling pathways that link regulation of the osteoclasts and osteoblasts have been identified. Osteoblast-lineage cells expressed Wnt5a, whereas osteoclast precursors expressed Ror2. Connection between these two cells leads to Wnt5a-Ror2 signaling between osteoblast-lineage cells and osteoclast precursors enhanced osteoclastogenesis, through increased RANK expression mediated by JNK signaling. A soluble form of Ror2 acted as a decoy receptor of Wnt5a and abrogated bone destruction in the mouse model, suggesting that the Wnt5a-Ror2 pathway is crucial for osteoclastogenesis in physiological and pathological environments and may represent a therapeutic target for bone diseases (Figure 3) [90]. Finally, a recent study reported that semaphorin 4D (Sema4D), previously shown to be an axon guidance molecule, expressed by osteoclasts and which potently inhibits bone formation [91]. Several studies have suggested that axon-guidance molecules, such as the semaphorins and ephrins, are involved in the cell-cell communication that occurs between osteoclasts and osteoblasts. The Binding of Sema4D to its receptor Plexin-B1 in osteoblasts resulted in the activation of the small GTPase RhoA, which inhibits bone formation by suppressing insulinlike growth factor-1 IGF-1 signaling and by modulating osteoblast motility. Notably, the suppression of Sema4D using a specific antibody was found to markedly prevent bone loss in a model of postmenopausal osteoporosis (Table 2) [91]. This finding identifies a new link between osteoclasts and osteoblast signaling, and suggests that suppression of the Sema4D-Plexin-B1-RhoA signaling axis may provide a new therapeutic target for reducing bone loss

Several lines of evidence have established that the cells that remodel the skeleton under physiological conditions are the same cells that mediate these processes in pathologic states. Mature bone consists of: an organic matrix (osteoid) composed mainly of type 1 collagen formed by osteoblasts; a mineral phase which contains the bulk of the body's reserve of calcium and phosphorus in crystalline form (hydroxyapatite) and deposited in close relation to the collagen fibers; bone cells; and a blood supply with sufficient levels of calcium and phosphate to mineralize the osteoid matrix. Bone turnover and remodeling occurs throughout life and involves the two-coupled processes of bone formation by osteoblasts and bone resorption by osteoclasts and perhaps osteolytic osteocytes. Abnormalities of bone remodeling can produce a variety of skeletal disorders (Table 3). The metabolic bone diseases may reflect disturbances in the organic matrix, the mineral phase, the cellular processes of remodeling, and the endocrine, nutritional, and other factors that regulate skeletal and mineral homeostasis. These disorders may be hereditary or acquired and usually affect the entire bony skeleton. The acquired metabolic bone diseases are the more common and include: osteoporosis, osteoma‐

relevance between GDS and bone mass in their mouse model system [89].

**4.11. New signals in bone remodeling**

and development of bone-increasing drugs.

**5. Pathophysiology of bone remodeling (diseases)**

### **4.10. Leptin–serotonin system pathway regulation of bone formation through gut-derived serotonin**

A new regulation mode of osteoblastic bone formation controlled by leptin-serotonin (BDS) sympathetic nervous system pathway has emerged in recent years. Leptin is a hormone produced by adipocytes that, besides its function in regulating body weight and gonadal function, can also act as an inhibitor of bone formation (Figure 3) [84]. Latest data indicates that these leptin functions require brainstem-derived serotonin [85]. Serotonin is a bioamine produced by neurons of the brainstem (brainstem-derived serotonin, BDS) and enterochro‐ maffin cells of the duodenum (gut-derived serotonin, GDS). BDS acts as a neurotransmitter, while GDS as an autocrine/paracrine signal that regulates mammary gland biogenesis, liver regeneration, and gastrointestinal tract motility [86]. There are two Tph genes that catalyze the rate-limiting step in serotonin biosynthesis: Tph1 expressed mostly, but not only, in entero‐ chromaffin cells of the gut and is responsable for the production of peripheral serotonin [86]. Tph2 is expressed exclusively in raphe neurons of the brainstem and is responsible for the production of serotonin in the brain [87]. Leptin inhibits BDS synthesis by decreasing the expression of Tph2, a major enzyme involved in serotonin synthesis in brain [85]. In addition, other data indicate, the key role of GDS in regulating bone formation as well as the relationship between GDS, Lrp5, and bone remodeling. Lrp5 controls bone formation by inhibiting GDS synthesis in the duodenum, and GDS directly acts on the osteoblast cells to inhibit osteoblast proliferation and suppress bone formation (Table 2) [88]. However, recent data to argue that Lrp5 affect bone mass mainly through local Wnt signaling pathway, and that the experiments did not support the Lrp5-GDS-osteoblast model because they found that there was no relevance between GDS and bone mass in their mouse model system [89].

### **4.11. New signals in bone remodeling**

**4.9. Insulin-like growth factor-I**

14 Topics in Osteoporosis

**serotonin**

stimulate osteoblast function and bone formation locally.

The Insulin-like growth factor-I (IGF-I) signaling through its type 1 receptor generates a complex signaling pathway that stimulates cell proliferation, function, and survival in osteoblasts (Table 2) [81]. Accordingly, mice lacking functional IGF-I exhibit severe deficiency in bone formation and a 60% deficit in peak bone mineral density (BMD) [82]. IGF-I can act in an endocrine, paracrine or autocrine manner and is regulated by a family of six IGF binding proteins (IGFBPs). The IGFBPs, have received considerable attention as regulators of IGF actions. The IGFBPs have been reported to have stimulatory or inhibitory actions on the IGFs in bone, and recent experiments have provided evidence that some of IGFBPs function independently of IGF to increase parameters of bone formation. The IGFBPs are often found bound to IGF-I in the circulation or complexed with IGF-I in osteoblasts. IGFBP-3 and -5 are known stimulators of IGF-I actions, whereas IGFBP-1, -2, -4 and -6 are known inhibitors of IGF-I action in bone. Once IGF-I binds to its receptor (type 1 IGF receptor) it initiates a complex signaling pathway including the phosphoinositol 3-kinase (PI3-K)/3-PI-dependent kinase (PDK)-1/Akt pathway and the Ras/Raf/mitogen-activated protein (MAP) kinase pathway which stimulate cell function and/or survival (Figure 3) [83]. Recent findings indicate that many of the IGFBPs and specific proteins in the IGF-I signaling pathways are also potent anabolic factors in regulating osteoblast function and may serve as potential targets to

**4.10. Leptin–serotonin system pathway regulation of bone formation through gut-derived**

A new regulation mode of osteoblastic bone formation controlled by leptin-serotonin (BDS) sympathetic nervous system pathway has emerged in recent years. Leptin is a hormone produced by adipocytes that, besides its function in regulating body weight and gonadal function, can also act as an inhibitor of bone formation (Figure 3) [84]. Latest data indicates that these leptin functions require brainstem-derived serotonin [85]. Serotonin is a bioamine produced by neurons of the brainstem (brainstem-derived serotonin, BDS) and enterochro‐ maffin cells of the duodenum (gut-derived serotonin, GDS). BDS acts as a neurotransmitter, while GDS as an autocrine/paracrine signal that regulates mammary gland biogenesis, liver regeneration, and gastrointestinal tract motility [86]. There are two Tph genes that catalyze the rate-limiting step in serotonin biosynthesis: Tph1 expressed mostly, but not only, in entero‐ chromaffin cells of the gut and is responsable for the production of peripheral serotonin [86]. Tph2 is expressed exclusively in raphe neurons of the brainstem and is responsible for the production of serotonin in the brain [87]. Leptin inhibits BDS synthesis by decreasing the expression of Tph2, a major enzyme involved in serotonin synthesis in brain [85]. In addition, other data indicate, the key role of GDS in regulating bone formation as well as the relationship between GDS, Lrp5, and bone remodeling. Lrp5 controls bone formation by inhibiting GDS synthesis in the duodenum, and GDS directly acts on the osteoblast cells to inhibit osteoblast proliferation and suppress bone formation (Table 2) [88]. However, recent data to argue that Lrp5 affect bone mass mainly through local Wnt signaling pathway, and that the experiments More recently, other signaling pathways that link regulation of the osteoclasts and osteoblasts have been identified. Osteoblast-lineage cells expressed Wnt5a, whereas osteoclast precursors expressed Ror2. Connection between these two cells leads to Wnt5a-Ror2 signaling between osteoblast-lineage cells and osteoclast precursors enhanced osteoclastogenesis, through increased RANK expression mediated by JNK signaling. A soluble form of Ror2 acted as a decoy receptor of Wnt5a and abrogated bone destruction in the mouse model, suggesting that the Wnt5a-Ror2 pathway is crucial for osteoclastogenesis in physiological and pathological environments and may represent a therapeutic target for bone diseases (Figure 3) [90]. Finally, a recent study reported that semaphorin 4D (Sema4D), previously shown to be an axon guidance molecule, expressed by osteoclasts and which potently inhibits bone formation [91]. Several studies have suggested that axon-guidance molecules, such as the semaphorins and ephrins, are involved in the cell-cell communication that occurs between osteoclasts and osteoblasts. The Binding of Sema4D to its receptor Plexin-B1 in osteoblasts resulted in the activation of the small GTPase RhoA, which inhibits bone formation by suppressing insulinlike growth factor-1 IGF-1 signaling and by modulating osteoblast motility. Notably, the suppression of Sema4D using a specific antibody was found to markedly prevent bone loss in a model of postmenopausal osteoporosis (Table 2) [91]. This finding identifies a new link between osteoclasts and osteoblast signaling, and suggests that suppression of the Sema4D-Plexin-B1-RhoA signaling axis may provide a new therapeutic target for reducing bone loss and development of bone-increasing drugs.

### **5. Pathophysiology of bone remodeling (diseases)**

Several lines of evidence have established that the cells that remodel the skeleton under physiological conditions are the same cells that mediate these processes in pathologic states. Mature bone consists of: an organic matrix (osteoid) composed mainly of type 1 collagen formed by osteoblasts; a mineral phase which contains the bulk of the body's reserve of calcium and phosphorus in crystalline form (hydroxyapatite) and deposited in close relation to the collagen fibers; bone cells; and a blood supply with sufficient levels of calcium and phosphate to mineralize the osteoid matrix. Bone turnover and remodeling occurs throughout life and involves the two-coupled processes of bone formation by osteoblasts and bone resorption by osteoclasts and perhaps osteolytic osteocytes. Abnormalities of bone remodeling can produce a variety of skeletal disorders (Table 3). The metabolic bone diseases may reflect disturbances in the organic matrix, the mineral phase, the cellular processes of remodeling, and the endocrine, nutritional, and other factors that regulate skeletal and mineral homeostasis. These disorders may be hereditary or acquired and usually affect the entire bony skeleton. The acquired metabolic bone diseases are the more common and include: osteoporosis, osteoma‐ lacia, the skeletal changes of hyperparathyroidism and chronic renal failure (renal osteodys‐ trophy), and Paget's disease [48,50].

nisms by which estrogens exert their bone-sparing effects. Since the discovery of the RANKL/ RANK/OPG axis, it has become clear that estrogen also exerts bone-sparing effects by targeting this regulatory axis. Specifically, estrogen stimulates the expression of OPG in mouse osteo‐ blasts and stromal cells [94]. Moreover, the expression of RANKL was elevated on the surface of bone marrow cells, such as osteoblasts and lymphocytes, from postmenopausal women with osteoporosis compared with cells from premenopausal controls [94]; this finding indicates that RANKL plays an important role in the pathogenesis of postmenopausal osteoporosis.

Molecular Aspects of Bone Remodeling http://dx.doi.org/10.5772/54905 17

As the global population ages, the prevalence of age-related osteoporosis (e.g., postmeno‐ pausal osteoporosis, male osteoporosis) and related fractures is likely to increase consider‐ ably (Table 3). Recent studies indicate that significant trabecular bone loss begins as early as the twenties in men and women long before any major hormonal changes [95]. In women, however, bone loss accelerates for 5 to 10 years after menopause due to the rapid decline in estrogen levels; after this phase, bone loss continues at approximately the same rate as in elderly males. Thus, the pathogenesis of osteoporosis in women involves primarily osteoclasts (bone resorption) and results from changes in estrogen and FSH levels at menopause and age related, is centered on osteoblasts (bone formation), and engages a number of distinct factors associated with the aging process in both men and women. Thus, age-related changes in the activity of either cell type may lead to bone loss [96]. Agerelated osteoporosis in men also has a multifactorial etiology. The decreased bone forma‐ tion caused by changes in ROS, IGF-1, and PTH levels associated with aging plays a predominant role in the pathogenesis of age-related osteoporosis in men. However, agerelated changes in the levels of sex steroids, including both estrogen and androgen, also

Glucocorticoids (GCs) are potent immunomodulatory drugs that are commonly used to treat a variety of inflammatory conditions and autoimmune disorders. GCs increase bone resorption and reduce bone formation (Table 3) [98]. Pharmacological doses of GCs induce osteoporosis primarily by altering normal bone remodeling. GCs exert deleterious effects on the differen‐ tiation, function, and survival of multiple cell types involved in the remodeling process. GCs have profound effects on osteoblast differentiation and function. As in other target tissues, glucocorticoids exert their effects on gene expression via cytoplasmic glucocorticoid type 2 receptors. In adult bone, functional glucocorticoid receptors are found in pre-osteoblast/ stromal cells, osteoblasts (the cells that produce bone matrix), but not in osteoclasts [99]. Instead, glucocorticoids stimulate osteoclast proliferation by suppressing synthesis of osteo‐ protegerin, an inhibitor of osteoclast differentiation from hematopoietic cells of the macro‐ phage lineage, and by stimulating production of the receptor activator of nuclear factor kappa-B (RANK), which is required for osteoclastogenesis. High glucocorticoid levels also stimulate RANKL synthesis by pre-osteoblast/stromal cells, supporting osteoclast differentiation and

contribute to the pathogenesis of age-related osteoporosis in men [97].

**5.4. Glucocorticoid-induced osteoporosis**

net bone resorption [100].

**5.3. Age-related osteoporosis**

### **5.1. Osteoporosis**

Osteoporosis is a common disease of bone remodeling characterized by low bone mass and defects in the microarchitecture of bone tissue; it causes bone fragility and an increased vulnerability to fractures. The loss of bone mass and strength can be contributed to by (a) failure to reach an optimal peak bone mass as a young adult, (b) excessive resorption of bone after peak mass has been achieved, or (c) an impaired bone formation response during remodeling. Osteoporosis, is traditionally classified into primary and secondary types. Primary osteopo‐ rosis is the most common metabolic disorder of the skeleton and has been divided into two subtypes: type I osteoporosis and type II osteoporosis, on the basis of possible differences in etiology. Type I osteoporosis or postmenopausal osteoporosis is a common bone disorder in postmenopausal women and is caused primarily by estrogen deficiency resulting from menopause, whereas type II osteoporosis or age-related osteoporosis is associated primarily with aging in both women and men (Table 3). In contrast, secondary osteoporosis refers to bone disorders that are secondary complications of various other medical conditions, conse‐ quences of changes in physical activity, or adverse results of therapeutic interventions for certain disorders [92].


**Table 3.** Diseases of bone remodeling.

### **5.2. Postmenopausal osteoporosis**

Postmenopausal osteoporosis is a common disease with a spectrum ranging from asympto‐ matic bone loss to disabling hip fracture (Table 3). The pathogenesis of postmenopausal osteoporosis is caused primarily by the decline in estrogen levels associated with menopause [93]. Since the establishment of a central role for estrogen deficiency in the pathogenesis of postmenopausal osteoporosis, enormous effort has been focused on elucidating the mecha‐ nisms by which estrogens exert their bone-sparing effects. Since the discovery of the RANKL/ RANK/OPG axis, it has become clear that estrogen also exerts bone-sparing effects by targeting this regulatory axis. Specifically, estrogen stimulates the expression of OPG in mouse osteo‐ blasts and stromal cells [94]. Moreover, the expression of RANKL was elevated on the surface of bone marrow cells, such as osteoblasts and lymphocytes, from postmenopausal women with osteoporosis compared with cells from premenopausal controls [94]; this finding indicates that RANKL plays an important role in the pathogenesis of postmenopausal osteoporosis.

### **5.3. Age-related osteoporosis**

lacia, the skeletal changes of hyperparathyroidism and chronic renal failure (renal osteodys‐

Osteoporosis is a common disease of bone remodeling characterized by low bone mass and defects in the microarchitecture of bone tissue; it causes bone fragility and an increased vulnerability to fractures. The loss of bone mass and strength can be contributed to by (a) failure to reach an optimal peak bone mass as a young adult, (b) excessive resorption of bone after peak mass has been achieved, or (c) an impaired bone formation response during remodeling. Osteoporosis, is traditionally classified into primary and secondary types. Primary osteopo‐ rosis is the most common metabolic disorder of the skeleton and has been divided into two subtypes: type I osteoporosis and type II osteoporosis, on the basis of possible differences in etiology. Type I osteoporosis or postmenopausal osteoporosis is a common bone disorder in postmenopausal women and is caused primarily by estrogen deficiency resulting from menopause, whereas type II osteoporosis or age-related osteoporosis is associated primarily with aging in both women and men (Table 3). In contrast, secondary osteoporosis refers to bone disorders that are secondary complications of various other medical conditions, conse‐ quences of changes in physical activity, or adverse results of therapeutic interventions for

> **Osteoporosis** Primary Menopause Associated **Age related** Secondary Glucocorticoid induced Immobilization induced Renal osteodystrophy Paget's disease Osteopetrosis

Postmenopausal osteoporosis is a common disease with a spectrum ranging from asympto‐ matic bone loss to disabling hip fracture (Table 3). The pathogenesis of postmenopausal osteoporosis is caused primarily by the decline in estrogen levels associated with menopause [93]. Since the establishment of a central role for estrogen deficiency in the pathogenesis of postmenopausal osteoporosis, enormous effort has been focused on elucidating the mecha‐

trophy), and Paget's disease [48,50].

**5.1. Osteoporosis**

16 Topics in Osteoporosis

certain disorders [92].

**Table 3.** Diseases of bone remodeling.

**5.2. Postmenopausal osteoporosis**

As the global population ages, the prevalence of age-related osteoporosis (e.g., postmeno‐ pausal osteoporosis, male osteoporosis) and related fractures is likely to increase consider‐ ably (Table 3). Recent studies indicate that significant trabecular bone loss begins as early as the twenties in men and women long before any major hormonal changes [95]. In women, however, bone loss accelerates for 5 to 10 years after menopause due to the rapid decline in estrogen levels; after this phase, bone loss continues at approximately the same rate as in elderly males. Thus, the pathogenesis of osteoporosis in women involves primarily osteoclasts (bone resorption) and results from changes in estrogen and FSH levels at menopause and age related, is centered on osteoblasts (bone formation), and engages a number of distinct factors associated with the aging process in both men and women. Thus, age-related changes in the activity of either cell type may lead to bone loss [96]. Agerelated osteoporosis in men also has a multifactorial etiology. The decreased bone forma‐ tion caused by changes in ROS, IGF-1, and PTH levels associated with aging plays a predominant role in the pathogenesis of age-related osteoporosis in men. However, agerelated changes in the levels of sex steroids, including both estrogen and androgen, also contribute to the pathogenesis of age-related osteoporosis in men [97].

### **5.4. Glucocorticoid-induced osteoporosis**

Glucocorticoids (GCs) are potent immunomodulatory drugs that are commonly used to treat a variety of inflammatory conditions and autoimmune disorders. GCs increase bone resorption and reduce bone formation (Table 3) [98]. Pharmacological doses of GCs induce osteoporosis primarily by altering normal bone remodeling. GCs exert deleterious effects on the differen‐ tiation, function, and survival of multiple cell types involved in the remodeling process. GCs have profound effects on osteoblast differentiation and function. As in other target tissues, glucocorticoids exert their effects on gene expression via cytoplasmic glucocorticoid type 2 receptors. In adult bone, functional glucocorticoid receptors are found in pre-osteoblast/ stromal cells, osteoblasts (the cells that produce bone matrix), but not in osteoclasts [99]. Instead, glucocorticoids stimulate osteoclast proliferation by suppressing synthesis of osteo‐ protegerin, an inhibitor of osteoclast differentiation from hematopoietic cells of the macro‐ phage lineage, and by stimulating production of the receptor activator of nuclear factor kappa-B (RANK), which is required for osteoclastogenesis. High glucocorticoid levels also stimulate RANKL synthesis by pre-osteoblast/stromal cells, supporting osteoclast differentiation and net bone resorption [100].

### **5.5. Immobilization-induced osteoporosis**

One of the major functions of bone remodeling is to adapt bone material and structural properties to the mechanical demands that are placed on the skeleton, including mechanical loading and weight bearing (Table 3). The importance of the mechanical balance of bone has been more recently stressed by the research on the effect of weightlessness on bone, and by the introduction of the concept of "mechanostat" in the pathogenesis of osteoporotic conditions. Immobilization osteoporosis has clinical (fractures, sometimes hypercalcemia, urinary lithiasis) and radiological features. Immobilization has an effect on bone modeling and remodeling, through an increased activation of remodeling loci, and a decrease of the osteo‐ blastic stimulus. For ordinary individuals, the skeleton is developed in childhood and then constantly remodeled throughout adulthood to maintain mechanical strength that can sufficiently support normal weight bearing and routine physical activities. However, for individuals such as athletes, the mechanical needs increase for certain regions of the skeleton; consequently, bone modeling results in the formation of stronger bone to replace old bone that could not adequately meet the increased mechanical demands [101].

The cause of Paget's disease is not entirely known, but it is thought to be caused in part from a childhood virus. A virus particle, known as a paramyxovirus nucleocapsid, has been identified within the bone cells of individuals with Paget's disease. This virus particle is not found in normal bone. Genetics plays a role, several genes have been implicated; however, the most commonly described mutation is a gene that encodes an ubiquitin-binding protein that

Molecular Aspects of Bone Remodeling http://dx.doi.org/10.5772/54905 19

There are several syndromes of osteopetrosis or osteosclerosis in which bone resorption is defective because of impaired formation of osteoclasts or loss of osteoclast function (Table 3). In these disorders, bone modeling as well as remodeling are impaired, and the architecture of the skeleton can be quite abnormal [105]. Osteopetrosis is a congenital disease that interferes with the formation of the bone marrow, and causes abnormal bone development, blindness, rickets, abnormal tooth development and fragile bones. It results from a defect in cells called osteoclasts, which are necessary for the formation of the bone marrow. In patients with osteopetrosis, osteoclasts not function properly, and no cavity is formed to the bone marrow [106]. The subclassification of these disorders is based upon the mode of inheritance, age of onset, severity, and clinical symptoms [107]. The pathophysiology of osteopetrosis involves mutations that affect osteoclast function. The three most important mutations are: carbonic

Bone is a specialized and dynamic tissue, in constant change. It has a complex structure and undergoes constant remodeling. The basic multicellular unit of bone, which comprises osteocytes, osteoclasts and osteoblasts, conducts the remodeling process. In the last years, more knowledge in bone cell biology and genetic studies, have been helped in our understanding of the essential signaling pathways that control bone remodeling and bone mass. They act in a coordinated manner to form or resorb bone. Recent advances in molecular biology and a thorough understanding of the remodeling process bone, many molecules have been discov‐ ered that have important roles in bone biology and novel signaling pathways regulating bone remodeling have also been identified. Now understand how PTH, Wnt signaling, and growth factors may trigger anabolic effects in bone. The explosion of this knowledge may serve as a basis for the development of novel therapeutic approaches targeted on the identified signaling molecules enable us to define the abnormalities in cells of the osteoblastic and osteoclastic lineages that lead to bone disease with the hope to the diagnosis and treatment of bone remodeling disorders. With this knowledge, can expect the development of even more therapies to evolve from a better understanding of the complex molecular aspects of bone

plays a role in NF-κB signaling [104].

anhydrase II, proton pump, and chloride channel [48].

**5.8. Osteopetrosis**

**6. Conclusions**

remodeling.

### **5.6. Renal osteodystrophy**

Renal osteodystrophy the term used to describe a heterogeneous group of metabolic bone diseases that accompany chronic kidney disease, is a multifactorial disorder of bone remod‐ eling (Table 3). The bone disorders in renal osteodystrophy include: osteomalacia of adults and rickets of children (so-called "renal rickets"); osteitis fibrosa and other bone changes of secondary hyperparathyroidism; osteopenia; and osteosclerosis. Renal osteodystrophy is an alteration of bone morphology in patients with CKD (Chronic Kidney Disease). The patho‐ physiology of renal osteodystrophy is complex and clearly reflects the importance of PTH and vitamin D on bone turnover and related pathological abnormalities. The bone changes are brought about by the abnormal metabolism of vitamin D, the overproduction of parathyroid hormone (PTH), and chronic metabolic acidosis. The diminished renal mass leads to a decreased renal conversion of 25-hydroxyvitamin D into 1,25-dihydroxyvitamin D, the active metabolite of vitamin D, resulting in diminished intestinal absorption of calcium, hypocalce‐ mia, and defective bone mineralization characterized by the presence of wide osteoid seams, osteomalacia in adults, and rickets in children [102].

### **5.7. Paget's disease**

Paget's disease is known as a bone remodeling disorder and that involves abnormal bone destruction and regrowth, which results in deformity. In Paget's disease, the bone remodeling process is disregulated (Table 3). New bone is placed where it is not needed, and old bone is removed where it is needed. This disregulation can distort the normal skeletal architecture [103]. Paget's disease is most commonly diagnosed in the sixth decade, and increases in prevalence as age increases. Paget's disease is very uncommon in individuals under 40 years of age. The most common bones affected by Paget's disease are the pelvis, femur, spine, skull, and tibia. Paget's disease is believed to be a primary disorder of increased osteoclast bone resorption with a secondary marked increase in osteoblast activity and new bone formation. The cause of Paget's disease is not entirely known, but it is thought to be caused in part from a childhood virus. A virus particle, known as a paramyxovirus nucleocapsid, has been identified within the bone cells of individuals with Paget's disease. This virus particle is not found in normal bone. Genetics plays a role, several genes have been implicated; however, the most commonly described mutation is a gene that encodes an ubiquitin-binding protein that plays a role in NF-κB signaling [104].

### **5.8. Osteopetrosis**

**5.5. Immobilization-induced osteoporosis**

18 Topics in Osteoporosis

**5.6. Renal osteodystrophy**

**5.7. Paget's disease**

One of the major functions of bone remodeling is to adapt bone material and structural properties to the mechanical demands that are placed on the skeleton, including mechanical loading and weight bearing (Table 3). The importance of the mechanical balance of bone has been more recently stressed by the research on the effect of weightlessness on bone, and by the introduction of the concept of "mechanostat" in the pathogenesis of osteoporotic conditions. Immobilization osteoporosis has clinical (fractures, sometimes hypercalcemia, urinary lithiasis) and radiological features. Immobilization has an effect on bone modeling and remodeling, through an increased activation of remodeling loci, and a decrease of the osteo‐ blastic stimulus. For ordinary individuals, the skeleton is developed in childhood and then constantly remodeled throughout adulthood to maintain mechanical strength that can sufficiently support normal weight bearing and routine physical activities. However, for individuals such as athletes, the mechanical needs increase for certain regions of the skeleton; consequently, bone modeling results in the formation of stronger bone to replace old bone that

Renal osteodystrophy the term used to describe a heterogeneous group of metabolic bone diseases that accompany chronic kidney disease, is a multifactorial disorder of bone remod‐ eling (Table 3). The bone disorders in renal osteodystrophy include: osteomalacia of adults and rickets of children (so-called "renal rickets"); osteitis fibrosa and other bone changes of secondary hyperparathyroidism; osteopenia; and osteosclerosis. Renal osteodystrophy is an alteration of bone morphology in patients with CKD (Chronic Kidney Disease). The patho‐ physiology of renal osteodystrophy is complex and clearly reflects the importance of PTH and vitamin D on bone turnover and related pathological abnormalities. The bone changes are brought about by the abnormal metabolism of vitamin D, the overproduction of parathyroid hormone (PTH), and chronic metabolic acidosis. The diminished renal mass leads to a decreased renal conversion of 25-hydroxyvitamin D into 1,25-dihydroxyvitamin D, the active metabolite of vitamin D, resulting in diminished intestinal absorption of calcium, hypocalce‐ mia, and defective bone mineralization characterized by the presence of wide osteoid seams,

Paget's disease is known as a bone remodeling disorder and that involves abnormal bone destruction and regrowth, which results in deformity. In Paget's disease, the bone remodeling process is disregulated (Table 3). New bone is placed where it is not needed, and old bone is removed where it is needed. This disregulation can distort the normal skeletal architecture [103]. Paget's disease is most commonly diagnosed in the sixth decade, and increases in prevalence as age increases. Paget's disease is very uncommon in individuals under 40 years of age. The most common bones affected by Paget's disease are the pelvis, femur, spine, skull, and tibia. Paget's disease is believed to be a primary disorder of increased osteoclast bone resorption with a secondary marked increase in osteoblast activity and new bone formation.

could not adequately meet the increased mechanical demands [101].

osteomalacia in adults, and rickets in children [102].

There are several syndromes of osteopetrosis or osteosclerosis in which bone resorption is defective because of impaired formation of osteoclasts or loss of osteoclast function (Table 3). In these disorders, bone modeling as well as remodeling are impaired, and the architecture of the skeleton can be quite abnormal [105]. Osteopetrosis is a congenital disease that interferes with the formation of the bone marrow, and causes abnormal bone development, blindness, rickets, abnormal tooth development and fragile bones. It results from a defect in cells called osteoclasts, which are necessary for the formation of the bone marrow. In patients with osteopetrosis, osteoclasts not function properly, and no cavity is formed to the bone marrow [106]. The subclassification of these disorders is based upon the mode of inheritance, age of onset, severity, and clinical symptoms [107]. The pathophysiology of osteopetrosis involves mutations that affect osteoclast function. The three most important mutations are: carbonic anhydrase II, proton pump, and chloride channel [48].

### **6. Conclusions**

Bone is a specialized and dynamic tissue, in constant change. It has a complex structure and undergoes constant remodeling. The basic multicellular unit of bone, which comprises osteocytes, osteoclasts and osteoblasts, conducts the remodeling process. In the last years, more knowledge in bone cell biology and genetic studies, have been helped in our understanding of the essential signaling pathways that control bone remodeling and bone mass. They act in a coordinated manner to form or resorb bone. Recent advances in molecular biology and a thorough understanding of the remodeling process bone, many molecules have been discov‐ ered that have important roles in bone biology and novel signaling pathways regulating bone remodeling have also been identified. Now understand how PTH, Wnt signaling, and growth factors may trigger anabolic effects in bone. The explosion of this knowledge may serve as a basis for the development of novel therapeutic approaches targeted on the identified signaling molecules enable us to define the abnormalities in cells of the osteoblastic and osteoclastic lineages that lead to bone disease with the hope to the diagnosis and treatment of bone remodeling disorders. With this knowledge, can expect the development of even more therapies to evolve from a better understanding of the complex molecular aspects of bone remodeling.

### **Author details**

Alma Y. Parra-Torres1,2, Margarita Valdés-Flores3 , Lorena Orozco4 and Rafael Velázquez-Cruz2\*

\*Address all correspondence to: rvelazquez@inmegen.gob.mx

1 Program in Biomedical Sciences-UNAM, Mexico

2 Genomics of Bone Metabolism Laboratory, National Institute of Genomic Medicine, Mexi‐ co City, Mexico

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4 Immunogenomics and Metabolic Diseases Laboratory, National Institute of Genomic Med‐ icine, Mexico City, Mexico


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, Lorena Orozco4

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**Chapter 2**

**Genetic Diseases Related with Osteoporosis**

Osteoporosis is a disease entity characterized by the progressive loss of bone mineral density (BMD) and the deterioration of bone microarchitecture, leading to the development of frac‐ tures. Its classification encompasses two large groups, primary and secondary osteoporosis [1]. Primary osteoporosis is the disease's most common form and results from the progressive loss of bone mass related to aging and unassociated with other illness, a natural process in adult life; its etiology is considered multifactorial and polygenic. This form currently represents a growing worldwide health problem due in part, to the contemporary environmental condi‐ tions of modern civilization. Risk factors that are considered as "modifiable" also play an important role and include physical activity, dietary habits and eating disorders. Furthermore, there is another group of associated risk factors that are considered "non-modifiable", including gender, age, race, a personal and/or family history of fractures that in turn, indirectly reflect the degree of genetic susceptibility to this disease [2-4]. Secondary osteoporosis encompasses a large heterogeneous group of primary conditions favoring osteoporosis development. Table 1 summarizes some of the disease entities associated to primary and

This form of osteoporosis results from the interaction of several environmental and genetic factors, leading to difficulties in its study. It is not easy to define the magnitude of the effect of genetic susceptibility since it is a trait determined by multiple genes whose products affect the bone phenotype; moreover, the environmental factors compromising bone mineral density are also difficult to analyze. However, in spite of these barriers, research suggests that inherited factors affect BMD in ranges between 40 – 70% in the spine, 70 – 85% in the hip and 50 – 60%

and reproduction in any medium, provided the original work is properly cited.

© 2013 Valdés-Flores et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

Margarita Valdés-Flores, Leonora Casas-Avila and

Additional information is available at the end of the chapter

Valeria Ponce de León-Suárez

http://dx.doi.org/10.5772/55546

**1. Introduction**

secondary osteoporosis.

**1.1. Genetic aspects of primary osteoporosis**

### **Genetic Diseases Related with Osteoporosis**

Margarita Valdés-Flores, Leonora Casas-Avila and Valeria Ponce de León-Suárez

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55546

**1. Introduction**

Osteoporosis is a disease entity characterized by the progressive loss of bone mineral density (BMD) and the deterioration of bone microarchitecture, leading to the development of frac‐ tures. Its classification encompasses two large groups, primary and secondary osteoporosis [1].

Primary osteoporosis is the disease's most common form and results from the progressive loss of bone mass related to aging and unassociated with other illness, a natural process in adult life; its etiology is considered multifactorial and polygenic. This form currently represents a growing worldwide health problem due in part, to the contemporary environmental condi‐ tions of modern civilization. Risk factors that are considered as "modifiable" also play an important role and include physical activity, dietary habits and eating disorders. Furthermore, there is another group of associated risk factors that are considered "non-modifiable", including gender, age, race, a personal and/or family history of fractures that in turn, indirectly reflect the degree of genetic susceptibility to this disease [2-4]. Secondary osteoporosis encompasses a large heterogeneous group of primary conditions favoring osteoporosis development. Table 1 summarizes some of the disease entities associated to primary and secondary osteoporosis.

### **1.1. Genetic aspects of primary osteoporosis**

This form of osteoporosis results from the interaction of several environmental and genetic factors, leading to difficulties in its study. It is not easy to define the magnitude of the effect of genetic susceptibility since it is a trait determined by multiple genes whose products affect the bone phenotype; moreover, the environmental factors compromising bone mineral density are also difficult to analyze. However, in spite of these barriers, research suggests that inherited factors affect BMD in ranges between 40 – 70% in the spine, 70 – 85% in the hip and 50 – 60%

© 2013 Valdés-Flores et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


osteoporosis development or the risk of fractures; these risks may vary according to the

Genetic Diseases Related with Osteoporosis http://dx.doi.org/10.5772/55546 31

Hormones and their receptors ESR1, ESR2, AR, VDR, PTHR1, CASR, PTH, CYP1A1, PRL, LEP,

Participants in osteoblastogenic proccesses ALOX12, ALOX15, BMP4, BMP7, IGF-1 LRP5, LRP6, SOST

The description in the literature of some genetic diseases of monogenic inheritance and whose phenotype includes the loss or increase in bone mineral density and even fractures, has suggested and even proved that bone phenotype has an important genetic component. These diseases include idiopathic osteoporosis, osteogenesis imperfecta in all its variants, osteopet‐ rosis, pycnodysostosis and the osteoporosis syndrome associated to pseudoglioma, among others. In some cases of severe osteoporosis, mutations in the estrogen and even the androgen

This is an unusual variety of osteoporosis whose frequency has not been precisely determined. This disease may develop in females and males, usually around 7 – 10 years of age; children present difficulty in gait, pain in the lower extremities, ankles, knees, occasionally in the hip and fractures tend to develop particularly in long bones. Radiologically, it is characterized by diffuse osteopenia, metaphyseal fractures – especially of the femur -, and vertebral collapse that may lead to severe kyphoscoliosis or collapse of the thoracic cage. This disease is consid‐ ered potentially reversible whereby in most cases, there is almost complete recovery of the

In these patients, it is important to exclude other disease entities or conditions manifest‐ ing secondarily as osteoporosis. A differential diagnosis must be made with other genetic diseases, particularly the different variants of osteogenesis imperfecta; this is relatively easy

LEPR, INS, INSR

fractures' anatomic location [3, 4, 24-30]

**Product Function Genes**

Matrix components COL1A1, COL1A2, OPN

Participants in osteoclastogenic proccesses P53, RANK, RANK-L

Other MTHFR, APOE

**2. Mendelian diseases and osteoporosis**

**Table 2.** Genes involved in bone metabolism.

receptor genes have been detected.

**2.1. Idiopathic juvenile osteoporosis**

bone tissue; growth, however, may be compromised.

Citokines and their receptors IL1α, IL1β, IL6, TNF, TNFR2

**Table 1.** Osteoporosis classification.

in the wrist. Bone density studies in monozygotic (MZ) and dizygotic (DZ) twins suggest that spinal and femoral neck BMD concordance is higher (6-8:1) in MZ versus DZ twins. Other studies have estimated that fracture predisposition heritability per se ranges between 25 – 35% and up to 40% of patients with osteoporotic fractures have a positive family history of fractures, thus reflecting the great influence of genetic factors in this disease. On the other hand, the geometry and length of the femoral neck, the bone's properties on ultrasound, growth speed and bone remodeling variations are also dependent on genetic factors. The genes associated with the bone phenotype are distributed throughout the human genome and located in practically all chromosomes; their products fulfill specific functions and contribute in different manners to the genetic control of the bone tissue phenotype [5-12]. Some of these genes and their products are presented in Table 2 [13-23].

It is important to mention that the mechanisms conditioning the hereditary susceptibility to osteoporosis are determined, among other factors, by the presence of mutations or genetic polymorphisms (natural genomic variations) in one or several genes involved in bone phenotype genetic control. These polymorphisms follow a well-defined inheritance pattern and their distribution is different among racial groups and populations. There are several reports in the world literature, of associations between specific genetic variants and osteoporosis development or the risk of fractures; these risks may vary according to the fractures' anatomic location [3, 4, 24-30]


**Table 2.** Genes involved in bone metabolism.

### **2. Mendelian diseases and osteoporosis**

The description in the literature of some genetic diseases of monogenic inheritance and whose phenotype includes the loss or increase in bone mineral density and even fractures, has suggested and even proved that bone phenotype has an important genetic component. These diseases include idiopathic osteoporosis, osteogenesis imperfecta in all its variants, osteopet‐ rosis, pycnodysostosis and the osteoporosis syndrome associated to pseudoglioma, among others. In some cases of severe osteoporosis, mutations in the estrogen and even the androgen receptor genes have been detected.

### **2.1. Idiopathic juvenile osteoporosis**

in the wrist. Bone density studies in monozygotic (MZ) and dizygotic (DZ) twins suggest that spinal and femoral neck BMD concordance is higher (6-8:1) in MZ versus DZ twins. Other studies have estimated that fracture predisposition heritability per se ranges between 25 – 35% and up to 40% of patients with osteoporotic fractures have a positive family history of fractures, thus reflecting the great influence of genetic factors in this disease. On the other hand, the geometry and length of the femoral neck, the bone's properties on ultrasound, growth speed and bone remodeling variations are also dependent on genetic factors. The genes associated with the bone phenotype are distributed throughout the human genome and located in practically all chromosomes; their products fulfill specific functions and contribute in different manners to the genetic control of the bone tissue phenotype [5-12]. Some of these genes and

It is important to mention that the mechanisms conditioning the hereditary susceptibility to osteoporosis are determined, among other factors, by the presence of mutations or genetic polymorphisms (natural genomic variations) in one or several genes involved in bone phenotype genetic control. These polymorphisms follow a well-defined inheritance pattern and their distribution is different among racial groups and populations. There are several reports in the world literature, of associations between specific genetic variants and

their products are presented in Table 2 [13-23].

**Table 1.** Osteoporosis classification.

**Type of osteoporosis Causes**

30 Topics in Osteoporosis

Primary Multifactorial, polygenic. Senile/Involutional

alcoholism.

Secondary Drugs compromising bone quality: anticonvulsants, antidepressants,

phenobarbital, phenothiazines, among others.

leading to decreased mobility or prolonged immobility.

Hypogonadism: Turner and Klinefelter syndromes.

anticoagulants, antacids with aluminum, aromatase inhibitors, barbiturates, cimetidine, corticosteroids, glucocorticoids, birth control pills, cancer drugs, gonadotropin releasing hormone (GnRH), loop diuretics, methotrexate,

Other entities: nephropathies, malabsorption syndromes, neoplasias, rheumatoid arthritis, ankylosing spondylitis, multiple sclerosis, any process

Metabolic diseases: diabetes, hyperthyroidism, hyperparathyroidism.

Behavioral disorders: anorexia nervosa, depression, prolonged physical inactivity, malnutrition, high caffeine intake, smoking and/or chronic

Monogenic diseases: osteogenesis imperfecta, glioma syndrome, osteoporosis.

This is an unusual variety of osteoporosis whose frequency has not been precisely determined. This disease may develop in females and males, usually around 7 – 10 years of age; children present difficulty in gait, pain in the lower extremities, ankles, knees, occasionally in the hip and fractures tend to develop particularly in long bones. Radiologically, it is characterized by diffuse osteopenia, metaphyseal fractures – especially of the femur -, and vertebral collapse that may lead to severe kyphoscoliosis or collapse of the thoracic cage. This disease is consid‐ ered potentially reversible whereby in most cases, there is almost complete recovery of the bone tissue; growth, however, may be compromised.

In these patients, it is important to exclude other disease entities or conditions manifest‐ ing secondarily as osteoporosis. A differential diagnosis must be made with other genetic diseases, particularly the different variants of osteogenesis imperfecta; this is relatively easy due to its clinical characteristics, lacking in idiopathic osteoporosis. The genetic basis of this disease has of yet, not been established but it is possible that genetic mutations with preferential tissue expression in bone and with great impact on the tissue's phenotype, may explain some of these cases [31, 32].

patients, there is increased difficulty and limitation in walking and periods of immobility become progressively more prolonged leading to the gradual loss of the mechanical stimuli that bone needs to maintain its strength and hence, favoring the development of osteoporosis. As all Mendelian diseases, these neuromuscular abnormalities follow different inheritance

This group of genetic diseases encompasses a great number of inborn defects with repercus‐ sions in several aspects of carbohydrate, amino acid, protein, vitamin, mineral, complex molecule, neurotransmitter and energy metabolism. The genetic basis of most of these entities hinges on gene mutations encoding proteins, particularly enzymes, leading to partial or complete blockade of one or several metabolic processes. In these diseases, symptoms arise for different reasons, including: a deficit of the products generated by the compromised enzymatic reaction, accumulation of the precursor immediate to the defect, an increase in alternative products due to increased activation of alternate metabolic pathways or inhibition of these alternate pathways due to the accumulated substrate. In most cases, inheritance of

In cases of metabolic errors, osteoporosis tends to develop for different reasons: in some cases, it is secondary to nutritional deficiencies, progressive neurologic or muscular impairment or as a consequence of the therapeutic measures taken in the management of the primary disease: their secondary effects directly compromise bone quality (steroids, antiseizure drugs, etc.). The number of monogenic diseases whose phenotype may include osteoporosis is large and are

LMNA Prelamin-A/C

precursor (LMNA)

1 (COL1A1)

1 (COL1A1)

2 (COL1A2)

1 (COL1A1)

2 (COL1A2)

COL1A2 Collagen, type I, alpha

COL1A2 Collagen, type I, alpha

Marfan syndrome; MFS FBN1 Fibrillin 1 (FBN1) 15q21.1 61, 62

**Location**

7q21.3

7q21.3

1q22 57, 58

Genetic Diseases Related with Osteoporosis http://dx.doi.org/10.5772/55546 33

17q21.33 33, 34

17q21.33 33, 59

17q21.33 33, 60

**Reference**

these diseases is autosomal recessive and less frequently, X-linked recessive.

shown in Tables 3-5, according to their Mendelian inheritance pattern [45-56].

**Disease Gene Product Genomic**

Osteogenesis imperfecta, Type I; OI1 COL1A1 Collagen, type I, alpha

Osteogenesis imperfecta, Type II; OI2 COL1A1 Collagen, type I, alpha

Osteogenesis imperfecta, Type III; OI3 COL1A1 Collagen, type I, alpha

patterns and present phenotypic variability [42-44].

**2.5. Inborn errors of metabolism**

Hutchinson-Gilford progeria

syndrome; HGPS

### **2.2. Osteogenesis imperfecta**

Osteogenesis imperfecta, also known as "brittle bone disease", has an estimated incidence of approximately 1 in 20 000 births. It has great phenotypic variability, different patterns of inheritance and a wide clinical spectrum ranging from very mild forms of the disease to severe cases with an unfavorable prognosis. It is caused by the defective synthesis of one of the two alpha chains of type I collagen (COL1A1 and COL1A2), leading to anomalies in these protein's structure; it is normally constituted by 3 coiled sub-units, two α1 chains and one α2 chain. This type of collagen is considered the most abundant component of structural protein in bone as well as in ligaments, tendons, sclerae and skin. Quantitative or qualitative defects in this protein lead to bone fragility and hence, to an increased risk of fractures.

The genes encoding the α1 and α2 chains are located in the 17q21.31-q22 and 7q22.1 chromo‐ somes, respectively. Aside from brittle bones, these patients may also present long bones with no curvatures, severe deformities preventing appropriate gait and even standing, conductive deafness due to malformations of the auditory canal, dentinogenesis imperfecta, joint hyper‐ laxity and intervertebral disc herniation. Patients with severe forms of the disease have a long history of fractures on mild impact and variable bone deformities. The most severe variants may even lead to fractures in utero and pre or perinatal death. Tables 3 and 4 shows different forms of the disease [33-35].

### **2.3. Osteoporosis – Pseudoglioma Syndrome (OPPG)**

This syndrome is an autosomal recessive disease characterized by bone and visual abnormalities including short stature, osteoporosis development during infancy, spontane‐ ous fractures, scoliosis, platyspondyly and long bone deformities. A crucial associated finding is the presence of pseudoglioma that may be associated to microcephaly, blind‐ ness during childhood, cataracts and iris atrophy. Occasionally, some patients present interventricular septal defects and mental retardation. This disease is conditioned by mutations of the LRP5 gene, located on chromosome 11q13.4 and that encodes the lowdensity lipoprotein receptor-related protein 5 (LRP5). It was initially believed that this entity was another variant of osteogenesis imperfecta (OI) but the study of collagen in patients with OPPG established that this protein was normal and the hypothesis was discarded; however, this is still the most relevant differential diagnosis [36-41].

### **2.4. Neuromuscular disorders**

Muscular dystrophies, peripheral neuropathies and muscle atrophies of hereditary origin, represent broad groups of diseases that aside from their characteristic clinical stigmata, can be associated with osteoporosis as one of their complications. As the disease progresses in these patients, there is increased difficulty and limitation in walking and periods of immobility become progressively more prolonged leading to the gradual loss of the mechanical stimuli that bone needs to maintain its strength and hence, favoring the development of osteoporosis. As all Mendelian diseases, these neuromuscular abnormalities follow different inheritance patterns and present phenotypic variability [42-44].

### **2.5. Inborn errors of metabolism**

due to its clinical characteristics, lacking in idiopathic osteoporosis. The genetic basis of this disease has of yet, not been established but it is possible that genetic mutations with preferential tissue expression in bone and with great impact on the tissue's phenotype,

Osteogenesis imperfecta, also known as "brittle bone disease", has an estimated incidence of approximately 1 in 20 000 births. It has great phenotypic variability, different patterns of inheritance and a wide clinical spectrum ranging from very mild forms of the disease to severe cases with an unfavorable prognosis. It is caused by the defective synthesis of one of the two alpha chains of type I collagen (COL1A1 and COL1A2), leading to anomalies in these protein's structure; it is normally constituted by 3 coiled sub-units, two α1 chains and one α2 chain. This type of collagen is considered the most abundant component of structural protein in bone as well as in ligaments, tendons, sclerae and skin. Quantitative or qualitative defects in this

The genes encoding the α1 and α2 chains are located in the 17q21.31-q22 and 7q22.1 chromo‐ somes, respectively. Aside from brittle bones, these patients may also present long bones with no curvatures, severe deformities preventing appropriate gait and even standing, conductive deafness due to malformations of the auditory canal, dentinogenesis imperfecta, joint hyper‐ laxity and intervertebral disc herniation. Patients with severe forms of the disease have a long history of fractures on mild impact and variable bone deformities. The most severe variants may even lead to fractures in utero and pre or perinatal death. Tables 3 and 4 shows different

This syndrome is an autosomal recessive disease characterized by bone and visual abnormalities including short stature, osteoporosis development during infancy, spontane‐ ous fractures, scoliosis, platyspondyly and long bone deformities. A crucial associated finding is the presence of pseudoglioma that may be associated to microcephaly, blind‐ ness during childhood, cataracts and iris atrophy. Occasionally, some patients present interventricular septal defects and mental retardation. This disease is conditioned by mutations of the LRP5 gene, located on chromosome 11q13.4 and that encodes the lowdensity lipoprotein receptor-related protein 5 (LRP5). It was initially believed that this entity was another variant of osteogenesis imperfecta (OI) but the study of collagen in patients with OPPG established that this protein was normal and the hypothesis was

discarded; however, this is still the most relevant differential diagnosis [36-41].

Muscular dystrophies, peripheral neuropathies and muscle atrophies of hereditary origin, represent broad groups of diseases that aside from their characteristic clinical stigmata, can be associated with osteoporosis as one of their complications. As the disease progresses in these

protein lead to bone fragility and hence, to an increased risk of fractures.

may explain some of these cases [31, 32].

**2.2. Osteogenesis imperfecta**

32 Topics in Osteoporosis

forms of the disease [33-35].

**2.4. Neuromuscular disorders**

**2.3. Osteoporosis – Pseudoglioma Syndrome (OPPG)**

This group of genetic diseases encompasses a great number of inborn defects with repercus‐ sions in several aspects of carbohydrate, amino acid, protein, vitamin, mineral, complex molecule, neurotransmitter and energy metabolism. The genetic basis of most of these entities hinges on gene mutations encoding proteins, particularly enzymes, leading to partial or complete blockade of one or several metabolic processes. In these diseases, symptoms arise for different reasons, including: a deficit of the products generated by the compromised enzymatic reaction, accumulation of the precursor immediate to the defect, an increase in alternative products due to increased activation of alternate metabolic pathways or inhibition of these alternate pathways due to the accumulated substrate. In most cases, inheritance of these diseases is autosomal recessive and less frequently, X-linked recessive.

In cases of metabolic errors, osteoporosis tends to develop for different reasons: in some cases, it is secondary to nutritional deficiencies, progressive neurologic or muscular impairment or as a consequence of the therapeutic measures taken in the management of the primary disease: their secondary effects directly compromise bone quality (steroids, antiseizure drugs, etc.). The number of monogenic diseases whose phenotype may include osteoporosis is large and are shown in Tables 3-5, according to their Mendelian inheritance pattern [45-56].



SNRPN /PWCR

SLC9A3R1/ NHERF

Hajdu-Cheney syndrome; HJCYS NOTCH2 Neurogenic locus

Prader-Willi syndrome; PWS NDN

Nephrolithiasis/osteoporosis, hypophosphatemic, 1; NPHLOP1

Nephrolithiasis/osteoporosis, hypophosphatemic, 2; NPHLOP2

Cardiomyopathy, dilated, with hypergonadotropic hypogonadism

dominant, 1; DKCA1

dominant, 2; DKCA2

dominant, 3; DKCA3

Dyskeratosis congenita, autosomal

Dyskeratosis congenita, autosomal

Dyskeratosis congenita, autosomal

Pigmented nodular adrenocortical disease, primary, 1; PPNAD1

Pigmented nodular adrenocortical disease, primary, 2; PPNAD2

Hyperostosis corticalis generalisata, benign form of worth, with torus

palatinus

**Location**

Genetic Diseases Related with Osteoporosis http://dx.doi.org/10.5772/55546

> 15q11.2 15q11.2

1p12-p11 80, 81

5q35.3 82, 83

17q25.1 84-86

1q22 87, 88

3q26.2 87, 88

5p15.33 89, 90

14q12 91, 92

17q24.2 93, 94

2q31.2 95, 96

11q13.2 97, 98

Necdin homolog (mouse) (NDN) Small nuclear ribonucleoproteinassociated protein N (SNRPN/PWCR)

Notch homolog protein 2 (NOTCH2)

phosphate transport

Na(+)/H(+) exchange regulatory cofactor NHE-RF1 (SLC9A3R1/

precursor (LMNA)

component (TERC)

transcriptase (TERT)

protein kinase type Ialpha regulatory subunit (PRKAR1A/

lipoprotein receptor-

nuclear factor 2 (TINF2)

protein 2A (SLC34A1/ .NPT2A)

NHERF)

(RNA)

LMNA Prelamin-A/C

TERC Telomerase RNA

TERT Telomerase reverse

TINF2 TERF1-interacting

PRKAR1A cAMP-dependent

TSE1)

and -GMP phosphodiesterase 11A (PDE11A)

PDE11A Dual 3',5'-cyclic-AMP

LRP5 Low density

SLC34A1 Sodium-dependent

**Reference**

35

78, 79


SMAD3

Ehlers-Danlos syndrome, Type I COL5A2 Collagen, type V,

Ehlers-Danlos syndrome, Type II COL5A1 Collagen, type V,

TGFBR1 Transforming growth

TGFBR2 Transforming growth

TGFBR2 Transforming growth

COL5A1 Collagen, type V,

COL5A2 Collagen, type V,

GNAS GNAS complex locus (GNAS)

GNAS GNAS complex locus (GNAS)

GNAS GNAS complex locus (GNAS)

COMP Cartilage oligomeric

COL1A1 Collagen, type I, alpha

factor-beta receptor, Type I (TGFBR1)

factor-beta receptor, Type II (TGFBR2)

factor-beta receptor, Type II (TGFBR2)

(Drosophila) (SMAD3)

alpha 2 (COL5A2)

alpha 1 (COL5A1)

alpha 1 (COL5A1)

alpha 2 (COL5A2)

[Gs, alpha subunit, included]

[Gs, alpha subunit, included]

[Gs, alpha subunit, included]

matrix protein (COMP)

1 (COL1A1)

Mothers against decapentaplegic homolog 3

Loeys-Dietz syndrome, Type 1A; LDS1A

34 Topics in Osteoporosis

Loeys-Dietz syndrome, Type 1B; LDS1B

Loeys-Dietz syndrome, Type 2B; LDS2B

Pseudohypoparathyroidism,

Pseudohypoparathyroidism,

Pseudopseudohypopara-thyroidism;

Epiphyseal dysplasia, multiple, 1;

Type IA; PHP1A

Type IC; PHP1C

PPHP

EDM1

Loeys-Dietz syndrome, Type 3; LDS3 MADH3/

**Location**

9q22.33 63, 64

3p24.1 65, 66

3p24.1 63, 65

15q22.33 67, 68

2q32.2 69, 70

9q34.3 70, 71

20q13.32 72, 73

20q13.32 73, 74

20q13.32 73, 75

19p13.11 76, 77

9q34.3

17q21.33

2q32.2


**Disease Gene Product Genomic**

H63D)

BMP2 [HFE hemochromatosi s, modifier of]

Thalassemia:HBB

LRP5 Low density

Thalassemia, Hispanic gammadelta-beta: LCRB

Homocysteinemia MTHFR (C677T) Methylenetetrahydro

Cleidocranial dysostosis; CLCD RUNX2 Runt-related

**Table 3.** Autosomal dominant diseases with bone mineral density loss.

Trichorhinophalangeal syndrome,

Vitamin D hydroxylation-deficient rickets, Type 1A; VDDR1A

Hemochromatosis; HFE HFE (C282Y y

Beta-Thalassemia beta-

Osteoporosis-pseudoglioma

beta-synthase deficiency

Homocystinuria due to cystathionine

syndrome; OPPG

type I; TRPS1

**Location**

**location**

20p12.3

11p15.5

21q22.3

1q23

6p21.1 119, 120

Genetic Diseases Related with Osteoporosis http://dx.doi.org/10.5772/55546

8q23.3 121, 122

12q13 123, 124

6p22.2 125, 126

11p15.4 47, 48

11q13.2 127, 128

21q22.3 45, 46

1p36.6 129, 130

transcription factor 2

transcription factor Trps1(TRPS1)

(RUNX2)

TRPS1 Zinc finger

CYP27B1 25-hydroxy-vitamin

D-1 alpha hydroxylase, mitochondrial (CYP27B1)

Hereditary hemochromatosis protein (HFE)

beta (HBB)

beta (LCRB)

(LRP5)

synthase (CBS)

folate reductase (MTHFR)

synthase (CBS)

(MTR/METH)

CBS/HIP4 Cystathionine beta-

CBS Cystathionine beta-

MS/MTR Methionine synthase

Bone morphogenetic protein 2 (BMP2)

Hemoglobin subunit

Locus control region,

lipoprotein receptorrelated protein 5

**Reference**

37


**Table 3.** Autosomal dominant diseases with bone mineral density loss.

**Disease Gene Product Genomic**

Van Buchem disease, Type 2; HVB2

36 Topics in Osteoporosis

1; OPTA1

2; OPTA2

Osteopetrosis, autosomal dominant

Osteopetrosis, autosomal dominant

ACTH-independent macronodular adrenal hyperplasia; AIMAH

Hyper-IgE recurrent infection syndrome, autosomal dominant

Coronary artery disease, autosomal dominant 2; ADCAD2 or CADO

Avascular necrosis of femoral head,

Spondyloepimetaphyseal dysplasia with joint laxity Type 2; SEMDJL2

Spondyloepiphyseal dysplasia, Maroteaux type (pseudo-Morquio

primary; ANFH

syndrome, Type 2)

**Location**

11q13.3 99, 100

11q13.3 101, 102

16p13.3 103, 104

20q13.32 105, 106

17q21.2 107, 108

12p13.2 109, 110

12q13.11 111, 112

16p11.2 113, 114

12q24.11 115, 116

1p36.12 117, 118

related protein 5

lipoprotein receptorrelated protein 5

lipoprotein receptorrelated protein 5

transporter 7 (CLCN7)

[Gs, alpha subunit, included]

lipoprotein receptorrelated protein 6

alpha 1 (COL2A1)

KIF22 (KIF22)

potential cation channel, subfamily V, member 4 (TRPV4)

liver/bone/kidney or alkaline phosphatase, tissue-nonspecific isozyme (ALPL)

(LRP5)

(LRP5)

(LRP5)

CLCN7 H(+)/Cl(-) exchange

GNAS GNAS complex locus (GNAS)

STAT3 Signal transducer and

LRP6 Low density

COL2A1 Collagen, type II,

KIF22 Kinesin-like protein

TRPV4 Transient receptor

Hypophosphatasia, adult ALPL Alkaline phosphatase,

activator of transcription 3 (STAT3)

(LRP6)

LRP5 Low density

LRP5 Low density



Niemann-Pick disease, Type A SMPD1 Sphingomyelin

Niemann-Pick disease, Type B SMPD1 Sphingomyelin

Lathosterolosis SC5DL Lathosterol oxidase

Fibromatosis, juvenile hyaline; JHF ANTXR2 Anthrax toxin

Aromatase deficiency CYP19A1 Cytochrome P450

Diastrophic dysplasia SLC26A2 Sulfate transporter 2

Desbuquois dysplasia; DBQD CANT1 Soluble calcium-

Torg-winchester syndrome MMP2 72 kDa type IV

Geroderma osteodysplasticum; GO GORAB RAB6-interacting

Lysinuric protein intolerance; LPI SLC7A7 Y+L amino acid

Exudative vitreoretinopathy 4; EVR4 LRP5 Low density

Mucopolysaccharidosis Type IVA

Mucopolysaccharidosis Type IVB

Cerebroretinal microangiopathy with calcifications and cysts; CRMCC

Nestor-Guillermo progeria syndrome;

NGPS

(Morquio syndrome A)

(Morquio syndrome B)

**location**

11p15.4 147, 148

Genetic Diseases Related with Osteoporosis http://dx.doi.org/10.5772/55546

11p15.4 147, 149

11q23.3 150, 151

16q24.3 152-154

4q21 155, 156

15q21.2 157, 158

5q32 159, 160

17q25.3 161, 162

16q12.2 163, 164

1q24.2 165, 166

14q11.2 167, 168

17p13.1 169, 170

11q13.2 171, 172

11q13.1 173, 174

3p22.3

phosphodiesterase 1, acid lysosomal (SMPD1/ASM)

phosphodiesterase 1, acid lysosomal (SMPD1/ASM)

(SC5DL)

receptor 2 (ANTXR2)

(S26A2)

activated nucleotidase 1 (CANT1)

19A1 (CYP19A1)

collagenase (MMP2)

transporter 1 (YLAT1)

lipoprotein receptorrelated protein 5

autointegration factor 1 (BANF1)

golgin (GORAB)

CTC1 CST complex subunit CTC1

(LRP5)

BANF1 Barrier to

GLB1 Beta-galactosidase1 (BGAL)

galactosamine-6 sulfatase (GALNS)

GALNS N-acetyl-

**Reference**

39


Homocysteinemia MTHFR (C677T) Methylenetetrahydro

Propionic acidemia PCCA Propionyl-CoA

Gaucher disease, Type I; GDI GBA Glucosylceramidase

Paget disease, juvenile; JPD TNFRSF11B Tumor necrosis factor

Osteogenesis imperfecta, Type IX;

Ehlers-Danlos syndrome, type VI;

Hypertrophic osteoarthropathy, primary, autosomal recessive, 1;

Pituitary adenoma, ACTH-secreting;

Lipodystrophy, congenital generalized, type 4; CGL4

[Osteogenesis imperfecta type II-B, III

OI9

EDS6

PHOAR1

CUDP

or IV PPIB related]

38 Topics in Osteoporosis

**location**

21q22.3

1q23

3q22.3

folate reductase (MTHFR)

synthase (CBS)

(MTR/METH)

trans isomerase B

carboxylase alpha chain, mitochondrial

carboxylase beta chain, mitochondrial

oxoglutarate 5 dioxygenase 1 (PLOD1)

prostaglandin dehydrogenase [NAD

interacting protein

receptor superfamily, member 11b (TNFRSF11B)

transcript release factor (PTRF)

(GLCM/GBA)

+] (HPGD)

(AIP)

CBS Cystathionine beta-

MS/MTR Methionine synthase

PPIB Peptidyl-prolyl cis-

(PPIB)

(PCCA)

(PCCB)

PLOD1 Procollagen-lysine,2-

HPGD 15-hydroxy-

AIP AH receptor-

Pycnodysostosis; PKND CTSK Cathepsin K 1q21.3 143, 144

PTRF Polymerase I and

PCCB Propionyl-CoA

**Reference**

1p36.6 33, 131, 132

15q22.31 35, 133

13q32.3 134, 135

1p36.22 69, 136

4q34.1 137, 138

11q13.2 139, 140

1q22 49, 50

8q24.12 141, 142

17q21.2 145, 146


Perrault syndrome; prlts HSD17B4 Peroxisomal

Glycogen storage disease Ia; GSD1A G6PC Glucose-6-

Cranioectodermal dysplasia 1; CED1 IFT122 Intraflagellar

Genitopatellar syndrome; GTPTS KAT6B Histone

Niemann-Pick disease, Type B SMPD1 Sphingomyelin

Cerebrotendinous xanthomatosis;

Congenital disorder of glycosylation,

Cutis laxa, autosomal recessive, Type

Cutis laxa, autosomal recessive, Type

Cutis laxa, autosomal recessive, Type

Trichothiodystrophy, photosensitive;

Arthropathy, progressive pseudorheumatoid, of childhood;

CTX

PPAC

Type IIk; CDG2K

IA; ARCL1A

IIB; ARCL2B

IIIB; ARCL3B

TTDP

Glycogen storage disease Ib; GSD1B SLC37A4 Glucose-6-phosphate

**location**

5q23.1 198, 199

Genetic Diseases Related with Osteoporosis http://dx.doi.org/10.5772/55546

17q21.31 200, 201

11q23.3 200, 201

3q21.3 202, 203

2q35 204, 205

6q21 206, 207

10q22.2 208, 209

4q12 210, 211

17q25.3 166, 214

17q25.3 212, 215

11p15.4 149, 216

2q14.3 217, 218

multifunctional enzyme type 2 (HSD17B4)

phosphatase, catalytic subunit

(G6PC)

CYP27A1 Sterol 26-hydroxylase,

WISP3 WNT1-inducible-

TMEM165 Transmembrane

PYCR1 Pyrroline-5-

PYCR1 Pyrroline-5-

ERCC3 TFIIH basal

translocase (SLC37A4)

transport protein 122 homolog (IFT122)

mitochondrial (CYP27A1/CP27A)

signaling pathway protein 3 (WISP3)

acetyltransferase

FBLN5 Fibulin-5 (FBLN5) 14q32.12 212, 213

carboxylate reductase 1, mitochondrial (PYCR1/P5CR1)

carboxylate reductase 1, mitochondrial (PYCR1/P5CR1)

phosphodiesterase

transcription factor

(SMPD1)

KAT6B

protein 165 (TMEM165/TM165) **Reference**

41


NOLA3 / NOP10 H/ACA

RIN2 Ras and Rab

HPGD 15-

Hyalinosis, infantile systemic; ISH ANTXR2 Anthrax toxin

Ovarian dysgenesis 1; ODG1 FSHR Follicle stimulating

Wilson disease; WND ATP7B Copper-transporting

Werner syndrome; WRN WRN/RECQL2 Werner syndrome

Rothmund-thomson syndrome; RTS RECQL4 ATP-dependent DNA

ribonucleoprotein complex subunit 3 (NOP10/ NOLA3)

hydroxyprostaglandin dehydrogenase [NAD+] (PGDH)

ylosylprotein 3-betaglucuronosyltransfera

interactor 2 (RIN2)

B3GAT3 Galactosylgalactosylx

se 3 (B3GAT3)

receptor 2 (ANTXR2)

(FSHR)

EIF2AK3 Eukaryotic translation

ERCC6 DNA excision repair

HSPG2 Basement

hormone receptor

initiation factor 2 alpha kinase 3 (EIF2AK3)

protein ERCC-6

ATPase 2 (ATP7B)

ATP-dependent helicase (WRN / RECQL2)

helicase Q4 (RECQL4)

membrane-specific heparan sulfate proteoglycan core protein (HSPG2)

Dyskeratosis congenita, autosomal

Macrocephaly, alopecia, cutis laxa,

Hypertrophic osteoarthropathy, primary, autosomal recessive, 1;

Multiple joint dislocations, short stature, craniofacial dysmorphism, and congenital heart defects

Epiphyseal dysplasia, multiple, with early-onset diabetes mellitus

Cerebrooculofacioskeletal syndrome

Schwartz-Jampel syndrome, Type 1;

recessive, 1; DKCB1

40 Topics in Osteoporosis

and scoliosis

PHOAR1

1; COFS1

SJS1

**location**

15q14 175, 176

20p11.23 177, 178

4q34.1 137, 179

11q12.3 180, 181

4q21.21 182, 183

2p16.3 184, 185

2p11.2 186, 187

10q11.23 188, 189

13q14.3 190, 191

8p12 192, 193

8q24.3 194, 195

1p36.12 196, 197


**Disease Gene Product Genomic**

Androgen insensitivity syndrome; AIS AR Androgen receptor

Fabry disease GLA Galactosidase, alpha

Occipital horn syndrome; OHS ATP7A Copper-transporting

Menkes disease ATP7A Copper-transporting

POF1B

CYP21A2 Steroid 21-

hydroxylase (CYP21A2)

PHEX Phosphate-regulating neutral endopeptidase (PHEX/PEX)

(AR)

(FMR1)

(AGAL)

(DKC1)

Within the different categories of genetic diseases, we can include numeric or structural chromosomal abnormalities. Two of the most common chromosomal diseases are Turner's syndrome and Klinefelter's syndrome, both associated to X chromosome aneuploidy; in the first case, there is complete or partial absence of an X chromosome and less frequently, it can be caused by structural anomalies in the short arms of the X chromosome. In Klinefelter's syndrome, there is an additional X chromosome and occasionally, there may be more than one

retardation protein 1

ATPase 1 (ATP7A)

ATPase 1 (ATP7A)

ribonucleoprotein complex subunit 4

GK Glycerol kinase (GK) Xp21.2 253, 254

FLNA Filamin-A (FLNA) Xq28 257, 258

Protein POF1B Xq21.1-

q21.2

FMR1 Fragile X mental

DKC1 H/ACA

Adrenal hyperplasia, congenital, due to 21-hydroxylase deficiency

Hypophosphatemic rickets, X-linked

dominant; XLHR or HYP

Fragile X mental retardation

Dyskeratosis congenita, X-linked;

(glycerol kinase deficiency; GKD)

Terminal osseous dysplasia; TOD or

Premature ovarian failure 2B; POF2B FLJ22792 /

**Table 5.** X-linked recessive diseases with bone mineral density loss.

**2.6. Genetic diseases of chromosomal origin and osteoporosis**

syndrome

DKCX

ODPF

Hyperglycerolemia

**Table 4.** Autosomal recessive diseases with bone mineral density loss.

**location**

**location**

6p21.33 239, 240

Genetic Diseases Related with Osteoporosis http://dx.doi.org/10.5772/55546

Xp22.11 241, 242

Xq12 243, 244

Xq27.3 245, 246

Xq22.1 51, 52

Xq21.1 247, 248

Xq21.1 249, 250

Xq28 251, 252

255, 256

**Reference**

43


**Table 4.** Autosomal recessive diseases with bone mineral density loss.

**Disease Gene Product Genomic**

Weill-Marchesani syndrome 1; WMS1 ADAMTS10 A disintegrin and

Laron syndrome GHR Growth hormone

Keutel syndrome MGP Matrix Gla protein

Hypophosphatasia, childhood ALPL Alkaline phosphatase,

Fanconi-Sickel syndrome; FBS SLC2A2 Solute carrier family

Lactose intolerance, adult type MCM6 DNA replication

Costello syndrome HRAS GTPase HRas (HRAS/

Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy; CARASIL

42 Topics in Osteoporosis

Mandibuloacral dysplasia with type A

Trichohepatoenteric syndrome 1;

THES1

lipodystrophy; MADA

**location**

6q25.3

19q13.32

10q26.13 219, 220

19p13.2 221, 222

5p13-p12 223, 224

1q22 225, 226

12p12.3 227, 228

1p36.12 229, 230

3q26.2 231, 232

2q21.3 233, 234

5q15 235, 236

11p15.5 237, 238

complex helicase XPB subunit (ERCC3)

factor IIH, subunit 5

transcription factor complex helicase XPD subunit (ERCC2)

metalloproteinase with thrombospondin

(ADAMTS10/ATS10)

receptor (GHR)

precursor (LMNA)

liver/bone/kidney or alkaline phosphatase, tissue-nonspecific isozyme (ALPL / PPBT)

2, facilitated glucose transporter member 2 (SLC2A2 / GTR2)

licensing factor MCM6

repeat domain 37

RASH) (HRAS / RASH)

(TTC37)

TTC37 Tetratricopeptide

GTF2H5 General transcription

ERCC2 TFIIH basal

HTRA1 Serine protease

LMNA Prelamin-A/C

(GTF2H5)

HTRA1

motifs 10

(MGP)

**Reference**


**Table 5.** X-linked recessive diseases with bone mineral density loss.

### **2.6. Genetic diseases of chromosomal origin and osteoporosis**

Within the different categories of genetic diseases, we can include numeric or structural chromosomal abnormalities. Two of the most common chromosomal diseases are Turner's syndrome and Klinefelter's syndrome, both associated to X chromosome aneuploidy; in the first case, there is complete or partial absence of an X chromosome and less frequently, it can be caused by structural anomalies in the short arms of the X chromosome. In Klinefelter's syndrome, there is an additional X chromosome and occasionally, there may be more than one extra X chromosome. In both syndromes, the phenotypic spectrum includes gonadal dysgen‐ esis, in Turner's syndrome there are fibrous bands instead of ovaries and in Klinefelter's, the testicles are hypoplastic, leading in both cases to hypogonadism and a partial or complete deficit in the sex hormones that would normally be produced by the ovaries and testicles. Due to their lack, the development of normal secondary sexual characteristics is stunted and the various metabolic processes dependent on the hormones are also compromised. One of these metabolic processes occurs in bone [259-262].

**Nomenclature**

OPN-Osteopontin

ESR1-Estrogen Receptor Alpha ESR2-Estrogen Receptor Beta

PTHR1-Parathohormone Receptor

CASR-Calcium Sensing Receptor

CYP1A1-Cytochrome P450, Subfamily A, Polypeptide 1

Genetic Diseases Related with Osteoporosis http://dx.doi.org/10.5772/55546 45

AR-Androgen Receptor VDR-Vitamin D Receptor

PTH-Parathormone

LEPR-Leptin Receptor

INSR-Insulin Receptor

ALOX12-Arachidonate 12-Lipoxygenase ALOX15-Arachidonate 15-Lipoxygenase

BMP4-Bone Morphogenetic Protein 4 BMP7-Bone Morphogenetic Protein 7

RANK-Receptor Activator Of Nf-Kb2

TNFR2-Tumor Necrosis Factor Receptor

RANK-L.-Receptor Activator Of Nf-Kb2 Ligand

IGF-1-Insulin-Like Growth Factor 1 (Somatomedin C)

PRL-Prolactin

LEP-Leptin

INS-Insulin

SOST-Sclerostin P53-Protein 53

IL1β-Interleucin 1 Beta

TNF-Tumor Necrosis Factor

APOE-Apolipoprotein E

IL6-Interleucin 6

Undoubtedly, bone metabolism is complex and the processes of osteoblastogenesis, osteo‐ clastogenesis and remodeling must occur in a balanced manner; it is important to mention that the entire family of steroid hormone receptors (estrogen, androgen, vitamin D and retinoids), are expressed in bone, both in osteoblasts and osteoclasts as well as in chondrocytes. Within this microenvironment, the action of these hormones on their receptors is key to appropriate skeletal development; as a matter of fact, individuals with genetic mutations encoding any of these receptors develop, among other manifestations, bad quality bone mass. These hormones and their receptors play a pivotal role in female and male bone growth and may also favor epiphyseal closure at the end of the growth period. It is known that one of effects of steroid hormones on bone metabolism is resorption inhibition since they promote osteoclast apoptosis and decrease the frequency of remodeling unit activation. Therefore, the integral treatment of both entities includes hormone replacement that to a certain extent, will improve bone mass and will prevent or delay the development of osteoporosis [263, 264].

### **3. Conclusion**

Bone metabolism and the large amount of processes that it involves, such as osteoblastogen‐ esis, osteoclastogenesis and bone remodeling, must be kept in constant balance. Each one of these aspects of the physiology of bone shows a particular gene expression patterns, which may even differ according to conditions and tissue needs. As previously men‐ tioned the number of genes involved is very large and sometimes their expression might be modified by multiple environmental conditions. It is important to mention that the expression of these genes is ubiquitous and is not restricted to the bone tissue, which explains why the phenotypic characteristics of a large number of monogenic and some polygenic entities include alterations on bone mineral density and on the microarchitec‐ ture of this tissue; this includes several degrees of osteopenia,osteoporosis or increased bone mineral density. Even a good number of these genes have been identified through the study of human disease whose phenotype includes altered bone mineral density. Without a doubt, the investigation of several processes that regulate bone metabolism will continue generat‐ ing new knowledge that will allow better understanding of bone physiology and physiopa‐ thology of multiple diseases and possibly new therapeutic options in diseases which compromise the quality and function of the bone.

### **Nomenclature**

extra X chromosome. In both syndromes, the phenotypic spectrum includes gonadal dysgen‐ esis, in Turner's syndrome there are fibrous bands instead of ovaries and in Klinefelter's, the testicles are hypoplastic, leading in both cases to hypogonadism and a partial or complete deficit in the sex hormones that would normally be produced by the ovaries and testicles. Due to their lack, the development of normal secondary sexual characteristics is stunted and the various metabolic processes dependent on the hormones are also compromised. One of these

Undoubtedly, bone metabolism is complex and the processes of osteoblastogenesis, osteo‐ clastogenesis and remodeling must occur in a balanced manner; it is important to mention that the entire family of steroid hormone receptors (estrogen, androgen, vitamin D and retinoids), are expressed in bone, both in osteoblasts and osteoclasts as well as in chondrocytes. Within this microenvironment, the action of these hormones on their receptors is key to appropriate skeletal development; as a matter of fact, individuals with genetic mutations encoding any of these receptors develop, among other manifestations, bad quality bone mass. These hormones and their receptors play a pivotal role in female and male bone growth and may also favor epiphyseal closure at the end of the growth period. It is known that one of effects of steroid hormones on bone metabolism is resorption inhibition since they promote osteoclast apoptosis and decrease the frequency of remodeling unit activation. Therefore, the integral treatment of both entities includes hormone replacement that to a certain extent, will improve bone mass

Bone metabolism and the large amount of processes that it involves, such as osteoblastogen‐ esis, osteoclastogenesis and bone remodeling, must be kept in constant balance. Each one of these aspects of the physiology of bone shows a particular gene expression patterns, which may even differ according to conditions and tissue needs. As previously men‐ tioned the number of genes involved is very large and sometimes their expression might be modified by multiple environmental conditions. It is important to mention that the expression of these genes is ubiquitous and is not restricted to the bone tissue, which explains why the phenotypic characteristics of a large number of monogenic and some polygenic entities include alterations on bone mineral density and on the microarchitec‐ ture of this tissue; this includes several degrees of osteopenia,osteoporosis or increased bone mineral density. Even a good number of these genes have been identified through the study of human disease whose phenotype includes altered bone mineral density. Without a doubt, the investigation of several processes that regulate bone metabolism will continue generat‐ ing new knowledge that will allow better understanding of bone physiology and physiopa‐ thology of multiple diseases and possibly new therapeutic options in diseases which

and will prevent or delay the development of osteoporosis [263, 264].

compromise the quality and function of the bone.

metabolic processes occurs in bone [259-262].

**3. Conclusion**

44 Topics in Osteoporosis

OPN-Osteopontin ESR1-Estrogen Receptor Alpha ESR2-Estrogen Receptor Beta AR-Androgen Receptor VDR-Vitamin D Receptor PTHR1-Parathohormone Receptor PTH-Parathormone CASR-Calcium Sensing Receptor CYP1A1-Cytochrome P450, Subfamily A, Polypeptide 1 PRL-Prolactin LEP-Leptin LEPR-Leptin Receptor INS-Insulin INSR-Insulin Receptor ALOX12-Arachidonate 12-Lipoxygenase ALOX15-Arachidonate 15-Lipoxygenase BMP4-Bone Morphogenetic Protein 4 BMP7-Bone Morphogenetic Protein 7 IGF-1-Insulin-Like Growth Factor 1 (Somatomedin C) SOST-Sclerostin P53-Protein 53 RANK-Receptor Activator Of Nf-Kb2 RANK-L.-Receptor Activator Of Nf-Kb2 Ligand IL1β-Interleucin 1 Beta IL6-Interleucin 6 TNF-Tumor Necrosis Factor TNFR2-Tumor Necrosis Factor Receptor APOE-Apolipoprotein E

### **Author details**

Margarita Valdés-Flores\* , Leonora Casas-Avila and Valeria Ponce de León-Suárez

\*Address all correspondence to: mvaldes@inr.gob.mx

Genetics Unit. National Rehabilitation Institute. Ministry of Health, Mexico

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**Chapter 3**

**Serum Leptin and Bone Turnover Markers in**

Osteoporosis is a very important health problem worldwide. It is defined as a disease charac‐ terized by low bone mass and micro-architectural deterioration of bone tissue, leading to enhanced bone fragility and consequent increase in fracture risk [1]. Osteoporosis is a silent disease and the health and financial impact of the disease result from fracture, particularly hip fracture, for which subjects with osteoporosis are at an increased risk [2]. In the UK, one in two women and one in five men suffer a fracture after the age of 50, with an annual cost to the

In 1990, the number of osteoporotic fractures estimated in Europe was 2.7 million, with an estimated direct cost in 2004 of €36 billion (£24.5 billion), of which €24.3 billion (£16.6 billion) were accounted for by hip fracture. Costs are expected to rise to €76.8 billion (£52.4 billion) by the year 2050 [5]. Similar projections are made for many other regions of the world because of the increasing numbers of the elderly. In the USA, the annual cost of incident fractures due to osteoporosis or low bone mass is predicted to rise from \$16.9 billion in 2006 to around \$25.3 billion by the year 2025 [4]. The direct costs of medical care of hip fractures were over \$65

There are many hormones involved in bone and mineral metabolism, such as oestrogens, testosterone and parathormone (PTH). The adipocyte also plays an important role in regulat‐ ing bone metabolism by releasing estrogens, and the adipokines, like leptin, resistin, adipo‐ nectin, and many others. After the discovery of leptin receptors in bone many studies have been done to explore its involvement in bone metabolism. Some studies have shown that leptin is expressed and secreted from primary cultures of human osteoblasts during the mineraliza‐ tion period, and that it may stimulate osteogenesis in human bone marrow in vitro [7,8].

> © 2013 Lateef et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

**Postmenopausal Osteoporosis**

Mehreen Lateef, Mukhtiar Baig and Abid Azhar

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54527

health services of around £2 billion [3,4].

**1. Introduction**

million in 2004 [6].

### **Serum Leptin and Bone Turnover Markers in Postmenopausal Osteoporosis**

Mehreen Lateef, Mukhtiar Baig and Abid Azhar

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54527

### **1. Introduction**

Osteoporosis is a very important health problem worldwide. It is defined as a disease charac‐ terized by low bone mass and micro-architectural deterioration of bone tissue, leading to enhanced bone fragility and consequent increase in fracture risk [1]. Osteoporosis is a silent disease and the health and financial impact of the disease result from fracture, particularly hip fracture, for which subjects with osteoporosis are at an increased risk [2]. In the UK, one in two women and one in five men suffer a fracture after the age of 50, with an annual cost to the health services of around £2 billion [3,4].

In 1990, the number of osteoporotic fractures estimated in Europe was 2.7 million, with an estimated direct cost in 2004 of €36 billion (£24.5 billion), of which €24.3 billion (£16.6 billion) were accounted for by hip fracture. Costs are expected to rise to €76.8 billion (£52.4 billion) by the year 2050 [5]. Similar projections are made for many other regions of the world because of the increasing numbers of the elderly. In the USA, the annual cost of incident fractures due to osteoporosis or low bone mass is predicted to rise from \$16.9 billion in 2006 to around \$25.3 billion by the year 2025 [4]. The direct costs of medical care of hip fractures were over \$65 million in 2004 [6].

There are many hormones involved in bone and mineral metabolism, such as oestrogens, testosterone and parathormone (PTH). The adipocyte also plays an important role in regulat‐ ing bone metabolism by releasing estrogens, and the adipokines, like leptin, resistin, adipo‐ nectin, and many others. After the discovery of leptin receptors in bone many studies have been done to explore its involvement in bone metabolism. Some studies have shown that leptin is expressed and secreted from primary cultures of human osteoblasts during the mineraliza‐ tion period, and that it may stimulate osteogenesis in human bone marrow in vitro [7,8].

© 2013 Lateef et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Furthermore, leptin may reduce ovariectomy-induced osteoporosis in rats and may also be involved in foetal and growing bone metabolism [9, 10].

port which has shown that when leptin injected into the brain of animals it will inhibit bone formation at doses lower than those that cause loss of body weight [24]. A lot of studies have been done to explore the relationship of serum leptin with bone mass den‐ sity and biochemical bone markers in osteoporosis. In this chapter, the mechanism of ac‐ tion of leptin on bone is reviewed and role of serum leptin in postmenopausal females is discussed with respect to its relation with bone mass density and biochemical bone

Serum Leptin and Bone Turnover Markers in Postmenopausal Osteoporosis

http://dx.doi.org/10.5772/54527

69

Leptin acts on bone by two different mechanisms. The first is the indirect mechanism revealed by Ducy et al (2000) in mutant mice and rats that either cannot produce or cannot respond to leptin [25]. Leptin secreted from fat cells is carried by the ObRa receptors of vascular endo‐ thelial cells across the blood-brain-barrier where it activates ObRb receptors in the hypothal‐ amus. These signals then stimulate expression of HOBIF (hypothalamic osteoblast inhibitory factor) which when released, lowers the matrix-making ability of osteoblasts [25-29] and because of this reason obese Ob (Lep)–/– mice, which should have low bone mass due to lack

The second direct mechanism of leptin action is by promoting differentiation of bone marrow stromal cells into osteoblasts [8] and by inhibiting osteoclast generation [30]. Bone marrow stromal cells (BMSC) can differentiate into either adipocytes or osteoblast cell lineage. Bone marrow adipocytes may serve as a direct source of leptin, which can inhibits adipogenesis differentiation of BMSC and stimulates differentiation of osteoblasts [31] while Kim et al. (2003) have shown that very high leptin levels led to BMSC apopto‐ sis [32]. Reseland et al. (2001) have found that human osteoblasts start making and se‐ creting leptin when they are either in the late, matrix-mineralizing stage or when changing to osteocytes [7]. Leptin has also stimulates the proliferation of cultured human osteoblasts [33], and it has been shown to cause human bone marrow stromal cells to ex‐ press alkaline phosphatase, collagen-I, and osteocalcin and to mineralize matrix [8]. These tissue culture experiments support the dual effect of leptin within the bone micro‐

In the last decade Ducy et al. (2000a,b) [25,27] have not found any long isoforms of the leptin receptors (Ob-R) on osteoblasts, so they assumed that leptin acted centrally as a very potent inhibitor of bone formation. Although the long isoform of Ob-R is abundant‐ ly expressed in the hypothalamus, and in a large number of peripheral tissues [14]. BMSC, osteoblasts, osteoclasts and chondrocytes also express leptin receptors [8]. In os‐ teoblasts, leptin acts through the osteoprotegerin (OPG)/RANKL (Receptor Activator for Nuclear factor κB Ligand) signaling pathway. Treatment with leptin changes the OPG/ RANKL expression profile favoring OPG [30]. Consequently, osteoclastogenesis is very likely suppressed by leptin through the OPG/RANKL pathway. In agreement with the previous findings, Burguera et al [10] have also confirmed the previous findings that ad‐

**2. Leptin and its mechanism of action on bone**

environment depending on the local leptin concentration.

of leptin, and thus estrogen, actually have an abnormally high bone mass.

markers.

Leptin, a fat-derived cytokine-like hormone, was discovered in 1994 by Friedman and colleagues [11]. A 16-kDa hormone, encoded by the OB gene, is predominantly expressed in adipose tissue [12] and circulates as a free and as a protein-bound entity. According to structural studies leptin belongs to the growth hormone four-helical cytokine subfamily. The leptin receptor was identified in the db locus of mouse chromosome 4. As a member of the IL-6 receptor family, the leptin receptor contains an extracellular-binding domain, a single transmembrane domain and a cytoplasmatic signaling domain [13]. Intracellular signaling is mediated through a non-covalently associated tyrosine kinase of the JAK kinase family [14]. Alternate gene splicing results in five known isoforms of the leptin receptor. The longest form of the receptor (ObR) is the only isoform capable of complete signal transduction. Conversely, the shorter isoforms of the leptin receptor have been suggested to participate in leptin clearance and/or to facilitate transport of leptin across the blood-brain barrier [14]. Circulating levels of leptin correlate with BMI and the content of fat mass. After crossing the blood–brain barrier, leptin reaches the hypothalamus, where it acts as a crucial regulator of feeding. Leptin is mainly regarded as a ''starvation-hormone'' signaling from the adipose tissue (AT) to the brain, indicating the size of the AT-stores [15].

Food intake and energy expenditure are controlled by leptin through an interaction with various neuropeptides in the hypothalamus. Neuropeptide Y (NPY) and agouti-related peptide (AGRP) expressions are inhibited, whereas pro-opiomelanocortin (POMC) expression is stimulated by leptin with increased food intake [16, 17]. Moreover, leptin interacts with neuromedin U (NMU); a novel and recently identified hypothalamic neuropeptide involved also in the regulation of appetite and locomotor activity [18]. Besides energy metabolism, leptin demonstrates pleiotropic effects in such areas as hematopoesis, blood pressure, T lymphocyte function, reproduction and bone mass regulation [14, 19]. Several endocrine and paracrine factors play a role in the fat-bone relationship. A number of local cytokines secreted by the adipose tissue, including leptin, have also been related to BMD variations [20]. Leptin because of its diverse role in bone is being considered one of the main functional connections between fat and bone.

Leptin, known to regulate appetite & energy expenditure may also contribute to mediate the effects of fat mass on bone. Interestingly, obesity seems to protect from osteoporosis. This observation led to researchers at bone formation in mouse models of obesity. Much effort has been dedicated to the relationship between leptin and bone. This interest stems from the well-founded knowledge that body weight is a major determinant of bone den‐ sity [21]. It is known that obesity is generally accompanied by increased bone strength. Obese persons have stronger bones and lose bone tissue at a slower pace [22]. Leptin has been proposed to regulate increased body weight as well as increased bone density. Mice that have congenital absence of leptin (ob/ob) have been shown to be obese and have very high bone density. Leptin makes them lose both fat and bone [23]. The bone mass phenotype of ob/ob mice can be rescued by intracerebroventricular (ICV) infusion of leptin, suggesting that leptin exerts an indirect influence on bone mass. There is a re‐ port which has shown that when leptin injected into the brain of animals it will inhibit bone formation at doses lower than those that cause loss of body weight [24]. A lot of studies have been done to explore the relationship of serum leptin with bone mass den‐ sity and biochemical bone markers in osteoporosis. In this chapter, the mechanism of ac‐ tion of leptin on bone is reviewed and role of serum leptin in postmenopausal females is discussed with respect to its relation with bone mass density and biochemical bone markers.

### **2. Leptin and its mechanism of action on bone**

Furthermore, leptin may reduce ovariectomy-induced osteoporosis in rats and may also be

Leptin, a fat-derived cytokine-like hormone, was discovered in 1994 by Friedman and colleagues [11]. A 16-kDa hormone, encoded by the OB gene, is predominantly expressed in adipose tissue [12] and circulates as a free and as a protein-bound entity. According to structural studies leptin belongs to the growth hormone four-helical cytokine subfamily. The leptin receptor was identified in the db locus of mouse chromosome 4. As a member of the IL-6 receptor family, the leptin receptor contains an extracellular-binding domain, a single transmembrane domain and a cytoplasmatic signaling domain [13]. Intracellular signaling is mediated through a non-covalently associated tyrosine kinase of the JAK kinase family [14]. Alternate gene splicing results in five known isoforms of the leptin receptor. The longest form of the receptor (ObR) is the only isoform capable of complete signal transduction. Conversely, the shorter isoforms of the leptin receptor have been suggested to participate in leptin clearance and/or to facilitate transport of leptin across the blood-brain barrier [14]. Circulating levels of leptin correlate with BMI and the content of fat mass. After crossing the blood–brain barrier, leptin reaches the hypothalamus, where it acts as a crucial regulator of feeding. Leptin is mainly regarded as a ''starvation-hormone'' signaling from the adipose tissue (AT) to the brain,

Food intake and energy expenditure are controlled by leptin through an interaction with various neuropeptides in the hypothalamus. Neuropeptide Y (NPY) and agouti-related peptide (AGRP) expressions are inhibited, whereas pro-opiomelanocortin (POMC) expression is stimulated by leptin with increased food intake [16, 17]. Moreover, leptin interacts with neuromedin U (NMU); a novel and recently identified hypothalamic neuropeptide involved also in the regulation of appetite and locomotor activity [18]. Besides energy metabolism, leptin demonstrates pleiotropic effects in such areas as hematopoesis, blood pressure, T lymphocyte function, reproduction and bone mass regulation [14, 19]. Several endocrine and paracrine factors play a role in the fat-bone relationship. A number of local cytokines secreted by the adipose tissue, including leptin, have also been related to BMD variations [20]. Leptin because of its diverse role in bone is being considered one of the main functional connections between

Leptin, known to regulate appetite & energy expenditure may also contribute to mediate the effects of fat mass on bone. Interestingly, obesity seems to protect from osteoporosis. This observation led to researchers at bone formation in mouse models of obesity. Much effort has been dedicated to the relationship between leptin and bone. This interest stems from the well-founded knowledge that body weight is a major determinant of bone den‐ sity [21]. It is known that obesity is generally accompanied by increased bone strength. Obese persons have stronger bones and lose bone tissue at a slower pace [22]. Leptin has been proposed to regulate increased body weight as well as increased bone density. Mice that have congenital absence of leptin (ob/ob) have been shown to be obese and have very high bone density. Leptin makes them lose both fat and bone [23]. The bone mass phenotype of ob/ob mice can be rescued by intracerebroventricular (ICV) infusion of leptin, suggesting that leptin exerts an indirect influence on bone mass. There is a re‐

involved in foetal and growing bone metabolism [9, 10].

indicating the size of the AT-stores [15].

fat and bone.

68 Topics in Osteoporosis

Leptin acts on bone by two different mechanisms. The first is the indirect mechanism revealed by Ducy et al (2000) in mutant mice and rats that either cannot produce or cannot respond to leptin [25]. Leptin secreted from fat cells is carried by the ObRa receptors of vascular endo‐ thelial cells across the blood-brain-barrier where it activates ObRb receptors in the hypothal‐ amus. These signals then stimulate expression of HOBIF (hypothalamic osteoblast inhibitory factor) which when released, lowers the matrix-making ability of osteoblasts [25-29] and because of this reason obese Ob (Lep)–/– mice, which should have low bone mass due to lack of leptin, and thus estrogen, actually have an abnormally high bone mass.

The second direct mechanism of leptin action is by promoting differentiation of bone marrow stromal cells into osteoblasts [8] and by inhibiting osteoclast generation [30]. Bone marrow stromal cells (BMSC) can differentiate into either adipocytes or osteoblast cell lineage. Bone marrow adipocytes may serve as a direct source of leptin, which can inhibits adipogenesis differentiation of BMSC and stimulates differentiation of osteoblasts [31] while Kim et al. (2003) have shown that very high leptin levels led to BMSC apopto‐ sis [32]. Reseland et al. (2001) have found that human osteoblasts start making and se‐ creting leptin when they are either in the late, matrix-mineralizing stage or when changing to osteocytes [7]. Leptin has also stimulates the proliferation of cultured human osteoblasts [33], and it has been shown to cause human bone marrow stromal cells to ex‐ press alkaline phosphatase, collagen-I, and osteocalcin and to mineralize matrix [8]. These tissue culture experiments support the dual effect of leptin within the bone micro‐ environment depending on the local leptin concentration.

In the last decade Ducy et al. (2000a,b) [25,27] have not found any long isoforms of the leptin receptors (Ob-R) on osteoblasts, so they assumed that leptin acted centrally as a very potent inhibitor of bone formation. Although the long isoform of Ob-R is abundant‐ ly expressed in the hypothalamus, and in a large number of peripheral tissues [14]. BMSC, osteoblasts, osteoclasts and chondrocytes also express leptin receptors [8]. In os‐ teoblasts, leptin acts through the osteoprotegerin (OPG)/RANKL (Receptor Activator for Nuclear factor κB Ligand) signaling pathway. Treatment with leptin changes the OPG/ RANKL expression profile favoring OPG [30]. Consequently, osteoclastogenesis is very likely suppressed by leptin through the OPG/RANKL pathway. In agreement with the previous findings, Burguera et al [10] have also confirmed the previous findings that ad‐ ministration of leptin reduced ovariectomy induced bone loss in rats by increasing osteo‐ protegerin mRNA in osteoblasts. Cornish et al in 2001 have found that leptin given peripherally increased bone strength in mice and also increased proliferation of osteo‐ blasts in vitro [34]. The results of these studies showed that direct peripheral action of leptin on bone is to enhance the strength of the bone in contrast to its central effect.

in women but not in men [31]. Sato et al. (2001) have found a positive correlation between serum leptin and calcaneus BMD in men, but the relationship became inverse when adjusted for body weight [18]. Pasco et al. (2001) have demonstrated a significant positive association between BMD and serum leptin in non-obese women [55]. Results of Blain et al (2002) reported that leptin is an independent predictor of whole body and femoral neck BMD in postmeno‐ pausal women [56]. Nagy et al. (2001) found a negative correlation between serum leptin levels and radial and femoral BMD in postmenopausal women [57]. Hadji et al. (2001) reported that bone mass is not correlated with the serum leptin level in pre or postmenopausal women [58]. Rauch et al. (1998) also reported no correlation between bone mass and serum leptin levels by examining total and trabecular bone density at the distal radius in adult women [59]. In leptin literature, several studies have examined the relationship between serum leptin and BMD in various cohorts, but the results remain contradictory. This fact reflects the intricacy of the

Serum Leptin and Bone Turnover Markers in Postmenopausal Osteoporosis

http://dx.doi.org/10.5772/54527

71

A study by Hamrick and Ferrari (2008) has documented that the effect of leptin is re‐ duced with increased age and higher BMI in both humans and laboratory animals in spite of high serum leptin levels [60]. It has been postulated that the under-responsive‐ ness to leptin, or leptin resistance, is mediated either by impaired transport of leptin through the blood-brain barrier, lower expression of leptin receptors and/or by the inhib‐ ition of the intracellular leptin signaling [61]. In plasma, leptin is bound to soluble lep‐ tin receptor (SLR), the product of an alternate splicing of leptin receptor mRNA or proteolytic cleavage [14]. Whereas serum leptin levels correlate positively with BMI, SLR is correlated negatively [14,62]. The inverse relationship between SLR and BMI reflects a feedback regulation between the body weight and leptin or leptin receptor expressions. It is observed in a study by Welt et al. (2004) when low serum leptin levels in women with hypothalamic amenorrhea (induced either by exercise or by low body weight) were treated with recombinant human leptin for three months, it led to an increase of osteo‐ calcin, bone alkaline phosphates and IGF-1, whereas urinary N-telopeptide did not change [63].They have demonstrated that leptin administration in individuals with lep‐ tin deficiency appeared to improve the growth hormone axis and markers of bone for‐

Bone markers are product derived from the bone remodeling process. During this process, compounds are released either from bone or from the cells involved in the bone remodeling process (osteoblasts and osteoclasts.) Markers of bone turnover are biochemical products measured usually in blood or urine that reflect the metabolic activity of bone but which themselves have no function in controlling skeletal metabolism [64]. Biochemical markers of bone turnover are broadly divided into two categories: markers of bone resorption, which reflect osteoclast activity and are for the most part degradation products of type I collagen; markers of bone formation, which reflect osteoblast activity and are byproducts of collagen

**4. Osteoporosis and bone turnover markers (BTMs)**

synthesis, matrix proteins or osteoblastic enzymes (Table 1) [65].

relationship of leptin and bone.

mation [63].

In order to evaluate its central effect, leptin was injected into the brain in the form of an intra-cerebelo-ventricular infusion [25, 27, 35]. Bone loss occurred in both wild-type mice and leptin deficient mice confirming that bone mass is partly regulated via the central, hypothalamic relay [25]. However, bone formation was inhibited at lower doses of leptin than those necessary to cause the loss of body weight. Ob/ob mice have low sympathetic activity, which led to the assumption that the central effect of leptin on bone is mediated by the sympathetic nervous system (SNS) [36]. The effect of leptin on the sympathetic nervous system is an important aspect in the regulation of energy homeostasis as well as several other physiological functions [37].

### **3. Serum leptin and Bone Mass Density (BMD)**

It is widely recognized that BMD measurement can predict fracture risk in the same way as cholesterol predicts cardiovascular disease [38]. In fact, the strength of BMD measurement to predict fracture is approx 3 fold higher than strength of serum cholesterol to predict cardio‐ vascular disease [39]. Bone mass measurement has been found to be a single best predictor of fracture risk and is required to the early diagnosis of osteoporosis [37,38,39].

It is observed in a study by Lateef et al 2010 that BMD is found to be significantly lower in postmenopausal females with and without osteoporosis as compared to premenopausal females [40] and there is a negative correlation between age and BMD found in post meno‐ pausal ostreoporotic females indicating bone loss with age and menopause [41]. A rapid bone loss is commonly seen in elderly individuals and tends to worsen with advancing age. The aging population is inevitably proven to be more osteoporotic unless it is intervened first with diagnostic tools and after preventive therapy [42].

Another study of Lateef et al 2011 showed that plasma leptin levels were positively correlated with bone mineral density (BMD) values in osteoporotic females [43]. Some reports have suggested a correlation between serum leptin concentration and BMD while other showed no such association [44-48]. It has been shown that plasma leptin levels are positively correlated with BMD at all skeleton sites measured in postmenopausal osteoporosis [49]. It is interesting to note that the obese are usually protected against osteoporosis and have increased bone mineral density [50]. This has been attributed to the mechanical effects of their excessive weight on bone tissue. It has been shown that obese postmenopausal women have a tendency to have increased bone mineral density compared with lean women [51-53]. The study of Di Carlo et al. (2007) documented a significant correlation between serum leptin and BMD in early postmenopausal women but the correlation was lost during progression of the postmeno‐ pausal period [54]. Thomas et al. (2001) have observed that serum leptin correlated with BMD in women but not in men [31]. Sato et al. (2001) have found a positive correlation between serum leptin and calcaneus BMD in men, but the relationship became inverse when adjusted for body weight [18]. Pasco et al. (2001) have demonstrated a significant positive association between BMD and serum leptin in non-obese women [55]. Results of Blain et al (2002) reported that leptin is an independent predictor of whole body and femoral neck BMD in postmeno‐ pausal women [56]. Nagy et al. (2001) found a negative correlation between serum leptin levels and radial and femoral BMD in postmenopausal women [57]. Hadji et al. (2001) reported that bone mass is not correlated with the serum leptin level in pre or postmenopausal women [58]. Rauch et al. (1998) also reported no correlation between bone mass and serum leptin levels by examining total and trabecular bone density at the distal radius in adult women [59]. In leptin literature, several studies have examined the relationship between serum leptin and BMD in various cohorts, but the results remain contradictory. This fact reflects the intricacy of the relationship of leptin and bone.

ministration of leptin reduced ovariectomy induced bone loss in rats by increasing osteo‐ protegerin mRNA in osteoblasts. Cornish et al in 2001 have found that leptin given peripherally increased bone strength in mice and also increased proliferation of osteo‐ blasts in vitro [34]. The results of these studies showed that direct peripheral action of leptin on bone is to enhance the strength of the bone in contrast to its central effect.

In order to evaluate its central effect, leptin was injected into the brain in the form of an intra-cerebelo-ventricular infusion [25, 27, 35]. Bone loss occurred in both wild-type mice and leptin deficient mice confirming that bone mass is partly regulated via the central, hypothalamic relay [25]. However, bone formation was inhibited at lower doses of leptin than those necessary to cause the loss of body weight. Ob/ob mice have low sympathetic activity, which led to the assumption that the central effect of leptin on bone is mediated by the sympathetic nervous system (SNS) [36]. The effect of leptin on the sympathetic nervous system is an important aspect in the regulation of energy homeostasis as well as

It is widely recognized that BMD measurement can predict fracture risk in the same way as cholesterol predicts cardiovascular disease [38]. In fact, the strength of BMD measurement to predict fracture is approx 3 fold higher than strength of serum cholesterol to predict cardio‐ vascular disease [39]. Bone mass measurement has been found to be a single best predictor of

It is observed in a study by Lateef et al 2010 that BMD is found to be significantly lower in postmenopausal females with and without osteoporosis as compared to premenopausal females [40] and there is a negative correlation between age and BMD found in post meno‐ pausal ostreoporotic females indicating bone loss with age and menopause [41]. A rapid bone loss is commonly seen in elderly individuals and tends to worsen with advancing age. The aging population is inevitably proven to be more osteoporotic unless it is intervened first with

Another study of Lateef et al 2011 showed that plasma leptin levels were positively correlated with bone mineral density (BMD) values in osteoporotic females [43]. Some reports have suggested a correlation between serum leptin concentration and BMD while other showed no such association [44-48]. It has been shown that plasma leptin levels are positively correlated with BMD at all skeleton sites measured in postmenopausal osteoporosis [49]. It is interesting to note that the obese are usually protected against osteoporosis and have increased bone mineral density [50]. This has been attributed to the mechanical effects of their excessive weight on bone tissue. It has been shown that obese postmenopausal women have a tendency to have increased bone mineral density compared with lean women [51-53]. The study of Di Carlo et al. (2007) documented a significant correlation between serum leptin and BMD in early postmenopausal women but the correlation was lost during progression of the postmeno‐ pausal period [54]. Thomas et al. (2001) have observed that serum leptin correlated with BMD

fracture risk and is required to the early diagnosis of osteoporosis [37,38,39].

several other physiological functions [37].

70 Topics in Osteoporosis

**3. Serum leptin and Bone Mass Density (BMD)**

diagnostic tools and after preventive therapy [42].

A study by Hamrick and Ferrari (2008) has documented that the effect of leptin is re‐ duced with increased age and higher BMI in both humans and laboratory animals in spite of high serum leptin levels [60]. It has been postulated that the under-responsive‐ ness to leptin, or leptin resistance, is mediated either by impaired transport of leptin through the blood-brain barrier, lower expression of leptin receptors and/or by the inhib‐ ition of the intracellular leptin signaling [61]. In plasma, leptin is bound to soluble lep‐ tin receptor (SLR), the product of an alternate splicing of leptin receptor mRNA or proteolytic cleavage [14]. Whereas serum leptin levels correlate positively with BMI, SLR is correlated negatively [14,62]. The inverse relationship between SLR and BMI reflects a feedback regulation between the body weight and leptin or leptin receptor expressions. It is observed in a study by Welt et al. (2004) when low serum leptin levels in women with hypothalamic amenorrhea (induced either by exercise or by low body weight) were treated with recombinant human leptin for three months, it led to an increase of osteo‐ calcin, bone alkaline phosphates and IGF-1, whereas urinary N-telopeptide did not change [63].They have demonstrated that leptin administration in individuals with lep‐ tin deficiency appeared to improve the growth hormone axis and markers of bone for‐ mation [63].

### **4. Osteoporosis and bone turnover markers (BTMs)**

Bone markers are product derived from the bone remodeling process. During this process, compounds are released either from bone or from the cells involved in the bone remodeling process (osteoblasts and osteoclasts.) Markers of bone turnover are biochemical products measured usually in blood or urine that reflect the metabolic activity of bone but which themselves have no function in controlling skeletal metabolism [64]. Biochemical markers of bone turnover are broadly divided into two categories: markers of bone resorption, which reflect osteoclast activity and are for the most part degradation products of type I collagen; markers of bone formation, which reflect osteoblast activity and are byproducts of collagen synthesis, matrix proteins or osteoblastic enzymes (Table 1) [65].


**5.2. Bone specific alkaline phosphatase**

indicate estrogen deficiency [69,70].

**5.3. Osteocalcin**

have been reported [72].

Bone specific alkaline phosphatase (BALP) is one of several isoenzymes of the alkaline phosphatase (ALP) family. The entire family is encoded by four gene loci, three tissue specific genes (bone,kidney,liver and other tissues). Although the nonspecific ALPs are the products of a single gene, the ioenzymes present in tissues such as bone, kidney or liver vary greatly because of variations in their carbohydrate side chains. These post translational modifications are exploited to distinguish the various ALP-isoforms from each other, employing methods including gel electrophoresis, heat denaturation, chemical inhibition or binding through specific monoclonal antibodies [68]. For therapeutic monitoring of patients, B-ALP measure‐ ments are good indicators of the metabolic activity of bone. Rising ALP concentrations may

Serum Leptin and Bone Turnover Markers in Postmenopausal Osteoporosis

http://dx.doi.org/10.5772/54527

73

Osteocalcin (OC) is a small, hydroxyapetite-binding protein synthesized by osteoblasts and to a lesser extent by hypertrophic chondrocytes. It contains three gamma- carboxyglutamic acid (Gla) residues which are responsible for calcium binding properties of protein. The precise function of osteocalcin has yet to be determined but recent studies suggest that OC is involved in bone remodeling via a negative mechanism. Serum osteocalcin is considered as a specific marker of osteoblast function, as its levels correlate with bone formation rates. However, the peptide is rapidly degraded in serum and both intact peptides and OC fragments of various sizes coexist in the circulation [70]. Osteocalcin is normally considered as bone formation marker. However, because it is released during bone formation from bone forming cells and during bone resorption from bone matrix, it reflects the overall turnover of bone. Assays have been developed to detect the intact protein and or the main breakdown product called N-mid fragment. OC serum levels follow a circadian rhythm with high values in early morning, but usually not influenced by food intake. Serum osteocalcin levels reportedly vary significantly

during the menstrual cycle with the highest level observerd during luteal phase [71].

The amino and carboxy terminal procollagen propeptides of type I collagen (PINP, PICP) are cleaved by specific extracellular endopeptidases from newly translated collagen type I pepetide. As these extension peptides are generated in a stoichiometric relationship with collagen biosynthesis, they are considered quantitative measures of newly formed type I collagen. However, because type I collagen is also a component of several soft tissues (fibro‐ cartilage, tendon, skin, gingival, intestine, heart valve, and large vessels) there is potential contribution to circulating procollagens from soft tissue synthesis of type I collagen. Both PICP and PINP demonstrate a cardian rhythm with peak values in the early morning, and are usually not influenced by food intake. Serum levels of amino and carboxy terminal procollagen propeptides of type I collagen (PINP, PICP) are measured by type and site specific immuno‐ assays [66]. Moderate correlations between serum PICP levels and the rate of bone formation

**5.4. Amino & carboxyterminal procollagen propeptides of type I collagen**

**Table 1.** List of important biochemical markers of bone turnover

### **5. Markers of bone formation**

Bone formation markers are direct or indirect products of active osteoblasts expressed during different phases of osteoblast development and reflecting different aspects of osteoblast function and bone formation. All markers are measured in serum or plasma [66].

### **5.1. Alkaline phosphatase**

Alkaline phosphatase (ALP) is a ubiquitous enzyme that plays an important role in osteoid formation and mineralization. The total ALP serum pool consists of several dimeric isoforms which originate from various tissues such as liver, bone, intestine, spleen, kidney and placenta. In adults with normal liver function, approximately 50% of the total ALP activity in serum arises from liver and 50% arise from bone [67].

### **5.2. Bone specific alkaline phosphatase**

Bone specific alkaline phosphatase (BALP) is one of several isoenzymes of the alkaline phosphatase (ALP) family. The entire family is encoded by four gene loci, three tissue specific genes (bone,kidney,liver and other tissues). Although the nonspecific ALPs are the products of a single gene, the ioenzymes present in tissues such as bone, kidney or liver vary greatly because of variations in their carbohydrate side chains. These post translational modifications are exploited to distinguish the various ALP-isoforms from each other, employing methods including gel electrophoresis, heat denaturation, chemical inhibition or binding through specific monoclonal antibodies [68]. For therapeutic monitoring of patients, B-ALP measure‐ ments are good indicators of the metabolic activity of bone. Rising ALP concentrations may indicate estrogen deficiency [69,70].

### **5.3. Osteocalcin**

Bone formation Detected in

Procollagen type I C-terminal propeptides (P1CP) Serum

Procollagen type I N-terminal propeptides (PINP) Serum/Plasma

Osteocalcins (OC) Serum/Plasma

Bone alkaline phosphatases (BALP) Serum

Hydroxyprolin Urine Pyridinolin(PYD) Urine Deoxypyridinoline (DPD) Urine

N-terminal cross-linked telopeptide (NTX) Urine and serum/plasma C-terminal cross-linked telopeptide (CTX) Urine and serum/plasma

Bone formation markers are direct or indirect products of active osteoblasts expressed during different phases of osteoblast development and reflecting different aspects of osteoblast

Alkaline phosphatase (ALP) is a ubiquitous enzyme that plays an important role in osteoid formation and mineralization. The total ALP serum pool consists of several dimeric isoforms which originate from various tissues such as liver, bone, intestine, spleen, kidney and placenta. In adults with normal liver function, approximately 50% of the total ALP activity in serum

Tartrate-resistant acid phosphatases (TRACP) Serum

function and bone formation. All markers are measured in serum or plasma [66].

Bone resorption Detected in

Byproducts of collagen synthesis

**Collagen degradation products**

Cross-linked telopeptides of type I collagen

Matrix protein

72 Topics in Osteoporosis

Osteoblast enzyme

Osteoclast enzymes

**5. Markers of bone formation**

**5.1. Alkaline phosphatase**

**Table 1.** List of important biochemical markers of bone turnover

arises from liver and 50% arise from bone [67].

Osteocalcin (OC) is a small, hydroxyapetite-binding protein synthesized by osteoblasts and to a lesser extent by hypertrophic chondrocytes. It contains three gamma- carboxyglutamic acid (Gla) residues which are responsible for calcium binding properties of protein. The precise function of osteocalcin has yet to be determined but recent studies suggest that OC is involved in bone remodeling via a negative mechanism. Serum osteocalcin is considered as a specific marker of osteoblast function, as its levels correlate with bone formation rates. However, the peptide is rapidly degraded in serum and both intact peptides and OC fragments of various sizes coexist in the circulation [70]. Osteocalcin is normally considered as bone formation marker. However, because it is released during bone formation from bone forming cells and during bone resorption from bone matrix, it reflects the overall turnover of bone. Assays have been developed to detect the intact protein and or the main breakdown product called N-mid fragment. OC serum levels follow a circadian rhythm with high values in early morning, but usually not influenced by food intake. Serum osteocalcin levels reportedly vary significantly during the menstrual cycle with the highest level observerd during luteal phase [71].

### **5.4. Amino & carboxyterminal procollagen propeptides of type I collagen**

The amino and carboxy terminal procollagen propeptides of type I collagen (PINP, PICP) are cleaved by specific extracellular endopeptidases from newly translated collagen type I pepetide. As these extension peptides are generated in a stoichiometric relationship with collagen biosynthesis, they are considered quantitative measures of newly formed type I collagen. However, because type I collagen is also a component of several soft tissues (fibro‐ cartilage, tendon, skin, gingival, intestine, heart valve, and large vessels) there is potential contribution to circulating procollagens from soft tissue synthesis of type I collagen. Both PICP and PINP demonstrate a cardian rhythm with peak values in the early morning, and are usually not influenced by food intake. Serum levels of amino and carboxy terminal procollagen propeptides of type I collagen (PINP, PICP) are measured by type and site specific immuno‐ assays [66]. Moderate correlations between serum PICP levels and the rate of bone formation have been reported [72].

### **5.5. Markers of bone resorption**

Most biochemical markers of bone resorption are degradation products of bone collagen, but noncollagenous proteins such as bone sialoprotein or tartarate resistant acid phosphatase have also been investigated [73].

The use of biochemical markers of bone remodeling in the monitoring of patients on treatment for osteoporosis is generally well-recognized [81,82]. However, optimum treatment targets specific to various therapies and the benefits of monitoring in terms of improvement in fracture

Serum Leptin and Bone Turnover Markers in Postmenopausal Osteoporosis

http://dx.doi.org/10.5772/54527

75

The changes in BMD and BTMs following the initiation of osteoporosis treatment independ‐ ently correlate with fracture risk reduction [84]. The advantage of BTM over BDM is that the change in BTMs following treatment explains a greater proportion of treatment as compared to BMD does, in terms of fracture risk reduction [85,86]. Also, the change in BMD is small and slow whereas the changes in BTMs are large and occur early after initiation of therapy. Repeat BMD is not advocated within 12 months after initiation of therapy as the changes do not generally attain significance within that time, and in fact 18-24 months may be appropriate for repeat BMD measurements [87]. BTMs on the other hand show significant change by 3-6 months. For example, bone resorption markers can be measured 3 months after initiation of oral bisphosphonates, and bone formation markers 6 months after start of therapy [88, 89). Changes in BTMs may be useful in monitoring osteoporosis treatment to confirm compliance

There are many studies which have demonstrated BTMs and their contribution to fracture risk, but the results of these studies have been inconsistent [91-95]. Many studies which have shown positive results with BTMs included bone resorption markers, with increased resorption marker predicting an increased fracture risk. While for BTMs to predict fracture risk inde‐ pendently of BMD, it is needed to clarify their relationships to other established risk factors.

The changes in BTMs following therapy are well documented. There is a decrease in BTMs following initiation of anti-resorptive therapy, reflecting inhibition of osteoclastic activity [96-100]. For example, with bisphosphonate treatment, there is a decrease in bone resorption markers within days following intravenous therapy, and within weeks following oral therapy.

Vasikaran et al [83] supports the role of BTMs in the management of patients with osteoporosis and also emphasized on the adoption of international reference standards for enhancing

The problem in BTMs use is their wide biological and analytical variability, Glover et al [101] emphasized that reference ranges should be defined and standardized with emphasis on sample size and age range of the population. Sandhu & Hampson (2011) describe that the best established clinical use for BTMs is in monitoring treatment efficacy and compliance [102]. In a study by Kim et al observed that BMT can be used to determine BMD response to antire‐

The Scientific Advisory Council of Osteoporosis Canada including multidisciplinary working group stated about the bone turnover markers in the management of postmenopausal osteoporosis that as far as potential uses of bone turn over markers (BTMs) are concerned, they can be used to predict bone loss and fracture in untreated postmenopausal women. They can also be used to monitor osteoporosis therapy, and up to some extent enhance the adherence to therapy but BTMs should not be used for diagnosis of osteoporosis as s separate and

laboratory consistency and to facilitate their inclusion in routine clinical practice.

sorptive therapy in Korean postmenopausal osteoporotic females [103].

outcomes or in adherence to oral therapies are not established [83].

with oral therapies and efficacy of treatment [90].

### **5.6. Hydroxy proline**

Hydroxyproline is an amino acid common to and characteristic of all forms of collagen, and urinary hydroxyproline excretion is the oldest test of bone resorption. However, this test lacks specificity for bone resorption because excreted hydroxyproline also comes from other tissues, particularly from skin collagen (which can turn over rapidly in certain disorders), from newly synthesized collagen that is not incorporated into tissue, and from dietary collagen and gelatin. Because it is less specific than newer tests, it is no longer widely used [74].

### **5.7. Pyridinoline (Pyr) and Deoxypyridinoline (DPD)**

The pyridinum crosslinks pyridinoline (PYD) and deoxypyridinolin (DPD) are the main crosslinks in skeletal tissues but act as stabilizers of mature croslinks in type I, II & III collagens of all major connective tissues (bone, dentin, ligaments, tendons, vascular walls, muscle and intestine) except skin. While PYD predominates in most tissues, DPD is most abundant in bone and therefore is considered the more specific marker [75].

### **5.8. Crosslinked Telopeptides**

The term "crosslinked telopeptides" refers to the measurement of collagen degradation products associated with the crosslink regions in type I collagen. Fragments derived from the C terminus are also released into circulation as a result of the osteoclast-mediated degradation of type I collagen and can be measured by various assays [76, 77]. The immuno reactive epitopes are located on peptide fragments derived from the N terminal (NTX-1) and C terminal (CTX-1 and ICTP) telopeptides of the collagen type I molecule. The NTX-1 and CTX-1 epitopes can be measured in both serum and in urine [78, 79].

### **6. Tatartarate Resistant Acid Phosphatase (TRACP)**

Tartarate resistant acid phosphate is synthesized and secreted by osteoclasts during active bone resorption. The process of resorption occurs after the attachment of osteoclasts to the bone surface and follows the secretion of acid and enzymes into a space created between the osteoclast and the bone. The acidic environment is produced by the action of carbonic anhydrase and an H-ATPase proton pump. TRACP, one of the enzymes secreted into this space, has been located in the adjacent osteoclast membrane (known as the ruffled border) [80]. Its activity in serum reflects bone resorption rates and more recently it has been possible to measure the isoenzyme by very specific immunoassays

The use of biochemical markers of bone remodeling in the monitoring of patients on treatment for osteoporosis is generally well-recognized [81,82]. However, optimum treatment targets specific to various therapies and the benefits of monitoring in terms of improvement in fracture outcomes or in adherence to oral therapies are not established [83].

**5.5. Markers of bone resorption**

also been investigated [73].

**5.8. Crosslinked Telopeptides**

**5.6. Hydroxy proline**

74 Topics in Osteoporosis

Most biochemical markers of bone resorption are degradation products of bone collagen, but noncollagenous proteins such as bone sialoprotein or tartarate resistant acid phosphatase have

Hydroxyproline is an amino acid common to and characteristic of all forms of collagen, and urinary hydroxyproline excretion is the oldest test of bone resorption. However, this test lacks specificity for bone resorption because excreted hydroxyproline also comes from other tissues, particularly from skin collagen (which can turn over rapidly in certain disorders), from newly synthesized collagen that is not incorporated into tissue, and from dietary collagen and gelatin.

The pyridinum crosslinks pyridinoline (PYD) and deoxypyridinolin (DPD) are the main crosslinks in skeletal tissues but act as stabilizers of mature croslinks in type I, II & III collagens of all major connective tissues (bone, dentin, ligaments, tendons, vascular walls, muscle and intestine) except skin. While PYD predominates in most tissues, DPD is most abundant in bone

The term "crosslinked telopeptides" refers to the measurement of collagen degradation products associated with the crosslink regions in type I collagen. Fragments derived from the C terminus are also released into circulation as a result of the osteoclast-mediated degradation of type I collagen and can be measured by various assays [76, 77]. The immuno reactive epitopes are located on peptide fragments derived from the N terminal (NTX-1) and C terminal (CTX-1 and ICTP) telopeptides of the collagen type I molecule. The NTX-1 and CTX-1 epitopes

Tartarate resistant acid phosphate is synthesized and secreted by osteoclasts during active bone resorption. The process of resorption occurs after the attachment of osteoclasts to the bone surface and follows the secretion of acid and enzymes into a space created between the osteoclast and the bone. The acidic environment is produced by the action of carbonic anhydrase and an H-ATPase proton pump. TRACP, one of the enzymes secreted into this space, has been located in the adjacent osteoclast membrane (known as the ruffled border) [80]. Its activity in serum reflects bone resorption rates and more recently it has been possible to

Because it is less specific than newer tests, it is no longer widely used [74].

**5.7. Pyridinoline (Pyr) and Deoxypyridinoline (DPD)**

and therefore is considered the more specific marker [75].

can be measured in both serum and in urine [78, 79].

measure the isoenzyme by very specific immunoassays

**6. Tatartarate Resistant Acid Phosphatase (TRACP)**

The changes in BMD and BTMs following the initiation of osteoporosis treatment independ‐ ently correlate with fracture risk reduction [84]. The advantage of BTM over BDM is that the change in BTMs following treatment explains a greater proportion of treatment as compared to BMD does, in terms of fracture risk reduction [85,86]. Also, the change in BMD is small and slow whereas the changes in BTMs are large and occur early after initiation of therapy. Repeat BMD is not advocated within 12 months after initiation of therapy as the changes do not generally attain significance within that time, and in fact 18-24 months may be appropriate for repeat BMD measurements [87]. BTMs on the other hand show significant change by 3-6 months. For example, bone resorption markers can be measured 3 months after initiation of oral bisphosphonates, and bone formation markers 6 months after start of therapy [88, 89). Changes in BTMs may be useful in monitoring osteoporosis treatment to confirm compliance with oral therapies and efficacy of treatment [90].

There are many studies which have demonstrated BTMs and their contribution to fracture risk, but the results of these studies have been inconsistent [91-95]. Many studies which have shown positive results with BTMs included bone resorption markers, with increased resorption marker predicting an increased fracture risk. While for BTMs to predict fracture risk inde‐ pendently of BMD, it is needed to clarify their relationships to other established risk factors.

The changes in BTMs following therapy are well documented. There is a decrease in BTMs following initiation of anti-resorptive therapy, reflecting inhibition of osteoclastic activity [96-100]. For example, with bisphosphonate treatment, there is a decrease in bone resorption markers within days following intravenous therapy, and within weeks following oral therapy.

Vasikaran et al [83] supports the role of BTMs in the management of patients with osteoporosis and also emphasized on the adoption of international reference standards for enhancing laboratory consistency and to facilitate their inclusion in routine clinical practice.

The problem in BTMs use is their wide biological and analytical variability, Glover et al [101] emphasized that reference ranges should be defined and standardized with emphasis on sample size and age range of the population. Sandhu & Hampson (2011) describe that the best established clinical use for BTMs is in monitoring treatment efficacy and compliance [102]. In a study by Kim et al observed that BMT can be used to determine BMD response to antire‐ sorptive therapy in Korean postmenopausal osteoporotic females [103].

The Scientific Advisory Council of Osteoporosis Canada including multidisciplinary working group stated about the bone turnover markers in the management of postmenopausal osteoporosis that as far as potential uses of bone turn over markers (BTMs) are concerned, they can be used to predict bone loss and fracture in untreated postmenopausal women. They can also be used to monitor osteoporosis therapy, and up to some extent enhance the adherence to therapy but BTMs should not be used for diagnosis of osteoporosis as s separate and independent factor. Similarly it must not be used to select the most appropriate type of osteoporotic therapy for the treatment. [104].

occurring in responses to weight loss and weight regain. The drop in leptin levels was strongly related to the increase in bone resorption marker occurring in response to weight loss. Similarly, after weight regain the rise in leptin levels was associated with a concomitant

Serum Leptin and Bone Turnover Markers in Postmenopausal Osteoporosis

http://dx.doi.org/10.5772/54527

77

The relationship of leptin and bone turnover markers in post menopausal osteoporosis has not yet been clarified. Although several studies have been done but still there is need to explore their exact connection. Many studies have recommended that in the treatment of the post-men‐ opausal women, biochemical markers of bone turnover may be useful as adjuncts to BMD and other diagnostic tests. They can be mainly used to monitor response to treatment and also used as relatively economical tools for studying bone metabolism. The exact roles of BTM need to be established in clinical practice. It is suggested that repeated measurements of bone markers during anti-resorptive therapy may help to improve the management of osteoporotic patients. Both a peripheral and a central action of leptin on bone metabolism have been suggested. Pe‐ ripherally, leptin is thought to exert positive effects on bone formation, whereas it is thought to reduce bone formation via a central control mechanism when binding to its specific receptors located on the hypothalamic nuclei [26]. It has been suggested that circulating leptin may act positively to maintain bone mass but these effects of serum leptin are not mediated due to these biochemical markers. Despite a preliminary understanding of leptin–bone mass interactions,

The role of BTMs in monitoring osteoporosis treatment to confirm compliance with oral therapies, and efficacy of treatment has been established. Further studies with reference to serum leptin and BTMs in post menopausal osteoporotic females are needed to clarify their association and significance of that association in treatment targets for various therapies and

and Abid Azhar3

\*Address all correspondence to: meher\_khan555@hotmail.com; drmukhtiarbaig@yahoo.com;

1 Pharmaceutical Research Centre, Pakistan Council of Scientific and Industrial Research

2 Department of Biochemistry, Bahria University Medical and Dental College (BUMDC),

3 Karachi Institute of Biotechnology and Genetic Engineering (KIBGE), Karachi, Pakistan

decrease in bone resorption. The reasons for these discrepancies need to be clarified.

the exact roles of leptin on bone metabolism have not yet been elucidated.

**8. Conclusion**

optimal monitoring regimes.

Mehreen Lateef1\*, Mukhtiar Baig2

Complex Laboratories Complex, PCSIR, Karachi, Pakistan

abid.azhar@kibge.edu.pk

**Author details**

Karachi, Pakistan

### **7. Relationship of leptin with bone markers in post menopausal osteoporotic females**

Data in the literature are inconsistent and conflicting about the relationship of leptin with bone markers in post menopausal osteoporotic females. The study of Goulding & Taylor (1998) was the first to examine relationships among plasma levels of leptin, and dynamic bi‐ ochemical markers of bone cell activity in postmenopausal women [46]. This study demon‐ strated no association between circulating plasma levels of leptin and biochemical markers of either osteoclastic or osteoblastic activity. They concluded that leptin itself does not play any significant direct role in controlling bone cell activity in postmenopausal women.

Scariano et al reported positive association between serum leptin and bone specific alkaline phosphatase in postmenopausal women and elderly men after adjustment for BMD, age and BMI [105]. The association of circulating leptin levels with bAP, a specific marker of osteo‐ blast activity suggests that leptin levels influence osteoblast activity in vivo in elderly wom‐ en and men. In a cross sectional study by Filip R & Raszewski G (2009) a positive association between leptin and osteocalcin in older patients with hip fracture [106]. Rauch et al. and La‐ teef et al also found no relationship between plasma leptin level and bone turnover markers in adult women and postmenopausal osteoporotic females respectively [40,59]. Filip & Ras‐ zewski et al, found no correlations of serum leptin with lumber spine BMD, femoral neck BMD, biochemical markers of bone turnover with leptin, in overweight and obese postme‐ nopausal women, even after stratification of the study group by BMI ratio value (25–29 9, 30–39 9 and ≥ 40), or by waist: hip ratio (WHR), ratio value (< 0 85 and ≥ 0 85) [106]. In a small study, Iwamoto et al. (2000) found correlations between serum leptin and bone re‐ modelling markers only in premenopausal women [107]. Peng et al (2008) reported no asso‐ ciation between serum leptin and bone turnover biochemical markers in men [108].

In postmenopausal osteoporotic patients with increased bone turnover, serum leptin con‐ centration is not correlated with BMD or with the biomarkers of bone formation or bone re‐ sorption [109]. According to few studies performed in China no correlation found between serum leptin and bone turnover biochemical markers in post-menopausal Chinese women [110-112]. Similarly, no correlation observed between leptin and bone turnover markers in Chinese adolescent dancers and control group in one more study (101) Blain, et al (2002) re‐ ported that serum leptin level was positively correlated with weight, fat mass, BMI, E2, crea‐ tinine clearance, and BAP level and inversely correlated with urine CTx [56]. They supported the suggestion that circulating leptin exerts its protective effect on bone through limiting the excessive bone resorption coupled with bone formation that is associated with bone loss after menopause.

Prouteau et al (2006) suggested a regulatory role of leptin on type I collagen metabolism [113]. The negative association between bone resorption (CTx levels) and serum leptin levels observed at baseline (stable body weight) was further confirmed by the biochemical changes occurring in responses to weight loss and weight regain. The drop in leptin levels was strongly related to the increase in bone resorption marker occurring in response to weight loss. Similarly, after weight regain the rise in leptin levels was associated with a concomitant decrease in bone resorption. The reasons for these discrepancies need to be clarified.

### **8. Conclusion**

independent factor. Similarly it must not be used to select the most appropriate type of

Data in the literature are inconsistent and conflicting about the relationship of leptin with bone markers in post menopausal osteoporotic females. The study of Goulding & Taylor (1998) was the first to examine relationships among plasma levels of leptin, and dynamic bi‐ ochemical markers of bone cell activity in postmenopausal women [46]. This study demon‐ strated no association between circulating plasma levels of leptin and biochemical markers of either osteoclastic or osteoblastic activity. They concluded that leptin itself does not play any significant direct role in controlling bone cell activity in postmenopausal women.

Scariano et al reported positive association between serum leptin and bone specific alkaline phosphatase in postmenopausal women and elderly men after adjustment for BMD, age and BMI [105]. The association of circulating leptin levels with bAP, a specific marker of osteo‐ blast activity suggests that leptin levels influence osteoblast activity in vivo in elderly wom‐ en and men. In a cross sectional study by Filip R & Raszewski G (2009) a positive association between leptin and osteocalcin in older patients with hip fracture [106]. Rauch et al. and La‐ teef et al also found no relationship between plasma leptin level and bone turnover markers in adult women and postmenopausal osteoporotic females respectively [40,59]. Filip & Ras‐ zewski et al, found no correlations of serum leptin with lumber spine BMD, femoral neck BMD, biochemical markers of bone turnover with leptin, in overweight and obese postme‐ nopausal women, even after stratification of the study group by BMI ratio value (25–29 9, 30–39 9 and ≥ 40), or by waist: hip ratio (WHR), ratio value (< 0 85 and ≥ 0 85) [106]. In a small study, Iwamoto et al. (2000) found correlations between serum leptin and bone re‐ modelling markers only in premenopausal women [107]. Peng et al (2008) reported no asso‐

ciation between serum leptin and bone turnover biochemical markers in men [108].

In postmenopausal osteoporotic patients with increased bone turnover, serum leptin con‐ centration is not correlated with BMD or with the biomarkers of bone formation or bone re‐ sorption [109]. According to few studies performed in China no correlation found between serum leptin and bone turnover biochemical markers in post-menopausal Chinese women [110-112]. Similarly, no correlation observed between leptin and bone turnover markers in Chinese adolescent dancers and control group in one more study (101) Blain, et al (2002) re‐ ported that serum leptin level was positively correlated with weight, fat mass, BMI, E2, crea‐ tinine clearance, and BAP level and inversely correlated with urine CTx [56]. They supported the suggestion that circulating leptin exerts its protective effect on bone through limiting the excessive bone resorption coupled with bone formation that is associated with

Prouteau et al (2006) suggested a regulatory role of leptin on type I collagen metabolism [113]. The negative association between bone resorption (CTx levels) and serum leptin levels observed at baseline (stable body weight) was further confirmed by the biochemical changes

**7. Relationship of leptin with bone markers in post menopausal**

osteoporotic therapy for the treatment. [104].

**osteoporotic females**

76 Topics in Osteoporosis

bone loss after menopause.

The relationship of leptin and bone turnover markers in post menopausal osteoporosis has not yet been clarified. Although several studies have been done but still there is need to explore their exact connection. Many studies have recommended that in the treatment of the post-men‐ opausal women, biochemical markers of bone turnover may be useful as adjuncts to BMD and other diagnostic tests. They can be mainly used to monitor response to treatment and also used as relatively economical tools for studying bone metabolism. The exact roles of BTM need to be established in clinical practice. It is suggested that repeated measurements of bone markers during anti-resorptive therapy may help to improve the management of osteoporotic patients.

Both a peripheral and a central action of leptin on bone metabolism have been suggested. Pe‐ ripherally, leptin is thought to exert positive effects on bone formation, whereas it is thought to reduce bone formation via a central control mechanism when binding to its specific receptors located on the hypothalamic nuclei [26]. It has been suggested that circulating leptin may act positively to maintain bone mass but these effects of serum leptin are not mediated due to these biochemical markers. Despite a preliminary understanding of leptin–bone mass interactions, the exact roles of leptin on bone metabolism have not yet been elucidated.

The role of BTMs in monitoring osteoporosis treatment to confirm compliance with oral therapies, and efficacy of treatment has been established. Further studies with reference to serum leptin and BTMs in post menopausal osteoporotic females are needed to clarify their association and significance of that association in treatment targets for various therapies and optimal monitoring regimes.

### **Author details**

Mehreen Lateef1\*, Mukhtiar Baig2 and Abid Azhar3

\*Address all correspondence to: meher\_khan555@hotmail.com; drmukhtiarbaig@yahoo.com; abid.azhar@kibge.edu.pk

1 Pharmaceutical Research Centre, Pakistan Council of Scientific and Industrial Research Complex Laboratories Complex, PCSIR, Karachi, Pakistan

2 Department of Biochemistry, Bahria University Medical and Dental College (BUMDC), Karachi, Pakistan

3 Karachi Institute of Biotechnology and Genetic Engineering (KIBGE), Karachi, Pakistan

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[108] Peng XD, Xie H, Zhao Q, et al.Relationships between serum adiponectin, leptin, re‐ sistin, visfatin levels and bone mineral density, and bone biochemical markers in Chinese men. ClinicaChimicaActa 2008; 387: 31-35.

**Chapter 4**

**Modification of Sex Hormones with RGD-Peptide: A**

In the time of transition from premenopausal state to postmenopausal state the capacity of ovary producing sex hormones including estrogens, progesterone and testosterone cuts down [1]. Due to the menopause the level of serum oestrogen dramatically decreases, which increases the production of bone-resorbing cytokines and osteoblasts and then increases the number and activity of osteoclast, thereby increasing the bone loss [2]. Hormonal replacement therapy (HRT) is able to prevent bone loss for sex hormones-deficient menopausal women and consequently is of clinical importance for the treatment of osteoporosis. [1-3] In Europe and USA the osteoporosis prevention of 25-50% of the post-menopausal women rely on HRT [2,5, 6]. In past years, however, the large international studies, such as the randomized Woman Health Initiative, the observational Million Women Study and the Women's International Study of long Duration, discussed both of the adverse and beneficial effects of post-menpausal HRT [7]. In respect of the adverse effects, the discussion was focused on HRT induced risk of breast cancer [8-11], venous thromboembolism [12], stroke and myocardial infarction [13], as well as coronary heart diseases [14]. To limit these adverse effects a series of regimens of HRT, such as continuous combination of oestrogen and progestogen or continuous oestrogen and interruptted progestogen [15], and with dehydroepiandrosterone as a new strategic tool [16], were developed. In general these regimens confer no positive result, and thus new strategies

Osteoporosis relates to both the decrease of the formation of osteoblast-modulated bone and the increase of the resorption osteoclast-modulated bone. Estrogen directly up-modulates the activity and the proliferation of osteoblasts, and/or regulats the gene expression in osteoblasts

and reproduction in any medium, provided the original work is properly cited.

© 2013 Zhao et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

**Strategy of Improving HRT and Other Secondary**

Ming Zhao, Yuji Wang, Jianhui Wu and Shiqi Peng

Additional information is available at the end of the chapter

**Osteoporosis Therapy**

http://dx.doi.org/10.5772/54361

**1. Introduction**

are still needed.


## **Modification of Sex Hormones with RGD-Peptide: A Strategy of Improving HRT and Other Secondary Osteoporosis Therapy**

Ming Zhao, Yuji Wang, Jianhui Wu and Shiqi Peng

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54361

### **1. Introduction**

[108] Peng XD, Xie H, Zhao Q, et al.Relationships between serum adiponectin, leptin, re‐ sistin, visfatin levels and bone mineral density, and bone biochemical markers in

[109] Shaarawy M, Abassi AE, Hassan H, et al.Relationship between serumleptinconcen‐ trations and bonemineraldensity as well as biochemicalmarkers of boneturnover in women with postmenopausalosteoporosis. Fertility & Sterility 2003; 79: 919-924. [110] Zhang H, Xie H, Zhao Q et al. Relationships between serum adiponectin, apelin, lep‐ tin, resistin, visfatin levels and bone mineral density, and bone biochemical markers in post-menopausal Chinese women. Journal of Endocrinological Investigation 2010;

[111] Wu N, Wang QP, Li H, et al. Relationships between serum adiponectin, leptin con‐ centrations and bone mineral density, and bone biochemical markers in Chinese

[112] Yang LC, Lan Y, Hu J et al. Correlation of serumleptinlevel with bonemineraldensity and boneturnovermarkers in Chineseadolescentdancers. Biomedicaland Enviromen‐

[113] Prouteau S, L Benhamou L, Courteix D. Relationships between serum leptin and bone markers during stable weight, weight reduction and weight regain in male and

female judoists. European Journal of Endocrinology 2006; 154: 389–395.

Chinese men. ClinicaChimicaActa 2008; 387: 31-35.

women. ClinicaChemicaActa 2010; 411:771-775.

talScience; 2009; 22:369-373.

33:707-711.

86 Topics in Osteoporosis

In the time of transition from premenopausal state to postmenopausal state the capacity of ovary producing sex hormones including estrogens, progesterone and testosterone cuts down [1]. Due to the menopause the level of serum oestrogen dramatically decreases, which increases the production of bone-resorbing cytokines and osteoblasts and then increases the number and activity of osteoclast, thereby increasing the bone loss [2]. Hormonal replacement therapy (HRT) is able to prevent bone loss for sex hormones-deficient menopausal women and consequently is of clinical importance for the treatment of osteoporosis. [1-3] In Europe and USA the osteoporosis prevention of 25-50% of the post-menopausal women rely on HRT [2,5, 6]. In past years, however, the large international studies, such as the randomized Woman Health Initiative, the observational Million Women Study and the Women's International Study of long Duration, discussed both of the adverse and beneficial effects of post-menpausal HRT [7]. In respect of the adverse effects, the discussion was focused on HRT induced risk of breast cancer [8-11], venous thromboembolism [12], stroke and myocardial infarction [13], as well as coronary heart diseases [14]. To limit these adverse effects a series of regimens of HRT, such as continuous combination of oestrogen and progestogen or continuous oestrogen and interruptted progestogen [15], and with dehydroepiandrosterone as a new strategic tool [16], were developed. In general these regimens confer no positive result, and thus new strategies are still needed.

Osteoporosis relates to both the decrease of the formation of osteoblast-modulated bone and the increase of the resorption osteoclast-modulated bone. Estrogen directly up-modulates the activity and the proliferation of osteoblasts, and/or regulats the gene expression in osteoblasts

© 2013 Zhao et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and osteoclasts [17-20]. Bone resorption is regulated by the adhesion of osteoclasts to the surface of the bone, which is mediated by the receptor αvβ3 integrin and its recognition to RGD (Arg-Gly-Asp) containing protein of osteoclasts [21]. These suggest that the activity and proliferation of osteoblasts and the adhesiveness of osteoclasts can be simultaneously upregulated with estrogen and down-regulated with RGD peptide, respectively. On the other hand, it was explored that the covalent modifications of hydrocortisone and estrone with kyotorphin (a dipeptide, Tyr-Arg) may increase the analgesic activities of hydrocortisone and estrone [22], as well as the covalent modifications of hydrocortisone and prednisolone with urotoxins (Gly-Asp-Gly, His-Gly-Gly, His-Gly-Lys and His-Gly-Lys-NHNH2) may increase the immunosuppressive activities of hydrocortisone and prednisolone [23]. Similarly, the antiosteoporosis activities of estrone and estradiol were enhanced by growth hormone releasing peptides (GHRPs: Tyr-Gly-Gly-Phe-Met-NH2, Tyr-Gly-Gly-Phe-Met, Tyr-Gly-Gly-Phe-Leu-NH2, Tyr-Gly-Gly-Phe-Leu, Tyr-Gly-Gly-Phe-Gly-NH2 and Tyr-Gly-Gly-Phe-Gly) [24-26]. In this context a strategy to enhance anti-osteoporosis potency and reduce adverse effects of HRT was practiced by covalent modifications of sex hormone with RGD-peptides.

**2.1. Covalent modification of estradiol with RGD-tetrapeptides decreasing bone turnover**

Modification of Sex Hormones with RGD-Peptide: A Strategy of Improving HRT and Other Secondary…

http://dx.doi.org/10.5772/54361

89

Using succinyl group as the linker the covalent modifications of the 17β-hydroxy of estradiol with RGD-tetrapeptides provided conjugates **1-3**, and using carbonylmethyl group as the linker the covalent modifications of the 3-hydroxy of estradiol or estrone with RGD-tetrapep‐ tides provided conjugates **4**-**9** (Figure 1). The changes of the levels of the serum calcium and serum alkaline phosphatase (ALP) of the mice receiving ip injection of **1-6** for 4 weeks are shown in Figure 2. After the treatments of conjugates **1-6** the levels of serum calcium and serum ALP of the treated mice are significantly lower than that of ovariotomy and estradiol treated mice. This means that the ip injection efficacy of conjugates **1-6** in decreasing the serum calcium and serum ALP is significantly higher than that of estradiol. Due to serum ALP been the biomarker of bone turnover low serum ALP means conjugates **1-6** benefits the inhibition of

**Figure 2.** Serum calcium and serum ALP of **1-6** treated mice. Dose = 110.3 μmol/kg, n=12, a) Compared to ovariotomy P<0.05; b) Compared to ovariotomy P<0.01; c) Compared to ovariotomy and estradiol P<0.01. The statistical analysis

The effects of ip injection of **1-6** for 4 weeks on the bone loss of the mice are shown in Figure 3. The level of bone loss is represented with the weight of dry femur and the weight of femur ash. The data indicate that the weight of dry femur and the weight of femur ash of **1-6** treated mice are significantly higher than those of ovariotomy and estradiol treated mice. This means that ip injection efficacy of **1-6** in inhibiting the bone loss is significantly higher than that of estradiol, and the covalent modification of estradiol with RGD-tetrapeptides benefits the

**2.3. Covalent modification of estrone with RGD-tetrapeptides inhibiting bone turnover**

Using carbonylmethyl group as the linker the covalent modifications of the 3-hydroxy of estrone with RGD-tetrapeptides provided conjugates **7**-**9** (Figure 4). The effects of ip injection

**2.2. Covalent modification of estradiol with RGD-tetrapeptides inhibiting bone loss**

of the data was carried out by use of an ANOVA test and p<0.05 was considered significant.

bone turnover.

inhibition of bone loss.

### **2. Covalent modifications of estrogen with RGD-peptides and ip treated ovariectomy mice**

Estrogens including estrone, estradiol, estriol, conjugated estrogen and tibolone have been widely used in HRT. Upon the promotion of the enzyme both estrone and estradiol can be converted to ertriol. Conjugated estrogen is an oral estrogen isolated from the urine of gravid horse and contains estrone monosodium sulfate (50.0% - 63.0%), equilin monosodium sulfate (22.5% - 32.5%), a few of 17α-estradiol monosodium sulfate and equilenin monosodium sulfate. Tibolone is an analog of norethynodrel. Of these estrogens, estrone and estradiol are the common parents and estradiol is the major agents of HRT. Thus estradiol and estrone were covalently modified by RGD-peptides (**1-9**, Figure 1) and evaluated with ip treated ovariec‐ tomy mice [27].

**Figure 1.** Structures of conjugates of estradiol-RGD-tetrapeptides. In **1**, **4,7** AA = Ser, in **2**, **5, 8** AA = Val, in **3**, **6, 9** AA = Phe.

### **2.1. Covalent modification of estradiol with RGD-tetrapeptides decreasing bone turnover**

and osteoclasts [17-20]. Bone resorption is regulated by the adhesion of osteoclasts to the surface of the bone, which is mediated by the receptor αvβ3 integrin and its recognition to RGD (Arg-Gly-Asp) containing protein of osteoclasts [21]. These suggest that the activity and proliferation of osteoblasts and the adhesiveness of osteoclasts can be simultaneously upregulated with estrogen and down-regulated with RGD peptide, respectively. On the other hand, it was explored that the covalent modifications of hydrocortisone and estrone with kyotorphin (a dipeptide, Tyr-Arg) may increase the analgesic activities of hydrocortisone and estrone [22], as well as the covalent modifications of hydrocortisone and prednisolone with urotoxins (Gly-Asp-Gly, His-Gly-Gly, His-Gly-Lys and His-Gly-Lys-NHNH2) may increase the immunosuppressive activities of hydrocortisone and prednisolone [23]. Similarly, the antiosteoporosis activities of estrone and estradiol were enhanced by growth hormone releasing peptides (GHRPs: Tyr-Gly-Gly-Phe-Met-NH2, Tyr-Gly-Gly-Phe-Met, Tyr-Gly-Gly-Phe-Leu-NH2, Tyr-Gly-Gly-Phe-Leu, Tyr-Gly-Gly-Phe-Gly-NH2 and Tyr-Gly-Gly-Phe-Gly) [24-26]. In this context a strategy to enhance anti-osteoporosis potency and reduce adverse effects of HRT

was practiced by covalent modifications of sex hormone with RGD-peptides.

**ovariectomy mice**

88 Topics in Osteoporosis

tomy mice [27].

Phe.

**2. Covalent modifications of estrogen with RGD-peptides and ip treated**

Estrogens including estrone, estradiol, estriol, conjugated estrogen and tibolone have been widely used in HRT. Upon the promotion of the enzyme both estrone and estradiol can be converted to ertriol. Conjugated estrogen is an oral estrogen isolated from the urine of gravid horse and contains estrone monosodium sulfate (50.0% - 63.0%), equilin monosodium sulfate (22.5% - 32.5%), a few of 17α-estradiol monosodium sulfate and equilenin monosodium sulfate. Tibolone is an analog of norethynodrel. Of these estrogens, estrone and estradiol are the common parents and estradiol is the major agents of HRT. Thus estradiol and estrone were covalently modified by RGD-peptides (**1-9**, Figure 1) and evaluated with ip treated ovariec‐

**Figure 1.** Structures of conjugates of estradiol-RGD-tetrapeptides. In **1**, **4,7** AA = Ser, in **2**, **5, 8** AA = Val, in **3**, **6, 9** AA =

Using succinyl group as the linker the covalent modifications of the 17β-hydroxy of estradiol with RGD-tetrapeptides provided conjugates **1-3**, and using carbonylmethyl group as the linker the covalent modifications of the 3-hydroxy of estradiol or estrone with RGD-tetrapep‐ tides provided conjugates **4**-**9** (Figure 1). The changes of the levels of the serum calcium and serum alkaline phosphatase (ALP) of the mice receiving ip injection of **1-6** for 4 weeks are shown in Figure 2. After the treatments of conjugates **1-6** the levels of serum calcium and serum ALP of the treated mice are significantly lower than that of ovariotomy and estradiol treated mice. This means that the ip injection efficacy of conjugates **1-6** in decreasing the serum calcium and serum ALP is significantly higher than that of estradiol. Due to serum ALP been the biomarker of bone turnover low serum ALP means conjugates **1-6** benefits the inhibition of bone turnover.

**Figure 2.** Serum calcium and serum ALP of **1-6** treated mice. Dose = 110.3 μmol/kg, n=12, a) Compared to ovariotomy P<0.05; b) Compared to ovariotomy P<0.01; c) Compared to ovariotomy and estradiol P<0.01. The statistical analysis of the data was carried out by use of an ANOVA test and p<0.05 was considered significant.

#### **2.2. Covalent modification of estradiol with RGD-tetrapeptides inhibiting bone loss**

The effects of ip injection of **1-6** for 4 weeks on the bone loss of the mice are shown in Figure 3. The level of bone loss is represented with the weight of dry femur and the weight of femur ash. The data indicate that the weight of dry femur and the weight of femur ash of **1-6** treated mice are significantly higher than those of ovariotomy and estradiol treated mice. This means that ip injection efficacy of **1-6** in inhibiting the bone loss is significantly higher than that of estradiol, and the covalent modification of estradiol with RGD-tetrapeptides benefits the inhibition of bone loss.

### **2.3. Covalent modification of estrone with RGD-tetrapeptides inhibiting bone turnover**

Using carbonylmethyl group as the linker the covalent modifications of the 3-hydroxy of estrone with RGD-tetrapeptides provided conjugates **7**-**9** (Figure 4). The effects of ip injection

**Figure 3.** Weight of dry femur and femur ash of **1-6** treated mice. Dose =110.3 μmol/kg, n=12; a) Compared to ovar‐ iotomy P<0.01; b) Compared to ovariotomy and estradiol P<0.01; c) Compared to ovariotomy and estradiol P<0.05; d) Compared to ovariotomy P<0.01, to estradiol P<0.05. The statistical analysis of the data was carried out by use of an ANOVA test and p<0.05 was considered significant.

**Figure 5.** Weight of dry femur and femur ash of conjugates **7-9** treated mice. Dose =110.3 μmol/kg, n=12; a) Com‐ pared to ovariotomy P<0.01; b) Compared to ovariotomy P<0.05; c) Compared to ovariotomy P<0.01, to estrone P<0.05. The statistical analysis of the data was carried out by use of an ANOVA test and p<0.05 was considered signifi‐

Modification of Sex Hormones with RGD-Peptide: A Strategy of Improving HRT and Other Secondary…

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91

**2.5. Covalent modification of estrogen with RGD-tetrapeptides inducing no endometrial**

The effects of ip injection of **1-9** for 4 weeks on endometrial cell hyperplasia of the mice were also observed. The weight of the uteri of ovariotomy, estradiol and estrone treated mice is significantly higher than that of **1**-**9** treated mice. Due to the weight of the uteri reflecting the level of endometrial cell hyperplasia of treated mice this comparison means that ip injection efficacy of **1-9** in inducing endometrial cell hyperplasia is significantly lower than that of estradiol and estrone and the covalent modification induces no observable endometrial cell

With RGD-tetrapeptides modifying one hydroxyl group of estradiol and estrone resulted in 9 conjugates. On ovariotomy mouse model and at 110.3 μmol/kg of ip dose their anti-osteopo‐ rosis activities were significantly higher than that of estradiol and estrone themselves. In contrast to estradiol and estrone themselves, the anti-osteoporosis therapy of these conjugates induced no endometrial cell hyperplasia. It is commonly accepted that osteoporosis relates to both the decrease in bone formation modulated by osteoblasts and the increase in bone resorption modulated by osteoclasts. In HRT, estradiol and estrone are used to treat the decrease in skeletal muscle and bone by the direct modulation of osteoblastic activity and proliferation or by the regulation of gene expression in osteoblasts and osteoclasts. Bone resorption is regulated by the binding of osteoclasts to the bone surface and, therefore, depends upon osteoclast adhesiveness. This bone adhesion process is mediated by RGD-tetrapeptides binding integrin receptor on cell surface. This action of RGD-tetrapeptides should be respon‐ sible for both the increased anti-osteoporosis activity and the decreased endometrial cell hyperplasia of the conjugates. Due to ovariotomy mouse model simulates the bone loss

**2.6. Summary of covalent modification of estrogen with RGD-tetrapeptides**

cant.

**cell hyperplasia**

hyperplasia.

of **7-9** for 4 weeks on serum calcium and serum ALP of the mice are shown in Figure 4. The serum calcium and serum ALP of **7-9** treated mice are significantly lower than that of ovar‐ iotomy and estrone treated mice. This means that the ip injection efficacy of conjugates **7-9** in decreasing the serum calcium and serum ALP is significantly higher than that of estrone. Due to serum ALP reflecting the level of bone turnover and low serum ALP corresponding with low bone turnover, **7-9** benefits the inhibition of bone turnover.

**Figure 4.** Serum calcium and ALP of **7-9** treated mice. Dose = 110.3 μmol/kg, n=12, a) Compared to ovariotomy P<0.05; b) Compared to ovariotomy P<0.01; c) Compared to ovariotomy and estrone P<0.01. The statistical analysis of the data was carried out by use of an ANOVA test and p<0.05 was considered significant.

### **2.4. Covalent modification of estrone with RGD-tetrapeptides preventing bone loss**

The effect of ip injection of **7-9** for 4 weeks on the bone loss of the mice is shown in Figure 5. The weight of dry femur and the weight of femur ash of **7-9** treated mice are significantly higher than that of ovariotomy and estrone treated mice. Due to the weight of dry femur and the weight of femur ash reflecting the level of bone loss of osteoporosis mice this comparison means that ip injection efficacy of **7-9** in inhibiting bone loss is significantly higher than that of estrone and the covalent modification enhances the inhibition of estrone in bone loss.

Modification of Sex Hormones with RGD-Peptide: A Strategy of Improving HRT and Other Secondary… http://dx.doi.org/10.5772/54361 91

**Figure 5.** Weight of dry femur and femur ash of conjugates **7-9** treated mice. Dose =110.3 μmol/kg, n=12; a) Com‐ pared to ovariotomy P<0.01; b) Compared to ovariotomy P<0.05; c) Compared to ovariotomy P<0.01, to estrone P<0.05. The statistical analysis of the data was carried out by use of an ANOVA test and p<0.05 was considered signifi‐ cant.

### **2.5. Covalent modification of estrogen with RGD-tetrapeptides inducing no endometrial cell hyperplasia**

The effects of ip injection of **1-9** for 4 weeks on endometrial cell hyperplasia of the mice were also observed. The weight of the uteri of ovariotomy, estradiol and estrone treated mice is significantly higher than that of **1**-**9** treated mice. Due to the weight of the uteri reflecting the level of endometrial cell hyperplasia of treated mice this comparison means that ip injection efficacy of **1-9** in inducing endometrial cell hyperplasia is significantly lower than that of estradiol and estrone and the covalent modification induces no observable endometrial cell hyperplasia.

### **2.6. Summary of covalent modification of estrogen with RGD-tetrapeptides**

of **7-9** for 4 weeks on serum calcium and serum ALP of the mice are shown in Figure 4. The serum calcium and serum ALP of **7-9** treated mice are significantly lower than that of ovar‐ iotomy and estrone treated mice. This means that the ip injection efficacy of conjugates **7-9** in decreasing the serum calcium and serum ALP is significantly higher than that of estrone. Due to serum ALP reflecting the level of bone turnover and low serum ALP corresponding with

**Figure 3.** Weight of dry femur and femur ash of **1-6** treated mice. Dose =110.3 μmol/kg, n=12; a) Compared to ovar‐ iotomy P<0.01; b) Compared to ovariotomy and estradiol P<0.01; c) Compared to ovariotomy and estradiol P<0.05; d) Compared to ovariotomy P<0.01, to estradiol P<0.05. The statistical analysis of the data was carried out by use of an

**Figure 4.** Serum calcium and ALP of **7-9** treated mice. Dose = 110.3 μmol/kg, n=12, a) Compared to ovariotomy P<0.05; b) Compared to ovariotomy P<0.01; c) Compared to ovariotomy and estrone P<0.01. The statistical analysis of

The effect of ip injection of **7-9** for 4 weeks on the bone loss of the mice is shown in Figure 5. The weight of dry femur and the weight of femur ash of **7-9** treated mice are significantly higher than that of ovariotomy and estrone treated mice. Due to the weight of dry femur and the weight of femur ash reflecting the level of bone loss of osteoporosis mice this comparison means that ip injection efficacy of **7-9** in inhibiting bone loss is significantly higher than that of estrone and the covalent modification enhances the inhibition of estrone in bone loss.

**2.4. Covalent modification of estrone with RGD-tetrapeptides preventing bone loss**

low bone turnover, **7-9** benefits the inhibition of bone turnover.

ANOVA test and p<0.05 was considered significant.

90 Topics in Osteoporosis

the data was carried out by use of an ANOVA test and p<0.05 was considered significant.

With RGD-tetrapeptides modifying one hydroxyl group of estradiol and estrone resulted in 9 conjugates. On ovariotomy mouse model and at 110.3 μmol/kg of ip dose their anti-osteopo‐ rosis activities were significantly higher than that of estradiol and estrone themselves. In contrast to estradiol and estrone themselves, the anti-osteoporosis therapy of these conjugates induced no endometrial cell hyperplasia. It is commonly accepted that osteoporosis relates to both the decrease in bone formation modulated by osteoblasts and the increase in bone resorption modulated by osteoclasts. In HRT, estradiol and estrone are used to treat the decrease in skeletal muscle and bone by the direct modulation of osteoblastic activity and proliferation or by the regulation of gene expression in osteoblasts and osteoclasts. Bone resorption is regulated by the binding of osteoclasts to the bone surface and, therefore, depends upon osteoclast adhesiveness. This bone adhesion process is mediated by RGD-tetrapeptides binding integrin receptor on cell surface. This action of RGD-tetrapeptides should be respon‐ sible for both the increased anti-osteoporosis activity and the decreased endometrial cell hyperplasia of the conjugates. Due to ovariotomy mouse model simulates the bone loss condition of postmenopausal women these RGD-tetrapeptides modified estradiol and estrone should be promising candidates for HRT use.

### **3. Covalent modification of estrogen with RGD-octapeptides and orally treated ovariectomy mice**

It was explored that the modification of RGD-tetrapeptides with oligopeptides usually increased their bioactivities [28, 29], suggesting the modification of RGD-tetrapeptides with RGD-tetrapeptides may result in increase of the activity of down-regulating proliferation of osteoblasts and the adhesiveness of osteoclasts. In this context estradiol and estrone were modified with RGD-octapeptides (**10-21**, Figure 6) to evaluate the oral activity on ovariectomy mice [30, 31].

**Figure 7.** Serum calcium and ALP of **10-15** treated mice. Dose = 110.3 nmol/kg, n=12, a) Compared to ovariotomy and estradiol P<0.01; b) Compared to ovariotomy P<0.01; c) Compared to ovariotomy P<0.01, to estradiol P<0.05. The statistical analysis of the data was carried out by use of an ANOVA test and p<0.05 was considered significant.

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93

The effect of orally administration of **10-15** for 4 weeks on the bone loss of the treated mice is shown in Figure 8, of which the activity is represented with dry femur weight and femur ash weight. The data indicate that both the weights of dry femur and femur ash of **10-15** treated mice are significantly higher than that of ovariotomy and estradiol treated mice. This means that when orally dosed **10-15** effectively inhibit the mice to lose femur and their efficacy is significantly higher than that of estradiol, and the covalent modification of estradiol benefits

**Figure 8.** Weight of dry femur and femur ash of conjugates **10-15** treated mice. Dose =110.3 nmol/kg, n=12; a) Com‐ pared to ovariotomy P<0.05; b) Compared to ovariotomy and estradiol P<0.01. The statistical analysis of the data was

**3.3. Covalent modification of estrone with RGD-octapeptides inhibiting bone turnover**

Using carbonylmethyl group as the linker the 3-hydroxy of estrone was modified with RGDoctapeptides and provided **16**-**18** (Figure 6). The effects of oral administration of **16-18** for 4 weeks on serum calcium and serum ALP of the mice are shown in Figure 9. The data indicate

carried out by use of an ANOVA test and p<0.05 was considered significant.

**3.2. Covalent modification of estradiol with RGD-octapeptides preventing bone loss**

the inhibition of bone loss.

**Figure 6.** Structures of conjugates of RGD-octapeptides and estradiol. In **10**, **13, 16, 19** AA = Ser, in **11**, **14**, **17, 20** AA = Val, in **12**, **15, 18, 21** AA = Phe.

#### **3.1. Covalent modification of estradiol with RGD-octapeptides inhibiting bone turnover**

Using succinyl group as the linker the 17β-hydroxy of estradiol was modified with RGDoctapeptides and provided **10-12**, using carbonylmethyl group as the linker the 3-hydroxy of estradiol was modified with RGD-octapeptides and provided **13**-**15** (Figure 6). The effect of oral administration of **10-15** for 4 weeks on serum calcium and serum ALP of the mice are shown in Figure 7. The data indicate that the serum calcium and serum ALP of **10-15** treated mice are significantly lower than that of ovariotomy and estradiol treated mice. This means that the frequency of bone turnover of **10-15** orally treated mice is significantly lower than that of estradiol treated mice, the efficacy of oral **10-15** in inhibiting bone turnover is significantly higher than that of estradiol.

Modification of Sex Hormones with RGD-Peptide: A Strategy of Improving HRT and Other Secondary… http://dx.doi.org/10.5772/54361 93

condition of postmenopausal women these RGD-tetrapeptides modified estradiol and estrone

**3. Covalent modification of estrogen with RGD-octapeptides and orally**

It was explored that the modification of RGD-tetrapeptides with oligopeptides usually increased their bioactivities [28, 29], suggesting the modification of RGD-tetrapeptides with RGD-tetrapeptides may result in increase of the activity of down-regulating proliferation of osteoblasts and the adhesiveness of osteoclasts. In this context estradiol and estrone were modified with RGD-octapeptides (**10-21**, Figure 6) to evaluate the oral activity on ovariectomy

**Figure 6.** Structures of conjugates of RGD-octapeptides and estradiol. In **10**, **13, 16, 19** AA = Ser, in **11**, **14**, **17, 20** AA

**3.1. Covalent modification of estradiol with RGD-octapeptides inhibiting bone turnover**

Using succinyl group as the linker the 17β-hydroxy of estradiol was modified with RGDoctapeptides and provided **10-12**, using carbonylmethyl group as the linker the 3-hydroxy of estradiol was modified with RGD-octapeptides and provided **13**-**15** (Figure 6). The effect of oral administration of **10-15** for 4 weeks on serum calcium and serum ALP of the mice are shown in Figure 7. The data indicate that the serum calcium and serum ALP of **10-15** treated mice are significantly lower than that of ovariotomy and estradiol treated mice. This means that the frequency of bone turnover of **10-15** orally treated mice is significantly lower than that of estradiol treated mice, the efficacy of oral **10-15** in inhibiting bone turnover is significantly

should be promising candidates for HRT use.

**treated ovariectomy mice**

mice [30, 31].

92 Topics in Osteoporosis

= Val, in **12**, **15, 18, 21** AA = Phe.

higher than that of estradiol.

**Figure 7.** Serum calcium and ALP of **10-15** treated mice. Dose = 110.3 nmol/kg, n=12, a) Compared to ovariotomy and estradiol P<0.01; b) Compared to ovariotomy P<0.01; c) Compared to ovariotomy P<0.01, to estradiol P<0.05. The statistical analysis of the data was carried out by use of an ANOVA test and p<0.05 was considered significant.

### **3.2. Covalent modification of estradiol with RGD-octapeptides preventing bone loss**

The effect of orally administration of **10-15** for 4 weeks on the bone loss of the treated mice is shown in Figure 8, of which the activity is represented with dry femur weight and femur ash weight. The data indicate that both the weights of dry femur and femur ash of **10-15** treated mice are significantly higher than that of ovariotomy and estradiol treated mice. This means that when orally dosed **10-15** effectively inhibit the mice to lose femur and their efficacy is significantly higher than that of estradiol, and the covalent modification of estradiol benefits the inhibition of bone loss.

**Figure 8.** Weight of dry femur and femur ash of conjugates **10-15** treated mice. Dose =110.3 nmol/kg, n=12; a) Com‐ pared to ovariotomy P<0.05; b) Compared to ovariotomy and estradiol P<0.01. The statistical analysis of the data was carried out by use of an ANOVA test and p<0.05 was considered significant.

#### **3.3. Covalent modification of estrone with RGD-octapeptides inhibiting bone turnover**

Using carbonylmethyl group as the linker the 3-hydroxy of estrone was modified with RGDoctapeptides and provided **16**-**18** (Figure 6). The effects of oral administration of **16-18** for 4 weeks on serum calcium and serum ALP of the mice are shown in Figure 9. The data indicate that the serum calcium and serum ALP of **16-18** treated mice are significantly lower than that of ovariotomy and estrone treated mice. This means that the frequency of bone turnover of **16-18** orally treated mice is significantly lower than that of estrone treated mice, the efficacy of oral **16-18** in inhibiting bone turnover is significantly higher than that of estrone.

**3.5. Covalent modification of estradiol with two RGD-octapeptides inhibiting bone**

in inhibiting bone turnover is significantly higher than that of estradiol.

Using succinyl group as the linker of the 17β-hydroxy and using carbonylmethyl group as the linker of the 3-hydroxy estradiol was simultaneously modified with RGD-tetrapeptides and provided **19**-**21** (Figure 6). The effects of oral administration of **19-21** for 4 weeks on serum calcium and serum ALP of the mice are shown in Figure 11. The data indicate that the serum calcium and serum ALP of **19-21** treated mice are significantly lower than that of ovariotomy and estradiol treated mice. This means that the frequency of bone turnover of **19-21** orally treated mice is significantly lower than that of estradiol treated mice, the efficacy of oral **19-21**

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**Figure 11.** Serum calcium and serum ALP of **19-21** treated mice. Dose = 110.3 nmol/kg, n=12. The statistical analysis of the data was carried out by use of an ANOVA test and p<0.05 was considered significant. For serum calcium a)

**3.6. Covalent modification of estradiol with two RGD-octapeptides preventing bone loss**

The effect of orally administration of **19-21** for 4 weeks on the bone loss of the treated mice is shown in Figure 12, their activities are represented with dry femur weight and femur ash weight. The data indicate that both the weights of dry femur and femur ash of **19-21** treated mice are significantly higher than that of ovariotomy and estradiol treated mice. This means that upon oral administration **19-21** effectively inhibit the mice losing femur, their efficacies are significantly higher than that of estradiol, and the covalent modifica‐

**3.7. Covalent modification of estradiol with RGD-octapeptides inducing no endometrial**

The effect of orally administration of **10-21** for 4 weeks on the endometrial cell hyperplasia of the mice was observed, of which the inhibition is represented with uteri weight. The data indicate that the weight of the uteri of **10**-**21** treated mice is significantly lower than that of ovariotomy and estradiol treated mice. This means that, in contrast to estradiol and estrone, oral administration of **10-21** induces no observable endometrial cell hyperplasia, and the covalent modification of estradiol and estrone with RGD-octapeptides limits the dose-related

Compared to ovariotomy P<0.05; For serum ALP b) compared to ovariotomy and estradiol P<0.01.

tion of estrone prevents the bone loss.

**cell hyperplasia**

adverse effects of estradiol.

**turnover**

**Figure 9.** Serum calcium and serum ALP of **16-18** treated mice. Dose = 110.3 nmol/kg, n=12, a) Compared to ovariot‐ omy P<0.01, to estrone P<0.05; b) Compared to ovariotomy P<0.05; c) Compared to ovariotomy and estrone P<0.01. The statistical analysis of the data was carried out by use of an ANOVA test and p<0.05 was considered significant.

### **3.4. Covalent modification of estrone with RGD-octapeptides preventing bone loss**

The effect of orally administration of **16-18** for 4 weeks on the bone loss of the treated mice is shown in Figure 10, their activities are represented with dry femur weight and femur ash weight. The data indicate that both the weights of dry femur and femur ash of **16-18** treated mice are significantly higher than that of ovariotomy and estradiol treated mice. This means that upon oral administration **16-18** effectively inhibit the mice losing femur, their efficacies are significantly higher than that of estrone, and the covalent modification of estrone prevents the bone loss.

**Figure 10.** Weight of dry femur and femur ash of conjugates **16-18** treated mice. Dose =110.3 nmol/kg, n=12; a) Compared to ovariotomy and estrone P<0.01; b) Compared to ovariotomy P<0.01, to estrone P<0.05. The statistical analysis of the data was carried out by use of an ANOVA test and p<0.05 was considered significant.

### **3.5. Covalent modification of estradiol with two RGD-octapeptides inhibiting bone turnover**

that the serum calcium and serum ALP of **16-18** treated mice are significantly lower than that of ovariotomy and estrone treated mice. This means that the frequency of bone turnover of **16-18** orally treated mice is significantly lower than that of estrone treated mice, the efficacy

**Figure 9.** Serum calcium and serum ALP of **16-18** treated mice. Dose = 110.3 nmol/kg, n=12, a) Compared to ovariot‐ omy P<0.01, to estrone P<0.05; b) Compared to ovariotomy P<0.05; c) Compared to ovariotomy and estrone P<0.01. The statistical analysis of the data was carried out by use of an ANOVA test and p<0.05 was considered significant.

The effect of orally administration of **16-18** for 4 weeks on the bone loss of the treated mice is shown in Figure 10, their activities are represented with dry femur weight and femur ash weight. The data indicate that both the weights of dry femur and femur ash of **16-18** treated mice are significantly higher than that of ovariotomy and estradiol treated mice. This means that upon oral administration **16-18** effectively inhibit the mice losing femur, their efficacies are significantly higher than that of estrone, and the covalent modification of estrone prevents

**Figure 10.** Weight of dry femur and femur ash of conjugates **16-18** treated mice. Dose =110.3 nmol/kg, n=12; a) Compared to ovariotomy and estrone P<0.01; b) Compared to ovariotomy P<0.01, to estrone P<0.05. The statistical

analysis of the data was carried out by use of an ANOVA test and p<0.05 was considered significant.

**3.4. Covalent modification of estrone with RGD-octapeptides preventing bone loss**

the bone loss.

94 Topics in Osteoporosis

of oral **16-18** in inhibiting bone turnover is significantly higher than that of estrone.

Using succinyl group as the linker of the 17β-hydroxy and using carbonylmethyl group as the linker of the 3-hydroxy estradiol was simultaneously modified with RGD-tetrapeptides and provided **19**-**21** (Figure 6). The effects of oral administration of **19-21** for 4 weeks on serum calcium and serum ALP of the mice are shown in Figure 11. The data indicate that the serum calcium and serum ALP of **19-21** treated mice are significantly lower than that of ovariotomy and estradiol treated mice. This means that the frequency of bone turnover of **19-21** orally treated mice is significantly lower than that of estradiol treated mice, the efficacy of oral **19-21** in inhibiting bone turnover is significantly higher than that of estradiol.

**Figure 11.** Serum calcium and serum ALP of **19-21** treated mice. Dose = 110.3 nmol/kg, n=12. The statistical analysis of the data was carried out by use of an ANOVA test and p<0.05 was considered significant. For serum calcium a) Compared to ovariotomy P<0.05; For serum ALP b) compared to ovariotomy and estradiol P<0.01.

### **3.6. Covalent modification of estradiol with two RGD-octapeptides preventing bone loss**

The effect of orally administration of **19-21** for 4 weeks on the bone loss of the treated mice is shown in Figure 12, their activities are represented with dry femur weight and femur ash weight. The data indicate that both the weights of dry femur and femur ash of **19-21** treated mice are significantly higher than that of ovariotomy and estradiol treated mice. This means that upon oral administration **19-21** effectively inhibit the mice losing femur, their efficacies are significantly higher than that of estradiol, and the covalent modifica‐ tion of estrone prevents the bone loss.

### **3.7. Covalent modification of estradiol with RGD-octapeptides inducing no endometrial cell hyperplasia**

The effect of orally administration of **10-21** for 4 weeks on the endometrial cell hyperplasia of the mice was observed, of which the inhibition is represented with uteri weight. The data indicate that the weight of the uteri of **10**-**21** treated mice is significantly lower than that of ovariotomy and estradiol treated mice. This means that, in contrast to estradiol and estrone, oral administration of **10-21** induces no observable endometrial cell hyperplasia, and the covalent modification of estradiol and estrone with RGD-octapeptides limits the dose-related adverse effects of estradiol.

**4. Direct covalent modification of androgen with RGD-tetrapeptides**

tetrapeptides (**22-24**, Figure 13) [32].

Phe.

**turnover**

In the improvements of the efficacy of HRT, the anti-osteoporosis efficacy of androgen is found to be higher than that of estrogen, inducing no endometrial cell hyperplasia and having no thrombosis risk. Particularly in the research of androgen, 17*β*-amino-11*α*-hydroxyandrost-1,4 diene-3-one is disclosed as a new androgen. Comparing to estrone and estrogen 17*β*-ami‐ no-11*α*-hydroxyandrost-1,4-diene-3-one has higher anti-osteoporosis activity and raises no endometrial cell hyperplasia and thrombosis risk. Thus 17*β*-amino-11*α*-hydroxyandrost-1,4 diene-3-one is selected as the androgen and directly and covalently modified with RGD-

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97

**Figure 13.** Structures of conjugates of androgen and RGD-tetrapeptides. In **22** AA = Ser, in **23** AA = Val, in **24** AA =

**4.1. Direct covalent modification of androgen with RGD-tetrapeptides inhibiting bone**

of oral **22**-**24** in inhibiting bone turnover is significantly higher than that of estradiol.

**4.2. Direct covalent modification of androgen with RGD-tetrapeptides preventing bone loss**

The effect of oral administration of **22-24** plus intramuscular prednisone for 4 weeks on the bone loss of the treated mice is shown in Figure 15, their activities are represented with dry femur weight and femur ash weight. The data indicate that both the weights of dry femur and femur ash of oral adminis-tration of **22-24** plus intramuscular prednisone treated mice are significantly higher than that of intramuscular prednisone alone and oral administration of estradiol plus intramuscular prednisone treated mice. This means that upon oral administra‐

The direct covalent modification of the 17*β*-amino of 17*β*-amino-11*α*-hydroxyandrost-1,4 diene-3-one (androgen) with RGD-tetrapeptides provided **22**-**24** (Figure 13). The effect of oral administration of **22**-**24** plus intramuscular prednisone for 4 weeks on serum calcium and serum ALP of the mice is shown in Figure 14. The data indicate that the serum calcium and serum ALP of oral administration of **22**-**24** plus intramuscular prednisone treated mice are significantly lower than that of prednisone alone and oral administration of estradiol plus intramuscular prednisone treated mice. This means that the frequency of bone turnover of **22**-**24** orally treated mice is significantly lower than that of androgen treated mice, the efficacy

**Figure 12.** Weight of dry femur and femur ash of conjugates **19-21** treated mice. Dose =110.3 nmol/kg, n=12, weights of dry femurs and femur ashes are represented as X±SD mg; a) Compared to ovariotomy, estradiol P<0.01; b) Compared to ovariotomy P<0.01, to estradiol P<0.05.

### **3.8. Covalent modification of estradiol with RGD-octapeptides having no thrombosis risk**

The effect of orally administration of **10-21** for 4 weeks on thrombosis risk of the mice was observed, of which the risk is represented with tail bleeding time. The data indicate that the tail bleeding time of **10**-**21** treated mice is significantly longer than that of ovariotomy, estradiol and estrone treated mice. This means that, in contrast to estradiol and estrone, oral adminis‐ tration of **10-21** induces no observable thrombosis risk, and the covalent modification of estradiol and estrone with RGD-octapeptides limits the dose-related adverse effects of estradiol.

### **3.9. Summary of covalent modification of estrogen with RGD-octapeptides**

With RGD-octapeptides modifying one hydroxyl group of estradiol and estrone or with RGD-tetrapeptides simultaneously modifying two hydroxyl groups of estradiol resulted in 12 conjugates. On ovariotomy mouse model and at 110.3 nmol/kg of oral dose their antiosteoporosis activities were significantly higher than that of estradiol and estrone them‐ selves. In contrast to estradiol and estrone themselves, the anti-osteoporosis therapy of these conjugates induced no endometrial cell hyperplasia and thrombosis risk. Comparing to RGD-tetrapeptide modified estradiol and estrone the effective dose of RGD-octapeptide modified estradiol and estrone is 1000 folds lower. This means that the anti-osteoporosis efficacy of RGD-octapeptide modified estradiol and estrone is 1000 folds higher than that of RGD-tetrapeptide modified estradiol and estrone. Reasonably, this dramatically en‐ hanced efficacy could attitude to the introduction of RGD-octapeptides. Furthermore, due to ovariotomy mouse model simulates the bone loss condition of postmenopausal women and high activity these RGD-octapeptides modified estradiol and estrone should be preferentially promising candidates for HRT use.

### **4. Direct covalent modification of androgen with RGD-tetrapeptides**

In the improvements of the efficacy of HRT, the anti-osteoporosis efficacy of androgen is found to be higher than that of estrogen, inducing no endometrial cell hyperplasia and having no thrombosis risk. Particularly in the research of androgen, 17*β*-amino-11*α*-hydroxyandrost-1,4 diene-3-one is disclosed as a new androgen. Comparing to estrone and estrogen 17*β*-ami‐ no-11*α*-hydroxyandrost-1,4-diene-3-one has higher anti-osteoporosis activity and raises no endometrial cell hyperplasia and thrombosis risk. Thus 17*β*-amino-11*α*-hydroxyandrost-1,4 diene-3-one is selected as the androgen and directly and covalently modified with RGDtetrapeptides (**22-24**, Figure 13) [32].

**Figure 13.** Structures of conjugates of androgen and RGD-tetrapeptides. In **22** AA = Ser, in **23** AA = Val, in **24** AA = Phe.

**3.8. Covalent modification of estradiol with RGD-octapeptides having no thrombosis risk**

**Figure 12.** Weight of dry femur and femur ash of conjugates **19-21** treated mice. Dose =110.3 nmol/kg, n=12, weights of dry femurs and femur ashes are represented as X±SD mg; a) Compared to ovariotomy, estradiol P<0.01; b)

The effect of orally administration of **10-21** for 4 weeks on thrombosis risk of the mice was observed, of which the risk is represented with tail bleeding time. The data indicate that the tail bleeding time of **10**-**21** treated mice is significantly longer than that of ovariotomy, estradiol and estrone treated mice. This means that, in contrast to estradiol and estrone, oral adminis‐ tration of **10-21** induces no observable thrombosis risk, and the covalent modification of estradiol and estrone with RGD-octapeptides limits the dose-related adverse effects of

With RGD-octapeptides modifying one hydroxyl group of estradiol and estrone or with RGD-tetrapeptides simultaneously modifying two hydroxyl groups of estradiol resulted in 12 conjugates. On ovariotomy mouse model and at 110.3 nmol/kg of oral dose their antiosteoporosis activities were significantly higher than that of estradiol and estrone them‐ selves. In contrast to estradiol and estrone themselves, the anti-osteoporosis therapy of these conjugates induced no endometrial cell hyperplasia and thrombosis risk. Comparing to RGD-tetrapeptide modified estradiol and estrone the effective dose of RGD-octapeptide modified estradiol and estrone is 1000 folds lower. This means that the anti-osteoporosis efficacy of RGD-octapeptide modified estradiol and estrone is 1000 folds higher than that of RGD-tetrapeptide modified estradiol and estrone. Reasonably, this dramatically en‐ hanced efficacy could attitude to the introduction of RGD-octapeptides. Furthermore, due to ovariotomy mouse model simulates the bone loss condition of postmenopausal women and high activity these RGD-octapeptides modified estradiol and estrone should be

**3.9. Summary of covalent modification of estrogen with RGD-octapeptides**

preferentially promising candidates for HRT use.

Compared to ovariotomy P<0.01, to estradiol P<0.05.

estradiol.

96 Topics in Osteoporosis

### **4.1. Direct covalent modification of androgen with RGD-tetrapeptides inhibiting bone turnover**

The direct covalent modification of the 17*β*-amino of 17*β*-amino-11*α*-hydroxyandrost-1,4 diene-3-one (androgen) with RGD-tetrapeptides provided **22**-**24** (Figure 13). The effect of oral administration of **22**-**24** plus intramuscular prednisone for 4 weeks on serum calcium and serum ALP of the mice is shown in Figure 14. The data indicate that the serum calcium and serum ALP of oral administration of **22**-**24** plus intramuscular prednisone treated mice are significantly lower than that of prednisone alone and oral administration of estradiol plus intramuscular prednisone treated mice. This means that the frequency of bone turnover of **22**-**24** orally treated mice is significantly lower than that of androgen treated mice, the efficacy of oral **22**-**24** in inhibiting bone turnover is significantly higher than that of estradiol.

### **4.2. Direct covalent modification of androgen with RGD-tetrapeptides preventing bone loss**

The effect of oral administration of **22-24** plus intramuscular prednisone for 4 weeks on the bone loss of the treated mice is shown in Figure 15, their activities are represented with dry femur weight and femur ash weight. The data indicate that both the weights of dry femur and femur ash of oral adminis-tration of **22-24** plus intramuscular prednisone treated mice are significantly higher than that of intramuscular prednisone alone and oral administration of estradiol plus intramuscular prednisone treated mice. This means that upon oral administra‐

**Figure 14.** Serum calcium and ALP of **22-24** treated mice. ip Dose of prednisone (PDN): 6.3 mg/kg, twice a week; oral dose of **22-24**: 110 nmol/kg, once a day; oral dose of estradiol (E2): 110 nmol/kg, once a day; n = 12. a) Compared to NS + PND and E2 + PND p< 0.01; b) Compared to NS + PND p< 0.01, to E2 + PND p< 0.05; c) Compared to NS + PND p< 0.05; d) Compared to NS + PND and E2 + PND p< 0.01. The statistical analysis of the data was carried out by use of an ANOVA test and p<0.05 was considered significant.

tion **22-24** effectively inhibit the mice losing femur, their efficacies are significantly higher than that of estradiol, and the direct covalent modification of androgen prevents the bone loss.

femurs of oral administration of **22-24** plus intramuscular prednisone treated mice are significantly higher than that of the femurs of NS plus intramuscular prednisone treated mice. This means that upon oral administration **22-24** effectively prevent intramuscular adminis‐

**Figure 16.** Total vBMD and images of pQCT scanning at a distance from the proximal femur growth palate corre‐ sponding to < 6 % of the total length of the femur of **22-24** treated mice. ip Dose of prednisone (PDN): 6.3 mg/kg, twice a week; oral dose of **22-24**: 110 nmol/kg, once a day; n = 12. a) Compared to NS alone , NS + PND, 23 + PDN and 22 + PDN p< 0.01; b) Compared to NS alone , NS + PND and 22 + PDN c) Compared to NS alone and NS + PND p< 0.01. The statistical analysis of the data was carried out by use of an ANOVA test and p<0.05 was considered significant.

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**4.4. Direct covalent modification of androgen with RGD-tetrapeptides increasing trabecular**

Figure 17 indicates that the trabecular vBMD of the femurs of NS plus intramuscular admin‐ istration of prednisone treated mice is significantly lower than that of the femurs of NS alone treated mice. This means that prednisone effectively induces the mice to decrease trabecular vBMD. The trabecular vBMDs of the femurs of oral administration of **22-24** plus intramuscular administration of prednisone treated mice are significantly higher than those of the femurs of NS plus intramuscular administration of prednisone treated mice. This means that upon oral administration **22-24** effectively prevent intra-muscular administration of prednisone treated

**4.5. Direct covalent modification of androgen with RGD-tetrapeptides inducing no**

with RGD-tetrapeptides induces no dose-related adverse effects of estradiol.

The effect of oral administration of **22-24** plus intramuscular administration of prednisone for 4 weeks on the endometrial cell hyperplasia of the mice was observed, and their inhibition activities are represented with uteri weight. The data indicate that the weight of the uteri of oral administration of **22**-**24** plus intramuscular administration of prednisone treated mice is significantly lower than that of intramuscular administration of prednisone alone and oral administration of estradiol plus intramuscular administration of prednisone treated mice. This means that, in contrast to oral administration of estradiol, oral administration of **19-21** induces no observable endometrial cell hyperplasia, and the direct covalent modification of androgen

tration of prednisone treated mice decreasing total vBMD.

mice decreasing trabecular vBMD.

**endomtrial cell hyperplasia**

**vBMD**

**Figure 15.** Weight of dry femur and femur ash of conjugates **22-24** treated mice. ip Dose of prednisone (PDN): 6.3 mg/kg, twice a week; oral dose of **22-24**: 110 nmol/kg, once a day; oral dose of estradiol (E2): 110 nmol/kg, once a day; n = 12. a) Compared to NS + PND and E2 + PND p< 0.01; b) Compared to NS + PND and E2 + PND p< 0.05. The statistical analysis of the data was carried out by use of an ANOVA test and p<0.05 was considered significant.

### **4.3. Direct covalent modification of androgen with RGD-tetrapeptides increasing total vBMD**

CT measured 3D bone geometry and the size-independent vBMD, as well as pQCT quantita‐ tively measured 3D bone geometry and size-independent vBMD were used to represent the anti-osteoporosis efficacy of **22-24** and are shown in Figure 16. The data indicates that the total vBMD of the femurs of NS plus intramuscular prednisone treated mice is significantly lower than that of the femurs of NS alone treated mice. This means that intramuscular administration of prednisone effectively induces the mice to decrease the total vBMD. The total vBMDs of the Modification of Sex Hormones with RGD-Peptide: A Strategy of Improving HRT and Other Secondary… http://dx.doi.org/10.5772/54361 99

**Figure 16.** Total vBMD and images of pQCT scanning at a distance from the proximal femur growth palate corre‐ sponding to < 6 % of the total length of the femur of **22-24** treated mice. ip Dose of prednisone (PDN): 6.3 mg/kg, twice a week; oral dose of **22-24**: 110 nmol/kg, once a day; n = 12. a) Compared to NS alone , NS + PND, 23 + PDN and 22 + PDN p< 0.01; b) Compared to NS alone , NS + PND and 22 + PDN c) Compared to NS alone and NS + PND p< 0.01. The statistical analysis of the data was carried out by use of an ANOVA test and p<0.05 was considered significant.

femurs of oral administration of **22-24** plus intramuscular prednisone treated mice are significantly higher than that of the femurs of NS plus intramuscular prednisone treated mice. This means that upon oral administration **22-24** effectively prevent intramuscular adminis‐ tration of prednisone treated mice decreasing total vBMD.

tion **22-24** effectively inhibit the mice losing femur, their efficacies are significantly higher than that of estradiol, and the direct covalent modification of androgen prevents the bone loss.

ANOVA test and p<0.05 was considered significant.

98 Topics in Osteoporosis

**Figure 14.** Serum calcium and ALP of **22-24** treated mice. ip Dose of prednisone (PDN): 6.3 mg/kg, twice a week; oral dose of **22-24**: 110 nmol/kg, once a day; oral dose of estradiol (E2): 110 nmol/kg, once a day; n = 12. a) Compared to NS + PND and E2 + PND p< 0.01; b) Compared to NS + PND p< 0.01, to E2 + PND p< 0.05; c) Compared to NS + PND p< 0.05; d) Compared to NS + PND and E2 + PND p< 0.01. The statistical analysis of the data was carried out by use of an

**Figure 15.** Weight of dry femur and femur ash of conjugates **22-24** treated mice. ip Dose of prednisone (PDN): 6.3 mg/kg, twice a week; oral dose of **22-24**: 110 nmol/kg, once a day; oral dose of estradiol (E2): 110 nmol/kg, once a day; n = 12. a) Compared to NS + PND and E2 + PND p< 0.01; b) Compared to NS + PND and E2 + PND p< 0.05. The statistical analysis of the data was carried out by use of an ANOVA test and p<0.05 was considered significant.

**4.3. Direct covalent modification of androgen with RGD-tetrapeptides increasing total**

CT measured 3D bone geometry and the size-independent vBMD, as well as pQCT quantita‐ tively measured 3D bone geometry and size-independent vBMD were used to represent the anti-osteoporosis efficacy of **22-24** and are shown in Figure 16. The data indicates that the total vBMD of the femurs of NS plus intramuscular prednisone treated mice is significantly lower than that of the femurs of NS alone treated mice. This means that intramuscular administration of prednisone effectively induces the mice to decrease the total vBMD. The total vBMDs of the

**vBMD**

### **4.4. Direct covalent modification of androgen with RGD-tetrapeptides increasing trabecular vBMD**

Figure 17 indicates that the trabecular vBMD of the femurs of NS plus intramuscular admin‐ istration of prednisone treated mice is significantly lower than that of the femurs of NS alone treated mice. This means that prednisone effectively induces the mice to decrease trabecular vBMD. The trabecular vBMDs of the femurs of oral administration of **22-24** plus intramuscular administration of prednisone treated mice are significantly higher than those of the femurs of NS plus intramuscular administration of prednisone treated mice. This means that upon oral administration **22-24** effectively prevent intra-muscular administration of prednisone treated mice decreasing trabecular vBMD.

### **4.5. Direct covalent modification of androgen with RGD-tetrapeptides inducing no endomtrial cell hyperplasia**

The effect of oral administration of **22-24** plus intramuscular administration of prednisone for 4 weeks on the endometrial cell hyperplasia of the mice was observed, and their inhibition activities are represented with uteri weight. The data indicate that the weight of the uteri of oral administration of **22**-**24** plus intramuscular administration of prednisone treated mice is significantly lower than that of intramuscular administration of prednisone alone and oral administration of estradiol plus intramuscular administration of prednisone treated mice. This means that, in contrast to oral administration of estradiol, oral administration of **19-21** induces no observable endometrial cell hyperplasia, and the direct covalent modification of androgen with RGD-tetrapeptides induces no dose-related adverse effects of estradiol.

ogy, respirology, neurology, hematology, dermatology, gastroenterology, and transplant medicine. Chronic exposure to pharmacological doses of glucocorticoids causes multiple deleterious effects on osteopenia, osteoporosis and bone fracture. Prostate cancer is one of the most common diseases in the older men. After the surgery or radiation therapy the male patients with localized or metastatic prostate cancer are generally given ADT. Though male patients on ADT usually have good prognosis, osteoporosis is a very common consequence of this therapy and up to 20% of the patients will fracture within 5 years. To prevent osteoporotic fracture in the female patients treated with glucocorticoids and the male patients receiving ADT novel effective agents are needed. The ability of these RGDoctapeptides modified 17*β*-amino-11*α*-hydroxyandrost-1,4-diene-3-one to prevent predni‐ sone treated mouse developing osteoporosis suggests that these conjugates should be

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**5. Indirect covalent modification of androgen with RGD-tetrapeptides**

For androgen a parallel covalent modification with the direct covalent modification is an indirect strategy. In brief, between the 17*β*-amino group of 17*β*-amino-11*α*-hydroxyan‐ drost-1,4-diene-3-one and RGD-tetrapeptides as a linker, succinyl group, is inserted to provide

**Figure 18.** Structures of conjugates of androgen, succinyl group and RGD-tetrapeptides. In **25** AA = Ser, in **26** AA =

**5.1. Indirect covalent modification of androgen with RGD-tetrapeptides inhibiting bone**

The effect of oral administration of **25-27** plus intramuscular administration of prednisone for 4 weeks on serum calcium and serum ALP of the mice is shown in Figure 19. The data indicate that the serum calcium and serum ALP of oral administration of **25-27** plus intramuscular administration of prednisone treated mice are significantly lower than those of intramuscular administration of prednisone alone and oral administration of estradiol plus intramuscular administration of prednisone treated mice. This means that the frequency of bone turnover of oral administration of **25-27** treated mice is significantly lower than that of oral administration of estradiol treated mice, the efficacy of oral administration of **25-27** in inhibiting bone turnover

promising candidates of secondary osteoporosis therapies.

RGD-tetrapeptides indirectly modified androgen (Figure 18) [33].

is significantly higher than that of oral administration of estradiol.

Val, in **27** AA = Phe.

**turnover**

**Figure 17.** Trabecular vBMD of the femurs of the treated mice at a distance from the proximal femur growth palate corresponding with < 6 % of the total length of the femurs of **22-24** treated mice. a) Trabecular vBMD is represented with mean ± SD mg/cm3, n = 12, PDN = prednisone, dose = 110 nmol/kg. b) Compared to NS + PDN, **22 +** PDN and **23 +** PDN p< 0.01; c) Compared to NS + PDN and **22 +** PDN p< 0.01; d) Compared to NS + PDN p< 0.01.

### **4.6. Direct covalent modification of androgen with RGD-tetrapeptides having no thrombosis risk**

The effect of oral administration of **22-24** plus intramuscular administration of prednisone for 4 weeks on thrombosis risk of the mice was observed, and the risk is represented with tail bleeding time. The data indicate that the tail bleeding time of oral administration of **22**-**24** plus intramuscular administration of prednisone treated mice is significantly longer than that of ntramuscular administration of prednisone alone and oral administration of estradiol plus intramuscular administration of prednisone treated mice. This means that, in contrast to oral administration of estradiol, oral administration of **22-24** induces no observable thrombosis risk, and the direct covalent modification of androgen with RGD-tetrapeptides induces no doserelated adverse effects of estradiol.

#### **4.7. Summary of direct covalent modification of androgen with RGD-tetrapeptides**

RGD-octapeptides directly modifying the 17*β*-amino group of 17*β*-amino-11*α*-hydroxyan‐ drost-1,4-diene-3-one was performed by amidation and resulted in 3 conjugates. On prednisone treated mouse model and at 110 nmol/kg of oral dose their anti-osteoporosis activities were significantly higher than that of estradiol. In contrast to estradiol, the antiosteoporosis therapy of these conjugates induced no endometrial cell hyperplasia and thrombosis risk. Comparing to RGD-tetrapeptide modified estradiol the effective dose of RGD-octapeptide modified 17*β*-amino-11*α*-hydroxyandrost-1,4-diene-3-one is 1000 folds lower. This means that the anti-osteoporosis efficacy of RGD-octapeptide modified 17*β*amino-11*α*-hydroxyandrost-1,4-diene-3-one is 1000 folds higher than that of RGD-tetrapep‐ tide modified estradiol. Reasonably, this dramatically enhanced efficacy could attitude to the introduction of 17*β*-amino-11*α*-hydroxyandrost-1,4-diene-3-one. In addition to premeno‐ pausal women and in older men, secondary osteoporosis is common in the patients treated with glucocorticoids and in prostate cancer patients receiving androgen deprivation therapy (ADT) in particular. Glucocorticoids are ubiquitously prescribed in the fields of rheumatol‐ ogy, respirology, neurology, hematology, dermatology, gastroenterology, and transplant medicine. Chronic exposure to pharmacological doses of glucocorticoids causes multiple deleterious effects on osteopenia, osteoporosis and bone fracture. Prostate cancer is one of the most common diseases in the older men. After the surgery or radiation therapy the male patients with localized or metastatic prostate cancer are generally given ADT. Though male patients on ADT usually have good prognosis, osteoporosis is a very common consequence of this therapy and up to 20% of the patients will fracture within 5 years. To prevent osteoporotic fracture in the female patients treated with glucocorticoids and the male patients receiving ADT novel effective agents are needed. The ability of these RGDoctapeptides modified 17*β*-amino-11*α*-hydroxyandrost-1,4-diene-3-one to prevent predni‐ sone treated mouse developing osteoporosis suggests that these conjugates should be promising candidates of secondary osteoporosis therapies.

### **5. Indirect covalent modification of androgen with RGD-tetrapeptides**

**4.6. Direct covalent modification of androgen with RGD-tetrapeptides having no**

**+** PDN p< 0.01; c) Compared to NS + PDN and **22 +** PDN p< 0.01; d) Compared to NS + PDN p< 0.01.

**4.7. Summary of direct covalent modification of androgen with RGD-tetrapeptides**

RGD-octapeptides directly modifying the 17*β*-amino group of 17*β*-amino-11*α*-hydroxyan‐ drost-1,4-diene-3-one was performed by amidation and resulted in 3 conjugates. On prednisone treated mouse model and at 110 nmol/kg of oral dose their anti-osteoporosis activities were significantly higher than that of estradiol. In contrast to estradiol, the antiosteoporosis therapy of these conjugates induced no endometrial cell hyperplasia and thrombosis risk. Comparing to RGD-tetrapeptide modified estradiol the effective dose of RGD-octapeptide modified 17*β*-amino-11*α*-hydroxyandrost-1,4-diene-3-one is 1000 folds lower. This means that the anti-osteoporosis efficacy of RGD-octapeptide modified 17*β*amino-11*α*-hydroxyandrost-1,4-diene-3-one is 1000 folds higher than that of RGD-tetrapep‐ tide modified estradiol. Reasonably, this dramatically enhanced efficacy could attitude to the introduction of 17*β*-amino-11*α*-hydroxyandrost-1,4-diene-3-one. In addition to premeno‐ pausal women and in older men, secondary osteoporosis is common in the patients treated with glucocorticoids and in prostate cancer patients receiving androgen deprivation therapy (ADT) in particular. Glucocorticoids are ubiquitously prescribed in the fields of rheumatol‐

The effect of oral administration of **22-24** plus intramuscular administration of prednisone for 4 weeks on thrombosis risk of the mice was observed, and the risk is represented with tail bleeding time. The data indicate that the tail bleeding time of oral administration of **22**-**24** plus intramuscular administration of prednisone treated mice is significantly longer than that of ntramuscular administration of prednisone alone and oral administration of estradiol plus intramuscular administration of prednisone treated mice. This means that, in contrast to oral administration of estradiol, oral administration of **22-24** induces no observable thrombosis risk, and the direct covalent modification of androgen with RGD-tetrapeptides induces no dose-

**Figure 17.** Trabecular vBMD of the femurs of the treated mice at a distance from the proximal femur growth palate corresponding with < 6 % of the total length of the femurs of **22-24** treated mice. a) Trabecular vBMD is represented with mean ± SD mg/cm3, n = 12, PDN = prednisone, dose = 110 nmol/kg. b) Compared to NS + PDN, **22 +** PDN and **23**

**thrombosis risk**

100 Topics in Osteoporosis

related adverse effects of estradiol.

For androgen a parallel covalent modification with the direct covalent modification is an indirect strategy. In brief, between the 17*β*-amino group of 17*β*-amino-11*α*-hydroxyan‐ drost-1,4-diene-3-one and RGD-tetrapeptides as a linker, succinyl group, is inserted to provide RGD-tetrapeptides indirectly modified androgen (Figure 18) [33].

### **5.1. Indirect covalent modification of androgen with RGD-tetrapeptides inhibiting bone turnover**

The effect of oral administration of **25-27** plus intramuscular administration of prednisone for 4 weeks on serum calcium and serum ALP of the mice is shown in Figure 19. The data indicate that the serum calcium and serum ALP of oral administration of **25-27** plus intramuscular administration of prednisone treated mice are significantly lower than those of intramuscular administration of prednisone alone and oral administration of estradiol plus intramuscular administration of prednisone treated mice. This means that the frequency of bone turnover of oral administration of **25-27** treated mice is significantly lower than that of oral administration of estradiol treated mice, the efficacy of oral administration of **25-27** in inhibiting bone turnover is significantly higher than that of oral administration of estradiol.

**5.3. Indirect covalent modification of androgen with RGD-tetrapeptides increasing total**

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CT measured 3D bone geometry and the size-independent vBMD, as well as pQCT quantita‐ tively measured 3D bone geometry and size-independent vBMD were used to represent the anti-osteoporosis efficacy of **25-27** and are shown in Figure 21. The data indicates that the total vBMD of the femurs of NS plus intramuscular administration of prednisone treated mice is significantly lower than that of the femurs of NS alone treated mice. This means that intra‐ muscular administration of prednisone effectively induces the mice to decrease the total vBMD. The total vBMDs of the femurs of oral administration of **25-27** plus intramuscular administration of prednisone treated mice are significantly higher than that of the femurs of NS plus intramuscular administration of prednisone treated mice. This means that upon oral administration **25-27** effectively inhibit intramuscular administration of prednisone treated

**Figure 21.** Total vBMD and images of pQCT scanning at a distance from the proximal femur growth palate corre‐

Figure 22 indicates that the trabecular vBMD of the femurs of NS plus intramuscular admin‐ istration of prednisone treated mice is significantly lower than that of the femurs of NS alone treated mice. This means that prednisone effectively induces the mice to decrease trabecular vBMD. The trabecular vBMDs of the femurs of oral administration of **25-27** plus intramuscular administration of prednisone treated mice are significantly higher than that of the femurs of NS plus intramuscular administration of prednisone treated mice. This means that upon oral administration **25-27** effectively inhibit intramuscular administration of prednisone treated

**5.4. Indirect covalent modification of androgen with RGD-tetrapeptides increasing**

sponding to < 6 % of the total length of the femur of **25-27** treated mice.

**vBMD**

mice decreasing total vBMD.

**trabecular vBMD**

mice decreasing trabecular vBMD.

**Figure 19.** Serum calcium and ALP of **25-27** treated mice. ip Dose of prednisone (PDN): 6.3 mg/kg, twice a week; oral dose of **25-27**: 110 nmol/kg, once a day; oral dose of estradiol (E2): 110 nmol/kg, once a day; n = 12. **For serum Ca+2:** a) Compared to NS + PND p< 0.01, to E2 + PND p< 0.05; b) Compared to NS + PND p< 0.05. **For serum ALP:** a) Com‐ pared to NS + PND and E2 + PND p< 0.01; b) Compared to NS + PND p< 0.05.

#### **5.2.IndirectcovalentmodificationofandrogenwithRGD-tetrapeptidespreventingboneloss**

The effect of oral administration of **25-27** plus intramuscular administration of prednisone for 4 weeks on the bone loss of the treated mice is shown in Figure 20, their activities are repre‐ sented with dry femur weight and femur ash weight. The data indicate that both the weights of dry femur and femur ash of oral administration of **25-27** plus intramuscular administration of prednisone treated mice are significantly higher than those of intramuscular administration of prednisone alone and oral administration of estradiol plus intramuscular administration of prednisone treated mice. This means that upon oral administration **25-27** effectively inhibit the mice to losing femur, their efficacies are significantly higher than that of oral administration of estradiol, and the direct covalent modification of androgen prevents the bone loss.

**Figure 20.** Weight of dry femur and femur ash of conjugates **25-27** treated mice. ip Dose of prednisone (PDN): 6.3 mg/kg, twice a week; oral dose of **25-27**: 110 nmol/kg, once a day; oral dose of estradiol (E2): 110 nmol/kg, once a day; n = 12. **For dry femur:** a) Compared to NS + PND and E2 + PND p< 0.05; b) Compared to NS + PND and E2 + PND p< 0.05. **For femur ash:** a) Compared to NS + PND and E2 + PND p< 0.01.

### **5.3. Indirect covalent modification of androgen with RGD-tetrapeptides increasing total vBMD**

CT measured 3D bone geometry and the size-independent vBMD, as well as pQCT quantita‐ tively measured 3D bone geometry and size-independent vBMD were used to represent the anti-osteoporosis efficacy of **25-27** and are shown in Figure 21. The data indicates that the total vBMD of the femurs of NS plus intramuscular administration of prednisone treated mice is significantly lower than that of the femurs of NS alone treated mice. This means that intra‐ muscular administration of prednisone effectively induces the mice to decrease the total vBMD. The total vBMDs of the femurs of oral administration of **25-27** plus intramuscular administration of prednisone treated mice are significantly higher than that of the femurs of NS plus intramuscular administration of prednisone treated mice. This means that upon oral administration **25-27** effectively inhibit intramuscular administration of prednisone treated mice decreasing total vBMD.

**Figure 19.** Serum calcium and ALP of **25-27** treated mice. ip Dose of prednisone (PDN): 6.3 mg/kg, twice a week; oral dose of **25-27**: 110 nmol/kg, once a day; oral dose of estradiol (E2): 110 nmol/kg, once a day; n = 12. **For serum Ca+2:** a) Compared to NS + PND p< 0.01, to E2 + PND p< 0.05; b) Compared to NS + PND p< 0.05. **For serum ALP:** a) Com‐

**5.2.IndirectcovalentmodificationofandrogenwithRGD-tetrapeptidespreventingboneloss**

The effect of oral administration of **25-27** plus intramuscular administration of prednisone for 4 weeks on the bone loss of the treated mice is shown in Figure 20, their activities are repre‐ sented with dry femur weight and femur ash weight. The data indicate that both the weights of dry femur and femur ash of oral administration of **25-27** plus intramuscular administration of prednisone treated mice are significantly higher than those of intramuscular administration of prednisone alone and oral administration of estradiol plus intramuscular administration of prednisone treated mice. This means that upon oral administration **25-27** effectively inhibit the mice to losing femur, their efficacies are significantly higher than that of oral administration

of estradiol, and the direct covalent modification of androgen prevents the bone loss.

**Figure 20.** Weight of dry femur and femur ash of conjugates **25-27** treated mice. ip Dose of prednisone (PDN): 6.3 mg/kg, twice a week; oral dose of **25-27**: 110 nmol/kg, once a day; oral dose of estradiol (E2): 110 nmol/kg, once a day; n = 12. **For dry femur:** a) Compared to NS + PND and E2 + PND p< 0.05; b) Compared to NS + PND and E2 + PND

p< 0.05. **For femur ash:** a) Compared to NS + PND and E2 + PND p< 0.01.

pared to NS + PND and E2 + PND p< 0.01; b) Compared to NS + PND p< 0.05.

102 Topics in Osteoporosis

**Figure 21.** Total vBMD and images of pQCT scanning at a distance from the proximal femur growth palate corre‐ sponding to < 6 % of the total length of the femur of **25-27** treated mice.

### **5.4. Indirect covalent modification of androgen with RGD-tetrapeptides increasing trabecular vBMD**

Figure 22 indicates that the trabecular vBMD of the femurs of NS plus intramuscular admin‐ istration of prednisone treated mice is significantly lower than that of the femurs of NS alone treated mice. This means that prednisone effectively induces the mice to decrease trabecular vBMD. The trabecular vBMDs of the femurs of oral administration of **25-27** plus intramuscular administration of prednisone treated mice are significantly higher than that of the femurs of NS plus intramuscular administration of prednisone treated mice. This means that upon oral administration **25-27** effectively inhibit intramuscular administration of prednisone treated mice decreasing trabecular vBMD.

**5.7. Summary of indirect covalent modification of androgen with RGD-tetrapeptides**

**6. Nano-structures of RGD-peptides modified estrogen and androgen**

**6.1. Nano-aggregators from modification of 17β-hydroxy of estradiol with RGD-**

As explained by Figure 6, using succinyl group and RGD-octapeptides modifying the 17βhydroxy of estradiol provides **10-12**. Figure 23 demonstrates that in water **10** forms stick like nano-aggregators of 161.1 nm in diameter and 222.2-888.9 nm in length, **11** forms maize like nano-aggregator of 388.9 nm in length, **12** forms solid pipe like nano-aggregator of 3.6 nm in

the nano-feature with the pharmacological activity.

Self-organization or self-assembly practically leads to the formation of various ordered nanostructures in solution, at bulk state, and on a solid surface [34,35]. Numerous selfassembling substances, such as highly fluorinated amphiphilic molecules[36], amphiphilic triblock copolymers with polyrotaxane as a central block [37], amphiphilic dodecyl ester derivatives from aromatic amino acids [38], dendritic molecules [39], the shape anisotropy of non-spherical colloidal building blocks [40], alkylated polycyclic aromatic hydrocarbons [41], porphyrins, graphenes and fullerenes [42], were designed. Of the self-assembling molecules, peptides have been considered a set of particular substance [43-51]. In respect of the selfassembly the formation of nano-structure is an inherent property of organic compounds. In this context, the nano-structures of **10**-**15** and **22-27** in aqueous are given below to explore the relationships between the nano-structure and the concentration or pH, as well as to correlate

sis therapies.

**octapeptides**

diameter and 263.9 nm in length.

RGD-tetrapeptides indirectly modifying the 17*β*-amino group of 17*β*-amino-11*α*-hydroxyan‐ drost-1,4-diene-3-one was performed by inserting a succinyl functional group and result‐ ed in 3 conjugates. On prednisone treated mouse model and at 110 nmol/kg of oral dose their anti-osteoporosis activities were significantly higher than that of estradiol. In con‐ trast to estradiol, the anti-osteoporosis therapy of these conjugates induced no endometri‐ al cell hyperplasia and thrombosis risk. In respect to inhibiting the prednisone treated mice to lose total vBMD, trabecular vBMD, femur ash weight, femur Ca2+ and bone mineral content 110 nmol/kg of RGD-tetrapeptides indirectly modified 17*β*-amino-11*α*-hydroxyan‐ drost-1,4-diene-3-one was more effective than 110 nmol/kg of RGD-tetrapeptides directly modified 17*β*-amino-11*α*-hydroxyandrost-1,4-diene-3-one, and this increased efficacy could be attributed to the insertion of a succinyl group. Similarly, the ability of these RGDoctapeptides indirectly modified 17*β*-amino-11*α*-hydroxyandrost-1,4-diene-3-one to pre‐ vent prednisone treated mouse developing osteoporosis and high activity suggests that these conjugates should be preferentially promising candidates for secondary osteoporo‐

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**Figure 22.** Trabecular vBMD in the femurs of treated mice at a distance from the proximal femur. Growth plate corre‐ sponds with < 6% of the total length of the femur of the treated mice. Trabecular vBMD is represented with mean ± SD mg/cm3, n = 12, PDN = prednisone, dose of **25-27** = 110 nmol/kg. a) Compared to NS + PDN, **25 +** PDN and **26 +** PDN p< 0.01; b) Compared to NS + PDN and **27 +** PDN p< 0.01; c) Compared to NS + PDN p< 0.01.

### **5.5. Indirect covalent modification of androgen with RGD-tetrapeptides inducing no endomtrial cell hyperplasia**

The effect of oral administration of **25-27** plus intramuscular administration of prednisone for 4 weeks on the endometrial cell hyperplasia of the mice was observed, and their inhibition activities are represented with uteri weight. The data indicate that the weight of the uteri of oral administration of **25**-**27** plus intramuscular administration of prednisone treated mice is significantly lower than that of intramuscular administration of prednisone alone and oral administration of estradiol plus intramuscular administration of prednisone treated mice. This means that, in contrast to oral administration of estradiol, upon oral administration **25-27** induces no observable endometrial cell hyperplasia, and the direct covalent modification of androgen with RGD-octapeptides induces no dose-related adverse effects of estradiol.

### **5.6. Indirect covalent modification of androgen with RGD-tetrapeptides having no thrombosis risk**

The effect of oral administration of **25-27** plus intramuscular administration of prednisone for 4 weeks on thrombosis risk of the mice was observed, and the risk is represented with tail bleeding time. The data indicate that the tail bleeding time of oral administration of **25**-**27** plus intramuscular administration of prednisone treated mice is significantly longer than that of ntramuscular administration of prednisone alone and oral administration of estradiol plus intramuscular administration of prednisone treated mice. This means that, in contrast to oral administration of estradiol, upon oral administration **25-27** induces no observable thrombosis risk, and the indirect covalent modification of androgen with RGD-octapeptides induces no dose-related adverse effects of estradiol.

### **5.7. Summary of indirect covalent modification of androgen with RGD-tetrapeptides**

RGD-tetrapeptides indirectly modifying the 17*β*-amino group of 17*β*-amino-11*α*-hydroxyan‐ drost-1,4-diene-3-one was performed by inserting a succinyl functional group and result‐ ed in 3 conjugates. On prednisone treated mouse model and at 110 nmol/kg of oral dose their anti-osteoporosis activities were significantly higher than that of estradiol. In con‐ trast to estradiol, the anti-osteoporosis therapy of these conjugates induced no endometri‐ al cell hyperplasia and thrombosis risk. In respect to inhibiting the prednisone treated mice to lose total vBMD, trabecular vBMD, femur ash weight, femur Ca2+ and bone mineral content 110 nmol/kg of RGD-tetrapeptides indirectly modified 17*β*-amino-11*α*-hydroxyan‐ drost-1,4-diene-3-one was more effective than 110 nmol/kg of RGD-tetrapeptides directly modified 17*β*-amino-11*α*-hydroxyandrost-1,4-diene-3-one, and this increased efficacy could be attributed to the insertion of a succinyl group. Similarly, the ability of these RGDoctapeptides indirectly modified 17*β*-amino-11*α*-hydroxyandrost-1,4-diene-3-one to pre‐ vent prednisone treated mouse developing osteoporosis and high activity suggests that these conjugates should be preferentially promising candidates for secondary osteoporo‐ sis therapies.

### **6. Nano-structures of RGD-peptides modified estrogen and androgen**

**Figure 22.** Trabecular vBMD in the femurs of treated mice at a distance from the proximal femur. Growth plate corre‐ sponds with < 6% of the total length of the femur of the treated mice. Trabecular vBMD is represented with mean ± SD mg/cm3, n = 12, PDN = prednisone, dose of **25-27** = 110 nmol/kg. a) Compared to NS + PDN, **25 +** PDN and **26 +**

The effect of oral administration of **25-27** plus intramuscular administration of prednisone for 4 weeks on the endometrial cell hyperplasia of the mice was observed, and their inhibition activities are represented with uteri weight. The data indicate that the weight of the uteri of oral administration of **25**-**27** plus intramuscular administration of prednisone treated mice is significantly lower than that of intramuscular administration of prednisone alone and oral administration of estradiol plus intramuscular administration of prednisone treated mice. This means that, in contrast to oral administration of estradiol, upon oral administration **25-27** induces no observable endometrial cell hyperplasia, and the direct covalent modification of androgen with RGD-octapeptides induces no dose-related adverse effects of estradiol.

**5.5. Indirect covalent modification of androgen with RGD-tetrapeptides inducing no**

**5.6. Indirect covalent modification of androgen with RGD-tetrapeptides having no**

The effect of oral administration of **25-27** plus intramuscular administration of prednisone for 4 weeks on thrombosis risk of the mice was observed, and the risk is represented with tail bleeding time. The data indicate that the tail bleeding time of oral administration of **25**-**27** plus intramuscular administration of prednisone treated mice is significantly longer than that of ntramuscular administration of prednisone alone and oral administration of estradiol plus intramuscular administration of prednisone treated mice. This means that, in contrast to oral administration of estradiol, upon oral administration **25-27** induces no observable thrombosis risk, and the indirect covalent modification of androgen with RGD-octapeptides induces no

PDN p< 0.01; b) Compared to NS + PDN and **27 +** PDN p< 0.01; c) Compared to NS + PDN p< 0.01.

**endomtrial cell hyperplasia**

104 Topics in Osteoporosis

**thrombosis risk**

dose-related adverse effects of estradiol.

Self-organization or self-assembly practically leads to the formation of various ordered nanostructures in solution, at bulk state, and on a solid surface [34,35]. Numerous selfassembling substances, such as highly fluorinated amphiphilic molecules[36], amphiphilic triblock copolymers with polyrotaxane as a central block [37], amphiphilic dodecyl ester derivatives from aromatic amino acids [38], dendritic molecules [39], the shape anisotropy of non-spherical colloidal building blocks [40], alkylated polycyclic aromatic hydrocarbons [41], porphyrins, graphenes and fullerenes [42], were designed. Of the self-assembling molecules, peptides have been considered a set of particular substance [43-51]. In respect of the selfassembly the formation of nano-structure is an inherent property of organic compounds. In this context, the nano-structures of **10**-**15** and **22-27** in aqueous are given below to explore the relationships between the nano-structure and the concentration or pH, as well as to correlate the nano-feature with the pharmacological activity.

### **6.1. Nano-aggregators from modification of 17β-hydroxy of estradiol with RGDoctapeptides**

As explained by Figure 6, using succinyl group and RGD-octapeptides modifying the 17βhydroxy of estradiol provides **10-12**. Figure 23 demonstrates that in water **10** forms stick like nano-aggregators of 161.1 nm in diameter and 222.2-888.9 nm in length, **11** forms maize like nano-aggregator of 388.9 nm in length, **12** forms solid pipe like nano-aggregator of 3.6 nm in diameter and 263.9 nm in length.

**Figure 23.** TEM images of **10** - **12** formed nano-aggregators. A) Stick like nano-aggregators of **10**; B) Maize like nanoaggregators of **11**; C) Solid pipe like nano-aggregator of 12.

**Figure 25.** TEM images of 1.1 mM of **22** in ultrapure water. A) Numerous smaller globes aggregated nano-globe of 400 nm in diameter; B) Dispersing globes of 55 - 200 nm in diameter; C) Dispersing globes of 18 - 146 nm in diameter.

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**Figure 26.** TEM images of 1.1 mM of **23** in ultrapure water. A) Nano-globe of 312.5 nm in diameter having small globes, blocks and awls on surface; B) Nano-globes of 21.9 - 82.9 nm in diameter; C) Nano-globes of 22.9 - 194.3 nm

**Figure 27.** TEM images of 1.1 mM of **24** in ultrapure water. A) Nano-globe of 183 nm in diameter having a number of nano-particles on surface; B) A hemisphere of 275 nm in diameter having some smaller globes on incomplete surface;

The nano-structures of **22**-**24** were explained with SEM nano-images and are shown with Figures 28-30. Figure 29 indicates that in solid state **22** exists as globes of 3.3 - 14.2 μm in diameter. Figure 44 indicates that in solid state **23** exists as eggs of 9.6 × 11.5 μm to 19.5 × 27.0 μm in diameter, of which surfaces have small eggs, and one egg remains its tail been incom‐ plete. Figure 30 indicates that in solid state **24** exists as beads of 9.2 × 10.0 μm to 21.4 × 22.8 μm

**6.4. SEM image of nano-globes of androgen having RGD-tetrapeptides modified 17β-**

in diameter.

**hydroxy**

C) Nano-globes of 48 - 188 nm in diameter.

in diameter, and beads remain been incomplete.

### **6.2. Nano-aggregators from modification of 3-hydroxy of estradiol with RGDoctapeptides**

As seen in Figure 6, carbonylmethyl and RGD-octapeptides modifying the 3-hydroxy of estradiol provides **13-15**. Figure 24 demonstrates that in water **13** forms porous nano-aggre‐ gators of 133.3-430.6 nm in length, **14** forms maize like nano-aggregator of 111.1-600.0 nm in length, **15** forms nano-globes of 66.7-237.5 nm in diameter.

**Figure 24.** TEM images of **13** - **15** formed nano-aggregators. A) Porous nano-aggregators of **13**; B) Porous nano-ag‐ gregators of **14**; C) Nano-globes of **15**.

### **6.3. TEM image of nano-globes of androgen having RGD-tetrapeptides modified 17βhydroxy**

The nano-structures of **22**-**24** were explained with TEM nano-images and are shown with Figures 25-27. Figure 26 indicates that in 1.1 mM aqueous solution **22** forms numerous smaller globes aggregated nano-globe of 400 nm in diameter, dispersing globes of 55 - 200 nm in diameter, and dispersing globes of 18 - 146 nm in diameter. Figure 41 indicates that in 1.1 mM aqueous solution **23** forms nano-globe of 312.5 nm in diameter having small globes, blocks and awls on surface, nano-globes of 21.9 - 82.9 nm in diameter and nanoglobes of 22.9 - 194.3 nm in diameter. Figure 27 indicates that in 1.1 mM aqueous solu‐ tion **24** forms nano-globe of 183 nm in diameter having a number of nano-particles on surface, hemisphere of 275 nm in diameter having some smaller globes on incomplete surface and nano-globes of 48 - 188 nm in diameter.

Modification of Sex Hormones with RGD-Peptide: A Strategy of Improving HRT and Other Secondary… http://dx.doi.org/10.5772/54361 107

**Figure 25.** TEM images of 1.1 mM of **22** in ultrapure water. A) Numerous smaller globes aggregated nano-globe of 400 nm in diameter; B) Dispersing globes of 55 - 200 nm in diameter; C) Dispersing globes of 18 - 146 nm in diameter.

**Figure 23.** TEM images of **10** - **12** formed nano-aggregators. A) Stick like nano-aggregators of **10**; B) Maize like nano-

As seen in Figure 6, carbonylmethyl and RGD-octapeptides modifying the 3-hydroxy of estradiol provides **13-15**. Figure 24 demonstrates that in water **13** forms porous nano-aggre‐ gators of 133.3-430.6 nm in length, **14** forms maize like nano-aggregator of 111.1-600.0 nm in

**Figure 24.** TEM images of **13** - **15** formed nano-aggregators. A) Porous nano-aggregators of **13**; B) Porous nano-ag‐

The nano-structures of **22**-**24** were explained with TEM nano-images and are shown with Figures 25-27. Figure 26 indicates that in 1.1 mM aqueous solution **22** forms numerous smaller globes aggregated nano-globe of 400 nm in diameter, dispersing globes of 55 - 200 nm in diameter, and dispersing globes of 18 - 146 nm in diameter. Figure 41 indicates that in 1.1 mM aqueous solution **23** forms nano-globe of 312.5 nm in diameter having small globes, blocks and awls on surface, nano-globes of 21.9 - 82.9 nm in diameter and nanoglobes of 22.9 - 194.3 nm in diameter. Figure 27 indicates that in 1.1 mM aqueous solu‐ tion **24** forms nano-globe of 183 nm in diameter having a number of nano-particles on surface, hemisphere of 275 nm in diameter having some smaller globes on incomplete

**6.3. TEM image of nano-globes of androgen having RGD-tetrapeptides modified 17β-**

**6.2. Nano-aggregators from modification of 3-hydroxy of estradiol with RGD-**

aggregators of **11**; C) Solid pipe like nano-aggregator of 12.

length, **15** forms nano-globes of 66.7-237.5 nm in diameter.

surface and nano-globes of 48 - 188 nm in diameter.

**octapeptides**

106 Topics in Osteoporosis

gregators of **14**; C) Nano-globes of **15**.

**hydroxy**

**Figure 26.** TEM images of 1.1 mM of **23** in ultrapure water. A) Nano-globe of 312.5 nm in diameter having small globes, blocks and awls on surface; B) Nano-globes of 21.9 - 82.9 nm in diameter; C) Nano-globes of 22.9 - 194.3 nm in diameter.

**Figure 27.** TEM images of 1.1 mM of **24** in ultrapure water. A) Nano-globe of 183 nm in diameter having a number of nano-particles on surface; B) A hemisphere of 275 nm in diameter having some smaller globes on incomplete surface; C) Nano-globes of 48 - 188 nm in diameter.

### **6.4. SEM image of nano-globes of androgen having RGD-tetrapeptides modified 17βhydroxy**

The nano-structures of **22**-**24** were explained with SEM nano-images and are shown with Figures 28-30. Figure 29 indicates that in solid state **22** exists as globes of 3.3 - 14.2 μm in diameter. Figure 44 indicates that in solid state **23** exists as eggs of 9.6 × 11.5 μm to 19.5 × 27.0 μm in diameter, of which surfaces have small eggs, and one egg remains its tail been incom‐ plete. Figure 30 indicates that in solid state **24** exists as beads of 9.2 × 10.0 μm to 21.4 × 22.8 μm in diameter, and beads remain been incomplete.

**Figure 28.** SEM images of **22** in solid state. A) Globes of 3.3 - 6.6 µm in diameter; B) Globes of 3.3 - 14.2 µm in diame‐ ter; C) Globes of 3.3 - 11.7 µm in diameter.

**Figure 31.** TEM images of 1.1 μM of **25** in ultrapure water. A) Dispersing globes of 8 - 150 nm in diameter; B) Dispers‐

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**Figure 32.** TEM images of 1.1 μM of **26** in ultrapure water. A)Dispersing globes of 29 - 69 nm in diameter; B) Dispers‐

**Figure 33.** TEM images of 1.1 μM of **27** in ultrapure water. A) Dispersing globes of 320 - 343 nm in diameter; B) Dis‐

The SEM image (Figures 34-36) demonstrates that in solid state **25-27** exist as nano-globes of 15 nm - 6.4 μm in diameter, nano-pine seeds of 286 nm - 2.7 μm in length, nano-eggs of 1.3 - 12.9 μm in length, nano-pinecones of 5.0 - 5.6 μm in length, nano-gear of 10 μm in diameter, nano-calabash of 4 μm in length, and uncompleted nano-calabash of 11.3 μm in length. The coexistence of nano-globe having nano-egg, nano-pine seed having nano-pinecone, and uncompleted nano-calabash having nano-calabash implies that the nano-egg, nano-pinecone and nano-calabash are built by nano-globes, nano-pine seeds and uncompleted nano-calabash. The correlation of the molecular constitutions and the nano-structures gave us an impression

**6.6. SEM image of nano-globes of androgen having RGD-tetrapeptides and succinyl**

ing globes of 17 - 94 nm in diameter; C) Dispersing globes of 27 - 82 nm in diameters.

ing globes of 70 - 120 nm in diameter; C) Dispersing globes of 67 - 150 nm in diameter.

persing globes of 76 - 139 nm in diameter; C) Dispersing globes of 120 - 171 nm in diameter.

**modified 17β-hydroxy**

**Figure 29.** SEM images of **23** in solid state. A) Egg of 12.9 × 14.4 µm in diameter; B) Egg of 19.5 × 27.0 µm in diame‐ ter; C) Egg of 9.6 × 11.5 µm in diameter and egg remains its tail been incomplete.

**Figure 30.** SEM images of **24** in solid state. A) Bead of 9.2 × 10.0 µm in diameters, and bead remains been incomplete; B) Hollow bead of 21.4 × 22.8 µm in diameters; C) Bead of 12.6 × 21.1 µm in diameter, and bead of 15.3 × 22.1 µm in diameter.

### **6.5. TEM image of nano-globes of androgen having RGD-tetrapeptides and succinyl modified 17β-hydroxy**

The TEM images (Figures 31-33) demonstrate that in water **25-27** consistently form nanoglobes with porous surface. The comparison of the nano-globes of **22**-**24** having no porous surface and the nano-globes of **25-27** having porous surface gave us an impression that the insertion of succinyl was a key to form the nano-globes with porous surface, 17β-ethyl-car‐ bonylaminoandrost-1,4-diene-3-one was responsible for forming nano-globe, and RGD-tet‐ rapeptide was responsible for characterizing the surface feature and size of the nano-globes, in particular. For instance, RGDS causes **25** to form dispersing nano-globes of 8 - 150 nm in diameter and having porous surfaces, RGDV causes **26** to form dispersing nano-globes of 29 - 150 nm in diameter and having porous surfaces, and RGDF causes **27** to form dispersing nano-globes of 76 - 343 nm in diameter and having porous surfaces.

Modification of Sex Hormones with RGD-Peptide: A Strategy of Improving HRT and Other Secondary… http://dx.doi.org/10.5772/54361 109

**Figure 31.** TEM images of 1.1 μM of **25** in ultrapure water. A) Dispersing globes of 8 - 150 nm in diameter; B) Dispers‐ ing globes of 17 - 94 nm in diameter; C) Dispersing globes of 27 - 82 nm in diameters.

**Figure 28.** SEM images of **22** in solid state. A) Globes of 3.3 - 6.6 µm in diameter; B) Globes of 3.3 - 14.2 µm in diame‐

**Figure 29.** SEM images of **23** in solid state. A) Egg of 12.9 × 14.4 µm in diameter; B) Egg of 19.5 × 27.0 µm in diame‐

**Figure 30.** SEM images of **24** in solid state. A) Bead of 9.2 × 10.0 µm in diameters, and bead remains been incomplete; B) Hollow bead of 21.4 × 22.8 µm in diameters; C) Bead of 12.6 × 21.1 µm in diameter, and bead of 15.3 × 22.1 µm in

The TEM images (Figures 31-33) demonstrate that in water **25-27** consistently form nanoglobes with porous surface. The comparison of the nano-globes of **22**-**24** having no porous surface and the nano-globes of **25-27** having porous surface gave us an impression that the insertion of succinyl was a key to form the nano-globes with porous surface, 17β-ethyl-car‐ bonylaminoandrost-1,4-diene-3-one was responsible for forming nano-globe, and RGD-tet‐ rapeptide was responsible for characterizing the surface feature and size of the nano-globes, in particular. For instance, RGDS causes **25** to form dispersing nano-globes of 8 - 150 nm in diameter and having porous surfaces, RGDV causes **26** to form dispersing nano-globes of 29 - 150 nm in diameter and having porous surfaces, and RGDF causes **27** to form dispersing

**6.5. TEM image of nano-globes of androgen having RGD-tetrapeptides and succinyl**

nano-globes of 76 - 343 nm in diameter and having porous surfaces.

ter; C) Egg of 9.6 × 11.5 µm in diameter and egg remains its tail been incomplete.

ter; C) Globes of 3.3 - 11.7 µm in diameter.

108 Topics in Osteoporosis

diameter.

**modified 17β-hydroxy**

**Figure 32.** TEM images of 1.1 μM of **26** in ultrapure water. A)Dispersing globes of 29 - 69 nm in diameter; B) Dispers‐ ing globes of 70 - 120 nm in diameter; C) Dispersing globes of 67 - 150 nm in diameter.

**Figure 33.** TEM images of 1.1 μM of **27** in ultrapure water. A) Dispersing globes of 320 - 343 nm in diameter; B) Dis‐ persing globes of 76 - 139 nm in diameter; C) Dispersing globes of 120 - 171 nm in diameter.

### **6.6. SEM image of nano-globes of androgen having RGD-tetrapeptides and succinyl modified 17β-hydroxy**

The SEM image (Figures 34-36) demonstrates that in solid state **25-27** exist as nano-globes of 15 nm - 6.4 μm in diameter, nano-pine seeds of 286 nm - 2.7 μm in length, nano-eggs of 1.3 - 12.9 μm in length, nano-pinecones of 5.0 - 5.6 μm in length, nano-gear of 10 μm in diameter, nano-calabash of 4 μm in length, and uncompleted nano-calabash of 11.3 μm in length. The coexistence of nano-globe having nano-egg, nano-pine seed having nano-pinecone, and uncompleted nano-calabash having nano-calabash implies that the nano-egg, nano-pinecone and nano-calabash are built by nano-globes, nano-pine seeds and uncompleted nano-calabash. The correlation of the molecular constitutions and the nano-structures gave us an impression that for **25** - **27** 17β-ethylcarbonyl-amino-androst-1,4-diene-3-one was responsible for forming a globe-like body, and RGD-tetrapeptide was responsible for characterizing globe-like body.

correlated with their anti-osteoporosis activities. Therefore by selecting the concentration and by modifying the chemical structure we are able to optionally get the desirable nano-structure

Modification of Sex Hormones with RGD-Peptide: A Strategy of Improving HRT and Other Secondary…

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111

Secondary osteoporosis is common in premenopausal women with osteoporosis and in older men, and is a major problem in clinical practice. More than one third of women with postme‐ nopausal osteoporosis have identifiable secondary causes that contribute to bone loss. The secondary causes of osteoporosis in older men account for 50% - 80% of the cases of bone loss leading to fracture. Besides, secondary osteoporosis is common in the patients treated with glucocorticoids and in prostate cancer patients receiving ADT in particular. Glucocorticoids are ubiquitously prescribed in the fields of rheumatology, respirology, neurology, hematology, dermatology, gastroenterology, and transplant medicine. Chronic exposure to pharmacolog‐ ical doses of glucocorticoids causes multiple deleterious effects on osteopenia, osteoporosis and bone fracture. Prostate cancer is one of the most common diseases in the older men. After the surgery or radiation therapy the male patients with localized or metastatic prostate cancer are generally given ADT. Though male patients on ADT usually have good prognosis, osteoporosis is a very common consequence of this therapy and up to 20% of the patients will fracture within 5 years. To prevent osteoporotic fracture in premenopausal women with osteoporosis, the female patients treated with glucocorticoids and the male patients receiving ADT RGD-peptides modified sex hormones were provided. On ovariotomy and prednisone induced osteoporosis mice either ip injection or orally dosed the modified hormones were able to enhance the efficacy and minimize the adverse effects. By forming nano-species their

This work was finished in Beijing Area Major Laboratory of Peptide and Small Molecular Drugs, supported by Innovation Platform Project of Education Committee of Beijing, Special Project (2011ZX09302-007-01), and Natural Scientific Foundation of China (81072522 and

College of Pharmaceutical Sciences, Capital Medical University, Beijing, PR China

and consequently to optionally get desirable anti-osteoporosis activity.

**7. Conclusions**

therapy could be further improved.

Ming Zhao, Yuji Wang, Jianhui Wu and Shiqi Peng\*

\*Address all correspondence to: sqpeng@bjmu.edu.cn

**Acknowledgements**

81273379).

**Author details**

**Figure 34.** SEM images of **25** in solid state. A) Nano-globes of 15 nm - 2 µm in diameter and nano-calabash of 4 µm in length; B) Nano-globes of 600 nm - 1.3 µm in diameter and nano-egg of 1.3 µm in length; C) Nano-globes of 3.0 - 5.0 µm in diameter and uncompleted nano-calabash of 11.3 µm in length.

**Figure 35.** SEM images of **26** in solid state. A) Globe of 2.9 µm in diameter; B) Globes of 5.0 - 6.4 µm in diameter and eggs of 5.7 - 12.9 µm in length; C) Globes of 2.8 - 3.5 µm in diameter.

**Figure 36.** SEM images of **27** in solid state. A) Pine seeds of 7.1 - 9.4 µm in length; B) Globe of 8.1 µm in diameter; C) Gear of 10 µm in diameter.

### **6.7. Summary of the nano-structures of RGD-peptides modified sex hormones**

In water RGD-peptides modified sex hormones generally formed diverse nano-species via selfassembly. Due to all non-covalent bond interactions could be involved into the self-assembly the size and the feature of the nano-species of RGD-peptides modified sex hormones clearly depend on the concentration of their aqueous solution. Similarly, due to all non-covalent bond interactions could be involved into the self-assembly the size and the feature of the nanospecies usually depend on the chemical structures of the sex hormones and the sequence of the RGD-peptides. In addition, the RGD-peptides modified sex hormones possessed various anti-osteoporosis activities. Thus the feature and the size of their nano-species could be correlated with their anti-osteoporosis activities. Therefore by selecting the concentration and by modifying the chemical structure we are able to optionally get the desirable nano-structure and consequently to optionally get desirable anti-osteoporosis activity.

### **7. Conclusions**

that for **25** - **27** 17β-ethylcarbonyl-amino-androst-1,4-diene-3-one was responsible for forming a globe-like body, and RGD-tetrapeptide was responsible for characterizing globe-like body.

**Figure 34.** SEM images of **25** in solid state. A) Nano-globes of 15 nm - 2 µm in diameter and nano-calabash of 4 µm in length; B) Nano-globes of 600 nm - 1.3 µm in diameter and nano-egg of 1.3 µm in length; C) Nano-globes of 3.0 - 5.0

**Figure 35.** SEM images of **26** in solid state. A) Globe of 2.9 µm in diameter; B) Globes of 5.0 - 6.4 µm in diameter and

**Figure 36.** SEM images of **27** in solid state. A) Pine seeds of 7.1 - 9.4 µm in length; B) Globe of 8.1 µm in diameter; C)

In water RGD-peptides modified sex hormones generally formed diverse nano-species via selfassembly. Due to all non-covalent bond interactions could be involved into the self-assembly the size and the feature of the nano-species of RGD-peptides modified sex hormones clearly depend on the concentration of their aqueous solution. Similarly, due to all non-covalent bond interactions could be involved into the self-assembly the size and the feature of the nanospecies usually depend on the chemical structures of the sex hormones and the sequence of the RGD-peptides. In addition, the RGD-peptides modified sex hormones possessed various anti-osteoporosis activities. Thus the feature and the size of their nano-species could be

**6.7. Summary of the nano-structures of RGD-peptides modified sex hormones**

µm in diameter and uncompleted nano-calabash of 11.3 µm in length.

eggs of 5.7 - 12.9 µm in length; C) Globes of 2.8 - 3.5 µm in diameter.

Gear of 10 µm in diameter.

110 Topics in Osteoporosis

Secondary osteoporosis is common in premenopausal women with osteoporosis and in older men, and is a major problem in clinical practice. More than one third of women with postme‐ nopausal osteoporosis have identifiable secondary causes that contribute to bone loss. The secondary causes of osteoporosis in older men account for 50% - 80% of the cases of bone loss leading to fracture. Besides, secondary osteoporosis is common in the patients treated with glucocorticoids and in prostate cancer patients receiving ADT in particular. Glucocorticoids are ubiquitously prescribed in the fields of rheumatology, respirology, neurology, hematology, dermatology, gastroenterology, and transplant medicine. Chronic exposure to pharmacolog‐ ical doses of glucocorticoids causes multiple deleterious effects on osteopenia, osteoporosis and bone fracture. Prostate cancer is one of the most common diseases in the older men. After the surgery or radiation therapy the male patients with localized or metastatic prostate cancer are generally given ADT. Though male patients on ADT usually have good prognosis, osteoporosis is a very common consequence of this therapy and up to 20% of the patients will fracture within 5 years. To prevent osteoporotic fracture in premenopausal women with osteoporosis, the female patients treated with glucocorticoids and the male patients receiving ADT RGD-peptides modified sex hormones were provided. On ovariotomy and prednisone induced osteoporosis mice either ip injection or orally dosed the modified hormones were able to enhance the efficacy and minimize the adverse effects. By forming nano-species their therapy could be further improved.

### **Acknowledgements**

This work was finished in Beijing Area Major Laboratory of Peptide and Small Molecular Drugs, supported by Innovation Platform Project of Education Committee of Beijing, Special Project (2011ZX09302-007-01), and Natural Scientific Foundation of China (81072522 and 81273379).

### **Author details**

Ming Zhao, Yuji Wang, Jianhui Wu and Shiqi Peng\*

\*Address all correspondence to: sqpeng@bjmu.edu.cn

College of Pharmaceutical Sciences, Capital Medical University, Beijing, PR China

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[35] Glotzer S.C. Horsch M.A. Iacovella C.R. Zhang Z. Chan E.R. Zhang X. Self- assembly of anisotropic tethered nanoparticle shape amphiphiles. Current Opinion in Colloid

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114 Topics in Osteoporosis


**Chapter 5**

**Oxidative Stress and Antioxidants in the Risk of**

**Polyphenols**

**1. Introduction**

L.G. Rao and A.V. Rao

http://dx.doi.org/10.5772/54703

Additional information is available at the end of the chapter

**Osteoporosis — Role of the Antioxidants Lycopene and**

Osteoporosis is a metabolic bone disease known as "the silent thief" because the gradual loss of bone associated with this disease usually occurs over the years, and there are usually no noticeable symptoms until the bones are so fragile that a fracture occurs [1]. Although most statistics on the prevalence of osteoporosis quoted in the literature are from those published in 1991 to 2004 [2,3] the projection is nevertheless very consistent. Thus, osteoporosis is estimated to affect over 200 million people worldwide and 75 million people in Europe, the United States, and Japan [4]. Approximately 1 in 2 women and 1 in 5 men older than 50 years will eventually experience osteoporotic fractures [5] An increase in the worldwide incidence of hip fracture by 240% in women and 310% in men is projected by the year 2050 [6]. Osteo‐ porosis is "a major public health threat" that is projected to results to 8.1 million fractures (78 % women, 22 % men) during the period between 2010 and 2050 [7]. The condition costs our healthcare system \$18 billion per year [8]. Records show that Osteoporosis has been known to exist since the Egyptian mummies have been found with suspected dowager's hump [9]. Newer findings on all aspects of osteoporosis have increased exponentially. The more importantly ones are the introduction and improvement in more sensitive diagnostic instru‐ ments, discovering an ever increasing number of risk factors including oxidative stress, opening up new knowledge on the involvement of the bone forming cells osteoblasts and the bone resorbing cells osteoclasts in the development of osteoporosis and finding new drugs and the nutritional alternatives for the prevention and treatment of osteoporosis. Advances in knowledge on osteoporosis is not without pitfalls. Hormone Replacement Therapy (HRT), once a first line of treatment for osteoporosis has been discontinued due to side effects [10]. It is becoming more evident that the drugs known as bisphosphonates, although effective in

> © 2013 Rao and Rao; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

## **Oxidative Stress and Antioxidants in the Risk of Osteoporosis — Role of the Antioxidants Lycopene and Polyphenols**

L.G. Rao and A.V. Rao

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54703

### **1. Introduction**

Osteoporosis is a metabolic bone disease known as "the silent thief" because the gradual loss of bone associated with this disease usually occurs over the years, and there are usually no noticeable symptoms until the bones are so fragile that a fracture occurs [1]. Although most statistics on the prevalence of osteoporosis quoted in the literature are from those published in 1991 to 2004 [2,3] the projection is nevertheless very consistent. Thus, osteoporosis is estimated to affect over 200 million people worldwide and 75 million people in Europe, the United States, and Japan [4]. Approximately 1 in 2 women and 1 in 5 men older than 50 years will eventually experience osteoporotic fractures [5] An increase in the worldwide incidence of hip fracture by 240% in women and 310% in men is projected by the year 2050 [6]. Osteo‐ porosis is "a major public health threat" that is projected to results to 8.1 million fractures (78 % women, 22 % men) during the period between 2010 and 2050 [7]. The condition costs our healthcare system \$18 billion per year [8]. Records show that Osteoporosis has been known to exist since the Egyptian mummies have been found with suspected dowager's hump [9]. Newer findings on all aspects of osteoporosis have increased exponentially. The more importantly ones are the introduction and improvement in more sensitive diagnostic instru‐ ments, discovering an ever increasing number of risk factors including oxidative stress, opening up new knowledge on the involvement of the bone forming cells osteoblasts and the bone resorbing cells osteoclasts in the development of osteoporosis and finding new drugs and the nutritional alternatives for the prevention and treatment of osteoporosis. Advances in knowledge on osteoporosis is not without pitfalls. Hormone Replacement Therapy (HRT), once a first line of treatment for osteoporosis has been discontinued due to side effects [10]. It is becoming more evident that the drugs known as bisphosphonates, although effective in

© 2013 Rao and Rao; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

stopping the resoption of bone and preventing osteoporosis in women, are associated with a number of side effects [11,12]. The side effects have been alarming a number of women with osteoporosis in such a way that they are now resorting to other mode of treatment, including that from natural food components. Our laboratory has carried out studies on the use of antioxidants such as lycopene and polyphenols as possible alternatives and/or complementa‐ ries to drugs in the treatment and prevention of osteoporosis. This chapter will include an overview on osteoporosis, the role of oxidative stress in bone cells osteoclasts and osteoblasts, oxidative stress as a risk factor in the development of osteoporosis and a review of studies on the use of antioxidants in counteracting oxidative stress in the prevention of osteoporosis. These topics should put our research in perspective and offer a rationale to our study ap‐ proaches. Finally we will highlight our pioneering studies on the effects of the lipid-soluble antioxidant lycopene and the water-soluble antioxidant polyphenols present in a nutritional supplement in an *in vitro* cultures of osteoblasts and osteoclasts and our clinical studies in the prevention of risk for osteoporosis in postmenopausal women.

"woman's disease." This is because men in their fifties do not experience the rapid loss of bone mass that women do in the years following menopause, and therefore men osteoporosis does not set in until later in life [21,22]. However, by age 65 to 70, men and women are losing bone mass at the same rate [23]. The World Health Organization (WHO) aptly defined osteoporosis as a systemic disease that is characterized by low bone mass and deterioration of the micro‐ architecture of bone, resulting in an increased risk of fracture. Bone mass or bone mineral density (BMD) is measured using a dual-energy x-ray absorptiometry, or DXA, at various

Oxidative Stress and Antioxidants in the Risk of Osteoporosis — Role of the Antioxidants Lycopene and Polyphenols

http://dx.doi.org/10.5772/54703

119

BMD is expressed as T-score which is a value compared to the expected value for young adults of the same sex and race. WHO has established that for normal BMD, the T-score is between standard deviation of +2.5 and -1.0; for osteopenia/low BMD, T-score is between -1.0 and -2.5, inclusive; for osteoporosis, T-score is lower than -2.5 and for severe osteoporosis,T-score is lower than -2.5 with the presence of one or more fragility fractures [25]. Thus BMD values can identify osteoporosis, determine the risk for fractures (broken bones), and measure the response to osteoporosis treatment [26]. In the case of severe osteoporosis, minimal trauma such as a minor fall or just a hug from a loved one can result to fragility fracture. Fragility fracture is defined by WHO as "a fracture caused by injury that would be insufficient to cause fracture normally. The spine, hip and distal forearm are the most common sites of fragility fracture [27]. Some doctors recommend that people be tested on a regular basis for bone loss. For women, those tests should begin after menopause. For men, they should begin after the age of sixty-five. Such tests are important since there are seldom other signs of osteoporosis. Therefore, those who have a higher rate of bone loss and are at higher risk for a fracture need a better diagnostic tool. Recently, the WHO introduced a prognostic tool to evaluate fracture risk of patients called the FRAX® [28]. The FRAX tool takes into account country, bone mineral density of the hip (when available), age, sex, and 8 clinical risk factors to calculate the 10-year probability of a major osteoporotic fracture and the 10-year probability of a hip fracture [29]. It assesses the 10-year risk of osteoporosis based on individual patient models that combines

During bone remodeling in healthy young adult, bone formation by osteoblasts equals bone resorption by osteoclasts. However In postmenopausal bone loss, the remodeling process becomes significantly more active with a primary increase in bone resorption and a corre‐ sponding, but an insufficient increase in bone formation [31]. Enzymes and/or other proteins are released into the blood that are considered to reflect either bone formation or bone resorption [32] and are termed as bone turnover markers. Molecular markers of bone turnover have been developed as a product of bone remodeling [31] in the diagnostic and therapeutic assessment of metabolic bone disease [33]. They are now used for the individual monitoring of osteoporotic patients treated with antiresorptive agents [34]. Specific and sensitive assess‐ ment of the rate of bone formation and bone resorption and prediction of fracture [35] can now

skeletal sites, including the spine, hip and wrist [24].

**2.3. Bone mineral density as predictor of osteoporosis**

clinical risk factors (CRF) as well as BMD at the femoral neck [30].

**2.4. Bone turnover markers for detecting osteoporosis**

### **2. Osteoporosis — Overview**

### **2.1. Bone cells involved in the development of osteoporosis**

Bone as a dynamic tissue continuously renews itself throughout life by the process of bone remodeling carried out by a functional and anatomic structure known as the basic multicellular unit (BMU) that requires the coordinated action of three major types of bone cells: osteoclasts, osteoblasts and osteocytes [13,14]. The remodeling process is the result of interactions between these cells and multiple molecular agents, including hormones, growth factors, and cytokines. Bone remodeling is a physiological process that follows a time sequence lasting approximately six months wherein osteoclasts eliminate old or damaged bone which is subsequently replaced with new bone formed by osteoblasts, while the osteocytes functions in the transduction of signals necessary to sustain mechanical loads. The coupled process of bone formation and bone resorption in mature, healthy bone is tightly regulated and maintained in order to prevent a significant alterations in bone mass or mechanical strength after each remodeling cycle [14,15]. At menopause when estrogen production is decreased, the increase in resorption cavities due to increased bone resorption, but insufficient increase in bone formation, results to incomplete filling of resorption cavities with new bone leading to a permanent loss of bone mass. Distur‐ bances in the remodeling process of this nature can lead to metabolic bone diseases. One such disturbance caused by oxidative stress, shown to control the functions of both osteoclasts and osteoblasts, may contribute to the pathogenesis of skeletal system including osteoporosis, the most prevalent metabolic bone disease [16].

### **2.2. Prevalence of osteoporosis**

Women over the age of 50 become susceptible to osteoporosis because of the loss of estrogen at menopause [17]. As well, men's susceptibility to osteoporosis is due to low levels of the sex hormone testosterone [18-20]. In the past, a majority of men view osteoporosis as solely a "woman's disease." This is because men in their fifties do not experience the rapid loss of bone mass that women do in the years following menopause, and therefore men osteoporosis does not set in until later in life [21,22]. However, by age 65 to 70, men and women are losing bone mass at the same rate [23]. The World Health Organization (WHO) aptly defined osteoporosis as a systemic disease that is characterized by low bone mass and deterioration of the micro‐ architecture of bone, resulting in an increased risk of fracture. Bone mass or bone mineral density (BMD) is measured using a dual-energy x-ray absorptiometry, or DXA, at various skeletal sites, including the spine, hip and wrist [24].

### **2.3. Bone mineral density as predictor of osteoporosis**

stopping the resoption of bone and preventing osteoporosis in women, are associated with a number of side effects [11,12]. The side effects have been alarming a number of women with osteoporosis in such a way that they are now resorting to other mode of treatment, including that from natural food components. Our laboratory has carried out studies on the use of antioxidants such as lycopene and polyphenols as possible alternatives and/or complementa‐ ries to drugs in the treatment and prevention of osteoporosis. This chapter will include an overview on osteoporosis, the role of oxidative stress in bone cells osteoclasts and osteoblasts, oxidative stress as a risk factor in the development of osteoporosis and a review of studies on the use of antioxidants in counteracting oxidative stress in the prevention of osteoporosis. These topics should put our research in perspective and offer a rationale to our study ap‐ proaches. Finally we will highlight our pioneering studies on the effects of the lipid-soluble antioxidant lycopene and the water-soluble antioxidant polyphenols present in a nutritional supplement in an *in vitro* cultures of osteoblasts and osteoclasts and our clinical studies in the

Bone as a dynamic tissue continuously renews itself throughout life by the process of bone remodeling carried out by a functional and anatomic structure known as the basic multicellular unit (BMU) that requires the coordinated action of three major types of bone cells: osteoclasts, osteoblasts and osteocytes [13,14]. The remodeling process is the result of interactions between these cells and multiple molecular agents, including hormones, growth factors, and cytokines. Bone remodeling is a physiological process that follows a time sequence lasting approximately six months wherein osteoclasts eliminate old or damaged bone which is subsequently replaced with new bone formed by osteoblasts, while the osteocytes functions in the transduction of signals necessary to sustain mechanical loads. The coupled process of bone formation and bone resorption in mature, healthy bone is tightly regulated and maintained in order to prevent a significant alterations in bone mass or mechanical strength after each remodeling cycle [14,15]. At menopause when estrogen production is decreased, the increase in resorption cavities due to increased bone resorption, but insufficient increase in bone formation, results to incomplete filling of resorption cavities with new bone leading to a permanent loss of bone mass. Distur‐ bances in the remodeling process of this nature can lead to metabolic bone diseases. One such disturbance caused by oxidative stress, shown to control the functions of both osteoclasts and osteoblasts, may contribute to the pathogenesis of skeletal system including osteoporosis, the

Women over the age of 50 become susceptible to osteoporosis because of the loss of estrogen at menopause [17]. As well, men's susceptibility to osteoporosis is due to low levels of the sex hormone testosterone [18-20]. In the past, a majority of men view osteoporosis as solely a

prevention of risk for osteoporosis in postmenopausal women.

**2.1. Bone cells involved in the development of osteoporosis**

**2. Osteoporosis — Overview**

118 Topics in Osteoporosis

most prevalent metabolic bone disease [16].

**2.2. Prevalence of osteoporosis**

BMD is expressed as T-score which is a value compared to the expected value for young adults of the same sex and race. WHO has established that for normal BMD, the T-score is between standard deviation of +2.5 and -1.0; for osteopenia/low BMD, T-score is between -1.0 and -2.5, inclusive; for osteoporosis, T-score is lower than -2.5 and for severe osteoporosis,T-score is lower than -2.5 with the presence of one or more fragility fractures [25]. Thus BMD values can identify osteoporosis, determine the risk for fractures (broken bones), and measure the response to osteoporosis treatment [26]. In the case of severe osteoporosis, minimal trauma such as a minor fall or just a hug from a loved one can result to fragility fracture. Fragility fracture is defined by WHO as "a fracture caused by injury that would be insufficient to cause fracture normally. The spine, hip and distal forearm are the most common sites of fragility fracture [27]. Some doctors recommend that people be tested on a regular basis for bone loss. For women, those tests should begin after menopause. For men, they should begin after the age of sixty-five. Such tests are important since there are seldom other signs of osteoporosis. Therefore, those who have a higher rate of bone loss and are at higher risk for a fracture need a better diagnostic tool. Recently, the WHO introduced a prognostic tool to evaluate fracture risk of patients called the FRAX® [28]. The FRAX tool takes into account country, bone mineral density of the hip (when available), age, sex, and 8 clinical risk factors to calculate the 10-year probability of a major osteoporotic fracture and the 10-year probability of a hip fracture [29]. It assesses the 10-year risk of osteoporosis based on individual patient models that combines clinical risk factors (CRF) as well as BMD at the femoral neck [30].

### **2.4. Bone turnover markers for detecting osteoporosis**

During bone remodeling in healthy young adult, bone formation by osteoblasts equals bone resorption by osteoclasts. However In postmenopausal bone loss, the remodeling process becomes significantly more active with a primary increase in bone resorption and a corre‐ sponding, but an insufficient increase in bone formation [31]. Enzymes and/or other proteins are released into the blood that are considered to reflect either bone formation or bone resorption [32] and are termed as bone turnover markers. Molecular markers of bone turnover have been developed as a product of bone remodeling [31] in the diagnostic and therapeutic assessment of metabolic bone disease [33]. They are now used for the individual monitoring of osteoporotic patients treated with antiresorptive agents [34]. Specific and sensitive assess‐ ment of the rate of bone formation and bone resorption and prediction of fracture [35] can now be possible using commercially available biochemical markers [36]. It remained to be seen whether bone turnover markers might contribute a useful independent risk factor for inclusion in FRAX [30]. The bone turnover markers we used for our clinical studies were crosslinked Ntelopeptide of type I collagen (NTx) [37-39] and crosslinked C-telopeptide of type I collagen (CTx) [40,41] as bone resorption markers and bone alkaline phosphatase (BAP) [37-39] and Procollagen type I N-terminal propeptide (PINP) [40,41] for a measure of bone formation in the serum of participants.

bles such as ibandronate and Zoledronate [51] Other drugs available include calcitonin, strontium renalate and the Selective Estrogen Receptor Modulator (SERM), Raloxifene (Evista) [52]. Parathyroid hormone, PTH1-34 or teriparatide (Forteo), is the only anabolic agent current‐ ly approved for use by the FDA [24,53]. The new class of osteoporosis medications now ap‐ proved for use is a fully human monoclonal antibody (Denosumab) which bind to RANKL, imitating the effects of OPG and acting as an inhibitor of RANKL [54]. A number of other drugs

Oxidative Stress and Antioxidants in the Risk of Osteoporosis — Role of the Antioxidants Lycopene and Polyphenols

**Unmodifiable Modifiable**

None of the drugs are without side effects. Side effects that emerged in clinical trials include esophageal irritation with oral administration and acute phase response with iv treatment or high-dose oral therapy. Uncommon side effects that have been noted with wide clinical use in‐ clude osteonecrosis of the jaw, musculoskeletal complaints, and atypical fractures. The num‐ bers of events are small, and a clear cause-and-effect relationship between these events and bisphosphonate treatment has not been established. Because Bisphosphonates accumulate in the bone, they create a reservoir leading to continued release from bone for months or years and provide some residual antifracture reduction when treatment is stopped. For this reason, there is a recommendation for a drug holiday after 5 –10 yr of bisphosphonate treatment [12,55]. The length of the holiday is based on fracture risk and previous duration of treatment and BMD sta‐ tus. Studies with risedronate and alendronate suggest that if treatment is stopped after 3–5 yr, there is persisting antifracture efficacy, at least for 1–2 yr. For those who are not on holiday, the consensus from expert panels [12] suggest not stopping the use of drug since the side effects are often rare, and that the benefits outweigh the side effects. In the balance, most individuals who

Considering the possible adverse side effects of HRT and the ever increasing reports on the side effects of bisphosphonates in the management of postmenopausal osteoporosis, there is

have osteoporosis are much better taking an osteoporosis medication [11].

**2.7. Alternative approach to prevention and treatment of osteoporosis**

Chronic inactivity Low body weight

Medication used

Low lifetime calcium intake

Oxidative stress-related factors Smoking Alcohol intake Low antioxidant status Nutrition deficiency Excessive sports activity Excessive caffeine intake

http://dx.doi.org/10.5772/54703

121

are being tested clinically for osteoporotic treatment and prevention.[24].

Race Sex Age Genetics Body size Family History Previous Fractures

**Table 1.** Risk Factors for Osteoporosis

Although BMD is considered the best parameter for determining the osteoporotic status of men and women, BMD is static and cannot predict changes that may occur post-measurement [42]. As well, changes in BMD occur slowly and can take up to one to two years to be detected during the course of therapy [43,44]. An alternative or additional parameters now measured clinically as either formation or resorption markers in the urine or serum of participants are bone turnover markers which can reveal changes much earlier in the course of therapy compared to changes in BMD [34]. When combined with BMD measurement, changes in bone turnover markers have been significantly linked to fracture risk due to a significant positive correlation between high bone turnover markers and loss of BMD [35]. Bone turnover markers are therefore very useful in assessing treatment protocol for a short duration period, e.g., 3 to 6 months. Measurement of bone turnover markers was therefore utilized in our clinical study during which postmenopausal women were given the antioxidant lycopene for 3 months [37-39] and in another study during which nutritional supplement greens+bone builderTM were administered for a period of eight weeks [40,41]. As will be reviewed in later sections, during the short period of treatment, positive changes were measured that correlated decreased bone resorption markers with decreases in oxidative stress parameters and thereby to decrease of risk for osteoporosis in postmenopausal women.

### **2.5. Risk factors of osteoporosis**

Some of the risk factors for osteoporosis [45,46] are presented in Table 1 [47]. The risk factors that are of interest in our studies are oxidative stress-generating factors, including smoking, alcohol intake, low antioxidant status, nutrition deficiency, excessive sports activity and excessive caffeine intake. Oxidative stress will be reviewed in detail below.

### **2.6. Prevention and treatment of osteoporosis**

Up until 10 years ago, the first line of treatment for women who have gone through menopause and was diagnosed with osteoporosis was hormone replacement therapy (HRT). However, re‐ sults of the Women's Health Initiative (WHI) warned women that HRT leads to higher risks for breast cancer, cardiovascular events, blood clots, cognitive decline, and more [10]. This treat‐ ment for osteoporosis has since been discontinued and is prescribed only for a short period of time to alleviate hot flashes in menopausal women [48]. A wide range of pharmaceuticals are available for the treatment of osteoporosis. The current antiresorptive treatments approved by the Food and Drug Administration (FDA) include a number of bisphosphonates under specific trademarks which inhibit bone resorption [49]. Some are taken daily while others are formulat‐ ed for weekly, monthly or intermittent oral use [50,51]. The newer bisphophonates are injecta‐ bles such as ibandronate and Zoledronate [51] Other drugs available include calcitonin, strontium renalate and the Selective Estrogen Receptor Modulator (SERM), Raloxifene (Evista) [52]. Parathyroid hormone, PTH1-34 or teriparatide (Forteo), is the only anabolic agent current‐ ly approved for use by the FDA [24,53]. The new class of osteoporosis medications now ap‐ proved for use is a fully human monoclonal antibody (Denosumab) which bind to RANKL, imitating the effects of OPG and acting as an inhibitor of RANKL [54]. A number of other drugs are being tested clinically for osteoporotic treatment and prevention.[24].


**Table 1.** Risk Factors for Osteoporosis

be possible using commercially available biochemical markers [36]. It remained to be seen whether bone turnover markers might contribute a useful independent risk factor for inclusion in FRAX [30]. The bone turnover markers we used for our clinical studies were crosslinked Ntelopeptide of type I collagen (NTx) [37-39] and crosslinked C-telopeptide of type I collagen (CTx) [40,41] as bone resorption markers and bone alkaline phosphatase (BAP) [37-39] and Procollagen type I N-terminal propeptide (PINP) [40,41] for a measure of bone formation in

Although BMD is considered the best parameter for determining the osteoporotic status of men and women, BMD is static and cannot predict changes that may occur post-measurement [42]. As well, changes in BMD occur slowly and can take up to one to two years to be detected during the course of therapy [43,44]. An alternative or additional parameters now measured clinically as either formation or resorption markers in the urine or serum of participants are bone turnover markers which can reveal changes much earlier in the course of therapy compared to changes in BMD [34]. When combined with BMD measurement, changes in bone turnover markers have been significantly linked to fracture risk due to a significant positive correlation between high bone turnover markers and loss of BMD [35]. Bone turnover markers are therefore very useful in assessing treatment protocol for a short duration period, e.g., 3 to 6 months. Measurement of bone turnover markers was therefore utilized in our clinical study during which postmenopausal women were given the antioxidant lycopene for 3 months [37-39] and in another study during which nutritional supplement greens+bone builderTM were administered for a period of eight weeks [40,41]. As will be reviewed in later sections, during the short period of treatment, positive changes were measured that correlated decreased bone resorption markers with decreases in oxidative stress parameters and thereby to decrease of

Some of the risk factors for osteoporosis [45,46] are presented in Table 1 [47]. The risk factors that are of interest in our studies are oxidative stress-generating factors, including smoking, alcohol intake, low antioxidant status, nutrition deficiency, excessive sports activity and

Up until 10 years ago, the first line of treatment for women who have gone through menopause and was diagnosed with osteoporosis was hormone replacement therapy (HRT). However, re‐ sults of the Women's Health Initiative (WHI) warned women that HRT leads to higher risks for breast cancer, cardiovascular events, blood clots, cognitive decline, and more [10]. This treat‐ ment for osteoporosis has since been discontinued and is prescribed only for a short period of time to alleviate hot flashes in menopausal women [48]. A wide range of pharmaceuticals are available for the treatment of osteoporosis. The current antiresorptive treatments approved by the Food and Drug Administration (FDA) include a number of bisphosphonates under specific trademarks which inhibit bone resorption [49]. Some are taken daily while others are formulat‐ ed for weekly, monthly or intermittent oral use [50,51]. The newer bisphophonates are injecta‐

excessive caffeine intake. Oxidative stress will be reviewed in detail below.

the serum of participants.

120 Topics in Osteoporosis

risk for osteoporosis in postmenopausal women.

**2.6. Prevention and treatment of osteoporosis**

**2.5. Risk factors of osteoporosis**

None of the drugs are without side effects. Side effects that emerged in clinical trials include esophageal irritation with oral administration and acute phase response with iv treatment or high-dose oral therapy. Uncommon side effects that have been noted with wide clinical use in‐ clude osteonecrosis of the jaw, musculoskeletal complaints, and atypical fractures. The num‐ bers of events are small, and a clear cause-and-effect relationship between these events and bisphosphonate treatment has not been established. Because Bisphosphonates accumulate in the bone, they create a reservoir leading to continued release from bone for months or years and provide some residual antifracture reduction when treatment is stopped. For this reason, there is a recommendation for a drug holiday after 5 –10 yr of bisphosphonate treatment [12,55]. The length of the holiday is based on fracture risk and previous duration of treatment and BMD sta‐ tus. Studies with risedronate and alendronate suggest that if treatment is stopped after 3–5 yr, there is persisting antifracture efficacy, at least for 1–2 yr. For those who are not on holiday, the consensus from expert panels [12] suggest not stopping the use of drug since the side effects are often rare, and that the benefits outweigh the side effects. In the balance, most individuals who have osteoporosis are much better taking an osteoporosis medication [11].

### **2.7. Alternative approach to prevention and treatment of osteoporosis**

Considering the possible adverse side effects of HRT and the ever increasing reports on the side effects of bisphosphonates in the management of postmenopausal osteoporosis, there is an increasing demand for complementary and alternative medicine (CAM) for the prevention and treatment of osteoporosis [56]. CAM is the term for medical practices, services and products that are not a part of standard care. Some of the approaches include exercise, acupuncture, diet, herbs rich in polyphenols and nutritional supplements including calcium, zinc, magnesium boron and other vitamins and minerals. Recent dietary guidelines for the prevention of chronic diseases have recommended an increase in the consumption of fruits and vegetables worldwide [57] that are good sources of dietary antioxidants [58]. The beneficial effects of antioxidants in bone health and osteoporosis are demonstrated epidemiologically and through clinical intervention. Given that many nutrients have been identified as being beneficial to bone health [59,60], there is strong scientific support for the potential benefits of incorporating therapeutic nutritional interventions with contemporary pharmaceutical treatments [61]. Diet is now recognized as an important life-style factor in the management of bone health [62]. As will be reviewed in this chapter, our clinical studies on lycopene treatment and nutritional supplements containing polyphenols and other nutritional components showed positive results on bone health.

pollution and toxins [79]. ROS production increases with age [80,81] and is associated with

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123

Under normal physiological conditions, the cells can fight free radical attack or oxidative stress by promoting antioxidant defenses. A number of endogenous defense mechanisms are present in the body, including the metal chelating proteins and the endogenous antioxidant enzymes catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD). [82]. Exoge‐ nous antioxidants come from dietary sources present in fruits and vegetables containing several phytonutrient antioxidants such as the carotinoids potent antioxidant lipid-soluble lycopene; the water-soluble antioxidant polyphenols; and vitamins such as C and E [83]. In cases where the endogenous antioxidants or antioxidants from diet fail to prevent oxidative damage, the repair antioxidants come into play which include DNA repair enzymes, lipase, protease and transferase [69]. When antioxidants loses its fight with oxidative stress, diseases associated with oxidative stress develop, which include cardiovascular disease, cancer,

The phytochemical antioxidants that are naturally present in plant- and animal-derived foods include the carotenoids, which are lipid-soluble, to which the potent antioxidant lycopene belongs and the water-soluble antioxidants such as polyphenols [85]. Figure 1 is a cartoon depicting the production of oxidative stress from ROS, the damaging effects they exert on DNA, lipid and protein which subsequently leads to chronic diseases and the protection

several chronic diseases including osteoporosis.

diabetes, neurological diseases and osteoporosis [84].

**Figure 1.** Oxidative Stress/Antioxidants & Chronic Diseases

**4. Antioxidants**

afforded by antioxidants.

### **3. Oxidative stress**

Oxidative stress is caused by reactive oxygen species (ROS) which are the main by-products formed in the cells of aerobic organisms that can initiate autocatalytic reactions in such a way that the target molecules gets converted into free radicals causing a chain of damage [63]. There is ample evidence to show that oxidative stress induced by ROS increases the rate of bone loss and is therefore a risk factor for osteoporosis. Epidemiological evidence in humans and studies in animals indicate that aging and the associated increase in ROS are responsible for bone loss [64]. As will be reviewed in later sections, oxidative stress is associated with the activity and function of both the osteoblasts and osteoclasts cells, the two major bone cells involved in the pathogenesis of osteoporosis.

Oxidative stress results from the weakening of antioxidant defense or an over production of ROS in the body. ROS contains one or more unpaired electrons, a state that makes them highly reactive as they seek out another electron to fill their orbital and stabilize their electron balance [65]. Therefore, ROS are a family of highly reactive, oxygen-containing molecules and free radicals, including hydroxyl (OH –), superoxide radicals (O2 –), hydrogen peroxide (H2O2), singlet oxygen, and lipid peroxides [66]. ROS have an extremely short half-life and are difficult to measure in humans, but it is possible to measure the damage they cause to protein, lipids, and DNA and the damage is manifested as chronic diseases including osteoporosis [67]. This can occur by the induction of apoptosis, reduction of cellular proliferation, cell cycle arrest and modulation of cellular differentiation [68]. The major intracellular sites for the generation of ROS are via electron transport chains in the mitochondria, endoplasmic reticulum and nuclear membranes [69]. Oxidative stress may result from normal metabolic activity [70]; during acute or chronic immune responses [71]; lifestyle factors such as cigarette smoke [72,73], high alcohol intake [74,75], low antioxidant status [76], nutrition deficiency [74] excessive sports activity [77], excessive caffeine [78]; and environmental factors such as ultraviolet radiation, chemicals, pollution and toxins [79]. ROS production increases with age [80,81] and is associated with several chronic diseases including osteoporosis.

### **4. Antioxidants**

an increasing demand for complementary and alternative medicine (CAM) for the prevention and treatment of osteoporosis [56]. CAM is the term for medical practices, services and products that are not a part of standard care. Some of the approaches include exercise, acupuncture, diet, herbs rich in polyphenols and nutritional supplements including calcium, zinc, magnesium boron and other vitamins and minerals. Recent dietary guidelines for the prevention of chronic diseases have recommended an increase in the consumption of fruits and vegetables worldwide [57] that are good sources of dietary antioxidants [58]. The beneficial effects of antioxidants in bone health and osteoporosis are demonstrated epidemiologically and through clinical intervention. Given that many nutrients have been identified as being beneficial to bone health [59,60], there is strong scientific support for the potential benefits of incorporating therapeutic nutritional interventions with contemporary pharmaceutical treatments [61]. Diet is now recognized as an important life-style factor in the management of bone health [62]. As will be reviewed in this chapter, our clinical studies on lycopene treatment and nutritional supplements containing polyphenols and other nutritional components

Oxidative stress is caused by reactive oxygen species (ROS) which are the main by-products formed in the cells of aerobic organisms that can initiate autocatalytic reactions in such a way that the target molecules gets converted into free radicals causing a chain of damage [63]. There is ample evidence to show that oxidative stress induced by ROS increases the rate of bone loss and is therefore a risk factor for osteoporosis. Epidemiological evidence in humans and studies in animals indicate that aging and the associated increase in ROS are responsible for bone loss [64]. As will be reviewed in later sections, oxidative stress is associated with the activity and function of both the osteoblasts and osteoclasts cells, the two major bone cells involved in the

Oxidative stress results from the weakening of antioxidant defense or an over production of ROS in the body. ROS contains one or more unpaired electrons, a state that makes them highly reactive as they seek out another electron to fill their orbital and stabilize their electron balance [65]. Therefore, ROS are a family of highly reactive, oxygen-containing molecules and free radicals, including hydroxyl (OH –), superoxide radicals (O2 –), hydrogen peroxide (H2O2), singlet oxygen, and lipid peroxides [66]. ROS have an extremely short half-life and are difficult to measure in humans, but it is possible to measure the damage they cause to protein, lipids, and DNA and the damage is manifested as chronic diseases including osteoporosis [67]. This can occur by the induction of apoptosis, reduction of cellular proliferation, cell cycle arrest and modulation of cellular differentiation [68]. The major intracellular sites for the generation of ROS are via electron transport chains in the mitochondria, endoplasmic reticulum and nuclear membranes [69]. Oxidative stress may result from normal metabolic activity [70]; during acute or chronic immune responses [71]; lifestyle factors such as cigarette smoke [72,73], high alcohol intake [74,75], low antioxidant status [76], nutrition deficiency [74] excessive sports activity [77], excessive caffeine [78]; and environmental factors such as ultraviolet radiation, chemicals,

showed positive results on bone health.

**3. Oxidative stress**

122 Topics in Osteoporosis

pathogenesis of osteoporosis.

Under normal physiological conditions, the cells can fight free radical attack or oxidative stress by promoting antioxidant defenses. A number of endogenous defense mechanisms are present in the body, including the metal chelating proteins and the endogenous antioxidant enzymes catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD). [82]. Exoge‐ nous antioxidants come from dietary sources present in fruits and vegetables containing several phytonutrient antioxidants such as the carotinoids potent antioxidant lipid-soluble lycopene; the water-soluble antioxidant polyphenols; and vitamins such as C and E [83]. In cases where the endogenous antioxidants or antioxidants from diet fail to prevent oxidative damage, the repair antioxidants come into play which include DNA repair enzymes, lipase, protease and transferase [69]. When antioxidants loses its fight with oxidative stress, diseases associated with oxidative stress develop, which include cardiovascular disease, cancer, diabetes, neurological diseases and osteoporosis [84].

The phytochemical antioxidants that are naturally present in plant- and animal-derived foods include the carotenoids, which are lipid-soluble, to which the potent antioxidant lycopene belongs and the water-soluble antioxidants such as polyphenols [85]. Figure 1 is a cartoon depicting the production of oxidative stress from ROS, the damaging effects they exert on DNA, lipid and protein which subsequently leads to chronic diseases and the protection afforded by antioxidants.

**Figure 1.** Oxidative Stress/Antioxidants & Chronic Diseases

### **4.1. Lycopene, a carotinoid lipid-soluble antioxidant**

Lycopene is a potent antioxidant that is not synthesized in the body. It is a carotenoid acyclic isomer of ß-carotene, with no vitamin A activity [86]. It is a highly unsaturated, straightchained hydrocarbon containing a total of 13 double bonds, of which 11 are conjugated, making it one of the most potent antioxidants [84,87]. The singlet oxygen-quenching ability of lycopene is twice that of ß-carotene and 10 times that of α-tocopherol [88]. The chemistry and antioxidant properties of lycopene have been comprehensively reviewed [87]. Other than from tomatoes and processed tomato, the dietary lycopene source of 85% of North Americans, lycopene can al‐ so be obtained from watermelon, pink guavas, and pink grapefruit [85]. Lycopene in an alltrans configuration such as that found in raw tomatoes, is not readily absorbed. Lycopene is absorbed more efficiently from processed tomato products than from raw tomatoes because it is converted from the all-trans to the cis-isomeric configuration with heat processing [89,90]. Since lycopene is a lipid-soluble compound that is absorbed via a chylomicron-mediated mech‐ anism [91], the presence of small amounts of lipids further enhance its absorption [92]. The health benefits of lycopene may be due to its potent antioxidant property, although there is evi‐ dence for other mechanisms such as its effects on gap junction communication [93] and cell cy‐ cling [94]. The reported average daily intake levels of lycopene vary considerably from country to country, from 0.7 mg per day in Finland to 25 mg per day in Canada. However, a generally ac‐ cepted universal level of daily intake is 2.5 mg. There is no official recommended daily intake of lycopene, but based on published research, a daily intake of 7 mg is suggested [95].

polyphenol antioxidants they contain [59,104,105]. The polyphenols of interest in our study are a mixture of flavonoids such as quercetin, apigenin, kaempferol and luteolin present in the supplement greens+TM [106]. greens+TM in combination with another supplement, bone builderTM, were used in our study on osteoblasts cells and in clinical intervention studies on the prevention of risk of osteoporosis in postmenopausal women. Studies on polyphenols and

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125

**5. Studies on the damaging effects of oxidative stress and the beneficial**

The evidence being reported on the role of oxidative stress in osteoblasts has increased exponentially. Up until 2002, only a few studies were reported. Thus, it was reported that treatment of rat osteosarcoma ROS 17/2.8 cells with tumor necrosis factor-alpha (TNF-a) suppressed bone sialoprotein (BSP) gene transcription through a tyrosine kinase-dependent pathway that generates ROS [107]. Osteoblasts can be induced to produce intracellular ROS [108,109], which can cause a decrease in alkaline phosphatase (ALP) activity that is partially inhibited by vitamin E and cause cell death [108,109]. The intracellular calcium (Ca++) activity in osteoblasts is modulated by H2O2 by increasing Ca++ release from the intracellular Ca++ stores [110]. High concentrations of ROS can damage osteoblast cells to prevent normal growth and development [111] and have been shown to induce osteoblast death [112]. In osteoblasts, H2O2 has been shown to decrease cell growth, ALP activity, calcification, mineralization and gene expression of osteogenic markers such as ALP, bone sailoprotein (BSP) and runt-related transcription factor 2 (Runx2) [113,114]. More recently, Ueno et al induced oxidative stress by adding 100 microM H2O2 to osteoblasts cultured from rat bone marrow, and showed that this treatment substantially impaired the proliferation, differentiation, and mineralization and that addition of the antioxidant N-acetyl cysteine into the culture restored these damages to a near normal level [115]]. With their study on hydrogen sulphide, Xu et al concluded that hydrogen sulfide (H2S) protected MC3T3-E1 osteoblastic cells via a MAPK (p38 and ERK1/2)-dependent mechanism against hydrogen peroxide (H2O2)-induced oxidative injury that cause the

More recently reported inducers of oxidative stress include Arsenic trioxide [117], Cobalt and

*In vitro* studies suggest an important role for antioxidants in abrogating the effects of oxidative stress on bone. Trolox, a water soluble vitamin E analogue, was shown to enhance ALP activity in MC3T3-E1 osteoblast-like cells, thus enhancing osteoblast differentiation by decreasing the generation of ROS [111]. The addition of metallothionein, a metal-chelating preventative anti‐ oxidant, to primary mouse bone marrow stromal cells impaired H2O2-stimulated NfκΒ signal‐

(Studies involving lycopene and polyphenols will be reviewed at a later section)

suppression of proliferation and differentiation of the cells [116].

Chromium ion [118] and Vanadium Compounds [119].

bone will be reviewed in later sections.

**effects of antioxidants**

**5.1. Studies on osteoblasts**

The role of lycopene in the prevention of human diseases is supported by a number of evidence [96]. Giovannicci was the first to publish the initial epidemiological observations suggesting an inverse relationship between the intake of tomatoes and lycopene and the incidence of prostate cancer [97]. Since then, there have been several epidemiological as well as clinical intervention studies showing the relationship between lycopene intake and the prevention of cancers at other sites, as well as coronary heart disease, hypertension, diabetes, macular degenerative disease, male infertility, and neurodegenerative disease [84]. The role of lycopene in bone health has so far been based on its potent antioxidant properties, the well known role of oxidative stress in bone health, and the limited studies on the effects of lycopene in bone cells in culture (see below) and more recently, the results of epidemiological studies [39,98]. To date our clinical intervention studies at St. Michael's Hospital on the role of lycopene and elucidation of its mechanism in lowering the risk for osteoporosis in postmenopausal women (aged 50 to 60 years) are so far the only studies reported in the literature.

### **4.2. Polyphenols, the water-soluble antioxidant**

Polyphenols are a class of water-soluble molecules naturally found in plants. They are defined as compounds having molecular masses ranging from 500 to 3000–4000 Da and possessing 12 to 16 phenolic hydroxy groups on five to seven aromatic rings per 1000 Da of relative molecular mass [99]. It is estimated that there are 10,000 different phytonutrients (phyto, meaning from plants). To date, over 8000 polyphenols have been identified [100]. Polyphenols can be divided into 2 main groups: flavonoids and non-flavonoids [101-103]. The health benefits associated with fruits, vegetables, red wine, tea, and Mediterranean diets are probably linked to the polyphenol antioxidants they contain [59,104,105]. The polyphenols of interest in our study are a mixture of flavonoids such as quercetin, apigenin, kaempferol and luteolin present in the supplement greens+TM [106]. greens+TM in combination with another supplement, bone builderTM, were used in our study on osteoblasts cells and in clinical intervention studies on the prevention of risk of osteoporosis in postmenopausal women. Studies on polyphenols and bone will be reviewed in later sections.

### **5. Studies on the damaging effects of oxidative stress and the beneficial effects of antioxidants**

(Studies involving lycopene and polyphenols will be reviewed at a later section)

### **5.1. Studies on osteoblasts**

**4.1. Lycopene, a carotinoid lipid-soluble antioxidant**

124 Topics in Osteoporosis

Lycopene is a potent antioxidant that is not synthesized in the body. It is a carotenoid acyclic isomer of ß-carotene, with no vitamin A activity [86]. It is a highly unsaturated, straightchained hydrocarbon containing a total of 13 double bonds, of which 11 are conjugated, making it one of the most potent antioxidants [84,87]. The singlet oxygen-quenching ability of lycopene is twice that of ß-carotene and 10 times that of α-tocopherol [88]. The chemistry and antioxidant properties of lycopene have been comprehensively reviewed [87]. Other than from tomatoes and processed tomato, the dietary lycopene source of 85% of North Americans, lycopene can al‐ so be obtained from watermelon, pink guavas, and pink grapefruit [85]. Lycopene in an alltrans configuration such as that found in raw tomatoes, is not readily absorbed. Lycopene is absorbed more efficiently from processed tomato products than from raw tomatoes because it is converted from the all-trans to the cis-isomeric configuration with heat processing [89,90]. Since lycopene is a lipid-soluble compound that is absorbed via a chylomicron-mediated mech‐ anism [91], the presence of small amounts of lipids further enhance its absorption [92]. The health benefits of lycopene may be due to its potent antioxidant property, although there is evi‐ dence for other mechanisms such as its effects on gap junction communication [93] and cell cy‐ cling [94]. The reported average daily intake levels of lycopene vary considerably from country to country, from 0.7 mg per day in Finland to 25 mg per day in Canada. However, a generally ac‐ cepted universal level of daily intake is 2.5 mg. There is no official recommended daily intake of

lycopene, but based on published research, a daily intake of 7 mg is suggested [95].

(aged 50 to 60 years) are so far the only studies reported in the literature.

**4.2. Polyphenols, the water-soluble antioxidant**

The role of lycopene in the prevention of human diseases is supported by a number of evidence [96]. Giovannicci was the first to publish the initial epidemiological observations suggesting an inverse relationship between the intake of tomatoes and lycopene and the incidence of prostate cancer [97]. Since then, there have been several epidemiological as well as clinical intervention studies showing the relationship between lycopene intake and the prevention of cancers at other sites, as well as coronary heart disease, hypertension, diabetes, macular degenerative disease, male infertility, and neurodegenerative disease [84]. The role of lycopene in bone health has so far been based on its potent antioxidant properties, the well known role of oxidative stress in bone health, and the limited studies on the effects of lycopene in bone cells in culture (see below) and more recently, the results of epidemiological studies [39,98]. To date our clinical intervention studies at St. Michael's Hospital on the role of lycopene and elucidation of its mechanism in lowering the risk for osteoporosis in postmenopausal women

Polyphenols are a class of water-soluble molecules naturally found in plants. They are defined as compounds having molecular masses ranging from 500 to 3000–4000 Da and possessing 12 to 16 phenolic hydroxy groups on five to seven aromatic rings per 1000 Da of relative molecular mass [99]. It is estimated that there are 10,000 different phytonutrients (phyto, meaning from plants). To date, over 8000 polyphenols have been identified [100]. Polyphenols can be divided into 2 main groups: flavonoids and non-flavonoids [101-103]. The health benefits associated with fruits, vegetables, red wine, tea, and Mediterranean diets are probably linked to the

The evidence being reported on the role of oxidative stress in osteoblasts has increased exponentially. Up until 2002, only a few studies were reported. Thus, it was reported that treatment of rat osteosarcoma ROS 17/2.8 cells with tumor necrosis factor-alpha (TNF-a) suppressed bone sialoprotein (BSP) gene transcription through a tyrosine kinase-dependent pathway that generates ROS [107]. Osteoblasts can be induced to produce intracellular ROS [108,109], which can cause a decrease in alkaline phosphatase (ALP) activity that is partially inhibited by vitamin E and cause cell death [108,109]. The intracellular calcium (Ca++) activity in osteoblasts is modulated by H2O2 by increasing Ca++ release from the intracellular Ca++ stores [110]. High concentrations of ROS can damage osteoblast cells to prevent normal growth and development [111] and have been shown to induce osteoblast death [112]. In osteoblasts, H2O2 has been shown to decrease cell growth, ALP activity, calcification, mineralization and gene expression of osteogenic markers such as ALP, bone sailoprotein (BSP) and runt-related transcription factor 2 (Runx2) [113,114]. More recently, Ueno et al induced oxidative stress by adding 100 microM H2O2 to osteoblasts cultured from rat bone marrow, and showed that this treatment substantially impaired the proliferation, differentiation, and mineralization and that addition of the antioxidant N-acetyl cysteine into the culture restored these damages to a near normal level [115]]. With their study on hydrogen sulphide, Xu et al concluded that hydrogen sulfide (H2S) protected MC3T3-E1 osteoblastic cells via a MAPK (p38 and ERK1/2)-dependent mechanism against hydrogen peroxide (H2O2)-induced oxidative injury that cause the suppression of proliferation and differentiation of the cells [116].

More recently reported inducers of oxidative stress include Arsenic trioxide [117], Cobalt and Chromium ion [118] and Vanadium Compounds [119].

*In vitro* studies suggest an important role for antioxidants in abrogating the effects of oxidative stress on bone. Trolox, a water soluble vitamin E analogue, was shown to enhance ALP activity in MC3T3-E1 osteoblast-like cells, thus enhancing osteoblast differentiation by decreasing the generation of ROS [111]. The addition of metallothionein, a metal-chelating preventative anti‐ oxidant, to primary mouse bone marrow stromal cells impaired H2O2-stimulated NfκΒ signal‐ ling, consequently preventing any inhibition of osteoblast differentiation [114]. Osteoblasts have been shown to produce antioxidants such as GPx which can protect against the damaging effects of ROS [120]. In MC3T3-E1 osteoblast-like cells, treatment with H2O2 to induce oxidative stress was associated with the prolonged up-regulation in gene expression of the transcription factor nuclear factor E2 p45-related factor 2 (Nrf2), which regulates antioxidant enzymes by as‐ sisting with recognition of the antioxidant-response element [113]. Using xanthine/xanthine oxidase to generate ROS, Fatokun *et al*.[120] showed that damage induced by ROS, as evi‐ denced by decreased cell viability, was prevented by CAT in MC3T3-E1 osteoblast-like cells. This effect is attributed to the ability of CAT to neutralize H2O2 [120]. Newer mechanism of ac‐ tion of ROS is beginning to come to light. It has been shown that increased ROS production di‐ verts the limited pool of β-catenin from TCF/LEF to FOXO-mediated transcription, converting the beneficial effects of Wnt/β-catenin on bone, eventually leading to decrease osteoblasts num‐ ber and activity and eventually leading to osteoporosis [121-124].

Antioxidants also play a role in osteoclast activity. Osteoclasts produce the antioxidant enzyme SOD in the plasma membrane [152]. ROS production in osteoclasts was inhibited after treating the cells with antioxidant enzymes such as SOD [142] and catalase [146]. ROS production in osteoclasts was also inhibited by estrogen [149], the superoxide scavenger deferoxamine mesylatemanganese complex [153], pyrrolidine dithiocarbamate (PDTC), and N-acetyl

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Other more recent antioxidant shown to affect osteoclasts include polyphenol extracts from dried plums [155], curcumerin [156], ascorbic acid [157], salvia miltorrhiza [158], coffee diterpene Kahweol [159], delthametrin [160], to name a few. The use of antioxidants from natural sources, such as fruits and vegetables, could be another way of inhibiting ROS. The

OVXed rats were treated with Strontium ranelate and at the end of the treatment, oxidative parameters including malondialdehyde (MDA) level, superoxide dismutase (SOD), gluta‐ thione peroxidase (GSH-Px) and catalase (CAT) activities were determined by biochemical analysis methods. Their results showed that Sr has preventive effect on oxidative damage in ovariectomized rats [161]. Yin et al showed that protection against osteoporosis by statins is linked to a reduction of oxidative stress and restoration of NO formation in aged and ovar‐ iectomized rats [162]. To investigate the anti-osteoporosis effect of Rhizoma Drynariae (RD), an effectively traditional Chinese medicine and its action mechanism, Liu et al administered with or without RD extract at a therapeutic dose to a group of rats for 12 weeks and showed that the anti-osteoporosis effect of RD has been reliably confirmed by the metabonomics method and that the osteoporosis might be prevented by RD via, among other things, through intervening antioxidant-oxidation balance in vivo in rats [163]. Treatment of OVXed rats with Salvia miltiorrhiza ethanol extract significantly ameliorated the decrease in BMD and trabec‐ ular bone mass according to DEXA and trabecular bone architecture analysis of trabecular bone structural parameters by μ-CT scanning. As well, SM decreased the released TRAP-5b, an osteoclast activation marker and oxidative stress parameters including MDA and NO induced by OVX [164]. Oxidative stress (OS) was assessed 100 days postovariectomy by measuring the activity of several enzymes, including catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase, as well as the concentrations of malondialdehyde (MDA), nitric oxide (NO), and total sulfhydryl groups in plasma and bone homogenates of OVXed rats treated with or without vitamin C. Their results suggest that ovariectomy may produce osteoporosis and oxidative stress in females, and vitamin C supplementation may provide alterations regarding improvement in OS and BMD values [165]. Curcumin was shown to inhibit OVX-induced bone loss, at least in part by reducing osteoclastogenesis as a result of increased antioxidant activity and impaired RANKL signaling [166]. In order to investigate the pathologic significance of oxidative stress in bones, Nojiri et al showed that mice deficient in cytoplasmic copper/zinc superoxide dismutase exhibited a distinct weakness in bone stiffness and decreased BMD, aging-like changes in collagen cross-linking, and transcriptional alterations in the genes associated with osteogenesis. They further demonstrated that intra‐

use of lycopene and polyphenols in this regard is reviewed in a later section.

cysteine (NAC) [154].

**5.3. Studies of on animal**

In recent years, the number of antioxidants reported to prevent oxidative stress in osteoblasts are as follows: Tetrahydrostibene [125,126], Curculigoside [127], Green tea [128], Simvastatin [129], N-acetylcysteine [115], flavonoids from parsimmon [130], prevastatin [131], Linarin [132], Panaxnotaginseng saponin [133], crysoeriol from surya cilliata leaves [134], quercetin [135] Drynaria fortunei [136], cathamus tinctorium flower extract [137], estrogen [138], diazoxide, atractylodes japonica root extract [139]. and Myrcetin, a naturally occurring flavonoid [140]. The mechanism of osteblastic defense against oxidative stress was shown to involve β-Catenin which serves as a cofactor of the forkhead box O (FOXO) transcription factors [121].

### **5.2. Studies on osteoclasts**

The mechanisms involved in the differentiation of osteoclasts and their ability to resorb bone is beginning to be unraveled, and evidence shows that ROS may be involved in this process [141]. Superoxide was detected both at the osteoclast-bone interface and intracellularly using nitroblue tetrazolium (NBT), which is reduced to purple-colored formazan by ROS, suggesting the participation of superoxide in bone resorption [142]. Both the H2O2 produced by endothelial cells [143] intimately associated with osteoclasts and the H2O2 that is produced by osteoclasts [144] increase osteoclastic activity and bone resorption. H2O2 may also be involved in osteoclast motility [144], differentiation of osteoclast precursors [145] and the regulation of osteoclast formation [146]. Osteoclastic superoxide is produced by NADPH oxidase [147]. The degrada‐ tion of collagen and other proteins is caused by highly destructive ROS as a result of the reaction of H2O2 with tartrate-resistant acid phosphatase (TRAP), found on the surface of osteoclasts [148]. 1,25-Dihydroxyvitamin D3 had a direct nongenomic effect on the generation of superoxide anion (O2\_), which was inhibited by estrogen [149]. Estrogen has been reported to have an antioxidant property [150]. Hormones known to stimulate bone resorption, such as parathyroid hormone (PTH) [151] and 1,25(OH)2D3, have stimulatory effects on ROS produc‐ tion in osteoclasts [149] and hormones known to have inhibitory effects on bone resorption, such as calcitonin, inhibit ROS production [151].

Antioxidants also play a role in osteoclast activity. Osteoclasts produce the antioxidant enzyme SOD in the plasma membrane [152]. ROS production in osteoclasts was inhibited after treating the cells with antioxidant enzymes such as SOD [142] and catalase [146]. ROS production in osteoclasts was also inhibited by estrogen [149], the superoxide scavenger deferoxamine mesylatemanganese complex [153], pyrrolidine dithiocarbamate (PDTC), and N-acetyl cysteine (NAC) [154].

Other more recent antioxidant shown to affect osteoclasts include polyphenol extracts from dried plums [155], curcumerin [156], ascorbic acid [157], salvia miltorrhiza [158], coffee diterpene Kahweol [159], delthametrin [160], to name a few. The use of antioxidants from natural sources, such as fruits and vegetables, could be another way of inhibiting ROS. The use of lycopene and polyphenols in this regard is reviewed in a later section.

### **5.3. Studies of on animal**

ling, consequently preventing any inhibition of osteoblast differentiation [114]. Osteoblasts have been shown to produce antioxidants such as GPx which can protect against the damaging effects of ROS [120]. In MC3T3-E1 osteoblast-like cells, treatment with H2O2 to induce oxidative stress was associated with the prolonged up-regulation in gene expression of the transcription factor nuclear factor E2 p45-related factor 2 (Nrf2), which regulates antioxidant enzymes by as‐ sisting with recognition of the antioxidant-response element [113]. Using xanthine/xanthine oxidase to generate ROS, Fatokun *et al*.[120] showed that damage induced by ROS, as evi‐ denced by decreased cell viability, was prevented by CAT in MC3T3-E1 osteoblast-like cells. This effect is attributed to the ability of CAT to neutralize H2O2 [120]. Newer mechanism of ac‐ tion of ROS is beginning to come to light. It has been shown that increased ROS production di‐ verts the limited pool of β-catenin from TCF/LEF to FOXO-mediated transcription, converting the beneficial effects of Wnt/β-catenin on bone, eventually leading to decrease osteoblasts num‐

In recent years, the number of antioxidants reported to prevent oxidative stress in osteoblasts are as follows: Tetrahydrostibene [125,126], Curculigoside [127], Green tea [128], Simvastatin [129], N-acetylcysteine [115], flavonoids from parsimmon [130], prevastatin [131], Linarin [132], Panaxnotaginseng saponin [133], crysoeriol from surya cilliata leaves [134], quercetin [135] Drynaria fortunei [136], cathamus tinctorium flower extract [137], estrogen [138], diazoxide, atractylodes japonica root extract [139]. and Myrcetin, a naturally occurring flavonoid [140]. The mechanism of osteblastic defense against oxidative stress was shown to involve β-Catenin which serves as a cofactor of the forkhead box O (FOXO) transcription

The mechanisms involved in the differentiation of osteoclasts and their ability to resorb bone is beginning to be unraveled, and evidence shows that ROS may be involved in this process [141]. Superoxide was detected both at the osteoclast-bone interface and intracellularly using nitroblue tetrazolium (NBT), which is reduced to purple-colored formazan by ROS, suggesting the participation of superoxide in bone resorption [142]. Both the H2O2 produced by endothelial cells [143] intimately associated with osteoclasts and the H2O2 that is produced by osteoclasts [144] increase osteoclastic activity and bone resorption. H2O2 may also be involved in osteoclast motility [144], differentiation of osteoclast precursors [145] and the regulation of osteoclast formation [146]. Osteoclastic superoxide is produced by NADPH oxidase [147]. The degrada‐ tion of collagen and other proteins is caused by highly destructive ROS as a result of the reaction of H2O2 with tartrate-resistant acid phosphatase (TRAP), found on the surface of osteoclasts [148]. 1,25-Dihydroxyvitamin D3 had a direct nongenomic effect on the generation of superoxide anion (O2\_), which was inhibited by estrogen [149]. Estrogen has been reported to have an antioxidant property [150]. Hormones known to stimulate bone resorption, such as parathyroid hormone (PTH) [151] and 1,25(OH)2D3, have stimulatory effects on ROS produc‐ tion in osteoclasts [149] and hormones known to have inhibitory effects on bone resorption,

ber and activity and eventually leading to osteoporosis [121-124].

factors [121].

126 Topics in Osteoporosis

**5.2. Studies on osteoclasts**

such as calcitonin, inhibit ROS production [151].

OVXed rats were treated with Strontium ranelate and at the end of the treatment, oxidative parameters including malondialdehyde (MDA) level, superoxide dismutase (SOD), gluta‐ thione peroxidase (GSH-Px) and catalase (CAT) activities were determined by biochemical analysis methods. Their results showed that Sr has preventive effect on oxidative damage in ovariectomized rats [161]. Yin et al showed that protection against osteoporosis by statins is linked to a reduction of oxidative stress and restoration of NO formation in aged and ovar‐ iectomized rats [162]. To investigate the anti-osteoporosis effect of Rhizoma Drynariae (RD), an effectively traditional Chinese medicine and its action mechanism, Liu et al administered with or without RD extract at a therapeutic dose to a group of rats for 12 weeks and showed that the anti-osteoporosis effect of RD has been reliably confirmed by the metabonomics method and that the osteoporosis might be prevented by RD via, among other things, through intervening antioxidant-oxidation balance in vivo in rats [163]. Treatment of OVXed rats with Salvia miltiorrhiza ethanol extract significantly ameliorated the decrease in BMD and trabec‐ ular bone mass according to DEXA and trabecular bone architecture analysis of trabecular bone structural parameters by μ-CT scanning. As well, SM decreased the released TRAP-5b, an osteoclast activation marker and oxidative stress parameters including MDA and NO induced by OVX [164]. Oxidative stress (OS) was assessed 100 days postovariectomy by measuring the activity of several enzymes, including catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase, as well as the concentrations of malondialdehyde (MDA), nitric oxide (NO), and total sulfhydryl groups in plasma and bone homogenates of OVXed rats treated with or without vitamin C. Their results suggest that ovariectomy may produce osteoporosis and oxidative stress in females, and vitamin C supplementation may provide alterations regarding improvement in OS and BMD values [165]. Curcumin was shown to inhibit OVX-induced bone loss, at least in part by reducing osteoclastogenesis as a result of increased antioxidant activity and impaired RANKL signaling [166]. In order to investigate the pathologic significance of oxidative stress in bones, Nojiri et al showed that mice deficient in cytoplasmic copper/zinc superoxide dismutase exhibited a distinct weakness in bone stiffness and decreased BMD, aging-like changes in collagen cross-linking, and transcriptional alterations in the genes associated with osteogenesis. They further demonstrated that intra‐ cellular oxidative resulted in the decrease in osteoblast number and accompanied by suppres‐ sion of RANKL/M-CSF osteoclastogenic signaling in bone; treatment with an antioxidant, vitamin C, effectively improved bone fragility and osteoblastic survival [167].

otic controls [183]. Supplementation of ascorbic acid and alpha-tocopherol was found useful

Oxidative Stress and Antioxidants in the Risk of Osteoporosis — Role of the Antioxidants Lycopene and Polyphenols

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129

Postmenopausal women with osteoporosis have been shown to have markedly reduced serum concentrations of retinol, β-cryptoxanthin, zeaxanthin and α- and β-carotene, compared to healthy postmenopausal women [175]. Overall carotenoid intake has been found to be inversely associated with risk of fracture [184]. Sadly, the effect of β-carotene on the risk of osteoporosis is still controversial. There are studies which suggest that β-carotene has benefi‐ cial effect on bone [98,185,186], while other studies suggest a null or even detrimental effect,

In summary, the studies presented above provide evidence of the detrimental effects of

The direct role of lycopene in osteoblasts and osteoclasts, the cells involved in the pathogenesis of osteoporosis is now being unraveled. This involvement is further supported by both epidemiological and clinical intervention with lycopene in postmenopausal women men who

Only few studies on the effects of lycopene in osteoblasts have been reported. This is most likely because lycopene is not soluble in the culture medium and needed to be solubilized in organic solvent before it can be added to the cell culture. In our study, we used lyc-o-mato preparation that is partially dispersed in micelle form in water. When added to the human osteoblast-like SaOS-2 cells, lycopene had a stimulatory effect on cell proliferation as well as a stimulatory effect on alkaline phosphatase activity, a marker of osteoblastic differentiation in more mature cells but, depending on the time of addition, it had an inhibitory or no effect on younger SaOS-Dex cells. These findings comprised the first report on the effect of lycopene on human osteoblasts [188]. In another study, the effect of lycopene on MC3T3 cells (the osteoblastic cells of mice) was contrary to the findings of Kim et al. [188] in that lycopene had an inhibitory effect on cell proliferation [189]. The discrepancy in the effects of lycopene on cell proliferation could be a result of species differences, age of the cells when lycopene was added or experimental conditions. Both studies, however, reported an effect of lycopene on the differentiation of the cells by stimulating the alkaline phosphatase activity [188,189] and gene expression of BSP [189]. The lycopene used in our study is the trans-configuration (95% trans, 5% cis). Subsequently, we studied which configuration of lycopene will prevent the damaging effect of oxidative stress as well as repair this damage in human osteoblast cultures. Lycopene with varying content of cis- and trans- configuration (45:55, 28:72 or 5:95 *cis*:*trans* lycopene) were added to cell cultures before and after challenging with H2O2 and the effect on the generation of ROS and stimulation of mineralized bone nodule were assessed. Our results demonstrated that the addition of H2O2 resulted in significant increase in generation of ROS

oxidative stress and beneficial effects of antioxidants on the risk of osteoporosis.

in preventing bone loss linked to oxidative stress in elderly [181].

most probably due to its association with vitamin A [187].

**6. Studies on lycopene**

are at risk of osteoporosis.

**6.1.** *In vitro* **studies of lycopene in osteoblasts**

### **5.4. Epidemiological and clinical studies on osteoporosis**

The detrimental effect of oxidative stress and the beneficial role of antioxidant in osteoporosis have been reviewed [58,83,168,169]. There is now ample evidence to suggest that ROS-induced oxidative stress is associated with the pathogenesis of osteoporosis. Thus, epidemiological studies demonstrated the adverse effect on bone of oxidative stress produced during strenuous exercise [170]; among heavy smokers [171] and that antioxidants including vitamin C, E and β-carotene may counteract these adverse effects and reduce the risk of osteoporosis [170-173]. A study of severe osteoporotic syndrome in relatively young males showed evidence linking osteoporosis to an increase in oxidative stress [174]. Maggio et al [175] demonstrated that women with osteoporosis had markedly decreased plasma antioxidants. A biochemical link between reduced bone density and increased oxidative stress biomarker 8-iso-prostaglandin F alpha (8-iso-PGF) has been reported [176,177]. Positive correlation was found between the severity of osteoporosis and the level of oxidative stress marker lactic acid in 2 men with mitochondrial deletion (mtDNA) [178].

Evidence points to the fact that Postmenopausal women are more prone to osteoporosis due to reduction in estrogen, but there is also ample evidence to support the theory that oxidative stress which accompanies the reduction in estrogen level may be the cause of osteoporosis [179]. Indeed, estrogen has been shown to have antioxidant properties [150]. Earlier reports on the association of oxidative stress with osteoporosis were confined mainly to epidemio‐ logical studies. Vitamins C, E, and A, uric acid, the antioxidant enzymes SOD in plasma and erythrocytes and GPx in plasma were consistently lower in osteoporotic than in control subjects. An epidemiological study by Hahn *et al*[180] found that GPx activity was significantly higher in postmenopausal women with osteopenia than that of postmenopausal women with a normal BMD, likely as a primary defense against the high levels of H2O2 in osteopenic women [180]. Osteoporotic women were found to have significantly depressed activities of CAT, GPx and SOD, compared to those found in healthy control women [177,181,182]. Furthermore, concentrations of the antioxidant enzymes SOD and CAT have been positively correlated with BMD which demonstrates a link between antioxidant status and BMD in postmenopausal women[182]. Surprisingly, a cross sectional analysis in healthy postmenopausal women aged 60-78 years revealed a negative association between 8-OH-dG levels and BMD of the lumbar spine, total hip, femoral neck, and trochanter and positive association with type I collagen Ctelopeptide (ICTP) levels, showing that oxidative stress is associated with increased bone resorption and low bone mass even in otherwise healthy women. Medications used to treat postmenopausal osteoporosis such as HRT and Raloxifene [183] may act in part to decrease oxidative stress by acting in part as antioxidants. A 3-month course of Raloxifene therapy significantly decreased the lipid peroxidation and increased the CAT activity in women with postmenopausal osteoporosis [177,181,182]. Raloxifene treatment for 12 months significantly decreased protein oxidation in osteoporotic participants compared to matched, non-osteopor‐ otic controls [183]. Supplementation of ascorbic acid and alpha-tocopherol was found useful in preventing bone loss linked to oxidative stress in elderly [181].

Postmenopausal women with osteoporosis have been shown to have markedly reduced serum concentrations of retinol, β-cryptoxanthin, zeaxanthin and α- and β-carotene, compared to healthy postmenopausal women [175]. Overall carotenoid intake has been found to be inversely associated with risk of fracture [184]. Sadly, the effect of β-carotene on the risk of osteoporosis is still controversial. There are studies which suggest that β-carotene has benefi‐ cial effect on bone [98,185,186], while other studies suggest a null or even detrimental effect, most probably due to its association with vitamin A [187].

In summary, the studies presented above provide evidence of the detrimental effects of oxidative stress and beneficial effects of antioxidants on the risk of osteoporosis.

### **6. Studies on lycopene**

cellular oxidative resulted in the decrease in osteoblast number and accompanied by suppres‐ sion of RANKL/M-CSF osteoclastogenic signaling in bone; treatment with an antioxidant,

The detrimental effect of oxidative stress and the beneficial role of antioxidant in osteoporosis have been reviewed [58,83,168,169]. There is now ample evidence to suggest that ROS-induced oxidative stress is associated with the pathogenesis of osteoporosis. Thus, epidemiological studies demonstrated the adverse effect on bone of oxidative stress produced during strenuous exercise [170]; among heavy smokers [171] and that antioxidants including vitamin C, E and β-carotene may counteract these adverse effects and reduce the risk of osteoporosis [170-173]. A study of severe osteoporotic syndrome in relatively young males showed evidence linking osteoporosis to an increase in oxidative stress [174]. Maggio et al [175] demonstrated that women with osteoporosis had markedly decreased plasma antioxidants. A biochemical link between reduced bone density and increased oxidative stress biomarker 8-iso-prostaglandin F alpha (8-iso-PGF) has been reported [176,177]. Positive correlation was found between the severity of osteoporosis and the level of oxidative stress marker lactic acid in 2 men with

Evidence points to the fact that Postmenopausal women are more prone to osteoporosis due to reduction in estrogen, but there is also ample evidence to support the theory that oxidative stress which accompanies the reduction in estrogen level may be the cause of osteoporosis [179]. Indeed, estrogen has been shown to have antioxidant properties [150]. Earlier reports on the association of oxidative stress with osteoporosis were confined mainly to epidemio‐ logical studies. Vitamins C, E, and A, uric acid, the antioxidant enzymes SOD in plasma and erythrocytes and GPx in plasma were consistently lower in osteoporotic than in control subjects. An epidemiological study by Hahn *et al*[180] found that GPx activity was significantly higher in postmenopausal women with osteopenia than that of postmenopausal women with a normal BMD, likely as a primary defense against the high levels of H2O2 in osteopenic women [180]. Osteoporotic women were found to have significantly depressed activities of CAT, GPx and SOD, compared to those found in healthy control women [177,181,182]. Furthermore, concentrations of the antioxidant enzymes SOD and CAT have been positively correlated with BMD which demonstrates a link between antioxidant status and BMD in postmenopausal women[182]. Surprisingly, a cross sectional analysis in healthy postmenopausal women aged 60-78 years revealed a negative association between 8-OH-dG levels and BMD of the lumbar spine, total hip, femoral neck, and trochanter and positive association with type I collagen Ctelopeptide (ICTP) levels, showing that oxidative stress is associated with increased bone resorption and low bone mass even in otherwise healthy women. Medications used to treat postmenopausal osteoporosis such as HRT and Raloxifene [183] may act in part to decrease oxidative stress by acting in part as antioxidants. A 3-month course of Raloxifene therapy significantly decreased the lipid peroxidation and increased the CAT activity in women with postmenopausal osteoporosis [177,181,182]. Raloxifene treatment for 12 months significantly decreased protein oxidation in osteoporotic participants compared to matched, non-osteopor‐

vitamin C, effectively improved bone fragility and osteoblastic survival [167].

**5.4. Epidemiological and clinical studies on osteoporosis**

mitochondrial deletion (mtDNA) [178].

128 Topics in Osteoporosis

The direct role of lycopene in osteoblasts and osteoclasts, the cells involved in the pathogenesis of osteoporosis is now being unraveled. This involvement is further supported by both epidemiological and clinical intervention with lycopene in postmenopausal women men who are at risk of osteoporosis.

### **6.1.** *In vitro* **studies of lycopene in osteoblasts**

Only few studies on the effects of lycopene in osteoblasts have been reported. This is most likely because lycopene is not soluble in the culture medium and needed to be solubilized in organic solvent before it can be added to the cell culture. In our study, we used lyc-o-mato preparation that is partially dispersed in micelle form in water. When added to the human osteoblast-like SaOS-2 cells, lycopene had a stimulatory effect on cell proliferation as well as a stimulatory effect on alkaline phosphatase activity, a marker of osteoblastic differentiation in more mature cells but, depending on the time of addition, it had an inhibitory or no effect on younger SaOS-Dex cells. These findings comprised the first report on the effect of lycopene on human osteoblasts [188]. In another study, the effect of lycopene on MC3T3 cells (the osteoblastic cells of mice) was contrary to the findings of Kim et al. [188] in that lycopene had an inhibitory effect on cell proliferation [189]. The discrepancy in the effects of lycopene on cell proliferation could be a result of species differences, age of the cells when lycopene was added or experimental conditions. Both studies, however, reported an effect of lycopene on the differentiation of the cells by stimulating the alkaline phosphatase activity [188,189] and gene expression of BSP [189]. The lycopene used in our study is the trans-configuration (95% trans, 5% cis). Subsequently, we studied which configuration of lycopene will prevent the damaging effect of oxidative stress as well as repair this damage in human osteoblast cultures. Lycopene with varying content of cis- and trans- configuration (45:55, 28:72 or 5:95 *cis*:*trans* lycopene) were added to cell cultures before and after challenging with H2O2 and the effect on the generation of ROS and stimulation of mineralized bone nodule were assessed. Our results demonstrated that the addition of H2O2 resulted in significant increase in generation of ROS (p<0.001), which long-term resulted in a decreased number and area of mineralized bone nodules (both: p<0.001). Pre- and post-treatment with 45:55 or 28:72 *cis*:*trans* lycopene resulted in significantly lower ROS generation (p<0.001) and higher mineralized bone nodule area (p<0.05), compared to treatment with H2O2 alone, vehicle or 5:95 *cis*:*trans* lycopene. These findings support the hypothesis that the *cis* isomers of lycopene are capable of preventing and repairing the damaging effects of H2O2-induced oxidative stress on the formation of mineral‐ ized bone nodules (unpublished observation) [169,190].

the beneficial effects of tomatoes and tomato products in the prevention of osteoporosis in the

Oxidative Stress and Antioxidants in the Risk of Osteoporosis — Role of the Antioxidants Lycopene and Polyphenols

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131

The role of lycopene in the prevention of risk for osteoporosis has recently been reviewed [58, 83,168,169]. Maggio et al [175] and Yang et al [198] both demonstrated that serum lycopene concentrations are lower in women with osteoporosis than in healthy women of the same age. The antioxidant mechanistic effect of lycopene is demonstrated by Misra *et al.* who have shown that HRT has the same antioxidant effects as lycopene in postmenopausal women by demon‐ strating that lipid peroxidation was significantly decreased while GSH significantly increased by both [199]. Epidemiological studies revealed the relationship between lycopene and BMD [185,186,200]. A cross-sectional and longitudinal analyses in men and women were carried out to evaluate the associations between total and individual carotenoid intakes (a-carotene, βcarotene, β-cryptoxanthin, lycopene, lutein and zeaxanthin) with BMD at the hip, spine, and radial shaft and the 4-y change in BMD. Their analyses showed significant associations between lycopene intake and 4-y change in lumbar spine BMD for women [200] protective associations by total carotenoids against 4-y loss in trochanter BMD in men and in lumbar spine in women [98]. On the other hand, radial BMD was not correlated with serum lycopene in postmenopausal Japanese participants, while there was a weak correlation between radial BMD and β-cryptoxanthin and β-carotene [185]. These discrepancy maybe resolved by further in-depth study into the effect of lycopene on BMD. On the positive note, lycopene was shown

to contribute to a decrease in the risk of fragility fracture related to osteoporosis.

We carried out a cross-sectional study in which 33 postmenopausal women aged 50–60 years provided seven-day dietary records and blood samples for analysis of oxidative stress parameters and bone turnover markers. Our results showed that postmenopausal women who consumed an average of 7.4 mg of lycopene per day had significantly higher serum lycopene. Our finding that the estimated dietary lycopene had a significant and direct correlation with serum lycopene suggests that lycopene from the diet is bioavailable. Our finding that a higher serum lycopene was associated with a low NTx (*p*<0.005) and lower protein oxidation (*p*<0.05). supports the antioxidative properties of lycopene involvement in its mechanisms of action in

The overrall conclusions from the epidemiological studies support the beneficial role of lycopene in the prevention of risk for osteoporosis. Further clinical studies described below

Since our laboratory is the only one to this date that reported clinical intervention studies with lycopene, this section will focus on reviewing our studies on the role of lycopene in the

We carried out 4 different clinical studies. In the first study, the objective was to determine the effects of a lycopene-restricted diet on oxidative stress parameters and bone turnover markers in postmenopausal women [38]. To avoid the effects of compounding factors with antioxidants, women who smoked or were on medications which may affect bone metabolism or have anti‐

Mediterranean population [104].

bone [39].

support this conclusion.

**6.5. Clinical intervention studies on lycopene**

prevention of risk for osteoporosis in postmenopausal women.

### **6.2.** *In vitro* **studies of lycopene in osteoclasts**

To date, there have been only 2 studies on the effects of lycopene in osteoclasts [191,192]. Rao et al. cultured cells from bone marrow prepared from rat femur in 16 well, calcium phosphatecoated OsteologicTM multi-test slides. Lycopene was added to the cells in the absence or presence of the resorbing agent parathyroid hormone (PTH) (1-34) and mineral resorption, TRAP+ multinucleated osteoclast formation, and NBT-staining were measured. Lycopene inhibited TRAP+ multinucleated cell formation in both vehicle- and PTH-treated cultures. The cells that were stained with the NBT reduction product formazan were decreased in number after treatment with lycopene, indicating that lycopene inhibited the formation of ROSsecreting osteoclasts [192]. The effect of lycopene on osteoclast formation and bone resorption was also reported by Ishimi et al in murine osteoclasts formed in co-culture with calvarial osteoblasts [191]. Their results differed from those of Rao et al [192] in that they found that lycopene inhibited PTH-induced, but not basal, TRAP+ multinucleated cell formation. Furthermore, they could not demonstrate any effect of lycopene on bone resorption. They also did not study the effect of lycopene on ROS production.

### **6.3. Lycopene intervention studies in animals**

Other than our intervention studies to be discussed in the next section, most of the intervention studies with lycopene were carried out in animals. Liang et al investigated the beneficial effect of lycopene on bone biomarkers in ovariectomized (OVX) rats. Their results showed that administration of lycopene (20, 30 and 40 mg/kg b.w.) for 8 weeks to OVXed rats significantly enhanced BMD, concluding that the consumption of lycopene may have the most protective effect on bone in OVX [193]. Ke et al fed OVXed rats for 3 months with EM-X, an antioxidant beverage derived from ferment of unpolished rice, sea weeds and papaya with combinations of microorganisms and contains, among other things, lycopene. Results showed that rats receiving EM-X for 3 months after sham operation or ovariectomy had increased bone density of the middle of femur that was statistically significantly different from unreated rats [194].

### **6.4. Epidemiological studies on lycopene**

A systematic review of the experimental studies on Mediterranean diet and disease prevention was made and analyzed [195]. Although the Mediterrenean diet comprised of many different food components, it is striking that one of the components is abundance of plant foods including fruits, vegetables [195]. The two possible active components in its properties to prevent diseases are lycopene [196] and polypenols [197]. Epidemiological evidence support the beneficial effects of tomatoes and tomato products in the prevention of osteoporosis in the Mediterranean population [104].

The role of lycopene in the prevention of risk for osteoporosis has recently been reviewed [58, 83,168,169]. Maggio et al [175] and Yang et al [198] both demonstrated that serum lycopene concentrations are lower in women with osteoporosis than in healthy women of the same age. The antioxidant mechanistic effect of lycopene is demonstrated by Misra *et al.* who have shown that HRT has the same antioxidant effects as lycopene in postmenopausal women by demon‐ strating that lipid peroxidation was significantly decreased while GSH significantly increased by both [199]. Epidemiological studies revealed the relationship between lycopene and BMD [185,186,200]. A cross-sectional and longitudinal analyses in men and women were carried out to evaluate the associations between total and individual carotenoid intakes (a-carotene, βcarotene, β-cryptoxanthin, lycopene, lutein and zeaxanthin) with BMD at the hip, spine, and radial shaft and the 4-y change in BMD. Their analyses showed significant associations between lycopene intake and 4-y change in lumbar spine BMD for women [200] protective associations by total carotenoids against 4-y loss in trochanter BMD in men and in lumbar spine in women [98]. On the other hand, radial BMD was not correlated with serum lycopene in postmenopausal Japanese participants, while there was a weak correlation between radial BMD and β-cryptoxanthin and β-carotene [185]. These discrepancy maybe resolved by further in-depth study into the effect of lycopene on BMD. On the positive note, lycopene was shown to contribute to a decrease in the risk of fragility fracture related to osteoporosis.

We carried out a cross-sectional study in which 33 postmenopausal women aged 50–60 years provided seven-day dietary records and blood samples for analysis of oxidative stress parameters and bone turnover markers. Our results showed that postmenopausal women who consumed an average of 7.4 mg of lycopene per day had significantly higher serum lycopene. Our finding that the estimated dietary lycopene had a significant and direct correlation with serum lycopene suggests that lycopene from the diet is bioavailable. Our finding that a higher serum lycopene was associated with a low NTx (*p*<0.005) and lower protein oxidation (*p*<0.05). supports the antioxidative properties of lycopene involvement in its mechanisms of action in bone [39].

The overrall conclusions from the epidemiological studies support the beneficial role of lycopene in the prevention of risk for osteoporosis. Further clinical studies described below support this conclusion.

### **6.5. Clinical intervention studies on lycopene**

(p<0.001), which long-term resulted in a decreased number and area of mineralized bone nodules (both: p<0.001). Pre- and post-treatment with 45:55 or 28:72 *cis*:*trans* lycopene resulted in significantly lower ROS generation (p<0.001) and higher mineralized bone nodule area (p<0.05), compared to treatment with H2O2 alone, vehicle or 5:95 *cis*:*trans* lycopene. These findings support the hypothesis that the *cis* isomers of lycopene are capable of preventing and repairing the damaging effects of H2O2-induced oxidative stress on the formation of mineral‐

To date, there have been only 2 studies on the effects of lycopene in osteoclasts [191,192]. Rao et al. cultured cells from bone marrow prepared from rat femur in 16 well, calcium phosphatecoated OsteologicTM multi-test slides. Lycopene was added to the cells in the absence or presence of the resorbing agent parathyroid hormone (PTH) (1-34) and mineral resorption, TRAP+ multinucleated osteoclast formation, and NBT-staining were measured. Lycopene inhibited TRAP+ multinucleated cell formation in both vehicle- and PTH-treated cultures. The cells that were stained with the NBT reduction product formazan were decreased in number after treatment with lycopene, indicating that lycopene inhibited the formation of ROSsecreting osteoclasts [192]. The effect of lycopene on osteoclast formation and bone resorption was also reported by Ishimi et al in murine osteoclasts formed in co-culture with calvarial osteoblasts [191]. Their results differed from those of Rao et al [192] in that they found that lycopene inhibited PTH-induced, but not basal, TRAP+ multinucleated cell formation. Furthermore, they could not demonstrate any effect of lycopene on bone resorption. They also

Other than our intervention studies to be discussed in the next section, most of the intervention studies with lycopene were carried out in animals. Liang et al investigated the beneficial effect of lycopene on bone biomarkers in ovariectomized (OVX) rats. Their results showed that administration of lycopene (20, 30 and 40 mg/kg b.w.) for 8 weeks to OVXed rats significantly enhanced BMD, concluding that the consumption of lycopene may have the most protective effect on bone in OVX [193]. Ke et al fed OVXed rats for 3 months with EM-X, an antioxidant beverage derived from ferment of unpolished rice, sea weeds and papaya with combinations of microorganisms and contains, among other things, lycopene. Results showed that rats receiving EM-X for 3 months after sham operation or ovariectomy had increased bone density of the middle of femur that was statistically significantly different from unreated rats [194].

A systematic review of the experimental studies on Mediterranean diet and disease prevention was made and analyzed [195]. Although the Mediterrenean diet comprised of many different food components, it is striking that one of the components is abundance of plant foods including fruits, vegetables [195]. The two possible active components in its properties to prevent diseases are lycopene [196] and polypenols [197]. Epidemiological evidence support

ized bone nodules (unpublished observation) [169,190].

did not study the effect of lycopene on ROS production.

**6.3. Lycopene intervention studies in animals**

**6.4. Epidemiological studies on lycopene**

**6.2.** *In vitro* **studies of lycopene in osteoclasts**

130 Topics in Osteoporosis

Since our laboratory is the only one to this date that reported clinical intervention studies with lycopene, this section will focus on reviewing our studies on the role of lycopene in the prevention of risk for osteoporosis in postmenopausal women.

We carried out 4 different clinical studies. In the first study, the objective was to determine the effects of a lycopene-restricted diet on oxidative stress parameters and bone turnover markers in postmenopausal women [38]. To avoid the effects of compounding factors with antioxidants, women who smoked or were on medications which may affect bone metabolism or have anti‐ oxidant properties were excluded from participating. Twenty-three healthy postmenopausal women, 50-60 years old, provided blood samples at baseline and after a one-month lycopenedepletion period. Serum samples were analyzed for carotenoids; the oxidative stress parame‐ ters protein thiols and lipid peroxidation TBARS; the antioxidant enzymes SOD, CAT and GPx, and the bone turnover markers BAP and NTX. Results revealed that lycopene restriction result‐ ed in significant decrease in serum lycopene, lutein/zeaxanthin and α-/β-carotene as shown in Table 2, However, the overall percent change in these serum carotenoids was not as high as that seen for lycopene. Figure 2 demonstrates that all configurations of lycopene (all trans, 5-cis- and other cis lycopene) were all decreased after lycopene restriction. The antioxidant enzymes CAT and SOD were significantly depressed (data not shown). These changes were accompanied by a significant increase in the bone resorption marker NTx [Figure 3]. antioxidant enzymes CAT and SOD were significantly depressed (data not shown). These changes were accompanied by a significant increase in the bone resorption marker NTx [**Figure 3**]**. Table 2**: Change in serum carotenoid concentrations after postmenopausal women were assigned to Lycopene-restricted diet for a period of 1 month. **Carotenoid Concentration in serum (nM) Results of** 

**paired t-test**<sup>1</sup>

**Average % change** 

after lycopene restriction.

negative health consequences to bone health.

the risk of osteoporosis in postmenopausal women [37].

daily diet may result in negative health consequences to bone health.

serum of postmenopausal women after lycopene restriction.

**0**

**10**

**20**

**NTx (nM BCE)**

**30**

serum of postmenopausal women after lycopene restriction.

1

a

**0**

**200**

**400**

**Serum lycopene (nM)**

**\***

**600**

**800**

**Formatted:** Not Highlight

12

In a **second study [37]**, clinical intervention was carried out to investigate directly the effects of supplementation with lycopene on decreasing the risk for osteoporosis. Sixty postmenopausal women, 50-60 years old, were recruited for a fully randomized controlled intervention. Following a one-month washout without lycopene consumption, participants consumed either (N=15/group): (1) regular tomato juice, (2) lycopene-rich tomato juice, (3) tomato lycopene capsules or (4) placebo capsules, twice daily for total lycopene intakes of 30, 70, 30 and 0 mg/day, respectively for 4 months. Serum collected was

 To our knowledge, this is the first study on the effects of dietary lycopene restriction on increasing the risk for osteoporosis in postmenopausal women which proves that lycopene may be beneficial in reducing this risk. It can be speculated that this significant increase in the bone resorption marker NTx may lead to a long-term decrease in BMD and increased fracture risk as was observed by Brown et al [42], and that a longer restriction period may be detrimental to a group of postmenopausal women who were already at high risk for osteoporosis. It can also mean that shorter wash-out periods of no lycopene consumption is all that is needed in clinical trials examining the effects of lycopene on bone health. In addition, lycopene is present in a select number of foods; therefore not consuming these products as a part of the regular daily diet may result in

133

antioxidant enzymes CAT and SOD were significantly depressed (data not shown). These changes were accompanied by a

**(mean ± SEM) Baseline (mean ±** 

**lycopene restricted ,** 

**(mean ± SEM)**  -carotene 408.4 ± 131.4 334.4 ± 110.6 p<0.05 -13.03 ± 6.88



Lycopene 1171.0 ± 111.1 494.9 ± 48.46 p<0.0001 -54.86 ± 3.59<sup>a</sup>

Lutein/zeaxanthin 516.4 ± 49.57 443.0 ± 47.04 p<0.01 -12.77 ± 5.03

**paired t-test**<sup>1</sup>

**Average % change** 

**Formatted:** Not Highlight

**Formatted:** Not Highlight

**Formatted:** Not Highlight

**Formatted:** Not Highlight

**Table 2**: Change in serum carotenoid concentrations after postmenopausal women were assigned to

**Carotenoid Concentration in serum (nM) Results of** 

Wilcoxon matched pairs test used for these non-normally distributed data sets.

**\* \***

**\***

Average percent change in lycopene was significantly higher than that seen for all

the other carotenoids (p<0.0001), as determined by unpaired t-test or Mann-Whitney test.

Baseline

Lycopene restricted

http://dx.doi.org/10.5772/54703

significant increase in the bone resorption marker NTx [**Figure 3**]**.**

**SEM)**

**all-***trans* **5-***cis* **other-***cis*

Oxidative Stress and Antioxidants in the Risk of Osteoporosis — Role of the Antioxidants Lycopene and Polyphenols

**Figure 2:** Decrease in all configurations of lycopene (all trans, 5-cis- and other cis lycopene) in the

**Figure 3:** Increase in the concentrations of the bone resorption marker, NTx, in the

**Baseline Lycopene restricted**

**Figure 3.** Increase in the concentrations of the bone resorption marker, NTx, in the serum of postmenopausal women

To our knowledge, this is the first study on the effects of dietary lycopene restriction on increasing the risk for osteoporosis in postmenopausal women which proves that lycopene may be beneficial in reducing this risk. It can be speculated that this significant increase in the bone resorption marker NTx may lead to a long-term decrease in BMD and increased fracture risk as was observed by Brown et al [42], and that a longer restriction period may be detrimental to a group of postmenopausal women who were already at high risk for osteoporosis. It can also mean that shorter wash-out periods of no lycopene consumption is all that is needed in clinical trials examining the effects of lycopene on bone health. In addition, lycopene is present in a select number of foods; therefore not consuming these products as a part of the regular

In a second study [37], clinical intervention was carried out to investigate directly the effects of supplementation with lycopene on decreasing the risk for osteoporosis. Sixty postmeno‐ pausal women, 50-60 years old, were recruited for a fully randomized controlled intervention. Following a one-month washout without lycopene consumption, participants consumed either (N=15/group): (1) regular tomato juice, (2) lycopene-rich tomato juice, (3) tomato lycopene capsules or (4) placebo capsules, twice daily for total lycopene intakes of 30, 70, 30 and 0 mg/day, respectively for 4 months. Serum collected was assayed for oxidative stress parameters and bone turnover markers. Lycopene-supplementation for 4 months significantly increased serum lycopene compared to placebo (p<0.001). Since the increase in serum lycopene was similar for all three supplements, the participants were pooled into a "LYCOPENEsupplemented" and PLACEBO-supplement group for further statistical analyses. LYCO‐ PENE-supplementation for 4 months resulted in significant increase in total antioxidant capacity as shown in Figure 4, decreased in oxidative stress parameters protein oxidation [Figure 5] and lipid peroxidation [Figure 6] which correlated to a decrease in NTx [Figure 7] in the LYCOPENE-supplemented group; all changes were significantly different from the PLACEBO group. These findings suggest that it did not matter whether lycopene was in the form of tomato juice or capsule to exert its potent antioxidant properties beneficial in reducing

Lycopene-restricted diet for a period of 1 month.

**Formatted:** Not Highlight

**Formatted:** Not Highlight

**Formatted:** Not Highlight


<sup>1</sup> Wilcoxon matched pairs test used for these non-normally distributed data sets. Average percent change in lycopene was significantly higher than that seen for all

negative health consequences to bone health.

serum of postmenopausal women after lycopene restriction.

**0**

**10**

**20**

**NTx (nM BCE)**

**30**

<sup>a</sup> Average percent change in lycopene was significantly higher than that seen for all the other carotenoids (p<0.0001), as determined by unpaired t-test or Mann-Whitney test. the other carotenoids (p<0.0001), as determined by unpaired t-test or Mann-Whitney test.

**Table 2.** Change in serum carotenoid concentrations after postmenopausal women were assigned to Lycopenerestricted diet for a period of 1 month.

**Figure 2:** Decrease in all configurations of lycopene (all trans, 5-cis- and other cis lycopene) in the serum of postmenopausal women after lycopene restriction. **Figure 2.** Decrease in all configurations of lycopene (all trans, 5-cis- and other cis lycopene) in the serum of postmeno‐ pausal women after lycopene restriction.

**\***

**Figure 3:** Increase in the concentrations of the bone resorption marker, NTx, in the

**Baseline Lycopene restricted**

12

In a **second study [37]**, clinical intervention was carried out to investigate directly the effects of supplementation with lycopene on decreasing the risk for osteoporosis. Sixty postmenopausal women, 50-60 years old, were recruited for a fully randomized controlled intervention. Following a one-month washout without lycopene consumption, participants consumed either (N=15/group): (1) regular tomato juice, (2) lycopene-rich tomato juice, (3) tomato lycopene capsules or (4) placebo capsules, twice daily for total lycopene intakes of 30, 70, 30 and 0 mg/day, respectively for 4 months. Serum collected was

 To our knowledge, this is the first study on the effects of dietary lycopene restriction on increasing the risk for osteoporosis in postmenopausal women which proves that lycopene may be beneficial in reducing this risk. It can be speculated that this significant increase in the bone resorption marker NTx may lead to a long-term decrease in BMD and increased fracture risk as was observed by Brown et al [42], and that a longer restriction period may be detrimental to a group of postmenopausal women who were already at high risk for osteoporosis. It can also mean that shorter wash-out periods of no lycopene consumption is all that is needed in clinical trials examining the effects of lycopene on bone health. In addition, lycopene is present in a select number of foods; therefore not consuming these products as a part of the regular daily diet may result in serum of postmenopausal women after lycopene restriction. Oxidative Stress and Antioxidants in the Risk of Osteoporosis — Role of the Antioxidants Lycopene and Polyphenols http://dx.doi.org/10.5772/54703 133

**Figure 2:** Decrease in all configurations of lycopene (all trans, 5-cis- and other cis lycopene) in the

**all-***trans* **5-***cis* **other-***cis*

significant increase in the bone resorption marker NTx [**Figure 3**]**.**

**SEM)**

Lycopene-restricted diet for a period of 1 month.

oxidant properties were excluded from participating. Twenty-three healthy postmenopausal women, 50-60 years old, provided blood samples at baseline and after a one-month lycopenedepletion period. Serum samples were analyzed for carotenoids; the oxidative stress parame‐ ters protein thiols and lipid peroxidation TBARS; the antioxidant enzymes SOD, CAT and GPx, and the bone turnover markers BAP and NTX. Results revealed that lycopene restriction result‐ ed in significant decrease in serum lycopene, lutein/zeaxanthin and α-/β-carotene as shown in Table 2, However, the overall percent change in these serum carotenoids was not as high as that seen for lycopene. Figure 2 demonstrates that all configurations of lycopene (all trans, 5-cis- and other cis lycopene) were all decreased after lycopene restriction. The antioxidant enzymes CAT and SOD were significantly depressed (data not shown). These changes were accompanied by a

significant increase in the bone resorption marker NTx [**Figure 3**]**.**

significant increase in the bone resorption marker NTx [Figure 3].

Lycopene-restricted diet for a period of 1 month.

**SEM)**

<sup>1</sup> Wilcoxon matched pairs test used for these non-normally distributed data sets.

**\***

negative health consequences to bone health.

serum of postmenopausal women after lycopene restriction.

**0**

**10**

**20**

**NTx (nM BCE)**

**30**

serum of postmenopausal women after lycopene restriction.

as determined by unpaired t-test or Mann-Whitney test.

**0**

**200**

**400**

**Serum lycopene (nM)**

pausal women after lycopene restriction.

**600**

**800**

restricted diet for a period of 1 month.

**Concentration in serum (nM)**

**Results of paired t-test1**

**(mean ± SEM)** 

**lycopene restricted ,** 

**Table 2**: Change in serum carotenoid concentrations after postmenopausal women were assigned to

**(mean ± SEM) Baseline (mean ±**

Wilcoxon matched pairs test used for these non-normally distributed data sets.

**\* \***

**\***

 Average percent change in lycopene was significantly higher than that seen for all the other carotenoids (p<0.0001), as determined by unpaired t-test or Mann-Whitney test.

> Baseline Lycopene restricted

**Carotenoid Concentration in serum (nM) Results of** 

**lycopene restricted , (mean ± SEM)** α-carotene 408.4 ± 131.4 334.4 ± 110.6 p<0.05 -13.03 ± 6.88 β-carotene 1443.0 ± 278.9 1035.0 ± 221.7 p<0.0005 -22.84 ± 5.09 β-cryptoxanthin 403.2 ± 58.4 367.6 ± 47.3 p = 0.229 -1.29 ± 8.21 Lycopene 1171.0 ± 111.1 494.9 ± 48.46 p<0.0001 -54.86 ± 3.59a Lutein/zeaxanthin 516.4 ± 49.57 443.0 ± 47.04 p<0.01 -12.77 ± 5.03

**SEM)**

<sup>a</sup> Average percent change in lycopene was significantly higher than that seen for all the other carotenoids (p<0.0001),

**Table 2.** Change in serum carotenoid concentrations after postmenopausal women were assigned to Lycopene-

**all-***trans* **5-***cis* **other-***cis*

**Figure 2:** Decrease in all configurations of lycopene (all trans, 5-cis- and other cis lycopene) in the

**Figure 2.** Decrease in all configurations of lycopene (all trans, 5-cis- and other cis lycopene) in the serum of postmeno‐

**Figure 3:** Increase in the concentrations of the bone resorption marker, NTx, in the

**Baseline Lycopene restricted**

**Average % change**

**(mean ± SEM) Baseline (mean ±** 

**paired t-test**<sup>1</sup>

**Average % change** 

1

a

**0**

**200**

**400**

**Serum lycopene (nM)**

**\***

**600**

**800**

12

In a **second study [37]**, clinical intervention was carried out to investigate directly the effects of supplementation with lycopene on decreasing the risk for osteoporosis. Sixty postmenopausal women, 50-60 years old, were recruited for a fully randomized controlled intervention. Following a one-month washout without lycopene consumption, participants consumed either (N=15/group): (1) regular tomato juice, (2) lycopene-rich tomato juice, (3) tomato lycopene capsules or (4) placebo capsules, twice daily for total lycopene intakes of 30, 70, 30 and 0 mg/day, respectively for 4 months. Serum collected was

 To our knowledge, this is the first study on the effects of dietary lycopene restriction on increasing the risk for osteoporosis in postmenopausal women which proves that lycopene may be beneficial in reducing this risk. It can be speculated that this significant increase in the bone resorption marker NTx may lead to a long-term decrease in BMD and increased fracture risk as was observed by Brown et al [42], and that a longer restriction period may be detrimental to a group of postmenopausal women who were already at high risk for osteoporosis. It can also mean that shorter wash-out periods of no lycopene consumption is all that is needed in clinical trials examining the effects of lycopene on bone health. In addition, lycopene is present in a select number of foods; therefore not consuming these products as a part of the regular daily diet may result in

**Carotenoid**

132 Topics in Osteoporosis

1

a



> consumption is all that is needed in clinical trials examining the effects of lycopene on bone health. In addition, lycopene is present in a select number of foods; therefore not consuming these products as a part of the regular daily diet may result in negative health consequences to bone health. In a **second study [37]**, clinical intervention was carried out to investigate directly the effects of supplementation with lycopene on decreasing the risk for osteoporosis. Sixty postmenopausal women, 50-60 years old, were recruited for a fully randomized controlled intervention. Following a one-month washout without lycopene consumption, participants consumed either (N=15/group): (1) regular tomato juice, (2) lycopene-rich tomato juice, (3) tomato lycopene capsules or (4) placebo capsules, twice daily for total lycopene intakes of 30, 70, 30 and 0 mg/day, respectively for 4 months. Serum collected was In a second study [37], clinical intervention was carried out to investigate directly the effects of supplementation with lycopene on decreasing the risk for osteoporosis. Sixty postmeno‐ pausal women, 50-60 years old, were recruited for a fully randomized controlled intervention. Following a one-month washout without lycopene consumption, participants consumed either (N=15/group): (1) regular tomato juice, (2) lycopene-rich tomato juice, (3) tomato lycopene capsules or (4) placebo capsules, twice daily for total lycopene intakes of 30, 70, 30 and 0 mg/day, respectively for 4 months. Serum collected was assayed for oxidative stress parameters and bone turnover markers. Lycopene-supplementation for 4 months significantly increased serum lycopene compared to placebo (p<0.001). Since the increase in serum lycopene was similar for all three supplements, the participants were pooled into a "LYCOPENEsupplemented" and PLACEBO-supplement group for further statistical analyses. LYCO‐ PENE-supplementation for 4 months resulted in significant increase in total antioxidant capacity as shown in Figure 4, decreased in oxidative stress parameters protein oxidation [Figure 5] and lipid peroxidation [Figure 6] which correlated to a decrease in NTx [Figure 7] in the LYCOPENE-supplemented group; all changes were significantly different from the PLACEBO group. These findings suggest that it did not matter whether lycopene was in the form of tomato juice or capsule to exert its potent antioxidant properties beneficial in reducing the risk of osteoporosis in postmenopausal women [37].

> > **Formatted:** Not Highlight

12

antioxidant enzymes CAT and SOD were significantly depressed (data not shown). These changes were accompanied by a

**(mean ± SEM) Baseline (mean ±** 

**lycopene restricted ,** 

**(mean ± SEM)**  -carotene 408.4 ± 131.4 334.4 ± 110.6 p<0.05 -13.03 ± 6.88



Lycopene 1171.0 ± 111.1 494.9 ± 48.46 p<0.0001 -54.86 ± 3.59<sup>a</sup>

Lutein/zeaxanthin 516.4 ± 49.57 443.0 ± 47.04 p<0.01 -12.77 ± 5.03

**paired t-test**<sup>1</sup>

**Average % change** 

**Formatted:** Not Highlight

**Formatted:** Not Highlight

**Formatted:** Not Highlight

**Formatted:** Not Highlight

**Table 2**: Change in serum carotenoid concentrations after postmenopausal women were assigned to

**Carotenoid Concentration in serum (nM) Results of** 

Wilcoxon matched pairs test used for these non-normally distributed data sets.

**\* \***

Average percent change in lycopene was significantly higher than that seen for all

the other carotenoids (p<0.0001), as determined by unpaired t-test or Mann-Whitney test.

Baseline

Lycopene restricted

**Formatted:** Not Highlight

**Formatted:** Not Highlight

**Formatted:** Not Highlight

women [37].

In a third study [169]. Serum lycopene, bone turnover markers and oxidative stress parame‐ ter data were compared between postmenopausal women who were supplemented with ly‐ copene and those who obtained lycopene from both a low and high daily food intake of lycopene to determine whether the elevated dose obtained through supplementation was more beneficial in reducing bone turnover markers than intakes typically obtained from the usual daily diet. Table 3 showed that women supplemented with lycopene had significantly lower TBARS and marginally significant lower NTx values than participants who obtained a low intake (or high intake lycopene, data not show) through their usual daily diets. These differences in NTx and TBARS may be attributed to a significantly higher concentration of serum 5-*cis* in lycopene-supplemented participants compared to low or high usual daily in‐ take participants. This suggests that it is the 5-*cis* isomer, with the most potent antioxidant capacity which, at higher concentrations, decreases bone turnover markers due to its ability to provide the greatest protection against oxidative stress. It also appears to show that sup‐ plementation with lycopene may be necessary in spite of the daily intake of lycopene. **In a third study** [169]. Serum lycopene, bone turnover markers and oxidative stress parameter data were compared between postmenopausal women who were supplemented with lycopene and those who obtained lycopene from both a low and high daily food intake of lycopene to determine whether the elevated dose obtained through supplementation was more beneficial in reducing bone turnover markers than intakes typically obtained from the usual daily diet. **Table 3** showed that women supplemented with lycopene had significantly lower TBARS and marginally significant lower NTx values than participants who obtained a low intake (or high intake lycopene, data not show) through their usual daily diets. These differences in NTx and TBARS may be attributed to a significantly higher concentration of serum 5-*cis* in lycopenesupplemented participants compared to low or high usual daily intake participants. This suggests that it is the 5-*cis* isomer, with the most potent antioxidant capacity which, at higher concentrations, decreases bone turnover markers due to its ability to provide the greatest protection against oxidative stress. It also appears to show that supplementation with lycopene may be necessary in spite of the daily intake of lycopene. women [37]. **In a third study** [169]. Serum lycopene, bone turnover markers and oxidative stress parameter data were compared between postmenopausal women who were supplemented with lycopene and those who obtained lycopene from both a low and high daily food intake of lycopene to determine whether the elevated dose obtained through supplementation was more beneficial in reducing bone turnover markers than intakes typically obtained from the usual daily diet. **Table 3** showed that women supplemented with lycopene had significantly lower TBARS and marginally significant lower NTx values than participants who obtained a low intake (or high intake lycopene, data not show) through their usual daily diets. These differences in NTx and TBARS may be attributed to a significantly higher concentration of serum 5-*cis* in lycopenesupplemented participants compared to low or high usual daily intake participants. This suggests that it is the 5-*cis* isomer, with the most potent antioxidant capacity which, at higher concentrations, decreases bone turnover markers due to its ability

assayed for oxidative stress parameters and bone turnover markers. Lycopene-supplementation for 4 months significantly increased serum lycopene compared to placebo (p<0.001). Since the increase in serum lycopene was similar for all three supplements, the participants were pooled into a "LYCOPENE-supplemented" and PLACEBO-supplement group for further statistical analyses. LYCOPENE-supplementation for 4 months resulted in significant increase in total antioxidant capacity as shown in **Figure 4**, decreased in oxidative stress parameters protein oxidation **[Figure 5]** and lipid peroxidation **[Figure 6]** which correlated to a decrease in NTx **[Figure 7]** in the LYCOPENE-supplemented group; all changes were significantly different from the PLACEBO group. These findings suggest that it did not matter whether lycopene was in the form of tomato juice or capsule to exert its potent antioxidant properties beneficial in reducing the risk of osteoporosis in postmenopausal

assayed for oxidative stress parameters and bone turnover markers. Lycopene-supplementation for 4 months significantly increased serum lycopene compared to placebo (p<0.001). Since the increase in serum lycopene was similar for all three supplements, the participants were pooled into a "LYCOPENE-supplemented" and PLACEBO-supplement group for further statistical analyses. LYCOPENE-supplementation for 4 months resulted in significant increase in total antioxidant capacity as shown in **Figure 4**, decreased in oxidative stress parameters protein oxidation **[Figure 5]** and lipid peroxidation **[Figure 6]**

juice or capsule to exert its potent antioxidant properties beneficial in reducing the risk of osteoporosis in postmenopausal

to provide the greatest protection against oxidative stress. It also appears to show that supplementation with lycopene may

**Figure 4**. Increase in the serum total antioxidant capacity of postmenopausal women supplemented with LYCOPENE compared to placebo capsules for 4 months. Values are mean SEM. Values compared within supplement group was determined to be statistically significant using repeated-measures ANOVA (\*p<0.05). **Figure 4.** Increase in the serum total antioxidant capacity of postmenopausal women supplemented with LYCOPENE compared to placebo capsules for 4 months. Values are mean ± SEM. Values compared within supplement group was determined to be statistically significant using repeated-measures ANOVA (\*p<0.05). **Time period for intervention (mos.) Figure 4**. Increase in the serum total antioxidant capacity of postmenopausal women supplemented with LYCOPENE compared to placebo capsules for 4 months. Values are mean SEM. Values compared within supplement group was determined to be statistically significant using repeated-measures ANOVA (\*p<0.05).

LYCOPENE-supplemented Placebo-supplemented

**Protein thiols (M)**

**Figure 5.** Increase the serum concentration of thiol (meaning decreased protein oxidation) in postmenopausal women supplemented with LYCOPENE compared to placebo capsules for a period of 4 months. Values are mean SEM. Values compared within supplement group was determined to be statistically significant using repeated-measures ANOVA (\*p<0.001). supplemented with LYCOPENE compared to placebo capsules for a period of 4 months. Values are mean SEM. Values compared within supplement group was determined to be statistically significant using repeated-measures ANOVA (\*p<0.001). **Figure 5.** Increase the serum concentration of thiol (meaning decreased protein oxidation) in postmenopausal wom‐ en supplemented with LYCOPENE compared to placebo capsules for a period of 4 months. Values are mean ± SEM. Values compared within supplement group was determined to be statistically significant using repeated-measures ANOVA (\*p<0.001).

**Figure 5.** Increase the serum concentration of thiol (meaning decreased protein oxidation) in postmenopausal women

13

13

**Formatted:** Not Highlight

**Formatted:** Not Highlight

Oxidative Stress and Antioxidants in the Risk of Osteoporosis — Role of the Antioxidants Lycopene and Polyphenols

**Formatted:** Not Highlight

**Formatted:** Not Highlight **Formatted:** Not Highlight

**Formatted:** Not Highlight **Formatted:** Not Highlight

**\* \*\***

**\* \*\***

**Formatted:** Not Highlight

**\***

**\***

**0 2 4 0 2 4**

LYCOPENE-supplemented Placebo-supplemented **Time period for intervention (mos.)**

**Figure 6.** Decrease in the serum concentration of TBARS or lipid peroxidation in postmenopausal women supplemented with LYCOPENE compared to placebo capsules for a period of 4 months. Values are mean SEM. Values compared within supplement group was determined to be statistically significant using repeated-measures

**Figure 6.** Decrease in the serum concentration of TBARS or lipid peroxidation in postmenopausal women supplemented with LYCOPENE compared to placebo capsules for a period of 4 months. Values are mean SEM. Values compared within supplement group was determined to be statistically significant using repeated-measures

**0 2 4 0 2 4**

LYCOPENE-supplemented Placebo-supplemented **Time period for intervention (mos.)**

**Figure 6.** Decrease in the serum concentration of TBARS or lipid peroxidation in postmenopausal women supple‐ mented with LYCOPENE compared to placebo capsules for a period of 4 months. Values are mean ± SEM. Values com‐ pared within supplement group was determined to be statistically significant using repeated-measures ANOVA

**0 2 4 0 2 4**

LYCOPENE-supplemented Placebo-supplemented **Time period for intervention (mos.)**

**0 2 4 0 2 4**

LYCOPENE-supplemented Placebo-supplemented **Time period for intervention (mos.)**

**Figure 7.** Decrease in the serum concentration of bone resorption marker NTx in postmenopausal women supple‐ mented with LYCOPENE compared to placebo capsules for a period of 4 months. Values are mean ± SEM. Values com‐ pared within supplement group was determined to be statistically significant at 2 and 4 months using repeated-

In a fourth study, we investigated whether the 172T→A or 584A→G polymorphisms of the paraoxonase 1 (PON 1) modulated the effects of serum lycopene on bone turnover markers, oxidative stress parameters and antioxidant capacity in women between the ages of 25-70 years. We showed that the PON1 polymorphism modified the association between lycopene and NTx and BAP (p<0.02 and p<0.05 for interaction). In the combined 172TT and 584G genotype, high serum lycopene was associated with decreased BAP (p<0.01) and NTx (p<0.05). Among those with the combined 172A and 584G genotype, however, increased serum lycopene was associated with increased BAP (p<0.05) and NTx (p<0.05). These findings show that PON1 polymorphisms modified the association between serum concentrations of lycopene and oxidative stress parameters and bone turnover markers and may, therefore,

**Figure 7.** Decrease in the serum concentration of bone resorption marker NTx in postmenopausal women supplemented with LYCOPENE compared to placebo capsules for a period of 4 months. Values are mean SEM. Values compared within supplement group was determined to be statistically significant at 2 and 4 months using repeated-measures

**Figure 7.** Decrease in the serum concentration of bone resorption marker NTx in postmenopausal women supplemented with LYCOPENE compared to placebo capsules for a period of 4 months. Values are mean SEM. Values compared within supplement group was determined to be statistically significant at 2 and 4 months using repeated-measures

**Table 3.** Comparison of lycopene values, oxidative stress parameters and bone turnover markers between women who were supplemented with lycopene with those who obtained a low lycopene (not shown) intake from their usual daily diet (unpaired t-

**Table 3.** Comparison of lycopene values, oxidative stress parameters and bone turnover markers between women who were supplemented with lycopene with those who obtained a low lycopene (not shown) intake from their usual daily diet (unpaired t-

> **group (N=48)**

**group (N=48)** 

**Lycopene intake (mg/day)** 2.59 ± 0.32a 43.33 ± 2.84 <0.0001 **Serum lycopene (nM)** Total 1094 ± 80.24 2012 ± 88.56 <0.0001

**Total antioxidant capacity (mM)** 1.65 ± 0.03 1.66 ± 0.05 0.190 **Bone turnover markers** NTx (nM BCE) 21.97 ± 1.11 19.19 ± 0.79 0.047

**Lycopene intake (mg/day)** 2.59 ± 0.32a 43.33 ± 2.84 <0.0001 **Serum lycopene (nM)** Total 1094 ± 80.24 2012 ± 88.56 <0.0001

**Antioxidant enzymes** CAT (K/g Hb) 81.50 ± 4.13 58.59 ± 2.06 <0.0001

**Antioxidant enzymes** CAT (K/g Hb) 81.50 ± 4.13 58.59 ± 2.06 <0.0001

**Total antioxidant capacity (mM)** 1.65 ± 0.03 1.66 ± 0.05 0.190 **Bone turnover markers** NTx (nM BCE) 21.97 ± 1.11 19.19 ± 0.79 0.047

 Data that were not normally distributed were compared using the Mann-Whitney test a The range of lycopene intake for the low usual daily intake group is 0.0-6.07 mg/day.

 Data that were not normally distributed were compared using the Mann-Whitney test a The range of lycopene intake for the low usual daily intake group is 0.0-6.07 mg/day.

GPx (U/g Hb) 15.44 ± 1.40 32.82 ± 2.85 <0.0001 1

GPx (U/g Hb) 15.44 ± 1.40 32.82 ± 2.85 <0.0001 1

**0**

**10**

**0**

**10**

**20**

**20**

**30**

**NTx (nM BCE)**

**NTx (nM BCE)**

ANOVA (\*p<0.01 and \*\*p<0.001).

ANOVA (\*p<0.01 and \*\*p<0.001).

measures ANOVA (\*p<0.01 and \*\*p<0.001).

**Parameter measured** 

**Parameter measured** 

moderate the risk of osteoporosis [201].

Protein thiols (M)

Protein thiols (M)

TBARS (nmol/mL)

TBARS (nmol/mL)

test).

test).

**Oxidative stress parameters** 

**Oxidative stress parameters** 

ANOVA (\*p<0.001).

ANOVA (\*p<0.001).

(\*p<0.001).

**30**

**TBARS (nmol/ml)**

**TBARS (nmol/ml)**

14

14

**In a fourth study,** we investigated whether the 172TA or 584AG polymorphisms of the paraoxonase 1 (PON 1) modulated the effects of serum lycopene on bone turnover markers, oxidative stress parameters and antioxidant capacity in women between the ages of 25-70 years. We showed that the PON1 polymorphism modified the association between lycopene and NTx and BAP (p<0.02 and p<0.05 for interaction). In the combined 172TT and 584G genotype, high serum lycopene was associated with decreased BAP (p<0.01) and NTx (p<0.05). Among those with the combined 172A and 584G genotype, however, increased serum lycopene was associated with increased BAP (p<0.05) and NTx (p<0.05). These

**In a fourth study,** we investigated whether the 172TA or 584AG polymorphisms of the paraoxonase 1 (PON 1) modulated the effects of serum lycopene on bone turnover markers, oxidative stress parameters and antioxidant capacity in women between the ages of 25-70 years. We showed that the PON1 polymorphism modified the association between lycopene and NTx and BAP (p<0.02 and p<0.05 for interaction). In the combined 172TT and 584G genotype, high serum lycopene was associated with decreased BAP (p<0.01) and NTx (p<0.05). Among those with the combined 172A and 584G genotype, however, increased serum lycopene was associated with increased BAP (p<0.05) and NTx (p<0.05). These

454.7 ± 15.37 455.2 ± 15.50 0.982

9.06 ± 0.35 6.80 ± 0.35 <0.0001

454.7 ± 15.37 455.2 ± 15.50 0.982

9.06 ± 0.35 6.80 ± 0.35 <0.0001

**Mean ± SEM p** 

**Mean ± SEM p** 

**Lycopene-supplemented group (N=45)** 

**Lycopene-supplemented group (N=45)** 

http://dx.doi.org/10.5772/54703

135

**value**<sup>1</sup> **Low usual daily intake** 

**value**<sup>1</sup> **Low usual daily intake** 

all-*trans* 539.0 ± 40.07 979.0 ± 48.48 <0.0001 5-*cis* 342.4 ± 26.13 685.0 ± 30.22 <0.0001 other-*cis* 212.6 ± 15.96 347.7 ± 17.36 <0.0001

BAP (U/L) 24.42 ± 1.40 23.56 ± 1.02 0.900

all-*trans* 539.0 ± 40.07 979.0 ± 48.48 <0.0001 5-*cis* 342.4 ± 26.13 685.0 ± 30.22 <0.0001 other-*cis* 212.6 ± 15.96 347.7 ± 17.36 <0.0001

SOD (U/mg Hb) 47.60 ± 2.44 39.22 ± 4.72 0.001

SOD (U/mg Hb) 47.60 ± 2.44 39.22 ± 4.72 0.001

BAP (U/L) 24.42 ± 1.40 23.56 ± 1.02 0.900

**Formatted:** Not Highlight which correlated to a decrease in NTx **[Figure 7]** in the LYCOPENE-supplemented group; all changes were significantly different from the PLACEBO group. These findings suggest that it did not matter whether lycopene was in the form of tomato Oxidative Stress and Antioxidants in the Risk of Osteoporosis — Role of the Antioxidants Lycopene and Polyphenols http://dx.doi.org/10.5772/54703 135

**Formatted:** Not Highlight

In a third study [169]. Serum lycopene, bone turnover markers and oxidative stress parame‐ ter data were compared between postmenopausal women who were supplemented with ly‐ copene and those who obtained lycopene from both a low and high daily food intake of lycopene to determine whether the elevated dose obtained through supplementation was more beneficial in reducing bone turnover markers than intakes typically obtained from the usual daily diet. Table 3 showed that women supplemented with lycopene had significantly lower TBARS and marginally significant lower NTx values than participants who obtained a low intake (or high intake lycopene, data not show) through their usual daily diets. These differences in NTx and TBARS may be attributed to a significantly higher concentration of serum 5-*cis* in lycopene-supplemented participants compared to low or high usual daily in‐ take participants. This suggests that it is the 5-*cis* isomer, with the most potent antioxidant capacity which, at higher concentrations, decreases bone turnover markers due to its ability to provide the greatest protection against oxidative stress. It also appears to show that sup‐ plementation with lycopene may be necessary in spite of the daily intake of lycopene.

**0 2 4 0 2 4**

LYCOPENE-supplemented Placebo-supplemented **Time period for intervention (mos.)**

**<sup>0</sup> <sup>2</sup> <sup>4</sup> <sup>0</sup> <sup>2</sup> <sup>4</sup> 0.0**

LYCOPENE-supplemented Placebo-supplemented **Time period for intervention (mos.)**

**\***

**Figure 4.** Increase in the serum total antioxidant capacity of postmenopausal women supplemented with LYCOPENE compared to placebo capsules for 4 months. Values are mean ± SEM. Values compared within supplement group was

**Figure 4**. Increase in the serum total antioxidant capacity of postmenopausal women supplemented with LYCOPENE compared to placebo capsules for 4 months. Values are mean SEM. Values compared within supplement group was

**\***

**Figure 4**. Increase in the serum total antioxidant capacity of postmenopausal women supplemented with LYCOPENE compared to placebo capsules for 4 months. Values are mean SEM. Values compared within supplement group was

**0.5 1.0 1.5 2.0**

**500 \***

**Trolox (mM)**

**0 2 4 0 2 4**

**<sup>0</sup> <sup>2</sup> <sup>4</sup> <sup>0</sup> <sup>2</sup> <sup>4</sup> <sup>0</sup>**

**Figure 5.** Increase the serum concentration of thiol (meaning decreased protein oxidation) in postmenopausal women supplemented with LYCOPENE compared to placebo capsules for a period of 4 months. Values are mean SEM. Values compared within supplement group was determined to be statistically significant using repeated-measures

**Figure 5.** Increase the serum concentration of thiol (meaning decreased protein oxidation) in postmenopausal wom‐ en supplemented with LYCOPENE compared to placebo capsules for a period of 4 months. Values are mean ± SEM. Values compared within supplement group was determined to be statistically significant using repeated-measures

LYCOPENE-supplemented Placebo-supplemented **Time period for intervention (mos.)**

LYCOPENE-supplemented Placebo-supplemented **Time period for intervention (mos.)**

**Figure 5.** Increase the serum concentration of thiol (meaning decreased protein oxidation) in postmenopausal women supplemented with LYCOPENE compared to placebo capsules for a period of 4 months. Values are mean SEM. Values compared within supplement group was determined to be statistically significant using repeated-measures

women [37].

women [37].

134 Topics in Osteoporosis

be necessary in spite of the daily intake of lycopene.

**0.0**

**500 \***

**Protein thiols (M)**

determined to be statistically significant using repeated-measures ANOVA (\*p<0.05).

determined to be statistically significant using repeated-measures ANOVA (\*p<0.05).

**0.5**

**1.0**

**Trolox (mM)**

determined to be statistically significant using repeated-measures ANOVA (\*p<0.05).

**Protein thiols (M)**

ANOVA (\*p<0.001).

ANOVA (\*p<0.001).

ANOVA (\*p<0.001).

**1.5**

**2.0**

be necessary in spite of the daily intake of lycopene.

assayed for oxidative stress parameters and bone turnover markers. Lycopene-supplementation for 4 months significantly increased serum lycopene compared to placebo (p<0.001). Since the increase in serum lycopene was similar for all three supplements, the participants were pooled into a "LYCOPENE-supplemented" and PLACEBO-supplement group for further statistical analyses. LYCOPENE-supplementation for 4 months resulted in significant increase in total antioxidant capacity as shown in **Figure 4**, decreased in oxidative stress parameters protein oxidation **[Figure 5]** and lipid peroxidation **[Figure 6]** which correlated to a decrease in NTx **[Figure 7]** in the LYCOPENE-supplemented group; all changes were significantly different from the PLACEBO group. These findings suggest that it did not matter whether lycopene was in the form of tomato juice or capsule to exert its potent antioxidant properties beneficial in reducing the risk of osteoporosis in postmenopausal

assayed for oxidative stress parameters and bone turnover markers. Lycopene-supplementation for 4 months significantly increased serum lycopene compared to placebo (p<0.001). Since the increase in serum lycopene was similar for all three supplements, the participants were pooled into a "LYCOPENE-supplemented" and PLACEBO-supplement group for further statistical analyses. LYCOPENE-supplementation for 4 months resulted in significant increase in total antioxidant capacity as shown in **Figure 4**, decreased in oxidative stress parameters protein oxidation **[Figure 5]** and lipid peroxidation **[Figure 6]**

juice or capsule to exert its potent antioxidant properties beneficial in reducing the risk of osteoporosis in postmenopausal

**In a third study** [169]. Serum lycopene, bone turnover markers and oxidative stress parameter data were compared between postmenopausal women who were supplemented with lycopene and those who obtained lycopene from both a low and high daily food intake of lycopene to determine whether the elevated dose obtained through supplementation was more beneficial in reducing bone turnover markers than intakes typically obtained from the usual daily diet. **Table 3** showed that women supplemented with lycopene had significantly lower TBARS and marginally significant lower NTx values than participants who obtained a low intake (or high intake lycopene, data not show) through their usual daily diets. These differences in NTx and TBARS may be attributed to a significantly higher concentration of serum 5-*cis* in lycopene-

differences in NTx and TBARS may be attributed to a significantly higher concentration of serum 5-*cis* in lycopene-

13

13

supplemented participants compared to low or high usual daily intake participants. This suggests that it is the 5-*cis* isomer, with the most potent antioxidant capacity which, at higher concentrations, decreases bone turnover markers due to its ability to provide the greatest protection against oxidative stress. It also appears to show that supplementation with lycopene may supplemented with LYCOPENE compared to placebo capsules for a period of 4 months. Values are mean SEM. Values compared within supplement group was determined to be statistically significant using repeated-measures ANOVA (\*p<0.001). **Figure 6.** Decrease in the serum concentration of TBARS or lipid peroxidation in postmenopausal women supple‐ mented with LYCOPENE compared to placebo capsules for a period of 4 months. Values are mean ± SEM. Values com‐ pared within supplement group was determined to be statistically significant using repeated-measures ANOVA (\*p<0.001). **Time period for intervention (mos.) Figure 6.** Decrease in the serum concentration of TBARS or lipid peroxidation in postmenopausal women supplemented with LYCOPENE compared to placebo capsules for a period of 4 months. Values are mean SEM.

**Figure 6.** Decrease in the serum concentration of TBARS or lipid peroxidation in postmenopausal women

Values compared within supplement group was determined to be statistically significant using repeated-measures

**0 2 4 0 2 4**

LYCOPENE-supplemented Placebo-supplemented

**30**

ANOVA (\*p<0.01 and \*\*p<0.001).

ANOVA (\*p<0.001).

**Oxidative stress parameters** 

**Oxidative stress parameters** 

**Figure 7.** Decrease in the serum concentration of bone resorption marker NTx in postmenopausal women supplemented with LYCOPENE compared to placebo capsules for a period of 4 months. Values are mean SEM. Values compared within supplement group was determined to be statistically significant at 2 and 4 months using repeated-measures ANOVA (\*p<0.01 and \*\*p<0.001). **Figure 7.** Decrease in the serum concentration of bone resorption marker NTx in postmenopausal women supple‐ mented with LYCOPENE compared to placebo capsules for a period of 4 months. Values are mean ± SEM. Values com‐ pared within supplement group was determined to be statistically significant at 2 and 4 months using repeatedmeasures ANOVA (\*p<0.01 and \*\*p<0.001).

**Table 3.** Comparison of lycopene values, oxidative stress parameters and bone turnover markers between women who were supplemented with lycopene with those who obtained a low lycopene (not shown) intake from their usual daily diet (unpaired ttest). **Parameter measured Mean ± SEM p value**<sup>1</sup> **Low usual daily intake group (N=48) Lycopene-supplemented group (N=45) Lycopene intake (mg/day)** 2.59 ± 0.32a 43.33 ± 2.84 <0.0001 **Serum lycopene (nM)** Total 1094 ± 80.24 2012 ± 88.56 <0.0001 all-*trans* 539.0 ± 40.07 979.0 ± 48.48 <0.0001 5-*cis* 342.4 ± 26.13 685.0 ± 30.22 <0.0001 other-*cis* 212.6 ± 15.96 347.7 ± 17.36 <0.0001 **Table 3.** Comparison of lycopene values, oxidative stress parameters and bone turnover markers between women who were supplemented with lycopene with those who obtained a low lycopene (not shown) intake from their usual daily diet (unpaired ttest). **Parameter measured Mean ± SEM p value**<sup>1</sup> **Low usual daily intake group (N=48) Lycopene-supplemented group (N=45) Lycopene intake (mg/day)** 2.59 ± 0.32a 43.33 ± 2.84 <0.0001 **Serum lycopene (nM)** Total 1094 ± 80.24 2012 ± 88.56 <0.0001 In a fourth study, we investigated whether the 172T→A or 584A→G polymorphisms of the paraoxonase 1 (PON 1) modulated the effects of serum lycopene on bone turnover markers, oxidative stress parameters and antioxidant capacity in women between the ages of 25-70 years. We showed that the PON1 polymorphism modified the association between lycopene and NTx and BAP (p<0.02 and p<0.05 for interaction). In the combined 172TT and 584G genotype, high serum lycopene was associated with decreased BAP (p<0.01) and NTx (p<0.05). Among those with the combined 172A and 584G genotype, however, increased serum lycopene was associated with increased BAP (p<0.05) and NTx (p<0.05). These findings show that PON1 polymorphisms modified the association between serum concentrations of lycopene and oxidative stress parameters and bone turnover markers and may, therefore, moderate the risk of osteoporosis [201].

> Protein thiols (M)

Protein thiols (M)

TBARS (nmol/mL)

TBARS (nmol/mL)

**Total antioxidant capacity (mM)** 1.65 ± 0.03 1.66 ± 0.05 0.190 **Bone turnover markers** NTx (nM BCE) 21.97 ± 1.11 19.19 ± 0.79 0.047

**Antioxidant enzymes** CAT (K/g Hb) 81.50 ± 4.13 58.59 ± 2.06 <0.0001

**Antioxidant enzymes** CAT (K/g Hb) 81.50 ± 4.13 58.59 ± 2.06 <0.0001

**Total antioxidant capacity (mM)** 1.65 ± 0.03 1.66 ± 0.05 0.190 **Bone turnover markers** NTx (nM BCE) 21.97 ± 1.11 19.19 ± 0.79 0.047

 Data that were not normally distributed were compared using the Mann-Whitney test a The range of lycopene intake for the low usual daily intake group is 0.0-6.07 mg/day.

 Data that were not normally distributed were compared using the Mann-Whitney test a The range of lycopene intake for the low usual daily intake group is 0.0-6.07 mg/day.

GPx (U/g Hb) 15.44 ± 1.40 32.82 ± 2.85 <0.0001 1

GPx (U/g Hb) 15.44 ± 1.40 32.82 ± 2.85 <0.0001 1

BAP (U/L) 24.42 ± 1.40 23.56 ± 1.02 0.900

all-*trans* 539.0 ± 40.07 979.0 ± 48.48 <0.0001 5-*cis* 342.4 ± 26.13 685.0 ± 30.22 <0.0001 other-*cis* 212.6 ± 15.96 347.7 ± 17.36 <0.0001

SOD (U/mg Hb) 47.60 ± 2.44 39.22 ± 4.72 0.001

SOD (U/mg Hb) 47.60 ± 2.44 39.22 ± 4.72 0.001

BAP (U/L) 24.42 ± 1.40 23.56 ± 1.02 0.900

14

14

**In a fourth study,** we investigated whether the 172TA or 584AG polymorphisms of the paraoxonase 1 (PON 1) modulated the effects of serum lycopene on bone turnover markers, oxidative stress parameters and antioxidant capacity in women between the ages of 25-70 years. We showed that the PON1 polymorphism modified the association between lycopene and NTx and BAP (p<0.02 and p<0.05 for interaction). In the combined 172TT and 584G genotype, high serum lycopene was associated with decreased BAP (p<0.01) and NTx (p<0.05). Among those with the combined 172A and 584G genotype, however, increased serum lycopene was associated with increased BAP (p<0.05) and NTx (p<0.05). These

**In a fourth study,** we investigated whether the 172TA or 584AG polymorphisms of the paraoxonase 1 (PON 1) modulated the effects of serum lycopene on bone turnover markers, oxidative stress parameters and antioxidant capacity in women between the ages of 25-70 years. We showed that the PON1 polymorphism modified the association between lycopene and NTx and BAP (p<0.02 and p<0.05 for interaction). In the combined 172TT and 584G genotype, high serum lycopene was associated with decreased BAP (p<0.01) and NTx (p<0.05). Among those with the combined 172A and 584G genotype, however, increased serum lycopene was associated with increased BAP (p<0.05) and NTx (p<0.05). These

454.7 ± 15.37 455.2 ± 15.50 0.982

9.06 ± 0.35 6.80 ± 0.35 <0.0001

454.7 ± 15.37 455.2 ± 15.50 0.982

9.06 ± 0.35 6.80 ± 0.35 <0.0001


**6.6. Concluding remark**

**7. Studies on polyphenols**

There is now ample evidence to show that oxidative stress brought about by the accumulation of ROS in the body is one of the causes of the development of several chronic diseases including osteoporosis and that antioxidants such as lycopene can counteract this damaging effect. The evidence includes studies on their role in osteoclastic resorption and osteoblastic bone formation, animal intervention studies, epidemiological studies and, more recently, clinical intervention studies. Considering the possible adverse side effects of the conventional therapy (eg, HRT and bisphosphonates) in the management of postmenopausal osteoporosis, there is an increasing demand for the use of antioxidants naturally present in foods. The results of these studies indicate that lycopene maybe useful either as a dietary alternative to drug therapy

Oxidative Stress and Antioxidants in the Risk of Osteoporosis — Role of the Antioxidants Lycopene and Polyphenols

http://dx.doi.org/10.5772/54703

137

or as a complement to the drugs presently used by women at risk for osteoporosis.

effects of green tea polyphenols has been reviewed [207,208].

rosis [209] and only a few studies will be reviewed here.

**7.1.** *In vitro* **studies on polyphenols in bone cells**

Polyphenols have long been known to have a role in the prevention of chronic diseases such as cardiovascular diseases, cancers, neurodegenerative diseases, diabetes, or osteoporosis. Only in the last 10 years has there been an increase in the interest on polyphenols and bone health [203-206]. Horcajada [204] has recently reviewed the anabolic role of phytonutrients and especially polyphenols in bone while Trzeciakiewicz [205] reviewed the mechanisms of action of polyphenol in osteoblast function and its interaction with osteoclasts. The beneficial

Currently, most of the research on polyphenols and their effects have emerged from *in vitro* and *in vivo* animal studies with only a few clinical studies available. In our recent review, we have included tables listing all the studies on polyphenols *in vitro* bone cell culture and the epidemiologic studies on the protective effects of polyphenol consumption against osteopo‐

The most commonly studied polyphenol abundant in green tea is epigallocatechin-3-Gallate (EGCG). We have shown that epigallocathechin-3-gallate (EGCG) increased the formation of mineralized bone nodules by human osteoblast-like cells [210]. EGCG has been shown to inhibit the expression of matrix metalloproteinase 9 (MMP-9) and the formation of osteoclasts [211]. H2O2-induced alterations of osteoblast viability and reduction in alkaline phosphatase activity were prevented by pre-incubating the osteoblasts with green tea polyphenol [212]. Green tea was shown to protect human osteoblasts from cigarette smoke-induced injury [128]. EGCG was shown to inhibit thyroid hormone-stimulated osteocalcin synthesis in osteoblasts [213], suppressed the differentiation of murine osteoblastic MC3T3-E1 cells [214], inhibit rat osteoclast formation and differentiation [215] and induces apoptosis via caspase activation in osteoclasts differentiated from RAW 264.7 cells [216]. Horcajada suggested that most studies investigating the effects of polyphenols on osteoblast cells have reported involvement of complex networks of anabolic signaling pathways such as BMPs or estrogen receptor mediated

1 Data that were not normally distributed were compared using the Mann-Whitney test

a The range of lycopene intake for the low usual daily intake group is 0.0-6.07 mg/day.

**Table 3.** Comparison of lycopene values, oxidative stress parameters and bone turnover markers between women who were supplemented with lycopene with those who obtained a low lycopene (not shown) intake from their usual daily diet (unpaired t-test).

A similar investigation was carried out in a fifth study to assesses whether the PON1 172T→A polymorphism affects the response to dietary intervention with lycopene. We showed that supplementation in the TT genotype and carriers of the A allele significantly increased serum lycopene (both: p<0.0001) while decreasing protein oxidation (p<0.005 and p<0.05, respective‐ ly) and lipid peroxidation (p<0.005 and p<0.0005). However, participants with the TT genotype responded more favourably to lycopene, with corresponding significant increase in total antioxidant capacity (TAC) (p<0.01) and significant decrease in NTx (p<0.001); this effect was not significant in carriers of the A allele. Further analyses showed that there was a significant interaction between PON1 genotype and change in TBARS (p<0.05) suggesting that supple‐ mentation with lycopene resulted in decreased lipid peroxidation, which interacted with the PON1 genotype to decrease bone resorption markers in postmenopausal women. These findings provide mechanistic evidence of how intervention with lycopene may act to decrease lipid peroxidation and thus the risk of osteoporosis in postmenopausal women [169,202].

### **6.6. Concluding remark**

**Mean ± SEM**

Total 1094 ± 80.24 2012 ± 88.56 <0.0001 all-*trans* 539.0 ± 40.07 979.0 ± 48.48 <0.0001 5-*cis* 342.4 ± 26.13 685.0 ± 30.22 <0.0001 other-*cis* 212.6 ± 15.96 347.7 ± 17.36 <0.0001

NTx (nM BCE) 21.97 ± 1.11 19.19 ± 0.79 0.047 BAP (U/L) 24.42 ± 1.40 23.56 ± 1.02 0.900

TBARS (nmol/mL) 9.06 ± 0.35 6.80 ± 0.35 <0.0001

CAT (K/g Hb) 81.50 ± 4.13 58.59 ± 2.06 <0.0001 SOD (U/mg Hb) 47.60 ± 2.44 39.22 ± 4.72 0.001 GPx (U/g Hb) 15.44 ± 1.40 32.82 ± 2.85 <0.0001

**group (N=48)**

**Lycopene intake (mg/day)** 2.59 ± 0.32a 43.33 ± 2.84 <0.0001

**Total antioxidant capacity (mM)** 1.65 ± 0.03 1.66 ± 0.05 0.190

**Table 3.** Comparison of lycopene values, oxidative stress parameters and bone turnover markers between women who were supplemented with lycopene with those who obtained a low lycopene (not shown) intake from their usual

A similar investigation was carried out in a fifth study to assesses whether the PON1 172T→A polymorphism affects the response to dietary intervention with lycopene. We showed that supplementation in the TT genotype and carriers of the A allele significantly increased serum lycopene (both: p<0.0001) while decreasing protein oxidation (p<0.005 and p<0.05, respective‐ ly) and lipid peroxidation (p<0.005 and p<0.0005). However, participants with the TT genotype responded more favourably to lycopene, with corresponding significant increase in total antioxidant capacity (TAC) (p<0.01) and significant decrease in NTx (p<0.001); this effect was not significant in carriers of the A allele. Further analyses showed that there was a significant interaction between PON1 genotype and change in TBARS (p<0.05) suggesting that supple‐ mentation with lycopene resulted in decreased lipid peroxidation, which interacted with the PON1 genotype to decrease bone resorption markers in postmenopausal women. These findings provide mechanistic evidence of how intervention with lycopene may act to decrease lipid peroxidation and thus the risk of osteoporosis in postmenopausal women [169,202].

**Serum lycopene (nM)**

136 Topics in Osteoporosis

**Bone turnover markers**

Protein thiols (μM)

1 Data that were not normally distributed were compared using the Mann-Whitney test

The range of lycopene intake for the low usual daily intake group is 0.0-6.07 mg/day.

**Oxidative stress parameters**

**Antioxidant enzymes**

daily diet (unpaired t-test).

a

**p value1 Low usual daily intake**

454.7 ± 15.37 455.2 ± 15.50 0.982

**Lycopene-supplemented group (N=45)**

There is now ample evidence to show that oxidative stress brought about by the accumulation of ROS in the body is one of the causes of the development of several chronic diseases including osteoporosis and that antioxidants such as lycopene can counteract this damaging effect. The evidence includes studies on their role in osteoclastic resorption and osteoblastic bone formation, animal intervention studies, epidemiological studies and, more recently, clinical intervention studies. Considering the possible adverse side effects of the conventional therapy (eg, HRT and bisphosphonates) in the management of postmenopausal osteoporosis, there is an increasing demand for the use of antioxidants naturally present in foods. The results of these studies indicate that lycopene maybe useful either as a dietary alternative to drug therapy or as a complement to the drugs presently used by women at risk for osteoporosis.

### **7. Studies on polyphenols**

Polyphenols have long been known to have a role in the prevention of chronic diseases such as cardiovascular diseases, cancers, neurodegenerative diseases, diabetes, or osteoporosis. Only in the last 10 years has there been an increase in the interest on polyphenols and bone health [203-206]. Horcajada [204] has recently reviewed the anabolic role of phytonutrients and especially polyphenols in bone while Trzeciakiewicz [205] reviewed the mechanisms of action of polyphenol in osteoblast function and its interaction with osteoclasts. The beneficial effects of green tea polyphenols has been reviewed [207,208].

Currently, most of the research on polyphenols and their effects have emerged from *in vitro* and *in vivo* animal studies with only a few clinical studies available. In our recent review, we have included tables listing all the studies on polyphenols *in vitro* bone cell culture and the epidemiologic studies on the protective effects of polyphenol consumption against osteopo‐ rosis [209] and only a few studies will be reviewed here.

### **7.1.** *In vitro* **studies on polyphenols in bone cells**

The most commonly studied polyphenol abundant in green tea is epigallocatechin-3-Gallate (EGCG). We have shown that epigallocathechin-3-gallate (EGCG) increased the formation of mineralized bone nodules by human osteoblast-like cells [210]. EGCG has been shown to inhibit the expression of matrix metalloproteinase 9 (MMP-9) and the formation of osteoclasts [211]. H2O2-induced alterations of osteoblast viability and reduction in alkaline phosphatase activity were prevented by pre-incubating the osteoblasts with green tea polyphenol [212]. Green tea was shown to protect human osteoblasts from cigarette smoke-induced injury [128]. EGCG was shown to inhibit thyroid hormone-stimulated osteocalcin synthesis in osteoblasts [213], suppressed the differentiation of murine osteoblastic MC3T3-E1 cells [214], inhibit rat osteoclast formation and differentiation [215] and induces apoptosis via caspase activation in osteoclasts differentiated from RAW 264.7 cells [216]. Horcajada suggested that most studies investigating the effects of polyphenols on osteoblast cells have reported involvement of complex networks of anabolic signaling pathways such as BMPs or estrogen receptor mediated pathways [204]. Trzeciakiewicz describing a more detailed mechanisms, suggested that polyphenols modulate the expression of transcription factors in osteoblasts such as runtrelated transcription factor-2 (Runx2) and Osterix, NFkappaB and activator protein-1 (AP-1) [205]. In agreement with Hocajada (2012), Trzeciakiewicz (2009) stated in his review that polyphenol may act on cellular signaling such as mitogen-activated protein kinase (MAPK), bone morphogenetic protein (BMP), oestrogen receptor and osteoprotegerin/receptor activator of NF-kappaB ligand (OPG/RANKL) and thus may affect osteoblast functions. The two reviews complement each other and paint a better understanding of the mechanisms of action of polyphenols in bone cells, with the warning that it is also important to take into account the possible interaction of these compounds on osteoblasts metabolism.

+*TM* and bone builder*TM*, were tested as combination, the effects were six times more effective

Oxidative Stress and Antioxidants in the Risk of Osteoporosis — Role of the Antioxidants Lycopene and Polyphenols

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139

**Figure 8.** Effect of continuous addition of greens+TM extract on the number of SaOS-2 cells cultured in the presence of EC, or varying dilutions of greens+TM at an early time points and number of nodules analyzed at the indicated time

**Figure 9.** Dose-dependent inhibitory effects of phenolic extracts of greens+TM on intracellular ROS levels stimulated by

20 uM H2O2 in SaOS-2 cells. Data are mean ± SEM of 6 replicates. \*p = < 0.05.

points. An asterix, \*, on a bar indicates statistical significance (p *<.*05) between a treatment and the control.

than either one alone in stimulating bone formation in osteoblast culture [226]*.*

Other polyphenols/sources of polyphenols which were found to have beneficial effects on bone cells include the dried plum polyphenols found to attenuate the detrimental effects of TNFalpha on osteoblast function coincident with up-regulation of Runx2, Osterix and IGF-I and increasing lysyl oxidase expression, and at the same time attenuate osteoclastogenesis signalling [217]; black tea polyphenol which affects the MMP activity and osteoclast formation and differentiation in vitro [215]; phenolic leaf extract of Heimia myrtifolia (Lythraceae) found to stimulate mineralization of SaOS-2 osteosarcoma cells) [218]; Oleuropein which enhances osteoblastogenesis and inhibits adipogenesis and the effects on differentiation in stem cells derived from bone marrow [219] and the polyphenol component of red wine resveratrol which promotes osteogenic differentiation and protects against dexamethasone damage in murineinduced pluripotent stem cells [220] and facilitates in vitro mineralization and in vivo bone regeneration [221]. A number of animal studies have been reported and this was reviewed by Rao et al [209].

Other good sources of polyphenols that are frequently studied are extracts containing combinations of polyphenols. One such source is the nutritional supplement greens+TM, a blend of several herbal and botanical products containing a substantial amount of polyphenols including quercetin, apigenin and luteolin [106] which act as antioxidants and therefore should be able to counteract oxidative stress. Our laboratory has shown that the polyphenolic extracts from greens+TM have stimulatory effect on mineralized bone nodule formation in human osteoblast cells in a dose- and time- dependent manner and is more effective than epicatechin (EC) as shown in Figure 8 [222]. We have further shown that this stimulatory effect is accom‐ panied by decreases in the reactive oxygen species H2O2 shown in Figure 9 [223], thus proving that greens+TM is able to counteract oxidative stress in human osteoblastic cells and may therefore be a good candidate as a nutritional supplement to prevent the risk of osteoporosis.

Two additional nutritional supplements have since been formulated which may prove to be good for bone health. These are the bone builderTM and the greens+bone builderTM ; the latter is the original greens+TM product that has been supplemented with the bone builderTM formula containing several compounds including vitamins, minerals, and antioxidants. These various components have been separately shown to have some beneficial effect on bone [224]. Using the human osteoblast SaOS-2 cells, we showed that similarly to the greens+*TM*, the watersoluble bone-builderTM extract had a significant dose-dependent stimulatory effect on bone nodules formation (Figure 10) [225]. Figure 11 shows that when the two supplements, greens +*TM* and bone builder*TM*, were tested as combination, the effects were six times more effective than either one alone in stimulating bone formation in osteoblast culture [226]*.*

pathways [204]. Trzeciakiewicz describing a more detailed mechanisms, suggested that polyphenols modulate the expression of transcription factors in osteoblasts such as runtrelated transcription factor-2 (Runx2) and Osterix, NFkappaB and activator protein-1 (AP-1) [205]. In agreement with Hocajada (2012), Trzeciakiewicz (2009) stated in his review that polyphenol may act on cellular signaling such as mitogen-activated protein kinase (MAPK), bone morphogenetic protein (BMP), oestrogen receptor and osteoprotegerin/receptor activator of NF-kappaB ligand (OPG/RANKL) and thus may affect osteoblast functions. The two reviews complement each other and paint a better understanding of the mechanisms of action of polyphenols in bone cells, with the warning that it is also important to take into account the

Other polyphenols/sources of polyphenols which were found to have beneficial effects on bone cells include the dried plum polyphenols found to attenuate the detrimental effects of TNFalpha on osteoblast function coincident with up-regulation of Runx2, Osterix and IGF-I and increasing lysyl oxidase expression, and at the same time attenuate osteoclastogenesis signalling [217]; black tea polyphenol which affects the MMP activity and osteoclast formation and differentiation in vitro [215]; phenolic leaf extract of Heimia myrtifolia (Lythraceae) found to stimulate mineralization of SaOS-2 osteosarcoma cells) [218]; Oleuropein which enhances osteoblastogenesis and inhibits adipogenesis and the effects on differentiation in stem cells derived from bone marrow [219] and the polyphenol component of red wine resveratrol which promotes osteogenic differentiation and protects against dexamethasone damage in murineinduced pluripotent stem cells [220] and facilitates in vitro mineralization and in vivo bone regeneration [221]. A number of animal studies have been reported and this was reviewed by

Other good sources of polyphenols that are frequently studied are extracts containing combinations of polyphenols. One such source is the nutritional supplement greens+TM, a blend of several herbal and botanical products containing a substantial amount of polyphenols including quercetin, apigenin and luteolin [106] which act as antioxidants and therefore should be able to counteract oxidative stress. Our laboratory has shown that the polyphenolic extracts from greens+TM have stimulatory effect on mineralized bone nodule formation in human osteoblast cells in a dose- and time- dependent manner and is more effective than epicatechin (EC) as shown in Figure 8 [222]. We have further shown that this stimulatory effect is accom‐ panied by decreases in the reactive oxygen species H2O2 shown in Figure 9 [223], thus proving that greens+TM is able to counteract oxidative stress in human osteoblastic cells and may therefore be a good candidate as a nutritional supplement to prevent the risk of osteoporosis.

Two additional nutritional supplements have since been formulated which may prove to be good for bone health. These are the bone builderTM and the greens+bone builderTM ; the latter is the original greens+TM product that has been supplemented with the bone builderTM formula containing several compounds including vitamins, minerals, and antioxidants. These various components have been separately shown to have some beneficial effect on bone [224]. Using the human osteoblast SaOS-2 cells, we showed that similarly to the greens+*TM*, the watersoluble bone-builderTM extract had a significant dose-dependent stimulatory effect on bone nodules formation (Figure 10) [225]. Figure 11 shows that when the two supplements, greens

possible interaction of these compounds on osteoblasts metabolism.

Rao et al [209].

138 Topics in Osteoporosis

**Figure 8.** Effect of continuous addition of greens+TM extract on the number of SaOS-2 cells cultured in the presence of EC, or varying dilutions of greens+TM at an early time points and number of nodules analyzed at the indicated time points. An asterix, \*, on a bar indicates statistical significance (p *<.*05) between a treatment and the control.

**Figure 9.** Dose-dependent inhibitory effects of phenolic extracts of greens+TM on intracellular ROS levels stimulated by 20 uM H2O2 in SaOS-2 cells. Data are mean ± SEM of 6 replicates. \*p = < 0.05.

and it was found that catechin was negatively associated with bone-resorption markers, association between energy-adjusted total flavonoid intakes and BMD at the femoreal neck and lumbar spine while annual percent change in BMD was associated with intakes of

Oxidative Stress and Antioxidants in the Risk of Osteoporosis — Role of the Antioxidants Lycopene and Polyphenols

Other than these clinical studies in the last two years, there has not been not been anymore

Our results on the in vitro effects of greens+TM, bone builderTM and greens+bone build‐ erTM on bone formation in osteoblasts encouraged us formed the rationale for our clinical studies to test whether these products can prevent the risk of osteoporosis in postmeno‐ pausal women. We chose to study the greens+bone builderTM since of the three products, it gave the greatest stimulatory effect on bone formation, being six times more effective than the other two. The first randomized cross-sectional clinical intervention study was carried out to test whether a daily supplementation with greens+ bone builderTM may be important in reducing oxidative damage in postmenopausal women at risk for osteo‐ porosis. [40]. Forty-seven postmenopausal women, 50-60 years old were randomized to either Treatment group consuming 1 scoop (equivalent to ¼ cup) daily of greens+ bone builder*TM* (N=23) or Placebo (N=24) group for a period of 8 weeks. Blood samples were collected at 0, 4 and 8 weeks of supplementation, processed and assayed for serum total antioxidant capacity (TAC), lipid peroxidation and protein oxidation as markers of oxida‐ tive stress. Results revealed that there was an increase in total antioxidant capacity (Fig‐ ure 12, as well as a decrease in both protein oxidation (Figure 13) and lipid peroxidation (Figure 14) over a 4 and 8-weeks of intervention with greens+ bone builder*TM* compared to placebo. This suggests that the nutritional supplement may have a beneficial effect on

Placebo

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141

Supplement **\***

**\*\***

reported clinical studies on polyphenols in human subjects except our studies.

bone health by counteracting the effects of oxidative stress [40].

4 weeks 8 weeks

**Figure 12.** Change relative to baseline in serum concentrations of trolox, a measure of total antioxidant capacity, in greens+bone builderTM-treated postmenopausal women was significantly increased after 4 and 8 weeks while that in the placebo-treated control was marginally decreased. Treated values were also higher than the placebo [unpaired t-

**-5**

test (\*p<0.01, \*\*p<0.0001)]. Values are mean ± SEM.

**0**

**5**

**Change in Trolox**

**from baseline (%)**

**10**

procyanidins and catechins [228].

**Figure 10.** Time and Dose-dependent Effects of bone builderTM on mineralized bone nodule area in SaOS-2 cells. indi‐ cates significant difference from vehicle: p<0.0001; p<0.0005; p<0.005, #, p<0.01 and ##, p< 0.05. There is a signifi‐ cant dose-dependent effect both at day 17 and day 20, according to One-way ANOVA; p<0.0001.

**Figure 11.** Dose dependent effect of greens + (g+) with and without 0.5 mg/ml of bone builder (bb) on the area of mineralized bone nodules in osteoblasts SaOS-2 Cells. Significant differences were found compared to respective con‐ trols. g+bb was more effective than either g+ or bb alone.

#### **7.2. Clinical intervention studies of polyphenol**

We have recently reviewed earlier clinical studies on polyphenols and osteoporosis [209]. Only the more recent reports, as well as our own clinical studies will be reviewed here. Shen et al [227] have extended their studies in osteopenic women and showed that dietary supplement in the form of green tea combined with tai-chi, a mind-body exercise, can alleviating bone loss in osteopenic women. The effect of catechin was studied in perimenopausal Scottish women and it was found that catechin was negatively associated with bone-resorption markers, association between energy-adjusted total flavonoid intakes and BMD at the femoreal neck and lumbar spine while annual percent change in BMD was associated with intakes of procyanidins and catechins [228].

Other than these clinical studies in the last two years, there has not been not been anymore reported clinical studies on polyphenols in human subjects except our studies.

Our results on the in vitro effects of greens+TM, bone builderTM and greens+bone build‐ erTM on bone formation in osteoblasts encouraged us formed the rationale for our clinical studies to test whether these products can prevent the risk of osteoporosis in postmeno‐ pausal women. We chose to study the greens+bone builderTM since of the three products, it gave the greatest stimulatory effect on bone formation, being six times more effective than the other two. The first randomized cross-sectional clinical intervention study was carried out to test whether a daily supplementation with greens+ bone builderTM may be important in reducing oxidative damage in postmenopausal women at risk for osteo‐ porosis. [40]. Forty-seven postmenopausal women, 50-60 years old were randomized to either Treatment group consuming 1 scoop (equivalent to ¼ cup) daily of greens+ bone builder*TM* (N=23) or Placebo (N=24) group for a period of 8 weeks. Blood samples were collected at 0, 4 and 8 weeks of supplementation, processed and assayed for serum total antioxidant capacity (TAC), lipid peroxidation and protein oxidation as markers of oxida‐ tive stress. Results revealed that there was an increase in total antioxidant capacity (Fig‐ ure 12, as well as a decrease in both protein oxidation (Figure 13) and lipid peroxidation (Figure 14) over a 4 and 8-weeks of intervention with greens+ bone builder*TM* compared to placebo. This suggests that the nutritional supplement may have a beneficial effect on bone health by counteracting the effects of oxidative stress [40].

**0.0 0.1 0.3 0.5 0.7 0.8 0.9 1.0**

concentration of bonebuilder (mg/ml)

**Figure 10.** Time and Dose-dependent Effects of bone builderTM on mineralized bone nodule area in SaOS-2 cells. indi‐ cates significant difference from vehicle: p<0.0001; p<0.0005; p<0.005, #, p<0.01 and ##, p< 0.05. There is a signifi‐

**Figure 11.** Dose dependent effect of greens + (g+) with and without 0.5 mg/ml of bone builder (bb) on the area of mineralized bone nodules in osteoblasts SaOS-2 Cells. Significant differences were found compared to respective con‐

We have recently reviewed earlier clinical studies on polyphenols and osteoporosis [209]. Only the more recent reports, as well as our own clinical studies will be reviewed here. Shen et al [227] have extended their studies in osteopenic women and showed that dietary supplement in the form of green tea combined with tai-chi, a mind-body exercise, can alleviating bone loss in osteopenic women. The effect of catechin was studied in perimenopausal Scottish women

 

**# #**

cant dose-dependent effect both at day 17 and day 20, according to One-way ANOVA; p<0.0001.

**#**

**#**

day 17 day 20

**0**

trols. g+bb was more effective than either g+ or bb alone.

**7.2. Clinical intervention studies of polyphenol**

**500**

**1000**

area of mineralized bone nodules

140 Topics in Osteoporosis

(arbitrary unit in pixels/well)

**1500**

**2000**

**Figure 12.** Change relative to baseline in serum concentrations of trolox, a measure of total antioxidant capacity, in greens+bone builderTM-treated postmenopausal women was significantly increased after 4 and 8 weeks while that in the placebo-treated control was marginally decreased. Treated values were also higher than the placebo [unpaired ttest (\*p<0.01, \*\*p<0.0001)]. Values are mean ± SEM.

in CTX correlated to the increase in their serum total antioxidant capacity [Figure 12] and decreases in oxidative parameters protein oxidation [Figure 13] lipid peroxidation [Figure 14]. These results suggest that a daily supplementation with polyphenols and micronutrients may be important in reducing oxidative damage by reducing bone resorption, thereby

Oxidative Stress and Antioxidants in the Risk of Osteoporosis — Role of the Antioxidants Lycopene and Polyphenols

**\***

**Figure 15.** Change relative to baseline in serum concentrations of CTX in greens+bone builderTM-treated postmeno‐ pausal women was significantly decreased after 8 weeks (meaning decreased bone resorption marker) compared to

Studies reported in the literature on the role of polyphenols in bone health have explod‐ ed in the last 10 years, but most of the reports involved in vitro studies in osteoclasts and osteoblasts, animal studies and epidemiologicai studies. There is little doubt from the excellent studies reported that oxidative stress is one of the primary culprits responsi‐ ble for the pathogenesis of osteoporosis via its role in osteoclastic resoption and the detri‐ mental effects on the bone-forming osteoblasts. To date, only four clinical intervention studies have been reported, including ours. It is easy to see why it is very difficult to evaluate the role of polyphenols since, as we learned from this review, there are at least 8,000 different polyphenols identified to date, and each one probably having different ef‐ fects on humans. Additionally, polyphenols are present in food with other constituents that may also be beneficial to bone health. In our clinical study, we combined the effects of a combination of polyphenols present in the nutritional supplement from greens+TM with the nutritional components present in bone builderTM such as minerals, vitamins and other nutrients. It is possible that the effects of greens+bone buildertm in increasing total antioxidant capacity, decreasing the oxidative stress markers protein oxidation and lipid peroxidation which correlated to the decrease in bone turnover marker for bone re‐ sorption is a result of the combined effects of the different polyphenols it contained with those of the other nutritional components present in the bone builderTM. It remained for future studies to zero in on specific component that is responsible for its beneficial effect.

Placebo Supplement

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143

reducing the risk of osteoporosis in postmenopausal women [41].

that of the placebo-treated control (a paired t-test (\*p<0.05).. Values are mean ± SEM.

**-20 -15 -10 -5 0 5 10**

**Change in CTX from**

**7.3. Concluding remarks**

**baseline at 8 weeks (%)**

**Figure 13.** Change relative to baseline in serum concentrations of thiol in greens+bone builder-treated postmeno‐ pausal women was significantly increased after 4 and 8 weeks (meaning decreased protein oxidation) while that in the placebo-treated control was unchanged; treated values were also higher than the placebo. Mann-Whitney test (\*p<0.05, \*\*p<0.001). Values are mean ± SEM.

**Figure 14.** Change relative to baseline in serum concentrations of TBARS in greens+bone builderTM-treated postmeno‐ pausal women was significantly decreased after 4 and 8 weeks (meaning decreased lipid peroxidation) while that in the placebo-treated control was unchanged; treated values were also lower than the placebo. Mann-Whitney test (\*p<0.05, \*\*p<0.001). Values are mean ± SEM.

In order to test whether the antioxidant properties of greens+bone builderTM can prevent the risk of osteoporosis in postmenopausal women, we also measured the serum bone turnover markers, C-terminal telopeptide of type I collagen (CTX) as indicator of bone resorption, and procollagen type I N-terminal propeptide (PINP) as indicator of bone formation, in addition to the serum antioxidant capacity, and the oxidative stress parameters lipid peroxidation, protein oxidation. As shown in Figure 15, statistical analysis showed that at 8 weeks, the greens +bone builderTM supplement group significantly decreased the bone resorption marker CTX, while the Placebo group showed no significant changes. The supplement group was also significantly different from that of the Placebo group in all parameters measured. This decrease in CTX correlated to the increase in their serum total antioxidant capacity [Figure 12] and decreases in oxidative parameters protein oxidation [Figure 13] lipid peroxidation [Figure 14]. These results suggest that a daily supplementation with polyphenols and micronutrients may be important in reducing oxidative damage by reducing bone resorption, thereby reducing the risk of osteoporosis in postmenopausal women [41].

**Figure 15.** Change relative to baseline in serum concentrations of CTX in greens+bone builderTM-treated postmeno‐ pausal women was significantly decreased after 8 weeks (meaning decreased bone resorption marker) compared to that of the placebo-treated control (a paired t-test (\*p<0.05).. Values are mean ± SEM.

### **7.3. Concluding remarks**

**Figure 13.** Change relative to baseline in serum concentrations of thiol in greens+bone builder-treated postmeno‐ pausal women was significantly increased after 4 and 8 weeks (meaning decreased protein oxidation) while that in the placebo-treated control was unchanged; treated values were also higher than the placebo. Mann-Whitney test

**\***

Placebo Supplement

(\*p<0.05, \*\*p<0.001). Values are mean ± SEM.

142 Topics in Osteoporosis

**Change in TBARS**

(\*p<0.05, \*\*p<0.001). Values are mean ± SEM.

**from baseline (%)**

**-15 -10 -5 0 5 10**

**\***

4 weeks 8 weeks

**Figure 14.** Change relative to baseline in serum concentrations of TBARS in greens+bone builderTM-treated postmeno‐ pausal women was significantly decreased after 4 and 8 weeks (meaning decreased lipid peroxidation) while that in the placebo-treated control was unchanged; treated values were also lower than the placebo. Mann-Whitney test

In order to test whether the antioxidant properties of greens+bone builderTM can prevent the risk of osteoporosis in postmenopausal women, we also measured the serum bone turnover markers, C-terminal telopeptide of type I collagen (CTX) as indicator of bone resorption, and procollagen type I N-terminal propeptide (PINP) as indicator of bone formation, in addition to the serum antioxidant capacity, and the oxidative stress parameters lipid peroxidation, protein oxidation. As shown in Figure 15, statistical analysis showed that at 8 weeks, the greens +bone builderTM supplement group significantly decreased the bone resorption marker CTX, while the Placebo group showed no significant changes. The supplement group was also significantly different from that of the Placebo group in all parameters measured. This decrease

Studies reported in the literature on the role of polyphenols in bone health have explod‐ ed in the last 10 years, but most of the reports involved in vitro studies in osteoclasts and osteoblasts, animal studies and epidemiologicai studies. There is little doubt from the excellent studies reported that oxidative stress is one of the primary culprits responsi‐ ble for the pathogenesis of osteoporosis via its role in osteoclastic resoption and the detri‐ mental effects on the bone-forming osteoblasts. To date, only four clinical intervention studies have been reported, including ours. It is easy to see why it is very difficult to evaluate the role of polyphenols since, as we learned from this review, there are at least 8,000 different polyphenols identified to date, and each one probably having different ef‐ fects on humans. Additionally, polyphenols are present in food with other constituents that may also be beneficial to bone health. In our clinical study, we combined the effects of a combination of polyphenols present in the nutritional supplement from greens+TM with the nutritional components present in bone builderTM such as minerals, vitamins and other nutrients. It is possible that the effects of greens+bone buildertm in increasing total antioxidant capacity, decreasing the oxidative stress markers protein oxidation and lipid peroxidation which correlated to the decrease in bone turnover marker for bone re‐ sorption is a result of the combined effects of the different polyphenols it contained with those of the other nutritional components present in the bone builderTM. It remained for future studies to zero in on specific component that is responsible for its beneficial effect.

### **8. General summary and conclusion**

In conclusion, we showed that oxidative stress due to ROS that are shown to cause the development of osteoporosis may be prevented by supplementation with the antioxidants lycopene and polyphenols. Results of in vitro studies in osteoblasts and osteoclasts, animal intervention studies, epidemiological studies and clinical intervention studies on lycopene and polyphenols are evidence for their potential use as alternative or complementary agent with other established drugs approved for the prevention or treatment of osteoporosis in postme‐ nopausal women.

[2] Hopkins, R, Pullenayegum, E, Goeree, R, Adachi, J, Papaioannou, A, Leslie, W, et al. Estimation of the lifetime risk of hip fracture for women and men in Canada. Osteo‐

Oxidative Stress and Antioxidants in the Risk of Osteoporosis — Role of the Antioxidants Lycopene and Polyphenols

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145

[3] Tarride, J, Hopkins, R, Leslie, W, Morin, S, & Adachi, J. Papaioannou Aea. The burden of illness of osteoporosis in Canada. Osteoporos Int 2012. (2012). Epub Mar 8. [Epub

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### **Acknowledgements**

Funding for this research into Oxidative Stress, Antioxidants and Bone Health is shared by Genuine Health Ltd (Canada), the H.J. Heinz Co (Canada), Millenium Biologix Inc. (Canada), Kagome Co. (Japan) and LycoRed Natural Product Industries, Ltd. (Israel) and matched by the Canadian Institutes of Health Research (CIHR). We sincerely thanked the valuable contributions to this research by the following students/graduate students and staff at the Calcium Research Laboratory, Department of Medicine at St Michael's Hospital and the University of Toronto and Department of Nutritional Sciences, University of Toronto: Dr. Bala Balachandran, Jaclyn Beca, Dawn Snyder, Loren Chan, Honglei Shen, Salva Sadeghi, Ayesha Quireshi, Dr. Erin Mackinnon and Nancy Kang. Their contributions were based on their experimental data, written reports published/in press manuscripts. We would also like to thank to Dr. R.G. Josse for providing us with his medical expertice as well as allowing us access to his list of patients we were able to recruit. Special thanks to Dr. H. Vandenberghe for carrying out the CTX assay and for her valuable suggestions.

### **Author details**


### **References**

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**8. General summary and conclusion**

out the CTX assay and for her valuable suggestions.

\*Address all correspondence to: leticia.rao@utoronto.ca

1 Department of Medicine, St Michael's Hospital and University of Toronto, Canada

[1] Ahmed, S, & Elmantaser, M. Secondary osteoporosis. Endocr Dev. (2009). , 16, 170-90.

2 Department of Nutritional Sciences, University of Toronto, Canada

nopausal women.

144 Topics in Osteoporosis

**Author details**

**References**

L.G. Rao1\* and A.V. Rao2

**Acknowledgements**

In conclusion, we showed that oxidative stress due to ROS that are shown to cause the development of osteoporosis may be prevented by supplementation with the antioxidants lycopene and polyphenols. Results of in vitro studies in osteoblasts and osteoclasts, animal intervention studies, epidemiological studies and clinical intervention studies on lycopene and polyphenols are evidence for their potential use as alternative or complementary agent with other established drugs approved for the prevention or treatment of osteoporosis in postme‐

Funding for this research into Oxidative Stress, Antioxidants and Bone Health is shared by Genuine Health Ltd (Canada), the H.J. Heinz Co (Canada), Millenium Biologix Inc. (Canada), Kagome Co. (Japan) and LycoRed Natural Product Industries, Ltd. (Israel) and matched by the Canadian Institutes of Health Research (CIHR). We sincerely thanked the valuable contributions to this research by the following students/graduate students and staff at the Calcium Research Laboratory, Department of Medicine at St Michael's Hospital and the University of Toronto and Department of Nutritional Sciences, University of Toronto: Dr. Bala Balachandran, Jaclyn Beca, Dawn Snyder, Loren Chan, Honglei Shen, Salva Sadeghi, Ayesha Quireshi, Dr. Erin Mackinnon and Nancy Kang. Their contributions were based on their experimental data, written reports published/in press manuscripts. We would also like to thank to Dr. R.G. Josse for providing us with his medical expertice as well as allowing us access to his list of patients we were able to recruit. Special thanks to Dr. H. Vandenberghe for carrying


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**Chapter 6**

**Pathogenesis, Clinical Diagnosis and Treatment, and**

People with chronic kidney disease (CKD) develop changes in circulating blood levels of calcium and phosphorus. The kidney gradually loses the ability to remove phosphorus from the blood and cannot produce adequate amounts of active vitamin D to maintain normal levels of calcium. This occurs mainly because of decreased renal excretion of phosphate and diminished renal hydroxylation of 25-hydroxyvitamin D to calcitriol due to low expression of alpha-1-hydroxylase in the failed kidneys [1]. Further compensation to maintain normal serum calcium and phosphorus homeostasis includes increased production and release of parathyroid hormone (PTH) and potentially other phosphaturic factors, such as fibroblast

Two main complications follow to the above mentioned molecular responses namely secon‐ dary hyperparathyroidism (sHPT) and vascular calcification (VC), which occur in a high percentage of CKD patients [1]. These molecular disorders alter bone metabolism which leads to bone abnormalities including altered bone production and resorption. In turn, bony changes may result in bone deformation, bone pain, and more risks of fracture [3]. All of the above biochemical abnormalities (calcium, phosphorus, vitamin D and PTH disturbances) and vascular calcification as well as changes in bone metabolism such as variation in turn‐ over and bone mineralization can be included under the descriptions for CKD-associated

This review explains the main pathological causes and mechanisms of CKD-MBD and the possible animal models for basic research on this disease. It also describes some clinically applicable diagnosis techniques and treatment methods with their advantages and side ef‐

and reproduction in any medium, provided the original work is properly cited.

© 2013 Zhang and Gebru; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

**Animal Models for Ckd-Mbd**

Yan Zhang and Yoseph Asmelash Gebru

http://dx.doi.org/10.5772/54238

growth factor-23 (FGF23) [2].

fects for CKD-MBD.

mineral and bone disorders (CKD-MBD) [4].

**1. Introduction**

Additional information is available at the end of the chapter

### **Pathogenesis, Clinical Diagnosis and Treatment, and Animal Models for Ckd-Mbd**

Yan Zhang and Yoseph Asmelash Gebru

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54238

### **1. Introduction**

People with chronic kidney disease (CKD) develop changes in circulating blood levels of calcium and phosphorus. The kidney gradually loses the ability to remove phosphorus from the blood and cannot produce adequate amounts of active vitamin D to maintain normal levels of calcium. This occurs mainly because of decreased renal excretion of phosphate and diminished renal hydroxylation of 25-hydroxyvitamin D to calcitriol due to low expression of alpha-1-hydroxylase in the failed kidneys [1]. Further compensation to maintain normal serum calcium and phosphorus homeostasis includes increased production and release of parathyroid hormone (PTH) and potentially other phosphaturic factors, such as fibroblast growth factor-23 (FGF23) [2].

Two main complications follow to the above mentioned molecular responses namely secon‐ dary hyperparathyroidism (sHPT) and vascular calcification (VC), which occur in a high percentage of CKD patients [1]. These molecular disorders alter bone metabolism which leads to bone abnormalities including altered bone production and resorption. In turn, bony changes may result in bone deformation, bone pain, and more risks of fracture [3]. All of the above biochemical abnormalities (calcium, phosphorus, vitamin D and PTH disturbances) and vascular calcification as well as changes in bone metabolism such as variation in turn‐ over and bone mineralization can be included under the descriptions for CKD-associated mineral and bone disorders (CKD-MBD) [4].

This review explains the main pathological causes and mechanisms of CKD-MBD and the possible animal models for basic research on this disease. It also describes some clinically applicable diagnosis techniques and treatment methods with their advantages and side ef‐ fects for CKD-MBD.

© 2013 Zhang and Gebru; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **2. Potential pathological mechanisms**

### **2.1. sHPT-related bone disorders**

The first changes that usually occur with the decline of renal function involve the deficiency of active vitamin D and decrease in phosphorus excretion by the remaining functional neph‐ rons [5]. In CKD, the failed kidney is inefficient in alpha-1-hydroxylase expression resulting in low synthesis of calcitriol. Simultaneously the kidney with lower function has reduced ability to reabsorb calcium from urine [6]. Therefore, low serum calcium level, high serum phosphorus level and impaired renal 1, 25-dihydroxyvitamin D synthesis with attendant re‐ duction in serum calcitriol concentration and decrease in vitamin D receptor expression in the parathyroid glands contribute to excess PTH secretion in patients with CKD [7].

There are a large number of promoters and inhibitors involved in vascular calcification and there are more vascular calcification inhibitors circulating in the blood under normal condi‐ tions [1]. Phosphorus is the most significant studied vascular calcification promoter which is available at higher level in patients with low renal function. Hyperphosphatemia reverses the normal process in which calcification inhibitors are down-regulated, while promoters

Pathogenesis, Clinical Diagnosis and Treatment, and Animal Models for Ckd-Mbd

http://dx.doi.org/10.5772/54238

165

On the other hand, many bone-associated proteins including osteocalcin, osteopontin and osteoprotegerin, and many bone morphogenetic proteins are involved in the process of VC [16]. Previous studies have proven that active mineralization mechanisms clearly re‐ sembling those of skeletal endochondral and membranous ossification participate in vas‐ cular calcium accumulation [17]. The findings of bone-related factors in the vasculature and the vascular calcification observed in several gene-knockout mouse models imply that CKD-MBD is an actively regulated process that may be preventable or even re‐ versed [18]. The most striking among these mouse models is the matrix gamma-carboxy‐ glutamic acid (Gla) protein (MGP) knockout mouse, which exhibits extensive and lethal calcification and cartilaginous metaplasia of the media of all elastic arteries as early as 2 weeks after birth [19, 20], indicating that this protein may be of primary importance in

According to the standardized diagnostic criteria for CKD-MBD developed and published by the international expert consensus group, kidney disease improving global outcomes (KDIGO), monitoring serum levels of calcium, phosphorus, PTH, and alkaline phosphatase is strongly recommended, and the frequency of monitoring is based on the occurrence and extent of abnormalities as well as the rate of CKD progression [21]. Phosphorus level equal to the upper phosphorus level of 5.5 mg/dL and calcium level more than 9.5 mg/dL have been suggested to be associated with increased mortality in CKD patients [22]. The com‐ bined use of second- and third-generation PTH assays allows to measure PTH (1–84) and PTH (7–84) as PTH (7-84) interacts with distinct receptors and thereby may have important roles in the regulation of bone resorption and serum calcium concentration [23]. The fre‐ quency of measurement on alkaline phosphatases is very similar to that of PTH and can pro‐ vide additional information on bone turnover. The recent more KDIGO guidelines recommend that the measurement on alkaline phosphatase levels should commence in stage 3 of CKD, and that in patients with stage 4-5 of CKD, alkaline phosphatase should be meas‐

The first measurable biomarker in urine is the decline of secreted Klotho expression (as de‐ tected by western blotting of concentrated urine samples, normalized to the same creatinine content) occurs as early as stage 1 of CKD [25], therefore, Kuro-O contends that decreased Klotho expression is the initiator of CKD-MBD pathophysiology and is potentially an early

are up-regulated [1].

human vascular calcification [18].

ured at least every 12 months [24].

**3. Clinical diagnosis**

**3.1. Biomarkers**

PTH strongly influences the exchange of calcium to and from bone through its involvement in bone cell apoptosis mechanisms and effects on the receptor activator of NF-kappa B (RANK)/receptor activator of NF-kappa B ligand (RANKL)/osteoprotegerin (OPG) axis. Continuously elevated PTH could upregulate RANKL expression, leading to an increase in the formation rate and survival time of bone-resorbing osteoclasts and net bone loss [8, 9]. Excess PTH also leads to high bone turnover, a condition characterized by accelerated rates of bone formation and bone resorption [1]. The high bone turnover due to sHPT is accompa‐ nied by about 5% (up to 10%) lower bone mass, which is partly reversible (low mineral bone, increased remodeling space) and partly irreversible (cortical thinning) [10]. The new formed bone in the course of sHPT is structurally inferior and fragile, and carries an in‐ creased risk of fractures.

Another main molecular mechanism underlying sHPT is attributed to Klotho-fibroblast growth factor-23 (FGF-23) system. Humans with CKD experience decreased Klotho expres‐ sion as early as stage 1 CKD. Klotho continues to decline as CKD progresses, causing FGF-23 resistance and provoking large FGF-23 and parathyroid hormone increases [11]. FGF-23 is a novel bone-derived hormone, in conjunction with its co-receptor, Klotho, acti‐ vates FGF receptor 1 (FGFR1) and acts on the kidney to induce renal phosphate wasting and to suppress 1,25-dihydroxyvitamin D synthesis [12]. In patients with CKD, circulating FGF23 levels are progressively increased to compensate for persistent phosphate retention, but this result in reduced renal production of 1, 25-dihydroxyvitamin D through suppress‐ ing 1α-hydroxylase activity, which leads to sHPT [13].

### **2.2. VC-related bone disorders**

Vascular calcification is very common in patients with CKD, appearing in 30-65% of patients with stage 3-5 CKD, 50-80% of patients with stage 5 CKD [14]. Calcium, a divalent cation, and phosphate, a trivalent anion, have a high binding affinity for one another and as the concentration of one or both ions increases in serum, there is an increased risk for an ionic bond to form, creating an insoluble complex which leads to vascular calcification [5]. Clini‐ cally, when the serum calcium-phosphate product exceeds 60 mg2 /dl2 , widespread tissue deposition of amorphous calcium phosphate occurs [15].

There are a large number of promoters and inhibitors involved in vascular calcification and there are more vascular calcification inhibitors circulating in the blood under normal condi‐ tions [1]. Phosphorus is the most significant studied vascular calcification promoter which is available at higher level in patients with low renal function. Hyperphosphatemia reverses the normal process in which calcification inhibitors are down-regulated, while promoters are up-regulated [1].

On the other hand, many bone-associated proteins including osteocalcin, osteopontin and osteoprotegerin, and many bone morphogenetic proteins are involved in the process of VC [16]. Previous studies have proven that active mineralization mechanisms clearly re‐ sembling those of skeletal endochondral and membranous ossification participate in vas‐ cular calcium accumulation [17]. The findings of bone-related factors in the vasculature and the vascular calcification observed in several gene-knockout mouse models imply that CKD-MBD is an actively regulated process that may be preventable or even re‐ versed [18]. The most striking among these mouse models is the matrix gamma-carboxy‐ glutamic acid (Gla) protein (MGP) knockout mouse, which exhibits extensive and lethal calcification and cartilaginous metaplasia of the media of all elastic arteries as early as 2 weeks after birth [19, 20], indicating that this protein may be of primary importance in human vascular calcification [18].

### **3. Clinical diagnosis**

### **3.1. Biomarkers**

**2. Potential pathological mechanisms**

The first changes that usually occur with the decline of renal function involve the deficiency of active vitamin D and decrease in phosphorus excretion by the remaining functional neph‐ rons [5]. In CKD, the failed kidney is inefficient in alpha-1-hydroxylase expression resulting in low synthesis of calcitriol. Simultaneously the kidney with lower function has reduced ability to reabsorb calcium from urine [6]. Therefore, low serum calcium level, high serum phosphorus level and impaired renal 1, 25-dihydroxyvitamin D synthesis with attendant re‐ duction in serum calcitriol concentration and decrease in vitamin D receptor expression in

PTH strongly influences the exchange of calcium to and from bone through its involvement in bone cell apoptosis mechanisms and effects on the receptor activator of NF-kappa B (RANK)/receptor activator of NF-kappa B ligand (RANKL)/osteoprotegerin (OPG) axis. Continuously elevated PTH could upregulate RANKL expression, leading to an increase in the formation rate and survival time of bone-resorbing osteoclasts and net bone loss [8, 9]. Excess PTH also leads to high bone turnover, a condition characterized by accelerated rates of bone formation and bone resorption [1]. The high bone turnover due to sHPT is accompa‐ nied by about 5% (up to 10%) lower bone mass, which is partly reversible (low mineral bone, increased remodeling space) and partly irreversible (cortical thinning) [10]. The new formed bone in the course of sHPT is structurally inferior and fragile, and carries an in‐

Another main molecular mechanism underlying sHPT is attributed to Klotho-fibroblast growth factor-23 (FGF-23) system. Humans with CKD experience decreased Klotho expres‐ sion as early as stage 1 CKD. Klotho continues to decline as CKD progresses, causing FGF-23 resistance and provoking large FGF-23 and parathyroid hormone increases [11]. FGF-23 is a novel bone-derived hormone, in conjunction with its co-receptor, Klotho, acti‐ vates FGF receptor 1 (FGFR1) and acts on the kidney to induce renal phosphate wasting and to suppress 1,25-dihydroxyvitamin D synthesis [12]. In patients with CKD, circulating FGF23 levels are progressively increased to compensate for persistent phosphate retention, but this result in reduced renal production of 1, 25-dihydroxyvitamin D through suppress‐

Vascular calcification is very common in patients with CKD, appearing in 30-65% of patients with stage 3-5 CKD, 50-80% of patients with stage 5 CKD [14]. Calcium, a divalent cation, and phosphate, a trivalent anion, have a high binding affinity for one another and as the concentration of one or both ions increases in serum, there is an increased risk for an ionic bond to form, creating an insoluble complex which leads to vascular calcification [5]. Clini‐

/dl2

, widespread tissue

the parathyroid glands contribute to excess PTH secretion in patients with CKD [7].

**2.1. sHPT-related bone disorders**

164 Topics in Osteoporosis

creased risk of fractures.

**2.2. VC-related bone disorders**

ing 1α-hydroxylase activity, which leads to sHPT [13].

cally, when the serum calcium-phosphate product exceeds 60 mg2

deposition of amorphous calcium phosphate occurs [15].

According to the standardized diagnostic criteria for CKD-MBD developed and published by the international expert consensus group, kidney disease improving global outcomes (KDIGO), monitoring serum levels of calcium, phosphorus, PTH, and alkaline phosphatase is strongly recommended, and the frequency of monitoring is based on the occurrence and extent of abnormalities as well as the rate of CKD progression [21]. Phosphorus level equal to the upper phosphorus level of 5.5 mg/dL and calcium level more than 9.5 mg/dL have been suggested to be associated with increased mortality in CKD patients [22]. The com‐ bined use of second- and third-generation PTH assays allows to measure PTH (1–84) and PTH (7–84) as PTH (7-84) interacts with distinct receptors and thereby may have important roles in the regulation of bone resorption and serum calcium concentration [23]. The fre‐ quency of measurement on alkaline phosphatases is very similar to that of PTH and can pro‐ vide additional information on bone turnover. The recent more KDIGO guidelines recommend that the measurement on alkaline phosphatase levels should commence in stage 3 of CKD, and that in patients with stage 4-5 of CKD, alkaline phosphatase should be meas‐ ured at least every 12 months [24].

The first measurable biomarker in urine is the decline of secreted Klotho expression (as de‐ tected by western blotting of concentrated urine samples, normalized to the same creatinine content) occurs as early as stage 1 of CKD [25], therefore, Kuro-O contends that decreased Klotho expression is the initiator of CKD-MBD pathophysiology and is potentially an early clinical marker of CKD [11]. The study, performed on sixty pre-dialysis patients with CKD 1-5, showed that the changing of serum OPG level happened at the earliest time (CKD 3) and its correlation coefficient with estimated glomerular filtration rate (eGFR) and BMD of Ward's triangle was statistically high, suggesting serum OPG may be a useful biomarker for early diagnosis of CKD-MBD [26], additionally, the multivariate analysis demonstrated that OPG was associated with aortic stiffness in patients with CKD stages 3-4, indicating OPG is also a marker to evaluate the cardiomyocyte dysfunction of CKD-MBD [27].

reducing serum phosphorus and PTH levels [32]. Therefore, the use of phosphate binders might be a promising and most practical strategy for the prevention of VC and sHPT which are the main pathological manifestations of the metabolic bone disease in CKD patients. The

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167

**1.** Aluminum-based phosphate binders are the first type of phosphate binders to be used. They are very effective at controlling phosphorus. The most common binder of this type is aluminum hydroxide. However, aluminum has toxic effects on bone and nervous system. For this reason, aluminum-based phosphate binders are not often used much

**2.** Calcium-based phosphate binders are effective in binding phosphates and can be source of calcium. Common types of calcium-based binders include calcium acetate and calcium carbonate, both of which could cause the elevation of free calcium cation level in the gastrointestinal tract and the subsequent increase of intestinal calcium absorption [34]. The Japanese Society of Dialysis Therapy (JSDT) clinical practice guideline has rec‐ ommended a higher level of these oral phosphate binders as the upper limit for clinical use [35]. These binders can also serve as calcium supplements if the calcium is low. However, if the patient is taking vitamin D supplements, he/she may already have high calcium levels, and these types of phosphate binders may provide more calcium than the normal level (i.e., excess calcium load). Therefore using calcium based phosphate binders should be accompanied with monitoring calcium levels and it should be pre‐

**3.** Aluminum-free, calcium-free phosphate binders are newer binders that are effective at controlling phosphorus. Because they do not contain aluminum or calcium, they do not cause problems with excess aluminum or calcium load. Lanthanum carbonate is a novel non-calcium, non-aluminum phosphate-binding agent, and has been approved for clini‐ cal use in patients on hemodialysis in Japan on March in 2009 [33]. Sevelamer is a poly‐ meric amine, which is the only non-absorbed, non-calcium-based phosphate binder currently indicated for phosphate control. The first formulation of sevelamer to be ap‐ proved was sevelamer hydrochloride, while a newer formulation, sevelamer carbonate, has more recently become available [36]. Sevelamer carbonate was developed to offer phosphorus lowering while eliminating the risk of worsening metabolic acidosis associ‐ ated with sevelamer hydrochloride and the consequent need to monitor for changes in

Vitamin D analogues suppress PTH synthesis and secretion in patients with sHPT. Reple‐ tion with native vitamin D may lead to improved control of secondary hyperparathyroidism in patients with CKD which reduces the risk of bone mineral disease. It has been demon‐ strated that treatment with vitamin D analogues can decrease mortality in dialysis patients [38]. There might be some differences in clinical outcomes among vitamin D compounds with fewer calcemic and phosphatemic effects [39], such as paricalcitol, doxercalciferol, and

following categories of phosphate binders are being applied clinically so far:

anymore [33].

scribed while limiting total calcium intake.

serum chloride or bicarbonate levels [37].

**4.2. Vitamin D compounds**

### **3.2. Imaging**

Histomorphometry remains the gold standard to evaluate bone, but it is rarely performed in clinical practice. Areal measurement of bone mineral density by dual-energy x-ray absorpti‐ ometry (DEXA) is routinely performed to evaluate bone mass. However, this technique presents some limitations. In 2000, the United States National Institutes of Health defined new "quality" criteria for the diagnosis of osteoporosis in addition to decreased bone mass. Bone strength actually integrates two concepts: bone quantity and bone quality (i.e., micro‐ architectural organization, bone turnover, bone material properties such as mineralization, collagen traits, and micro-damage) that cannot be evaluated by DEXA. New three-dimen‐ sional, noninvasive bone-imaging techniques have thus been developed, e.g., high-resolu‐ tion peripheral quantitative computed tomography (HR-pQCT). HR-pQCT allows evaluation of both volumetric density and microarchitecture in different compartments of bone [28]. Bacchetta reported for the first time an early impairment of trabecular microarchi‐ tecture in stage 2-4 CKD patients using a noninvasive bone-imaging device, HR-pQCT [29].

Physicians usually use a variety of noninvasive imaging tools to identify VC, some with merely qualitative and others with both qualitative and quantitative capabilities. Plain xrays and ultra-sonography can be used to identify macroscopic calcification of aorta and pe‐ ripheral arteries, and computed tomography technologies constitute the gold standard for quantification of cardiovascular calcification [30].

### **4. Clinical treatment strategy**

The clinical treatment for CKD-MBD targets the possible pathological mechanisms of main‐ ly sHPT and VC in patients with kidney failure as treating these abnormalities will have a direct positive impact on preventing the metabolic bone disease. However, the heterogenei‐ ty of CKD-MBD makes strict protocol-driven therapeutic approaches difficult. Accordingly, considerable individualized therapy is required [31]. The followings are currently the most common and effective intervening methods.

### **4.1. Phosphate binders**

In patients at stage 3-5 of CKD, multiple studies from different parts of the world have shown that higher levels of serum phosphorus have been associated with an increased rela‐ tive risk of mortality [24]. Many clinical trials show that phosphate binders are effective in reducing serum phosphorus and PTH levels [32]. Therefore, the use of phosphate binders might be a promising and most practical strategy for the prevention of VC and sHPT which are the main pathological manifestations of the metabolic bone disease in CKD patients. The following categories of phosphate binders are being applied clinically so far:


### **4.2. Vitamin D compounds**

clinical marker of CKD [11]. The study, performed on sixty pre-dialysis patients with CKD 1-5, showed that the changing of serum OPG level happened at the earliest time (CKD 3) and its correlation coefficient with estimated glomerular filtration rate (eGFR) and BMD of Ward's triangle was statistically high, suggesting serum OPG may be a useful biomarker for early diagnosis of CKD-MBD [26], additionally, the multivariate analysis demonstrated that OPG was associated with aortic stiffness in patients with CKD stages 3-4, indicating OPG is

Histomorphometry remains the gold standard to evaluate bone, but it is rarely performed in clinical practice. Areal measurement of bone mineral density by dual-energy x-ray absorpti‐ ometry (DEXA) is routinely performed to evaluate bone mass. However, this technique presents some limitations. In 2000, the United States National Institutes of Health defined new "quality" criteria for the diagnosis of osteoporosis in addition to decreased bone mass. Bone strength actually integrates two concepts: bone quantity and bone quality (i.e., micro‐ architectural organization, bone turnover, bone material properties such as mineralization, collagen traits, and micro-damage) that cannot be evaluated by DEXA. New three-dimen‐ sional, noninvasive bone-imaging techniques have thus been developed, e.g., high-resolu‐ tion peripheral quantitative computed tomography (HR-pQCT). HR-pQCT allows evaluation of both volumetric density and microarchitecture in different compartments of bone [28]. Bacchetta reported for the first time an early impairment of trabecular microarchi‐ tecture in stage 2-4 CKD patients using a noninvasive bone-imaging device, HR-pQCT [29]. Physicians usually use a variety of noninvasive imaging tools to identify VC, some with merely qualitative and others with both qualitative and quantitative capabilities. Plain xrays and ultra-sonography can be used to identify macroscopic calcification of aorta and pe‐ ripheral arteries, and computed tomography technologies constitute the gold standard for

The clinical treatment for CKD-MBD targets the possible pathological mechanisms of main‐ ly sHPT and VC in patients with kidney failure as treating these abnormalities will have a direct positive impact on preventing the metabolic bone disease. However, the heterogenei‐ ty of CKD-MBD makes strict protocol-driven therapeutic approaches difficult. Accordingly, considerable individualized therapy is required [31]. The followings are currently the most

In patients at stage 3-5 of CKD, multiple studies from different parts of the world have shown that higher levels of serum phosphorus have been associated with an increased rela‐ tive risk of mortality [24]. Many clinical trials show that phosphate binders are effective in

also a marker to evaluate the cardiomyocyte dysfunction of CKD-MBD [27].

quantification of cardiovascular calcification [30].

**4. Clinical treatment strategy**

common and effective intervening methods.

**4.1. Phosphate binders**

**3.2. Imaging**

166 Topics in Osteoporosis

Vitamin D analogues suppress PTH synthesis and secretion in patients with sHPT. Reple‐ tion with native vitamin D may lead to improved control of secondary hyperparathyroidism in patients with CKD which reduces the risk of bone mineral disease. It has been demon‐ strated that treatment with vitamin D analogues can decrease mortality in dialysis patients [38]. There might be some differences in clinical outcomes among vitamin D compounds with fewer calcemic and phosphatemic effects [39], such as paricalcitol, doxercalciferol, and maxacalcitol. Therefore, it is important for desirable active vitamin D compounds to achieve optimal vitamin D receptor (VDR) activation without inducing hypercalcemia. It is likely that the elevated calcium levels caused by calcitriol may be directly and/or indirectly re‐ sponsible for the relative risk of the cardiovascular diseases that are aggravated by hypercal‐ cemia in patient populations.

The 2009 KDIGO guideline suggested parathyroidectomy to patients who are at CKD stages 3-5 with severe hyperparathyroidism and fail to respond to medical/pharmacologi‐ cal therapy [46]. There are two main surgical procedures which are generally used, namely subtotal parathyroidectomy and total parathyroidectomy with immediate auto‐ transplantation. The number and size of affected parathyroid glands are the most impor‐ tant factors for selecting the treatment procedure [47, 48]. Clinical studies proved that parathyroidectomy with autotransplantation from forearm was significantly effective and safe in patients in whom medical treatment had failed, particularly in terms of improv‐ ing calcium and phosphate control [49, 50]. The procedure need to be performed as early as possible to avoid the adverse, irreversible effects of prolonged hyperparathyroidism, and to improve osteoarticular symptoms. Future strategies may focus on the stimulation

Pathogenesis, Clinical Diagnosis and Treatment, and Animal Models for Ckd-Mbd

http://dx.doi.org/10.5772/54238

169

Experimental model of 5/6 nephrectomy or the remnant kidney model represents one of the most used animal models of progressive renal failure by reducing nephron number, best-characterized in rats [52]. The reduction of renal mass is achieved by either infarc‐ tion or surgical excision of both poles, with removal of the contra-lateral kidney. The 5/6 nephroctomy model has been found to produce serum creatinine level which is on aver‐ age 2.2-fold higher than control animals, and thereafter, if without the concurrent use of vitamin D, the phosphorus level after 8 weeks of surgery would range up to 2.6-fold higher than control animals [53]. Increased fibrosis, increased number of osteoblasts and osteoclasts as well as a mineralization defect (increased osteoid volumes and osteoid sur‐ face), those of which are typical bone changes upon sHPT, have been observed in 5/6

The operation of 5/6 nephrectomy, combining with a diet containing 1.2% P plus 0.6% Ca, could effectively induce sHPT in rats [54]. Additionally, the progressive partial nephrecto‐ my with thyroparathyroidectomy (TPTx-Nx) reduced the storage modulus, which is a me‐ chanical factor, in CKD model rats as compared with controls that underwent thyroparathyroidectomy alone (TPTx). Moreover, the TPTx-Nx rats exerted different cortical bone chemical composition and increased enzymatic crosslinks ratio and pentosidine to ma‐

As concerned as VC associated with CKD-MBD, it can be induced in 5/6 nephrectomy rat model by feeding a high-phosphorus, high-lactose diet (1.2% P, 1% Ca, and 20% lactose) af‐ ter 10 weeks follow up for the reason that lactose increases calcium and phosphorus absorp‐

of apoptotic activity of hyperplastic parathyroid cells [51].

**5. Animal models**

**5.1. 5/6 nephrectomy model**

nephrectomy animal models [14].

trix ratio [55].

tion in intestine [56].

### **4.3. Calcimimetics**

Calcimimetics bind to the calcium sensing receptor (CaSR) in parathyroid gland and mimic the effect of an elevated extracellular ionized calcium concentration. These molecules reduce serum levels of PTH and calcium, with a leftward shift in the set-point for calcium-regulated PTH secretion [40]. Cinacalcet is the only clinically available calcimimetic and has been shown to be a very effective therapeutic compound in the metabolic bone disease associated with CKD. Many clinical trials with cinacalcet in hemodialysis patients have shown a reduc‐ tion in parathyroid hormone, calcium, phosphate and calcium × phosphate product levels, allowing far greater success in reaching therapeutic goals as recommended by international guidelines [41]. In addition to effective control of secondary hyperparathyroidism, treatment with cinacalcet may improve the mineral balance in patients with dialysis who have serum phosphate/calcium disequilibrium, and furthermore helps treating the vascular calcification as well. While, calcimimetics are not approved for use in paediatric patients with CKD and long-term data on their effects on bone, growth and biochemical parameters in children are lacking. Thus, further studies are warranted to determine the optimal strategy for control‐ ling secondary hyperparathyroidism in the paediatric CKD population [42].

### **4.4. Administering BMP-7**

One of the bone morphogenetic proteins, BMP-7, also known as osteogenic protein 1, is highly expressed in the adult kidney, and circulates in the bloodstream [43]. Therefore, it is apparent that the decrease of renal mass results in the decreased production of BMP-7, causing mineral bone disease in CKD patients [44]. One may expect an accumulation of osteoblast precursors as stimulated by PTH in CKD. While, these progenitors may be un‐ able to differentiate mature osteoblasts because of BMP-7 deficiency considering it is im‐ portant in osteoblast development and function. In this situation, the subsequent accumulation of fibrous cells could then offer an explanation for the marrow fibrosis ob‐ served in secondary hyperparathyroidism in the setting of CKD and applying BMP-7 ex‐ ternally can heal the disorder. There are an increasing number of recent clinical trials that provide supportive evidence for the use of BMP-7 in the treatment of fractures and bone nonunions [45]. It is not yet started to use BMP-7 as a routine clinical treatment tool except for trials in patients even though many of the studies have shown the bone healing efficacy of this molecule.

### **4.5. Surgery on thyroid gland**

A surgical correction in the parathyroid gland is the final, symptomatic therapy for the most severe forms of sHPT which cannot be controlled by the above medical treatments. The 2009 KDIGO guideline suggested parathyroidectomy to patients who are at CKD stages 3-5 with severe hyperparathyroidism and fail to respond to medical/pharmacologi‐ cal therapy [46]. There are two main surgical procedures which are generally used, namely subtotal parathyroidectomy and total parathyroidectomy with immediate auto‐ transplantation. The number and size of affected parathyroid glands are the most impor‐ tant factors for selecting the treatment procedure [47, 48]. Clinical studies proved that parathyroidectomy with autotransplantation from forearm was significantly effective and safe in patients in whom medical treatment had failed, particularly in terms of improv‐ ing calcium and phosphate control [49, 50]. The procedure need to be performed as early as possible to avoid the adverse, irreversible effects of prolonged hyperparathyroidism, and to improve osteoarticular symptoms. Future strategies may focus on the stimulation of apoptotic activity of hyperplastic parathyroid cells [51].

### **5. Animal models**

maxacalcitol. Therefore, it is important for desirable active vitamin D compounds to achieve optimal vitamin D receptor (VDR) activation without inducing hypercalcemia. It is likely that the elevated calcium levels caused by calcitriol may be directly and/or indirectly re‐ sponsible for the relative risk of the cardiovascular diseases that are aggravated by hypercal‐

Calcimimetics bind to the calcium sensing receptor (CaSR) in parathyroid gland and mimic the effect of an elevated extracellular ionized calcium concentration. These molecules reduce serum levels of PTH and calcium, with a leftward shift in the set-point for calcium-regulated PTH secretion [40]. Cinacalcet is the only clinically available calcimimetic and has been shown to be a very effective therapeutic compound in the metabolic bone disease associated with CKD. Many clinical trials with cinacalcet in hemodialysis patients have shown a reduc‐ tion in parathyroid hormone, calcium, phosphate and calcium × phosphate product levels, allowing far greater success in reaching therapeutic goals as recommended by international guidelines [41]. In addition to effective control of secondary hyperparathyroidism, treatment with cinacalcet may improve the mineral balance in patients with dialysis who have serum phosphate/calcium disequilibrium, and furthermore helps treating the vascular calcification as well. While, calcimimetics are not approved for use in paediatric patients with CKD and long-term data on their effects on bone, growth and biochemical parameters in children are lacking. Thus, further studies are warranted to determine the optimal strategy for control‐

ling secondary hyperparathyroidism in the paediatric CKD population [42].

One of the bone morphogenetic proteins, BMP-7, also known as osteogenic protein 1, is highly expressed in the adult kidney, and circulates in the bloodstream [43]. Therefore, it is apparent that the decrease of renal mass results in the decreased production of BMP-7, causing mineral bone disease in CKD patients [44]. One may expect an accumulation of osteoblast precursors as stimulated by PTH in CKD. While, these progenitors may be un‐ able to differentiate mature osteoblasts because of BMP-7 deficiency considering it is im‐ portant in osteoblast development and function. In this situation, the subsequent accumulation of fibrous cells could then offer an explanation for the marrow fibrosis ob‐ served in secondary hyperparathyroidism in the setting of CKD and applying BMP-7 ex‐ ternally can heal the disorder. There are an increasing number of recent clinical trials that provide supportive evidence for the use of BMP-7 in the treatment of fractures and bone nonunions [45]. It is not yet started to use BMP-7 as a routine clinical treatment tool except for trials in patients even though many of the studies have shown the bone

A surgical correction in the parathyroid gland is the final, symptomatic therapy for the most severe forms of sHPT which cannot be controlled by the above medical treatments.

cemia in patient populations.

**4.4. Administering BMP-7**

healing efficacy of this molecule.

**4.5. Surgery on thyroid gland**

**4.3. Calcimimetics**

168 Topics in Osteoporosis

### **5.1. 5/6 nephrectomy model**

Experimental model of 5/6 nephrectomy or the remnant kidney model represents one of the most used animal models of progressive renal failure by reducing nephron number, best-characterized in rats [52]. The reduction of renal mass is achieved by either infarc‐ tion or surgical excision of both poles, with removal of the contra-lateral kidney. The 5/6 nephroctomy model has been found to produce serum creatinine level which is on aver‐ age 2.2-fold higher than control animals, and thereafter, if without the concurrent use of vitamin D, the phosphorus level after 8 weeks of surgery would range up to 2.6-fold higher than control animals [53]. Increased fibrosis, increased number of osteoblasts and osteoclasts as well as a mineralization defect (increased osteoid volumes and osteoid sur‐ face), those of which are typical bone changes upon sHPT, have been observed in 5/6 nephrectomy animal models [14].

The operation of 5/6 nephrectomy, combining with a diet containing 1.2% P plus 0.6% Ca, could effectively induce sHPT in rats [54]. Additionally, the progressive partial nephrecto‐ my with thyroparathyroidectomy (TPTx-Nx) reduced the storage modulus, which is a me‐ chanical factor, in CKD model rats as compared with controls that underwent thyroparathyroidectomy alone (TPTx). Moreover, the TPTx-Nx rats exerted different cortical bone chemical composition and increased enzymatic crosslinks ratio and pentosidine to ma‐ trix ratio [55].

As concerned as VC associated with CKD-MBD, it can be induced in 5/6 nephrectomy rat model by feeding a high-phosphorus, high-lactose diet (1.2% P, 1% Ca, and 20% lactose) af‐ ter 10 weeks follow up for the reason that lactose increases calcium and phosphorus absorp‐ tion in intestine [56].

### **5.2. Electrocautery models**

In the mouse electrocautery model, CKD is induced by surgical ablation of the kidneys. This is a two-step procedure. Initially the cortex of one kidney is electrocauterized paying careful attention to avoid destroying the adrenals and the hilum of kidney. One week later, once the animals have recovered, the second kidney is nephrectomized [44]. This procedure appears to produce variable severity of CKD with blood urea levels ranging from 1.5- to 4.8-fold higher than normal animals [53]. This murine model displayed an increase in osteoblast sur‐ face and osteoid accumulation as well as increased activation frequency and increased os‐ teoclast surface consistent with high turnover renal osteodystrophy [44]. Lund developed a standard CKD rat models by involving electrocauthery of the right kidney followed by nephroctomy of the left kidney, and found that there was a significant hyperosteoidosis pro‐ duced in this model as a result of the secondary hyperparathyroidism [57].

**5.5. Obstructive nephropathy**

sin II and its type 2 receptor in tibia of UUO mice (Fig. 4) [62].

The mouse with unilateral ureteral obstruction (UUO) is a well-established model of tubuloin‐ terstitial fibrosis of the kidney as the interstitial fibrosis is a hallmark of chronic renal failure [60]. We previously reported the vitamin D signaling attenuates renal fibrosis in obstructive nephropathy by suppressing the renin-angiotensin system (RAS) [61], furthermore, we found the mice developed hypocalcaemia and hyperparathyroidism after 7 days of ureteric obstruc‐ tion [62], and the down-regulation of *Cbfa1* and *Col* mRNA expression (Fig. 1) and the up-regu‐ lation of *Tgf-β*, *CtsK*, *CaII*, *Opg* and *Rankl* mRNA expression (Fig. 2) in tibia of UUO mice as well as the microarchitectural changes in the proximal tibia, likely to be precursors of the early stage during CKD-MBD [62]. The pathological alterations of proximal tibia in UUO group were char‐ acterized by a marked expansion of hypertrophic zone of chondrocytes and a dramatic de‐ crease in osteoid content of the primary spongiosa zone, where the immature, poorly mineralized woven bones were present, indicating impaired mineralization of the newly formed bones (Fig. 3B). Above all, in addition to established genetic pathways, we suggest that the local skeletal renin-angiotensin system may be involved in the bone deteriorations associat‐ ed with CKD as demonstrated by the marked up-regulation of protein expression of angioten‐

Sham UUO

Sham UUO

Sham UUO

Sham UUO

**Figure 1.** mRNA expression of osteoblast-specific genes in tibia of sham-operated and UUO mice

**Figure 2.** mRNA expression of osteoclast-specific genes in tibia of sham-operated and UUO mice

11

11

nephropathy by suppressing the renin-angiotensin system (RAS) [61], furthermore, we found the mice

171

http://dx.doi.org/10.5772/54238

nephropathy by suppressing the renin-angiotensin system (RAS) [61], furthermore, we found the mice

developed hypocalcaemia and hyperparathyroidism after 7 days of ureteric obstruction [62], and the

developed hypocalcaemia and hyperparathyroidism after 7 days of ureteric obstruction [62], and the

down-regulation of *Cbfa1* and *Col* mRNA expression (Fig. 1) and the up-regulation of *Tgf-β*, *CtsK*,

down-regulation of *Cbfa1* and *Col* mRNA expression (Fig. 1) and the up-regulation of *Tgf-β*, *CtsK*,

*CaII*, *Opg* and *Rankl* mRNA expression (Fig. 2) in tibia of UUO mice as well as the microarchitectural

*CaII*, *Opg* and *Rankl* mRNA expression (Fig. 2) in tibia of UUO mice as well as the microarchitectural

changes in the proximal tibia, likely to be precursors of the early stage during CKD-MBD [62]. The

changes in the proximal tibia, likely to be precursors of the early stage during CKD-MBD [62]. The

pathological alterations of proximal tibia in UUO group were characterized by a marked expansion of

pathological alterations of proximal tibia in UUO group were characterized by a marked expansion of

hypertrophic zone of chondrocytes and a dramatic decrease in osteoid content of the primary spongiosa

hypertrophic zone of chondrocytes and a dramatic decrease in osteoid content of the primary spongiosa

zone, where the immature, poorly mineralized woven bones were present, indicating impaired

zone, where the immature, poorly mineralized woven bones were present, indicating impaired

mineralization of the newly formed bones (Fig. 3B). Above all, in addition to established genetic

mineralization of the newly formed bones (Fig. 3B). Above all, in addition to established genetic

pathways, we suggest that the local skeletal renin-angiotensin system may be involved in the bone

pathways, we suggest that the local skeletal renin-angiotensin system may be involved in the bone

deteriorations associated with CKD as demonstrated by the marked up-regulation of protein expression

deteriorations associated with CKD as demonstrated by the marked up-regulation of protein expression

Figure 1 mRNA expression of osteoblast-specific genes in tibia of sham-operated and UUO mice

Figure 1 mRNA expression of osteoblast-specific genes in tibia of sham-operated and UUO mice

Figure 2 mRNA expression of osteoclast-specific genes in tibia of sham-operated and UUO mice

Figure 2 mRNA expression of osteoclast-specific genes in tibia of sham-operated and UUO mice

of angiotensin II and its type 2 receptor in tibia of UUO mice (Fig. 4) [62].

of angiotensin II and its type 2 receptor in tibia of UUO mice (Fig. 4) [62].

*Tgf-β*

Pathogenesis, Clinical Diagnosis and Treatment, and Animal Models for Ckd-Mbd

*Tgf-β*

*Alp*

*Alp*

*Cbfa1*

*Cbfa1*

*Col*

*Col*

*β2m*

*β2m*

*CtsK*

*CtsK*

*CaII*

*CaII*

*β2m*

*β2m*

### **5.3. Adenine-contained diet**

Normally, adenine is efficiently salvaged by adenine phosphoribosyltransferase (APRT) and is present at very low level in blood and urine. APRT is involved in the conversion of adenine to adenosine monophosphate. When adenine is administered in high level, APRT activity is satu‐ rated and adenine is oxidized to 2,8-dihydroxyadenine. Adenine and 2,8-dihydroxyadenine are excreted in the urine. However, the very low solubility of 2,8-dihydroxyadenine results in its precipitation in the kidney. The accumulation of insoluble 2,8-dihydroxyadenine results in nephrolithiasis and renal failure with permanent kidney damage. Induction of chronic renal failure (CRF) in mice by dietary administration of 0.75% adenine for 4 weeks results in irrever‐ sible renal dysfunction and then CKD [53]. High-adenine feeding in rats results in the forma‐ tion of crystals in the renal tubules, with subsequent tubular injury and inflammation, obstruction, and marked fibrosis [56]. Future investigations of the biochemical basis for the link between vascular calcification and bone resorption will be facilitated by the present discovery that a synthetic, 2.5% protein diet containing 0.75% adenine produces consistent and dramatic medial calcification in adult rats within just 4 weeks [58].

### **5.4. Gene knockout mice**

JCK mouse is a genetic model of polycystic kidney disease. At 6 weeks of age, the mice have normal renal function and no evidence of bone disease but exhibit continual de‐ cline in renal function and death by 20 weeks of age, when approximately 40% to 60% of them have vascular calcification. Temporal changes in serum parameters of JCK mice relative to wild-type mice from 6 through 18 weeks of age were shown to largely mirror serum changes commonly associated with clinical CKD-MBD. Bone histomorphometry revealed progressive changes associated with increased osteoclast activity and elevated bone formation [59].

Klotho null mice display premature aging and CKD-MBD-like phenotypes mediated by hyper‐ phosphatemia and remediated by phosphate-lowering interventions (diets low in phosphate or vitamin D; knockouts of 1α-hydroxylase, vitamin D receptor, or NaPi cotransporter) [11].

#### **5.5. Obstructive nephropathy** nephropathy by suppressing the renin-angiotensin system (RAS) [61], furthermore, we found the mice nephropathy by suppressing the renin-angiotensin system (RAS) [61], furthermore, we found the mice

**5.2. Electrocautery models**

170 Topics in Osteoporosis

**5.3. Adenine-contained diet**

**5.4. Gene knockout mice**

bone formation [59].

In the mouse electrocautery model, CKD is induced by surgical ablation of the kidneys. This is a two-step procedure. Initially the cortex of one kidney is electrocauterized paying careful attention to avoid destroying the adrenals and the hilum of kidney. One week later, once the animals have recovered, the second kidney is nephrectomized [44]. This procedure appears to produce variable severity of CKD with blood urea levels ranging from 1.5- to 4.8-fold higher than normal animals [53]. This murine model displayed an increase in osteoblast sur‐ face and osteoid accumulation as well as increased activation frequency and increased os‐ teoclast surface consistent with high turnover renal osteodystrophy [44]. Lund developed a standard CKD rat models by involving electrocauthery of the right kidney followed by nephroctomy of the left kidney, and found that there was a significant hyperosteoidosis pro‐

Normally, adenine is efficiently salvaged by adenine phosphoribosyltransferase (APRT) and is present at very low level in blood and urine. APRT is involved in the conversion of adenine to adenosine monophosphate. When adenine is administered in high level, APRT activity is satu‐ rated and adenine is oxidized to 2,8-dihydroxyadenine. Adenine and 2,8-dihydroxyadenine are excreted in the urine. However, the very low solubility of 2,8-dihydroxyadenine results in its precipitation in the kidney. The accumulation of insoluble 2,8-dihydroxyadenine results in nephrolithiasis and renal failure with permanent kidney damage. Induction of chronic renal failure (CRF) in mice by dietary administration of 0.75% adenine for 4 weeks results in irrever‐ sible renal dysfunction and then CKD [53]. High-adenine feeding in rats results in the forma‐ tion of crystals in the renal tubules, with subsequent tubular injury and inflammation, obstruction, and marked fibrosis [56]. Future investigations of the biochemical basis for the link between vascular calcification and bone resorption will be facilitated by the present discovery that a synthetic, 2.5% protein diet containing 0.75% adenine produces consistent and dramatic

JCK mouse is a genetic model of polycystic kidney disease. At 6 weeks of age, the mice have normal renal function and no evidence of bone disease but exhibit continual de‐ cline in renal function and death by 20 weeks of age, when approximately 40% to 60% of them have vascular calcification. Temporal changes in serum parameters of JCK mice relative to wild-type mice from 6 through 18 weeks of age were shown to largely mirror serum changes commonly associated with clinical CKD-MBD. Bone histomorphometry revealed progressive changes associated with increased osteoclast activity and elevated

Klotho null mice display premature aging and CKD-MBD-like phenotypes mediated by hyper‐ phosphatemia and remediated by phosphate-lowering interventions (diets low in phosphate or vitamin D; knockouts of 1α-hydroxylase, vitamin D receptor, or NaPi cotransporter) [11].

duced in this model as a result of the secondary hyperparathyroidism [57].

medial calcification in adult rats within just 4 weeks [58].

The mouse with unilateral ureteral obstruction (UUO) is a well-established model of tubuloin‐ terstitial fibrosis of the kidney as the interstitial fibrosis is a hallmark of chronic renal failure [60]. We previously reported the vitamin D signaling attenuates renal fibrosis in obstructive nephropathy by suppressing the renin-angiotensin system (RAS) [61], furthermore, we found the mice developed hypocalcaemia and hyperparathyroidism after 7 days of ureteric obstruc‐ tion [62], and the down-regulation of *Cbfa1* and *Col* mRNA expression (Fig. 1) and the up-regu‐ lation of *Tgf-β*, *CtsK*, *CaII*, *Opg* and *Rankl* mRNA expression (Fig. 2) in tibia of UUO mice as well as the microarchitectural changes in the proximal tibia, likely to be precursors of the early stage during CKD-MBD [62]. The pathological alterations of proximal tibia in UUO group were char‐ acterized by a marked expansion of hypertrophic zone of chondrocytes and a dramatic de‐ crease in osteoid content of the primary spongiosa zone, where the immature, poorly mineralized woven bones were present, indicating impaired mineralization of the newly formed bones (Fig. 3B). Above all, in addition to established genetic pathways, we suggest that the local skeletal renin-angiotensin system may be involved in the bone deteriorations associat‐ ed with CKD as demonstrated by the marked up-regulation of protein expression of angioten‐ sin II and its type 2 receptor in tibia of UUO mice (Fig. 4) [62]. developed hypocalcaemia and hyperparathyroidism after 7 days of ureteric obstruction [62], and the down-regulation of *Cbfa1* and *Col* mRNA expression (Fig. 1) and the up-regulation of *Tgf-β*, *CtsK*, *CaII*, *Opg* and *Rankl* mRNA expression (Fig. 2) in tibia of UUO mice as well as the microarchitectural changes in the proximal tibia, likely to be precursors of the early stage during CKD-MBD [62]. The pathological alterations of proximal tibia in UUO group were characterized by a marked expansion of hypertrophic zone of chondrocytes and a dramatic decrease in osteoid content of the primary spongiosa zone, where the immature, poorly mineralized woven bones were present, indicating impaired mineralization of the newly formed bones (Fig. 3B). Above all, in addition to established genetic pathways, we suggest that the local skeletal renin-angiotensin system may be involved in the bone deteriorations associated with CKD as demonstrated by the marked up-regulation of protein expression of angiotensin II and its type 2 receptor in tibia of UUO mice (Fig. 4) [62]. developed hypocalcaemia and hyperparathyroidism after 7 days of ureteric obstruction [62], and the down-regulation of *Cbfa1* and *Col* mRNA expression (Fig. 1) and the up-regulation of *Tgf-β*, *CtsK*, *CaII*, *Opg* and *Rankl* mRNA expression (Fig. 2) in tibia of UUO mice as well as the microarchitectural changes in the proximal tibia, likely to be precursors of the early stage during CKD-MBD [62]. The pathological alterations of proximal tibia in UUO group were characterized by a marked expansion of hypertrophic zone of chondrocytes and a dramatic decrease in osteoid content of the primary spongiosa zone, where the immature, poorly mineralized woven bones were present, indicating impaired mineralization of the newly formed bones (Fig. 3B). Above all, in addition to established genetic pathways, we suggest that the local skeletal renin-angiotensin system may be involved in the bone deteriorations associated with CKD as demonstrated by the marked up-regulation of protein expression of angiotensin II and its type 2 receptor in tibia of UUO mice (Fig. 4) [62].

Figure 1 mRNA expression of osteoblast-specific genes in tibia of sham-operated and UUO mice **Figure 1.** mRNA expression of osteoblast-specific genes in tibia of sham-operated and UUO mice Figure 1 mRNA expression of osteoblast-specific genes in tibia of sham-operated and UUO mice

11

11

Figure 2 mRNA expression of osteoclast-specific genes in tibia of sham-operated and UUO mice

Figure 2 mRNA expression of osteoclast-specific genes in tibia of sham-operated and UUO mice

**Figure 2.** mRNA expression of osteoclast-specific genes in tibia of sham-operated and UUO mice

was shown in A (Sham) and B (UUO) and it was visually separated into two areas, proliferative zone (PZ) and hypertrophic zone (HZ). Calcified cartilage with overlying newly bone underneath growth plate is known as the primary spongiosa (PS). Magnification, ×100. **Figure 3.** Hematoxylin and Eosin staining of the proximal tibia. The chondrocyte zone at growth plate was shown in A (Sham) and B (UUO) and it was visually separated into two areas, proliferative zone (PZ) and hypertrophic zone (HZ). Calcified cartilage with overlying newly bone underneath growth plate is known as the primary spongiosa (PS). Mag‐ nification, ×100. Figure 3 Hematoxylin and Eosin staining of the proximal tibia. The chondrocyte zone at growth plate was shown in A (Sham) and B (UUO) and it was visually separated into two areas, proliferative zone (PZ) and hypertrophic zone (HZ). Calcified cartilage with overlying newly bone underneath growth

plate is known as the primary spongiosa (PS). Magnification, ×100.

Sham UUO

PS HZ PZ

Figure 4 Protein expression of RAS components in mice tibia

12

12

Figure 3 Hematoxylin and Eosin staining of the proximal tibia. The chondrocyte zone at growth plate

**6. Conclusion**

**Author details**

Shanghai , P.R.China

**References**

514-519.

novel drugs with less adverse effects.

Yan Zhang1,2\* and Yoseph Asmelash Gebru1

Miner Res 2007;22(S2) S91–94.

Nephrol 2010;21(8) 1371-1380.

\*Address all correspondence to: medicineyan@yahoo.com.cn

University, Hung Hom, Kowloon, Hong Kong, P.R.China

In summary, the present review demonstrates that the main pathological mechanisms in‐ volved in CKD-MBD are secondary hyperparathyroidism and vascular calcification. There‐ fore the main focus of the therapeutic research and the further molecular investigations should be on these main abnormalities associated with CKD. This can be achieved by em‐ ploying the proper animal models for each of these complications including genetically modified mouse models. The animal models can play a great role in understanding the un‐ derlying mechanisms for CKD-MBD. The clinical treatment approaches should depend on the specific levels of the biomarkers of CKD patients. Further studies are needed to discover other pathological mechanisms for CKD-MBD other than those explained here. More ad‐ vanced basic medical sciences should also be performed on the research and development of

Pathogenesis, Clinical Diagnosis and Treatment, and Animal Models for Ckd-Mbd

http://dx.doi.org/10.5772/54238

173

This work was sponsored by Shanghai Pujiang Program (10PJ1407700) and Innovation Pro‐

1 Center for Systems Biomedical Sciences, University of Shanghai for Science and Technology,

2 Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic

[1] Mejía N, Roman-García P, Miar AB, Tavira B, Cannata-Andía JB. Chronic Kidney Disease – Mineral and Bone Disorder: A Complex Scenario. Nefrologia 2011;31(5)

[2] Anca G, Stuart MS. Role of Vitamin D Deficiency in Chronic Kidney Disease. J Bone

[3] Nickolas TL, Stein E, Cohen A, Thomas V, Staron RB, McMahon DJ, Leonard MB, Shane E. Bone Mass and Microarchitecture in CKD Patients with Fracture. J Am Soc

gram of Shanghai Municipal Education Commission (11ZZ137) for Yan Zhang.

**Figure 4.** Protein expression of RAS components in mice tibia

### **6. Conclusion**

In summary, the present review demonstrates that the main pathological mechanisms in‐ volved in CKD-MBD are secondary hyperparathyroidism and vascular calcification. There‐ fore the main focus of the therapeutic research and the further molecular investigations should be on these main abnormalities associated with CKD. This can be achieved by em‐ ploying the proper animal models for each of these complications including genetically modified mouse models. The animal models can play a great role in understanding the un‐ derlying mechanisms for CKD-MBD. The clinical treatment approaches should depend on the specific levels of the biomarkers of CKD patients. Further studies are needed to discover other pathological mechanisms for CKD-MBD other than those explained here. More ad‐ vanced basic medical sciences should also be performed on the research and development of novel drugs with less adverse effects.

This work was sponsored by Shanghai Pujiang Program (10PJ1407700) and Innovation Pro‐ gram of Shanghai Municipal Education Commission (11ZZ137) for Yan Zhang.

### **Author details**

Yan Zhang1,2\* and Yoseph Asmelash Gebru1

\*Address all correspondence to: medicineyan@yahoo.com.cn

1 Center for Systems Biomedical Sciences, University of Shanghai for Science and Technology, Shanghai , P.R.China

2 Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong, P.R.China

### **References**

12

12

Figure 3 Hematoxylin and Eosin staining of the proximal tibia. The chondrocyte zone at growth plate was shown in A (Sham) and B (UUO) and it was visually separated into two areas, proliferative zone (PZ) and hypertrophic zone (HZ). Calcified cartilage with overlying newly bone underneath growth

Figure 3 Hematoxylin and Eosin staining of the proximal tibia. The chondrocyte zone at growth plate was shown in A (Sham) and B (UUO) and it was visually separated into two areas, proliferative zone (PZ) and hypertrophic zone (HZ). Calcified cartilage with overlying newly bone underneath growth

Ang II

AT1

AT1

Ang II

AT2

AT2

Renin

Renin



plate is known as the primary spongiosa (PS). Magnification, ×100.

plate is known as the primary spongiosa (PS). Magnification, ×100.

**Figure 3.** Hematoxylin and Eosin staining of the proximal tibia. The chondrocyte zone at growth plate was shown in A (Sham) and B (UUO) and it was visually separated into two areas, proliferative zone (PZ) and hypertrophic zone (HZ). Calcified cartilage with overlying newly bone underneath growth plate is known as the primary spongiosa (PS). Mag‐

PS PZ

PS PZ

HZ

HZ

PS HZ PZ

PS HZ PZ

Sham UUO

Sham UUO

Figure 4 Protein expression of RAS components in mice tibia

Figure 4 Protein expression of RAS components in mice tibia

**Figure 4.** Protein expression of RAS components in mice tibia

**A**

172 Topics in Osteoporosis

**A**

**B**

**B**

nification, ×100.


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[58] Price PA, Roublick AM, Williamson MK. Artery Calcification in Uremic Rats is In‐ creased by a Low Protein Diet and Prevented by Treatment with Ibandronate. Kid‐ ney Int 2006;70(9) 1577-1583.

**Chapter 7**

**Osteoporosis and Nutrition — Nutrition,**

Silvija Lukanović, Nenad Bićanić, Robert Domitrović,

Olga Cvijanović, Sandra Pavičić Žeželj,

http://dx.doi.org/10.5772/54433

and increased bone brittleness [1].

**1. Introduction**

fractures in female [2].

Dragica Bobinac and Željka Crnčević Orlić

Additional information is available at the end of the chapter

**Anthropometry and Bone Mineral Density in Women**

Osteoporosis affects millions of people all around the world and it is the most common metabolic bone disease, characterized by low bone mass, disrupted bone micro architecture

Interaction of numerous factors, such as: genetic, medical, anthropometric, pharmacological, lifestyle and nutrition, lead to loss of bone mass and to increased risk for the osteoporotic

The most of the studies which have explored the effect of calcium on bone mass in females, demonstrated that high calcium intake is related to greater bone mass, compared to smaller bone mass in respondents who had less dietary calcium intake [3]. Besides calcium, sufficient dietary intake of other micronutrients, such as: zinc, magnesium, potassium, dietary fibers as well as vitamin C are believed to have favorable effect on the bone metabolism too [4].

The study of osteoporotic fractures reports that higher intake of animal proteins compared to vegetable proteins is associated to increased risk of loss of the bone mass and occurrence of the osteoporotic fractures [5]. High protein and sodium intake increases calcium excretion in urine, which increases the need for dietary calcium. It has been also found that a high dietary total protein intake, increases production of endogenous acid, which results in accelerated bone resorption and reduced bone formation. This is especially expressed in diets high in animal proteins [6]. It is believed that unfavorable effect of the animal proteins on the bone

and reproduction in any medium, provided the original work is properly cited.

© 2013 Cvijanović et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

metabolism can be repaired by higher fruit and vegetable intake [7].


**Chapter 7**

### **Osteoporosis and Nutrition — Nutrition, Anthropometry and Bone Mineral Density in Women**

Olga Cvijanović, Sandra Pavičić Žeželj, Silvija Lukanović, Nenad Bićanić, Robert Domitrović, Dragica Bobinac and Željka Crnčević Orlić

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54433

### **1. Introduction**

[58] Price PA, Roublick AM, Williamson MK. Artery Calcification in Uremic Rats is In‐ creased by a Low Protein Diet and Prevented by Treatment with Ibandronate. Kid‐

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Osteoporosis affects millions of people all around the world and it is the most common metabolic bone disease, characterized by low bone mass, disrupted bone micro architecture and increased bone brittleness [1].

Interaction of numerous factors, such as: genetic, medical, anthropometric, pharmacological, lifestyle and nutrition, lead to loss of bone mass and to increased risk for the osteoporotic fractures in female [2].

The most of the studies which have explored the effect of calcium on bone mass in females, demonstrated that high calcium intake is related to greater bone mass, compared to smaller bone mass in respondents who had less dietary calcium intake [3]. Besides calcium, sufficient dietary intake of other micronutrients, such as: zinc, magnesium, potassium, dietary fibers as well as vitamin C are believed to have favorable effect on the bone metabolism too [4].

The study of osteoporotic fractures reports that higher intake of animal proteins compared to vegetable proteins is associated to increased risk of loss of the bone mass and occurrence of the osteoporotic fractures [5]. High protein and sodium intake increases calcium excretion in urine, which increases the need for dietary calcium. It has been also found that a high dietary total protein intake, increases production of endogenous acid, which results in accelerated bone resorption and reduced bone formation. This is especially expressed in diets high in animal proteins [6]. It is believed that unfavorable effect of the animal proteins on the bone metabolism can be repaired by higher fruit and vegetable intake [7].

© 2013 Cvijanović et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Recent researches indicate a risk of excessive fat intake, which leads to metabolic bone disorders. High fat intake is considered to be a risk factor for osteoporosis, because it reduces the calcium absorption, since calcium forms insoluble compounds with fatty acids [7].

**2.3. Anthropometric, biochemical and bone mineral status measurements**

calculated as bodyweight divided by body height squared, BMI (kg/m²) [10].

nosorbent Assay, ELISA), according to manufacturer's protocol [11,12,13].

mineral density (BMD, mg/cm2

**2.4. Statistical analysis**

**2.5. Results**

was 27 kg/m².

expressed in standard deviations) [14].

Bodyweight was measured on a portable electronic scale (SECA, Hamburg, Germany), with accuracy of ± 0,1 kg. Body height was measured on a portable stadiometer, which is a part of a specified scale (SECA, Hamburg, Germany), to the nearest ± 0,5 cm. Body mass index was

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181

Biochemical indicator of bone resorption, deoxypyridinolin (DPD) and biochemical indicator of bone formation, bone alkaline phosphatase (ALP) and vitamin D were determined from urine and blood of the respondents by immune-enzymatic method (Enzyme Linked Immu‐

Bone density in the anterior-posterior images of the spine and hip was measured using the device for bone densitometry (Hologic, Bedford, MA, USA). The obtained values were quantified according to the following parameters: bone mineral content (BMC, mg), bone

values of peak bone mass of young people expressed in standard deviations) and Z-score (deviation of the measured values BMD of the average bone mass of persons of the same age,

Statistical analysis of data was performing by using Statistica for Windows, release 9.1 (Stasoft, INC, Tulsa, USA). Normality of distribution for the data interval scale (quantitative data), was tested using the Kolmogorov- Smirinov test. The results were shown as arithmetic mean and standard deviation. Results were distributed normally and in the analytical statistics, one-way analysis of variance (one-way ANOVA) was used. To determine the significance of the contribution of the percentage of nutrients on the metabolic bone status, multiple regression analysis was used. All statistical values were considered significant at the level P<0,05 [15].

Age, anthropometric characteristics, values of the bone densitometry parameters and concen‐ trations of the bone remodeling markers are presented in Table 1. Women of generative age are significantly taller than women in menopause (P=0,01) and have significantly higher body weight than women in menopause (P<0,001). The average value of Body Mass Index (BMI)

Subjects of generative age have significantly higher values of BMD and BMC of the spine (P<0,001, P<0,001), as well as the values of T-score and Z-score (P<0,001, P<0,001), than menopausal women, respectively. Values of BMD and BMC of the hip (P<0,001, P<0,001) and the value of T-score (P<0,001) were also significantly higher in women of generative age.

Regarding the bone remodeling markers, significantly lower values of DPD (P<0,001) and bone

ALP (P=0,004) were found in fertile women compared to menopausal women.

) and T-score (represents a deviation from the BMD measured

The aim of this study was to quantify the intake of trace elements in fruit and vegetable: zinc, magnesium, potassium and dietary fats as well as fat derivatives intake in examinees and to explore their relation to the bone mass. The aim was to examine the extent to which these nutritional parameters are predictors of values of bone mineral density.

### **2. Patients and methods**

### **2.1. Subjects**

The study population consisted of women with sedentary occupations in age ranged from 40 to 67 years. Women are inhabitants of the down town Rijeka, Croatia. Exclusion criteria for further participation in the survey were: smoking and any medical therapy which can alter bone metabolism, including food supplements with added calcium. Dietary habits, anthropo‐ metric characteristics, serum concentration of the biochemical markers and values of the bone densitometry parameters were comprised by this study. 200 women were included in this investigation, of which 120 menopausal women constituted experimental group, and 80 fertile women represents the control group.

### **2.2. Dietary intakes**

Participants completed an anonymous, encrypted questionnaire, conducted in accordance with ethical and bioethical principles and their privacy and protection of confidential infor‐ mation was ensured.

For the assessment of dietary habits and the average daily energy and nutrients intake, we used data obtained from semi- quantitative Food Frequency Questionnaire- sq-FFQ, the main method for collecting data about a foodstuff choice, as well as the type and quantity of food intake in the study population. This method of identifying the dietary habits is a questionnaire validated by the Department of Nutrition, Harvard School of Public Health [8], from which are obtained informations about daily intake of energy and nutrients. Women were asked to note the frequency and the quantity of offered food items. The amount of each food item was offered as one portion and declared as small, medium and large. This method quantified the values of nutritional parameters that are essential for bone health, such as: calcium, phospho‐ rus, vitamin D, proteins, zinc, magnesium, potassium, dietary fibers and vitamin C. We also determined a total fat intake and the emphasis was placed on the intake of total fat, saturated fatty acids, monounsaturated fatty acids and polyunsaturated fatty acids. The nutritive and energy values of each food noted were calculated using the composition tables of raw and cooked food [9].

### **2.3. Anthropometric, biochemical and bone mineral status measurements**

Bodyweight was measured on a portable electronic scale (SECA, Hamburg, Germany), with accuracy of ± 0,1 kg. Body height was measured on a portable stadiometer, which is a part of a specified scale (SECA, Hamburg, Germany), to the nearest ± 0,5 cm. Body mass index was calculated as bodyweight divided by body height squared, BMI (kg/m²) [10].

Biochemical indicator of bone resorption, deoxypyridinolin (DPD) and biochemical indicator of bone formation, bone alkaline phosphatase (ALP) and vitamin D were determined from urine and blood of the respondents by immune-enzymatic method (Enzyme Linked Immu‐ nosorbent Assay, ELISA), according to manufacturer's protocol [11,12,13].

Bone density in the anterior-posterior images of the spine and hip was measured using the device for bone densitometry (Hologic, Bedford, MA, USA). The obtained values were quantified according to the following parameters: bone mineral content (BMC, mg), bone mineral density (BMD, mg/cm2 ) and T-score (represents a deviation from the BMD measured values of peak bone mass of young people expressed in standard deviations) and Z-score (deviation of the measured values BMD of the average bone mass of persons of the same age, expressed in standard deviations) [14].

### **2.4. Statistical analysis**

Recent researches indicate a risk of excessive fat intake, which leads to metabolic bone disorders. High fat intake is considered to be a risk factor for osteoporosis, because it reduces the calcium absorption, since calcium forms insoluble compounds with fatty

The aim of this study was to quantify the intake of trace elements in fruit and vegetable: zinc, magnesium, potassium and dietary fats as well as fat derivatives intake in examinees and to explore their relation to the bone mass. The aim was to examine the extent to which these

The study population consisted of women with sedentary occupations in age ranged from 40 to 67 years. Women are inhabitants of the down town Rijeka, Croatia. Exclusion criteria for further participation in the survey were: smoking and any medical therapy which can alter bone metabolism, including food supplements with added calcium. Dietary habits, anthropo‐ metric characteristics, serum concentration of the biochemical markers and values of the bone densitometry parameters were comprised by this study. 200 women were included in this investigation, of which 120 menopausal women constituted experimental group, and 80 fertile

Participants completed an anonymous, encrypted questionnaire, conducted in accordance with ethical and bioethical principles and their privacy and protection of confidential infor‐

For the assessment of dietary habits and the average daily energy and nutrients intake, we used data obtained from semi- quantitative Food Frequency Questionnaire- sq-FFQ, the main method for collecting data about a foodstuff choice, as well as the type and quantity of food intake in the study population. This method of identifying the dietary habits is a questionnaire validated by the Department of Nutrition, Harvard School of Public Health [8], from which are obtained informations about daily intake of energy and nutrients. Women were asked to note the frequency and the quantity of offered food items. The amount of each food item was offered as one portion and declared as small, medium and large. This method quantified the values of nutritional parameters that are essential for bone health, such as: calcium, phospho‐ rus, vitamin D, proteins, zinc, magnesium, potassium, dietary fibers and vitamin C. We also determined a total fat intake and the emphasis was placed on the intake of total fat, saturated fatty acids, monounsaturated fatty acids and polyunsaturated fatty acids. The nutritive and energy values of each food noted were calculated using the composition tables of raw and

nutritional parameters are predictors of values of bone mineral density.

acids [7].

180 Topics in Osteoporosis

**2.1. Subjects**

**2. Patients and methods**

women represents the control group.

**2.2. Dietary intakes**

mation was ensured.

cooked food [9].

Statistical analysis of data was performing by using Statistica for Windows, release 9.1 (Stasoft, INC, Tulsa, USA). Normality of distribution for the data interval scale (quantitative data), was tested using the Kolmogorov- Smirinov test. The results were shown as arithmetic mean and standard deviation. Results were distributed normally and in the analytical statistics, one-way analysis of variance (one-way ANOVA) was used. To determine the significance of the contribution of the percentage of nutrients on the metabolic bone status, multiple regression analysis was used. All statistical values were considered significant at the level P<0,05 [15].

### **2.5. Results**

Age, anthropometric characteristics, values of the bone densitometry parameters and concen‐ trations of the bone remodeling markers are presented in Table 1. Women of generative age are significantly taller than women in menopause (P=0,01) and have significantly higher body weight than women in menopause (P<0,001). The average value of Body Mass Index (BMI) was 27 kg/m².

Subjects of generative age have significantly higher values of BMD and BMC of the spine (P<0,001, P<0,001), as well as the values of T-score and Z-score (P<0,001, P<0,001), than menopausal women, respectively. Values of BMD and BMC of the hip (P<0,001, P<0,001) and the value of T-score (P<0,001) were also significantly higher in women of generative age.

Regarding the bone remodeling markers, significantly lower values of DPD (P<0,001) and bone ALP (P=0,004) were found in fertile women compared to menopausal women.


**Parameters Fertile women**

kJ

Energetic food equivalent kcal

\* statistical significance on the level P < 0,05

LS T-score, LS Z-score, LH BMC, LH T-score.

eters was observed for menstrual status and diet (Table 3).

**(n =80)**

**Menopausal women (n = 120)**

Osteoporosis and Nutrition — Nutrition, Anthropometry and Bone Mineral Density in Women

Proteins (total) (g) 90,76 ± 65,4 60,52 ± 28,9 75,64 ± 50,7 <0,001\* Proteins (vegetable) (g) 21,95 ± 22,5 19,70 ± 9,2 20,71 ±16,7 <0,001\* Proteins (animal) (g) 67,88 ± 45,5 40,20 ± 24,7 54,04 ± 37,0 <0,001\* Total fat (g) 101,74 ± 63,9 74,76 ± 45,37 87,30 ± 56,3 <0,001\* Saturated fatty acids (g) 45,35 ± 28,3 31,41 ± 22,6 38,38 ± 25,8 <0,001\* Monounsaturated fatty acids (g) 34,40 ± 25,32 26,37 ± 18,2 30,39 ± 22,1 <0,001\* Polyunsaturated fatty acids (g) 20,89 ± 13,05 16,98 ± 8,2 18,94 ± 11,5 <0,001\* Carbohydrates (g) 180,16 ± 215,9 152,05 ± 90,2 166,10 ± 163,8 <0,001\* Vegetable fibers (g) 26,72 ± 24,1 14,27 ± 11,5 20,50 ± 18,7 <0,001\* Vitamin D (μg) 9,91 ± 5,1 6,32 ± 7,6 7,76 ± 6,9 <0,001\* Vitamin C (mg) 131,62 ± 111,1 118,36 ± 112,0 123,66 ± 166,8 <0,001\*

Calcium (mg) 953,91 ± 316,32 918,79 ± 232,0 932,74 ± 268,7 0,366

Phosphorus (mg) 1012,23 ± 315,23 1132,21 ± 235,25 1072,22 ± 235,2 <0,001\* Potassium (mg) 6441,99 ± 3231,4 4453,70 ± 1362,0 5294,02 ± 2482,4 <0,001\* Magnesium (mg) 546,16 ± 245,9 404,70 ± 136,1 461,28 ± 199,8 <0,001\* Zinc (mg) 17,38 ± 8,0 13,02 ± 4,2 14,77 ± 6,4 <0,001\*

DXA parameters which were extracted by ROC analyses as excellent predictors of bone metabolism were included in multiple regression analyses. Those include: LS BMC, LS BMD,

The results of multiple regression analysis by which are defined total shares and significance of contributions of menstrual status, age, anthropometry and nutrition on the bone densitom‐ etry parameters. The largest total share of contributions to all the bone densitometry param‐

**Table 2.** The average daily nutrient intake in fertile and in menopausal women (*X*¯± SD)

2851,91 ± 1034,4 2448,65 ± 716,37 2609,95 ± 877,9 <0,001\* 11932,38 ± 4327,8 10,245 ± 2997,3 10920,04 ± 3673,5 <0,001\*

**Total (n = 200)** **P- values**

183

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\* statistical significance on level P < 0,05

LS – lumbar spine

LH – left hip

**Table 1.** Age, anthropometry, bone densitometry parameters, bone remodeling markers and vitamin D (*X*¯± SD)

Dietary habits of the study participants are presented in the Table 2. One-way analysis of variance (ANOVA) showed that women of generative age have significantly higher average daily intake of vitamin D, vitamin C, potassium, magnesium and zinc, while menopausal women have significantly higher average daily phosphorus intake (P<0,001).


**Table 2.** The average daily nutrient intake in fertile and in menopausal women (*X*¯± SD)

**Parameters Fertile women**

182 Topics in Osteoporosis

\*

LS – lumbar spine LH – left hip

statistical significance on level P < 0,05

**(n =80)**

**Menopausal women**

**Total (n = 200)** **P-value**

**(n = 120)**

Age 47,6 ±4,1 59,9 ± 5,1 54,9 ± 7,7 <0,001\*

Body height (cm) 74,0 ± 6,4 71,7 ± 13,3 72,6 ± 11,1 0,001\*

Body weight (kg) 166,8 ± 0,05 161,9 ± 0,06 163,8 ± 0,06 <0,001\*

BMI (kg/m2) 26,6 ± 2,3 27,3 ± 4,7 27,0 ±3,9 0,210

BMD LS (g/cm2) 1,074 ± 0,1 0,897 ± 0,1 0,968 ± 0,2 <0,001\*

BMC LS (g) 67,49 ± 9,4 52,84 ± 9,7 58,70 ± 11,9 <0,001\*

T-score 0,400 ± 1,3 -1,325 ± 1,3 -0,635 ± 1,9 <0,001\*

Z-score 0,835 ± 1,3 0,033 ± 1,4 0,354 ± 1,4 <0,001\*

BMD LH (g/cm2) 0,944 ± 0,1 0,860 ± 0,1 0,893 ± 0,1 <0,001\*

BMC LH (g) 37,29 ± 5,9 30,83 ± 5,5 33,41 ± 6,5 <0,001\*

T-score 0,122 ± 0,8 -0,647 ± 1,1 -0,339 ± 1,1 <0,001\*

Z-score 0,453 ± 0,9 0,298 ± 1,0 0,360 ± 1,0 0,269

DPD (nmol/l) 5,26 ± 1,4 6,85 ± 2,5 6,22 ± 2,2 <0,001\*

ALP (ng/ml) 22,77 ± 8,1 26,0 ± 7,4 24,71 ± 7,8 0,004\*

Vitamin D (nmol/l) 62,03 ± 25,8 68,90 ± 29,1 66,16 ± 27,9 0,09

**Table 1.** Age, anthropometry, bone densitometry parameters, bone remodeling markers and vitamin D (*X*¯± SD)

women have significantly higher average daily phosphorus intake (P<0,001).

Dietary habits of the study participants are presented in the Table 2. One-way analysis of variance (ANOVA) showed that women of generative age have significantly higher average daily intake of vitamin D, vitamin C, potassium, magnesium and zinc, while menopausal

DXA parameters which were extracted by ROC analyses as excellent predictors of bone metabolism were included in multiple regression analyses. Those include: LS BMC, LS BMD, LS T-score, LS Z-score, LH BMC, LH T-score.

The results of multiple regression analysis by which are defined total shares and significance of contributions of menstrual status, age, anthropometry and nutrition on the bone densitom‐ etry parameters. The largest total share of contributions to all the bone densitometry param‐ eters was observed for menstrual status and diet (Table 3).


**Parameters Milkand milk products Fish Vegetables Fruit LS BMD β P β P β P β P**

LS BMC 0,109 0,128 0,171 0,014\* 0,006 0,346 0,016 0,645 LS T-score 0,077 0,301 0,043 0,577 0,089 0,150 0,047 0,556 LS Z-score 0,105 0,163 0,008 0,024\* 0,127 0,135 0,092 0,256 LH BMC 0,039 0,598 0,086 0,005\* 0,214 0,002\* 0,156 0,050 LH T-score 0,178 0,015\* 0,135 <0,001\* 0,178 0,004\* 0,107 0,178

**Table 5.** Interactions of predictors (milk and milk products, fish, vegetables and fruit) to categorical variables (LS BMD,

Milk and milk products, fish, vegetables and fruit are exactly proportional to the bone

**Parameters LS BMC LS BMD LS T-score LS Z-score LH BMC LH T-score**

Menstrual status 40,1 29,6 29,6 20,8 27,8 24,5 Age 8,4 3,3 6,1 7,2 12,0 0,5 Energy 2,7 0,5 2,8 3,2 1,4 0,2 Proteins (g) 0,2 0,1 0,2 0 5,8 4,8 Total fat (g) 5,8 0,9 2,4 1,8 1,5 2,4

Calcium (mg) 0,9 0,7 1,0 1,8 0,2 0 Potassium (mg) 8,8 5,9 3,4 4,1 7,3 5,1 Phosphorus (mg) 5,3 0,6 3,5 4,5 0 0,9 Magnesium (mg) 10,1 0,2 3,1 2,4 2,8 0,8

**Table 6.** Total shares of contributions of menstrual status, age and nutrition on DXA (%)

**share of contribution (%)**

0,5 1,6 3,3 0,2 1,7 0,7

1,4 0,2 2,2 0,3 1,5 0,7

1,6 0,5 1,6 0,1 0,2 0,6

**share of contribution (%)**

**share of contribution (%)**

**share of contribution (%)**

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185

**share of contribution (%)**

\*statistical significance on level P < 0,05

LS BMC, LS T-score, LS Z-score, LH BMC, LH T-score) are shown

**share of contribution (%)**

β – regression coefficient

densitometry parameters.

Saturated fatty acids

Monounsaturated fatty acids (g)

Polyunsaturated fatty acids (g)

(g)

LS – lumbar spine LH – left hip

0,008 0,169 0,031 0,677 0,034 0,742 0,027 0,733

Osteoporosis and Nutrition — Nutrition, Anthropometry and Bone Mineral Density in Women

LS – lumbar spine

LH – left hip

**Table 3.** Total shares of contributions of menstrual status, age, anthropometry and nutrition on DXA (%)


\*statistical significance on level P < 0,05

β – regression coefficient

LS – lumbar spine

LH – left hip

**Table 4.** Statistically significant interactions of predictors (menstrual status, age, and nutrients) to categorical variables (LS BMC, LS BMD, LS T-score, LS Z-score, LH BMC, LH T-score) are shown

Menstrual status, age, total fat, saturated fatty acids, are inversely proportional to the values of the bone densitometry parameters, while energy, proteins, monounsaturated fatty acids, polyunsaturated fatty acids, calcium, potassium and magnesium are exactly proportional to the values of the bone densitometry parameters.

Osteoporosis and Nutrition — Nutrition, Anthropometry and Bone Mineral Density in Women http://dx.doi.org/10.5772/54433 185


\*statistical significance on level P < 0,05

β – regression coefficient

LS – lumbar spine

LH – left hip

**Parameters Menstrual**

184 Topics in Osteoporosis

LS – lumbar spine LH – left hip

Nutrition

Saturated fatty acids (g)

Monounsaturated fatty acids (g)

Polyunsaturated fatty acids (g)

\*statistical significance on level P < 0,05

(LS BMC, LS BMD, LS T-score, LS Z-score, LH BMC, LH T-score) are shown

the values of the bone densitometry parameters.

β – regression coefficient

LS – lumbar spine LH – left hip

**status**

**Share of contributions (%)** **Share of contributions (%)**

LS BMC 40,1 8,4 8,5 44,8 LS BMD 29,6 3,3 16,6 12,7 LS T-score 29,6 6,1 19,3 27,4 LS Z-score 20,8 7,2 13,0 12,7 LH BMC 27,8 12,0 24,2 23,2 LH T-score 24,5 0,5 27,0 18,6

**Table 3.** Total shares of contributions of menstrual status, age, anthropometry and nutrition on DXA (%)

**Parameters LS BMC LS BMD LS T-score LS Z-score LH BMC LH T-score**

Menstrual status -0,632 <0,001\* -0,560 <0,001\* -0,632 <0,001\* -0,529 <0,001\* -0,568 <0,001\* -0,389 <0,001\* Age -0,156 0,057 -0,097 0,251 -0,156 0,057 -0,365 0,001\* -0,210 0,010\* -0,017 0,831

Energy 0,346 0,014\* 0,238 0,150 0,667 0,016\* 0,627 0,002\* 0,400 0,048\* 0,063 0,529 Proteins (g) 0,008 0,580 0,010 0,445 0,081 0,481 0,004 0,978 0,349 <0,001\* 0,786 0,082 Total fat (g) -0,273 0,036\* -0,103 0,386 -0,098 0,016\* -0,191 0,127 -0,427 0,016\* -0,500 0,054

Calcium (mg) 2,045 0,256 0,538 0,168 0,007 0,145 -0,219 0,032\* 0,034 0,626 0,023 0,748 Potassium (mg) 0,211 0,004\* 0,536 0,006\* 0,213 0,005\* 0,103 0,004\* 0,089 0,001\* 0,750 0,414 Phosphorus (mg) -0,623 0,277 -0,078 0,053 0,139 0,031\* 0,071 0,033\* -0,078 0,851 -0,234 0,588 Magnesium (mg) 0,073 0,002\* 0,054 0,053 0,133 0,036\* 0,031 0,010\* 0,157 0,002\* 0,422 0,187

**Table 4.** Statistically significant interactions of predictors (menstrual status, age, and nutrients) to categorical variables

Menstrual status, age, total fat, saturated fatty acids, are inversely proportional to the values of the bone densitometry parameters, while energy, proteins, monounsaturated fatty acids, polyunsaturated fatty acids, calcium, potassium and magnesium are exactly proportional to

**ß P ß P ß P ß P ß P ß P**


0,079 0,551 0,029 0,875 0,144 0,019\* 0,031 0,145 0,328 0,182 0,258 0,312

0,086 0,546 0,044 0,782 0,209 0,049\* 0,013 0,104 0,310 0,069 0,329 0,063

**Age Anthropometry Nutrition**

**Share of contributions (%)** **Share**

**of contributions (%)**

**Table 5.** Interactions of predictors (milk and milk products, fish, vegetables and fruit) to categorical variables (LS BMD, LS BMC, LS T-score, LS Z-score, LH BMC, LH T-score) are shown

Milk and milk products, fish, vegetables and fruit are exactly proportional to the bone densitometry parameters.


**Table 6.** Total shares of contributions of menstrual status, age and nutrition on DXA (%)

### **3. Discussion**

It is considered that menopausal women have the highest risk of osteoporotic fractures and the incidence of osteoporosis in this group is increased by 25% compared to fertile women [3]. Furthermore, the frequency of osteoporotic vertebral and hip fractures in both genders increases exponentially with age [1]. Results obtained by this study coin‐ cide with the majority of results of similar studies, showing that menopausal women have significantly lower values of the bone densitometry parameters [17,18,19]. The latter is additionally confirmed by our finding of inversely proportional relationship between menopause and bone mineral density parameters. Our result of inversely proportional relationship between age and bone mineral density parameters, corresponds to a study conducted on 450 000 participants from Sweden [20].

(Table 3). A positive relationship between increased body weight or body mass index (BMI) to bone mass has been already reported [28,29]. Some authors have considered that increased body weight can improve bone mass, by stimulation of the osteoblast dif‐ ferentiation. Body weight increase in postmenopausal period is correlated to increased number of adipocytes. Adipocytes are an important source of estrogen, a hormone which stimulates bone formation [30]. The opposite theories to afore mentioned have been re‐ ported too [31] and amongst those is a research of Kroke et al. [32], who did not find strong influence of anthropometric parameters on bone mineral density neither in wom‐

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It has been proposed that high energy intake, leads to body weight increase and finally to increased values of the bone mineral density [33,34]. Similarly to results obtained by Kumar et al. [35], our results indicate that daily energy intake is exactly proportional to the values of

Total daily protein intake is directly proportional to the values of the bone densitometry parameters, which was significant for the LH BMC (P<0,001) (Table 4). Such result is in agreement to results obtained by Misra et al. [36]. These researchers have documented a positive correlation between total protein intake and increased bone mineral density. Fur‐ ther protein analysis revealed a positive influence of proteins of vegetable origin and negative influence of proteins of animal origin on the bone mass. SOF study (Study of osteoporotic fractures) found that women with increased animal proteins intake have low values of the bone mineral density and increased risk of osteoporotic fractures [5].

Total fat and saturated fatty acids are inversely proportional to DXA parameters, while monounsaturated and polyunsaturated fatty acids are exactly proportional to DXA pa‐ rameters (Table 4). Greater shares of contribution of all four types of fat were found for DXA parameters of the lumbar spine than of the left hip (Table 6). Significance was ob‐ served for the T-score of the lumbar spine (Table 4). Corwin et al., conducted the survey on menopausal women, and found a negative correlation between total fat intake and bone mineral density [37]. Another research of Hogstroma et al. corresponds to our re‐ sults since they have also found positive correlation between monounsaturated fatty

Regarding daily calcium intake, total shares of calcium contribution are greater in lum‐ bar spine than in the left hip. Interestingly calcium does not contribute at all to the values of the LH T-score (Table 6). Calcium is directly proportional to the values of DXA parameters, but the only statistical significance relates to LS Z-score (Table 4). Similarly was found in the study of H. F. Saadi et al., where dietary calcium intake of fertile women and postmenopausal women is positively, but not significantly correlat‐ ed to bone mineral density [39]. Average daily calcium intake amounted 932,74 g, which are adequate amounts of dietary calcium according to existing recommendations

en of generative age, nor in postmenopausal women.

the bone densitometry parameters (Table 4).

acids intake and bone mineral density [38].

in Croatia (Table 2).

Bone remodeling markers provide information about the dynamic state of bone metab‐ olism, and those are very useful tools to predict early changes in the bone metabo‐ lism. Along with the bone densitometry, bone remodeling markers are needy in diagnosis and follow up of diseases of the bone mass deficit [21]. We have demon‐ strated that women of generative age have significantly lower values of DPD (P<0,001) than women in menopause. This is consistent to increased excretion of DPD in post‐ menopausal women [22]. Menopausal women had significantly higher levels of bone ALP (P=0,004) compared to women in generative age. The latter could be explained by the fact that high serum concentration of bone formation markers is associated with greater bone loss [23]. Average concentration of vitamin D in all study participants amounted 66,16 nmol/l, out of which average value in fertile women amounted 62,03 nmol/l, and 68,9 nmol/l in menopausal women (Table 2). Similar values of vitamin D concentrations were observed in a study of postmenopausal women of nine European countries [24]. By adding our results of low vitamin D concentrations to similar results published by Kraljević et al. [25] and by Žerjavić et al. [26], we can summarize that some action should be done by Croatian Health Care system, such as food-based strat‐ egies, to prevent vitamin D deficiency in Croatia.

Nutrition has a unique role in processes of growth and modeling of the human skeleton, as well as in maintaining the peak bone mass in adulthood [2]. The most of the studies are focused on dietary calcium intake [19], some of the researchers have analyzed the impact of some other nutrients such as proteins, carbohydrates, fat and energy intake [27], and only a few studies have explored the influence of vitamins and minerals on bone mass [7]. Calcium in a form of the calcium phosphate or calcium carbonate is the major mineral constituent of the bone tissue. High calcium intake, within the normal di‐ et, does not protect against fractures, but low calcium intake represents a risk factor for the osteoporosis [19].

By means of the multiple regression analysis we have determined that the greatest con‐ tribution of the anthropometry is to values of the BMC and the T-score of the left hip (Table 3). A positive relationship between increased body weight or body mass index (BMI) to bone mass has been already reported [28,29]. Some authors have considered that increased body weight can improve bone mass, by stimulation of the osteoblast dif‐ ferentiation. Body weight increase in postmenopausal period is correlated to increased number of adipocytes. Adipocytes are an important source of estrogen, a hormone which stimulates bone formation [30]. The opposite theories to afore mentioned have been re‐ ported too [31] and amongst those is a research of Kroke et al. [32], who did not find strong influence of anthropometric parameters on bone mineral density neither in wom‐ en of generative age, nor in postmenopausal women.

**3. Discussion**

186 Topics in Osteoporosis

It is considered that menopausal women have the highest risk of osteoporotic fractures and the incidence of osteoporosis in this group is increased by 25% compared to fertile women [3]. Furthermore, the frequency of osteoporotic vertebral and hip fractures in both genders increases exponentially with age [1]. Results obtained by this study coin‐ cide with the majority of results of similar studies, showing that menopausal women have significantly lower values of the bone densitometry parameters [17,18,19]. The latter is additionally confirmed by our finding of inversely proportional relationship between menopause and bone mineral density parameters. Our result of inversely proportional relationship between age and bone mineral density parameters, corresponds to a study

Bone remodeling markers provide information about the dynamic state of bone metab‐ olism, and those are very useful tools to predict early changes in the bone metabo‐ lism. Along with the bone densitometry, bone remodeling markers are needy in diagnosis and follow up of diseases of the bone mass deficit [21]. We have demon‐ strated that women of generative age have significantly lower values of DPD (P<0,001) than women in menopause. This is consistent to increased excretion of DPD in post‐ menopausal women [22]. Menopausal women had significantly higher levels of bone ALP (P=0,004) compared to women in generative age. The latter could be explained by the fact that high serum concentration of bone formation markers is associated with greater bone loss [23]. Average concentration of vitamin D in all study participants amounted 66,16 nmol/l, out of which average value in fertile women amounted 62,03 nmol/l, and 68,9 nmol/l in menopausal women (Table 2). Similar values of vitamin D concentrations were observed in a study of postmenopausal women of nine European countries [24]. By adding our results of low vitamin D concentrations to similar results published by Kraljević et al. [25] and by Žerjavić et al. [26], we can summarize that some action should be done by Croatian Health Care system, such as food-based strat‐

Nutrition has a unique role in processes of growth and modeling of the human skeleton, as well as in maintaining the peak bone mass in adulthood [2]. The most of the studies are focused on dietary calcium intake [19], some of the researchers have analyzed the impact of some other nutrients such as proteins, carbohydrates, fat and energy intake [27], and only a few studies have explored the influence of vitamins and minerals on bone mass [7]. Calcium in a form of the calcium phosphate or calcium carbonate is the major mineral constituent of the bone tissue. High calcium intake, within the normal di‐ et, does not protect against fractures, but low calcium intake represents a risk factor for

By means of the multiple regression analysis we have determined that the greatest con‐ tribution of the anthropometry is to values of the BMC and the T-score of the left hip

conducted on 450 000 participants from Sweden [20].

egies, to prevent vitamin D deficiency in Croatia.

the osteoporosis [19].

It has been proposed that high energy intake, leads to body weight increase and finally to increased values of the bone mineral density [33,34]. Similarly to results obtained by Kumar et al. [35], our results indicate that daily energy intake is exactly proportional to the values of the bone densitometry parameters (Table 4).

Total daily protein intake is directly proportional to the values of the bone densitometry parameters, which was significant for the LH BMC (P<0,001) (Table 4). Such result is in agreement to results obtained by Misra et al. [36]. These researchers have documented a positive correlation between total protein intake and increased bone mineral density. Fur‐ ther protein analysis revealed a positive influence of proteins of vegetable origin and negative influence of proteins of animal origin on the bone mass. SOF study (Study of osteoporotic fractures) found that women with increased animal proteins intake have low values of the bone mineral density and increased risk of osteoporotic fractures [5].

Total fat and saturated fatty acids are inversely proportional to DXA parameters, while monounsaturated and polyunsaturated fatty acids are exactly proportional to DXA pa‐ rameters (Table 4). Greater shares of contribution of all four types of fat were found for DXA parameters of the lumbar spine than of the left hip (Table 6). Significance was ob‐ served for the T-score of the lumbar spine (Table 4). Corwin et al., conducted the survey on menopausal women, and found a negative correlation between total fat intake and bone mineral density [37]. Another research of Hogstroma et al. corresponds to our re‐ sults since they have also found positive correlation between monounsaturated fatty acids intake and bone mineral density [38].

Regarding daily calcium intake, total shares of calcium contribution are greater in lum‐ bar spine than in the left hip. Interestingly calcium does not contribute at all to the values of the LH T-score (Table 6). Calcium is directly proportional to the values of DXA parameters, but the only statistical significance relates to LS Z-score (Table 4). Similarly was found in the study of H. F. Saadi et al., where dietary calcium intake of fertile women and postmenopausal women is positively, but not significantly correlat‐ ed to bone mineral density [39]. Average daily calcium intake amounted 932,74 g, which are adequate amounts of dietary calcium according to existing recommendations in Croatia (Table 2).

Daily intake of the minerals magnesium and potassium has the greatest contribution of all the given minerals to DXA parameters (Table 6). Both of the minerals are directly proportional to DXA parameters (Table 4). Studies published so far argue that sufficient magnesium and potassium intake is related to increased bone mineral density [40,41], or as contrast opinions state, to reduced bone mass and increased risk of wrist fracture [43]. Magnesium is important in processes of bone mineralization, and potassium has impor‐ tant role in systemic acid-base (pH) homeostasis. Potassium salts neutralize bone-deplet‐ ing metabolic acids, and therefore conditions that require drain of alkalizing compounds from bone lead to loss of bone tissue. Positive influence of potassium on bone health has been reported [3,4,40,45].

**Author details**

Robert Domitrović<sup>4</sup>

**References**

7, 227-243.

Olga Cvijanović1\*, Sandra Pavičić Žeželj<sup>2</sup>

Faculty of Medicine, Rijeka, Croatia

\*Address all correspondence to: olgac@medri.hr

County, Rijeka Faculty of Medicine, Rijeka, Croatia

al women. Osteoporos Int (2004). , 15, 439-446.

Am J Clin Nutr (2001). , 73, 118-22.

tion and bone health. Am J Clin Nutr (2000). , 71, 142-51.

, Dragica Bobinac<sup>1</sup>

1 Department of Anatomy, Rijeka Faculty of Medicine, Rijeka, Croatia

, Silvija Lukanović<sup>1</sup>

2 Department of Ecology Health, Teaching Institute of Public Health Mountain-Littoral

3 Department of Endocrinology, Clinics for Internal Medicine Rijeka Clinical Centre, Rijeka

[1] Prentice, A. Diet, nutrition and prevention of osteoporosis. Pub Health Nutr (2004). ,

[2] Bainbridge, K. E, Sowers, M, Lin, X, & Harlow, S. D. Risk factors for low bone miner‐ al density and the 6-year rate of bone loss among premenopausal and perimenopaus‐

[3] New, S. A, Robins, S. P, Campbell, M. K, Martin, J. C, Gorton, M. J, Bolton-smith, C, Grubb, D. A, Lee, S. J, & Reid, D. M. Dietary influences on bone mass and bone me‐ tabolism: further evidence of a positive link between fruit and vegetable consump‐

[4] Nieves, J. W. Osteoporosis: the role of micronutrients. Am J Clin Nutr (2005). S-9S.

[5] Sellmeyer, D. E, Stone, K. L, Sebastian, A, & Cummings, S. R. For the Study of Osteo‐ porotic Fractures Research Group. A high ratio of dietary animal to vegetable protein increases the rate of bone loss and the risk of fracture in postmenopausal women.

[6] Weikert, C, Dietmar, W, Hoffman, K, Kroke, A, Bergmann, M. M, & Boeing, H. The Relation between Dietary Protein, Calcium and Bone Health in Women: Results from

[7] Mecdonald, H. M, New, S. A, Golden, M. H, Cambel, M. K, & Reid, D. M. Nutritional associations with bone loss during the menopausal transition: evidence of a benefi‐

the EPIC-Postdam Cohort. Ann Nutr Metab (2005). , 49, 312-318.

4 Department of Chemistry and Biochemistry, Rijeka Faculty of Medicine, Rijeka, Croatia

and Željka Crnčević Orlić<sup>3</sup>

Osteoporosis and Nutrition — Nutrition, Anthropometry and Bone Mineral Density in Women

, Nenad Bićanić<sup>3</sup>

,

http://dx.doi.org/10.5772/54433

189

Zinc is a cofactor for alkaline phosphatase, an enzyme, necessary for bone mineraliza‐ tion. Low concentration of the zinc in serum and its increased secretion in urine is asso‐ ciated with osteoporosis [44]. Considering that calcification of the bone is reduced with insufficient zinc intake, we analyzed influence of the dietary zinc on bone health. Results revealed that daily zinc intake is directly proportional to DXA parameters, but the influ‐ ence was not significant (data not shown).

Analyzing the impact of fruit and vegetables on DXA parameters in women, we have found that vegetables are directly proportional to the parameters of the left hip, which is statistically significant (table 5). Similar results were obtained by New et al. [3] who have that bone mineral density in premenopausal women was positively related to fruit and vegetable intake, as well as to magnesium, calcium, zinc and plant fibers. Similarly, Tucker et al. have found better bone mass in women who consumed more fruits, vegetables, potassium and magnesium [41]. However, other study which has in‐ cluded postmenopausal women, showed no relationship between bone mass to fruit or vegetable intake [7,46].

Regarding the mechanisms fruit and vegetables influence bone metabolism, it is impor‐ tant to mention that these nutrients create an alkaline environment and therefore reduce urinary calcium excretion. Besides, fruits and vegetables are rich in vitamins with antiox‐ idant properties such as vitamin C and beta-carotene. Vegetables are an important source of vitamin K, which also has a role in the mineralization of bone since it induces carbox‐ ylation of osteocalcin [40].

### **4. Conclusion**

Analyses of the impact of age, anthropometric parameters, menstrual status and nutrition on the bone status, represents the age and menstrual status as predictors with the highest influence on the bone mineral density in women.

Fruits and vegetables have pleiotropic effects on bone metabolism, which include: alkalinity, antioxidant properties of vitamins and as it was determined by this study, beneficial influence of minerals magnesium, potassium and zinc.

### **Author details**

Daily intake of the minerals magnesium and potassium has the greatest contribution of all the given minerals to DXA parameters (Table 6). Both of the minerals are directly proportional to DXA parameters (Table 4). Studies published so far argue that sufficient magnesium and potassium intake is related to increased bone mineral density [40,41], or as contrast opinions state, to reduced bone mass and increased risk of wrist fracture [43]. Magnesium is important in processes of bone mineralization, and potassium has impor‐ tant role in systemic acid-base (pH) homeostasis. Potassium salts neutralize bone-deplet‐ ing metabolic acids, and therefore conditions that require drain of alkalizing compounds from bone lead to loss of bone tissue. Positive influence of potassium on bone health has

Zinc is a cofactor for alkaline phosphatase, an enzyme, necessary for bone mineraliza‐ tion. Low concentration of the zinc in serum and its increased secretion in urine is asso‐ ciated with osteoporosis [44]. Considering that calcification of the bone is reduced with insufficient zinc intake, we analyzed influence of the dietary zinc on bone health. Results revealed that daily zinc intake is directly proportional to DXA parameters, but the influ‐

Analyzing the impact of fruit and vegetables on DXA parameters in women, we have found that vegetables are directly proportional to the parameters of the left hip, which is statistically significant (table 5). Similar results were obtained by New et al. [3] who have that bone mineral density in premenopausal women was positively related to fruit and vegetable intake, as well as to magnesium, calcium, zinc and plant fibers. Similarly, Tucker et al. have found better bone mass in women who consumed more fruits, vegetables, potassium and magnesium [41]. However, other study which has in‐ cluded postmenopausal women, showed no relationship between bone mass to fruit or

Regarding the mechanisms fruit and vegetables influence bone metabolism, it is impor‐ tant to mention that these nutrients create an alkaline environment and therefore reduce urinary calcium excretion. Besides, fruits and vegetables are rich in vitamins with antiox‐ idant properties such as vitamin C and beta-carotene. Vegetables are an important source of vitamin K, which also has a role in the mineralization of bone since it induces carbox‐

Analyses of the impact of age, anthropometric parameters, menstrual status and nutrition on the bone status, represents the age and menstrual status as predictors with the highest

Fruits and vegetables have pleiotropic effects on bone metabolism, which include: alkalinity, antioxidant properties of vitamins and as it was determined by this study, beneficial influence

been reported [3,4,40,45].

188 Topics in Osteoporosis

vegetable intake [7,46].

ylation of osteocalcin [40].

influence on the bone mineral density in women.

of minerals magnesium, potassium and zinc.

**4. Conclusion**

ence was not significant (data not shown).

Olga Cvijanović1\*, Sandra Pavičić Žeželj<sup>2</sup> , Silvija Lukanović<sup>1</sup> , Nenad Bićanić<sup>3</sup> , Robert Domitrović<sup>4</sup> , Dragica Bobinac<sup>1</sup> and Željka Crnčević Orlić<sup>3</sup>

\*Address all correspondence to: olgac@medri.hr

1 Department of Anatomy, Rijeka Faculty of Medicine, Rijeka, Croatia

2 Department of Ecology Health, Teaching Institute of Public Health Mountain-Littoral County, Rijeka Faculty of Medicine, Rijeka, Croatia

3 Department of Endocrinology, Clinics for Internal Medicine Rijeka Clinical Centre, Rijeka Faculty of Medicine, Rijeka, Croatia

4 Department of Chemistry and Biochemistry, Rijeka Faculty of Medicine, Rijeka, Croatia


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**Chapter 8**

**Bone Mineral Density and High-Performance Aerobic**

Population aging has exhibited a significant increase in the last decades. For instance, in Brazil, recent projections by the IBGE 1 (Instituto Brasileiro de Geografia e Estatística/Brazilian Institute of Geography and Statistics) forecast a threefold increase of the elderly population by 2050 from the current 10.8% to 29.7% of the country's total population, corresponding to almost 65 million people. The life expectancy at birth of the overall population increased to 73 years in the last decade (1999-2009), ranging from 73.9 to 77 years among females and 66.3 to 69 years among males. Such aging of the Brazilian population will pose increasing challenges to the national public health system, SUS (Sistema Único de Saúde/Unified Health System), as older adults exhibit a larger number of chronic diseases, which contribute to loss of function‐

The expected and, indeed, already occurring consequences include the progressive increase of the demand for public healthcare services, higher numbers of hospital admissions, and the use of long-term care facilities [2, 3]. The growth of the elderly population may cause a significant increase of the prevalence of chronic diseases, frailty syndrome, and femoral head

In a Brazilian population-based study involving more than 2,400 individuals older than 40 years, Pinheiro et al. 4 reported incidences of frailty fractures of 15.1% among females and 12.8% among males. A study conducted by the National Institute of Traumatology and Orthopedics (Instituto Nacional de Traumato-Ortopedia - INTO) in Rio de Janeiro reported that the incidence of osteoporosis was 36.4% among men older than 80 years 5. Cost studies indicate that the average cost of in-hospital intervention and surgery per patient is approxi‐

and reproduction in any medium, provided the original work is properly cited.

© 2013 Leme and Sitta; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

fractures, which are frequent occurrences in developed countries.

**Activity in Older Adults Experience in Brazil**

Luiz Eugênio Garcez Leme and

ality and decline of the quality of life.

mately BRL 24,000.00 (USD 11,700.00) 6.

Additional information is available at the end of the chapter

Maria do Carmo Sitta

http://dx.doi.org/10.5772/55661

**1. Introduction**


### **Bone Mineral Density and High-Performance Aerobic Activity in Older Adults Experience in Brazil**

Luiz Eugênio Garcez Leme and Maria do Carmo Sitta

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55661

### **1. Introduction**

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[41] Tucker, K. L, Hannan, M. T, Chen, H, Cupples, L. A, Wilson, P. W, & Kiel, D. P. Po‐ tassium, magnesium and fruit and vegetable intakes are associated with grater bone mineral density in elderly men and women. Am J Clin Nutr (1999). , 69, 727-36.

[42] Schaafsma, A. Vries PJF, Saris WHM. Delay of natural bone loss by higher intakes of specific minerals and vitamins. Crit Rev Food Sci Nutr (2001). , 41(3), 225-249.

[43] Jackson, R. D. LaCroix AZ, Cauley JA, McGowan J. The impact of magnesium intake on fractures: results from the women's health initiative observational study (WHI-

[44] Ilich, Z. J, & Kerstetter, J. E. Nutrition in bone health revisited: A story beyond calci‐

[45] Kaptoge, S, Welch, A, Mctaggart, A, Mulligan, A, Dalzell, N, Day, N. E, Birgham, S, Knaw, K. T, & Reeve, J. Effects of dietary nutrients and food groups on bone loss from the proximal femur in men and women in 7th and 8th decades of age. Osteo‐

[46] Macdonald, H. M, New, S. A, Fraser, W. D, Cambell, M. K, & Reid, D. V. Low dietary po‐ tassium intakes and high dietary estimates of net endogenous acid production are asso‐ ciated with low bone mineral density in premenopausal women and increased markers of bone resorption in postmenopausal women. Am J Clin Nutr (2005). , 81, 923-33.

tional study in 5 age and sex cohorts. Am J Clin Nutr (2006). , 83, 1420-1428.

mingham Osteoporosis Study. Osteoporos Int (2011). , 22(1), 345-349.

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Nutr (2007). , 85(2), 803-7.

OS). ASBMR (2003). abstr.

poros Int (2003). , 14, 418-28.

um. J Am Coll Nutr (2000). , 19, 715-37.

Population aging has exhibited a significant increase in the last decades. For instance, in Brazil, recent projections by the IBGE 1 (Instituto Brasileiro de Geografia e Estatística/Brazilian Institute of Geography and Statistics) forecast a threefold increase of the elderly population by 2050 from the current 10.8% to 29.7% of the country's total population, corresponding to almost 65 million people. The life expectancy at birth of the overall population increased to 73 years in the last decade (1999-2009), ranging from 73.9 to 77 years among females and 66.3 to 69 years among males. Such aging of the Brazilian population will pose increasing challenges to the national public health system, SUS (Sistema Único de Saúde/Unified Health System), as older adults exhibit a larger number of chronic diseases, which contribute to loss of function‐ ality and decline of the quality of life.

The expected and, indeed, already occurring consequences include the progressive increase of the demand for public healthcare services, higher numbers of hospital admissions, and the use of long-term care facilities [2, 3]. The growth of the elderly population may cause a significant increase of the prevalence of chronic diseases, frailty syndrome, and femoral head fractures, which are frequent occurrences in developed countries.

In a Brazilian population-based study involving more than 2,400 individuals older than 40 years, Pinheiro et al. 4 reported incidences of frailty fractures of 15.1% among females and 12.8% among males. A study conducted by the National Institute of Traumatology and Orthopedics (Instituto Nacional de Traumato-Ortopedia - INTO) in Rio de Janeiro reported that the incidence of osteoporosis was 36.4% among men older than 80 years 5. Cost studies indicate that the average cost of in-hospital intervention and surgery per patient is approxi‐ mately BRL 24,000.00 (USD 11,700.00) 6.

© 2013 Leme and Sitta; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

As the number of hospital admissions due to bone fractures among the Brazilian population older than 40 years is higher than 250,000 per year [7], a total cost of approximately 3 billion dollars (BRL 6.5 billion) might be estimated. This estimate comprises only hospital expenses; the cost of home and outpatient care and the loss of patient and caregiver productivity must be added to this estimate together with the incalculable costs associated with loss of quality of life and health. Consequently population aging is an emerging and serious problem.

There is a clear correlation between the criteria used in the definition of frailty and the factors related with fall risk and osteoporosis. From the syndromic perspective, a strong correlation exists indicating that frailty as such might be considered as a predisposing factor for falls,

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Falls are most likely the main health problem among the elderly population, frail people in particular. The risks of fractures and their complications increase together with osteoporosis. A cohort study conducted in the city of São Paulo and involving more than 1,500 participants [13] found that 35% to 40% of the elderly individuals aged 60 years or older fall at least once

In the abovementioned study, the variables that independently and significantly correlated with increased probability of falls were female gender, previous history of fractures, difficulty

Upon investigating falls, another cohort study in São Paulo involving more than 2,000 older adults [14] reported that 33.5% of the studied population reported falls in the past year, whereby 20.2% of the participants reported one single episode, 5.9% reported two episodes,

That same study clearly established a direct correlation between frailty and number and

Osteoporosis and falls represent the main risk factors for the occurrence of fractures, which

A reduction of bone mass associated with deterioration of its microarchitecture predisposes an individual to fractures. The bone is a metabolically active tissue, undergoing constant remodeling through the action of the cells responsible for bone resorption (osteoclasts – derived from the monocyte lineage) and formation (osteoblasts – derived from the fibroblast

The control of bone resorption and formation is coordinated and synchronized by a system known as RANK-RANKL-OPG, which allows for a better understanding of bone physiology

.

The skeleton performs a double function related with metabolism and body support.

per year, and this rate grows to 50% among individuals older than 80 years.

to perform physical activity, and reported poor or very poor vision.

and 7.4% reported three or more episodes.

possibly are the main health problem of older adults.

and paves the way for the development of novel treatments1

**4. Osteoporosis and aging**

fractures, and their complications among the elderly population.

**3. Falls**

severity of falls.

**4.1. Physiopathology**

lineage).

### **2. Frailty**

Aging is associated with progressive manifestations of frailty, poorer capacity of adaptation, and less resilience, which is defined as the ability of individuals to address problems, overcome obstacles, or resist the pressure imposed by adverse conditions.

The conference on frailty in older adults sponsored by the American Geriatrics Society (AGS) and the National Institute on Aging (NIA) defined frailty as a "state of greater vulnerability to stressors due to age and declines related with the neuromuscular, metabolic and immune physiological reserve [8].

The correlation between frailty and orthopedic risks is patent among older adults.

Fried et al. [9] created a definition of the phenotype of frailty that is widely used in research protocols and was validated in the Cardiovascular Health Study (CHS), which was conducted with more than 5,000 men and women aged 65 years or older. According to this study, frailty corresponds to the presence of three or more of the following criteria (pre-frailty corresponds to the presence of less than three):


In a study on the index of osteoporotic fractures, Ensrud et al. [10] applied a simpler index to define frailty, whereby it corresponds to the presence of at least two out of the three following criteria:


Several studies [11, 12] found that the two abovementioned indices were comparable in the prediction of the risk of fall, deficiency, fracture, hospital admission, and death.

There is a clear correlation between the criteria used in the definition of frailty and the factors related with fall risk and osteoporosis. From the syndromic perspective, a strong correlation exists indicating that frailty as such might be considered as a predisposing factor for falls, fractures, and their complications among the elderly population.

### **3. Falls**

As the number of hospital admissions due to bone fractures among the Brazilian population older than 40 years is higher than 250,000 per year [7], a total cost of approximately 3 billion dollars (BRL 6.5 billion) might be estimated. This estimate comprises only hospital expenses; the cost of home and outpatient care and the loss of patient and caregiver productivity must be added to this estimate together with the incalculable costs associated with loss of quality of

Aging is associated with progressive manifestations of frailty, poorer capacity of adaptation, and less resilience, which is defined as the ability of individuals to address problems, overcome

The conference on frailty in older adults sponsored by the American Geriatrics Society (AGS) and the National Institute on Aging (NIA) defined frailty as a "state of greater vulnerability to stressors due to age and declines related with the neuromuscular, metabolic and immune

Fried et al. [9] created a definition of the phenotype of frailty that is widely used in research protocols and was validated in the Cardiovascular Health Study (CHS), which was conducted with more than 5,000 men and women aged 65 years or older. According to this study, frailty corresponds to the presence of three or more of the following criteria (pre-frailty corresponds

**2.** Exhaustion (positive answers to questions on the effort needed for physical activity)

In a study on the index of osteoporotic fractures, Ensrud et al. [10] applied a simpler index to define frailty, whereby it corresponds to the presence of at least two out of the three following

Several studies [11, 12] found that the two abovementioned indices were comparable in the

prediction of the risk of fall, deficiency, fracture, hospital admission, and death.

**5.** Low physical activity (Kcal per week: men < 383 Kcal, women < 270 Kcal)

The correlation between frailty and orthopedic risks is patent among older adults.

life and health. Consequently population aging is an emerging and serious problem.

obstacles, or resist the pressure imposed by adverse conditions.

**1.** Weight loss (≥ 5% of the body weight in the past year)

**4.** Slow walking speed (> 6 to 7 minutes to walk 15 m)

**1.** Loss of 5% of the body weight in the past year

**2.** Inability to rise from a chair five times without using the arms

**3.** Answering "no" to the question, "Do you feel full of energy?"

**2. Frailty**

194 Topics in Osteoporosis

criteria:

physiological reserve [8].

to the presence of less than three):

**3.** Weakness (reduced grip strength)

Falls are most likely the main health problem among the elderly population, frail people in particular. The risks of fractures and their complications increase together with osteoporosis.

A cohort study conducted in the city of São Paulo and involving more than 1,500 participants [13] found that 35% to 40% of the elderly individuals aged 60 years or older fall at least once per year, and this rate grows to 50% among individuals older than 80 years.

In the abovementioned study, the variables that independently and significantly correlated with increased probability of falls were female gender, previous history of fractures, difficulty to perform physical activity, and reported poor or very poor vision.

Upon investigating falls, another cohort study in São Paulo involving more than 2,000 older adults [14] reported that 33.5% of the studied population reported falls in the past year, whereby 20.2% of the participants reported one single episode, 5.9% reported two episodes, and 7.4% reported three or more episodes.

That same study clearly established a direct correlation between frailty and number and severity of falls.

### **4. Osteoporosis and aging**

Osteoporosis and falls represent the main risk factors for the occurrence of fractures, which possibly are the main health problem of older adults.

### **4.1. Physiopathology**

The skeleton performs a double function related with metabolism and body support.

A reduction of bone mass associated with deterioration of its microarchitecture predisposes an individual to fractures. The bone is a metabolically active tissue, undergoing constant remodeling through the action of the cells responsible for bone resorption (osteoclasts – derived from the monocyte lineage) and formation (osteoblasts – derived from the fibroblast lineage).

The control of bone resorption and formation is coordinated and synchronized by a system known as RANK-RANKL-OPG, which allows for a better understanding of bone physiology and paves the way for the development of novel treatments1 .

The cytokine RankL, a member of the TNF (tumor necrosis factor) superfamily, is expressed and secreted by osteoblasts. Interaction between RankL (expressed on the osteoblast surface) and RanK (expressed on the surface of the osteoclast precursors) mediates differentiation and activation of osteoclasts in the presence of M-CSF (macrophage colony-stimulating factor). Mature osteoclasts initiate the process of bone resorption. The interaction between RankL and its receptor on osteoclasts is controlled by osteoprotegerin (OPG). OPG is a soluble receptor belonging to the TNF family that inhibits the binding of RankL to RanK, thus preventing the recruitment, proliferation, and activation of osteoclasts, and this receptor also exerts inhibitory effects on the osteoclast precursor cells. The balance between OPG and RankL controls bone remodeling.

**5. Physical activity**

osteoporosis.

intended purposes.

**6. The rule of aerobic exercises**

the inflammatory activity is still unknown.

Given the growth of the elderly population, the establishment of health promotion measures to reduce the prevalence of chronic diseases, improve functionality, and control multimor‐ bidity is notably important. The goal in this regard is to improve the quality of life of older adults and to reduce healthcare expenses. Among such health-promoting measures, physical activity is one of the main factors associated with control of comorbidities and the reduction of the risk of morbimortality by cardiovascular diseases [15], diabetes [16], obesity [17], and osteoporosis [18]. Physical activity has also been correlated with improved cognition [19, 20,

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197

Regular exercise is important for healthy aging because it has an influence on chronic diseases and functionality. Exercise seems to be a protective factor against genetic and molecular aging and is associated with longevity [24]. Exercise protects [25] the organism against oxidative stress [26] and inflammation [27], which cause damage to the deoxyribonucleic acid (DNA) and other cell structures, resulting in progressive loss of metabolic and physiological functions and greater propensities for cardiovascular, neurodegenerative, and oncological diseases.

The beneficial effects of physical exercise have been demonstrated in the prevention and control of cardiovascular and osteomuscular diseases and diabetes and in the prevention of neoplasias. [28] In recent years, research has focused on the beneficial effects of physical

Together with nutritional measures, hormone and calcium replacement, and use of bisphosph‐ onates, programmed physical exercise has been reported as a protective factor against osteoporosis in older adults. Programmed physical exercise is an acknowledged source of countless benefits in all population sectors, including the elderly. Several authors have correlated the absence or reduction of such physical activity with a higher prevalence of

Nevertheless, the prescription of physical activity involves a heterogeneous range of inter‐ ventions, with each one possessing particular risks and benefits. Therefore, in addition to stimulating the practice of physical activities by their patients, healthcare professionals must carefully and thoroughly analyze the types of activity that are most appropriate for their

Aerobic exercises and, more particularly, walking and running, are the activities most often recommendedbyhealthcareprofessionalsandmostwidelypracticedbytheelderlypopulation. However, overly intense exercise (ultramarathon, running > 64 km per week) is associated with a larger number of osteoarticular lesions and immunosuppression. In addition, the ideal level of physical activity promoting cognitive benefits and modulating neuroprotectors and

21, 22] and reduced risk of the incidence of Alzheimer's disease [23].

activity on cognitive functions and prevention of dementias [29].

The balance of the RANK/RANKL/OPG system is regulated by cytokines and hormones.

Parathormone (PTH), glucocorticoids, and E2 prostaglandins increase the activity of RANKL and reduce the activity of OPG. However, transforming growth factor beta (TGF-β), 17 βestradiol, interleukin 1 (IL-1), and TNF-α exhibit the opposite actions, i.e., they reduce the activity of RANKL and activate OPG. [3]

During growth and aging, the predominance of formation and resorption alternate in the development of bone. Formation is greater until the age of 25 years, stabilizes until the age of 35 years, then decreases progressively, exhibiting a greater decline starting at the age of 70 years. Resorption predominates starting at the age of 35 years and accelerates during the postmenopausal period and until the age of 70 years.

Osteoporosis can be classified as primary or secondary. In turn, primary osteoporosis is subdivided into


Secondary osteoporosis, resulting from other pathologies, may be triggered by


Osteoporosis is universal among the elderly population, exhibits progressive incidence, and is directly correlated with age and lifestyle, including the practice of sports.

<sup>1</sup> RANK – is the abbreviation of receptor activator of nuclear factor kappa B; RANKL – receptor activator of nuclear factor kappa-B ligand; and OPG –osteoprotegerin.

### **5. Physical activity**

The cytokine RankL, a member of the TNF (tumor necrosis factor) superfamily, is expressed and secreted by osteoblasts. Interaction between RankL (expressed on the osteoblast surface) and RanK (expressed on the surface of the osteoclast precursors) mediates differentiation and activation of osteoclasts in the presence of M-CSF (macrophage colony-stimulating factor). Mature osteoclasts initiate the process of bone resorption. The interaction between RankL and its receptor on osteoclasts is controlled by osteoprotegerin (OPG). OPG is a soluble receptor belonging to the TNF family that inhibits the binding of RankL to RanK, thus preventing the recruitment, proliferation, and activation of osteoclasts, and this receptor also exerts inhibitory effects on the osteoclast precursor cells. The balance between OPG and RankL controls bone

The balance of the RANK/RANKL/OPG system is regulated by cytokines and hormones.

Parathormone (PTH), glucocorticoids, and E2 prostaglandins increase the activity of RANKL and reduce the activity of OPG. However, transforming growth factor beta (TGF-β), 17 βestradiol, interleukin 1 (IL-1), and TNF-α exhibit the opposite actions, i.e., they reduce the

During growth and aging, the predominance of formation and resorption alternate in the development of bone. Formation is greater until the age of 25 years, stabilizes until the age of 35 years, then decreases progressively, exhibiting a greater decline starting at the age of 70 years. Resorption predominates starting at the age of 35 years and accelerates during the

Osteoporosis can be classified as primary or secondary. In turn, primary osteoporosis is

**•** Endocrine disorders: Hyperthyroidism, Diabetes, Hyperparathyroidism, Hypercortisolism,

**•** Malabsorption syndromes, Inflammatory Bowel Disease, Coeliac Disease, Post-Gastrecto‐

Osteoporosis is universal among the elderly population, exhibits progressive incidence, and

1 RANK – is the abbreviation of receptor activator of nuclear factor kappa B; RANKL – receptor activator of nuclear factor

**•** Type I or postmenopausal, which is characterized by increased bone resorption.

Secondary osteoporosis, resulting from other pathologies, may be triggered by

**•** Type II or senile, which is characterized by decreased bone formation.

**•** Drugs: corticosteroids, anticonvulsants, alcohol, thyroid hormone.

is directly correlated with age and lifestyle, including the practice of sports.

remodeling.

196 Topics in Osteoporosis

subdivided into

Hypogonadism

**•** Kidney Failure

**•** Neoplasias: Myeloma, Lymphoma

kappa-B ligand; and OPG –osteoprotegerin.

my

activity of RANKL and activate OPG. [3]

postmenopausal period and until the age of 70 years.

**•** Rheumatic disorders: Rheumatoid arthritis, Spondylitis

Given the growth of the elderly population, the establishment of health promotion measures to reduce the prevalence of chronic diseases, improve functionality, and control multimor‐ bidity is notably important. The goal in this regard is to improve the quality of life of older adults and to reduce healthcare expenses. Among such health-promoting measures, physical activity is one of the main factors associated with control of comorbidities and the reduction of the risk of morbimortality by cardiovascular diseases [15], diabetes [16], obesity [17], and osteoporosis [18]. Physical activity has also been correlated with improved cognition [19, 20, 21, 22] and reduced risk of the incidence of Alzheimer's disease [23].

Regular exercise is important for healthy aging because it has an influence on chronic diseases and functionality. Exercise seems to be a protective factor against genetic and molecular aging and is associated with longevity [24]. Exercise protects [25] the organism against oxidative stress [26] and inflammation [27], which cause damage to the deoxyribonucleic acid (DNA) and other cell structures, resulting in progressive loss of metabolic and physiological functions and greater propensities for cardiovascular, neurodegenerative, and oncological diseases.

The beneficial effects of physical exercise have been demonstrated in the prevention and control of cardiovascular and osteomuscular diseases and diabetes and in the prevention of neoplasias. [28] In recent years, research has focused on the beneficial effects of physical activity on cognitive functions and prevention of dementias [29].

Together with nutritional measures, hormone and calcium replacement, and use of bisphosph‐ onates, programmed physical exercise has been reported as a protective factor against osteoporosis in older adults. Programmed physical exercise is an acknowledged source of countless benefits in all population sectors, including the elderly. Several authors have correlated the absence or reduction of such physical activity with a higher prevalence of osteoporosis.

Nevertheless, the prescription of physical activity involves a heterogeneous range of inter‐ ventions, with each one possessing particular risks and benefits. Therefore, in addition to stimulating the practice of physical activities by their patients, healthcare professionals must carefully and thoroughly analyze the types of activity that are most appropriate for their intended purposes.

### **6. The rule of aerobic exercises**

Aerobic exercises and, more particularly, walking and running, are the activities most often recommendedbyhealthcareprofessionalsandmostwidelypracticedbytheelderlypopulation.

However, overly intense exercise (ultramarathon, running > 64 km per week) is associated with a larger number of osteoarticular lesions and immunosuppression. In addition, the ideal level of physical activity promoting cognitive benefits and modulating neuroprotectors and the inflammatory activity is still unknown.

Data in the literature regarding the benefits of long-distance running in the prevention of osteoporosis among older adults are controversial. Novotny et al. [30] assessed an Olympic and world champion long-distance runner 35 years after the end of his racing career and found that his joints were free of signs of arthrosis but that he presented with exceptionally advanced osteoporosis. Conversely, Maud et al. [31] studied a similar case of a long-distance runner older than 70 years who had more than 50 years of training and did not find any alterations in any system (including musculoskeletal). Additionally, regarding resistance exercise, the consensus seems to point to reduction of falls [32] and thus of fractures [33], although not necessarily of osteoporosis [34].

To analyze those parameters, 44 male athletes older than 60 years (mean 64 years) who regularly run more than 15,000 meters were compared; this group included several marathon runners and a number of super-marathon runners. The control group included 18 non-athlete individuals older than 60 years (mean 66.72 years) who had positive self-perceptions of their health and were independent in their everyday life activities. The groups were comparable

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Data were collected by means of double-absorption densitometry and were subjected to descriptive statistical analysis and Student's t-test for paired samples; the chi-square test was used in calculating sport activity as an intervention factor in controls and study subjects in a

Comparisons of bone density, measured at the femoral neck and lumbar spine, between

As a complementary measure, comparative analysis of the bone density in a subgroup of athletes over a 6-year period was performed. Although that group maintained its physical

**Athletes –Femoral Neck Density**

Mean 0.958 -0.898

SD 0.152 1.175

Median 0.933 -1.100

Mean 0.90 -1.22

SD 0.10 0.77

Median 0.88 -1.45

BMD – Bone Mass Density in g/cm2 /T-Score or Young Adults compared in standard deviations

**Table 1.** Density of the femoral neck in athletes and controls

**p=0.169**

**Controls –Femoral Neck Density**

BMD T - VALUE

BMD T - VALUE

activity, no significant differences were identified among the measurements (table 3).

athletes and controls did not reveal any statistical significance (tables 1 and 2).

with *p*=0,419.

2 x 2 table.

Several authors reported increases in bone density among high-performance runners, mainly in the femoral neck [35], whereas according to other authors, similarly to what appears to be the case of women [36], such runners exhibited reductions in bone mass 37 with debatable physiopathology but with a possible association with the metabolism of PTH. Finally, a third group of investigators did not identify any significant differences in bone density among the various groups. [38, 39]

When aerobic activity is combined with resistance training, the increase in bone mass becomes more evident, at least at the experimental level. [40]

### **6.1. Metanalyses**

As the literature data concerning the benefits of aerobic activity in the elderly are conflicting, conducting metanalysis can be a real benefit in his assessment.

Metanalyses of studies on anaerobic exercise and osteoporosis in women produced notably modest results [41]. Several meta-analyses studies published by the Cochrane Collaboration [42] report that both resistance and aerobic exercises might improve bone density among women, and even walking might increase bone density at the hip. It is worth noting that the authors of the abovementioned study stated that the quality of the articles included in the review was modest, whereby the reliability of the results is limited. Still, in this regard, Yamasaki et al. reported that walking improved bone density at the lumbar spine and hip in postmenopausal women. [43]

### **7. Our experience**

To analyze the effects of high-performance physical activity among older Brazilian adults, a cohort of senior athletes from IOTFMUSP (Instituto de Ortopedia e Traumatologia do Hospital das Clínicas da USP/Institute of Orthopedics and Traumatology of the Clinical Hospital of USP) was established in 2001. During the last 11 years, athletes older than 60 years and a control group consisting of healthy non-athlete individuals older than 60 years have been followed periodically by the assessment of several parameters, including bone density and body composition.

To analyze those parameters, 44 male athletes older than 60 years (mean 64 years) who regularly run more than 15,000 meters were compared; this group included several marathon runners and a number of super-marathon runners. The control group included 18 non-athlete individuals older than 60 years (mean 66.72 years) who had positive self-perceptions of their health and were independent in their everyday life activities. The groups were comparable with *p*=0,419.

Data in the literature regarding the benefits of long-distance running in the prevention of osteoporosis among older adults are controversial. Novotny et al. [30] assessed an Olympic and world champion long-distance runner 35 years after the end of his racing career and found that his joints were free of signs of arthrosis but that he presented with exceptionally advanced osteoporosis. Conversely, Maud et al. [31] studied a similar case of a long-distance runner older than 70 years who had more than 50 years of training and did not find any alterations in any system (including musculoskeletal). Additionally, regarding resistance exercise, the consensus seems to point to reduction of falls [32] and thus of fractures [33], although not necessarily of

Several authors reported increases in bone density among high-performance runners, mainly in the femoral neck [35], whereas according to other authors, similarly to what appears to be the case of women [36], such runners exhibited reductions in bone mass 37 with debatable physiopathology but with a possible association with the metabolism of PTH. Finally, a third group of investigators did not identify any significant differences in bone density among the

When aerobic activity is combined with resistance training, the increase in bone mass becomes

As the literature data concerning the benefits of aerobic activity in the elderly are conflicting,

Metanalyses of studies on anaerobic exercise and osteoporosis in women produced notably modest results [41]. Several meta-analyses studies published by the Cochrane Collaboration [42] report that both resistance and aerobic exercises might improve bone density among women, and even walking might increase bone density at the hip. It is worth noting that the authors of the abovementioned study stated that the quality of the articles included in the review was modest, whereby the reliability of the results is limited. Still, in this regard, Yamasaki et al. reported that walking improved bone density at the lumbar spine and hip in

To analyze the effects of high-performance physical activity among older Brazilian adults, a cohort of senior athletes from IOTFMUSP (Instituto de Ortopedia e Traumatologia do Hospital das Clínicas da USP/Institute of Orthopedics and Traumatology of the Clinical Hospital of USP) was established in 2001. During the last 11 years, athletes older than 60 years and a control group consisting of healthy non-athlete individuals older than 60 years have been followed periodically by the assessment of several parameters, including bone density and body

osteoporosis [34].

198 Topics in Osteoporosis

various groups. [38, 39]

postmenopausal women. [43]

**7. Our experience**

composition.

**6.1. Metanalyses**

more evident, at least at the experimental level. [40]

conducting metanalysis can be a real benefit in his assessment.

Data were collected by means of double-absorption densitometry and were subjected to descriptive statistical analysis and Student's t-test for paired samples; the chi-square test was used in calculating sport activity as an intervention factor in controls and study subjects in a 2 x 2 table.

Comparisons of bone density, measured at the femoral neck and lumbar spine, between athletes and controls did not reveal any statistical significance (tables 1 and 2).

As a complementary measure, comparative analysis of the bone density in a subgroup of athletes over a 6-year period was performed. Although that group maintained its physical activity, no significant differences were identified among the measurements (table 3).


**Table 1.** Density of the femoral neck in athletes and controls


In addition, several studies demonstrated that the incidence of osteoporosis among men increases quickly and progressively with age; in the studied area, osteoporosis may affect up

Bone Mineral Density and High-Performance Aerobic Activity in Older Adults Experience in Brazil

http://dx.doi.org/10.5772/55661

201

Our data showed that predominantly aerobic activity, such as high-performance running, did not exhibit a statistically significant correlation with increased bone density; however, the density also did not decrease over a 6-year period. Therefore, our data agree with the findings

However,this findingplacesus atthe centerofthedebatesonaerobic exercise andosteoporosis. The participants of this study belonged to a group of senior athletes with good athletic performance. An average running distance of 15 km and the fact that those athletes exhibited statistically significant improvement over the years denote effective training and follow-up. As a measurement of bone mineral density (BMD), the femoral neck was preferentially used because it is location one most commonly tested. However, our data show that there is a statistically significant difference between the femoral neck and total body bone density that

In regard to the incidence of osteoporosis among older adults, analysis of the participants' bone densities in 2001 and 2007 (table 3) did not reveal any statistically significant differences. Despite the small number of controls in this group, which may compromise its reliability, the data couldn't show us any perceptible difference between athletes and controls in respect to

In the other hand we could not find alterations in bone density between comparisons with seven years of interval in the athletes group. This find may indicate that, if there were no gain in bone mass, on the other hand there were no losses, which might lead us to imagine a

These data lead us to conclude that regarding the prevention or treatment of osteoporosis in older adults, the practice of aerobic physical activity alone is controversial. In the best of cases, physical activity leads to reduced bone loss, although this finding is also poorly supported by

We are currently studying a subgroup of our cohort consisting of senior athletes practicing high-performance aerobic activity and simultaneously being subjected to parallel resistance training. We believe that the results obtained from this group might shed new light on this currently unclear aspect of the prevention and treatment of osteoporosis in older adults.

This chapter was partially funded by FAPESP - Fundação de Amparo à Pesquisa do Estado de São Paulo, Brasil (Foundation for Research Support of the State of São Paulo, Brazil), by

incidence of osteoporosis in concordance with international refereed data.

protective effect of bone loss in this group, confirming literature data.

to 40% of the male population older than 80 years [5].

by Kemmler [37] and Wisswell [38].

prevents their undifferentiated use.

evidence.

**Acknowledgements**

process number 2013/00480-2.

BMD – Bone Mass Density in g/cm2 /T-Score or Young Adults compared in standard deviations

**Table 2.** Density of the lumbar spine in athletes and controls


T-Score or Young Adults compared in standard deviations

**Table 3.** Progression of bone density in athletes, 2001-2007

### **8. Discussion and conclusions**

The first noteworthy aspect of our study is that only men were included, whereas most studies on osteoporosis, including those addressing physical activity, focus on women. This condition, resulting from the overall design of our cohort, was employed because although osteoporosis is less frequent among men, the consequences of its major complication, i.e., fractures, are more severe, resulting in higher indices of morbimortality among men compared to women [44].

In addition, several studies demonstrated that the incidence of osteoporosis among men increases quickly and progressively with age; in the studied area, osteoporosis may affect up to 40% of the male population older than 80 years [5].

Our data showed that predominantly aerobic activity, such as high-performance running, did not exhibit a statistically significant correlation with increased bone density; however, the density also did not decrease over a 6-year period. Therefore, our data agree with the findings by Kemmler [37] and Wisswell [38].

However,this findingplacesus atthe centerofthedebatesonaerobic exercise andosteoporosis.

The participants of this study belonged to a group of senior athletes with good athletic performance. An average running distance of 15 km and the fact that those athletes exhibited statistically significant improvement over the years denote effective training and follow-up.

As a measurement of bone mineral density (BMD), the femoral neck was preferentially used because it is location one most commonly tested. However, our data show that there is a statistically significant difference between the femoral neck and total body bone density that prevents their undifferentiated use.

In regard to the incidence of osteoporosis among older adults, analysis of the participants' bone densities in 2001 and 2007 (table 3) did not reveal any statistically significant differences.

Despite the small number of controls in this group, which may compromise its reliability, the data couldn't show us any perceptible difference between athletes and controls in respect to incidence of osteoporosis in concordance with international refereed data.

In the other hand we could not find alterations in bone density between comparisons with seven years of interval in the athletes group. This find may indicate that, if there were no gain in bone mass, on the other hand there were no losses, which might lead us to imagine a protective effect of bone loss in this group, confirming literature data.

These data lead us to conclude that regarding the prevention or treatment of osteoporosis in older adults, the practice of aerobic physical activity alone is controversial. In the best of cases, physical activity leads to reduced bone loss, although this finding is also poorly supported by evidence.

We are currently studying a subgroup of our cohort consisting of senior athletes practicing high-performance aerobic activity and simultaneously being subjected to parallel resistance training. We believe that the results obtained from this group might shed new light on this currently unclear aspect of the prevention and treatment of osteoporosis in older adults.

### **Acknowledgements**

**Athletes –Lumbar Spine Density**

**Controls –Lumbar Spine Density**

**p=0.501**

Year *2001 2007* Mean 1.02 1.02 SD 0.20 0.20 Median 1.03 1.03 *p=0.464*

The first noteworthy aspect of our study is that only men were included, whereas most studies on osteoporosis, including those addressing physical activity, focus on women. This condition, resulting from the overall design of our cohort, was employed because although osteoporosis is less frequent among men, the consequences of its major complication, i.e., fractures, are more severe, resulting in higher indices of morbimortality among men compared to women [44].

BMD – Bone Mass Density in g/cm2 /T-Score or Young Adults compared in standard deviations

**Athlete Year**

**Table 2.** Density of the lumbar spine in athletes and controls

200 Topics in Osteoporosis

T-Score or Young Adults compared in standard deviations

**Table 3.** Progression of bone density in athletes, 2001-2007

**8. Discussion and conclusions**

Mean 1.15 -0.89 SD 0.17 1.69 Median 1.12 -0.95

Mean 1.01 -0.21 SD 0.57 1.54 Median 1.23 -0.02

BMD T Score Value

BMD T Score Value

T – VALUE T Score Value

This chapter was partially funded by FAPESP - Fundação de Amparo à Pesquisa do Estado de São Paulo, Brasil (Foundation for Research Support of the State of São Paulo, Brazil), by process number 2013/00480-2.

### **Author details**

Luiz Eugênio Garcez Leme and Maria do Carmo Sitta

Faculty of Medicine of University of São Paulo, Universidade de São Paulo – USP, Brazil

rics Society/National Institute on Aging Research Conference on Frailty in Older

Bone Mineral Density and High-Performance Aerobic Activity in Older Adults Experience in Brazil

http://dx.doi.org/10.5772/55661

203

[9] Fried LP, Tangen CM, Walston J, Newman AB, Hirsch C, Gottdiener J, Seeman T, Tracy R, Kop WJ, Burke G, McBurnie MA: Frailty in older adults: evidence for a phe‐ notype Cardiovascular Health Study Collaborative Research Group. J Gerontol A Bi‐

[10] Ensrud KE, Ewing SK, Taylor BC, Fink HA, Cawthon PM, Stone KL, Hillier TA, Cau‐ ley JA, Hochberg MC, Rodondi N, Tracy JK, Cummings Comparison of 2 frailty in‐ dexes for prediction of falls, disability, fractures, and death in older women. SR. Arch

[11] Ensrud KE, Ewing SK, Cawthon PM, Fink HA, Taylor BC, Cauley JA, Dam TT, Mar‐ shall LM, Orwoll ES, Cummings SR. A comparison of frailty indexes for the predic‐ tion of falls, disability, fractures, and mortality in older men. Osteoporotic Fractures

[12] Kiely DK, Cupples LA, Lipsitz LA: Validation and comparison of two frailty indexes:

[13] PERRACINI, Monica Rodrigues; RAMOS, Luiz Roberto. Fatores associados a quedas em uma coorte de idosos residentes na comunidade [Factors associated with falls in a cohort of community-residing older adults]. Rev. Saúde Pública, São Paulo, v. 36, n. 6, Dec. 2002. Available at <http://www.scielo.br/scielo.php?script=sci\_art‐ text&pid=S0034-89102002000700008&lng=en&nrm=iso>. Accessed on 12 Nov. 2012.

[14] Lebrão, M.L. et al. Saúde, bem-estar e envelhecimento: o estudo SABE no Município de Sã Paulo [Health, wellbeing and aging: SABE study at the São Paulo Municipali‐

[15] Berlin JA, Colditz GA. A meta-analysis of physical activity in the prevention of coro‐

[16] Albright A, Franz M, Hornsby G. American College of Sports Medicine position stand: exercise and type 2 diabetes. Med Sci Sports Exerc. 2000; 32(7): 1345-1360. [17] Warburton DE, Nicol CW, Bredin SS. Health benefits of physical activity: the evi‐

[18] Layne JE, Nelson ME. The effects of progressive resistance training on bone density:

[19] Yaffe K, Barnes D, Nevitt M, Lui LY, Covinsky K. A prospective study of physical activity and cognitive decline in elderly women: women who walk. Arch Intern Med

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dence. CMAJ. 2006; 174(6):801-809.

2001; 161:1703-1708.

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The MOBILIZE Boston Study. J Am Geriatr Soc. 2009; 57(9):1532.

Adults. J. Am Geriatr Soc. 2006; 54(6):991.

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[9] Fried LP, Tangen CM, Walston J, Newman AB, Hirsch C, Gottdiener J, Seeman T, Tracy R, Kop WJ, Burke G, McBurnie MA: Frailty in older adults: evidence for a phe‐ notype Cardiovascular Health Study Collaborative Research Group. J Gerontol A Bi‐ ol Sci Med Sci. 2001; 56(3):M146.

**Author details**

202 Topics in Osteoporosis

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**Chapter 9**

**Influence of the Nutrition on Bone Health of Children**

Maintaining adequate nutritional status, is an essential factor for bone growth and minerali‐ zation. Both processes, both the increase in size and development through the deposit of min‐ erals pass alongside and under the regulation by different factors [1]. In this sense, acquired during childhood and adolescence adequate bone mass will be a prime target for complica‐

Currently, in developed countries, osteoporosis is a serious public health problem that affects mainly children and adolescents as a consequence of sedentary lifestyle and unhealthy eating

Recent studies suggest that osteoporosis prevention should begin in childhood. Children and adolescents should achieve an adequate peak bone mass (PMO) before the end of its growth. Otherwise, they may develop osteopenia or osteoporosis early, with a high risk of fractures

Given that the risk of developing osteoporosis depends in 60-80% of genetic factors on which we can not intervene, we must act on those other environmental factors involved which if capable of being corrected, mainly food [5]. It is known that bone mineral den‐ sity (BMD) is modifiable by diet and exercise as much as 20%. Nutrients ingested daily through food involved in the development and maintenance of adequate bone minerali‐ zation by means of different processes, favoring the differentiation of bone tissue func‐ tional cells (osteoblasts and osteoclasts) and acting as plastics elements. In addition, provide adequate nutrition essential vitamins involved in bone matrix synthesis and cal‐ cium absorption at intestinal level, also allowing the synthesis of certain hormones and

and reproduction in any medium, provided the original work is properly cited.

© 2013 González-Jiménez; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

tions, not only in childhood and adolescence but during adulthood [2].

**and Adolescents**

Emilio González-Jiménez

http://dx.doi.org/10.5772/54773

**1. Introduction**

habits [3].

Additional information is available at the end of the chapter

and consequently a lower quality of life [4].

growth factors involved [6].

## **Influence of the Nutrition on Bone Health of Children and Adolescents**

Emilio González-Jiménez

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54773

### **1. Introduction**

Maintaining adequate nutritional status, is an essential factor for bone growth and minerali‐ zation. Both processes, both the increase in size and development through the deposit of min‐ erals pass alongside and under the regulation by different factors [1]. In this sense, acquired during childhood and adolescence adequate bone mass will be a prime target for complica‐ tions, not only in childhood and adolescence but during adulthood [2].

Currently, in developed countries, osteoporosis is a serious public health problem that affects mainly children and adolescents as a consequence of sedentary lifestyle and unhealthy eating habits [3].

Recent studies suggest that osteoporosis prevention should begin in childhood. Children and adolescents should achieve an adequate peak bone mass (PMO) before the end of its growth. Otherwise, they may develop osteopenia or osteoporosis early, with a high risk of fractures and consequently a lower quality of life [4].

Given that the risk of developing osteoporosis depends in 60-80% of genetic factors on which we can not intervene, we must act on those other environmental factors involved which if capable of being corrected, mainly food [5]. It is known that bone mineral den‐ sity (BMD) is modifiable by diet and exercise as much as 20%. Nutrients ingested daily through food involved in the development and maintenance of adequate bone minerali‐ zation by means of different processes, favoring the differentiation of bone tissue func‐ tional cells (osteoblasts and osteoclasts) and acting as plastics elements. In addition, provide adequate nutrition essential vitamins involved in bone matrix synthesis and cal‐ cium absorption at intestinal level, also allowing the synthesis of certain hormones and growth factors involved [6].

© 2013 González-Jiménez; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Therefore, based on the above premises, childhood and adolescence are important periods in which an adequate nutrition will firmly prevent the development of osteopenia or osteoporosis at an early age [7].

plenty of eggs, meat, fish and dairy products which guarantees a significant amount of protein [14]. Moreover, low consumption of protein (less than 45-55 g/day in men or 45 g/day in wom‐ en) are usually accompanied by a lower muscle mass and bone, this mainly due to a reduction in bone structure protein. Meanwhile, a high protein intake (greater than 2 grams/kilogram/ day) may also cause a loss of bone mass, since for each gram of protein intake in excess through

Influence of the Nutrition on Bone Health of Children and Adolescents

http://dx.doi.org/10.5772/54773

209

Both nutrients are the substances most studied for their influence on the prevention and treat‐ ment of osteoporosis. Calcium is the most abundant mineral in the human skeleton. 99% is deposited in the bone and passing about 30 grams at birth [10 grams per kilogram of body weight) to about 1,300 grams in adult (19 grams per kilogram of body weight) [15]. Now, by weight, calcium accounts for 40% of bone mineral and 60% phosphorus. From a metabolic point of view, the absorption of calcium from food depends on several factors. First of its bioavailability in the diet, the deposits of vitamin D, the calcium / phosphorus (Ca/P) and the presence in food of substances that facilitate or interfere with its absorption [16]. In the case of phosphorus (P), this also deposits in the bone by 85%. The high presence among foods in our diet (60-70%), guaranteed to meet the needs of our food without difficulty, especially among the young. While its excessive intake may alter the Ca/P thus hindering the absorption of

The study of calcium-phosphorus balance is complex when you consider its high fecal excretion. In the case of calcium, their daily loss (through excretion in the urine, fecal and dermal) are estimated at 420 mg/day. Calcium is absorbed in the intestine not ever exceeding 30% of the total amount ingested [18]. For absorption, calcium must compete with other substances such as phytates and oxalates act by inhibiting its absorption. The solubilized calcium in foods like milk or juice is absorbed supplemented with ease. In the case of phosphates, these are found in a wide variety of foods thereby showing a higher bioavailability than calcium. His diet recommendations are estimated at 700-800 mg / day in adults compared to 1200 mg/day estimated as necessary during adoles‐ cence. For its part, has a phosphorus antiosificante effect by increasing the secretion of

In the case of calcium, recommendations vary depending on the stage of life where we are. For children under 1 year and for both sexes, the recommended intake of between 400-600 mg/day of calcium. During adolescence, the recommendations are found up to 1200 mg/day for both sexes [19]. For adult males, the recommendations provide a daily intake of between 800 to 1000 mg/day. For adult women the recommendations are higher, between 1200 and 1500 mg/day for those postmenopausal women in gestation and during lactation [20]. According to recent studies, 60% of the calcium in our diet comes from foods such as dairy products, followed by 13% from foods such as cereals, 15% from fruits, vegetables, legumes, while not as only 6% comes from foods like meat, fish or eggs. This entails risks if it is very sedentary children and

the diet, results in a loss of 1 mg of calcium [14].

**Calcium and phosphorus**

calcium [17].

**Calcium-phosphorus balance**

PTH and reduced intestinal calcium absorption [18].

**Recommendations for calcium intake**

### **2. Nature of bone tissue and skeletal development**

Bone consists of cells (2-5%) and in a largely inert matter (95-98%), ie basically protein and minerals [8]. From a structural standpoint, the protein component is composed of fibers of collagen type I and gla protein (osteocalcin, osteonectin, fibronectin, osteopontin and bone sialoprotein). Its component consists mainly of hydroxyapatite mineral-rich carbonates (37-40% calcium and phosphate 50-58%) and to a lesser extent sodium, potassium, magnesium and citrate [8].

At birth, the newborn already has 70 to 95 grams of bone mass approximately equiva‐ lent to 4% by weight. During adolescence, girls have 2,400 grams of bone [9]. The boys, meanwhile, are at increased volume estimated at approximately 3,300 grams. Both quan‐ tities correspond to a 85% to development of the cortical bone and 15% of the cancel‐ lous bone development [9].

In this sense, it is considered that the girls get their peak bone mass at age 18 as opposed to boys who reach it some years later, at the age of 23 years or so [9]. The gain of this peak is mediated by the action of sex hormones on growth factor 1 (IGF-1), which is stimulated in parallel mode by proteins [10].

Once acquired peak bone mass, it will tend to stabilize. After bone mass will be reduced only to certain pathological processes involving a state of prostration of the patient or following the administration of certain drugs such as glucocorticoids [11]. Although the bone loss does not occur evenly across the skeleton. Normally, women aged between 20 and 30 begin to develop a reduced bone loss (<1%) level of the vertebrae [12]. It will be during the first 5 years post‐ menopause when the loss increases between 2 and 6% annually. Among men, the loss occurs at older ages, ie from 50 years of age [12].

### **3. Nutritional factors involved in the process of bone mineralization**

### **Energy intake**

Maintain adequate caloric intake permitted during childhood and adolescence to ensure growth, maturation and bone mineralization process [13].

### **Protein intake**

Protein intake, is another important factor for the formation of bone matrix. However, protein intake appears to be important risk factor among our child population as they often exceed recommendations [14]. This mainly because the food in Western countries usually contain plenty of eggs, meat, fish and dairy products which guarantees a significant amount of protein [14]. Moreover, low consumption of protein (less than 45-55 g/day in men or 45 g/day in wom‐ en) are usually accompanied by a lower muscle mass and bone, this mainly due to a reduction in bone structure protein. Meanwhile, a high protein intake (greater than 2 grams/kilogram/ day) may also cause a loss of bone mass, since for each gram of protein intake in excess through the diet, results in a loss of 1 mg of calcium [14].

### **Calcium and phosphorus**

Therefore, based on the above premises, childhood and adolescence are important periods in which an adequate nutrition will firmly prevent the development of osteopenia or osteoporosis

Bone consists of cells (2-5%) and in a largely inert matter (95-98%), ie basically protein and minerals [8]. From a structural standpoint, the protein component is composed of fibers of collagen type I and gla protein (osteocalcin, osteonectin, fibronectin, osteopontin and bone sialoprotein). Its component consists mainly of hydroxyapatite mineral-rich carbonates (37-40% calcium and phosphate 50-58%) and to a lesser extent sodium, potassium, magnesium

At birth, the newborn already has 70 to 95 grams of bone mass approximately equiva‐ lent to 4% by weight. During adolescence, girls have 2,400 grams of bone [9]. The boys, meanwhile, are at increased volume estimated at approximately 3,300 grams. Both quan‐ tities correspond to a 85% to development of the cortical bone and 15% of the cancel‐

In this sense, it is considered that the girls get their peak bone mass at age 18 as opposed to boys who reach it some years later, at the age of 23 years or so [9]. The gain of this peak is mediated by the action of sex hormones on growth factor 1 (IGF-1), which is stimulated in

Once acquired peak bone mass, it will tend to stabilize. After bone mass will be reduced only to certain pathological processes involving a state of prostration of the patient or following the administration of certain drugs such as glucocorticoids [11]. Although the bone loss does not occur evenly across the skeleton. Normally, women aged between 20 and 30 begin to develop a reduced bone loss (<1%) level of the vertebrae [12]. It will be during the first 5 years post‐ menopause when the loss increases between 2 and 6% annually. Among men, the loss occurs

**3. Nutritional factors involved in the process of bone mineralization**

Maintain adequate caloric intake permitted during childhood and adolescence to ensure

Protein intake, is another important factor for the formation of bone matrix. However, protein intake appears to be important risk factor among our child population as they often exceed recommendations [14]. This mainly because the food in Western countries usually contain

**2. Nature of bone tissue and skeletal development**

at an early age [7].

208 Topics in Osteoporosis

and citrate [8].

**Energy intake**

**Protein intake**

lous bone development [9].

parallel mode by proteins [10].

at older ages, ie from 50 years of age [12].

growth, maturation and bone mineralization process [13].

Both nutrients are the substances most studied for their influence on the prevention and treat‐ ment of osteoporosis. Calcium is the most abundant mineral in the human skeleton. 99% is deposited in the bone and passing about 30 grams at birth [10 grams per kilogram of body weight) to about 1,300 grams in adult (19 grams per kilogram of body weight) [15]. Now, by weight, calcium accounts for 40% of bone mineral and 60% phosphorus. From a metabolic point of view, the absorption of calcium from food depends on several factors. First of its bioavailability in the diet, the deposits of vitamin D, the calcium / phosphorus (Ca/P) and the presence in food of substances that facilitate or interfere with its absorption [16]. In the case of phosphorus (P), this also deposits in the bone by 85%. The high presence among foods in our diet (60-70%), guaranteed to meet the needs of our food without difficulty, especially among the young. While its excessive intake may alter the Ca/P thus hindering the absorption of calcium [17].

### **Calcium-phosphorus balance**

The study of calcium-phosphorus balance is complex when you consider its high fecal excretion. In the case of calcium, their daily loss (through excretion in the urine, fecal and dermal) are estimated at 420 mg/day. Calcium is absorbed in the intestine not ever exceeding 30% of the total amount ingested [18]. For absorption, calcium must compete with other substances such as phytates and oxalates act by inhibiting its absorption. The solubilized calcium in foods like milk or juice is absorbed supplemented with ease. In the case of phosphates, these are found in a wide variety of foods thereby showing a higher bioavailability than calcium. His diet recommendations are estimated at 700-800 mg / day in adults compared to 1200 mg/day estimated as necessary during adoles‐ cence. For its part, has a phosphorus antiosificante effect by increasing the secretion of PTH and reduced intestinal calcium absorption [18].

### **Recommendations for calcium intake**

In the case of calcium, recommendations vary depending on the stage of life where we are. For children under 1 year and for both sexes, the recommended intake of between 400-600 mg/day of calcium. During adolescence, the recommendations are found up to 1200 mg/day for both sexes [19]. For adult males, the recommendations provide a daily intake of between 800 to 1000 mg/day. For adult women the recommendations are higher, between 1200 and 1500 mg/day for those postmenopausal women in gestation and during lactation [20]. According to recent studies, 60% of the calcium in our diet comes from foods such as dairy products, followed by 13% from foods such as cereals, 15% from fruits, vegetables, legumes, while not as only 6% comes from foods like meat, fish or eggs. This entails risks if it is very sedentary children and adolescents and women with menopause. In some cases this will be indicated calcium sup‐ plementation, especially through the diet is not achieved optimal calcium intake. In the study by Johsnton et al (1992) [21], from a population of prepubertal twins who are supplemented with a dose of 700 mg/day of calcium, managed to increase its process of mineralization and bone mass in a 5%.

**Alcohol consumption, snuff and caffeine**

iron overload and decreased testosterone [31].

lower bone mineral density in these subjects.

**4. In conclusion**

order.

**Author details**

Melilla, Spain

Emilio González-Jiménez\*

Address all correspondence to: emigoji@ugr.es

Excessive alcohol consumption, is an important risk factor for osteoporosis, especially among young males. Ingestion, causes a decrease in bone mass through an alteration of the formation and bone remodeling [29]. During adolescence, a high intake of alcohol reduces bone mass peak. This circumstance enables the development of osteopenia or osteoporosis at an early age [30]. In addition, alcohol intake is associated with dietary disorders, which adversely affect the bone metabolism. Thus, their intake is related to a deficiency of vitamin D and parathyroid hormone (PTH), hypoproteinemia, liver, hypomagnesemia, deficit B vitamins and folic acid,

Influence of the Nutrition on Bone Health of Children and Adolescents

http://dx.doi.org/10.5772/54773

211

Regarding the consumption of snuff in adolescents, it has been associated with a significant reduction in bone mineral density (BMD). It has been shown that adolescent smokers, espe‐ cially girls, have a lower bone mineral density and increased rate of bone loss that girls do not

Regarding caffeine intake, it increases urinary calcium excretion during the first 3 hours after ingestion. It has been found that a daily intake of two or more cups of coffee, is correlated with

Among all the factors involved in the mineralization of bone mass, maintaining a balanced diet is a key factor. A balanced and varied diet will be the best procedure to ensure proper bone development among young people. Therefore, an adequate energy and protein intake, coupled with a contribution provided essential nutrients such as calcium, phosphorus or flu‐ orine and certain vitamins such as D and K Assume the basic nutritional elements to ensure adequate bone mass during the later stages early in life. Given that bone development achieved during childhood and adolescence will influence the health status and bone mass in adulthood and that this depends largely on food, care for the food of the young should be a prime target

Department of Nursing. Faculty of Nursing (Campus of Melilla), University of Granada,

smoke. This has been demonstrated also among male adolescents [32].

### **Sodium intake**

Excessive sodium intake in the diet may be accompanied by a reduction in bone mineral den‐ sity. This decrease in bone mass is mediated by a renal calcium excretion [22]. In this regard, given an approximate intake of 450 mg of sodium in the diet, the kidney is capable of removing in parallel up to 10 mg of calcium. Accordingly, the recommendations established for calcium intake in adolescents, they must not exceed a daily sodium intake greater than 2000 mg or what is just 5 grams of salt [22].

### **Intake of vitamin D and K and its importance in the process of bone mineralization**

Vitamin D belongs to the group of so-called fat-soluble vitamins. Their presence in food is by way of cholecalciferol (D3) and ergocalciferol. Usually has its origin in cholesterol or ergosterol derivative which is converted to ergocalciferol (D2) the effect of ultraviolet radiation. However, the active form of this vitamin is called calcitriol [23]. Its synthesis is closely associated with sun exposure, that is, with a daily sun exposure is insufficient to meet the physiological needs of the vitamin in our body [24]. Their presence facili‐ tates the absorption of calcitriol in the intestine. In this regard, serum levels of this vita‐ min have been correlated with bone density in certain locations such as the lumbar spine and femoral neck. Another vitamin involved in the process of bone mineralization during childhood and adolescence is vitamin K. Participates in the process of carboxyla‐ tion of osteocalcin and thereby cause a deficit decreased and carboxylation of osteocal‐ cin synthesis [25]. *Fluorine* Within the elementary ions, fluoride in nature and chemical behavior is the most active of all elementary ions. Its concentration is high in mineral water, fish, tea and certain meals [26]. Fluoride ingested through the diet, is rapidly ab‐ sorbed from the gastrointestinal tract to blood from which will be distributed to tissues and organs by simple diffusion. Among its benefits to bone level highlights its ability to stimulate osteoblast activity, increasing the mainly trabecular bone. In this regard, it has been demonstrated that administration of 25 mg/day slow-release fluoride supplied for 4 years reduces the incidence of vertebral fractures [26]. *Consumption of carbonated drinks*

The increased consumption of carbonated beverages is associated with a progressive decrease in milk intake, has led to high consumption of phosphoric acid associated with calcium defi‐ ciency [27]. This eating pattern will have consequences for bone health, because when a diet is high in phosphorus and low in calcium, bone resorption increases to recover the serum levels of this mineral. There are epidemiological studies that linked the consumption of carbonated beverages to an increased risk of fracture in children and young girls. However, there is some controversy about this relationship [28]. Many authors, conclude that the major effect of car‐ bonated beverages is mainly due to displacement of milk in the diet, especially among young people.

### **Alcohol consumption, snuff and caffeine**

adolescents and women with menopause. In some cases this will be indicated calcium sup‐ plementation, especially through the diet is not achieved optimal calcium intake. In the study by Johsnton et al (1992) [21], from a population of prepubertal twins who are supplemented with a dose of 700 mg/day of calcium, managed to increase its process of mineralization and

Excessive sodium intake in the diet may be accompanied by a reduction in bone mineral den‐ sity. This decrease in bone mass is mediated by a renal calcium excretion [22]. In this regard, given an approximate intake of 450 mg of sodium in the diet, the kidney is capable of removing in parallel up to 10 mg of calcium. Accordingly, the recommendations established for calcium intake in adolescents, they must not exceed a daily sodium intake greater than 2000 mg or what

**Intake of vitamin D and K and its importance in the process of bone mineralization**

Vitamin D belongs to the group of so-called fat-soluble vitamins. Their presence in food is by way of cholecalciferol (D3) and ergocalciferol. Usually has its origin in cholesterol or ergosterol derivative which is converted to ergocalciferol (D2) the effect of ultraviolet radiation. However, the active form of this vitamin is called calcitriol [23]. Its synthesis is closely associated with sun exposure, that is, with a daily sun exposure is insufficient to meet the physiological needs of the vitamin in our body [24]. Their presence facili‐ tates the absorption of calcitriol in the intestine. In this regard, serum levels of this vita‐ min have been correlated with bone density in certain locations such as the lumbar spine and femoral neck. Another vitamin involved in the process of bone mineralization during childhood and adolescence is vitamin K. Participates in the process of carboxyla‐ tion of osteocalcin and thereby cause a deficit decreased and carboxylation of osteocal‐ cin synthesis [25]. *Fluorine* Within the elementary ions, fluoride in nature and chemical behavior is the most active of all elementary ions. Its concentration is high in mineral water, fish, tea and certain meals [26]. Fluoride ingested through the diet, is rapidly ab‐ sorbed from the gastrointestinal tract to blood from which will be distributed to tissues and organs by simple diffusion. Among its benefits to bone level highlights its ability to stimulate osteoblast activity, increasing the mainly trabecular bone. In this regard, it has been demonstrated that administration of 25 mg/day slow-release fluoride supplied for 4 years reduces the incidence of vertebral fractures [26]. *Consumption of carbonated drinks*

The increased consumption of carbonated beverages is associated with a progressive decrease in milk intake, has led to high consumption of phosphoric acid associated with calcium defi‐ ciency [27]. This eating pattern will have consequences for bone health, because when a diet is high in phosphorus and low in calcium, bone resorption increases to recover the serum levels of this mineral. There are epidemiological studies that linked the consumption of carbonated beverages to an increased risk of fracture in children and young girls. However, there is some controversy about this relationship [28]. Many authors, conclude that the major effect of car‐ bonated beverages is mainly due to displacement of milk in the diet, especially among young

bone mass in a 5%.

is just 5 grams of salt [22].

**Sodium intake**

210 Topics in Osteoporosis

people.

Excessive alcohol consumption, is an important risk factor for osteoporosis, especially among young males. Ingestion, causes a decrease in bone mass through an alteration of the formation and bone remodeling [29]. During adolescence, a high intake of alcohol reduces bone mass peak. This circumstance enables the development of osteopenia or osteoporosis at an early age [30]. In addition, alcohol intake is associated with dietary disorders, which adversely affect the bone metabolism. Thus, their intake is related to a deficiency of vitamin D and parathyroid hormone (PTH), hypoproteinemia, liver, hypomagnesemia, deficit B vitamins and folic acid, iron overload and decreased testosterone [31].

Regarding the consumption of snuff in adolescents, it has been associated with a significant reduction in bone mineral density (BMD). It has been shown that adolescent smokers, espe‐ cially girls, have a lower bone mineral density and increased rate of bone loss that girls do not smoke. This has been demonstrated also among male adolescents [32].

Regarding caffeine intake, it increases urinary calcium excretion during the first 3 hours after ingestion. It has been found that a daily intake of two or more cups of coffee, is correlated with lower bone mineral density in these subjects.

### **4. In conclusion**

Among all the factors involved in the mineralization of bone mass, maintaining a balanced diet is a key factor. A balanced and varied diet will be the best procedure to ensure proper bone development among young people. Therefore, an adequate energy and protein intake, coupled with a contribution provided essential nutrients such as calcium, phosphorus or flu‐ orine and certain vitamins such as D and K Assume the basic nutritional elements to ensure adequate bone mass during the later stages early in life. Given that bone development achieved during childhood and adolescence will influence the health status and bone mass in adulthood and that this depends largely on food, care for the food of the young should be a prime target order.

### **Author details**

Emilio González-Jiménez\*

Address all correspondence to: emigoji@ugr.es

Department of Nursing. Faculty of Nursing (Campus of Melilla), University of Granada, Melilla, Spain

### **References**

[1] Jones, G. Early life nutrition and bone development in children. Nestle Nutr Workshop Ser Pediatr Program (2011). , 68, 227-33.

[19] Basabe, B, Mena, M. C, Faci, M, Aparicio, A, López-sobaler, A. M, & Ortega, R. M. Influencia de la ingesta de calcio y fósforo sobre la densidad mineral ósea en mujeres

Influence of the Nutrition on Bone Health of Children and Adolescents

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[20] Jackson, R. D. LaCroix AZ, Gass M, Wallace RB, Robbins J, Lewis CE, et al. Calcium plus vitamin D supplementation and the risk of fractures. N Engl J Med (2006). , 354,

[21] Johsnton, C. C, Miller, J. Z, Siemenda, C. W, Reister, T. K, Hui, S, & Christian, J. C. Calcium supplementation and increases in bone mineral in children. N Engl J Med

[22] Chan, R, Woo, J, Lau, W, Leung, J, Xu, L, Zhao, X, Yu, W, Lau, E, & Pocock, N. Effects of lifestyle and diet on bone health in young adult Chinese women living in Hong Kong

[23] Holick, M. F, & Chen, T. C. Vitamin D deficiency: A world wide problem with health

[25] Ahmadieh, H, & Arabi, A. Vitamins and bone health: beyond calcium and vitamin D.

[26] Grajeta, H. Nutrition in prevention and treatment of osteoporosis. Przegl Lek (2003). ,

[27] Ma, D, & Jones, G. Soft drink and milk consumption, physical activity, bone mass, and upper limb fractures in children: a population-based case-control study. Calcif Tissue

[28] Wyshak, G. Teenaged girls, carbonated beverage consumption, and bone fractures.

[29] Chakkalakal, D. A. Alcohol-induced bone loss and deficiente bone repair. Alcohol Clin

[30] Turner, R. T. Skeletal response to alcohol. Alcohol Clin Exp Res (2000). , 24, 1693-701. [31] Kim, M. J, Shim, M. S, Kim, M. K, Lee, Y, Shing, Y. G, Churg, C. H, et al. Effect of chronic alcohol ingestión on bone mineral density in males without liver cirrosis. Korean J

[32] Vogt, M. T, Hanscom, B, Lauerman, W. C, & Kang, I. D. Influence of smoking on the health patients. The National Spine network data base. Spine (2002). , 27, 313-19.

jóvenes. Arch Latinoam Nutr (2004). , 54, 203-8.

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Arch Pediatr Adolesc Med (2000). , 154(6), 610-13.

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[24] Holick, M. F. Vitamin D deficiency. N Engl J Med (2007). , 357, 266-81.

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[19] Basabe, B, Mena, M. C, Faci, M, Aparicio, A, López-sobaler, A. M, & Ortega, R. M. Influencia de la ingesta de calcio y fósforo sobre la densidad mineral ósea en mujeres jóvenes. Arch Latinoam Nutr (2004). , 54, 203-8.

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[1] Jones, G. Early life nutrition and bone development in children. Nestle Nutr Workshop

[2] Tristán Fernández JMRuiz Santiago F, Pérez de la Cruz A, Lobo Tañer G, Aguilar Cor‐ dero MJ, Collado Torreblanca F. Influencia de la nutrición y del entorno social en la

[3] Quesada Gómez JMSosa Henríquez M. Nutrición y osteoporosis. Calcio y vitamina D.

[4] Adami, S, Isaia, G, Luisetto, G, Minisola, S, Sinigaglia, L, Silvestri, S, et al. ICARO Study Group. Osteoporosis treatment and fracture incidence: the ICARO longitudinal study.

[5] Bechtold-Dalla Pozza SBone density measurements on growing skeletons and the clin‐

[7] Hirota, T, & Hirota, K. Nutrition in bone growth and development. Clin Calcium

[8] Qiu, Z. Y, Li, G, Zhang, Y. Q, Liu, J, Hu, W, Ma, J, & Zhang, S. M. Fine structure analysis and sintering properties of Si-doped hydroxyapatite. Biomed Mater (2012).

[9] Mora, S, & Gilsanz, V. Establishment of peak bone mass. Endocrinol Metab Clin N Am

[10] Zofková, I. Soft tissues, hormones and the skeleton. Vnitr Lek (2012). , 58(2), 135-39. [11] Krall, E. A, & Dawson-hughes, B. Osteoporosis: En: Shils ME, Olson JA, Ross AC, ed‐ itores. Nutrición en Salud y Enfermedad. México, Interamericana, (2002).

[12] Zatonski, T, Temporale, H, & Krecicki, T. Hearing and balance in metabolic bone dis‐

[13] Zagarins, S. E, Ronnenberg, A. G, Gehlbach, S. H, Lin, R, & Bertone-johnson, E. R. Are existing measures of overall diet quality associated with peak bone mass in young

[15] Caroli, A, Poli, A, Ricotta, D, Banfi, G, & Cocchi, D. Invited review: Dairy intake and bone health: a viewpoint from the state of the art. J Dairy Sci (2011). , 94(11), 5249-62. [16] Food and Nutritional BoardDietary Reference Intakes (DRI) for calcium, phosphorus, magnesium, vitamin D and fluoride. Washington: National Academy of Sciences. Na‐

[17] Bonjour, J. P. Bone mineral adquisition in adolescente. En: Markus R, Felman D, Kesley

[18] Loui, A, Raab, A, Obladen, M, & Brätter, P. Calcium, phosphorus and magnesium bal‐ ance: FM 85 fortification of human milk does not meet mineral needs of extremely low

J, editores. Osteoporosis. San Diego: Academic Press; (1996). , 465-476.

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[6] Cashman, K. D. Diet, Nutrition, and Bone Health. J Nutr (2007). S, 2507-12.

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premenopausal women? J Hum Nutr Diet (2012). , 25(2), 172-79. [14] Bonjour, J. P. Protein intake and bone health. Int J Vitam Nutr Res (2011).

Rev Osteoporos Metab Miner (2011). , 4, 165-82.


**Chapter 10**

**The Effectiveness of Progressive Load Training**

**of Falls in Women with Osteoporosis**

Lucas Teixeira, Stella Peccin, Kelson Silva,

Joelma Magalhães and Virgínia Trevisani

Additional information is available at the end of the chapter

Tiago Teixeira, Aline Mizusaki Imoto,

Greater losses are in the osteoporosis area.

http://dx.doi.org/10.5772/54554

**1. Introduction**

**Associated to the Proprioceptive Training for Prevention**

In most of the cases, osteoporosis is a related condition to aging. It can be seen in both genders, but it especially manifests in women after menopause due to estrogen production rate fall. For understanding what happens, it is necessary to bear in mind that the bones are com‐ pounded of a matrix in which mineral complexes such as calcium are laid up. Another important feature is that they are in constant renewal process, since they are formed by cells called osteoclasts which are responsible for reabsorbing the aged areas and others, the osteoblasts, which is responsible for producing new bones. This permanent and constant process makes possible the bone reconstitution when fractures happen and it explains why around every ten years the human skeleton is entirely renewed. Along the time, however, the old cells absorption increases and the bone new cells formation decreases. The outcome is that the bones become more porous, losing resistance. Bone mass lighter loss features osteopenia.

If it is not early prevented, or if it is not treated, the bone mass loss is progressively increasing, in an asymptomatic fashion, without any manifestation, until a fracture occurrence. What features the osteoporotic fractures, is when they take place with a minimum trauma, what would not cause fractures in a normal bone. Therefore, they are also called fragility fractures. The incidence of osteoporotic fractures is strictly related to the individual bone mass that depends on the speed of loss throughout life as well as the amount of bone tissue in the end

and reproduction in any medium, provided the original work is properly cited.

© 2013 Teixeira et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

## **The Effectiveness of Progressive Load Training Associated to the Proprioceptive Training for Prevention of Falls in Women with Osteoporosis**

Lucas Teixeira, Stella Peccin, Kelson Silva, Tiago Teixeira, Aline Mizusaki Imoto, Joelma Magalhães and Virgínia Trevisani

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54554

**1. Introduction**

In most of the cases, osteoporosis is a related condition to aging. It can be seen in both genders, but it especially manifests in women after menopause due to estrogen production rate fall.

For understanding what happens, it is necessary to bear in mind that the bones are com‐ pounded of a matrix in which mineral complexes such as calcium are laid up. Another important feature is that they are in constant renewal process, since they are formed by cells called osteoclasts which are responsible for reabsorbing the aged areas and others, the osteoblasts, which is responsible for producing new bones. This permanent and constant process makes possible the bone reconstitution when fractures happen and it explains why around every ten years the human skeleton is entirely renewed. Along the time, however, the old cells absorption increases and the bone new cells formation decreases. The outcome is that the bones become more porous, losing resistance. Bone mass lighter loss features osteopenia. Greater losses are in the osteoporosis area.

If it is not early prevented, or if it is not treated, the bone mass loss is progressively increasing, in an asymptomatic fashion, without any manifestation, until a fracture occurrence. What features the osteoporotic fractures, is when they take place with a minimum trauma, what would not cause fractures in a normal bone. Therefore, they are also called fragility fractures.

The incidence of osteoporotic fractures is strictly related to the individual bone mass that depends on the speed of loss throughout life as well as the amount of bone tissue in the end

© 2013 Teixeira et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

of puberty and beginning of adulthood. The great variation in bone mass peak is explained not only by hereditary factors but also by gender, race, eating habits, several hormone influence, body composition of lean mass and body fat, intercurrent diseases, chronic use of medications and physical activity (Brandão & Vieira, 1999).

Falls are multifactorial (Cathleen et al., 2006), (Tinetti et al., 1989) and their intrinsic causes include altered balance, gait, muscle strength, visual acuity, cognition and the presence of

The Effectiveness of Progressive Load Training Associated to the Proprioceptive Training…

http://dx.doi.org/10.5772/54554

217

The participation of environmental risk factors might reach up to 50% of the falls in elderly that live in the community. These factors include poor lighting, slippery surfaces, loose or folded rugs, high or narrow stairs, obstacles in the way (low furniture, small objects, wires), lack of rails in halls and bathrooms, extremely low or high shelves, inadequate shoes and clothes, poorly maintained streets with holes or irregularities and inappropriate orthosis.

A reduction of approximately 30% in the strength is found in individual ages ranging from 50 to 70. These changes are more common in women than in men, more prevalent in lower limbs than in upper limbs and a great amount of this reduction in strength is caused by a selective

Evidences have shown that specific exercises might reduce the risk factors for falls and the

The purpose of our study was to evaluate the efficacy of the resistance training associated to a proprioceptive training in the prevention of falls and reduction of the respective risk factors

Prevention in individuals older than 60 years has an important role in avoiding adverse

The work to prevent fractures related to osteoporosis should focus the prevention or increase of material and structural properties of the bone, the prevention of falls and improvement of

**1.** physical activity of transporting weight is essential to the normal development and maintenance of a health skeleton. Activities that focus the increase of muscle strength

**2.** a sedentary woman might progressively increase her bone mass by becoming active, but the primary benefit of increasing the activity is to prevent a future bone reduction that

**4.** the optimal program for an older woman might include activities that improve the strength, flexibility and coordination which might indirectly, but effectively decrease the incidence of osteoporotic fractures by reducing the probability of falls. Therefore, the treatment of osteoporosis should aim the prevention of falls and fractures and preserva‐

might also be beneficial, particularly for bones that do not support weight;

**3.** exercise should not be recommended as a replacement to medications treatment;

atrophy of Type IIB fibers (American College of Sports Medicine Position, 1998).

chronic diseases (van Schoor et al., 2002).

number of falls in the elderly.

in postmenopausal women with osteoporosis.

**2. Physical exercise to prevent falls**

resulting from the lack of activity;

tion or improvement of bone mineral density.

consequences resulting from falls (Weatherall, 2004).

total mass of lean tissue (American College of Sports Medicine, 1995).

The American College of Sports Medicine recommends that:

Like any other chronic disease, the ethiology of osteoporosis is multifactorial. Genetic factors contribute approximately with 46% to 62% of bone mineral density (BMD) whereas other causes include lifestyle, diet and physical exercise (Neto et al., 2002).

Osteoporosis clinical symptoms do not usually occur before a fracture occurrence. Osteopo‐ rosis is considered an asymptomatic disease. Indeed, during the disease progression, the bones become progressively more fragile without affecting the individuals. This characteristic of being a silent disease exposes the population to even a greater risk of suffering a fracture.

Osteoporosis is considered a "silent disease" until a fracture occurs. Approximately 1.5 million fractures per year are attributable to this disease. Only in the USA, these fractures result in 500.000 hospitalizations, 800.000 emergency room visits, 2.6 million physician visits. The treatment cost is high. In 2002, 12 billion dollars to 18 billion dollars were spent (Gass & Huges, 2006). In 1998, cost management of osteoporosis fractures in the UK recorded 942 million pounds per year (Szejnfeld et al., 2007). Osteoporosis has become one of the major public health problems. Nowadays, the impact of osteoporosis is compared to the impact caused by most important health problems, such as cardiovascular diseases and cancer (Froes et al., 2002).

It exposes the fallers to a high risk of fractures (Johnell et al., 2005; Siris et al., 2006). The first hip fracture is associated to 2.5-fold increased risk of subsequent fracture (Cólon-Emeric et al., 2003) with a high level of morbidity and mortality (Cathleen et al., 2006).

It is believed that about 25% of menopausal women in the USA will exhibit some kind of fracture as a consequence of osteoporosis. The most severe fractures are the fractures of femur and they are associated with higher medical expenses than all other osteoporotic fractures together (Moreira & Xaxier, 2001). The incidence of these fractures has doubled in the last 25 years and it is estimated that six million people in the world will suffer fracture of the proximal femur in 2050. Fractures resulted from the decrease of bone mineral loss are considered an orthopedic epidemic leading to an increase in costs for several countries and consequently representing a big social and economic problem (Ramalho et al., 2001).

There have been a significant number of evidences showing that the decrease in bone quality, from generation to generation, is caused by a change in life style, having as a main determinant the lack of physical activity. This evidence varies with the biology of the basic bone. However, epidemiological studies indicate that physical activity is the most important factor to maintain bone mass and prevent fractures (Mosekilde, 1995).

Almost all hip fractures (more than 90%) occur as a result of a fall and these fractures are related not only to the decreased bone mass, but also to other factors such as reduction of balance, muscle strength and power in the lower extremities (American College of Sports Medicine [ACSM], 1995; Parkkari et al. 1999). Therefore, aging and alterations in balance and muscle strength, as well as sensorial changes, predispose patients with osteoporosis to a higher risk of having fractures due to falls.

Falls are multifactorial (Cathleen et al., 2006), (Tinetti et al., 1989) and their intrinsic causes include altered balance, gait, muscle strength, visual acuity, cognition and the presence of chronic diseases (van Schoor et al., 2002).

The participation of environmental risk factors might reach up to 50% of the falls in elderly that live in the community. These factors include poor lighting, slippery surfaces, loose or folded rugs, high or narrow stairs, obstacles in the way (low furniture, small objects, wires), lack of rails in halls and bathrooms, extremely low or high shelves, inadequate shoes and clothes, poorly maintained streets with holes or irregularities and inappropriate orthosis.

A reduction of approximately 30% in the strength is found in individual ages ranging from 50 to 70. These changes are more common in women than in men, more prevalent in lower limbs than in upper limbs and a great amount of this reduction in strength is caused by a selective atrophy of Type IIB fibers (American College of Sports Medicine Position, 1998).

Evidences have shown that specific exercises might reduce the risk factors for falls and the number of falls in the elderly.

The purpose of our study was to evaluate the efficacy of the resistance training associated to a proprioceptive training in the prevention of falls and reduction of the respective risk factors in postmenopausal women with osteoporosis.

### **2. Physical exercise to prevent falls**

of puberty and beginning of adulthood. The great variation in bone mass peak is explained not only by hereditary factors but also by gender, race, eating habits, several hormone influence, body composition of lean mass and body fat, intercurrent diseases, chronic use of

Like any other chronic disease, the ethiology of osteoporosis is multifactorial. Genetic factors contribute approximately with 46% to 62% of bone mineral density (BMD) whereas other

Osteoporosis clinical symptoms do not usually occur before a fracture occurrence. Osteopo‐ rosis is considered an asymptomatic disease. Indeed, during the disease progression, the bones become progressively more fragile without affecting the individuals. This characteristic of being a silent disease exposes the population to even a greater risk of suffering a fracture. Osteoporosis is considered a "silent disease" until a fracture occurs. Approximately 1.5 million fractures per year are attributable to this disease. Only in the USA, these fractures result in 500.000 hospitalizations, 800.000 emergency room visits, 2.6 million physician visits. The treatment cost is high. In 2002, 12 billion dollars to 18 billion dollars were spent (Gass & Huges, 2006). In 1998, cost management of osteoporosis fractures in the UK recorded 942 million pounds per year (Szejnfeld et al., 2007). Osteoporosis has become one of the major public health problems. Nowadays, the impact of osteoporosis is compared to the impact caused by most important health problems, such as cardiovascular diseases and cancer (Froes et al., 2002). It exposes the fallers to a high risk of fractures (Johnell et al., 2005; Siris et al., 2006). The first hip fracture is associated to 2.5-fold increased risk of subsequent fracture (Cólon-Emeric et al.,

It is believed that about 25% of menopausal women in the USA will exhibit some kind of fracture as a consequence of osteoporosis. The most severe fractures are the fractures of femur and they are associated with higher medical expenses than all other osteoporotic fractures together (Moreira & Xaxier, 2001). The incidence of these fractures has doubled in the last 25 years and it is estimated that six million people in the world will suffer fracture of the proximal femur in 2050. Fractures resulted from the decrease of bone mineral loss are considered an orthopedic epidemic leading to an increase in costs for several countries and consequently

There have been a significant number of evidences showing that the decrease in bone quality, from generation to generation, is caused by a change in life style, having as a main determinant the lack of physical activity. This evidence varies with the biology of the basic bone. However, epidemiological studies indicate that physical activity is the most important factor to maintain

Almost all hip fractures (more than 90%) occur as a result of a fall and these fractures are related not only to the decreased bone mass, but also to other factors such as reduction of balance, muscle strength and power in the lower extremities (American College of Sports Medicine [ACSM], 1995; Parkkari et al. 1999). Therefore, aging and alterations in balance and muscle strength, as well as sensorial changes, predispose patients with osteoporosis to a higher risk

medications and physical activity (Brandão & Vieira, 1999).

216 Topics in Osteoporosis

causes include lifestyle, diet and physical exercise (Neto et al., 2002).

2003) with a high level of morbidity and mortality (Cathleen et al., 2006).

representing a big social and economic problem (Ramalho et al., 2001).

bone mass and prevent fractures (Mosekilde, 1995).

of having fractures due to falls.

Prevention in individuals older than 60 years has an important role in avoiding adverse consequences resulting from falls (Weatherall, 2004).

The work to prevent fractures related to osteoporosis should focus the prevention or increase of material and structural properties of the bone, the prevention of falls and improvement of total mass of lean tissue (American College of Sports Medicine, 1995).

The American College of Sports Medicine recommends that:


### **2.1. Exercises for postural control**

Postural control is a result of the combination of several types of sensorial information, such as visual, vestibular and somatosensorial information, and passive and active properties of the nervous system and skeletomuscle system that composes the human postural control system (Figure 2), (Shumway-Cook et al., 2000).

Exercises to stimulate proprioception and dynamic stabilization should be performed in closed-chain activities and with small movements, since the compression stimulates the articular receptors and the changes in the curve length-tension stimulate the muscle receptors. Limbs repositioning exercises should also be performed to stimulate the sense of joint position

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219

The improvement of dynamic stiffness is another important aspect. It is suggested that muscle receptors increase its sensitivity through the increase of dynamic stiffness (Adler et al., 2008).

Exercises that involve eccentric training, like going down the stairs and landing after jumps, are the most efficient to increase anticipatory and reactive muscular stiffness (Bas‐

The reactive neuromuscular control is reached through exercises that create unexpected situations, such as perturbations in unstable surfaces in unipodal support and during gait. Apparently, this kind of training improves the preparatory and reactive muscle activation

**1.** 5 – 10 minutes of warm-up, with stretching movements for upper and lower limbs, 03 repetitions for each movement being kept for 30 seconds, with 30-second intervals among the series. After stretching, movements of fast gait as previous warm-up were performed

**2.** Proprioceptive exercises followed an evolution sequence based on the use of stable surfaces to unstable, walking straight forward progressing to changes in direction, from gait with no obstacles to gait with obstacles, alteration in the support base (from open to closed), exercises with eyes open to closed eyes, always respecting the functional capacity of each patient and progressively increasing the difficulty of each exercise. To aid the training, cones, balance boards, sticks, mats and trampolines were used. According to the

Stretching should be performed during the warm up and in the last phase. A great joint range of motion (ROM) increases the muscle, reduces the risk of lesion and increases the cartilage nutrition. Painful joints should not be stretched excessively to a point that will result in more pain; all movements should be made in order to get the maximum pain-free ROM. The use of heat before stretching reduces pain and increases the range. At least three sessions of stretching might be performed a week. In the beginning, three to five repetitions and a gradual increase up to 10 repetitions is the ideal. The muscle should be stretched during 10 to 30 seconds.

Muscle strengthening should be acquired with weights or elastic bands which will give endurance to the movement. The training protocols should include the following principles:

and in the end of the session, slow gait movements and stretching.

patient's evolution, the exercises were combined creating the circuits.

and neuromuscular control (Lephart & Henry, 1995).

tian et al., 2006).

(Swanik et al., 2002).

**2.2. Stretching**

**2.3. Muscle strengthening**

The training protocol might include:

The postural control system use three functions that are required to maintain balance: support, stabilization and balance. The body should contract the adequate muscles to sustain the body against gravity; the articular segments should be stabilized and the body should be stabilized in the body's support base (Rothwell, 1994). This way the treatment program which aims at falls prevention should contain strength and resistance increasing components for promoting the articular coaptation and to face the gravity, it also should prevent the posture reorganiza‐ tion aligning the body gravity center.

Currently, proprioception is defined as a set of afferent information provided by joints, muscles, tendons and other tissues that reaches the Central Nervous System (CNS) where it is processed, having an influence on reflex responses and voluntary motor control. Propriocep‐ tion contributes to postural control, joint stability and several conscious sensations (Lephart & Fu, 2000).

Therefore the sensorial-motor training becomes indispensable for an appropriate falls pre‐ vention program, since it potentializes the propriocetive information captation and transmis‐ sion providing to SNC information regarding the contraction speed, movement speed, articular position and angle, which are fundamental for a good motor control.

It is extremely important to understand that proprioception is only limited to the acquisition of the mechanical stimulus and its transduction in neural stimuli, not having any influence on the CNS processing and its motor response (Lephart & Fu, 2000).

Proprioception is part of a system denominated somatosensorial system. This includes all mechanical information provided by the mechanoreceptors. The feeling of pain is provid‐ ed by the nociceptors and the thermal information provided by thermoreceptors (Guyton & Hall, 2006).

All propriocetive information are originated at the muscular and tendon receptors called muscular fusion and Golgi tendon organ and receptors located in ligaments, articular capsule, meniscus and cutaneous tissues (Guyton & Hall, 2006).

Four elements should be focused to reestablish the sensorimotor deficits: proprioception, stabilization, reactive neuromuscular control and functional motor patterns (Lephart & Henry, 1995).

The proprioceptive mechanism comprises both conscious and unconscious pathways. Therefore, the prescribed exercises need to include conscious exercises to stimulate the cognition as well as sudden and unexpected alterations of joint position that initiate reflex muscle contraction. These exercises should involve balance in an unstable surface while the individual perform functional activities. The purpose of the dynamic stabilization training is to improve the co-activation between the antagonist muscles (Hurd et al., 2006)

Exercises to stimulate proprioception and dynamic stabilization should be performed in closed-chain activities and with small movements, since the compression stimulates the articular receptors and the changes in the curve length-tension stimulate the muscle receptors. Limbs repositioning exercises should also be performed to stimulate the sense of joint position and neuromuscular control (Lephart & Henry, 1995).

The improvement of dynamic stiffness is another important aspect. It is suggested that muscle receptors increase its sensitivity through the increase of dynamic stiffness (Adler et al., 2008).

Exercises that involve eccentric training, like going down the stairs and landing after jumps, are the most efficient to increase anticipatory and reactive muscular stiffness (Bas‐ tian et al., 2006).

The reactive neuromuscular control is reached through exercises that create unexpected situations, such as perturbations in unstable surfaces in unipodal support and during gait. Apparently, this kind of training improves the preparatory and reactive muscle activation (Swanik et al., 2002).

The training protocol might include:


### **2.2. Stretching**

**2.1. Exercises for postural control**

218 Topics in Osteoporosis

(Figure 2), (Shumway-Cook et al., 2000).

tion aligning the body gravity center.

& Fu, 2000).

& Hall, 2006).

Henry, 1995).

Postural control is a result of the combination of several types of sensorial information, such as visual, vestibular and somatosensorial information, and passive and active properties of the nervous system and skeletomuscle system that composes the human postural control system

The postural control system use three functions that are required to maintain balance: support, stabilization and balance. The body should contract the adequate muscles to sustain the body against gravity; the articular segments should be stabilized and the body should be stabilized in the body's support base (Rothwell, 1994). This way the treatment program which aims at falls prevention should contain strength and resistance increasing components for promoting the articular coaptation and to face the gravity, it also should prevent the posture reorganiza‐

Currently, proprioception is defined as a set of afferent information provided by joints, muscles, tendons and other tissues that reaches the Central Nervous System (CNS) where it is processed, having an influence on reflex responses and voluntary motor control. Propriocep‐ tion contributes to postural control, joint stability and several conscious sensations (Lephart

Therefore the sensorial-motor training becomes indispensable for an appropriate falls pre‐ vention program, since it potentializes the propriocetive information captation and transmis‐ sion providing to SNC information regarding the contraction speed, movement speed,

It is extremely important to understand that proprioception is only limited to the acquisition of the mechanical stimulus and its transduction in neural stimuli, not having any influence on

Proprioception is part of a system denominated somatosensorial system. This includes all mechanical information provided by the mechanoreceptors. The feeling of pain is provid‐ ed by the nociceptors and the thermal information provided by thermoreceptors (Guyton

All propriocetive information are originated at the muscular and tendon receptors called muscular fusion and Golgi tendon organ and receptors located in ligaments, articular capsule,

Four elements should be focused to reestablish the sensorimotor deficits: proprioception, stabilization, reactive neuromuscular control and functional motor patterns (Lephart &

The proprioceptive mechanism comprises both conscious and unconscious pathways. Therefore, the prescribed exercises need to include conscious exercises to stimulate the cognition as well as sudden and unexpected alterations of joint position that initiate reflex muscle contraction. These exercises should involve balance in an unstable surface while the individual perform functional activities. The purpose of the dynamic stabilization training is

to improve the co-activation between the antagonist muscles (Hurd et al., 2006)

articular position and angle, which are fundamental for a good motor control.

the CNS processing and its motor response (Lephart & Fu, 2000).

meniscus and cutaneous tissues (Guyton & Hall, 2006).

Stretching should be performed during the warm up and in the last phase. A great joint range of motion (ROM) increases the muscle, reduces the risk of lesion and increases the cartilage nutrition. Painful joints should not be stretched excessively to a point that will result in more pain; all movements should be made in order to get the maximum pain-free ROM. The use of heat before stretching reduces pain and increases the range. At least three sessions of stretching might be performed a week. In the beginning, three to five repetitions and a gradual increase up to 10 repetitions is the ideal. The muscle should be stretched during 10 to 30 seconds.

### **2.3. Muscle strengthening**

Muscle strengthening should be acquired with weights or elastic bands which will give endurance to the movement. The training protocols should include the following principles:


within the age group proposed by the present research and 80 of them were included in the

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221

Patients were from 65 to 75 years old and only individuals with a postmenopausal osteopo‐ rosis, according to the OMS, with a bone mineral density (BMD) T-score of −2.5 standard deviation (SD), in the lumbar spine, femoral neck or total femur region (Lewiecki et al., 2004)

The following women were excluded: those with secondary osteoporosis, visual deficiency with no possibilities of previous corrections; severe auditive deficiency; with vestibular alteration of important clinical status; as well as women who used assisted walking devices (orthesis or prosthesis); those who planned to be out of town for two consecutive weeks during the 18-week study and also women who presented absolute contraindications for physical

All patients selected according to the inclusion and exclusion criteria signed an informed consent (IC). The randomization was performed by a technical assistant not involved in the research using a computer program and the sequential numbers were kept in opaque, not translucent and sealed envelope being given to one of the two groups based on the Consort

The volunteers were included in two groups: the first group (G1) comprised 40 patients who underwent 18-week proprioceptive and progressive muscular strength training associated to a drug treatment of Osteoporosis; and the second called G2 also included 40 patients that only

The registration of patients was made during the medical evaluation in order to include or exclude the individuals in the research and their personal and clinical data were also registered.

All patients were evaluated by a physical therapist who was blinded to the group to which the patient belonged. The quality of life, functional skills and the risk and number of falls were

The quality of life was evaluated using the Short Form Health Survey (SF-36), a questionnaire displayed in a scale from 0 to 100, where 0 means the worst quality of life and 100 points corresponds to the best quality of life, according to what is proposed by the survey (Ciconelli

The Berg Balance Scale, a test where the maximum score that can be achieved is 56 and where each item has an ordinal scale of five alternatives which varied from 0 to 4 points, was used to

The functional mobility was evaluated by the Timed "Up & Go" Test which measures the time an individual takes to get up of a chair, walks to a line on the floor 3 meters away as fast and safe as possible, turn around, walk back to the chair and sits down again allowing the buttocks and lumbar region to touch the seat surface (Podsiadlo et al., 1991, Shumway-Cook et al.,

evaluate the balance (Berg et al., 1996), Miyamoto et al., 2004).

study, since they met the required inclusion criteria (Fig. 1).

exercise, according to the American College of Sports Medicine.

were included.

recommendations.

**3.1. Evaluation**

evaluated.

et al., 1999).

underwent a conventional drug treatment.


Isometric exercises are indicated for unstable or swollen joints. On the other hand, isometric contractions result in a low articular pressure and are well tolerated by older patients. It should start with contractions with an intensity of approximately 30% of maximal strength, slowly increasing to 80%. The contraction should not be kept for more than 6-10 seconds and the repetitions should be increased from 8 to 10, if tolerated by the patient. It should be performed twice a day during the inflammatory period and after the inflammation is over, it should be increased from 5 to 10 times a day.

Isotonic exercises should include from 8 to 10 exercises involving the major muscle groups (four exercises for the upper limbs and from four to six for the lower limbs). At first, patients should use weights with 40% of the individual's maximal load, increasing up to 80%. Gener‐ ally, a series of four to six repetitions should be made, avoiding the muscle fatigue. At first, the frequency should be at most twice a week but in case of individuals with advanced age or significant fragility the exercises should be made only once a week. Between the sessions, there might be at least one full day of rest.

Changes in strength after resistance exercise training RET are assessed using a variety of methods, including isometric, isokinetic, one-repetition maximum (1-RM), and multiplerepe‐ tition (e.g., 3-RM) maximum-effort protocols. In general, strength increases after RET in older adults seem to be greater with measures of 1-RM or 3-RM performance compared with isometric or isokinetic measures. Older adults can substantially increase their strength after RET—with reported increases ranging from less than 25% to greater than 100% (American College of Sports Medicine, 2009).

### **3. Material and methods**

The present research was approved by the Research Ethics Committee of the Federal Univer‐ sity of São Paulo. The clinical trial was registered in the Australian New Zealand Clinical Trials Registry (ANZCTR).

Among the 758 bone densitometries tests made in the Image Diagnosis Service at the Ambu‐ latório de Especialidades de Interlagos, São Paulo - Brazil, 284 were found positive for Osteoporosis, where 162 of these densitometries tests were from patients which ages were within the age group proposed by the present research and 80 of them were included in the study, since they met the required inclusion criteria (Fig. 1).

Patients were from 65 to 75 years old and only individuals with a postmenopausal osteopo‐ rosis, according to the OMS, with a bone mineral density (BMD) T-score of −2.5 standard deviation (SD), in the lumbar spine, femoral neck or total femur region (Lewiecki et al., 2004) were included.

The following women were excluded: those with secondary osteoporosis, visual deficiency with no possibilities of previous corrections; severe auditive deficiency; with vestibular alteration of important clinical status; as well as women who used assisted walking devices (orthesis or prosthesis); those who planned to be out of town for two consecutive weeks during the 18-week study and also women who presented absolute contraindications for physical exercise, according to the American College of Sports Medicine.

All patients selected according to the inclusion and exclusion criteria signed an informed consent (IC). The randomization was performed by a technical assistant not involved in the research using a computer program and the sequential numbers were kept in opaque, not translucent and sealed envelope being given to one of the two groups based on the Consort recommendations.

The volunteers were included in two groups: the first group (G1) comprised 40 patients who underwent 18-week proprioceptive and progressive muscular strength training associated to a drug treatment of Osteoporosis; and the second called G2 also included 40 patients that only underwent a conventional drug treatment.

### **3.1. Evaluation**

**•** muscle contraction exercises should be made in a moderate speed;

**•** muscles should not be exercised to fatigue;

**•** exercise endurance should be submaximal;

include few repetitions;

220 Topics in Osteoporosis

increased from 5 to 10 times a day.

might be at least one full day of rest.

College of Sports Medicine, 2009).

**3. Material and methods**

Registry (ANZCTR).

**•** exercises should be chosen according to joint stability and degree of pain and edema;

**•** inflamed articular joints should be strengthen with isometric exercises and at first it should

Isometric exercises are indicated for unstable or swollen joints. On the other hand, isometric contractions result in a low articular pressure and are well tolerated by older patients. It should start with contractions with an intensity of approximately 30% of maximal strength, slowly increasing to 80%. The contraction should not be kept for more than 6-10 seconds and the repetitions should be increased from 8 to 10, if tolerated by the patient. It should be performed twice a day during the inflammatory period and after the inflammation is over, it should be

Isotonic exercises should include from 8 to 10 exercises involving the major muscle groups (four exercises for the upper limbs and from four to six for the lower limbs). At first, patients should use weights with 40% of the individual's maximal load, increasing up to 80%. Gener‐ ally, a series of four to six repetitions should be made, avoiding the muscle fatigue. At first, the frequency should be at most twice a week but in case of individuals with advanced age or significant fragility the exercises should be made only once a week. Between the sessions, there

Changes in strength after resistance exercise training RET are assessed using a variety of methods, including isometric, isokinetic, one-repetition maximum (1-RM), and multiplerepe‐ tition (e.g., 3-RM) maximum-effort protocols. In general, strength increases after RET in older adults seem to be greater with measures of 1-RM or 3-RM performance compared with isometric or isokinetic measures. Older adults can substantially increase their strength after RET—with reported increases ranging from less than 25% to greater than 100% (American

The present research was approved by the Research Ethics Committee of the Federal Univer‐ sity of São Paulo. The clinical trial was registered in the Australian New Zealand Clinical Trials

Among the 758 bone densitometries tests made in the Image Diagnosis Service at the Ambu‐ latório de Especialidades de Interlagos, São Paulo - Brazil, 284 were found positive for Osteoporosis, where 162 of these densitometries tests were from patients which ages were

**•** pain or edema in a joint after an hour of exercise indicates excessive activity.

The registration of patients was made during the medical evaluation in order to include or exclude the individuals in the research and their personal and clinical data were also registered.

All patients were evaluated by a physical therapist who was blinded to the group to which the patient belonged. The quality of life, functional skills and the risk and number of falls were evaluated.

The quality of life was evaluated using the Short Form Health Survey (SF-36), a questionnaire displayed in a scale from 0 to 100, where 0 means the worst quality of life and 100 points corresponds to the best quality of life, according to what is proposed by the survey (Ciconelli et al., 1999).

The Berg Balance Scale, a test where the maximum score that can be achieved is 56 and where each item has an ordinal scale of five alternatives which varied from 0 to 4 points, was used to evaluate the balance (Berg et al., 1996), Miyamoto et al., 2004).

The functional mobility was evaluated by the Timed "Up & Go" Test which measures the time an individual takes to get up of a chair, walks to a line on the floor 3 meters away as fast and safe as possible, turn around, walk back to the chair and sits down again allowing the buttocks and lumbar region to touch the seat surface (Podsiadlo et al., 1991, Shumway-Cook et al., 1997). The TUG was performed along with other balance and functional mobility tests (Bohannon et al., 2006) since it is a sensitive and specific measurement of the fall probability among elderly adults (Large et al., 2006, Kristensen et al., 2007).

schedule their re-evaluations, as illustrated in the chart based on the Consort recommendations

Stability exercises Unipodal or bipodal support / open or

758 bone densitometries tests evaluated

80 randomized

33 analysed 32 analysed

**Figure 1.** Organizational chart (based on Consort recommendations) including the inclusion and exclusion analysis,

center

contact

member

**Options of Exercises Evolution of Exercises Time or # of repetitions**

Dyna disc) Eyes open or closed / stable or unstable 10 rep / 30s

Anteroposterior and latero-lateral gait With or without obstacle and

Exercises with sticks With or without arm

Mat exercises Go up/down: 1 to 3 mats 10 rep / 3 series Exercises on the stairs Variation in speed 10 rep / 3 series Exercises with sticks With or without arm movements 10 rep / 3 series

With or without obstacle and Variation

After evaluating 758 patients, 80 were randomized and only 65 concluded the study, being 33 patients from G1 and 32 from G2. Three patients included in the G1 group did not complete the study because they did not have appropriate means of transportation, two others due to financial conditions, another one moved to a different city and the last one abandoned the study due to personal reasons. Two patients from G2 group did not complete the study for personal reasons, three started exercising regularly in another place, one quit due to illness of a family member and two others because we could not contact them by phone in order to schedule their re-evaluations, as illustrated in the chart based on the Consort recommendations (Moher et al., 2001) (Fig.

> 40 allocated into the control group

8 didn't complete the study 3 started exercising in another

2 for personal reasons 2 due to loss of telephone

1 due to illness of a family

658 were excluded 374 didn't have osteoporosis 122 weren't in the age

162 didn't meet the

group

Mat exercises Go up/down: 1 to 3 mats 10 rep / 3 series Exercises on the stairs Variation in speed 10 rep / 3 series

The Effectiveness of Progressive Load Training Associated to the Proprioceptive Training…

close base 10 rep / 30s

movements 10 rep / 3 series

Variation in speed 10 rep (3 m)

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223

in speed 10 rep (3 m)

(Moher et al., 2001) (Fig. 1).

**Table 1.** Examples of exercises

1).

Balance exercises (balance board, mini-trampoline.

Anteroposterior and latero-lateral gait

**3.3. Data analysis** 

Table 1. Examples of exercises

40 allocated into the exercise group

> 7 didn't complete the study 3 no means of transportation 2 due to financial conditions 1 moved to a different city 1 for personal reasons

randomization, group allocation, losses and patients who concluded the study.

The dynamic strength of the quadriceps muscle was evaluated by the One Repetition Maxi‐ mum (1 RM) Test that measures the maximum weight a subject can lift with one repetition when making a standard weight lifting exercise. Three attempts were made to reach the plateau in the 1-RM score with 3-minute intervals between each attempt (Weier 1997, Hortobagyi et al., 1998).

The number of falls was evaluated by monitoring the immediate report of falls from patients of both groups during 24 weeks. The patients were also questioned if they experienced falls six months preceding the study.

### **3.2. Treatment protocol of Teixeira & Silva et al., 2010**

The protocol consisted of a routine where: 1) the patients participated in a 5-10 minutes warmup in a treadmill, static stretching exercises (global and segmentary) for the upper and lower limbs, lumbar, cervical and thoracic region with 3 repetitions for each muscle or muscular group, maintaining the stretching for 30 seconds between the 2 series of exercises. 2) The functional exercises (proprioception and balance) were performed in a routine that follows a progressive order beginning with stable surface and changing to unstable surfaces, gait training without obstacles and then performance of gait training with obstacles, exercises first with eyes opened and then eyes closed, first low speed exercises and according to the patient performance high speed exercises, bipedal training and then unipedal, also using resources such as balance, trampoline and proprioceptive boards always using the same progressive order (table 1). 3) Strengthening exercises included leg extensions with a load up to 80% of 1- RM, following a protocol of two weeks of adjustment wearing 1 to 2 kilos ankle weight, progressing for 50%, 60%, 70% up to 80% of 1-RM (American College of Sports Medicine, 2002).

Examples of exercises: ten repetitions with one-minute intervals for antero-posterior and latero-lateral gait; gait with obstacles (20 cm high); gait over mattress; going up and down the stairs; change in direction according to the sound stimulus; balance exercises lasting 30 seconds and with one-minute interval for unipodal and bipodal support on the floor with eyes open and/or closed; change in floor for a more unstable surface such as a trampoline and balance board; exercises with dissociation of waist and use of a stick (Table 1).

### **3.3. Data analysis**

After evaluating 758 patients, 80 were randomized and only 65 concluded the study, being 33 patients from G1 and 32 from G2. Three patients included in the G1 group did not complete the study because they did not have appropriate means of transportation, two others due to financial conditions, another one moved to a different city and the last one abandoned the study due to personal reasons. Two patients from G2 group did not complete the study for personal reasons, three started exercising regularly in another place, one quit due to illness of a family member and two others because we could not contact them by phone in order to transportation, two others due to financial conditions, another one moved to a different city and the last one abandoned the study

in order to schedule their re-evaluations, as illustrated in the chart based on the Consort recommendations (Moher et al., 2001) (Fig.

schedule their re-evaluations, as illustrated in the chart based on the Consort recommendations (Moher et al., 2001) (Fig. 1).


**Table 1.** Examples of exercises due to personal reasons. Two patients from G2 group did not complete the study for personal reasons, three started exercising regularly in another place, one quit due to illness of a family member and two others because we could not contact them by phone

1).

1997). The TUG was performed along with other balance and functional mobility tests (Bohannon et al., 2006) since it is a sensitive and specific measurement of the fall probability

The dynamic strength of the quadriceps muscle was evaluated by the One Repetition Maxi‐ mum (1 RM) Test that measures the maximum weight a subject can lift with one repetition when making a standard weight lifting exercise. Three attempts were made to reach the plateau in the 1-RM score with 3-minute intervals between each attempt (Weier 1997, Hortobagyi et

The number of falls was evaluated by monitoring the immediate report of falls from patients of both groups during 24 weeks. The patients were also questioned if they experienced falls

The protocol consisted of a routine where: 1) the patients participated in a 5-10 minutes warmup in a treadmill, static stretching exercises (global and segmentary) for the upper and lower limbs, lumbar, cervical and thoracic region with 3 repetitions for each muscle or muscular group, maintaining the stretching for 30 seconds between the 2 series of exercises. 2) The functional exercises (proprioception and balance) were performed in a routine that follows a progressive order beginning with stable surface and changing to unstable surfaces, gait training without obstacles and then performance of gait training with obstacles, exercises first with eyes opened and then eyes closed, first low speed exercises and according to the patient performance high speed exercises, bipedal training and then unipedal, also using resources such as balance, trampoline and proprioceptive boards always using the same progressive order (table 1). 3) Strengthening exercises included leg extensions with a load up to 80% of 1- RM, following a protocol of two weeks of adjustment wearing 1 to 2 kilos ankle weight, progressing for 50%, 60%, 70% up to 80% of 1-RM (American College of Sports Medicine, 2002).

Examples of exercises: ten repetitions with one-minute intervals for antero-posterior and latero-lateral gait; gait with obstacles (20 cm high); gait over mattress; going up and down the stairs; change in direction according to the sound stimulus; balance exercises lasting 30 seconds and with one-minute interval for unipodal and bipodal support on the floor with eyes open and/or closed; change in floor for a more unstable surface such as a trampoline and balance

After evaluating 758 patients, 80 were randomized and only 65 concluded the study, being 33 patients from G1 and 32 from G2. Three patients included in the G1 group did not complete the study because they did not have appropriate means of transportation, two others due to financial conditions, another one moved to a different city and the last one abandoned the study due to personal reasons. Two patients from G2 group did not complete the study for personal reasons, three started exercising regularly in another place, one quit due to illness of a family member and two others because we could not contact them by phone in order to

board; exercises with dissociation of waist and use of a stick (Table 1).

among elderly adults (Large et al., 2006, Kristensen et al., 2007).

**3.2. Treatment protocol of Teixeira & Silva et al., 2010**

al., 1998).

222 Topics in Osteoporosis

**3.3. Data analysis**

six months preceding the study.

**Figure 1.** Organizational chart (based on Consort recommendations) including the inclusion and exclusion analysis, randomization, group allocation, losses and patients who concluded the study.

### **3.4. Statistical analysis**

In order to verify the presumed normality in the data distribution, the Shapiro-Wilk test was used, as well as the Q-Q plot. Since the studied variable distribution could not be rounded up by the normal distribution the median and quartile 1 and 3 were calculated to describe the variables in the study.

**Variables Moment Control (N=32) Intervention (N=33) ∆ (intervention-control) p-value**

t0 54,4(26,42) 63,95(22,56) — —

t0 44,05(33,95) 36,05(34,63) — —

t0 38,88(20,63) 38,21(19,94) — —

t0 52(22,38) 51,09(17,72) — —

t0 50,71(22,4) 58,26(20,26) — —

t0 63,62(29,4) 69,4(26,69) — —

t0 52,4(44,29) 55,79(40,38) — —

t0 48,29(21,08) 64,3(20,52) — —

**Variables Moment Control (N=32) Intervention (N=33) ∆(intervention-control) p-value**

Maximum load (kg) t0 7.6(2.27) 8.02(1.81) — — Maximum load (kg) t18 8.1(2.81) 14.81(3.14) 3.65[2.74;4.57] < 0.0001 Time up and go (s) t0 11.35(2.88) 10.74(2.23) — — Time up and go (s) t18 11.15(2.55) 6.9(1.11) -3.96[-4.63;-3.29] < 0.0001

**Table 3.** Pre and Post-Training Values for the Time Up and Go Test (s), maximum load (Kg) and Berg Balance Scale

The data were expressed as the mean (standard deviation) or average [95% confidence interval].

The data were expressed as the mean (standard deviation) or average [95% confidence interval].

t18 50,6(29,45) 82,44(17,3) 25,11[17,7;32,52] < 0.0001

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225

t18 43,57(37,42) 92,44(17,71) 51,62[39,97;63,28] < 0.0001

t18 44,93(21,51) 65,58(23,2) 20,98[12,29;29,68] < 0.0001

t18 55,88(23,81) 73,67(17,54) 18,38[11,29;25,47] < 0.0001

t18 54,05(23,51) 74,88(15,49) 16,55[9,61;23,48] < 0.0001

t18 68,05(28,51) 93,12(13,68) 23,06[14,32;31,81] < 0.0001

t18 61,12(44,15) 85,28(28,49) 22,7[8,69;36,7] < 0.0018

t18 52,86(21,05) 78,84(17,41) 15,26[9,03;21,48] < 0.0001

Functional skills

Physical aspects

Pain

General Health Status

Vitality

Social aspects

Emotional aspects

Mental health

(scores).

**Table 2.** Pre and post-training values for SF-36 scores

The chi-square test was employed to evaluate the epidemiological data in the baseline.

The significance of the influence of the time the treatment was performed (pre and postintervention) and influence of the groups (control and experimental) was evaluated by using the nonparametric hypothesis test (Robertson et al., 2005).

The statistical significance was set at P ≤ 0.05. All the statistical process was performed with the statistical language R (version, 2.6.2; R Foundation for Statistical Computing, Vienna, Austria).

### **3.5. Results**

The basal characteristics of the patients of both groups were similar in relation to age, bone mineral density, history of fractures, osteoporosis treatment, use of diuretics, hypnotics, and antidepressants, other rheumatic diseases and number of individuals that fell 6 months prior to the study.

According to data described in Table 2, it is possible to conclude that the scores for SF-36 in the intervention group were better in all eight sub-scales after the rehabilitation period compared to the admission time, as well as the control group. These changes were statistically (p ≤ 0.007) and clinically significant (a change of at least 13.5 points in each sub-scale of the SF-36) for all sub-scales.

According to what was described in Table 3, there was a significant difference in the results of the Timed Up & Go Test in the pre and post- training (p < 0.001) for the experimental group. Furthermore, the post-training values for the experimental group were significantly greater than the ones shown by the control group (p < 0.001). In terms of maximum dynamic load, a significant increase between pre and post-training in the experimental group (p < 0.001) was observed. Besides that, the post-training values in the experimental group were significantly greater than the ones of the control group (p < 0.001).

Variables such as physical activity, rotational component and decreased base showed a significant increase when compared to the admission data and the control group (p ≤ 0.003). The general score of the Berg Scale (TABLE 4) showed a significant increase in the experimental group (p < 0.001), where the post-training values were significantly greater in the experimental group compared to the control (p < 0.001). No significant differences were found in the items Transference and Static Tests. Although the changes in numbers are not huge they are consistent. A lot of people in the experimental group showed increased scores; therefore the possibility of a small score may not be great. No statistical reduction in the number of falls per patient was observed.

The Effectiveness of Progressive Load Training Associated to the Proprioceptive Training… http://dx.doi.org/10.5772/54554 225


The data were expressed as the mean (standard deviation) or average [95% confidence interval].

**Table 2.** Pre and post-training values for SF-36 scores

**3.4. Statistical analysis**

224 Topics in Osteoporosis

variables in the study.

Vienna, Austria).

**3.5. Results**

to the study.

SF-36) for all sub-scales.

patient was observed.

In order to verify the presumed normality in the data distribution, the Shapiro-Wilk test was used, as well as the Q-Q plot. Since the studied variable distribution could not be rounded up by the normal distribution the median and quartile 1 and 3 were calculated to describe the

The significance of the influence of the time the treatment was performed (pre and postintervention) and influence of the groups (control and experimental) was evaluated by using

The statistical significance was set at P ≤ 0.05. All the statistical process was performed with the statistical language R (version, 2.6.2; R Foundation for Statistical Computing,

The basal characteristics of the patients of both groups were similar in relation to age, bone mineral density, history of fractures, osteoporosis treatment, use of diuretics, hypnotics, and antidepressants, other rheumatic diseases and number of individuals that fell 6 months prior

According to data described in Table 2, it is possible to conclude that the scores for SF-36 in the intervention group were better in all eight sub-scales after the rehabilitation period compared to the admission time, as well as the control group. These changes were statistically (p ≤ 0.007) and clinically significant (a change of at least 13.5 points in each sub-scale of the

According to what was described in Table 3, there was a significant difference in the results of the Timed Up & Go Test in the pre and post- training (p < 0.001) for the experimental group. Furthermore, the post-training values for the experimental group were significantly greater than the ones shown by the control group (p < 0.001). In terms of maximum dynamic load, a significant increase between pre and post-training in the experimental group (p < 0.001) was observed. Besides that, the post-training values in the experimental group were significantly

Variables such as physical activity, rotational component and decreased base showed a significant increase when compared to the admission data and the control group (p ≤ 0.003). The general score of the Berg Scale (TABLE 4) showed a significant increase in the experimental group (p < 0.001), where the post-training values were significantly greater in the experimental group compared to the control (p < 0.001). No significant differences were found in the items Transference and Static Tests. Although the changes in numbers are not huge they are consistent. A lot of people in the experimental group showed increased scores; therefore the possibility of a small score may not be great. No statistical reduction in the number of falls per

The chi-square test was employed to evaluate the epidemiological data in the baseline.

the nonparametric hypothesis test (Robertson et al., 2005).

greater than the ones of the control group (p < 0.001).


The data were expressed as the mean (standard deviation) or average [95% confidence interval].

**Table 3.** Pre and Post-Training Values for the Time Up and Go Test (s), maximum load (Kg) and Berg Balance Scale (scores).


Table 3. Pre and Post-Training Values for the Time Up and Go Test (s), maximum load (Kg) and Berg Balance Scale (scores).

**Control (n=32) Intervention (n=33)**

The Effectiveness of Progressive Load Training Associated to the Proprioceptive Training…

T0 0,5625 (18/32) 0,6060 (20/33) T18 0,6250 (20/32) 0,1515 (5/33) Razão T18/T0 1,111 0,25

Evidences have shown that specific exercises might reduce the risk factors for falls and number

**Muscle Strength Training and Balance Training**

We identified women 65 to 75 years of age in whom osteoporosis had been diagnosed by dual-energy X-ray absorptiometry in our hospital between 1996 and 2000 and who were not engaged in regular weekly programs of moderate or hard exercise. Women who agreed to participate were randomly assigned to participate in a twice-weekly exercise class or to not participate in the class. We measured baseline data and, 20 weeks later, changes in static balance (by dynamic posturography), dynamic balance (by a timed figure-eight run) and knee extension strength (by dynamometry).

Sixty-six consecutive elderly women were selected from the Osteometabolic Disease

Of 93 women who began the trial, 80 completed it. Before adjustment for covariates, the intervention group tended to have greater, although nonsignificant, improvements in static balance (mean difference 4.8%, 95% confidence interval [CI] –1.3% to 11.0%), dynamic balance (mean difference 3.3%, 95% CI –1.7% to 8.4%) and knee extension strength (mean difference 7.8%, 95% CI – 5.4% to 21.0%).

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227

Sixty women completed the study and were analyzed. The BBS difference was significant

**Autor Objective Desing Results**

**Table 5.** Comparison of odds ratio falling between the control and intervention groups.

The data were expressed as ratio and odds ratio.

of falls in older people (Table 6, 7).

Exercise programs improve balance, strength and agility in elderly people and thus may prevent falls. However, specific exercise programs that might be widely used in the community and that might be "prescribed" by physicians, especially for patients with osteoporosis, have not been evaluated. We conducted a randomized controlled trial of such a program designed specifically for women with osteoporosis.

The purpose of this study was to investigate the effect of a 12 month Balance Training

**4. Discussion**

**Carter**, et al, 2002

Madureira et al; 2007

General score t0 51.71(4.1) 52.07(3.63) — —

2) was observed. We could also observe a significant reduction between the pre and post-training in the experimental group (p <

Besides that, the post-training values in the experimental group were significantly lower than the ones shown by the control group

T0 0,5625 (18/32) 0,6060 (20/33) T18 0,6250 (20/32) 0,1515 (5/33) Razão T18/T0 1,111 0,25

**Table 4.** Table 4. Pre and Post-Training Values for the Berg Balance Scale (scores). t18 51.26(4.66) 55.12(1.73) 3.58[2.75;4.42] < 0.0001

0.001).

Based on the positive results of the protocol used for the physical status, an expressive reduction in the number of total falls (Figure 2) was observed. We could also observe a significant reduction between the pre and post-training in the experimental group (p < 0.001). The data were expressed as the mean (standard deviation) or average [95% confidence interval]. Based on the positive results of the protocol used for the physical status, an expressive reduction in the number of total falls (Figure

Table 4. Table 4. Pre and Post-Training Values for the Berg Balance Scale (scores).

Figure 2. Number of falls 24 weeks preceding the treatment (Before) and 24 weeks after the treatment (After), in the Intervention (G1) and Control Groups (G2) **Figure 2.** Number of falls 24 weeks preceding the treatment (Before) and 24 weeks after the treatment (After), in the Intervention (G1) and Control Groups (G2)

(p < 0.001), confirmed by the odds ratio (TABLE 5). **Control (n=32) Intervention (n=33)**  Besides that, the post-training values in the experimental group were significantly lower than the ones shown by the control group (p < 0.001), confirmed by the odds ratio (TABLE 5).

Table 5. Comparison of odds ratio falling between the control and intervention groups.


The data were expressed as ratio and odds ratio.

**Table 5.** Comparison of odds ratio falling between the control and intervention groups.

### **4. Discussion**

**Variables Moment Control (N=32) Intervention (N=33) ∆(intervention-control) p-value** Decreased base t0 9.98(2.31) 10.05(1.4) — —

The data were expressed as the mean (standard deviation) or average [95% confidence interval].

Static Tests t0 11.88(0.33) 11.95(0.21) — —

Rotational component t0 11.21(1.14) 11.37(1.75) — —

Transference t0 11.24(1.16) 11.53(1.05) — —

**Variables Moment Control** 

General score t0 51.71(4.1) 52.07(3.63) — —

Based on the positive results of the protocol used for the physical status, an expressive reduction in the number of total falls (Figure 2) was observed. We could also observe a significant reduction between the pre and post-training in the experimental group (p < 0.001).

The data were expressed as the mean (standard deviation) or average [95% confidence interval].

**0 5 10 15 20 25**

**Number of falls**

**Figure 2.** Number of falls 24 weeks preceding the treatment (Before) and 24 weeks after the treatment (After), in the

Besides that, the post-training values in the experimental group were significantly lower than the ones shown by the control group (p < 0.001), confirmed by the odds ratio (TABLE 5).

Table 4. Table 4. Pre and Post-Training Values for the Berg Balance Scale (scores).

The data were expressed as the mean (standard deviation) or average [95% confidence interval].

**Falls**

Rotational

**Table 4.** Table 4. Pre and Post-Training Values for the Berg Balance Scale (scores).

0.001).

226 Topics in Osteoporosis

Groups (G2)

Intervention (G1) and Control Groups (G2)

**G1**

 **G2**

**T 18**

**T 0**

**T 18**

**T0**

(p < 0.001), confirmed by the odds ratio (TABLE 5).

Table 5. Comparison of odds ratio falling between the control and intervention groups.

t18 9.67(2.2) 11.28(1.44) 1.56[1.04;2.08] < 0.0001

Variables such as physical activity, rotational component and decreased base showed a significant increase when compared to the admission data and the control group (p ≤ 0.003). The general score of the Berg Scale (TABLE 4) showed a significant increase in the experimental group (p < 0.001), where the post-training values were significantly greater in the experimental group compared to the control (p < 0.001). No significant differences were found in the items Transference and Static Tests. Although the changes in numbers are not huge they are consistent. A lot of people in the experimental group showed increased scores; therefore the

Decreased base t0 9.98(2.31) 10.05(1.4) — —

Static Tests t0 11.88(0.33) 11.95(0.21) — t18 11.71(0.99) 12(0) 0.19[-0.07;0.45] 0.1537

component t0 11.21(1.14) 11.37(1.75) — —

Transference t0 11.24(1.16) 11.53(1.05) — t18 11.26(1.21) 11.81(0.93) 0.35[0.01;0.69] 0.0533 General score t0 51.71(4.1) 52.07(3.63) — —

Based on the positive results of the protocol used for the physical status, an expressive reduction in the number of total falls (Figure 2) was observed. We could also observe a significant reduction between the pre and post-training in the experimental group (p <

Figure 2. Number of falls 24 weeks preceding the treatment (Before) and 24 weeks after the treatment (After), in the Intervention (G1) and Control

Besides that, the post-training values in the experimental group were significantly lower than the ones shown by the control group

T0 0,5625 (18/32) 0,6060 (20/33) T18 0,6250 (20/32) 0,1515 (5/33) Razão T18/T0 1,111 0,25

**Control (n=32) Intervention (n=33)** 

**Intervention** 

t18 9.67(2.2) 11.28(1.44) 1.56[1.04;2.08] < 0.0001

t18 11.17(099) 11.91(0.37) 0.7[0.43;0.97] < 0.0001

t18 51.26(4.66) 55.12(1.73) 3.58[2.75;4.42] < 0.0001

**(N=33) Δ(intervention-control) p-value** 

Table 3. Pre and Post-Training Values for the Time Up and Go Test (s), maximum load (Kg) and Berg Balance Scale (scores).

t18 11.71(0.99) 12(0) 0.19[-0.07;0.45] 0.1537

possibility of a small score may not be great. No statistical reduction in the number of falls per patient was observed.

t18 11.17(099) 11.91(0.37) 0.7[0.43;0.97] < 0.0001

**(N=32)** 

t18 11.26(1.21) 11.81(0.93) 0.35[0.01;0.69] 0.0533

t18 51.26(4.66) 55.12(1.73) 3.58[2.75;4.42] < 0.0001

Evidences have shown that specific exercises might reduce the risk factors for falls and number of falls in older people (Table 6, 7).



**Muscle Strength Training and Balance Training**

measure was fall rate, measured by using monthly fall calendars for 1 year. Secondary outcomes were balance confidence (Activity-specific Balance Confidence Scale), quality of life (QOL), and activity level (LASA), assessed posttreatment subsequent to the program and after 1 year of follow-up.

The Effectiveness of Progressive Load Training Associated to the Proprioceptive Training…

Sample consisted of 33 women with osteoporosis, randomized into one of two groups: intervention group, in which exercises for balance and improvement of muscular strength of the inferior members were performed for 8 wks (n = 17, age 72.8 +/- 3.6 yrs); control group, which was women not practicing exercises (n = 16, age 74.4 +/- 3.7 yrs).At baseline and after 8 wks of treatment, postural control was assessed using a force plate (Balance Master, Neurocom), and muscular strength during ankle dorsiflexion, knee extension, and flexion was assessed by dynamometry.

One hundred sedentary postmenopausal women with osteoporosis, ages ranging from 55 to 75, were randomized into two groups: the intervention group comprised of 50 patients who underwent a 18-week of progressive load training for the quadriceps and proprioception training and the control group that included 50 patients of osteoporosis. The muscular strength, balance, functional

61; 95% confidence interval, . 40-.94). Balance confidence in the exercise group increased by 13.9% (P=.001). No group differences were observed in QOL and activity levels.

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229

When compared with the control group, individuals in the intervention group significantly improved the center of pressure velocity (P = 0.02) in the modified clinical test of sensory interaction for balance test, center of pressure velocity (P < 0.01), and directional control (P < 0.01) in limits of stability test, isometric force during ankle dorsiflexion (P = 0.01), knee extension (P < 0.01), and knee flexion (P < 0.01).

Eighty-five patients concluded the research. The program promoted a significant difference among the groups for SF-36 in the eight subscales (p ≤ 0.007), Timed Up & Go Test (p < 0.001), 1-RM test (p < 0.001), Berg Balance Scale (p < 0.001) and also a decrease in the total number of falls in the intervention group compared to control (p < 0.001).

**Autor Objective Desing Results**

history in a randomized controlled trial.

To assess the efficacy of an exercise program aiming to improve balance and muscular strength, for postural control and muscular strength of women with osteoporosis.

To evaluate the effect of a progressive muscular strength and proprioception training program on the muscle strength of the quadriceps, balance, quality of life and reduction in the risk of falls in postmenopausal women with osteoporosis.

Burke TN et al; 2010

Teixeira, et al, 2010

**Table 6.** Studies that used different methods of muscle strength training and balance training



**Muscle Strength Training and Balance Training**

Outpatient Clinic and randomized into 2 groups: the 'Intervention', submitted for balance training; and the 'Control', without intervention. Balance, mobility and falling frequency were evaluated before and at the end of the trial, using the Berg Balance Scale (BBS), the Clinical Test Sensory Interaction Balance (CTSIB) and the Timed "Up & Go" Test (TUGT). Intervention used techniques to improve balance consisting of a 1-hour session each week and a home-based exercise program.

Sixty-one independently living elderly females aged 65 years and older with low bone. Subjects were recruited and randomly assigned to 24 weeks of tai chi (60 minutes/session, three sessions/week, n = 30) or a control group (n = 31). Computerized dynamic posturography, gait, 'timed up and go', five-chair sit-to-stand and quality of life assessed at baseline, 12 and 24 weeks.

higher in the Intervention group compared to Control (5.5±5.67 vs differences between the TUGT were reduced in the Intervention group compared to Control (−3.65±3.61 vs 2.27±7.18 seconds, p< 0.001). Notably, this improvement was paralleled by a reduction in the number of falls/patient in the Intervention group compared to Control (−0.77 ± 1.76 vs 0.33 ± 0.96, p=0.018).

After 24 weeks, subjects in the tai chi group demonstrated an increase in stride width (P = 0.05) and improvement in general health (P = 0.008), vitality (P = 0.02) and bodily pain (P = 0.03) compared with those in the control group.

The fall rate in the exercise group was 39% lower than for the control group (.72 vs 1.18 falls/person-year; risk ratio, .

**Autor Objective Desing Results**

Program on balance, mobility and falling frequency in women with osteoporosis

To evaluate the effects of tai chi exercise on risk factors for falls in postmenopausal women with osteopaenia through measurements of balance, gait, physical function and quality of life.

To evaluate the efficacy of the Nijmegen Falls Prevention Program (NFPP) for persons with osteoporosis and a fall

**Table 6.** Studies that used different methods of muscle strength training and balance training

**Muscle Strength Training and Balance Training**

Persons with osteoporosis and a fall history (N=96; mean ± SD age, 71.0±4.7y; 90 women). Randomized in two groups. Primary outcome

**Autor Objective Desing Results**

Chyu, et al, 2010

228 Topics in Osteoporosis

Smulders E et al; 2010


This relationship becomes even more important when the muscle strengthening program aims

The Effectiveness of Progressive Load Training Associated to the Proprioceptive Training…

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231

Despite the knowledge on muscular strength power and the proprioception for a good motor control, and consequently a lower unbalance and fall risk, previous studies to this one ignore

The significant results found in the present research might be explained by the concern in following the ACSM recommendations when prescribing exercises, respecting the basic

Additionally, one should take into consideration that the skill to develop muscle strength decreases with aging (Hakkinen et al, 1998) explaining the importance of the gradual progres‐ sion (Adams et al, 1999). With sedentary elderly people, a period of adaptation and low working load for two weeks should be applied for further implementation of a loading

Data combined from three studies conducted by Gillespie et al., 2006 (Cochrane Library review) with a total of 556 women aged 80 years or older, who underwent to the same progressive muscular strengthening program, balance training and gait training indicate that this intervention decreased the number of individuals that fell during a year, having also reduced the number of injurious falls. Although the studies had methodological limitations, there is a determined consistency as for the decrease of falls in multiple interventions exercises (Gillespie et al., 2009). As for the physical exercise, we only know that it improves balance without a direct association with the decrease in the number of falls (Howe Tracey et al., 2009) and that although the decline in muscle strength is a risk factor for falls, the muscle strength training could not be associated to the reduced number of falls (Sherrington et al, 2009),

During strength training elderly people respond positively presenting exponential gains in muscular strength, on explosion as well in muscular resistance. This is explained due to muscular mass decreases in approximately 50% between twenty and ninety years old and the number of muscular fibers in an elder person is around 20% less than in an adult person, being

In this study, after a 18-week training, an average increase of 89.5% in the maximum dynamic strength of the quadriceps muscle (1RM) in the intervention group was observed, being within the values described by Humphries et al., 2000 which shows increases of 20 to 200% in the dynamic muscle strength of the quadriceps depending on the initial values and duration of the training. This increase in the knee extension force is significantly important because this force is an independent risk factor for falls and fractures caused by osteoporosis (Nguyen et al., 1993). The increase in the force occurs as a result of neural changes and muscle adjustments

The body balance depends on information appropriate receiving through sensorial, cognitive components from the nervous system and from the musculoskeletal system in an integrated manner by the proprioception. The association of muscle strengthening and proprioceptive

clear the latent capability for recovery of a strength pattern nearly to an adult.

the association importance of sensorial-motor training to strength training.

progression protocol (American College of Sports Medicine, 2002).

to improve the functional balance and prevention of falls.

concepts of prescription exercises.

(Gillespie, et al., 2009).

(Resende et al, 2006).

**Table 7.** Studies that used different methods of muscle strength training and balance training

Because of the strong interaction between osteoporosis and falls, the selection of participants in protocols for the prevention of fractures should be based on factors related to bones and falls (Pfeifer et al., 2004).

The German Society of Sport Medicine and the American College of Sport Medicine also recommend that the ideal program for women with osteoporosis should include activities that improve strength, flexibility and coordination that might indirectly and more effectively decrease the incidence of osteoporotic fractures by the reduction in the probability of falls (Lange et al., 2005).

Few studies take into consideration the importance of the proprioceptive training as a fundamental and unseparable part of a muscular strengthening program. Mechanoreceptors located in the joints, tendons, muscles and neighbor tissue provide information to the Nervous System about the position and articular movements and about the forces generated in the muscles (Huntlei, 2003) (van der Esch, 2007).

The knee proprioception is essential for the modulation and accurate activation of the muscle contraction, once the functional skill and muscular balance are strongly affected by the proprioceptive inaccuracy and muscle weakness (van der Esch, 2007). Studies including patients with knee ligament lesions show that the proprioceptive training promotes additional sensorial information that contributes to the improvement in postural control (Bonfin, 2008). This relationship becomes even more important when the muscle strengthening program aims to improve the functional balance and prevention of falls.

**Muscle Strength Training and Balance Training**

mobility, quality of life were evaluated in the beginning and end of the research. The number of falls was evaluated 24 weeks post treatment.

In a pragmatic randomized trial, 86 post-menopausal osteopenic women, aged 45-70, were recruited. Primary outcomes were changes between baseline and nine months of bone mineral density (BMD) of the proximal femur and lumbar spine (dual-energy X-ray absorptiometry) and serum markers of bone resorption and formation. Secondary outcomes included quality of life.

Changes in sway parameters were significantly improved by TC vs. UC (average sway velocity, P = 0.027; anteriorposterior sway range, P = 0.014). Clinical measures of balance and function showed non-significant trends in favor of TC.

**Autor Objective Desing Results**

**Table 7.** Studies that used different methods of muscle strength training and balance training

Because of the strong interaction between osteoporosis and falls, the selection of participants in protocols for the prevention of fractures should be based on factors related to bones and

The German Society of Sport Medicine and the American College of Sport Medicine also recommend that the ideal program for women with osteoporosis should include activities that improve strength, flexibility and coordination that might indirectly and more effectively decrease the incidence of osteoporotic fractures by the reduction in the probability of falls

Few studies take into consideration the importance of the proprioceptive training as a fundamental and unseparable part of a muscular strengthening program. Mechanoreceptors located in the joints, tendons, muscles and neighbor tissue provide information to the Nervous System about the position and articular movements and about the forces generated in the

The knee proprioception is essential for the modulation and accurate activation of the muscle contraction, once the functional skill and muscular balance are strongly affected by the proprioceptive inaccuracy and muscle weakness (van der Esch, 2007). Studies including patients with knee ligament lesions show that the proprioceptive training promotes additional sensorial information that contributes to the improvement in postural control (Bonfin, 2008).

Wayne, et al, 2012

230 Topics in Osteoporosis

falls (Pfeifer et al., 2004).

(Lange et al., 2005).

muscles (Huntlei, 2003) (van der Esch, 2007).

Tai Chi (TC) is a mind-body exercise that shows potential as an effective and safe intervention for preventing fall-related fractures in the elderly.

Despite the knowledge on muscular strength power and the proprioception for a good motor control, and consequently a lower unbalance and fall risk, previous studies to this one ignore the association importance of sensorial-motor training to strength training.

The significant results found in the present research might be explained by the concern in following the ACSM recommendations when prescribing exercises, respecting the basic concepts of prescription exercises.

Additionally, one should take into consideration that the skill to develop muscle strength decreases with aging (Hakkinen et al, 1998) explaining the importance of the gradual progres‐ sion (Adams et al, 1999). With sedentary elderly people, a period of adaptation and low working load for two weeks should be applied for further implementation of a loading progression protocol (American College of Sports Medicine, 2002).

Data combined from three studies conducted by Gillespie et al., 2006 (Cochrane Library review) with a total of 556 women aged 80 years or older, who underwent to the same progressive muscular strengthening program, balance training and gait training indicate that this intervention decreased the number of individuals that fell during a year, having also reduced the number of injurious falls. Although the studies had methodological limitations, there is a determined consistency as for the decrease of falls in multiple interventions exercises (Gillespie et al., 2009). As for the physical exercise, we only know that it improves balance without a direct association with the decrease in the number of falls (Howe Tracey et al., 2009) and that although the decline in muscle strength is a risk factor for falls, the muscle strength training could not be associated to the reduced number of falls (Sherrington et al, 2009), (Gillespie, et al., 2009).

During strength training elderly people respond positively presenting exponential gains in muscular strength, on explosion as well in muscular resistance. This is explained due to muscular mass decreases in approximately 50% between twenty and ninety years old and the number of muscular fibers in an elder person is around 20% less than in an adult person, being clear the latent capability for recovery of a strength pattern nearly to an adult.

In this study, after a 18-week training, an average increase of 89.5% in the maximum dynamic strength of the quadriceps muscle (1RM) in the intervention group was observed, being within the values described by Humphries et al., 2000 which shows increases of 20 to 200% in the dynamic muscle strength of the quadriceps depending on the initial values and duration of the training. This increase in the knee extension force is significantly important because this force is an independent risk factor for falls and fractures caused by osteoporosis (Nguyen et al., 1993). The increase in the force occurs as a result of neural changes and muscle adjustments (Resende et al, 2006).

The body balance depends on information appropriate receiving through sensorial, cognitive components from the nervous system and from the musculoskeletal system in an integrated manner by the proprioception. The association of muscle strengthening and proprioceptive training was fundamental to the increase of functional mobility and skills, which can be related to the reduction of 36% in the time spent to the performance of the TUG. In this case, the lower the time spent to make the exercise, the better the balance (Resende et al, 2006).

Madureira et. al (2006) conducted a randomized clinical trial that included 66 postmenopausal women with osteoporosis assigned to two groups. One of the groups underwent a 12-month of balance training once a week combined with oriented training at home showing significant

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Swanenburg et. al (2007) studied 24 women (65 years old or older) with osteoporosis or osteopeny who underwent three months of strength, balance and coordination training. After twelve months, they observed a reduction in the risk of fall (Berg Scale) and increase in the muscle strength of lower limbs. They also found a decrease in the number of falls in the intervention group (89%), showing a significant number although it was a pilot study.

Our figures concerning the reduction of number of falls are similar to the ones found in other studies, although an average of 40% (Barnett et al., 2003) is still not well-substantiated, which can be explained by the differences in the population and mainly in the interventions used in

The programmed answers execution by the central nervous system is performed by the musculoskeletal system and the reflex answers, voluntary motor control, postural control and articular stability influence it, fundamental components for falls risk decrease. Therefore the proposed and performed program in this study took in consideration the effector system optimization importance and the neural components, thus, by associating the strength training to sensorial-motor training we obtained effective outcomes and even more vigorous than those that only use muscular strength or balance without taking in consideration the integrated action among the central nervous system, peripheral nervous system (through proprioceptors)

As we could observe, several studies have shown to be effective to increase the strength, balance and functional skills, decreasing the risk of falls. Only the research conducted by Madureira et al. 2007 and Swanenburg et al. 2007 direct related these outcomes with the number of falls. However, it is difficult to compare the studies because the training programs

The possible limitations of the present study include the tests and functional scales used, that are validated but are not so accurate as the lab tests considered the gold pattern. On the other side, we used the BBS, TUG and 1-RM Test which are highly reproducible in the daily clinic

A high adherence rate to the exercises, the thorough evaluation made by a blinded physical therapist, the size of the sample and also the strict methodology used when prescribing the

The purpose of this study was to implement a muscle and proprioceptive training program that would follow the recommendations stated by ACSM, promoting a program that would

results concerning balance, mobility and decrease in the number of falls.

the different researches.

and the effecting organs.

and the evaluation methods are different.

practice, where the access to lab tests is not very often.

exercises might have contributed to the outcomes in this present study.

be strictly followed and prescribed, but easy to use and reproduce.

Although the changes in the numbers were small, the improvement in the balance evaluated by the BBS was consistent, and they are in agreement with the results found by Madureira et al, 2007.

Bemben (2000) compared the effects of high and low-intensity training in 25 postmenopausal women (41 to 60 years old) using a high repetition (40% 1-RM, 16 repetitions) and high load (80 % 1-RM, 8 repetitions) protocols for six months showing increases from 30 to 40%, respectively in the dynamic strength in quadriceps.

In a randomized controlled trial of 10 weeks of strength, balance and stretching training in 53 postmenopausal women with osteoporosis, Malmros and colleagues (1998) showed that strength and muscle mass and also the static balance improved significantly.

In another randomized clinical trial, physiotherapy-directed exercise in 30 patients with osteoporosis significantly improved static balance measured by functional reach and increased quadriceps dynamic strength (Mitchell et al, 1998).

These two studies indicate that the exercises programs improved the profile of fall risk but showed limitations because of the small number of samples and short time of the interventions.

Hartard et al. (1996) studied the effects of muscle strength training in 16 postmenopausal women with osteopenia, where fifteen belonged to the control group. Although they used a small group, a proper load protocol for 6 months, twice a week at 70% 1RM was applied demonstrating a considerable increase in muscle strength ranging from 44 to 76%, with results similar to the ones found in the present investigation.

Kemmler et. al (2002) evaluated the dynamic force (1RM tests) in 137 postmenopausal women with osteopenia divided in two groups and observed a significant increase of 43% in the leg press in the intervention group training at 70% of 1-RM for fourteen months.

Carter et al. (2002) in a program that trains instructors to work with the community selected 93 postmenopausal women with osteoporosis who were randomized and underwent physical exercises of balance and muscle strength for twenty weeks. No improvement in the quality of life was found, which might be explained by the high quality of life at baseline. Researchers observed an improvement of 6.3% in the dynamic balance and an increase of 12.8% in the muscular strength.

On the other hand, this study contradicts other researches since it shows a significant im‐ provement in the quality of life evaluated by the SF-36, where the values for the physical aspects and mental aspects were considerably higher than the ones found in the control group and the values in the baseline. These outcomes might be related to the systemic physiologic benefits provided by the exercises, which improves the capability of performing daily activities. The results can also be explained by the psychological effects the physical exercise provides, the socialization with other patients and the low levels of quality of life the patients had in the beginning of this study.

Madureira et. al (2006) conducted a randomized clinical trial that included 66 postmenopausal women with osteoporosis assigned to two groups. One of the groups underwent a 12-month of balance training once a week combined with oriented training at home showing significant results concerning balance, mobility and decrease in the number of falls.

training was fundamental to the increase of functional mobility and skills, which can be related to the reduction of 36% in the time spent to the performance of the TUG. In this case, the lower

Although the changes in the numbers were small, the improvement in the balance evaluated by the BBS was consistent, and they are in agreement with the results found by Madureira et

Bemben (2000) compared the effects of high and low-intensity training in 25 postmenopausal women (41 to 60 years old) using a high repetition (40% 1-RM, 16 repetitions) and high load (80 % 1-RM, 8 repetitions) protocols for six months showing increases from 30 to 40%,

In a randomized controlled trial of 10 weeks of strength, balance and stretching training in 53 postmenopausal women with osteoporosis, Malmros and colleagues (1998) showed that

In another randomized clinical trial, physiotherapy-directed exercise in 30 patients with osteoporosis significantly improved static balance measured by functional reach and increased

These two studies indicate that the exercises programs improved the profile of fall risk but showed limitations because of the small number of samples and short time of the interventions. Hartard et al. (1996) studied the effects of muscle strength training in 16 postmenopausal women with osteopenia, where fifteen belonged to the control group. Although they used a small group, a proper load protocol for 6 months, twice a week at 70% 1RM was applied demonstrating a considerable increase in muscle strength ranging from 44 to 76%, with results

Kemmler et. al (2002) evaluated the dynamic force (1RM tests) in 137 postmenopausal women with osteopenia divided in two groups and observed a significant increase of 43% in the leg

Carter et al. (2002) in a program that trains instructors to work with the community selected 93 postmenopausal women with osteoporosis who were randomized and underwent physical exercises of balance and muscle strength for twenty weeks. No improvement in the quality of life was found, which might be explained by the high quality of life at baseline. Researchers observed an improvement of 6.3% in the dynamic balance and an increase of 12.8% in the

On the other hand, this study contradicts other researches since it shows a significant im‐ provement in the quality of life evaluated by the SF-36, where the values for the physical aspects and mental aspects were considerably higher than the ones found in the control group and the values in the baseline. These outcomes might be related to the systemic physiologic benefits provided by the exercises, which improves the capability of performing daily activities. The results can also be explained by the psychological effects the physical exercise provides, the socialization with other patients and the low levels of quality of life the patients

the time spent to make the exercise, the better the balance (Resende et al, 2006).

strength and muscle mass and also the static balance improved significantly.

press in the intervention group training at 70% of 1-RM for fourteen months.

respectively in the dynamic strength in quadriceps.

quadriceps dynamic strength (Mitchell et al, 1998).

similar to the ones found in the present investigation.

al, 2007.

232 Topics in Osteoporosis

muscular strength.

had in the beginning of this study.

Swanenburg et. al (2007) studied 24 women (65 years old or older) with osteoporosis or osteopeny who underwent three months of strength, balance and coordination training. After twelve months, they observed a reduction in the risk of fall (Berg Scale) and increase in the muscle strength of lower limbs. They also found a decrease in the number of falls in the intervention group (89%), showing a significant number although it was a pilot study.

Our figures concerning the reduction of number of falls are similar to the ones found in other studies, although an average of 40% (Barnett et al., 2003) is still not well-substantiated, which can be explained by the differences in the population and mainly in the interventions used in the different researches.

The programmed answers execution by the central nervous system is performed by the musculoskeletal system and the reflex answers, voluntary motor control, postural control and articular stability influence it, fundamental components for falls risk decrease. Therefore the proposed and performed program in this study took in consideration the effector system optimization importance and the neural components, thus, by associating the strength training to sensorial-motor training we obtained effective outcomes and even more vigorous than those that only use muscular strength or balance without taking in consideration the integrated action among the central nervous system, peripheral nervous system (through proprioceptors) and the effecting organs.

As we could observe, several studies have shown to be effective to increase the strength, balance and functional skills, decreasing the risk of falls. Only the research conducted by Madureira et al. 2007 and Swanenburg et al. 2007 direct related these outcomes with the number of falls. However, it is difficult to compare the studies because the training programs and the evaluation methods are different.

The possible limitations of the present study include the tests and functional scales used, that are validated but are not so accurate as the lab tests considered the gold pattern. On the other side, we used the BBS, TUG and 1-RM Test which are highly reproducible in the daily clinic practice, where the access to lab tests is not very often.

A high adherence rate to the exercises, the thorough evaluation made by a blinded physical therapist, the size of the sample and also the strict methodology used when prescribing the exercises might have contributed to the outcomes in this present study.

The purpose of this study was to implement a muscle and proprioceptive training program that would follow the recommendations stated by ACSM, promoting a program that would be strictly followed and prescribed, but easy to use and reproduce.

### **5. Conclusion**

The association of progressive strength training for the quadriceps and the proprioceptive training is effective for the prevention of falls, increasing the muscle power, the static and dynamic balance and increasing the speed of the motor responses, therefore improving the performance of daily activities.

[5] American College of Sports Medicine. (1998). Position Exercise and physical activity

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http://dx.doi.org/10.5772/54554

235

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### **Acknowledgements**

The authors would like to thank Federal University of São Paulo, University of Santo Amaro and Federal University of Amazonas for all the support given when developing this project.

### **Author details**

Lucas Teixeira1,3, Stella Peccin1,4, Kelson Silva1 , Tiago Teixeira2 , Aline Mizusaki Imoto1 , Joelma Magalhães5 and Virgínia Trevisani1,2

1 Department of Internal and Therapeutic Medicine – Federal University of São Paulo, Brazil

2 Department of Rheumatology - University of Santo Amaro, Brazil

3 Department of Physical Therapy - Federal University of Amazonas, Brazil

4 Department of Physical Therapy - Federal University of São Paulo, Brazil

5 Department of Physical Therapy - Uninorte Laureat University, Brazil


[5] American College of Sports Medicine. (1998). Position Exercise and physical activity for older adults. *Med Sci Sports Exerc*. 30: 992-1008.

**5. Conclusion**

234 Topics in Osteoporosis

performance of daily activities.

Lucas Teixeira1,3, Stella Peccin1,4, Kelson Silva1

and Virgínia Trevisani1,2

2 Department of Rheumatology - University of Santo Amaro, Brazil

3 Department of Physical Therapy - Federal University of Amazonas, Brazil

4 Department of Physical Therapy - Federal University of São Paulo, Brazil

5 Department of Physical Therapy - Uninorte Laureat University, Brazil

bilitation patient. J Cardiopulm Rehabil 19:209-215.

*scription*. 5th Ed. Baltimore: Williams and Wilkins, pp. 1–373.

**Acknowledgements**

**Author details**

Joelma Magalhães5

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The association of progressive strength training for the quadriceps and the proprioceptive training is effective for the prevention of falls, increasing the muscle power, the static and dynamic balance and increasing the speed of the motor responses, therefore improving the

The authors would like to thank Federal University of São Paulo, University of Santo Amaro and Federal University of Amazonas for all the support given when developing this project.

1 Department of Internal and Therapeutic Medicine – Federal University of São Paulo, Brazil

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**Chapter 11**

**Anabolic Agents as New Treatment Strategy in**

Osteoporosis is a systemic skeletal disease characterized by low bone mineral density (BMD), microarchitectural deterioration of bone tissue, and an increase in fracture risk [1]. Several drugs have been developed to treat osteoporosis: most of these are inhibitors of bone resorption. Effective treatment of osteoporosis requires not only resorption inhibitors, but al‐ so stimulators of bone formation especially in patients who already have lost a significant degree of bone. Although therapeutic alternatives are available for inhibiting bone resorp‐ tion, options of bone anabolic agents are much more limited with regard to the bone resorp‐

Although patients included in randomised controlled trials have osteoporosis defined ac‐ cording to the WHO criteria, i.e. a T score below -2.5 SD and/or prevalent fragility fractures, a large proportion of fractures occurs at T-scores above -2.5 SD and in patients without prior fractures [1]. Therefore, therapies with proven fracture risk reduction efficacy in patients with osteopenia and/or clinical risk factors may contribute to earlier and more effective in‐

The past decade has witnessed major advances in the diagnosis and treatment of osteoporo‐ sis. It would appear that anabolic drugs challenge prevailing paradigm by stimulating bone formation, therefore enhancing bone turnover. There is a great need to anabolic agents for reverse of osteoporosis. In this review, we summarize current informations about the ana‐

and reproduction in any medium, provided the original work is properly cited.

© 2013 Okman-Kilic and Sagiroglu; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

**Osteoporosis**

http://dx.doi.org/10.5772/54452

**1. Introduction**

tion inhibitors.

bolic agents.

tervention against fractures.

Tulay Okman-Kilic and Cengiz Sagiroglu

Additional information is available at the end of the chapter


## **Anabolic Agents as New Treatment Strategy in Osteoporosis**

Tulay Okman-Kilic and Cengiz Sagiroglu

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54452

### **1. Introduction**

postmenopausal women with osteoporosis: a randomized controlled trial. *Osteoporo‐*

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240 Topics in Osteoporosis

Osteoporosis is a systemic skeletal disease characterized by low bone mineral density (BMD), microarchitectural deterioration of bone tissue, and an increase in fracture risk [1]. Several drugs have been developed to treat osteoporosis: most of these are inhibitors of bone resorption. Effective treatment of osteoporosis requires not only resorption inhibitors, but al‐ so stimulators of bone formation especially in patients who already have lost a significant degree of bone. Although therapeutic alternatives are available for inhibiting bone resorp‐ tion, options of bone anabolic agents are much more limited with regard to the bone resorp‐ tion inhibitors.

Although patients included in randomised controlled trials have osteoporosis defined ac‐ cording to the WHO criteria, i.e. a T score below -2.5 SD and/or prevalent fragility fractures, a large proportion of fractures occurs at T-scores above -2.5 SD and in patients without prior fractures [1]. Therefore, therapies with proven fracture risk reduction efficacy in patients with osteopenia and/or clinical risk factors may contribute to earlier and more effective in‐ tervention against fractures.

The past decade has witnessed major advances in the diagnosis and treatment of osteoporo‐ sis. It would appear that anabolic drugs challenge prevailing paradigm by stimulating bone formation, therefore enhancing bone turnover. There is a great need to anabolic agents for reverse of osteoporosis. In this review, we summarize current informations about the ana‐ bolic agents.

© 2013 Okman-Kilic and Sagiroglu; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **2. Parathormon**

Parathyroid hormon (PTH) is released from the parathyroid glands and is an important reg‐ ulator in the bloodstream's levels of calcium phosphorus. It stimulates both bone formation and resorption [2,3]. Its intermittent low-dose using increases bone formation more than bone resorption, leading to increased bone mass. Intermittent PTH administration increases the number and activity of osteoblasts, enhances the mean wall thickness and trabecular bone volume, and improves bone microarchitecture by establishing trabecular connectivity and increasing cortical thickness [2,4].

Ingested strontium is distributed in the body in three compartments: plasma extracellular fluid; soft tissue and superficial zone of bone tissue; and bone itself, the greatest portion is the calcified tissues [13]. In bones, total amount of strontium relatively lower than amount of calcium. After its absorption, both strontium and calcium exhibit the same

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The strontium levels in bone vary according to the anatomical site. However, strontium levels at different skeletal sites are strongly correlated [14]. The strontium levels in bone also vary according to the bone structure and higher amounts of strontium are found in cancellous bone than in cortical bone. Strontium is mainly incorporated by exchange on‐ to the crystal surface. In new bone, only a few strontium atoms may be incorporated in‐ to the crystal by ionic substitution of calcium [12, 14]. Bone strontium content is highly correlated with plasma strontium levels. Mechanism of action of strontium ranelate is

Strontium ranelate decreases osteoclast differentiation and activity [15]. Also able to increase pre-osteoblast replication, collagen type I synthesis [16]. Therefore strontium ranelate has a dual affect on bone remodeling, being able to stimulate bone formation by osteoblasts, a property shared with bone-forming agents, and to inhibit bone resorption by osteoclasts, as

characteristics [12].

shown in figure 1.

**Figure 1.** Mechanism of action of strontium ranelate in bone [13]

do anti-resorptive agents [17,18].

Continuous infusions, which result in a persistent elevation of the serum parathyroid hor‐ mone concentration, lead to greater bone resorption than do daily injections, which cause only transient increases in the serum parathyroid hormone concentration [5,6]. The anabolic effects of PTH on bone formation are through the medium of PTH receptor-dependent mechanisms. Teriparatide (PTH 1-34 ) is the biological active, a recombinant form of PTH [7]. Patients with fractures of postmenopausal osteoporosis administered teriparatide 20 and 40 μg/d in FPT (Fracture Prevention Trial) [8]. After 18 months teriparatide 20 μg/d reduces the risk of spine fracture by %65 and non-spine fracture risk by %53. Over a median of 18 months spine fracture risk reduced by %69 and non-spine fracture risk reduced by %54 with the 40 μg/d regimen [8].

Subbiah et al. reported the second patient to develop osteosarcoma [9]. Although teripara‐ tide reduces osteoporosis related fractures in select patient populations, important contrain‐ dications, such as prior radiation exposure, Paget's disease of bone, unexplained elevations of serum alkaline phosphate, open epiphysis should be considered before use.

It has been suggested teriparatide could be useful for treatment of severe and resistant forms of osteoporosis to other medications [10].

In summary, we think that the clinical benefits of parathyroid hormone reflect its ability to stimulate bone formation and thereby increase bone mass and strength. This hormone ap‐ pears to be effective in preventing fractures in postmenopausal women. Hovewer, it should be used attention because of its important contraindications.

### **3. Stontium ranelate**

Strontium ranelate is composed of an organic molecule (ranelic acid) and of two atoms of stable non-radioactive strontium [11]. Strontium naturally present in trace amounts in hu‐ man body and has close similarities with calcium; act as calcium agonist in most of physio‐ logic process [12].

Strontium is similar to calcium in its absorption in the gastrointestinal track takes place in two ways: passive diffusion and carrier mediated absorption. Both calcium and strontium share the same carrier system, which tents to be greater affinity to calcium. High dietary in‐ take of calcium has been shown to reduce concurrent absorption of strontium [11].

Ingested strontium is distributed in the body in three compartments: plasma extracellular fluid; soft tissue and superficial zone of bone tissue; and bone itself, the greatest portion is the calcified tissues [13]. In bones, total amount of strontium relatively lower than amount of calcium. After its absorption, both strontium and calcium exhibit the same characteristics [12].

**2. Parathormon**

242 Topics in Osteoporosis

and increasing cortical thickness [2,4].

forms of osteoporosis to other medications [10].

be used attention because of its important contraindications.

the 40 μg/d regimen [8].

**3. Stontium ranelate**

logic process [12].

Parathyroid hormon (PTH) is released from the parathyroid glands and is an important reg‐ ulator in the bloodstream's levels of calcium phosphorus. It stimulates both bone formation and resorption [2,3]. Its intermittent low-dose using increases bone formation more than bone resorption, leading to increased bone mass. Intermittent PTH administration increases the number and activity of osteoblasts, enhances the mean wall thickness and trabecular bone volume, and improves bone microarchitecture by establishing trabecular connectivity

Continuous infusions, which result in a persistent elevation of the serum parathyroid hor‐ mone concentration, lead to greater bone resorption than do daily injections, which cause only transient increases in the serum parathyroid hormone concentration [5,6]. The anabolic effects of PTH on bone formation are through the medium of PTH receptor-dependent mechanisms. Teriparatide (PTH 1-34 ) is the biological active, a recombinant form of PTH [7]. Patients with fractures of postmenopausal osteoporosis administered teriparatide 20 and 40 μg/d in FPT (Fracture Prevention Trial) [8]. After 18 months teriparatide 20 μg/d reduces the risk of spine fracture by %65 and non-spine fracture risk by %53. Over a median of 18 months spine fracture risk reduced by %69 and non-spine fracture risk reduced by %54 with

Subbiah et al. reported the second patient to develop osteosarcoma [9]. Although teripara‐ tide reduces osteoporosis related fractures in select patient populations, important contrain‐ dications, such as prior radiation exposure, Paget's disease of bone, unexplained elevations

It has been suggested teriparatide could be useful for treatment of severe and resistant

In summary, we think that the clinical benefits of parathyroid hormone reflect its ability to stimulate bone formation and thereby increase bone mass and strength. This hormone ap‐ pears to be effective in preventing fractures in postmenopausal women. Hovewer, it should

Strontium ranelate is composed of an organic molecule (ranelic acid) and of two atoms of stable non-radioactive strontium [11]. Strontium naturally present in trace amounts in hu‐ man body and has close similarities with calcium; act as calcium agonist in most of physio‐

Strontium is similar to calcium in its absorption in the gastrointestinal track takes place in two ways: passive diffusion and carrier mediated absorption. Both calcium and strontium share the same carrier system, which tents to be greater affinity to calcium. High dietary in‐

take of calcium has been shown to reduce concurrent absorption of strontium [11].

of serum alkaline phosphate, open epiphysis should be considered before use.

The strontium levels in bone vary according to the anatomical site. However, strontium levels at different skeletal sites are strongly correlated [14]. The strontium levels in bone also vary according to the bone structure and higher amounts of strontium are found in cancellous bone than in cortical bone. Strontium is mainly incorporated by exchange on‐ to the crystal surface. In new bone, only a few strontium atoms may be incorporated in‐ to the crystal by ionic substitution of calcium [12, 14]. Bone strontium content is highly correlated with plasma strontium levels. Mechanism of action of strontium ranelate is shown in figure 1.

**Figure 1.** Mechanism of action of strontium ranelate in bone [13]

Strontium ranelate decreases osteoclast differentiation and activity [15]. Also able to increase pre-osteoblast replication, collagen type I synthesis [16]. Therefore strontium ranelate has a dual affect on bone remodeling, being able to stimulate bone formation by osteoblasts, a property shared with bone-forming agents, and to inhibit bone resorption by osteoclasts, as do anti-resorptive agents [17,18].

Strontiun ranelate shows affect by binding calcium receptor in bone. Strontium has lower affinity for calcium sensing receptor than calcium[19].

There are higher calcium ion concentrations within the bone microenvironment in case of osteoclastic resorption. Affect of calcium receptor increases in higher extracellular calcium concentrations. Strontium ranelate intake prevents bone loss with non-osteoporotic patients in early post-menopausal period [19].

In the PREVOS study (PREVention Of early postmenopausal bone loss by Strontium rane‐ late) usage of strontium ranelate 1g/d for period of 2 years resulted in significantly high‐ er increase of femur BMD (bone mineral density). There was a significant increase in the bone formation markers and concurrent increase of bone resorption markers has not been recorded [20].

In the Treatment of Peripheral Osteoporosis (TROPOS) study strontium ranelate in‐ creased bone mineral density throughout the study, reaching at 3-yr 8.2% (femoral neck) and 9.8% (total hip). Same study shows %36 decrease in hip fracture risk even in highrisk subgroup over 3-yr period [21].

The Spinal Osteoporosis Therapeutic Intervention (SOTI) study investigated the safety of strontium ranelate and its efficacy against vertebral fractures. In patients used strontium ra‐ nelate 2 g/d the risk of vertebral fractures was decreased by 41% over 3-yr [22].

In both studies strontium ranelate was well tolerated. The most common adverse events consisted of nausea and diarrhea was disappeared after third month of treatment [21,22].

Therefore, we suggest that strontium ranelate has been proving antifracture efficacy in pa‐ tients with osteopenia and/or clinical risk factors and very old elderly. Also, it may contrib‐ ute to earlier and more effective intervention against fractures because of well- tolerated.

**Figure 2.** The mechanism of action and place of prostaglandins in bone metabolism [24]

formation, therefore can be used in osteoporosis treatment [27].

been reported [28].

teoporosis prophylaxis [29,30].

It has been reported in certain studies that prostaglandins have anabolic effect on the bone

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245

It has been demonstrated that systemic PGE2 administration stimulates proliferation of osteoblast precursors or differentiation of osteoprogenitor cells in bone marrow and 4.7% increase in bone mass eventually was found in the same study [27]. Increase of total bone surface by means of osteoblast stimulation with PGE2 administration to rats has

Misoprostol is a methylene analogue of prostaglandin E1 (PGE1) has been administered to oophorectomized rats. Misoprostol is being used for treatment of gastric ulcer due to its cy‐ toprotective effect by inhibiting gastric acid and pepcin secretion [23]. Rats receiving miso‐ prostol had significantly reduced oophorectomy related bone loss at site of lumber spine. Thus, it has been proposed that misoprostol is choice for treatment of post-menopausal os‐

Misoprostol 800 μg/d had been administered for 6 moths to post-menopausal osteoporotic patients. At the end of the treatment increase by 8.1% in femur bone mineral density, by 5% increase in lumber spine bone mineral density and by 3.6% increase in Ward's triangle bone

### **4. Prostaglandins**

Prostaglandins act as locally acting hormones, developed as new therapeutic approach. They show the effect and are metabolized in the tissue where they are synthesized. Prostaglandins are synthesized from arachidonic acid, a polyunsaturated fatty acid with 20-carbon chain [23].

Prostaglandins are produced from bone cells by mediated cyclooxygenase. Prostaglandin production is regulated by mechanical stress, cytokines, growth factor and systemic hor‐ mones. Furthermore, prostaglandins are able to regulate their own production [24]. Prosta‐ glandins have both inhibitory and stimulatory effects on bone structuring. The most prominent effect of prostaglandin E2 (PGE2) is to stimulate bone resorption and formation [24]. PGE2 exerts its action through the cell surface receptors. Four subtypes of prostaglan‐ din E receptors (EP1, EP2, EP3 and EP4) [25,26] have been identified. PGE2 stimulates bone formation by EP4 receptor mediation [26]. The importance and impact of prostaglandins in bone metabolism is summarized in figure 2

**Figure 2.** The mechanism of action and place of prostaglandins in bone metabolism [24]

Strontiun ranelate shows affect by binding calcium receptor in bone. Strontium has lower

There are higher calcium ion concentrations within the bone microenvironment in case of osteoclastic resorption. Affect of calcium receptor increases in higher extracellular calcium concentrations. Strontium ranelate intake prevents bone loss with non-osteoporotic patients

In the PREVOS study (PREVention Of early postmenopausal bone loss by Strontium rane‐ late) usage of strontium ranelate 1g/d for period of 2 years resulted in significantly high‐ er increase of femur BMD (bone mineral density). There was a significant increase in the bone formation markers and concurrent increase of bone resorption markers has not been

In the Treatment of Peripheral Osteoporosis (TROPOS) study strontium ranelate in‐ creased bone mineral density throughout the study, reaching at 3-yr 8.2% (femoral neck) and 9.8% (total hip). Same study shows %36 decrease in hip fracture risk even in high-

The Spinal Osteoporosis Therapeutic Intervention (SOTI) study investigated the safety of strontium ranelate and its efficacy against vertebral fractures. In patients used strontium ra‐

In both studies strontium ranelate was well tolerated. The most common adverse events consisted of nausea and diarrhea was disappeared after third month of treatment [21,22].

Therefore, we suggest that strontium ranelate has been proving antifracture efficacy in pa‐ tients with osteopenia and/or clinical risk factors and very old elderly. Also, it may contrib‐ ute to earlier and more effective intervention against fractures because of well- tolerated.

Prostaglandins act as locally acting hormones, developed as new therapeutic approach. They show the effect and are metabolized in the tissue where they are synthesized. Prostaglandins are synthesized from arachidonic acid, a polyunsaturated fatty acid with

Prostaglandins are produced from bone cells by mediated cyclooxygenase. Prostaglandin production is regulated by mechanical stress, cytokines, growth factor and systemic hor‐ mones. Furthermore, prostaglandins are able to regulate their own production [24]. Prosta‐ glandins have both inhibitory and stimulatory effects on bone structuring. The most prominent effect of prostaglandin E2 (PGE2) is to stimulate bone resorption and formation [24]. PGE2 exerts its action through the cell surface receptors. Four subtypes of prostaglan‐ din E receptors (EP1, EP2, EP3 and EP4) [25,26] have been identified. PGE2 stimulates bone formation by EP4 receptor mediation [26]. The importance and impact of prostaglandins in

nelate 2 g/d the risk of vertebral fractures was decreased by 41% over 3-yr [22].

affinity for calcium sensing receptor than calcium[19].

in early post-menopausal period [19].

risk subgroup over 3-yr period [21].

recorded [20].

244 Topics in Osteoporosis

**4. Prostaglandins**

20-carbon chain [23].

bone metabolism is summarized in figure 2

It has been reported in certain studies that prostaglandins have anabolic effect on the bone formation, therefore can be used in osteoporosis treatment [27].

It has been demonstrated that systemic PGE2 administration stimulates proliferation of osteoblast precursors or differentiation of osteoprogenitor cells in bone marrow and 4.7% increase in bone mass eventually was found in the same study [27]. Increase of total bone surface by means of osteoblast stimulation with PGE2 administration to rats has been reported [28].

Misoprostol is a methylene analogue of prostaglandin E1 (PGE1) has been administered to oophorectomized rats. Misoprostol is being used for treatment of gastric ulcer due to its cy‐ toprotective effect by inhibiting gastric acid and pepcin secretion [23]. Rats receiving miso‐ prostol had significantly reduced oophorectomy related bone loss at site of lumber spine. Thus, it has been proposed that misoprostol is choice for treatment of post-menopausal os‐ teoporosis prophylaxis [29,30].

Misoprostol 800 μg/d had been administered for 6 moths to post-menopausal osteoporotic patients. At the end of the treatment increase by 8.1% in femur bone mineral density, by 5% increase in lumber spine bone mineral density and by 3.6% increase in Ward's triangle bone mineral density have been found. It has been reported that misoprostol can be an alternative on treatment of osteoporosis [31].

ration [41]. Maeda et al showed that hydrophobic statins such as simvastatin, atorvastatin, and cerivastatin stimulated VEGF expression by osteoblasts via reduced protein prenylation and the phosphatidylinositide-3 kinase pathway, promoting osteoblastic differentiation [42].

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In 2009 Pault et al revealed a dose-dependent effect and improved fracture healing under

Fukui et al reported that the effect of the systemic administration of statins was limited due to its metabolism in the liver and high-dose administration may cause adverse side effects. They locally applied with gelatin hydrogel to fracture sites at a dose similar to that used in clinical settings and shown to induced fracture union in a rat unhealing bone fracture model

We suggest that the results of studies also point to the need for more information in order to

It is known that growth hormone (GH) is important in the regulation of longitudinal bone growth [45]. Several *in vivo* and *in vitro* studies have demonstrated that GH is important in the regulation of both bone formation and bone resorption. In Figure 3 a model for the cellu‐

**Figure 3.** The mechanism of action at the cellular level for GH in regulation of bone remodeling. The left part of the figure represents osteoclast-mediated bone resorption. The right part represents osteoblast-mediated bone forma‐

tion. ? indicates that both stimulatory and inhibitory effects have been shown [45]

local application of simvastatin [43].

particularly gelatin hydrogel form.

**7. Growth hormone and IGF-I**

via its effect on both angiogenesis and osteogenesis [44].

lar effects of GH in the regulation of bone remodeling is showed [45].

We think that misoprostol may be an alternative therapy for patients with osteopenia and osteoporosis who are not suitable for hormone replacement therapy.

### **5. Sesamin**

Sesamin is a major lignan compound in sesame seeds. Its activity on bone cell function is unclear. Recently, it has been reported that sesamin has direct effects on osteoblasts by stimulating the expression of essential genes and key enzymes of the bone minerali‐ zation process [32,33].

Wanachewin et al suggested that sesamin had the ability to trigger osteoblast differantiation by activation of the p38 and ERK/MAPK (mitogen-activated protein kinase) signaling path‐ way and possibility indirectly regulate osteoclast development via the expression of OPG and RANKL in osteoblasts [32].

The **MAPK/ERK pathway** is a chain of proteins in the cell that transmits a signal from a re‐ ceptor on the surface of the cell to the DNA in the nucleus of the cell. MAPKs play impor‐ tant roles in cellular response to growth factors, cytokines, or environmental stress.

They are classified into four classes: extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinase or stress-activated protein kinase, p38 MAPKs, and ERK5 [34]. ERKs are involved in cell proliferation/transformation and survival. p38 MAPKs are involved in many cellular processes, such as inflammatory responses, osteoblast differentiation, apoptosis [35,36].

We think that sesamin which is a phytochemical agent, may be effective addition to osteo‐ porotic therapy. Future studies are needed.

### **6. Statins**

Statins are inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-COA) reductase. Usually, it has been known that they have efficacy and credibility on coronary artery diseases and hyperlipidemia [37,38]. First, Mundy et al showed that statins lead to in‐ crease 90 % in the trabecular bone volume by stimulating bone formation invitro and therefore it was started to studies investigating the place of statins for osteoporosis treat‐ ment [39]. Hamelin et al suggested that statins decrease the bone destruction by supress‐ ing the formation of the mevalonate that it is an important precursor on the control of osteoclastic activity as bisphosphonates [40].

It was reported that statins have efficacy on the bone metabolism, by increasing bone mor‐ phogenetic protein 2 (BMP2) activation stimulated osteoblastic cell proliferation and matu‐ ration [41]. Maeda et al showed that hydrophobic statins such as simvastatin, atorvastatin, and cerivastatin stimulated VEGF expression by osteoblasts via reduced protein prenylation and the phosphatidylinositide-3 kinase pathway, promoting osteoblastic differentiation [42].

In 2009 Pault et al revealed a dose-dependent effect and improved fracture healing under local application of simvastatin [43].

Fukui et al reported that the effect of the systemic administration of statins was limited due to its metabolism in the liver and high-dose administration may cause adverse side effects. They locally applied with gelatin hydrogel to fracture sites at a dose similar to that used in clinical settings and shown to induced fracture union in a rat unhealing bone fracture model via its effect on both angiogenesis and osteogenesis [44].

We suggest that the results of studies also point to the need for more information in order to particularly gelatin hydrogel form.

### **7. Growth hormone and IGF-I**

mineral density have been found. It has been reported that misoprostol can be an alternative

We think that misoprostol may be an alternative therapy for patients with osteopenia and

Sesamin is a major lignan compound in sesame seeds. Its activity on bone cell function is unclear. Recently, it has been reported that sesamin has direct effects on osteoblasts by stimulating the expression of essential genes and key enzymes of the bone minerali‐

Wanachewin et al suggested that sesamin had the ability to trigger osteoblast differantiation by activation of the p38 and ERK/MAPK (mitogen-activated protein kinase) signaling path‐ way and possibility indirectly regulate osteoclast development via the expression of OPG

The **MAPK/ERK pathway** is a chain of proteins in the cell that transmits a signal from a re‐ ceptor on the surface of the cell to the DNA in the nucleus of the cell. MAPKs play impor‐

They are classified into four classes: extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinase or stress-activated protein kinase, p38 MAPKs, and ERK5 [34]. ERKs are involved in cell proliferation/transformation and survival. p38 MAPKs are involved in many cellular processes, such as inflammatory responses, osteoblast differentiation,

We think that sesamin which is a phytochemical agent, may be effective addition to osteo‐

Statins are inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-COA) reductase. Usually, it has been known that they have efficacy and credibility on coronary artery diseases and hyperlipidemia [37,38]. First, Mundy et al showed that statins lead to in‐ crease 90 % in the trabecular bone volume by stimulating bone formation invitro and therefore it was started to studies investigating the place of statins for osteoporosis treat‐ ment [39]. Hamelin et al suggested that statins decrease the bone destruction by supress‐ ing the formation of the mevalonate that it is an important precursor on the control of

It was reported that statins have efficacy on the bone metabolism, by increasing bone mor‐ phogenetic protein 2 (BMP2) activation stimulated osteoblastic cell proliferation and matu‐

tant roles in cellular response to growth factors, cytokines, or environmental stress.

osteoporosis who are not suitable for hormone replacement therapy.

on treatment of osteoporosis [31].

**5. Sesamin**

246 Topics in Osteoporosis

zation process [32,33].

apoptosis [35,36].

**6. Statins**

and RANKL in osteoblasts [32].

porotic therapy. Future studies are needed.

osteoclastic activity as bisphosphonates [40].

It is known that growth hormone (GH) is important in the regulation of longitudinal bone growth [45]. Several *in vivo* and *in vitro* studies have demonstrated that GH is important in the regulation of both bone formation and bone resorption. In Figure 3 a model for the cellu‐ lar effects of GH in the regulation of bone remodeling is showed [45].

**Figure 3.** The mechanism of action at the cellular level for GH in regulation of bone remodeling. The left part of the figure represents osteoclast-mediated bone resorption. The right part represents osteoblast-mediated bone forma‐ tion. ? indicates that both stimulatory and inhibitory effects have been shown [45]

GH increases bone formation in two ways [46]:


**9. Other potential agents for anabolic treatment of osteoporosis**

**Figure 4.** Bzb's action on bone formation in the patients with multiple myeloma [60]

clearly defined.

ly Figure 4.

**Bortezomib:** There are multiple potential alternative agents for increasing bone formation. A potential treatment is to target the osteoblast proteasome. It was reported that the protea‐ some inhibitor bortezomib (Bzb) had bone forming effects in patients with multiple mye‐ lome [60]. The mechanism for Bzb's effects on osteoblastic differentiation has not been

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249

It was showed that Bzb with lenalidomide or thalidomide may increase bone formation by stimulating osteoblast activity and inhibiting osteoclastic bone destruction, respective‐

rhGH (recombinant human Growth Hormone) increases bone turnover in normal subjects and improves bone mineral metabolism in postmenopausal females [45]. GH treatment also results in increased bone resorption. It is still unknown whether osteoclasts express func‐ tional GHRs, but recent in vitro studies indicate that GH regulates osteoclast formation in bone marrow cultures [45, 46]. Possible modulations of the GH/IGF (Insulin like Growth Factor) axis by glucocorticoids and estrogens are also included in Fig. 3 [45].

Bone is the second richest source of IGF-I in the body. Locally this peptide promotes osteo‐ blast differentiation and growth [48]. Recently, studies show that low levels of IGF-I are as‐ sociated with a greater risk of hip and spine fractures [49–51]. Hence, there is a strong opinion for considering human GH or IGF-I as potential anabolic agents for the treatment of osteoporosis. There are potential advantages for using rhIGF-I (recombinant human Insulin like Growth Factor-1) compared with rhGH in the treatment of osteoporosis. These include


It was reported that low doses of rhIGF-I may directly increase osteoblastic function with only a minimal increase in bone resorption [52]. In 2008, it was suggested a potential role for IGF-1 in the early identification of women at risk for low bone mass and osteoporosis. They suggested measuring the serum level of IGF-1 in women around 40 years old. When its val‐ ue is 1.5 SD below the peak, BMD measurement by DXA could be considered [53]

There are limited number studies using rhIGF-I than rhGH. Therefore, these advantages have not been validated yet.

### **8. Sodium fluoride**

Sodium fluoride is the first anabolic agentsto be used in the treatment of postmenopausal osteoporosis. Side-effects, consisting of upper gastrointestinal symptoms and a lower ex‐ tremity pain syndrome, are common.

Using slow release formulation of sodium fluoride, it was showed a 50% reduction in verte‐ bral fracture incidence with impressive increases in bone mass [54-56]. More recently, it has been suggested that a different formulation of fluoride, monofluorophosphate when is used in lower dosages and more favorable formulations, gastrointestinal side-effects are reduced [57-59]. However, consensus about its clinical utility has still not been reached.

### **9. Other potential agents for anabolic treatment of osteoporosis**

GH increases bone formation in two ways [46]:

**1.** more direct stimulation of bone formation,

have not been validated yet.

tremity pain syndrome, are common.

**8. Sodium fluoride**

**2.** bypass of skeletal GH resistance that can be present, and

tor-1).

248 Topics in Osteoporosis

**1.** via a direct interaction with GHRs on osteoblasts

**2.** via an induction of endocrine and autocrine/paracrine IGF-I (Insulin like Growth Fac‐

rhGH (recombinant human Growth Hormone) increases bone turnover in normal subjects and improves bone mineral metabolism in postmenopausal females [45]. GH treatment also results in increased bone resorption. It is still unknown whether osteoclasts express func‐ tional GHRs, but recent in vitro studies indicate that GH regulates osteoclast formation in bone marrow cultures [45, 46]. Possible modulations of the GH/IGF (Insulin like Growth

Bone is the second richest source of IGF-I in the body. Locally this peptide promotes osteo‐ blast differentiation and growth [48]. Recently, studies show that low levels of IGF-I are as‐ sociated with a greater risk of hip and spine fractures [49–51]. Hence, there is a strong opinion for considering human GH or IGF-I as potential anabolic agents for the treatment of osteoporosis. There are potential advantages for using rhIGF-I (recombinant human Insulin like Growth Factor-1) compared with rhGH in the treatment of osteoporosis. These include

**3.** a reduction in GH-induced side-effects such as carpal tunnel and diabetes mellitus. [47] It was reported that low doses of rhIGF-I may directly increase osteoblastic function with only a minimal increase in bone resorption [52]. In 2008, it was suggested a potential role for IGF-1 in the early identification of women at risk for low bone mass and osteoporosis. They suggested measuring the serum level of IGF-1 in women around 40 years old. When its val‐

There are limited number studies using rhIGF-I than rhGH. Therefore, these advantages

Sodium fluoride is the first anabolic agentsto be used in the treatment of postmenopausal osteoporosis. Side-effects, consisting of upper gastrointestinal symptoms and a lower ex‐

Using slow release formulation of sodium fluoride, it was showed a 50% reduction in verte‐ bral fracture incidence with impressive increases in bone mass [54-56]. More recently, it has been suggested that a different formulation of fluoride, monofluorophosphate when is used in lower dosages and more favorable formulations, gastrointestinal side-effects are reduced

[57-59]. However, consensus about its clinical utility has still not been reached.

ue is 1.5 SD below the peak, BMD measurement by DXA could be considered [53]

Factor) axis by glucocorticoids and estrogens are also included in Fig. 3 [45].

**Bortezomib:** There are multiple potential alternative agents for increasing bone formation. A potential treatment is to target the osteoblast proteasome. It was reported that the protea‐ some inhibitor bortezomib (Bzb) had bone forming effects in patients with multiple mye‐ lome [60]. The mechanism for Bzb's effects on osteoblastic differentiation has not been clearly defined.

It was showed that Bzb with lenalidomide or thalidomide may increase bone formation by stimulating osteoblast activity and inhibiting osteoclastic bone destruction, respective‐ ly Figure 4.

**Figure 4.** Bzb's action on bone formation in the patients with multiple myeloma [60]

**Oxytocin:** An other approach is oxytocin (OT) that increases osteoblastic bone formation. It has been reported that OT may regulate maternal skeletal homeostasis during pregnancy and lactation. The fetal skeleton is unlikely to be mineralized effectively in the absence of calcium mobilized from the maternal skeleton [61]. It has been suggested that elevated OT levels during pregnancy and lactation not only enhance bone resorption by increasing the number of osteoclasts to make maternal calcium existing to the fetus, but also prevent unre‐ stricted bone removal by inhibiting the activity of mature osteoclasts. Therefore, it was re‐ ported that recombinant OT or its analogs because of its skeletal anabolic action, might have potential utility in therapy for human osteoporosis [61].

**Anti-sclerostin monoclonal antibody:** Sclerostin is a protein encoded by the SOST gene in osteocytes. It inhibits osteoblastic bone formation [66,67]. The binding of Wnt proteins to the LRP5/6-Frizzled co-receptor on the cell membrane of osteoblasts leads to stabilization of in‐ tracellular beta-catenin and regulation of gene transcription that promotes osteoblastic bone formation. Sclerostin is a modulator of osteoblast function. It antagonizes Wnt signaling and inhibits osteoblastic bone formation [68]. Recent studies reported that anti-sclerostin therapy enhances fracture healing and bone repair.[69-71]. AMG 785 is a humanized sclerostin mon‐ oclonal antibody, was first studied in humans. It enhances Wnt signaling and increase osteo‐

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251

Treatment with AMG 785 has been well tolerated. In postmenopausal women with low bone mineral density (BMD) after 12 months of AMG 785 administration, increase in BMD is determined. Although there is no evidence that AMG 785 increases the risk of osteosarcoma,

Aging is associated with impaired bone formation which is a principal pathogenetic cause mediating bone fragility in osteoporosis. Ideally, patients at high risk of fracture should be identified early and treated by a combination of lifestyle changes, correction of secondary causes of osteoporosis, and specific treatments to improve bone density and decrease frac‐ ture risk. By now, there were a limited number of therapeutic agent for activating bone for‐ mation and increasing bone mass and strength. More effective and better tolerated therapies will become available soon. We think that new treatments will be able to contribute to in‐ crease the currently low treatment rate of even severe osteoporosis by allowing approaches

1 Department of Obsterics and Gynecology, Trakya University, Medical Faculty, Edirne,

aimed at minimising fracture risk at the individual patient level.

and Cengiz Sagiroglu2

\*Address all correspondence to: ajlankilic@hotmail.com

2 Tasyapi Health Group, Istanbul, Turkey

blastic bone formation [72]. Figure 5

**10. Conclusion**

**Author details**

Tulay Okman-Kilic1

Turkey

new studies are needed to modify this risk.

**Beta-blocker:** Wiens et al found that beta-blocker use was associated with a significant de‐ crease in fracture risk [62]. However, in 2008, Reid determinated that there was no any evi‐ dence to support the hypothesis that beta'-blockers reduce fracture numbers [63]. In 2012, Yang et al reported that beta-blockers are associated with reduced risk of fracture in older adults, but the effect size is likely to be modes [64]t. In summary, there was no an adequate evidence to support using beta-blockers in the treatment of osteoporosis.

**Lithium:** The mean (+/-SD) bone density in lithium treated patients was reported that 4.5% higher at the spine (P<0.05), 5.3% higher at the femoral neck (P<0.05) and 7.5% higher at the trochanter (P<0.05). In addition, lithium treated patients had lower serum total ALP (P<0.005), lower serum osteocalcin (P<0.005) and lower serum CTX (P<0.05) but the totalcal‐ cium, PTH and urinary calcium excretion did not differ significantly between patients and controls. In conclusion, it was suggested that therapy with lithium carbonate may preserve or enhance bone mass [65].

**Figure 5.** Modulation of Wnt signaling by sclerostin [73]

**Anti-sclerostin monoclonal antibody:** Sclerostin is a protein encoded by the SOST gene in osteocytes. It inhibits osteoblastic bone formation [66,67]. The binding of Wnt proteins to the LRP5/6-Frizzled co-receptor on the cell membrane of osteoblasts leads to stabilization of in‐ tracellular beta-catenin and regulation of gene transcription that promotes osteoblastic bone formation. Sclerostin is a modulator of osteoblast function. It antagonizes Wnt signaling and inhibits osteoblastic bone formation [68]. Recent studies reported that anti-sclerostin therapy enhances fracture healing and bone repair.[69-71]. AMG 785 is a humanized sclerostin mon‐ oclonal antibody, was first studied in humans. It enhances Wnt signaling and increase osteo‐ blastic bone formation [72]. Figure 5

Treatment with AMG 785 has been well tolerated. In postmenopausal women with low bone mineral density (BMD) after 12 months of AMG 785 administration, increase in BMD is determined. Although there is no evidence that AMG 785 increases the risk of osteosarcoma, new studies are needed to modify this risk.

### **10. Conclusion**

**Oxytocin:** An other approach is oxytocin (OT) that increases osteoblastic bone formation. It has been reported that OT may regulate maternal skeletal homeostasis during pregnancy and lactation. The fetal skeleton is unlikely to be mineralized effectively in the absence of calcium mobilized from the maternal skeleton [61]. It has been suggested that elevated OT levels during pregnancy and lactation not only enhance bone resorption by increasing the number of osteoclasts to make maternal calcium existing to the fetus, but also prevent unre‐ stricted bone removal by inhibiting the activity of mature osteoclasts. Therefore, it was re‐ ported that recombinant OT or its analogs because of its skeletal anabolic action, might have

**Beta-blocker:** Wiens et al found that beta-blocker use was associated with a significant de‐ crease in fracture risk [62]. However, in 2008, Reid determinated that there was no any evi‐ dence to support the hypothesis that beta'-blockers reduce fracture numbers [63]. In 2012, Yang et al reported that beta-blockers are associated with reduced risk of fracture in older adults, but the effect size is likely to be modes [64]t. In summary, there was no an adequate

**Lithium:** The mean (+/-SD) bone density in lithium treated patients was reported that 4.5% higher at the spine (P<0.05), 5.3% higher at the femoral neck (P<0.05) and 7.5% higher at the trochanter (P<0.05). In addition, lithium treated patients had lower serum total ALP (P<0.005), lower serum osteocalcin (P<0.005) and lower serum CTX (P<0.05) but the totalcal‐ cium, PTH and urinary calcium excretion did not differ significantly between patients and controls. In conclusion, it was suggested that therapy with lithium carbonate may preserve

potential utility in therapy for human osteoporosis [61].

or enhance bone mass [65].

250 Topics in Osteoporosis

**Figure 5.** Modulation of Wnt signaling by sclerostin [73]

evidence to support using beta-blockers in the treatment of osteoporosis.

Aging is associated with impaired bone formation which is a principal pathogenetic cause mediating bone fragility in osteoporosis. Ideally, patients at high risk of fracture should be identified early and treated by a combination of lifestyle changes, correction of secondary causes of osteoporosis, and specific treatments to improve bone density and decrease frac‐ ture risk. By now, there were a limited number of therapeutic agent for activating bone for‐ mation and increasing bone mass and strength. More effective and better tolerated therapies will become available soon. We think that new treatments will be able to contribute to in‐ crease the currently low treatment rate of even severe osteoporosis by allowing approaches aimed at minimising fracture risk at the individual patient level.

### **Author details**

Tulay Okman-Kilic1 and Cengiz Sagiroglu2

\*Address all correspondence to: ajlankilic@hotmail.com

1 Department of Obsterics and Gynecology, Trakya University, Medical Faculty, Edirne, Turkey

2 Tasyapi Health Group, Istanbul, Turkey

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**Chapter 12**

**Osteoporosis in Spaceflight**

http://dx.doi.org/10.5772/54708

**1.1. Renal stones during spaceflight**

while in microgravity [Buckey, 2006].

**1. Introduction**

Satoshi Iwase, Naoki Nishimura and Tadaaki Mano

No major medical diffifulties were experienced during spaceflight in the era of Russian Vostok/ Voshot or spaceflight programs of Mercury and Gemini, however, when prolonged stays in space stations began in the 1980's, astronauts or Russian cosmonauts had an increased risk of suffering from renal stones, and resultant bone loss. The detailed mechanism behind this phenomenon is still unknown, but one explanation is that unloading of the skeleton that would normally bear the bodyweight led to calcium (Ca) leaving the bones for the bloodstream. The Ca entered the kidneys, filtered into the urine, causing hypercalciuria, and increased the risk of kidney stone formation. Such stones formed in the kidney break down and travel into the ureter, causing flank pain. Thus astronauts continually subjected to risks of bone and Ca loss

Factors that seem to affect the bone and Ca loss while in microgravity are as follows; low light levels, high environmental CO2 levels, and minimal skeletal loading. It is reported that urinary Ca excretion increases by 60-70 % within a few days of entering the microgravity. Data from the Skylab program in 1973-74, when nine astronauts stayed in the space station for 28 to 84 days, showed that the estimated rate of Ca loss from the bone per month was 0.3% of the total body Ca [Whedon et al. 1974].Data from the Mir program indicated that the bone losing the most Ca losing bone is the coxal bone estimated to lose 1.5% of the total body Ca per month [Le Blanc et al. 2000]. Skylab was a space station launched and operated by NASA (National Aeronautics and Space Administration of the USA) and was the America's first space station, which orbited the Earth from 1973 to 79, and included a workshop, a solar observatory, and other systems with the weight of 77 tons. Mir (мир, peace/world) was a space station that operated in low Earth orbit from 1986 to 2001, at first by Soviet Union and then by Russia. Assembled in orbit from 1986 to 96, Mir was the first modular space station and had a greater

> © 2013 Iwase et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

Additional information is available at the end of the chapter

### **Chapter 12**

### **Osteoporosis in Spaceflight**

Satoshi Iwase, Naoki Nishimura and Tadaaki Mano

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54708

### **1. Introduction**

### **1.1. Renal stones during spaceflight**

No major medical diffifulties were experienced during spaceflight in the era of Russian Vostok/ Voshot or spaceflight programs of Mercury and Gemini, however, when prolonged stays in space stations began in the 1980's, astronauts or Russian cosmonauts had an increased risk of suffering from renal stones, and resultant bone loss. The detailed mechanism behind this phenomenon is still unknown, but one explanation is that unloading of the skeleton that would normally bear the bodyweight led to calcium (Ca) leaving the bones for the bloodstream. The Ca entered the kidneys, filtered into the urine, causing hypercalciuria, and increased the risk of kidney stone formation. Such stones formed in the kidney break down and travel into the ureter, causing flank pain. Thus astronauts continually subjected to risks of bone and Ca loss while in microgravity [Buckey, 2006].

Factors that seem to affect the bone and Ca loss while in microgravity are as follows; low light levels, high environmental CO2 levels, and minimal skeletal loading. It is reported that urinary Ca excretion increases by 60-70 % within a few days of entering the microgravity. Data from the Skylab program in 1973-74, when nine astronauts stayed in the space station for 28 to 84 days, showed that the estimated rate of Ca loss from the bone per month was 0.3% of the total body Ca [Whedon et al. 1974].Data from the Mir program indicated that the bone losing the most Ca losing bone is the coxal bone estimated to lose 1.5% of the total body Ca per month [Le Blanc et al. 2000]. Skylab was a space station launched and operated by NASA (National Aeronautics and Space Administration of the USA) and was the America's first space station, which orbited the Earth from 1973 to 79, and included a workshop, a solar observatory, and other systems with the weight of 77 tons. Mir (мир, peace/world) was a space station that operated in low Earth orbit from 1986 to 2001, at first by Soviet Union and then by Russia. Assembled in orbit from 1986 to 96, Mir was the first modular space station and had a greater

© 2013 Iwase et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

mass than that of any previous spacecraft until its deorbit on March 21, 2001. Many experiments including biomedical sciences.

at an appropriate level to keep the strain within an appropriate range, showing that exceeding the setpoint of bone strain bone modeling intiates reduction of the strain back to the setpoint had been carried out. Surveys on athletes indicated that high weight bearing, which is observed in weightlifters and significant impact loading in gymnasts resulted in significantly high bone mass [Nilsson & Westlin, 1971, Uusi-Rasi et al. 1971, Taaffe et al. 1997, Huddleston et al., 1980]. As for the gravity-induced high bone mass density, evidence has shown that heavy-weight people exhibited higher bone density, and spinal injury patients with a wheelchair lose significantly bone mass at lower extremity while not in the lumbar spine [Biering-Sorensen et al., 1988, 1990]. The impact of contact with the ground on the bones of the coxa and the lower extremities is an important factor to maintain the bone mass density [Kreb et al., 1998].

Osteoporosis in Spaceflight http://dx.doi.org/10.5772/54708 261

Not only impacts or weight bearing on the bone, but also muscular contractions also play a role in strain bearing on the bone at 1 G [Kreb et al., 1998, Schulthesis et al., 1991]. However, 0 G conditions may influence the skeletal loading both through a loss of ground reaction forces

**Figure 1.** Regulation of energy balance and bone mass Leptin suppresses appetite and enhances sympathetic nerve activity through the hypothalamus elevating energy expenditure. At the same time higher activity of the sympathetic

Sex hormones, estrogens and androgens are associated with bone mass density; however, there seems to be no significant changes in sex hormones during spaceflight. Growth hormone increases the bone mass, but it does not seem to have a relationship with spaceflight. Insulin-

and through marked reduction in the forces needed to move the weightless limbs.

Adapted with permission from Flier JS: Nature 420(6916): 619, 621-622(2002)

nervous system inhibits bone formation of osteoblasts, inducing bone loss.

**3.2. Hormones**

In spite of rapid bone loss under conditions of weightlessness, a slow recovery rate is reported. Data from the Mir program showed that approximately 12% of bone was lost during 4.5 months in space while only 6% recovered in a year on the Earth [Linenger 2000]. Follow-up study of the Skylab crew members after 28-84 days of the microgravity exposure suggested that not all the bone lost on the station had been recovered [Tilton et al. 1980]. These findings indicate the seriousness of bone loss under conditions of microgravity, which could present significant problems, and may progress to osteoporosis, in long-duration spaceflight. Bone loss under conditions of weightlessness should be strictly monitored and controlled.

### **2. Calcium metabolism during spaceflight**

During spaceflight, several factors influence the Ca metabolism, including alterations in diet intake, low lighting, increased ambient CO2, and the most important factor is unloading of the bodyweight when considering the long duration spaceflight.

The recommended Ca2+ intake is 1,000 mg/day, and nutritionists prepare the space food to satisfy this criterion.

In spacecraft, where no sunlight or ultraviolet light exposure is occurs, vitamin D deficiency may develop without sufficient Ca intake, which causes poor mineralization of bone, dimin‐ ished intestinal Ca absorption, decreased serum Ca2+ levels. These situations may cause an increase in parathormone, but actually, sufficient Ca intake protects the bone loss, and serum Ca2+ level has been proved to be increased, and parathormone level is suppressed..

Compared with the low CO2 level on the Earth, which is 0.03% of the atmosphere, confined and isolated circumstances such as in spacecraft or a space station have increased CO2 levels of 0.7-1%, which affects the acid-base balance, and consequently the bone metabolism. Increased CO2 levels in inhaled air cause acidosis, and carbonates and phosphates in the bone play important roles in neutralizing the acidosis, which leads to bone resorption [Bushinsky et al., 1997]. Although CO2 levels >1% were reported to have some effects on urinary bone absorption markers [Drummer et al. 1998], respiratory acidosis may decrease the urinary pH, and increase the risk of kidney stone formation [Coe et al. 1992].

Bone remodeling and the remodeling changes during spaceflight are profoundly dependent on genetic factors in terms of the baseline level [Boyden et al. 2002, Judex et al. 2002]

### **3. Bone formation factors**

### **3.1. Mechanical loading**

The mechanism by which bone senses and responds to loading has yet to be fully clarified yet. Frost [1987] postulated that bone has a mechanostat that senses strain and maintains bone mass at an appropriate level to keep the strain within an appropriate range, showing that exceeding the setpoint of bone strain bone modeling intiates reduction of the strain back to the setpoint had been carried out. Surveys on athletes indicated that high weight bearing, which is observed in weightlifters and significant impact loading in gymnasts resulted in significantly high bone mass [Nilsson & Westlin, 1971, Uusi-Rasi et al. 1971, Taaffe et al. 1997, Huddleston et al., 1980].

As for the gravity-induced high bone mass density, evidence has shown that heavy-weight people exhibited higher bone density, and spinal injury patients with a wheelchair lose significantly bone mass at lower extremity while not in the lumbar spine [Biering-Sorensen et al., 1988, 1990]. The impact of contact with the ground on the bones of the coxa and the lower extremities is an important factor to maintain the bone mass density [Kreb et al., 1998].

Not only impacts or weight bearing on the bone, but also muscular contractions also play a role in strain bearing on the bone at 1 G [Kreb et al., 1998, Schulthesis et al., 1991]. However, 0 G conditions may influence the skeletal loading both through a loss of ground reaction forces and through marked reduction in the forces needed to move the weightless limbs.

Adapted with permission from Flier JS: Nature 420(6916): 619, 621-622(2002)

**Figure 1.** Regulation of energy balance and bone mass Leptin suppresses appetite and enhances sympathetic nerve activity through the hypothalamus elevating energy expenditure. At the same time higher activity of the sympathetic nervous system inhibits bone formation of osteoblasts, inducing bone loss.

### **3.2. Hormones**

mass than that of any previous spacecraft until its deorbit on March 21, 2001. Many experiments

In spite of rapid bone loss under conditions of weightlessness, a slow recovery rate is reported. Data from the Mir program showed that approximately 12% of bone was lost during 4.5 months in space while only 6% recovered in a year on the Earth [Linenger 2000]. Follow-up study of the Skylab crew members after 28-84 days of the microgravity exposure suggested that not all the bone lost on the station had been recovered [Tilton et al. 1980]. These findings indicate the seriousness of bone loss under conditions of microgravity, which could present significant problems, and may progress to osteoporosis, in long-duration spaceflight. Bone

loss under conditions of weightlessness should be strictly monitored and controlled.

During spaceflight, several factors influence the Ca metabolism, including alterations in diet intake, low lighting, increased ambient CO2, and the most important factor is unloading of the

The recommended Ca2+ intake is 1,000 mg/day, and nutritionists prepare the space food to

In spacecraft, where no sunlight or ultraviolet light exposure is occurs, vitamin D deficiency may develop without sufficient Ca intake, which causes poor mineralization of bone, dimin‐ ished intestinal Ca absorption, decreased serum Ca2+ levels. These situations may cause an increase in parathormone, but actually, sufficient Ca intake protects the bone loss, and serum

Compared with the low CO2 level on the Earth, which is 0.03% of the atmosphere, confined and isolated circumstances such as in spacecraft or a space station have increased CO2 levels of 0.7-1%, which affects the acid-base balance, and consequently the bone metabolism. Increased CO2 levels in inhaled air cause acidosis, and carbonates and phosphates in the bone play important roles in neutralizing the acidosis, which leads to bone resorption [Bushinsky et al., 1997]. Although CO2 levels >1% were reported to have some effects on urinary bone absorption markers [Drummer et al. 1998], respiratory acidosis may decrease the urinary pH,

Bone remodeling and the remodeling changes during spaceflight are profoundly dependent

The mechanism by which bone senses and responds to loading has yet to be fully clarified yet. Frost [1987] postulated that bone has a mechanostat that senses strain and maintains bone mass

on genetic factors in terms of the baseline level [Boyden et al. 2002, Judex et al. 2002]

Ca2+ level has been proved to be increased, and parathormone level is suppressed..

**2. Calcium metabolism during spaceflight**

bodyweight when considering the long duration spaceflight.

and increase the risk of kidney stone formation [Coe et al. 1992].

including biomedical sciences.

260 Topics in Osteoporosis

satisfy this criterion.

**3. Bone formation factors**

**3.1. Mechanical loading**

Sex hormones, estrogens and androgens are associated with bone mass density; however, there seems to be no significant changes in sex hormones during spaceflight. Growth hormone increases the bone mass, but it does not seem to have a relationship with spaceflight. Insulinlike growth factor (IGF-1) has also been shown to be an agent that can increase bone mass, and in animal experiments performed during the Space Shuttle experiment, administration of IGF-1 to rats during the 10 days of the Shuttle flight increased bone formation in the humerus.

**4. Bone resorption factors**

The unloading of weight bearing on bone on Earth is associated with prolonged bed rest, immobilization, or paralysis. Studies of patients with spinal cord injury have demonstrated that approximately 30–50% of lower extremity bone mass can be lost before reaching a plateau, which occurs an average of approximately 16 months after admission [Biering-Sorensen et al.

Chronic increase in parathormone levels enhances resorption, which causes osteoporosis. This increase in parathormone is sometimes produced by lower serum Ca2+ and vitamin D; however, intermittent administration of parathormone with enough Ca2+ levels with vitamin D can also be anabolic. Thyroid hormones and glucocorticoid can be a cause of osteoporosis,

protein also provides an acid load, which causes bone loss mediated by skeletal buffering.

Skylab missions were the first opportunity to study the Ca metabolism in space [Whendon et al., 1974]. These included the unmanned Skylab 1, Skylab 2 with 3 crew members staying 29 days of stay, Skylab 3 also with 3 crew members staying 59 days, and Skylab 4 again with 3 crew members staying 84 days. Thereafter, various Space Shuttle Programs have been

Urinary excretion of Ca2+ is enhanced promptly just after microgravity exposure, and remains elevated for several months throughout weightlessness exposure or eventually returns to normal depending on individual. The mechanism behind individual differences has not benn well clarified. Data from two Russian cosmonauts demonstrated no Ca2+ excretion increases were observed after 218 days of microgravity exposure [Grigoriev et al., 1994]. The reason for this lack of an increase in Ca2+ might be the effectiveness of countermeasure program.

Frozen urine samples from the Skylab mission were subsequently examined; the data showed that N-telopeptide was increased throughout the flight [Smith et al., 1998], and C-telopeptide also remained elevated throughout on a-180 day Mir flight [Caillot-Augusseau et al., 1998].

Therefore, it is favorable for space food to contain low salt as well as low protein.

conducted to examine the effects of space flights on Ca metabolism in humans.

excretion as well as urinary Ca2+ excretion. A high level of dietary

Osteoporosis in Spaceflight http://dx.doi.org/10.5772/54708 263

**4.1. Immobilization**

1990, Garland et al. 1992].

but it is unlikely to occur in space.

intake enhances Na+

**5.1. Bone loss markers during spaceflight**

**5. Bone loss and osteoporosis in spaceflight**

**4.2. Hormones**

**4.3. Dietary factors**

High Na+

Parathormone has a complex effect on the bone. Its ultimate goal is to increase the serum Ca2+ level, so that without enough Ca2+ intake or vitamin D deficiency, it acts on bone resorption and reduces the bone mass. The action sites of parathormone are the bone, the kidney, and the intestine.

The mechanism of parathormone action on the bone is indirect since osteoclasts have no receptor of parathormone, rather parathormone binds to osteoblasts to increase their expres‐ sion of RANKL (receptor activator of nuclear factor kappa-B ligand, osteoclast differentiation factor) and inhibits their expression of osteoprotegrin. Osteoprotegerin binds to RANKL and inhibits it from interacting with RANK (receptor activator of nuclear factor kappa-B, receptor of osteoclast differentiation factor). The binding of RANKL to RANK is facilitated by the decreased amount of osteoprotegerin, and stimulates osteoclast precursors to fuse. It resulted to form new osteoclasts, which ultimately enhances bone resorption.

Parathormone also acts on the kidney to enhance the active reabsorption of Ca2+ and Mg2+ from the distal tubules and the thick ascending limb, and maintains or increases the serum Ca2+ levels.

It also enhances the absorption of Ca2+ from the intestine indirectly, by increasing the produc‐ tion of activated vitamin D, which is activated in the kidney. The activated vitamin D increases the absorption of Ca2+ by the intestine.

Calcitonin is secreted from the thyroid gland by an increase in serum Ca2+ levels, and acts to lower the Ca2+ levels, which counteracts parathormone. Calcitonin lowers Ca2+ levels by 1) inhibiting Ca2+ absorption by the intestines, 2) inhibiting osteoclast activity in bones, 3) inhibiting renal tubular cell reabsorption of Ca2+ by allowing Ca2+ to be secreted in the urine. Therefore, calcitonin protects bone from Ca2+ loss, however, it has not proved to be effective in preventing bone loss in immobilization in either animals [Thomas et al., 1995] or humans [Hantman et al., 1973].

### **3.3. Dietary factors**

Ca, vitamin D, and vitamin K are the essential factors for bone formation, and their oral intake is recommended during spaceflight.

### **3.4. Electric fields and vibration**

Studies have shown exposure of marrow culture to low-frequency, low-intensity electric fields inhibited the recruitment of osteoclasts [Rubin et al., 1996]. Low magnitude (0.25 G) and high frequency (30-90 Hz) vibration has been proved to be effective in inhibiting bone loss and increasing bone formation in humans [Bosco et al., 1999, Rubin et al. 2004]. This vibration constitues a promising countermeasure against bone loss in spaceflight, and studies are now being conducted to clarify the optimal magnitude and frequency.

### **4. Bone resorption factors**

### **4.1. Immobilization**

like growth factor (IGF-1) has also been shown to be an agent that can increase bone mass, and in animal experiments performed during the Space Shuttle experiment, administration of IGF-1 to rats during the 10 days of the Shuttle flight increased bone formation in the humerus.

Parathormone has a complex effect on the bone. Its ultimate goal is to increase the serum Ca2+ level, so that without enough Ca2+ intake or vitamin D deficiency, it acts on bone resorption and reduces the bone mass. The action sites of parathormone are the bone, the kidney, and the

The mechanism of parathormone action on the bone is indirect since osteoclasts have no receptor of parathormone, rather parathormone binds to osteoblasts to increase their expres‐ sion of RANKL (receptor activator of nuclear factor kappa-B ligand, osteoclast differentiation factor) and inhibits their expression of osteoprotegrin. Osteoprotegerin binds to RANKL and inhibits it from interacting with RANK (receptor activator of nuclear factor kappa-B, receptor of osteoclast differentiation factor). The binding of RANKL to RANK is facilitated by the decreased amount of osteoprotegerin, and stimulates osteoclast precursors to fuse. It resulted

Parathormone also acts on the kidney to enhance the active reabsorption of Ca2+ and Mg2+ from the distal tubules and the thick ascending limb, and maintains or increases the serum Ca2+

It also enhances the absorption of Ca2+ from the intestine indirectly, by increasing the produc‐ tion of activated vitamin D, which is activated in the kidney. The activated vitamin D increases

Calcitonin is secreted from the thyroid gland by an increase in serum Ca2+ levels, and acts to lower the Ca2+ levels, which counteracts parathormone. Calcitonin lowers Ca2+ levels by 1) inhibiting Ca2+ absorption by the intestines, 2) inhibiting osteoclast activity in bones, 3) inhibiting renal tubular cell reabsorption of Ca2+ by allowing Ca2+ to be secreted in the urine. Therefore, calcitonin protects bone from Ca2+ loss, however, it has not proved to be effective in preventing bone loss in immobilization in either animals [Thomas et al., 1995] or humans

Ca, vitamin D, and vitamin K are the essential factors for bone formation, and their oral intake

Studies have shown exposure of marrow culture to low-frequency, low-intensity electric fields inhibited the recruitment of osteoclasts [Rubin et al., 1996]. Low magnitude (0.25 G) and high frequency (30-90 Hz) vibration has been proved to be effective in inhibiting bone loss and increasing bone formation in humans [Bosco et al., 1999, Rubin et al. 2004]. This vibration constitues a promising countermeasure against bone loss in spaceflight, and studies are now

being conducted to clarify the optimal magnitude and frequency.

to form new osteoclasts, which ultimately enhances bone resorption.

intestine.

262 Topics in Osteoporosis

levels.

the absorption of Ca2+ by the intestine.

is recommended during spaceflight.

**3.4. Electric fields and vibration**

[Hantman et al., 1973].

**3.3. Dietary factors**

The unloading of weight bearing on bone on Earth is associated with prolonged bed rest, immobilization, or paralysis. Studies of patients with spinal cord injury have demonstrated that approximately 30–50% of lower extremity bone mass can be lost before reaching a plateau, which occurs an average of approximately 16 months after admission [Biering-Sorensen et al. 1990, Garland et al. 1992].

### **4.2. Hormones**

Chronic increase in parathormone levels enhances resorption, which causes osteoporosis. This increase in parathormone is sometimes produced by lower serum Ca2+ and vitamin D; however, intermittent administration of parathormone with enough Ca2+ levels with vitamin D can also be anabolic. Thyroid hormones and glucocorticoid can be a cause of osteoporosis, but it is unlikely to occur in space.

### **4.3. Dietary factors**

High Na+ intake enhances Na+ excretion as well as urinary Ca2+ excretion. A high level of dietary protein also provides an acid load, which causes bone loss mediated by skeletal buffering. Therefore, it is favorable for space food to contain low salt as well as low protein.

### **5. Bone loss and osteoporosis in spaceflight**

Skylab missions were the first opportunity to study the Ca metabolism in space [Whendon et al., 1974]. These included the unmanned Skylab 1, Skylab 2 with 3 crew members staying 29 days of stay, Skylab 3 also with 3 crew members staying 59 days, and Skylab 4 again with 3 crew members staying 84 days. Thereafter, various Space Shuttle Programs have been conducted to examine the effects of space flights on Ca metabolism in humans.

Urinary excretion of Ca2+ is enhanced promptly just after microgravity exposure, and remains elevated for several months throughout weightlessness exposure or eventually returns to normal depending on individual. The mechanism behind individual differences has not benn well clarified. Data from two Russian cosmonauts demonstrated no Ca2+ excretion increases were observed after 218 days of microgravity exposure [Grigoriev et al., 1994]. The reason for this lack of an increase in Ca2+ might be the effectiveness of countermeasure program.

### **5.1. Bone loss markers during spaceflight**

Frozen urine samples from the Skylab mission were subsequently examined; the data showed that N-telopeptide was increased throughout the flight [Smith et al., 1998], and C-telopeptide also remained elevated throughout on a-180 day Mir flight [Caillot-Augusseau et al., 1998]. Elevation of these two markers demonstrated that the increased urinary Ca2+ was due to an increase in bone resorption. Osteocalcin, a bone formation marker, measured during a 180-day Mir flight showed a decrease.

Microneurographically, on the other hand, recorded neural traffic in humans is known to reflect muscle and skin sympathetic nerve activity (MSNA and SSNA), and MSNA controls the vasomotor function of the muscular bed, responding to blood pressure changes against gravitational stress [Iwase et al., 1987, Mano 1990, Mano et al., 2009]. MSNA was found to be suppressed during an exposure to short-term microgravity induced by parabolic flight [Iwase et al., 1999], to mild lower body positive pressure (10-20 mmHg LBPP) [Fu et al., 1998], and to thermoneutral head-out water immersion [Miwa et al., 1996] responding to the loading or unloading of cardiopulmonary receptor stimulated by cephalad fluid shift. Contrarily, MSNA was enhanced after an exposure to long-term microgravity in spaceflight and its simulation induced by dry immersion (Iwase et al., 2000), and 6°head-down bed rest (Kamiya et al., 2000), caused by various mechanisms including plasma volume loss, changes in baroreflex, and vascular compliance after the human body has acclimated to microgravity situation..

Osteoporosis in Spaceflight http://dx.doi.org/10.5772/54708 265

As for the sympathetic influence on bone metabolism, sympathetic stimulation facilitated bone resorption, while it inhibited ossification by osteoblasts mediated by hypothalamus and leptin in mouse. Loading of weak chronic stress in mouse reduced the osteoblastic activity with elevated noradrenaline, which was prevented by β-blocker [Kondo et al., 2005]. The beneficial effects of β-adrenergic blocker on bone mass and metabolism were reported in mice and rats [Minkowitz et al., 1991, Pierroz, et al., 2006]. Other studies were controversial, however, recent studies have indicated that there are two systems that regulate bone metabolism; one through β2 receptors in bone which facilitates osteolysis and inhibits osteogenesis, and the other that facililtates osteogenesis through a kind of neuropeptide called CART (cocaine amphetamine

From human studies, there have been reports that administration of β-blockers may reduce the risk of bone fracture as well as higher bone density (Graham et al., 2008, Levasseur et al., 2005, Pasco et al., 2004, 2005, Reid et al., 2005a, b, Reinmark et al., 2004, 2006, Schlienger et al.,

Prolonged exposure to microgravity in space for 14 days enhanced the sympathetic neural traffic in humans as evidenced by the Neurolab mission[Cox et al., 2002, Fu et al., 2002, Levine et al., 2002, Ertl et al., 2002], with comparable results in elevated noradrenaline spillover and clearance in space [Ertl et al., 2002]. Corresponding results were obtained from simulated microgravity including dry immersion [Iwase et al., 2000] or head-down bed rest [Kamiya et al., 2000]. Elderly people generally have low bone mass and density and high sympathetic neural traffic to muscles although response to gravitational stress becomes lowered (Iwase et al. 1991). Our preliminary data show that changes in sympathetic neural traffic to muscles after long-term bedrest of 20 days had a significant correlation with changes in the urinary secretion level of deoxypyridinoline (Mano et al., 2009, Nishimura et al., 2010) (Fig.2), which a specific marker for bone resorption (Robbins et al., 1994). On the basis of these findings, it is postulated that an exposure to prolonged microgravity enhances the sympathetic neural traffic to bone, which increases the noradrenaline level, inhibits osteogenesis and facilitates osteolysis through

**6.2. Space flight-related changes in sympathetic regulation on bone metabolism**

β-receptors to induce bone mineral loss; however, it is no better than hypothesis.

regulated transcript) [Elefteriou et al., 2005].

2004, Turker et al., 2006)

### **5.2. Bone loss location**

Bone mineral density was measured before and after flights on the Mir program. The changes per month were as follows; +0.6% in the skull, +0.1% in the arm, −1.07% in the spine, −1.35% in the pelvis, −1.16% in the femoral neck, −1.58% in the trochanter major, −1.25% in the tibia, and −1.50% in the calcaneus, all per month, with comparable results obtained from the International Space Station [Lang et al., 2004].

The bones that are most affected during spaceflight seems to be weight bearing bones, such as the pelvis (os coxae), the trochanter major of the femur, the femoral neck, the tibia, and the calcaneus.

### **5.3. Parathormone and Vitamin D**

Data from the Skylab program exhibited a slight increase in serum Ca2+ levels and decrease in parathormone. Spacelab Life Sciences 1 (9 days), and 2(14 days) flights and a 180-day Mir flight showed decreased serum parathormone levels in crew members, and active vitamin D levels were also decreased, which in turn reduced Ca absorption [Caillot-Augusseau et al., 1998].

### **5.4. Summary**

In summary, bone resorption is increased, bone formation is decreased, bone loss occurs in weight-bearing areas, and parathormone is suppressed during space flight, which are comparable to the data from immobilization under conditions of bedrest or spinal cord injury.

### **6. Sympathetic modulation of bone metabolism during spaceflight**

It has been reported that sympathetic neural traffic to bone inhibits the function of osteoblasts and enhances that of osteoclasts thus facilitating bone loss. Possible roles of the sympathetic nervous system in the mechanisms of bone loss in humans exposed to long-term space flight will be discussed [Mano et al., 2010].

### **6.1. Alterations in sympathetic neural traffic under microgravity**

Sympathetic neural traffic indirectly measured by plasma noradrenaline level has been reported to increase during spaceflight compared with the pre-flight control level [Christensen & Norsk, 1998, Ertl et al., 2002], and vagal activity estimated by power spectral analysis of heart rate variability has been shown to be reduced after long-term spaceflight [Cooke et al., 2000, Mano, 2005].

Microneurographically, on the other hand, recorded neural traffic in humans is known to reflect muscle and skin sympathetic nerve activity (MSNA and SSNA), and MSNA controls the vasomotor function of the muscular bed, responding to blood pressure changes against gravitational stress [Iwase et al., 1987, Mano 1990, Mano et al., 2009]. MSNA was found to be suppressed during an exposure to short-term microgravity induced by parabolic flight [Iwase et al., 1999], to mild lower body positive pressure (10-20 mmHg LBPP) [Fu et al., 1998], and to thermoneutral head-out water immersion [Miwa et al., 1996] responding to the loading or unloading of cardiopulmonary receptor stimulated by cephalad fluid shift. Contrarily, MSNA was enhanced after an exposure to long-term microgravity in spaceflight and its simulation induced by dry immersion (Iwase et al., 2000), and 6°head-down bed rest (Kamiya et al., 2000), caused by various mechanisms including plasma volume loss, changes in baroreflex, and vascular compliance after the human body has acclimated to microgravity situation..

Elevation of these two markers demonstrated that the increased urinary Ca2+ was due to an increase in bone resorption. Osteocalcin, a bone formation marker, measured during a 180-day

Bone mineral density was measured before and after flights on the Mir program. The changes per month were as follows; +0.6% in the skull, +0.1% in the arm, −1.07% in the spine, −1.35% in the pelvis, −1.16% in the femoral neck, −1.58% in the trochanter major, −1.25% in the tibia, and −1.50% in the calcaneus, all per month, with comparable results obtained from the

The bones that are most affected during spaceflight seems to be weight bearing bones, such as the pelvis (os coxae), the trochanter major of the femur, the femoral neck, the tibia, and the

Data from the Skylab program exhibited a slight increase in serum Ca2+ levels and decrease in parathormone. Spacelab Life Sciences 1 (9 days), and 2(14 days) flights and a 180-day Mir flight showed decreased serum parathormone levels in crew members, and active vitamin D levels were also decreased, which in turn reduced Ca absorption [Caillot-Augusseau et al., 1998].

In summary, bone resorption is increased, bone formation is decreased, bone loss occurs in weight-bearing areas, and parathormone is suppressed during space flight, which are comparable to the data from immobilization under conditions of bedrest or spinal cord injury.

It has been reported that sympathetic neural traffic to bone inhibits the function of osteoblasts and enhances that of osteoclasts thus facilitating bone loss. Possible roles of the sympathetic nervous system in the mechanisms of bone loss in humans exposed to long-term space flight

Sympathetic neural traffic indirectly measured by plasma noradrenaline level has been reported to increase during spaceflight compared with the pre-flight control level [Christensen & Norsk, 1998, Ertl et al., 2002], and vagal activity estimated by power spectral analysis of heart rate variability has been shown to be reduced after long-term spaceflight [Cooke et al., 2000,

**6. Sympathetic modulation of bone metabolism during spaceflight**

**6.1. Alterations in sympathetic neural traffic under microgravity**

Mir flight showed a decrease.

International Space Station [Lang et al., 2004].

**5.3. Parathormone and Vitamin D**

will be discussed [Mano et al., 2010].

**5.2. Bone loss location**

264 Topics in Osteoporosis

calcaneus.

**5.4. Summary**

Mano, 2005].

As for the sympathetic influence on bone metabolism, sympathetic stimulation facilitated bone resorption, while it inhibited ossification by osteoblasts mediated by hypothalamus and leptin in mouse. Loading of weak chronic stress in mouse reduced the osteoblastic activity with elevated noradrenaline, which was prevented by β-blocker [Kondo et al., 2005]. The beneficial effects of β-adrenergic blocker on bone mass and metabolism were reported in mice and rats [Minkowitz et al., 1991, Pierroz, et al., 2006]. Other studies were controversial, however, recent studies have indicated that there are two systems that regulate bone metabolism; one through β2 receptors in bone which facilitates osteolysis and inhibits osteogenesis, and the other that facililtates osteogenesis through a kind of neuropeptide called CART (cocaine amphetamine regulated transcript) [Elefteriou et al., 2005].

From human studies, there have been reports that administration of β-blockers may reduce the risk of bone fracture as well as higher bone density (Graham et al., 2008, Levasseur et al., 2005, Pasco et al., 2004, 2005, Reid et al., 2005a, b, Reinmark et al., 2004, 2006, Schlienger et al., 2004, Turker et al., 2006)

### **6.2. Space flight-related changes in sympathetic regulation on bone metabolism**

Prolonged exposure to microgravity in space for 14 days enhanced the sympathetic neural traffic in humans as evidenced by the Neurolab mission[Cox et al., 2002, Fu et al., 2002, Levine et al., 2002, Ertl et al., 2002], with comparable results in elevated noradrenaline spillover and clearance in space [Ertl et al., 2002]. Corresponding results were obtained from simulated microgravity including dry immersion [Iwase et al., 2000] or head-down bed rest [Kamiya et al., 2000]. Elderly people generally have low bone mass and density and high sympathetic neural traffic to muscles although response to gravitational stress becomes lowered (Iwase et al. 1991). Our preliminary data show that changes in sympathetic neural traffic to muscles after long-term bedrest of 20 days had a significant correlation with changes in the urinary secretion level of deoxypyridinoline (Mano et al., 2009, Nishimura et al., 2010) (Fig.2), which a specific marker for bone resorption (Robbins et al., 1994). On the basis of these findings, it is postulated that an exposure to prolonged microgravity enhances the sympathetic neural traffic to bone, which increases the noradrenaline level, inhibits osteogenesis and facilitates osteolysis through β-receptors to induce bone mineral loss; however, it is no better than hypothesis.

The Ca intake necessary to minimize a negative Ca balance is approximately 1,000 mg of elemental Ca (~40% of CaCO3), which is currently recommended for space station flights up to 360 days [Weaver, 2000]. Howver, excessive oral intake of Ca is associated with a risk of hypercalciuria due to skeletal unloading, which may lead to high risk of kidney stones. However, kidney stones may not develop because orally administered Ca may bind to oxalate in the intestine and reduce the oxalate absorption [Heller 1999]. Therefore, Ca intake during

Osteoporosis in Spaceflight http://dx.doi.org/10.5772/54708 267

Exercise upon exposure to weightlessness has been incorporated into some countermeasure programs, however, exercise alone cannot prevent the bone loss. The current exercise program for the ISS is a combination of aerobic and resistive exercises for 2.5 hrs, 6 days/week. Data from the space flight demonstrate that bone loss occurs mainly in the femur, tibia, calcaneus, and vertebrae. Therefore, exercise should be concentrated on these bones, and impact loading

Hip joint: Some of the larger bone losses observed in the space program have been concentrated in the hips (os coxae mainly) [Le Blanc et al., 1999]. A study using hip joint pressure sensors has demonstrated that peak joint forces can range from 3–4 times when walking, 5.5 times with jogging, and as high as 8.7 times bodyweight with stumbling [Hall, 1995], which are do not seem to be generated during the exercise in microgravity. The peak pressure in the articulatio coxae (hip joint) during supine isometric abduction was 3.78 mPa, which was as high as those during walking of 3.64 mPa [Strickland et al., 1992], and that during rising from a seated position, was 7.14 mPa [Hodge et al., 1986]. These data suggest that running on a treadmill generates an insufficient load to generate enough loads, but short periods of high loading using abduction, adduction and squatting would be necessary to load sufficient pressures on the hip

Lumbar spine: The bodyweight is loaded on the L3 vertebra when standing under conditions of 1 G state, but unloaded vertebrae would lose their bone mass density under microgravity. The concern is the minimum period of standing position required to maintain the bone mass, which has not been clarified. Moreover, the even if this duration is clarified; weight loading under weightlessness is difficult. Whether a shorter duration of a higher load can provide the

Femur: The femoral neck and the trochanter major are the principal sites of weight bearing under the condition of 1 G, and are site at which significant bone mass is lost during the spaceflight. The femoral shaft mainly consists of cortical bone and the loss of bone mass from the femoral shaft were −1.6% on average after 4–6 months of spaceflight [Oganov, 1996]. The kind of exercise that be most effective to prevent bone loss from the femur is not known.

Tibia: The proximal tibia consists of trabecular and cortical bone, and its loss of bone mass was reported to be −1.25% per month. The most effective exercise to prevent the bone loss from this

same bone protection as a longer duration of a lower load has also yet to be solved.

should primarily be provided rather than static loading [Taaffe et al., 1997].

spaceflight should be taken orally [Martini & Wood, 2000].

**7.2. Physical factors**

joint.

area seemed to be squatting exercise.

Adapted with permission from Nishimura N et al: Space Utiliz Res. 26: 122-124 (2010)

**Figure 2.** Correlation between muscle sympathetic nerve activity (MSNA) and urinary secretion of deoxypyridinoline in healthy humans exposed to long-term bed rest. A significant correlation was found between percent changes in burst numbers per minute of MSNA recorded microneurographically from the tibial nerve (abscissa) and percent changes in urinary secretion of deoxypyridinoline (ordinate) in 11 young healthy male subjects (24±5 years old) exposed to longterm head-down bed rest (20 days).

### **7. Countermeasures for space-related osteoporosis**

The bone has difficulty regaining its density and once it is lost, as shown by the studies of bed rest and spinal cord injury patients. Data from spaceflight suggest that this slow recovery is also exhibited by astronauts as well [Linenger, 2000, Tilton et al., 1980]. Bone can be recovered, but it takes a longer time to recover it than to lose it. This means that the prevention of bone loss is a more preferable than its recovery by aggressive rehabilitation. The strategy of "less loss to regain fast" might be an effective way to minimize the necessary amount of postflight rehabilitation needed and to extend the time people can remain in microgravity.

### **7.1. Vitamin D and calcium intake**

The low light levels in the spacecraft necessitate sufficient oral intake of vitamin D and Ca since parathormone levels are suppressed during spaceflight. Oral intake of 600−800 IU/day vitamin D is recommended in the absence of sunlight although 400 IU/day is usually adequate to maintain bone mass density [Holick, 1996].

The Ca intake necessary to minimize a negative Ca balance is approximately 1,000 mg of elemental Ca (~40% of CaCO3), which is currently recommended for space station flights up to 360 days [Weaver, 2000]. Howver, excessive oral intake of Ca is associated with a risk of hypercalciuria due to skeletal unloading, which may lead to high risk of kidney stones. However, kidney stones may not develop because orally administered Ca may bind to oxalate in the intestine and reduce the oxalate absorption [Heller 1999]. Therefore, Ca intake during spaceflight should be taken orally [Martini & Wood, 2000].

### **7.2. Physical factors**

Adapted with permission from Nishimura N et al: Space Utiliz Res. 26: 122-124 (2010)

**7. Countermeasures for space-related osteoporosis**

term head-down bed rest (20 days).

266 Topics in Osteoporosis

**7.1. Vitamin D and calcium intake**

maintain bone mass density [Holick, 1996].

**Figure 2.** Correlation between muscle sympathetic nerve activity (MSNA) and urinary secretion of deoxypyridinoline in healthy humans exposed to long-term bed rest. A significant correlation was found between percent changes in burst numbers per minute of MSNA recorded microneurographically from the tibial nerve (abscissa) and percent changes in urinary secretion of deoxypyridinoline (ordinate) in 11 young healthy male subjects (24±5 years old) exposed to long-

The bone has difficulty regaining its density and once it is lost, as shown by the studies of bed rest and spinal cord injury patients. Data from spaceflight suggest that this slow recovery is also exhibited by astronauts as well [Linenger, 2000, Tilton et al., 1980]. Bone can be recovered, but it takes a longer time to recover it than to lose it. This means that the prevention of bone loss is a more preferable than its recovery by aggressive rehabilitation. The strategy of "less loss to regain fast" might be an effective way to minimize the necessary amount of postflight

The low light levels in the spacecraft necessitate sufficient oral intake of vitamin D and Ca since parathormone levels are suppressed during spaceflight. Oral intake of 600−800 IU/day vitamin D is recommended in the absence of sunlight although 400 IU/day is usually adequate to

rehabilitation needed and to extend the time people can remain in microgravity.

Exercise upon exposure to weightlessness has been incorporated into some countermeasure programs, however, exercise alone cannot prevent the bone loss. The current exercise program for the ISS is a combination of aerobic and resistive exercises for 2.5 hrs, 6 days/week. Data from the space flight demonstrate that bone loss occurs mainly in the femur, tibia, calcaneus, and vertebrae. Therefore, exercise should be concentrated on these bones, and impact loading should primarily be provided rather than static loading [Taaffe et al., 1997].

Hip joint: Some of the larger bone losses observed in the space program have been concentrated in the hips (os coxae mainly) [Le Blanc et al., 1999]. A study using hip joint pressure sensors has demonstrated that peak joint forces can range from 3–4 times when walking, 5.5 times with jogging, and as high as 8.7 times bodyweight with stumbling [Hall, 1995], which are do not seem to be generated during the exercise in microgravity. The peak pressure in the articulatio coxae (hip joint) during supine isometric abduction was 3.78 mPa, which was as high as those during walking of 3.64 mPa [Strickland et al., 1992], and that during rising from a seated position, was 7.14 mPa [Hodge et al., 1986]. These data suggest that running on a treadmill generates an insufficient load to generate enough loads, but short periods of high loading using abduction, adduction and squatting would be necessary to load sufficient pressures on the hip joint.

Lumbar spine: The bodyweight is loaded on the L3 vertebra when standing under conditions of 1 G state, but unloaded vertebrae would lose their bone mass density under microgravity. The concern is the minimum period of standing position required to maintain the bone mass, which has not been clarified. Moreover, the even if this duration is clarified; weight loading under weightlessness is difficult. Whether a shorter duration of a higher load can provide the same bone protection as a longer duration of a lower load has also yet to be solved.

Femur: The femoral neck and the trochanter major are the principal sites of weight bearing under the condition of 1 G, and are site at which significant bone mass is lost during the spaceflight. The femoral shaft mainly consists of cortical bone and the loss of bone mass from the femoral shaft were −1.6% on average after 4–6 months of spaceflight [Oganov, 1996]. The kind of exercise that be most effective to prevent bone loss from the femur is not known.

Tibia: The proximal tibia consists of trabecular and cortical bone, and its loss of bone mass was reported to be −1.25% per month. The most effective exercise to prevent the bone loss from this area seemed to be squatting exercise.

Calcaneus: The calcaneus receives a reaction force from the ground of 2–3 times of body weight to the foot while running under 1 G. The fact that the calcaneus of gymnasts exhibits increased bone density suggests that not only the number of loading cycles but also peak loading is significant in increasing bone mass density in the calcaneus [Taaffe et al., 1999].

the healing of even physiological microinjuries within bone. Osteonecrosis of the maxilla and the mandible likely results from the inability of hypodynamic and hypovascular bone to meet an increased demand for repair and remodeling because several kinds of manipulation are

Osteoporosis in Spaceflight http://dx.doi.org/10.5772/54708 269

Thiazide diuretics and potassium citrate are not usually considered drugs for bone loss prevention, but are usually used for kidney stone prevention; they act by markedly reducing

Estrogen is effective for bone mass preservation in both men and women, but it has a side effect of thrombophlebitis, which would be a very significant for bedrest subjects or astronauts in

There is some evidence suggesting that statins might be effective to increase bone mass, in addition to their main role; however, no data from bedrest or immobilization studies have shown the effectiveness of their use. Therefore it might be too early to apply it to astronauts

Parathormone has anabolic effects on bone, and also acts on the kidney to stimulate the resorption of Ca2+ and enhance the synthesis of vitamin D. In this sense, parathormone may stimulate the bone formation, increase vitamin D synthesis, and stimulate Ca2+ resorption. Since the suppression of bone resorption is favorable for stimulating bone formation during

For human space voyages of several years duration, such as those envisioned for the explora‐ tion of Mars, astronauts would be at risk of catastrophic consequences should any of the systems that provide adequate air, water, food, or thermal protection fail. Beyond that, astronauts will face serious health and/or safety risks resulting from severe physiologic deconditioning associated with prolonged weightlessness [Buckey 1999]. The principal physiologic deconditioning risks are related to physical and functional deterioration of the loss of regulation of several systems including blood circulation, decreased aerobic capacity, musculo-skeletal systems, and altered sensory-motor system performance. These physiologic effects of weightlessness are generally adaptive to spaceflight and present a hazard only following G-transitions upon return to Earth or landing on another planet [Young 1999]. Among them, bone mineral metabolism would be greatly affected during prolonged space‐

spaceflight, the administration of parathormone appears strategically unfavorable.

associated with this necrosis.

the urinary Ca level.

the space.

*7.3.4. Statins*

in spaceflight.

*7.3.5. Parathormone*

**7.4. Artificial gravity**

flight.

*7.3.2. Thiazide diuretics and potassium citrate*

*7.3.3. Selective estrogen receptor modulators*

### **7.3. Pharmacological factors**

Since bone mass is adequate at the onset of spaceflight, the optimal strategy for pharmaco‐ therapy against bone loss is the prevention of bone loss, not the acceleration of bone formation, when the loading is removed during spaceflight. Several drugs have been proposed for the prevention of bone loss under microgravity.

### *7.3.1. Bisphosphonates*

Bisphosphonates have two phosphonate (PO3) groups and have a similar structure to pyro‐ phosphate. They bind to hydroxyapatite in the bone matrix, and prevent the bone loss by inhibiting the osteoclastic bone resorption. Bisphosphonates have been demonstrated to be effective in preventing bone loss during bed rest studies [Grigoriev et al., 1992, Rodan & Fleisch, 1996, Thompson et al., 1990, LeBlanc et al., 1998]. Among several kinds of bisphosph‐ onate, pamidronate has been proved to suppress bone mineral loss and to prevent the formation of renal stones during bedrest study (Watanabe et al., 2004).

In 2010, LeBlanc and Matsumoto proposed an experiment on the effectiveness of bisphosph‐ onate as a countermeasure to spaceflight-induced bone loss. The astronauts chose either oral administration of alendronate at 70 mg once per week or intravenous administration of zoledronate at 4 mg before flight, and were examined their bone density by DEXA (dual energy x-ray absorptiometry), QCT (quantitative computed tomography), and pQCT (peripheral qualitative computed tomography), bone metabolism markers including bone formation and resorption markers, and renal stone formation. One of the co-investigators, Ohshima reported successful results in suppressing the space flight-induced bone loss and renal stone formation [Ohshima, personal communication].

The disadvantages of bisphosphonates are local irritation of the upper gastrointestinal (GI) tract, and poor absorption from the GI tract. Therefore, the oral administration of bisphosph‐ onates requires the intake with 200 mL of water and for the subject to remain upright posture for at least 30 min, and until after consumption of the first food of the day to facilitate delivery to the stomach. The problem is that an upright posture cannot be achieved in space under conditions of microgravity. Another problem is the possibility of osteonecrosis of the maxilla and the mandible occurring although the incidence of this is low [Durie et al., 2005]. Since these osteonecroric or osteolytic phenomena are always accompanied by physiological stress (mastication), iatrogenic trauma (tooth extraction/denture injury), or tooth infection [Ruggiero et al. 2004, 2008], it is preferable to prevent such phenomena.

Bisphosphonates are hardly metabolized, and high concentrations of them are maintained in the bones for long periods. Because bone formation is closely coupled to bone turnover, longterm use of these compounds with the resultant suppression of bone turnover can compromise the healing of even physiological microinjuries within bone. Osteonecrosis of the maxilla and the mandible likely results from the inability of hypodynamic and hypovascular bone to meet an increased demand for repair and remodeling because several kinds of manipulation are associated with this necrosis.

### *7.3.2. Thiazide diuretics and potassium citrate*

Thiazide diuretics and potassium citrate are not usually considered drugs for bone loss prevention, but are usually used for kidney stone prevention; they act by markedly reducing the urinary Ca level.

### *7.3.3. Selective estrogen receptor modulators*

Estrogen is effective for bone mass preservation in both men and women, but it has a side effect of thrombophlebitis, which would be a very significant for bedrest subjects or astronauts in the space.

### *7.3.4. Statins*

Calcaneus: The calcaneus receives a reaction force from the ground of 2–3 times of body weight to the foot while running under 1 G. The fact that the calcaneus of gymnasts exhibits increased bone density suggests that not only the number of loading cycles but also peak loading is

Since bone mass is adequate at the onset of spaceflight, the optimal strategy for pharmaco‐ therapy against bone loss is the prevention of bone loss, not the acceleration of bone formation, when the loading is removed during spaceflight. Several drugs have been proposed for the

Bisphosphonates have two phosphonate (PO3) groups and have a similar structure to pyro‐ phosphate. They bind to hydroxyapatite in the bone matrix, and prevent the bone loss by inhibiting the osteoclastic bone resorption. Bisphosphonates have been demonstrated to be effective in preventing bone loss during bed rest studies [Grigoriev et al., 1992, Rodan & Fleisch, 1996, Thompson et al., 1990, LeBlanc et al., 1998]. Among several kinds of bisphosph‐ onate, pamidronate has been proved to suppress bone mineral loss and to prevent the

In 2010, LeBlanc and Matsumoto proposed an experiment on the effectiveness of bisphosph‐ onate as a countermeasure to spaceflight-induced bone loss. The astronauts chose either oral administration of alendronate at 70 mg once per week or intravenous administration of zoledronate at 4 mg before flight, and were examined their bone density by DEXA (dual energy x-ray absorptiometry), QCT (quantitative computed tomography), and pQCT (peripheral qualitative computed tomography), bone metabolism markers including bone formation and resorption markers, and renal stone formation. One of the co-investigators, Ohshima reported successful results in suppressing the space flight-induced bone loss and renal stone formation

The disadvantages of bisphosphonates are local irritation of the upper gastrointestinal (GI) tract, and poor absorption from the GI tract. Therefore, the oral administration of bisphosph‐ onates requires the intake with 200 mL of water and for the subject to remain upright posture for at least 30 min, and until after consumption of the first food of the day to facilitate delivery to the stomach. The problem is that an upright posture cannot be achieved in space under conditions of microgravity. Another problem is the possibility of osteonecrosis of the maxilla and the mandible occurring although the incidence of this is low [Durie et al., 2005]. Since these osteonecroric or osteolytic phenomena are always accompanied by physiological stress (mastication), iatrogenic trauma (tooth extraction/denture injury), or tooth infection [Ruggiero

Bisphosphonates are hardly metabolized, and high concentrations of them are maintained in the bones for long periods. Because bone formation is closely coupled to bone turnover, longterm use of these compounds with the resultant suppression of bone turnover can compromise

significant in increasing bone mass density in the calcaneus [Taaffe et al., 1999].

formation of renal stones during bedrest study (Watanabe et al., 2004).

et al. 2004, 2008], it is preferable to prevent such phenomena.

**7.3. Pharmacological factors**

268 Topics in Osteoporosis

*7.3.1. Bisphosphonates*

prevention of bone loss under microgravity.

[Ohshima, personal communication].

There is some evidence suggesting that statins might be effective to increase bone mass, in addition to their main role; however, no data from bedrest or immobilization studies have shown the effectiveness of their use. Therefore it might be too early to apply it to astronauts in spaceflight.

### *7.3.5. Parathormone*

Parathormone has anabolic effects on bone, and also acts on the kidney to stimulate the resorption of Ca2+ and enhance the synthesis of vitamin D. In this sense, parathormone may stimulate the bone formation, increase vitamin D synthesis, and stimulate Ca2+ resorption. Since the suppression of bone resorption is favorable for stimulating bone formation during spaceflight, the administration of parathormone appears strategically unfavorable.

### **7.4. Artificial gravity**

For human space voyages of several years duration, such as those envisioned for the explora‐ tion of Mars, astronauts would be at risk of catastrophic consequences should any of the systems that provide adequate air, water, food, or thermal protection fail. Beyond that, astronauts will face serious health and/or safety risks resulting from severe physiologic deconditioning associated with prolonged weightlessness [Buckey 1999]. The principal physiologic deconditioning risks are related to physical and functional deterioration of the loss of regulation of several systems including blood circulation, decreased aerobic capacity, musculo-skeletal systems, and altered sensory-motor system performance. These physiologic effects of weightlessness are generally adaptive to spaceflight and present a hazard only following G-transitions upon return to Earth or landing on another planet [Young 1999]. Among them, bone mineral metabolism would be greatly affected during prolonged space‐ flight.

### *7.4.1. Why artificial gravity?*

Space biomedical researchers have been working for many years to develop "countermeas‐ ures" to reduce or eliminate the deconditioning associated with prolonged weightlessness. Intensive and sustained aerobic exercise on a treadmill, bicycle, or rowing machine coupled with intensive resistive exercise has been used on U.S. and Russian spacecraft to minimize these problems. The procedures were uncomfortable and excessively time-consuming for many astronauts, and their effectiveness for maintaining bone, muscle, and aerobic fitness has not been demonstrated, owing, at least in part to the low reliability of the devices used to date. Furthermore, they have had inconsistent effects on postflight orthostatic hypotension or sensory-motor adaptive changes. With the exception of fluid loading before reentry, other kinds of countermeasures (e.g., diet, lower body negative pressure, or wearing a "penguin suit" to force joint extension against a resistive force) have been either marginally effective or present an inconvenience or hazard.

*7.4.2. Why artificial gravity with exercise?*

ameliorated by this protocol, but not completely.

prevention of bone loss in spaceflight (Fig. 3).

**8. Bone loss monitoring in space**

tion markers.

While short radius centrifuge has been proposed several times, only the loading of artificial gravity has not so effective to prevent spaceflight deconditioning. Also human-powered shortarm centrifuge is effective to load exercise to the astronaut. Considering the size of the

Osteoporosis in Spaceflight http://dx.doi.org/10.5772/54708 271

In 1999, Iwase proposed the manufacture of the facility of artificial gravity with ergomet‐ ric exercise, and it was subsequently installed at Nagoya University [Iwase 2005]. Several studies were performed using this short-radius centrifuge with an ergometer. In 2002, bedrest study was carried out to validate the effectiveness of artificial gravity with ergometric exercise. In 2005, the facility was moved to Aichi Medical University, and bedrest studies were performed to finalize the protocol. In 2006, this daily AG-EX stepup protocol (1.4 G of artificial gravity load with 60W of ergometric exercise, and the load was stepped up by 0.2 G and 15 W respectively) has been shown to be effective to prevent cardiovascular, musculoskeletal, and bone metabolism deconditioning, while an alternateday protocol failed to prevent this. In this experiment, bone metabolism was moderately

The authors proposed installing a short-radius centrifuge facility at the International Space Station, and using it to prevent this spaceflight deconditioning including bone loss. This project, Artificial GRavity with Ergometric Exercise (AGREE project) is promising for the

Most of the space medicine studies on bone metabolism have utilized the blood/urine samples collected before, during, and after spaceflight, and analyzed them in laboratories on Earth. However, during prolonged spaceflight now and in the future, the astronauts or spaceflight surgeons will necessitate to collect samples by themselves and to analyze them in the space station or spacecraft to assess the effects of any countermeasures. Although dual X-ray absorptiometry (DEXA) is effective to measure the bone mass, it cannot detect small changes in bone metabolism so may not provide timely information on the effects of countermeasures..

At least, serum/urinary Ca levels and blood/urinary markers of bone resorption should be determined and monitored, and additional information on bone mass (and/or density) and

Guidelines for bed rest standardization [2012] suggest the use of osteocalcin, bone-specific alkaline phosphatase (BSAP), N-terminal propeptide of type I procollagen (P1NP) as bone formation markers, and N-telopeptide, C-telopeptide, and deoxypyridinoline as bone resorp‐

Further measurements for bone loss using miniature mass spectrometers and ultrasound may be possible. In particular, ultrasound echography of the bone would be helpful to measure the

bone formation/resorption markers in blood and urine is desirable.

International Space Station, it is appropriate to employ the short-radius centrifuge

To succeed in the near-term goal of a human mission to Mars during the second quarter of this century, the human risks associated with prolonged weightlessness must be mitigated well beyond our current capabilities. Indeed, during nearly 45 years of human spaceflight experience, including numerous long-duration missions, research has not produced any single countermeasure or combination of countermeasures that is completely effective. Current operational countermeasures have not been rigorously validated and have not fully protected any long-duration (>3 months) astronauts in low-Earth orbit. Thus, it seems unlikely that they will adequately protect astronauts journeying to Mars and back over a three-year period.

Although improvements in exercise protocols, changes in diet, or pharmaceutical treatments of individual systems may be of value, they are unlikely to eliminate the full range of physio‐ logic deconditioning. Therefore, a complete research and development program aimed at substituting for the missing gravitational cues and loading in space is warranted.

The urgency for exploration-class countermeasures is compounded by the limited availability of flight resources for vallidating a large number of system-specific countermeasure ap‐ proaches. Furthermore, recent evidence of rapid degradation of pharmaceuticals flown aboard long-duration missions, putatively because of radiation effects, raises concerns regarding the viability of some promising countermeasure development research. Although the rotation of a Mars-bound spacecraft will not be a panacea for all the human risks of spaceflight (artificial gravity cannot solve the critical problems associated with radiation exposure, isolation, confinement, and environmental homeostasis), artificial gravity does offer significant promise as an effective, efficient, multi-system countermeasure against the physiologic deconditioning associated with prolonged weightlessness. Virtually all of the identified risks associated with cardiovascular deconditioning, myatrophy, bone loss, and neurovestibular disturbances, space anemia, immune compromise, neurovegetative might be alleviated by the appropriate application of artificial gravity.

### *7.4.2. Why artificial gravity with exercise?*

*7.4.1. Why artificial gravity?*

270 Topics in Osteoporosis

present an inconvenience or hazard.

three-year period.

application of artificial gravity.

Space biomedical researchers have been working for many years to develop "countermeas‐ ures" to reduce or eliminate the deconditioning associated with prolonged weightlessness. Intensive and sustained aerobic exercise on a treadmill, bicycle, or rowing machine coupled with intensive resistive exercise has been used on U.S. and Russian spacecraft to minimize these problems. The procedures were uncomfortable and excessively time-consuming for many astronauts, and their effectiveness for maintaining bone, muscle, and aerobic fitness has not been demonstrated, owing, at least in part to the low reliability of the devices used to date. Furthermore, they have had inconsistent effects on postflight orthostatic hypotension or sensory-motor adaptive changes. With the exception of fluid loading before reentry, other kinds of countermeasures (e.g., diet, lower body negative pressure, or wearing a "penguin suit" to force joint extension against a resistive force) have been either marginally effective or

To succeed in the near-term goal of a human mission to Mars during the second quarter of this century, the human risks associated with prolonged weightlessness must be mitigated well beyond our current capabilities. Indeed, during nearly 45 years of human spaceflight experience, including numerous long-duration missions, research has not produced any single countermeasure or combination of countermeasures that is completely effective. Current operational countermeasures have not been rigorously validated and have not fully protected any long-duration (>3 months) astronauts in low-Earth orbit. Thus, it seems unlikely that they will adequately protect astronauts journeying to Mars and back over a

Although improvements in exercise protocols, changes in diet, or pharmaceutical treatments of individual systems may be of value, they are unlikely to eliminate the full range of physio‐ logic deconditioning. Therefore, a complete research and development program aimed at

The urgency for exploration-class countermeasures is compounded by the limited availability of flight resources for vallidating a large number of system-specific countermeasure ap‐ proaches. Furthermore, recent evidence of rapid degradation of pharmaceuticals flown aboard long-duration missions, putatively because of radiation effects, raises concerns regarding the viability of some promising countermeasure development research. Although the rotation of a Mars-bound spacecraft will not be a panacea for all the human risks of spaceflight (artificial gravity cannot solve the critical problems associated with radiation exposure, isolation, confinement, and environmental homeostasis), artificial gravity does offer significant promise as an effective, efficient, multi-system countermeasure against the physiologic deconditioning associated with prolonged weightlessness. Virtually all of the identified risks associated with cardiovascular deconditioning, myatrophy, bone loss, and neurovestibular disturbances, space anemia, immune compromise, neurovegetative might be alleviated by the appropriate

substituting for the missing gravitational cues and loading in space is warranted.

While short radius centrifuge has been proposed several times, only the loading of artificial gravity has not so effective to prevent spaceflight deconditioning. Also human-powered shortarm centrifuge is effective to load exercise to the astronaut. Considering the size of the International Space Station, it is appropriate to employ the short-radius centrifuge

In 1999, Iwase proposed the manufacture of the facility of artificial gravity with ergomet‐ ric exercise, and it was subsequently installed at Nagoya University [Iwase 2005]. Several studies were performed using this short-radius centrifuge with an ergometer. In 2002, bedrest study was carried out to validate the effectiveness of artificial gravity with ergometric exercise. In 2005, the facility was moved to Aichi Medical University, and bedrest studies were performed to finalize the protocol. In 2006, this daily AG-EX stepup protocol (1.4 G of artificial gravity load with 60W of ergometric exercise, and the load was stepped up by 0.2 G and 15 W respectively) has been shown to be effective to prevent cardiovascular, musculoskeletal, and bone metabolism deconditioning, while an alternateday protocol failed to prevent this. In this experiment, bone metabolism was moderately ameliorated by this protocol, but not completely.

The authors proposed installing a short-radius centrifuge facility at the International Space Station, and using it to prevent this spaceflight deconditioning including bone loss. This project, Artificial GRavity with Ergometric Exercise (AGREE project) is promising for the prevention of bone loss in spaceflight (Fig. 3).

### **8. Bone loss monitoring in space**

Most of the space medicine studies on bone metabolism have utilized the blood/urine samples collected before, during, and after spaceflight, and analyzed them in laboratories on Earth. However, during prolonged spaceflight now and in the future, the astronauts or spaceflight surgeons will necessitate to collect samples by themselves and to analyze them in the space station or spacecraft to assess the effects of any countermeasures. Although dual X-ray absorptiometry (DEXA) is effective to measure the bone mass, it cannot detect small changes in bone metabolism so may not provide timely information on the effects of countermeasures..

At least, serum/urinary Ca levels and blood/urinary markers of bone resorption should be determined and monitored, and additional information on bone mass (and/or density) and bone formation/resorption markers in blood and urine is desirable.

Guidelines for bed rest standardization [2012] suggest the use of osteocalcin, bone-specific alkaline phosphatase (BSAP), N-terminal propeptide of type I procollagen (P1NP) as bone formation markers, and N-telopeptide, C-telopeptide, and deoxypyridinoline as bone resorp‐ tion markers.

Further measurements for bone loss using miniature mass spectrometers and ultrasound may be possible. In particular, ultrasound echography of the bone would be helpful to measure the

Fig. 3

**Author details**

**References**

293-301.

**9. Conclusion and summary**

Satoshi Iwase1\*, Naoki Nishimura1

tiveness of any countermeasure program against bone loss..

\*Address all correspondence to: s\_iwase@nifty.com

2 Gifu University of Medical Sciences, Seki, Gifu, Japan

injury. Eur J Clin Invest. (1990). , 20, 330-335.

tein 5. N Engl J Med. (2002). , 346, 1513-1521.

versity Press, New York, NY, , 1-31.

Physiol.(1999). , 19, 183-187.

Res (1999). , 4, 353-356.

In conclusion, it is favorable to administer bisphosphonate orally with artificial gravity with exercise in order to prevent the osteoporosis in space. Monitoring of the blood and urine samples in a space station or spacecraft by a simple method is necessary to assess the effec‐

Osteoporosis in Spaceflight http://dx.doi.org/10.5772/54708 273

and Tadaaki Mano2

1 Department of Physiology, Aichi Medical University, Yazako-Karimata, Nagakute, Japan

[1] Biering-sørensen, F, Bohr, H. H, & Schaadt, O. P. Bone mineral content of the lumbar spine and lower extremities years after spinal cord lesion. Paraplegia. (1988). , 26,

[2] Biering-sørensen, F, & Bohr, H. H. Schaadt OP Longitudinal study of bone mineral content in the lumbar spine, the forearm and the lower extremities after spinal cord

[3] Bosco, C, Colli, R, Introini, E, Cardinale, M, Tsarpela, O, Madella, A, Tihanyi, J, & Vi‐ ru, A. Adaptive responses of human skeletal muscle to vibration exposure. Clin

[4] Boyden, L. M, Mao, J, Belsky, J, Mitzner, L, Farhi, A, Mitnick, M. A, Wu, D, Insogna, K, & Lifton, R. P. High bone density due to a mutation in LDL-receptor-related pro‐

[5] Buckey JC JrPreparing for Mars: the physiologic and medical challenges. Eur J Med

[6] BuckeyJC Jr. Bone Loss, In: Space Physiology, by Buckey, JC Jr., (2006). Oxford Uni‐

[7] Bushinsky, D. A, Riordon, D. R, Chan, J. S, & Krieger, N. S. Decreased potassium

stimulates bone resorption. Am J Physiol. 272: FF780, (1997). , 774.

**Figure 3.** Short arm centrifuge to be installed in the International Space Station.

bone mass/density. Determination of bone mass/density at the hip joint or the calcaneus is helpful to assess the bone status and to validate the countermeasure programs in space [National Osteoporosis Society].

### **9. Conclusion and summary**

Fig. 3 In conclusion, it is favorable to administer bisphosphonate orally with artificial gravity with exercise in order to prevent the osteoporosis in space. Monitoring of the blood and urine samples in a space station or spacecraft by a simple method is necessary to assess the effec‐ tiveness of any countermeasure program against bone loss..

### **Author details**

Satoshi Iwase1\*, Naoki Nishimura1 and Tadaaki Mano2

\*Address all correspondence to: s\_iwase@nifty.com

1 Department of Physiology, Aichi Medical University, Yazako-Karimata, Nagakute, Japan

2 Gifu University of Medical Sciences, Seki, Gifu, Japan

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Rotating Structure Assembly

Support Structure

Motor Assembly

Assembly

**Figure 3.** Short arm centrifuge to be installed in the International Space Station.

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### *Edited by Margarita Valdés Flores*

Osteoporosis affects the osteo-articular system. However, there are hormonal, kidney related, gastrointestinal and neuromuscular factors among other, that can be involved in the etiopathogenesis of the disease. In the other hand, for osteoporosis prevention there are many lifestyle conditions that are very important, as dietary habits, physical activity, drugs and caffeine intake, smoking, associated diseases, etc. Based on the above, treatment and prevention of osteoporosis have to be addressed in a multidisciplinary and integral approach. The knowledge about bone metabolism and the related disorders represents an extensive field that is currently increasing through many investigations conducted in the world. The purpose of this book is to show several reviews and original investigations related with osteoporosis.

Topics in Osteoporosis

Topics in Osteoporosis

*Edited by Margarita Valdés Flores*

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