**3. Clinical‐related publications featuring K1 supplementation**

that vitamin K status affects the mineralization processes in both bone and atherosclerotic plaques in a healthy manner [16]. And, from the population‐based Rotterdam Study, the relation between low vitamin K status and development of coronary artery disease [17] is

The importance of vitamin D for bone health has been known since it was used in the treatment of rachitis in the 1930s. Vitamin D and calcium supplements have been recommended as a pillar in the treatment of osteoporosis over the last three decades. Vitamin D and calcium supplementation increases spinal BMD in healthy, postmenopausal women [18, 19], and vitamin D is crucial in the process of mobilizing the Ca2+ ions into the bone tissue [20]. Interestingly, vitamin D and calcium alone are not able to rebuild bone tissue and infrastructure that are being lost. The synergistic effect of vitamin D and retinoic acid on osteoblast produc‐ tion of osteocalcin was shown in 1993 [21], where Hara et al. demonstrated that MK‐4 was able to partly inhibit the bone resorption induced by inflammation, vitamin D loss and ensuing PTH induction, as seen in the calvariae and incubation medium in his mode *ex vivo* model system. This observation served later as the basis for the introduction and acknowledgement of menatetrenone (MK‐4) supplementation in the clinic [22]. And finally, in 1995, Hara et al. [23] also showed that the inhibition of bone resorption was related to vitamin K2's long side

**2. Vitamin K‐dependent proteins in the bone building process**

Osteocalcin is produced by osteoblasts during bone formation, and serves as the most abundant protein in bone after collagen. Furthermore, it is crucial for bone mineralization. Activated osteocalcin is located within hydroxyapatite crystals and binds calcium strongly to facilitate mineralization of the hydroxyapatite crystal grid [24]. Osteocalcin production is regulated by a plethora of factors including retinoic acid (RA), estrogens, glucocorticoids, as well as vitamin D [25–27]. In 1995, Douglas et al. showed the percentage of carboxylated osteocalcin (cOC), as calculated from total osteocalcin, was found to be less than 60% in osteoporotic postmenopausal women compared to 70–80% in young, healthy adults [28]. The osteocalcin production is increased by vitamin D but also increased by both MK‐4 and MK‐7 in a synergistic fashion. From 2007, as used by Knapen et al., osteocalcin has been employed as a marker for the deficiency of vitamin K in bone. Today, the specific osteocalcin molecules are total Osteocalcin (tOC), ucOsteocalcin (ucOC), and cOsteocalcin (cOC) [28, 44]. A secon‐ dary action of MK‐4 and MK‐7 is the ensuing increased collagen production by cells of the osteoblastic lineage. Collagen should make the structural fundament, on which calcium and other minerals are accumulated within the bone matrix. Increased deposition of collagen makes the bone more flexible and this is very important for the attainment of "higher" or better bone quality [24]. In 2001, Yamaguchi et al. unraveled the stimulatory effect of MK‐7 on osteoblastic bone formation *in vitro*, but they also discovered the suppressive effect of MK‐7 on osteoclast‐like cell formation and osteoclastic bone resorption in rat bone tissues *in vitro* [29]. Furthermore, in 2001, Yamaguchi and Ma [30] confirmed the dual effects of MK‐7 but also a significant decrease number of osteoclasts. Finally, in 2011, Yamaguchi and Weilzmann showed

indisputable.

104 Vitamin K2 - Vital for Health and Wellbeing

chain.

In relation to the loss of bone associated by deficient intake of vitamin K1, many observational studies have been conducted. However, few randomized studies have been able to reveal a significant positive rebuilding of bone mass and increased BMD. This chapter deals only with randomized clinical trials with a duration exceeding 1 year.

Schaafsma et al. from the Netherlands showed in a 1‐year long randomized study from 2000 [32] (featuring four groups with 400 IU vitamin D3; vitamin K1 supplementation 80 μg daily, vitamin K1 and D3, and placebo of Dutch postmenopausal women (with a patient total, *n* = 141) with either normal or low bone mineral densities (BMD). It was shown that women with low BMD had a lower %cOC at baseline than the women with normal BMD. However, this difference disappeared after 1 year of supplementation with vitamin K1 [(mean ± S.D.) 68 ± 11% (95% CI = 64.5 ± 71.2%) versus 72 ± 6% (95% CI = 70.1 ± 72.9%)], respectively. On the other hand, 1 year of supplementation with vitamin D3 showed maximum increases in 25(OH)D of 33 ± 29% (95% CI = 24.8 ± 41.8%) and 68 ± 58% (95% CI = 50.1 ± 84.6%) in women with normal and low BMD, respectively. No effect was observed on BMD [32].

In the Bram et al. study from 2003, three groups were examined; group K1D: the effect of vitamin K1 (1 mg/day) and vitamin D (8 μg/day including standard mineral supplementation), group D (vitamin D and minerals) and group 3, placebo on bone loss retardation in a random‐ ized, double‐blind, placebo‐controlled 3‐year intervention study. Of 181 healthy postmeno‐ pausal women between 50 and 60 years of age, 155 completed the observation period. The main outcomes of the study were significant changes in BMD‐values of the femoral neck and lumbar spine after 3 years. The group receiving the supplement with additional vitamin K1 showed a decline in the bone loss from the femoral neck. The difference in femoral neck bone mass between the K1D group and the placebo group was 1.7% (95% CI: 0.35–3.44). The difference between the K1D group and the D group was 1.3% (95% CI: 0.10–3.41). No significant differences were observed among the three groups with respect to changes in BMD at the site of the lumbar spine. It was therefore concluded that the minerals and vitamin D, coadminis‐ tered with the vitamin K1 supplement, substantially contributed to a significant reduction in postmenopausal bone loss at the site of the femoral neck [33].

In a systematic review and meta‐analysis of 700 patients, Cockayne et al. showed in 2006 that the MK‐4 intake in Japan yielded a powerful reduction in the incident of fractures. However, the authors would not recommend vitamin K supplementation until a new randomized clinical trial confirmed the results [34].

Boton‐Smith et al. performed a 2‐year, randomized, double‐blind, and placebo‐controlled study in 2007, scrutinizing the effect of dietary supplementation with either: (1) 200 μg vitamin K1 daily, (2) vitamin D 400 UI daily (3) and calcium 1000 mg daily, or (4) their combination on 244 healthy nonosteoporotic older women. Baseline and 6‐month measurements included DXA bone mineral scans of the hip and wrist, markers of bone turnover, and vitamin status. The results reported were the following: the combined vitamin K1 with vitamin D plus calcium treatment was associated with only a modest but significant increase in BMC at the ultradistal radius, however not at other sites in the hip or radius [35].

at that time, it seemed that the menaquinones (menatetrenone: MK‐4), might diminish the fracture risk incurred by the enrolled patients. Whether vitamin K is an important "by‐player" in maintaining skeletal health in females situated in the northern part of the US remains an unsolved issue. Furthermore, different entities of K vitamins (i.e., phylloquinone (K1) and MK‐ 4) may exert differing biological effects on the skeleton. The present study was designed to assess the efficacy of either vitamin K1 or MK‐4 exposure on biomarkers of skeletal health and bone mineral density (BMD) in postmenopausal nonosteoporotic, North American women. In the present, placebo‐controlled and double‐blind investigation, a total of 381 postmenopausal females were given either vitamin K1 (1 mg/day), MK‐4 (45 mg/day), or a placebo treatment. The whole observation period was 12 months. All enrolled patients/participants were given either Ca2+ or vitamin D3 supplementation. Bone‐specific BSALP (alkaline phosphatase) in blood samples, as well as the *n*‐telopeptide of collagen, type 1 (NTX) were measured at before the onset of "medication," and subsequent to 1, 3, 6, and 12 months, respectively. Both lumbar spine and proximal femur BMD values, as well as proximal femur geometry were assessed by DXA, before the onset of the trial, and after 6 and 12 months of treatment. At the onset of the trial, all treatment groups showed identical demographic parameters. The patients' compli‐ ance rates related to the intake of either calcium, vitamin K1, or MK‐4, were some 87–93%, respectively. Interestingly, K1 and MK‐4 treatments both diminished the patient levels of serum ucCO, however, neither BSALP nor NTX levels were changed. Lastly, no effects of K1, or MK‐ 4 on lumbar spine or proximal femur BMD or proximal femoral geometric parameters could be observed. This study does not support a role for vitamin K supplementation in osteoporosis prevention among healthy, postmenopausal, North American women, receiving calcium and

Vitamin K2 and Bone Health http://dx.doi.org/10.5772/64876 107

**4. Clinical randomized controlled osteopenia/osteoporosis studies with menatetrenone‐4 (MK‐4) from 1 year duration, in countries with different**

In 1998, Orimo et al from Japan evaluated the effects of menatetrenone‐4 (MK‐4) on bone and calcium metabolism in osteoporosis patients in a 24‐week double‐blind placebo‐controlled study, where 80 osteoporotic patients were included. Treatment was MK‐4, 90 mg/day (*n* = 39) or placebo (*n* = 41). Bone density was assessed on X‐ray films of the right, second metacarpal bone, using the microdensitometric method. In the MK‐4 group, bone density increased by about 2.2 ± 2.5% from the baseline; in the placebo group it decreased by about 7.3 ± 3.7% (*P* = 0.037, K2 treatment versus placebo). The excretion of γ‐carboxyglutamic acid (Gla) to the urine was markedly enhanced (i.e., from 72.6 ± 4.1 nmol/mg of creatinine before initiation of "medication," to 88.4 ± 5.4 during the 24th week subsequent to the sustained MK‐4 treatment (*P* = 0.008) period). In the group receiving placebo, no significant changes in the excretion of urinary Gla could be observed. However, during the 24‐week long treatment period, the urinary ratio of calcium over creatinine in the K2 treatment group was reduced from 0.14 ± 0.02 to 0.12 ± 0.02, respectively. However, in the placebo group it increased from 0.15 ± 0.02 to 0.19 ± 0.03. Accordingly, the 24‐week levels shown by members of both the MK‐4 and the placebo

vitamin D supplementation [38].

**background intake of vitamin K**

The Booth et al.'s study was published 1 year later, in 2008. The goal of the present investigation was to pinpoint the effect of a consecutive 3‐year administration of vitamin K1 on putative alterations in bone mineral density (BMD) of the femoral neck in elderly patients of both sexes, who presented upon inclusion with calcium and vitamin D repletion. In the present double‐ blind, controlled survey, 452 individuals (both men and women, 60–80 years of age) were evenly distributed, in a randomized fashion, each to receive a multivitamin containing either 500 μg/day of vitamin K1 or placebo, in addition to a daily, 600 mg elemental calcium with vitamin D (400 IU) supplement. Analyses of the femoral neck, spine (L2–L4), and total‐body BMD, turnover of bone mass, and indigenous status of both vitamins K and D was checked every 6–12 months. Results were as follows: one could not find any distinction in BMD values localized to any pertinent body sites, when comparing the two patient groups. Furthermore, the population receiving the vitamin K1 supplement showed a markedly higher vitamin K1 level, as well as a substantially lower degree (percentage) of ucCO concentrations, when matched with the patients not ingesting K1. Neither of the additional biochemical variables measured differed between the patient treatment groups. Hence, the authors concluded that vitamin K1 supplementation, in a dose attainable in the diet, does not confer any additional benefit for bone health at the spine or hip when taken with recommended amounts of calcium and vitamin D [36].

The emerging questions to ask were then: Was the dose of vitamin K1 supplement too low, or the follow up period too short to elicit an increase of BMD, or was it possible to use other sub‐ family vitamin K members, such as menaquinone‐4 (MK‐4)? Or, could vitamin K supplemen‐ tation potentially be harmful to the body?

Cheung et al. from Canada addressed the last of these questions in their 2–4‐year study from 2008. In this trial, 440 postmenopausal women with osteopenia were randomized into a placebo‐controlled double‐blind trial, and it was conducted, mainly to determine whether daily high‐dose vitamin K1 supplementation safely reduces bone loss, bone turnover, and fractures. The conclusions coming out of the study were: 5 mg of daily vitamin K1 supple‐ mentation for 2–4 years does not protect against age‐related decline in BMD, but may protect against fractures and cancers in postmenopausal women with osteopenia. Overall fracture rate was reduced by 50% (9 versus 20, *P* = 0.04) versus placebo. Interestingly, cancer incidence was reduced by 75% with vitamin K1 (3 versus 12, *P* = 0.02). However, more studies are needed to further examine the effect of vitamin K on fractures and cancers [37].

Brinkley et al. from USA conducted a 1‐year study in 2009 and found that low vitamin K status is associated with low BMD and an increased fracture risk. From the bulk of reports available at that time, it seemed that the menaquinones (menatetrenone: MK‐4), might diminish the fracture risk incurred by the enrolled patients. Whether vitamin K is an important "by‐player" in maintaining skeletal health in females situated in the northern part of the US remains an unsolved issue. Furthermore, different entities of K vitamins (i.e., phylloquinone (K1) and MK‐ 4) may exert differing biological effects on the skeleton. The present study was designed to assess the efficacy of either vitamin K1 or MK‐4 exposure on biomarkers of skeletal health and bone mineral density (BMD) in postmenopausal nonosteoporotic, North American women. In the present, placebo‐controlled and double‐blind investigation, a total of 381 postmenopausal females were given either vitamin K1 (1 mg/day), MK‐4 (45 mg/day), or a placebo treatment. The whole observation period was 12 months. All enrolled patients/participants were given either Ca2+ or vitamin D3 supplementation. Bone‐specific BSALP (alkaline phosphatase) in blood samples, as well as the *n*‐telopeptide of collagen, type 1 (NTX) were measured at before the onset of "medication," and subsequent to 1, 3, 6, and 12 months, respectively. Both lumbar spine and proximal femur BMD values, as well as proximal femur geometry were assessed by DXA, before the onset of the trial, and after 6 and 12 months of treatment. At the onset of the trial, all treatment groups showed identical demographic parameters. The patients' compli‐ ance rates related to the intake of either calcium, vitamin K1, or MK‐4, were some 87–93%, respectively. Interestingly, K1 and MK‐4 treatments both diminished the patient levels of serum ucCO, however, neither BSALP nor NTX levels were changed. Lastly, no effects of K1, or MK‐ 4 on lumbar spine or proximal femur BMD or proximal femoral geometric parameters could be observed. This study does not support a role for vitamin K supplementation in osteoporosis prevention among healthy, postmenopausal, North American women, receiving calcium and vitamin D supplementation [38].

Boton‐Smith et al. performed a 2‐year, randomized, double‐blind, and placebo‐controlled study in 2007, scrutinizing the effect of dietary supplementation with either: (1) 200 μg vitamin K1 daily, (2) vitamin D 400 UI daily (3) and calcium 1000 mg daily, or (4) their combination on 244 healthy nonosteoporotic older women. Baseline and 6‐month measurements included DXA bone mineral scans of the hip and wrist, markers of bone turnover, and vitamin status. The results reported were the following: the combined vitamin K1 with vitamin D plus calcium treatment was associated with only a modest but significant increase in BMC at the ultradistal

The Booth et al.'s study was published 1 year later, in 2008. The goal of the present investigation was to pinpoint the effect of a consecutive 3‐year administration of vitamin K1 on putative alterations in bone mineral density (BMD) of the femoral neck in elderly patients of both sexes, who presented upon inclusion with calcium and vitamin D repletion. In the present double‐ blind, controlled survey, 452 individuals (both men and women, 60–80 years of age) were evenly distributed, in a randomized fashion, each to receive a multivitamin containing either 500 μg/day of vitamin K1 or placebo, in addition to a daily, 600 mg elemental calcium with vitamin D (400 IU) supplement. Analyses of the femoral neck, spine (L2–L4), and total‐body BMD, turnover of bone mass, and indigenous status of both vitamins K and D was checked every 6–12 months. Results were as follows: one could not find any distinction in BMD values localized to any pertinent body sites, when comparing the two patient groups. Furthermore, the population receiving the vitamin K1 supplement showed a markedly higher vitamin K1 level, as well as a substantially lower degree (percentage) of ucCO concentrations, when matched with the patients not ingesting K1. Neither of the additional biochemical variables measured differed between the patient treatment groups. Hence, the authors concluded that vitamin K1 supplementation, in a dose attainable in the diet, does not confer any additional benefit for bone health at the spine or hip when taken with recommended amounts of calcium

The emerging questions to ask were then: Was the dose of vitamin K1 supplement too low, or the follow up period too short to elicit an increase of BMD, or was it possible to use other sub‐ family vitamin K members, such as menaquinone‐4 (MK‐4)? Or, could vitamin K supplemen‐

Cheung et al. from Canada addressed the last of these questions in their 2–4‐year study from 2008. In this trial, 440 postmenopausal women with osteopenia were randomized into a placebo‐controlled double‐blind trial, and it was conducted, mainly to determine whether daily high‐dose vitamin K1 supplementation safely reduces bone loss, bone turnover, and fractures. The conclusions coming out of the study were: 5 mg of daily vitamin K1 supple‐ mentation for 2–4 years does not protect against age‐related decline in BMD, but may protect against fractures and cancers in postmenopausal women with osteopenia. Overall fracture rate was reduced by 50% (9 versus 20, *P* = 0.04) versus placebo. Interestingly, cancer incidence was reduced by 75% with vitamin K1 (3 versus 12, *P* = 0.02). However, more studies are needed to

Brinkley et al. from USA conducted a 1‐year study in 2009 and found that low vitamin K status is associated with low BMD and an increased fracture risk. From the bulk of reports available

further examine the effect of vitamin K on fractures and cancers [37].

radius, however not at other sites in the hip or radius [35].

106 Vitamin K2 - Vital for Health and Wellbeing

and vitamin D [36].

tation potentially be harmful to the body?
