**2. Iron overload in thalassemia patients**

#### **2.1. Causes of iron overload**

Patients with β-thalassemia have a partial or complete lack of ability to synthesize the β-chains of hemoglobin [13]. This process of β-globin chain synthesis is controlled by gene that is found to be present in Chromosome 11. There are more than 200 points of mutation and rare deletions of this gene. The production of the β-globin chain can range from near normal to completely absent, leading to varying degrees of excess α-globins in the β-globin chain production. In the β-thalassemia trait, the one gene defect is asymptomatic and results in microcytosis and mild anemia. β-thalassemia major or Cooley anemia results from either severely reduced synthesis or the absence of synthesis in both genes. If the synthesis of the beta chain is less severely reduced, the person will have beta thalassemia intermedia. These persons experience symp‐ toms that are less severe and do not require lifelong transfusions to survive past the age of 20 years [14]. The amount of iron (20 - 30 mg) required for the daily production of 300 billion RBCs is provided mostly by the iron that is recycled by the macrophages [6]. Importantly, iron stored in the macrophages is safe and does not lead to oxidative damage [15]. Iron overload can be caused by an increase in dietary iron absorption in hereditary hemochromatosis patients [16] and by multiple blood transfusions in β-thalassemia patients. Duodenal iron absorption in normal persons is approximately 1 – 2 mg/day and balanced with iron excretion at 1 – 2 mg/ day. Though thalassemia intermedia patients do not receive transfusions, abnormal iron absorption produces an increase in the body's iron burden evaluated to 2 – 5 g/year (3 - 9 mg iron/day) [17]. Regular blood transfusions (420 ml/unit of donor blood equivalent to 200 mg of iron) lead to double iron accumulation. Increased iron absorption along with multiple blood transfusions in thalassemia patients can result in hemosiderosis, oxidative stress, hypercoa‐ gulability, liver inflammation, cardiomyopathy, endocrine dysfunctions and bone deformity, of which the complications can be meliorated by many drugs and agents (Figure 1).

7

 Consequently, iron overload and accumulation introduces progressive damage in the liver, heart and in the endocrine glands. Plasma NTBI occurs in patients who have had multiple blood transfusions. NTBI was originally identified in reference [18] for playing a major role in the pathological conditions of iron overload. Circulating NTBI, as well as LPI, is

Consequently, iron overload and accumulation introduces progressive damage in the liver, heart and in the endocrine glands. Plasma NTBI occurs in patients who have had multiple blood transfusions. NTBI was originally identified in reference [18] for playing a major role in the pathological conditions of iron overload. Circulating NTBI, as well as LPI, is detected whenever the capacity of transferrin to incorporate iron is derived either from the GI tract or when RES becomes a limiting factor. Both forms of toxic iron appear primarily in transfused patients where the TIBC has been surpassed [19]. Pathologically, the NTBI fraction seems to be translocated across the cell membrane irregularly, while the LPI is redox active and susceptible to chelation [20]. Chronic iron overload has been attributed to highly elevated levels of plasma iron and high accumulations of tissue iron. Excessive iron accumulation in

(hydroxyurea, hydroxycarbamide, decitabin and phenylbutyrate) **Hb stimulating agents** (erythropoietin, folic acid) **Bone marrow transplantation** 

**Figure 1** Complications and treatments of thalassemia patients with iron overload

**Hb F modifiers**

**Gene therapy HSC therapy**

**Figure 1.** Complications and treatments of thalassemia patients with iron overload

**Iron chelators** (DFO, DFP, DFX) **Inhibitors of iron absorption** (tea, Ca) **Foods** (phytate and oxalate)

**Hemosiderosis**

**Thalassemias with iron overload**  (From *Nature Genetics Review*, 2004)

**Endocrinopathy Ineffective erythropoiesis**

**Antioxidative compounds** (vitamin C, E, NAC)

Antioxidants as Complementary Medication in Thalassemia

**Antioxidative phytochemicals** (flavonoids, curcuminoids)

**Oxidative stress** 

**Liver inflammation and cirrhosis**

**Vitamin D Osteoclast supplement**

**Hydroxyurea Aspirin Coumadin**

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121

**Cardiomyopathy** 

**Bone deformity**

**Hypercoagulability** 

**Hormone supplement** 

(www.nctrallawblog.com) **Blood transfusion** (420 ml/unit 200 mg Fe)

> (http://saschina.org) **Duodenal iron absorption** (3 - 9 mg Fe/day in thalassemia intermedia)

**Figure 1** Complications and treatments of thalassemia patients with iron overload **Figure 1.** Complications and treatments of thalassemia patients with iron overload

directly onto the plasma iron as well as the cellular iron; afterwards, the complexes can be excreted from the body easily [8]. Serum oxidant activity in young β-thalassemia major patients with iron overload is directly correlated with the serum ceruloplasmin and copper concentra‐ tions, and with serum iron (SI) concentration and total iron-binding capacity (TIBC), but not with serum vitamin E concentration [9]. Levels of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) were greatly elevated in the RBC of β-thalassemia minor patients to fight cellular increased oxidant and are close to normal values in the RBC of β-thalassemia major patients due to the presence of transfused normal RBC [10]. Levels of antioxidant compounds such as serum retinol (vitamin A), carotenoids, α-tocopherol (vitamin E) were decreased in β-thalassemia major patients [11, 12]. Supplements of the antioxidant vitamins can prevent some of the damage in the thalassemic RBC membrane. Thus, antioxidant therapy can be a supplemental medical regime to meliorate

pathophysiological complications and improve quality of life of thalassemia patients.

Patients with β-thalassemia have a partial or complete lack of ability to synthesize the β-chains of hemoglobin [13]. This process of β-globin chain synthesis is controlled by gene that is found to be present in Chromosome 11. There are more than 200 points of mutation and rare deletions of this gene. The production of the β-globin chain can range from near normal to completely absent, leading to varying degrees of excess α-globins in the β-globin chain production. In the β-thalassemia trait, the one gene defect is asymptomatic and results in microcytosis and mild anemia. β-thalassemia major or Cooley anemia results from either severely reduced synthesis or the absence of synthesis in both genes. If the synthesis of the beta chain is less severely reduced, the person will have beta thalassemia intermedia. These persons experience symp‐ toms that are less severe and do not require lifelong transfusions to survive past the age of 20 years [14]. The amount of iron (20 - 30 mg) required for the daily production of 300 billion RBCs is provided mostly by the iron that is recycled by the macrophages [6]. Importantly, iron stored in the macrophages is safe and does not lead to oxidative damage [15]. Iron overload can be caused by an increase in dietary iron absorption in hereditary hemochromatosis patients [16] and by multiple blood transfusions in β-thalassemia patients. Duodenal iron absorption in normal persons is approximately 1 – 2 mg/day and balanced with iron excretion at 1 – 2 mg/ day. Though thalassemia intermedia patients do not receive transfusions, abnormal iron absorption produces an increase in the body's iron burden evaluated to 2 – 5 g/year (3 - 9 mg iron/day) [17]. Regular blood transfusions (420 ml/unit of donor blood equivalent to 200 mg of iron) lead to double iron accumulation. Increased iron absorption along with multiple blood transfusions in thalassemia patients can result in hemosiderosis, oxidative stress, hypercoa‐ gulability, liver inflammation, cardiomyopathy, endocrine dysfunctions and bone deformity,

of which the complications can be meliorated by many drugs and agents (Figure 1).

**2. Iron overload in thalassemia patients**

**2.1. Causes of iron overload**

7 the liver, heart and in the endocrine glands. Plasma NTBI occurs in patients who have had multiple blood transfusions. NTBI was originally identified in reference [18] for playing a major role in the pathological conditions of iron overload. Circulating NTBI, as well as LPI, is Consequently, iron overload and accumulation introduces progressive damage in the liver, heart and in the endocrine glands. Plasma NTBI occurs in patients who have had multiple blood transfusions. NTBI was originally identified in reference [18] for playing a major role in the pathological conditions of iron overload. Circulating NTBI, as well as LPI, is detected whenever the capacity of transferrin to incorporate iron is derived either from the GI tract or when RES becomes a limiting factor. Both forms of toxic iron appear primarily in transfused patients where the TIBC has been surpassed [19]. Pathologically, the NTBI fraction seems to be translocated across the cell membrane irregularly, while the LPI is redox active and susceptible to chelation [20]. Chronic iron overload has been attributed to highly elevated levels of plasma iron and high accumulations of tissue iron. Excessive iron accumulation in

Consequently, iron overload and accumulation introduces progressive damage in

the vital organs is the cause of various liver diseases (e.g. hepatitis, hepatic fibrosis and hepatocellular carcinoma), cardiomyopathies (e.g. cardiac arrhythmia and heart failure) and endocrine gland dysfunction (e.g. diabetes, growth retardation, hypogonardism and hypo‐ parathyroidism) [21, 22].  **Pharmacology and Nutritional Intervention in the Treatment of Disease** 

Another Fenton reagent can be hemichromes, which are a family of denatured methemoglo‐ bins [42]. Hydroxyl radicals might be most harmful to lipid, protein and DNA, which are the essential cell components (Figure 3). The •OH-induced membrane damage can be related directly to a membrane-associated Fenton reagent [43]. Oxidative cell damage has been attributed to the emergence of excessive levels of LPI that promote the production of ROS to

Antioxidants as Complementary Medication in Thalassemia

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123

a level that exceeds the cellular defense capacity [44].

**Figure 3.** Production and harmfulness of ROS (Reprinted with modification from [45])

In β-thalassemia major patients, an outpouring of catabolic iron overwhelms the capacity of iron-transporting protein transferrin, and generates NTBI and redox-active LPI in the plasma compartment. Cytosolic LIP has been shown to be comprised of transitory Fe (II) and Fe (III) forms which are possibly mediated by specific cellular iron reductases [20, 44]. In sub-cellular organelles, mitochondrial iron serves in the formation of protein iron-sulfur clusters and porphyrins. Additionally, endosomal iron provides the translocation of the endocytosed iron

tract or when RES becomes a limiting factor. Both forms of toxic iron appear primarily in

#### **2.2. Chemistry of catalytic iron** detected whenever the capacity of transferrin to incorporate iron is derived either from the GI

Iron acts as a cofactor within the active site of key enzymes in the biochemical pathways and as a chemical catalyst in the unique redox activity. The iron can cycle between two oxidation states, ferric ion [Fe(III) or Fe3+] and ferrous ion [Fe(II) or Fe2+], allowing it to act as an electron donor and acceptor [23]. The biochemical functions of iron in vital cells are dependent on its chemical properties. Limitation of iron bioavailability under aerobic conditions occurs when the Fe2+ is rapidly oxidized in the solution to insoluble Fe3+ at physiological pH [*K*free Fe(III) = 10−18 M] [24]. Fe(III) iron is the most stable state of the biological complexes at physiological oxygen concentrations. Many complexes of iron and biomolecules (protein or simpler mole‐ cules) are involved with reduction potential or redox potential (*Eh*). The *Eh* of complexed iron in the range of a biological oxidant is +820 mV to the reductant at −320 mV, the redox reactions are reversible whereas the reaction may be irreversible for the iron complexes when the reduction potential occurs outside this range [1]. transfused patients where the TIBC has been surpassed [19]. Pathologically, the NTBI fraction seems to be translocated across the cell membrane irregularly, while the LPI is redox active and susceptible to chelation [20]. Chronic iron overload has been attributed to highly elevated levels of plasma iron and high accumulations of tissue iron. Excessive iron accumulation in the vital organs is the cause of various liver diseases (e.g. hepatitis, hepatic fibrosis and hepatocellular carcinoma), cardiomyopathies (e.g. cardiac arrhythmia and heart failure) and endocrine gland dysfunction (e.g. diabetes, growth retardation, hypogonardism and hypoparathyroidism) [21, 22]. **2.2 Chemistry of catalytic iron**  Iron acts as a cofactor within the active site of key enzymes in the biochemical pathways and as a chemical catalyst in the unique redox activity. The iron can cycle between two oxidation states, ferric ion [Fe(III) or Fe3+] and ferrous ion [Fe(II) or Fe2+], allowing it to act as an electron donor and acceptor [23]. The biochemical functions of iron in vital cells are dependent on its chemical properties. Limitations of iron bioavailability under aerobic conditions is occurs when the Fe2+ is rapidly oxidized in the solution to insoluble

Iron is a crucial enzyme cofactor that acts in the reduction-oxidation reaction in the metabolism in the cells. Of outstanding interest, iron deposition in the heart cells can lead to oxidative stress and cellular damage [25-27]. Heart failure is the leading cause of death among hemosiderotic β-thalassemia patients, of whom, around 60% die of this cardiac failure [28-30]. The importance of metals to both enzymatic reactions and oxidative stress makes them the key players in mitochondria. Mitochondria are the primary energygenerating organelles of the cells that produce ATP through a chain of enzymatic com‐ plexes that require cytochromes iron and sulfur-iron, and are highly sensitive to oxidative damage. Moreover, the heart is one of the most mitochondria-rich tissues in the body, making metals of particular importance to cardiac function [31]. In cardiac cells, excess iron may result in oxidative stress and an alteration of myocardial function because of the DNA damage that is caused by hydrogen peroxide through the Fenton reaction [6, 32]. Harm‐ ful effects of iron overload on the hearts of patients with β-thalassemia major can be monitored [25, 33-38] and treatment with effective iron chelators can protect these pa‐ tients from cardiac arrhythmia [39, 40]. Iron catalyzes the production of ROS including O2 •-, H2O2 and •OH via Haber-Weiss and Fenton reactions (Figure 2). Fe3+ at physiological pH [*K*free Fe(III) = 10<sup>−</sup>18 M] [24]. Fe(III) iron is the most stable state of the biological complexes at physiological oxygen concentrations. Many complexes of iron and biomolecules (protein or simpler molecules) are involved with reduction potential or redox potential (*Eh*). The *Eh* of complexed iron in the range of a biological oxidant is +820 mV to the reductant at −320 mV, the redox reactions are reversible whereas the reaction may be irreversible for the iron complexes when the reduction potential occurs outside this range [1]. Iron is a crucial enzyme cofactor that acts in the reduction-oxidation reaction in the metabolism in the cells. Of outstanding interest, iron deposition in the heart cells can lead to oxidative stress and cellular damage [25-27]. Heart failure is the leading cause of death among hemosiderotic -thalassemia patients, of whom, around 60% die of this cardiac failure [28-30]. The importance of metals to both enzymatic reactions and oxidative stress makes them the key players in mitochondria. Mitochondria are the primary energygenerating organelles of the cells that produce ATP through a chain of enzymatic complexes that require cytochromes iron and sulfur-iron, and are highly sensitive to oxidative damage. Moreover, the heart is one of the most mitochondria-rich tissues in the body, making metals of particular importance to cardiac function [31]. In cardiac cells, excess iron may result in oxidative stress and an alteration of myocardial function because of the DNA damage that is caused by hydrogen peroxide through the Fenton reaction [6, 32]. Harmful effects of iron overload on the hearts of patients with -thalassemia major can be monitored [25, 33-38] and treatment with effective iron chelators can protect these patients from cardiac arrhythmia [39, 40]. Iron catalyzes the production of ROS) including O2 - , H2O2 and OH via

$$\begin{array}{cccc} \mathsf{H}\_{2}\mathsf{I}^{\star} + \mathsf{H}\_{2}\mathsf{O}\_{2} & \xrightarrow{\mathsf{Free}\mathsf{I}\mathsf{C}\mathsf{u}} & \mathsf{HO}^{\star} + \mathsf{HO}^{\star} + \mathsf{O}\_{2} & \mathsf{H}\mathsf{a}\mathsf{b}\mathsf{e}\mathsf{r}\text{-}\mathsf{We}\mathsf{iss}\mathsf{reaction} \\\\ \mathsf{Fe}^{2+} + \mathsf{H}\_{2}\mathsf{O}\_{2} & \xrightarrow{\mathsf{I}} & \mathsf{Fe}^{3+} + \mathsf{HO}^{\star}\mathsf{+} \mathsf{HO}^{\star} & \end{array} \qquad \begin{array}{cccc} \mathsf{H}\mathsf{a}\mathsf{b}\mathsf{e}\mathsf{r}\text{-}\mathsf{We}\mathsf{iss}\mathsf{reaction} \\\\ \mathsf{H}\mathsf{a}\mathsf{b}\mathsf{e}\mathsf{r}\text{-}\mathsf{We}\mathsf{ess}\mathsf{e}\mathsf{caction} \end{array}$$

8

**Figure 2** Hydroxyl radical formation in metals-catalyzed Haber-Weiss and Fenton reactions [41] **Figure 2.** Hydroxyl radical formation in metals-catalyzed Haber-Weiss and Fenton reactions [41]

Haber-Weiss and Fenton reactions (**Figure 2**).

Another Fenton reagent can be hemichromes, which are a family of denatured methemoglo‐ bins [42]. Hydroxyl radicals might be most harmful to lipid, protein and DNA, which are the essential cell components (Figure 3). The •OH-induced membrane damage can be related directly to a membrane-associated Fenton reagent [43]. Oxidative cell damage has been attributed to the emergence of excessive levels of LPI that promote the production of ROS to a level that exceeds the cellular defense capacity [44].

the vital organs is the cause of various liver diseases (e.g. hepatitis, hepatic fibrosis and hepatocellular carcinoma), cardiomyopathies (e.g. cardiac arrhythmia and heart failure) and endocrine gland dysfunction (e.g. diabetes, growth retardation, hypogonardism and hypo‐

 **Pharmacology and Nutritional Intervention in the Treatment of Disease** 

detected whenever the capacity of transferrin to incorporate iron is derived either from the GI tract or when RES becomes a limiting factor. Both forms of toxic iron appear primarily in transfused patients where the TIBC has been surpassed [19]. Pathologically, the NTBI fraction seems to be translocated across the cell membrane irregularly, while the LPI is redox active and susceptible to chelation [20]. Chronic iron overload has been attributed to highly elevated levels of plasma iron and high accumulations of tissue iron. Excessive iron accumulation in the vital organs is the cause of various liver diseases (e.g. hepatitis, hepatic fibrosis and hepatocellular carcinoma), cardiomyopathies (e.g. cardiac arrhythmia and heart failure) and endocrine gland dysfunction (e.g. diabetes, growth retardation, hypogonardism

Iron acts as a cofactor within the active site of key enzymes in the biochemical pathways and as a chemical catalyst in the unique redox activity. The iron can cycle between two oxidation states, ferric ion [Fe(III) or Fe3+] and ferrous ion [Fe(II) or Fe2+], allowing it to act as an electron donor and acceptor [23]. The biochemical functions of iron in vital cells are dependent on its chemical properties. Limitation of iron bioavailability under aerobic conditions occurs when the Fe2+ is rapidly oxidized in the solution to insoluble Fe3+ at physiological pH [*K*free Fe(III) = 10−18 M] [24]. Fe(III) iron is the most stable state of the biological complexes at physiological oxygen concentrations. Many complexes of iron and biomolecules (protein or simpler mole‐ cules) are involved with reduction potential or redox potential (*Eh*). The *Eh* of complexed iron in the range of a biological oxidant is +820 mV to the reductant at −320 mV, the redox reactions are reversible whereas the reaction may be irreversible for the iron complexes when the

Iron is a crucial enzyme cofactor that acts in the reduction-oxidation reaction in the metabolism in the cells. Of outstanding interest, iron deposition in the heart cells can lead to oxidative stress and cellular damage [25-27]. Heart failure is the leading cause of death among hemosiderotic β-thalassemia patients, of whom, around 60% die of this cardiac failure [28-30]. The importance of metals to both enzymatic reactions and oxidative stress makes them the key players in mitochondria. Mitochondria are the primary energygenerating organelles of the cells that produce ATP through a chain of enzymatic com‐ plexes that require cytochromes iron and sulfur-iron, and are highly sensitive to oxidative damage. Moreover, the heart is one of the most mitochondria-rich tissues in the body, making metals of particular importance to cardiac function [31]. In cardiac cells, excess iron may result in oxidative stress and an alteration of myocardial function because of the DNA damage that is caused by hydrogen peroxide through the Fenton reaction [6, 32]. Harm‐ ful effects of iron overload on the hearts of patients with β-thalassemia major can be monitored [25, 33-38] and treatment with effective iron chelators can protect these pa‐ tients from cardiac arrhythmia [39, 40]. Iron catalyzes the production of ROS including O2

 Iron acts as a cofactor within the active site of key enzymes in the biochemical pathways and as a chemical catalyst in the unique redox activity. The iron can cycle between two oxidation states, ferric ion [Fe(III) or Fe3+] and ferrous ion [Fe(II) or Fe2+], allowing it to act as an electron donor and acceptor [23]. The biochemical functions of iron in vital cells are dependent on its chemical properties. Limitations of iron bioavailability under aerobic conditions is occurs when the Fe2+ is rapidly oxidized in the solution to insoluble Fe3+ at physiological pH [*K*free Fe(III) = 10<sup>−</sup>18 M] [24]. Fe(III) iron is the most stable state of the biological complexes at physiological oxygen concentrations. Many complexes of iron and biomolecules (protein or simpler molecules) are involved with reduction potential or redox potential (*Eh*). The *Eh* of complexed iron in the range of a biological oxidant is +820 mV to the reductant at −320 mV, the redox reactions are reversible whereas the reaction may be irreversible for the iron complexes when the reduction potential occurs outside this range [1]. Iron is a crucial enzyme cofactor that acts in the reduction-oxidation reaction in the metabolism in the cells. Of outstanding interest, iron deposition in the heart cells can lead to oxidative stress and cellular damage [25-27]. Heart failure is the leading cause of death among hemosiderotic -thalassemia patients, of whom, around 60% die of this cardiac failure [28-30]. The importance of metals to both enzymatic reactions and oxidative stress makes them the key players in mitochondria. Mitochondria are the primary energygenerating organelles of the cells that produce ATP through a chain of enzymatic complexes that require cytochromes iron and sulfur-iron, and are highly sensitive to oxidative damage. Moreover, the heart is one of the most mitochondria-rich tissues in the body, making metals of particular importance to cardiac function [31]. In cardiac cells, excess iron may result in oxidative stress and an alteration of myocardial function because of the DNA damage that is caused by hydrogen peroxide through the Fenton reaction [6, 32]. Harmful effects of iron overload on the hearts of patients with -thalassemia major can be monitored [25, 33-38] and treatment with effective iron chelators can protect these patients from cardiac

8

**Figure 2** Hydroxyl radical formation in metals-catalyzed Haber-Weiss and Fenton reactions

+ HO-

+ HO-

•-,

OH via


+ O2 *Haber-Weiss reaction*

*Fenton reaction*

, H2O2 and

parathyroidism) [21, 22].

**2.2. Chemistry of catalytic iron**

reduction potential occurs outside this range [1].

and hypoparathyroidism) [21, 22].

122 Pharmacology and Nutritional Intervention in the Treatment of Disease

**2.2 Chemistry of catalytic iron** 

H2O2 and •OH via Haber-Weiss and Fenton reactions (Figure 2).

Haber-Weiss and Fenton reactions (**Figure 2**).

Fe2+ + H2O2 Fe3+ + HO

+ H2O2 HO

O2 -

Fe / Cu

arrhythmia [39, 40]. Iron catalyzes the production of ROS) including O2

[41] **Figure 2.** Hydroxyl radical formation in metals-catalyzed Haber-Weiss and Fenton reactions [41]

**Figure 3.** Production and harmfulness of ROS (Reprinted with modification from [45])

In β-thalassemia major patients, an outpouring of catabolic iron overwhelms the capacity of iron-transporting protein transferrin, and generates NTBI and redox-active LPI in the plasma compartment. Cytosolic LIP has been shown to be comprised of transitory Fe (II) and Fe (III) forms which are possibly mediated by specific cellular iron reductases [20, 44]. In sub-cellular organelles, mitochondrial iron serves in the formation of protein iron-sulfur clusters and porphyrins. Additionally, endosomal iron provides the translocation of the endocytosed iron into cytosol or mitochondria, while lysosomal iron is associated with the products of ironprotein degradation [46, 47]. LIP is a source of chelatable and redox-active transient iron in the cells that serves as a crossroads of the cellular iron metabolism. The nature of the LIP has been revealed by its capacity to promote ROS generation in its "rise and fall" patterns. LIP plays a role as a self-regulatory pool that is sensed by cytosolic iron-regulatory proteins (IRP) and its feedback is regulated by an IRP-dependent expression of iron import and storage. The LIP can also be modulated by biochemical mechanism that override the IRP regulatory loops and contributes to basic cellular functions.

revision suggested that iron-induced free radical formation in thalassemia patients might lead to lipid peroxidation, LDL oxidation, stimulation of apoptosis and other damaging processes. The essence in the chelating and antioxidant treatments of thalassemia patients has to be

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125

Afanas'ev identified the major routes of superoxide damaging effects in the mitochondria. These include initiation of apoptosis through a reduction of cytochrome c, activation of uncoupled proteins by superoxide and a competition between superoxide and nitric oxide at the Complex IV site (or cytochrome oxidase). The author suggests an application of effective free radicals scavengers (rutin and flavonoids) for the treatment of thalassemic patients [58, 59]. From a comprehensive study in Indonesia, non-transfused thalassemia intermedia patients showed mild signs of oxidative stress and increased hemoglobin degradation, but revealed no significant indication of tissue or cell damage. Transfusion-dependent β-thalas‐ semia major patients showed a highly significant decrease in antioxidants and thiols, and a tremendous iron overload along with cell damage. This situation was made even worse in long-term transfused patients [60]. ROS and lipid peroxidation were found to be higher, and GSH lower, in thalassemic RBC compared with normal RBC at the baseline as well as following the hydrogen peroxide treatment. These effects were reversed by treatment with antioxidative *N*-acetylcysteine (NAC) [61]. Platelets obtained from β-thalassemia patients contained higher ROS and lower GSH contents than those from normal donors, indicating a state of oxidative stress. Exposure of platelets to oxidants such as hydrogen peroxide and tert-butylhydroper‐ oxide, or to the platelet activators (e.g. thrombin), ionophore (e.g. valinomycin) or phorbol myristyl acetate (PMA), stimulated the platelets' oxidative stress. Iron, hemin and thalassemic RBC also stimulated the platelets' oxidative stress. Therefore, oxidative stress of the platelets can lead to the activation of thromboembolic events [62]. Basal ROS level expressed as mean fluorescent intensity (FI) was higher in thalassemia polymorphonuclear cells (PMN) (FI = 95.6±19.8) than in normal PMN (FI = 12.7±4.5). Treatment of thalassemia PMN with PMA markedly increased the basal ROS level; in comparison, treatment with antioxidants, such as NAC, vitamins C and vitamin E, reduced their basal ROS but enhanced their PMA response. Administration of effective antioxidants may compromise their antibacterial capacity and give

DNA damage can be caused by iron-induced free radicals in thalassemia patients. Lympho‐ cytes from a normalfemale did notrespond to ferric chloride or hemosiderin, but did to ferrous chloride and ferrous sulphate. Comparatively, lymphocytes from an Australian patient with Hb S/β-thalassemia (double heterozygote-sickle phenotype) were more sensitive to ferrous sulphate treatment. Interestingly, desferrioxamine (DFO) and deferiprone (DFP) reduced the response [64]. Erythrocyte GPx activity was significantly lower in non-chelated β-thalassemia major patients than those chelated with either DFO or DFP [65]. β-Thalassemia major patients were deficient in vitamin A, C, D, B and folic acid [66]. Levels of ceruloplasmin concentra‐ tions and its ferroxidase activity were significantly higher in the thalassemia patients than in the healthy controls. Interestingly, the levels were significantly higher in thalassemia patients with Hp2-2 phenotype than in patients with other phenotypes, suggesting that thalassemia patients with Hp 2-2 phenotype are under greater iron-driven oxidative stress [67]. Reduc‐ tion of delta-aminolevulinic acid synthase (δ-ALA) activity and an increase of peroxiredox‐ in-2 expression in thalassemic erythroid cells might represent two novel stress-response

considered within the context of free radical damage and its prevention [57].

prophylaxis for recurrent infections [63].

protective systems [68].

#### **2.3. Oxidative stress in thalassemia patients**

In one study, β-thalassemia children were found to have elevated levels of thiobarbituric acid reactive substances (TBARS), NAD(P)H oxidase (NOX) and SOD activity. Additionally, they were found to have decreased levels of CAT activity and reduced glutathione (GSH) concentra‐ tion, along withunchangedGPx activity intheirplasma comparedto theplasma ofthe controls' [48]. Seminal SODandCATactivities ofhomozygousβ-thalassemicpatientswithironoverload were increased, probably due to a compensatory reaction to the persistence of high levels of ROS. Increased seminal lipid peroxidation could have contributed to the impairment of sperm motility [49]. Erythrocyte free reactive (non-heme) iron was significantly higher in β-thalasse‐ mia patients with HbE (30% >controls), which was associated with a high level of serum TBARS (86% >controls) [50]. Elevated serum ferritin showed a positive correlation with elevated levels of suchliver enzymes as aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP), but not γ-glutamyl transferase (GGT), confirming hepatic iron overload. Serum ferritin also showed a positive correlation with elevated levels of plasma TBARS and SOD. Plasma TBARS concentrations were increased in patients with α-thalasse‐ mia trait, and increased to the highest level in Hb H disease patient. Plasma levels of vitamin A, C, and E were significantly decreased in α- and β-thalassemia patients [51, 52]. In addition, their RBC revealed lower levels of vitamin E, GSH, CAT and SOD [51].

Isolated Hb chains can behave as pro-oxidants that can trigger oxidation of low-density lipoprotein (LDL). Importantly, the descending order of the different Hb chains to the relative oxidation of LDL protein, Apoprotein B (ApoB) and lipid parts was: α-globin chains > β-globin chains > HbA. This indicates that the extracellular globin chains may be the trigger of the lipoprotein alterations observed in β-thalassemia patients [53] and suggests that iron overload should be involved in the oxidative stress shown in the cells [54]. In comparison with apopro‐ tein E2 (apoE2) and apoprotein E3 (apoE3) apoprotein E4 (apoE4) is considered the least efficient under conditions of oxidative stress in thalassemia patients and this imples that apoE4 is a genetic risk factor for left venticular dysfunction [55]. Mean triglyceride concentrations were not significantly different between thalassemia patients and the controls. Total choles‐ terol and LDL-cholesterol concentrations were found to be lower in β-thalassemia major and thalassemia intermediate patients than in the controls (*p* <0.001), while HDL-cholesterol concentrations were lower in thalassemia intermediate patients (*p* <0.03) [56]. This may account for increased erythropoiesis and cholesterol consumption in thalassemia intermediate patients, and iron overload and oxidative stress in β-thalassemia major patients. A previous revision suggested that iron-induced free radical formation in thalassemia patients might lead to lipid peroxidation, LDL oxidation, stimulation of apoptosis and other damaging processes. The essence in the chelating and antioxidant treatments of thalassemia patients has to be considered within the context of free radical damage and its prevention [57].

into cytosol or mitochondria, while lysosomal iron is associated with the products of ironprotein degradation [46, 47]. LIP is a source of chelatable and redox-active transient iron in the cells that serves as a crossroads of the cellular iron metabolism. The nature of the LIP has been revealed by its capacity to promote ROS generation in its "rise and fall" patterns. LIP plays a role as a self-regulatory pool that is sensed by cytosolic iron-regulatory proteins (IRP) and its feedback is regulated by an IRP-dependent expression of iron import and storage. The LIP can also be modulated by biochemical mechanism that override the IRP regulatory loops and

In one study, β-thalassemia children were found to have elevated levels of thiobarbituric acid reactive substances (TBARS), NAD(P)H oxidase (NOX) and SOD activity. Additionally, they were found to have decreased levels of CAT activity and reduced glutathione (GSH) concentra‐ tion, along withunchangedGPx activity intheirplasma comparedto theplasma ofthe controls' [48]. Seminal SODandCATactivities ofhomozygousβ-thalassemicpatientswithironoverload were increased, probably due to a compensatory reaction to the persistence of high levels of ROS. Increased seminal lipid peroxidation could have contributed to the impairment of sperm motility [49]. Erythrocyte free reactive (non-heme) iron was significantly higher in β-thalasse‐ mia patients with HbE (30% >controls), which was associated with a high level of serum TBARS (86% >controls) [50]. Elevated serum ferritin showed a positive correlation with elevated levels of suchliver enzymes as aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP), but not γ-glutamyl transferase (GGT), confirming hepatic iron overload. Serum ferritin also showed a positive correlation with elevated levels of plasma TBARS and SOD. Plasma TBARS concentrations were increased in patients with α-thalasse‐ mia trait, and increased to the highest level in Hb H disease patient. Plasma levels of vitamin A, C, and E were significantly decreased in α- and β-thalassemia patients [51, 52]. In addition,

Isolated Hb chains can behave as pro-oxidants that can trigger oxidation of low-density lipoprotein (LDL). Importantly, the descending order of the different Hb chains to the relative oxidation of LDL protein, Apoprotein B (ApoB) and lipid parts was: α-globin chains > β-globin chains > HbA. This indicates that the extracellular globin chains may be the trigger of the lipoprotein alterations observed in β-thalassemia patients [53] and suggests that iron overload should be involved in the oxidative stress shown in the cells [54]. In comparison with apopro‐ tein E2 (apoE2) and apoprotein E3 (apoE3) apoprotein E4 (apoE4) is considered the least efficient under conditions of oxidative stress in thalassemia patients and this imples that apoE4 is a genetic risk factor for left venticular dysfunction [55]. Mean triglyceride concentrations were not significantly different between thalassemia patients and the controls. Total choles‐ terol and LDL-cholesterol concentrations were found to be lower in β-thalassemia major and thalassemia intermediate patients than in the controls (*p* <0.001), while HDL-cholesterol concentrations were lower in thalassemia intermediate patients (*p* <0.03) [56]. This may account for increased erythropoiesis and cholesterol consumption in thalassemia intermediate patients, and iron overload and oxidative stress in β-thalassemia major patients. A previous

their RBC revealed lower levels of vitamin E, GSH, CAT and SOD [51].

contributes to basic cellular functions.

**2.3. Oxidative stress in thalassemia patients**

124 Pharmacology and Nutritional Intervention in the Treatment of Disease

Afanas'ev identified the major routes of superoxide damaging effects in the mitochondria. These include initiation of apoptosis through a reduction of cytochrome c, activation of uncoupled proteins by superoxide and a competition between superoxide and nitric oxide at the Complex IV site (or cytochrome oxidase). The author suggests an application of effective free radicals scavengers (rutin and flavonoids) for the treatment of thalassemic patients [58, 59]. From a comprehensive study in Indonesia, non-transfused thalassemia intermedia patients showed mild signs of oxidative stress and increased hemoglobin degradation, but revealed no significant indication of tissue or cell damage. Transfusion-dependent β-thalas‐ semia major patients showed a highly significant decrease in antioxidants and thiols, and a tremendous iron overload along with cell damage. This situation was made even worse in long-term transfused patients [60]. ROS and lipid peroxidation were found to be higher, and GSH lower, in thalassemic RBC compared with normal RBC at the baseline as well as following the hydrogen peroxide treatment. These effects were reversed by treatment with antioxidative *N*-acetylcysteine (NAC) [61]. Platelets obtained from β-thalassemia patients contained higher ROS and lower GSH contents than those from normal donors, indicating a state of oxidative stress. Exposure of platelets to oxidants such as hydrogen peroxide and tert-butylhydroper‐ oxide, or to the platelet activators (e.g. thrombin), ionophore (e.g. valinomycin) or phorbol myristyl acetate (PMA), stimulated the platelets' oxidative stress. Iron, hemin and thalassemic RBC also stimulated the platelets' oxidative stress. Therefore, oxidative stress of the platelets can lead to the activation of thromboembolic events [62]. Basal ROS level expressed as mean fluorescent intensity (FI) was higher in thalassemia polymorphonuclear cells (PMN) (FI = 95.6±19.8) than in normal PMN (FI = 12.7±4.5). Treatment of thalassemia PMN with PMA markedly increased the basal ROS level; in comparison, treatment with antioxidants, such as NAC, vitamins C and vitamin E, reduced their basal ROS but enhanced their PMA response. Administration of effective antioxidants may compromise their antibacterial capacity and give prophylaxis for recurrent infections [63].

DNA damage can be caused by iron-induced free radicals in thalassemia patients. Lympho‐ cytes from a normalfemale did notrespond to ferric chloride or hemosiderin, but did to ferrous chloride and ferrous sulphate. Comparatively, lymphocytes from an Australian patient with Hb S/β-thalassemia (double heterozygote-sickle phenotype) were more sensitive to ferrous sulphate treatment. Interestingly, desferrioxamine (DFO) and deferiprone (DFP) reduced the response [64]. Erythrocyte GPx activity was significantly lower in non-chelated β-thalassemia major patients than those chelated with either DFO or DFP [65]. β-Thalassemia major patients were deficient in vitamin A, C, D, B and folic acid [66]. Levels of ceruloplasmin concentra‐ tions and its ferroxidase activity were significantly higher in the thalassemia patients than in the healthy controls. Interestingly, the levels were significantly higher in thalassemia patients with Hp2-2 phenotype than in patients with other phenotypes, suggesting that thalassemia patients with Hp 2-2 phenotype are under greater iron-driven oxidative stress [67]. Reduc‐ tion of delta-aminolevulinic acid synthase (δ-ALA) activity and an increase of peroxiredox‐ in-2 expression in thalassemic erythroid cells might represent two novel stress-response protective systems [68].

#### **2.4. Iron chelation therapy**

Nowadays, DFO, DFP and deferasirox (DFX) are iron chelators of choice used for the treatment of β-thalassemia patients with iron overload. Their chemical structures are shown in Figure 4 [69-72]. DFO (Desferal®) is the first drug that was introduced in the 1970s to treat iron overload. The hexadentate chelator has an extremely high affinity for iron (III) (DFO : Fe = 1:1, Ka =1029) and a much lower affinity for other metal ions, such as zinc, calcium and magnesium [73]. DFO is poorly absorbed from the GI tract and rapidly excreted in the urine (plasma halflife of 5 - 10 minutes), it must be therefore administered parenterally; intravenously (*iv*), intramuscularly (*im*) or subcutaneously (*sc*) [8, 74]. However, the drug exhibits side effects including an elevated body iron burden, serious neurotoxicity and abnormalities of cartilage formation [75-78]. DFP (L1 or Ferriprox®), a synthetic bidentate chelator, has been the first orally active drug available for clinical use. A previous study demonstrated DFP decreased serum ferritin and liver iron concentrations in transfusion-dependent thalassemia patients [79]. Using magnetic resonance imaging (MRI) technique, DFP was able to reduce cardiac iron overload and improve cardiac function more effectively when compared to patients treated with DFO [80]. Recently, the Government Pharmaceutical Organization (GPO) of Thailand has manufactured and launched domestically produced DFP product (GPO-L-One®) for the treatment of Thai thalassemia patients with iron overload. This will make the in-house DFP cheaper and more available than the imported DFP. However, its side effects include nausea, vomiting, gastrointestinal disturbance, leucopenia and thrombocytopenia and zinc deficiency, as they are typically observed these side effects are being evaluated in the patients [81, 82]. DFX (ICL670 or Exjade®), a tridentate oral chelator with a high affinity and specificity for iron, has been clinically used for the treatment of transfusion-dependent thalassemia patients since 2003 [83-85]. Efficacy and safety of DFX uses have previously been evaluated and reported [83, 84, 86-88]. Common side effects of DFX are abdominal symptoms (usually diarrhea), skin exanthems, elevated serum creatinine levels and renal tubular dysfunction [70].  **Pharmacology and Nutritional Intervention in the Treatment of Disease**  and abnormalities of cartilage formation [75-78]. DFP (L1 or Ferriprox), a synthetic bidentate chelator, has been the first orally active drug available for clinical use. A previous study demonstrated DFP decreased serum ferritin and liver iron concentrations in transfusion-dependent thalassemia patients [79]. Using magnetic resonance imaging (MRI) technique, DFP was able to reduce cardiac iron overload and improve cardiac function more effectively when compared to patients treated with DFO [80]. Recently, the Government Pharmaceutical Organization (GPO) of Thailand has manufactured and launched domestically produced DFP product (GPO-L-One® ) for the treatment of Thai thalassemia patients with iron overload. This will make the in-house DFP cheaper and more available than the imported DFP. However, its side effects include nausea, vomiting, gastrointestinal disturbance, leucopenia and thrombocytopenia and zinc deficiency, as they are typically observed these side effects are being evaluated in the patients [81, 82]. DFX (ICL670 or Exjade), a tridentate oral chelator with a high affinity and specificity for iron, has been clinically used for the treatment of transfusion-dependent thalassemia patients since 2003 [83-85]. Efficacy and safety of DFX uses have previously been evaluated and reported [83, 84, 86-88]. Common side effects of DFX are abdominal symptoms (usually diarrhea), skin In the medical regimen, DFO must be subcutaneously infused in β-thalassemia patients for extensive periods in order to achieve a negative iron balance, ranging from 8 to 12 hours, five to seven times per week, and at a daily dosage of 20 to 60 mg/kg body weight [72]. The patients experienced pain and swelling at the injection site, cumulatively leading to poor patient compliance [89, 90]. DFO chelation along with DFP or DFX has been designed to improve the efficacy and to diminish the adverse effects in the treated patients [91, 92]. Ideally, the iron chelator should be orally active, cheap and highly specific for iron, but not for other metal ions, and should freely penetrate into the target tissues, so as to get the patients compliant and show minimal side effects. Chelators can act upon different iron pools, including transferrin-bound iron (TBI), NTBI and LPI in plasma compartment, and LIP in cytoplasm to form iron-chelate(s) which will then be excreted in the urine and feces [8]. Clinical efficacy of chelation therapy has been evaluated at different periods of time, generally by following up the levels of the biochemical markers, including ferritin, total iron, TIBC, transferrin saturation, NTBI and LPI in plasma with colorimetry [93, 94], and of tissue biopsied iron with pathological examination (Perl's staining), as well as in organ iron with a semi-quantum uncoupled inductive device (SQUID) and magnetic resonance imaging (MRI) techniques [95-98]. Encompassing forms of plasma NTBI are readily chelated by effective iron chelators [19]. However, the pathologically relevant fraction of NTBI is that which is seemingly translocated across cell membranes in a non-regulated manner and leads to excessive iron accumulation in the liver, heart, pancreas and endocrine organs [20]. The LPI fraction is not only an accessible diagnostic marker of iron overload and cell toxicity, but also a clinical parameter for assessing the mode and efficacy of

Antioxidants as Complementary Medication in Thalassemia

http://dx.doi.org/10.5772/57372

127

Antioxidants can be defined as compounds that inhibit or delay, but do not completely prevent oxidation. There are two basic categories of antioxidants, namely synthetic and natural

Mostly, the synthetic antioxidants that are widely used are phenolic compounds; for example, butylated hydroxyanisole, butylated hydroxytoluene, tertiary-butylhydroquinone and gallic

Natural antioxidants are found to be present in many sources such as plants, fungi, microor‐ ganism and even animal tissues. Phenolic compounds are also the majority group of natural antioxidants. The three important groups of antioxidant are tocopherols, flavonoids and phenolic acid. Natural antioxidants have been widely used in complementary and alternative medicines; in comparison, the synthetic antioxidants have reported signs of toxicological

chelation therapy.

**3. Antioxidants**

**3.1. Synthetic antioxidants**

acid (GA) derivatives.

**3.2. Natural antioxidants**

antioxidants.

exanthems, elevated serum creatinine levels and renal tubular dysfunction [70].

12

In the medical regimen, DFO must be subcutaneously infused in -thalassemia patients for extensive periods in order to achieve a negative iron balance, ranging from 8 to 12 hours, five to seven times per week, and at a daily dosage of 20 to 60 mg/kg body weight [72]. The patients experienced pain and swelling at the injection site, cumulatively leading to poor patient compliance [89, 90]. DFO chelation along with DFP or DFX has been designed to improve the efficacy and to diminish the adverse effects in the treated patients [91, 92]. Ideally, the iron chelator should be orally active, cheap and highly specific for iron, but not for other metal ions, and should freely penetrate into the target tissues, so as to get the

**Figure 4** Chemical structures of DFO, DFP and DFX (Redrawn with modification from [89]) **Figure 4.** Chemical structures of DFO, DFP and DFX (Redrawn with modification from [89])

In the medical regimen, DFO must be subcutaneously infused in β-thalassemia patients for extensive periods in order to achieve a negative iron balance, ranging from 8 to 12 hours, five to seven times per week, and at a daily dosage of 20 to 60 mg/kg body weight [72]. The patients experienced pain and swelling at the injection site, cumulatively leading to poor patient compliance [89, 90]. DFO chelation along with DFP or DFX has been designed to improve the efficacy and to diminish the adverse effects in the treated patients [91, 92]. Ideally, the iron chelator should be orally active, cheap and highly specific for iron, but not for other metal ions, and should freely penetrate into the target tissues, so as to get the patients compliant and show minimal side effects. Chelators can act upon different iron pools, including transferrin-bound iron (TBI), NTBI and LPI in plasma compartment, and LIP in cytoplasm to form iron-chelate(s) which will then be excreted in the urine and feces [8]. Clinical efficacy of chelation therapy has been evaluated at different periods of time, generally by following up the levels of the biochemical markers, including ferritin, total iron, TIBC, transferrin saturation, NTBI and LPI in plasma with colorimetry [93, 94], and of tissue biopsied iron with pathological examination (Perl's staining), as well as in organ iron with a semi-quantum uncoupled inductive device (SQUID) and magnetic resonance imaging (MRI) techniques [95-98]. Encompassing forms of plasma NTBI are readily chelated by effective iron chelators [19]. However, the pathologically relevant fraction of NTBI is that which is seemingly translocated across cell membranes in a non-regulated manner and leads to excessive iron accumulation in the liver, heart, pancreas and endocrine organs [20]. The LPI fraction is not only an accessible diagnostic marker of iron overload and cell toxicity, but also a clinical parameter for assessing the mode and efficacy of chelation therapy.
