**2. β-Thalassemia**

#### **2.1 Etiology and pathophysiology**

β-Thalassemia is a quantitative hemoglobinopathy which impairs the production of β-globin chains in Hb due to mutations of the gene located on the short arm of chromosome 11. Accordingly, a reduction of β<sup>+</sup> or an absence of β<sup>0</sup> in β-globin synthesis causes the precipitation of excessive unbound α-globin chains in erythroid precursors due to chain imbalances. The abnormal erythroid precursors are driven into apoptosis pathway during their differentiation and maturation in the bone marrow, consequently leading to erythroid expansion, accelerated extramedullary erythropoiesis, increased dietary iron absorption, and high turnover of RBC. Moreover, impaired β-globin synthesis and ineffective erythropoiesis result in microcytic anemia and progressive splenomegaly. There are three main types of β-thalassemia, in order of decreasing severity, homozygous β-thalassemia major (TM), β-thalassemia intermedia (TI), and heterozygous β-thalassemia minor. Hemoglobin E (HbE)/β-thalassemia is the most prevalent in Southeast Asia where the carrier frequency is around 50%. The interaction of HbE and β-thalassemia results in a clinical spectrum ranging from a severe condition that is indistinguishable from TM to a mild form of TI [1].

Nowadays, β-thalassemia is divided into transfusion-dependent β-thalassemia (TDT) and non-transfusion-dependent β-thalassemia (NTDT). In other mammals, mouse homozygous beta-globin knockout (BKO) thalassemia shows many clinical features of abnormal RBC indices including a decrease in blood Hb concentration,

**41**

to chelation [15].

*2.2.2 Iron overload*

*Nutraceutical Benefits of Green Tea in Beta-Thalassemia with Iron Overload*

hematocrit (Hct), RBC numbers, and osmotic fragility and an increase of reticulocyte count. Likely, increased degradation of abnormal RBC is an important consequence of unstable Hb and excessive membrane iron in patients with β-thalassemia. Invasive and noninvasive laboratory investigations reveal systemic and cellular iron overload in TDT and NTDT patients. Large amounts of the irons from enterocytes and reticuloendothelial (RE) macrophages in the spleen can get into plasma compartment and subsequently bind to transferrin (Tf). Accordingly, this can lead to a high saturation of Tf with iron, an appearance of NTBI and LPI, and high levels of ferritin in plasma compartment, together with high iron deposition ferritin (Ft) in several tissues in the body. Consequently, iron accumulation in the vital organs is the cause of susceptibility to infections and immunological abnormalities, 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, hypogonadism, and hypoparathyroidism) [2–4]. Evidentially, most of patients with β-thalassemia with iron overload die of heart failure, while some patients frequently die from infections and suffer from

In fact, abnormal iron absorption in patients with thalassemia produces an increase in the body iron burden evaluated at 2–5 g per year, and regular blood transfusion (420 mL/U, equivalent to 200 mg of iron) leads to double iron accumulation [5]. Normally, iron is bound to iron-transporting protein in plasma (called transferrin) and in milk (called lactoferrin), forms transferrin-bound iron (TBI), and is transported in blood circulation to target cells. The circulating TBI, which is low saturation in iron-deficiency persons, one-third saturation in normal people, and high saturation in iron overload patients, is taken up into iron-requiring target cells by using ATP-dependent transferrin receptor 1 (TfR1)-mediated endocytosis and delivered in cells for functions and storage in ferritin molecules (H- and L-subunits). When the capacity of transferrin to incorporate iron derived from gastrointestinal (GI) tract and RE cells becomes limited, the transferrin iron-binding capacity (TIBC) has been surpassed [6]. Afterward, two forms of redox-active iron such as NTBI and LPI appear primarily in plasma of patients with β-thalassemia. Pathologically, the NTBI fraction seems to translocate across plasma membrane via specific transporters on specific cell types. NTBI transporter which is originally called divalent metal ion transporter 1 (DMT1) or natural resistanceassociated macrophage protein 2 (Nramp2) is proposed to locate on intestinal epithelial cells, erythroid cells and astrocytes, L-type calcium channel (LTCC), T-type calcium channel (TTCC) on cardiomyocytes, T-type calcium channel (TTCC) on hepatocytes, pancreatic islets β cells and kidney tubular cells, anion-exchange protein 2 (AE2) on bronchial epithelial cells, ferrireductases on kidney proximal tubule, and zinc ion protein 18 (ZIP18) on kidney tubular cells and hippocampal neuronal cells [7–14]. Importantly, NTBI and LPI are redox-active and susceptible

The iron that cells do not require immediately for metabolic processes is stored in ferritin in the liver, enterocytes, and RE macrophages, representing the storage iron pool. The iron that mobilizes transiently inside the cells is called LIP that

*DOI: http://dx.doi.org/10.5772/intechopen.92970*

liver diseases and endocrinopathies.

*2.2.1 Iron transport into cells*

**2.2 Iron overload and oxidative stress**

*Nutraceutical Benefits of Green Tea in Beta-Thalassemia with Iron Overload DOI: http://dx.doi.org/10.5772/intechopen.92970*

hematocrit (Hct), RBC numbers, and osmotic fragility and an increase of reticulocyte count. Likely, increased degradation of abnormal RBC is an important consequence of unstable Hb and excessive membrane iron in patients with β-thalassemia. Invasive and noninvasive laboratory investigations reveal systemic and cellular iron overload in TDT and NTDT patients. Large amounts of the irons from enterocytes and reticuloendothelial (RE) macrophages in the spleen can get into plasma compartment and subsequently bind to transferrin (Tf). Accordingly, this can lead to a high saturation of Tf with iron, an appearance of NTBI and LPI, and high levels of ferritin in plasma compartment, together with high iron deposition ferritin (Ft) in several tissues in the body. Consequently, iron accumulation in the vital organs is the cause of susceptibility to infections and immunological abnormalities, 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, hypogonadism, and hypoparathyroidism) [2–4]. Evidentially, most of patients with β-thalassemia with iron overload die of heart failure, while some patients frequently die from infections and suffer from liver diseases and endocrinopathies.

#### **2.2 Iron overload and oxidative stress**

#### *2.2.1 Iron transport into cells*

*Beta Thalassemia*

**2. β-Thalassemia**

**2.1 Etiology and pathophysiology**

able from TM to a mild form of TI [1].

of chromosome 11. Accordingly, a reduction of β<sup>+</sup>

Human β-thalassemia is characterized by mutations of β-globin gene, resulting in deficient production of the β-globin chains of Hb molecule (ineffective erythropoiesis) and chronic anemia. Over 200 mutations have been identified in this gene, and the type of mutations can influence the severity of the disease. Blood transfusions aim to maintain normal Hb levels to prevent tissue hypoxia, whereas repeated blood transfusions lead to the inevitable consequence of iron accumulation in the body. Iron deposition occurs considerably in almost all tissues but is primarily located in the spleen, liver, heart, and endocrine glands. Besides ferritin iron and hemosiderin, the uncommon forms of iron including labile or transient iron pools (LIP) in the tissues, nonheme iron in RBC membrane, and non-transferrin-bound iron (NTBI) and labile iron pool (LPI) in plasma appear to be redox-active and subsequently generate reactive oxygen species (ROS) via Haber-Weiss and Fenton reactions. The ROS can oxidize biomolecules, causing oxidative tissue damage, organ dysfunctions, complications, and death. Effective iron chelation needs giving to counteract the resulting iron overload and prevent oxidative tissue damage. So far, monotherapy or combined therapy with iron chelators such as desferrioxamine (DFO), deferiprone (DFP), and deferasirox (DFX) has been used for the treatment of patients with β-thalassemia with iron overload, but they present some side effects. Modified medical regimens such as adjunctive iron chelator and antioxidant and drug cocktail are purported to increase the chelation efficacy, minimize the side effects, and achieve additive chelation efficacy. Moreover, commercially available antioxidants such as vitamin C, vitamin E, and *N*-acetylcysteine (NAC) are usually given together with the chelators to relieve the oxidative stress in patients with thalassemia. Herein, an interesting natural product such as green tea extract from tea leaves (*Camellia sinensis*) has been documented in terms of bifunctional antioxidant and iron-chelating properties in iron-overloaded cells and mouse and human thalassemia, rather than its general biological and pharmacological properties.

β-Thalassemia is a quantitative hemoglobinopathy which impairs the production of β-globin chains in Hb due to mutations of the gene located on the short arm

Nowadays, β-thalassemia is divided into transfusion-dependent β-thalassemia (TDT) and non-transfusion-dependent β-thalassemia (NTDT). In other mammals, mouse homozygous beta-globin knockout (BKO) thalassemia shows many clinical features of abnormal RBC indices including a decrease in blood Hb concentration,

synthesis causes the precipitation of excessive unbound α-globin chains in erythroid precursors due to chain imbalances. The abnormal erythroid precursors are driven into apoptosis pathway during their differentiation and maturation in the bone marrow, consequently leading to erythroid expansion, accelerated extramedullary erythropoiesis, increased dietary iron absorption, and high turnover of RBC. Moreover, impaired β-globin synthesis and ineffective erythropoiesis result in microcytic anemia and progressive splenomegaly. There are three main types of β-thalassemia, in order of decreasing severity, homozygous β-thalassemia major (TM), β-thalassemia intermedia (TI), and heterozygous β-thalassemia minor. Hemoglobin E (HbE)/β-thalassemia is the most prevalent in Southeast Asia where the carrier frequency is around 50%. The interaction of HbE and β-thalassemia results in a clinical spectrum ranging from a severe condition that is indistinguish-

or an absence of β<sup>0</sup>

in β-globin

**40**

In fact, abnormal iron absorption in patients with thalassemia produces an increase in the body iron burden evaluated at 2–5 g per year, and regular blood transfusion (420 mL/U, equivalent to 200 mg of iron) leads to double iron accumulation [5]. Normally, iron is bound to iron-transporting protein in plasma (called transferrin) and in milk (called lactoferrin), forms transferrin-bound iron (TBI), and is transported in blood circulation to target cells. The circulating TBI, which is low saturation in iron-deficiency persons, one-third saturation in normal people, and high saturation in iron overload patients, is taken up into iron-requiring target cells by using ATP-dependent transferrin receptor 1 (TfR1)-mediated endocytosis and delivered in cells for functions and storage in ferritin molecules (H- and L-subunits). When the capacity of transferrin to incorporate iron derived from gastrointestinal (GI) tract and RE cells becomes limited, the transferrin iron-binding capacity (TIBC) has been surpassed [6]. Afterward, two forms of redox-active iron such as NTBI and LPI appear primarily in plasma of patients with β-thalassemia. Pathologically, the NTBI fraction seems to translocate across plasma membrane via specific transporters on specific cell types. NTBI transporter which is originally called divalent metal ion transporter 1 (DMT1) or natural resistanceassociated macrophage protein 2 (Nramp2) is proposed to locate on intestinal epithelial cells, erythroid cells and astrocytes, L-type calcium channel (LTCC), T-type calcium channel (TTCC) on cardiomyocytes, T-type calcium channel (TTCC) on hepatocytes, pancreatic islets β cells and kidney tubular cells, anion-exchange protein 2 (AE2) on bronchial epithelial cells, ferrireductases on kidney proximal tubule, and zinc ion protein 18 (ZIP18) on kidney tubular cells and hippocampal neuronal cells [7–14]. Importantly, NTBI and LPI are redox-active and susceptible to chelation [15].

#### *2.2.2 Iron overload*

The iron that cells do not require immediately for metabolic processes is stored in ferritin in the liver, enterocytes, and RE macrophages, representing the storage iron pool. The iron that mobilizes transiently inside the cells is called LIP that

is potentially redox-active and increased when the cells are heavily loaded with a large number of extracellular irons, TBI, and NTBI. Iron distribution in the body is strictly regulated by two regulatory systems, systemic and cellular iron homeostasis. Systemic iron homeostasis strictly responds to ensure a balance of iron absorption and iron utilization, which relies on the hepatic hepcidin hormone and the ferroportin actions and occurs in enterocytes, hepatocytes, and splenic macrophages. Hepcidin levels found to decrease in primary hemochromatosis and secondary hemochromatosis such as TI patients due to an acceleration of erythropoietic activity driven by increases of erythropoietin (EPO) production and TfR1 expression. Inversely, hepcidin levels increased in TM patients due to blood transfusion that they do not need to increase erythropoiesis to compensate ineffective erythropoiesis [16]. The regulation will reduce iron efflux from ferroportin at the basolateral part of duodenal epithelial cells and from RE macrophage into the plasma, resulting in iron retention within the cells. Drugs or natural products that increase hepcidin expression and production would be beneficial for the supportive treatment of TI patients with iron overload. Cellular iron homeostasis is dependent on the expression and function of TfR1 and ferritin mediated by iron regulatory element (IRE)/iron regulatory protein (IRP) system. Once TBI is internalized into cells via TfR-mediated endocytosis, iron is mainly stored by ferritin inside the cells [17]. In patients with thalassemia, large amounts of iron from diet and degradation of effete RBC by RE macrophage drain into plasma transferrin and subsequently taken into cells. Consequently, this will result in iron overload, oxidative stress, and depletion of antioxidant defense systems in plasma compartment and many vital organs in the body.

### *2.2.2.1 Blood*

Thalassemia RBC containing large amounts of iron and low protective antioxidant system is prone to be damaged by ROS, leading to chronic hemolytic anemia. In young patients with β-thalassemia, plasma and RBC levels of thiobarbituric acid-reactive substances (TBARS), superoxide dismutase (SOD), and ferritin were increased, but the level of catalase (CAT) was decreased when compared with normal children [18]. Interestingly, Aphinives and colleagues have found decreased levels of antioxidants such as reduced glutathione (GSH) and vascular endothelial dysfunction in young Thai patients with β-thalassemia with HbE patients [19]. Additionally, blood levels of CAT, glutathione-*S*-transferase (GST), GSH, and vitamin C were found to decrease in β-thalassemia major patients while blood SOD level was increased [20]. Importantly, the levels of blood antioxidant system including GST, glutathione peroxidase (GPx), glutathione reductase (GR), peroxiredoxin 2 (Prx2), thioredoxin 1, and thioredoxin reductase were decreased in β-thalassemia major patients with regular blood transfusion and iron chelation therapy, whereas the blood levels of CAT and SOD were increased when compared to healthy subjects [21]. Consistently, the levels of red cell SOD, GPx, and CAT activities increased in hemoglobin H (HbH) disease (a moderately severe α-thalassemia) patients when compared to healthy control. However, the Chinese medicine "Yisui Shengxue" granules which have been officially prescribed and clinically used for the treatment of thalassemia for a long time effectively decreased the GPx and CAT activities but increased the SOD activity [22]. Prolonged bleeding times and abnormal platelet aggregation can be found in transfusion-dependent β-thalassemia major patients, possibly due to artifacts caused by in vitro manipulations, oxidative platelets, and circulating procoagulants such as microparticles (MP) tissue factors and platelet-derived MP [23, 24]. Platelet numbers were

**43**

*Nutraceutical Benefits of Green Tea in Beta-Thalassemia with Iron Overload*

tors such as DFO and antioxidants such as NAC and vitamin C [24].

approximately 1.5- and 4-fold increase in Thai non-splenectomized and splenectomized patients with HbE/β-thalassemia when compared with normal subjects [25]. In comparison, thrombocytosis, platelet hyperaggregation, and decreased levels of protein S, protein C, and antithrombin III were detected in NTDT (such as TI) patients (9.4% with splenectomy and 90.6% without splenectomy) [26]. Increased oxidative stress of thalassemia platelets was restored by the treatment of iron chela-

The spleen is an organ containing some of the RE cells that function to destroy RBC hemoglobin by macrophages and store the released iron in the form of ferritin and hemosiderin. The number of blood transfusions in β-thalassemia major patients seems to correlate with their splenic hemosiderosis and splenic weight [27]. Hemosiderin deposition was found to be greater in the iron-overloaded livers than in the iron-overloaded spleens. Ferritin and hemosiderin increased in hepatocytes and splenic RE cells [28]. Splenectomy is one of the therapeutic options in hyper-transfused β-thalassemia major patients to reduce hyperactivity of RE macrophages; nevertheless, it may increase the iron overload. As a consequence, complications in patients with splenectomized thalassemia have included hypercoagulability, an increased incidence of vascular thrombosis, and an increased risk of infection. Iron overload in the spleen can activate latent nuclear factor-kappa B (NFκB) in alveolar macrophage, reduce immunity, and increase susceptibility to infection [29]. Notably, Prx2, which is a typical-2 cysteine peroxiredoxin and a key antioxidant system, is upregulated during erythropoiesis in patients with β-thalassemia and contributes to the stress erythropoiesis in the patients. Nuclear factor erythroid 2 (Nrf2) is a redoxresponse transcriptional nuclear factor and cellular adaptive process in response to and protection of oxidative stress [30]. Therefore, the regulation of *Prx2* and *Nrf2* genes results in the upregulation of antioxidant (antioxidant responsive element, ARE) genes required to ensure the survival of iron-overloaded cells.

The liver is one of the main storage organs for iron. Iron overload is considered

to be when the ferritin level consistently exceeds 1000 ng/mL (normal range 20–200 ng/mL). Excess free radicals can cause progressive tissue injury and eventually cirrhosis or hepatocellular carcinoma in iron overload patients whose iron is sequestrated predominantly in ferritin or hemosiderin [31]. When plasma transferrin becomes highly saturated, NTBI is detectable and is rapidly transported across the hepatocyte membrane via a specific pathway. Likely, ferroportin 1 is the only protein that mediates the transport of iron out of hepatocytes and is then oxidized by ceruloplasmin and bound to transferrin. Iron deposition affects hepatic parenchymal cells (hepatocytes and bile duct cells) and mesenchymal cells (endothelial cells, macrophage, and Kupffer cells) and often distributes differently from one area to another [32]. As mentioned above, iron overload can induce ROS which can oxidize biomolecules. Lipid peroxidation activates tumor growth factor-beta1 (TGF-β1) expression which is the most potent pro-fibrogenic cytokine, and its expression is increased in almost all of fibrotic diseases. Type I collagen is induced by TGFβ resulting in uncontrolled collagen production and leads to tissue scarring and organ failure. The scar tissue replaces normal parenchyma, increases fibrosis, and blocks the liver portal blood flow consequently generating liver cirrhosis.

*DOI: http://dx.doi.org/10.5772/intechopen.92970*

*2.2.2.2 Spleen*

*2.2.2.3 Liver*

approximately 1.5- and 4-fold increase in Thai non-splenectomized and splenectomized patients with HbE/β-thalassemia when compared with normal subjects [25]. In comparison, thrombocytosis, platelet hyperaggregation, and decreased levels of protein S, protein C, and antithrombin III were detected in NTDT (such as TI) patients (9.4% with splenectomy and 90.6% without splenectomy) [26]. Increased oxidative stress of thalassemia platelets was restored by the treatment of iron chelators such as DFO and antioxidants such as NAC and vitamin C [24].
