Lipid Peroxidation and Antioxidant Status in β-Thalassemic patients: Effect of Iron Overload Iraqi J Pharm Sci, Vol.18(2) 2009 Antioxident in thalassemla 8 Lipid Peroxidation and Antioxidant Status in β-Thalassemic Patients: Effect of Iron Overload Bassm N. Aziz *,1 , Mohammad A. Al-Kataan ** and Wasan K. Ali *** * Department of Anaesthesis, Mosul Technical Institute, Mosul, Iraq. ** Department of Clinical Pharmacy, College of Pharmacy, Mosul University, Mosul, Iraq. *** Department of Chemistry, College of Science, Mosul University, Mosul, Iraq. Abstract To study the effect of iron overload due to continuous blood transfusions on peroxidation products, such as malondialdehyde (MDA) and peroxynitrite, with evaluation of some antioxidants like, glutathione (GSH), superoxide dismutase (SOD), vitamin A, vitamin C, vitamine E, Ceruloplasmin, uric acid and albumin in thalassemia patients. Forty patients with thalassemia major, aged 5 to 15 years, were carried out in Abn-Alatheer Teaching Hospital in Mosul city, during the period from October 2007 to April 2008. They were on Chelation therapy with desferrioxamine. They were divided into two groups, the first one without iron overload (90,97±12.92), and the second one with iron overload (157.75±7.57). All the patients were received whole blood. Blood samples were collected before and after blood transfusion. The results showed that there were significant increase in MDA and peroxynitrite in patients with iron overload five days before and after blood transfusion in compared with groups having normal iron level. On the other hand, glutathione, superoxide dismutase activity, Vitamin A, vitamin C, vitamin E, albumin and ceruloplasmin were significantly decreased whereas, uric acid was increased significantly. It is concluded that, Iron over load due to continuous blood transfusion in thalassemia causes increase in oxidative tissue damage with a changes in antioxidants status. Key Words: Beta-thalassemia, lipid peroxidation, antioxidants, Malondialdehyde, Iron الخالصة فزغ ذحًُم انحذَذ َرُدح إعطاء انذو انًسرًز فٍ َىاذح انثُزوكسذج انًرًثهح تانًانىَذَانذَهاَذ وَرزاخ انثُزوكسُذ, نذراسح ذأثُز Eوفُرايٍُ Cوفُرايٍُ Aعاداخ األكسذج كانكهىذاثاَىٌ وانسىتز اوكساَذ دسًُىذُش وكم يٍ فُرايٍُ يع قُاص تعط يٍ ي يزَعا يٍ انًصاتٍُ تانُىع انزئُسٍ نًزض ٨٤وانسُزوتالسيٍُ وحايط انُىرَك واألنثىيٍُ عُذ يزظً انثالسًُُا. أخرُز سُح ويٍ انزاقذٍَ فٍ يسرشفً أتٍ ٥٩−٩ذزاوحد أعًارهى يٍ انثالسًُُا, وانهذٍَ هى ذحد عالج عقار انذسفُزوكسايٍُ. حُث يدًىعرٍُ, األونً اذصفد تعذو إنً. ذى ذقسًُهى ٦٤٤8ونغاَح َُساٌ ٦٤٤٧األثُزانرعهًٍُ تانًىصم, خالل انفرزج يٍ ذشزٍَ األول عذها ذى إعطاء انذو نكم أفزاد (. ت٧٫٩٧±٥٩٧٫٧٩( وانثاَُح تىخىد فزغ ذحًُم نهحذَذ )٥٦٫٠٦±٠٤٫٠٧وخىد فزغ ذحًُم نهحذَذ ) انعُُح انًذروسح, وأخذخ عُُاخ انذو قثم وتعذ عًهُح َقم انذو. أظهزخ َرائح هذِ انذراسح وخىد سَادج يعُىَح فٍ يسرىَاخ حذَذ انًانىَذاَانذَهاَذ وَرزاخ انثُزوكسُذ فٍ انًزظً انذٍَ َعاَىٌ يٍ فزغ ذحًُم انحذَذ يقارَح تاِخزٍَ انذٍَ َكىٌ يسرىي ان Aعُذهى غثُعٍ. يٍ َاخُح أخزي, فقذ اَخفعد يعُىَا يسرىَاخ انكهىذاثاَىٌ وفعانُح أَشَى انسىتز اوكساَذ دسًُىذُش وكم يٍ فُرايٍُ فزغ وقذ أسرُرح يٍ انذراسح تأٌ واألنثىيٍُ وانسُزَىتالسيٍُ, تًُُا ارذفع يعُىَا يسرىي حايط انُىرَك. Eوفُرايٍُ Cوفُرايٍُ ٌ يع اخرالف فٍ يسرىَاخ يعاداخ ذَذ انُاذح يٍ َقم انذو انًرىاصم نًزظً انثالسًُُا قذ سثة سَادج شذج انكزب انراكسذحًُم انحذ . األكسذج Introduction Increased level of lipid peroxidation and decreased level of antioxidants play important roles in the pathogenesis of anemias 1 . It is well documented that disturbances of oxidant - antioxidant balance occur in hemoglobinopathies, especially in thalassemia 2 . In beta-thalassemia, decreased or impaired biosynthesis of beta-globin leads to accumulation of unpaired alpha globin chains 3 . Excess presence of the alpha-globin chains primarily 3 and also iron overload, as a result of multiple transfusions, are the main reasons for the cellular oxidative damage in thalassemias 4 . Iron overload is still a major concern in homozygous β-thalassemia. Under physiological conditions, iron ions are not available to catalyze the conversion of molecular oxygen to highly reactive radical species by Fenton reaction, because ferric iron is bound to proteins, preventing it from participating in reactions that could lead to cell injury 5 . Under various pathological conditions associated with iron overload, including thalassemia, due to blood transfusion used for treatment of thalassemia. There is evidence of an increase in iron in both serum and cells 6 . 1 Corresponding author E- mail : bassam_alwakeel@yahoo.com Received : 26 / 11 / 2008 Accepted : 23 / 6 / 2009 mailto:bassam_alwakeel@yahoo.com Iraqi J Pharm Sci, Vol.18(2) 2009 Antioxident in thalassemla ٠ This increases generation of free radicals 7 , and promotes peroxidative damage to cell and organelle membranes in organs that accumulate excess iron, including liver, pituitary gland, pancreas, and heart 8 . This study evaluates the total antioxidant potential and several individual antioxidants, as well as parameters of peroxidative stress, including malondialdehyde (MDA) (the breakdown product of lipid peroxidation), in serum of patients with β-thalassemia major, transfusion- dependent, and under regular iron chelation therapy with or without signs of iron overload, before and after transfusion. Subjects and Methods Experimental Design This study was conducted at Abn- Alatheer Teaching Hospital in Mosul city, from October 2007 to April 2008. Forty patients with β-thalassemia major, aged 5 to 15 years (mean, 7.3±3.7), were divided into two groups. The first group included twenty patients without signs of iron overload as revealed from the mean value of serum iron (90,97±12.92), referred as control, while the second group included twenty patients with clinical and biochemical signs of iron overload (157.75±7.57). Both groups were under continuous and regular blood transfusion program (one transfusion process/ month). Chelation therapy with desferrioxamine (DFO) was administered to each of the patients (by pump, five days a week, 40-60mg/kg/day, 12 hours infusion) 9 . All of the patients were examined regularly once a month in Pediatric Hematology Department of this hospital. Sample Collection and Clinical Chemistry Analysis Ten milliliters of blood samples were obtained from each patient five days before and after blood transfusion process. After clotting, serum was separated by centrifugation and divided in several aliquots. The analytical determinations described below were either performed immediately, or serum was stored at -20°C and used within 72 hours. The readings of the measured parameters were done at clinical biochemical laboratory in the College of Science. Clinical laboratory examinations on serum including, MDA and peroxynitrite as an oxidant indicator and the total antioxidant capacity like glutathione, SOD, vitamin A, vitamin C, vitamin E, ceruloplasmin, uric acid, and albumin levels were evaluated. The level of serum MDA was determined by a modified procedure using the thiobarbituric acid reaction substance (TBARS) methods 10 , and the activity of SOD levels in blood serum was determined using photochemical method described by Brown and Goldstein 11 . This methods depends on an indirect approach to determine the SOD activity through the change in formazene absorbance formed from the reduction of O2 •¯ , which is produced by radiating the sample of serum with light) for nitroblue tetrazolum (NBT) dye. Decreased difference in formazene absorbance means increased SOD activity. Serum glutathion is determined by a modified procedure utilizing Ellman`s reagent 12 .Serum vitamin A 13 , vitamin C 14 and vitamin E 15 were measured spec- trophotometrically. Ceruloplasmin, Peroxy nitrite activity were measured by modified method described by Menden et al. 16 and Vanuffelen et al. 17 respectively. The level of uric acid 18 and serum albumin 19 were measured. Statistical Analysis All data were compared by t-test between patient groups in SPSS 10.0 program. The values within the tables were given as mean ± standard deviation. Statistical significance was considered at p<0.05 20 . Results The effects of blood transfusion in thalassemic patients, without iron overload, represented with normal serum iron (90,97±12.92), on lipid peroxidation and antioxidant status are presented in table (1). Glutathione, vitamin A, vitamin C vitamin E, and albumin increased significantly, while MDA, peroxynitrite, SOD activity, ceruloplasmin, and uric acid did not changed significantly after blood transfusion in comparison with the control group (before transfusion). On the other hand, with the exception of peroxynitrite, SOD activity, albumin and ceruloplasmin other parameters like Glutathione, vitamin A, vitamin C, and vitamin E were increased significantly, while MDA and uric acid were decreased significantly in thalassemic patients, with iron overload, after receiving blood transfusion as shown in table (2). The effects of iron overload in thalassemic patients, represented with increased serum iron (157.75±7.57), before blood transfusion, on lipid peroxidation and antioxidant status are presented in table (3). MDA, peroxynitrite, and uric acid were found to be higher in thalassemic patients with iron overload when compared with non-iron overload patients. On the other hand, glutathione, SOD activity, vitamin A, vitamin C, vitamin E, ceruloplasmin, and albumin were significantly decreased in the same group. Moreover, after blood transfusion, all parameters were altered significantly in iron overload group in comparison with thalassemic Iraqi J Pharm Sci, Vol.18(2) 2009 Antioxident in thalassemla ٥٤ patient without iron overload as shown in table (4). Glutathione, SOD activity, vitamin A, vitamin C, vitamin E, albumin and ceruloplasmin were decreased significantly. In contrast, MDA, peroxynitrite, and uric acid were shown to be increased significantly. Table 1: Effects of blood transfusion in thalassemic patients, without iron overload, on lipid peroxidation and antioxidant status.  Values are expressed as means ± SD from 20 subjects per group.  (NS): Not significant *Significantly different from the control (p<0.05) ** Significantly different from the control (p<0.01) *** Significantly different from the control (p<0.001) Table 2: Effects of blood transfusion in thalassemic patients, with iron overload, on lipid peroxidation and antioxidant status.  Values are expressed as means ± SD from 20 subjects per group.  (NS): Not significant *Significantly different from the control (p<0.05) ** Significantly different from the control (p<0.01) *** Significantly different from the control (p<0.001) Table 3: Effects of iron overload in thalassemic patients, before blood transfusion, on lipid peroxidation and antioxidant status.  Values are expressed as means ± SD from 20 subjects per group.  (NS): Not significant *Significantly different from the control (p<0.05) ** Significantly different from the control (p<0.01) *** Significantly different from the control (p<0.001) Iraqi J Pharm Sci, Vol.18(2) 2009 Antioxident in thalassemla ٥٥ Table 4: Effects of iron overload in thalassemic patients, after blood transfusion, on lipid peroxidation and antioxidant status.  Values are expressed as means ± SD from 20 subjects per group.  (NS): Not significant *Significantly different from the control (p<0.05) ** Significantly different from the control (p<0.01) *** Significantly different from the control (p<0.001) Discussion It has been postulated that the biochemical and metabolic changes of thalassemic patients are associated with a constant oxidative stress within the red cell caused by precipitation of excess alpha-globin chains, and release of free iron 21 . The measurement of the peroxidation products, together with the evaluation of the antioxidants may be the simple measurement of iron overload due to blood transfusion in thalassemia 9 . Increased plasma MDA level, which is measured by the thiobarbituric acid reaction substance (TBARS) methods, was found in beta-thalassemia patients 22 , where MDA is a good indicator of oxidative damage. The increased in serum MDA levels in patient with thalassemia in our study, as shown in tables (3 and 4) can be compared with those obtained by other investigators 9 . In addition, Peroxynitrite, was measured in the present study. Serum levels of this pro-oxidants was increased significantly in thalassemic patient with iron overload compared with other without iron overload.. Nitric oxide (NO ∙ ) contains unpaired numbers of electrons and are therefore free radicals. It was first recognized as a distinct gas in 1772 by Joseph Priestley 23 . It can be produced by vascular endothelium. It can react with another endogenous free radical, superoxide, to produce a reactive intermediate, peroxynitrite (ONOO − ), which is a powerful oxidant, able to damage many biological molecules, and can decompose at acid pH to release small amounts of hydroxyl radicals. 24 ONOO − + H + ∙ OH + NO2 As a result of continuous blood transfusions, our patients might be subjected to peroxidative tissue injury by the secondary iron overload 4 . These finding might support the idea of iron overload in beta-thalassemia leads to an enhanced generation of reactive oxygen species and oxidative stress. Iron is also an important nutritional metal for many physiological functions 25 . However, persons receiving multiple transfusions as part of the treatment for thalassemia, are faced with problem of iron overload and consequent metabolic dearrangements. Under normal circumstances there is virtually no free iron. All irons are protein bound. In patients with thalassemia, the irons liberated from the haemoglobin saturate the transferrins, and the transferrins transfer the iron on to a storage iron protein called apoferritin. When the ferritin is saturated, another storage iron protein called hemosiderin is formed. 7 On this bases, iron overload, in the present study, was not created immediately until multiple blood transfusion were performed. For this reason, MDA level was not altered significantly in patients after blood transfusion was achieved immediately.During the 1984, the Cambridge chemist H.J.H. Fenton described a reaction between iron salts and H202 that caused oxidative damage to organic molecules such as tartaric acid 26 . The Fenton reaction is widely represented as in the following Eqs.. Fe 2+ + H2O2 Fe 3+ + OH − + ∙ OH Fe 3+ + H2O2 Fe 2+ + HO2 ∙ + H + overall reaction Iron salt + 2 H2O2 2H2O + O2 In the process of changing from the ferrous to the ferric state, an electron is transferred from iron to oxygen to make superoxide as shown in in the following Eq. 5. Fe 2+ + O2 Fe 2+ O2 ↔ Fe 3+ O2 − Fe 3+ + O2 ∙ − Iraqi J Pharm Sci, Vol.18(2) 2009 Antioxident in thalassemla ٥٦ Therefore, the presence of iron complexes stimulate peroxidation by peroxide decomposition of unsaturated fatty acids generating alkoxyl (LO ∙ ) and peroxyl (LO2 ∙ ) radicals 27 . Oxidative stress is a prominent contributor to the premature destruction of RBC as well as anemia in thalassemia. The oxidative status within red blood cells is maintained by the balance between oxidative systems, such as Reactive Oxygen Species (ROS), and antioxidative systems, like reduced glutathione (GSH) 28 . Glutathione is a low- molecular-mass, thiol-containing tripeptide, It is synthesized endogenously in the human cell, It acts to protect the body against the production of free radicals 29 . As a result, increased production of H2O2 in thalassemia major induces glutathione peroxidase, an enzyme that lead to decreased glutathione concentration 30 . On this basis, a significant decrease in serum glutathione concentration was noticed in the present work in patients with iron overload, serving as oxidative stress, versus those from non-iron overload group. During the course of metabolism, a superoxide anion is produced. Normally the superoxide anion is converted by the enzyme SOD to produce H2O2 31 , which in turn is converted to innocuous compounds by the action of catalase and peroxidase. But if free ferrous iron is available it reacts with H2O2 to produce hydroxyl radical which is an extremely reactive species leading to depolymerisation of polysaccharide 32 . The production of free radicals due to thalassemia was associated with a significant decrease in enzymatic antioxidants activity like SOD as shown in the present study in tables (3 and 4). It can be compared with other research of Şimşek, et al., 9 in which erythrocyte SOD (a preventive antioxidant) levels was found to be higher in beta-thalassemic patients than healthy children. Moreover, marked changes in the other antioxidant pattern were also observed in all patients. Evidence is presented of a net drop in the concentration of vitamin A, vitamin C, and vitamin E in all patients with iron overload when compared with those without iron overload, as shown in tables (3 and 4). On the other hand, a significant increase of nutrient antioxidants was observed in all patients received blood transfusion in comparison with the same patient before receiving blood transfusion as shown in tables (1 and 2). As vitamin C is essential to maintain vitamin E status and function, depletion of vitamin C, in turn, contributes to further exacerbate the depletion of vitamin E 33 . Although efficient antioxidants such as uric acid and bilirubin are high, they cannot compensate for lipid-soluble antioxidants, so that tissue lipid compartments are not suitably preserved. The observed depletion of serum levels of vitamin E and vitamin A can be explained by impairment of liver function and peroxidative processes causing a substantial reduction of serum lipids, producing a concurrent reduction of serum vitamin E and vitamin A 34 . A significant drop in nutrient antioxidants obtained in the present work can be compared with another research made by Livrea et al., 4 who observed a significant decline in the concentration of vitamin A, vitamin C, and vitamin E in all patients affected with thalassemia due to continuous blood transfusions. Another antioxidant parameter involves uric acid and albumin was included in the present study. Uric acid was increased significantly in opposite to albumin which decreased significantly in patients with iron overload. Similarly, Livrea et al., 4 showed an increase of uric acid whereas serum albumin was in the normal range in all thalassemic patients.Uric acid provides an excellent example of the adaptation of the organism to oxidative stress. It is a cellular waste product originating from the oxidation of hypoxanthine and xanthine by xanthine oxidase and dehydrogenase. High uric acid levels may provide efficient antioxidant activity for the organism. Urate, the physiological state of uric acid, reacts with hydroxyl radicals producing a stable urate radical that can be regenerated by ascorbate. This compound can act with peroxyl radicals, superoxide dismutase, ozone, nitrous oxide ∙ , and other nitrogen-oxygen radicals. Urate also protects protein from nitration; it can chelate metal ions, such as copper and iron, and prevent them from participating in redox cycling 35 . Ceruloplasmin, is a chain breaking antioxidant, it can protect the body against the deleterious effects of oxygen free radical (OFR) 36 . The antioxidant property of ceruloplasmin is through its oxidase activity, which is directed towards ferrous ions (ferroxidase activity). It also inhibits ferrous ion stimulated lipid peroxidation and is known to be involved in the decomposition of lipid peroxides 37 . Serum Ceruloplasmin was significantly lower in the iron-overload group compared to the non iron-overload patients, as shown in tables (3 and 4). The inverse relationship between serum ceruloplasmin and the level of iron is indicating the anti- thalassemic nature of this antioxidant. Conclusion These results point out that the iron- induced oxidative stress in thalassemia may play a major role in the depletion of most Iraqi J Pharm Sci, Vol.18(2) 2009 Antioxident in thalassemla ٥٧ antioxidants, including lipid-soluble antioxidants. Our results suggest that the measurement of peroxidation products, matched with evaluation of antioxidants, may be a simple measure of oxidative stress in thalessemia. References 1. MChiu D, Kuypers F, Lubin B: Lipid peroxidation in human red cells. Semin Hematol 1989; 26: 257-276. 2. Kattamis C, Kattamis AC. Oxidative stress disturbances in erythrocytes of β- thalassemia. Pediat Hematol Oncol 2001; 18: 85-88. 3. Scott MD, Van den Berg JJM, Repka T, et al. Effect of excess β-hemoglobin chains on cellular and membrane oxidation in model β-thalassemic erythrocytes. J Clin Invest 1993; 91: 1706-1712. (Cited by Şimşek et al., 2005). 4. Livrea MA, Tesoriere L, Pintaudi AM, Calabrese A, Maggio A, Freisleben HJ, D'Arpa D, D'Anna R, Bongiorno A. Oxidative stress and antioxidant status in beta-thalassemia major: iron overload and depletion of lipid soluble antioxidants. Blood 1996; 88: 3608-3614. 5. Deighton N, Hider RC: Intracellular low molecular weight iron. Biochem Soc Trans 1989; 17: 490. 6. Britton RS, Ferrali M, Magiera CJ, Recknagel RO, Bacon BR: Increased prooxidant action of hepatic cytosol low- molecular-weight iron in experimental iron overload. Hepatology 1990; 11: 1038. 7. Goswami K, Ghosh S, Bandyopadhyay M, Mukherjee KL. Iron store and free radicals in thalassemia. Indian J Clin. Bioch 2005; 20: 192-194. 8. Halliwell B, Gutteridge JMC: Role of free radicals and catalytic metal ions in human disease: An overview, in Packer L, Glazer AN (eds): Methods in Enzymology, vol 186. San Diego, CA, Academic, 1990, p 1 (cited by Livrea et al., 1996). 9. Şimşek F, Öztürk G, Kemahlı S, Erbaş D, Hasanoğlu A. Oxidant and antioxidant status in beta thalassemia major patients. J Ankara Univer Fac Med 2005; 58(1): 34 - 38. 10. Guidet B, Shah SV. Am J Physiol 1989; 257(26): F440. (Cited by Muslih RK, Al- Nimer MS, Al-Zamely OM. The level of malondialdehyde after activiation with H2O2 and CuSO4 and inhibition by desferoxamine and molsidomine in the serum of patients with acute myocardial infarction. Nation J Chem 2002; 5: 139- 148. 11. Brown MS and Goldstein. Ann Rev Biochem 1983; 52: 223. (cited by Al- Zamely OM, Al-Nimer MS, Muslish RK. "Detection the level of peroxynitrite and related with antioxidant status in the serum of patient with acute myocardial infarction". Nat J Chem 2001; 4: 625-637. 12. Sedlak J, Lindsay RH. Anal Biochem 1968; p: 192. 13. Wotton IDP. "Microanalysis in Medical Biochemistry". 6 th ed. Edinburge London pp. 1982; 236-237. 14. Stanley T, David T, Howerds S. "Selected method for the determination of ascorbic acid in Animal cells, tissues and fluids". 1979; Method in Enzymology, vol. 62. vitamins and coenzymes part D. 15. Varley H, Gowenlock AH, Bell M. “Practical clinical biochemistry”. 1980; Vol. (1), London, pp. 222-225, 553-555. 16. Menden EE, Boiano JM, Murthy L, petering HG. "Modification of phenylene diamine oxidase method to permit non- antomated ceruloplasmin determination in batches of rat serum or plasma micro samples. Analytical. 1977; 10: 197-204. 17. Vanuffelen BE, Van Derzec J, Dekoster BM. Biochem J. 1998; 330: 719. (Cited by Al-Zamely et al., 2001). 18. Al-Umary, Mohammad Ramzi. (1986). Practical Clinical Chemistry, Dar Al- Tikani for publishing and edition, Technical Institution. (In Arabic) 19. Varley H, Gowenlock AH, Bell M. “Practical clinical biochemistry” 1980; Vol. (1), London, pp. 222-225, 553-555. 20. Steel, R.G.D. and Torrie, J.H., 1980. Principles and Procedures of Statistics. 2 nd ed. New York: McGraw-Hill Book Company, Inc. 1960: pp.87-80, 107-109, 125-127. 21. Kattamis C, Kattamis AC. Oxidative stress disturbances in erythrocytes of β- thalassemia. Pediat Hematol Oncol 2001; 18: 85-88. 22. Tesoriero L, D’Arpa D, Butera D, et al. Oral supplements of vitamin E improve measures of oxidative stress in plasma and reduce oxidative damage to LDL and erythrocytes in beta-thalassemia intermedia patients. Free Radical Res 2001; 34(5): 529-540. (Cited by Şimşek et al., 2005) 23. Moncada S, Higgs EA. Endogenous nitric oxide: physiology, pathology and clinical relevance (Review). Eur J Clin Invest 1991; 21: 361-374. 24. Beckman JS, Beckman TW, Chen J, Marshall PA, Freeman BA. Apparent hydroxyl radical production by Iraqi J Pharm Sci, Vol.18(2) 2009 Antioxident in thalassemla ٥٨ peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc Natl Acad Sci U S A 1990; 87: 1620- 1624. 25. Conrad ME. Introduction: Iron overloading disorder and iron regulation. Seminars in Hematol. 1998; 35 (1): 1-4. (Cited by Goswami et al., 2005). 26. Fenton HJH. Oxidation of tartaric acid in presence of iron. J Chem Soc 1984; 65: 899-909. 27. Gutteridge JMC, Kerry PJ. Detection by fluorescence of peroxides and carbonyls in samples of arachidonic acid. Br J Pharmacol 1982; 76: 459-61. 28. Fibach E, Amer J, Rachmilewitz E, Guy E, Rivella S. Oxidative stress of RBC in a Murine model of Beta-Thalassemia can be reversed by treatment with antioxidants. Am Soc Hematol 2005; 106: 3643. (Abstract). 29. Kohen R and Nyska A. Invited Review: Oxidation of biological systems: Oxidativestress phenomena, antioxidants, redox reactions, and methods for their Quantification. Toxicol pathol 2002; 30 (6): 620–650. 30. Beutler E, Matsumoto F, Powars D and Warner J. Increased glutathione peroxidase activity in alpha-thalassemia. Am Soc Hematol 2008; 50: 647-655. From www.bloodjournal.org. 31. McCord JM. (1992). Superoxide production and human disease. In Jesaitis A and Dratz E. (eds.) : Molecular basis of oxidative damage by leukocytes. Boca Raton. FL. CRC, pp. 225-239. 32. McCord JM. Free radicals and inflammation: Protection of synovial fluid by superoxide dismutase. Science 1974; 185: 159-163. 33. Rice-Evans C, Baysal E: Iron-mediated oxidative stress in erythrocytes. Biochem J 1987; 244: 191. 34. Jordan P, Brubacher D, Moser U, Stahelin HB, Gey KF. Vitamin E and vitamin A concentrations in plasma adjusted for cholesterol and triglycerides by multiple regression. Clin Chem 1995; 41: 924. 35. Ames BN, Cathcart R, Schwiers E, Hochstein P. Uric acid provides an antioxidant defense in humans against oxidant- and radical-caused aging and cancer: A hypothesis. Proc Natl Acad Sci USA 1981; 78: 6858–6862. 36. Mateeseu M, Chahine R, Roger S, Atanasiu R. Protection of myocardical tissue against deleterious effects of oxygen free radicals by ceruloplasmin. Arzneim Forsch/drug Res 1995; 45, 476- 480. 37. Osaki S, Johnson D, Frieden E. The possible significance of the ferrous oxidase activity of ceruloplasmin in normal human serum. J Biol Chem 1966; 241: 2746-2751. . http://www.hematology.org/meetings/abstract_rights.cfm http://www.bloodjournal.org/