Oxidative Stress in Congenital and Acquired Hemolytic Anemias

1. Oxidative Stress in Congenital and Acquired Hemolytic Anemias

2. Oxidants Antioxidants Oxidative status The balance between oxidants and antioxidants

3. Oxidative Stress Oxidants Antioxidants

4. Reactive Oxygen Species O 2 - Superoxide radical OH* Hydroxyl radical ROO · Peroxyl radical H O Hydrogen peroxide 2 2 O Singlet oxygen 1 2 NO · Nitric oxide - ONOO Peroxynitrite HOCl Hypochlorous acid Themost important oxidants are those derived from Oxygen - Reactive Oxygen Species (ROS)

5. Anti-oxidant mechanism Fe 2+ Fe 3+ SOD Fenton Reaction 2 O2l H2O2 OHl 2 H + O2 GSH NADP+ Glutathione Peroxidase Glutathione Reductase Catalase G-6-PD GSSG NADPH + H+ 2 H2O + O2 2H2O

8. Physiological / Pathological Roles • The oxidative status modulates various normal physiological cell functions, e.g., signal transduction • Oxidative stress is cytotoxic – it oxidizes proteins, lipids and DNA - leading to apoptosis and organ damage • Oxidative stresscontributes to the pathogenesis of many diseases: cancer, atherosclerosis, diabetes, cardiovascular, thromboembolic, neurodegenerative pathologies as well as hemolytic anemias • We studied the role of oxidative stress in: • Hemoglobinopathies (thalassemia and sickle cell anemia) • Paroxysmal Nocturnal Hemoglobinuria (PNH) • G6PD deficiency • Myelodysplastic syndrome (MDS)

9. Oxidative Stress in Hemolytic Anemias Although oxidative stress is not the primary etiology of these diseases, it participates in causing damage to RBC (as well as neutrophils and platelets) • The Causes of Oxidative Stress • Hemoglobin instability (hemoglobinopathies) • Iron-overload (due to increased absorption and/or therapeutic blood transfusion • Hemolysis

18. Iron Overload Excess iron catalyzes hydroxyl radical generation from activated oxygen species by the Haber-Weiss reaction: Fe3+ + O2- Fe2+ + O2 Fe2+ + H2O2 Fe3+ + OH- + OH* (Fenton reaction) Net reaction: O2-+ H2O2 O2+ OH- + OH*

20. Non-transferrin bound iron • Increased plasma iron overwhelms binding capacity of transferrinNTBI • - fraction of iron pool implicated in oxidant damage to tissues • - possible marker for iron overload and chelation efficacy • NTBI Components: • DCI: Directly Chelatable Iron • LPI:Labile Plasma Iron • functionally active, chemically labile iron • engaged in redox cycling • Esposito et al, Blood 102: 2670, 2003

21. Iron Overload in Thalassemia Caused by: • Hb instability enhances intracellular iron release • Increased uptake of dietary iron • Frequent blood transfusions

22. ROS in RBC Normal Thalassemia Basal level MFC 66 MFC 16 MFC 282 MFC 681 H2O2- stimulated levels H2O2-stimulated

23. ROS Generation by Normal & Thalassemic RBC Unstimulated samples Normal 23.2 Thal 38.5 H2O2-stimulated samples Normal 194.8 Thal 304.8 Thalassemic patients Normal donors (p< 0.0001, by Student’s t-test)

26. Oxidative status in RBC

31. Effect of thalassemic plasma on ROS production by platelets Untreated Treated

32. Summary: Vit E supplement • Vitamin E supplement 200-300 mg/day • Normalize serum vitamin E • Decrease activity of anti-oxidant enz GPx • Decrease lipid peroxidation • Increase red cell survival (small series) • No change in Hb level or transfusion requirement • Vitamin E supplement 600 mg/day • Normalize MDA level at 3 months • Vit E in LDL remains low at 6 months • No change in Hb level

33. Turmeric Volatile oil Curcuminoids (2-20%) polyphenol compounds high anti-oxidant activity Tonnesen et al. Int J pharmaceutics. 1994; 161-167. Sharma OP. Biochem Pharmacol. 1976;25:1811-1812. Safe (FDA approved as food additives) Prevent primaquin- induced hemolysis in vitro Tonnesen et al. 1994 Decreases iron-catalyzed lipid peroxidation in vitro Grinberg et al, 1996 Anti-inflammatory & Anti-tumor effects Miquel 2001 Curcumin

34. Effect of Curcumin 21 adult b-thal HbE, 500 mg/day x 3 mo Treatment Withdrawal Siritanaratkul, Kalprawidth et al 2002

35. Result: Red cell survival

36. 24 -thalassemia/HbE patients (11 splenectomized) • No blood transfusion at least 3 months before donating their blood for this study • Hb level 4.7 – 9.5 g/l • Age 16 - 48 years • Receiving curcumin 500 mg/d for 6 months

37. Treatment of Curcumin for 6 months in -thalassemia / Hb E MDA 30.73% SOD 15.30% GSH-Px 18.91% GSH 19.48% NTBI No significant change in Hb and ferritin

41. Normal SCD 2005, 132:108-113 ROS

42. The oxidative status of blood cells and platelets from normal donors and sickle cell disease (SCD) patients Platelets (PLT), red blood cells (RBC) and polymorphonuclear neutrophils (PMN) from 25 normal donors and 15 SCD patients were analysed for reactive oxygen species (ROS) production (A) and glutathione (GSH) content (B). The average ± SD mean fluorescence channel (MFC) values are shown.

43. MDS Patients * Desferal – for 10 months** ; Desferal – for 2 years; RCMD = Refractory anemia with multilineage dysplasia; RARS = Refractory anemia with ringed Sideroblasts; RAEB1 = Refractory anemia with excess of blasts; GCSF = Granulocyte Colony Stimulating Factor; RHuEPO = Recombinant Human Erythropoietin; PC= Packed red blood cells

44. Platelets RBC PMN Evidence for Oxidative Stress in MDS Blood Cells ROS ROS (% of control) GSH GSH (% of control) H Ghoti et al, EJH 2007 Platelets RBC PMN

45. Changes in Oxidation Parameters in 14 patients with MDS (RA, RARS)* following 11 weeks treatment with ExJade 20mg/kg/day * > 20 units of blood serum ferritin > 1000ng/ml

46. RBC

47. RBC ±27 ±24

48. RBC ±2.3 ±2.1

49. Paroxysmal Nocturnal Hemoglobinuria (PNH) An acquired clonal stem cell disorder, due to a somatic mutation, associated with intravascular (C’-mediated) hemolysis (and thrombosis) as a result of deficiency in the surface glcosyl-phosphotidylinositol (GPI)-anchored antigens, CD55 and CD59.