Fe3+ as a potent accelerator for hemoglobin oxidation in vitro and prooxidant effects of reduced glutathione (GSH).

Authors: Mustafa, I. and Scott, M.D. Department of Pathology and Laboratory Medicine and the Centre for Blood Research at the University of British Columbia and the Canadian Blood Services.


Iron is an essential trace element for all living cells.Indeed, iron cores constitute the functional sites of many enzymes and proteins involved in generating energy, transporting oxygen, and DNA synthesis. Containing approximately 20 mM iron, normal erythrocytes are the most iron and oxygen rich somatic cell.While this important metal is vital, maintaining its biological balance in an organism is far more crucial than virtually any other trace element (with the possible exception of copper). Excess iron, due to its catalysis of one electron redox chemistry, plays a key role in the formation of toxic oxygen radicals.Indeed, this is readily observed in diseases such as thalassemia and sickle cell anemia. This potentially dangerous combination of oxygen and iron within the erythrocyte is kept in check by a number of endogenous mechanisms. These include the hemoglobin (Hb) tetramer itself, as well as a number of antioxidants such as superoxide dismutase (SOD), catalase, glutathione/glutathione peroxidase, and methemoglobin reductase.

Experiments were conducted in vitro using purified hemoglobin (HbA; a2 ß2) exposed to ferric (Fe3+) iron.Upon Fe3+ addition (0-250 µM ferric ammonium sulfate), spectrophotometric shifts in the absorption spectra (500 –700 nm) of purified hemoglobin (~10 µM HbA; pH 7.4) was determined. HAb concentration was determined by the cyanomethemoglobin (Drabkin’s) method. The inhibitory effects of glutathione (GSH), iron chelators (desferrioxamine, DFO; and Deferiprone, L1), SOD and catalase on Fe3+-driven oxidation was assessed.In the absence of Fe3+ no HbA oxidation was noted.Similarly, in contrast to erythrocyte hemolysates, simple addition of Fe3+ to purified HbA also had minimal oxidative effects.However, addition of the “anti-oxidant” GSH, resulted in a potent GSH (0-10 mM) and Fe3+ (0-250 µM) dose-dependent oxidation of the purified HbA to methemoglobin. Methemoglobin is characteristized by a peak (or shoulder) at 630 nm. This data demonstrates that a reducing agent is necessary for iron-driven oxidation.Notably the intact erythrocyte is rich in both GSH and ascorbate. This oxidation was further enhanced by the presence of hydrogen peroxide (H2O2), a normal byproduct of HbA auto-oxidation.Importantly, inclusion of the iron chelators (DFO or L1) inhibited Fe3+-mediated HbA oxidation in a dose-dependent manner.For example, >250 µM DFO, inhibited virtually all iron mediated HbA oxidation induced by the addition of 250 µM Fe3+ and 0. 4 mM GSH.Further experiments were carried out to see if Fe3+ mediated HbA oxidation could be inhibited by SOD or catalase. These studies demonstrated no protective effects, suggesting that Fe3+ – GSH degradation is not due to either O2 or H2O2, or even hydroxy radicals, because SOD or catalase would have blocked hydroxyl radical formation. Consequently this data implicates thiol radicals as the agent of injury.

These findings have significant implications in the mechanisms of injury in disease such as the thalassemias and sickle cell anemia.Furthermore, these results suggest that chelation of bioreactive iron within the abnormal erythrocyte may be an effective therapeutic intervention.My research is currently focusing on a novel iron-shuttle chelation therapy regime utilizing low and high molecular weight iron chelators.



Iron Shuttle Chelation Therapy: A Novel Approach To Treating Hemoglobinopathies

I. Mustafa, N. Rossi, J. N. Kizhakkedathu and M.D. Scott
Canadian Blood Services and the Centre for Blood Research and the Department of Pathology and Laboratory Medicine at the University of British Columbia, Vancouver, BC, Canada

Background: Thalassemias arise from deficiency of the globin subunits of adult hemoglobin (HbA) and results in ineffective erythropoiesis and the rapid destruction of RBC in the periphery. These iron-driven events give rise to anemia. While transfusion therapy corrects the anemia, it give rise to secondary iron overload. Thus, both the primary and secondary pathology of thalassemia arise from “misplaced” iron. Our previous studies suggest that chelation of bioreactive iron within the thalassemic/sickle RBC may be an effective therapeutic intervention. Our research is focused on a novel iron-shuttle chelation therapy utilizing both low (shuttle) and high (docking) molecular weight iron chelators.
Methods: The effect of shuttle and docking chelators on ferric iron (Fe3+) – driven Hb and lipid oxidation was assessed singularly and in combination. Experiments were conducted in vitro using HbA exposed to Fe3+ (0-175µM). HbA oxidation was quantitated spectrophotometrically. Shuttle chelators included DFO, L1, HBED and ICL-670.The docking chelators consisted of S-DFO (a starch conjugate of DFO) and P-DFO (a novel poly(ethylene glycol)-acrylate based copolymer of DFO).Lipid peroxidation was measured by thiobabutaric acid reactive substances (TBARS) formation
Results: In the absence of Fe3+ no HbA oxidation was noted. Addition of 175µM Fe3+ resulted in rapid methemoglobin formation. Importantly, inclusion of any of our chelators inhibited HbA oxidation in a dose-dependent manner. For example,175 or 200 µM DFO , P-DFO or S-DFO respectively, inhibited >90% HbA oxidation induced by 175 µM Fe3+. Similarly, lipid peroxidation was inhibited in a chelator dose dependent manner: 400 µM DFO or P-DFO equivalents resulted in a 58 or 70% reduction TBARS formation (respectively).
Conclusions: As shown, both shuttle and docking chelators can effectively bind and remove free and complexed iron /heme from aqueous and lipid environments and prevent redox-driven damage. These data suggest that a two component iron shuttle chelation system may effectively slow/prevent iron-driven damage and improve both effective erythropoiesis and the viability of abnormal RBC within the periphery. Thus this iron shuttle system may have therapeutic importance in the treatment of hemoglobinopathies.

Note: This abstract was published on AABB Transfusion journal 2008 special edition for the anual conference in Montreal.