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.

Abstract

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 O^-₂ or H₂O_2, 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.