Hydroxyl radical formation by sickle erythrocyte membranes: role of pathologic iron deposits and cytoplasmic reducing agents

Abstract

Sickle erythrocyte (RBC) membranes were previously shown to manifest increased Fenton activity (iron-dependent, peroxide-driven formation of hydroxyl radical [.OH]) compared with normal RBC membranes, but the nature of the catalytic iron was not defined. We now find that sickle membranes exposed to superoxide (.O2-) and hydrogen peroxide (H2O2) have three distinct iron compartments able to act as Fenton catalysts: preexisting free iron, free iron released during oxidant stress, and a component that cannot be chelated with deferoxamine (DF). In a model system, addition of iron compounds to normal ghosts showed that free heme, hemoglobin, Fe/adenosine diphosphate (ADP), and ferritin all catalyze .OH production; concurrent inhibition studies using DF documented that the unchelatable Fenton component is free heme or hemoglobin. During exposure to peroxide only, the iron in sickle membranes was unable to act as a Fenton catalyst without addition of a reducing agent. At physiologic concentrations, both ascorbate and glutathione restored Fenton activity. Lipid peroxidation studies showed that at physiologic levels ascorbate acts primarily as an antioxidant; however, as pharmacologic levels are reached, its pro-oxidant effects predominate. This study elucidates the catalytic ability of the iron compartments in the sickle cell membrane, the importance of which relates to the potential role of .OH in membrane damage. It also illustrates the potential participation of cytoplasmic reducing agents in this process, which may be especially relevant in the context of administration of supraphysiologic doses of ascorbate to sickle cell patients.

Oxidative stress and inflammation in iron‐overloaded patients with β‐thalassaemia or sickle cell disease

“…First, plasma MDA could be enhanced in thalassaemia patients because it may be dependent on the amount of circulating erythroid precursors and peripheral blood erythrocytes that have a high density of unpaired a-haemoglobin chains (Scott et al, 1993). The excess a-chains in thalassemic red blood cells are unstable and prone to denaturation and oxidation (Scott et al, 1993) and, in the present work, may have contributed more to enhanced plasma MDA than the SCD haemoglobin, which is also capable of producing oxidants (Repka & Hebbel, 1991). The a-chains in thalassemic red blood cells can autoxidise, release haem and generate superoxide (Scott et al, 1993), which can then increase lipid peroxidation and thus enhance MDA levels.…”

Oxidative stress, eryptosis and anemia: a pivotal mechanistic nexus in systemic diseases

“…Oxidative stress is ascribed an important role in the pathophysiology of SCD and can arise from different origins and sources in that condition. HbS, for example, is able to bind to the erythrocyte membrane and can act as a Fenton reagent, which augments the production of hydroxyl radicals (OH·), H 2 O 2 and superoxide anions (), leading to membrane damage (Fig. ).…”

Priapism in Sickle-Cell Disease: A Hematologist’s Perspective

“…Many publications present data implicating scavenging of NO by hemoglobin released from intravascular lysis of red blood cells in SCD [43,44], compounded by depletion of arginine, the obligate substrate for NO synthase, and by arginase‐1 also released from red cells during hemolysis [45]. NO may also be scavenged by reactive oxygen species that are abundantly produced in SCD [46], contributing to robust oxidative stress further exacerbated by the oxidant properties of free heme and iron‐catalyzed Fenton reactions [47,48]. Levels of endogenous inhibitors of NO synthase are also found in the plasma from patients with SCD at triple the level of healthy control subjects [49,50].…”