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It is hypothesized that membrane-associated iron in the sickle red cell is of pathophysiologic importance, but the actual existence of such iron in the intact cell has been questioned. Using a strategy whereby membrane iron can be detected through its bioavailability for catalyzing peroxidation, we used phospholipid exchange protein to load membranes of intact erythrocytes (RBC) with approximately 2% phosphatidylethanolamine hydroperoxide (PEOOH) and monitored the development of peroxidation by-products during subsequent incubation. Normal RBC loaded with PEOOH developed very little peroxidation, but vitamin E-replete sickle RBC showed an exuberant peroxidation response that was not seen in cells loaded with control nonoxidized phosphatidylethanolamine. Ancillary studies of sickle RBC revealed that the catalytic iron included both heme iron and free iron located at the bilayer inner leaflet. Significantly, these studies also revealed that peroxidation after PEOOH loading is promoted by cellular dehydration and inhibited by hydration, thus identifying a dynamic interaction between hemoglobin (sickle &gt;&gt; normal) and membrane lipid. High-reticulocyte control RBC and sickle trait RBC behaved exactly like normal RBC, while HbCC RBC and RBC having membranes gilded with hemoglobin iron because of prior exposure to acetylphenylhydrazine showed an abnormal peroxidation response like that of sickle RBC. Indeed, the peroxidation response of RBC loaded with PEOOH paralleled amounts of iron measured on inside-out membranes prepared from them (r = 0.783, P &lt; 0.01). These studies corroborate existence of membrane-associated heme and free iron in the intact sickle cell, and they document its bioavailability for participation in injurious peroxidative processes. That association of cytosolic sickle hemoglobin with membrane lipid is modulated by cell hydration status provides a mechanism that may help explain increased development of oxidative membrane lesions in abnormally dehydrated sickle RBC regardless of the mechanism underlying their formation.

Detection, characterization, and bioavailability of membrane-associated iron in the intact sickle red cell.