A role for ascorbate-derived electrons in protection against oxidative damage to membrane lipids was investigated in resealed human erythrocyte ghosts. Incubation of resealed ghosts with the membrane-impermeant oxidant ferricyanide doubled the ghost membrane concentration of F-isoprostanes, a sensitive marker of lipid peroxidation. Incorporation of ascorbate into ghosts during resealing largely prevented F-isoprostane formation due to extravesicular ferricyanide. This protection was associated with a rapid transmembrane oxidation of intravesicular ascorbate by extravesicular ferricyanide. Transmembrane electron transfer, which was measured indirectly as ascorbate-dependent ferricyanide reduction, correlated with the content of -tocopherol in the ghost membrane in several respects. First, ascorbate resealed within ghosts protected against ferricyanide-induced oxidation of endogenous -tocopherol in the ghost membrane. Second, when exogenous -tocopherol was incorporated into the ghost membrane during the resealing step, subsequent ferricyanide reduction was enhanced. Last, incubation of intact erythrocytes with soybean phospholipid liposomes, followed by resealed ghost preparation, caused a proportional decrease in both the membrane content of -tocopherol and in ferricyanide reduction. Incorporation of exogenous -tocopherol during resealing of ghosts prepared from liposome-treated cells completely restored the ferricyanide-reducing capacity of the ghosts. These results suggest that the transmembrane transfer of ascorbate-derived electrons in erythrocyte ghosts is dependent in part on -tocopherol and that such transfer may help to protect the erythrocyte membrane against oxidant stress originating outside the cell.

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