INTERACTION OF ASCORBATE AND ALPHA-TOCOPHEROL IN RESEALED HUMAN ERYTHROCYTE-GHOSTS - TRANSMEMBRANE ELECTRON-TRANSFER AND PROTECTION FROM LIPID-PEROXIDATION

A role for ascorbate-derived electrons in protection against oxidative damage to membrane lipids was investigated in resealed human erythroc yte ghosts, Incubation of resealed ghosts with the membrane-impermeant oxidant ferricyanide doubled the ghost membrane concentration of F-2- isoprostanes, a sensitive marker of lipid peroxidation, Incorporation of ascorbate into ghosts during resealing largely prevented F-2-isopro stane formation due to extravesicular ferricyanide. This protection wa s associated with a rapid transmembrane oxidation of intravesicular as corbate by extravesicular ferricyanide. Transmembrane electron transfe r, which was measured indirectly as ascorbate-dependent ferricyanide r eduction, correlated with the content of alpha-tocopherol in the ghost membrane in several respects, First, ascorbate resealed within ghosts protected against ferricyanide-induced oxidation of endogenous alpha- tocopherol in the ghost membrane, Second, when exogenous alpha-tocophe rol 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 r esealed ghost preparation, caused a proportional decrease in both the membrane content of alpha-tocopherol and in ferricyanide reduction, In corporation of exogenous alpha-tocopherol during resealing of ghosts p repared from liposome-treated cells completely restored the ferricyani de-reducing capacity of the ghosts. These results suggest that the tra nsmembrane transfer of ascorbate-derived electrons in erythrocyte ghos ts is dependent in part on alpha-tocopherol and that such transfer may help to protect the erythrocyte membrane against oxidant stress origi nating outside the cell.