Abstract
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 F2-isoprostanes, a sensitive marker of lipid peroxidation. Incorporation of ascorbate into ghosts during resealing largely prevented F2-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 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-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 alpha-tocopherol and in ferricyanide reduction. Incorporation of exogenous alpha-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 alpha-tocopherol and that such transfer may help to protect the erythrocyte membrane against oxidant stress originating outside the cell.
Highlights
Erythrocytes possess redundant and overlapping mechanisms for protection of their cytoplasmic contents against oxidative damage, including catalase [1], superoxide dismutase [2], and low molecular weight antioxidants such as GSH and ascorbate [3]
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
The protective effect of intravesicular ascorbate involves a transmembrane transfer of ascorbate-derived electrons, the extent of which depends on the membrane content of ␣-tocopherol
Summary
(Received for publication, September 11, 1995, and in revised form, February 7, 1996). 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. Ascorbic acid does not directly retard peroxidation of membrane lipids [11], it may do so indirectly by reducing the tocopheroxyl free radical in the lipid bilayer [12, 13] Beyond their ability to use intracellular reducing potential to protect the cell membrane from oxidant stress, erythrocytes have long been known to reduce extracellular oxidants such as ferricyanide (14 –16), methemoglobin [17], and ferricytochrome c [18]. The protective effect of intravesicular ascorbate involves a transmembrane transfer of ascorbate-derived electrons, the extent of which depends on the membrane content of ␣-tocopherol
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