Interaction of Ascorbate and α-Tocopherol in Resealed Human Erythrocyte Ghosts

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 F 2 -isoprostanes, a sensitive marker of lipid peroxidation. Incorporation of ascorbate into ghosts during resealing largely prevented F 2 -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.

Ascorbate Stimulates Ferricyanide Reduction in HL-60 Cells through a Mechanism Distinct from the NADH-dependent Plasma Membrane Reductase

“…In liposomes, it was found that ␣-tocopherol, a natural antioxidant present in membrane lipids, mediated ferricyanide reduction by ascorbate without intervention of an enzyme system (2). Also, for erythrocytes, it was observed that ␣-tocopherol could augment ascorbate-induced ferricyanide reduction, but the involvement of a membrane-localized enzyme system could not be excluded (2). Conversely, the present results show that, in HL-60 cells, the supplementation or depletion of ␣-tocopherol had no significant effect on the efficacy of ascorbate in the stimulation of ferricyanide reduction (Table II).…”

“…There have been reports suggesting a role for ␣-tocopherol in assisting the transport of electrons over the membrane (2,28). Under standard culture conditions, cells have a very low ␣-tocopherol content due to the low level of this compound in serum (29).…”

“…It could involve either direct chemical reactions or a membrane-based enzyme (2,28). There have been reports in the literature suggesting a nonenzymatic route of electron transport.…”

“…This is not the natural substrate for the redox system, but as yet, there has been no conclusive evidence for the substrates of this system or for its primary function. It has been suggested that the system is involved in the maintenance of the redox state of sulfhydryl residues in membrane proteins (1), the neutralization of oxidative stressors outside the cell (2), or the uptake of iron through a non-transferrin pathway (3,4).…”

Oxidative stress in Portuguese children with Down syndrome

“…Transmembrane reductase of ferricyanide (TMR) expresses the existence of an erythrocyte transmembrane redox system responsible for, among others, recycling antioxidants (Hatherill et al, 1991;May, Qu & Morrow, 1996;Orringer & Roer, 1979).…”