Protection and Recycling of α-Tocopherol in Human Erythrocytes by Intracellular Ascorbic Acid

Ascorbic acid can recycle α-tocopherol from the tocopheroxyl free radical in lipid bilayers and in micelles, but such recycling has not been demonstrated to occur across cell membranes. In this work the ability of intracellular ascorbate to protect and to recycle α-tocopherol in intact human erythrocytes and erythrocyte ghosts was investigated. In erythrocytes that were 80% depleted of intracellular ascorbate by treatment with the nitroxide Tempol, both 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) and ferricyanide oxidized α-tocopherol to a greater extent than in cells not depleted of ascorbate. In contrast, in erythrocytes in which the intracellular ascorbate concentration had been increased by loading with dehydroascorbate, loss of α-tocopherol was less with both oxidants than in control cells. Protection against AAPH-induced oxidation of α-tocopherol was not prevented by extracellular ascorbate oxidase, indicating that the protection was due to intracellular and not to extracellular ascorbate. Incubation of erythrocytes with lecithin liposomes also generated an oxidant stress, which caused lipid peroxidation in the liposomes and depleted erythrocyte α-tocopherol, leading to hemolysis. Ascorbate loading of the erythrocytes delayed liposome oxidation and decreased loss of α-tocopherol from both cells and from α-tocopherol-loaded liposomes. When erythrocyte ghosts were resealed to contain ascorbate and challenged with free radicals generated by AAPH outside the ghosts, intravesicular ascorbate was totally depleted over 1 h of incubation, whereas α-tocopherol decreased only after ascorbate was substantially oxidized. These results suggest that ascorbate within the erythrocyte protects α-tocopherol in the cell membrane by a direct recycling mechanism.