PROTECTION AND RECYCLING OF ALPHA-TOCOPHEROL IN HUMAN ERYTHROCYTES BYINTRACELLULAR ASCORBIC-ACID

Ascorbic acid can recycle cr-tocopherol from the tocopheroxyl free rad ical in lipid bilayers and in micelles, but such recycling has not bee n demonstrated to occur across cell membranes. In this work the abilit y of intracellular ascorbate to protect and to recycle Lu-tocopherol i n intact human erythrocytes and erythrocyte ghosts was investigated. I n erythrocytes that were 80% depleted of intracellular ascorbate by tr eatment with the nitroxide Tempol, both 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) and ferricyanide oxidized alpha-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 alpha-tocoph erol was less with both oxidants than in control cells. Protection aga inst AAPH-induced oxidation of alpha-tocopherol was not prevented by e xtracellular ascorbate oxidase, indicating that the protection was due to intracellular and not to extracellular ascorbate. Incubation of er ythrocytes with lecithin liposomes also generated an oxidant stress, w hich caused lipid peroxidation in the liposomes and depleted erythrocy te alpha-tocopherol, leading to hemolysis. Ascorbate loading of the er ythrocytes delayed liposome oxidation and decreased loss of alpha-toco pherol from both cells and from cu-tocopherol-loaded liposomes. When e rythrocyte ghosts were resealed to contain ascorbate and challenged wi th free radicals generated by AAPH outside the ghosts, intravesicular ascorbate was totally depleted over 1 h of incubation, whereas Lu-toco pherol decreased only after ascorbate was substantially oxidized. Thes e results suggest that ascorbate within the erythrocyte protects alpha -tocopherol in the cell membrane by a direct recycling mechanism. (C) 1998 Academic Press.