ASCORBIC-ACID RECYCLING ENHANCES THE ANTIOXIDANT RESERVE OF HUMAN ERYTHROCYTES

The role of ascorbate transport and metabolism in the response of huma n erythrocytes to an extracellular oxidant stress was investigated. Ra tes of entry and exit of [C-14]dehydroascorbate from erythrocytes were more than 10-fold greater than those of [C-14]ascorbate. Both the red uced and oxidized forms of the vitamin were transported largely by the glucose transporter. Inside erythrocytes, dehydroascorbate was conver ted to ascorbate, increasing intracellular ascorbate concentrations 2- 3-fold over those in the medium. In such ascorbate-loaded cells, the m embrane-impermeant oxidant ferricyanide induced a transmembrane oxidat ion of intracellular ascorbate to dehydroascorbate. The latter escaped the cells on the glucose transporter, which resulted in a halving of the net entry of [C-14]dehydroascorbate in, the presence of ferricyani de. Treatment of ascorbate-loaded cells with H2O2 and Cu2+ also oxidiz ed ascorbate and induced efflux of [C-14]dehydroascorbate. Ferricyanid e-dependent intracellular oxidation of ascorbate resulted in a corresp onding reduction of extracellular ferricyanide, which served as an int egrated measure of ascorbate recycling. Ferricyanide reduction was pro portional to the loading concentration of dehydroascorbate and was enh anced when loss of dehydroascorbate from cells was decreased, either b y blockade of the glucose transporter or by concentrating the cells. S elective depletion of cellular ascorbate lowered rates of ferricyanide reduction by two-thirds, suggesting that ascorbate rather than NADH i s the major donor for the transmembrane ferricyanide oxidoreductase ac tivity. On the basis of the ascorbate-dependent rate of ferricyanide r eduction, erythrocytes at a 45% hematocrit can regenerate the ascorbic acid present in whole blood every 3 min. Erythrocyte ascorbate recycl ing may thus contribute more to the antioxidant reserve of blood than is evident from plasma ascorbate concentrations alone.