ASCORBATE RECYCLING IN HUMAN ERYTHROCYTES - ROLE OF GSH IN REDUCING DEHYDROASCORBATE

Human erythrocytes regenerate ascorbate from its oxidized product, deh ydroascorbate. The extent to which such ascorbate recycling occurs by a GSH dependent mechanism was investigated. In the presence of glucose , erythrocytes took up over 90% of extracellular [C-14]dehydroascorbat e and rapidly converted it to [C-14]ascorbate, which was trapped withi n the cells. Dehydroascorbate uptake and reduction was not associated with generation of a monoascorbyl free radical intermediate. Uptake an d reduction of dehydroascorbate by glucose-depleted erythrocytes coord inately decreased GSH and raised GSSG concentrations in erythrocytes. This effect was reversed by D-glucose, but not by L-lactate. Conversel y, depletion of cellular GSH decreased the ability of cells to recycle dehydroascorbate to ascorbate, as reflected in the extent to which ce lls were able to reduce extracellular ferricyanide. Monoascorbyl free radical. was formed during the reduction of extracellular ferricyanide , indicating that one electron transfer steps were involved in this pr ocess. In GSH-depleted cells, addition of L-lactate as an energy sourc e for glycolysis-dependent NADH regeneration did cause a partial recov ery of the ability of cells to reduce ferricyanide. However, in reseal ed erythrocyte ghosts containing either 4 mM GSH or 400 mu M NADH, onl y the GSH-containing ghosts supported regeneration of ascorbate from a dded dehydroascorbate. These results suggest that in human erythrocyte s ascorbate regeneration from dehydroascorbate is largely GSH dependen t, and that it occurs through either enzymatic or nonenzymatic reactio ns not involving the monoascorbyl free radical.