ACCUMULATION OF VITAMIN-C (ASCORBATE) AND ITS OXIDIZED METABOLITE DEHYDROASCORBIC ACID OCCURS BY SEPARATE MECHANISMS

It is unknown whether ascorbate alone (vitamin C), its oxidized metabo lite dehydroascorbic acid alone, or both species are transported into human cells. This problem was addressed using specific assays for each compound, freshly synthesized pure dehydroascorbic acid, the speciall y synthesized analog 6-chloroascorbate, and a new assay for 6-chloroas corbate. Ascorbate and dehydroascorbic acid were transported and accum ulated distinctly; neither competed with the other, Ascorbate was accu mulated as ascorbate by sodium-dependent carrier-mediated active trans port, Dehydroascorbic acid transport and accumulation as ascorbate was at least 10-fold faster than ascorbate transport and was sodium-indep endent. Once transported, dehydroascorbic acid was immediately reduced intracellularly to ascorbate. The analog 6-chloroascorbate had no eff ect on dehydroascorbic acid transport but was a competitive inhibitor of ascorbate transport. The K-i for 6-chloroascorbate (2.9-4.4 mu M) w as similar to the K-m for ascorbate transport (9.8-12.6 mu M) 6-Chloro ascorbate was itself transported and accumulated in fibroblasts by a s odium-dependent transporter. These data provide new information that a scorbate and dehydroascorbic acid are transported into human neutrophi ls and fibroblasts by two distinct mechanisms and that the compound av ailable for intracellular utilization is ascorbate.