Ascorbic acid accumulation and transport in human fibroblasts.

As the initial step in the use of fibroblasts as a model system for 'in situ kinetics', ascorbic acid (vitamin C) accumulation in normal human fibroblasts was investigated for the first time. Ascorbic acid was transported into fibroblasts and accumulated against a concentration gradient up to 20-fold, as measured by h.p.l.c. with coulometric electrochemical detection. Ascorbic acid accumulation was mediated by two concentration-dependent transport activities. The first was a high-affinity activity with an apparent Km of 6 microM and an apparent Vmax. of 203 microM/h, and the second was a low-affinity activity with an apparent Km of 5 mM and an apparent Vmax. of 1.8 mM/h. Both activities were inhibited by metabolic inhibitors and inhibitors of ascorbic acid transport in human neutrophils. The low-affinity transporter could not be accounted for by diffusion. Although the high-affinity transport activity was comparable with that described for human neutrophils, the low-affinity transporter was different. These data provide the first evidence that two-component ascorbic acid transport may be a generalized mechanism for accumulation of this vitamin in humans.