2 DISTINCT UPTAKE MECHANISMS FOR ASCORBATE AND DEHYDROASCORBATE IN HUMAN LYMPHOBLASTS AND THEIR INTERACTION WITH GLUCOSE

In diabetes, a major cause of mortality is from cardiovascular causes, and low levels of antioxidants such as vitamin C have been associated with such complications. Leucocyte ascorbic acid status can reflect t otal body stores but the mechanisms that mediate the uptake of ascorbi c acid (AA) or dehydroascorbic acid (DHA) in human lymphoid cells are undefined. We have investigated the uptake of AA and DHA with mass ass ays in human lymphoblasts by using HPLC, with precautions to prevent t he oxidation of AA and to take into account the instability of DHA in buffers. Human lymphoblasts exhibit distinct uptake mechanisms for bot h AA and DHA, with V-max values of 1.35 +/- 0.14 and 29.0 +/- 5.8 nmol /h per 10(6) cells and K-m values of 23.5 +/- 6 and 104 +/- 84 mu M re spectively. The AA uptake was Na+-dependent and inhibitable with ouaba in, whereas DHA uptake was independent of Na+ and ouabain-insensitive. Both uptake mechanisms were inhibited by phloretin or cytochalasin B. AA uptake was decreased significantly (by 13 +/- 2%) only at extracel lular glucose concentrations of 20 mM (P < 0.05). In contrast, glucose competitively inhibited DHA uptake with a K-i of 2.2 mM so that DHA u ptake was decreased by glucose even in the physiological range. Phorbo l esters stimulated AA but not DHA uptake; this was abolished in the p resence of extracellular reductant, indicating that AA was converted t o DHA before uptake occurred. Prolonged increased glucose levels (20 m M) led to a decrease in the V-max of DHA uptake. At concentrations of plasma AA or DHA, the AA uptake mechanism might be nearly half-saturat ed but the DHA mechanism has enormous spare capacity. This allows for cellular uptake and regeneration of AA from DHA derived from oxidative stress. In diabetes, high glucose levels might impair DHA uptake acut ely by competitive inhibition or by down-regulation of uptake with chr onic glucose exposure, leading to an impaired ability to store and rec ycle oxidized AA.