Two 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 total body stores but the mechanisms that mediate the uptake of ascorbic acid (AA) or dehydroascorbic acid (DHA) in human lymphoid cells are undefined. We have investigated the uptake of AA and DHA with mass assays in human lymphoblasts by using HPLC, with precautions to prevent the oxidation of AA and to take into account the instability of DHA in buffers. Human lymphoblasts exhibit distinct uptake mechanisms for both AA and DHA, with Vmax values of 1.35±0.14 and 29.0±5.8 nmol/h per 106 cells and Km values of 23.5±6 and 104±84 μM respectively. The AA uptake was Na+-dependent and inhibitable with ouabain, 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 extracellular glucose concentrations of 20 mM (P < 0.05). In contrast, glucose competitively inhibited DHA uptake with a Ki of 2.2 mM so that DHA uptake was decreased by glucose even in the physiological range. Phorbol esters stimulated AA but not DHA uptake; this was abolished in the presence of extracellular reductant, indicating that AA was converted to DHA before uptake occurred. Prolonged increased glucose levels (20 mM) led to a decrease in the Vmax of DHA uptake. At concentrations of plasma AA or DHA, the AA uptake mechanism might be nearly half-saturated 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 acutely by competitive inhibition or by down-regulation of uptake with chronic glucose exposure, leading to an impaired ability to store and recycle oxidized AA.