Recommended dietary allowances (RDAs) for vitamin C (ascorbate) have been based on preventing the deficiency disease scurvy. We proposed that new RDAs for vitamin C and other vitamins could be determined using in situ kinetics, a concept developed by this laboratory. In situ kinetics has biochemical and clinical goals. The biochemical goals are to determine vitamin C molecular functions in relation to vitamin concentrations. For these studies vitamin C is investigated in human tissues such as fibroblasts and neutrophils. To determine how intracellular concentration is regulated, two mechanisms of vitamin transport were characterized. Ascorbate is transported as such by carriers that are sodium-dependent, saturable, energy dependent, and inhibited by newly synthesized ascorbate analogs. The two human transporters hSVCT1 and hSVCT2 were cloned and characterized, and genomic characterization and studies of nucleotide polymorphisms are underway. In contrast, the oxidized form of vitamin C, dehydroascorbic acid, is transported by glucose transporters GLUT I, III, and IV, and immediately reduced intracellularly to ascorbate. The protein responsible for reduction in neutrophils was isolated, sequenced, identified as thioltransferase (glutaredoxin), and cloned. The full-length human glutaredoxin gene was cloned and its promoter characterized. These studies indicate that vitamin C function in neutrophils may be to protect neutrophils from their own oxidants. Overall findings suggest that vitamin C function can be determined in relation to its concentration in living tissues. The clinical goals of in situ kinetics are to determine how vitamin concentrations are achieved in normal humans as a function of dose and whether concentrations humans achieve are those which regulate molecular functions of the vitamin. A clinical trial was completed in healthy male inpatients hospitalized at the Clinical Center. For the first time, the following were described: the relationship between vitamin C doses over a wide range and its concentration in plasma and tissues; true bioavailability of vitamin C; vitamin urinary excretion in relation to dose; and potential adverse effects in relation to dose. A clinical trial in healthy women is underway. Based on our data, RDAs for vitamin C were revised upward in 2000 by the National Academy of Sciences. For men the RDA was increased from 60 to 90 mg daily, and for women the RDA was increased from 60 to 75 mg daily. Based on our data, many European countries and Japan have also increased their RDAs for vitamin C. We recommend that vitamin C intake is from at least 5 servings of fruits and vegetables daily. Forthcoming data from our laboratory may have further impact on recommendations for healthy and ill people.
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