13C NMR Studies of Vitamin C Transport and Its Redox Cycling in Human Erythrocytes

Abstract

¹³C NMR spectra of labeled [1-¹³C]- and [2-¹³C]ascorbic acid were seen to contain resonances arising from the intra- and extracellular populations in suspensions of human erythrocytes; i.e., they displayed the “split-peak” phenomenon. This new observation enabled the ready determination of the location, whether inside or outside cells, of the redox reactions in which the vitamin C was involved and to monitor the transport of the compounds into and out of the cells. Thus, the membrane permeability of ascorbic acid and the apparent V_max and K_M for the reduction of dehydroascorbic acid were determined in a noninvasive manner. In contrast to other work, evidence was found of a transporter of dehydroascorbic acid which is different from the glucose transporter. This transport system also appeared to be involved in the simultaneous reduction of dehydroascorbic acid on its passage into the cells. A second reduction process appeared to occur extracellularly, by the passage of reducing equivalents through the plasma membrane, as occurs with the reduction of ferricyanide. Evidence is presented that the processes of vitamin C recycling rely on different cellular sources of reducing equivalents. Whereas the transport and reduction via the membrane appeared to be dependent on glycolysis (NADH), the reduction of intracellular dehydroascorbic acid, formed in the process of transmembrane electron transfer or by transport from the outside of the cell, is currently thought to depend on NADPH.