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 Vmax and KM 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.

Efflux of hepatic ascorbate: a potential contributor to the maintenance of plasma vitamin C

“…The activity of transplasma membrane electron transport in HepG2 cells was therefore determined by the rate of ferricyanide reduction, as described previously [11]. In contrast with its effects on DHA uptake, cytochalasin B had no effect on extracellular ferricyanide reduction by HepG2 cells (Table 2), as has been demonstrated for HL-60 cells [27] and erythrocytes [28].…”

Redox Reactions and Electron Transfer Across the Red Cell Membrane

“…Similarly, DHA efflux is observed with hydrogen peroxide as the oxidant. Greater DHA efflux is observed from ghosts than whole cells due to the lower ability of ghosts to recycle AA, possibly explaining the lack of DHA efflux reported by Himmelreich et al in their whole-cell NMR experiments (15). AA appears to be the main electron donor.…”

“…This recycling appears to play an important role in the protection of RBC from oxidative stress. DHA and AA enter the RBC via the GLUT1 glucose transporter, however the rate of entry for AA is substantially lower than that of DHA (15,23).…”

“…Nuclear magnetic resonance spectroscopy (NMR) enables real-time, non-invasive investigation of extra-and intracellular events simultaneously, hence providing direct insight into the metabolic effects of extracellular oxidative stress and the reduction of the extracellular oxidant ( Fig. 2) (8,15,16). Membrane potential measurements (17) and observations of free radicals with electron paramagnetic resonance spectroscopy (EPR) (18) provide strong evidence for electron transfer.…”

Evaluation of the antioxidant response in the plasma of healthy or hypertensive subjects after short-term exercise

“…34 Thus, the plasma level increase of this important antioxidant must result from its release from the liver, 35 or the regeneration of its oxidised forms (Asc free radical and dehydroascorbate) on hepatocyte and erythrocyte plasma membrane. [36][37][38] A possible interpretation of the lower Asc increase in hypertensive patients is that, in these patients, either more Asc is oxidised, or less oxidised Asc is recycled in the reduced form, or both circumstances occur. In these patients, an excess of hypoxantine, which is produced in the muscle during exercise, 2,39 has been suggested to be converted to urate by the enzyme xantine oxidase.…”