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

Abstract

Ascorbate (AH, the reduced form of vitamin C) is an important radical scavenger and antioxidant in human plasma; the resulting ascorbyl radical can disproportionate to AH and dehydroascorbic acid (DHA). Here we address potential maintenance mechanism(s) for extracellular AH by examining the ability of cells to convert extracellularly presented DHA to AH. DHA was rapidly transported into human liver (HepG2), endothelial and whole blood cells invitro by plasma membrane glucose transporters and reduced intracellularly. Liver cells displayed the highest capacity to release the intracellularly accumulated AH. The proteins responsible for DHA uptake and AH release could be distinguished by inhibitor studies. Thus, unlike DHA uptake, AH efflux was largely insensitive to cytochalasin B and thiol-reactive agents but was inhibited by phloretin, 4,4′-di-isothiocyanostilbene-2,2′-disulphonate and isoascorbate. Efflux of AH from cells was temperature-sensitive and saturable with a low affinity (millimolar, intracellular) for AH. In addition to isolated liver cells, perfusion of intact rat and guinea-pig liver with DHA resulted in AH in the circulating perfusate. Our results show that hepatocytes take up and reduce DHA and subsequently release part of the AH formed, probably via a membrane transporter. By converting extracellular DHA to extracellular AH, the liver might contribute to the maintenance of plasma AH, a process that could be important under conditions of oxidative stress.

Non-transferrin Iron Reduction and Uptake Are Regulated by Transmembrane Ascorbate Cycling in K562 Cells

“…Moreover, cytochalasin B was without significant effect on cell-mediated reduction of ferricyanide (data not shown). This indicates, as observed previously for HepG2 cells (44), that cytochalasin B-dependent inhibition of extracellular ascorbate accumulation from DHA cannot be attributed to potential inhibition of transplasma membrane electron transport to extracellular DHA. Thus, the inability of extracellularly restricted DHA to stimulate extracellular ascorbate production (Fig.…”

“…2A, squares) without affecting intracellular ascorbate (data not shown). Ascorbate release cannot be attributed to cellular lysis as the proportion of viable cells before and after the assays was unchanged (data not shown), as previously noted for HepG2 cells (44). Interestingly, the release of ascorbate by DHA-loaded K562 cells was partially inhibited by addition of the cell-impermeant, generic anion channel inhibitor, 4,4Ј-diisothiocyanatostilbene-2,2Ј-disulfonate (DIDS) (Fig.…”

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

“…It is known that Asc, which is dependent on dietary intake, cannot be synthesised in humans. 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.…”

Rapid uptake of oxidized ascorbate induces loss of cellular glutathione and oxidative stress in liver slices

“…Liver damage induced by ischemia-reperfusion is generally recognized as being mediated by oxidative stress (Layton et al, 1996;Cerwenka et al, 1999) but considerable uncertainty surrounds the mechanism of oxidative stress-induced damage. The liver is also considered to be a reservoir: maintaining homeostatic level of plasma ascorbate since it stores a large amount of ascorbate (Hornig, 1975;Upston et al, 1999). The physiological significance of high concentration of ascorbate in liver is, however, not well defined.…”