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 in vitro 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.

Protective effect of L-ascorbic acid against oxidative damage in the liver of rats with water-immersion restraint stress

“…34 In rats, the liver releases AA to the bloodstream to maintain the plasma level of the vitamin. 35 AA is known to be consumed by reaction with ROS, such as O 2…”

Transfer of ascorbic acid across the vascular endothelium: mechanism and self-regulation

“…Ascorbate is known to efflux from cultured endothelial cells (35), especially in response to increases in intracellular calcium (9). It follows that this efflux could reflect vectorial ascorbate transport from the abluminal or basolateral side of the cells (25).…”

“…Ascorbate efflux has been described in endothelial cells (9,35), as well as in hepatocytes (35) and brain cells under glutamate-induced excitotoxic stress (28). We (25) recently showed that cultured EA.hy926 endothelial cells have substantial rates of ascorbate efflux that are countered by reuptake on the SVCT transporter.…”

Ascorbic acid efflux and re-uptake in endothelial cells: maintenance of intracellular ascorbate

“…It diffuses slowly out of a simple cell such as an erythrocyte, which loses 10–20% of ascorbate over 40 min of incubation, depending on the initial intracellular concentration [14, 17, 29]. Efflux is more rapid from endothelial cells than from erythrocytes [14].…”

“…Of the two mechanisms, uptake of ascorbate on its specific transporter probably contributes much more to the trans-plasma membrane ascorbate gradient than does uptake and reduction of DHA, since concentrations of the latter are usually very low in plasma [12], since DHA competes with 5 mM D-glucose for glucose transporters, and since human erythrocytes, which lack ascorbate transporters [13], have the same intracellular ascorbate concentration as in plasma [1, 11]. Ascorbate appears to be trapped in most cells because of its negative charge at physiologic pH, although ascorbate efflux has been described in hepatocytes [14], in brain cells under glutamate-induced stress [15], and in endothelial cells [14, 16]. Loss of ascorbate from cells has also been documented following its oxidation as efflux of DHA on glucose transporters [17].…”