GSH is required to recycle ascorbic acid in cultured liver cell lines.
Liver is the site of ascorbic acid synthesis in most mammals. As human liver cannot synthesize ascorbate de novo, it may differ from liver of other species in the capacity or mechanism for ascorbate recycling from its oxidized forms. Therefore, we compared the ability of cultured liver-derived cells from humans (HepG2 cells) and rats (H4IIE cells) to take up and reduce dehydroascorbic acid (DHA) to ascorbate. Neither cell type contained appreciable amounts of ascorbate in culture, but both rapidly took up and reduced DHA to ascorbate. Intracellular ascorbate accumulated to concentrations of 10-20 mM following loading with DHA. The capacity of HepG2 cells to take up and reduce DHA to ascorbate was more than twice that of H4IIE cells. In both cell types, DHA reduction lowered glutathione (GSH) concentrations and was inhibited by prior depletion of GSH with diethyl maleate, buthionine sulfoximine, and phenylarsine oxide. NADPH-dependent DHA reduction due to thioredoxin reductase occurred in overnight-dialyzed extracts of both cell types. These results show that cells derived from rat liver synthesize little ascorbate in culture, that cultured human-derived liver cells have a greater capacity for DHA reduction than do rat-derived liver cells, but that both cell types rely largely on GSH- or NADPH-dependent mechanisms for ascorbate recycling from DHA.
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Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232-6303, USA.
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D000818:Animals; D001152:Arsenicals / Q000494:pharmacology; D001205:Ascorbic Acid / Q000378:metabolism*; D019328:Buthionine Sulfoximine / Q000494:pharmacology; D002460:Cell Line; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005978:Glutathione / Q000378:metabolism* / Q000502:physiology*; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000166:cytology* / Q000187:drug effects*; D008298:Maleates / Q000494:pharmacology; D009249:NADP / Q000378:metabolism; D051381:Rats; D013880:Thioredoxin-Disulfide Reductase / Q000378:metabolism / Q000494:pharmacology; D013997:Time Factors; D014867:Water / Q000737:chemistry
D001152:Arsenicals; D004791:Enzyme Inhibitors; D008298:Maleates; D014867:Water; C029341:oxophenylarsine; D019328:Buthionine Sulfoximine; D009249:NADP; D013880:Thioredoxin-Disulfide Reductase; C014476:diethyl maleate; D005978:Glutathione; D001205:Ascorbic Acid