title: Roles of superoxide and myeloperoxidase in ascorbate oxidation in stimulated neutrophils and H2O2-treated HL60 cells.
abstract: Ascorbate is present at high concentrations in neutrophils and becomes oxidized when the cells are stimulated. We have investigated the mechanism of oxidation by studying cultured HL60 cells and isolated neutrophils. Addition of H(2)O(2) to ascorbate-loaded HL60 cells resulted in substantial oxidation of intracellular ascorbate. Oxidation was myeloperoxidase-dependent, but not attributable to hypochlorous acid, and can be explained by myeloperoxidase (MPO) exhibiting direct ascorbate peroxidase activity. When neutrophils were stimulated with phorbol myristate acetate, about 40% of their intracellular ascorbate was oxidized over 20 min. Ascorbate loss required NADPH oxidase activity but in contrast to the HL60 cells did not involve myeloperoxidase. It did not occur when exogenous H(2)O(2) was added, was not inhibited by myeloperoxidase inhibitors, and was the same for normal and myeloperoxidase-deficient cells. Neutrophil ascorbate loss was enhanced when endogenous superoxide dismutase was inhibited by cyanide or diethyldithiocarbamate and appears to be due to oxidation by superoxide. We propose that in HL60 cells, MPO-dependent ascorbate oxidation occurs because cellular ascorbate can access newly synthesized MPO before it becomes packaged in granules: a mechanism not possible in neutrophils. In neutrophils, we estimate that ascorbate is capable of competing with superoxide dismutase for a small fraction of the superoxide they generate and propose that the superoxide responsible is likely to come from previously identified sites of intracellular NADPH oxidase activity. We speculate that ascorbate might protect the neutrophil against intracellular effects of superoxide generated at these sites.
authors.affiliation: Department of Pathology, University of Otago Christchurch, Christchurch, New Zealand.
authors.forename: Amber
authors.initials: A
authors.lastname: Parker
authors.forename: Sarah L
authors.initials: SL
authors.lastname: Cuddihy
authors.forename: Tae G
authors.initials: TG
authors.lastname: Son
authors.forename: Margreet C M
authors.initials: MC
authors.lastname: Vissers
authors.forename: Christine C
authors.initials: CC
authors.lastname: Winterbourn
mesh_terms: D000975:Antioxidants / Q000378:metabolism / Q000494:pharmacology; D001205:Ascorbic Acid / Q000378:metabolism / Q000494:pharmacology; D002374:Catalase / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005978:Glutathione / Q000032:analysis; D018922:HL-60 Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006997:Hypochlorous Acid / Q000494:pharmacology; D009504:Neutrophils / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D009928:Organ Specificity; D010084:Oxidation-Reduction / Q000187:drug effects; D018384:Oxidative Stress; D009195:Peroxidase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013482:Superoxide Dismutase / Q000037:antagonists & inhibitors / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D000975:Antioxidants; D004791:Enzyme Inhibitors; D013481:Superoxides; D006997:Hypochlorous Acid; D006861:Hydrogen Peroxide; D002374:Catalase; D009195:Peroxidase; D013482:Superoxide Dismutase; D005978:Glutathione; D013755:Tetradecanoylphorbol Acetate; D001205:Ascorbic Acid