Myeloperoxidase-dependent loss of malondialdehyde: a limitation for detecting neutrophil-mediated lipid peroxidation.
Lipid peroxidation is commonly measured using the thiobarbituric acid (TBA) assay. We have examined how this assay is affected by hypochlorite, which is generated by the neutrophil enzyme myeloperoxidase. The TBA reactivity of phospholipid liposomes that had been partially peroxidized with iron/ascorbate was destroyed by low concentrations of sodium hypochlorite. Since most of the TBA reactivity in peroxidized liposomes is due to malondialdehyde, its reactivity was investigated. Addition of sodium hypochlorite destroyed the uv absorbance of malondialdehyde with a 2:1 stoichiometry and eliminated its TBA reactivity. The TBA reactivity of malondialdehyde and peroxidized liposomes was also lost after treatment with myeloperoxidase. The reaction with myeloperoxidase required chloride and was inhibited by catalase and methionine, indicating the involvement of hypochlorite. Neutrophils stimulated with phorbol myristate acetate or N-formyl-methionyl-leucyl-phenylalanine destroyed the TBA reactivity of malondialdehyde and peroxidized liposomes by a hypochlorite-dependent mechanism. The ability of hypochlorite to break down malondialdehyde explains why lipid peroxidation by stimulated neutrophils, as measured with TBA, is apparently inhibited by myeloperoxidase. Myeloperoxidase may not, however, inhibit the peroxidation process. The TBA assay and other assays of malondialdehyde may be of limited value, therefore, for assessing lipid peroxidation in systems where neutrophils or myeloperoxidase are involved.
Winterbourn
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CC
Department of Pathology, Christchurch School of Medicine, New Zealand.
Carr
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AC
D002712:Chlorides / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D006997:Hypochlorous Acid / Q000494:pharmacology; D015227:Lipid Peroxidation / Q000187:drug effects*; D008081:Liposomes / Q000378:metabolism*; D008315:Malondialdehyde / Q000032:analysis; D008956:Models, Chemical; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D009195:Peroxidase / Q000378:metabolism* / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D017392:Thiobarbituric Acid Reactive Substances / Q000032:analysis; D014969:Xanthine Oxidase / Q000378:metabolism
D002712:Chlorides; D008081:Liposomes; D017392:Thiobarbituric Acid Reactive Substances; D008315:Malondialdehyde; D009240:N-Formylmethionine Leucyl-Phenylalanine; D006997:Hypochlorous Acid; D006861:Hydrogen Peroxide; D009195:Peroxidase; D014969:Xanthine Oxidase; D013755:Tetradecanoylphorbol Acetate