title: Absence of an effect of vitamin E on protein and lipid radical formation during lipoperoxidation of LDL by lipoxygenase.
abstract: Low-density lipoprotein (LDL) oxidation is the primary event in atherosclerosis, and LDL lipoperoxidation leads to modifications in apolipoprotein B-100 (apo B-100) and lipids. Intermediate species of lipoperoxidation are known to be able to generate amino acid-centered radicals. Thus, we hypothesized that lipoperoxidation intermediates induce protein-derived free radical formation during LDL oxidation. Using DMPO and immuno-spin trapping, we detected the formation of protein free radicals on LDL incubated with Cu(2+) or the soybean lipoxidase (LPOx)/phospholipase A2 (PLA2). With low concentrations of DMPO (1mM), Cu(2+) dose-dependently induced oxidation of LDL and easily detected apo B-100 radicals. Protein radical formation in LDL incubated with Cu(2+) showed maximum yields after 30 min. In contrast, the yields of apo B-100 radicals formed by LPOx/PLA2 followed a typical enzyme-catalyzed kinetics that was unaffected by DMPO concentrations of up to 50mM. Furthermore, when we analyzed the effect of antioxidants on protein radical formation during LDL oxidation, we found that ascorbate, urate, and Trolox dose-dependently reduced apo B-100 free radical formation in LDL exposed to Cu(2+). In contrast, Trolox was the only antioxidant that even partially protected LDL from LPOx/PLA2. We also examined the kinetics of lipid radical formation and protein radical formation induced by Cu(2+) or LPOx/PLA2 for LDL supplemented with α-tocopherol. In contrast to the potent antioxidant effect of α-tocopherol on the delay of LDL oxidation induced by Cu(2+), when we used the oxidizing system LPOx/PLA2, no significant protection was detected. The lack of protection of α-tocopherol on the apo B-100 and lipid free radical formation by LPOx may explain the failure of vitamin E as a cardiovascular protective agent for humans.
authors.affiliation: Free Radical Metabolism Section, Laboratory of Toxicology and Pharmacology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA. Electronic address: ganinidasilvad@niehs.nih.gov.
authors.forename: Douglas
authors.initials: D
authors.lastname: Ganini
authors.affiliation: Free Radical Metabolism Section, Laboratory of Toxicology and Pharmacology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
authors.forename: Ronald P
authors.initials: RP
authors.lastname: Mason
mesh_terms: D000975:Antioxidants / Q000494:pharmacology*; D053299:Apolipoprotein B-100 / Q000737:chemistry* / Q000378:metabolism; D003300:Copper / Q000494:pharmacology*; D005609:Free Radicals / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D015227:Lipid Peroxidation / Q000187:drug effects; D008077:Lipoproteins, LDL / Q000737:chemistry*; D008084:Lipoxygenase / Q000494:pharmacology*; D010084:Oxidation-Reduction; D019097:Spin Trapping; D014810:Vitamin E / Q000494:pharmacology*
chemical_list: D000975:Antioxidants; D053299:Apolipoprotein B-100; D005609:Free Radicals; D008077:Lipoproteins, LDL; C099252:oxidized low density lipoprotein; D014810:Vitamin E; D003300:Copper; D008084:Lipoxygenase
keywords: Atherosclerosis; Copper; DMPO; Free radicals; Immuno-spin trapping; LDL oxidation; Lipoperoxidation; Lipoxidase; Lipoxygenase; Vitamin E
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