Oxidation and Antioxidation of Human Low-Density Lipoprotein and Plasma Exposed to 3-Morpholinosydnonimine and Reagent Peroxynitrite

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Abstract
As peroxynitrite is implicated as an oxidant for low-density lipoprotein (LDL) in atherogenesis, we investigated this process using reagent peroxynitrite (ONOO-) and 3-morpholinosydnonimine (SIN-1, which produces peroxynitrite via generation of NO• and O2•-). LDL oxidation was assessed by the consumption of ubiquinol-10 (CoQ10H2) and α-tocopherol (α-TOH), the accumulation of cholesteryl ester hydro(pero)xides, the loss of lysine (Lys) and tryptophan (Trp) residues, and the change in relative electrophoretic mobility. Exposure to ONOO- or SIN-1 resulted in rapid (- or SIN-1 was used at oxidant-to-LDL ratios of 200:1, α-TOH enrichment decreased LDL lipid peroxidation for both SIN-1 and ONOO-. In contrast to lipid peroxidation, altering the α-TOH content of LDL did not affect Trp or Lys loss, independent of the amounts of either oxidant added. Aqueous antioxidants inhibited ONOO--induced lipid and protein oxidation with the order of efficacy: 3-hydroxyanthranilate (3-HAA) > urate > ascorbate. With SIN-1, these antioxidants inhibited Trp consumption, while only the co-antioxidants ascorbate and 3-HAA prevented α-TOH consumption and lipid peroxidation. Exposure of human plasma to SIN-1 resulted in the loss of ascorbate followed by loss of CoQ10H2 and bilirubin. Lipid peroxidation was inhibited during this period, though proceeded as a radical-chain process after depletion of these antioxidants and in the presence of α-TOH and urate. Bicarbonate at physiological concentrations decreased ONOO--induced lipid and protein oxidation, whereas it enhanced SIN-1-induced lipid peroxidation, Trp consumption, and α-tocopheroxyl radical formation in LDL. These results indicate an important role for tocopherol-mediated peroxidation and co-antioxidation in peroxynitrite-induced lipoprotein lipid peroxidation, especially when peroxynitrite is formed time-dependently by SIN-1. The studies also highlight differences between ONOO-- and SIN-1-induced LDL oxidation with regards to the effects of bicarbonate, ascorbate, and urate.