Plasma lipid oxidation induced by peroxynitrite, hypochlorite, lipoxygenase and peroxyl radicals and its inhibition by antioxidants as assessed by diphenyl-1-pyrenylphosphine

Abstract

Lipid oxidation has been implicated in the pathogenesis of many diseases. Lipids are oxidized in vivo by several different oxidants to give diverse products, in general lipid hydroperoxides as the major primary product. In the present study, the production of lipid hydroperoxides in the oxidation of mouse plasma induced by multiple oxidants was measured using diphenyl-1-pyrenylphosphine (DPPP) as a probe. DPPP itself is not fluorescent, but it reacts with lipid hydroperoxides stochiometrically to give highly fluorescent DPPP oxide and lipid hydroxides. The production of lipid hydroperoxides could be followed continuously in the oxidation of plasma induced by peroxynitrite, hypochlorite, 15-lipoxygenase, and peroxyl radicals with a microplate reader. A clear lag phase was observed in the plasma oxidation mediated by aqueous peroxyl radicals and peroxynitrite, but not in the oxidation induced by hypochlorite and lipoxygenase. The effects of several antioxidants against lipid oxidation induced by the above oxidants were assessed. The efficacy of antioxidants was dependent markedly on the type of oxidants. α-Tocopherol exerted potent antioxidant effects against peroxyl radical-mediated lipid peroxidation, but it did not inhibit lipid oxidation induced by peroxynitrite, hypochlorite, and 15-lipoxygenase efficiently, suggesting that multiple antioxidants with different selectivities are required for the inhibition of plasma lipid oxidation in vivo. This is a novel, simple and most high throughput method to follow plasma lipid oxidation induced by different oxidants and also to assess the antioxidant effects in biologically relevant settings.