Abstract

The involvement of lipid hydroperoxides in the oxidation of human low density lipoproteins (LDL) induced by Cu++ or azoperoxyl radicals was studied. Concurrent or pretreatment of LDL with the synthetic peroxidase Ebselen and its cofactor glutathione totally prevented Cu+(+)-dependent oxidation, as measured by the formation of thiobarbituric acid reactive substances and LDL mobility on agarose gels. LDL could be oxidized in the absence of metals with peroxy radicals generated via homolysis of azo compounds. With the azo initiator, 2,2'-azobis-2-amidinopropane hydrochloride, Ebselen plus glutathione prevented lipid oxidation when present during oxidation. However, if LDL were first pretreated with Ebselen plus glutathione and these reagents removed before oxidation, they did not prevent oxidation. Analysis by high-performance liquid chromatography of lipids extracted from Cu+(+)- or azoperoxyl radical-modified lipoproteins indicated that the major lipid oxidation products were derived from linoleate and that Ebselen-glutathione addition reduced linoleate hydroperoxides. Oxidation of LDL by human monocyte-derived macrophages was also prevented by coincubation with Ebselen-glutathione or the antioxidant, probucol. These results demonstrate that initiation of Cu+(+)-dependent oxidation absolutely requires the presence of trace amounts of lipid hydroperoxides in the lipoprotein and that hydroperoxides play a major role in the propagation reactions even in the absence of added transition metals.