Free radical-initiated lipid peroxidation has been implicated in many human diseases; the most studied of these has been atherosclerosis. Deposition of low density lipoprotein (LDL) in the vascular wall and the oxidation of its lipid components is believed to contribute importantly to the pathology of this disorder. Cholesterol esters containing either linoleate or arachidonate are a major component of LDL in humans. Up to 30 percent of cholesterol esters in human atherosclerotic lesions are oxidized. The oxidation products of cholesteryl linoleate have been defined and are primarily monooxygenated derivatives and their scission products. In contrast, the peroxidation of cholesteryl arachidonate in vivo has not been studied. Unlike cholesteryl linoleate, significantly more complex products containing cyclic structures should result from the oxidation of cholesteryl arachidonate since arachidonic acid can incorporate more than one molecule of oxygen. It is our hypothesis that the oxidation of cholesteryl arachidonate in vivo can be defined and that an important mechanism results in the formation of novel products containing endoperoxide (isoprostane), monocyclic peroxide, and serial cyclic peroxide moieties. Preliminary studies support this hypothesis. We have also found that one compound, a cholesteryl isoprostane, possesses potent bioactivity and modulates inflammatory mediator release in relevant cell types including macrophages and vascular endothelial cells. We will use the resources of the Research Center for Pharmacology and Drug Toxicology to study the formation, metabolism, and pharmacological and biological properties of these novel cholesteryl arachidonate oxygenation products. We will 1) provide definitive evidence employing mass spectrometry for the formation of serial cyclic peroxide- and endoperoxide-containing species in vivo in human and animal atherosclerotic tissue; 2) determine whether these compounds are present in circulating LDL and are substrates for lipoprotein metabolizing enzymes as evidence that lipoprotein oxidation occurs in the circulation; 3) examine factors that likely influence the formation of these compounds in vivo including vitamin E and oxygen tension; 4) determine if enzymes present in inflammatory cells that populate atherosclerotic lesions generate complex cyclic oxidation products; 5) examine whether the duration or extent of atherosclerosis in humans and animals influences the formation of cholesteryl arachidonate oxidation products; and 6) determine if cholesterol ester metabolizing enzymes utilize cholesteryl arachidonate oxidation species as substrates. These studies will yield important insights into the role of these compounds in diseases associated with oxidant stress and ultimately allow for the identification of targets for the development of drug therapies to modulate the formation of cholesteryl arachidonate oxidation products in vivo.
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