This study is designed to answer some unresolved questions about the metabolism of polyunsaturated fatty acids (PUFA) and of hydroxy fatty acids related to leukotrienes. In order to fully understand the function of the peroxisomal 3-hydroxyacyl-CoA epimerase in the degradation of PUFA, the rat liver enzyme will be purified and characterized. A specific inhibitor of 2,4-dienoyl-CoA reductase will be designed and an E. coli mutant lacking 2,4-dienoyl-CoA reductase will be isolated. Both the reductase inhibitor and the E. coli mutant will be used to determine the contribution of the old epimerase-dependent pathway to the degradation of PUFA in rat liver peroxisomes and E. coli. The inhibitor of 2,4-dienoyl-CoA reductase will also be used to determine, if the reaction catalyzed by the reductase is rate-limiting in the oxidaiton of PUFA. The presence or absence of cis-3-trans-2-enoyl-CoA isomerase in rat liver peroxisomes will be established and if present, the enzyme will be isolated and characterized. We will continue to study the hormonal regulation of 2,4-dienoyl-CoA reductase and of the oxidaiton of PUFA. This study necessitates the purification of mitochondrial 2,4-dienoyl-CoA reductase and the preparation of antireductase antibodies which will be used to determine the levels of reductase in mRNA. Additionally, differences between the rates of oxidation of cis and trans unsaturated fatty acids will be determined with rat heart mitochondria. The oxidation of hydroxy fatty acids including 5-hydroxy-6,8,11,13-eicosatetraenoic acid (5-HETE) in mitochondria and peroxisomes will be studied, as will be the inhibition of Beta-oxidation by these and some other potential inhibitory compounds. An assessment of the effectiveness of inhibitors of Beta-oxidation in liver and heart necessitates the purification and characterization of acyl-CoA synthetases present in the mitochondrial matrix. Finally, we will test 3-decynoic acid and other possible inhibitors of acyl-CoA oxidase for their effectiveness as inhibitors of peroxisomal fatty acid oxidation. If effective, such compounds will be used to evaluate the contribution of peroxisomal Beta-oxidation to the hepatic metabolism of unsaturated as well as saturated fatty acids. The results of this study will contriubte to a better understanding of PUFA metabolism and its regulation in normal and diseased tissues, e.g. in patients afflicted with Reye's syndrome.
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