Thrombosis, inflammation, lipoprotein biosynthesis and central nervous system function are in part regulated by the composition of polyunsaturated fatty acids (PUFA) in membrane lipids. The long-term objective of this project is to synthesize appropriate radioactive and stable isotopes for use in determining what integrated factors regulate PUFA and phospholipid biosynthesis. [1-14C]Labeled acyl-CoAs will be used to determine whether liver microsomes contain separate condensing enzymes for chain elongating 18- versus 20-carbon (n-3) and (n-6) PUFA. Deuterated 2-trans- and beta-hydroxy acids will be used to define the steriochemistry of the dehydrase reaction and to establish whether microsomes contain more than one fatty acyl-CoA dehydrase and 2-trans-enoyl-CoA reductase. The metabolism of these deuterated intermediates will be studied in isolated hepatocytes to determine whether products subsequent to the rate limiting condensation step are in part catabolized thus regulating overall chain elongation. The diabetic rat will be used as a model to determine whether the altered membrane lipid fatty acid composition is due to depressed 6-desaturase activity or enhanced release of endogenous linoleate thus leading to pool dilution of exogenous [1-14C]18:2(n-6)-CoA. This animal model will also be used to further determine how many acyl-CoA condensing enzymes are present in microsomes. The composition of the acyl-CoA pool in livers of rats fed corn oil or a corn oil-fish diet will be compared to determine whether altered membrane lipid PUFA composition is primarily regulated by acyl-CoA levels or the conversion of 18:2(n-6) to 20:4(n-6). A major aim is to define what incubation conditions must be used to assay the 4-desaturase and whether the true substrate is an acyl-CoA or an esterified acid. Parallel studies with isolated peroxisomes will determine what regulates the conversion of 22:4(n-6), 22:5(n-3) and 22:6(n-3) to 20-carbon acids which in turn may be chain elongated and subsequently desaturated at position-4. Related experiments will determine whether brain microsomes contain 4-desaturase activity and whether these preparations have a unique ability to chain elongate very long chain PUFA, thus explaining why their membrane lipids accumulate 24-36 carbon PUFA. Rat liver microsomes and cultured hepatocytes will be used to evaluate the hypothesis that esterified fatty acids are the principal substrates for fatty acyl-CoA desaturation and chain elongation.
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