There is little if any evidence to indicate that 22-carbon (n-3) and (n- 6) fatty acids are metabolized in vivo to bioactive fatty acids via cyclooxygenase and lipoxygenase pathways. The amounts of esterified 22- carbon acids and in particular 22:6 (n-3) have been correlated with central nervous System function and visual acuity. Patients with retinitis pigmentosa or Zellweger's disease contain low levels of esterified 22:6(n-3). We refitted the commonly accepted but untested hypothesis that 22:6(n-3) and 22:5(n-6) are made in the endoplasmic reticulum by an acyl-CoA-dependent 4-desaturase acting on 22:5(n-3) and 22:4(n-6). The latter two acids are precursors of 22:6(n-3) and 22:5(n- 6) but their synthesis requires intracellular communication between the endoplasmic reticulum and a site for 3-oxidation where the endoplasmic reticulum products, 24:5(n-6) and 24:6(n-3), are chain shortened to 22:5(n-6) and 22:6(n-3). The long-term objective of studies in this proposal is to determine what regulates intracellular communication as it relates to unsaturated fatty acid biosynthesis and membrane lipid biogenesis. Appropriately labeled unsaturated fatty acids will be incubated with rat liver peroxisomes to quantify their ability to totally 3-oxidize these compounds. The role played by acyl-CoA hydrolase, carnitine acyltransferase and the esterification of chain shortened metabolites into acceptors will be evaluated as to their role in terminating the 3-oxidation spiral. A major aim is to quantify what differences exist between the 3-oxidation and subsequent esterification of (n-6) versus (n-3) 20-and 22-carbon acids when they are generated from 24-carbon precursors and to determine whether the nutritional status of the animal affects these metabolic processes. The outer membrane of liver mitochondria will be used as an enzyme source to determine whether mitochondria can activate 24-carbon acids and convert acyl-CoAs to acylcarnitines. Intact mitochondria will be incubated with unsaturated acids to compare their rates of oxidation and determine whether chain shortened intermediates are esterified into mitochondria lipids or transported out of mitochondria as free acids or acylcamitines. Hepatocytes will be incubated with an inhibitor of carnitine palmitoyl-CoA transferase to determine how unsaturated acids distribute themselves between mitochondria and peroxisomes as they are 3-oxidized. An objective of the hepatocyte studies is to determine whether partial 3-oxidation products from mitochondria can be used for membrane lipid biosynthesis.
