DIHOMO-Y-LINOLENIC ACID METABOLITES LIPOXYGENASE PATHWAY

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Arachidonic acid is the major fatty acid precursor for the synthesis of biologically active eicosanoids. The critical role of the eicosanoids as regulators of physiologic and immune mechanisms is well documented and, therefore, control of their levels is of great interest in the treatment of prophylaxis of many diseases. One of the promising strategies in this context is the modulation of eicosanoid production by providing alternate substrates for oxidative metabolism through dietary manipulations. Recently attention has been focused on the metabolism of dihomo-gamma- linolenic acid (DHGLA) because it is thought to be the agent responsible for the apparent effectiveness of primrose oil in the treatment of a variety of diseases. These include inflammatory processes, skin diseases, immunological disorders, premenstrual pain, and malignancy. The overall objective of the proposed research is to understand the basic mechanisms underlying the beneficial nutritional effects of DHGLA. The research is based on the premise that substitution of membrane arachidonic acid by DHGLA results in an attenuation of arachidonic acid-derived eicosanoid levels. The overall hypothesis to be tested is that DHGLA acts as a competitive inhibitor of oxidative enzymes with respect to arachidonic acid substrate, that the oxidation products of DHGLA are biologically less potent than arachidonic acid metabolites, and that in some cases the former compounds act as antagonists to the cellular receptors of eicosanoids. The central goal of the proposed project will be to produce and characterize the metabolites derived from DHGLA via the 5-lipoxygenase pathway and to test the relative bioactivities of these molecules in assays of immune and physiologic functions which are known to be regulated by the lipoxygenase products of arachidonic acid. The hypothesis will be addressed by a number of in vitro experiments, the specific aims of which are as follows: 1) synthesis and characterization of primary and secondary oxygenation products of DHGLA catalyzed by electrophoretically pure 5-lipoxygenase, the main focus of which will be on stereochemical analysis of the metabolites produced as well as purification of quantities sufficient to test their biological activities; 2) systematic kinetic analysis of the competition between arachidonic acid (and its metabolites) and DHGLA (and its metabolites) as substrates for key enzymes of leukotriene synthesis, in this case 5-lipoxygenase, LTA hydrolase, and LTC synthase; 3) determination of the biological relevance of DHGLA-derived metabolites using a battery of physiological and immune assays which are known to be regulated by the lipoxygenase products of arachidonic acid, and 4) elucidation of the interrelationship between cytosine and eicosanoid-mediated immune mechanisms. Our long-range objective is to determine the in vivo relevance of the effects observed in the in vitro experimentation.