Project 2: Biochemical Pharmacology of Eicosapentaenoic Acid Oxidation

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The ?-3 polyunsaturated fatty acids (PUFAs) present in fish oil, particularly eicosapentaenoic acid (C20:5, ?-3, EPA), are used in the prevention and treatment of many diseases. The mechanisms by which these PUFAs are protective are not understood although they possess anti-inflammatory properties. Our hypothesis is that the anti-inflammatory effects of ?-3 fatty acids are due, in part, to the generation of bioactive oxidation products and data we have generated in the past two years support this hypothesis. Previously, we defined the free radical-initiated peroxidation of arachidonic acid and have proposed a unified mechanism for its oxidation. The major complex peroxidation products consist of novel compounds containing mono- and serial cyclic peroxide and endoperoxide (isoprostane, IsoP) moieties. These compounds possess potent pro-inflammatory bioactivity and likely mediate various pathophysiological processes. Unlike arachidonate, the oxidation of ?-3 PUFAs, and in particular EPA, is predicted to be significantly more complex since these compounds contain additional unsaturated carbon-carbon bonds. Work carried out in this grant over the past two years has, indeed, shown this to be the case. Studies proposed in this renewal application will extend our knowledge regarding the biochemistry and pharmacology of EPA peroxidation. We hypothesize the oxidation of EPA can be defined and results in the formation of compounds that contribute to the anti-inflammatory properties of the PUFA. The structural and mechanistic work proposed will use eicosapentaenoyl-glycerophosphatidylcholine (EPA-PC), because it is a major biologically relevant form of this PUFA in vivo and offers a unique opportunity to define the peroxidation of EPA in a physiologically relevant form. In addition, this will allow for the development of approaches to accurately characterize and quantify complex phosphatidylcholine oxidation products. A detailed study of the peroxidation of EPA esterified in phosphatidylcholine has not been undertaken. , In Specific Aims 1 and 2, we will define mechanistically the free radical-initiated peroxidation of EPA-PC and characterize novel oxidation products using mass spectrometric approaches that we will develop. In Specific Aim 3, we will determine the effect of various pro- and antioxidants on the formation of EPA-PC peroxidation products. In Specific Aim 4, we will determine mechanisms that account for the anti-inflammatory properties of EPA and its peroxidation products. We will study the extent to which EPA decreases the formation of proinflammatory arachidonate-derived IsoPs, prostaglandins and leukotrienes in humans and animals. We will also examine the anti-inflammatory and vasoactive properties of EPA-derived IsoPs that are formed in vivo. We believe that identifying novel oxidation products of EPA and examining mechanisms by which these compounds are formed will yield insights into the role of EPA in human biology.