Increased dietary intake of the ro-3 polyunsaturated fatty acids (PUFAs) in fish oil,eicosapentaenoic acid (EPA; C20:5, w-3) and docosahexaenoic acid (DHA; C22:6, co-3), reduces the incidence of diseases in which environmental and lifestyle factors play a role including atherosclerosis and neurodegeneration, among others. The mechanisms by which these PUFAs are protective have not been elucidated but they possess anti- inflammatory properties. It has been proposed that these anti-inflammatory effects are due,in part, to the generation of bioactive oxidation products although the identity of these compounds isunknown. We previously defined the free radical-initiated peroxidation of the co-6 PUFA arachidonic acid (C20:4,co- 6) in vitro and in vivo and have proposed a unified mechanism for its oxidation. Various biologically active products are generated, including endoperoxide-derived isoprostanes (IsoPs), that are pro-inflammatory and likely mediate sequelae of oxidant stress. The peroxidation of EPA and DHA has never been systematically studied but is predicted to be significantly more complex than arachidonic acid owing to the additional unsaturated carbon- carbon bond(s) in these PUFAs. The goal of proposed studies is to examine the biochemistry and pharmacology of EPA oxidation. Our hypothesis is that the peroxidation of EPA can be rationally defined and leads to the formation of compounds that contribute to its biological properties. In Specific Aims 1 and 2, we will define mechanistically the peroxidation of EPA in vitro and in vivo and structurally characterize the products predicted to form including monocyclic and serial cyclic peroxides, endoperoxides (IsoPs), and unique dioxolane-endoperoxides utilizing novel mass spectrometric approaches. To further support our proposed mechanism of EPA oxidation, we will characterize the major EPA-derived IsoP regioisomers generated. These latter studies will provide a rationale to study their biology. In Specific Aim 3, we will determine the effects of factors including molecular oxygen, oc-tocopherol, novel antioxidants synthesized in Project 1, and enzymatic oxidative pathways on the peroxidation of EPA. In Specific Aim 4, we will determine the inflammatory mediating properties of EPA and EPA oxidation products. We will study the extent to which to which fish oil and EPA decrease the formation ofpro- inflammatory F2-IsoPs in animals and humans. In addition, we will determine the extent to which EPA decreases atherosclerosis and IsoP formationin mouse models of the disease. Finally, we will define the biological activity of one EPA-derived IsoP formed in vivo, IS-Fat-IsoP.