Peroxidation of polyunsaturated fatty acids (PUFAs) and their biologically relevant phospholipid esters is acomplex reaction giving scores of possible products from a single molecular species. This process is ahallmark of diverse environmental chemical exposures, drug toxicities and oxidative stresses. In addition tothe many peroxide products that form from polyunsaturated lipids, a set of reactive electrophiles is alsogenerated. These electrophilic residuals of lipid peroxidation modify nucleic acids and proteins and in thisway, the consequence of lipid oxidative degradation is distributed to other important biomolecules. Thisproposal outlines experiments that probe the chemical mechanisms of lipid peroxidation, provides aframework for understanding the oxidation of highly unsaturated lipids present in fish oils, describes novelnew affinity-tags useful in the isolation of lipid electrophile-protein adducts and examines new phenolicantioxidants more potent than vitamin E. Highly unsaturated co-3 PUFAs are better reducing agents thanmore saturated co-6 lipids and we will look for consequences of this difference by analyzing peroxidationbiomarkers formed in model membrane oxidations and in tissues and fluids of stressed animals on fish oildiets. Our affinity-tag lipids are analogs of natural lipids having a terminal alkyne substituted at the 00position (co-yne) of fatty acid chains. This terminal alkyne undergoes 'click' cycloaddition with biotinsubstitutedazides, permitting 'pull-down' of any proteins covalently attached to lipid-derived electrophilesbearing an (co-yne). The proposed research is based on the hypothesis that the chemical mechanisms oflipid peroxidation and the formation of electrophilic byproducts that are a hallmark of this process can berationally defined. The affinity tags when coupled to powerful HPLC/MS/MS proteomics methods permit thestructural identification of individual lipid-protein adducts even though such species are only a small part of avery complex mixture. Profiling of human THP-1 cells exposed to an oxidative stress will include studies inwhich affinity tag (co-yne) lipids are incorporated into the cells, permitting isolation of lipid-protein adducts.The electrophiles identified from phospholipids will form the basis of a screening program in collaborationwith Projects 3 and 4 of the Program Project. New powerful pyridinol antioxidants will be studied in 'proof ofconcept' in vivo rodent experiments.
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