The overall objective of the project is to provide a fundamental mechanistic basis for the rational design of therapeutic measures to prevent or ameliorate the pathological consequences of lipid oxidation, for example, by blocking the formation of certain toxic products that damage the retina resulting in age-related macular degeneration. The immediate goal is to understand oxidative fragmentation reactions of polyunsaturated fatty acyl derivatives that generate a complex mixture of oxidatively truncated phospholipids and aliphatic lipid fragments. These fragmentation products exhibit diverse and often pathological biological activities. Of particular interest is the production of mutagenic epoxyalkenals and cytotoxic hydroxyalkenals. Knowledge of the chemistry of the reactive intermediates involved in their generation is important for understanding how environmental or genetic factors can promote their formation. For example, all of the intermediates that we have identified require catalytically active metal ions to undergo fragmentation under biological conditions. One type of intermediate that we have yet to study is believed to spontaneously fragment, without the need for catalysis. However, such intermediates have not been thoroughly characterized. We will prepare pure well characterized samples by chemical synthesis. If we confirm our hypothesis that metal ion catalysis is also required for such intermediates, then one therapeutic strategy would be to remove or detoxify such metal ions. Knowledge of the further transformations of the primary fragmentation products that occur under biological conditions, including oxidation, deacylation, and protein adduction is important because these processes produce biologically active secondary products. For example, we have shown that the protein adducts of hydroxyalkenal phospholipids derived from docosahexaenoic acid initiate macular degeneration and also promote the pathological sprouting of capillaries into the retina. The project focuses on studying key intermediates that are not stable under the oxidative fragmentation reaction conditions. Three basic questions are being addressed: (1) are the putative intermediates actually involved, and if so (2) what products are generated by their decomposition and (3) by what mechanism(s) do they fragment? In some cases, they will be trapped as stable derivatives to confirm their involvement. To provide ample quantities of some reactive intermediates, unambiguous total syntheses are designed and executed. The authentic samples are being used as standards for establishing methods for detecting and quantifying levels of the intermediates in oxidation reaction product mixtures. Their generation and conversion into toxic or innocuous end products is being investigated. We are determining the influences of (1) environments such as those found in different organelles or associated with pathological conditions, (2) levels of cofactors, or (3) oxidation initiating systems and inhibitors, on the production of the reactive intermediates and on the relative importance of various pathways for their subsequent transformations.
Phospholipids, major building blocks human tissue, are attacked and broken apart by oxygen producing fragments that can be toxic. For example, some of these fragments modify proteins in the retina causing the immune system to attack and destroy the retina. To provide a rational basis for the design of therapeutic strategies, this project will determine exactly how such oxidative fragmentation of phospholipids occurs, what the pieces are, and what are some of the chemical reactions involved in their toxicity.
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