One of the major targets of oxidant injury is lipids which undergo peroxidation. During the previous funding period, we made the intriguing discovery that a novel series of bioactive prostaglandin F2-like compounds, now termed F2-isoprostanes, are produced in abundance in vivo in humans by a cyclooxygenase-independent mechanism involving free radical catalyzed peroxidation of arachidonic acid. Endogenous formation of isoprostanes was shown to increase profoundly in animal models of oxidant injury and in certain human diseases. Interestingly, we also demonstrated that isoprostanes are initially formed in situ on phospholipids and subsequently released in free form. Studies are now proposed to investigate the possibility that other lipid products of potential biological importance are produced by this pathway, specifically prostaglandin D(2)/E(2)-like compounds, thromboxane-like compounds, and levuglandin-like compounds. Isoprostanes will be chemically synthesized and their bioactivity determined. The primary source from which urinary F(2)-isoprostanes derive will be established. The biological properties of isoprostane phospholipids will be investigated regarding their effect on uptake of lipoproteins by macrophages, their effects on membrane fluidity, and the possibility that these unique phospholipids may interact with receptors of platelet activating factor. The mammalian phospholipase(s) that is responsible for hydrolyzing isoprostanes from phospholipids will be determined and the role of glutathione peroxidases in reducing isoprostane endoperoxides to F(2)-isoprostanes will be investigated. We have obtained provocative data suggesting that overproduction of isoprostanes and oxidant stress may play a fundamental role in the development of hepatorenal syndrome in humans. Thus, studies are planned to further investigate the role of isoprostanes in the pathogenesis of hepatorenal syndrome in an animal model of this disorder. A mass spectrometric method for the analysis of oxidized damage products of DNA will be established and the degree of oxidant damage to DNA and lipids correlated in settings of oxidant injury. Finally, the role of oxidant stress in the pathogenesis of high altitude pulmonary edema, acetaminophen poisoning, and halothane hepatotoxicity in humans will be investigated. Thus, a series of studies are proposed that should provide valuable new insights into the role of isoprostanes specifically and oxidant injury in general in the pathophysiology of human disease. The results of these investigations may provide the rationale for developing new strategies for therapeutic intervention in human disorders associated with oxidant stress.
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