Free radicals are postulated to play an important role in human diseases as diverse as atherosclerosis and cancer. We previously reported that a series of novel bioactive prostaglandin-like compounds, termed isoprostanes (IsoPs), are produced in vivo by non-enzymatic free radical catalyzed peroxidation of arachidonic acid. Measurement of these compounds represents a major advance in our ability to assess of oxidative stress status in vivo. A series of studies are now proposed to further explore the biochemistry and toxicology of compounds formed as products of the IsoP pathway. We will structurally characterize compounds which preliminary data suggests are prostacylin-like molecules formed by rearrangement of IsoP endoperoxide intermediates. The ability of PGF-synthase and liver aldehyde reductase to reduce IsoP endoperoxides to F-ring IsoP's will also be examined. IsoP are initially formed in situ on phospholipids and released preformed. We will characterize the ability of two phospholipase to hydrolyze IsoP's from phospholipids. In addition, we will determine whether IsoP-phospholipids exert bioactivity. Preliminary evidence suggests that significant quantities of IsoPs exist as glucuronide conjugates. We will undertake studies to structurally characterize these conjugates and explore their biological activity. We have also established that IsoP endoperoxides ring cleavage in vitro to form keto-aldehyde compounds termed isolevuglandins (IsoLGs), which readily form adducts with proteins and DNA. Mass spectrometric methods will be developed for the detection of IsoLG-protein adducts in vivo. We will also examine the biological toxicity of IsoLGs. Methodology will also be developed utilizing immunohistochemistry and confocal microscopy to explore the subcellular sites of formation of IsoP's and IsoLG protein adducts. The role of free radicals and IsoP's in mediating the renal dysfunction in an animal model of the hepatorenal syndrome will also be explored. Radioligand binding studies will be undertaken to determine whether a novel 'IsoP receptor' is present in vascular smooth muscle. Studies will also be undertaken to structurally characterize novel IsoP-like compounds derived from docosahexenoic acid, a major fatty acid in neurons, and examine the utility of quantification of these compounds to assess oxidant stress in the brain of animal models and neurologic diseases in humans. The synthesis of new F-ring IsoP's will be also carried out and their biological activities explored.
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