We previously discovered that a series of novel bioactive prostaglandin (PG)-like compounds, termed isoprostanes (IsoPs), is produced in vivo. Quantification of IsoPs has greatly expanded our knowledge of the role of oxidant stress in human diseases. The first compounds characterized were the F2-IsoPs which possess a structure isomeric to PGF/2alpha. Subsequently, we found that bioactive IsoPs containing D-type and E-type prostane rings, analogous to PGD2 and PGE2, are formed in large amounts in vivo. These compounds are termed D2/E2-IsoPs. Our hypothesis is that IsoPs contribute importantly to the pathophysiological sequelae of oxidant stress. Studies are thus proposed to explore the biochemistry and pharmacology of D2/E2-IsoPs and related compounds. We will test the hypothesis that D-ring IsoPs are formed in vivo. We have found that abundant quantities of racemic PGE2 are formed in vivo from isomerization of the E-ring IsoP, racemic 15-E/2t-IsoP (8-iso- PGE2), suggesting that a signification portion of PG production occurs via this mechanism. We will definitely determine that D-ring IsoPs are formed by providing evidence that racemic PGD2 is generated from IsoP endoperoxides; the extent to which this pathway contributes to the formation of PGD2 in animals and humans will be studied. We will also explore whether the enantiomer of PGD2 is metabolized in a manner similar to PGD2. The biological activities of PGD2 and its enantiomer will be compared. Studies will be undertaken to identify novel D/3-IsoP-like compounds, D4/E4-neuroprostanes, derived from docosahexaenoic acid, a major fatty acid in the nervous system. We will examine their formation and metabolism in animal models of oxidant stress and in human neurodegenerative diseases associated with oxidative injury. D2/32-Isops dehydrate in vivo to cyclopentenone IsoPs, termed A2/J2- IsoPs. These compounds exert potent bioactivity and, unlike other IsoPs, are unique in that they readily adduct various biomolecules including glutathione (GSH). We have found that cyclopentenone IsoPs undergo GSH- transferase catalyzed conjugation with GSH. Studies are proposed to explore the relationship between conjugation of these compounds with GSH and their biological actions. Further, following infusion into a human of a rabiolabeled A2-IsoP, 15-A/2t-IsoP, virtually all excreted radioactivity in the urine is in the form of a polar conjugate. Thus, as a basis for the development of a means to assess the formation of A2/J2- IsoPs in vivo, the nature of this conjugate will be structurally elucidated.
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