Prostaglandin D2 (PGD2) is an unstable molecule that can undergo dehydration to form PGJ derivatives, delta 12-PGJ2 and 15-deoxy-delta12,14-PGJ2. There has been considerable interest in these PGJ2 compounds owing to the fact that they have been shown to modulate tumor cell proliferation, inhibit Nf kappaB activation, and exert anti-inflammatory activity in vitro. More recently there has been an upsurge in the interest in these compounds because they have been shown capable of activating the nuclear receptor, PPAR gamma. However, there is has been no convincing evidence to support the notion that these cyclopentenone derivatives of PGD2 are actually formed in relevant quantities in vivo. However, we recently convincingly demonstrated the formation of PGA2/J2-1ike compounds esterified in tissue phospholipids in rats as products of the isoprostane pathway in vivo but it remains to be established whether PGD2 produced by the cyclooxygenase also undergoes dehydration in vivo. We had previously demonstrated that alpha l2-PGJ2 rapidly conjugates to glutathione and cysteine in vitro. We also demonstrated that alpha 12-PGJ2 undergoes extensive conjugation with glutathione in CHO cells after which the C-11 carbonyl is reduced to an alcohol and further metabolized to cysteinylglycinyl and cysteinyl derivatives. Moreover, following intravenous administration of radiolabelled Alpha 12-PGJ2 to rats we found that all of the radioactivity excreted in to the bile and urine was in the form of a polar conjugate(s). This suggests that the detection of the formation of PGJ2 derivatives in vivo can only be accomplished by analysis of their conjugates. Therefore, we propose to determine the metabolic disposition of Alpha 12-PGJ2 and 15-deoxy-Alpha 12,14-PGJ2 in a non-human primate and develop mass spectrometric assays for the major species excreted into the urine. We will then assess whether these metabolites can be detected in normal human urine and whether levels increase in patients with systemic mastocytosis which is associated with a marked overproduction of PGD2 and in normal volunteers following administration of niacin, which induces a release of prodigious quantities of PGD2. We will also determine the relative contribution of the cyclooxygenase and isoprostane pathways to the levels of these metabolites in human urine by treatment of subjects with inhibitors of the cyclooxygenase enzyme.
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