Identification of the major urinary metabolite of the highly reactive cyclopentenone isoprostane 15-A(2t)-isoprostane in vivo.

Milne GL, Gao L, Porta A, Zanoni G, Vidari G, Morrow JD
J Biol Chem. 2005 280 (26): 25178-84

PMID: 15878849 · DOI:10.1074/jbc.M502891200

The cyclopentenone isoprostanes (A(2)/J(2)-IsoPs) are formed in significant amounts in humans and rodents esterified in tissue phospholipids. Nonetheless, they have not been detected unesterified in the free form, presumably because of their marked reactivity. A(2)/J(2)-IsoPs, similar to other electrophilic lipids such as 15-deoxy-Delta(12,14)-prostaglandin J(2) and 4-hydroxynonenal, contain a highly reactive alpha,beta-unsaturated carbonyl, which allows these compounds to react with thiol-containing biomolecules to produce a range of biological effects. We sought to identify and characterize in rats the major urinary metabolite of 15-A(2t)-IsoP, one of the most abundant A(2)-IsoPs produced in vivo, in order to develop a specific biomarker that can be used to quantify the in vivo production of these molecules. Following intravenous administration of 15-A(2t)-IsoP containing small amounts of [(3)H(4)]15-A(2t)-IsoP, 80% of the radioactivity excreted in the urine remained in aqueous solution after extraction with organic solvents, indicating the formation of a polar conjugate(s). Using high pressure liquid chromatography/mass spectrometry, the major urinary metabolite of 15-A(2t)-IsoP was determined to be the mercapturic acid sulfoxide conjugate in which the carbonyl at C9 was reduced to an alcohol. The structure was confirmed by direct comparison to a synthesized standard and via various chemical derivatizations. In addition, this metabolite was found to be formed in significant quantities in urine from rats exposed to an oxidant stress. The identification of this metabolite combined with the finding that these metabolites are produced in in vivo settings of oxidant stress makes it possible to use this method to quantify, for the first time, the in vivo production of cyclopentenone prostanoids.

MeSH Terms (18)

Acetylcysteine Animals Biomarkers Carbon Chromatography, High Pressure Liquid Cyclopentanes Dinoprost Isoprostanes Ligands Male Mass Spectrometry Models, Chemical Prostaglandins Rats Rats, Sprague-Dawley Scintillation Counting Sulfoxides Time Factors

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