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J. Biol. Chem., Vol. 280, Issue 26, 25178-25184, July 1, 2005

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Identification of the Major Urinary Metabolite of the Highly Reactive Cyclopentenone Isoprostane 15-A_2t-Isoprostane in Vivo^*

Ginger L. Milne, Ling Gao, Alessio Porta, Giuseppe Zanoni, Giovanni Vidari, and Jason D. Morrow¶

From the Departments of Medicine and Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 and the Department of Organic Chemistry, University of Pavia, Viale Taramelli, 10, 27100 Pavia, Italy

The cyclopentenone isoprostanes (A₂/J₂-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. A₂/J₂-IsoPs, similar to other electrophilic lipids such as 15-deoxy-^12,14-prostaglandin J₂ and 4-hydroxynonenal, contain a highly reactive ,-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 A₂-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 [³H₄]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.

Received for publication, March 16, 2005 , and in revised form, April 28, 2005.

^* This work is supported by National Institutes of Health Grants GM15431, CA77839, DK48831, HL07323, RR00095, and CA38079. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Recipient of a Burroughs Wellcome Fund Clinical Scientist Award in Translational Research. To whom correspondence should be addressed: Dept. of Medicine and Pharmacology, 526 RRB, 23rd and Pierce Ave., Vanderbilt University School of Medicine, Nashville, TN 37232-6602. Tel.: 615-343-1124: Fax: 615-322-3669; E-mail: jason.morrow{at}vanderbilt.edu.

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