Identification of the Major Urinary Metabolite of the Highly Reactive Cyclopentenone Isoprostane 15-A2t-Isoprostane in Vivo

Abstract

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-⌬ 12,14 -prostaglandin J 2 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 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.

Involvement of eicosanoids and surfactant protein D in extrinsic allergic alveolitis

“…The isoprostane pathway also contributes to the PGD 2 overproduction in acute EAA [2], although the current approach was unable to ascertain its relative contribution to PGD 2 production. It has recently been discovered that isoprostanes containing D-or E-type prostane rings are excreted into the urine as conjugates with N-acetyl cysteine sulfoxide, suggesting that these metabolites may be used as biomarkers to estimate whole-body production of D-or E-type isoprostanes [23]. Future experiments using this methodology will hopefully provide even more answers.…”

Recent advances in the biochemistry and clinical relevance of the isoprostane pathway

“…These cyclopentenone IsoPs are rapidly metabolized in vivo by GSH transferase enzymes to water-soluble modified GSH conjugates (25)(26)(27). The major urinary cyclopentenone IsoP metabolite in rats, a 15-A 2t -IsoP mercapturic acid sulfoxide conjugate, was recently identified in our laboratory (28). This metabolite can be readily quantified in urine by LC/MS/MS and was found to accumulate significantly over 24 h following induction of lipid peroxidation with CCl 4 , providing evidence that A 2 /J 2 -IsoPs are formed, released from membrane phospholipids, and metabolized in vivo (28).…”

Human Biochemistry of the Isoprostane Pathway

“…A 2 /J 2 -IsoPs are highly reactive electrophiles that readily form Michael adducts with cellular thiols, including those found on cysteine residues in proteins and GSH (35). These cyclopentenone IsoPs are rapidly metabolized in vivo by GSH transferase enzymes to water-soluble modified GSH conjugates (36). A major urinary cyclopentenone IsoP metabolite in rats, a 15-A 2t -IsoP mercapturic acid sulfoxide conjugate, was recently identified.…”