Formation of reactive cyclopentenone compounds in vivo as products of the isoprostane pathway

Cyclopentenone prostaglandins A(2) and J(2) are reactive compounds that possess unique biological activities. However, the extent to which they are formed in vivo remains unclear. In this study, we explored whether D-2/E-2-isoprostanes undergo dehydration in vivo to form A(2)/J(2)-isoprostanes. Oxidation of arachidonic acid in vitro generated a series of compounds that were confirmed to be A(2)/J(2)-isoprostanes by mass spectrometric analyses. A(2)/J(2)-isoprostanes were detected in vivo esterified to lipids in livers from normal rats at a level of 5.1 +/- 2.3 ng/g, and levels increased dramatically by a mean of 24-fold following administration of CCl4. An A(2)-isoprostane, 15-A(2t)-isoprostane, was obtained and found to readily undergo Michael addition with glutathione and to adduct covalently to protein. A(2)/J(2)-isoprostanes could not be detected in the circulation, even following CCl4 administration, which we hypothesized might be explained by rapid formation of adducts. This was supported by finding that essentially all the radioactivity excreted into the urine following infusion of radiolabaled 15-A(2t)-isoprostane into a human volunteer was in the form of a polar conjugate(s). These data identify a new class of reactive compounds that are produced in vivo as products of the isoprostane pathway that can exert biological effects relevant to the pathobiology of oxidant injury.