Abstract
Cyclopentenone prostaglandins A2 and J2 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 D2/E2-isoprostanes undergo dehydration in vivo to form A2/J2-isoprostanes. Oxidation of arachidonic acid in vitro generated a series of compounds that were confirmed to be A2/J2-isoprostanes by mass spectrometric analyses. A2/J2-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 A2-isoprostane, 15-A2t-isoprostane, was obtained and found to readily undergo Michael addition with glutathione and to adduct covalently to protein. A2/J2-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 radiolabeled 15-A2t-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.
Highlights
Cyclopentenone (CP)1 prostaglandins (PG) of the A and J series have been shown to be produced in vitro by dehydration of the cyclopentane ring of PGE2 and PGD2, respectively
This is of particular interest since the same authors recently reported that high levels of PGD synthase are present in human male reproductive organs and that seminal plasma greatly facilitates dehydration of PGD2 [21]. They recently reported that the level of PGD synthase in male urine is approximately twice that found in female urine [22]. These findings suggest that at least some of the ⌬12PGJ2 detected in urine may have arisen from dehydration of PGD2 in the genitourinary tract and may explain the higher levels of ⌬12-PGJ2 in urine from males
The amount of A2/J2-IsoPs analyzed as this derivative was calculated to be 118 Ϯ 32 ng/mg of arachidonic acid, which is less than the amount formed when analyzed as an O-methyloxime, TMS ether derivative. This discrepancy can be explained by the fact that we have found that, whereas treatment of PGA2 with BSTFA/piperidine efficiently converts it to the piperidyl-enol-TMS ether derivative, only trivial amounts of this derivative are formed with PGJ2
Summary
Cyclopentenone (CP)1 prostaglandins (PG) of the A and J series have been shown to be produced in vitro by dehydration of the cyclopentane ring of PGE2 and PGD2, respectively. We reported the discovery of PG-like compounds, termed isoprostanes (IsoPs), that are produced in vivo nonenzymatically as products of free radical-induced peroxidation of arachidonoyl lipids [25, 26]. 15-A2t[3H]IsoP was added prior to HPLC purification, and compounds that coeluted with 15-A2t-[3H]IsoP were converted to a PFB ester, O-methyloxime, TMS ether derivative and analyzed by GC/electron ionization/MS.
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