Identification and Measurement of Endogenous β-Oxidation Metabolites of 8-epi-Prostaglandin F2α

F2-isoprostanes are prostaglandin-like compounds derived from nonenzymatic free radical-catalyzed peroxidation of arachidonic acid. 8-epi-Prostaglandin (PG) F2alpha, a major component of the F2-isoprostane family, can be conveniently measured in urine to assess noninvasively lipid peroxidation in vivo. Measurement of major metabolites of endogenous 8-epi-PGF2alpha, in addition to the parent compound, may be useful to better define its formation in vivo. 2,3-Dinor-5,6-dihydro-8-epi-PGF2alpha is the only identified metabolite of 8-epi-PGF2alpha in man, but its endogenous levels are unknown. In addition to this metabolite, we have identified another major endogenous metabolite, 2,3-dinor-8-epi-PGF2alpha, in human and rat urine. The identity of these compounds, present at the pg/ml level in urine, was proven by a number of complementary approaches, based on: (a) immunoaffinity chromatography for selective extraction; (b) gas chromatography-mass spectrometry for structural analysis; (c) in vitro metabolism in isolated rat hepatocytes; and (d) chemical synthesis of the enantiomer of 2,3-dinor-5, 6-dihydro-8-epi-PGF2alpha as a reference standard. In humans, the urinary excretion rate of both dinor metabolites is comparable with that of 8-epi-PGF2alpha. Both metabolites increase in parallel with the parent compound in cigarette smokers, and they are not reduced during cyclooxygenase inhibition. Another beta-oxidation product, 2, 3,4,5-tetranor-8-epi-PGF2alpha, was identified as a major product of rat hepatocyte metabolism. In conclusion, at least two major beta-oxidation products of 8-epi-PGF2alpha are present in urine, which may be considered as additional analytical targets to evaluate 8-epi-PGF2alpha formation and degradation in vivo.