Identification and measurement of endogenous β-oxidation metabolites of 8-epi-prostaglandin F2α

F-2-isoprostanes are prostaglandin-like compounds derived from nonenzymatic free radical-catalyzed peroxidation of arachidonic acid. 8-epi-Prostaglandin (PG) F-2 alpha, a major component of the F-2-isoprostane family, can be conveniently measured in urine to assess noninvasively lipid peroxidation in vivo. Measurement of major metabolites of endogenous 8-epi PGF(2 alpha), in addition to the parent compound, may be useful to better define its formation in vivo. 2,3-dinor-5,6-dihydro-8-epi-PGF(2 alpha) is the only identified metabolite of 8-epi-PGF(2 alpha) 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 PGF(2 alpha), 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-PGF(2 alpha) as reference standard. In humans, the urinary excretion rate of both diner metabolites is comparable with that of 8-epi-PGF(2 alpha). 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-PGF(2 alpha), was identified as a major product of rat hepatocyte metabolism. In conclusion, at least two major beta-oxidation products of 8-epi-PGF(2 alpha) are present in urine, which may be considered as additional analytical targets to evaluate 8-epi-PGF(2 alpha) formation and degradation in vivo.