Abstract
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.
Highlights
F2-isoprostanes are prostaglandin-like compounds derived from nonenzymatic free radical-catalyzed peroxidation of arachidonic acid. 8-epi-Prostaglandin (PG) F2␣, a major component of the F2-isoprostane family, can be conveniently measured in urine to assess noninvasively lipid peroxidation in vivo
Search Strategy for -Oxidation Metabolites of Endogenous 8-epi-PGF2␣—Because polyclonal antibodies raised against prostanoids coupled to the carrier protein via the carboxyl group often cross-react with the corresponding -oxidation products [12, 13, 19], we tested three antibodies (A, B, and C) raised against 8-epi-PGF2␣ [16] for their ability to extract any of its possible ␣-chain-shortened (C18 and C16) metabolites
In the hepatocyte preparation incubated with PGF2␣, we confirmed the identity of 2,3dinor-5,6-dihydro-PGF2␣ and 2,3,4,5-tetranor-PGF2␣ but could not find 2,3-dinor-PGF2␣, either in solid phase extraction (SPE) or in IAC extracts examined by both electron impact (EI)- and negative ion chemical ionization (NICI)-MS, similar to what had been observed in rat urine
Summary
Materials—8-epi-PGF2␣ and 3,3Ј,4,4Ј-[2H4]-8-epi-PGF2␣ were purchased from Cayman Chemicals (Ann Arbor, MI). GC operating conditions were: NB-54 (5% phenyl, 95% dimethylpolysiloxane) or CP-Sil 19 CB (14% cyanopropylphenyl, 86% dimethylpolysiloxane) fused silica capillary columns (length, 25 m; inner diameter, 0.32 mm; film thickness, 0.12 m); solvent-split injection (50 to 300 °C); oven temperature, isothermal at 120 °C for 1 min, and programmed to 300 °C at 25°/min; helium was used as a carrier gas. The cell-free incubation medium was collected and stored at Ϫ20 °C until analyzed Metabolites were extracted both by IAC and by C18 solid phase extraction (SPE). The latter was performed by loading the sample at pH 3.5, washing with water and petroleum ether, and eluting with methyl formate. Aliquots of IAC and C18 SPE extracts were dried and derivatized to ME-TMS or PFB-TMS and analyzed by GC-EI-MS or GC-NICI-MS, respectively
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
