Quantification of the major urinary metabolite of 15-F-2t-isoprostane (8-iso-PGF(2 alpha)) by a stable isotope dilution mass spectrometric assay

The isoprostanes (IsoPs) are a series of novel prostaglandin (PG)-like comp ounds generated from the free radical-catalyzed peroxidation of arachidonic acid. The first series of IsoPs characterized contained F-type prostane ri ngs analogous to PGF(2 alpha). One F-ring IsoP, 15-F-2t-IsoP (8-iso-PGF(2 a lpha)) has been shown to be formed in abundance in vivo and to exert potent biological activity. As a means to assess the endogenous production of thi s compound, we developed a method to quantify the major urinary metabolite of 15-F-2t-IsoP, 2,3-dinor-5,6-dihydro-15-F-2t-IsoP (2,3-dinor-5,6-dihydro- 8-iso-PGF(2 alpha)), by gas chromotography/negative ion chemical ionization mass spectrometry. This metabolite was chemically synthesized and converte d to an O-18(2)-labeled derivative for use as an internal standard. After p urification, the compound was analyzed as a pentafluorobenzyl ester trimeth ylsilyl ether. Precision of the assay is +/-4% and accuracy is 97%, The low er limit of sensitivity is approximately 20 pg, Levels of the urinary excre tion of this metabolite in 10 normal adults were found to be 0.39 +/- 0.18 ng/mg creatinine (mean +/- 2 SD). Substantial elevations in the urinary exc retion of the metabolite were found in situations in which IsoP generation is increased and antioxidants effectively suppressed metabolite excretion. Levels of 2,3-dinor-5,6-dihydro-15-F-2t-IsoP were not affected by cyclooxyg enase inhibitors. Thus, this assay provides a sensitive and accurate method to assess endogenous production of 15-F-2t-IsoP as a means to explore the pathophysiological role of this compound in human disease. (C) 1999 Academi c Press.