Quantification of the major urinary metabolite of 15-F2t-isoprostane (8-iso-PGF2alpha) by a stable isotope dilution mass spectrometric assay.
The isoprostanes (IsoPs) are a series of novel prostaglandin (PG)-like compounds generated from the free radical-catalyzed peroxidation of arachidonic acid. The first series of IsoPs characterized contained F-type prostane rings analogous to PGF2alpha. One F-ring IsoP, 15-F2t-IsoP (8-iso-PGF2alpha) 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 this compound, we developed a method to quantify the major urinary metabolite of 15-F2t-IsoP, 2,3-dinor-5,6-dihydro-15-F2t-IsoP (2,3-dinor-5, 6-dihydro-8-iso-PGF2alpha), by gas chromotography/negative ion chemical ionization mass spectrometry. This metabolite was chemically synthesized and converted to an 18O2-labeled derivative for use as an internal standard. After purification, the compound was analyzed as a pentafluorobenzyl ester trimethylsilyl ether. Precision of the assay is +/-4% and accuracy is 97%. The lower limit of sensitivity is approximately 20 pg. Levels of the urinary excretion 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 excretion 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-F2t-IsoP were not affected by cyclooxygenase inhibitors. Thus, this assay provides a sensitive and accurate method to assess endogenous production of 15-F2t-IsoP as a means to explore the pathophysiological role of this compound in human disease.
Morrow
J D
JD
Departments of Medicine and Pharmacology, Vanderbilt University, Nashville, Tennessee 37232, USA.
Zackert
W E
WE
Yang
J P
JP
Kurhts
E H
EH
Callewaert
D
D
Dworski
R
R
Kanai
K
K
Taber
D
D
Moore
K
K
Oates
J A
JA
Roberts
L J
LJ
D000328:Adult; D000818:Animals; D015237:Dinoprost / Q000031:analogs & derivatives* / Q000737:chemistry / Q000652:urine; D028441:F2-Isoprostanes; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006801:Humans; D018384:Oxidative Stress; D010103:Oxygen Isotopes; D051381:Rats; D012016:Reference Values; D015203:Reproducibility of Results
D028441:F2-Isoprostanes; D010103:Oxygen Isotopes; C075750:8-epi-prostaglandin F2alpha; D015237:Dinoprost