An improved method for the measurement of urinary and plasma F2-isoprostanes using gas chromatography-mass spectrometry.
We have developed an improved method for the measurement of F2-isoprostanes using stable isotope dilution capillary gas chromatography/electron capture negative ionization mass spectrometry (GC-ECNI-MS). The F2-isoprostane family consists of a series of chemically stable prostaglandin F2 (PGF2)-like compounds generated during peroxidation of arachidonic acid in phospholipids. There is evidence that measurement of F2-isoprostanes represents a reliable and useful index of lipid peroxidation and oxidant stress in vivo. Furthermore, 8-epi-PGF2alpha, which is one of the more abundant F2-isoprostanes, is biologically active, being a potent mitogen and vasoconstrictor of rat and rabbit lung and kidney, as well as a partial agonist of platelet aggregation. Measurement of F2-isoprostanes in biological samples is complex and has involved methods which utilize multiple chromatographic steps, including separation by thin-layer chromatography, leading to poor sample recovery. We now present an improved method for the measurement of plasma and urinary F2-isoprostanes using a combination of silica and reverse-phase extraction cartridges, high-performance liquid chromatography (HPLC), and GC-ECNI-MS. Different approaches to the derivatization of the F2-isoprostanes prior to GC-ECNI-MS are also addressed. The overall recovery of F2-isoprostanes is improved (approx 70% for urine) and the within and between assay reproducibility is 6.7% (n = 23) and 3.7% (n = 3), respectively. The mean urinary excretion of F2-isoprostanes in eight healthy males was 365 +/- 5 pmol/mmol creatinine and in three smokers 981 +/- 138 pmol/mmol creatinine. The mean total (free + esterified) plasma F2-isoprostane concentration was 952 +/- 38 pmol/liter, with a within and between assay reproducibility of 8% (n = 13) and 5.6% (n = 3), respectively. This improved method for the measurement of F2-isoprostanes represents a significant advance in terms of the rapidity and yield in the purification of biological samples. The inclusion of HPLC separation enables improved analysis of F2-isoprostanes by GC-MS. This methodology will assist in defining the role of F2-isoprostanes as in vivo markers of oxidant stress in clinical and experimental settings.
Department of Medicine and the Western Australian Heart Research Institute, University of Western Australia, Perth, Perth, 6000, Western Australia.tmori@cyllene.uwa.edu.au
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Mori
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Croft
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Puddey
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Beilin
D000328:Adult; D015237:Dinoprost / Q000031:analogs & derivatives* / Q000097:blood / Q000302:isolation & purification / Q000652:urine; D028441:F2-Isoprostanes; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006801:Humans; D015227:Lipid Peroxidation; D008297:Male
D028441:F2-Isoprostanes; C075750:8-epi-prostaglandin F2alpha; D015237:Dinoprost