Mori T A, Croft K D, Puddey I B, Beilin L J
Department of Medicine and the Western Australian Heart Research Institute, University of Western Australia, Perth, Perth, 6000, Western
Anal Biochem. 1999 Mar 1;268(1):117-25. doi: 10.1006/abio.1998.3037.
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.
我们开发了一种改进的方法，用于使用稳定同位素稀释毛细管气相色谱/电子捕获负离子化质谱法（GC-ECNI-MS）测量F2-异前列腺素。F2-异前列腺素家族由一系列在磷脂中花生四烯酸过氧化过程中产生的化学稳定的前列腺素F2（PGF2）样化合物组成。有证据表明，F2-异前列腺素的测量是体内脂质过氧化和氧化应激的可靠且有用的指标。此外，8-表-PGF2α是含量较为丰富的F2-异前列腺素之一，具有生物活性，是大鼠和兔肺及肾脏的强效促有丝分裂剂和血管收缩剂，也是血小板聚集的部分激动剂。生物样品中F2-异前列腺素的测量很复杂，所涉及的方法需要多个色谱步骤，包括通过薄层色谱进行分离，导致样品回收率较低。我们现在提出一种改进的方法，使用硅胶和反相萃取柱、高效液相色谱（HPLC）和GC-ECNI-MS相结合来测量血浆和尿液中的F2-异前列腺素。还讨论了在GC-ECNI-MS之前对F2-异前列腺素进行衍生化的不同方法。F2-异前列腺素的总体回收率得到了提高（尿液约为70%），测定内和测定间的重现性分别为6.7%（n = 23）和3.7%（n = 3）。8名健康男性尿液中F2-异前列腺素的平均排泄量为365±5 pmol/mmol肌酐，3名吸烟者为981±138 pmol/mmol肌酐。血浆中F2-异前列腺素的平均总（游离+酯化）浓度为952±38 pmol/升，测定内和测定间的重现性分别为8%（n = 13）和5.6%（n = 3）。这种改进的F2-异前列腺素测量方法在生物样品纯化的速度和产率方面取得了显著进展。加入HPLC分离能够通过GC-MS对F2-异前列腺素进行更好的分析。这种方法将有助于确定F2-异前列腺素作为临床和实验环境中氧化应激体内标志物的作用。
