Elsevier

Analytical Biochemistry

Volume 268, Issue 1, 1 March 1999, Pages 117-125
Analytical Biochemistry

Regular Article
An Improved Method for the Measurement of Urinary and Plasma F2-Isoprostanes Using Gas Chromatography–Mass Spectrometry

https://doi.org/10.1006/abio.1998.3037Get rights and content

Abstract

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 stressin vivo.Furthermore, 8-epi-PGF2α, 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 asin vivomarkers of oxidant stress in clinical and experimental settings.

References (42)

  • B. Halliwell et al.

    Methods Enzymol.

    (1990)
  • J.D. Morrow et al.

    Anal. Biochem.

    (1990)
  • J.D. Morrow et al.

    Biochem. Pharmacol.

    (1996)
  • J.D. Morrow et al.

    Biochim. Biophys. Acta.

    (1994)
  • K. Hanasaki et al.

    Biochem. Pharmacol.

    (1990)
  • N.K. Gopaul et al.

    FEBS Lett.

    (1995)
  • D. Pratico et al.

    J. Biol. Chem.

    (1996)
  • J. Nourooz-Zadeh et al.

    Biochem. Biophys. Res. Commun.

    (1997)
  • D. Pratico et al.

    J. Biol. Chem.

    (1995)
  • S. Basu

    Prostaglandin Leuk. Essential Fatty Acids

    (1998)
  • A. Bachi et al.

    Free Radical Biol. Med.

    (1996)
  • R.J. Waugh et al.

    J. Am. Soc. Mass Spectrom.

    (1996)
  • R.J. Waugh et al.

    Free. Radical Biol. Med.

    (1997)
  • B. Halliwell

    Free Radical Res.

    (1996)
  • B. Halliwell et al.

    J. Lab. Clin. Med.

    (1992)
  • J.D. Morrow et al.

    Proc. Natl. Acad. Sci. USA

    (1990)
  • C. Patrono et al.

    Arterioscler. Thromb. Vasc. Biol.

    (1997)
  • K. Takabashi et al.

    J. Clin. Invest.

    (1992)
  • M. Banerjee et al.

    Am. J. Physiol.

    (1992)
  • K.H. Kang et al.

    J. Appl. Physiol.

    (1993)

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    1

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