Quantification of F2‐Isoprostanes by Gas Chromatography/Mass Spectrometry as a Measure of Oxidant Stress

Erik S. Musiek1, Jason D. Morrow1

1 Vanderbilt University School of Medicine, Nashville, Tennessee
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 17.6
DOI:  10.1002/0471140856.tx1706s24
Online Posting Date:  June, 2005
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Abstract

Oxidative stress has been implicated in a wide variety of disease processes. One method to quantify oxidative injury is to measure lipid peroxidation. Most methods to do this are fraught with problems particularly when utilized to assess oxidative stress in vivo. On the other hand, quantification of a group of prostaglandin F2‐like compounds, termed the F2‐isoprostanes (F2‐IsoPs) provides an accurate assessment of oxidative stress both in vitro and in vivo and has come to be regarded as the gold standard to quantify lipid peroxidation. This unit describes methods to assess lipid peroxidation associated with oxidant injury in vivo by quantifying concentrations of either esterified or free F2‐IsoPs in biological fluids and tissues. The techniques employed for the analysis of these compounds from biological sources that are detailed herein utilize mass spectrometric approaches. Measurement of F2‐IsoPs represents an important advance in the ability to assess the role of oxidative stress in human disease.

Keywords: isoprostane; oxidative stress; lipid peroxidation; eicosanoid

     
 
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Table of Contents

  • Basic Protocol 1: Quantification of F2‐Isoprostanes in Tissue Lipids
  • Alternate Protocol 1: Extraction of Lipids from Phospholipid‐Containing Biological Fluids
  • Basic Protocol 2: Quantification of F2‐IsoPs
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Quantification of F2‐Isoprostanes in Tissue Lipids

  Materials
  • Fresh or frozen tissue
  • Folch solution (see recipe)
  • Butylated hydroxytoluene (BHT; Aldrich)
  • Nitrogen or argon source
  • 0.9% (w/v) aqueous sodium chloride (NaCl; high purity; EMD Biosciences) prepared in ultrapure water
  • Methanol (high‐quality; Burdick and Jackson or VWR Scientific) containing 0.005% (v/v) BHT
  • 15% (w/v) aqueous potassium hydroxide (KOH pellets; high purity; EMD Biosciences) prepared in ultrapure water
  • 1 N HCl (American Chemical Society certified or equivalent grade)
  • 50‐ml conical, polypropylene centrifuge tubes
  • Blade homogenizer (PTA 10S generator, Brinkman Instruments)
  • Table‐top centrifuge
  • Analytical evaporation unit (Organomation Associates)
  • 37°C water bath

Alternate Protocol 1: Extraction of Lipids from Phospholipid‐Containing Biological Fluids

  • Biological fluid, e.g., plasma
  • Triphenylphosphine (TPP; Aldrich)
  • 0.043% (w/v) magnesium chloride (MgCl 2; high purity; EMD Biosciences) in ultrapure water

Basic Protocol 2: Quantification of F2‐IsoPs

  Materials
  • Fluid or tissue extract, hydrolyzed
  • 1 N HCl
  • [2H 4] 15‐F 2‐IsoP (8‐iso‐PGF ) internal standard (Cayman Chemical)
  • C18 Sep‐Pak column (Waters)
  • Methanol (high purity; Burdick and Jackson, VWR Scientific)
  • pH 3 water (ultrapure filtered, adjusted to pH 3 with ACS‐grade HCl)
  • Heptane (high purity; Burdick and Jackson, VWR Scientific)
  • Ethyl acetate (high purity; Burdick and Jackson, VWR Scientific)
  • Na 2SO 4 (anhydrous)
  • Silica Sep‐Pak column (Waters)
  • 10% (v/v) pentafluorobenzyl bromide (PFBB; Sigma‐Aldrich) in acetonitrile
  • 10% (v/v) N,N′‐Diisopropylethylamine (DIPE; Sigma‐Aldrich) in acetonitrile
  • Chloroform with ethanol (high purity; Burdick and Jackson, VWR Scientific)
  • Ethanol (high purity; Burdick and Jackson, VWR Scientific)
  • PGF methyl ester (Cayman Chemical)
  • 10% phosphomolybdic acid (Sigma) solution in ethanol
  • N,O‐bis(trimethylsilyl)trifluoroacetamide (BSTFA; Supelco)
  • Dimethylformamide (DMF; Aldrich)
  • Undecane (Aldrich)
  • 10‐ml disposable plastic syringes
  • 20‐ml glass scintillation vials
  • 5‐ml Reacti‐Vials (Pierce Scientific)
  • Analytical evaporation unit (Organomation Associates)
  • 37°C water bath
  • Glass TLC tank and TLC paper
  • TLC plates (LK6B silica; Whatman)
  • 90°C oven
  • Dessicator
  • 1.5‐ml microcentrifuge tubes
  • Gas chromatograph/mass spectrometer with capabilities for negative ion chemical ionization (NICI) mass spectrometry (model 6890N, Agilent Technologies or equivalent)
  • Capillary gas chromatography column (DB‐1701, Fisons)
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Figures

Videos

Literature Cited

   DeZwart, L.L., Meerman, J.H.N., Commandeur, J.N.M., and Vermeulen, P.E. 1999. Biomarkers of free radical damage applications in experimental animals and in humans. Free Rad. Biol. Med. 26:202‐226.
   Fam, S.S. and Morrow, J.D. 2003. The isoprostanes: Unique products of arachidonic acid oxidation. Curr. Med. Chem. 10:1723‐1740.
   Halliwell, B. and Grootveld, M. 1987. The measurement of free radical reactions in humans. Some thoughts for future experimentation. FEBS Lett. 213:9‐14.
   Halliwell, B. and Gutteridge, J.M.C. 1990. Role of free radicals and catalytic metal ions in human disease: An overview. Methods Enzymol. 186:1‐85.
   Lawson, J.A., Li, H., Rokach, J., Adiyaman, M., Hwang, S.W., Khanapure, S.P., and FitzGerald, G.A. 1998. Identification of two major F2 isoprostanes, 8,12‐iso‐ and 5‐epi‐8,12‐iso‐isoprostane F2αVI, in human urine. J. Biol. Chem. 293:29295‐29301.
   Liang, Y., Wei, P., Duke, R.W., Reaven, P.D., Harman, S.M., Cutler, R.G., and Heward, C.B. 2003. Quantification of 8‐iso‐prostaglandin F2α and 2,3‐dinor‐8‐iso‐prostaglandin F2α in human urine using liquid chromatography‐tandem mass spectrometry. Free Radic. Biol. Med. 34:409‐418.
   Morrow, J.D. and Roberts, L.J. 1997. The isoprostanes: Unique bioactive products of lipid peroxidation. Prog. Lipid Res. 36:1‐21.
   Morrow, J.D. and Roberts, L.J. 1999. Mass spectrometric quantification of F2‐isoprostanes in biological fluids and tissues. Meth. Enzymol. 300:3‐12.
   Morrow, J.D. and Roberts, L.J. 2002. Mass spectrometric quantification of F2‐isoprostanes as indicators of oxidant stress. Meth. Molec. Biol. 186:57‐66.
   Morrow, J.D., Hill, K.E., Burk, R.F, Nammour, T.M., Badr, K.F, and Roberts, L.J. 1990. A series of prostaglandin F2‐like compounds are produced in vivo in humans by a non‐cyclooxygenase, free radical catalyzed mechanism. Proc. Natl. Acad. Sci. U.S.A. 87:9383‐9387.
   Morrow, J.D., Awad, J.A., Boss, H.J., Blair, I.A., and Roberts, L.J. 1992. Non‐cyclooxygenase‐derived prostanoids (F2‐isoprostanes) are formed in situ on phospholipids. Proc. Natl. Acad. Sci. U.S.A. 89:10721‐10725.
   Morrow, J.D., Zackert, W.E., Van der Ende, D.S., Reich, E.E., Terry, E.S., Cox, B., Sanchez, S.C., Montine, T.J., and Roberts, L.J. 2001. Quantification of isoprostanes as indicators of oxidant stress in vivo. In Handbook of Antioxidants 2nd ed. (E. Cadenas and L. Packer, eds.) pp. 57‐74. Marcel Dekker, New York.
   Pratico, D., Barry, O.P., Lawson, J.A., Adiyaman, M., Hwang, S.W., Khanapure, S.P., Iuliano, L., Rokach, J., and FitzGerald, G.A. 1998. IPF2α‐1: An index of lipid peroxidation in humans. Proc. Natl. Acad. Sci. U.S.A. 95:3449‐3454.
   Roberts, L.J. and Morrow, J.D. 2000. Measurement of F2‐isoprostanes as an index of oxidative stress in vivo. Free Radic. Biol. Med. 28:505‐513.
Key References
   Fam and Morrow, 2003. See above.
  An up‐to‐date review of the isoprostane field.
   Morrow et al., 1990. See above.
  The initial report of isoprostane formation in vivo and the potential use of these compounds as an index of oxidant stress in vivo.
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