F3‐Isoprostanes as a Measure of in vivo Oxidative Damage in Caenorhabditis elegans

Thuy T. Nguyen1, Michael Aschner2

1 Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee, 2 Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 11.17
DOI:  10.1002/0471140856.tx1117s62
Online Posting Date:  November, 2014
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Oxidative stress has been implicated in the development of a wide variety of disease processes, including cardiovascular disease, cancer, and neurodegenerative diseases, as well as progressive and normal aging processes. Isoprostanes (IsoPs) are prostaglandin‐like compounds that are generated in vivo from lipid peroxidation of arachidonic acid (AA, C20:4, ω‐6) and other polyunsaturated fatty acids (PUFA). Since the discovery of IsoPs by Morrow and Roberts in 1990, quantification of IsoPs has been shown to be an excellent source of biomarkers of in vivo oxidative damage. Eicosapentaenoic acid (EPA, C20:5, ω‐3) is the most abundant PUFA in Caenorhabditis elegans and gives rise to F3‐IsoPs upon nonenzymatic free‐radical‐catalyzed lipid peroxidation. The protocol presented is the current methodology that our laboratory uses to quantify F3‐IsoPs in C. elegans using gas chromatography/mass spectrometry (GC/MS). The methods described herein have been optimized and validated to provide the best sensitivity and selectivity for quantification of F3‐IsoPs from C. elegans lysates. © 2014 by John Wiley & Sons, Inc.

Keywords: C. elegans; F3‐isoprostanes; oxidative stress; lipid peroxidation; reactive oxygen species

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Table of Contents

  • Introduction
  • Basic Protocol 1: Sample Collection and Preparation
  • Basic Protocol 2: Lipid Extraction and Hydrolysis of F3‐IsoP‐Containing Phospholipids in C. elegans Lysates
  • Basic Protocol 3: Sample Purification for Mass Spectrometric Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Sample Collection and Preparation

  • Nematodes (Caenorhabditis Genetics Center; http://www.cbs.umn.edu/research/resources/cgc/)
  • 85 mM sodium chloride (NaCl; see recipe)
  • Lysis buffer (see recipe)
  • Diluted bleach mixture (1:10 bleach/H 2O)
  • 15‐ml conical tubes
  • Table‐top centrifuge
  • 2‐ml sterile polypropylene screw‐cap tubes
  • 1.0‐mm zirconia beads (BioSpec Products, cat. no. 11079110zx)
  • High‐energy cell disruptor, (BioSpec Products, cat. no. 607)

Basic Protocol 2: Lipid Extraction and Hydrolysis of F3‐IsoP‐Containing Phospholipids in C. elegans Lysates

  • 15% KOH, w/v (see recipe)
  • 0.005% (w/v) butylated hydroxytoluene (BHT) in methanol (see recipe)
  • 0.001 M HCl (pH 3; see recipe)
  • 1 M HCl
  • 1.7‐ml microcentrifuge tube
  • Table‐top centrifuge
  • 15‐ml plastic tube (Denville, cat. no. T8173)

Basic Protocol 3: Sample Purification for Mass Spectrometric Analysis

  • [2H 4]‐15‐F 2t‐IsoP (8‐iso‐PGF2 α; Cayman Chemical, cat. no. 316351)
  • Methanol (Fisher, cat. no. A4544)
  • 0.001 M HCl (pH 3; see recipe)
  • 1 M HCl
  • Heptane (Fisher, cat. no. H3504)
  • Ethyl acetate (Fisher, cat. no. E1964)
  • Anhydrous sodium sulfate (Na 2SO 4)
  • Acetonitrile (Fisher, cat. no. A9984)
  • 10% pentafluorobenzyl bromide (PFBB) in acetonitrile (see recipe)
  • 10% N,N’‐diisopropylethylamine (DIPE) in acetonitrile (see recipe)
  • Chloroform (VWR, EM‐CX1059‐1)
  • Ethanol, absolute, 200 proof (Fisher, AC61509040)
  • Acetic acid
  • Prostaglandin F (PGF ), methyl ester
  • Phosphomolybdic acid solution (Sigma‐Aldrich, cat. no. P4869)
  • Bis(trimethylsilyl)trifluoroacetamide (BSTFA)
  • Anhydrous dimethylformamide (DMF)
  • Undecane
  • 10‐ml disposable plastic syringe (Laboratory Supply, cat. no. SMJ5512878)
  • 15‐ml plastic tube (Denville, cat. no. T8173)
  • 5‐ml plastic vial with cap (Denville, cat. no. T8200)
  • 1.7‐ml microcentrifuge tube
  • C18 Plus solid‐phase extraction (Sep‐Pak) cartridge (Waters Associates, cat. no. WAT036575)
  • Silica Plus solid‐phase extraction (Sep‐Pak) cartridge (Waters Associates, cat. no. WAT036580)
  • Supelco Visiprep SPE vacuum manifold (Sigma‐Aldrich, cat. no. 57265)
  • Analytical evaporation unit with water bath at 37°C (Organomation, cat. no. 11634‐P)
  • TLC filter paper (VWR, cat. no. 28298‐020)
  • TLC silica plates (Analtech, cat. no. 43931‐2)
  • Glass TLC tank (VWR, cat. no. 21432‐761)
  • Heat gun
  • Table‐top centrifuge
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Literature Cited

Literature Cited
  Back, P., De Vos, W.H., Depuydt, G.G., Matthijssens, F., Vanfleteren, J.R., and Braeckman, B.P. 2012. Exploring real‐time in vivo redox biology of developing and aging Caenorhabditis elegans. Free Radic. Biol. Med. 52:850‐859.
  Bonini, M.G., Rota, C., Tomasi, A., and Mason, R.P. 2006. The oxidation of 2′,7′‐dichlorofluorescin to reactive oxygen species: A self‐fulfilling prophesy? Free Radic. Biol. Med. 40:968‐975.
  Burkitt, M. J. and Wardman, P. 2001. Cytochrome C is a potent catalyst of dichlorofluorescin oxidation: implications for the role of reactive oxygen species in apoptosis. Biochem. Biophys. Res. Commun. 282:329‐333.
  Butterfield, D.A. 1997. β‐Amyloid‐associated free radical oxidative stress and neurotoxicity: Implications for Alzheimer's disease. Chem. Res. Toxicol. 10:495‐506.
  Chang, C.‐T., Patel, P., Kang, N., Lawson, J.A., Song, W.L., Powell, W.S., FitzGerald, G.A., and Rokach, J. 2008. Eicosapentaenoic‐acid‐derived isoprostanes: Synthesis and discovery of two major isoprostanes. Bioorg. Med. Chem. Lett. 18:5523‐5227.
  Chen, Q., Vazquez, E.J., Moghaddas, S., Hoppel, C.L., and Lesnefsky, E.J. 2003. Production of reactive oxygen species by mitochondria: Central role of complex III. J. Biol. Chem. 278:36027‐36031.
  Dingley, S., Polyak, E., Lightfoot, R., Ostrovsky, J., Rao, M., Greco, T., Ischiropoulos, H., and Falk, M.J. 2010. Mitochondrial respiratory chain dysfunction variably increases oxidant stress in Caenorhabditis elegans. Mitochondrion 10:125‐136.
  Folkes, L.K., Patel, K.B., Wardman, P., and Wrona, M. 2009. Kinetics of reaction of nitrogen dioxide with dihydrorhodamine and the reaction of the dihydrorhodamine radical with oxygen: Implications for quantifying peroxynitrite formation in cells. Arch. Biochem. Biophys. 484:122‐126.
  Gao, L., Yin, H., Milne, G.L., Porter, N., and Morrow, J.D. 2006. Formation of F‐ring isoprostane‐like compounds (F3‐isoprostanes) in vivo from eicosapentaenoic acid. J. Biol. Chem. 281:14092‐14099.
  Gardner, H.W. 1989. Oxygen radical chemistry of polyunsaturated fatty acids. Free Radic. Biol. Med. 7:65‐86.
  Gutteridge, J.M. 1995. Lipid peroxidation and antioxidants as biomarkers of tissue damage. Clin. Chem. 41:1819‐1828.
  Halliwell, B. and Gutteridge, J.M. 1990. Role of free radicals and catalytic metal ions in human disease: An overview. Methods Enzymol. 186:1‐85.
  Hempel, S.L., Buettner, G.R., O'Malley, Y.Q., Wessels, D.A., and Flaherty, D.M. 1999. Dihydrofluorescein diacetate is superior for detecting intracellular oxidants: Comparison with 2′,7′‐dichlorodihydrofluorescein diacetate, 5(and 6)‐carboxy‐2′,7′‐dichlorodihydrofluorescein diacetate, and dihydrorhodamine 123. Free Radic. Biol. Med. 27:146‐159.
  Kadiiska, M.B., Gladen, B.C., Baird, D.D., Germolec, D., Graham, L.B., Parker, C.E., Nyska, A., Wachsman, J.T., Ames, B.N., Basu, S., Brot, N., Fitzgerald, G.A., Floyd, R.A., George, M., Heinecke, J.W., Hatch, G.E., Hensley, K., Lawson, J.A., Marnett, L.J., Morrow, J.D., Murray, D.M., Plastaras, J., Roberts, L.J., II, Rokach, J., Shigenaga, M.K., Sohal, R.S., Sun, J., Tice, R.R., Van Thiel, D.H., Wellner, D., Walter, P.B., Tomer, K.B., Mason, R.P., and Barrett, J.C. 2005. Biomarkers of oxidative stress study II: Are oxidation products of lipids, proteins, and DNA markers of CCl4 poisoning? Free Radic. Biol. Med. 38:698‐710.
  Knoefler, D., Thamsen, M., Koniczek, M., Niemuth, N.J., Diederich, A.K., and Jakob, U. 2012. Quantitative in vivo redox sensors uncover oxidative stress as an early event in life. Mol. Cell 47:767‐776.
  Labuschagne, C.F., Stigter, E.C., Hendriks, M.M., Berger, R., Rokach, J., Korswagen, H.C., and Brenkman, A.B. 2013. Quantification of in vivo oxidative damage in Caenorhabditis elegans during aging by endogenous F3‐isoprostane measurement. Aging Cell 12:214‐223.
  Milne, G.L., Yin, H., Brooks, J.D., Sanchez, S., Roberts, L.J., II, and Morrow, J.D. 2007. Quantification of F2‐isoprostanes in biological fluids and tissues as a measure of oxidant stress. Methods Enzymol. 433:113‐126.
  Morrow, J.D. and Roberts, L.J., II. 1999. Mass spectrometric quantification of F2‐isoprostanes in biological fluids and tissues as measure of oxidant stress. Methods Enzymol. 300:3‐12.
  Morrow, J.D., Hill, K.E., Burk, R.F., Nammour, T.M., Badr, K.F., and Roberts, L.J., II. 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., II. 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., Yang, J.P., Kurhts, E.H., Callewaert, D., Dworski, R., Kanai, K., Taber, D., Moore, K., Oates, J.A., and Roberts, L.J. 1999. Quantification of the major urinary metabolite of 15‐F2t‐isoprostane (8‐iso‐PGF2α) by a stable isotope dilution mass spectrometric assay. Anal. Biochem. 269:326‐331.
  Muller, F.L., Liu, Y., and Van Remmen, H. 2004. Complex III releases superoxide to both sides of the inner mitochondrial membrane. J. Biol. Chem. 279:49064‐49073.
  Niki, E. 2009. Lipid peroxidation: Physiological levels and dual biological effects. Free Radic. Biol. Med. 47:469‐484.
  Schulz, T.J., Zarse, K., Voigt, A., Urban, N., Birringer, M., and Ristow, M. 2007. Glucose restriction extends Caenorhabditis elegans life span by inducing mitochondrial respiration and increasing oxidative stress. Cell Metab. 6:280‐293.
  Singh, G., Gutierrez, A., Xu, K., and Blair, I.A. 2000. Liquid chromatography/electron capture atmospheric pressure chemical ionization/mass spectrometry: Analysis of pentafluorobenzyl derivatives of biomolecules and drugs in the attomole range. Anal. Chem. 72:3007‐3013.
  Södergren, E., Vessby, B., and Basu, S. 2000. Radioimmunological measurement of F2‐isoprostanes after hydrolysis of lipids in tissues. Prostaglandins. Leukot. Essent. Fatty Acids 63:149‐152.
  Song, W.‐L. 2009. Novel eicosapentaenoic acid‐derived F3‐isoprostanes as biomarkers of lipid peroxidation. J. Biol. Chem. 284:23636‐23643.
  Stafforini, D.M., Sheller, J.R., Blackwell, T.S., Sapirstein, A., Yull, F.E., McIntyre, T.M., Bonventre, J.V., Prescott, S.M., and Roberts, L.J., II. 2006. Release of free F2‐isoprostanes from esterified phospholipids is catalyzed by intracellular and plasma platelet‐activating factor acetylhydrolases. J. Biol. Chem. 281:4616‐4623.
  Vanfleteren, J.R. 1993. Oxidative stress and ageing in Caenorhabditis elegans. Biochem. J. 292:605‐608.
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