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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Abstract

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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Materials

Basic Protocol 1: Sample Collection and Preparation

  Materials
  • 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

  Materials
  • 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

  Materials
  • [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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Figures

Videos

Literature Cited

Literature Cited
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