Measurement of Lipid Peroxidation

Christopher A. Reilly1, Steven D. Aust1

1 Utah State University, Logan, Utah
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 2.4
DOI:  10.1002/0471140856.tx0204s00
Online Posting Date:  May, 2001
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Abstract

There is currently considerable interest in what is termed “oxidative stress,” or the oxidation of biological macromolecules, with emphasis on its involvement in various diseases and toxicities and methods to limit either its occurrence or effects. This unit describes traditional methods to measure the extent or rate of lipid peroxidations, including assays for conjugated dienes, lipid hydroperoxides, the polyunsaturated lipid breakdown product malondialdehyde, and hemolysis, along with discussion of alternative methods.

     
 
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Table of Contents

  • Basic Protocol 1: Assay for Conjugated Dienes
  • Basic Protocol 2: Assay for Lipid Hydroperoxides
  • Basic Protocol 3: Measurement of Malondialdehyde
  • Basic Protocol 4: Red Blood Cell Lysis
  • Support Protocol 1: Anaerobic Extraction of Lipid
  • Support Protocol 2: Colorimetric Quantitation of Phosphorus
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Assay for Conjugated Dienes

  Materials
  • Aqueous lipid suspension (see recipe)
  • Argon‐purged 2:1 (v/v) chloroform/methanol (see recipe)
  • Argon
  • Cyclohexane
  • 45°C water bath

Basic Protocol 2: Assay for Lipid Hydroperoxides

  Materials
  • Aqueous lipid suspension (see recipe)
  • 1 mM EDTA
  • 3:2 (v/v) acetic acid/chloroform (see recipe)
  • 1.2 g/ml potassium iodide in argon‐purged H 2O (prepare immediately before use and keep on ice protected from light)
  • 0.5% (w/v) cadmium acetate in argon‐purged H 2O (prepare fresh)
  • 2:1 (v/v) chloroform/methanol (see recipe)
  • Argon
  • Cumene hydroperoxide or t‐butyl hydroperoxide
  • 45°C water bath
  • Additional reagents and equipment for extraction and recovery of lipids (see protocol 1)
NOTE: It is essential to minimize the exposure of solutions containing iodide to oxygen.

Basic Protocol 3: Measurement of Malondialdehyde

  Materials
  • Aqueous lipid suspension (see recipe)
  • 4× TBA/TCA/HCl reagent (see recipe)
  • 2% (w/v) butylated hydroxytoluene (BHT) in ethanol (store protected from light up to 1 month at room temperature)
  • 1,1,3,3‐Tetramethoxypropane (optional)
  • Boiling water bath
  • Spectrophotometer or spectrofluorometer

Basic Protocol 4: Red Blood Cell Lysis

  Materials
  • Erythrocytes (see recipe)
  • 0.9% NaCl, pH 6.8 to 7.5 ( appendix 2A; sterile filter and store up to 1 week at 4°C)

Support Protocol 1: Anaerobic Extraction of Lipid

  Materials
  • Tissue to be extracted (e.g. Pel‐Freez Biologicals, or live animal)
  • Argon gas
  • 2:1 (v/v) chloroform/methanol (see recipe)
  • Aqueous Folch salt (see recipe)
  • Erlenmeyer and vacuum flasks
  • Separatory funnel
  • Rotary evaporator
  • Water bath

Support Protocol 2: Colorimetric Quantitation of Phosphorus

  Materials
  • 2:1 (v/v) chloroform/methanol (see recipe)
  • Stock lipid (see protocol 5)
  • 1 mM phosphoric acid or 1 mM monobasic sodium phosphate
  • 10 N H 2SO 4
  • 30% (v/v) H 2O 2
  • 0.22% (w/v) ammonium molybdate
  • Fiske‐Subbarow reagent (see recipe)
  • 18 × 150–mm test tubes
  • Glass marbles
  • Boiling‐water bath
  • Oven heated to 150°C
  • Spectrophotometer
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Figures

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Literature Cited

Literature Cited
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