Measurement of Isoprostanes as Markers of Oxidative Stress in Neuronal Tissue

Dejan Milatovic1, Michael Aschner1

1 Vanderbilt University Medical Center, Nashville, Tennessee
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 12.14
DOI:  10.1002/0471140856.tx1214s39
Online Posting Date:  February, 2009
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Oxidative stress is implicated in the pathogenesis of a variety of human diseases, including neurodegenerative disease, atherosclerosis, and cancer, as well as progressive and even normal aging processes. Increased generation of free radicals derived primarily from molecular oxygen has also been associated with neuronal damage induced by a variety of environmental agents. However, measuring oxidative stress in biological systems is complex and requires accurate quantification of either free radicals or damaged biomolecules. One method for quantifying oxidative injury is to measure lipid peroxidation caused by free radicals. One group of these peroxidation products, F2‐isoprostanes (F2‐IsoPs), is derived by free‐radical peroxidation of arachidonic acid (AA). These prostaglandin F2‐like compounds are currently the most accurate measure of oxidative damage in vivo. This unit summarizes current methodology for quantifying F2‐IsoPs and discusses the utility of these and other prostaglandin (PG)‐like compounds as in vivo biomarkers for oxidative stress in neuronal tissues. Curr. Protoc. Toxicol. 39:12.14.1‐12.14.12. © 2009 by John Wiley & Sons, Inc.

Keywords: F2‐isoprostanes; oxidative damage; lipid peroxidation; neuroprostanes

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Table of Contents

  • Basic Protocol 1: Quantification of F2‐IsoPs
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Quantification of F2‐IsoPs

  • Tissue samples, fresh or frozen
  • Folch solution: 2:1 (v/v) chloroform/methanol, ice cold, containing 0.005% (w/v) butylated hydroxytoluene (BHT; Sigma‐Aldrich, cat. no. B1378)
  • 0.9% (w/v) NaCl
  • Methanol, with and without 0.005% (v/v) butylated hydroxytoluene (BHT)
  • 15% (w/v) KOH
  • 1 M HCl
  • pH 3 water: adjusted by adding 1 N HCl
  • Deuterated standard: deuterium‐labeled isoprostane, [2H 4]15‐F 2t‐IsoP (8‐iso‐PGF ; Cayman Chemical, cat. no. 316350)
  • Heptane
  • 50:50 (v/v) ethyl acetate/heptane
  • Na 2SO 4, anhydrous
  • Ethyl acetate
  • 50:50 (v/v) ethyl acetate/methanol
  • 10:90 (v/v) pentafluorobenzyl bromide (PFBB; Sigma‐Aldrich, cat. no. 10105‐2)/anhydrous acetonitrile
  • 10:90 (v/v) N,N′‐diisopropylethylamine (DIPE; Sigma‐Aldrich, cat. no. D3887)/anhydrous acetonitrile
  • 2:3 (v/v) chloroform/methanol
  • 90:10 (v/v) ethyl acetate/ethanol
  • 93:7 (v/v) chloroform/ethanol
  • Thin‐layer chromatography (TLC) standard: prostaglandin F (PGF ) methyl ester in methanol (Cayman Chemical, cat. no. 16011)
  • Phosphomolybdic acid in ethanol (Sigma Chemical Co., cat. no. P4869)
  • Dimethylformamide (DMF, Sigma‐Aldrich, cat. no. 6407), stored over calcium hydride to prevent water accumulation
  • Bis(trimethylsilyl)trifluoroacetamide (BSTFA, Supelco, cat. no. 33084)
  • Undecane, dried and stored over calcium hydride
  • Methane gas
  • 15‐ml polypropylene culture tube with cap
  • Blade homogenizer (e.g., PT 10‐35; Brinkmann Instruments)
  • Nitrogen gas tank
  • 25°C and 37°C water baths
  • Temperature‐controlled centrifuge, 25°C
  • Sonicator (e.g., Utrasonic Bath, Fisher Scientific)
  • Sep‐Pak Plus C18 cartridge (Waters, cat. no. WAT03657)
  • 10‐ml plastic syringe (Laboratory Supply; SMJ512878)
  • 20‐ml scintillation vial
  • Silica Sep‐Pak cartridge (Waters, cat. no. WAT036580)
  • 5‐ml glass Reacti‐Vial with Teflon‐lined cap (e.g., Supelco)
  • TLC plates: 5 × 20–cm glass plates covered with a 250‐µm layer of silica gel particles 60 Å in diameter (Partisil LK6D; Whatman, cat. no. WC486562IV)
  • 95°C oven
  • TLC developing chamber (e.g., VWR)
  • Hot plate (e.g., Corning, cat. no. 6795‐200)
  • 1.5‐ml microcentrifuge tube
  • Filter paper, P8 grade (Fisher Scientific)
  • Hair dryer
  • Autosampler vial
  • 15‐m, 0.25‐mm diameter, 0.25‐µm film thickness, DB1701 fused silica capillary GC column (Fisons)
  • Gas chromatography (GC)/mass spectroscopy (MS) system (e.g., Hewlett Packard 5982A interfaced with an IBM Pentium computer)
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Literature Cited

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