Measurement of Oxygen Radicals and Lipid Peroxidation in Neural Tissues

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

One of the most completely validated processes involved in secondary tissue damage following acute brain or spinal cord injury and in many chronic neurodegenerative diseases has to do with the pathological formation of reactive oxygen species (ROS) and reactive nitrogen species (RNS). These are generated by multiple mechanisms and give rise to highly reactive oxygen radicals that can damage neuronal, glial, and microvascular elements. Particular interest has centered upon oxygen radical?induced, iron?catalyzed lipid peroxidation (LP) as the principal mechanism of neuronal injury associated with oxygen radicals. Thus, there has been a growing interest in monitoring increased oxygen radical levels as an index of oxidative stress, as well as measuring markers of LP?associated oxidative injury in in vitro and in vivo model systems and neurological patient samples. Accordingly, the purpose of this unit is to provide a variety of methods for the measurement of hydroxyl radical formation and/or LP in nervous tissue or biofluids.Curr. Protoc. Neurosci. 48:7.17.1?7.17.51. © 2009 by John Wiley & Sons, Inc.

Keywords: reactive oxygen species; reactive nitrogen species; oxygen?free radicals; lipid peroxidation

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Introduction
Basic Protocol 1: Salicylate Trapping and HPLC Assay of Hydroxyl Radical
Basic Protocol 2: Spectrophotometric Assay of Lipid‐Conjugated Dienes
Basic Protocol 3: HPLC Assay of Vitamin E
Basic Protocol 4: HPLC Assay of Glutathione
Basic Protocol 5: HPLC‐Chemiluminescence Assay of Lipid Hydroperoxides
Support Protocol 1: Xylenol Orange Determination of Hydroperoxide Content in Standards
Basic Protocol 6: Thiobarbituric Acid Assay of Malondialdehyde
Basic Protocol 7: HPLC Assay of Malondialdehyde Using UV Detection
Alternate Protocol 1: HPLC Assay of TBA‐Malondialdehyde Adduct Using Fluorescence Detection
Basic Protocol 8: GC/MS Determination and Quantification of 15‐F2T Isoprostane
Alternate Protocol 2: Immunoassay of F2‐Isoprostanes
Basic Protocol 9: Instrumental Detection and Quantification of F4‐Neuroprostanes
Basic Protocol 10: Immunoassay for 4‐Hydroxynonenal
Alternate Protocol 3: Immunohistochemical Detection of 4‐Hydroxynonenal
Reagents and Solutions
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: Salicylate Trapping and HPLC Assay of Hydroxyl Radical

Materials

HPLC‐grade solvents (EM Science):
- Acetonitrile
- Tetrahydrofuran
- Water
- Ethanol
- 30% (v/v) methanol
Monochloroacetic acid (Aldrich)
3,4‐Dihydroxycinnamic acid (3,4‐DHCA; Aldrich)
Phenylalanine
Mannitol
Deferoxamine (Ciba)
2,3‐ and 2,5‐Dihydroxybenzoic acid (2,3‐ and 2,5‐DHBA; Aldrich)
Salicylate (Aldrich)
Tissue samples of interest: ∼500‐mg pieces, dissected and immediately frozen in liquid nitrogen, and stored at –80°C

Magnetic stir bar and plate
Vacuum
0.5‐ and 1.5‐ml microcentrifuge tubes
Balance
Sonicator fitted with a microprobe tip (e.g., VirSonic 60 Ultrasonicator; Virtis Company)
Refrigerated centrifuge (e.g., Tomy MTX 150 equipped with a TMA‐11 rotor)
250‐µl autosampler vials (e.g., Sun Brokers)
High‐performance liquid chromatograph (HPLC) equipped with:
- Autosampler (e.g., Perkin‐Elmer ISS‐100) with 150‐µl vials
- Refrigerated constant‐temperature circulating water bath capable of cooling to 4°C
- HPLC pump capable of delivering a flow rate of 1.0 ml/min at pressures up to 4000 psi (e.g., Waters 510 LC)
- Biophase ODS analytical HPLC column (4.6 × 250–mm, 5‐µm particle diameter, BAS)
- Electrochemical detector (e.g., Bioanalytical Systems)
- Programmable UV absorbance detector (e.g., Applied Biosystems 785A)
- Data acquisition software (e.g., Waters Maxima 820)

Basic Protocol 2: Spectrophotometric Assay of Lipid‐Conjugated Dienes

Materials

Rat liver, fresh or frozen
Sucrose/PBS/EDTA solution (see recipe ; containing 0.1 M EDTA)
Methanol
Chloroform
Oxygen‐free nitrogen
Cyclohexane, spectrophotometric‐grade

Tissue homogenizer (blade homogenizer)
40‐ml graduated, heavy‐walled, stoppered centrifuge tubes
40° to 50°C water bath
Spectrophotometer with 1‐cm path‐length cuvette

NOTE: Commercially available solvents in this protocol can be used without further purification.

Basic Protocol 3: HPLC Assay of Vitamin E

Materials

HPLC‐grade water
HPLC‐grade methanol (EM Science)
Pyridine (Mallinckrodt)
Sodium perchlorate (Sigma)
Helium
α‐Tocopherol (vitamin E; Aldrich)
HPLC‐grade ethyl acetate (EM Science)
Experimental animal
Liquid nitrogen

Vacuum filtration device
47‐mm, 0.5‐µm Teflon filter (Type FH; Millipore)
250‐µl autosampler vials (e.g., Sun Brokers)
1.5‐ml microcentrifuge tubes
1.5‐ml amber autosampler vials (e.g., National Scientific)
Homogenizer
Sonicator fitted with a microprobe tip (e.g., VirSonic 60 Ultrasonicator; Virtis Company)
Refrigerated centrifuge (e.g., Tomy MTX 150 equipped with a TMA‐11 rotor)
12 × 75–mm polypropylene culture tubes (e.g., Falcon)
High‐performance liquid chromatograph (HPLC) equipped with:
- Autosampler (e.g., Perkin‐Elmer ISS‐100)
- Refrigerated constant‐temperature circulating 4°C water bath
- Inertsil C8 column (3.0 × 250–mm, 5‐µm particle size; Metachem Technologies)
- Inertsil C8 guard column (10 × 4.3–mm, 5‐µm particle size; Metachem Technologies)
- 3‐mm precolumn inlet filter (Rheodyne model 7335)
- HPLC pump (e.g., Waters 510 LC pump or any pump capable of delivering a flow rate of 1.0 ml/min at pressures up to 4000 psi)
- Electrochemical detector with a glassy carbon electrode and Ag/AgCl reference electrode, or any equivalent detector capable of oxidation at 700 mV (e.g., Waters 460)
- Scanning fluorescence detector (Waters 470) or other detector capable of reading emission at 340 nm and excitation at 290 nm
- Data acquisition system (Waters Millennium 2010 version 2.1 or equivalent)

Basic Protocol 4: HPLC Assay of Glutathione

Materials

0.15 M sodium acetate, pH 7.0 (see recipe )
Methanol, reagent grade (EM Science)
Helium
Reduced glutathione (GSH)
10 mM HCl, ice cold
Oxidized glutathione (GSSG)
5 mg/ml OPA solution (see recipe )
100 mM sodium phosphate buffer, pH 7.0 ( appendix 2A )
Tissue of interest: 20‐ to 40‐mg samples stored at –70°C
25 mM sodium phosphate buffer, pH 6.0 ( appendix 2A ), ice cold
100 mM Tris⋅Cl, pH 8.5 ( appendix 2A )
25 and 50 mM dithiothreitol (DTT; Sigma)
2 mM N ‐ethylmaleimide (NEM; Sigma)
2.5% (w/v) 5‐sulfosalicylic acid (SSA; Sigma)

Vacuum filtration unit
0.5‐µm Teflon filter (Type FH; Millipore)
Sonicator fitted with a microprobe tip (e.g., VirSonic 60 Ultrasonicator; Virtis Company)
Refrigerated centrifuge (e.g., Tomy MTX 150 equipped with a TMA‐11 rotor)
High‐performance liquid chromatograph (HPLC) equipped with:
- Autosampler equipped with a heater/cooler to maintain constant sample compartment temperature at 4°C, and carousel capable of holding up to 96 autosampler vials (e.g., Waters 717)
- Analytical C18 reversed‐phase ODS column (25 cm × 4.6 mm; 5‐µm particle size)
- RP18 guard column
- HPLC pump (e.g., Waters 510 LC pump or any pump capable of delivering a flow rate of 1.0 ml/min at pressures up to 4000 psi)
- Scanning fluorescence detector or other detector capable of reading emission at 420 nm and of excitation at 340 nm (e.g., Waters 470)
- Data acquisition system (Waters Millennium 2010, version 2.1 or equivalent)
1‐ml autosampler vials containing low‐volume inserts with springs and caps (Waters)

Basic Protocol 5: HPLC‐Chemiluminescence Assay of Lipid Hydroperoxides

Materials

HPLC‐grade chloroform (EM Science)
HPLC‐grade methanol (EM Science)
40 mM monobasic potassium phosphate
25 mg/ml phosphocholine (PC; Avanti Polar Lipids)
25 mg/ml phosphoethanolamine (PE; Avanti Polar Lipids)
Methylene blue
Appropriate standards (e.g., 15(S)‐HPEPE, 13(S)‐HPODE, or 9(S)‐HPODE, Biomol Research Laboratories)
1% (v/v) butylated hydroxytoluene (BHT; Sigma)/methanol
Tissue samples, dissected and immediately frozen in liquid nitrogen, stored at –80°C
0.15 M saline: 0.9% (w/v) NaCl in HPLC‐grade water
Nitrogen
2:1 and 1:9 (v/v) chloroform/methanol, freshly prepared
Chemiluminescent (CL) cocktail (see recipe )

Vacuum
200‐ml beaker
UV light source
High‐performance liquid chromatograph (HPLC) equipped with:
- 3‐ml Supelclean LC‐Si column (Supelco)
- SIL‐LC‐Si analytical HPLC column (25 × 4.6–mm, 5‐µm particle size; Supelco)
- Guard column (e.g., Supelguard LC‐Si; Supelco)
- HPLC pump capable of delivering 1 ml/min at pressures up to 4000 psi (e.g., Waters 600)
- Autosampler at 4°C and containing a carousel capable of holding up to 96 autosampler vials (e.g., Waters 717)
- Postcolumn mixing tee (e.g., ISCO 500D syringe pump)
- Chemiluminescence (CL) detector (e.g., Jasco model no. 825‐CL)
- Data acquisition system (Waters Millennium 2010, or equivalent)
4‐ml amber glass vials with caps
1.5‐ml microcentrifuge tubes
Sonicator fitted with a microprobe tip (e.g., VirSonic 60 Ultrasonicator; Virtis Company)
Refrigerated centrifuge (e.g., Tomy MTX 150 equipped with a TMA‐11 rotor)
Pasteur pipets
Reacti‐therm heating module/evaporating unit (Pierce)
Autosampler vials containing low‐volume inserts with springs and caps (Waters)

Additional reagents and equipment for xylenol orange assay (see protocol 6 )

Support Protocol 1: Xylenol Orange Determination of Hydroperoxide Content in Standards

Materials

Degassed water
Ferrous ammonium sulfate (FAS; see recipe )
Argon
Methanol
Butylated hydroxytoluene (BHT; Sigma)
Concentrated (18 M) H₂ SO₄
Xylenol orange (Aldrich)
Standards to be tested (see protocol 5 )

1.5‐ml microcentrifuge tubes
Pasteur pipets
125‐ml container
Spectrophotometer and cuvettes

Basic Protocol 6: Thiobarbituric Acid Assay of Malondialdehyde

Materials

Biological sample: 1.0 to 2.0 mg/ml membrane protein or 0.1 to 0.2 mM lipid phosphate
TCA/TBA/HCl solution (see recipe )

Boiling water bath
Centrifuge
Spectrophotometer and cuvettes

Basic Protocol 7: HPLC Assay of Malondialdehyde Using UV Detection

Materials

Tris base
HPLC‐grade water (EM Science)
1 N HCl
HPLC‐grade acetonitrile (EM Science)
0.1 mM H₂ O₂
Malonaldehyde bis(diethylacetal) (also known as 1,1,3,3‐tetraethoxypropane or TEP; Aldrich)
10 mM potassium phosphate buffer, pH 7.0 ( appendix 2A )
Frozen tissue samples: dissected and immediately frozen in liquid nitrogen, stored at –80°C
40 mM Tris⋅Cl, pH 7.4 ( appendix 2A )
10 mM FAS solution (see recipe )
0.02% (v/v) butylated hydroxytoluene (BHT; Sigma) in acetonitrile

Vacuum filtration device
47‐mm, 0.5‐µm Teflon filter (Type FH; Millipore)
250‐ml glass‐stoppered flask
Parafilm
37° and 50°C water baths
10‐ml volumetric flask
UV spectrophotometer (e.g., Beckman model DU‐7500 diode array)
1.5‐ml microcentrifuge tubes
Sonicator fitted with a microprobe tip (e.g., VirSonic 60 Ultrasonicator; Virtis Company)
Refrigerated centrifuge (e.g., Tomy MTX 150 fitted with a TMA‐11 rotor)
1‐ml autosampler vials containing low‐volume inserts with springs and caps (Waters)
High‐performance liquid chromatograph (HPLC) equipped with:
- Autosampler configured with a thermostatted sample compartment held at 4°C and a carousel capable of holding up to 96 autosampler vials (e.g., Waters 717)
- Zorbax SB‐CN analytical HPLC column (4.6 × 25–cm, 5‐µm particle size; MAC‐MOD Analytical)
- Zorbax SB‐CN guard column (4.0 × 12.5–mm, 5‐µm particle size) fitted with PEEK‐encapsulated frit‐gasket (MAC‐MOD Analytical)
- HPLC pump capable of delivering 0.9 ml/min at pressures up to 4000 psi (e.g., Waters 600)
- Programmable UV absorbance detector (e.g., Applied Biosystems 785A)
- Data acquisition system (Waters Millennium 2010 version 2.1 or equivalent)

Alternate Protocol 1: HPLC Assay of TBA‐Malondialdehyde Adduct Using Fluorescence Detection

0.9% (w/v) NaCl
TBA/acetate/DTPA solution (see recipe )
5% (v/v) butylated hydroxytoluene (BHT) in 99% (v/v) ethanol

0.2‐ and 0.45‐µm filters (Millipore)
Water bath capable of maintaining temperatures up to 100°C
150 × 4.6–mm Inertsil ODS‐2 analytical HPLC column (MetaChem Tech)
50 × 4.0–mm guard column (MetaChem Tech)
HPLC pump capable of delivering a rate of 1 ml/min
Fluorescence detector with excitation at 515 nm and emission at 553 nm

Basic Protocol 8: GC/MS Determination and Quantification of 15‐F2T Isoprostane

Materials

15 F_2t ‐isoprostane‐d₄ (8‐iso prostaglandin F_α ‐d₄ ; Cayman Chemical cat. no. 316350) in methyl acetate
1 mg/ml 15 F_2t ‐isoprostane (8‐iso prostaglandin F_α ; Cayman Chemical cat. no. 16350) in absolute ethanol
Absolute ethanol USP
Sample fluid (e.g., serum, plasma, or cerebrospinal fluid)
0.1 M HCl or KOH
Butylhydroxytoluene (BHT) or 2,6‐di‐tert‐butyl‐4‐methylphenol (Sigma‐Aldrich cat. no. B1378)
Methanol, HPLC grade
pH 3.0 solution: prepare a 1 mM HCl solution and adjust pH to 3.0 with 0.1 M HCl or KOH
Ethyl acetate, HPLC grade
Heptane, HPLC grade
Nitrogen
Pentafluorobenzylbromide (PFBB; Sigma‐Aldrich, cat. no. 101052) made up to 10% solution in acetonitrile
10% diisopropylethylamine (N ,N ‐diisopropylethylamine; DIPE; Sigma‐Aldrich cat. no. D3887) in acetonitrile
Chloroform
TLC standard (prostaglandin F_2α , methyl ester)
Phosphomolybdic acid spray solution (Sigma‐Adrich cat. no. P4869)
N ,O ‐Bis[trimethylsilyl]trifluoroacetamide (BSTFA; Pierce Biotechnology cat. no. 38830)

13 × 100–mm tubes
C₁₈ solid phase extraction columns (Waters Corporation cat. no. WAT020805)
30‐ and 50‐ml beakers
Silica solid phase extraction columns (Waters Corporation cat. no. WAT020810)
Speedisk Positive Pressure Processor 48 (J.T. Baker), optional
10‐ml centrifuge tubes
37°C heating block
Whatman Partisil 4 channel TLC plates (Whatman cat. no. 4865‐621 or Fisher Scientific cat. no. 05‐713‐327)
150°C oven
Desiccator
Hair dryer
Chamber large enough to hold TLC plates vertically
Heating plate
Razor blades
1.5‐ml microcentrifuge tubes
10‐ml borosilicate glass centrifuge tubes
50°C water bath
GC/MS‐NICI instrument containing a 30‐m, DB‐5 column (0.25 mm × 0.25 µm)

Alternate Protocol 2: Immunoassay of F2‐Isoprostanes

Materials

Sample: cell cultures or tissues stored at –80°C
HPLC‐grade solvents (EM Science):
- Absolute ethanol, ice cold
- Hexane
- Ethyl acetate
- Methanol
100 mM sodium phosphate buffer, pH 7.2, prepared with deionized water
8‐Isoprostane EIA kit (8‐iso‐PGF_2α ; Cayman Chemical) containing:
- Precoated (mouse anti‐rabbit IgG) EIA 96‐well strip plates
- 96‐well cover sheets
- Tween 20
- EIA tracer dye
- EIA antiserum dye
- Blue EIA kit box (96 well)
- Ellman's reagent
- EIA buffer concentrate (10×)
- Wash buffer concentrate (400×)
- 8‐Isoprostane AChE tracer
- 8‐Isoprostane EIA antiserum
- 8‐Isoprostane EIA standard
Polytron tissue homogenizer
Refrigerated microcentrifuge
C₁₈ Sep‐Pak columns (Waters)
1.5‐ml microcentrifuge tubes
Vacuum centrifuge
Platform shaker
Microplate reader (e.g., Techan SLT Spectra) with microplate analysis software (e.g., DeltaSOFT II; Biometallics)

Basic Protocol 9: Instrumental Detection and Quantification of F4‐Neuroprostanes

Materials

15 F_2t ‐isoprostane‐d₄ (8‐iso prostaglandin F_α ‐d₄ ; Cayman Chemical, cat. no. 316350)
15 F_2t ‐isoprostane (8‐iso prostaglandin F_α ; Cayman Chemical, cat. no. 16350)
Tissue sample (fresh or ≤1 year at −80°C; >30 mg)
Folch solution: 3:1 (v/v) chloroform/methanol containing 0.005% BHT, stored up to 6 months at −20°C, ice cold
Nitrogen
0.9% NaCl
Methanol, HPLC grade
Butylated hydroxytoluene (BHT; Sigma)
Potassium hydroxide (KOH)
11.8 M formic acid
pH 3.0 solution: prepare 1 mM HCl and adjust pH to 3.0 with 0.1 M HCl or deionized water
C₁₈ solid phase extraction columns (Waters, cat. no. WAT020805)
Heptane, HPLC grade
Ethyl acetate, HPLC grade
Sodium sulfate
Silica solid phase extraction columns (Waters, cat. no. WAT020810)
Pentafluorobenzylbromide (PFBB; Sigma‐Aldrich, cat. no. 101052)
Diisopropylethylamine (N ,N ‐diisopropylethylamine; DIPE; Sigma‐Aldrich, cat. no. D3887)
Whatman Partisil 4 channel TLC plates (Whatman, cat. no. 4865‐621 or Fisher Scientific, cat. no. 05‐713‐327)
Ethanol USP, absolute
Chloroform, HPLC grade
TLC standard (prostaglandin F_2α , methyl ester)
Phosphomolybdic acid spray solution (Sigma‐Adrich, cat. no. P4869)
N ,O ‐Bis[trimethylsilyl]trifluoroacetamide (BSTFA; Pierce Biotechnology, cat. no. 38830)

10‐ and 40‐ml centrifuge tubes
Handheld tissue homogenizer (e.g., PowerGen model 35, Fisher Scientific)
Centrifuge
37°C incubator/heating block
Speedisk Positive Pressure Processor 48 (J.T. Baker), optional
30‐ and 50‐ml beakers
Hair dryer
Heating plate
Razor blades
1.5‐ml microcentrifuge tubes
Microcentrifuge
50°C water bath
Target vials
GC/MS‐NICI instrument containing a 30‐m DB‐5 column (0.25 mm × 0.25 µm)
Injector liner, e.g., Restek Open Top Drilled Uniliner (hole on top; Restek)

Basic Protocol 10: Immunoassay for 4‐Hydroxynonenal

Materials

Protease inhibitor (PI) buffer: add 1 protease inhibitor tablet (Roche Applied Science cat. no. 1 836 153) to every 10 ml of Triton lysis buffer (see recipe ), prepare immediately before use and keep chilled
Tissue
Bio‐Rad DC protein assay kit reagents (Bio‐Rad Laboratories Reagent A cat. no. 500‐0113; Reagent B cat. no. 500‐0114; Reagent S cat. no. 500‐0115)
TBS (see recipe )
Blocking buffer: dissolve 5 g of dry non‐fat milk in TBS (see recipe ) to a final volume of 100 ml
Primary antibody for 4‐HNE, rabbit anti‐hydroxynonenal (HNE) (Alpha Diagnostics International cat. no. HNE11‐S)
Tris‐buffered saline with Tween 20, pH 8.0 (TBST; Sigma‐Aldrich cat. no. T‐9039)
Secondary antibody for 4‐HNE and 3‐NT IRDye800CW goat anti‐rabbit IgG (Rockland Immunochemicals, cat. no. 611‐131‐122)

1.5‐ or 2.0‐ml centrifuge tubes
Sonicator
Refrigerated centrifuge
Slot blot apparatus
Nitrocellulose/filter paper sandwiches (Bio‐Rad Laboratories cat. no. 162‐0233)
Bio‐Dot SF filter paper (Bio‐Rad Laboratories cat. no. 162‐0161)
Blotting paper
40°C oven
LI‐COR Odyssey imager or equivalent

Alternate Protocol 3: Immunohistochemical Detection of 4‐Hydroxynonenal

Materials

35‐µm (or other, as appropriate) sections of brain or spinal cord tissue
10× (1 M) phosphate buffered saline (PBS; Fisher Scientific cat. no. BP399) dilute with deionized water to yield 0.2 M PBS
Sodium borohydride (NaBH₄ )
0.1 M 3‐(N ‐morpholino)propanesulfonic acid hemisodium salt, pH 8.0 (MOPS, Sigma‐Adrich cat. no. M0289)
0.3% H₂ O₂ in PBS: add 1 part 30% hydrogen peroxide to 99 parts 0.2 M PBS; prepare fresh just before use
Blocking buffer (see recipe ), prepared fresh
Primary antibody for HNE, rabbit anti‐4‐HNE (Calbiochem, EMD Chemicals cat. no. 393207)
Secondary antibody (biotinylated goat anti‐rabbit secondary antibody)
Secondary antibody kit, ABC‐AP kit (Vector Laboratories, cat. no. AK‐5001)
0.1 M Tris⋅Cl, pH 8.2 ( appendix 2A )
Vector Blue Kit (Vector Laboratories, cat. no. SK‐5300) containing:
- Reagent 1
- Reagent 2
- Reagent 3
Nuclear fast red (Vector Laboratories, cat. no. H‐3403)
Permount (Fisher Scientific, cat. no. SP15‐100)

12‐well plates and tissue wells (netwells, Corning cat. no. 07‐200‐211)
Paint brush
Platform rocker
Aluminum foil
Microscope slides
Coverslips

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Figures

Figure 7.17.1 Salicylate trapping of hydroxyl radical.

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Figure 7.17.2 HPLC chromatograms of 2,3‐DHBA, 2,5‐DHBA, salicylate (SAL), and internal standard (IS) using electrochemical (A,C ) and UV detection (B,D ). (A ) 2,3‐DHBA, 2,5‐DHBA, and IS measured in prepared standards; (B ) salicylate in prepared standards; (C ) 2,3‐DHBA, 2,5‐DHBA, and IS measured in striatum of gerbil brain; (D ) salicylate in striatum of gerbil brain.

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Figure 7.17.3 Lipid peroxidation followed by formation of conjugated dienes. Ethyl linoleate (an ester of linoleic acid and ethanol) was purified, and its light absorption was plotted at different wavelengths (A). Similar plots are shown for a sample oxidized by air for 8 hr at 30°C (B), and for a sample in which peroxidation was accelerated by addition of nitrogen dioxide (C). In both cases, the shoulder of UV absorbance due to conjugated diene formation is clearly visible. Reprinted from Halliwell and Gutteridge () with permission from Oxford University Press.

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Figure 7.17.4 Antioxidant role of vitamin E in the defense against lipid peroxidation (L = lipid).

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Figure 7.17.5 HPLC chromatograms of vitamin E and internal standard (85A). (A ) Standards. (B ) Vitamin E level in striatum of gerbil brain. Internal standard 85A is not commercially available. However, the concentration of vitamin E in samples can be determined using a vitamin E standard curve.

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Figure 7.17.6 Antioxidant roles of glutathione and the enzymes glutathione peroxidase and glutathione reductase (L = lipid).

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Figure 7.17.7 HPLC‐chemiluminescence measurement of lipid hydroperoxides (PCOOH, PEOOH, fatty acid‐OOH).

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Figure 7.17.8 HPLC chemiluminescence chromatograms of FFAOOH, PEOOH, and PCOOH in (A ) standards, (B ) control spinal neuron cultures, and (C ) cultures exposed to 200 µM ferrous iron for 40 min. Note dramatic increase in all three hydroperoxide species.

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Figure 7.17.9 Formation of malondialdehyde (MDA) and measurement by thiobarbituric acid (TBA) reaction. The product is measured by absorbance at 535 nm or fluorescence at 553 nm.

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Figure 7.17.10 HPLC‐UV chromatogram of MDA in gerbil brain extract. Machine output is in mV; actual output is absorbance.

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Figure 7.17.11 Chemistry of arachidonic acid peroxidation resulting in 5‐ and 15‐F₂ isoprostanes. See Morrow et al. (), Roberts et al. (), and Halliwell and Gutteridge () for further details.

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Figure 7.17.12 Representative ion chromatogram from human plasma sample exhibiting m/z 569.4 peak at 10.9 min. This peak represents endogenous, free 5 and 15‐F_2t ‐isoprostane present in the sample.

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Figure 7.17.13 Chemistry of docosahexaenoic acid (DHA) peroxidation resulting in F₄ neuroprostane. See Morrow et al. (), Roberts et al. (), and Halliwell and Gutteridge () for further details.

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Figure 7.17.14 Representative ion chromatogram from murine cortical tissue exhibiting m/z 593 area, representing total F₄ ‐neuroprostanes.

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Figure 7.17.15 Sources of superoxide and iron‐ and peroxynitrite‐derived oxygen‐free radicals.

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Figure 7.17.16 Initiation and propagation of membrane lipid peroxidation.

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Literature Cited

Literature Cited
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Measurement of Oxygen Radicals and Lipid Peroxidation in Neural Tissues

Abstract

Table of Contents

Materials

Basic Protocol 1: Salicylate Trapping and HPLC Assay of Hydroxyl Radical

Basic Protocol 2: Spectrophotometric Assay of Lipid‐Conjugated Dienes

Basic Protocol 3: HPLC Assay of Vitamin E

Basic Protocol 4: HPLC Assay of Glutathione

Basic Protocol 5: HPLC‐Chemiluminescence Assay of Lipid Hydroperoxides

Support Protocol 1: Xylenol Orange Determination of Hydroperoxide Content in Standards

Basic Protocol 6: Thiobarbituric Acid Assay of Malondialdehyde

Basic Protocol 7: HPLC Assay of Malondialdehyde Using UV Detection

Alternate Protocol 1: HPLC Assay of TBA‐Malondialdehyde Adduct Using Fluorescence Detection

Basic Protocol 8: GC/MS Determination and Quantification of 15‐F2T Isoprostane

Alternate Protocol 2: Immunoassay of F2‐Isoprostanes

Basic Protocol 9: Instrumental Detection and Quantification of F4‐Neuroprostanes

Basic Protocol 10: Immunoassay for 4‐Hydroxynonenal

Alternate Protocol 3: Immunohistochemical Detection of 4‐Hydroxynonenal

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