| 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) 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) H 2 SO 4 -
Xylenol orange (Aldrich) -
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