Measurement of Glutathione and Glutathione Disulfide

Debbie Mustacich1

1 Arizona Cancer Center, Tucson, Arizona
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 6.2
DOI:  10.1002/0471140856.tx0602s00
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Measurements of glutathione should include quantification of both the reduced and oxidized forms. HPLC‐based assays for glutathione and other cellular thiols and disulfides utilize a variety of detection methods, including ultraviolet, fluorescence, and electrochemical methods. This unit provides rapid, sensitive, highly versatile and reproducible HPLC methods for separating and quantifying sulfur‐containing amino acids and related derivatives from a variety of biological samples.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Derivatization and Analysis of GSH and GSSG by HPLC with UV Detection
  • Alternate Protocol 1: Derivatization and Analysis of GSH and GSSG by HPLC with Fluorescence Detection
  • Alternate Protocol 2: Derivatization and Analysis of GSH and GSSG by HPLC with Electrochemical Detection
  • Basic Protocol 2: Derivatization and Analysis of GSH and GSSG by Enzymatic Detection
  • Sample Preparation for Analysis of GSH and GSSG
  • Support Protocol 1: Preparation of Samples from Freshly Dissected Tissues
  • Support Protocol 2: Preparation of Samples from Cultured Cells
  • Support Protocol 3: Preparation of Sample from Hepatocytes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Derivatization and Analysis of GSH and GSSG by HPLC with UV Detection

  Materials
  • Perchloric acid (PCA) extract of freshly dissected tissue (see protocol 5), cultured cells (see protocol 6), or hepatocytes (see protocol 7)
  • 0.4 mM γ‐glutamyl glutamate (γ‐Glu‐Glu; see recipe)
  • 100 mM iodoacetic acid (IAA) in 0.2 mM m‐cresol purple (see recipe)
  • 1% (v/v) 1‐fluoro‐2,4‐dinitrobenzene (FDNB) in 100% HPLC grade ethanol (store at 4°C in a dark container covered with foil)
  • KOH/KHCO 3 solution (see recipe)
  • 0.4 mM GSH (see recipe)
  • 0.4 mM GSSG (see recipe)
  • Mobile phase A (see recipe)
  • Mobile phase B (see recipe)
  • HPLC column: 20‐cm × 4.6‐mm‐i.d. column with 5‐µm Exsil silica derivatized with 3‐aminopropyltriethoxy silane (CEL Associates; see Reed et al., )
  • HPLC system:
  •  Pump with gradient capability (Spectra‐Physics P200, Spectra‐Physics)
  •  UV detector (365 nm; Alltech 200, Alltech)
  •  Recording integrator (Hewlett Packard 3390, Alltech)
  •  Autosampler (optional; Labtronix)
    Table 6.2.1   Materials   Amino Acids and Related Compounds Measured by HPLC a   Amino Acids and Related Compounds Measured by HPLC

    Order of elution Compound Retention time (min)
    Sulfur‐containing compounds
    1 Homocystine  6.68
    3 Cystathionine  7.84
    4 Cystine (CySS)  8.31
    5 Homocysteic acid 12.13
    6 Homocysteine 13.18
    7 Cysteinylglycine 13.21
    8 Penicillamine 13.28
    9 γ‐Glutamylcysteine 13.49
    11 Cysteic acid 15.57
    12 Cysteine (CyS) 17.13
    13 γ‐Glutamyl glutamate (ISTD) 21.95
    14 Cysteinyl‐glutathione disulfide (CySSG) 22.95
    15 Glutathione (GSH) 25.95
    16 Glutathione disulfide (GSSG) 30.15
    Other compounds
    2 Glutamate (Glu)  7.51
    10 Aspartate (Asp) 14.74

     aRetention times vary with mobile‐phase gradient and length and age of column.
  • Hamilton gas‐tight syringe or equivalent (100 µl and 1.0 ml; e.g., Hamilton 1700 series)
  • Side‐arm Erlenmeyer filtration flask (PyrexPlus, 1 or 2 liter)
  • Millipore filtration apparatus to fit side‐arm flask
  • Millipore polypropylene filters, type HV, pore size 0.45 µm

Alternate Protocol 1: Derivatization and Analysis of GSH and GSSG by HPLC with Fluorescence Detection

  • PCA/DEPA indicator solution (see recipe)
  • 1 mg/ml dansyl chloride in acetonitrile
  • 2.0 M LiOH in metal‐free water
  • 50 mM IAA in metal‐free water (make fresh daily)
  • 0.1 mM γ‐Glu‐Glu (see recipe)
  • Fluorescence detector (Hewlett Packard model 1046A; 328 nm excitation and 541 nm emission)

Alternate Protocol 2: Derivatization and Analysis of GSH and GSSG by HPLC with Electrochemical Detection

  • Mobile phase: 50 mM NaH 2PO 4/0.05 mM octane sulfonic acid/2% (v/v) acetonitrile adjusted to pH 2.7 with phosphoric acid
  • 200 mM methane sulfonic acid (MSA) containing 5 mM diethylenetriaminepentaacetic acid (DTPA)
  • Coulometric electrochemical detector with porous graphite electrodes (ESA model 5200, ESA)
  • Glass Dounce homogenizer
  • 5‐µm, 0.4 × 20–cm ODS2 HPLC column

Basic Protocol 2: Derivatization and Analysis of GSH and GSSG by Enzymatic Detection

  Materials
  • 1% (w/v) picric acid
  • Stock buffer: 143 mM sodium phosphate and 6.3 mM sodium EDTA in metal‐free water, pH 7.5
  • 0.3 mM NADPH in stock buffer (prepare fresh daily and store at 4°C)
  • 6 mM DTNB in stock buffer (store for 2 weeks at −20°C)
  • 50 U/ml GSH reductase in stock buffer (store for 2 weeks at −20°C)
  • 2‐Vinylpyridine (Fluka; store at −20°C and use undiluted; replace if reagent becomes brown or viscous)
  • Triethanolamine
  • Spectrophotometer (412 nm; Alltech 200, Alltech)

Support Protocol 1: Preparation of Samples from Freshly Dissected Tissues

  Materials
  • Animal to be used for tissue studies
  • 1 g/ml sodium pentobarbital
  • 0.9% saline (NaCl; appendix 2A) in metal‐free water, prewarmed to 37°C
  • Liquid nitrogen
  • PCA/BPDS (see recipe)
  • Peristaltic pump (Barnant 72501‐000, VWR)
  • 5‐ml cryogenic specimen containers (Nalgene, Fisher)
  • Mortar and pestle
  • Ultrasonic cell disrupter (Cole Parmer 4710, Cole Parmer)

Support Protocol 2: Preparation of Samples from Cultured Cells

  Materials
  • Cultured adherent cells
  • PBS ( appendix 2A), ice cold
  • PCA/BPDS (see recipe)

Support Protocol 3: Preparation of Sample from Hepatocytes

  Materials
  • PCA/BPDS (see recipe)
  • Dibutyl phthalate (Sigma)
  • Isolated hepatocytes
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Anderson, M.E. 1985. Determination of glutathione and glutathione disulfide in biological samples. Methods Enzymol. 113:548‐555.
   Asensi, M., Sastre, J., Pallardó, F.V., García de la Asunción, J., Estrela, J.M., and Viña, J. 1994. A high‐performance liquid chromatography method for measurement of oxidized glutathione in biological samples. Anal. Biochem. 217:323‐328.
   Brigelius, R., Muckel, C., Akerboom, T.P.M., and Sies, H. 1983. Identification and quantitation of glutathione in hepatic protein mixed disulfides and its relationship to glutathione disulfide. Biochem. Pharmacol. 32:2529‐2534.
   Eady, J.J., Orta, T., Dennis, M.F., Tratford, M.R.L., and Peacock, J.H. 1995. Glutathione determination by the Tietze enzymatic recycling assay and its relationship to cellular radiation response. Br. J. Cancer 72:1089‐1095.
   Farris, M.W. and Reed, D.J. 1987. High‐performance liquid chromatography of thiols and disulfides: Dinitrophenol erivatives. Methods Enzymol. 143:101‐109.
   Farris, M.W., Brown, M.K., Schmitz, J.A., and Reed, D.J. 1985. Mechanism of chemical‐induced toxicity. I. Use of a rapid centrifugation technique for the separation of viable and nonviable hepatocytes. Toxicol. Appl. Pharm. 79:283‐295.
   Griffith, O.W. 1980. Determination of glutathione and glutathione disulfide using glutathione reductase and 2‐vinylpyridine. Anal. Biochem. 106:207‐212.
   Jones, D.P., Carlson, J.L., Samiec, P.S., Sternberg, P. Jr., Mody, V.C. Jr., Reed, R.L., and Brown, L.A. 1998. Glutathione measurement in human plasma. Evaluation of sample collection, storage and derivatization conditions for analysis of dansyl derivatives by HPLC. Clin. Chim. Acta. 275:175‐184.
   Krien, P.M., Margou, V., and Kermici, M. 1992. Electrochemical determination of femtomole amounts of free reduced and oxidized glutathione. Application to human hair follicles. J. Chromatogr. 576:255‐261.
   Lakritz, J., Plopper, C.G., and Buckpitt, A.R. 1997. Validated high‐performance liquid chromatography–electrochemical method for determination of glutathione and glutathione disulfide in small tissue samples. Anal. Biochem. 246:63‐68.
   Lowry, J.C., Rosebrough, N.J., Farr, A.L., and Radall, R.J. 1951. Protein measurement with the Folin phenol reagent. J. Biol.Chem. 193:265‐275.
   Martin, J. and White, I.N.H. 1991. Fluorimetric determination of oxidised and reduced glutathione in cells and tissues by high‐performance liquid chromatography following derivatization with dansyl chloride. J. Chromatogr. 568:219‐225.
   Potter, D.W. and Tran, T. 1993. Apparent rates of glutathione turnover in rat tissues. Toxicol. Appl. Pharm. 120:186‐192.
   Reed, D.J. 1994. A centrifuge filtration separation of cells, and the measurement of constituents. Methods Toxicol. 1(B):421‐430.
   Reed, D.J. and Ellis, W.W. 1982. Influence of γ‐glutamyl transpeptidase inactivation on the status of extracellular glutathione and glutathione conjugates. In Biological Reactive Intermediates IIA. (R. Snyder, D.V. Parke, J.J. Kocsis, D.J. Jollow, C.D. Gibson, and C.M. Witmer, eds.) pp. 75‐86. Plenum, New York.
   Reed, D.J., Babson, J.R., Beatty, P.W., Brodie, A.E., Ellis, W.W., and Potter, D.W. 1980. High‐performance liquid chromatography analysis of nanomole levels of glutathione, glutathione disulfide, and related thiols and disulfides. Anal. Biochem. 106:55‐62.
   Roberts, J.C. and Francetic, D.J. 1993. The importance of sample preparation and storage in glutathione analysis. Anal. Biochem. 211:183‐187.
   Rose, R.C. and Bode, A.M. 1995. Analysis of water‐soluble antioxidants by high‐pressure liquid chromatography. Biochem. J. 306:101‐105.
   Sian, J., Dexter, D.T., Cohen, G., Jenner, P.G., and Marsden, C.D. 1997. Comparison of HPLC and enzymatic recycling assays for measurement of oxidized glutathione in rat brain. J. Pharm. Pharmacol. 49:332‐335.
   Thioudellet, C., Oster, T., Leroy, P., Nicolas, A., and Wellman, M. 1995. Influence of sample preparation on cellular glutathione recovery from adherent cells in culture. Cell Biol. Toxicol. 11:103‐111.
   Tietze, F. 1969. Enzymatic method for quantitative determination of nanogram amounts of total and oxidized glutathione: Applications to mammalian blood and other tissues. Anal. Biochem. 27:502‐522.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library