Analysis of GSH and GSSG after derivatization with N-ethylmaleimide

Journal name:
Nature Protocols
Volume:
8,
Pages:
1660–1669
Year published:
DOI:
doi:10.1038/nprot.2013.095
Published online

Abstract

This protocol describes a procedure for determining glutathione (GSH) and glutathione disulfide (GSSG) concentrations in blood and other tissues. Artifactual oxidation to GSSG of 5–15% of the GSH found in a sample can occur during deproteination of biological samples with any of the commonly used acids, with consequent marked overestimation of GSSG. This can be prevented by derivatizing GSH with the alkylating agent N-ethylmaleimide (NEM) to form GS-NEM before acid deproteination, followed by back-extraction of excess NEM from the deproteinized samples with dichloromethane. GSSG concentration is then measured by spectrophotometry with the GSH recycling method, on the basis of conversion of GSSG to GSH by glutathione reductase and NADPH and reaction with 5,5′-dithiobis-(2-nitrobenzoic acid). GSH concentration is instead measured by either of two methods: by analysis of GS-NEM conjugates by HPLC in the same sample that is used to measure GSSG or, alternatively, by analysis of GSH by spectrophotometry (GSH recycling method) on one additional sample aliquot that has not been derivatized with NEM. The procedure can assay GSH and GSSG in blood and other tissues in 30 min or less.

At a glance

Figures

  1. Schematic representation of the mechanism of the GSH recycling method.
    Figure 1: Schematic representation of the mechanism of the GSH recycling method.

    The assay measures GSH and GSSG after its reduction to two molecules of GSH (2GSH) by GR in the presence of NADPH. GSH reacts with DTNB to form TNB and GS-TNB (glutathione adduct of TNB). GS-TNB is then reduced by GSH with the formation of GSSG and TNB or (more probably) by GR and NADPH with the generation of GSH and TNB. The formed TNB is measured at 412 nm. Yellow circles indicate the only compound that develops absorbance at 412 nm.

  2. Graphical representation of the preanalytical manipulation of blood samples for GSH and GSSG analyses.
    Figure 2: Graphical representation of the preanalytical manipulation of blood samples for GSH and GSSG analyses.

    The easier procedure consists in detecting GSH by HPLC and GSSG by spectrophotometry. In this case, only one blood sample is collected, and a tube containing both tripotassium EDTA and NEM is used. If HPLC is not available, tGSH is measured by spectrophotometry. In this case, an additional blood sample needs to be collected into a tube containing just tripotassium EDTA. The figure refers to typical amounts of blood and reagents used for human samples.

  3. Representative tracings obtained by applying the GSH recycling method for GSSG analysis in blood.
    Figure 3: Representative tracings obtained by applying the GSH recycling method for GSSG analysis in blood.

    (a) A blank analysis is carried out by adding into the cuvette all the reagents without the sample (substituted by TCA) and, after 1 min, 10 μl of 10 μM GSSG. Slope blank = 0.0048 ΔA.U. min−1; slope blank + GSSG standard = 0.0297 ΔA.U. min−1. (b) The measurement of GSSG in blood is performed by adding into the cuvette all the reagents and the acidified supernatant after NEM extraction. After 1 min, 10 μl of 10 μM GSSG is added and the reaction is registered for 1 additional minute. Slope sample = 0.015 ΔA.U. min−1; slope sample + GSSG standard = 0.0398 ΔA.U. min−1. Tracings must be linear.

  4. Representative chromatogram of the GS-NEM conjugate in blood samples.
    Figure 4: Representative chromatogram of the GS-NEM conjugate in blood samples.

    The GS-NEM conjugate was analyzed by reversed-phase HPLC with UV detection at 265 nm in supernatants obtained from NEM-treated blood after acidification. GS-NEM conjugate separation results in two peaks with the same area at 4.29 min and 4.95 min r.t. These two peaks form as a consequence of the generation of diastereomers, which are separable under achiral chromatographic conditions. The unreacted NEM gives a peak at an r.t. of 5.8 min. mA.U., milliabsorbance units.

  5. GSH titration by spectrophotometer
    Supplementary Fig. 1: GSH titration by spectrophotometer

    Spectrophotometer is set at 412 nm wavelength and a continuous reading is performed for a few minutes, until a plateau is reached. 1 mL PB50, 40 µM (final concentration) GSH and 0.1 mM (final concentration) DTNB are added into optical cuvettes in this order. A typical curve obtained by this procedure is shown in the figure. Other details are described in Supplementary Methods.

  6. Analysis of GSH by HPLC in rat tissues other than blood
    Supplementary Fig. 2: Analysis of GSH by HPLC in rat tissues other than blood

    Some tissues present a slight peak around 4.95 min r.t. that may overlap with one of the peaks of GS-NEM. An example is shown in the figure. Rat lungs were removed and rapidly washed. One lung was homogenized in BSAN buffer as described in PROCEDURE, the other one was homogenized in the same buffer omitting NEM. After deproteination with TCA15, both samples were analyzed by HPLC. The elution profile of the sample homogenized in BSAN buffer (blue tracing) is characterized by the presence of both the two symmetrical peaks corresponding to GS-NEM conjugate (4.29 min and 4.95 min r.t.) and the peak corresponding to the excess of NEM (5.8 min r.t.). In the absence of NEM an interfering peak in correspondence of the GS-NEM peak eluting at 4.95 min r.t. can be noted (red tracing). These data enhance the notion that even if, in theory, either peaks relative to GS-NEM conjugate can be used for calculations, the peak eluting at about 4.29 min r.t. is to be preferred since, in our experience, it is always free of interferences.

  7. Stability of GS-NEM conjugate
    Supplementary Fig. 3: Stability of GS-NEM conjugate

    Human blood was processed for GSH analysis by HPLC, i.e. collection into tubes containing K3EDTA/NEM310 and deproteination by addition of TCA15 (as described in PROCEDURE). The blue tracing shows the chromatogram obtained by freshly analyzing the sample. The same sample was then analyzed after 72 hours of storage at room temperature (red tracing). No significant difference was observed between the two tracings and the area of the peak eluting at 4.29 min r.t. was identical. These data indicate that acidified samples are stable for at less 48 hours at room temperature.

References

  1. Wu, G., Fang, Y.Z., Yang, S., Lupton, J.R. & Turner, N.D. Glutathione metabolism and its implications for health. J. Nutr. 134, 489492 (2004).
  2. Jones, D.P., Mody, V.C. Jr., Carlson, J.L., Lynn, M.J. & Sternberg, P. Jr. Redox analysis of human plasma allows separation of pro-oxidant events of aging from decline in antioxidant defenses. Free Radic. Biol. Med. 33, 12901300 (2002).
  3. Giustarini, D., Dalle-Donne, I., Lorenzini, S., Milzani, A. & Rossi, R. Age-related influence on thiol, disulfide, and protein-mixed disulfide levels in human plasma. J. Gerontol. A Biol. Sci. Med. Sci. 61, 10301038 (2006).
  4. Schafer, F.Q. & Buettner, G.R. Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple. Free Radic. Biol. Med. 30, 11911212 (2001).
  5. Ballatori, N. et al. Glutathione dysregulation and the etiology and progression of human diseases. Biol. Chem. 390, 191214 (2009).
  6. Dalle-Donne, I. et al. Molecular mechanisms and potential clinical significance of S-glutathionylation. Antioxid. Redox Signal. 10, 445473 (2008).
  7. Giustarini, D., Dalle-Donne, I., Tsikas, D. & Rossi, R. Oxidative stress and human diseases: origin, link, measurement, mechanisms, and biomarkers. Crit. Rev. Clin. Lab. Sci. 46, 241281 (2009).
  8. Giustarini, D., Dalle-Donne, I., Milzani, A. & Rossi, R. Low molecular mass thiols, disulfides and protein mixed disulfides in rat tissues: influence of sample manipulation, oxidative stress and ageing. Mech. Ageing Dev. 132, 141148 (2011).
  9. Bruce, M. et al. Multiple glutathione disulfide removal pathways mediate cytosolic redox homeostasis. Nat. Chem. Biol. 9, 119125 (2013).
  10. Kojer, K. et al. Glutathione redox potential in the mitochondrial intermembrane space is linked to the cytosol and impacts the Mia40 redox state. EMBO J. 31, 31693182 (2012).
  11. Rossi, R. et al. Blood glutathione disulfide: in vivo factor or in vitro artifact? Clin. Chem. 48, 742753 (2002).
  12. Srivastava, S.K. & Beutler, E. Oxidized glutathione levels in erythrocytes of glucose-6-phosphate-dehydrogenase–deficient subjects. Lancet 2, 2324 (1968).
  13. Tietze, F. Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal. Biochem. 27, 502522 (1969).
  14. Rahman, I., Kode, A. & Biswas, S.K. Assay for quantitative determination of glutathione and glutathione disulfide levels using enzymatic recycling method. Nat. Protoc. 1, 31593165 (2006).
  15. Akerboom, T.P.M. & Sies, H. Assay of glutathione, glutathione disulfide, and glutathione mixed disulfides in biological samples. Methods Enzymol. 77, 373382 (1981).
  16. Garcia-Saura, M.F. et al. Nitroso-redox status and vascular function in marginal and severe ascorbate deficiency. Antioxid. Redox Signal. 17, 937950 (2012).
  17. Lorente-Cantarero, F.J. et al. Prepubertal children with suitable fitness and physical activity present reduced risk of oxidative stress. Free Radic. Biol. Med. 53, 415420 (2012).
  18. Asensi, M. et al. A high-performance liquid chromatography method for measurement of oxidized glutathione in biological samples. Anal. Biochem. 217, 323328 (1994).
  19. Griffith, O.W. Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinylpyridine. Anal. Biochem. 106, 207212 (1980).
  20. Rossi, R., Dalle-Donne, I., Milzani, A. & Giustarini, D. Oxidized forms of glutathione in peripheral blood as biomarkers of oxidative stress. Clin. Chem. 52, 14061414 (2006).
  21. Giustarini, D., Dalle-Donne, I., Colombo, R., Milzani, A. & Rossi, R. An improved HPLC measurement for GSH and GSSG in human blood. Free Radic. Biol. Med. 35, 13651372 (2003).
  22. Giustarini, D. et al. Protein glutathionylation in erythrocytes. Clin. Chem. 49, 327330 (2003).
  23. Beutler, E. Effect of flavin compounds on glutathione reductase activity: in vivo and in vitro studies. J. Clin. Invest. 48, 19571966 (1969).
  24. Ellman, G.L. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82, 7077 (1959).
  25. Guntherberg, H. & Rost, J. The true oxidized glutathione content of red blood cells obtained by new enzymic and paper chromatographic methods. Anal. Biochem. 15, 205210 (1966).
  26. Dalle-Donne, I., Rossi, R., Colombo, R., Giustarini, D. & Milzani, A. Biomarkers of oxidative damage in human disease. Clin. Chem. 52, 601623 (2006).
  27. Reed, D.J. et al. High-performance liquid chromatography analysis of nanomole levels of glutathione, glutathione disulfide, and related thiols and disulfides. Anal. Biochem. 106, 5562 (1980).
  28. Giustarini, D., Dalle-Donne, I., Milzani, A. & Rossi, R. Detection of glutathione in whole blood after stabilization with N-ethylmaleimide. Anal. Biochem. 415, 8183 (2011).
  29. Colombo, G. et al. Oxidative damage in human gingival fibroblasts exposed to cigarette smoke. Free Radic. Biol. Med. 52, 15841596 (2012).
  30. Kuninori, T. & Nishiyama, J. Some properties of diastereomers formed in the reactions of N-ethylmaleimide with biological thiols. Agric. Biol. Chem. 49, 24532454 (1985).
  31. Khazim, K. et al. Glutathione redox potential is low and glutathionylated and cysteinylated hemoglobin levels are elevated in maintenance hemodialysis patients. Transl. Res. 162, 1625 (2013).
  32. Rossi, R. et al. Differential thiol status in blood of different mouse strains exposed to cigarette smoke. Free Radic. Res. 43, 538545 (2009).
  33. Guan, X., Hoffman, B., Dwivedi, C. & Matthees, D.P. A simultaneous liquid chromatography/mass spectrometric assay of glutathione, cysteine, homocysteine and their disulfides in biological samples. J. Pharm. Biomed. Anal. 31, 251261 (2003).
  34. Shaik, I.H. & Mehvar, R. Rapid determination of reduced and oxidized glutathione levels using a new thiol-masking reagent and the enzymatic recycling method: application to the rat liver and bile samples. Anal. Bioanal. Chem. 385, 105113 (2006).
  35. Rebrin, I. et al. Effect of antioxidant-enriched diets on glutathione redox status in tissue homogenates and mitochondria of the senescence-accelerated mouse. Free Radic. Biol. Med. 39, 549557 (2005).
  36. Papp, A., Németh, I., Karg, E. & Papp, E. Glutathione status in retinopathy of prematurity. Free Radic. Biol. Med. 27, 738743 (1999).

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Author information

Affiliations

  1. Department of Life Sciences, Laboratory of Pharmacology and Toxicology, University of Siena, Siena, Italy.

    • Daniela Giustarini &
    • Ranieri Rossi
  2. Department of BioSciences, University of Milan, Milan, Italy.

    • Isabella Dalle-Donne &
    • Aldo Milzani
  3. Division of Nephrology, Department of Medicine, The University of Texas Health Science Center, San Antonio, Texas, USA.

    • Paolo Fanti
  4. Audie L. Murphy Veterans Affairs (VA) Hospital, San Antonio, Texas, USA.

    • Paolo Fanti

Contributions

D.G. performed the experiments and wrote the manuscript; I.D-.D. and A.M. analyzed data; P.F. wrote the manuscript; and R.R. supervised the work and wrote the manuscript.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

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Supplementary information

Supplementary Figures

  1. Supplementary Figure 1: GSH titration by spectrophotometer (55 KB)

    Spectrophotometer is set at 412 nm wavelength and a continuous reading is performed for a few minutes, until a plateau is reached. 1 mL PB50, 40 µM (final concentration) GSH and 0.1 mM (final concentration) DTNB are added into optical cuvettes in this order. A typical curve obtained by this procedure is shown in the figure. Other details are described in Supplementary Methods.

  2. Supplementary Figure 2: Analysis of GSH by HPLC in rat tissues other than blood (102 KB)

    Some tissues present a slight peak around 4.95 min r.t. that may overlap with one of the peaks of GS-NEM. An example is shown in the figure. Rat lungs were removed and rapidly washed. One lung was homogenized in BSAN buffer as described in PROCEDURE, the other one was homogenized in the same buffer omitting NEM. After deproteination with TCA15, both samples were analyzed by HPLC. The elution profile of the sample homogenized in BSAN buffer (blue tracing) is characterized by the presence of both the two symmetrical peaks corresponding to GS-NEM conjugate (4.29 min and 4.95 min r.t.) and the peak corresponding to the excess of NEM (5.8 min r.t.). In the absence of NEM an interfering peak in correspondence of the GS-NEM peak eluting at 4.95 min r.t. can be noted (red tracing). These data enhance the notion that even if, in theory, either peaks relative to GS-NEM conjugate can be used for calculations, the peak eluting at about 4.29 min r.t. is to be preferred since, in our experience, it is always free of interferences.

  3. Supplementary Figure 3: Stability of GS-NEM conjugate (106 KB)

    Human blood was processed for GSH analysis by HPLC, i.e. collection into tubes containing K3EDTA/NEM310 and deproteination by addition of TCA15 (as described in PROCEDURE). The blue tracing shows the chromatogram obtained by freshly analyzing the sample. The same sample was then analyzed after 72 hours of storage at room temperature (red tracing). No significant difference was observed between the two tracings and the area of the peak eluting at 4.29 min r.t. was identical. These data indicate that acidified samples are stable for at less 48 hours at room temperature.

PDF files

  1. Supplementary Figure 1: GSH titration by spectrophotometer. (483 KB)

    The spectrophotometer is set at a 412-nm wavelength and a continuous reading is performed for a few minutes until a plateau is reached. 1 ml of PB50, 40 μM (final concentration) GSH and 0.1 mM (final concentration) DTNB are added into optical cuvettes in this order. A typical curve obtained by this procedure is shown. Other details are described in the Supplementary Methods.

  2. Supplementary Figure 2: Analysis of GSH by HPLC in rat tissues other than blood. (2.57 MB)

    Some tissues present a slight peak around an r.t. of 4.95 min that may overlap with one of the peaks of GS-NEM. An example is shown. Rat lungs were removed and rapidly washed. One lung was homogenized in BSAN buffer as described in the PROCEDURE; the other one was homogenized in the same buffer omitting NEM. After deproteination with TCA15, both samples were analyzed by HPLC. The elution profile of the sample homogenized in BSAN buffer (blue tracing) is characterized by the presence of both the two symmetrical peaks corresponding to the GS-NEM conjugate (4.29 min and 4.95 min r.t.) and the peak corresponding to the excess of NEM (5.8 min r.t.). In the absence of NEM, an interfering peak corresponding to he GS-NEM peak eluting at 4.95 min r.t. can be noted (red tracing). These data enhance the notion that even if, in theory, either peak relative to the GS-NEM conjugate can be used for calculations, the peak eluting at about 4.29 min r.t. is preferred, as in our experience, it is always free of interference.

  3. Supplementary Figure 3: Stability of GS-NEM conjugate. (560 KB)

    Human blood was processed for GSH analysis by HPLC (i.e., by collection into tubes containing K3EDTA/NEM310 and deproteination by addition of TCA15 (as described in the PROCEDURE)). The blue tracing shows the chromatogram obtained by freshly analyzing the sample. The same sample was then analyzed after 72 h of storage at room temperature (red tracing). No significant difference was observed between the two tracings, and the area of the peak eluting at 4.29 min r.t. was identical. These data indicate that acidified samples are stable for at least 48 h at room temperature.

  4. Supplementary Methods (506 KB)

    Titration of NADPH, activity of glutathione reductase and preparation of RBCs.

  5. Supplementary Table 1 (526 KB)

    Measured levels of GSH and GSSG in human whole blood by applying different methodological protocols.

  6. Supplementary Table 2 (483 KB)

    Recovery of GSH and GSSG added to blood.

Additional data