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Analytical and Bioanalytical Chemistry

, Volume 406, Issue 24, pp 5867–5876 | Cite as

Simultaneous determination of reduced and oxidized glutathione in tissues by a novel liquid chromatography-mass spectrometry method: application in an inhalation study of Cd nanoparticles

  • L. BláhováEmail author
  • J. Kohoutek
  • J. Lebedová
  • L. Bláha
  • Z. Večeřa
  • M. Buchtová
  • I. Míšek
  • K. Hilscherová
Research Paper

Abstract

The paper presents the development of an advanced extraction and fast analytical LC MS/MS method for simultaneous analyses of reduced and oxidized glutathione (GSH and GSSG, respectively) in different animal tissues. The simultaneous determination of GSH and GSSG is crucial because the amount and ratio of both GSH and GSSG may be altered in response to oxidative stress, an important mechanism of toxicity. The method uses the derivatization of free thiol groups in GSH. Its performance was demonstrated for less explored tissues (lung, brain, and liver) in mouse. The combined extraction and analytical method has very low variability and good reproducibility, maximum coefficients of variance for within-run and between-run analyses under 8 %, and low limits of quantification; for GSH and GSSG, these were 0.2 nM (0.06 ng/mL) and 10 nM (6 ng/mL), respectively. The performance of the method was further demonstrated in a model experiment addressing changes in GSH and GSSG concentrations in lung of mice exposed to CdO nanoparticles during acute 72 h and chronic 13-week exposures. Inhalation exposure led to increased GSH concentrations in lung. GSSG levels were in general not affected; nonsignificant suppression occurred only after the longer 13-week period of exposure. The developed method for the sensitive detection of both GSH and GSSG in very low tissue mass enables these parameters to be studied in cases where only a little sample is available, i.e. in small organisms or in small amounts of tissue.

Keywords

Glutathione LC MS/MS Tissues Oxidative stress Nanoparticle Cadmium oxide 

Notes

Acknowledgments

We thank Ing. Filip Mika, PhD, from the Institute of Scientific Instruments, CAS v.v.i., Brno, Czech Republic for performing the STEM analysis. The work was supported by the Czech Science Foundation grant No. P503/11/2315, project LO1214 (Research Centre for Toxic Compounds in the Environment) funded by the National Sustainability Programme of the Czech Republic, European Social Fund and the state budget of the Czech Republic (OPVK—operational programme education for competitiveness).

References

  1. 1.
    Guan X, Hoffman B, Dwivedi C, Matthees DP (2003) A simultaneous liquid chromatography/mass spectrometric assay of glutathione, cysteine, homocysteine and their disulfides in biological samples. J Pharm Biomed Anal 31:251–261CrossRefGoogle Scholar
  2. 2.
    Iwasaki Y, Saito Y, Nakano Y, Mochizuki K, Sakata O, Ito R, Saito K, Nakazawa H (2009) Chromatographic and mass spectrometric analysis of glutathione in biological samples. J Chromatogr B 877:3309–3317CrossRefGoogle Scholar
  3. 3.
    Liu J, Qu W, Kadiiska MB (2009) Role of oxidative stress in cadmium toxicity and carcinogenesis. Toxicol Appl Pharm 238:209–214CrossRefGoogle Scholar
  4. 4.
    Mika A, Skorkowski E, Stepnowski P (2013) The use of different MS techniques to determine glutathione levels in marine tissues. Food Anal Methods 6(3):789–802CrossRefGoogle Scholar
  5. 5.
    Sass JO, Endres J (1997) Quantitation of total homocysteine in human plasma by derivatization to its N(O, S)-propoxycarbonyl propyl ester and gas chromatography mass spectrometry analysis. J Chromatogr A 776:342–347CrossRefGoogle Scholar
  6. 6.
    Maeso N, Garcia-Martinez D, Ruperez FJ, Cifuentes A, Barbas C (2005) Capillary electrophoresis of glutathione to monitor oxidative stress and response to antioxidant treatments in an animal model. J Chromatogr B Analyt Technol Biomed Life Sci 822:61–69CrossRefGoogle Scholar
  7. 7.
    Mansoor MA, Svardal AM, Ueland PM (1992) Determination of the in vivo redox status of cysteine, cysteinylglycine, mohocysteine, and glutathione in human plasma. Anal Biochem 200:218–229CrossRefGoogle Scholar
  8. 8.
    Martin J, White INH (1991) Fluorometric-determination of oxidized and reduced glutathione in cells and tissues by high-performance liquid chromatography following derivatization with dansyl chloride. J Chromatogr 568:219–225CrossRefGoogle Scholar
  9. 9.
    Loughlin AF, Skiles GL, Alberts DW, Schaefer WH (2001) An ion exchange liquid chromatography/mass spectrometry method for the determination of reduced and oxidized glutathione and glutathione conjugates in hepatocytes. J Pharm Biomed Anal 26:131–142CrossRefGoogle Scholar
  10. 10.
    Norris RL, Eaglesham GK, Shaw GR, Smith MJ, Chiswell RK, Seawright AA, Moore MR (2001) A sensitive and specific assay for glutathione with potential application to glutathione disulphide, using high-performance liquid chromatography-tandem mass spectrometry. J Chromatogr B Biomed Sci Appl 762:17–23CrossRefGoogle Scholar
  11. 11.
    Santori G, Domenicotti C, Bellocchio A, Pronzato MA, Marinari UM, Cottalasso D (1997) Different efficacy of iodoacetic acid and N-ethylmaleimide in high-performance liquid chromatographic measurement of liver glutathione. J Chromatogr B Biomed Sci Appl 695:427–433CrossRefGoogle Scholar
  12. 12.
    Bouligand J, Deroussent A, Paci A, Morizet J, Vassal G (2006) Liquid chromatography–tandem mass spectrometry assay of reduced and oxidized glutathione and main precursors in mice liver. J of Chromatogr B 832:67–74CrossRefGoogle Scholar
  13. 13.
    Chang YL, Hsieh CL, Huang YM, Chiou WL, Kuo YH, Tseng MH (2013) Modified method for determination of sulfur metabolites in plant tissues by stable isotope dilution-based liquid chromatography electrospray ionization tandem mass spectrometry. Anal Biochem 442(1):24–33CrossRefGoogle Scholar
  14. 14.
    Camera E, Rinaldi M, Briganti S, Picardo M, Fanali S (2001) Simultaneous determination of reduced and oxidized glutathione in peripheral blood mononuclear cells by liquid chromatography-electro spray mass spectrometry. J Chromatogr B 757:69–78CrossRefGoogle Scholar
  15. 15.
    Reinbold J, Koehler P, Rychlik M (2014) Quantitation of glutathione and its oxidation products in erythrocytes by multiple-label stable-isotope dilution. Anal Biochem 445:41–48CrossRefGoogle Scholar
  16. 16.
    Moore T, Le A, Niemi AK, Kwan T, Cusmano-Ozog K, Enns GM, Cowan TM (2013) A new LC-MS/MS method for the clinical determination of reduced and oxidized glutathione from whole blood. J Chromatogr B Analyt Technol Biomed Life Sci 929:51–55CrossRefGoogle Scholar
  17. 17.
    Squellerio I, Caruso D, Porro B, Veglia F, Tremoli E, Cavalca V (2012) Direct glutathione quantification in human blood by LC-MS/MS: comparison with HPLC with electrochemical detection. J Pharm Biomed Anal 71:111–118CrossRefGoogle Scholar
  18. 18.
    Rhieu SY, Urbas AA, Lippa KA, Reipa V (2013) Quantitative measurements of glutathione in yeast cell lysate using 1H NMR. Anal Bioanal Chem 405:4963–4968CrossRefGoogle Scholar
  19. 19.
    Zhu P, Oe T, Blair IA (2008) Determination of cellular redox status by stable isotope dilution liquid chromatography/mass spectrometry analysis of glutathione and glutathione disulfide. Rapid Commun Mass Spectrom 22:432–440CrossRefGoogle Scholar
  20. 20.
    Monostori P, Wittmann G, Karg E, Túri S (2009) Determination of glutathione and glutathione disulfide in biological samples: an in-depth review. J Chromatogr B Analyt Technol Biomed Life Sci 877:3331–3346CrossRefGoogle Scholar
  21. 21.
    Lee GB, Brandt EB, Xiao C, Gibson AM, Le Cras TD, Brown LAS, Fitzpatrick AM, Hershey GKK (2013) Diesel exhaust particles induce cysteine oxidation and S-glutathionylation in house dust mite induced murine asthma. PLoS ONE 8(3):e60632. doi: 10.1371/journal.pone.0060632 CrossRefGoogle Scholar
  22. 22.
    Nair PMG, Park SY, Chung JW, Choi J (2013) Transcriptional regulation of glutathione biosynthesis genes, gamma-glutamyl-cysteine ligase and glutathione synthetase in response to cadmium and nonylphenol in Chironomus riparius. Environ Toxicol Phar 36(2):265–273CrossRefGoogle Scholar
  23. 23.
    Srinivas A, Jaganmohan Rao P, Selvam G, Balakrishna Murthy P, Neelakanta Reddy P (2011) Acute inhalation toxicity of cerium oxide nanoparticles in rats. Toxicol Lett 205:105–115CrossRefGoogle Scholar
  24. 24.
    Fall M, Guerbet M, Park B, Gouriou F, Dionnet F, Morin JP (2007) Evaluation of cerium oxide and cerium oxide based fuel additive safety on organotypic cultures of lung slices. Nanotoxicology 1(3):227–234CrossRefGoogle Scholar
  25. 25.
    Shvedova AA, Kisin E, Murray AR, Johnson VJ, Gorelik O, Arepalli S, Hubbs AF, Mercer RR, Keohavong P, Sussman N, Jin J, Yin J, Stone S, Chen BT, Deye G, Maynard A, Castranova V, Baron PA, Kagan VE (2008) Inhalation vs. aspiration of single-walled carbon nanotubes in C57BL/6 mice: inflammation, fibrosis, oxidative stress, and mutagenesis. Am J Physiol Lung Cell Mol Physiol 295:L552–L565CrossRefGoogle Scholar
  26. 26.
    Raemy OD, Grass RN, Stark WJ, Schumacher CM, Clift MJD, Gehr P, Rothen-Rutishauseret B (2012) Effects of flame made zinc oxide particles in human lung cells—a comparison of aerosol and suspension exposures. Part Fibre Toxicol 9:33CrossRefGoogle Scholar
  27. 27.
    Sauvageau JA, Jumarie C (2013) Different mechanisms for metal-induced adaptation to cadmium in the human lung cell lines A549 and H441. Cell Biol Toxicol 29(3):159–173CrossRefGoogle Scholar
  28. 28.
    Zhao YZ, Chen LJ, Gao S, Toselli P, Stone P, Li WD (2010) The critical role of the cellular thiol homeostasis in cadmium perturbation of the lung extracellular matrix. Toxicology 267:60–69CrossRefGoogle Scholar
  29. 29.
    Hart BA, Gong Q, Eneman JD (1996) Pulmonary metallothionein expression in rats following single and repeated exposure to cadmium aerosols. Toxicology 112:205–218CrossRefGoogle Scholar
  30. 30.
    Kirschvink N, Martin N, Fievez L, Smith N, Marlin D, Gustin P (2006) Airway inflammation in cadmium-exposed rats is associated with pulmonary oxidative stress and emphysema. Free Radic Res 40(3):241–250CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • L. Bláhová
    • 1
    Email author
  • J. Kohoutek
    • 1
  • J. Lebedová
    • 1
  • L. Bláha
    • 1
  • Z. Večeřa
    • 2
  • M. Buchtová
    • 3
  • I. Míšek
    • 3
  • K. Hilscherová
    • 1
  1. 1.Masaryk University, Faculty of Science, RECETOXBrnoCzech Republic
  2. 2.Academy of Sciences of the Czech RepublicInstitute of Analytical Chemistry, v.v.i.BrnoCzech Republic
  3. 3.Academy of Sciences of the Czech RepublicInstitute of Animal Physiology and Genetics, v.v.i.BrnoCzech Republic

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