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
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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.
KeywordsGlutathione LC MS/MS Tissues Oxidative stress Nanoparticle Cadmium oxide
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).
- 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
- 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