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Analysis of glutathione in supernatants and lysates of a human proximal tubular cell line from perfusion culture upon intoxication with cadmium chloride by HPLC and LC-ESI-MS

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Abstract

A simple and highly effective reversed-phase (RP) high-performance liquid chromatography (HPLC) method is described for analysing glutathione (GSH) and glutathione disulfide (GSSG) in out-flowing supernatants and lysates of perfusion cell cultures of human kidney cells (HK-2 cells) continuously exposed to cadmium chloride (CdCl2), which is a well-known nephrotoxin. The developed linear liquid chromatographic gradient employs monolithic poly(styrene-co-divinylbenzene) (PS/DVB) as a stationary phase and is adaptable for coupling to mass spectrometry via an electrospray ionisation interface (LC-ESI/MS), which is carried out in case of co-eluting peaks. This study presents a quantitative assay of glutathione over the time of experiment and cell lysates at the end of the experiment. The assay of out-flowing supernatants has the potential to be applied as an online assay in high time resolution. Glutathione (reduced and oxidised, GSH and GSSG) is chosen as an indicator for toxic effects in the cultured cells. In principle it is possible to show the concentration of glutathione as a function of time in an investigation of exposure of the HK-2 cell line to CdCl2. In addition to glutathione analysis, well-established assays of cell death such as enzyme release and cell viability are performed to obtain information about the number of living cells. Toxicity of 5 μM CdCl2 is manifested in all of the assays applied. Fast (<7 min) and highly reproducible (max. aberration 4.7%) determination of glutathione could be achieved.

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Acknowledgements

This work was part of a contract study (21471-2003-12 F1ED ISP AT) performed for the European Centre for the Validation of Alternative Methods (ECVAM), Joint Research Centre of the European Commission, Ispra, Italy. Support was also given by the Austrian Science Foundation (FWF), SFB-Project 021 (Vienna, Austria), the Genome Research in Austria GEN-AU (Federal Ministry for Education, Science and Culture, Vienna, Austria) and by the West Austrian Initiative for Nano Networking (WINN).

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Authors and Affiliations

  1. Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens University, Innrain 52a, 6020, Innsbruck, Austria

    Hans Hahn, Christian W. Huck, Matthias Rainer, Muhammad Najam-ul-Haq, Rania Bakry & Günther K. Bonn

  2. Department of Physiology, Medical University, Fritz-Pregl-Str. 3/1, 6020, Innsbruck, Austria

    Thomas Abberger, Paul Jennings & Walter Pfaller

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  1. Hans Hahn
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  2. Christian W. Huck
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  3. Matthias Rainer
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  4. Muhammad Najam-ul-Haq
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  5. Rania Bakry
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  6. Thomas Abberger
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  7. Paul Jennings
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  9. Günther K. Bonn
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Correspondence to Christian W. Huck.

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Hahn, H., Huck, C.W., Rainer, M. et al. Analysis of glutathione in supernatants and lysates of a human proximal tubular cell line from perfusion culture upon intoxication with cadmium chloride by HPLC and LC-ESI-MS. Anal Bioanal Chem 388, 1763–1769 (2007). https://doi.org/10.1007/s00216-007-1401-1

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  • DOI: https://doi.org/10.1007/s00216-007-1401-1

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