Determination of intracellular glutathione and glutathione disulfide using high performance liquid chromatography with acidic potassium permanganate chemiluminescence detection

doi:10.1039/c1an00004g

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Determination of intracellular glutathione and glutathione disulfide using high performance liquid chromatography with acidic potassium permanganate chemiluminescence detection

McDermott, Geoffrey P., Francis, Paul S., Holt, Kayla J., Scott, Kristen L., Martin, Sheree D., Stupka, Nicole, Barnett, Neil W. and Conlan, Xavier A. 2011, Determination of intracellular glutathione and glutathione disulfide using high performance liquid chromatography with acidic potassium permanganate chemiluminescence detection, Analyst, vol. 136, no. 12, pp. 2578-2585, doi: 10.1039/c1an00004g.

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Title	Determination of intracellular glutathione and glutathione disulfide using high performance liquid chromatography with acidic potassium permanganate chemiluminescence detection
Author(s)	McDermott, Geoffrey P. Francis, Paul S. orcid.org/0000-0003-4165-6922 Holt, Kayla J. Scott, Kristen L. Martin, Sheree D. Stupka, Nicole orcid.org/0000-0002-1000-1707 Barnett, Neil W. Conlan, Xavier A. orcid.org/0000-0003-0829-0551
Journal name	Analyst
Volume number	136
Issue number	12
Start page	2578
End page	2585
Total pages	8
Publisher	Royal Society of Chemistry
Place of publication	Cambridge, United Kingdom
Publication date	2011
ISSN	0003-2654 1364-5528
Summary	Measurement of glutathione (GSH) and glutathione disulfide (GSSG) is a crucial tool to assess cellular redox state. Herein we report a direct approach to determine intracellular GSH based on a rapid chromatographic separation coupled with acidic potassium permanganate chemiluminescence detection, which was extended to GSSG by incorporating thiol blocking and disulfide bond reduction. Importantly, this simple procedure avoids derivatisation of GSH (thus minimising auto-oxidation) and overcomes problems encountered when deriving the concentration of GSSG from ‘total GSH’. The linear range and limit of detection for both analytes were 7.5 × 10−7 to 1 × 10−5 M, and 5 × 10−7 M, respectively. GSH and GSSG were determined in cultured muscle cells treated for 24 h with glucose oxidase (0, 15, 30, 100, 250 and 500 mU mL−1), which exposed them to a continuous source of reactive oxygen species (ROS). Both analyte concentrations were greater in myotubes treated with 100 or 250 mU mL−1 glucose oxidase (compared to untreated controls), but were significantly lower in myotubes treated with 500 mU mL−1 (p < 0.05), which was rationalised by considering measurements of H2O2 and cell viability. However, the GSH/GSSG ratio in myotubes treated with 100, 250 and 500 mU mL−1 glucose oxidase exhibited a dose-dependent decrease that reflected the increase in intracellular ROS.
Language	eng
DOI	10.1039/c1an00004g
Field of Research	030108 Separation Science
Socio Economic Objective	970103 Expanding Knowledge in the Chemical Sciences
HERDC Research category	C1 Refereed article in a scholarly journal
Related work	DU:30042598
Copyright notice	©2011, Royal Society of Chemistry
Free to Read?	Yes
Persistent URL	http://hdl.handle.net/10536/DRO/DU:30040441

Document type:	Journal Article
Collections:	Faculty of Science, Engineering and Built Environment School of Life and Environmental Sciences Institute for Technology Research and Innovation Open Access Collection GTP Research


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