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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.

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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.
Holt, Kayla J.
Scott, Kristen L.
Martin, Sheree D.
Stupka, Nicole
Barnett, Neil W.
Conlan, Xavier A.
Journal name Analyst
Volume number 136
Issue number 12
Start page 2578
End page 2585
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
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
Persistent URL http://hdl.handle.net/10536/DRO/DU:30040441

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Every reasonable effort has been made to ensure that permission has been obtained for items included in DRO. If you believe that your rights have been infringed by this repository, please contact drosupport@deakin.edu.au.