Faisal Nuhu
Measurement of Glutathione as a Tool for Oxidative Stress Studies by High Performance Liquid Chromatography
Nuhu, Faisal; Gordon, Andrew; Sturmey, Roger; Seymour, Anne Marie; Bhandari, Sunil
Authors
Andrew Gordon
Professor Roger Sturmey R.Sturmey@hull.ac.uk
Professor of Reproductive Medicine
Anne Marie Seymour
Sunil Bhandari
Abstract
Background: Maintenance of the ratio of glutathione in the reduced (GSH) and oxidised (GSSG) state in cells is important in redox control, signal transduction and gene regulation, factors that are altered in many diseases. The accurate and reliable determination of GSH and GSSG simultaneously is a useful tool for oxidative stress determination. Measurement is limited primarily to the underestimation of GSH and overestimation GSSG as a result of auto-oxidation of GSH. The aim of this study was to overcome this limitation and develop, optimise and validate a reverse-phase high performance liquid chromatographic (HPLC) assay of GSH and GSSG for the determination of oxidant status in cardiac and chronic kidney diseases. Methods: Fluorescence detection of the derivative, glutathione-O-pthaldialdehyde (OPA) adduct was used. The assay was validated by measuring the stability of glutathione and glutathione-OPA adduct under conditions that could affect the reproducibility including reaction time and temperature. Linearity, concentration range, limit of detection (LOD), limit of quantification (LOQ), recovery and extraction efficiency and selectivity of the method were assessed. Results: There was excellent linearity for GSH (r2 = 0.998) and GSSG (r2 = 0.996) over concentration ranges of 0.1 μM–4 mM and 0.2 μM–0.4 mM respectively. The extraction of GSH from tissues was consistent and precise. The limit of detection for GSH and GSSG were 0.34 μM and 0.26 μM respectively whilst their limits of quantification were 1.14 μM and 0.88 μM respectively. Conclusion: These data validate a method for the simultaneous measurement of GSH and GSSG in samples extracted from biological tissues and offer a simple determination of redox status in clinical samples.
Citation
Nuhu, F., Gordon, A., Sturmey, R., Seymour, A. M., & Bhandari, S. (2020). Measurement of Glutathione as a Tool for Oxidative Stress Studies by High Performance Liquid Chromatography. Molecules, 25(18), Article 4196. https://doi.org/10.3390/molecules25184196
Journal Article Type | Article |
---|---|
Acceptance Date | Sep 8, 2020 |
Online Publication Date | Sep 13, 2020 |
Publication Date | Sep 2, 2020 |
Deposit Date | Sep 14, 2020 |
Publicly Available Date | Sep 15, 2020 |
Journal | Molecules |
Electronic ISSN | 1420-3049 |
Publisher | MDPI |
Peer Reviewed | Peer Reviewed |
Volume | 25 |
Issue | 18 |
Article Number | 4196 |
DOI | https://doi.org/10.3390/molecules25184196 |
Keywords | Glutathione analysis; Oxidative stress; HPLC; GSH; GSSG |
Public URL | https://hull-repository.worktribe.com/output/3605197 |
Publisher URL | https://www.mdpi.com/1420-3049/25/18/4196 |
Files
Published article
(2.8 Mb)
PDF
Copyright Statement
©2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open accessarticle distributed under the terms and conditions of the Creative Commons Attribution(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
You might also like
Practical considerations of dissolved oxygen levels for platelet function under hypoxia
(2021)
Journal Article
Metabolic remodeling in the aging heart
(2005)
Journal Article
Semiquantitative analysis of collagen types in the hypertrophied left ventricle
(2001)
Journal Article
Downloadable Citations
About Repository@Hull
Administrator e-mail: repository@hull.ac.uk
This application uses the following open-source libraries:
SheetJS Community Edition
Apache License Version 2.0 (http://www.apache.org/licenses/)
PDF.js
Apache License Version 2.0 (http://www.apache.org/licenses/)
Font Awesome
SIL OFL 1.1 (http://scripts.sil.org/OFL)
MIT License (http://opensource.org/licenses/mit-license.html)
CC BY 3.0 ( http://creativecommons.org/licenses/by/3.0/)
Powered by Worktribe © 2024
Advanced Search