Comparison of three methods for in vivo quantification of glutathione in tissues of hypertensive rats

Abstract

Glutathione (γ-L-glutamyl-L-cysteinyl-glycine, GSH) is a tripeptide that is part of the antioxidant defense system and contributes to numerous redox-regulatory processes. In vivo, reduced GSH and oxidized glutathione disulfide (GSSG) are present in redox equilibrium and their ratio provides important information on the cellular redox state. Here, we compared three different methods for in vivo quantification of glutathione in tissues of hypertensive rats, an accepted animal model of oxidative stress. In the present study, we used hypertensive rats (infusion of 1 mg/kg/d angiotensin-II for 7 days) to determine the levels of reduced GSH and/or GSH/GSSG ratios in different tissue samples. We used an HPLC-based method with direct electrochemical detection (HPLC/ECD) and compared it with Ellman’s reagent (DTNB) dependent derivatization of reduced GSH to the GS-NTB adduct and free NTB (UV/Vis HPLC) as well as with a commercial GSH/GSSG assay (Oxiselect). Whereas all three methods indicated overall a decreased redox state in hypertensive rats, the assays based on HPLC/ECD and DTNB derivatization provided the most significant differences. We applied a direct, fast and sensitive method for electrochemical GSH detection in tissues from hypertensive animals, and confirmed its reliability for in vivo measurements by head-to-head comparison with two other established assays. The HPLC/ECD but not DTNB and Oxiselect assays yielded quantitative GSH data but all three assays reflected nicely the qualitative redox changes and functional impairment in hypertensive rats. However, especially our GSH/GSSG values are lower than reported by others pointing to problems in the work-up protocol.

Keywords:

• Redox state
• glutathione
• electrochemical HPLC detection
• Ellman’s reagent
• oxidative stress
• hypertension

Acknowledgments

The authors thank Jacek Zielonka (Medical College of Wisconsin, Milwaukee, WI 53226, USA) for helping to establish the electrochemical HPLC method for GSH detection in our laboratory and his helpful comments for improvement of the manuscript. The authors are indebted to Angelica Karpi, Nicole Glas and Jörg Schreiner for expert technical assistance.

Disclosure statement

The authors declare that they have no conflicts of interest.

Additional information

Funding

S.K. holds a stipend from the TransMed PhD Program of the University Medical Center Mainz funded by the Boehringer Ingelheim Foundation. P.S. holds a stipend from “Stiftung Mainzer Herz”. T.M. is PI of the DZHK (German Center for Cardiovascular Research), Partner Site Rhine-Main, Mainz, Germany. A.D. and T.M. were supported by vascular biology research grants from the Boehringer Ingelheim Foundation for the collaborative research group “Novel and neglected cardiovascular risk factors: molecular mechanisms and therapeutics”. Further support was provided by the Else-Kröner Fresenius Foundation (“CD40(L) and arterial hypertension” 2019_A110 to S.D. and “Noise and arterial hypertension” 2017_A106 to S.S.). The work exclusively contains parts of the thesis of Sanela Kalinovic.