Addressing Glutathione Redox Status in Clinical Samples by Two-Step Alkylation with N-ethylmaleimide Isotopologues.

Tamara Tomin,Matthias Schittmayer,Ruth Birner-Gruenberger

doi:10.3390/metabo10020071

Tamara Tomin, Matthias Schittmayer + Show 1 more

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https://doi.org/10.3390/metabo10020071

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Journal: Metabolites	Publication Date: Feb 16, 2020
Citations: 23	License type: CC BY 4.0

Affiliation: Medical University of Graz, TU Wien, BioTechMed-Graz

Abstract

Determination of the ratio of reduced to oxidized glutathione is of profound clinical interest in assessing the oxidative status of tissues and body fluids. However, this ratio is not yet a routine clinical parameter due to the analytically challenging interconversion of reduced (free) glutathione to oxidized (bound) glutathione. We aimed to facilitate this ratio determination in order to aid its incorporation as a routine clinical parameter. To this end, we developed a simple derivatization route that yields different isotopologues of N-ethylmaleimide alkylated glutathione from reduced and oxidized glutathione (after its chemical reduction) for mass spectrometric analysis. A third isotopologue can be used as isotopic standard for simultaneous absolute quantification. As all isotopologues have similar chromatographic properties, matrix effects arising from different sample origins can only impact method sensitivity but not quantification accuracy. Robustness, simplified data analysis, cost effectiveness by one common standard, and highly improved mass spectrometric sensitivity by conversion of oxidized glutathione to an alkylated glutathione isotopologue are the main advantages of our approach. We present a method fully optimized for blood, plasma, serum, cell, and tissue samples. In addition, we propose production of N-ethylmaleimide customized blood collection tubes to even further facilitate the analysis in a clinical setting.

Highlights

Glutathione (γ-L-glutamyl-L-cysteinyl-glycine) is the main endogenous, thiol-based antioxidant existing in reduced (GSH) and oxidized (GSSG) forms in mammalian cells
The method is suitable for samples acquired by minimally invasive techniques such as fine needle aspiration biopsies (FNAB) (~500,000 cells per FNAB [11])
Complete GSH derivatization in tissue was achieved by vortexing 3–4 mg of skeletal or cardiac muscle tissue cut into small pieces in NEM containing phosphate-buffered saline (PBS), without the need for additional lysis steps, greatly simplifying sample handling

Summary

Introduction

Glutathione (γ-L-glutamyl-L-cysteinyl-glycine) is the main endogenous, thiol-based antioxidant existing in reduced (GSH) and oxidized (GSSG) forms in mammalian cells. More than 98% of the total glutathione pool consists of GSH in the concentration range of 1–10 mM, while GSSG accounts for the residual 1–% [1,2,3]. Its predominant role is to remove hydrogen peroxides by acting as a cofactor for the selenium-based enzyme family of glutathione peroxidases (GPx) [3], a reaction during which it oxidizes to GSSG [4]. While GSSG is recycled by glutathione reductase (GR) to GSH in an NADPH-dependent manner [5], extensive oxidative stress can alter the GSH/GSSG ratio as a consequence of either insufficient capacity of GR or redox imbalance [6]. The GSH-to-GSSG ratio is used as a readout of tissue redox state [7]

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