Rapid Method for the Quantification of Reduced and Oxidized Glutathione in Human Plasma and Saliva

Abstract

A new method is developed to determine the concentrations of reduced (GSH) and oxidized glutathione (GSSG), and it enables the calculation of the GSH:GSSG ratios in human plasma and saliva samples. The assay is based on the masking of GSH in a GSH and GSSG mixture via a 1,4-addition reaction with p-benzoquinone (BQ), followed by enzymatic kinetic measurement. The enzyme, glutathione reductase, is highly specific to glutathione. Excess BQ can thus be easily removed by the addition of non-GSH thiols. The assay takes less than 2 min, is suitable for a short-time-scale study, and minimizes the in vitro underestimation of the GSH:GSSG ratio arising from the degradation of GSH and formation of GSSG. We further show in this paper that the stability of the total glutathione content (GSH + GSSG) and GSH in saliva is significantly greater than in plasma, encouraging the development of noninvasive saliva sensing.

Chemical analysis in saliva and the search for salivary biomarkers – a tutorial review

“…30 min) due to the activity of γ-glutamyltransferase enzyme and metal-catalyzed oxidation of GSH to GSSG. 148 On the other hand, salivary glutathione displays significantly greater stability. At the temperature of -20 °C, saliva GSH has been demonstrated to be stable for 11 days, 169 while at room temperature, saliva GSH is stable for at least 90 min.…”

“…At the temperature of -20 °C, saliva GSH has been demonstrated to be stable for 11 days, 169 while at room temperature, saliva GSH is stable for at least 90 min. 148 The stability of saliva GSH offers a great advantage in diagnostic/clinical applications in addition to the non-invasive nature of saliva collection.…”

Chemical Monitoring in Clinical Settings: Recent Developments toward Real-Time Chemical Monitoring of Patients

“…Advances have been made to improve the detection of molecules in saliva, such as an antifouling bio-interface plasmonic sensor for HepB, 158 and a salivary cortisol sensor using fibre optics. 159 Additionally, advances include a new spectrophotometric assay method of measuring glutathione (a marker for oxidative stress), 160 and a non-enzymatic method of detecting glucose in saliva demonstrating low detection limit and good resolution in low glucose concentration human saliva. 161 Patients can willingly give a sample of saliva on demand.…”

Three-component working electrode micron-sized Ag particles/tiO2 layer/Ti: template electrochemical synthesis and potential use as electrochemical sensor for glutathione detection

“…Currently, a very important task of biomaterial chemistry is the development of new sensory materials for the analysis of biological markers that indicate the presence of certain metabolic [1] or pathological [2] processes. Glutathione (GSH) determination is of the great importance in the diagnosis [3][4][5]. This is due to the fact that GSH is responsible for antioxidant activity in the body, and its content in saliva can serve as a criterion for the diagnosis of a number of diseases [3,6,7].…”