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

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.