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 GSHGSSG 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 GSHGSSG 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.