Enzymatic method for rapid determination of oxidized and reduced glutathione

摘要

The present invention relates to a method for the silmultaneous quantitation of oxidized and reduced glutathione (GSSG.GSH) by introducing a step of treating a sample with a GSH trapping agent, to the conventional enzymatic assay method of glutathione employing oxidizing agent and glutathione reductase. The method for rapid quantitation of oxidized and reduced glutathione comprises the steps of: (i) obtaining a tissue sample and then preparing a homogenate of the tissue whose metabolism is blocked; (ii) preparing equal volume of the homogenate fractions with and without a GSH trapping agent, and adding phosphate buffer, distilled water and NADPH solution to each of the fractions; (iii) adding glutathione reductase and oxidizing agent to each of the fractions to initiate reaction; and, (iv) measuring absorbances of the reaction mixtures containing the fractions with and without a GSH trapping agent to quantitate oxidized glutathione (GSSG) and total glutathione, respectively, then,substrating the amount of GSSG from that of total glutathione to determine the amount of reduced glutathione (GSH). In accordance with the invention, the amount of oxidized and reduced glutathione can be simultaneously measured in a simple and accurate manner, which makes possible its practical application in the diagnoses and treatments of diseases caused by the abnormality of oxidized/reduced state of the cells.