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BIOXYTECH GSH/GSSG-412 Colorimetric Determination of Reduced and Oxidized Glutathione

Source: OXIS Research a division of OXIS Health Products, Inc.
Reduced glutathione (GSH), a tripeptide (g-glutamylcysteinylglycine) with a free thiol group
Introduction:

Reduced glutathione (GSH), a tripeptide (g-glutamylcysteinylglycine) with a free thiol group, is a major antioxidant in human tissues that provides reducing equivalents for the glutathione peroxidase (GPx) catalyzed reduction of hydrogen peroxide and lipid hydroperoxides to water and the respective alcohol. During this process GSH becomes oxidized glutathione (GSSG). The GSSG is then recycled to GSH by reduction by the reduced form of ยง-nicotinamide adenine dinucleotide phosphate (NADPH), catalyzed by glutathione reductase (GR). When mammalian cells are exposed to increased oxidative stress, the ratio of GSH/GSSG will decrease as a consequence of GSSG accumulation. The measure of the GSSG level, or determining the GSH/GSSG ratio, is a useful measure of oxidative stress and to monitor the effectiveness of antioxidant intervention strategies.

PRINCIPLE OF THE PROCEDURE

The accurate measurement of GSSG levels has proved very difficult due to the lower amount of GSSG in tissues and because of the absence of effective methods to prevent oxidation of GSH to GSSG during sample preparation. To measure the GSSG in tissues, Guntherberg and Rost (2) first introduced N-ethylmaleimide (NEM) to eliminate the GSH. Although NEM can react with GSH to form a stable complex and prevent the participation of the reduced form in the enzymatic assay, NEM also inhibits GR. For this reason, Griffith (3) first introduced 2-vinylpyridine (2-VP), which does not inhibit GR significantly, to derivatize GSH. However, the 2-VP reaction is relatively slow and the reagent has little solubility in an aqueous medium.

The GSH/GSSG-412 assay uses the thiol-scavenging reagent, 1-methyl-2-vinylpyridinium trifluoromethanesulfonate (M2VP) at a level that rapidly scavenges GSH but does not interfere with the GR assay.

For Research Use Only. Not For Use In Diagnostic Procedures.

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