Automated continuous-flow colorimetric determination of glutathione peroxidase with dichloroindophenol

Automation of the glutathione peroxidase enzyme assay has been problematical. Although such methods have been reported, they do not give equivalent results to the standard manual assay, wherein glutathione oxidation is coupled to NADPH oxidation via glutathione reductase. We report here the development of a fully automated, continuous-flow, colorimetric method for glutathione peroxidase assays in which glutathione oxidation is monitored by its effect on the reaction of glutathione with the colorimetric reagent 2,6-dichloroindophenol. This method has a linear response to glutathione peroxidase over an 800-fold range of enzyme concentrations. Results of assays done by this method in erythrocyte and plasma samples correlate well with the standard manual coupled assay (r = 0.997 and 0.923, respectively), with no evidence of systematic errors. The assay works equally well with hydrogen peroxide or cumene hydroperoxide as substrate and shows the same selectivity toward glutathione S-transferases as the standard coupled assay. The within-day repeatability and the between-day reproducibility were estimated as 1.1 to 6.4% and 1.3 to 7.1% (relative standard deviation), respectively. This method is suitable for enzyme determinations in whole blood, erythrocytes, plasma, and serum from rats, rabbits, monkeys, and humans.