Development of an Inhibition-Based Colorimetric Method For Glutathione Determination

Abstract

Aim: Glutathione (GSH, L-γ-glutamyl-L-cysteinyl-glycine), one of the major cellular antioxidants, is an important non-protein intracellular physiological antioxidant with sulphhydryl groups for detoxification of reactive oxygen species (ROS) in all living organisms. GSH deficiency has been shown to be associated with many human diseases, including cardiovascular, immune and ageing diseases, arthritis and diabetes. Therefore, the development of an accurate, reliable and sensitive method for the determination of GSH in biological fluids is essential for the understanding of GSH homeostasis in medicine and biochemical research. Materials and Methods : In this study, a very inexpensive, practical, rapid, sensitive and highly specific colourimetric method for the determination of glutathione (GSH) was developed that can be detected by the naked eye. The method is based on the inhibition of horseradish peroxidase (HRP) by GSH. As the concentration of glutathione increases, a pink coloured compound consisting of 4-chlorophenol, H2O2 and 4-aminoantipyrine (4-AAP) decomposes as a result of the reaction catalysed by HRP, thus reducing the intensity of the colour. Results: While the linear range of the developed method was found to be between 15.6-1000 M, the intra- and inter-day repeatability % coefficient of variation values of the method were less than 15%. The effect of potential interfering substances on the developed method was tested and no interference was found except for cysteine. Cysteine increased the response for GSH by 10%. The developed method was used for the determination of GSH in commercial serum samples and results were obtained between 91-106%. Conclusion: In conclusion, this study has developed a very simple, inexpensive and unique colourimetric method for the determination of GSH.