Quantifying Reduced Glutathione by Square-wave Voltammetry

Marcos Vergilio Corrãªa-Da-Silva

doi:10.4172/2155-6210.1000133

Abstract

This study examines the use of square wave voltammetry (SWV) to quantify reduced glutathione (GSH) dissolved in phosphate buffer (pH 7.5) using a static mercury drop electrode (working), Ag/AgCl electrode (standard) and a platinum wire (auxiliary). The applied voltage ranged from -0.7 to -0.2 V. Increasing concentrations of GSH (13-188 μmol/L) correlated with the voltammogram peak area (R2=0.99) and with the current at peak potential (Ip) (R=0.99). The reaction of GSH with diamide was monitored for validation of the method. Addition of increasing concentrations of diamide (13.3-50.8 μmol/L) to a fixed concentration of GSH (120 μmol/L) decreased the Ip, and the results obtained presented a relative deviation (RD) ≤ 14.5% (compared with expected concentrations by stoichiometry) for GSH concentrations above 33.8 μmol/L, whereas the spectrophotometric method (Elman’s reagent) presented RD ≤ 25.6%. These data indicate that SWV method is more accurate and presented equal precision (SD<8%) as compared to the commonly used spectrophotometric method. This method seems suitable for measuring GSH concentrations at room temperature and pH 7.5 (near biological conditions). Other advantages of this method that make it highly desirable for rapid diagnostic purposes include low cost, simplicity, sensitivity, rapid response and no prior sample preparation.

Highlights

The reduced glutathione/glutathione disulfide ratio (GSH/GSSG) is crucially important for maintaining an appropriate redox state necessary for cellular metabolic functions
A typical SW voltammetric response of GSH (61.5 μmol/L) at a static mercury drop electrode recorded in phosphate buffer at pH 7.5 is shown in figure 1
The concentration of GSH directly correlates with the peak area (R2=0.99) obtained by integrating the curves of the voltammograms and the results are presented in figure 3

Summary

Introduction

The reduced glutathione/glutathione disulfide ratio (GSH/GSSG) is crucially important for maintaining an appropriate redox state necessary for cellular metabolic functions. Many proteins, including enzymes of cell metabolism and regulation [1,2,3,4,5,6], transcription factors and regulators of cellular cycle [7,8,9] are potentially influenced by the formation of glutathione adducts [6]. In addition to regulating these processes, GSH participates in the antioxidant system as a preferred target of radical species. It chelates metal ions and is a substrate for the enzyme glutathione peroxidase, which maintains adequate levels of hydroperoxides (H2O2 or ROOH) [10,11,12]. Even small changes in GSH concentrations are related to oxidative stress, which, depending on its intensity, has been associated with the onset or progression of many diseases such as atherosclerosis, cancer, psoriasis, Alzheimer's, hypertension, heart and liver disease and with important physiological processes such as aging and physical exercise [12,14,15,16,17,18,19,20,21]

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