Determination of glutathione in biological material by flow-injection analysis using an enzymatic recycling reaction

Abstract

A sensitive and specific assay for glutathione using a recycling reaction followed by spectrophotometric detection in a flow-injection analysis system is presented. The proposed method provides specific amplification of the response to glutathione by combined use of the enzyme GSSG reductase and the chromogenic reagent 5,5′-dithiobis(2-nitrobenzoic acid). Both oxidized (GSSG) and reduced (GSH) glutathione are detected, so that GSSG must be determined separately after alkylation of the GSH with N-ethylmaleimide. The sensitivity is controlled by the number of times the cycle occurs and therefore by the residence time of the sample in the reactor. This time depends on the reactor length and the flow rate. The influence of residence time, temperature, and enzyme concentration on the response has been studied and the optimum reaction conditions have been selected. The sample throughput is as high as 30 h−1 and the detection limit is 1 pmol GSH at a signal-to-noise ratio of 3. The method has been evaluated by the quantification of GSH and GSSG in isolated hepatocytes. A high correlation between the new flow-injection analysis method and the original spectrophotometric batch assay has been found (slope = 1.039, INTERCEPT = 0.6, n = 216, r = 0.977). The main advantages of the proposed method are high sample throughput, high sensitivity, and good reproducibility.