Dynamic monitoring of glutathione in erythrocytes, without a separation step, in the presence of an oxidant insult

A method for the quantitative determination of the antioxidant form of glutathione (GSH) in red blood cells (RBCs) is described that does not require separation of the analyte of interest from the complex cellular matrix. The measurement portion of the analysis is performed using fluorescence spectrophotometry after monochlorobimane ( a recognized probe for GSH) is added to a mixture containing RBCs and glutathione transferase (GST). This method was employed to determine the GSH concentration (0.042 +/- 0.002 mM) in a solution of 1% RBCs obtained from rabbits (n = 6). When spiked with authentic GSH (0.50 mu mol), 99.8% of the GSH was recovered. Addition of GST to the sample mixture enabled most measurements to be made after 5-10 min of reaction time. Importantly, a decrease in GSH was measured upon the addition of a recognized oxidant ( diamide) to the RBC sample followed by a subsequent return to normal levels of GSH. The ability of the GSH to recover from the oxidant attack occurred in a dose-dependent manner, requiring 30 and 90 min to recover from oxidant insults of 20 and 40 mu M diamide, respectively. The antioxidant capabilities of the GSH were able to be monitored in real time, thus providing a method to dynamically monitor the ability of the RBC to maintain homeostasis in a complex matrix.