125 mM Na-Phosphate (J.T. Baker Inc.), 6.3 mM Na-EDTA (Sigma Chemical Co., U.S.) buffer solution (pH 7.5) was prepared.  Five solutions were made from the buffer:  50 ml of 0.3 mM NADPH (Sigma Chemical Co., U.S.), 10 ml of 6 mM DTNB (Sigma Chemical Co., U.S.), 1.12 ml of 50 units/ml glutathione reductase (Sigma Chemical Co., U.S.) (all three solutions are stable for at least two weeks at 0^oC ) 50 ml of 120 mM GSH (Sigma Chemical Co., U.S.) 50 ml of 60 mM GSSG (Sigma Chemical Co., U.S.)  Five 1 ml samples of various concentrations of glutathione were produced by serial dilution.

Total Glutathione Assay

700 µl NADPH, 190 µl of glutathione sample, and 10 µl glutathione reductase were added to a one ml cuvette.  The cuvette with the sample was incubated at 30^o C for one minute.  After careful drying, 100 µl of DTNB substrate was added and the mixture was shaken five times to initiate the reaction.  Blank cuvettes contained 700 µl NADPH, 100µlDTNB, 10µl glutathione reductase, and 190 µl buffer. The kinetics of the reaction was monitored at 412 nm for a period of 300 secs at time intervals of 15 secs. 

2-VP Method

2-vinyl-pyridine was added to the glutathione solutions, 2 ml 2-VP per 100 ml solution.  The solutions were incubated for one hour at room temperature (25^o C).  Procedure steps 1-2 (Total Glutathione Assay) were repeated.