2.6. Determination of the Reduced Glutathione (GSH) Content in the PBMC Lysate

The GSH content in PBMC lysate was determined spectrophotometrically by using HT Glutathione Assay Kit (Trevigen, Gaithersburg, MD, USA). In this assay, sulfhydryl groups of GSH reacts with 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) to produce both a yellow colored 5-thio-2-nitrobenzoic acid (TNB), that absorbs at 405 nm or 414 nm, and the mixed disulfide, GSTNB, that is reduced by glutathione reductase to recycle the GSH and produce more TNB. The rate of TNB production is proportional to the concentration of detected glutathione in the sample. This method allows to measure the total glutathione because the oxidized glutathione (GSSG) present in the sample is reduced by glutathione reductase, present in the reaction mixture, to two molecules of GSH. To measure GSSG content, free thiols present in the reaction must be masked with 2 M of 4-Vinylpyridine. The concentration of GSH is calculated by subtracting the GSSG levels from the total (GSH plus GSSG) glutathione value.

After incubation PBMCs were collected, washed once with PBS, deproteinized with 5% (w/v) metaphosphoric acid and lysed by the addition of 0.1% Triton X-100 and inhibitor protease cocktail to the cell suspension. A Multiskan Ascent reader (Thermo Labsystem) and 96-well plates were used for measurements. The reaction medium (final volume of each well: 0.2 mL) consisted of 1X assay buffers and samples (untreated PBMC lysates or 4-Vinylpyridine-treated PBMC lysates for the determination of total glutathione and GSSG, respectively). The reaction was started by adding 150 μL of freshly-prepared reaction mix, containing DTNB and the glutathione reductase, and monitored by recording the absorbance increase at 414 nm, due to TNB production, at 1 min intervals over a 10 min period. The samples were analyzed in quadruplicate (replicates of the measurements), and the slope from the maximum linearity portion of each curve was determined. Glutathione (total and GSSG) concentrations for each sample, were determined by comparing the slope of the samples with that of the standard curve obtained by using a GSSG standard. GSH content was thus calculated and expressed in nmol/mg of protein. The protein content in PBMC lysate was determined by Bradford’s method using BSA as a standard as described for the Glo1 activity. In the statistical test the averages of those replicates were compared, thus a total of 8 values per participant and per incubation time were compared in the statistical test.