Evaluation of Glutathione (GSH) System in Porcine Saliva: Validation and Application of Colorimetric Method

Abstract

(1) Reduced glutathione (GSH) is considered the first line of antioxidant defense. During oxidative stress, it is oxidized to glutathione disulphide (GSSG). (2) A simple and quick spectrophotometric method based on sodium borohydride (NaBH₄) as a reductant to measure the total and reduced GSH in porcine saliva was analytically validated and evaluated in two situations in this species: (a) in a physiological situation, involving sows during the late lactation and post-weaning periods, and (b) in a situation of sepsis in pigs experimentally induced by LPS administration. (3) The results of the analytical validation showed that the assay was precise and accurate in the porcine saliva samples. Higher total GSH and GSSG and lower reduced GSH were observed in the saliva of sows during the post-weaning period, as well as in pigs with experimentally induced sepsis. (4) In conclusion, the validated assay showed adequate analytical results and could be used to evaluate the GSH system of porcine saliva, as demonstrated during the clinical performance.

Keywords: antioxidants; biomarkers; glutathione system; saliva; validation.

Figure 1

Regression line showing the reduced glutathione (GSH) and glutathione disulphide (GSSG) concentrations in pig saliva samples spiked with reduced GSH and GSSG solutions, respectively. The coefficient of determination (R2) is shown.

Figure 2

Total glutathione (GSH), reduced GSH, oxidized GSH (GSSG), and GSH reductase (GSH red) in saliva of sows during late lactation and post-weaning periods. Sows in conventional farrowing system are represented by blue squares, and sows housed in temporary crating system are represented by orange circles. L12, 12th day of lactation; L23, 23rd or last day of lactation; W1, 1st day post-weaning; W2, 2nd day post-weaning. The plots show individual values, median (black line), and 25th and 75th percentiles (colored lines) for each group of sows. * Statistically significant difference (p ≤ 0.05) between time points considering the data from all sows in the study.

Figure 3

Total glutathione (GSH), reduced GSH, oxidized GSH (GSSG) and GSH reductase (GSH red) in the saliva of 5 control pigs (blue box) and 5 pigs with endotoxin lipopolysaccharide (LPS)-induced experimental sepsis (purple box). * p ≤ 0.05.