Induced sputum methodology: Validity and reproducibility of total glutathione measurement in supernatant of healthy and asthmatic individuals

Glutathione (GSH), a major antioxidant, has repeatedly been linked to the pathogenesis of pulmonary disease. The measurement of GSH in induced sputum (IS) offers a noninvasive tool for the study and monitoring of oxidative stress in airway diseases. In this study we assessed the validity and reproducibility of GSH quantification in IS from healthy subjects and individuals with mild asthma. We spectrophotometrically quantified total GSH in the IS of 31 healthy nonsmoking volunteers and 12 individuals with mild asthma. IS was processed with varying concentrations of dithiothreitol (DTT) in an effort to evaluate the effect of DTT on GSH measurements. We performed spiking experiments with defined concentrations of GSH and quantified the percentage of recovery and also analyzed the effect of induction time on GSH levels through sequential sampling of sputum portions (15, 30, and 45 minutes' induction). Finally we tested the reproducibility of GSH measurements at 2 separate time points (0 and 72 hours) and expressed it as an intraclass correlation coefficient (R-i) with a coefficient of reliability (CR). Processing with DTT increased GSH values in IS (P < .05 for each DTT concentration > 0.001%). Recovery of GSH after spiking was complete, with a mean recovery of 102% +/- 4.8%. Increasing duration of induction led to an increase in sputum GSH (15 minutes, 10.2 +/- 2.3 mumol/L; 30 minutes, 18.4 +/- 3.5 mumol/L; 45 minutes, 26.1 +/- 4 mumol/L; P < .05 for all comparisons). Reproducibility of sputum GSH both in healthy subjects and asthmatic individuals was good (R-i = .78, P < .001; and R-i = .51, P = .003, respectively). With the use of standardized protocols for duration of induction and sample processing, sputum GSH measurement in healthy subjects and asthmatic individuals is valid and reproducible.