52 Evaluation of marker proteins for quantitation of CFTR

Abstract

Objectives: P. aeruginosa, a common pathogen in airways of patients with CF is sensitive to NOand nitrite-mediated killing. L-arginine is a substrate for nitric oxide (NO) synthase and arginase and both substitution of L-arginine and/or inhibition of arginase could have positive effects on Pseudomonas infection. We investigated the effect of L-arginine and of arginase inhibitor ABH on NOmetabolite formation in a novel CF sputum assay. Methods: Expectorated sputum was homogenized with the use of sputulysin and DPBS following standardized protocols before incubation with different concentrations of L-arginine or ABH. Nitrate and nitrite were measured using Griess reagent after 1 hour of incubation time at 37oC. Results: Total NO-metabolites (NOx) and nitrite concentrations were unaffected by incubation with buffer alone. L-arginine (10, 30, 100 and 300mmol) resulted in a concentration-dependent increase in both total NOx (1.7, 2.9, 3.9 and 3.9fold, p< 0.01, ANOVA) and nitrite (2.0, 3.4, 4.9 and 5.4-fold, p< 0.01). ABH (10, 30, and 100mmol) also resulted in a significant increase in NOx and nitrite (1.5fold, p< 0.01). The combination of ABH 1mmol with increasing concentrations of L-arginine (0, 0.33, 1, 3, and 10mmol) (n = 21) resulted in additive effects with significant increase in sputum nitrite observed for L-arginine of 3mmol and higher (p< 0.001) compared to ABH 1 mmol, respectively. Conclusions: Arginase inhibitor added to CF sputum alone or in combination with L-arginine significantly increases NO production. Studies to assess the effects of arginase inhibition on NO-mediated killing of bacteria in CF sputum are currently under way. 52 Evaluation of marker proteins for quantitation of CFTR