Myeloperoxidase-dependent oxidative metabolism of nitric oxide in the cystic fibrosis airway  Anna L.P. Chapman, Brian M. Morrissey, Vihas T. Vasu, Maya M. Juarez, Jessica S. Houghton, Chin-Shang Li, Carroll E. Cross, Jason P. Eiserich  Journal of Cystic Fibrosis  Volume 9, Issue 2, Pages 84-92 (March 2010) DOI: 10.1016/j.jcf.2009.10.001 Copyright © 2009 European Cystic Fibrosis Society. Terms and Conditions

Fig. 1 High peroxidase activity is associated with decreased eNO in CF patients. Shown are eNO levels measured at two different expiration flow rates and plotted against sputum heme peroxidase activity (A, C). Symbols denoted with an asterisk (*) indicate CF patients (n=6), those without an asterisk indicate normal control subjects (n=7). Data are also represented as the ratio of eNO to heme peroxidase activity for normal controls and CF patients (B, D). A two-sided Wilcoxon–Mann–Whitney test revealed that there was a statistically significant difference in the ratio of eNO (40ml/s) and eNO (100ml/s) to peroxidase activity (p=0.001). More specifically, the ratio of eNO (at both 40ml/s and 100ml/s) to peroxidase activity was statistically significantly higher in the normal group compared to the CF group. Journal of Cystic Fibrosis 2010 9, 84-92DOI: (10.1016/j.jcf.2009.10.001) Copyright © 2009 European Cystic Fibrosis Society. Terms and Conditions

Fig. 2 Expectorated sputum NOx (NO2−+NO3−) levels are not different between normal and CF patients. A scatter plot is shown for 6 normal controls and 12 CF patients. The average±SEM for controls and CF patients was 133±30µM and 132±30µM, respectively. A two-sided Wilcoxon–Mann–Whitney test revealed that there was no statistically significant difference in sputum NOx between the two groups (p=1.0). Journal of Cystic Fibrosis 2010 9, 84-92DOI: (10.1016/j.jcf.2009.10.001) Copyright © 2009 European Cystic Fibrosis Society. Terms and Conditions

Fig. 3 Relationship between ex vivo NO consumption rates and sputum peroxidase activity in CF patients. Data represent sputum from 16 individual adult CF patients. Whereas there was a trend toward a direct relationship, the two-sided p-value using Spearman's rank correlation coefficient (ρ=0.366; ASE=0.222) to assess the association between NO consumption rate and peroxidase activity was 0.163, indicating no statistically significant association. Journal of Cystic Fibrosis 2010 9, 84-92DOI: (10.1016/j.jcf.2009.10.001) Copyright © 2009 European Cystic Fibrosis Society. Terms and Conditions

Fig. 4 Ex vivo NO consumption rates are directly proportional to sputum MPO protein levels in CF patients. MPO protein levels in CF sputum, determined by ELISA (A), were generally confirmed by Western blotting (B). Low, medium, and high in panel B refer to representative CF patients who had MPO levels determined by ELISA in the lowest third, the middle third, and the highest third, respectively (two independent patients per category). The two-sided p-value using Spearman's rank correlation coefficient (ρ=0.697; ASE=0.164) to assess the association between NO consumption rate and MPO protein was 0.004, which indicates that there was a statistically significant association between NO consumption rate and MPO protein. Journal of Cystic Fibrosis 2010 9, 84-92DOI: (10.1016/j.jcf.2009.10.001) Copyright © 2009 European Cystic Fibrosis Society. Terms and Conditions

Fig. 5 MPO is the major heme peroxidase in CF sputum responsible for NO consumption. Immunodepletion of MPO from sputum of 4 CF patients decreases ex vivo NO consumption rates (open bars) and MPO protein as determined by ELISA (filled bars). Values are represented as a percentage of control values (set at 100%) in identical specimens treated and processed identically, but without addition of the anti-MPO antibody. Each bar indicates the average of two independent determinations. The p-value of the two-sided Wilcoxon signed-rank test was 0.125, which reveals that there was no statistically significant difference between MPO protein and NO consumption. Journal of Cystic Fibrosis 2010 9, 84-92DOI: (10.1016/j.jcf.2009.10.001) Copyright © 2009 European Cystic Fibrosis Society. Terms and Conditions

Fig. 6 Bidirectional modulation of peroxidase-dependent NO consumption in CF sputum. Traces indicating real-time NO levels are shown in A, B, D and E. Arrows indicate additions of H2O2 (10µM). Shown are peroxidase/H2O2-dependent NO consumption rates in CF sputum (A) or CF sputum supplemented with 500µM l-tyrosine (B). Tyrosine increases peroxidase/H2O2-dependent NO consumption in a concentration-dependent manner (C). Also shown are peroxidase/H2O2-dependent NO consumption rates in CF sputum (D) and CF sputum supplemented with 500µM Trolox (E). Trolox decreases peroxidase/H2O2-dependent consumption of NO in a concentration-dependent manner (F). Values in C and F represent the average±SD. Journal of Cystic Fibrosis 2010 9, 84-92DOI: (10.1016/j.jcf.2009.10.001) Copyright © 2009 European Cystic Fibrosis Society. Terms and Conditions

Fig. 7 Schematic illustrating potential pathways for modulating MPO-dependent NO metabolism in airway secretions of CF patients. Second order rate constants are given with units of M−1 s−1 for reactions. Tyrosine enhances MPO-dependent consumption of NO, whereas Trolox inhibits MPO-dependent consumption of NO. Journal of Cystic Fibrosis 2010 9, 84-92DOI: (10.1016/j.jcf.2009.10.001) Copyright © 2009 European Cystic Fibrosis Society. Terms and Conditions