© 2002 American Thoracic Society
Monitoring reactive nitrogen species in biological milieuA difficult journeyTo the Editor :Corradi and colleagues report that the concentration of S-nitrosothiols (RSNO), nitrite, and exhaled nitric oxide (eNO) are increased in some inflammatory airway diseases. The chemistry of reactive nitrogen species (RNS) in biological systems is complex (2) and it is still unclear whether point estimates of concentration of RNS in blood and urine provide information on endogenous NO metabolism (3). Although collection of exhaled breath condensate (EBC) and eNO measurements may offer attractive alternative methods for measuring RNS, great care in sample handling and interpretation of results is mandatory. EBC was collected using a glass condensing device, by cooling of the exhalate during tidal breathing for 15 minutes while wearing a nose clip. Contamination by trace transition metals, leaching from any glass containers, and exposure to light may have accelerated RSNO decomposition. Total RSNO content in EBC was measured by spectrophotometry, using a method adapted from Saville and Griess that involves cleavage of the NO group with mercuric chloride followed by quantitative detection of nitrite in the diazotation assay (the Griess assay). Artifactual loss of nitrite due to its disproportionation to NO or nitrosation of the remaining thiol-containing compounds in the EBC during the necessary sample acidification step in the Griess assay, would also be a possibility. Exhaled breath measurements in inflammatory disorders are prone to additional confounding factors. For example, we have recently confirmed that nitrite releases free NO even at physiological pH (4). Moreover, recent advances by Hunt and Gaston (5) suggest that endogenous airway acidification is a pathological feature of asthma. This would accelerate decomposition of nitrite to NO producing eNO (4, 5). The cellular origin of exhaled RNS varies according to the disease. The elevated concentrations of eNO in asthma could be from epithelial NOS II (inducible NOS) or of neural origin (6). Identification of the isoform of NOS responsible for RNS production in inflammatory disorders will have to await the introduction of selective inhibitors of the different isoforms of the enzyme. Monitoring RNS in biological milieu is a challenging endeavour, and selective methods to measure endogenous NO production in vivo will certainly help unravel the complex interactions between nitrogen oxides and their derivatives.
University of Sheffield and Sheffield Hallam University Sheffield, United Kingdom REFERENCES
From the Authors We thank Dr. Demoncheaux and coworkers for their insightful comments on our article raising concerns about the measurement of reactive nitrogen species in exhaled breath condensate (1). Demoncheaux and colleagues suggest that exhaled breath condensate samples may be contaminated by trace transition metals from any glass containers or tubing, and that exposure to light may accelerate the decomposition of S-nitrosothiols. There is currently no evidence that either of these factors have any influence on the concentrations of S-nitrosothiols in exhaled breath condensate, but systematic studies are needed. We have shown that the concentrations of S-nitrosothiols are identical to those reported in our manuscript when using a condenser system that does not contain any glass component and is protected from light, suggesting that these factors are unlikely to have any significant effect on measurements. Furthermore, we looked at concentrations of S-nitrosothiols in different groups of subjects, and are more interested in the differences between these groups than the absolute concentrations in the samples. Demoncheaux and colleagues are concerned that airway acidification may reduce the concentrations of S-nitrosothiols in patients with asthma. In fact, the pH of exhaled breath condensate is no different than normal in stable asthma and is lower only during acute exacerbations (2, 3). We agree that selective NOS inhibitors will help us to identify the isoforms of NOS responsible for the production of reactive nitrogen species in inflammatory diseases of the lungs. We have previously demonstrated that inhaled aminoguanidine, which has some selectivity in inhibiting inducible NOS, causes a greater reduction in exhaled NO in patients with asthma compared with normal control subjects (4). Our recent unpublished observations have demonstrated that a more potent and specific iNOS inhibitor was capable of reducing exhaled NO by over 90% in patients with asthma, confirming that iNOS accounts for the majority of exhaled NO in asthma. Monitoring of markers in exhaled breath condensate in patients with pulmonary disease has enormous potential as a noninvasive means of monitoring airway inflammation and oxidative stress (5), and represents a clear way forward rather than a difficult journey.
National Heart and Lung Institute, Imperial College London, United Kingdom REFERENCES
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