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Seeking the meaning of exhaled NO
In this issue of Thorax, Ojoo et al1 conclude their paper with a sentence that is worthy of direct quotation. Their data “draw attention to the complexity of NO [nitric oxide] metabolism, where multiple pathways of NO synthesis and clearance are likely to have variable relevance in different circumstances”. Exhaled NO measurement is now a clinically approved test and, with approximately 1000 articles so far published, there are sufficient data to support its utility as an objectively measurable biomarker relevant to lung disease. However, despite all of the publications, how the multiple intracorporeal biochemical pathways interact and determine NO exhalation remains unclear.
In order to avert confusion, it is necessary to assure that vocabulary is shared. “Exhaled NO” has become the vernacular for the fractional exhalation of NO (FENO). This is not the amount of NO produced in the lung. Indeed, only a tiny fraction of NO produced in the lung ends up being exhaled. Increased production of NO in the airway can occur from increased nitric oxide synthase (NOS) activity, from inorganic acidification of nitrite (NO2−),2–4 and from homolytic cleavage of nitrosothiols.5 Increased exhalation of NO can occur from enhanced action of any of these aforementioned processes or, importantly, from decreases in various NO consumptive pathways that also are abundantly active in the airway. Such consumptive pathways include reaction with haemoglobin and various thiols, reduction by bacteria, and oxidation reactions that form the higher oxides of nitrogen (NOx)—specifically NO2−, S-nitrosothiols, and nitrate (NO3−)—as products.5
Ojoo et al1 report that, while high acidity (low pH) of exhaled breath condensate (EBC) was identified in subjects with cystic fibrosis (CF), exhaled NO levels were lower than in controls. These data suggest that airway acid acting on NO2− is neither the sole nor the dominant determinant of exhaled NO in this patient group. Additionally, their data reveal that, in contrast to NO, NOx are exhaled in greater amounts from subjects with CF than controls. If we roughly back calculate from Ojoo’s data we find that, in their healthy control subjects, mole for mole, the amount of NOx exhaled per hour as identified in EBC (90 nmol/h) is about half the amount of NO exhaled when measured in gas phase (180 nmol/h). In patients with CF, however, NOx are exhaled at the rate of 240 nmol/h compared with only 80 nmol/h exhaled as NO. One can see that the sum total exhalation of NO plus NOx is somewhat higher in CF than in controls, while the ratios of NO to NOx between the groups differ by a factor of 6. If we consider the original source of all the nitrogen oxides in the lung to be from NOS (a somewhat risky assumption), then these data suggest that NO production is actually enhanced in the CF airway, not decreased as has at times been supposed. The fact that NO exhalation is low in CF then appears to result from overactive oxidative processes consuming NO.
A little more caution is needed here, as it is not clear how much of the gas phase NO available for exhaled measurements also contributes to the EBC NO2− and NO3− levels. That work has yet to be done. But, as the authors note, it seems clear that NOx in EBC are not derived solely from NO gas dissolving and oxidising in the EBC ex vivo, as there are not enough available moles of NO gas in the exhaled air to provide for the levels of NOx seen in the EBC of CF patients.
Oxidation is one key sump for NO in the airway. Many believe oxidative processes to be a key mechanism by which “inflammation”—a generic term of uncertain meaning beyond the classical “rubor”, “tumor”, “calor” and “dolor”—dutifully accomplishes its various missions. In general we consider NOS to be upregulated, acidity increased, and oxidation enhanced in inflammatory conditions. Inflammation might lead to higher or lower exhaled NO levels depending on the relative effects of the inflammation on NOS activity and acidic and oxidative stresses.
Ojoo et al have shown how these three chemical aspects of inflammation (NO, acid, and oxidants) can be monitored and distinguished. By measuring exhaled NO, exhaled acids, and exhaled NOx all together, one can make the following suppositions. If exhaled NO is high while exhaled NOx are low, then that particular airway inflammatory process has a relatively high ratio of airway NO production to oxidant activity. If exhaled NO is low while exhaled NOx are high, then the inflammation is relatively more dominated by oxidant stress than by NO production. When acidification is present, then NO will be both produced from NO2− and consumed by the higher oxidative activity that occurs in acidic conditions.3 Thus, EBC acidity stands by itself as an indicator of airway acid stress but not of NO production, and exhaled NO and NOx measured concurrently begin to elucidate the relative—not absolute—activities of the NO production pathways and oxidative processes.
The same acidity that can convert aqueous NO2− into NO will also enhance oxidative activity in general, including the reaction of NO2− with the reductant, glutathione. This reaction forms S-nitrosoglutathione, an endogenous bronchodilator.5 To complicate matters, glutathione stores themselves can be depleted by excessive oxidative processes. Furthermore, the CF airway is commonly colonised with bacteria capable of reducing NO to ammonia, a process which can consume NO.6 Despite these complexities, efforts at measurement—and conscientious interpretation—of the chemistry of the airways helps us to recognise how acidic and oxidative processes, as well as NO chemistry, behave in various lung diseases. These processes are key mechanisms of inflammatory effect and are relatively easy therapeutic targets.
Another point is worth noting. Spirometric values in CF in this study were not significantly lower on average during acute respiratory exacerbations. This statement reminds us that “no statistically significant difference” is not synonymous with “no difference”. Given that EBC pH was significantly lower in the exacerbating group relative to the stable group, the lay reader might conclude that EBC pH is more useful than spirometry at identifying CF exacerbations. A more cautious and probably wiser interpretation might be that—together—spirometry, EBC pH, exhaled NO, and NO2− and NO3− assays provide a more comprehensive and true picture of airway disease than any single measurement. EBC pH informs us about airway acid stress. NO and NOx, when measured together, inform us about NO production and oxidative burden. Spirometry provides physiological data. Of course, none of these tests delineates perfectly the amount of “inflammation”. But then, in the absence of a unifying definition of inflammation, no one test possibly could.
Seeking the meaning of exhaled NO
Competing interest statement: (1) The University of Virginia owns intellectual property relating to measurement of exhaled breath condensate pH. (2) The author is a founder of Respiratory Research Inc, a company that builds exhaled breath condensate collection equipment. (3) The author is an inventor on US and international patents relating to measurement of exhaled breath condensate nitrogen oxides, but has no financial interest in these patents.
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