INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Measurement of the levels of nitric oxide (NO) in exhaled air has been recently proposed as a noninvasive means of assessing the degree of airway inflammation in asthmatic patients (1). Although inhaled NO is rapidly absorbed by the lungs, environmental levels of NO are a cause of concern in interpreting the results of studies in which levels of exhaled NO are measured. These environmental levels may indeed vary considerably from one location to another and over short periods in the same location, particularly during the cold season.

In a recent position document by the European Respiratory Society (ERS) task force on measurement of nitric oxide in exhaled air (4), the panel of experts contributing to the document excluded a contributory factor of inhaled NO to exhaled NO when the latter is measured during slow exhalation against a low resistance. However, they recommended that NO-free air should be used when ambient NO levels are higher than 40 ppb (4). Because changes in ambient NO in the range from 0 to 3 ppb to 150 ppb are frequently observed in our laboratory, we evaluated whether environmental levels of NO in the range of 0 to 150 ppb significantly affect levels of exhaled NO.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

In our study, we made repeated evaluations in two groups of subjects of exhaled NO with ambient NO at 0 to 3 ppb and at higher concentrations.

In the first group of 16 subjects (Group 1), consisting of 12 children with mild asthma and four normal adults, exhaled NO was initially measured when environmental levels of NO were in the 20- to 60-ppb range, and a second time when they were in the 0- to 3-ppb range. In a further group of 30 volunteers (Group 2), we measured exhaled NO both at environmental NO levels of 0 to 3 ppb and at higher levels. Of these latter subjects, 21 also underwent testing when environmental levels of NO were in the 80- to 100-ppb range, and 24 underwent testing when these levels were in the 120- to 150-ppb range. Eighteen subjects underwent repeated measurement under both of these environmental NO conditions. For each of the different environmental concentrations of NO, all tests were done on the same day on all of the subjects tested. Assessments under the different NO conditions were done on different days but within 1 wk from the day of the first determination for the subjects in Group 1 and within 2 wk for the subjects in Group 2. The asthmatic children in Group 1 were studied at the Istituto Pio XII, a residential home for asthmatic children located 1,756 m above sea level in the Italian Dolomites, in an environment free of the most common allergens. It is therefore reasonable to speculate that the degree of airway inflammation in these children was unchanged during the time of the study. No infection was observed in this group during the study period. Likewise, no known event that could interfere with the level of exhaled NO occurred between the two measurements in the second group, except in the case of three patients who presented with a single episode each of upper respiratory infection. These subjects were therefore excluded from the data analysis.

The measurements were made according to a previously described method (3, 5) involving a Logan chemiluminescence analyzer (LR2149; Logan, Rochester, UK) (3). Briefly, subjects were asked to perform a single slow exhalation through a mouthpiece, against a resistance, and with biofeedback used to maintain steady flow at 5 to 6 L/min. This method leads to isolation of the nasopharynx from the oropharynx by the soft palate, thus preventing the contamination of exhaled NO with nasal NO, and has proved effective when used both with adults and with children. The values of NO considered in the data analysis were always measured in the final phase of exhalation (plateau exhaled NO), taking the plateau of the end-exhaled CO₂ reading as representative of an alveolar sample (3).

The results of the study were compared through a paired Student's t test and analysis of variance (ANOVA) for repeated measures. Because the small number of subjects in the study could have affected the normal distribution of the data, analysis was also verified with nonparametric tests, consisting of Wilcoxon's signed-rank test and Friedman's test, as appropriate to the situation. Differences were considered statistically significant at p < 0.05. Correlations between results were analyzed by simple regression. The influence of different levels of ambient NO with regard to the full range of test conditions (i.e., 0 to 150 ppb) was also analyzed by the Bland-Altman method (6).

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

In Group 1, exhaled NO levels (mean ± SEM) were 9.81 ± 1.43 ppb and 9.78 ± 1.47 ppb at the higher (20 to 60 ppb) and lower (0 to 3 ppb) environmental ranges of NO, respectively, with no significant difference in these exhaled levels. Figure 1 shows the data distribution and correlation of the results for this group (r = 0.937, p < 0.0001). Considering the 12 asthmatic children independently of the four normal adults, there was no significant change in exhaled NO at higher and lower environmental NO concentrations. In Group 2, the mean levels of exhaled NO for subjects who were tested at environmental NO concentrations of 0 to 3 ppb and 80 to 100 ppb were 9.75 ± 1.83 ppb and 9.07 ± 2.00 ppb, respectively (p = ns). Figure 2 shows the data distribution and correlation of the results for this group (r = 0.983, p < 0.0001). Those subjects who were tested at 0 to 3 ppb and 120 to 150 ppb of environmental NO had exhaled NO concentrations of 9.15 ± 1.21 and 9.26 ± 1.23 ppb, respectively (p = ns). The data distribution and correlation of results for this group are shown in Figure 3 (r = 0.966, p < 0.0001). The Bland-Altman method confirmed the repeatability of the measurement in this second group of patients when taking into consideration our extreme experimental conditions (Figure 4). For the 18 subjects tested at all of the NO concentrations investigated (i.e., 0 to 3 ppb, 80 to 100 ppb, and 120 to 150 ppb NO), the mean levels of exhaled NO were, respectively, 9.23 ± 1.51 ppb, 7.78 ± 1.19 ppb, and 9.33 ± 1.55 ppb. No significant difference was found for the values observed under the three different conditions. The data distribution for these subjects is shown in Figure 5. When all results were compared through nonparametric tests, the differences were also found to be nonsignificant.

View larger version (14K): [in this window] [in a new window]   Figure 1.   Data distribution and correlation for the 16 subjects whose exhaled NO was tested when environmental levels of NO were from 20 to 60 ppb and 0 to 3 ppb.

View larger version (14K): [in this window] [in a new window]   Figure 2.   Data distribution and correlation for the 24 subjects whose exhaled NO was tested when environmental levels of NO were from 80 to 100 ppb and 0 to 3 ppb.

View larger version (16K): [in this window] [in a new window]   Figure 3.   Data distribution and correlation for the 21 subjects whose exhaled NO was tested when environmental levels of NO were from 120 to 150 ppb and 0 to 3 ppb.

View larger version (17K): [in this window] [in a new window]   Figure 4.   Difference from mean for exhaled NO when environmental levels of NO were 0 to 3 ppb and 120 to 150 ppb.

View larger version (12K): [in this window] [in a new window]   Figure 5.   Data distribution for the 18 subjects whose exhaled NO was tested when environmental levels of NO were from 0 to 3 ppb, 80 to 100 ppb, and 120 to 150 ppb.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The results of this study suggest that significantly different ambient levels of NO in the range of 0 to 150 ppb do not influence levels of exhaled NO tested by slow expiration against a resistance. A previous observation by Borland and coworkers (7) was that different environmental concentrations of NO, in the range of 0 to 7 ppb, did not affect exhalation test results. Our results extend this observation to significantly higher concentrations of environmental NO, and show that these levels do not influence the amount of NO measured in exhaled air. It has also been shown that inhalation of NO calibration gas at concentrations from 113 to 800 ppb does not affect the peak exhaled NO in normal subjects (2, 8). However, these results may be influenced by the unnatural model of short exposure of subjects to high levels of inhaled NO from a calibration gas reservoir. By contrast, our study investigated what happens under real conditions, when subjects are naturally exposed to high environmental levels of NO. In urban areas, especially during cold seasons, significant increases in the environmental concentration of NO are frequently observed. For this reason, one previous study postponed measurements of exhaled NO when ambient NO concentrations were higher than 10 ppb (9). In another study, the levels of NO measured in the environment at the time of testing were subtracted from the values measured in air exhaled by patients (10).

The results of the present study suggest that no correction of exhaled NO values is necessary when environmental levels of NO are within the range of 0 to 150 ppb and exhaled NO is measured with a slow exhalation against a low resistance. This observation can be explained by the rapid combination of NO with hemoglobin (4, 11) and its absorption by the lungs when inhaled. Nevertheless, because environmental NO concentrations exceeding 150 ppb have been reported, it is always essential to know the actual level of NO in the air when testing for exhaled NO, and studies at concentrations of inhaled NO above 150 ppb are needed to test the hypothesis of their possibly having an influence on exhaled NO. Furthermore, our conclusions apply only to the on-line measurement of exhaled NO levels when considering plateau values of environmental NO; our results do not permit an analysis of the influence of ambient NO levels on peak values of exhaled NO. In fact, in our experimental design, patients were asked to start exhalation after the environmental level of NO had reached a plateau, so as to provide a clear idea of the factors exerted by the ambient NO concentration at the time of each single test. The peak value generated by the first exhalation of NO was not distinguishable when high ambient NO concentrations were present and were read by the analyzer. The present data also do not permit drawing any conclusions about methods other than on-line testing, which may depend on the specific gas collection and storage techniques.