Exhaled nitric oxide (eNO) is elevated in patients with inflammatory pulmonary diseases and it has attracted increasing interest as a simple, noninvasive marker of airway inflammation. Little is known, however, about factors that might affect eNO in healthy subjects. We measured eNO in 157 healthy 7- to 13-yr-old children (mean 9.7 yr, 77 girls), with no history of respiratory tract disease, using a recently validated, single-breath technique. Measurements of eNO were obtained at driving (mouth) pressures of 10, 15, and 20 cm H2O and 3 eNO plateaux were achieved for each child at each pressure. Exhaled NO decreased with increasing pressure (increasing expiratory flow) (p < 0.001) and increased with age (p < 0.001). Concentrations were greater in children with a positive skin prick test (p < 0.0001). Geometric mean eNO levels were 7.2 ppb in children with no positive skin prick tests (n = 116), 10.9 ppb in children with one positive reaction (n = 24), and 20.1 ppb in children with two or more skin reactions (n = 17). Age and immunological reactions to common allergens are associated with increased eNO in children and should be controlled for in studies of eNO. The mechanisms responsible for these associations require further study.
Recent studies in adults and children strongly suggest that measurements of exhaled nitric oxide (eNO) reflect airway inflammation (1-9). Indeed, in asthmatic children eNO is a more sensitive indicator of disease severity than other markers of inflammation such as serum eosinophil cationic protein (ECP) and soluble interleukin-2 (IL-2) receptor (10). Endogenous NO is synthesized from l-arginine by isoenzymes of NO synthetase. Some isoenzymes are induced by inflammatory cytokines, and inducible forms of NO synthetase (iNOS) are inhibited by glucocorticoids (11). Nitric oxide is produced by several classes of pulmonary cells including inflammatory, endothelial, and airway epithelial cells and is easily detected in exhaled air (11). Nitric oxide is raised in exhaled air from asthmatics compared with healthy control subjects in both adults (1, 12-13) and children (14-16) and is reduced in expired air after administration of a specific nitric oxide synthetase inhibitor (9). Corticosteroids reduce eNO in asthmatic but not healthy subjects (9, 17). Thus, it is thought that eNO is raised in asthmatics because of induction of iNOS as a result of airway inflammation (9, 17). Inducible NO synthetase is found in the bronchial epithelial cells, leading some to speculate that they are the cells responsible in asthmatics for raised eNO (9).
Different measurement conditions and techniques can have significant effects on absolute values of eNO. Factors that seem particularly important are expiratory flow, flow profile, lung volume, and contamination by nasal NO (18, 19). Silkoff and colleagues (19) have demonstrated in adults that expiratory flow has a significant effect on eNO and must be constant in order to obtain reliable estimates of eNO during a single expiration. Recently efforts have been made to standardize measurement procedures (19, 20).
A number of other factors, not related to methodology, have also been shown to affect eNO levels within a healthy population. These include smoking (12, 21), physical exercise (22), and menstrual cycle variations (23). There is no evidence for differences in eNO concentrations based on gender (24, 25), and the data with respect to age (10, 15, 24) and body size (20) are equivocal.
In the present study we used a validated, single expiratory breath technique (19) to measure eNO levels in 157 healthy children. This method involves measuring eNO during a constant expiratory flow and eliminates NO contamination from the nasopharynx. The maneuver is simple and can be performed adequately by both adults (19) and children (10). The aim of this study was to establish reference eNO values and to investigate variables that might affect concentrations in population studies.
A total of 188 children (91 girls) were recruited for the study from local primary schools. A modified respiratory health questionnaire (26) was distributed through the schools and only children without reported upper or lower respiratory tract disease were included in the study. The parents of the children gave written informed consent for their children to undergo a medical assessment at the Respiratory Medicine Department at Princess Margaret Hospital. The assessment included a skin prick test, lung function (spirometry), and measurements of eNO. The protocol was approved by both the King Edward Memorial and Princess Margaret Hospitals Ethics Committee and Murdoch University Human Research Ethics Committee.
The purpose of this study was to measure eNO in healthy children. During the visit to the hospital the parents were questioned further regarding the respiratory health of their child. As a result of this, 28 children were excluded from the study—two children had mild respiratory symptoms on the day of the assessment, 12 had undiagnosed symptoms that had not been previously reported, and 14 had a history of wheeze during infancy. A further three children refused the skin prick test and were also not included in the study. The remaining 157 children (77 girls) had no history of chronic upper or lower respiratory tract disease and no acute upper or lower respiratory symptoms for at least 2 wk prior to the test.
Exhaled NO was measured using a fast response (0.02 s) chemiluminescence analyzer (NOA 280; Seivers Instruments Inc., Boulder, CO). The sensitivity of the analyzer for measurement of gas-phase NO is less than 1 ppb by volume. The sampling flow is 200 ml · min−1. Measurements of eNO were made using the technique described by Silkoff and colleagues (19). The children were seated and breathed through a mouthpiece attached to a one-way valve. The valve had two sampling ports near the mouthpiece. Nitric oxide was sampled directly into the analyzer through a Teflon side-arm tube attached to one of the sampling ports. Exhalation (mouth) pressure was measured by a pressure transducer in the analyzer via the second sampling port. Both pressure and NO were displayed simultaneously on the front panel of the analyzer and on a computer attached to the RS-232 output. Data were stored and analyzed on the computer using NOAnalysis software (Seivers Instruments Inc., Boulder, CO).
If ambient NO concentrations were high (> 30 ppb) the subjects inspired medical air containing < 1 ppb NO. Otherwise the children inspired ambient air. In our laboratory ambient NO ranged from < 10 ppb to a maximum of 200 ppb. With the technique employed in this study NO levels in inspired air up to 30 ppb do not influence exhaled concentrations (20).
Nitric oxide measurements were made for each child using three different mouth pressures (10, 15, and 20 cm H2O) corresponding to expiratory flows of 50, 75, and 100 ml · s−1. After inhalation to total lung capacity (TLC) the children immediately exhaled into the mouthpiece. Mouth pressure was displayed on a computer screen as a prompt for the children to maintain a steady flow. Nitric oxide values were recorded as the plateau at the last part of the exhalation. Three measurements that varied by less than 10% were taken for each of the different mouth pressures and NO concentrations were recorded as the average of the three plateaux.
The 24-h repeatability of the test was determined in 10 subjects comprising healthy and asthmatic children age 7 to 13 yr. Measurements were obtained at each mouth pressure on two occasions 24 h apart and the coefficient of repeatability was calculated according to the method of Bland and Altman (27).
Lung function variables (forced vital capacity [FVC] and forced expiratory volume in one second [FEV1]) were measured using a hand-held spirometer (Pneumocheck Spirometer 6100; Welch Allyn, Skaneateles Falls, NY).
Skin prick (SP) reactions to seven common allergens were tested on the forearm of the children. The allergens were egg, cow's milk, cat hair, dog hair, grass mix, Dermatophagoides pteronyssinus, and Alternaria tenuis (Miles Inc., Elkhart, IN). Histamine hydrochloride (1.0 mg/ml) and a saline solution were used as positive and negative controls. Weal sizes were measured after 15 min as the diameter of the long axis of the raised area. Results were interpreted in relation to the size of the reaction to the histamine and only a weal of equal or greater diameter was accepted as a positive result. The children were grouped according to the number of positive SP reactions as follows: no positive reactions (Group 1), one reaction only (Group 2), and two or more reactions (Group 3).
The original respiratory health questionnaire completed by the parents of the children included questions on smoking in the home. These were: who smoked; the number of cigarettes smoked, on average, per day; and where, inside the home, smoking took place. In order to determine the effects of passive smoking on eNO the children were grouped into those who lived with a smoker and those who did not.
Exhaled NO concentrations were skewed to the right so all values were transformed to their natural logarithm (ln) to achieve a normal distribution. Two-way analysis of variance (ANOVA) was used to compare eNO values for the three different mouth pressures. Comparisons between males and females for age, eNO levels, and lung function (percentage of predicted FVC and FEV1) were made using the Student's t test and between the three SP groups using one-way analysis of variance (ANOVA). The effect of age on eNO was determined by simple linear regression. A multivariate linear regression model was performed with variables that were found to have a significant relationship with eNO (ln transformed data) from the univariate analyses. These variables were age, skin prick reactivity, reported history of eczema, and FVC. Gender and height were also included in the model although they were not significant in the univariate analyses. All univariate analysis was done using Statview (version 4.5) and the multivariate regression model was performed on Minitab 12.11. Exhaled NO values are reported as the geometric mean with 95% confidence intervals (95% CI). Other variables are expressed as the arithmetic mean with 95% CI.
We measured eNO at three separate mouth pressures (10, 15, and 20 cm H2O). A mouth pressure of 15 cm H2O was found to be the best compromise between reported ease of performance by subjects and the calculated between-test repeatability of eNO measurements in the 10 subjects studied on two occasions. The mean concentrations of eNO (range) at 10, 15, and 20 cm H2O in the 10 subjects were 23.9 ppb (5.0 to 88.8 ppb), 20.9 ppb (3.9 to 79.3 ppb), and 18.8 ppb (3.4 to 66.4 ppb). The coefficients of repeatability of measurements at 10, 15, and 20 cm H2O were 8.3 ppb, 5.2 ppb, and 6.3 ppb which represents 9.9%, 6.9%, and 10.0% of the range of eNO levels at each pressure. The difference between measurements was not dependent upon measurement size (Figure 1).
Results presented below are for mean levels of eNO at a mouth pressure of 15 cm H2O except where the effect of flow on eNO concentrations is presented. Associations between eNO and reported covariables were similar at each mouth pressure.
In the final analyses, 157 children (77 girls) were included. They were all asymptomatic with no history of upper or lower respiratory tract disease. The age range of the children was 7 to 13 yr (mean 9.7 yr). There were no differences between boys and girls for age or lung function (percentage of predicted FVC and FEV1).
The mean (95% CI) values for eNO at mouth pressures of 10, 15, and 20 cm H2O were: 10.3 (9.2 to 11.5), 8.5 (7.6 to 9.5), and 7.4 ppb (6.6 to 8.3), respectively. The decrease in exhaled NO values with increasing mouth pressures (increasing expiratory flow) was significant (p < 0.001) (Figure 2).
Univariate analyses of individual variables revealed that eNO increased with age (p = 0.0002), FVC (p = 0.007), and FEV1 (p = 0.009). There was no difference in eNO concentrations between boys (mean 8.8 ppb, 95% CI 7.5 to 10.4 ppb) and girls (mean 8.2 ppb, 95% CI 6.9 to 9.6 ppb) and no association between eNO and height. Mean eNO levels were higher in children who had positive skin reactions than children who did not react to any allergens (p < 0.0001). A number of variables were included in a multiple linear regression model. These included gender, age, SP reactivity, presence of eczema, FVC, and height. Only age (p = 0.04) and SP reactivity (p < 0.001) were significant predictors of eNO in this model.
From the multiple regression analysis age had a β coefficient of 0.1106. This corresponds to a multiplicative factor of 1.12 or approximately a 12% increase in eNO with each year of age.
The differences in eNO concentrations between the three SP groups are illustrated in Figure 3. Of the 157 children tested, 116 (73%) had no reaction to any of the seven allergens (Group 1), 24 (16%) had one positive reaction only (Group 2), and 17 (11%) reacted to two or more allergens (Group 3). There were no differences in age or lung function (percentage of predicted FVC and FEV1) between the three groups (Table 1).
|Group 1 (n = 116 ) no +ve SPTs||Group 2 (n = 24) 1 +ve SPT||Group 3 (n = 17) ⩾ 2 +ve SPTs|
|Age, yr*||9.5 (7-13)||9.9 (7-12)||10.4 (8-13)|
|FVC, % pred†||99.0 (97.1–100.9)||98.4 (94–102.8)||105.0 (100.7–109.3)|
|FEV1, % pred†||96.5 (93.7–97.7)||97.4 (92.7–102.1)||100.0 (94.7–105.3)|
|eNO 15 cm H2O, ppb‡||7.2 (6.4–8)||10.9 (7.8–15.3)||20.1 (14.8–27.4)|
There was no significant difference in eNO concentrations between children who lived with a smoker (n = 20) and those who did not (n = 137). Concentrations were 8.2 ppb (95% CI 3.4 to 17 ppb) and 8.6 ppb (95% CI 4.1 to 18.4 ppb) respectively.
Exhaled NO concentrations can vary considerably with different measurement techniques and conditions (18). In the present study we used a validated, single-breath technique to measure eNO in healthy children (19). With this procedure expiratory flow is controlled and nasal NO is excluded by maintaining a positive mouth pressure (ensuring vellum closure). The results of this study have highlighted a number of factors that affect eNO concentrations and need to be considered when measuring eNO.
A flow dependence of eNO concentrations has been reported previously in adults (18, 19). In this study we have demonstrated a similar relationship in children. Expiratory flow is an important factor for measuring eNO and consideration should be given to the ease of the maneuver while employing an expiratory flow that results in a range of eNO values that can discriminate between individuals. At low flows the distribution of values is increased; however, longer time is required to reach a plateau (19). Silkoff and colleagues (19) suggest using an expiratory flow between 10 and 40 ml · s−1 with a high expiratory resistance, whereas Kharitonov and coworkers (20) recommend flows in the range of 80 to 250 ml · s−1 against a low resistance. We used three flows of 50, 75, and 100 ml · s−1 corresponding to mouth pressures of 10, 15, and 20 cm H2O. Generally, the children found the procedure relatively easy to accomplish, however, some had difficulty maintaining a steady flow at 50 ml · s−1 and others found 100 ml · s−1 difficult to achieve. We found that for children in this study the most comfortable expiratory flow was 75 ml · s−1 with a driving pressure of 15 cm H2O. At this flow the 24-h repeatability was best.
We observed a number of factors unrelated to methodology that affect eNO. In our study group there was an increase in eNO concentrations with age. Other studies have been unable to demonstrate a similar relationship in either adults (25) or children (10, 28). However, Dineravic and coworkers (25) found that eNO was higher in adults than children. Mean nasal NO increases with age, reaching a plateau at about 10 yr, and it was suggested that this was consistent with the development and pneumatization of the paranasal sinuses (29). Our data indicate that there may also be an increase in orally expired NO concentrations at least until the age of 13 yr. The mechanism for this is not known but one plausible explanation is changes in lung volume with age.
In adults, Silkoff and colleagues (19) demonstrated that eNO values were lower if expiratory maneuvers were performed at lung volumes below TLC, indicating that eNO falls as lung volume decreases. In the multiple regression analysis of our data neither FVC nor height were associated with eNO after adjusting for all other variables. Age, however, remained a significant predictor of eNO. This suggests that mechanisms other than increased lung volume are responsible for the association between age and eNO observed in this study.
An intriguing observation from the present study was an association between skin prick reactivity and exhaled NO concentrations. This association remained highly significant even after controlling for all other variables. The degree of reactivity seems to be important as there was a significant difference in eNO levels between children with one positive SP reaction and those with two or more. The reason for this finding is unknown.
Only one other study has reported exhaled NO concentrations from an atopic (more than 2 positive allergen prick tests and a history of atopy) nonasthmatic group (10). Mean levels in this group did not appear to be significantly different from nonatopic healthy subjects (16 ± 2 ppb and 14 ± 2 ppb, respectively). However, the study by Lanz and coworkers (10) only included six atopic children and seven nonatopic children and we believe there was insufficient power to detect differences of exhaled NO between the two groups.
The source of the elevated NO concentrations in the SP-positive groups is not known. One possibility is that it reflects the presence of subclinical inflammation and induction of iNOS by inflammatory cytokines. This explanation is supported by data from histopathological studies that demonstrate the presence of airway inflammation in atopic subjects without asthma (30). Inhaled allergens are thought to be an important cause of ongoing inflammation in the lungs of sensitized children who have developed asthma (31), but little is known about the effect of allergen exposure on sensitized children without clinical signs of asthma. Exposure to inhaled allergens in the SP-positive groups may have contributed to elevated eNO.
The most common reactions in the study population were to house dust mite (Dermatophagoides pteronyssinus) (74%) and mixed grass pollen (54%). House dust mite concentrations were not measured in this study but are currently being investigated as part of a follow-up study. Most of the testing for this study was conducted during the peak pollen season (September to November) and airborne pollen counts were obtained from a local monitoring station. Five of the children with a positive skin reaction to pollen and high exhaled NO levels (> 20 ppb) were tested in a week when there was at least one day with a high to extreme pollen count; however, five pollen-sensitive children who were tested during a week with high pollen counts did not have elevated exhaled NO. Furthermore four children with high NO levels were tested prior to the pollen season. These data do not support an association between sensitization, current exposure, and exhaled NO level. A follow-up study will be conducted to test all pollen-sensitive children outside of the pollen season.
Alternative explanations for the association between skin reactivity and concentrations of eNO are (1) an independent effect of atopy on eNO production, (2) linkage between genes associated with atopy and mutations responsible for elevated NO production, or (3) mutations of NO synthetase genes that result in atopy. Genes for iNOS are found on chromosome 17 (32). To date there have not been any studies that demonstrate linkage between atopy and chromosome 17 or mutations of iNOS genes. More information is required about whether inducible or constitutive isoforms of NO synthetase are upregulated in atopy and which cell or cells are responsible for producing increased amounts of NO. Until these questions are answered the mechanism responsible for the association between eNO and skin reactivity will remain elusive.
Exhaled NO concentrations are lower in smokers than nonsmokers (12, 21, 33) and are reduced, transiently, immediately after the consumption of a single cigarette (33). There are no data, however, on the effects of passive smoking on eNO concentrations in adults or children. In this study we were unable to find any significant difference in eNO levels between children who lived with a smoker and those who did not. Reasons why we may not have been able to detect a significant effect of passive smoking in this study are: (1) the small number of children who lived with a smoker (n = 20), and (2) the use of questionnaire data to determine environmental tobacco smoke exposure. LeSeuof and coworkers (34) found that after the age of 2 yr there are only weak or no associations between reported parental smoking and children's urinary cotinine levels. This suggests that reported parental smoking is a poor indicator of environmental tobacco smoke exposure in the age group we studied.
In this study we have found that increasing age and immunological reactions to common allergens are associated with elevated levels of NO in the expirate of healthy children. These factors should be considered when measuring and reporting concentrations of expired NO in future studies. Furthermore, the mechanism underlying these relationships requires further study.
Dr. Hannes Wildhaber provided valuable technical assistance for this study.
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Peter Franklin was supported by the Asthma Foundation of Western Australia. The Seivers NOA 280 nitric oxide analyzer was bought by Glaxo-Wellcome, Australia.