As part of a large exposure assessment in Seattle, the author collected exhaled breath in children’s homes. Preliminary findings suggest that eNO may be an effective, noninvasive method for estimating lung inflammation in studies of health effects of air pollution.

 Author Jane Q. Koenig, PhD, right, with Karen Jansen, MS, holding the device used in the study.

Asthma is the most common chronic disease of childhood. It is the primary cause of school absenteeism and has been increasing in prevalence since 1980. One of the hallmarks of asthma is airway inflammation, which is associated with hyperreactive responses. Asthma onset and progression are influenced by both genetic and environmental factors; one of the environmental factors known to be a trigger for asthma aggravation is air pollution.1 Several studies2-4 have found decrements in lung function associated with community air-pollution exposures. Airway inflammation has been reported in controlled laboratory exposures to ozone and nitrogen dioxide. Less is known about associations between airway inflammation and daily, community air-pollution exposure. Recently, testing for exhaled nitric oxide has been suggested as a easily performed, noninvasive measure of airway inflammation. At the University of Washington, Seattle, we conducted a study5 designed to evaluate whether indoor, outdoor, and/or personal exposure to fine particles is associated with airway inflammation, as measured using exhaled nitric oxide, in a group of children with asthma in Seattle. Our hypothesis was that exposure to fine particles would be associated with increases in exhaled nitric oxide and that the use of inhaled corticosteroids would modify the effect.

Study Structure
This study was part of an intensive exposure assessment and health-effects study6 conducted in Seattle from October 1999 through May 2001. Three groups of adult subjects and a group of children with asthma were recruited. The children with asthma formed the group discussed here. The monitoring sessions for each subject consisted of 10 consecutive days, starting at 4 pm on Tuesdays and ending at 4 pm on Fridays. Up to nine subjects were monitored in each session. The children were monitored between November 2000 and May 2001. The health status of the children was assessed using spirometry, symptom questionnaires, and exhaled–nitric oxide testing. Only the exhaled–nitric oxide results are discussed here.

Nitric oxide is a ubiquitous molecule in the body and is involved in many physiological functions. In the lung, nitric oxide is involved in regulation of vasodilation, in neurotransmission, and as an agent of inflammation and cell-mediated immunity.7 There is considerable evidence that nitric oxide is elevated in the lungs of children with asthma, especially during an asthma episode. Therefore, nitric oxide has been suggested as a sensitive measurement of airway inflammation in asthma.8,9 Several studies7,10 have reported a relationship between the lung’s exhaled–nitric oxide levels and the use of inhaled corticosteroid therapy, with exhaled–nitric oxide levels decreasing in association with inhaled corticosteroid treatment.

Nineteen subjects, aged 6 to 13 years, participated. All had physician-diagnosed asthma; nine were prescribed inhaled corticosteroid therapy and 10 were not. The children were recruited from a local asthma and allergy clinic; 14 of the 19 subjects were male. The percent of predicted forced expiratory volume in 1 second achieved by these subjects ranged from 43% to 100%. All children were prescribed bronchodilator medication (n=15), inhaled corticosteroids (n=9), or other anti-inflammatory medications (montelukast or cromolyn, n=6).

Exhaled breath measurements were collected off-line daily for up to 10 consecutive days in the children’s homes by having the subjects breathe into a nitric oxide–inert, impermeable Mylar® balloon. Samples were collected in the afternoon or early evening. Children were asked to forego food intake for an hour prior to collection of exhaled breath, as some foods may elevate nitrate levels. The children were instructed to inhale nearly to total lung capacity and to exhale through a Teflon® straw with an inner diameter of 3.5 mm and length of 35 cm until the Mylar bag (46 cm in diameter) was half filled.11 Exhalation through a small-diameter straw creates sufficient pressure (at least 6 cm H2O) to close the epiglottis and prevent contamination of the airway nitric oxide sample by nasal nitric oxide. Nitric oxide concentrations were measured the next day using a nitrogen oxides chemiluminescence analyzer. While we did not measure flow directly, the straw technique has been shown to restrict flow to within the range desired (50 to 500 mL/sec).9

Measurements of particulate matter sized 2.5 µm (PM2.5) were taken inside and outside subjects’ residences.6

Personal environmental monitors were worn by all children daily and placed by their beds at night. These integrated fixed-site and personal measurements were collected over 24 hours for 10 consecutive session days. PM2.5 concentrations also were monitored continuously at three central Seattle sites using tapered element oscillating microbalance monitors operated by the Puget Sound Clean Air Agency.

We used a linear mixed-effects model with random intercept and interaction terms for inhaled corticosteroid use. Our interest was in the within-subject/within-session data. Confounding variables were temperature and outdoor nitric oxide measured at a central site by the local air agency. Analyses were conducted for all children from both winter and spring sessions using commercially available statistical-analysis software.

Results
The average exhaled–nitric oxide values in the study were 19.9+12.4 parts per billion (ppb) during the winter sessions and 12.7+6.7 ppb during the spring sessions. The mean PM2.5 values from monitors outside the residences over the 10 days of the study were 13.3+1.4 mg/m3. During the study period, based on timed activity diaries, the children spent an average of 66% of their time indoors at home and 21% indoors at school. The rest of their time was spent outdoors or in transit.

The analyses showed that exhaled nitric oxide was associated with outdoor PM2.5 for the children who were not using inhaled corticosteroids. There was no association between exhaled nitric oxide and PM2.5 in the children using inhaled corticosteroid medications. The association was expressed as a 10 µg/m3 increase in PM2.5 concentration relative to the session average for each subject and for zero day lag (exposure on the same day as the exhaled–nitric oxide measurement). The association with PM2.5 values from the monitor outside the home for nonusers of inhaled corticosteroids was a 4.3-ppb increase in exhaled nitric oxide (with a confidence interval of 1.4 to 7.2). The other associations between PM2.5 and exhaled nitric oxide were with indoor-monitor (4.2 ppb), personal-monitor (4.5 ppb), and the average central-site–monitor values (3.8 ppb).

Discussion
This study found a significant association between exhaled nitric oxide and exposure to fine particles in children with asthma who were not using inhaled corticosteroid medication. It is not surprising that the use of inhaled corticosteroids modified the exhaled–nitric oxide response, as it has been shown in clinical studies10 that exhaled–nitric oxide levels in subjects with asthma decrease at a rate of about 2 ppb per week of treatment when they are prescribed inhaled corticosteroids. Our data agree with those of several other studies that looked at associations between exhaled nitric oxide and community air pollution (PM10, black smoke, and nitrogen dioxide).13 Our results, however, contrast with those of studies14,15 of controlled exposures to ozone; these, to date, have shown no association between ozone exposure and exhaled–nitric oxide levels.

The concentration of off-line exhaled nitric oxide reported here is consistent with values reported in the literature. Beck-Ripp et al10 reported an average value of 14.9±1.9 ppb in a group of 34 children with asthma who were not prescribed corticosteroids.

Outdoor sources of PM2.5 in Seattle are wood smoke from residential wood stoves, diesel exhaust particles from trucks and buses, and automotive traffic. Seattle is one of the most traffic-congested cities in the United States. One source-apportionment study evaluated speciated data from one site in Seattle using positive matrix factorization.16 The authors estimated the major sources contributing to PM2.5 in Seattle to be vegetative burning (35%), mobile sources (22%), and sulfate (20%), with road dust, nitrate, and marine aerosol making up the balance. Our study found consistent associations between PM2.5 and exhaled nitric oxide from children studied during both winter and spring; there was no significant seasonal effect. PM2.5 values were, however, considerably higher during winter, when fine particles from wood stoves predominate.

We conclude that these data add documentation that fine particles in Seattle are associated with aggravation of asthma. Previous studies have reported lung function decrements during winter months in children with asthma associated with fine particles,2 increased emergency-department visits for asthma associated with PM2.517 and an association between the risk of asthma symptoms (cough and shortness of breath) in children and PM2.5 concentrations18 in Seattle.

Acknowledgments
The study described here was the collaborative work of 10 investigators from the Departments of Environmental Health, Biostatistics, Pediatrics, and Civil and Environmental Engineering, University of Washington, Seattle. This research was supported by grants from the US Environmental Protection Agency and the US National Institutes of Health. The author and the other investigators thank the children and their parents for participation in the study.

Jane Q. Koenig, PhD, is professor, Department of Environmental Health, University of Washington, Seattle.

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