INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

There is increasing evidence that nitric oxide (NO) may play a role in the pathogenesis of asthma. Exhaled NO (ENO) appears to be increased in patients with asthma, and it may reflect disease activity (1). NO is generated from L-arginine by the enzyme NO synthase (NOS), which is found in many cells of the lung. The constitutive isoform of NOS seems to release NO under physiologic conditions, whereas it has been suggested that the increased ENO levels found in asthmatic subjects may be due to an increase of inducible NOS expression in the respiratory tract (4). ENO has been shown to decrease after glucocorticoid treatment, and has been suggested to be a marker of airway inflammation in asthma (2, 5, 6). However, even though exercise represents an important physiologic trigger for asthma, the effect of exercise on ENO has so far been studied only in healthy adults (7), and there are no data on the relationship between exercise-induced bronchoconstriction (EIB) and ENO.

The pathogenesis of EIB has been a source of debate for many years, and the precise mechanisms through which exercise causes airway obstruction in asthmatic patients remain unknown. EIB is used as a test of airway hyperresponsiveness (AHR), and has been found to be very sensitive in the detection of bronchial hyperreactivity in asthmatic children (10).

The aim of this study was to assess the effect of an exercise challenge on ENO concentration, and the relationship between ENO values and bronchial reactivity to exercise in asthmatic children. For this purpose, we measured ENO and spirometric parameters before and after an exercise test in children with mild to moderate asthma.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Study Subjects

The study included 24 asthmatic children (17 males, and 7 females) aged 11.2 ± 0.4 yr (mean ± SEM) (range: 6 to 13 yr). They were recruited from patients attending the outpatient clinic at the Department of Pediatrics of the University of Padua. The diagnosis of asthma was based on clinical history and examination, and on pulmonary function parameters, according to international guidelines (11). Eighteen of the 24 subjects were under continuous treatment: 17 with inhaled steroids at low doses (200 to 400 µg/d beclomethasone dipropionate, or equivalent doses of fluticasone or budesonide), and one with cromones. All subjects used inhaled albuterol on demand. The presence of allergy to common allergens (cat, house dust mite, Parietaria, grass pollen, Artemisia, Alternaria, milk, and eggs) was demonstrated in 20 of the subjects by positive skin prick tests or radioallergosorbent testing. Patients were excluded from the study if they had experienced an upper respiratory tract infection within 3 wk before the first prestudy visit.

We also measured ENO levels in 18 healthy children aged 10.8 ± 0.6 yr (range: 7 to 15 yr) with negative skin prick tests and normal pulmonary function parameters, who served as a control group. They had no history of allergy, respiratory disease, or recent respiratory tract infections. Nine of these control subjects underwent exercise testing.

Study Design

We measured ENO, FEV₁, and heart rate (HR) in asthmatic and healthy children before (baseline) and repeatedly after the end of a standardized exercise test of 6 min on a treadmill. To assess the effect of spirometry on ENO levels, we measured ENO before and 18 min after forced expiratory maneuvers in 11 asthmatic children (age 11 ± 0.5 yr; range: 7 to 13 yr) who did not undergo the exercise test.

Exercise Testing and Spirometry

Exercise testing was performed on an electrically driven treadmill (PK Morgan Ltd., Gillingham, Kent, UK) for 6 min under stable environmental conditions. The speed and the incline set of the treadmill were adjusted to produce a workload that would generate a target HR of 170 beats/min or greater during the first minute of exercise. All subjects were studied in the afternoon. The asthmatic subjects refrained from taking any medication for at least 24 h before the study.

Spirometric measurements were made with the subjects at rest and at 1, 6, 12, and 18 min after the end of exercise, using a 10-L bell spirometer (Biomedin, Padua, Italy). The best of three FEV₁ values was expressed as a percentage of predicted reference values (12). In order to investigate subjects with a significant decrease in FEV₁ after exercise, we considered as a suitable cutoff a 12% postexercise decrease in FEV₁, which is generally accepted to define EIB (13).

HR was recorded continuously during exercise and recovery with a cardiofrequency meter (Sport Tester TM PE 3000; Polar Electro, Kempele, Finland).

Measurement of ENO

ENO measurements were made with the subjects at rest (baseline) and at 1, 6, 12, and 18 min after the exercise challenge. ENO concentration was measured with a single-breath reservoir technique, using a device developed for exhaled air collection (Figure 1). Subjects were in an upright position during the measurements, and were asked to inhale orally to TLC and to perform a slow VC maneuver. They exhaled through a restrictor connected directly to a T valve (Quintron, Milwaukee, WI) that allowed the separation of exhaled air into two fractions: the first portion of exhaled air (200 ml), which was discarded, and the remainder (lung air), which was collected. Subjects did not wear nose clips, and were instructed not to hold their breath before exhalation. The sample gas was collected in an NO-impermeable Mylar reservoir. In order to keep the soft palate closed and to isolate the nasopharynx, the mouthpiece of the collecting device had an internal restrictor (diameter 5 mm) that during exhalation created a pressure in the mouth of 5 to 7 cm H₂O. At this mouth pressure the expiratory flow, measured with a heated pneumotachograph (Hans Rudolf, Kansas City, MO), was 180 to 200 ml/s. Ambient NO levels were always recorded before the test. If the NO content of ambient air was > 5 ppb, the subjects breathed for 30 s before the collection of exhaled air from a reservoir filled with air containing < 5 ppb NO, to eliminate contamination by ambient air. NO-depleted air was obtained by passing ambient air through a Purafill converter (Maihak, Milan, Italy). Gas samples were immediately analyzed.

View larger version (27K): [in this window] [in a new window]   Figure 1.   Schematic diagram of the equipment used for collection of ENO with a single-breath reservoir technique. Subjects exhaled through a mouthpiece connected to a T-valve, allowing separation of exhaled air into a first portion, which was discarded, and the remainder, which was collected into a Mylar reservoir. To keep the soft palate closed, the mouthpiece of the collecting device had an internal restrictor that, during exhalation, created a pressure in the mouth of 5 to 7 cm H2O.

The measurement of ENO was done with a chemiluminescence analyzer (CLD 700 Al-Med; Ecophysics, Durnten, Switzerland) that has a sampling rate of 0.7 L/min. Before each study, the chemiluminescence analyzer was checked with a certified calibration mixture (300 ppb) of NO in nitrogen (SIAD, Bergamo, Italy) with guaranteed stability.

Statistical Analysis

ENO concentrations are reported in parts per billion. Results are expressed as mean ± SEM. A computer package (Statistica; Microsoft Corp., Redmond, WA) was utilized for statistical analysis. Analysis of variance (ANOVA) with Scheffe's post hoc test was used to compare baseline ENO values and the time course of ENO, HR, and FEV₁ in the EIB, non-EIB, and control groups. Correlation between baseline ENO levels and percent decrease in FEV₁ was evaluated with Spearman's rank correlation test. ENO measurements before and 18 min after spirometry were compared through Wilcoxon's matched-pairs test. Results were considered significant at a value of p < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The asthmatic subjects were divided into groups with (EIB group) (n = 10; age: 12.2 ± 0.2 yr) and without (non-EIB group) (n = 14; age: 10.5 ± 0.5 yr) EIB. The main results are presented in Table 1.

Baseline ENO values were higher in the EIB group (12.3 ± 1.6 ppb) than in the total control group (6.1 ± 0.2 ppb, p < 0.01). No significant differences were found between the non-EIB group (9.1 ± 1.0 ppb) and the control group (p = NS), or between the EIB and non-EIB groups (p = NS). There was no difference between the EIB (88 ± 2%) and the non-EIB groups (91 ± 3%) (p = NS) in baseline FEV₁ expressed as a percent of predicted values.

After the exercise test, the mean maximal decrease in FEV₁ was 34 ± 5% (range: 12 to 64%) in the EIB group and 5 ± 1% (range: 2 to 9%) in the non-EIB group. The time course of ENO was similar in the EIB, non-EIB, and control groups (p = NS), with no significant changes induced by exercise (Figure 2).

View larger version (16K): [in this window] [in a new window]   Figure 2.   Baseline and postexercise ENO (left vertical axis) and FEV1 values (right vertical axis) in the EIB group (closed squares), in the non-EIB group (open circles), and in the control group (closed circles). Postexercise FEV1 is expressed as % of the baseline value. The time course of ENO after the exercise test was similar in all three groups, without significant changes from baseline (p = NS, ANOVA).

In the 24 asthmatic subjects we found a significant correlation (r = 0.61, p < 0.01) between baseline ENO and the magnitude of the maximal postexercise decrease in FEV₁ (Figure 3). After the test, there was no correlation between variation of FEV₁ and variations in ENO levels (p = NS) in any of the three study groups. Similarly, we found no correlation between variation of ENO and variation of HR (p = NS).

View larger version (11K): [in this window] [in a new window]   Figure 3.   Relationship between baseline ENO and maximal postexercise decrease in FEV1 in the 24 asthmatic subjects.

Of the 17 patients taking inhaled steroids, nine had EIB. There was no difference between the steroid-treated and steroid-naive subjects (p = NS) in the time course of ENO after the exercise test.

In the group of asthmatic subjects who did not undergo exercise testing, we found no difference between the values of ENO measured before and 18 min after spirometry (11.7 ± 2.2 ppb and 10.6 ± 2.0 ppb, respectively; p = NS).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Our study shows that ENO concentrations do not change after exercise challenge in either healthy controls or asthmatic children with and without EIB. Baseline ENO levels are higher in asthmatic children with EIB than in healthy subjects, and correlate with the magnitude of the postexercise decrease in FEV₁.

There is increasing evidence that ENO levels are significantly increased in exhaled air of patients with asthma (2, 4- 6). Despite much work on ENO and asthma, no studies have so far been reported of ENO and EIB, which represents an interesting model of asthma induced by a physiologic stimulus.

The role played by NO during physical exercise remains controversial. Several studies that have examined the effect of exercise on ENO in healthy adult subjects have found that during exercise, ENO decreases whereas NO output increases (7). The duration of this effect after exercise is unknown.

We measured ENO with a single-breath reservoir technique that is known to be well correlated with on-line measurements (14). In our study, acute airway obstruction induced by exercise challenge (a mean decrease in FEV₁ of 34% in the EIB group) was not associated with acute changes in ENO measured sequentially after exercise. Previous studies of the effect of airway caliber on ENO reached different conclusions. De Gouw and colleagues (18) showed that acute bronchoconstriction induced by hypertonic saline and challenge with adenosine monosphosphate was associated with a significant reduction in ENO. Other authors, on the other hand, found that acute changes in airway caliber following administration of bronchodilators or methacholine or histamine challenge did not affect ENO concentrations (5, 19).

A similar result (i.e., a lack of change in ENO levels) has been reported by Kharitonov and associates (20) during the early response to allergen challenge, which is likely to be due to release of bronchoconstrictor mediators such as histamine and leukotrienes from airway mast cells. An increase in ENO, on the other hand, was observed during the late asthmatic response to allergen, and was attributed to induction of an acute inflammatory response in the asthmatic airway.

Deykin and coworkers (21) found that relatively few spirometric efforts reduced NO values in expired air for at least 8 h after spirometry. In order to assess whether our data could have been affected by spirometry, we measured ENO in 11 of our asthmatic subjects before and 18 min after spirometry. We found no changes after spirometry, and therefore believe that spirometric efforts cannot have masked a possible effect of exercise on ENO. In order to avoid peaks in ENO caused by accumulation of NO in the upper airways, we used a reservoir collection technique, discarding dead-space gas in an attempt to exclude gas not derived from the lower airway. In addition, our subjects did not hold their breath before expiring into the reservoir (16).

EIB can be used as a diagnostic test of bronchial hyperresponsiveness (10). AHR is one of the hallmarks of asthma, and seems also to be correlated with airway inflammation. We found a significant correlation between baseline ENO levels and magnitude of the decline in FEV₁ in response to exercise. The correlation between ENO and AHR that had been previously reported in asthmatic adults during methacholine and histamine challenge tests (22, 23), indirectly suggests a relationship between NO and airway inflammation. However, in contrast to the finding of Dupont and colleagues, who used a histamine challenge, we also found a significant relationship between ENO and AHR in steroid-treated patients. This discrepancy could be explained by the different mechanisms that might be responsible for AHR to different stimuli (24).

In conclusion, our study shows that ENO levels do not change during acute airway obstruction induced by exercise challenge in asthmatic children. In addition, baseline ENO values correlate with the magnitude of post-exercise bronchoconstriction. This relationship suggests that baseline ENO may contribute to AHR precipitated by nonsensitizing bronchoconstrictor stimuli. However, further studies are needed to clarify whether NO has a specific role as a causative factor in EIB, or merely reflects airway inflammation.