asthma, and were correlated with the degree of airway hyperresponsiveness (AHR), ... fore, the noninvasive measurement of NOexh allowed us to discriminate, ...
Exhaled Nitric Oxide Correlates with Airway Hyperresponsiveness in Steroid-naive Patients with Mild Asthma LIEVEN J. DUPONT, FELICIEN ROCHETTE, MAURITS G. DEMEDTS, and GEERT M. VERLEDEN Department of Pulmonary Medicine, University Hospital Gasthuisberg, Katholieke Universiteit Leuven, Leuven, Belgium
Endogenously released nitric oxide (NO) has been detected in the exhaled air of humans. Exhaled NO (NOexh) levels have been significantly increased in patients with inflammatory airways disorders such as asthma, and NOexh has been suggested to be a usable marker of airway inflammation. In the present study, NOexh levels were measured both in steroid-treated and untreated subjects with mild asthma, and were correlated with the degree of airway hyperresponsiveness (AHR), measured as the dose of histamine that produced a 20% decrease in FEV1 (PC20histamine). NOexh levels, which were significantly increased in steroid-naive patients (Group A1: NOexh 5 21 6 11 ppb; n 5 56) in comparison with levels in control subjects (Group B: NOexh 5 10 6 2 ppb; n 5 20; p , 0.001), correlated significantly with the PC20histamine (r 5 20.65; p , 0.0001). The NOexh level was significantly lower in patients with chronic cough of other causes than bronchial asthma (Group A2: NOexh 5 11 6 3 ppb; n 5 18) when compared with the level in subjects with mild asthma (Group A1: p , 0.001). Therefore, the noninvasive measurement of NOexh allowed us to discriminate, among patients with respiratory complaints, between those with and without AHR. In asthmatic subjects treated with inhaled steroids, the NOexh levels were significantly lower (Group A3: NOexh 5 13 6 5 ppb; n 5 25) than in untreated subjects (Group A1; p , 0.01), and there was no relationship with the PC20histamine (r 5 20.18, p 5 NS). These findings confirm that NOexh reflects AHR in patients with mild asthma who have not already been treated with inhaled steroids. Patients treated with inhaled steroids had an NOexh level comparable to levels in control subjects, although AHR could still be demonstrated. Dupont LJ, Rochette F, Demedts MG, Verleden GM. Exhaled nitric oxide correlates with airway hyperresponsiveness in steroid-naive patients with mild asthma. AM J RESPIR CRIT CARE MED 1998;157:894–898.
The importance of the highly reactive molecule nitric oxide (NO), which acts as an intracellular messenger in many biologic processes, has been widely recognized. In the respiratory system, NO is known as an inflammatory mediator (1), as well as a vasodilator (2) and nonadrenergic, noncholinergic neurotransmitter (3). Under physiologic conditions NO is released by the action of the constitutive, calcium-dependent enzyme nitric oxide synthase (NOS) in endothelial cells and neurons. In response to immunologic stimulation or inflammation, NO is also synthesized by an inducible calcium-independent NOS in several cell types within the respiratory tract (monocytes, macrophages, epithelial cells). Glucocorticoids, which control inflammation efficiently, potently inhibit the expression of iNOS (4). Release of endogenous NO in the respiratory tract may play an important signaling role in the physiologic control of airway function and in the pathophysiology of airway diseases. (Received in original form September 16, 1997 and in revised form November 17, 1997) Correspondence and requests for reprints should be addressed to Prof. Dr. G. M. Verleden, Department of Pulmonary Medicine, University Hospital Gasthuisberg, 49 Herestraat, B-3000 Leuven, Belgium. Am J Respir Crit Care Med Vol 157. pp 894–898, 1998
NO has been detected in low concentrations in the exhaled air of humans and animals through the use of sensitive chemiluminescence analyzers (5). In healthy subjects, exhaled NO seems to originate mainly in the upper airways (paranasal sinuses) (6), whereas the contribution from the lower respiratory tract is small. However, NO levels are significantly increased in orally exhaled air of patients with inflammatory airways disorders such as asthma (7, 8), respiratory-tract infections (9), bronchiectasis (10), and chronic rejection following lung transplantation (11). It is likely that the increased NO levels are due to induction of iNOS, since increased NOS activity has been demonstrated in lung tissue of patients with inflammatory lung diseases (12). Treatment with inhaled glucocorticoids reduced these high NO levels in asthmatic individuals in a dose-dependent manner (13). This suggests that NO in exhaled air may be used as a marker of antiinflammatory treatment, and possibly also as a marker of airways inflammation. Airway hyperresponsiveness (AHR) is one of the hallmarks of asthma, and has been attributed to and also correlated with the characteristic asthmatic airway inflammation (14). Since AHR is considered a parameter of airway inflammation, we were interested in further investigating a possible relationship between NO in exhaled air and AHR in patients with mild asthma.
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Dupont, Rochette, Demedts, et al.: NO Correlates with AHR in Mild Asthma TABLE 1 ASTHMA SEVERITY SCORE ASSESSED BY MEANS OF A STANDARDIZED QUESTIONNAIRE* Symptoms
Score
Symptoms of bronchial reactivity to nonspecific stimuli Fog Smoke Smells Change in temperature Other None Hyperventilation asthma Laughing, crying Sneezing, coughing Talking None Exercise-induced asthma Mild exercise Moderate exercise Heavy exercise None Nocturnal asthma , 1/wk 1–3/wk 3–6/wk . 6/wk None Frequency of morning dip symptoms , 1/wk 1–3/wk 3–6/wk . 6/wk None Frequency of inhaled symptomatic treatment , 1/24 h 1–4/24 h . 4/24 h None
1 1 1 1 1 0 1 1 1 0 3 2 1 0 1 2 3 4 0 1 2 3 4 0 1 2 3 0
* Questionnaire described the listed symptoms over the preceding 14 d. The score for each symptom was noted, and the maximum total score was 22.
METHODS Subjects were recruited from among outpatients who had symptoms suggestive of asthma (e.g., cough, wheezing, episodic dyspnea) and who were referred for a histamine challenge test to document the potential of variable airways obstruction. We recruited 104 patients (Group A: 49 females and 55 males, mean age 40 6 17 yr), from whom informed consent was obtained. Subjects had to be nonsmokers with an FEV1 . 80% of predicted. All of them completed a questionnaire regarding their respiratory complaints and current medication use (Table 1) (15). All patients underwent spirometry according to Amer-
ican Thoracic Society (ATS) guidelines (16). Subsequently, exhaled NO was measured and patients underwent a histamine challenge according to the method of Cockcroft and colleagues (17). The provocative concentration of histamine causing a 20% decrease in FEV1 (PC20histamine) was calculated by linear interpolation. Twenty healthy subjects, recruited from among the staff of the University hospital Gasthuisberg (Leuven, Belgium), were used as a control group (Group B: eight females and 12 males, mean age 42 6 12 yr). All were nonsmokers, had no history of cardiovascular or respiratory disease, and were receiving no regular medication. They had absolutely no respiratory complaints, and had normal spirometric results together with normal bronchial responsiveness (PC20histamine > 8 mg/ml). Neither the healthy volunteers nor the patients had a history of upper-respiratory-tract infection for at least 4 wk prior to the study. They had also no active complaints of rhinoconjunctivitis for at least 3 mo prior to the study. Neither patients nor subjects consumed any alcohol-containing or caffeinated beverages, nor did they use inhaled short-acting b2mimetics in the 8 h prior to NO and PC20histamine measurement. Because it is known that steroid treatment reduces the level of exhaled NO (8) and ameliorates the PC20histamine (21), patients were subdivided for further analysis into several subgroups according to their PC20histamine and current treatment. Group A1 consisted of 56 patients with a PC20histamine , 8 mg/ml and who were diagnosed as having asthma. Some of these patients were treated with inhaled b2agonists as required, but none of them was taking inhaled glucocorticoids, nor had they received any kind of steroids (including nasal steroids) within the previous 3 mo. Group A2 consisted of 18 subjects who had a PC20histamine > 8 mg/ml and who were not treated with inhaled steroids. Because of the absence of variable airways obstruction, these 18 patients could not be diagnosed as having asthma. A review of their medical records demonstrated the following diagnoses in this subgroup: chronic cough due to postnasal drip (seven patients), chronic cough due to gastroesophageal reflux (five patients), angiotensin converting enzyme (ACE)-inhibitor-induced cough (three patients), hyperventilation syndrome (one patient), and chronic cough of unknown origin (two patients). Group A3 consisted of 25 patients with a PC20histamine , 8 mg/ml and who were treated with inhaled steroids for at least 6 wk (between 500 and 1000 mg beclomethasone dipropionate, or equivalent). Patients who had both a PC20histamine > 8 mg/ml and who were treated with steroids were excluded (n 5 5), since a diagnosis of asthma could not be confirmed in these patients. Exhaled NO was measured during a single breath exhalation by means of an Ecophysics CLD 700 AL MED (Dürnten, Switzerland) chemiluminescence analyzer adapted for online recording. Measurements were made according to European Respiratory Society (ERS) guidelines for the measurement of exhaled NO (18), which allow a reproducible measurement of the NO level (19). In brief, subjects performed a slow VC maneuver over a 30-s period against a fixed expiratory resistance with a target gauge of 20 cm H2O, which prevented autoinhalation of NO from the nasopharynx (20) and resulted in a constant flow of 20 ml/s. Exhaled air was led via a Hans Rudolph nonrebreathing valve into a Teflon tubing system connected to the analyzer. Air was continuously sampled from the exhalation limb of the system at a sampling rate of 0.7 L/min and a response time of 1 s. Results were
TABLE 2 PATIENT CHARACTERISTICS AND EXHALED NO LEVEL IN STUDY GROUPS*
Number of patients Age, yr Inhaled steroids Symptom score Number of atopics FEV1, % FVC, % PC20histamine, mg/ml Exhaled NO
A1
A2
A3
B
56 40 6 17 no 463 34/56 102 6 12 107 6 12 1.8 6 2.2 21 6 11 ppb†‡
18 42 6 18 no 362 1/18 107 6 12 107 6 13 .8 11 6 3 ppb
25 39 6 18 yes 564 15/25 108 6 13 109 6 13 2.0 6 2.2 13 6 5 ppb
20 34 6 14 no 0 0/20 111 6 9 106 6 9 .8 10 6 2 ppb
* NO level in mean 6 standard deviation. † p , 0.001 compared with Group A2 or Group B. ‡ p , 0.01 compared with Group A3.
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displayed on a chart recorder, and the plateau level was noted. Three reproducible recordings (i.e., , 15% variation) were made at 2-min intervals, and the highest of three readings was used for analysis. All exhaled NO levels are reported in parts per billion (ppb). The detection limit for NO was 1 ppb, and measurements were made in the same chamber under constant conditions. Before each measurement, NO zeroing was performed, using NO-free air. Twopoint calibration was done with a certified concentration of NO (100 ppb) balanced with N2, in accordance with the ERS guidelines for the measurement of exhaled NO (18). Day-to-day reproducibility was assessed in preliminary experiments. The variability between NO levels measured at the same time of day on three consecutive days was less than 10%. The measurements of exhaled NO were compared by means of the Mann-Whitney U test for unpaired data. Logarithmic transformation was applied to exhaled NO levels and to values of PC20histamine before analysis. Correlations were made with Spearman’s rank test; linear regression was done with the least-squares method.
RESULTS The characteristics of the patients and control subjects, and their NO levels (mean 1 SD), are depicted in Table 2. There was no difference in mean FEV1, FVC, symptom score (15), or mean age between the four study groups. Mean PC20histamine values and the number of atopic subjects were not significantly different in groups A1 and A3. There was no sex difference in the levels of exhaled NO in any of the four study groups. Exhaled NO was significantly greater in steroid-naive patients with a PC20histamine , 8 mg/ml than in those with a PC20histamine > 8 mg/ml (21 6 11 ppb versus 11 6 3 ppb, respectively, p , 0.001) and in control subjects (21 6 11 ppb versus 10 6 2 ppb, respectively, p , 0.001). Patients with a PC20histamine , 8 mg/ml and who were treated with inhaled steroids had a significantly lower exhaled NO level than did steroid-naive patients (13 6 5 ppb versus 21 6 11 ppb, respectively, p , 0.01). In the steroid-naive patients (Group A1), exhaled NO levels were significantly correlated with the PC20histamine value (r 5 20.65, p , 0.0001; Figure 1). This correlation between the PC20histamine and exhaled NO was lost in patients who were being treated with inhaled steroids (Group A3: r 5 20.18, p 5 NS; Figure 2).
Figure 1. Relationship between exhaled NO (ppb) and PC 20histamine (mg/mI) in steroid-naive patients with mild asthma (Group A1: r 5 20.65; p , 0.0001).
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DISCUSSION We have shown for the first time that the increased concentration of NO in the exhaled air of patients with mild asthma correlates with the degree of AHR, as measured with PC20histamine. The easy-to-perform, noninvasive measurement of NO further allowed us to discriminate patients with and without airway responsiveness. However, we failed to demonstrate a correlation between exhaled NO and PC20histamine in patients with mild asthma who were being treated with steroids. The exhaled NO level was significantly lower in these patients than in steroid-naive patients. These findings are compatible with the hypothesis that the increase in exhaled NO reflects airway inflammation in asthma. This study was originally undertaken to determine the value of the measurement of exhaled NO in the diagnosis of asthma in a population of patients who presented with complaints suggestive of obstructive airway disease but who had a normal FEV1 (e.g., . 80% predicted). This makes it obviously difficult to document a bronchodilator-induced variability in FEV1 of > 15%. In the absence of this bronchodilator-induced variability, histamine responsiveness, defined as a PC20histamine < 8 mg/dl, was used to document reversible airway obstruction in these patients. Exhaled NO was measured at the time of the histamine challenge, and was correlated with the PC20histamine value. To control for the effects of inhaled steroids on exhaled NO levels and on histamine responsiveness, patients were subdivided into different groups. Group A1 represented a group of patients who were labeled as steroidnaive, mildly asthmatic patients based on their symptoms and a positive histamine challenge. Group A2 consisted of patients who, although presenting with respiratory complaints, could not be diagnosed as having asthma due to the absence of variable airway obstruction and a normal histamine challenge test. These patients were diagnosed as having chronic cough caused by other diagnosed conditions such as postnasal drip or gastroesophageal reflux. The patients in Group A2 did not have an increased exhaled NO level, in contrast to the patients in Group A1. This suggests that the level of exhaled NO, which is much more easily measured, correlates well with the presence or absence of AHR in a steroid-naive population of patients with normal FEV1 and with respiratory complaints possibly indicative of obstructive airways disease.
Figure 2. Relationship between exhaled NO (ppb) and PC 20histamine (mg/ml) in steroid-treated patients with mild asthma (Group A3: r 5 20.18, p 5 NS).
Dupont, Rochette, Demedts, et al.: NO Correlates with AHR in Mild Asthma
Group A3 represented a group of patients with mild asthma (normal FEV1 and positive histamine challenge test) who were treated with inhaled steroids. As demonstrated previously, the exhaled NO level of these patients was significantly lower than that of the steroid-naive asthmatic patients. Asthmatic patients have repeatedly been found to exhale exaggerated levels of NO (7, 8). An increase in the expression of iNOS in epithelial cells, induced by proinflammatory cytokines, may account for the increase in exhaled NO (12). Treatment with inhaled steroids, which inhibits the production of endogenous NO, significantly and dose-dependently reduced NO levels (13). There was also an increase in exhaled NO during exacerbations of asthma (22). On the basis of these findings, measurement of exhaled NO has been suggested as a noninvasive method to monitor airway inflammation. However, a more direct correlation of exhaled NO with other parameters of airway inflammation in asthma has not been clearly demonstrated up to now. Kharitonov and colleagues reported an increase in exhaled NO during the late-phase reaction of asthmatic subjects after antigen challenge. Moreover, there was a significant relationship between the extent of the late response and the increase in exhaled NO (23). The late asthmatic reaction after antigen challenge has been associated with AHR through induction of an acute inflammatory response in asthmatic airways (24), which also upregulates the expression and activity of iNOS (25). The correlation between the extent of the late response and the NO level provides more direct evidence that exhaled NO reflects allergic inflammation in asthmatic airways. Recently, a significant correlation has also been demonstrated between the concentration of nitrite and nitrate, the stable end products of NO metabolism, in induced sputum of asthmatic individuals, and the percentage of eosinophils and extracellular protein (ECP) level in sputum (26). Patients with bronchiectasis, another lung disease characterized by chronic inflammation in the airways, have also been shown to exhale elevated levels of NO. The exhaled NO levels of patients who were not treated with inhaled steroids were significantly correlated with disease severity, as determined by thin-section computed tomography (CT). This relationship could not be established in patients who were treated with regular inhaled steroids (10). These conclusions are similar to our findings in a group of mildly asthmatic patients. We have not only found that exhaled NO levels were increased in patients with AHR, but have also shown a significant correlation between the exhaled NO level and the degree of AHR measured as PC20histamine in those patients who were not treated with steroids. AHR has been defined as an increase in the ease and degree of airways narrowing in response to bronchoconstrictor stimuli (27). The presence and severity of hyperresponsiveness to chemical bronchoconstrictor mediators such as histamine and metacholine has been correlated, in mildly asthmatic subjects, with the number and state of activation of inflammatory cells within the airways (28, 29). A correlation between exhaled NO and AHR indirectly suggests a relation with airways inflammation. Additional research, however, is needed to correlate exhaled NO levels more directly with measures of inflammation. Barnes and Juniper and colleagues showed that inhaled glucocorticoids reduce airways inflammation and hyperresponsiveness, although AHR rarely returns to normal values, and the change with inhaled steroids is often small (21, 30). These investigators therefore concluded that AHR correlates well with airway inflammation (28, 29), although it might not be a useful means of monitoring the response to antiinflammatory treatment, such as with inhaled steroids. However, the
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measured level of exhaled NO, which correlates with AHR in steroid-naive patients with mild asthma, was significantly higher in subjects with untreated mild asthma than in steroidtreated patients, and comparable to the levels of exhaled NO of control subjects, although AHR could still be demonstrated in these patients. This confirms previous findings that bronchial responsiveness to inhaled steroids tends to be smaller than that to systemic steroids (21, 29), and suggests that the exhaled NO level reflects the activity of steroids more directly than does the measurement of airways responsiveness or the FEV1. However, in this study, only patients with mild asthma with normal valves of FEV1 were included. The results of the study therefore do not exclude the possibility of a correlation between exhaled NO and bronchial hyperresponsiveness in a population of patients with more severe asthma (FEV1 , 80%), or with less well-controlled asthma, in whom NO levels could be persistently increased. References 1. Barnes, P. J., and F. Y. Liew. 1995. Nitric oxide and asthmatic inflammation. Immunol. Today 16:128–130. 2. Barnes, P. J. 1995. Nitric oxide and airway disease. Ann. Med. 27:389– 393. 3. Belvisi, M. G., C. D. Stretton, M. Miura, G. M. Verleden, S. Tadjkarimi, M. H. Yacoub, and P. J. Barnes. 1992. Inhibitory NANC nerves in human tracheal smooth muscle: a quest for the neurotransmitter. J. Appl. Physiol. 73:2505–2510. 4. Forstermann, U., and H. Kleinert. 1995. Nitric oxide synthase: expression and expressional control of the three isoforms. Naunyn. Schmiedebergs Arch. Pharmacol. 352:351–364. 5. Gustafsson, L. E., A. M. Leone, M. G. Persson, N. P. Wiklund, and S. Moncada. 1991. Endogenous nitric oxide is present in the exhaled air of rabbits, guinea pigs and humans. Biochem. Biophys. Res. Commun. 181:852–857. 6. Lundberg, J. O., T. Farkas-Szallasi, E. Weitzberg, J. Rinder, J. Lidholm, A. Anggaard, T. Hokfelt, J. M. Lundberg, and K. Alving. 1995. High nitric oxide production in human paranasal sinuses. Nature Medicine 1:370–373. 7. Persson, M. G., O. Zetterstrom, V. Agrenius, E. Ihre, and L. E. Gustafsson. 1994. Single-breath nitric oxide measurements in asthmatic patients and smokers. Lancet 343:146–147. 8. Kharitonov, S. A., D. Yates, R. A. Robbins, R. Logan-Sinclair, E. A. Shinebourne, and P. J. Barnes. 1994. Increased nitric oxide in exhaled air of asthmatic patients. Lancet 343:133–135. 9. Kharitonov, S. A., D. Yates, and P. J. Barnes. 1995. Increased nitric oxide in exhaled air of normal human subjects with upper respiratory tract infections. Eur. Respir. J. 8:295–297. 10. Kharitonov, S. A., A. U. Wells, B. J. O’Connor, P. J. Cole, D. M. Hansell, R. B. Logan-Sinclair, and P. J. Barnes. 1995. Elevated levels of exhaled nitric oxide in bronchiectasis. Am. J. Respir. Crit. Care Med. 151:1889–1893. 11. Verleden, G. M., L. J. Dupont, L. Lamont, B. Buyse, M. Delcroix, D. Van Raemdonck, T. Lerut, J. Vanhaecke, and M. G. Demedts. 1997. Is there a role for measuring exhaled NO in lung transplant recipients with chronic rejection? J. Heart Lung Transplant. (In press) 12. Hamid, Q., D. R. Springall, V. Riveros-Moreno, P. Chanez, P. Howarth, A. Redington, J. Bousquet, P. Godard, S. Holgate, and J. M. Polak. 1993. Induction of nitric oxide synthase in asthma. Lancet 342:1510– 1513. 13. Kharitonov, S. A., D. H. Yates, K. F. Chung, and P. J. Barnes. 1996. Changes in the dose of inhaled steroid affect exhaled nitric oxide levels in asthmatic patients. Eur. Respir. J. 9:196–201. 14. Ohashi, Y., S. Motojima, T. Fukuda, and S. Makino. 1992. Airway hyperresponsiveness, increased intracellular spaces of bronchial epithelium and increased infitration of eosinophils and lymphocytes in bronchial mucosa in asthma. Am. Rev. Respir. Dis. 145:1469–1476. 15. Nackaerts, K., G. M. Verleden, and M. G. Demedts. 1993. Asthma severity score and hyperreactivity (abstract). Eur. Respir. J. 6:469S. 16. American Thoracic Society. 1987. Standardization of Spirometry. Am. Rev. Respir. Dis. 136:1285–1298. 17. Cockcroft, D. W., D. N. Killian, J. J. Mellon, and F. E. Hargreave. 1977. Bronchial reactivity to inhaled histamine: a method and clinical sur-
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vey. Clin. Allergy 7:235–243. 18. Kharitonov, S. A., K. Alving, and P. J. Barnes. 1997. Exhaled and nasal nitric oxide measurements: recommendations (ERS Task Force Report). Eur. Respir. J. 10:1683–1693. 19. Gabbay, E., A. J. Fisher, T. Small, A. J. Leonard, and P. A. Corris. 1997. Exhaled single breath nitric oxide measurements are reproducible, repeatable and reflect levels of nitric oxide found in the lower airways. Eur. Respir. J. (In press) 20. Silkoff, P. E., P. A. McClean, A. S. Slutsky, H. G. Furlott, E. Hoffstein, S. Wakita, K. R. Chapman, J. P. Szalai, and N. Zamel. 1997. Marked flow-dependence of exhaled Nitric Oxide using a new technique to exclude nasal nitric oxide. Am. J. Respir. Crit. Care Med. 155:260–267. 21. Barnes, P. J. 1990. Effect of corticosteroids on airway hyperresponsiveness. Am. Rev. Respir. Dis. 141:S70–S76. 22. Massaro, A. F., B. Gaston, D. Kita, C. Fanta, J. S. Stamler, and J. M. Drazen. 1995. Expired nitric oxide levels during treatment of acute asthma. Am. J. Respir. Crit. Care Med. 152:800–803. 23. Kharitonov, S. A., B. J. O’Connor, D. J. Evans, and P. J. Barnes. 1995. Allergen-induced late asthmatic reactions are associated with elevation of exhaled nitric oxide. Am. J. Respir. Crit. Care Med. 151:1894– 1899. 24. O’Byrne, P. M., J. Dolovich, and F. E. Hargreave. 1987. Late asthmatic
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