Effects of inhaled versus systemic corticosteroids on exhaled nitric oxide in severe acute asthma

Transcription

1 Respiratory Medicine (2009) 103, 614e620 available at journal homepage: Effects of inhaled versus systemic corticosteroids on exhaled nitric oxide in severe acute asthma See Meng Khoo*,a, T.K. Lim a Division of Respiratory and Critical Care Medicine, National University Hospital, Lower Kent Ridge Road, Singapore , Singapore Received 14 May 2008; accepted 10 October 2008 Available online 20 November 2008 KEYWORDS Acute asthma; Airway inflammation; Exhaled nitric oxide; Inhaled corticosteroids; Systemic corticosteroids Summary Background: There is a paucity of information on the differential effects of systemic versus inhaled corticosteroids on airway inflammation in patients with acute asthma. This study aimed to evaluate the effects of stopping systemic corticosteroids while maintaining the inhaled corticosteroids (ICS) on airway inflammation, lung function and asthma symptoms in patients who had been discharged from hospital after treatment for severe acute asthma. Methods: Twenty-four adult patients with severe exacerbations of asthma were treated with both oral and inhaled corticosteroids after discharge from hospital. Oral corticosteroids were stopped after 1 week. Spirometry, asthma quality of life questionnaire (AQLQ) score and exhaled nitric oxide (NO) were measured at discharge, 1 week, and 2 weeks after discharge. Results: Withdrawal of oral corticosteroids resulted in significant rebound in mean exhaled NO by 11.0 ppb (95% CI, 4.9e17.1 ppb, p < 0.001) or 47.7% (95% CI, 22.4e73.1%) despite uninterrupted ICS treatment. The rebound in exhaled NO occurred despite significant improvement in the mean AQLQ score (p Z 0.006) and frequency of reliever use (p Z 0.003) and was not associated with significant change in the mean FEV 1 (p Z 0.64). Conclusions: In patients discharged from hospital after treatment for asthma exacerbations, withdrawal of oral corticosteroids resulted in increase in exhaled NO levels despite continued ICS treatment. ª 2008 Elsevier Ltd. All rights reserved. Introduction Abbreviations: NO, nitric oxide; FEV 1, forced expiratory volume in 1 s; AQLQ, asthma quality of life questionnaire; ICS, inhale corticosteroids. * Corresponding author. Tel.: þ ; fax: þ address: KhooSM@nuh.com.sg (S.M. Khoo). a Both authors were involved in the planning, data collection, data analysis and writing up of the study. Measurement of exhaled nitric oxide (NO) is a non-invasive test for detection of endogenous inflammatory signals in patient with asthma. 1 Elevated levels of exhaled NO have been documented in patients with asthma. 2,3 Exhaled NO levels are further increased during asthma exacerbations, 4,5 and fall following treatment with inhaled 6 and systemic corticosteroids. 4 Oral prednisolone reduces exhaled NO in /$ - see front matter ª 2008 Elsevier Ltd. All rights reserved. doi: /j.rmed

2 Effects of inhaled versus systemic corticosteroids on exhaled NO 615 infants and young children with wheezing exacerbations. 5 In children with more severe asthma, oral corticosteroids shift their exhaled NO down to the levels of mild-to-moderate asthma with accompanying improvement in lung function. 7 A combination of oral prednisolone and inhaled corticosteroids (ICS) reduces exhaled NO by 65% in children with acute asthma. 8 However, there is an absence of prospective studies that evaluate the differential effects of inhaled versus systemic corticosteroids on exhaled NO, lung function and asthma symptoms in patients with acute asthma. Sippel and coworkers, in a cross-sectional study, showed that exhaled NO levels were significantly correlated with markers of asthma control such as asthma symptoms, dyspnoea score, use of rescue medication and reversibility of airflow obstruction. 9 Jones and colleagues showed that elevation in exhaled NO levels was a useful early indicator of loss of asthma control following withdrawal of ICS treatment. 10 A similar trend has been observed after withdrawal of leukotriene receptor antagonists. 11 Furthermore, exhaled NO appears to be sensitive to changes in corticosteroids treatment, even in the absence of lung function responses. 12 These findings suggest that exhaled NO may be related to asthma control and corticosteroids treatment, and that serial exhaled NO measurements in individual patients over time may be useful in the adjustment of anti-inflammatory treatment. Although exhaled NO has been used to monitor the effect of anti-inflammatory treatment in asthma 6,13 and asthma exacerbations, 4,12 there is a lack of prospective serial studies of exhaled NO, together with lung function, asthma symptoms and asthma relapse. The utility of serial exhaled NO measurements in patients with acute asthma after completion of systemic corticosteroids treatment, when they are at risk of relapse and re-admissions, has not been studied previously. The aim of our study was to evaluate the effects of stopping systemic corticosteroids while maintaining the ICS on the exhaled NO levels, lung function and asthma symptoms in patients who have been discharged from hospital after treatment for acute severe asthma. Materials and methods Study subjects Adult patients admitted to the Medical Department of a university hospital with acute exacerbations of asthma were invited to participate in the study. All patients were non-smokers, had prior diagnosis of asthma and had presented with symptoms consistent with acute exacerbations to the Emergency Department. Asthma was diagnosed based upon a history of episodic respiratory symptoms, a prior physician s diagnosis of asthma, the use of inhaled asthma therapy and pulmonary function testing prior to the exacerbation which demonstrated at least 15% improvement in FEV 1 following bronchodilator therapy. Acute exacerbations were defined as increasing symptoms requiring emergency treatment and presentation to the hospital. Ethical approval was obtained from the National Healthcare Group (NHG) Ethics Committee and informed consent was obtained from all study participants. Figure 1 Measurements done at discharge, visit 1 (1 week after discharge from hospital) and visit 2 (2 weeks after discharge from hospital). Definition of abbreviations: ENO Z exhaled nitric oxide; FEV 1 Z forced expiratory volume in 1 s; AQLQ Z asthma quality of life questionnaire score. Study design Patients who did not respond adequately to therapy instituted in the Emergency Department were admitted to hospital for inpatient treatment of acute asthma. Research nurse was contacted to screen for eligibility, obtain informed consent and complete clinical asthma questionnaire. During the inpatient period, all patients received standard treatment for acute asthma, which consisted of 5 mg of nebulized salbutamol and 500 mg of ipratropium bromide every 6 h for the first 24 h and systemic corticosteroids in the form of oral prednisolone of 0.5 mg/kg. Patients were also maintained on their usual dose of ICS. Decision to discharge patients from hospital was made by the attending physicians based on symptom resolution and lung function. On the day of discharge, all patients were continued on ICS and prescribed a non-tapering course of prednisolone of 0.5 mg/kg per day for 1 week. Spirometry and exhaled NO were measured on the day of discharge. Spirometry, exhaled NO and asthma quality of life questionnaire (AQLQ) were measured at visit 1 (1 week after discharge, when the patients had been on both oral prednisolone and inhaled corticosteroids) and at visit 2 (2 weeks after discharge, when the patients had been on maintenance ICS only). Measurements obtained at each visit are shown in Figure 1. Patients were instructed to keep diaries on the frequency of reliever use. All patients were contacted by a research nurse by telephone after hospital discharge to reinforce compliance with asthma medications. They were also contacted monthly for 3 months after hospital discharge to obtain information relating to relapses which required commencement of systemic corticosteroids, increase in maintenance dose of inhaled corticosteroids, unscheduled visits to doctors or hospital admissions. Exhaled NO level was measured according to the 1999 American Thoracic Guidelines, 14 using a nitric oxide

3 616 S.M. Khoo, T.K. Lim Figure 2 Exhaled nitric oxide (ENO) measurements at discharge, visit 1 and visit 2. ENO decreased significantly after treatment with oral and inhaled steroids (visit 1). ENO increased significantly after withdrawal of oral steroids despite the uninterrupted inhaled steroids treatment (visit 2). Definition of abbreviations: ENO Z exhaled nitric oxide; ICS Z inhaled corticosteroids. analyzer (NIOX, Aerocrine, Stockholm, Sweden) at an expiratory flow rate of 50 ml/s. Forced expiratory volume in 1 s (FEV 1 ) and forced midexpiratory flow rate (FEF 25e75% ) were measured according to American Thoracic Society standards 15 and the best value of three maneuvers was expressed as a percentage of the predicted value for the local population. 16 A disease-specific quality of life measure, the Asthma Quality of Life Questionnaire (AQLQ), 17 was used in this study. The instrument has been validated and demonstrated to be responsive to change. 18 The minimal clinically important difference is a change in score of 0.5 (on a scale of 7). 19 Figure 4 Asthma quality of life questionnaire (AQLQ) scores at visit 1 and visit 2. With uninterrupted inhaled steroids treatment, AQLQ scores improved significantly despite withdrawal of oral steroids. Statistical analysis Continuous variables are expressed as mean standard deviation unless otherwise specified. One-factor repeated measures ANOVA with adjustment for multiple comparison (Bonferroni) was used to compare the exhaled NO levels, FEV 1 and FEF 25e75% at discharge, visit 1 and visit 2. Student s paired t-test was used to compare AQLQ scores between visit 1 and visit 2. Wilcoxon Signed Ranks test was used to compare the paired data on frequency of reliever use between visit 1 and visit 2. P values of <0.05 were considered statistically significant. Results Twenty-four patients entered the study (a total of 30 patients were approached for the study, of whom six declined to participate). Demographic data are given in Table 1. None of the patients were on nasal corticosteroids. Figure 3 FEV 1 (% predicted) at discharge, visit 1 and visit 2. FEV 1 improved significantly after treatment with oral and inhaled steroids (visit 1). No significant change in FEV 1 after withdrawal of oral steroids (visit 2). Definition of abbreviations: FEV 1 Z forced expiratory volume in 1 s; ICS Z inhaled corticosteroids. Figure 5 Average daily frequency of reliever use in the weeks prior to visit 1 and visit 2. With uninterrupted inhaled steroids treatment, frequency of reliever use decreased significantly despite withdrawal of oral steroids.

4 Effects of inhaled versus systemic corticosteroids on exhaled NO 617 Table 1 Subjects characteristics Age, mean (range) 39 (18e65) year Male/female 9/15 Duration of asthma, mean (range) 15.9 (4e33) year Asthma therapy n (%) Budesonide 800 mg per day and 11 (46) short-acting b 2 -agonist Beclometasone 400 mg per day 7 (29) and short-acting b 2 -agonist Fluticasone 500 mg per day 5 (21) and short-acting b 2 -agonist Fluticasone 500 mg per day 1 (4) and long-acting b 2 -agonist Length of stay, mean (SD) 2.0 (1.2) days FEV 1 at discharge, mean (SD) 73.9 (20.0) % predicted ENO at discharge, mean (SD) 60.3 (46.3) ppb The mean (SD) exhaled NO level and FEV 1 on the day of discharge from hospital were ppb and % predicted respectively. Average length of stay was 2.0 days (range, 1e5 days). All patients had been treated with ICS for persistent asthma prior to the exacerbations. None of the patients reported relapse of asthma exacerbations which resulted in commencement of systemic corticosteroids, increase in maintenance dose of inhaled corticosteroids, unscheduled visits to doctors or hospital admissions in the 3 months after discharge from hospital. Exhaled nitric oxide The mean exhaled NO level on the day of discharge from hospital was ppb. Combined treatment with systemic and inhaled corticosteroids over 7 days was associated with a significant decrease in the mean exhaled NO levels by 24.2 ppb (95% CI, 6.5e41.8 ppb, p Z 0.005) or 31.3% (95% CI, 16.3e46.2%) from baseline. Withdrawal of systemic corticosteroids resulted in a rebound of the mean exhaled NO level by 11.0 ppb (95% CI, 4.9e17.1 ppb, p < 0.001) or 47.7% (95% CI, 22.4e73.1%) despite the continued ICS treatment (Fig. 2). Pulmonary function, quality of life and frequency of reliever use At discharge from hospital, the mean FEV 1 was % predicted. At visit 1, the mean FEV 1 improved significantly by 13.6% predicted (95% CI, 5.8e21.4%, p < 0.001) after treatment with both systemic and inhaled corticosteroids for 7 days (Fig. 3). At visit 2, 1 week after the withdrawal of the systemic corticosteroids, the mean FEV 1 showed a continued but non-significant improvement of 2.3% predicted (95% CI, 2.3 to 6.9, p Z 0.64). FEF 25e75% measurements showed a trend that was similar to that of FEV 1. The mean FEF 25e75% improved by 17.8% predicted (95% CI, 5.8e29.9%, p Z 0.003) between discharge and visit 1, but did not show any significant change between visit 1 and visit 2 ( 0.12% predicted; 95% CI, 8.0 to 7.7%, p Z 1.00). AQLQ scores improved significantly by 0.53 (95% CI, 0.17e0.88, p Z 0.006) between visit 1 and visit 2 (Fig. 4). Frequency of reliever use (Fig. 5) also decreased significantly despite the withdrawal of systemic corticosteroids and rebound in exhaled NO levels (p Z 0.003). Discussion The novel finding of this study was that in patients discharged from hospital after treatment for severe acute asthma, withdrawal of oral corticosteroids resulted in rebound of exhaled NO levels despite continued ICS treatment. These changes in exhaled NO were not accompanied by worsening of lung function or asthma symptoms. There are a few possible explanations for our observations. First, the increase in exhaled NO levels may be a reflection of a peripheral component of airway inflammation that is less influential or has a delayed effect on lung function and asthma symptoms. The rebound in exhaled NO levels despite uninterrupted ICS treatment may be a result of the inability of ICS to penetrate beyond smaller distal airways. It is possible that ICS are able to suppress inducible NO synthase in the larger airways but unable to reach the distal airways to have effects on the peripheral parenchymal inflammation. Lehtimaki et al. had used the two-compartment model described by Tsoukias and George, 20,21 and found that alveolar NO concentration was increased in asthmatic patients with nocturnal symptoms. 22 The same group also observed that inhaled fluticasone decreased bronchial NO flux but had no effect on alveolar NO concentration. 23 Gelb et al. reported increased alveolar NO concentration in patients with clinically stable asthma despite treatment with ICS. 24 Five days of 30 mg prednisone resulted in isolated significant decrease in alveolar NO concentration. Results from these studies suggest the presence of inflammation in very distal gas exchanging respiratory bronchioles and alveoli in asthmatic patients. 24 There is an increasing body of evidence that there may be a concomitant lung parenchymal component in asthma. Kraft et al. reported the presence of inflammatory cells in the lung parenchyma obtained by fiberoptic bronchoscopy biopsy in patients with nocturnal asthma. 25 Gelb et al. reported unexplained loss of lung parenchymal elastic recoil properties not due to emphysema in stable, nonsmoking, patients with chronic persistent asthma with abnormal expiratory airflow limitation. 26,27 Berry et al. found that alveolar NO were elevated in patients with refractory asthma and reduced by oral prednisolone treatment. 28 Belda et al. reported that in patients with acute asthma, oral prednisone reduced blood eosinophil counts, while inhaled fluticasone reduced airway eosinophil counts, suggesting that the anti-inflammatory performance of inhaled fluticasone is exerted locally. 29 The findings of the present study may be related to the presence of a separate component of peripheral inflammation in patients with acute exacerbation of asthma that is not suppressed by ICS. Second, changes in exhaled NO level may precede other parameters such as lung function and may therefore serve as an early warning of loss of control. Previous studies have yielded inconsistent data regarding the correlation

5 618 S.M. Khoo, T.K. Lim between exhaled NO and the conventional measures of asthma control. 4,8,30 Kharitonov and colleagues have described exhaled NO as a rapid response marker, which is extremely sensitive to corticosteroid treatment as it may be significantly reduced even after 6 h following a single treatment with nebulized corticosteroids, or within 2e 3 days after inhaled corticosteroids, reaching maximal effect after 2e4 weeks of treatment. 31 A possible explanation for our finding is, therefore, that our patients had indeed had early sub-clinical airway inflammation, which was detected only by exhaled NO measurements following oral corticosteroids withdrawal. However, the absence of exacerbations in the 3 months after hospital discharge in all twenty-four patients suggests that these early signals of occult airway inflammation may be of doubtful clinical significance. Third, our observations may be due to the dose-response effects of corticosteroids on airway inflammation and exhaled NO. It is possible that the rebound in exhaled NO had resulted from the reduction in the total dose of corticosteroids received by the study subjects after withdrawal of the oral corticosteroids. Previous studies have suggested a dose-response relationship between exhaled NO and ICS. 32 However, these studies also found that a plateau effect on exhaled NO was seen at a dose of 400 mg of inhaled budesonide. 32,33 In the present study, all the subjects were maintained on ICS doses that were either equivalent to or above 400 mg of budesonide, putting them on the upper end of the dose-response curve. The rebound in exhaled NO was more likely to be due to the differential effect of oral corticosteroids withdrawal than the doseresponse effect of corticosteroids on exhaled NO since all the subjects were maintained on the upper end of the doseresponse curve throughout the study period. In addition, commencement of oral prednisone has been shown to result in a decrease in exhaled NO in patients with asthma who were already on ICS, suggesting a separate effect of oral corticosteroids that may not be related to the doseresponse relationship. 24,28 The differential effects of inhaled versus systemic corticosteroids on the three components of asthma: airway inflammation, lung function and clinical symptoms in patients with acute asthma are still unclear. Finally, it is possible that our observations had resulted from the isolated effects of corticosteroids on exhaled NO and were unrelated to airway inflammation. Corticosteroids have been shown to reduce exhaled NO by directly inhibiting the induction of nitric oxide synthase 2 (NOS2). 34 The increase in exhaled NO levels after withdrawal of oral corticosteroids may have been the result of a loss of direct inhibition of NOS2 by systemic corticosteroids and not the removal of suppression of pro-inflammatory cytokines. Results of the present study have two important clinical implications. First, exhaled NO may have limited clinical utility as a marker to guide anti-inflammatory therapy in the period immediately after asthma exacerbations when patients are at risk of relapse. The lack of concordant response between exhaled NO and other measures of asthma control suggests that exhaled NO level is highly sensitive to variations in oral corticosteroid treatment and may not be a useful indicator of response to treatment during recovery from acute exacerbations of asthma in patients who are on ICS. Recommencement of oral corticosteroids or escalation in the dose of ICS in our patients in response to the increase in exhaled NO levels may have resulted in over-treatment with corticosteroids. Second, the presence of a component of inflammation that is not ICS responsive, if confirmed by further studies, challenges the role of ICS in the management of acute asthma. 23,24,28 An emerging area of study is the role of ICS in the treatment of acute asthma. Trials in which ICS were compared with placebo demonstrated clear beneficial effects of ICS. In contrast, the evidence was conflicting in trials where ICS were compared with systemic corticosteroids. 35 These findings may be related to the presence of peripheral inflammation that is suppressed by systemic corticosteroids but not ICS. The characteristics, anatomic site and clinical significance of this component of airway inflammation need clarification with further studies. Although our findings suggest a lack of concordant response between exhaled NO and other measures of asthma control in patients treated for acute asthma, the results must be interpreted with caution. First, the discordant relationship observed may have been due to the small sample size. However, the consistent trend of improvement observed in lung function and asthma symptoms suggests the presence of a genuine discordant relationship between the inflammatory signals and lung function. Second, a parallel study design with a control arm may have provided a better design to investigate the effect of oral corticosteroids withdrawal. However, a parallel design would have required some of the study subjects to be continued on oral prednisolone beyond 1 week despite improvement in all the conventional measures of asthma control (lung function, clinical symptoms and frequency of reliever use). The fact that the study subjects were maintained on relatively high doses of ICS (upper end of the dose-response curve) throughout the study period and the consistent trend of rebound in exhaled NO suggest the presence of a significant exhaled NO signal associated with oral corticosteroids withdrawal. Third, exhaled NO levels, lung function and asthma symptoms were monitored in the setting of uninterrupted maintenance ICS treatment in the present study. This may hence limit generalization of the results to patients who are not on maintenance ICS after acute exacerbations. Fourth, we did not perform sputum eosinophils count or bronchial biopsy to ascertain the presence of airway inflammation. However, the observed improvement in lung function, asthma symptoms and the absence of relapse in the follow up period suggest the absence of clinically important airway inflammation that requires escalation of anti-inflammatory treatment. In conclusion, we found that in patients discharged from hospital after treatment for severe asthma exacerbations, withdrawal of oral corticosteroids resulted in an increase in exhaled NO levels despite continued ICS treatment. The increase in exhaled NO was not accompanied by reduction in lung function and occurred despite improvement in AQLQ scores and rescue medication use. The differential effects of inhaled versus systemic corticosteroids on different sources of exhaled NO and their relationships with different components of airway inflammation need further investigations and clarifications.

6 Effects of inhaled versus systemic corticosteroids on exhaled NO 619 Funding Funded by NHG research fund (NHG-RPR/02059). Disclosure Both authors do not have financial interest in the subject matter to declare. Originality and clinical relevance There is a paucity of data on the differential effects of inhaled versus systemic corticosteroids on airway inflammation, lung function and asthma symptoms in patients with severe acute asthma. Our study found that withdrawal of oral corticosteroids resulted in an increase in exhaled NO levels despite continued inhaled corticosteroid treatment. There was also a discordant relationship between exhaled NO, lung function and clinical symptoms. Our results may have important clinical implications relating to: (1) the clinical utility of exhaled NO in patients with acute asthma; and (2) the effects of inhaled corticosteroids on airway inflammation in acute asthma. Acknowledgements We are grateful to our research and asthma nurses Ms. K.E. Thein, L.K. Tan, N. Said for their assistance in data collection and the care of our patients, Ms. W.J. Ngerng for her help in data entry, and Dr. Y.H. Chan for his advice on biostatistics. References 1. Kharitonov SA, Barnes PJ. Exhaled biomarkers. Chest 2006; 130:1541e6. 2. Kharitonov SA, Yates D, Robbins RA, Logan-Sinclair R, Shinebourne EA, Barnes PJ. Increased nitric oxide in exhaled air of asthmatic patients. Lancet 1994;343:133e5. 3. Persson MG, Zetterstrom O, Agrenius V, Ihre E, Gustafsson LE. Single-breath nitric oxide measurements in asthmatic patients and smokers. Lancet 1994;343:146e7. 4. Massaro AF, Gaston B, Kita D, Fanta C, Stamler JS, Drazen JM. Expired nitric oxide levels during treatment of acute asthma. Am J Respir Crit Care Med 1995;152:800e3. 5. Baraldi E, Dario C, Ongaro R, et al. Exhaled nitric oxide concentrations during treatment of wheezing exacerbation in infants and young children. Am J Respir Crit Care Med 1999; 159:1284e8. 6. Kharitonov SA, Yates DH, Barnes PJ. Inhaled glucocorticoids decrease nitric oxide in exhaled air of asthmatic patients. Am J Respir Crit Care Med 1996;153:454e7. 7. Baraldi E, Azzolin NM, Zanconato S, Dario C, Zacchello F. Corticosteroids decrease exhaled nitric oxide in children with acute asthma. J Pediatr 1997;131:381e5. 8. Lanz MJ, Leung DY, White CW. Comparison of exhaled nitric oxide to spirometry during emergency treatment of asthma exacerbations with glucocorticoids in children. Ann Allergy Asthma Immunol 1999;82:161e4. 9. Sippel JM, Holden WE, Tilles SA, et al. Exhaled nitric oxide levels correlate with measures of disease control in asthma. J Allergy Clin Immunol 2000;106:645e Jones SL, Kittelson J, Cowan JO, et al. The predictive value of exhaled nitric oxide measurements in assessing changes in asthma control. Am J Respir Crit Care Med 2001;164:738e Montuschi P, Mondino C, Koch P, Ciabattoni G, Barnes PJ, Baviera G. Effects of montelukast treatment and withdrawal on fractional exhaled nitric oxide and lung function in children with asthma. Chest 2007;132:1876e Kharitonov SA, Yates DH, Chung KF, Barnes PJ. Changes in the dose of inhaled steroid affect exhaled nitric oxide levels in asthmatic patients. Eur Respir J 1996;9:196e Gustafsson LE. Exhaled nitric oxide as a marker in asthma. Eur Respir J Suppl 1998;26:49Se52S. 14. Recommendations for standardized procedures for the on-line and off-line measurement of exhaled lower respiratory nitric oxide and nasal nitric oxide in adults and children This official statement of the American Thoracic Society was adopted by the ATS Board of Directors, July Am J Respir Crit Care Med 1999;160:2104e Standardization of spirometryd1987 update. Statement of the American Thoracic Society. Am Rev Respir Dis 1987;136: 1285e da Costa JL. Pulmonary function studies in healthy Chinese adults in Singapore. Am Rev Respir Dis 1971;104:128e Juniper EF, Guyatt GH, Ferrie PJ, Griffith LE. Measuring quality of life in asthma. Am Rev Respir Dis 1993;147:832e Juniper EF, Guyatt GH, Epstein RS, Ferrie PJ, Jaeschke R, Hiller TK. Evaluation of impairment of health related quality of life in asthma: development of a questionnaire for use in clinical trials. Thorax 1992;47:76e Juniper EF, Guyatt GH, Willan A, Griffith LE. Determining a minimal important change in a disease-specific Quality of Life Questionnaire. J Clin Epidemiol 1994;47:81e Tsoukias NM, George SC. A two-compartment model of pulmonary nitric oxide exchange dynamics. J Appl Physiol 1998;85:653e Tsoukias NM, Shin HW, Wilson AF, George SC. A single-breath technique with variable flow rate to characterize nitric oxide exchange dynamics in the lungs. J Appl Physiol 2001;91: 477e Lehtimaki L, Kankaanranta H, Saarelainen S, Turjanmaa V, Moilanen E. Increased alveolar nitric oxide concentration in asthmatic patients with nocturnal symptoms. Eur Respir J 2002;20:841e Lehtimaki L, Kankaanranta H, Saarelainen S, Turjanmaa V, Moilanen E. Inhaled fluticasone decreases bronchial but not alveolar nitric oxide output in asthma. Eur Respir J 2001;18: 635e Gelb AF, Taylor CF, Nussbaum E, et al. Alveolar and airway sites of nitric oxide inflammation in treated asthma. Am J Respir Crit Care Med 2004;170:737e Kraft M, Djukanovic R, Wilson S, Holgate ST, Martin RJ. Alveolar tissue inflammation in asthma. Am J Respir Crit Care Med 1996;154:1505e Gelb AF, Zamel N. Unsuspected pseudophysiologic emphysema in chronic persistent asthma. Am J Respir Crit Care Med 2000; 162:1778e Gelb AF, Licuanan J, Shinar CM, Zamel N. Unsuspected loss of lung elastic recoil in chronic persistent asthma. Chest 2002; 121:715e Berry M, Hargadon B, Morgan A, et al. Alveolar nitric oxide in adults with asthma: evidence of distal lung inflammation in refractory asthma. Eur Respir J 2005;25:986e Belda J, Margarit G, Martinez C, et al. Anti-inflammatory effects of high-dose inhaled fluticasone versus oral prednisone in asthma exacerbations. Eur Respir J 2007;30:1143e Stirling RG, Kharitonov SA, Campbell D, et al. Increase in exhaled nitric oxide levels in patients with difficult asthma and correlation with symptoms and disease severity despite

7 620 S.M. Khoo, T.K. Lim treatment with oral and inhaled corticosteroids. Asthma and Allergy Group. Thorax 1998;53:1030e Kharitonov SA, Barnes PJ. Exhaled markers of pulmonary disease. Am J Respir Crit Care Med 2001;163:1693e Jatakanon A, Kharitonov S, Lim S, Barnes PJ. Effect of differing doses of inhaled budesonide on markers of airway inflammation in patients with mild asthma. Thorax 1999;54:108e Aziz I, Wilson AM, Lipworth BJ. Effects of once-daily formoterol and budesonide given alone or in combination on surrogate inflammatory markers in asthmatic adults. Chest 2000;118: 1049e Guo FH, Comhair SA, Zheng S, et al. Molecular mechanisms of increased nitric oxide (NO) in asthma: evidence for transcriptional and post-translational regulation of NO synthesis. J Immunol 2000;164:5970e Edmonds ML, Camargo Jr CA, Pollack Jr CV, Rowe BH. Early use of inhaled corticosteroids in the emergency department treatment of acute asthma. Cochrane Database Syst Rev 2003;(3). CD