Exhaled Nitric Oxide in Asthma: Relationship to Pulmonary Function, Disease Severity and Steroid Use

Transcription

1 Original Article 42 Copyright University of Medicine, Tirana AJMHS Vol. 47 (1): Exhaled Nitric Oxide in Asthma: Relationship to Pulmonary Function, Disease Severity and Steroid Use Eugerta Dilika 1, Artan Simaku 2, Eritjan Tashi 1, Perlat Kapisyzi 1 1 Service of Pneumology, Faculty of Medicine, University of Medicine, Tirana and University Hospital Shefqet Ndroqi, Tirana, Albania. 2 Institute of Public Health, Albania. Abstract Background: Asthma is considered a heterogeneous disease, requiring multiple biomarkers for diagnosis and management. Fractional exhaled nitric oxide (FeNO) in exhaled breath was the first useful non-invasive marker of airway inflammation in asthma and still is the most widely used. There are many studies describing the levels of FeNO in asthma patients and some of the published reports are conflicting in their conclusions. Aims: The purpose of this study was to determine the practical utility of FeNO values in the diagnosis and management of asthma. Study design: This is a prospective study. Methods: This study is focused on the relationship of FeNO levels with lung function, disease severity, total IgE and steroid use in asthma patients. FeNO was measured by an electrochemical nitric oxide analyzer (NIOX MINO). All data were collected from medical records. Results: Results showed that the mean value of FeNO in asthma is 53.51ppb ± 5.66SD. Among the 67 subjects with asthma there is significant change of the mean FeNO according to the severity of bronchial obstruction (p=.2). There is no significant change of the FeNO mean according to the stages of asthma: p=.7 (near significant). There is no significant correlation between FeNO and FEV1, TLC, RV, RV/TLC and there is significant correlation between FeNO and Raw) (p=.2) and MEF 5 ml (p =.4) in patients with asthma with small airways obstruction. The mean of FeNO with normal value of total IgE (<2UI/ml) is 31,6 ± 3,2 SD. The mean of FeNO with high value of IgE (> 2 UI/ml) is 74,5 ± 64,4 SD. The difference between values of FeNO in diseases with SMA is significant (p<.1).the mean of FeNO before treatment was ± SD and after treatment is ± SD. There is statistically significant change (p=.2). Conclusions: The measurement of FeNO is helpful in the diagnosis of asthma. FeNO is a promising marker for the diagnosis and therapy of adults asthma patients. Keywords: FeNO, asthma, lung function, severity, total IgE. Address for correspondence: Eugerta Dilika, Service of Pneumology, Faculty of Medicine, University of Medicine, Tirana and University Hospital Shefqet Ndroqi Tirana, Albania. eugerta@yahoo.com Phone:

2 Dilika E. et al. Exhaled Nitric Oxide in Asthma: Relationship to Pulmonary Function 43 INTRODUCTION Asthma is a chronic disease characterized by inflammation of the airways (1). Monitoring the severity of the disease and treatment response by evaluating airway inflammation may enable a better control of the disease (2). In the presence of compatible respiratory symptoms and airflow limitation, an elevated FeNO has high sensitivity and specificity for diagnosing of asthma (3,4). Further studies are needed to identify the subgroups that benefit from FeNO-based management of asthma and to develop a correct algorithm for this purpose (5). Although FeNO measurement can provide information about the disease activity characterized by eosinophilic airway inflammation, it is not useful in more severe disease forms especially in the presence of neutrophilic inflammation (6). Measurement of FeNO may therefore be a useful screening procedure for patients with chronic cough and would readily identify those patients with cough caused by asthma (7). There are conflicting reports on the significance of the various factors such as age, gender, height, and total IgE that complicate the definition of normal values of FeNO (8). It is shown to be a strong association between elevated FeNO and total IgE (9). The increase in exhaled NO (nitric oxide) does not appear to be related to asthma severity (1). There is little correlation between FeNO levels and pulmonary function tests in asthma patients (11). There is no significant relationship between FeNO and FEV1 in patients with asthma (12,13). The level of FeNO decreases after the administration of ICS (14) and systemic corticosteroids (15). One of the most useful features of measuring the FeNO is its ability to predict the response to therapy with oral corticosteroids (16) and ICS in patients with asthma (17, 18). AIM OF THE STUDY The aim of this study was to assess the correlation between FeNO levels and conventional measures of lung function and to study the practical utility of FeNO values in the diagnosis and management of asthma; the correlation of FeNO values with total IgE; the practical utility of FeNO values in the diagnosis and management of diseases with obstruction of SMA; the correlation of FeNO values with the degree of obstruction; the correlation of FeNO values with disease severity and to determine the relationship between FeNO levels in asthma and steroid use. MATERIAL AND METHODS This prospective diagnostic study was performed between June 212 and August 213 in University Hospital Shefqet Ndroqi, Tirana, Albania. Two groups of patient were studied: first group, 67 subjects, nonsmoking with intermittent to severe asthma (Table 1) and second one 71 subjects with obstruction of SMA, etiologic diagnosis of which were 31 with asthma, 14 with GERD, 11 with COPD, 9 with bronchiectasis and 6 with sarcoidosis (Table 2).

3 44 Dilika E. et al. Exhaled Nitric Oxide in Asthma: Relationship to Pulmonary Function Table 1. Summary baseline characteristics of the eligible study asthma patients. Age BMI Baseline characteristics Frequency Percent Mean SD Mean SD Sex male 36 53, female 31 46, total 67, Residence city 43 64, country 23 34, Stages of asthma intermittent 17 25, mild 18 26, moderate 8 11, severe 24 35, The degree of small airways 19 28, bronchial obstruction normal mild moderate severe very severe Total IgE UI/ml < >= Table 2. Summary baseline characteristics of the patients with obstruction of SMA. Diagnosis Age BMI Gender N/ (%) COPD Mean 58,99 29,7727 Male 7 (63.6) Std. Deviation 9,4696 5,9681 Female 4 (36.4) Sarcoidosis Mean 4, , Male Std. Deviation 12, ,52982 Female 6 () Asthma Mean 45, 28,3594 Male 9 (4.9) Std. Deviation 15, ,48934 Female 22 (59.1) Bronchiectasis Mean 52, ,7222 Male 4 (44.4) Std. Deviation 11, ,3551 Female 5 (55.6) GERD Mean 48, ,8571 Male 1 (7.1) Std. Deviation 12,2942 5,51721 Female 13 (92.9)

4 Ne fillim Dilika E. et al. Exhaled Nitric Oxide in Asthma: Relationship to Pulmonary Function 45 Moderate to severe persistent asthma treated with inhaler budesonide 2 mcg twice a day and oral prednisone 25 mg /day. For patients recruited to the asthma group, inclusion criteria were a diagnosis of asthma as defined by the criteria of Global Initiative for Asthma (GINA) guidelines. Spirometry was performed according to the standards of the ATS. Lung volumes were measured by body plethysmography. FeNO was measured by an electrochemical nitric oxide analyzer (NIOX MINO); Aerocrine AB, Solna, Sweden). FeNO5 was measured according to the ATS/ERS recommendations (19). STATISTICAL ANALYSIS Statistical analyses were performed using SPSS16. Comparisons of mean FeNO values between the groups were performed by ANOVA and unpaired t tests. Paired samples t-test. Pearson s correlation coefficients were calculated to determine the correlation between the FeNO values and continuous data. All data were expressed as mean ± SD and significance was defined as a p value of less than.5. RESULTS The mean of FeNO in asthma is ± 5.66SD,(y = x) F-ratio=6.6, p=.1. When BMI (body mass index) increased with one unit, FeNO decreased with 3.7 units, demonstrating significant correlation (Fig 1) BMI Figure 1. The correlation of FeNO with me BMI in asthma patients.

5 FeNO 46 Dilika E. et al. Exhaled Nitric Oxide in Asthma: Relationship to Pulmonary Function Among the 67 subjects with asthma the results according to the degrees of bronchial obstruction are: normal 44,25 ± 22,54SD, small airways ± SD, mild ± 8.33 SD, moderate 49.7±42.88 SD, severe 52.44±29.24 SD, very severe ± 9.32 SD. There is significant change of the mean of FeNO according of severity of bronchial obstruction. ANOVA F-ratio=2.9 p=.2. The mean of FeNO in: intermittent is 31.6±24.2SD, mild 73.6±74.9SD, moderate 68.±44.25SD, severe 49. ±37.9SD. There is no significant change of the FeNO mean according to stages of asthma ANOVA F- ratio=2.4 p=.7 (near significant). (y= x) F-ratio=3.2, p=.8. When FEV1 ml increase with one unit, FeNO increased with 15.8 unites. There is no significant correlation (small case) (Fig.2) FEV1ml Figure 2. The correlation of FeNO with FEV1 ml.

6 FeNO Dilika E. et al. Exhaled Nitric Oxide in Asthma: Relationship to Pulmonary Function TLC% Figure 3. The correlation of FeNO with TLC % in asthma. (y= x) F-ratio=.6, p=.4 When TLC ml increased with one unit, FeNO decreased with 6.3 units, there is no significant correlation. (y = x) F- ratio=.1, p=.9. When TLC % increased with one unit, FeNO decreased with.1 units, there is no significant correlation (Fig.3). (y = x) F- ratio=.2, p=.6. When RV % increased with one unit, FeNO decreased with.6 units. There is no significant correlation (Fig.4). (y= x) F-ratio=.7, p=.3. When RV/TLC % increased with one unit, FeNO decreased with.4 units, there is no significant correlation. (y = x) F-ratio=5.4, p=.2. When Raw % increased with one unit, FeNO decreased with.1 units, there is significant correlation (Fig.5). (y= x) F-ratio =., p =.9 When bronchial reversibility (ml) increased with one unit, FeNO decreased with.2 units, there is no significant correlation. The mean of FeNO before salbutamol is ± SD after salbutamol is 45.6 ± SD. There is no significant correlation between them t=.4 p=.6 (Fig.6).

7 FeNO FeNO 48 Dilika E. et al. Exhaled Nitric Oxide in Asthma: Relationship to Pulmonary Function RV% Figure 4. The correlation of FeNO with RV% Raw% Figure 5. The correlation of FeNO with Raw %.

8 Dilika E. et al. Exhaled Nitric Oxide in Asthma: Relationship to Pulmonary Function FeNO before salbutamol FeNO after salbutamol Figure 6. The mean of e FeNO before and after salbutamol in patients with short- term bronchial reversibility in asthma. The total patients according the value of total IgE in this study were: 31 patients with total IgE >=2 UI/ml and 36 patients with total IgE < 2 UI/ml. F=37.3 p<.1. When total IgE increased with one unit, FeNO increased with.8 units. There is significant correlation between FeNO and total IgE (Fig.7). The mean of FeNO with normal value of total IgE (<2 UI/ml) is 31,6 ± 3,2SD. The mean of FeNO with high value of total IgE (>= 2 UI/ml) is 74,5 ± 64,4 SD. There is significant difference between values of FeNO in asthma with normal total IgE and high total IgE (t-test = -3,7 p=,4).

9 FeNO 5 Dilika E. et al. Exhaled Nitric Oxide in Asthma: Relationship to Pulmonary Function IgE Figure 7. The correlation between value of FeNO and total IgE. The mean of FeNO before treatment is ± SD. The mean of FeNO after treatment is ± SD t =3.5 p=.2. There is statistically significant change (Paired samples t-test) (Fig.8) The mean of FeNO in diseases with obstruction of SMA is: in asthma 37.8 ± 35.8 SD, in bronchiectasis 11.2 ± 4.73 SD, in COPD 11.3 ± 8.7 SD, in GERD 12 ± 5.5 SD, in sarcoidosis 8 ± 3.5 SD. The difference between values of FeNO in diseases with obstruction of SMA is significant. ANOVA F -ratio= 5.2 p<.1 (Fig. 9) y = x) F-ratio=4.6 p=.59. When MEF 75 ml increased with one unit, FeNO increased with 1.6 units, but the relationship is not significant [very close but no significance ( small cases)]. (y= x) F-ratio=5.2 p=.4. When MEF 5 ml increased with one unit, FeNO increased with 19 units, the relationship is significant. (y= x) F-ratio=.8 p=.7. When MEF 25 ml increased with one unit, FeNO increased with 1,8 units, the relationship is not significant.

10 Dilika E. et al. Exhaled Nitric Oxide in Asthma: Relationship to Pulmonary Function Before tratment After treatment Figure 8. Changing of value of FeNO after treatment with steroid in asthma. Figure 9. Diseases with obstruction of SMA.

11 52 Dilika E. et al. Exhaled Nitric Oxide in Asthma: Relationship to Pulmonary Function DISCUSSION In the present study the value of FeNO in patients with asthma is ± 5.66 SD. The mean of FeNO in: intermittent is 31.6±24.2SD, mild 73.6±74.9SD, moderate 68.±44.25SD, severe 49. ±37.9SD. These data are consistent with other studies where elevated FeNO levels have been widely documented in adults with asthma, even in mild and asymptomatic conditions (12, 2-22). This correlation might be explained by the fact that the severe obstruction causes decrease of ventilation FeNO. This argument is supported by negative correlation between Raw and FeNO. There is significant correlation between FeNO and Raw (p=.2). Though many studies while evaluated the relationship between FeNO and obesity in asthmatic patients showed no significant relationship between FeNO and body mass index in asthmatic subjects (23-26). In the present study there is significant inverse correlation: decreasing of FeNO associated with increase of BMI (p=.1). No clear relationship has been established between obesity and airway inflammation (27). Obesity induces systemic oxidative stress. It is hypothesized that the lung serves as a target organ for this oxitative stress. This is manifested as increased oxidation of airway NO into nitrate and reactive nitrogen species and hence reduction of NO bioavailability and FeNO levels (28). There is significant change of the mean FeNO according to the severity of bronchial obstruction (p =.2), but there is no significant change of the FeNO values according to the stages of asthma (p =.7). The correlation between FeNO and severity of bronchial obstruction can be explained not only by the degree of inflammation but also the severity of obstruction. FeNO correlates strongly with atopy as demonstrated by skin prick test scores (9, 29-31) and specific and total IgE (9, 31). We did not study the correlation of FeNO with specific IgE but only total IgE. We found that there is significant difference between values of FeNO in asthma with normal total IgE and high total IgE (t-test= -3,7 p=,4). There is significant correlation between FeNO and total IgE (p<.1). We investigated the relationship between FeNO values, pulmonary functions and the alterations of these parameters after using B2-agonist and treatment with steroid. There is no significant correlation between FeNO and FEV1, TLC, RV, RV/TLC. The missing of correlation between FeNO and FEV1 can be explained that FEV1 is not directly related to airway inflammation (22), while the missing of correlation between FeNO and TLC, RV, RV/TLC can be explained by the fact that air trapping and FENO are independent determinants for lung hyperinflation in asthma (32). There is significant correlation between FeNO and MEF 5 ml in patients with asthma with small airways obstruction. The positive correlation between MEF 5 and FeNO supports

12 Dilika E. et al. Exhaled Nitric Oxide in Asthma: Relationship to Pulmonary Function 53 the correlation between the severity of bronchial obstruction with FeNO. Chatkin and colleagues have previously demonstrated in a population of subjects with chronic cough that FENO discriminated well between those with and those without asthma (7). The finding of an elevated FeNO is helpful in establishing the correct diagnosis of asthma in patients (3, 4, 33, 34) and in our study the difference between values of FeNO in asthma and other diseases with SMA obstruction was significant, p<.1. High levels of FeNO is highly suggestive of asthma and a good predictor of response to treatment with corticosteroids (35) In this study, we investigated the effect of treatment with corticosteroids. Treatment with corticosteroids reduces exhaled NO. There is statistically significant change between FeNO value before and after treatment (p=.2). It can be postulated that asthma treatment with corticosteroids results in a reduction in expired NO levels due to both the reducing effects of steroids on the underlying airways inflammation in asthma and inhibitory effects on inos expression itself (16). Although anti-inflammatory treatment in asthma usually reduces FeNO, some patients continue to have elevated FeNO levels, despite steroid treatment. This is most frequently a result of non-compliance, but may also arise from poor inhalation technique, inadequate steroid dosage, chronic exposure to allergen, or noneosinophilic airway inflammation as the cause of the asthma, only rarely will an asthma patient be truly steroid resistant (36,37,38), the multifaceted nature of the asthma disease with many different phenotypes within the asthma population (39,4). According to our result the exhaled NO is related more to asthma control than to asthma severity. CONCLUSIONS High levels of FeNO is highly suggestive of asthma. The measurement of FeNO is helpful in the diagnosis and management of asthma. FeNO may provide useful information about airway inflammation as a complementary tool to lung function tests. We found out a significant difference between mean values of FeNO and severity of bronchial obstruction in asthma. There is significant correlation between FeNO and Raw. There is a strong correlation of FeNO values with total IgE. In asthma with severe obstruction the FeNO values do not really represent the degree of inflammation. In bronchial small airway obstruction, high values of FeNO support the diagnosis of eosinophilic inflammation. Patients with chronic cough that is not attributable to asthma have lower NO values as compared to patients with asthma. An elevation of exhaled NO is not specific for asthma, but an increased level may be useful in differentiating asthma from other causes of chronic cough. FeNO is an important biomarker in differential

13 54 Dilika E. et al. Exhaled Nitric Oxide in Asthma: Relationship to Pulmonary Function diagnosis of diseases associated with obstruction of the small airways, because the etiologic diagnosis of small airway diseases is often very difficult. Levels of FeNO are a good predictor of response to treatment with corticosteroids. FeNO is a promising marker as a guide for diagnosis and treatment of patients with asthma identifying patients requiring changes in therapy. Acknowledgements: Not available Conflict of interest disclosure: Not available REFERENCES 1. Buse WW, Lemanske RF Jr. Asthma. N Engl J Med 21;344: Pizzichini E, Pizzichini MM, Hargreave FE. Induced sputum in the management of asthma. Seminars in Respiratory and Critical Care Medicine1998;19: Dupont LJ, Demedts MG, Verleden GM. Prospective evaluation of the validity of exhaled nitric oxide for the diagnosis of asthma. Chest 23;123: Smith AD, Cowan JO, Filsell S. Diagnosing asthma: comparisons between exhaled nitric oxide measurements and conventional tests. Am J Respir Crit Care Med 24;169: Rouhos A. Clinical aspects of exhaled nitric oxide in adults: Associations with atopy, bronchial hyperresponsiveness, smoking and chronic obstruction [dissertation]. Helsinki (Finland): Central Helsinki University; Reddel HK, Taylor DR, Bateman ED, Boulet LP, Boushey HA, Busse WW. American Thoracic Society/ European Respiratory Society Task Force on Asthma Control and Exacerbations. American Thoracic Society/European Respiratory Society Task Force on Asthma Control and Exacerbations. An official American Thoracic Society/European Respiratory Society statement: asthma control and exacerbations: standardizing endpoints for clinical asthma trials and clinical practice. Am J Respir Crit Care Med 29;18: Chatkin JM, Ansarin K, Silkoff PE, McClean P, Gutierrez C, Zamel N, Chapman KR. Exhaled nitric oxide as a noninvasive assessment of chronic cough. Am J Respir Crit Care Med 1999; 159: Abba, Abdullah A. Exhaled nitric oxide in diagnosis and management of respiratory diseases. Ann Thorac Med 29;4: Ho LP, Wood FT, Robson A, Innes JA, Greening AP. Atopy influences exhaled nitric oxide levels in adult asthmatics. Chest 2;118: Van den Toorn LM, Overbeek SE, de Jongste JC, Leman K, Hoogsteden HC. Airway inflammation is present during clinical remission of atopic asthma. Am J Respir Critical Care Medical 21; 164: Aerocrine. Improving Diagnosis and Management of Asthma through FeNO Measurement. Eur Respir J 25; 26: Kharitonov SA, Yates DH, Robbins RA, Logan-Sinclair R, Shinebourne EA, Barnes PJ. Increased nitric oxide in exhaled air of asthmatic patients. Lancet 1994;343: Ansarin K, Chatkin JM, Ferreira IM, Gutierrez CA, Zamel N. Exhaled nitric oxide in chronic obstructive pulmonary disease: relationship to pulmonary function. Eur Respir J 21; 17: Silkoff P, McClean P, Slutsky A. Exhaled nitric oxide and bronchial reactivity during and after inhaled beclomethasone in mild asthma. J Asthma 1998;35: Yates D, Kharitonov S, Robbins R.. Effect of a nitric oxide synthase inhibitor and a glucocorticoid on

14 Dilika E. et al. Exhaled Nitric Oxide in Asthma: Relationship to Pulmonary Function 55 exhaled nitric oxide. Am J Respir Crit Care Med 1995;152: 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: Szefler S, Phillips BR, Martinez F. Characterization of within-subject responses to fluticasone and montelukast in childhood asthma. J Allergy Clin Immunol 25;115: Zeiger RS, Szefler SJ, Phillips BR. Response profiles to fluticasone and montelukast in mild-to-moderate persistent childhood asthma. J Allergy Clin Immunol 26;117: American Thoracic Society; European Respiratory Society. ATS/ERS recommendations for standardized procedures for the online and offline measurement of exhaled lower nitric oxide and nasal nitric oxide. Am J Respir Crit Med 25;171: Persson MG, Zetterstöm O, Agrenius V, Ihre E, Gustafsson LE. Single-breath nitric oxide measurement in asthmatic patients and smokers. Lancet 1994;343: Alving K, Weitzberg E, Lundberg JM: Increased amount of nitric oxide in exhaled air of asthmatics. Eur Respir J 1993;6: Kharitonov SA, Barnes PJ. Exhaled markers of pulmonary disease. Am J Respir Crit Care Med 21;163(7): Buchvald F, Baraldi E, Carraro S, Gaston B, De Jongste J. Measurements of exhaled nitric oxide in healthy subjects age 4 to 17 years. J Allergy Clin Immunol 25;115: Cibella F, Cuttitta G, La Grutta S, Passalacqua G, Viegi G. Factors that influence exhaled nitric oxide in italian schoolchildren. Ann Allergy Asthma Immunol 28,11: Santamaria F, Montella S, De Stefano S, Sperlì F, Barbarano F, Spadaro R, Franzese A. Asthma, atopy, and airway inflammation in obese children. J Allergy Clin Immunol 27,12: Verhulst SL, Aerts L, Jacobs S, Schrauwen N, Haentjens D, Claes R, Vaerenberg H, Van Gaal LF, De Backer WA, Desager KN. Sleep-disordered breathing, obesity, and airway inflammation in children and adolescents. Chest 28;134: Sutherland TJ, Cowan JO,Young S, Goulding A, Grant AM, Williamsan A. The association between obesity and asthma interactions between systemic and airway inflammation. Am J Respir Crit Care Med 28;178: Habib Syed S, Abba Abdullah A, Al- Zoghaibi Muhammed A, Subhan Mirza M. Reference ranfe values of fractional exhaled nitric oxide in healthy Arab adult males. Saudi Med J 29;3(11): Van Amsterdam JG, Janssen NA, de Meer G, Fischer PH, Nierkens S et al. The relationship between exhaled nitric oxide and allergic sensitization in a random sample of school children. Clin Exp Allergy 23;33: Sandrini A, Ferreira IM, Jardim JR, Zamel N, Chapman KR. Effect of nasal triamcinolone acetonide on lower airway inflammatory markers in patients with allergic rhinitis. J. Allergy Clin Immunol 23;111: Saito J, Inoue K, Sugawara A, Yoshikawa M, Watanabe K. Exhaled nitric oxide as a marker of airway inflammation for an epidemiologic study in schoolchildren. J Allergy Clin Immunol 24; 114: Dweik RA, Sorkness RL, Wenzel S, Hammel J, Curran-Everett D, Comhair SA. Use of exhaled nitric oxide measurement to identify a reactive, atrisk phenotype among patients with asthma. Am J Respir Crit Care Med 21;181(1): Deykin A, Massaro AF, Drazen JM, et al. Exhaled nitric oxide as a diagnostic test for asthma: online versus offline techniques and effect of flow rate. Am J

15 56 Dilika E. et al. Exhaled Nitric Oxide in Asthma: Relationship to Pulmonary Function Respir Crit Care Med 22;165: Malmberg LP, Pelkonen AS, Haahtela T. Exhaled nitric oxide rather than lung function distinguishes preschool children with probable asthma. Thorax 23;58: Smith AD, Cowan JO, Brassett KP, et al. Exhaled nitric oxide: a predictor of steroid response. Am J Respir Crit Care Med 25;172: Stirling RG, Kharitonov SA, Campbell D. Increase in exhaled nitric oxide levels in patients with difficult asthma and correlation with symptoms and disease severity despite treatment with oral and inhaled corticosteroids. Asthma and Allergy Group. Thorax 1998;53: Jones SL, Kittelson J, Cowan JO. The predictive value of exhaled nitric oxide measurements in assessing changes in asthma control. Am J Respir Crit Care Med 21;164: Payne DN, Wilson NM, James A, Hablas H, Agrafioti C, Bush A. Evidence for different subgroups of difficult asthma in children. Thorax 21;56: Wenzel SE. Asthma: defining of the persistent adult phenotypes. Lancet 26;368: Bush A, Menzies-Gow A. Phenotypic differences between pediatric and adult asthma. Proc Am Thorac Soc 29;6: