Increased Leukotriene E 4 in the Exhaled Breath Condensate of Children With Mild Asthma*

CHEST Original Research Increased Leukotriene E 4 in the Exhaled Breath Condensate of Children With Mild Asthma* Atsushi Shibata, MD; Toshio Katsunuma, MD, PhD; Morimitsu Tomikawa, MD; Aiko Tan, MD; Keisuke Yuki, PhD; Kenichi Akashi, MD; and Yoshikatsu Eto, MD, PhD ASTHMA Background: Chronic airway inflammation is a feature of asthma. Increased levels of cysteinyl leukotrienes (cys-lts; leukotriene [LT]C 4, LTD 4, LTE 4 ) have been shown in the exhaled breath condensate (EBC) of children with moderate-to-severe asthma. The aim of this study was to examine the relationship between EBC cys-lts (LTE 4 ) levels and bronchial hyperreactivity in children with mild asthma in order to evaluate the clinical utility of measuring EBC cys-lts levels. Methods: We measured LTE 4 levels in the EBC of children aged 8 to 18 years, including healthy nonasthmatic children (n 6) and children with mild asthma (n 37). Patients with mild asthma were classified into the following three groups: group 1, participants who had been asymptomatic (no wheezing/symptoms of asthma) for > 6 months prior to examination (n 12); group 2, participants who were asymptomatic but had had wheezing/symptoms of asthma within 6 months before examination (n 18); and group 3, patients with current wheeze and/or mild symptoms of asthma exacerbation at the time of examination. Results: Exhaled LTE 4 levels were increased in all children with mild asthma compared with nonasthmatic control subjects (5.69 9.62 pg/20 min vs 0.74 0.79 pg/20 min, p < 0.05) [mean SD]. In particular, the EBC LTE 4 levels in group 2 (4.99 6.70 pg/20 min) and group 3 (14.66 17.11 pg/20 min) were increased compared with control subjects and group 1 (1.50 1.69 pg/20 min). The EBC LTE 4 levels negatively correlated with the provocative concentration of methacholine causing a 15% fall in FEV 1 (r 0.454, p 0.012). Conclusion: EBC cys-lts may be useful as a noninvasive marker assessing airway inflammation and hyperreactivity in children with asthma. (CHEST 2006; 130:1718 1722) Key words: childhood asthma; exhaled breath condensate; leukotriene E 4 ; mild asthma Abbreviations: cys-lt cysteinyl leukotriene; EBC exhaled breath concentrate; eno exhaled nitric oxide; LT leukotriene; PC 15 provocative concentration of methacholine causing a 15% fall in FEV 1 Asthma is characterized by chronic airway inflammation that is evident even in patients with mild disease. 1 Several studies 2 6 describing biopsy patterns of inflammation in children with asthma have been published in the last few years. The safety of the endobronchial biopsy procedure has also been *From the Department of Pediatrics, The Jikei University School of Medicine, Tokyo, Japan. The authors have no financial or other potential conflicts of interest associated with this study. Manuscript received September 23, 2005; revision accepted June 28, 2006. documented. 7 In addition, the measurement of levels of exhaled nitric oxide (eno), reflecting the activity of airway eosinophil, mast cells, and epithelial cells, has been found to be easy, noninvasive, and useful in asthmatic patients. 8 10 Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (www.chestjournal. org/misc/reprints.shtml). Correspondence to: Toshio Katsunuma, MD, PhD, Department of Pediatrics, The Jikei University School of Medicine, 3-25-8, Nishi-Shinbashi, Minato-ku, Tokyo, 105-8461, Japan; e-mail: tkatsunuma@jikei.ac.jp DOI: 10.1378/chest.130.6.1718 1718 Original Research

Performing bronchoscopy-guided biopsy, however, is still likely to be difficult in children, at least as a common clinical diagnostic tool. Moreover, elevated eno levels are not specific for asthma, given that increased eno is found in other inflammatory respiratory disorders, such as sinus disease and viral upper respiratory tract infection. 11,12 Increased levels of cysteinyl leukotrienes (cys- LTs) have been detected in the exhaled breath condensate (EBC) of children with moderate-tosevere persistent asthma. 13,14 The measurement of cys-lts in the EBC may be useful as a noninvasive way to assess airway inflammation. Bronchial hyperresponsiveness to methacholine, histamine, or exercise is a key feature of asthma, and may be an indicator of asthma severity. Bronchial hyperreactivity relates closely to asthma severity of asthma and frequency of symptoms, as well as to need for treatment. 15 The aim of the present study was to examine the relationship between leukotriene (LT) E 4 levels and bronchial hyperreactivity in mild asthma in children. Such a correlation would allow us to evaluate the clinical utility of measuring EBC cys-lts levels as a marker of airway inflammation in childhood asthma. Finally, we hope to apply the results to the diagnosis of asthma in infants and children 6 years old, in whom the correct diagnosis of asthma is not easy. Patients Materials and Methods Children (age range, 8 to 18 years) with asthma were recruited from the pediatric clinic of the Jikei University Hospital, Tokyo, Japan. We measured EBC and urinary LTE 4 levels of all participants, including healthy nonasthmatic children (n 6) and children with mild asthma (n 37). Eosinophil counts and serum IgE were determined from the blood samples collected from the asthmatic children. Patients with mild asthma were classified into three groups: group 1, subjects who had been asymptomatic (no wheezing/ symptoms of asthma) for 6 months prior to examination (n 12); group 2, subjects who were asymptomatic but had had wheezing/symptoms of asthma within 6 months before examination (n 18); and group 3, subjects with current wheeze and/or mild symptoms of asthma exacerbation at the time of examination (n 7). EBC collection could be performed in group 3 members because their dyspnea was not severe. However, the methacholine provocation test was not performed to prevent inducing serious bronchoconstriction. Patient characteristics are summarized in Table 1. There was no significant difference in age between the groups. The number of subjects treated with fluticasone propionate is also summarized in Table 1. No child had been treated with a dose 200 g/d (mean daily dose in group 1, 120.8 g; group 2, 140.6 g; group 3: 158.3 g). No patient was taking aspirin before or during EBC collection. The diagnosis of bronchial asthma was based on the criteria of the American Thoracic Society, 16 and asthma severity was classified according to the Global Initiative for Asthma guidelines. 17 When severity was based on clinical features present and the step of the daily medication with fluticasone ( 200 g/d), all the subjects were considered to have mild intermittent or persistent asthma. Healthy nonasthmatic control children were recruited from among the children of one of the authors (T.K.) and his friends. It was confirmed by the parent(s), using a questionnaire regarding medical history, that the children in the control group had no history wheezing episode or of allergic disease. The protocol was approved by the Ethics Committee of the Jikei University, and informed consent was obtained from all parents and children recruited into the study. Study Design Baseline spirometry followed by collection of EBC and bronchial responsiveness to methacholine were measured. All medications were withheld for at least 12 h, and montelukast and salmeterol were withheld for 36 h before testing. Measurements of Provocative Concentration of Methacholine Causing a 15% Fall in FEV 1 Spirometry (Autospiro AS500; Minato; Osaka, Japan) was performed using standard techniques. Aerosol was delivered using a nebulizer (model 646; Devilbis; Somerset, PA). Subjects Table 1 Patient Characteristics* Characteristics Control Group (n 6) Group 1, No Wheeze 6mo (n 12) Group 2, Wheeze 6mo (n 18) Group 3, Mild Exacerbation (n 7) Age, yr 10.3 (9 12) 10.9 (8 16) 12.2 (8 18) 10.2 (8 14) Male/female gender, No. 5/1 11/1 14/4 5/2 PEFR, % predicted 94.3 (32 132) 98.0 (70 112) 100.4 (70 137) 97.6 (79 120) FEV 1, % predicted 91.3 (82 108) 94.8 (82 111) 90.6 (79 106) 82.4 (58 116) FEV 1 /FVC 89.6 (85 92) 84.5 (76 91) 83.3 (73 91) 83.1 (59 89) MMF, % predicted 91.3 (70 115) 88.4 (66 118) 72.3 (51 99) 65.7 (26 91) Eosinophil, % Not done 9.8 (1.1 33.5) 7.8 (0.3 19.8) 5.3 (0.4 10.4) Total IgE, IU/mL Not done 2,018 (64 14,674) 1,414 (180 6484) 676 (33 1,816) FP, yes/no 0/6 9/3 11/7 6/1 LT1RA, yes/no 0/6 4/8 10/8 5/2 *Data are presented as mean (range) unless otherwise indicated. PEFR peak expiratory flow rate; MMF maximum midexpiratory flow rate; FP fluticasone propionate; LT1RA cys-lt1 receptor antagonist. www.chestjournal.org CHEST / 130 / 6/ DECEMBER, 2006 1719

inhaled normal saline solution at first as a control. Then increasing doses of methacholine, starting from 0.032 mg/ml and then progressively doubling the concentration to reach 16 mg/ml, were administered. The provocative concentration of methacholine causing a 15% fall in FEV 1 (PC 15 ) was calculated. EBC EBC was collected by using a handcrafted condenser constructed as per a report by Scheideler et al. 18 The validity of measuring LT levels in EBC using our handcrafted condenser was confirmed by the Bland-Altman plot in comparison with Ecoscreen (Jaeger; Hoechberg, Germany). 19 Amylase in the EBC was measured by the 2-chloro-4-nitrophenyl maltotrioside method (Kanto Chemical; Tokyo, Japan), with a lower limit of detection of 1.4 IU/L. Amylase was not detected in the EBC samples. Subjects were asked to breathe through a mouthpiece at a normal frequency and tidal volume for a period of 20 min with a nose clip. Condensate of least 1 ml in volume was collected and immediately stored at 80 C. The mean collected volume of EBC was 4.5 ml. LT Measurement The samples were freeze-dried with a freeze-drier (EYELA FDU-2200; Tokyo Rika Kikai; Tokyo, Japan). After freeze-drying, the samples were resolved with 100 L of water, and LTE 4 concentrations were measured with a specific enzyme immunoassay (Cayman Chemical; Ann Arbor, MI). We initially concentrated the EBC samples by freeze-drying in order to improve the sensitivity for detection of LTE 4 concentrations. The lower limit of detection was 30 pg/ml. LTE 4 values in EBC were expressed as the total amount (in picograms) expired in 20 min of breath. 20 to 2.2 pg/20 min; p 0.05) [Fig 1]. In particular, EBC LTE 4 levels in group 2 (4.99 6.70 pg/20 min; range, 0.49 to 28.7 pg/20 min) and in group 3 (14.66 17.11 pg/20 min; range, 0.41 to 49.4 pg/20 min) were increased compared with control subjects and with group 1 (1.50 1.69 pg/20 min; range, 0.17 to 6.4 pg/20 min). There were no significant differences in urinary LTE 4 levels among the groups (control subjects, 2,610 2,326 pg/mg creatinine; group 1, 1,523 2,140 pg/mg creatinine; group 2, 982 1,628 pg/mg creatinine; group 3, 1,679 1,105 pg/mg creatinine) [Fig 2]. EBC LTE 4 levels were negatively correlated with PC 15 (r 0.454, p 0.012) [Fig 3]. Among group 1 members, EBC LTE 4 levels were significantly lower (p 0.05) and PC 15 levels were significantly higher (p 0.05) than in group 2 members. Discussion We found a significant increase in EBC LTE 4 concentrations in children with mild asthma. In group 2 (those who were asymptomatic but had had wheezing/symptoms of asthma within 6 months prior to examination) and in group 3 (children with current wheeze and/or mild symptoms of asthma exacerbation at the time of examination), EBC LTE 4 levels were significantly increased compared with control subjects or group 1 (children who had been asymptomatic for 6 months prior to the examina- Statistical Analysis Statistical analysis was performed using the statistical software (SAS version 8.2; SAS Institute; Cary, NC). A Student t test was used to confirm the difference between the control and mild asthma groups for the primary end point of this study. After confirmation of the primary end point, a Tukey multiple comparison tests was used to compare the mean values between control and each asthma groups. Data were expressed as mean SD. The correlation between the LTE 4 levels and the PC 15 was determined by nonparametric Spearman correlation analysis. Statistical significance was defined at p 0.05. Results There were no significant differences among the groups in serum IgE levels or peripheral blood eosinophil counts (Table 1). All the subjects in groups 1 to 3 were atopic, as confirmed by positive levels of serum IgE to at least one of the following antigens: house dust mite, Japanese cedar pollen, or cat dander. EBC LTE 4 levels, however, were increased in all patients with mild asthma (5.69 9.62 pg/20 min; range, 0.41 to 49.4 pg/20 min) vs nonasthmatic control subjects (0.74 0.79 pg/20 min; range, 0.31 Figure 1. Exhaled LTE 4 levels in children with mild asthma. Exhaled LTE 4 levels were increased in all patients with mild asthma compared with nonasthmatic control (cont) subjects. EBC LTE 4 levels in group 2 patients (children who were asymptomatic but had had wheezing/symptoms of asthma within 6 months before examination) and group 3 (children with current wheeze and mild symptoms of asthma at the time of examination) were increased compared with control subjects and with group 1 patients (children who had been asymptomatic, with no wheezing/symptoms of asthma, for 6 months prior to examination). *p 0.05; p 0.05 (vs group 1 patients). 1720 Original Research

Figure 2. Urinary LTE 4 levels. There were no significant differences in urinary (U) LTE 4 levels among the groups. Cr creatinine. See Figure 1 for expansion of abbreviation. tion), suggesting that EBC LTE 4 levels may reflect a clinical instability of asthmatic symptoms based on the latent activity of airway inflammation. Csoma et al 21 found that the concentrations of EBC cys-lts of asymptomatic children with mild intermittent asthma were not significantly elevated compared to normal subjects, even though EBC cys-lts of patients with mild persistent, and moderate-to-severe persistent asthma were elevated. One probable explanation for this difference between those results and ours is that Csoma et al 21 might have primarily studied patients with mild intermittent asthma and very stable symptoms. In this study, we could not find a significant difference in the EBC LTE 4 levels of nonasthmatic control subjects and patients with mild asthma who had been asymptomatic for 6 months prior to examination. We also found a significant correlation between EBC LTE 4 levels and PC 15 in our subjects (r 0.454, p 0.012) [Fig 3]. It is known that Figure 3. EBC LTE 4 levels were negatively correlated with methacholine (r 0.454, p 0.012). airway hyperresponsiveness is a key feature of asthma and can be used as an indicator of asthma severity. This correlation suggests that LT itself may contribute to airway hyperresponsiveness. 22 A few previous studies 23,24 have reported no correlation between the activation of airway inflammatory cells, such as eosinophils and mast cells, and the degree of airway hyperresponsiveness in asthmatic subjects; however, there are also a similar number of studies 25 28 that do show a significant correlation. Our result, depicting a significant correlation between EBC LTE 4 levels and airway hyperreactivity, suggests that airway hyperreactivity is affected by the extent of airway inflammation. Measurement of EBC LTE 4 can be a marker reflecting airway inflammation and hyperreactivity, which may be useful in infants and young children in whom the objective estimation of asthma can be difficult to perform. However, further longitudinal study must be necessary to more thoroughly investigate the clinical utility of EBC LTs. Cys-LTs are mainly produced by airway mast cells and eosinophils. In this study, we could not determine the exact source of the cys-lts because there were no significant differences in serum IgE levels and in peripheral eosinophil counts among the groups. This would imply that the function of mast cells and/or eosinophils, rather than the actual number of cells, may more greatly affect EBC LTE 4 levels. Some of the subjects had been treated with inhaled fluticasone propionate ( 200 g/d in all users) prior to examination (Table 1). There were no significant differences in frequency of fluticasone users between group 1 and group 2. However, the frequency in group 3 was significantly higher than group 1 and group 2. Mondino et al 13 showed that EBC LTE 4 levels in children with asthma were reduced by 18% after 4 weeks of treatment with fluticasone. Nevertheless, we found that EBC LTE 4 levels in group 3 were increased, not decreased, compared with control subjects and group 1. Therefore, although the higher number of children treated with fluticasone in group 3 might have affected the results, we would predict the effect of fluticasone to be the opposite of what our results show. In fact, we may have found even higher levels of LTE 4 if fewer children in group 3 had been treated with fluticasone. Our results suggest that airway cys-lts may play a role in the pathogenesis of asthma even in children with mild, asymptomatic asthma, and that levels of airway cys-lts may reflect degree of airway hyperreactivity, probably based on chronic airway inflammation. Given these perspectives, the current asthma treatment recommendations, as stated in www.chestjournal.org CHEST / 130 / 6/ DECEMBER, 2006 1721

many guidelines, that suggest early intervention with inhaled corticosteroid 29 31 seem reasonable. EBC cys-lts may be useful as a noninvasive marker assessing airway inflammation and hyperreactivity in children with asthma. In the future, we would expect that measuring EBC LTs will become a part of the clinical practice in asthma management in combination with other useful markers such as eno, especially in infants and young children. References 1 Synek M, Beasley R, Holgate ST, et al. Cellular infiltration of the airways in asthma of varying severity. Am J Respir Crit Care Med 1996; 154:224 230 2 Barbato A, Turato G, Baraldo S, et al. Airway inflammation in childhood asthma. Am J Respir Crit Care Med 2003; 168: 798 803 3 Van Den Toorn LM, Overbeek SE, Prins JB, et al. Airway inflammation is present during clinical remission of atopic asthma. Am J Respir Crit Care Med 2001; 164:2107 2113 4 Cokugras H, Akcakaya N, Aksoy F, et al. Ultrastructural examination of bronchial biopsy specimens from children with moderate asthma. Thorax 2001; 56:25 29 5 Payne DNR, Qiu Y, Jeffery PK, et al. Airway inflammation in children with difficult asthma: relationships with airflow limitation and persistent symptoms. Thorax 2004; 59:862 869 6 Payne DNR, Rogers AV, Jeffery PK, et al. Early thickening of the reticular basement membrane in children with difficult asthma. Am J Respir Crit Care Med 2003; 167:78 82 7 Salva PS, Theroux C, Schwartz D. Safety of ronchial biopsy in 170 children with chronic respiratory symptoms. Thorax 2003; 58:1058 1060 8 Jatakanon A, Lim S, Barnes PJ, et al. Correlation between exhaled nitric oxide, sputum eosinophils, and methacholine responsiveness in patients with mild asthma. Thorax 1998; 53:91 95 9 Lim S, Jatakanon A, Barnes PJ, et al. Relationship between exhaled nitric oxide and mucosal eosinophilic inflammation in mild to moderately severe asthma. Thorax 2000; 55:184 188 10 Strunk RC, Szefler SJ, Lemanske RF Jr, et al. Relationship of exhaled nitric oxide to clinical and inflammatory markers of persistent asthma in children. J Allergy Clin Immunol 2003; 112:883 892 11 Al-Ali MK, Howarth PH. Nitric oxide and the respiratory system in health and disease. Respir Med 1998; 92:701 715 12 Kharitonov SA, Yates D, Barnes PJ. Increased nitric oxide in exhaled air of normal human subjects with upper respiratory tract infections. Eur Respir J 1995; 8:295 297 13 Mondino C, Ciabattoni G, Koch P, et al. Effects of inhaled corticosteroids on exhaled leukotrienes and prostanoids in asthmatic children: exhaled leukotrienes and prostaglandins in asthma. J Allergy Clin Immunol 2004; 114:761 767 14 Zanconato S, Carraro S, Baraldi E, et al. Leukotrienes and 8-isoprostane in exhaled breath condensate of children with stable and unstable asthma. J Allergy Clin Immunol 2004; 113:257 263 15 Juniper EF, Frith PA, Hargreave FE. Airway responsiveness to histamine and methacholine: relationship to minimum treatment to control symptoms of asthma. Thorax 1981; 36:575 579 16 American Thoracic Society. Standards for the diagnosis and care of patients with chronic obstructive pulmonary disease (COPD) and asthma. Am Rev Respir Dis 1987; 136:225 244 17 Global Initiative for Asthma. Workshop Report, global strategy for asthma management and prevention. Available at: http://www.ginasthma.com/guidelineitem.asp??/11 2&/2 1& intid 60. Accessed August 1, 2006 18 Scheideler L, Manke HG, Hammerle H, et al. Detection of nonvolatile macromolecules in breath: a possible diagnostic tool? Am Rev Respir Dis 1993; 148:778 784 19 Bland JM, Altman DG. Measuring agreement in method comparison studies. Stat Method Med Res 1999; 8:135 160 20 Montuschi P, Barnes PJ. Exhaled leukotrienes and prostaglandins in asthma. J Allergy Clin Immunol 2002; 109:615 620 21 Csoma Z, Kharitonov SA, Barnes PJ, et al. Increased leukotrienes in exhaled breath condensate in childhood asthma. Am J Respir Crit Care Med 2002; 166:1345 1349 22 Leff AR. Role of leukotrienes in bronchial hyperresponsiveness and cellular responses in airways. Thorax 2000; 55(suppl2):S32 S37 23 Ollerenshaw SL, Woolcock AJ. Characteristics of the inflammation in biopsies from large airways of subjects with asthma and subjects with chronic airflow limitation. Am Rev Respir Dis 1992; 145:922 927 24 Crimi E, Spanevello A, Brusasco V, et al. Dissociation between airway inflammation and airway hyperresponsiveness in allergic asthma. Am J Respir Crit Care Med 1998; 157:4 9 25 Jeffery PK, Wardlaw AJ, Kay AB, et al. Bronchial biopsies in asthma: an ultrastructural, quantitative study and correlation with hyperreactivity. Am Rev Respir Dis 1989; 140:1745 1753 26 Kelly C, Ward C, Walters EH, et al. Number and activity of inflammatory cells in bronchoalveolar lavage fluid in asthma and their relation to airway responsiveness. Thorax 1988; 43:684 692 27 Laprise C, Laviolette M, Boulet LP, et al. Asymptomatic airway hyperresponsiveness: relationships with airway inflammation and remodelling. Eur Respir J 1999; 14:63 73 28 Chetta A, Foresi A, Olivieri D, et al. Bronchial responsiveness to distilled water and methacholine and its relationship to inflammation and remodeling of the airways in asthma. Am J Respir Crit Care Med 1996; 153:910 917 29 Warner JO, Marguet C, Pohunek P, et al. Inflammatory mechanisms in childhood asthma. Clin Exp Allergy 1998; 28:S71 S75 30 Martinez FD. Present and future treatment of asthma in infants and young children. J Allergy Clin Immunol 1999; 104:S169 S174 31 Spahn JD, Szefler SJ. Childhood asthma: new insights into management. J Allergy Clin Immunol 2002; 109:3 13 1722 Original Research