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Effects of a leukotriene receptor antagonist on exhaled leukotriene E4 and prostanoids in children with asthma
Paolo Montuschi, MD,a Chiara Mondino, MD,b Pierluigi Koch, MD,c Peter J. Barnes, DM,d and Giovanni Ciabattoni, MDe Rome, Palidoro, and Chieti, Italy, and London, United Kingdom
Background: Leukotriene (LT) E4 and 8-isoprostane concentrations are elevated in exhaled breath condensate in children with asthma. The effects of leukotriene receptor antagonists (LTRAs) on exhaled leukotriene and prostanoids in children with asthma are unknown. Objective: (1) To study the effect of montelukast, a LTRA, on exhaled LTE4, 8-isoprostane, and prostaglandin E2 in children with asthma and atopic children; (2) to measure exhaled nitric oxide. Methods: An open-label study with oral montelukast (5 mg once daily for 4 weeks) was undertaken in 17 atopic children with asthma and 16 atopic children without asthma. Results: Pretreatment exhaled LTE4 (P < .0001) and 8isoprostane (P < .0001) values were higher in atopic children with asthma than in atopic children without asthma. In atopic children with asthma, montelukast reduced exhaled LTE4 by 33% (P < .001), and this reduction was correlated with pretreatment LTE4 values (r 5 20.90; P 5 .0001). Posttreatment exhaled LTE4 levels in children with asthma were higher than pretreatment LTE4 values in atopic children without asthma (P < .004). Montelukast had no effect on exhaled LTE4 in atopic children without asthma (P 5 .74), or on exhaled 8-isoprostane (atopic children with asthma, P 5 .94; atopic children without asthma, P 5 .55) and PGE2 (atopic children with asthma, P 5 .56; atopic children without asthma, P 5 .93) in both groups. In atopic children with asthma, exhaled nitric oxide concentrations were reduced by 27% (P < .05) after montelukast. Conclusion: Leukotriene receptor antagonists decrease exhaled LTE4 in atopic children with asthma. This reduction is dependent on baseline exhaled LTE4 values.
From athe Department of Pharmacology, Faculty of Medicine, Catholic University of the Sacred Heart, Rome; bthe Department of Immunodermatology, Istituto Dermopatico dell’Immacolata, IRCCS, Rome; cthe Department of Pulmonology, Ospedale Pediatrico Bambino Gesu`, Palidoro; dthe Department of Thoracic Medicine, Imperial College, School of Medicine, National Heart and Lung Institute, London; and ethe Department of Drug Sciences, School of Pharmacy, University ‘‘G. d’Annunzio,’’ Chieti. Supported by the Catholic University of the Sacred Heart, Rome, Italy academic grant 2004-2005. Disclosure of potential conflict of interest: P. J. Barnes has received grant support from GlaxoSmithKline, AstraZeneca, Pfizer, Novartis, and Boehringer-Ingelheim, and is on the advisory board for GlaxoSmithKline, Pfizer, Boehringer-Ingelheim, and Altana. The rest of the authors have declared that they have no conflict of interest. Received for publication July 5, 2005; revised April 7, 2006; accepted for publication April 13, 2006. Available online July 5, 2006. Reprint requests: Paolo Montuschi, MD, Department of Pharmacology, Faculty of Medicine, Catholic University of the Sacred Heart, Largo F. Vito, 1, 00168 Rome, Italy. E-mail: [email protected]. 0091-6749/$32.00 Ó 2006 American Academy of Allergy, Asthma and Immunology doi:10.1016/j.jaci.2006.04.010
Clinical implications: Measurement of exhaled LTE4 might help identify children with asthma most likely to benefit from LTRAs. (J Allergy Clin Immunol 2006;118:347-53.) Key words: Leukotriene E4, prostanoids, exhaled breath condensate, exhaled nitric oxide, childhood asthma, airway inflammation, noninvasive markers, leukotriene receptor antagonists
Lipid mediators including leukotrienes and prostanoids play an important pathophysiological role in asthma.1 Measurement of leukotrienes and prostanoids in exhaled breath condensate (EBC) is an in vivo approach for studying the role of eicosanoids in asthma.2,3 EBC is a completely noninvasive method to sample secretions from the airways.4 Using immunoassays, cysteinyl-leukotrienes (CysLTs) and 8-isoprostane, a marker of oxidative stress,5 were detected in EBC in healthy children and found increased in children with stable asthma6-9 and asthma exacerbations.10 We have confirmed the presence of leukotriene (LT) E4, the most chemically stable CysLT, in EBC in healthy children using gas chromatography/mass spectrometry.11 Compared with those in healthy children, exhaled LTE4 concentrations are increased in steroid-naive children with asthma.11 Montelukast, a leukotriene receptor antagonist (LTRA),12 inhibits CysLTs production in allergen-stimulated PBMCs from adults with asthma.13 In an experimental model of allergic asthma, montelukast decreased the production of CysLTs in the lungs both directly and by inhibiting the recruitment of inflammatory cells.14 In addition to its antagonist effect on CysLT1 receptor, 1 study reported that montelukast has a selective direct inhibitory effect of 5-lipoxygenase activity in human mast cells in vitro at concentrations of potential therapeutic relevance.15 Oral montelukast at a dose of 10 mg once daily for 4 weeks reduces exhaled CysLTs concentrations in adults with asthma.16 However, other exhaled eicosanoids were not measured in this study, and the effects of LTRAs on exhaled LTs and prostanoids in children with asthma are unknown. Considering its mechanism of action, receptor blockade by montelukast can be clinically more important than decreasing LTE4 production. A multicenter study has shown that an increased likelihood of pulmonary response to montelukast is associated with higher baseline urinary LTE4 levels.17 Measurement of exhaled LTE4 might help identify children with asthma who need CysLT1 receptor blocking and are most likely to benefit from LTRAs. This might 347
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TABLE I. Subject characteristicsy Healthy children
Asthma diagnosis and treatment
N Age, y Sex, F/M FEV1, L FEV1, % predicted FVC, L FVC, % predicted FEV1/FVC, % predicted FEF25%-75% Atopyà Steroids LTRAs
15 10 6 7/8 2.2 6 101.5 6 2.3 6 100.8 6 102.5 6 104.5 6 No No No
1 0.1 2.7 0.2 2.5 1.5 4.2
Atopic children without asthmaz
16 96 9/7 2.2 6 99.7 6 2.5 6 102.8 6 101.5 6 102.8 6 Yes No No
1 0.2 3.1 0.1 2.4 1.8 3.9
Atopic children with asthmaz
17 10 6 1 9/8 2.0 6 0.1 94.1 6 2.8 2.4 6 0.2 103.6 6 2.9 85.3 6 1.6* 73.7 6 4.6** Yes No No
F, Female; M, male. *P < .05 and **P < .01 compared with atopic children without asthma and healthy children. Data are expressed as numbers or means 6 SEMs. àPresence of atopy was confirmed by skin prick testing for common aeroallergens; a positive skin test response was defined as a wheal with a mean diameter (mean of maximum and 90° midpoint diameters) of at least 3 mm greater than that produced with a saline control. All atopic children had a clinical history of atopy. Children had no corticosteroid or LTRA treatment within 4 weeks.
Abbreviations used CysLTs: Cysteinyl-leukotrienes EBC: Exhaled breath condensate FEF25%-75%: Forced expiratory flow between 25% and 75% of the vital capacity FVC: Forced vital capacity LT: Leukotriene LTRAs: Leukotriene receptor antagonists NO: Nitric oxide PGE2: Prostaglandin E2
have relevant clinical implications given the importance of considering an individualized approach to asthma management and assessment rather than a strategy directed to the best outcome in a group of patients.18 In the current study, we sought (1) to investigate the effect of montelukast on exhaled LTE4 and prostanoids in atopic children with asthma and atopic children without asthma, (2) to measure exhaled nitric oxide (NO), a wellestablished marker of airway inflammation in patients with asthma who are steroid-naive,19 and (3) to compare concentrations of exhaled markers in children with asthma and atopic children without asthma enrolled in the montelukast study with those in healthy control children.
METHODS Study subjects Two groups of children were included in the montelukast study: 18 white atopic children with stable mild intermittent asthma and 18 atopic children without asthma. Seventeen atopic children with asthma and 16 atopic children without asthma completed the study (Table I; see the Methods section in the Online Repository at www. jacionline.org). Atopic children were recruited from the Allergy Outpatient Clinic of the Istituto Dermopatico dell’Immacolata, Rome, Italy, and the Asthma and Allergy Clinic of the Pediatric
Hospital Bambino Gesu`, Palidoro, Italy. The diagnosis and classification of asthma were based on clinical history and examination and pulmonary function parameters according to the guidelines issued by the National Heart, Lung, and Blood Institute of the National Institutes of Health.20 Atopic children had mild intermittent asthma with symptoms less often than twice a week, FEV1 of 80% or greater of predicted value and reversibility of 12% or greater to salbutamol, or a positive provocation test result with methacholine or exercise. They were not taking any regular medication, but used inhaled short-acting b2-agonists as needed for symptom relief. Sixteen atopic children without asthma were included in the montelukast study as control population without asthma (Table I; see the Methods section in the Online Repository at www. jacionline.org). Fifteen healthy nonatopic children without asthma who were recruited from children of staff were included in the cross-sectional study (Table I). They had no history of asthma and atopic disease, negative skin prick test results, and normal spirometry results. Study group children had no upper respiratory tract infections in the previous 3 weeks. Children were excluded from the study if they had used corticosteroids or LTRAs in the previous 4 weeks or nonsteroidal anti-inflammatory drugs in the last 2 weeks.
Study design Two studies were performed: an interventional study with montelukast and a cross-sectional study. A pilot uncontrolled open-label study with montelukast was undertaken in 17 atopic children with asthma and 16 atopic children without asthma. Children attended the Asthma Outpatient Clinic of the University Hospital A. Gemelli and the Department of Pharmacology of the Catholic University of the Sacred Heart, Rome, Italy, on 3 occasions for clinical examination, measurement of exhaled NO, collection of EBC, and lung function tests. After a screening visit (visit 1) and 1-week run-in period (visit 2), children were given oral montelukast (5 mg as tablet [Singulair; Merck & Co, Whitehouse Station, NJ]) once daily for 4 weeks. After treatment with montelukast, all of these tests were repeated (visit 3). Skin testing was performed on visit 1. In the cross-sectional study, concentrations of exhaled markers and lung function tests in atopic children with asthma and atopic children without asthma at baseline (visit 1) were compared with those in nonatopic children without asthma. Healthy children
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TABLE II. Absolute exhaled eicosanoid and NO values in the montelukast study group childreny
Atopic children with asthma (n 5 17) LTE4 8-Isoprostane PGE2 Exhaled NO (ppb) Atopic children without asthma (n 5 16) LTE4 8-Isoprostane PGE2 Exhaled NO (ppb)
Baseline (day 27)
Pretreatment (day 0)
Posttreatment (day 28)
44 31 28 45
(30-50) (26-35) (25-31) (40-61)
43 31 30 45
(31-48) (25-34) (23-32) (36-65)
29 33 28 32
(24-32)** (26-39) (25-34) (27-46)*
15 16 26 17
(13-18) (15-20) (23-29) (12-22)
17 16 26 16
(14-18) (15-19) (25-28) (12-21)
16 17 25 16
(13-19) (14-21) (23-29) (10-19)
*P < .05 and **P < .001 compared with pretreatment values. Values are medians and interquartile ranges (25th-75th percentiles). Values at baseline (day –7), before (day 0), and after treatment with oral montelukast (5 mg once daily for 4 weeks; day 28) are reported. Eicosanoid values are expressed as picograms produced during 15 minutes of breathing.
attended on 1 occasion for measurement of exhaled NO, collection of EBC, spirometry, and skin prick testing. Repeatability for eicosanoid measurement was assessed in all children with asthma and atopic children without asthma collecting EBC at screening visit and after 1-week run-in period. Informed consent was obtained from parents, and the study was approved by the Ethics Committee of the Catholic University of the Sacred Heart, Rome, Italy.
Pulmonary function FEV1, forced vital capacity (FVC), and forced expiratory flow between 25% and 75% of vital capacity (FEF25%-75%) were measured with a 10-L bell spirometer (Biomedin, Padova, Italy) and the best of 3 maneuvers, expressed as percentage of predicted values, chosen.
EBC Exhaled breath condensate was collected by using a condensing chamber (Echoscreen; Jaeger, Hoechberg, Germany), as described previously21 (see the Methods section in the Online Repository at www.jacionline.org). Children were instructed to breath tidally through a mouthpiece connected to the condenser for 15 minutes and not to inhale through their nose. An average of 1.5 mL EBC per child was collected and stored at 280°C before eicosanoid measurements. a-Amylase concentrations in all EBC samples were measured with a functional enzyme assay to exclude gross salivary contamination (Roche Diagnostics, Basel, Switzerland). However, activity assays for amylase are not sufficient to rule out salivary contamination definitively, because they have not been validated in EBC, and the duration that amylase activity persists in EBC when spiked at low concentration is currently unknown.
Measurement of exhaled LTE4 and prostanoids Leukotriene E4 in EBC was measured with a specific enzyme immunoassay kit (Cayman Chemical, Ann Arbor, Mich)7,21 (see the Methods section in the Online Repository at www.jacionline. org). The detection limit was 8 pg/mL. However, because of the reported variability at these concentrations, results below 16 pg/mL should be interpreted cautiously. The intra-assay and interassay coefficients of variation for LTE4 assay were 10% and 15%, respectively. 8-Isoprostane and prostaglandin E2 (PGE2) in EBC were measured with specific RIAs developed in our laboratory22-24 and validated by reverse-phase HPLC.23 The detection limit for 8-isoprostane and
PGE2 was 10 pg/mL. The intra-assay and interassay coefficients of variation for prostanoid RIAs were as follows: 8-isoprostane, <2% and <3%, respectively; PGE2, <4% and <5%, respectively. Because differences in the composition of enzyme immunoassay buffer (usually containing 1 mg/mL albumin) and EBC samples, which often have a lower protein concentration,25 can influence the analytical results, we compared LTE4 standard curves obtained with the kit immunoassay buffer (n 5 4) with those obtained with a more EBC-like buffer (phosphate buffer 0.025 mol/L, pH 7.5, not containing proteins; n 5 3; see this article’s Fig E1 in the Online Repository at www.jacionline.org). Because the 2 types of standard curves were different, immunoassays for LTE4 were performed by using a buffer not containing proteins. 8-Isoprostane and PGE2 standard curves were performed by using phosphate buffer 0.025 mol/L, pH 7.5, not containing proteins. Because dilution of respiratory droplets by water vapor may explain part of the variation in concentrations of nonvolatile compounds in EBC,25 LTE4 and 8-isoprostane results were presented as a change in ratio of LTE4 and 8-isoprostane to PGE2, another eicosanoid. Absolute LTE4 and prostanoid values in EBC, expressed as total amount (in picograms) of eicosanoids expired in the 15-minute breath test (eicosanoid concentrations 3 volume of EBC), are shown in Table II. For samples below the lowest detection limits, the data were expressed as half the detection limit.8,9
Exhaled NO measurement Exhaled NO was measured with the NIOX system (Aerocrine, Stockholm, Sweden) with a single breath online method at a constant flow of 50 mL/s according to American Thoracic Society guidelines19 (see the Methods section in the Online Repository at www.jacionline.org).
Skin testing Atopy was assessed by skin prick tests for common aeroallergens (Stallergenes, Antony, France)7 (see the Methods section in the Online Repository at www.jacionline.org).
Sample size The study sample was calculated26 considering exhaled LTE4 values as the primary outcome. Sample size was estimated to be 18 children after having considered a SD of 9 pg/15 min, a dropout of 15%, and identified the minimal difference of biological significance (10 pg/15 min) corresponding to a 33% reduction in median exhaled LTE4 values7 with a power of 90% (a value of 5% and b value of 10%).
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Exhaled eicosanoid (pg/15 min)
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Asthma diagnosis and treatment FIG 1. LTE4 in 17 atopic children with asthma (A) and 16 atopic children without asthma (B) at baseline (day 27), after 1-week run-in period (day 0), and after treatment with oral montelukast (5 mg once a day for 4 weeks; day 28). Values are expressed as LTE4/ PGE2 ratio.
Statistical analysis Exhaled eicosanoid and NO values were expressed as medians and interquartile ranges (25th and 75th percentiles). Kruskal-Wallis tests followed by Mann-Whitney U tests were used to compare groups. Eicosanoid values within the groups and comparison before and after treatment were analyzed by using Friedman repeated measures of ANOVA. Spirometry values were expressed as means 6 SEMs. Correlation was expressed as Spearman coefficient. Significance was defined as a value of P < .05.Within-subject repeatability of exhaled eicosanoid and exhaled NO measurements was expressed as limits of agreement (mean difference 6 2 SDs of the differences) for measurements taken at the baseline and after a 1-week run-in period.27 Limits of agreement estimate the largest likely size of difference between 2 measurements on the same subject27 (see the Methods section in the Online Repository at www.jacionline.org).
RESULTS No a-amylase concentrations were detected in any study sample, excluding gross salivary contamination.
Repeatability of measurements Limits of agreement in children with asthma (eicosanoids values expressed as picograms of eicosanoids expired in the 15-minute breath test) were as follows: LTE4, 5 pg and 26 pg; 8-isoprostane, 6 pg and 25 pg; PGE2, 8 pg and 28 pg; exhaled NO, 10 ppb and 210 ppb (see this article’s Figs E2E5 in the Online Repository at www.jacionline.org). For each biomarker, all of the differences between the 2 measurements were within its limits of agreement. Regarding exhaled LTE4, most of the children had variations (1-3 pg) smaller than their estimated limits of agreement (see
FIG 2. 8-Isoprostane (IP) (A) and PGE2 (B) in EBC in 17 atopic children with asthma at baseline (day 27), after 1-week run-in period (day 0), and after treatment with oral montelukast (5 mg once a day for 4 weeks; day 28). 8-Isoprostane values are expressed as 8-isoprostane/PGE2 ratio. PGE2 values are expressed as picograms produced during 15 minutes of breathing.
this article’s Fig E2 and Results section in the Online Repository at www.jacionline.org). Limits of agreement in atopic children without asthma are shown in this article’s Figs E6-E9 and values are provided in the Results section in the Online Repository at www.jacionline.org.
Exhaled LTE4 and prostanoids In the montelukast study, values of exhaled eicosanoids at baseline and after 1-week run-in period were similar in both groups of children (see this article’s Figs E2-E4 and E6-E8 and the Results section in the Online Repository at www.jacionline.org). Children with asthma had higher pretreatment exhaled LTE4/PGE2 (1.35 [1.15-1.51] vs 0.63 [0.53-0.72]; P < .0001) and 8-isoprostane/PGE2 (1.06 [0.93-1.14] vs 0.64 [0.59-0.71]; P < .0001) than atopic children without asthma. Pretreatment exhaled PGE2 values were similar in the 2 groups of children (P 5 .44). In children with asthma, median exhaled LTE4/ PGE2 was reduced by 33% after montelukast (pretreatment: 1.35 [1.15-1.51]; posttreatment: 0.90 [0.80-1.25]; P < .001; Fig 1, A), whereas exhaled 8-isoprostane/ PGE2 (pretreatment: 1.06 [0.93-1.14]; posttreatment: 0.96 [0.85-1.20]; P 5 .94; Fig 2, A) and PGE2 values (pretreatment: 30[23-32] pg; posttreatment: 28[25-34] pg; P 5 .56; Fig 2, B) were unaffected. The reduction in exhaled LTE4 after treatment was correlated with pretreatment LTE4 values (r 5 20.90; P < .0001; Fig 3). Posttreatment exhaled LTE4/PGE2 in children with asthma was higher than pretreatment exhaled LTE4/PGE2 in atopic children without asthma (0.90 [0.80-1.25] vs 0.63 [0.53-0.72]; P < .004). Montelukast had no effect on exhaled LTE4 (P 5 .74), 8-isoprostane (P 5 .55), and
FIG 3. Correlation between changes in LTE4 values in EBC after treatment with oral montelukast (5 mg once daily for 4 weeks) in 17 atopic children with asthma and pretreatment exhaled LTE4 levels (r 5 –0.90, P < .0001). Values are expressed as picograms produced during 15 minutes of breathing.
PGE2 (P 5 .93) in atopic children without asthma (Fig 1, B; see this article’s Fig E10 in the Online Repository at www.jacionline.org). In the cross-sectional study, atopic children with asthma had higher exhaled LTE4 (P < .001) and 8-isoprostane (P < .01) than atopic children without asthma, and there was no difference in exhaled LTE4 (P 5 .30) and 8-isoprostane values (P 5 .20) between atopic children without asthma and healthy children (see this article’s Fig E11, Fig E12, A, and Table E1 in the Online Repository at www.jacionline.org). Exhaled PGE2 values in the 3 groups of children were similar (P 5 .18; see this article’s Fig E12, B, and Table E1 in the Online Repository at www.jacionline.org). There was no correlation between exhaled eicosanoids or exhaled NO and age, sex, and lung function. There was no correlation between the different exhaled markers.
Exhaled NO In atopic children with asthma, exhaled NO was reduced by 27% (pretreatment: 45 [36-65] ppb; posttreatment: 32 [27-46] ppb; P < .05) after montelukast (Fig 4). Posttreatment reduction in exhaled NO was correlated with pretreatment exhaled NO concentrations (r 5 20.84; P < .0001; see this article’s Fig E13 in the Online Repository at www.jacionline.org). Montelukast had no effect on exhaled NO in atopic children without asthma (P 5 .65; see this article’s Fig E14 in the Online Repository at www.jacionline.org). Compared with healthy children, exhaled NO was increased in atopic children without asthma (P < .05) and, to a greater extent, in children with asthma (P < .001; see this article’s Fig E15 and Table E1 in the Online Repository at www.jacionline.org). Pulmonary function Median spirometry values before and after montelukast were similar in atopic children with and without asthma (see this article’s Table E2 in the Online Repository at www.jacionline.org). One child with asthma had a significant response to montelukast defined as improvement
FIG 4. Exhaled NO concentrations in 17 atopic children with asthma at baseline (day 27), after 1-week run-in period (day 0), and after treatment with oral montelukast (5 mg once a day for 4 weeks; day 28).
in FEV1 of 7.5% or greater with a concomitant reduction in exhaled LTE4 of 25 pg. There was no association between the effect of montelukast on exhaled LTE4 values and the effect of montelukast on lung function in the children with asthma.
DISCUSSION We sought to investigate the effect of montelukast on exhaled LTE4, 8-isoprostane, and PGE2 in atopic children with and without asthma. In the current study, median exhaled LTE4 values were decreased by 33% after montelukast in children with asthma, whereas treatment had no effect on exhaled LTE4 in atopic children without asthma. Montelukast had no effect on exhaled 8-isoprostane and PGE2 in both groups of children. In children with asthma, there was a strong correlation between reduction in exhaled LTE4 after treatment and pretreatment LTE4 levels, indicating that children with higher baseline LTE4 levels might be the most likely to have a significant reduction in exhaled LTE4 from LTRAs. Reduction in leukotriene production might be functionally relevant, because in adults with nocturnal asthma, 5-lipoxygenase inhibition decreases urinary LTE4 levels with a concomitant trend for improving nocturnal FEV1.28 However, studies aimed to measure urinary LTE4 for confirmation of the reduced LTE4 production and to establish the relationships between urinary and exhaled LTE4 are warranted. In our study, a pulmonary response to montelukast (defined as improvement in FEV1 of 7.5% or greater) was observed in 1 out of 17 children with asthma. Future studies should be performed to clarify whether baseline exhaled LTE4 values are associated with a change in more sensitive spirometry parameters, particularly those reflecting peripheral airway function such as FEF25%-75% values, or in asthma symptoms or course. Exhaled 8-isoprostane, a specific marker of lipid peroxidation, was elevated in children with asthma, indicating that asthma is associated with increased oxidative stress.7,8 Reduced production of PGE2, an endogenous bronchodilating compound that may have anti-inflammatory effects in the airways, has been implicated in the pathophysiology
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of asthma.29 Similar exhaled PGE2 concentrations in children with asthma and control children observed in our study do not seem to support this hypothesis. There was no effect of montelukast on exhaled 8-isoprostane and PGE2 in children with asthma, indicating that the antiinflammatory effects of this drug within the airways are unlikely to be explained by inhibition of lipid peroxidation or COX interactions. In atopic children with asthma, exhaled NO was decreased after montelukast consistent with previous studies.30,31 Exhaled LTE4 and exhaled NO values were not correlated in line with the lack of correlation between urinary LTE4 and exhaled NO32 (see the Discussion section in the Online Repository at www.jacionline.org). These findings suggest that exhaled LTE4 and exhaled NO have a different biological significance as markers of airway inflammation in children with asthma and cannot be used interchangeably when assessing the antiinflammatory response to antiasthmatic drugs. Our study with montelukast was open-label, was uncontrolled, and involved a limited number of children with asthma. This limits the strength of our trial and precludes definitive conclusions. Larger controlled studies are required to definitively establish the effect of LTRAs on exhaled leukotrienes and prostanoids in children with asthma, to ascertain whether there are subgroups of children with asthma with a different response to LTRAs in terms of reduction in exhaled eicosanoids, and to clarify the effect of these findings on management of children with asthma. Studies on the relationship between exhaled LTE4 levels and polymorphisms in the 5-lipoxygenase promoter could contribute to explain the wide interindividual variability in the pretreatment exhaled LTE4 values observed in our study. Finally, a drug withdrawal study would likely reveal the predicted value of LTE4 in asthma subpopulations. We were unable to ascertain the mechanisms by which LTRAs decrease exhaled LTE4 in children with asthma. EBC analysis cannot provide information on the cellular origin of mediators for which invasive techniques such as bronchoscopy and bronchial biopsies are required.33 Several types of airway cells can synthesize CysLTs, including mast cells and eosinophils.34 Montelukast inhibits CysLTs production in allergen-stimulated PBMCs from adults with asthma.13 In an experimental animal model of allergic asthma, montelukast decreased the production of CysLTs in the lungs both directly and by inhibiting the infiltration of inflammatory cells into the bronchial submucosa.14 However, whether montelukast has similar effects in children with asthma is currently unknown, as well as its cellular targets. In the cross-sectional study, baseline exhaled LTE4 and 8-isoprostane were elevated in atopic children with asthma, but not in atopic children without asthma, in contrast with the 1 airway hypothesis, which suggests that nasal inflammation reflects lower airway inflammation. By contrast, exhaled NO was increased in atopic children without asthma and, to a greater extent, in atopic children with asthma. These findings indicate that LTE4 and 8-
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isoprostane in EBC might be considered as markers of asthma rather than reflecting the different degree of inflammation within the respiratory tract in atopic children with and without asthma.7 Currently, because of the lack of a standardized procedure and validated analytical methods, EBC analysis is more reliable for relative measures than for determining absolute levels of inflammatory mediators. Collection and/or storage of EBC samples are possible sources of variability in exhaled eicosanoid measurements.35 However, part of this variability could be related to analytical methods, particularly at LT concentrations close to the detection limit of the immunoassay. These data should be interpreted cautiously. However, in our study, day-today repeatability for exhaled eicosanoid measurements was acceptable, as indicated by their limits of agreement, consistent with previous findings.7,8,10 Matrix effects as well as sample pretreatment may play a role, because LTE4 standard curves performed using the kit immunoassay buffer and those performed with a more EBC-like buffer such as phosphate buffer were different (see the Discussion section in the Online Repository at www. jacionline.org). However, further investigation to address this issue formally is required. Another limitation of the current study is that a reference dilution indicator was not measured.25 However, the fact that LTE4 results expressed as both absolute values and LTE4/PGE2 ratio were similar indicates that dilution of respiratory droplets has little, if any, effect on LTE4 levels in EBC. In conclusion, exhaled LTE4 and exhaled NO values, but not exhaled 8-isoprostane and PGE2, are reduced by LTRAs in atopic children with asthma. Reduction in exhaled LTE4 after LTRAs is dependent on baseline pretreatment exhaled LTE4 values. Identification of children with asthma who are most likely to benefit from LTRAs based on exhaled LTE4 levels might lead to a more rational and targeted use of these drugs. REFERENCES 1. Bochner BS, Busse WW. Allergy and asthma. J Allergy Clin Immunol 2005;115:953-9. 2. Montuschi P. Indirect monitoring of lung inflammation. Nat Rev Drug Discov 2002;1:238-42. 3. Hunt JF. Exhaled breath condensate: an evolving tool for noninvasive evaluation of lung disease. J Allergy Clin Immunol 2002;110:28-34. 4. Kharitonov SA, Barnes PJ. Exhaled markers of pulmonary diseaae. Am J Respir Crit Care Med 2001;163:1693-722. 5. Montuschi P, Barnes PJ, Roberts LJ II. Isoprostanes: markers and mediators of oxidative stress. FASEB J 2004;18:1791-800. 6. Csoma Z, Kharitonov SA, Balint B, Bush A, Wilson NM, Barnes PJ. Increased leukotrienes in exhaled breath condensate in childhood asthma. Am J Respir Crit Care Med 2002;166:1345-9. 7. Mondino C, Ciabattoni G, Koch P, Pistelli R, Trove´ A, Barnes PJ, et al. Effects of inhaled corticosteroids on exhaled leukotrienes and prostanoids in asthmatic children. J Allergy Clin Immunol 2004;114:761-7. 8. Zanconato S, Carraro S, Corradi M, Alinovi R, Pasquale MF, Piacentini G, et al. Leukotrienes and 8-isoprostane in exhaled breath condensate of children with stable and unstable asthma. J Allergy Clin Immunol 2004; 113:257-63. 9. Carraro S, Corradi M, Zanconato S, Alinovi R, Pasquale MF, Zacchello F, et al. Exhaled breath condensate cysteinyl-leukotrienes are increased
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in children with exercise-induced bronchoconstriction. J Allergy Clin Immunol 2005;115:764-70. Baraldi E, Carraro S, Alinovi R, Pesci A, Ghiro L, Bosini A, et al. Cysteinyl-leukotrienes and 8-isoprostane in exhaled breath condensate of children with asthma exacerbations. Thorax 2003;58:505-9. Cap P, Chladek J, Pehal F, Marek M, Petru V, Barnes PJ, et al. Gas-chromatography mass spectrometry analysis of exhaled leukotrienes in asthmatic patients. Thorax 2004;59:465-70. Leff AR. Antileukotrienes Working Group. Discovery of leukotrienes and development of antileukotrienes agents. Ann Allergy Asthma Immunol 2001;86(suppl 1):4-8. Frieri M, Therattil J, Wang SF, Huang CY, Wang YC. Montelukast inhibits interleukin-5 mRNA expression and cysteinyl leukotriene production in ragweed and mite-stimulated peripheral blood mononuclear cells from patients with asthma. Allergy Asthma Proc 2003;24:359-66. Murai A, Abe M, Hayashi Y, Sakata N, Katsuragi T, Tanaka K. Comparison study between the mechanisms of allergic asthma amelioration by a cysteinyl-leukotriene type 1 receptor antagonist montelukast and methylprednisolone. J Pharmacol Exp Ther 2005;312:432-40. Ramires R, Caiaffa MF, Tursi A, Haeggstrom JZ, Macchia L. Novel inhibitory effect on 5-lipoxygenase activity by the anti-asthma drug montelukast. Biochem Biophys Res Commun 2004;324:815-21. Biernacki WA, Kharitonov SA, Biernacka HM, Barnes PJ. Effect of montelukast on exhaled leukotrienes and quality of life in asthmatic patients. Chest 2005;128:1958-63. Szefler SJ, Phillips BR, Martinez FD, Chinchilli VM, Lemanske RF, Strunk RC, et al. Characterization of within-subject responses to fluticasone and montelukast in childhood asthma. J Allergy Clin Immunol 2005;115:233-42. Szefler SJ, Apter A. Advances in pediatric and adult asthma. J Allergy Clin Immunol 2005;115:470-7. 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—1999: official statement of the American Thoracic Society 1999. Am J Respir Crit Care Med 1999;160:2104-17. National Asthma Education and Prevention Program Expert Panel report: guidelines for the diagnosis and management of asthma update on selected topics—2002. J Allergy Clin Immunol 2003;111:S141-219. Montuschi P, Barnes PJ. Exhaled leukotrienes and prostaglandins in asthma. J Allergy Clin Immunol 2002;109:615-20.
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22. Wang Z, Ciabattoni G, Creminon C, Lawson J, FitzGeral GA, Patrono C, et al. Immunochemical characterization of urinary 8-epi-prostaglandin F2a excretion in man. J Pharmacol Exp Ther 1995;275:94-100. 23. Montuschi P, Ragazzoni E, Valente S, Corbo G, Mondino C, Ciappi G, et al. Validation of 8-isoprostane and prostaglandin E2 measurements in exhaled breath condensate. Inflamm Res 2003;52:502-6. 24. Ciabattoni G, Pugliese F, Spaldi M, Cinotti GA, Patrono C. Radioimmunoassay measurement of prostaglandin E2 and F2a in human urine. J Endocrinol Invest 1979;2:173-82. 25. Effros RM, Hoagland KW, Bosbous M, Castello D, Foss B, Dunning M, et al. Dilution of respiratory solutes in exhaled condensates. Am J Respir Crit Care Med 2002;165:663-9. 26. Florey CV. Sample size for beginners. BMJ 1993;306:1181-4. 27. Bland JM, Altman DG. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1986;1:307-10. 28. Wenzel SE, Trudeau JB, Kaminsky DA, Colin J, Martin RJ, Wescott JY. Effect of 5-lipoxygenase inhibition on bronchoconstriction and airway inflammation in nocturnal asthma. Am J Respir Crit Care Med 1995; 152:897-905. 29. Pavord I, Tattersfield AE. Bronchoprotective role for endogenous prostaglandin E2. Lancet 1995;336:436-8. 30. Bisgaard H, Loland L, Oj JA. NO in exhaled air of asthmatic children is reduced by leukotriene receptor antagonist montelukast. Am J Respir Crit Care Med 1999;160:1227-31. 31. Straub DA, Minocchieri S, Moeller A, Hamacher J, Wildhaber JH. The effect of montelukast on exhaled nitric oxide and lung function in asthmatic children 2 to 5 years old. Chest 2005;127:509-14. 32. Strunk RC, Szefler SJ, Phillips BR, Zeiger RS, Chinchilli V, Larsen G, et al. Relationships of exhaled nitric oxide to clinical and inflammatory markers of persistent asthma in children. J Allergy Clin Immunol 2003;112:883-92. 33. Montuschi P, Barnes PJ. Analysis of exhaled breath condensate for monitoring airway inflammation. Trends Pharmacol Sci 2002;23:232-7. 34. Leff AR. Regulation of leukotrienes in the management of asthma: biology and clinical therapy. Annu Rev Med 2001;52:1-14. 35. Montuschi P. Analysis of exhaled breath condensate: methodological issues. In: Montuschi P, editor. New perspectives in monitoring lung inflammation: analysis of exhaled breath condensate. Boca Raton: CRC Press; 2005. p. 11-29.
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Background: Leukotriene (LT) E4 and 8-isoprostane concentrations are elevated in exhaled breath condensate in children with asthma. The effects of leukotriene receptor antagonists (LTRAs) on exhaled leukotriene and prostanoids in children with asthma are unknown. Objective: (1) To study the effect of montelukast, a LTRA, on exhaled LTE4, 8-isoprostane, and prostaglandin E2 in children with asthma and atopic children; (2) to measure exhaled nitric oxide. Methods: An open-label study with oral montelukast (5 mg once daily for 4 weeks) was undertaken in 17 atopic children with asthma and 16 atopic children without asthma. Results: Pretreatment exhaled LTE4 (P < .0001) and 8isoprostane (P < .0001) values were higher in atopic children with asthma than in atopic children without asthma. In atopic children with asthma, montelukast reduced exhaled LTE4 by 33% (P < .001), and this reduction was correlated with pretreatment LTE4 values (r 5 20.90; P 5 .0001). Posttreatment exhaled LTE4 levels in children with asthma were higher than pretreatment LTE4 values in atopic children without asthma (P < .004). Montelukast had no effect on exhaled LTE4 in atopic children without asthma (P 5 .74), or on exhaled 8-isoprostane (atopic children with asthma, P 5 .94; atopic children without asthma, P 5 .55) and PGE2 (atopic children with asthma, P 5 .56; atopic children without asthma, P 5 .93) in both groups. In atopic children with asthma, exhaled nitric oxide concentrations were reduced by 27% (P < .05) after montelukast. Conclusion: Leukotriene receptor antagonists decrease exhaled LTE4 in atopic children with asthma. This reduction is dependent on baseline exhaled LTE4 values.
From athe Department of Pharmacology, Faculty of Medicine, Catholic University of the Sacred Heart, Rome; bthe Department of Immunodermatology, Istituto Dermopatico dell’Immacolata, IRCCS, Rome; cthe Department of Pulmonology, Ospedale Pediatrico Bambino Gesu`, Palidoro; dthe Department of Thoracic Medicine, Imperial College, School of Medicine, National Heart and Lung Institute, London; and ethe Department of Drug Sciences, School of Pharmacy, University ‘‘G. d’Annunzio,’’ Chieti. Supported by the Catholic University of the Sacred Heart, Rome, Italy academic grant 2004-2005. Disclosure of potential conflict of interest: P. J. Barnes has received grant support from GlaxoSmithKline, AstraZeneca, Pfizer, Novartis, and Boehringer-Ingelheim, and is on the advisory board for GlaxoSmithKline, Pfizer, Boehringer-Ingelheim, and Altana. The rest of the authors have declared that they have no conflict of interest. Received for publication July 5, 2005; revised April 7, 2006; accepted for publication April 13, 2006. Available online July 5, 2006. Reprint requests: Paolo Montuschi, MD, Department of Pharmacology, Faculty of Medicine, Catholic University of the Sacred Heart, Largo F. Vito, 1, 00168 Rome, Italy. E-mail: [email protected]. 0091-6749/$32.00 Ó 2006 American Academy of Allergy, Asthma and Immunology doi:10.1016/j.jaci.2006.04.010
Clinical implications: Measurement of exhaled LTE4 might help identify children with asthma most likely to benefit from LTRAs. (J Allergy Clin Immunol 2006;118:347-53.) Key words: Leukotriene E4, prostanoids, exhaled breath condensate, exhaled nitric oxide, childhood asthma, airway inflammation, noninvasive markers, leukotriene receptor antagonists
Lipid mediators including leukotrienes and prostanoids play an important pathophysiological role in asthma.1 Measurement of leukotrienes and prostanoids in exhaled breath condensate (EBC) is an in vivo approach for studying the role of eicosanoids in asthma.2,3 EBC is a completely noninvasive method to sample secretions from the airways.4 Using immunoassays, cysteinyl-leukotrienes (CysLTs) and 8-isoprostane, a marker of oxidative stress,5 were detected in EBC in healthy children and found increased in children with stable asthma6-9 and asthma exacerbations.10 We have confirmed the presence of leukotriene (LT) E4, the most chemically stable CysLT, in EBC in healthy children using gas chromatography/mass spectrometry.11 Compared with those in healthy children, exhaled LTE4 concentrations are increased in steroid-naive children with asthma.11 Montelukast, a leukotriene receptor antagonist (LTRA),12 inhibits CysLTs production in allergen-stimulated PBMCs from adults with asthma.13 In an experimental model of allergic asthma, montelukast decreased the production of CysLTs in the lungs both directly and by inhibiting the recruitment of inflammatory cells.14 In addition to its antagonist effect on CysLT1 receptor, 1 study reported that montelukast has a selective direct inhibitory effect of 5-lipoxygenase activity in human mast cells in vitro at concentrations of potential therapeutic relevance.15 Oral montelukast at a dose of 10 mg once daily for 4 weeks reduces exhaled CysLTs concentrations in adults with asthma.16 However, other exhaled eicosanoids were not measured in this study, and the effects of LTRAs on exhaled LTs and prostanoids in children with asthma are unknown. Considering its mechanism of action, receptor blockade by montelukast can be clinically more important than decreasing LTE4 production. A multicenter study has shown that an increased likelihood of pulmonary response to montelukast is associated with higher baseline urinary LTE4 levels.17 Measurement of exhaled LTE4 might help identify children with asthma who need CysLT1 receptor blocking and are most likely to benefit from LTRAs. This might 347
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TABLE I. Subject characteristicsy Healthy children
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N Age, y Sex, F/M FEV1, L FEV1, % predicted FVC, L FVC, % predicted FEV1/FVC, % predicted FEF25%-75% Atopyà Steroids LTRAs
15 10 6 7/8 2.2 6 101.5 6 2.3 6 100.8 6 102.5 6 104.5 6 No No No
1 0.1 2.7 0.2 2.5 1.5 4.2
Atopic children without asthmaz
16 96 9/7 2.2 6 99.7 6 2.5 6 102.8 6 101.5 6 102.8 6 Yes No No
1 0.2 3.1 0.1 2.4 1.8 3.9
Atopic children with asthmaz
17 10 6 1 9/8 2.0 6 0.1 94.1 6 2.8 2.4 6 0.2 103.6 6 2.9 85.3 6 1.6* 73.7 6 4.6** Yes No No
F, Female; M, male. *P < .05 and **P < .01 compared with atopic children without asthma and healthy children. Data are expressed as numbers or means 6 SEMs. àPresence of atopy was confirmed by skin prick testing for common aeroallergens; a positive skin test response was defined as a wheal with a mean diameter (mean of maximum and 90° midpoint diameters) of at least 3 mm greater than that produced with a saline control. All atopic children had a clinical history of atopy. Children had no corticosteroid or LTRA treatment within 4 weeks.
Abbreviations used CysLTs: Cysteinyl-leukotrienes EBC: Exhaled breath condensate FEF25%-75%: Forced expiratory flow between 25% and 75% of the vital capacity FVC: Forced vital capacity LT: Leukotriene LTRAs: Leukotriene receptor antagonists NO: Nitric oxide PGE2: Prostaglandin E2
have relevant clinical implications given the importance of considering an individualized approach to asthma management and assessment rather than a strategy directed to the best outcome in a group of patients.18 In the current study, we sought (1) to investigate the effect of montelukast on exhaled LTE4 and prostanoids in atopic children with asthma and atopic children without asthma, (2) to measure exhaled nitric oxide (NO), a wellestablished marker of airway inflammation in patients with asthma who are steroid-naive,19 and (3) to compare concentrations of exhaled markers in children with asthma and atopic children without asthma enrolled in the montelukast study with those in healthy control children.
METHODS Study subjects Two groups of children were included in the montelukast study: 18 white atopic children with stable mild intermittent asthma and 18 atopic children without asthma. Seventeen atopic children with asthma and 16 atopic children without asthma completed the study (Table I; see the Methods section in the Online Repository at www. jacionline.org). Atopic children were recruited from the Allergy Outpatient Clinic of the Istituto Dermopatico dell’Immacolata, Rome, Italy, and the Asthma and Allergy Clinic of the Pediatric
Hospital Bambino Gesu`, Palidoro, Italy. The diagnosis and classification of asthma were based on clinical history and examination and pulmonary function parameters according to the guidelines issued by the National Heart, Lung, and Blood Institute of the National Institutes of Health.20 Atopic children had mild intermittent asthma with symptoms less often than twice a week, FEV1 of 80% or greater of predicted value and reversibility of 12% or greater to salbutamol, or a positive provocation test result with methacholine or exercise. They were not taking any regular medication, but used inhaled short-acting b2-agonists as needed for symptom relief. Sixteen atopic children without asthma were included in the montelukast study as control population without asthma (Table I; see the Methods section in the Online Repository at www. jacionline.org). Fifteen healthy nonatopic children without asthma who were recruited from children of staff were included in the cross-sectional study (Table I). They had no history of asthma and atopic disease, negative skin prick test results, and normal spirometry results. Study group children had no upper respiratory tract infections in the previous 3 weeks. Children were excluded from the study if they had used corticosteroids or LTRAs in the previous 4 weeks or nonsteroidal anti-inflammatory drugs in the last 2 weeks.
Study design Two studies were performed: an interventional study with montelukast and a cross-sectional study. A pilot uncontrolled open-label study with montelukast was undertaken in 17 atopic children with asthma and 16 atopic children without asthma. Children attended the Asthma Outpatient Clinic of the University Hospital A. Gemelli and the Department of Pharmacology of the Catholic University of the Sacred Heart, Rome, Italy, on 3 occasions for clinical examination, measurement of exhaled NO, collection of EBC, and lung function tests. After a screening visit (visit 1) and 1-week run-in period (visit 2), children were given oral montelukast (5 mg as tablet [Singulair; Merck & Co, Whitehouse Station, NJ]) once daily for 4 weeks. After treatment with montelukast, all of these tests were repeated (visit 3). Skin testing was performed on visit 1. In the cross-sectional study, concentrations of exhaled markers and lung function tests in atopic children with asthma and atopic children without asthma at baseline (visit 1) were compared with those in nonatopic children without asthma. Healthy children
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TABLE II. Absolute exhaled eicosanoid and NO values in the montelukast study group childreny
Atopic children with asthma (n 5 17) LTE4 8-Isoprostane PGE2 Exhaled NO (ppb) Atopic children without asthma (n 5 16) LTE4 8-Isoprostane PGE2 Exhaled NO (ppb)
Baseline (day 27)
Pretreatment (day 0)
Posttreatment (day 28)
44 31 28 45
(30-50) (26-35) (25-31) (40-61)
43 31 30 45
(31-48) (25-34) (23-32) (36-65)
29 33 28 32
(24-32)** (26-39) (25-34) (27-46)*
15 16 26 17
(13-18) (15-20) (23-29) (12-22)
17 16 26 16
(14-18) (15-19) (25-28) (12-21)
16 17 25 16
(13-19) (14-21) (23-29) (10-19)
*P < .05 and **P < .001 compared with pretreatment values. Values are medians and interquartile ranges (25th-75th percentiles). Values at baseline (day –7), before (day 0), and after treatment with oral montelukast (5 mg once daily for 4 weeks; day 28) are reported. Eicosanoid values are expressed as picograms produced during 15 minutes of breathing.
attended on 1 occasion for measurement of exhaled NO, collection of EBC, spirometry, and skin prick testing. Repeatability for eicosanoid measurement was assessed in all children with asthma and atopic children without asthma collecting EBC at screening visit and after 1-week run-in period. Informed consent was obtained from parents, and the study was approved by the Ethics Committee of the Catholic University of the Sacred Heart, Rome, Italy.
Pulmonary function FEV1, forced vital capacity (FVC), and forced expiratory flow between 25% and 75% of vital capacity (FEF25%-75%) were measured with a 10-L bell spirometer (Biomedin, Padova, Italy) and the best of 3 maneuvers, expressed as percentage of predicted values, chosen.
EBC Exhaled breath condensate was collected by using a condensing chamber (Echoscreen; Jaeger, Hoechberg, Germany), as described previously21 (see the Methods section in the Online Repository at www.jacionline.org). Children were instructed to breath tidally through a mouthpiece connected to the condenser for 15 minutes and not to inhale through their nose. An average of 1.5 mL EBC per child was collected and stored at 280°C before eicosanoid measurements. a-Amylase concentrations in all EBC samples were measured with a functional enzyme assay to exclude gross salivary contamination (Roche Diagnostics, Basel, Switzerland). However, activity assays for amylase are not sufficient to rule out salivary contamination definitively, because they have not been validated in EBC, and the duration that amylase activity persists in EBC when spiked at low concentration is currently unknown.
Measurement of exhaled LTE4 and prostanoids Leukotriene E4 in EBC was measured with a specific enzyme immunoassay kit (Cayman Chemical, Ann Arbor, Mich)7,21 (see the Methods section in the Online Repository at www.jacionline. org). The detection limit was 8 pg/mL. However, because of the reported variability at these concentrations, results below 16 pg/mL should be interpreted cautiously. The intra-assay and interassay coefficients of variation for LTE4 assay were 10% and 15%, respectively. 8-Isoprostane and prostaglandin E2 (PGE2) in EBC were measured with specific RIAs developed in our laboratory22-24 and validated by reverse-phase HPLC.23 The detection limit for 8-isoprostane and
PGE2 was 10 pg/mL. The intra-assay and interassay coefficients of variation for prostanoid RIAs were as follows: 8-isoprostane, <2% and <3%, respectively; PGE2, <4% and <5%, respectively. Because differences in the composition of enzyme immunoassay buffer (usually containing 1 mg/mL albumin) and EBC samples, which often have a lower protein concentration,25 can influence the analytical results, we compared LTE4 standard curves obtained with the kit immunoassay buffer (n 5 4) with those obtained with a more EBC-like buffer (phosphate buffer 0.025 mol/L, pH 7.5, not containing proteins; n 5 3; see this article’s Fig E1 in the Online Repository at www.jacionline.org). Because the 2 types of standard curves were different, immunoassays for LTE4 were performed by using a buffer not containing proteins. 8-Isoprostane and PGE2 standard curves were performed by using phosphate buffer 0.025 mol/L, pH 7.5, not containing proteins. Because dilution of respiratory droplets by water vapor may explain part of the variation in concentrations of nonvolatile compounds in EBC,25 LTE4 and 8-isoprostane results were presented as a change in ratio of LTE4 and 8-isoprostane to PGE2, another eicosanoid. Absolute LTE4 and prostanoid values in EBC, expressed as total amount (in picograms) of eicosanoids expired in the 15-minute breath test (eicosanoid concentrations 3 volume of EBC), are shown in Table II. For samples below the lowest detection limits, the data were expressed as half the detection limit.8,9
Exhaled NO measurement Exhaled NO was measured with the NIOX system (Aerocrine, Stockholm, Sweden) with a single breath online method at a constant flow of 50 mL/s according to American Thoracic Society guidelines19 (see the Methods section in the Online Repository at www.jacionline.org).
Skin testing Atopy was assessed by skin prick tests for common aeroallergens (Stallergenes, Antony, France)7 (see the Methods section in the Online Repository at www.jacionline.org).
Sample size The study sample was calculated26 considering exhaled LTE4 values as the primary outcome. Sample size was estimated to be 18 children after having considered a SD of 9 pg/15 min, a dropout of 15%, and identified the minimal difference of biological significance (10 pg/15 min) corresponding to a 33% reduction in median exhaled LTE4 values7 with a power of 90% (a value of 5% and b value of 10%).
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Exhaled eicosanoid (pg/15 min)
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Asthma diagnosis and treatment FIG 1. LTE4 in 17 atopic children with asthma (A) and 16 atopic children without asthma (B) at baseline (day 27), after 1-week run-in period (day 0), and after treatment with oral montelukast (5 mg once a day for 4 weeks; day 28). Values are expressed as LTE4/ PGE2 ratio.
Statistical analysis Exhaled eicosanoid and NO values were expressed as medians and interquartile ranges (25th and 75th percentiles). Kruskal-Wallis tests followed by Mann-Whitney U tests were used to compare groups. Eicosanoid values within the groups and comparison before and after treatment were analyzed by using Friedman repeated measures of ANOVA. Spirometry values were expressed as means 6 SEMs. Correlation was expressed as Spearman coefficient. Significance was defined as a value of P < .05.Within-subject repeatability of exhaled eicosanoid and exhaled NO measurements was expressed as limits of agreement (mean difference 6 2 SDs of the differences) for measurements taken at the baseline and after a 1-week run-in period.27 Limits of agreement estimate the largest likely size of difference between 2 measurements on the same subject27 (see the Methods section in the Online Repository at www.jacionline.org).
RESULTS No a-amylase concentrations were detected in any study sample, excluding gross salivary contamination.
Repeatability of measurements Limits of agreement in children with asthma (eicosanoids values expressed as picograms of eicosanoids expired in the 15-minute breath test) were as follows: LTE4, 5 pg and 26 pg; 8-isoprostane, 6 pg and 25 pg; PGE2, 8 pg and 28 pg; exhaled NO, 10 ppb and 210 ppb (see this article’s Figs E2E5 in the Online Repository at www.jacionline.org). For each biomarker, all of the differences between the 2 measurements were within its limits of agreement. Regarding exhaled LTE4, most of the children had variations (1-3 pg) smaller than their estimated limits of agreement (see
FIG 2. 8-Isoprostane (IP) (A) and PGE2 (B) in EBC in 17 atopic children with asthma at baseline (day 27), after 1-week run-in period (day 0), and after treatment with oral montelukast (5 mg once a day for 4 weeks; day 28). 8-Isoprostane values are expressed as 8-isoprostane/PGE2 ratio. PGE2 values are expressed as picograms produced during 15 minutes of breathing.
this article’s Fig E2 and Results section in the Online Repository at www.jacionline.org). Limits of agreement in atopic children without asthma are shown in this article’s Figs E6-E9 and values are provided in the Results section in the Online Repository at www.jacionline.org.
Exhaled LTE4 and prostanoids In the montelukast study, values of exhaled eicosanoids at baseline and after 1-week run-in period were similar in both groups of children (see this article’s Figs E2-E4 and E6-E8 and the Results section in the Online Repository at www.jacionline.org). Children with asthma had higher pretreatment exhaled LTE4/PGE2 (1.35 [1.15-1.51] vs 0.63 [0.53-0.72]; P < .0001) and 8-isoprostane/PGE2 (1.06 [0.93-1.14] vs 0.64 [0.59-0.71]; P < .0001) than atopic children without asthma. Pretreatment exhaled PGE2 values were similar in the 2 groups of children (P 5 .44). In children with asthma, median exhaled LTE4/ PGE2 was reduced by 33% after montelukast (pretreatment: 1.35 [1.15-1.51]; posttreatment: 0.90 [0.80-1.25]; P < .001; Fig 1, A), whereas exhaled 8-isoprostane/ PGE2 (pretreatment: 1.06 [0.93-1.14]; posttreatment: 0.96 [0.85-1.20]; P 5 .94; Fig 2, A) and PGE2 values (pretreatment: 30[23-32] pg; posttreatment: 28[25-34] pg; P 5 .56; Fig 2, B) were unaffected. The reduction in exhaled LTE4 after treatment was correlated with pretreatment LTE4 values (r 5 20.90; P < .0001; Fig 3). Posttreatment exhaled LTE4/PGE2 in children with asthma was higher than pretreatment exhaled LTE4/PGE2 in atopic children without asthma (0.90 [0.80-1.25] vs 0.63 [0.53-0.72]; P < .004). Montelukast had no effect on exhaled LTE4 (P 5 .74), 8-isoprostane (P 5 .55), and
FIG 3. Correlation between changes in LTE4 values in EBC after treatment with oral montelukast (5 mg once daily for 4 weeks) in 17 atopic children with asthma and pretreatment exhaled LTE4 levels (r 5 –0.90, P < .0001). Values are expressed as picograms produced during 15 minutes of breathing.
PGE2 (P 5 .93) in atopic children without asthma (Fig 1, B; see this article’s Fig E10 in the Online Repository at www.jacionline.org). In the cross-sectional study, atopic children with asthma had higher exhaled LTE4 (P < .001) and 8-isoprostane (P < .01) than atopic children without asthma, and there was no difference in exhaled LTE4 (P 5 .30) and 8-isoprostane values (P 5 .20) between atopic children without asthma and healthy children (see this article’s Fig E11, Fig E12, A, and Table E1 in the Online Repository at www.jacionline.org). Exhaled PGE2 values in the 3 groups of children were similar (P 5 .18; see this article’s Fig E12, B, and Table E1 in the Online Repository at www.jacionline.org). There was no correlation between exhaled eicosanoids or exhaled NO and age, sex, and lung function. There was no correlation between the different exhaled markers.
Exhaled NO In atopic children with asthma, exhaled NO was reduced by 27% (pretreatment: 45 [36-65] ppb; posttreatment: 32 [27-46] ppb; P < .05) after montelukast (Fig 4). Posttreatment reduction in exhaled NO was correlated with pretreatment exhaled NO concentrations (r 5 20.84; P < .0001; see this article’s Fig E13 in the Online Repository at www.jacionline.org). Montelukast had no effect on exhaled NO in atopic children without asthma (P 5 .65; see this article’s Fig E14 in the Online Repository at www.jacionline.org). Compared with healthy children, exhaled NO was increased in atopic children without asthma (P < .05) and, to a greater extent, in children with asthma (P < .001; see this article’s Fig E15 and Table E1 in the Online Repository at www.jacionline.org). Pulmonary function Median spirometry values before and after montelukast were similar in atopic children with and without asthma (see this article’s Table E2 in the Online Repository at www.jacionline.org). One child with asthma had a significant response to montelukast defined as improvement
FIG 4. Exhaled NO concentrations in 17 atopic children with asthma at baseline (day 27), after 1-week run-in period (day 0), and after treatment with oral montelukast (5 mg once a day for 4 weeks; day 28).
in FEV1 of 7.5% or greater with a concomitant reduction in exhaled LTE4 of 25 pg. There was no association between the effect of montelukast on exhaled LTE4 values and the effect of montelukast on lung function in the children with asthma.
DISCUSSION We sought to investigate the effect of montelukast on exhaled LTE4, 8-isoprostane, and PGE2 in atopic children with and without asthma. In the current study, median exhaled LTE4 values were decreased by 33% after montelukast in children with asthma, whereas treatment had no effect on exhaled LTE4 in atopic children without asthma. Montelukast had no effect on exhaled 8-isoprostane and PGE2 in both groups of children. In children with asthma, there was a strong correlation between reduction in exhaled LTE4 after treatment and pretreatment LTE4 levels, indicating that children with higher baseline LTE4 levels might be the most likely to have a significant reduction in exhaled LTE4 from LTRAs. Reduction in leukotriene production might be functionally relevant, because in adults with nocturnal asthma, 5-lipoxygenase inhibition decreases urinary LTE4 levels with a concomitant trend for improving nocturnal FEV1.28 However, studies aimed to measure urinary LTE4 for confirmation of the reduced LTE4 production and to establish the relationships between urinary and exhaled LTE4 are warranted. In our study, a pulmonary response to montelukast (defined as improvement in FEV1 of 7.5% or greater) was observed in 1 out of 17 children with asthma. Future studies should be performed to clarify whether baseline exhaled LTE4 values are associated with a change in more sensitive spirometry parameters, particularly those reflecting peripheral airway function such as FEF25%-75% values, or in asthma symptoms or course. Exhaled 8-isoprostane, a specific marker of lipid peroxidation, was elevated in children with asthma, indicating that asthma is associated with increased oxidative stress.7,8 Reduced production of PGE2, an endogenous bronchodilating compound that may have anti-inflammatory effects in the airways, has been implicated in the pathophysiology
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of asthma.29 Similar exhaled PGE2 concentrations in children with asthma and control children observed in our study do not seem to support this hypothesis. There was no effect of montelukast on exhaled 8-isoprostane and PGE2 in children with asthma, indicating that the antiinflammatory effects of this drug within the airways are unlikely to be explained by inhibition of lipid peroxidation or COX interactions. In atopic children with asthma, exhaled NO was decreased after montelukast consistent with previous studies.30,31 Exhaled LTE4 and exhaled NO values were not correlated in line with the lack of correlation between urinary LTE4 and exhaled NO32 (see the Discussion section in the Online Repository at www.jacionline.org). These findings suggest that exhaled LTE4 and exhaled NO have a different biological significance as markers of airway inflammation in children with asthma and cannot be used interchangeably when assessing the antiinflammatory response to antiasthmatic drugs. Our study with montelukast was open-label, was uncontrolled, and involved a limited number of children with asthma. This limits the strength of our trial and precludes definitive conclusions. Larger controlled studies are required to definitively establish the effect of LTRAs on exhaled leukotrienes and prostanoids in children with asthma, to ascertain whether there are subgroups of children with asthma with a different response to LTRAs in terms of reduction in exhaled eicosanoids, and to clarify the effect of these findings on management of children with asthma. Studies on the relationship between exhaled LTE4 levels and polymorphisms in the 5-lipoxygenase promoter could contribute to explain the wide interindividual variability in the pretreatment exhaled LTE4 values observed in our study. Finally, a drug withdrawal study would likely reveal the predicted value of LTE4 in asthma subpopulations. We were unable to ascertain the mechanisms by which LTRAs decrease exhaled LTE4 in children with asthma. EBC analysis cannot provide information on the cellular origin of mediators for which invasive techniques such as bronchoscopy and bronchial biopsies are required.33 Several types of airway cells can synthesize CysLTs, including mast cells and eosinophils.34 Montelukast inhibits CysLTs production in allergen-stimulated PBMCs from adults with asthma.13 In an experimental animal model of allergic asthma, montelukast decreased the production of CysLTs in the lungs both directly and by inhibiting the infiltration of inflammatory cells into the bronchial submucosa.14 However, whether montelukast has similar effects in children with asthma is currently unknown, as well as its cellular targets. In the cross-sectional study, baseline exhaled LTE4 and 8-isoprostane were elevated in atopic children with asthma, but not in atopic children without asthma, in contrast with the 1 airway hypothesis, which suggests that nasal inflammation reflects lower airway inflammation. By contrast, exhaled NO was increased in atopic children without asthma and, to a greater extent, in atopic children with asthma. These findings indicate that LTE4 and 8-
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isoprostane in EBC might be considered as markers of asthma rather than reflecting the different degree of inflammation within the respiratory tract in atopic children with and without asthma.7 Currently, because of the lack of a standardized procedure and validated analytical methods, EBC analysis is more reliable for relative measures than for determining absolute levels of inflammatory mediators. Collection and/or storage of EBC samples are possible sources of variability in exhaled eicosanoid measurements.35 However, part of this variability could be related to analytical methods, particularly at LT concentrations close to the detection limit of the immunoassay. These data should be interpreted cautiously. However, in our study, day-today repeatability for exhaled eicosanoid measurements was acceptable, as indicated by their limits of agreement, consistent with previous findings.7,8,10 Matrix effects as well as sample pretreatment may play a role, because LTE4 standard curves performed using the kit immunoassay buffer and those performed with a more EBC-like buffer such as phosphate buffer were different (see the Discussion section in the Online Repository at www. jacionline.org). However, further investigation to address this issue formally is required. Another limitation of the current study is that a reference dilution indicator was not measured.25 However, the fact that LTE4 results expressed as both absolute values and LTE4/PGE2 ratio were similar indicates that dilution of respiratory droplets has little, if any, effect on LTE4 levels in EBC. In conclusion, exhaled LTE4 and exhaled NO values, but not exhaled 8-isoprostane and PGE2, are reduced by LTRAs in atopic children with asthma. Reduction in exhaled LTE4 after LTRAs is dependent on baseline pretreatment exhaled LTE4 values. Identification of children with asthma who are most likely to benefit from LTRAs based on exhaled LTE4 levels might lead to a more rational and targeted use of these drugs. REFERENCES 1. Bochner BS, Busse WW. Allergy and asthma. J Allergy Clin Immunol 2005;115:953-9. 2. Montuschi P. Indirect monitoring of lung inflammation. Nat Rev Drug Discov 2002;1:238-42. 3. Hunt JF. Exhaled breath condensate: an evolving tool for noninvasive evaluation of lung disease. J Allergy Clin Immunol 2002;110:28-34. 4. Kharitonov SA, Barnes PJ. Exhaled markers of pulmonary diseaae. Am J Respir Crit Care Med 2001;163:1693-722. 5. Montuschi P, Barnes PJ, Roberts LJ II. Isoprostanes: markers and mediators of oxidative stress. FASEB J 2004;18:1791-800. 6. Csoma Z, Kharitonov SA, Balint B, Bush A, Wilson NM, Barnes PJ. Increased leukotrienes in exhaled breath condensate in childhood asthma. Am J Respir Crit Care Med 2002;166:1345-9. 7. Mondino C, Ciabattoni G, Koch P, Pistelli R, Trove´ A, Barnes PJ, et al. Effects of inhaled corticosteroids on exhaled leukotrienes and prostanoids in asthmatic children. J Allergy Clin Immunol 2004;114:761-7. 8. Zanconato S, Carraro S, Corradi M, Alinovi R, Pasquale MF, Piacentini G, et al. Leukotrienes and 8-isoprostane in exhaled breath condensate of children with stable and unstable asthma. J Allergy Clin Immunol 2004; 113:257-63. 9. Carraro S, Corradi M, Zanconato S, Alinovi R, Pasquale MF, Zacchello F, et al. Exhaled breath condensate cysteinyl-leukotrienes are increased
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J ALLERGY CLIN IMMUNOL VOLUME 118, NUMBER 2