Basek, Pavel

Inci, Demet

Moeller, Alexander

Wildhaber, Johannes H.

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Clin Chem Lab Med 2012;50(3):593–594 © 2012 by Walter de Gruyter • Berlin • Boston. DOI 10.1515/CCLM.2011.801 Letter to the Editor  Leptin levels in exhaled breath condensate from asthmatic children: a pilot study  Demet  Inci 1, *,  Pavel  Basek 1 ,  Johannes H.  Wildhaber 2 and  Alexander  Moeller 1  1  Division of Respiratory Medicine ,  University Children ’ s Hospital, Z ü rich ,  Switzerland  2  Department of Paediatrics ,  Cantonal Hospital Fribourg ,  Switzerland  A cross-sectional analysis of the Third National Health and Nutrition Examination Survey (NHANES III) found that overweight children aged between 6 and 16 years had three times more asthma risk than their healthy weight peers  (1) . Another cross-sectional study supported the relationship between asthma and being overweight in childhood and showed that the prevalence of asthma was greater in over-weight children  (2) .  Leptin, a 167-amino-acid peptide, is a recently discov-ered hormone which is believed to play a major role in the regulation of body weight. Recently, it has been reported that serum leptin levels are increased in asthmatic children  (3) . Leptin has also been reported to have a specifi c effect on T-lymphocyte responses. It has been hypothesized that lep-tin is involved in the regulation of asthmatic airway infl am-mation levels by regulating T-cell responses by polarizing Th cells toward a Th1 phenotype and by inhibiting immune responses, such as Th2 activation and may have a role in asthma  (4) . Mai et al.  (5) found twice as high serum lev-els of leptin in overweight asthmatic children compared to overweight children without current asthma. Serum leptin levels are associated with asthma in adult women, indepen-dent of BMI  (6) . In children with asthma close associations have been shown between serum adiponectin and leptin and pulmonary function, but it has been suggested that they may play a regulatory role rather than being an etiologic mecha-nism of asthma development  (7) .  As very little is known about the role of leptin within the airways and lung tissue and asthma, non-invasive tools to investigate, such associations would allow epidemiological and longitudinal studies to be performed on a larger scale. Such studies are needed to confi rm the relationships of leptin and asthma and improve our understanding of associations between asthma and being overweight.  Exhaled breath condensates (EBC) contain aerosolized air-way lining fl uid and volatile compounds from the airways and the lung periphery. The assessment of EBC compounds is rec-ognized to be a potentially useful research tool in children for the non-invasive quantifi cation of airway infl ammation and metabolic processes in asthma and other airway diseases.  The aim of this pilot study was to evaluate whether leptin can be found in EBC and to investigate differences between condensate leptin levels in overweight and healthy weight children.  A total of 20 children (fi ve overweight asthmatics, fi ve healthy weight asthmatics, fi ve overweight healthy and fi ve healthy weight healthy controls), aged 6 – 15 years, were included in this study. We obtained data on weight and length (body mass index), lung function, fractional exhaled nitric oxide (FE NO ) and allergic sensitization by the means of skin prick testing using a panel of common allergens including house dust mite, mold mix, tree mix, grass mix, weed mix, cat and dog hair. Informed consent was taken from the parents.  Exhaled breath condensates were collected using the commercial EcoScreen breath condenser system (Jaeger, Wuerzburg, Germany). Approximately 1.5 mL of condensate was collected in 15 min and immediately stored at  – 70 ° C for later analysis. We standardized possible variables during and after EBC collection, according to the methodological recom-mendations of the ATS/ERS task force  (8) .  Condensate leptin levels were measured according to the instruction by the manufacturer using a commercially available ChemoKine TM Human leptin EIA kit (Chemicon International Inc., CA, USA) with a lower detection limit of 0.488 ng/mL. Leptin levels were measured in un-concentrated, native EBC and after concentrating the breath condensate 3-fold by freeze drying.  The patient characteristics, results of spirometry, FE NO, skin prick test and EBC leptin levels are summarized in Table  1 .  Leptin levels were below the detection limit of the assay (0.488 ng/mL) in the native, un-concentrated EBC and in 3-fold concentrated samples and were therefore considered as non-detectable in any of the EBC samples.  We were not able to detect leptin in the EBC from asth-matic, healthy overweight and healthy weight children, even after 3-fold concentration of the EBC samples. We strictly followed the methodological recommendations of the ATS/ERS task force  (8) and the instructions of the manufactur-ers and standardized possible variables during and after EBC  *Corresponding author: Demet Inci, MD, University Children ’ s Hospital Steinwiesstrasse 75 8032 Zurich, Switzerland Phone:  + 41 44 266 7111, Fax:  + 41 44 266 7670 , E-mail:  demet.inci@kispi.uzh.ch Received September 21, 2011; accepted November 7, 2011; previously published online November 23, 2011 594  Inci et al.: Leptin in exhaled breath condensatecollection. We therefore are confi dent that the non-detectable leptin levels in EBC were not due to a collection or measure-ment error. The assay we used is established for the mea-surement of leptin in biological fl uids and is not restricted to serum.  There is still some controversy about the extent of proteins in EBC, but it is accepted that they are generally very low and often close to the detection limit of the assay used. More sensitive and/or specifi c assays, such as radio immunoassay or mass spectrometry may allow the measurement of leptin within EBC, but levels are expected to be extremely low.  A wide range of constituents in EBC have previously been measured and the analysis of EBC components has the poten-tial to provide a non-invasive means of monitoring airway infl ammation and cellular metabolism of the lung and air-ways. However, a range of important methodological issues need to be addressed for any EBC compound. These include repeatability, dilution, environmental contamination, effects of breathing patterns coating of the collector system and assay sensitivity  (9, 10) .  No leptin was detected in the EBC in native and 3-fold concentrated EBC. Exhaled breath condensates are not a use-ful tool to assess leptin metabolism in overweight and healthy weight asthmatic children.  Acknowledgments  The authors would like to thank Susanne Loeliger for her technical assistance with leptin assays.  Confl ict of interest statement  Authors ’ confl ict of interest disclosure: The authors stated that there are no confl icts of interest regarding the publication of this article.  Research funding: None declared.  Employment or leadership: None declared.  Honorarium: None declared.  References  1.  Romieu I, Mannino DM, Redd SC, McGeehin MA. Dietary intake, physical activity, body mass index, and childhood asthma in the Third National Health and Nutrition Survey (NHANES III). Pediatr Pulmonol 2004;38:31 – 42.  2.  Stingone JA, Ramirez OF, Svensson K, Claudio L. Prevalence, demographics, and health outcomes of comorbid asthma and overweight in urban children. J Asthma 2011;48:876 – 85.  3.  Guler N, Kirerleri E, Ones U, Tamay Z, Salmayenli N, Darendeliler F. Leptin: does it have any role in childhood asthma ? J Allergy Clin Immunol 2004;114:254 – 9.  4.  Gurkan F, Atamer Y, Ece A, Kocyigit Y. Serum leptin levels in asthmatic children treated with an oral corticosteroid. Ann Allergy Asthma Immunol 2004;93:277 – 80.  5.  Mai XM, B ö ttcher MF, Leijon I. Leptin and asthma in over-weight children at 12 years of age. Pediatr Allergy Immunol 2004;15:523 – 30.  6.  Sood A, Ford ES, Camargo CA Jr. Association between leptin and asthma in adults. Thorax 2006;61:300 – 5.  7.  Kim KW, Shin YH, Lee KE, Kim ES, Sohn MH, Kim KE. Relationship between adipokines and manifestations of child-hood asthma. Pediatr Allergy Immunol 2008;19:535 – 40.  8.  Horv á th I, Hunt J, Barnes PJ, Alving K, Antczak A, Baraldi E, et al. ATS/ERS Task Force on Exhaled Breath Condensate. Exhaled breath condensate: methodological recommendations and unresolved questions. Eur Respir J 2005;26:523 – 48.  9.  Rosias PP, Dompeling E, Hendriks HJ, Heijnens JW, Donckerwolcke RA, J ö bsis Q. Exhaled breath condensate in children: pearls and pitfalls. Pediatr Allergy Immunol 2004;15:4 – 19.  10.  Hunt J. Exhaled breath condensate: an evolving tool for non-invasive evaluation of lung disease. J Allergy Clin Immunol 2002;110:28 – 34.  Table 1  Patient characteristics and results of assessments.  Asthmatics HealthyHealthy weight Overweight Healthy weight Overweightn (male/female) 4/1 3/2 4/1 3/2Age 9.6 ± 3.3 13.2 ± 3.03 12.4 ± 0.8 11.4 ± 2.8Sensitization  a  5.8 ± 4.0 3.4 ± 3.3 NA NABMI, kg/m 2 18.7 ± 3.1 28.2 ± 4.3 17.5 ± 1.9 29.3 ± 6.9FEV 1 ,  % pred 92.4 ± 15.75 91.6 ± 10.35 90.8 ± 5.4 90.0 ± 13.0FE NO , ppb 6.3 ± 2.0 12.48 ± 9.39 NA NAEBC leptin, ng/mL  < 0.488  < 0.488  < 0.488  < 0.488 All data in mean ± standard deviation. Overweight: BMI-for-age  ≥ 95th percentile. NA, not available.  a Number of allergens. ppb, parts per billion. 

Leptin levels in exhaled breath condensate from asthmatic children: a pilot study

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