Hermans, PWM

Hop, Wim C.J.

Jobsis, Q (Quirijn)

Jongste, Johan

Raatgeep, Rolien

Schellekens, SL

Undetermined

EUR Research Repository

EUR Research Information PortalHydrogen peroxide in exhaled air of healthy children: reference valuesPublished in:European Respiratory JournalPublication status and date:Published: 01/01/1998Document VersionPublisher's PDF, also known as Version of recordCitation for the published version (APA):Jobsis, Q., Raatgeep, R., Schellekens, SL., Hop, Hermans, PWM., & Jongste, J. (1998). Hydrogen peroxide in exhaled air ofhealthy children: reference values. European Respiratory Journal, 12, 483-485. http://hdl.handle.net/1765/8896Link to publication on the EUR Research Information PortalTerms and Conditions of UseExcept as permitted by the applicable copyright law, you may not reproduce or make this material available to any third partywithout the prior written permission from the copyright holder(s). 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In case of a legitimate complaint,we will make the material inaccessible and/or remove it from the website.Eur Respir J 1998; 12: 483–485DOI: 10.1183/09031936.98.12020483Printed in UK - all rights reservedCopyright ©ERS Journals Ltd 1998European Respiratory JournalISSN 0903 - 1936SHORT REPORTHydrogen peroxide in exhaled air of healthy children:reference valuesQ. Jöbsis*, H.C. Raatgeep*, S.L. Schellekens*, W.C.J. Hop**,P.W.M. Hermans*, J.C. de Jongste*aaA noninvasive method to assess the presence and activ-ity of airway inflammation would be valuable in the earlydiagnosis and monitoring of inflammatory airway dis-eases [1]. Exhaled air condensate can be collected withminimal risk and inconvenience and its content may reflectthe com-position of the lower airway fluids [2, 3]. Hydro-gen per-oxide (H2O2) in the exhaled air condensate is apotential marker of airway inflammation [4–10]. The onlytwo studies to suggest elevated levels of exhaled H2O2 inasthmatic children have used small numbers of healthyadults as controls [4, 10]. The aim of this study was, there-fore, to establish reference values of [H2O2] in the exhaledair con-densate of a large group of healthy school-agedchildren.Patients and methodsOne hundred and twenty-nine school children, pupils ofa primary school, were interviewed with questionnaires onasthma, rhinitis and eczema, translated and validated fromthe core questionnaires of the International Study of As-thma and Allergy in Childhood (ISAAC). Of these chil-dren, 93 had negative questionnaires and were included inthe study. Their mean age was 10 yrs (range 8–13 yrs) and45 were male (table 1). All were term born, Caucasian life-long nonsmokers, within the normal range for height, andused no medication. None of the subjects reported symp-toms of acute respiratory infection within the previousmonth. Maximal expiratory flow-volume measurementswere performed in all children (Vicatest P2A, Mijnhardt,The Netherlands); forced vital capacity (FVC) and forcedexpiratory volume in one second (FEV1) were expressed aspercentage predicted [11]. Condensate was collected whilethe children, wearing a noseclip, breathed quietly througha mouthpiece and a two-way nonrebreathing valve (Rudolph,Hydrogen peroxide in exhaled air of healthy children: reference values. Q. Jöbsis, H.C.Raatgeep, S.L. Schellekens, W.C.J. Hop, P.W.M. Hermans, J.C. de Jongste. ERS JournalsLtd 1998.ABSTRACT: An increased content of hydrogen peroxide (H2O2), a marker of inflam-mation, has been described in the condensate of exhaled air from adults and childrenwith inflammatory lung disorders, including asthma. However, the normal range of[H2O2] in the exhaled air condensate from healthy children has not been established.Therefore, the aim of this study was to determine the reference range of exhaled[H2O2] in healthy school-aged children.Ninety-three healthy nonsmoking children (48 female and 45 male, mean age 10yrs, range 8–13 yrs), with a negative history for allergy, eczema or respiratory diseaseand with a normal lung function, participated. Exhaled air condensate was examinedfluorimetrically for the presence of H2O2. In addition, the reproducibility of [H2O2]within subjects and between days and the stability of [H2O2] during storage at -20°Cwere assessed.The median [H2O2] in the exhaled air condensate of all children was 0.13 µM, witha 2.5–97.5% reference range of <0.01–0.48 µM. No significant difference existed bet-ween males and females. There was no correlation between exhaled [H2O2] and age orlung function. Repeated [H2O2] measurements on 2 consecutive days showed satisfac-tory within-subject reproducibility and [H2O2] in stored samples remained stable forat least 1 month at -20°C.In conclusion, this study provides reference data for exhaled hydrogen peroxide ina large group of healthy children. The observed levels were lower than those reportedpreviously for healthy adults and were independent of age, sex and lung function.Eur Respir J 1998; 12: 483–485.*Dept of Paediatrics, Division of Paediat-ric Respiratory Medicine, and **Dept ofBiostatistics, Erasmus University and Uni-versity Hospital/Sophia Children's Hospi-tal,  Rotterdam, The Netherlands.Correspondence: J.C. de JongsteDept of Paediatric Respiratory MedicineSophia Children's HospitalRoom Sp-2465Dr Molewaterplein 603015 GJ RotterdamThe NetherlandsFax: 31 10 4636801Keywords: Childrenexhaled airhydrogen peroxideinflammatory markerreference valuesReceived: October 30 1997Accepted after revision April 21 1998Supported by grant 94.14 from the NetherlandsAsthma Fund.Table 1.  –  Characteristics of the study populationHealthy children (n=93)*Age  yrs†Sex  male/femaleFVC  % pred‡FEV1  % pred‡10 (8–13)45/4898±12100±12*: all were lifelong nonsmokers, had no symptoms of asthma,eczema or rhinitis, used no medication, and had no symptoms ofrespiratory infection in the 4 weeks before the study. †: mean(range); ‡: mean±SD.484 Q. JÖBSIS ET AL.Kansas City, KS, USA), which also served as a saliva trap.At least 1.5 mL of condensate was obtained by passingexpired air through a 50 cm double jacketed glass tubecooled to 0°C and collected on ice. The concentration ofH2O2 in the condensate was determined in duplicate with afluorimetric assay based on the reaction of H2O2 withhorseradish peroxidase to form a compound which oxi-dizes p-hydroxyphenylacetic acid to a fluorescent product,as described previously [4, 12]. As saliva is a source ofH2O2, the  equipment was designed to avoid such contami-nation, as verified previously by measuring amylase insaliva and breath condensates. Amylase was present insaliva in high concentrations (20,000–300,000 U·L-1), andcould never be demonstrated in condensate (invariably <10U·L-1, unpublished data); therefore, it could be stated withconfidence that no contamination with saliva had takenplace. To examine the reproducibility of repeated H2O2 mea-surements within subjects, condensate was collected from10 healthy subjects on 2 consecutive days in the morning.To assess the stability of [H2O2] in the frozen condensate,4 mL of condensate was collected from 5 subjects. Thesewere divided into 1 mL aliquots, in which H2O2 concentra-tions were determined immediately after collection, after48 h, 1 week and 1 month of storage at -20°C.Analysis of dataResults of H2O2 measurements are expressed as medianand range because of an asymmetrical distribution and be-cause some values were below the dection limit. Leastsquares regression analysis was used to determine possi-ble effects of age or lung function on exhaled H2O2. Dif-ferences between males and females were evaluated withthe Mann-Whitney test. Reproducibility was assessed bycalculating the mean within-subject difference of two H2O2measurements obtained on separate days from 10 subjectsand the SD. Stability of H2O2 in frozen samples was esti-mated by comparing mean concentrations using a two-sided paired t-test, and by calculating mean differencesand SD of the differences of immediately determined valuesand values after a given period of storage. Significancewas assumed at p<0.05.ResultsAll subjects had a normal lung function with a meanFVC of 98±12% and an FEV1 of 100±12%. The H2O2concentration in the exhaled air condensate ranged frombelow the detection limit of 0.01 µM to 0.50 µM, with amedian value of 0.13 µM. The 2.5 and 97.5 percentileswere <0.01 and 0.48 µM, respectively. Median [H2O2] frommales and females were similar (0.13 µM and 0.14 µM,respectively, p=0.98). Individual data of all children areshown in figure 1. There was no effect of age on [H2O2] (r=-0.04, p=0.68). Likewise, no correlation was found betweenabsolute values of FEV1 and [H2O2] in the condensate(r=0.07, p=0.48). Repeated measurements on 2 consecu-tive days showed a mean within-subject difference of 0.02µM (SD 0.04 µM). The stability of [H2O2] in frozen conden-sate was satisfactory, with no significant difference bet-ween the [H2O2] immediately after collection (0.13±0.03µM) and after storage at -20°C for 48 h (0.11± 0.02 µM,p=0.14), 1 week (0.12±0.05 µM, p=0.55) and 1 month(0.13±0.02 µM, p=0.59).DiscussionIn this study the reference range of H2O2 in exhaled aircondensate obtained from a large group of healthy school-aged children has been defined. The methodology was non-invasive and well-tolerated and gave reproducible results.Samples could be stored for at least 1 month and analysedlater without changes in [H2O2].Until now, published data on normal values of H2O2concentration in exhaled air were obtained from smallnumbers of adults [4–6, 8–10]. Peroxide concentrationswere observed previously in healthy adults that were high-er than the upper limit of the reference range in the presentstudy [4]. This suggests either that healthy children pro-duce less peroxide than adults or that subclinical airwayinflammation may be more common in asymptomatic ad-ults than in healthy children. On the basis of these resultsit can be stated that earlier observations in asthmatic chil-dren have indeed shown H2O2 concentrations in exhaledair that were substantially higher than those found in healthychildren [4, 10].In conclusion, this study defined the reference range ofhydrogen peroxide, a putative marker of airway inflam-mation, in exhaled air condensates from a large group ofhealthy school-aged children. Longitudinal studies are need-ed to examine the potential value of this inflammatory markerin the management of childhood asthma.Acknowledgement: The authors are indebted to thechildren and teachers of De Wilgenstam school in Rotter-dam who participated in this study.References1. O'Byrne PM, Hargreave FE. Non-invasive monitoring ofairway inflammation. Am J Respir Crit Care Med 1994;150: s100–s102.[H2O2]  µM0.500.400.300.200.10     08 9 10 11 12 13Age  yrs●●●●●●●●●●●●●●●●● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●● ●●●●●●● ●●●●●●●●●●●●●●●●●●●●▲▲▲▲▲Fig. 1.  –  Concentration of hydrogen peroxide (H2O2) in the exhaled aircondensate of 93 healthy children. The results were independent ofage.        : represents the 97.5 percentile upper reference limit (0.48µM); and             : represents the median (0.13 µM). ▲ : values below thedetection limit (0.01 µM). Each symbol represents one subject. HYDROGEN PEROXIDE IN EXHALED AIR 4852. Scheideler L, Manke HG, Schwulera U, Inacker O, Häm-merle H. Detection of nonvolatile macromolecules inbreath; a possible diagnostic tool? Am Rev Respir Dis1993; 148: 778–784.3. Becher G, Beck E, Winsel K. Leukotriene C4, D4, E4, F4 inthe breathing condensate of asthmatics in relation tobronchial challenge test. Am J Respir Crit Care Med1995; 151: A679.4. Jöbsis Q, Raatgeep HC, Hermans PWM, de Jongste JC.Hydrogen peroxide in exhaled air is increased in stableasthmatic children. Eur Respir J 1997; 10: 519–521.5. Nowak D, Antczak A, Krol M, et al. Increased content ofhydrogen peroxide in the expired breath of cigarette smok-ers. Eur Respir J 1996; 9: 652–657.6. Dekhuijzen PNR, Aben KKH, Dekker I, et al. Increasedexhalation of hydrogen peroxide in patients with stableand un stable chronic obstructive pulmonary disease. AmJ Respir Crit Care Med 1996; 154: 813–816.7. Baldwin SR, Grum CM, Boxer LA, Simon RH, Ketai LH,Devall LJ. Oxidant activity in expired breath of patientswith adult respiratory distress syndrome. Lancet 1986; 1:11–14.8. Kietzmann D, Kahl R, Müller M, Burchardi H, Kettler D.Hydrogen peroxide in expired breath condensate of pat-ients with acute respiratory failure and with ARDS. IntenCare Med 1993; 19: 78–81.9. Sznajder JI, Fraiman A, Hall JB, et al. Increased hy-drogen peroxide in the expired breath of patients withacute hypoxemic respiratory failure. Chest 1989; 96: 606–612.10. Dohlman AW, Black HR, Royall JA. Expired breath hy-drogen peroxide is a marker of acute airway inflamma-tion in pediatric patients with asthma. Am Rev Respir Dis1993; 148: 955–960.11. Zapletal A, Samanek M, Paul T. Lung function in chil-dren and adolescents. Methods, reference values. Basel,Karger, 1987.12. Hyslop PA, Sklar LA. A quantitative fluorimetric assayfor the determination of oxidant production by poly-morphonuclear leukocytes: its use in the simultaneousfluorimetric assay of cellular activation processes. 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Hydrogen peroxide in exhaled air of healthy children: reference values

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Hydrogen peroxide in exhaled air of healthy children: reference values

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