Exhaled Breath Condensate: Technical and Diagnostic Aspects · 2016. 6. 13. · ReviewArticle Exhaled Breath Condensate: Technical and Diagnostic Aspects EfstathiaM.Konstantinidi,1,2

Review ArticleExhaled Breath Condensate Technical and Diagnostic Aspects

Efstathia M Konstantinidi12 Andreas S Lappas12

Anna S Tzortzi1 and Panagiotis K Behrakis12

1Smoking and Lung Cancer Research Centre Hellenic Cancer Society 8 Doryleou Street 11521 Athens Greece2Biomedical Research Foundation Academy of Athens 4 Soranou Ephessiou Street 11527 Athens Greece

Correspondence should be addressed to Efstathia M Konstantinidi feikon04gmailcom

Received 20 January 2015 Accepted 21 April 2015

Academic Editor Bruno Balbi

Copyright copy 2015 Efstathia M Konstantinidi et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Purpose The aim of this study was to evaluate the 30-year progress of research on exhaled breath condensate in a disease-basedapproach Methods We searched PubMedMedline ScienceDirect and Google Scholar using the following keywords exhaledbreath condensate (EBC) biomarkers pH asthma gastroesophageal reflux (GERD) smoking COPD lung cancer NSCLCmechanical ventilation cystic fibrosis pulmonary arterial hypertension (PAH) idiopathic pulmonary fibrosis interstitial lungdiseases obstructive sleep apnea (OSA) and drugs Results We found 12600 related articles in total in Google Scholar 1807 inScienceDirect and 1081 in PubMedMedline published from 1980 to October 2014 228 original investigation and review articleswere eligible Conclusions There is rapidly increasing number of innovative articles covering all the areas of modern respiratorymedicine and expanding EBC potential clinical applications to other fields of internal medicine However themajority of publishedpapers represent the results of small-scale studies and thus current knowledge must be further evaluated in large cohorts In regardto the potential clinical use of EBC-analysis several limitations must be pointed out including poor reproducibility of biomarkersand absence of large surveys towards determination of reference-normal values In conclusion contemporary EBC-analysis is anintriguing achievement but still in early stage when it comes to its application in clinical practice

1 Introduction

Exhaled breath condensate (EBC) analysis is a recent non-invasive method of detecting biomarkers mainly comingfrom the lower respiratory tract EBC is collected duringquiet breathing as a product of cooling and condensa-tion of the exhaled aerosol [1] It is a unique techniqueamong lung function tests in terms of identifying molecularpathways which reflect the airway epithelial function Anadditional advantage is the easy subject-friendly samplingprocedure requiring only tidal breathing in contrast withrelative invasive and technically challenging methods such asbronchoalveolar lavage (BAL) [2]

There is detailed literature focused on detection of inflam-matory markers which reflect the state of chronic airwaysdiseases such as chronic obstructive pulmonary disease(COPD) asthma and cystic fibrosis (CF) [1] However con-temporary methodology focus on EBC throughout analysis

with a view to identification ofmetabolic [3] proteomic [4 5]and genomic [6] fingerprints of breathing aiming for an earlydiagnosis of not only respiratory [4 6] but also systemicdiseases [7ndash9]

There is undoubtedly an increasing trend of biomedi-cal research towards development of noninvasive subject-friendly respiratory function testing Furthermore thereis rapidly increasing number of original research articlesregarding potential clinical applications of EBC-analysisusing various methodologies Therefore we aimed to presenta systematic review of the literature describing the 30-yearprogress of research on exhaled breath condensate in adisease-based approach with special reference to the poten-tial clinical applications and limitations of the contemporaryEBC-analysis

We searched the PubMedMedline ScienceDirect andGoogle Scholar databases using the following keywordsexhaled breath condensate EBC biomarkers pH asthma

Hindawi Publishing Corporatione Scientific World JournalVolume 2015 Article ID 435160 25 pageshttpdxdoiorg1011552015435160

2 The Scientific World Journal

Figure 1 EcoScreen 2 device for exhaled breath condensate (EBC) collection (FILT Lungen-amp Thorax Diagnostik GmbH) EcoScreen 2electrically refrigerates exhaled air conducted through a lamellar PFTE coated aluminum double lumen system EBC is formatted in adisposable polyethylene bag (at approximately minus10∘C) It also allows the fractionated collection of EBC from different areas of the bronchialtree into two disposable polyethylene bags so that the dead space condensate which contains biomarkers of no clinical relevance can bediscarded The device is not portable and weighs 20 kg (published with permission from FILT Lungen-ampThorax Diagnostik GmbH)

gastroesophageal reflux GERD chronic obstructive pul-monary disease smoking COPD lung cancer mechanicalventilation non small lung cancer NSCLC cystic fibro-sis pulmonary arterial hypertension PAH idiopathic pul-monary fibrosis interstitial lung diseases obstructive sleepapnea OSA and drugs

We found 12600 related articles in total in Google Scholar1807 in ScienceDirect and 1081 in PubMedMedline pub-lished from 1980 to October 2014 We used the followingas exclusion criteria (i) studies available only in languagesother than English (ii) studies published only in the formof abstract (iii) studies published in journals without impactfactor and (iv) studies applying EBC-analysis in veterinarymedicine Furthermore we preferably used original investi-gation articles instead of review articles when possible

207 original investigation papers and 21 review articleswhere eligible after applying the abovementioned criteria 47of them refer tomethodological and technical aspects and 181refer to the diagnostic aspects of EBC-analysis

2 Methodology of EBC Collection andAnalysis

21 EBC Collection Devices EBC collection is a simplenoninvasive technique requiring only quiet breathing of thesubject via a system of exhaled air cooling After applying anose-clip the subject breaths quietly for 10minutes through aspecialmouthpiece with a salivary trap and a single-way valveattached diverting the exhaled airflow through a Teflon orpolypropylene tube inside a cooling containerThere exhaledair in the form of droplets is converted to exhaled breathcondensate (EBC) [1 10]

Various house-built devices have been described basedon two simple layouts (a) a Teflon tube dipped in abucket filled with ice and (b) a double glass layer containerwhere exhaled air condensation takes place between the twolayers [1] Commercially available devices are EcoScreen

Turbodeccs Rtube andAnacon glass condenser based on theabovementioned layouts

The EcoScreen device electrically refrigerates exhaledair conducted through a lamellar PFTE coated aluminumdouble lumen system EBC is then formatted in a dispos-able polypropylene collecting cup (at approximately minus10∘C)EcoScreen 1 device has been extensively used in researchprotocols However it is not currently manufactured due toseveral technical drawbacks including lack ofmanual controlof condensing temperature and cleaning requirements ofthe device between consequent trials [11] Therefore theEcoScreen 1 has been replaced by EcoScreen 2 device whichallows the fractionated collection of EBC from different areasof the bronchial tree into two disposable polyethylene bags sothat the dead space condensate which contains biomarkers ofno clinical relevance may be discarded EcoScreen 1 and 2 arenot portable and weight 20 kg (Figure 1)

TurboDECCS consists of a portable TurboUnit and a dis-posable DECCS collection system DECCS is equipped witha mouthpiece a one-way valve a tube and a collection cellinserted in a Peltier-type electrical cooling system (Figure 2)

RTube is also portable and can be used by unsupervisedsubjects at home RTube disposable collection system consistsof a large Tee section (made of polypropylene (PP)) whichseparates saliva from the exhaled breath a one-way valve(made of silicone rubber) and a PP collection tube whichis cooled by a cooling-sleeve placed around (Figure 3)

ANACON condenser has been used by many researchgroups especially in mechanically ventilated patients [1012] It is a condensing device which can be attached to theexpiratory branch of the ventilator circuit Condensationtemperature is constantly monitored within the limits ofminus15∘C to minus5∘C (Figure 4)

22 Sampling

221 Sampling Duration After 10min of quiet breathing 1ndash3mL EBC is collected from adult subjects (1198811015840 = 100 Lmin)

The Scientific World Journal 3

DECCS

DECCS

Turbo

Figure 2 TurboDECCS device for exhaled breath condensate (EBC) collection (Medivac SRL Italy) TurboDECCS consists of a portableTurbo Unit and a disposable DECCS collection system DECCS is equipped with a mouthpiece a one-way valve a tube and a collection cellinserted in a Peltier-type electrical cooling system (published with permission from Medivac SRL Italy)

Figure 3 RTube device for exhaled breath condensate (EBC) collection (RespiratoryResearch Inc USA) RTube disposable collection systemconsists of a large Tee section (from polypropylene (PP)) which separates saliva from the exhaled breath an one-way valve (from siliconerubber) and a PP collection tube which is cooled by a cooling-sleeve placed around RTube is portable and can be used by unsupervisedsubjects at home (published with permission from Respiratory Research Inc USA)

[1] Sampling duration varies among studies from 10 to30min but collection of up to 60min has also been described[13 14] In EcoScreen device total EBC volume is propor-tional with total exhaled volume breathing frequency andtotal EBC protein and urea concentrations [15] Howeverwhen using Rtube where the temperature of cooling chamberis increasing with condensation notmuch EBC is added after10min of sampling

10min collection is usually applied on adults and childrenover 4 years of age as it is an easily tolerated period fromthe majority of subjects and provides sufficient EBC volumeDuring this time released particles range between 01 and 4particles times cm3 which have a mean diameter of 03 120583m [16]

222 Sampling Conditions There are many studies inves-tigating the effect of environmental conditions and differ-ent breathing patterns on the volume and composition of

EBC Increased 1198811015840119864(ventilation per minute) resulted in

significantly increased volume of EBC [17ndash19] In particularMcCafferty et al [17] demonstrated that 1198811015840

119864of 75 Lmin

15 Lmin and 225 Lmin resulted in EBC volume of 627120583L1019 120583L and 1358 120583L respectively whereas lower 119881

119879(tidal

volume) resulted in smaller EBC volumes Proteins nitritesand pH had no change with different breathing patterns Inthe same study EBC volume was also reduced when cooland dry air was inspired When an additional resistance of5 cmH

2O(Res5)was applied to the outflow tract of EcoScreen

device by Davidsson et al [20] an increased EBC volumewascollected due to the recruitment of additional alveoli

Several studies investigated the effect of different col-lection conditions on certain EBC biomarkers In a studyby Vaughan et al [14] temperature (minus44∘C to +13∘C) andduration of collection (2ndash7min) acute airway obstruction(after methacholine challenge) profound hypoventilationand hyperventilation were proved to have no effect on


3

To the ventilator

Expiratory branch Inspiratory branch

1313

21

13 13

5

9966

4

Patient

Filter191

∘Cminus111

∘C

Figure 4 ANACON (Biostec Valencia Spain) condenser integrated in the mechanical ventilation circuit The condenser is inserted inthe expiratory branch of the ventilation circuit via 2 adaptors (9) and 2 elastomeric connectors (6) The exhaled air passes towards thecondensation tubes (13) that pass through the body of the condenser (1) A Y piece (5) closes the circuit with the collection tube for theexhaled breath condensate (4) A thermometer (2) allows the condensation temperature to be monitored The apparatus also contains acooling switch (3) (reproduced with permission from [12])

EBC pH Contrarily in a study by Kullmann et al [21]environmental temperature and relative humidity were foundto contribute to the variability of EBC-pH and it was sug-gested that these factors should be controlled as part of thestandardization of EBC collection Regarding H

2O2levels in

EBC Schleiss et al [22] demonstrated that they were flow-dependent at expiratory flow rates between 48 and 140mLs

223 EBC Origin The dominant component of EBC iscondensed vapour (999 of EBC volume) Therefore watersoluble volatile compounds (acids and bases) are highlydiluted Daily 350mL of water is lost through breathing andreleased as vapor from the airways surface [23] Total amountof water lost is determined from ventilation perminute (1198811015840

119864)

as exhaled breath is almost completely saturated with watervapor [24]

Apart from water soluble volatile compounds theremaining liquid (01) which consists of exhaled dropletshas entrapped nonvolatile compounds at extremely lowconcentration Each 1mL of EBC contains lt01120583L of airwaylining fluid (ALF) droplets [25] Droplets are detached fromALF covering the surface of the respiratory tract due tothe turbulent flow of the exhaled air through the airways[16 26] However during exhalation droplets are detachednot only from the bronchi and trachea but also from larynxpharynx and upper gastrointestinal nasal and oral cavityand therefore it is impossible to identify the exact origin ofthe biomarkers [2] Volume and number of exhaled dropletsincrease because of cough and increased secretion while nocorrelation between ventilation and the amount of exhaleddroplets has been described yet [24] According to a recenthypothesis nonvolatile compounds coming from the ALFundergo aerosolization during inhalation when bronchioles

and alveoli burst to open (bronchiole fluid film burst (BFFB))[27]This fluid has previously been accumulated and trappedin the collapsed alveoli during exhalation [25 28]

The concentration of nonvolatile compounds diluted inEBC is expressed by the equation of Effros et al [24]

[119883]EBC = [119883]ALF119881ALF119881EBC= [119883]ELF

119899119881ALF

119899119881ALF + 119881vapor (1)

where 119881ALF = volume of exhaled droplets 119881EBC = volume ofcollected EBC 119881vapor = volume of condensed vapor in EBC119899119881ALF = number of respiratory droplets and 119881ALF = meanvolume

According to Effros et al [24] the following parametersare responsible for the increase of substances in EBC

(i) increased concentration of substances in the ALF(ii) increased number and volume of droplets(iii) reduced volume of water

Based on the above changes in levels of EBC biomarkersreflect changes in cellular composition and function [29ndash31]

224 Dilution of EBC EBC is a matrix containing largeamount of water vapour [31] and thus most biomarkersare highly diluted near the lower assay sensitivity limitsGiven the fact that EBC dilution is higher than ALF dilution[32] it is necessary to establish an EBC dilution indicatorable to reflect the actual levels of each biomarker in theairways According to Effros et al [24] an ideal dilutionfactor would have a known and stable plasma concentrationand high diffusing capacity through the cellular membraneand would not be a product of the respiratory tract Until


now no gold standard dilution factor has been defined andused extensively in studies Nevertheless multiple dilutionindicators have been proposed such as urea [30 34ndash36]total protein [36] total cations (Na+ K+ Ca2+ Mg2+) andconductivity of lyophilized and vacuum-evaporated samples[30 34]

225 Sample Contamination Since the main target is todetect compounds coming from the lower respiratory tractexclusion of sample contamination bymediators and proteinsoriginating from saliva oral cavity and upper airways isnecessary [37] EBC contamination can be avoided with theuse of various methods including saliva trap swan-neckshaped tube sodium bicarbonate 45 mouthwash prior tosampling and periodic swallowing by the subject duringEBC collection [1 25] To exclude salivary contaminationafter sampling completion measurement of salivary amylasein EBC has been proposed [30 38] When the abovemen-tioned precautionary measures are taken amylase is barelydetectable in EBC found in 10000-fold lower concentrationcompared to the saliva [30 38]

23 Prior to Sampling Exercise withdrawal is recommended1 hour prior to the sample collection since it may beresponsible for changes in EBC compounds concentration[1] Additionally subjects are recommended not to smoke for3 hours prior to EBC collection as smoking has immediateeffects on EBC-H

2O2 8-isoprostane and NO-metabolites

concentration [39 41] Caffeinated drinks must also beavoided when measuring EBC-adenosine levels [41] Con-sumption of 1 L carbonated beverage or water prior to sam-pling causes significant reduction of EBC pH [42] Finallyfood consumption has not been shown to have any influenceon EBC biomarkers concentration [41]

24 Sample Maintenance Collected EBC must undergoinstant analysis or it must be preserved at minus70∘C Mainte-nance duration is determined by the stability time of chemicalcompounds concerned Furthermore repetitive freezing-defreezing cycles must be avoided since this procedureresults in loss of unstable chemical compounds [43]

25 Sample Processing

251 pH Measurements EBC pH is not stable due to thesignificant pH reduction caused by both the atmosphericand the exhaled dissolved carbon dioxide (CO

2) Therefore

avoidance of CO2(gas standardization) is recommended in

order to achieve accurate and comparable pH measurements[44] CO

2deaeration is achieved either through bubbling

free gas (usually argon) in order to displace CO2or through

bubbling a knownquantity of CO2into EBC samples in order

to standardize partial CO2pressure to 40mmHg In both

methods pHmeasurement is recommended to be performedtwice before and after gas standardization in order toconfirm the pH change after CO

2deaeration [45 46] During

argon deaeration pH increases along with decreased pCO2

whereas during CO2loading pH is expected to decrease with

a simultaneous increase in pCO2[44]

252 Prechromatographic Sample Processing Chemical com-pounds extraction and concentration are required in orderto analyze EBC sample with chromatographic methods Thisprocedure is achieved through various techniques includ-ing (i) immunoaffinity extraction (IAE) in which specialantibodies are used [47] (ii) solid phase extraction (SPE)which is based on chemical and natural properties of thechemical compounds [48] (iii) solvent extraction (SE) whichis based on the relative water or organic solubility [49] and(iv) lyophiliosis which requires exsiccation of the sampleachieved through cooling [50] Lyophiliosis provides bothvolatile and semivolatile compounds concentration but alsoremoval of volatile compounds such as ammonia (NH

3)

[51] Lyophilized EBC gives 20 times the concentrations ofbiomarkers in comparison to the other techniques men-tioned above [52]

253 EBC-Analysis Methods Various methods able to detectdifferent compounds are available for EBC-analysis Com-mercial ELISA kits are of low sensitivity and specificity sincethey are not oriented to detect compounds at the extremelylow concentrations identified in EBC Furthermore EBCitself is not an appropriate matrix for such commercial kits[53]

Therefore other traditional techniques including spec-trophotometry [54] spectrofluorimetry [33] and enzymebased techniques [55] and immunoassay methods suchas ELISA radioimmunoassay (RIA) immune sensors andmultiple immunoassay (MIA) [56] are frequently usedHowever analysis based on the abovementioned techniquesmust be validated using analytical methods such as massspectrometry and high-performance liquid chromatographyable to provide quantitative analysis of EBC compounds [57]

Recently introduced methods such as 2-dimensionalprotein gel electrophoresis (2D PEG) [57] followed bychromatographic proteomic microanalysis are able to pro-vide both qualitative and quantitative EBC-analysis Con-temporary chromatographic methods include gas chro-matography (GC) high-performance liquid chromatography(HPLC) mass spectrometry (MS) and combinations ofthe abovementioned techniques including LC-MS GC-MS[58] HPLC-MS [59] Chromatography-Differential MobilitySpectrometry (GC-DMS) [60] and Electrospray Ionization-Differential Mobility Spectrometry (ESI-DMS) [61]

3 Clinical Applications of EBC-Analysis

31 Asthma Numerous pioneering studies enrolling rela-tively small number of subjects detected significantly lowerEBC pH in patients with stable asthma in comparison tohealthy controls [62ndash65] and further pH reduction wasidentified during asthmatic exacerbation [62 66ndash68] Fur-thermore EBC pH has been found significantly increasedafter treatment with inhaled corticosteroids [66 69 70]However it must be pointed out that in big cohorts nodifference was demonstrated in EBC-pH between patientswith stable asthma and healthy controls [45 71ndash73] cominginto conclusion that older studies demonstrated misleadingfindings


Another marker of oxidative stress hydrogen peroxide(H2O2) has been found in increased levels in EBC of

asthmatic patients and significant correlation between H2O2

increase and forced expiratory volume in 1 sec FEV1reduc-

tion has been detected [74] According to a meta-analysisby Teng et al [75] based on 8 studies involving 500 patientswith asthma and 228 healthy subjects EBC-H

2O2levels were

significantly higher in nonsmokers with asthma comparedto healthy subjects In particular adults with asthma hadhigher EBC-H

2O2levels than healthy subjects However

no significant difference was found between EBC-H2O2

concentration in children with asthma and control groupMoreover EBC-H

2O2levels correlated with disease severity

and control status whereas an inverse correlation with FEV1

was found Finally treatment with corticosteroids resulted ina reduction of H

2O2levelsThese findings indicate that H

2O2

has a potential as a biomarker for guiding asthma treatmentLeukotrienes (LTs) are well-studied inflammatory medi-

ators produced by leukocytes through arachidonic acid oxi-dationmediated by 5-lipoxygenase [76] CysLTs (LTD

4 LTE4

and LTC4) are produced frommast cells and eosinophils and

they have been found increased in atopic patients [77] andpatients with exercise-induced bronchoconstriction (EIB)[77 78] More specifically preexercise levels of CysLTs havebeen found elevated in EBC of asthmatic patients withEIB (median concentration 422 pgmL associated with thedecrease in FEV

1after exercise) compared to asthmatics

without EIB (117 pgmL) and healthy controls (58 pgmL)[78] Furthermore another study demonstrated increasedlevels of CysLTs in asthmatic patients with EIB 10min afterexercise [79] This fact supports the concept that CysLTsrelease is involved in the development of EIB LTs arereleased from the airway inflammatory cells after exposureto allergens promoting bronchoconstriction increase ofvascular permeability and mucus production [80] Increasedconcentrations of LTs in EBC of asthmatic patients have beenidentified in numerous studies [15 77 81ndash88] and EBC-LTs levels increase is correlated with severity of the disease[89] Moreover treatment with oral corticosteroids duringasthmatic exacerbation is followed by significant reductionof EBC-cysLTs (minus60) [90] However it has been shown thatEBC-LTE

4levels are not significantly reduced after inhaled

corticosteroid treatment in patients with stable asthma [83]Additionally increased levels of LTB

4have also been

identified in patients withCOPD [91] CF [92] and non-smallcell lung cancer (NSCLC) [93] and thus it is not an asthma-specific biomarker

EBC-concentrations of other eicosanoids such as 8-isoprostane and prostaglandin E

2(PGE2) have also been

studied in asthmatic populations 8-isoprostane levels havebeen found increased in adult and pediatric populationswith stable asthma but they were not significantly reducedafter inhaled or oral corticosteroid treatment [83 94 95]EBC-PGE

2concentration was not increased significantly

among both children and adult nonsmokers with stableasthma [15 83 95] However levels of EBC-PGE

2have been

found significantly increased in adult smokers with stableasthma [96]Therefore increasedEBC-PGE

2possibly reflects

smoking status in asthmatic patients

There is also detailed literature regarding EBC-interleukins (ILs) concentration in patients with asthmadetecting increased levels of IL-4 [97ndash102] IL-5 [103] IL-6[98ndash101 104] IL-8 [105] IL-10 [45] and IL17 [105] In astudy of Shahid et al [97] increased levels of EBC-IL-4with simultaneous reduction of interferon-120574 were identifiedamong pediatric asthmatic population changes indicative ofTh2-mediated inflammation

Another interesting biomarker involved in bronchocon-striction mechanism is RANTES It is a chemotactic agentfor eosinophils T-lymphocytes and mast cells whichinduce histamine and Cys-LTs secretion from mast cellsand eosinophilic cationic protein from eosinophils causingbronchospasm in patients with asthma [106] In a study ofMatsunaga et al [105] simultaneous increase of IL-4 IL-6 IL-8 IL-10 TNF-120572 TGF-120573 MIP1120572 (Macrophage Inflam-matory Protein 1120572 CCL3) MIP1120573 (Macrophage Inflam-matory 1120573 CCL4) and RANTES was identified amongadult steroid-naıve asthmatic patients Furthermore EBC-RANTES increase was significantly correlated with bothFEV1

reduction and airways resistance increase whileincreased levels of EBC-TNF-120572 and TGF-120573were significantlycorrelated with nonspecific bronchial hyperresponsiveness(BHR methacholine challenge)

In other studies by Zietkowski et al [107 108] significantlyincreased levels of EBC-RANTES and endothelin-1 (ET-1)were identified in adult patients with stable asthma comparedto the healthy controls Moreover both RANTES and ET-1 levels were significantly higher in subjects with unstableasthma compared to stabilized asthmatic subjectsThereforeit seems that EBC-RANTES levels are correlatedwith severityof the disease

Extensive proteomic analysis of EBC has also revealedother biomarkers related with pathophysiological mecha-nisms involved in asthma In a study of Bloemen et al[109] significantly higher levels of EBC-Cytokeratine 1 weredetected among asthmatic children compared to healthycontrols whereas no significant differences were identifiedin concentrations of EBC-cytokeratines 2 5 6 8 9 10 14and 16 albumin actin haemoglobin lysozyme calgranulinB and desmin In a study of Bartoli et al [110] EBC-Malondialdehyde (MDA) levels were found significantlyhigher among asthmatic patients in comparison to healthycontrols (26 nM versus 156 nM resp) and inhaled corti-costeroid treatment was related to lower EBC-MDA levels(21 nM) in comparison to untreated patients (32 nM) How-ever EBC-MDA levels were not significantly correlated withseverity of the disease in this study

Asymmetric Dimethylarginine (ADMA) is an L-arginineanalog which inhibits nitric oxide (NO) synthesis con-tributing to asthma pathogenesis (airways inflammation andremodeling oxidative stress and BHR) In a study of Carraroet al [111] significantly increased levels of EBC-ADMATyrratio (median value = 012 interquartile range 005ndash032)have been identified among pediatric population comparedto healthy controls (median value = 007 interquartile range005ndash012)

Eotaxin-1 (CCL11) is a chemokine for the eosinophilsinducing both their accumulation to the airway wall and


their activation and degranulation In a study by Zietkowskiet al [112] significantly increased EBC-CCL11 levels wereidentified among patients with stable asthma in compari-son to healthy controls and patients with unstable asthmademonstrated significantly higher CCL11 levels comparedto stabilized asthmatic patients Furthermore significantreduction of CCL11 levels was identified after omalizumabtreatment among patients with severe asthma reflectingremission of eosinophilic inflammation [113]

In another study by Zietkowski et al [114] significantlyincreased levels of EBC-high-sensitivity c-reactive protein(hs-CRP) a well-studied inflammatory mediator were iden-tified among patients with stable asthma compared to healthycontrols and significantly higher levels of EBC-hs-CRP weredetected in patients with unstable asthma in comparisonwith stabilized asthmatics Moreover not only serum hs-CRP but also Fractional Exhaled Nitric Oxide (FeNO) levelswere significantly correlated with EBC-hs-CRP levels in allsubjects

EBC genomic analysis is also becoming an intriguingarea of research especially when combined with proteomicanalysis In a prospective study of Klaassen et al [115]intercellular adhesionmolecule-1 (ICAM-1) genes expressionin terms of mRNA and proteins was related to asthma man-ifestation among preschool children Significantly increasedEBC-solute-ICAM-1 as well as EBC-1L-4 IL-10 and IL-13was found among children with asthma

EBC has been also studied in asthmatic patients withGERD (gastroesophageal reflux disease) in order to assess theairway acidification When the acid refluxes up to the phar-ynx it enters the airways causing epithelial damage due toacid injury and therefore worsening of asthma symptoms Ina study investigating the determinants of EBC-pH in a largepopulation with asthma GERD symptoms were associatedwith low EBC-pH [116] However Niimi et al [63] enrollingpatients with chronic cough suffering from various diseases(asthma rhinitis GER bronchiectasis) demonstrated thatlow EBC-pH was associated with chronic cough regardlessof the underlying medical conditionTherefore low EBC-pHis not a specific finding for GER

Banovic et al [117] demonstrated that children withuncontrolled asthma had significantly lower mean EBC-pHthan children with GERD without asthma (6791 plusmn 0374versus 7002 plusmn 0361 119901 = 0006) Moreover both asthmaticand GERD children had lower total magnesium (10 120583molLand 20120583molL resp) with the later demonstrating a negativecorrelation (119903 = minus0404 119901 = 0003) between mag-nesium levels and pH values Additionally in a study byFitzpatrick et al [118] EBC-pH failed to distinguish patientswith asthma depending on the presence of asymptomaticGERD Furthermore EBC-pH did not alter significantly aftertreatment with lansoprazole failing to act as a marker forthe response to PPIs treatment However in a former studyby Shimizu et al [119] patients with moderate asthma andGERD responded to a 2-month PPI therapy (lansoprazole30mgday) by increasing pH (from 72 plusmn 01 to 73 plusmn 01)and significantly reducing 8-isoprostane levels (from 327 plusmn34 to 192 plusmn 34) along with the improvement of GERDsymptoms

Pepsin was also used as a marker of GER-related aspira-tion in patients with GERD and GERD-induced respiratoryproblems Pepsin is normally present in gastric secretions butnot in bronchial tree so an increase of EBC-pepsin wouldbe expected in GERD patients However in a study by Soyeret al [120] pepsin levels in EBC of GERD patients werebelow detection limits failing to verify the abovementionedhypothesis Nevertheless in this study EBC-NO

119909(nitrites

and nitrates) levels were lower in GERD patients comparedwith non-GERD patients (137 120583molL versus 21 120583molL) Anegative correlation between reflux index and NO

119909levels

in EBC was also demonstrated (119903 = minus0331 119901 = 0023)This finding indicates that longer periods of reflux may causemore epithelial damage and subsequently a drop in NO

119909

levels Based on the above there is no specific EBC biomarkerindicative of airway acidification due to GERD in asthmaticpatients

Summarizing EBC alterations in asthma it can beobserved that themain problem of existing studies is the poorreproducibility of resultsThis could be attributable to the lackof specific analysis of the different inflammatory pathwaysin the vast majority of studies Asthmatics with neutrophilicpattern of inflammation would possibly have mediators intheir EBC different from asthmatics with an eosinophilic pat-tern and probably would have a different response to therapyTherefore existing knowledge is insufficient regarding thedetection of specific markers for asthma

An additional methodological issue is that in the greatpart of studies biomarkers have been studied only in stableasthma (Table 1) More studies concerning biomarkers inunstable asthma are needed aiming to predict exacerba-tion and treatment response especially to corticosteroidswhich is of paramount importance in daily clinical practice(Table 1 summarizes the changes of exhaled breath conden-sate biomarkers in asthma)

32 Chronic Obstructive Pulmonary Disease (COPD)

321 Immediate Effects of Tobacco Smoking on EBC Imme-diate effects of smoking a single cigarette (CS) have beenextensively studied Some early studies detected nonsignifi-cant changes of EBC-pH among healthy smokers [121 122]whereas in a recent study immediate increase of EBC-pHwas identified lasting for 2 hours after CS [41] Interestinglythe opposite change (significant reduction) in EBC-pH wasidentified immediately after CS among patients with allergicrhinitis [123] and asthma [121 124] which is indicative of adifferent acute response of the respiratory system after smok-ing probably reflecting the additive effects of smoking andatopic airway inflammation expressed as increased oxidativestress

Significant increase of EBC-nitrates concentration (from202120583M to 298 120583M) [39] EBC-8-isoprostane levels (+50)[40] and EBC-electric conductivity (from 40120583Scm to90 120583Scm) [122] immediately afterCShas also been identifiedHowever a recent study demonstrates nonsignificant 8-isoprostane change [121]

In regard to EBC-nitrites S-nitrosothiols nitrotyrosine[39] ammonium and IL-8 [122] levels no significant change


Table 1 Changes of exhaled breath condensate biomarkers in asthma

Biomarkers Stable asthma Unstable asthma

pH

harr [45 71ndash73]darr [62ndash65]uarr after ICS treatment [66 69 72]darr in GERD [63 116 117] uarr after lansoprazoleharr in GERD [118]harr after lansoprazole

darrdarr [62 66ndash68]

H2O2uarr [74] correlated with darr FEV1uarr [75] correlated with severity darr FEV1 and darr after treatment with corticosteroids

LTsuarr [15 76 80ndash87]

Cys-LTs uarr in EIB before exercise [78] Cys-LTs darr after OCStreatment [89]

uarr in EIB 10min after exercise [78]LTE4 uarr butharr after ICS treatment [82]

8-Isoprostaneuarr butharr after ICS OCS treatment [82 93 94]

PGE2Nonsmokersharr [15 82 94]smokers uarr [95]

ILsIL-4

uarr [96ndash101]IL-5

uarr [102]IL-6

uarr [97ndash100 103]IL-8

uarr [104]IL-10

uarr [70]IL-17

uarr [104]INF-120574

darr in paediatric population [96]

RANTES uarr [104 106 107]correlated with darr FEV1 and uarr Raw [104] uarruarr [106 107]

MIP1120572 MIP1120573uarr in steroid naive adults [104]

TNF-120572 TGF-120573uarr correlated with nonspecific BHR(PC) [104]

ET-1uarr [106 107] uarruarr [106 107]

Cytokeratine 1uarr in paediatric population [109]

MDAuarr darr after ICS treatment [110]

ADMAuarr In paediatric population [111]

CCL11uarr [112]

uarruarr [112]darr after omalizumabtherapy in severeasthmatic patients[113]

hs-CRPuarr correlated with uarr FeNO and uarr serum hs-CRP [114]

uarruarr correlated with uarrFeNO and uarr serumhs-CRP [114]

sICAM-1uarr related to bronchial asthma manifestation in paediatric population [115]

uarr = increase darr = reductionharr = no significant change and [119909] = corresponding referenceICS = inhaled corticosteroids GERD = gastroesophageal reflux H2O2 = hydrogen peroxide LTs = leukotrienes PGE2 = prostaglandin-E2 EIB = exercise-induced bronchoconstriction OCS = oral corticosteroids ILs = interleukins INF-120574 = interferon-120574 MIP =macrophage inflammatory proteins TNF-a = tumornecrosis factor-a TGF-120573 = transforming growth factor-120573 BHR(PC) = bronchial hyperresponsiveness(provocative concentration) ET-1 = endothelin-1 SPs =surfactant proteins MDA = malondialdehyde ADMA = asymmetric dimethylarginine CCL11 = eotaxin-1 hs-CRP = high sensitivity c-reactive protein andsICAM-1 = solute intercellular adhesion molecule-1

has been demonstrated among healthy smokers immediatelyafter CS

A recent study investigating the immediate effects of 30-minute waterpipe smoking (WPS) on EBC [125] detectedsignificant reduction of EBC-IL-4 IL-5 IL-10 IL-17 and INF-120574 due to either decreased production or decreased delivery

as an effect of high levels of carbon monoxide (CO) which isa potent anti-inflammatory agent

322 Long-Term Effects of Tobacco Smoking on EBC It hasbeen shown that there is no significant difference in EBC-pHamong mild healthy smokers (lt10 pack-years) and healthy


never-smokers (median values 817 versus 816 resp) Con-trarily EBC-pH of asymptomatic smokers with cigaretteconsumption higher than 10 pack-years has been foundsignificantly reduced presenting values close to the ones ofCOPD patients (median values 74 and 736 resp) [122 126]Furthermore pH reduction is significantly correlated withsmoking history (005-degree EBC-pH reduction for everyyear of regular cigarette smoking was identified) [126]

Inflammatory agents including EBC-IL-6 LTB4[116] IL-

8 [127 128] and TNF-120572 [129] have been found significantlyincreased in chronic asymptomatic smokers MoreoverEBC-IL-6 and TNF-120572 levels are significantly correlated withForced Vital Capacity (FVC) and FEV

1values [127 129]

while levels of exhaled CO (eCO) and smoking history aresignificantly correlated only with IL-6 levels [129] Addition-ally it has been shown that smokers with allergic rhinitisdemonstrate increased baseline EBC-LTB

4levels [130] sug-

gesting that rhinitic smokers are more susceptible to thedeleterious effects of smoking

323 Secondhand Smoking (SHS) Effects Little is knownregarding SHS effects on EBC In a recent study [131] sig-nificant reduction of EBC-pH was identified among healthynonsmokers immediately after 1 hour of exposure to SHSlasting for 180 minutes Moreover significantly increasedlevels of EBC-H

2O2were identified at 120 minutes after

exposure and remained increased for the next 120 minutesThere are still a limited number of studies investigating

the effects of cigarette smoking on EBC However it has beenproved that even a single cigarette smoking causes significantalterations involving inflammatory pathways Furthermorethere is a different inflammatory response (both short- andlong-term) of the respiratory system to cigarette smokingamong smokers with and without allergic rhinitis Thisfinding indicates that there is a high possibility that the samecould apply to smokers with and without asthma Carefullydesigned studies are needed in order to investigate thispossibility and the susceptibility of these patients to developCOPD in the future (Table 2 summarizes the short- and long-term effects of tobacco smoking on EBC)

324 Stable COPD EBC-pH of COPD patients has beenfound acidic in numerous studies [57 132 134] reflecting theendogenous airway acidification Furthermore significantcorrelation between pH reduction and FEV

1decrease has

been identified [57] but this finding has not been con-firmed by other investigators [132] Influence of smoking wasdetermined in a large cohort by MacNee et al [133] whodemonstrated that EBC pH was lower in COPD than inhealthy control nonsmokers but did not differentiate COPDfrom smokers without COPD According to these findingsEBC pH does not appear to be a useful biomarker in smokerswith COPD

In another study Papaioannou et al [134] detectedsignificantly reduced EBC-pH in COPDpatients both smok-ers and ex-smokers in comparison to healthy controls(both smokers and ex-smokers) Interestingly though ex-smokers of COPD patients demonstrated significantly lower

EBC-pH values than their still-smoking counterparts Addi-tionally ex-smokers with GOLD stages III and IV COPDdemonstrated significantly reduced EBC-pH compared toex-smokers with GOLD stages I and II COPD whereasno significant difference was detected among smokers ofCOPD patients with increasing GOLD stage FurthermoreEBC-pH levels were correlated with static hyperinflation(ICTLC (Inspiratory CapacityTotal Lung Capacity)) airtrapping (RVTLC (Residual VolumeTotal Lung Capacity))and diffusing capacity for carbon monoxide in ex-smokerswhereas no corresponding significant correlations were iden-tified among current smokers

Based on the above current data regarding EBC-pH inCOPD is conflictingThe biomarkermay be used as a COPD-severity index only in ex-smokers of COPD patients sinceit seems that the inflammatory effects of current smokingbursting the airway oxidative stress cover up the ongoinginflammatory process of the disease

In regard to airway oxidative stress EBC-H2O2has

been found significantly increased among COPD patientsin numerous studies [135ndash137] but there was no significantcorrelation between EBC-H

2O2increase and FEV

1reduction

[135]Furthermore in a study of Corradi et al [138] EBC-

concentrations of various aldehydes (MDA hexanal hep-tanal) well-characterized biomarkers of the attack of ReactiveOxygen Species (ROS) on unsaturated lipids in cell mem-branes were found significantly increased in COPD patientscompared to healthy nonsmokers However only EBC-MDAconcentration (significantly increased in COPD) served as adistinguishing factor between smoking controls and COPDpatients suggesting that MDA could be a potentially usefulbiomarker in COPD patients follow-up Another study ofCorradi et al [139] demonstrated increased levels of aldehy-des (mainly MDA) in EBC of COPD patients compared tocontrol groups whereas in a more recent research by Antuset al [140] difference in MDA levels between COPD patientsand healthy controls was found in sputum but not in EBC

In regard to classic inflammatory markers EBC-8-isoprostane levels have been found equally increased in bothsmokers and ex-smokers of COPD patients compared tohealthy controls [136 141 142] (18-fold in COPD versushealthy smokers and 22-fold in COPD versus healthy non-smokers [136]) However no significant correlation amongEBC-8-isoprostane increase and FEV

1reduction was iden-

tified Significant increase of EBC-LTB4has been detected

among smokers with COPD in comparison to healthysmokers [16 141] and significant correlation among EBC-LTB4increase and CO diffusing capacity reduction has been

identified whereas EBC-LTB4increase was not correlated

with FEV1reduction [141] Additionally no significant dif-

ference in levels of EBC-LTB4among COPD smokers and

ex-smokers has been detected [142] Thus it seems thatincreased levels of 8-isoprostane and LTB

4may be indicative

of increased airway inflammatory stress regardless of smok-ing status However Borrill et al [143] observed considerablewithin- and between-day variability of these biomarkersposing limitations to the value of the abovementionedfindings


Table 2 Short and long-term effects of tobacco smoking on exhaled breath condensate

Biomarkers Immediate effect Long-term effect

pH

harr in healthy smokers [121 122]

harr in healthy smokers with lt10 py [122 126]darr in healthy smokers with gt10 py [122 126]

uarr in healthy smokers [41]darr in smokers with allergic rhinitis [123]darr in smokers with bronchial asthma[121 124]darr after SHS in healthy nonsmokers [130]

H2O2 uarr after SHS in healthy nonsmokers [130]

8-Isoprostane harr [121]uarr [46]

ILsIL-4 5 10 17 darr after waterpipe smoking [125]

IL-6 uarr [116 127] correlated with FVC FEV1 and eCO levelsand smoking duration [127 128]

IL-8 harr [122] uarr [127 128]TNF-120572 uarr correlated with FVC and FEV1 values [129]INF-120574 darr after waterpipe smoking [125]Nitrates uarr [45]LTB4 uarr [116] uarruarr in smokers with allergic rhinitis [130]EBC electric conductivity (120583S) uarr [122]Haemoglobin 120573-chain Detected [144]Data represent changes after cigarette smoking When changes refer to secondhand cigarette smoking or other tobacco product effects there is a specialreference inside the tableuarr = increase darr = reductionharr = no significant change and [119909] = corresponding referencepy = pack-years SHS = secondhand cigarette smoking H2O2 = hydrogen peroxide ILs = interleukins TNF-a = tumor necrosis factor-a INF-120574 = interferon-120574and LTB4 = leukotriene-B4

Increased levels of EBC-PGE2and PGF

2have also

been identified in COPD patients compared with asthmaticpatients However significantly increased levels of EBC-LTE

4

and TxB2have been detected in asthmatic patients whereas

the abovementioned biomarkers were undetectable in COPDpatients [16]

A special characteristic of airway inflammation in COPDis its mediation by neutrophils activated through interactionwith cytokines especially interleukins (ILs) Indeed signif-icantly increased levels of IL1120573 and IL-12 have been foundin patients with stable COPD [52] Furthermore in a recentstudy of Fumagalli et al [144] where proteomic EBC-analysiswas applied different EBC proteomic profiles were identifiedamong COPD patients and healthy controls regardless ofsmoking status More specifically signaling and regulatoryproteins including cytokeratins I and II (CK-1 CK-5 CK-9CK-14 and CK-26) were dominant (34) in EBC of healthycontrols (smokers and nonsmokers) In COPD patientsincluding patients with 120572

1-antitrypsin deficiency (A

1AD)

cytokines (IL-1120572 IL-1120573 IL-2 IL-12120572 IL-12120573 and IL-15) wereidentified as the dominant protein type (62) but INF-120572INF-120574 TNF-120572 andC3 complement fractionwere detected Inhealthy controls levels of the abovementioned inflammatorymediators were negligible and INF-120572 and IL-12 were notdetected Interestingly though haemoglobin 120573-chain wasdetected in healthy smokers probably reflecting haemoly-sis due to smoking induced oxidative stress Additionally

considerable amount of enzymes (8) which were identifiedin healthy controls was absent in COPD patients Fur-thermore COPD versus A

1AD comparative EBC proteomic

analysis demonstrated significant differences In A1AD-

patients 1205721-antitrypsin was not detected but IL-1120572 and

lysozyme-C which were undetectable in the other COPDpatients were identified Other types of proteins includingsurfactant proteins (SP-A1 SP-A2) and S100 calgranulin Aand calgranulin B (calcium-binding proteins) were equallyidentified in both healthy controls and COPD patients butnot in A

1AD-patients The abovementioned differences in

proteomic profile may prove to be a useful tool in regardto identification of pathogenetic mechanisms involved inCOPD

325 Acute COPD Exacerbation (AECOPD) EBC-pH hasbeen found significantly reduced during AECOPD (557 plusmn007) and significant increase following treatment has alsobeen identified (604 plusmn 008) [145] Additionally increasedlevels of EBC-CysLts [3] LTB

4[146 147] 8-isoprostane

and H2O2

have been detected followed by significantdecrease after antibiotic treatment with cephalosporins andmacrolides [146]

In regard to classic inflammatory markers significantlyincreased levels of EBC-IL-6 [52 148] IL-1120573 IL-8 IL-10 IL-12 TNF-120572 [52] and EBC-nitrites [149] have been identified in


patients with AECOPD in comparison to patients with stableCOPD

EBC proteomic analysis in patients with infectiousAECOPD revealed significant increase of 120572

1-antitrypsin-

antiprotease (AAT 2-fold in comparison to stable COPD)[150] metalloproteinase-9 (MMP-9) and metalloproteinase-1-inhibitor (TIMP-1) [151] reflecting the acute phaseresponse In a study of Corhay et al [152] which enrolled ex-smokers with COPD EBC-neutrophil chemotactic activity(NCA assessed by the use of Boyden microchambers) wasfound increased in outpatients with AECOPD followedby significant reduction 6 weeks after AECOPD recoveryHowever no significant increase of EBC-NCA was detectedin hospitalized patients with AECOPD and no significantdifference on EBC-NCA was identified between patientswith stable COPD and outpatients with AECOPD Inregard to neutrophil chemoattractants EBC-LTB

4was found

significantly increased in patients with both stable COPD andAECOPD but EBC-GRO-120572 (growth related oncogene-120572)was found significantly increased only in AECOPD patientsMoreover EBC-LTB

4and EBC-GRO-120572 reduced significantly

after recovery from AECOPD Based on the above GRO-120572 astrong neutrophil chemoattractant may be pathogeneticallyinvolved in AECOPD and further investigation is necessaryin order to evaluate its role in the follow-up of such patients

Other agents have been also proposed as prognosticfactors of AECOPD including increased levels of solute-HLA-1 and solute-CD95 [153] Contrarily in other stud-ies demonstrating increased levels of EBC-secretory leuko-cyte protease inhibitor-1 (SLP-1) and myeloperoxidase theauthors concluded that such changes probably reflect thepulmonary immune response to antibiotic treatment ratherthan a pathogenetic mechanism involved in manifestation ofAECOPD [154]

EBC genomic analysis has also given interesting resultsNucleic acids of both viral and bacterial agents includinginfluenza virus and respiratory syncytial virus but alsoLegionella pneumophila have been detected in EBC ofAECOPD patients while EBC analysis has been provedmoresensitive in detection of Legionella pneumophila compared toinduced sputum [155]

Summarizing current knowledge in COPD there arenumerous studies investigating a variety of EBC biomarkersin both stable and exacerbated COPD without promisingresults regarding a reliable predictor of exacerbation Further-more no study demonstrates specific inflammatory pathwaysthat can differentiate between and ideally predict COPD clin-ical phenotypes (ie chronic bronchitis emphysema overlapsyndrome of asthma and COPD) Further research is neededin order to identify the specific fingerprint of each phenotypewhich would lead to different treatment approaches (Table 3summarizes the changes of EBC biomarkers in COPD)

33 Lung Cancer There is a lot of potential regarding therole of EBC-analysis in early diagnosis prognosis as well asfollow-up of patients with lung cancer since a plethora oftumor-specific EBC biomarkers have been identified to date

In regard to oxidative stress Lases et al [156] detectedsignificantly higher increase of EBC-H

2O2and urine-MDA

in patients with lung carcinoma who had undergone lobec-tomyin comparison to pneumonectomy but also a strongcorrelation was found between EBC-H

2O2and urine-MDA

In regard to inflammatory markers Carpagnano et al[157] first detected increased levels of EBC-IL-6 in patientswith non-small cell lung cancer (NSCLC) compared tohealthy controls In another study of Carpagnano et al [158]increased levels of EBC-IL-2 TNF-120572 and leptin were foundin patients with stages I and IINSCLC comparedwith healthyex-smokers In a study of Brussino et al [159] increased EBC-IL-6 IL-17 and TNF-120572 were associated with increased levelsof EBC-Vascular Endothelial Growth Factor (VEGF) and allthe abovementioned biomarkers were significantly correlatedwith the tumor diameter estimated throughCTAdditionallyCarpagnano et al [160] have also detected increased levels ofEBC-endothelin-1 (ET-1) in NSCLC patients and Dalaveriset al [161] demonstrated increased TNF-120572 and VEGF inpatients with T3 and T4 NSCLC

However many of the abovementioned inflammatorymarkers have also been found in increased levels amongpatients with COPD and asthma as previously mentioned

Kullmann et al [162] demonstrated that the cytokineprofile in EBC of patients with lung cancer was differentfrom that of controls regardless of smoking habits lungfunction and airway inflammation More specifically EBC-analysis of lung cancer patients showed 3-fold increase ofCCL-28 (lymphocyte chemoattractant) 2-fold increase ofMIP-3 (granulocyte activator) and GRo-a (monocyte adhe-sion molecule) 3-fold decrease of FGF-6 (fibroblast growthfactor) 25-fold decrease of eotaxin-2 eotaxin-3 (eosinophilchemoattractants) FGF-7 (fibroblast growth factor) and IL-10 in comparison to control group However in a more recentstudy of Barta et al [163] no difference was found betweenpatients with squamous cell lung carcinoma and healthysmokers based on EBC cytokine signals

Furthermore significantly increased levels of proinflam-matory mediators including EBC-IL-1120573 IL-6 IL-8 TNF-120572and sICAM-1 have been measured on postoperative days(days 1 3 and 7) in patients who have undergone lobectomyor pneumonectomy [164] sICAM-1 was significantly higheron day 1 after resection (sICAM-1 is associated with malig-nant tumours) TNF-120572 remained increased from day 1 to day7 IL-8 peaked on day 3 IL-6 peaked on day 1 and IL-1120573 levelsincreased after day 1 There was no significant associationhowever between levels of EBC-IL-1120573 IL-6 IL-8 TNF-120572 andsICAM-1 and the extent of lung cancer resection (lobectomyversus pneumonectomy) [164]

In a study by Gessner et al [165] significantly increasedlevels of EBC-VEGF and bFGF (basic fibroblast growthfactor) were identified in patients recently diagnosed withNSCLC compared to healthy controls patients with stableCOPD and patients with AECOPD Furthermore signif-icantly lower EBC-levels of the abovementioned growthfactors were identified among patients who underwent 2-cycle chemotherapy and who responded with 25 reductionof the tumor size compared to recently diagnosed untreatedpatients

Further EBC proteomic analysis has given numerouspromising markers still requiring further evaluation


Table 3 Changes of EBC biomarkers in chronic obstructive pulmonary disease (COPD)

Biomarkers Stable COPD AECOPD

pHharr [132]

darrdarr and uarr after treatment [145]darr [57 132 134] correlated with darr FEV1 [57]darr correlated with GOLD stage ICTLC RVTLCand CO diffusing capacity only in ex-smokers [134]

H2O2 uarr [135ndash137] uarr and darr after treatment [146]

Aldehydes

(MDA hexanal heptanal) uarr [138 139]harr [140]

8-Isoprostane uarr [136 141 142]uarr and darr after treatment [146]

High variability within day and between days [143]

LTs

LTB4uarr [16 141 152] correlated with darr CO diffusingcapacity

uarr [146 147] and darr after treatment [146]uarr and darr after recovery [152]

LTE4Undetectable [16]High variability within day and between days [143]

Cys-LTs uarr [3]

PGsPGE2 PGF2 uarr (compared to asthmatic patients) [16]

Acute phase responseAAT MMP-9 TIMP-1 uarr [150 151]

Neutrophilic inflammationIL-1120573 IL-12 uarr[52]IL-1a IL-1120573

Dominant protein type (62) [144]IL-2 IL-12a IL-1120573 IL-6IL-12120573 IL-15 IL-8 IL-10 uarruarr[52 148 149]INF-120572 INF-120574 IL-12 TNF-aTNF-a C3

NCA GRO-a uarr and darr after treatment (only in outpatients)[152]

Nitrites uarr [149]1205721-antitrypsinSP-A1 SP-A2S-100 calgranulins

Detected in COPD but not in A1AD patients [144]

IL-1120572lysozyme-C

Detected in A1AD but not in the other COPDpatients [144]

sHLA-1 sCD95SLP-1 MPO uarr [153 154]

Nucleic acids ofinfluenza virus RSV L pneumophila Detected [155]uarr = increase darr = reductionharr = no significant change and [119909] = corresponding referenceAECOPD = acute exacerbation of COPD FEV1 = forced expiratory volume at 1 sec IC = inspiratory capacity TLC = total lung capacity RV = residualvolume CO = carbon monoxide MDA = malondialdehyde H2O2 = hydrogen peroxide LTs = leukotrienes PGs = prostaglandins ILs = interleukins INF =interferon TNF-a = tumour necrosis factor-a C3 = complement fraction 3 NCA = neutrophil chemotactic activity AAT = a1-antitrypsin antiprotease MMP-9 =metalloproteinase-9 TIMP-1 = metalloproteinase-1 inhibitor SPs = surfactant proteins s = solute SLP-1 = secretory leukocyte protease inhibitor-1 MPO =myeloperoxidase RSV = respiratory syncytial virus and L pneumophila = Legionella pneumophila


Carpagnano et al [166] suggested that EBC-COX-2(cyclooxygenase-2) and survivin an apoptosis inhibitorcould be used as markers for early lung cancer diagnosisamong high risk smokers since significantly increased levelsof EBC-COX-2 and survivin were identified in healthysmokers compared to nonsmokers but also in NSCLCpatients compared to healthy smokers Moreover increasedlevels of the abovementioned biomarkers were associatedwith increasing stage of NSCLC

In another study by Carpagnano et al [167] high con-centration of EBC-matrix metalloproteinase-9 (MMP-9) hasbeen found in NSCLC patients and quantitative correlationwas identified between EBC-MMP-9 levels and smokinghistory but also with the stage of the disease

Cheng et al [168] first identified the Growth HormoneRegulated TBC Protein 1 (GRTP-1) in EBC of a single patientwith NSCLC a finding that warrants further evaluation assuggested by the authors themselves Moreover out of a totalnumber of 29 proteins detected 18 were common amonghealthy nonsmokers healthy smokers and NSCLC patients1 was common among healthy smokers and NSCLC patientsand 4 were identified only in ex-smokers

Zou et al [169] suggested that EBC-CEA (carcinoembry-onic antigen) SCC (squamous cell carcinoma antigen) andNSE (neuron-specific enolase) were more sensitive indexesfor diagnosis of early stage NSCLC and more stronglycorrelated to the histological type compared with the serum-CEA SCC and NSE The authors concluded that EBC-CEAwas the parameter with the best diagnostic accuracy in regardto NSCLC especially for adenocarcinoma (Se = 838Sp = 679) EBC-SCC and EBC-NSE also demonstratedacceptable distinction capacity (Se = 381 Sp = 929Se = 552 and Sp = 661 resp)

Increasing number of studies demonstrated impressiveresults after applying genomic analysis of EBC Gessner et al[170] detected somatic mutations of the p53 gene in patientswith lung cancer which were undetectable in healthycontrols Carpagnano et al [171] described microsatelliteDNA instability located in 3p chromosome (locus D3S2338D3S1266 D3S1304 and D3S1289) but also loss of heterozy-gosity both in healthy smokers and NSCLC patients Inanother study conducted by the same team [172] comparativeanalysis of mutations in DNA isolated from EBC peripheralblood and tumor tissue detected superiority of EBC as amatrix for identification of DNA mutations in patients withNSCLC In this study [172] the same microsatellite EBC-DNA changes were identified both in heavy smokers andpatients with NSCLC

An additional conclusion of the same authors was that theamount of detected 3p mutations was of negative prognosticvalue since it was associated with poor survival of patientswith lung cancer More specifically loss of heterozygosityin D3S1289 demonstrated negative prognostic value for lungadenocarcinoma but it was also related with poor prognosisof patients with NSCLC regardless of the stage of the diseaseMoreover microsatellite instability and loss of heterozygosityin D3S2338 were associated with poor survival of patientswith squamous cell lung cancer [173]

Microsatellite EBC-DNA mutations have also beendetected in chromosome 19q resulting in ERCC-1 and ERCC-2 genes which were present in 25 of NSCLC patientsand 16 of smoking subjects but they were undetectable innonsmokers [174]

Mozzoni et al [175] detected increased levels of miRNA-21 and particularly reduced levels of miRNA-486 in patientswith NSCLC and proposed their use as first-line screeningtest for diagnosis of NSCLC

Another impressive finding regarding lung cancer patho-genesis has been identified in a study of Kordiak et al [176]Specifically levels of mutated KRAS oncogene (mutationlocated in codon 12) were significantly reduced in EBC ofpatients withNSCLC after tumor resection by 13 times at 7thpostoperative day and by 37 times at 30th postoperative dayIn 10 patients the specificKRASmutation was undetectable at30th postoperative day

DNA methylation is a common biochemical processessential for normal development through various genomicfunctions including X-chromosome inactivation but alsosuppression of repetitive elements However aberrant DNAmethylation patterns (hyper and hypomethylation) havebeen associated with numerous human malignancies In astudy of Han et al [177] ex-smokers had higher density ofmethylatedRASSF-1A gene compared to current smokers andnonsmokers and methylation of theDAPK and PAX5120573 geneswas significantly correlated with NSCLC In a latter studyXiao et al [6] detected methylation of p16 gene in EBC ofpatients with NSCLC

HPV (human papillomavirus) infection has been pro-posed as the main infectious agent involved in the patho-genesis of lung cancer but it has been demonstrated thatfungal infections could also be involved through productionof carcinogenic mycotoxins In a study by Carpagnano et al[178] HPV genome was identified in EBC of 164 of a totalpopulationwithNSCLCwhile it was undetectable in the totalhealthy control population Furthermore the same authorsusing the technique of EBC cultivation for first timewere ableto identify that 279 of a total population with NSCLC wascolonized with Aspergillus niger A Ochraceus or Penicilliumspp whereas fungal colonization was absent in EBC of thehealthy control population [179]

In regard to lung cancer there is rapidly increasing num-ber of studies on EBC biomarkers applying contemporarytechniques especially proteomic and genomic analysis Themain goal of these studies was to demonstrate biomarkersfor early diagnosis of LC The most promising results camefrom genomic analysis in studies enrolling heavy smokersandNSCLCpatients inwhich commonDNAalterationswereidentified This finding is promising and indicates that suchalterations may be of prognostic value However method-ological issues should be addressed current knowledge isbased on studies enrolling patients with NSCLC and there isa relative lack of studies enrolling patients with small cell lungcarcinoma which is also a prevalent disease Additionallylarge scale screening and prospective studies are neededenrolling asymptomatic patients with all histological typesof LC in order to evaluate if EBC markers could identify


a preclinical stage of the disease (Table 4 summarizes theexhaled breath condensate biomarkers in lung cancer)

34 EBC in Mechanically Ventilated Patients EBC has beenwidely studied in mechanically ventilated (MV) patients asa noninvasive alternative to evaluate airway inflammationpredict the development of VAP (ventilator associated pneu-monia) and assess the ventilator associated damage andlung distention EBC biomarkers have been studied not onlyin MV patients with ALIARDS and VAP but also in MVpatients without respiratory failure and ALIARDS (ie braininjured)

Acidic pH has been found in MV patients both with[180 181] and without lung injury [182 183] comparedto healthy controls Interestingly in MV patients withoutALI EBC pH has been proved to inversely correlate withventilation time (119903 = minus0636 119901 = 0048) supportingthe hypothesis that positive-pressure ventilation inducespulmonary inflammation and physical disruption of tissuesand cells resulting in airway acidification [184] AdditionallyEBC pH shows further reduction when MV patientsrsquo clinicalcondition deteriorates [181] or lung injury progresses [180]and returns to higher levels after recovery [181] EBC pHvalues also elevate after salbutamol administration in MVpatients with ALI (from 766 to 783) [185] However in astudy by Nannini et al [186] EBC pH failed to distinguishMV patients according to whether they eventually developedVAP died or met criteria for weaning

A compound formed by in vivo peroxidation of arachi-donic acid membrane 8-iso-PGF

2a was found significantlyhigher in MV patients with ALIARDS (87 plusmn 28 pgmL)compared to healthy subjects intubated duringminor surgicalprocedures (7 plusmn 4 pgmL) [187] Additionally reduction of8-iso-PGF2a was described after salbutamol inhalation alongwith a tendency to decrease nitrosative species [185] Thesefindings indicate that salbutamol might prevent the lipidperoxidation that characterizes ALI and ARDS In a laterstudy by Roca et al [182] 8-isoprostane levels were notsignificantly different between MV patients without ALI andhealthy nonsmokers

The hypothesis that neutrophil activation and oxidantproduction are involved in the pathogenesis of ARDS issupported by several studies MV patients who eventuallydeveloped ARDS demonstrated higher levels of H

2O2(168 plusmn

035mumolL) compared to others who did not (034 plusmn008mumolL) [188] Other inflammatory biomarkers suchas IL-1120573 IL-10 IL-12p70 and TNF-120572 are also elevated in EBCof MV patients both with and without ALIARDS revealingthe airway inflammation in such patients [183 189] withthe former having also increased IL-6 and IL-8 [189] LTB4and H

2O2have been found significantly elevated in MV

patients who underwent lobectomy but this change was notsignificant in MV patients after coronary artery by-pass graftsurgery probably due to the higher risk of ALI developmentafter lobectomy [190] In a study by Gessner et al [191] EBC-NO2minus increased linearly with 119881

119879and correlated well with

expiratory minute volume possibly due to mechanical stressof the remaining open lung units in injured lungs

NO2minusNO3

minus were found elevated inMV patients withoutALI (mean 6622 120583M) compared to healthy subjects (mean1506 120583M) and their levels had negative correlation withdynamic compliance (119903 = minus0952 119901 lt 0001) and positivecorrelation with respiratory rate (119903 = 0683 119901 = 0029) [182]This finding could be related to themechanical stress inducedby positive-pressure mechanical ventilation

Cytokeratins 2 9 and 10 were also detected in EBCof MV patients with respiratory failure PEEP PIP andLISS (lung injury score) were positively correlated withthe levels of EBC-cytokeratins (2 9 and 10) revealing arelationship between these biomarkers and the ventilatorassociated damage to the lung parenchyma [192] sTREM-1(soluble triggering receptor expressed on myeloid cell-1) wasdetected both in BAL and EBC of MV patients with VAPindicating that the patients respond to bacterial stimuli withthe triggering of the secretion of inflammatory cytokines bysTREM-1 [193]

35 Other Lung and Systemic Diseases

351 Idiopathic Pulmonary Fibrosis (IPF) Chow et al [194]demonstrated significantly elevated pH and increased levelsof 8-isoprostane H

2O2 3-nitrotyrosine and nitric oxides

(NO119909) in patients with IPF compared to healthy controls

Additionally in a study by Montesi et al [195] 9 differenttypes of lysophosphatidic acid (LPA) which is an importantmediator of fibroblast requirement were detected in EBCof IPF patients Moreover docosatetraenoyl-LPA was foundsignificantly increased in comparison to healthy controls(918 pM versus 034 pM resp)

352 Cystic Fibrosis (CF) In a study by Tate et al [196]significantly reduced EBC-pH was detected in patients withCF compared to healthy controls but also further pH reduc-tion was identified during CF exacerbation However in arecent research by Antus et al [197] pH does not appear tobe a useful biomarker in CF since it does not discriminatebetween CF patients and healthy controls

A significant increase of EBC-nitrotyrosinewas identifiedin patients with stable CF but there was no significant cor-relation between FeNO and EBC-nitrotyrosine levels [198]However this finding was not confirmed by a later studyby Celio et al [199] who showed no difference in EBC 3-nitrotyrosine levels betweenCF patients and healthy controls

In a study by Horak et al [200] increased levels of EBC-nitrite were not associated with either lung function or chestX-ray findingsThose results are in contrast with a latter studyof Robroeks et al [201] who proposed increased EBC-nitritesand 8-isoprostane as markers of CF exacerbation They alsofound a reduction of EBC-nitric oxide and pH which wascorrelated with severity of the disease

In regard to classic inflammatory markers Cunninghamet al [202] demonstrated no significant difference in EBC-IL-8 levels among CF patients and healthy controls Howeverin a latter study by Carpagnano et al [203] significantlyincreased levels of EBC-IL-6 IL-8 and LTE

4were identified

during CF exacerbation especially when it was inducedby Pseudomonas aeruginosa infection Moreover significant


Table 4 Exhaled breath condensate biomarkers in lung cancer

Biomarkers Lung CancerH2O2 uarr correlated with uarr urine-MDA [156]Inflammatory markers

ILsIL-2 uarr [158]

NSCLCIL-6 uarr [157 169]IL-17 uarr [169]

TNF-120572 uarr [158 159 161]IL-1120573 IL-6

uarr after lobectomy or pneumonectomy [164]IL-8 TNF-a sICAM-1Leptin uarr in NSCLC [158]ET-1 uarr in NSCLC [160]

VEGFuarr [159 161 165] correlated with uarr IL-6 IL-17 TNF-120572and with tumor diameter in CT [159] NSCLC

darr after chemotherapy [165]bFGF uarr in NSCLC and darr after chemotherapy [165]COX-2

uarr in NSCLC patients along with the disease stage [160]survivinMMP-9 uarr in NSCLC patients along with the disease stage [158]GRTP-1 Detected in a single NSCLC patient [159]CCL28

uarr in lung cancer [162]harr no difference in cytokine signals [163]MIP-3

GRo-aEotaxin-2

darr in lung cancer [162]harr no difference in cytokine signals [163]

Eotaxin-3FGF-6FGF-7IL-10Tumor markers

CEAuarr in NSCLC and strongly correlated with histologicaltype [169]SCC

NSEGenomic analysis

Somatic mutations of p53 Detected [170]Microsatelite DNA instability and loss of heterozygosity3p chromosome (locus D3S2338 D3S1266 D3S1304 D3S1289) Detected in NSCLC patients [171]

loss of heterozygosity 3p chromosome (locus D3S1289) Correlated with negative prognostic value inadenocarcinoma and poor prognosis in NSCLC [172]

Microsatelite DNA instability and loss of heterozygosity3p chromosome (locus D3S2338)

Correlated with poor survival among patients withsquamous cell lung carcinoma [173]

ERCC1 and ERCC2 genes (chromosome 19q) Increased risk of NSCLC among smokers [174]

miRNA uarrmiRNA-21 with darrdarrmiRNA-486 proposed asscreening test for NSCLC diagnosis [175]

KRAS mutation in codon 12 darr after NSCLC resection surgery [176]Methylation of DAPK PAX5120573 and p16 genes Detected in patients with NSCLC [6 177]HPV genome Detected in patients with NSCLC [178]uarr = increase darr = reductionharr = no significant change and [119909] = corresponding referenceH2O2 = hydrogen peroxide MDA = malondialdehyde NSCLC = non-small cell lung cancer ILs = interleukins TNF-a = tumour necrosis factor-a ET-1 =endothelin-1 VEGF = vascular endothelial growth factor bFGF = basic fibroblast growth factor COX-2 = cyclooxygenase-2 MMP-9 = metalloproteinase-9GRTP-1 = growth hormone regulated TBC protein-1 CCL-28 = mucosae-associated epithelial chemokine (MEC) MIP = macrophage inflammatory proteinsGRO-a = growth related oncogene-a FGF-6 = fibroblast growth factor-6 FGF-7 = fibroblast growth factor-7 CEA = carcinoembryonic antigen SCC =squamous cell carcinoma antigen NSE = neuron-specific enolase miRNA = micro-RNA and HPV = human papillomavirus


reduction of the abovementioned inflammatory markers wasdetected after treatment with antibiotics [203 204] In a studybyRobroeks et al [205] no significant differencewas detectedin EBC-IL-1 IL-4 IL-10 and INF-120574 concentration amongasthmatic and CF paediatric populations whereas EBC-IL-5and TNF-120572 were detectable only in the CF-population

353 Pulmonary Arterial Hypertension- (PAH-) Influence ofCOPD In a study by Warwick et al [206] significantlyhigher levels of EBC-natriuretic peptide pro-BNP and ET-1 were identified in patients with idiopathic PAH (IPAH)compared to patients with COPD-induced PAH whereasin the latter significantly higher levels of 6-keto-PGF

1120572

were detected Additionally He et al [207] demonstrated asignificant raise of EBC-8-isoprostane and IL-6 in COPDpatients with PAH in comparison to patients with IPAH

In a study by Carratu et al [208] significantly higherEBC-ET-1 levels were identified in COPD patients with PAHand significant correlation was detected between EBC-ET-1levels and Pulmonary Artery Systolic Pressure (PASP) values

Mansoor et al [209] applied EBC proteomic analysisusingGMMS in order to detect volatile compounds (VOCs)which revealed a unique EBC-metabolic fingerprint for IPAHpatients 32 VOCs were detectable only in patients withIPAH and 6 of them were significantly associated withhaemodynamic variables of the pulmonary circulation

354 Sarcoidosis Piotrowski et al [9] applied comparativeanalysis of EBC and BAL in patients with sarcoidosis whichdetected high levels of 8-isoprostane and Cys-LTs in bothbiological materials as well as significant correlation amongtheir levels

In a study by Ahmadzai et al [210] significantly higherlevels of EBC-neopterin and TGF-1205731 were identified inpatients with sarcoidosis compared to healthy controlswhereas no significant difference in levels of EBC-angiotensinconverting enzyme was detected between the two popula-tions

355 Obstructive Sleep Apnea Syndrome (OSA) Studies inpediatric populations with OSA (clinical score gt 40) detectedsignificantly increased EBC-IL-6 and 8-isoprostane concen-trations compared to healthy controls The abovementionedbiomarkers were significantly correlated with clinical scoreand cardiac dysfunction [211] Additionally increased levelsof EBC-LTB

4and Cys-LTs were found significantly increased

in children with Apnea-Hypopnea Index (AHI) gt 5h andthey were associated with severity of the disease [212] Fur-thermore elevated morning-EBC-H

2O2levels were found

in pediatric population with severe OSA This finding isindicative of the intense night long oxidative stress [213]which is also expressed by the significant increase of EBC-uric salts [214]

Studies enrolling adult population with OSA have alsodetected a significant raise of EBC markers of oxidativestress More specifically significantly increased levels of 8-isoprostane [215ndash217] TNF-120572 IL-6 [216 217] H

2O2[218]

and reduced pH [217] have been identified compared to both

healthy and obese controls EBC-8-isoprostane concentra-tion has been found significantly higher immediately aftermorning awakening and it has been correlated to AHI scoreand neck periphery Furthermore a significant reduction ofEBC-8-isoprostane levels has been found after CPAP therapy[215] In addition significantly reduced EBC-H

2O2levels

have been identified after 6 weeks of CPAP therapy (from 450plusmn 130 nmolL to 294 plusmn 110 nmolL) [218]

It must be pointed out that EBC-ICAM-1 and IL-8 havebeen found equally increased both in healthy obese adultsand in obese adults with OSA [219] implying that theabovementioned inflammatorymarkers are notOSA specific

356 Systemic Lulus Erythematosus (SLE) Nielepkowicz-Gozdzinska et al detected significantly increased IL-8 [220]IL-6 and IL-10 [221] both in EBC and in BAL of SLE patientsFurthermore the abovementioned changes were significantlycorrelated with the disease activity [222]

357 Chronic Renal Disease (CRD) Significantly increasedEBC-pH nitrites and nitrates have been demonstratedin patients with end stage CRD Moreover levels of theabovementioned biomarkers decreased after haemodialysis[222] Additionally 22-fold EBC-H

2O2concentration has

been demonstrated in patients with severe uremia whohave undergone haemodialysis compared to healthy controls[223]

358 Pharmacokinetics EBC proteomic analysis has alsobeen applied in toxicology for detection of several com-pounds including drugs More specifically EBC analysisthrough mass spectrometry can potentially be used fordetection of drug metabolites concentrations during patientfollow-up [224] A well-studied example is the detection ofEBC-methadone through LC-TMS analysis [225] (Table 5refers to EBC biomarkers detected in all the abovementionedrespiratory and systemic diseases)

4 Discussion and Conclusion

This study is a systematic review of the literature presentingboth the theoretical principles and the potential clinical appli-cations and limitations of the contemporary EBC analysis

Bibliographic investigation detected a prominentresearch activity on that field resulting in rapidly increasingnumber of innovative papers published every day concerningalmost all the areas of modern respiratory medicine

Study of exhaled air has been an area that has garneredinterest since the beginning of medical science and investiga-tion From the Hippocratic medicine (uremic breath) [226]to the modern applications of e-nose [227] the study ofexhaled breath aiming to reveal clinically useful pathogeneticmechanisms of various diseases has undergone a remarkableevolution

EBC collection and analysis are the most complete prod-uct of that long-term research It is undoubtedly an advanta-geous technique when noninvasive effortless and painlesscollection of respiratory biological material is concerned


Table 5 Exhaled breath condensate biomarkers in several lung and systemic diseases

Disease Biomarkers Changes

Idiopathic pulmonary fibrosis (IPF)

8-IsoprostaneH2O23-nitrotyrosineNO119909

docosatetraenoyl-LPA

uarr [194 195]

Cystic fibrosis (CF)

pH darr and darrdarr during exacerbation [196 201]harr [197]Nitrotyrosine uarr [198]harr 3-nitrotyrosine [199]Nitrites uarr [200 201]Nitric oxide darr correlated with disease severity [201]8-Isoprostane uarr marker of CF exacerbation [201]IL-6 uarr during exacerbation and darr after treatment [203 204]

IL-8 harr in stable CF [202] uarr in CF exacerbation and darr aftertreatment [203 204]

IL-5TNF-120572 Detected in paediatric CF population [208]

LTE4 uarr during exacerbation and darr after treatment [203 204]

Pulmonary arterial hypertension (PAH)

Natriuretic peptidepro-BNPET-1

Higher levels in IPAH versus COPD-PAH [206]

ET-1 uarr in COPD-PAH correlated with PASP [208]6-keto-PGF

1120572

8-isoprostaneIL-6

Higher levels in COPD-PAH versus IPAH [207]

Sarcoidosis

8-IsoprostaneCys-LTs uarr in both EBC and BAL [9]

NeopterinTGF-1205731 uarr [210]

Obstructive Sleep Apnea Syndrome (OSA)

Pediatric patients

8-IsoprostaneIL-6

uarr in patients with clinical score gt40 correlated with cardiacdysfunction [211]

LTB4Cys-LTs uarr in patients with AHI gt5h [212]

H2O2 uarr [213]Uric salts uarr [214]

Adult patients

8-Isoprostane uarr [215ndash217] correlated with AHI and neck periphery and darrafter CPAP therapy [215]

IL-6 uarr [216 217]TNF-120572 uarr [216 217]pH darr [217]H2O2 uarr and darr after after CPAP therapy [218]ICAM-1IL-8 uarr in both healthy obese and obese OSA-patients [219]

Systemic Lupus Erythematosus (SLE) IL-6 IL-8 IL-10 uarr in both EBC and BAL [220 221] significantly correlatedwith the disease activity

Chronic Renal Disease (CRD)

pHNitritesNitrates

uarr and darr after haemodialysis [222]

H2O2uarr in patients with severe uremia who underwenthaemodialysis [223]

uarr = increase darr = reductionharr = no significant change and [119909] = corresponding referenceH2O2 = hydrogen peroxide NO

119909= nitric oxides ILs = interleukins TNF-a = tumor necrosis factor-a LTE4 = leukotriene-E4 pro-BNP = pro-brain natriuretic

peptide ET-1 = endothelin-1 LTs = leukotrienes TGF-1205731 = transforming growth factor-1205731 LTB4 = leukotriene-B4 and ICAM-1 = intercellular adhesionmolecule-1


Furthermore it is innovative since EBC is proved to be a wellmanageable matrix for application of all the technologicallyadvanced methods of contemporary biomedical researchIn this way EBC-analysis produces findings expressing aconsiderable part of proteomic metabolic and genomicfingerprint

However in order for a new technique to be introduced inclinical practice high quality of standardization is requiredboth in sampling and in interpretation In regard to samplingand EBC collection procedures there are reliable guidelines[1] which have been applied in the vast majority of theoriginal research studies mentioned in the present reviewbut they already count 10 years from their publication(2005)Therefore they lack updated knowledge coming fromproteomic and genomic analysis especially in lung cancerand systemic diseases Many promising biomarkers havebeen detected especially in the genomics area creating newperspectivesMoreover the guidelines focus on inflammatoryand oxidative stress biomarkers detected in early pilot studiesthe results of which are questioned since they are contradic-torywith recent data demonstrating that such biomarkers arenonspecific and with low diagnostic accuracy Thus recentstudies should be added to an updated version of ATSERStask force dedicated to EBC

In regard to standardization of EBC-analysis it mustbe pointed out that there is absence of large cohortstowards determination of normal-reference values of EBC-biomarkers [228] and therefore normal proteomicmetabolicand genomic ldquofingerprintsrdquo of exhaled breath have not beenadequately characterized yet Furthermore existing knowl-edge is based on data provided by small scale studies oftendemonstrating conflicting results possibly attributable to thepoor reproducibility of EBC biomarkers [137] There is amethodological limitation of existing studies that predisposesto such poor repeatability In the vast majority of studiesespecially ones on asthma and COPD the different inflam-matory pathways are not distinguished and thereby inflam-matory heterogeneity of enrolled patients could possibly bea factor associated with poor reproducibility Patients witha neutrophilic pattern of inflammation for example willpossibly have different EBC-mediators from patients withan eosinophilic or a paucigranular inflammatory patternand a different response to treatment Therefore despite theintensive research on asthma COPD and lung cancer thereis still no specific marker useful for diagnosis follow-upresponse to treatment and prediction of exacerbation

There is also a lack of screening and prospective studiesenrolling asymptomatic patients in order to evaluate if EBCmarkers could identify a preclinical lung disease conditionSuch studies demonstrating results of clinical significancecould potentially become the brink of a major breakthroughin respiratory medicine Such results combined with existingportable EBC collection equipment and the easy noninva-sive sampling could make EBC an exceptionally promisingscreening method for early detection of lung disease

Summarizing it seems that EBC-analysis is an intrigu-ing achievement of biomedical research but still in earlystage when it comes to its application in clinical practiceCurrent knowledge however creates optimism regarding

the potential of EBC-analysis to reveal disease-specific fin-gerprints as the sum of many mediators evaluated with newtechnologies able to characterize a disease or its exacerba-tion

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

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[2] A S Jackson A Sandrini C Campbell S Chow P S Thomasand D H Yates ldquoComparison of biomarkers in exhaled breathcondensate and bronchoalveolar lavagerdquo American Journal ofRespiratory and Critical Care Medicine vol 175 no 3 pp 222ndash227 2007

[3] P Montuschi D Paris D Melck et al ldquoNMR spectroscopymetabolomic profiling of exhaled breath condensate in patientswith stable and unstable cystic fibrosisrdquo Thorax vol 67 no 3pp 222ndash228 2012

[4] R Aebersold and M Mann ldquoMass spectrometry-based pro-teomicsrdquo Nature vol 422 no 6928 pp 198ndash207 2003

[5] J E Wiktorowicz and M Jamaluddin ldquoProteomics analysis ofthe asthmatic airwayrdquo in Heterogeneity in Asthma vol 795 ofAdvances in Experimental Medicine and Biology pp 221ndash232Springer New York NY USA 2014

[6] P Xiao J-R Chen F Zhou et al ldquoMethylation of P16 in exhaledbreath condensate for diagnosis of non-small cell lung cancerrdquoLung Cancer vol 83 no 1 pp 56ndash60 2014

[7] P M-L Sinues R Zenobi and M Kohler ldquoAnalysis of theexhalome a diagnostic tool of the futurerdquo Chest vol 144 no3 pp 746ndash749 2013

[8] M Hakim S Billan U Tisch et al ldquoDiagnosis of head and neckcancer from exhaled breathrdquo British Journal of Cancer vol 104no 10 pp 1649ndash1655 2011

[9] W J Piotrowski A Antczak J Marczak A Nawrocka ZKurmanowska and P Gorski ldquoEicosanoids in exhaled breathcondensate and BAL fluid of patients with sarcoidosisrdquo Chestvol 132 no 2 pp 589ndash596 2007

[10] K Czebe I Barta B Antus M Valyon I Horvath and T Kull-mann ldquoInfluence of condensing equipment and temperature onexhaled breath condensate pH total protein and leukotrieneconcentrationsrdquo Respiratory Medicine vol 102 no 5 pp 720ndash725 2008

[11] S R Carter C S Davis and E J Kovacs ldquoExhaled breathcondensate collection in the mechanically ventilated patientrdquoRespiratory Medicine vol 106 no 5 pp 601ndash613 2012

[12] P V Romero B Rodrigeuz S Martinez R Canizares DSepulveda and F Manresa ldquoAnalysis of oxidative stress inexhaled breath condensate from patients with severe pul-monary infectionsrdquo Archivos de Bronconeumologia vol 42 no3 pp 113ndash119 2006

[13] L Scheideler H-G Manke U Schwulera O Inacker andH Hammerle ldquoDetection of nonvolatile macromolecules inbreath a possible diagnostic toolrdquo American Review of Respi-ratory Disease vol 148 no 3 pp 778ndash784 1993


[14] J Vaughan L Ngamtrakulpanit T N Pajewski et al ldquoExhaledbreath condensate pH is a robust and reproducible assay ofairway acidityrdquo European Respiratory Journal vol 22 no 6 pp889ndash894 2003

[15] P Montuschi and P J Barnes ldquoExhaled leukotrienes andprostaglandins in asthmardquo Journal of Allergy and ClinicalImmunology vol 109 no 4 pp 615ndash620 2002

[16] C I Fairchild and J F Stampfer ldquoParticle concentrationin exhaled breathrdquo American Industrial Hygiene AssociationJournal vol 48 no 11 pp 948ndash949 1987

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[18] J S Debley A S Ohanian C F Spiekerman M L Aitkenand T S Hallstrand ldquoEffects of bronchoconstriction minuteventilation and deep inspiration on the composition of exhaledbreath condensaterdquo Chest vol 139 no 1 pp 16ndash22 2011

[19] J Liu and P S Thomas ldquoRelationship between exhaled breathcondensate volume and measurements of lung volumesrdquo Respi-ration vol 74 no 2 pp 142ndash145 2007

[20] A Davidsson K N Sjosward L Lundman and B SchmekelldquoQuantitative assessment and repeatability of chlorine inexhaled breath condensate comparison of two types of conden-satorsrdquo Respiration vol 72 no 5 pp 529ndash536 2005

[21] T Kullmann I Barta B Antus M Valyon and I HorvarthldquoEnvironmental temperature and relative humidity influenceexhaled breath condensate pHrdquo European Respiratory Journalvol 31 no 2 pp 474ndash475 2008

[22] M B Schleiss O Holz M Behnke K Richter H Magnussenand R A Jorres ldquoThe concentration of hydrogen peroxidein exhaled air depends on expiratory flow raterdquo EuropeanRespiratory Journal vol 16 no 6 pp 1115ndash1118 2000

[23] R W Hoyt and A Honig Body Fluid Balance Exercise andSport CRC Press Boca Raton Fla USA 1996

[24] R M Effros M B Dunning III J Biller and R Shaker ldquoThepromise and perils of exhaled breath condensatesrdquo AmericanJournal of PhysiologymdashLung Cellular and Molecular Physiologyvol 287 no 6 pp L1073ndashL1080 2004

[25] P P R Rosias E Dompeling H J E Hendriks J W C MHeijnens R A M G Donckerwolcke and Q Jobsis ldquoExhaledbreath condensate in children pearls and pitfallsrdquo PediatricAllergy and Immunology vol 15 no 1 pp 4ndash19 2004

[26] R S Papineni and F S Rosenthal ldquoThe size distribution ofdroplets in the exhaled breath of healthy human subjectsrdquoJournal of Aerosol Medicine Deposition Clearance and Effectsin the Lung vol 10 no 2 pp 105ndash116 1997

[27] G R Johnson and L Morawska ldquoThe mechanism of breathaerosol formationrdquo Journal of Aerosol Medicine and PulmonaryDrug Delivery vol 22 no 3 pp 229ndash237 2009

[28] J Hunt ldquoExhaled breath condensate an evolving tool fornoninvasive evaluation of lung diseaserdquo Journal of Allergy andClinical Immunology vol 110 no 1 pp 28ndash34 2002

[29] B Balbi P Pignatti M Corradi et al ldquoBronchoalveolar lavagesputum and exhaled clinically relevant inflammatory markersvalues in healthy adultsrdquo European Respiratory Journal vol 30no 4 pp 769ndash781 2007

[30] R M Effros K W Hoagland M Boasbous et al ldquoDilution ofrespiratory solutes in exhaled condensatesrdquoAmerican Journal ofRespiratory and Critical Care Medicine vol 165 no 5 pp 663ndash669 2002

[31] P Rosias C Robroeks JHendriks EDompelingQ Jobsis andI Rahman ldquoExhaled breath condensate a space odessey whereno one has gone beforerdquo European Respiratory Journal vol 24no 1 pp 189ndash190 2004

[32] I Cepelak and S Dodig ldquoExhaled breath condensate a newmethod for lung disease diagnosisrdquo Clinical Chemistry andLaboratory Medicine vol 45 no 8 pp 945ndash952 2007

[33] P Montuschi ldquoAnalysis of exhaled breath condensate in respi-ratory medicine methodological aspects and potential clinicalapplicationsrdquo Therapeutic Advances in Respiratory Disease vol1 no 1 pp 5ndash23 2007

[34] RM Effros J Biller B Foss et al ldquoA simplemethod for estimat-ing respiratory solute dilution in exhaled breath condensatesrdquoAmerican Journal of Respiratory and Critical Care Medicine vol168 no 12 pp 1500ndash1505 2003

[35] T M Dwyer ldquoSampling airway surface liquid non-volatiles inthe exhaled breath condensaterdquo Lung vol 182 no 4 pp 241ndash250 2004

[36] C Gessner H Kuhn H-J Seyfarth et al ldquoFactors influencingbreath condensate volumerdquo Pneumologie vol 55 no 9 pp 414ndash419 2001

[37] J T ZakrzewskiNC Barnes J F Costello andP J Piper ldquoLipidmediators in cystic fibrosis and chronic obstructive pulmonarydiseaserdquo American Review of Respiratory Disease vol 136 no 3pp 779ndash782 1987

[38] E Huszar G Vass E Vizi et al ldquoAdenosine in exhaled breathcondensate in healthy volunteers and in patients with asthmardquoEuropean Respiratory Journal vol 20 no 6 pp 1393ndash1398 2002

[39] B Balint L E Donnelly T Hanazawa S A Kharitonov and PJ Barnes ldquoIncreased nitric oxide metabolites in exhaled breathcondensate after exposure to tobacco smokerdquo Thorax vol 56no 6 pp 456ndash461 2001

[40] P Montuschi J V Collins G Ciabattoni et al ldquoExhaled 8-isoprostane as an in vivo biomarker of lung oxidative stress inpatients with COPD and healthy smokersrdquo American Journal ofRespiratory and Critical Care Medicine vol 162 no 3 pp 1175ndash1177 2000

[41] A L Calusic V M Varnai and J Macan ldquoAcute effects ofsmoking and food consumption on breath condensate pH inhealthy adultsrdquo Experimental Lung Research vol 37 no 2 pp92ndash100 2011

[42] T Kullmann I Barta B Antus and I Horvath ldquoDrinkinginfluences exhaled breath condensate acidityrdquo Lung vol 186 no4 pp 263ndash268 2008

[43] American Thoracic Society ldquoRecommendations for standard-ized procedures for the online and offline measurement ofexhaled lower respiratory nitric oxide and nasal nitric oxide inadults and childrenmdash1999rdquoAmerican Journal of Respiratory andCritical Care Medicine vol 160 no 6 pp 2104ndash2117 1999

[44] T Kullman I Barta Z Lazar et al ldquoExhaled breath condensatepH standardised for CO

2

partial pressurerdquo European Respira-tory Journal vol 29 no 3 pp 496ndash501 2007

[45] J C Ojoo S A Mulrennan J A Kastelik A H Morice andA E Redington ldquoExhaled breath condensate pH and exhalednitric oxide in allergic asthma and in cystic fibrosisrdquo Thoraxvol 60 no 1 pp 22ndash26 2005

[46] ldquoATS Workshop Proceedings exhaled breath condensate nitricoxide and nitric oxide oxidative metabolism in exhaled breathcondensaterdquo Proceedings of the American Thoracic Society vol3 pp 131ndash145 2006


[47] K Syslova P Kacer B Vilhanova et al ldquoDetermination ofcysteinyl leukotrienes in exhaled breath condensate methodcombining immunoseparation with LC-ESI-MSMSrdquo Journalof Chromatography B Analytical Technologies in the Biomedicaland Life Sciences vol 879 no 23 pp 2220ndash2228 2011

[48] J S Debley T S Hallstrand T Monge A Ohanian G J Red-ding and J J Zimmerman ldquoMethods to improve measurementof cysteinyl leukotrienes in exhaled breath condensate fromsubjects with asthma and healthy controlsrdquo Journal of Allergyand Clinical Immunology vol 120 no 5 pp 1216ndash1217 2007

[49] J D Pleil H F Hubbard J R Sobus K Sawyer and MC Madden ldquoVolatile polar metabolites in exhaled breathcondensate (EBC) collection and analysisrdquo Journal of BreathResearch vol 2 no 2 Article ID 026001 2008

[50] M Janicka P Kubica A Kot-Wasik J Kot and J NamiesnikldquoSensitive determination of isoprostanes in exhaled breathcondensate samples with use of liquid chromatography-tandemmass spectrometryrdquo Journal of Chromatography B AnalyticalTechnologies in the Biomedical and Life Sciences vol 893-894pp 144ndash149 2012

[51] R M Effros R Casaburi J Su et al ldquoThe effects of volatilesalivary acids and bases on exhaled breath condensate pHrdquoTheAmerican Journal of Respiratory and Critical Care Medicine vol173 no 4 pp 386ndash392 2006

[52] C Gessner R Scheibe M Wotzel et al ldquoExhaled breathcondensate cytokine patterns in chronic obstructive pulmonarydiseaserdquo Respiratory Medicine vol 99 no 10 pp 1229ndash12402005

[53] P P Rosias ldquoMethodological aspects of exhaled breath conden-sate collection and analysisrdquo Journal of Breath Research vol 6no 2 Article ID 027102 2012

[54] Z Vlasic S Dodig I Cepelak et al ldquoIron and ferritin concen-trations in exhaled breath condensate of children with asthmardquoJournal of Asthma vol 46 no 1 pp 81ndash85 2009

[55] R M Effros B Peterson R Casaburi et al ldquoEpithelial liningfluid solute concentrations in chronic obstructive lung diseasepatients and normal subjectsrdquo Journal of Applied Physiology vol99 no 4 pp 1286ndash1292 2005

[56] P Montuschi New Perspectives in Monitoring Lung Inflamma-tion Analysis of Exhaled Breath Condensate CRC Press BocaRaton Fla USA 2005

[57] G I Sidorenko E I Zborovskiı and D I Levina ldquoSurface-active properties of the exhaled air condensate (a new methodof studying lung function)rdquo Terapevticheskii Arkhiv vol 52 no3 pp 65ndash68 1980

[58] M Janicka A Kot-Wasik J Kot and J NamiesnikldquoIsoprostanes-biomarkers of lipid peroxidation their utility inevaluating oxidative stress and analysisrdquo International Journalof Molecular Sciences vol 11 no 11 pp 4631ndash4659 2010

[59] E Glowacka U Jedynak-Wasowicz M Sanak and G LisldquoExhaled eicosanoid profiles in children with atopic asthma andhealthy controlsrdquo Pediatric Pulmonology vol 48 no 4 pp 324ndash335 2013

[60] M A Molina W Zhao S Sankaran M Schivo N JKenyon and C E Davis ldquoDesign-of-experiment optimizationof exhaled breath condensate analysis using a miniature differ-ential mobility spectrometer (DMS)rdquo Analytica Chimica Actavol 628 no 2 pp 155ndash161 2008

[61] C E Davis M J Bogan S Sankaran et al ldquoAnalysis of volatileand non-volatile biomarkers in human breath using differentialmobility spectrometry (DMS)rdquo IEEE Sensors Journal vol 10 no1 pp 114ndash122 2010

[62] L Brunetti R Francavilla R Tesse et al ldquoExhaled breathcondensate pH measurement in children with asthma allergicrhinitis and atopic dermatitisrdquo Pediatric Allergy and Immunol-ogy vol 17 no 6 pp 422ndash427 2006

[63] A Niimi L T Nguyen O Usmani B Mann and K F ChungldquoReduced pH and chloride levels in exhaled breath condensateof patients with chronic coughrdquoThorax vol 59 no 7 pp 608ndash612 2004

[64] G E Carpagnano P J Barnes J Francis N Wilson A Bushand S A Kharitonov ldquoBreath condensate pH in children withcystic fibrosis and asthma a new noninvasive marker of airwayinflammationrdquo Chest vol 125 no 6 pp 2005ndash2010 2004

[65] P P R Rosias E Dompeling M A Dentener et al ldquoChildhoodasthma exhaled markers of airway inflammation asthma con-trol score and lung function testsrdquo Pediatric Pulmonology vol38 no 2 pp 107ndash114 2004

[66] B Antus I Barta T Kullmann et al ldquoAssessment of exhaledbreath condensate pH in exacerbations of asthma and chronicobstructive pulmonary disease a longitudinal studyrdquo AmericanJournal of Respiratory and Critical Care Medicine vol 182 no12 pp 1492ndash1497 2010

[67] K Kostikas A I Papaioannou K Tanou et al ldquoExhaled NOand exhaled breath condensate pH in the evaluation of asthmacontrolrdquo Respiratory Medicine vol 105 no 4 pp 526ndash532 2011

[68] J F Hunt K Fang R Malik et al ldquoEndogenous airway acid-ification Implications for asthma pathophysiologyrdquo AmericanJournal of Respiratory and Critical Care Medicine vol 161 no 3pp 694ndash699 2000

[69] K Kostikas G Papatheodorou K Ganas K Psathakis PPanagou and S Loukides ldquopH in expired breath condensate ofpatients with inflammatory airway diseasesrdquo American Journalof Respiratory and Critical Care Medicine vol 165 no 10 pp1364ndash1370 2002

[70] L Brunetti R Francavilla R Tesse et al ldquoExhaled breathcondensate cytokines and pH in pediatric asthma and atopicdermatitisrdquo Allergy and Asthma Proceedings vol 29 no 5 pp461ndash467 2008

[71] N C Nicolaou L A Lowe C S Murray A Woodcock ASimpson and A Custovic ldquoExhaled breath condensate pH andchildhood asthma unselected birth cohort studyrdquo AmericanJournal of Respiratory and Critical Care Medicine vol 174 no3 pp 254ndash259 2006

[72] R Koczulla S Dragonieri R Schot et al ldquoComparison ofexhaled breath condensate pH using two commercially avail-able devices in healthy controls asthma and COPD patientsrdquoRespiratory Research vol 10 article 78 2009

[73] G MacGregor S Ellis J Andrews et al ldquoBreath condensateammonium is lower in childrenwith chronic asthmardquoEuropeanRespiratory Journal vol 26 no 2 pp 271ndash276 2005

[74] A Antczak D Nowak B Shariati M Krol G Piasecka andZ Kurmanowska ldquoIncreased hydrogen peroxide and thiobar-bituric acid-reactive products in expired breath condensate ofasthmatic patientsrdquo European Respiratory Journal vol 10 no 6pp 1235ndash1241 1997

[75] Y Teng P Sun J Zhang et al ldquoHydrogen peroxide inexhaled breath condensate in patients with asthma a promisingbiomarkerrdquo Chest vol 140 no 1 pp 108ndash116 2011

[76] B Samuelsson ldquoLeukotrienes mediators of immediate hyper-sensitivity reactions and inflammationrdquo Science vol 220 no4597 pp 568ndash575 1983

[77] E Ono H Mita M Taniguchi et al ldquoIncrease in inflam-matory mediator concentrations in exhaled breath condensate


after allergen inhalationrdquo The Journal of Allergy and ClinicalImmunology vol 122 no 4 pp 768e1ndash773e1 2008

[78] S Carraro M Corradi S Zanconato et al ldquoExhaled breathcondensate cysteinyl leukotrienes are increased in childrenwithexercise-induced bronchoconstrictionrdquo The Journal of Allergyand Clinical Immunology vol 115 no 4 pp 764ndash770 2005

[79] A Bikov R Gajdocsi E Huszar et al ldquoExercise increasesexhaled breath condensate cysteinyl leukotriene concentrationin asthmatic patientsrdquo Journal of Asthma vol 47 no 9 pp 1057ndash1062 2010

[80] S T Holgate M Peters-Golden R A Panettieri et al ldquoRoles ofcysteinyl leukotrienes in airway inflammation smooth musclefunction and remodelingrdquo The Journal of Allergy and ClinicalImmunology vol 111 no 1 pp S18ndashS36 2003

[81] A Antczak P Montuschi S Kharitonov P Gorski and PJ Barnes ldquoIncreased exhaled cysteinyl-leukotrienes and 8-isoprostane in aspirin-induced asthmardquo American Journal ofRespiratory and Critical Care Medicine vol 166 no 3 pp 301ndash306 2002

[82] Z Csoma S A Kharitonov B Balint A Bush N M Wilsonand P J Barnes ldquoIncreased leukotrienes in exhaled breath con-densate in childhood asthmardquo American Journal of Respiratoryand Critical Care Medicine vol 166 no 10 pp 1345ndash1349 2002

[83] CMondinoGCiabattoni P Koch et al ldquoEffects of inhaled cor-ticosteroids on exhaled leukotrienes and prostanoids in asth-matic childrenrdquoThe Journal of Allergy and Clinical Immunologyvol 114 no 4 pp 761ndash767 2004

[84] P Cap J Chladek F Pehal et al ldquoGas chromatographymassspectrometry analysis of exhaled leukotrienes in asthmaticpatientsrdquoThorax vol 59 no 6 pp 465ndash470 2004

[85] S Zanconato S Carraro M Corradi et al ldquoLeukotrienes and8-isoprostane in exhaled breath condensate of children withstable and unstable asthmardquoThe Journal of Allergy and ClinicalImmunology vol 113 no 2 pp 257ndash263 2004

[86] P Montuschi C Mondino P Koch P J Barnes and G Cia-battoni ldquoEffects of a leukotriene receptor antagonist on exhaledleukotriene E

4

and prostanoids in children with asthmardquo TheJournal of Allergy and Clinical Immunology vol 118 no 2 pp347ndash353 2006

[87] A Shibata T Katsunuma M Tomikawa et al ldquoIncreasedleukotriene E

4

in the exhaled breath condensate of childrenwithmild asthamardquo Chest vol 130 no 6 pp 1718ndash1722 2006

[88] B Kielbasa A Moeller M Sanak et al ldquoEicosanoids in exhaledbreath condensates in the assessment of childhood asthmardquoPediatric Allergy and Immunology vol 19 no 7 pp 660ndash6692008

[89] T Hanazawa S A Kharitonov and P J Barnes ldquoIncreasednitrotyrosine in exhaled breath condensate of patients withasthmardquo American Journal of Respiratory and Critical CareMedicine vol 162 no 4 pp 1273ndash1276 2000

[90] E Baraldi S Carraro R Alinovi et al ldquoCysteinyl leukotrienesand 8-isoprostane in exhaled breath condensate of childrenwithasthma exacerbationsrdquoThorax vol 58 no 6 pp 505ndash509 2003

[91] P Montuschi S A Kharitonov G Ciabattoni and P J BarnesldquoExhaled leukotrienes and prostaglandins in COPDrdquo Thoraxvol 58 no 7 pp 585ndash588 2003

[92] A Bodini C DrsquoOrazio D Peroni et al ldquoBiomarkers of neu-trophilic inflammation in exhaled air of cystic fibrosis childrenwith bacterial airway infectionsrdquo Pediatric Pulmonology vol 40no 6 pp 494ndash499 2005

[93] G E Carpagnano G P Palladino D Lacedonia A KoutelouS Orlando and M P Foschino-Barbaro ldquoNeutrophilic airwaysinflammation in lung cancer the role of exhaled LTB-4 and IL-8rdquo BMC Cancer vol 11 article 226 2011

[94] P Montuschi M Corradi G Ciabattoni J Nightingale S AKharitonov and P J Barnes ldquoIncreased 8-isoprostane amarkerof oxidative stress in exhaled condensate of asthma patientsrdquoAmerican Journal of Respiratory and Critical Care Medicine vol160 no 1 pp 216ndash220 1999

[95] E Baraldi L Ghiro V Piovan et al ldquoIncreased exhaled 8-isoprostane in childhood asthmardquo Chest vol 124 no 1 pp 25ndash31 2003

[96] K Kostikas G Papatheodorou K Psathakis P Panagou and SLoukides ldquoProstaglandin E

2

in the expired breath condensateof patients with asthmardquo European Respiratory Journal vol 22no 5 pp 743ndash747 2003

[97] S K Shahid S A Kharitonov N M Wilson A Bush and P JBarnes ldquoIncreased interleukin-4 and decreased interferon-120574 inexhaled breath condensate of children with asthmardquo AmericanJournal of Respiratory and Critical Care Medicine vol 165 no 9pp 1290ndash1293 2002

[98] G E Carpagnano O Resta M Gelardi et al ldquoExhaledinflammatory markers in aspirin-induced asthma syndromerdquoAmerican Journal of Rhinology vol 21 no 5 pp 542ndash547 2007

[99] G E Carpagnano O Resta M T Ventura et al ldquoAirwayinflammation in subjects with gastro-oesophageal reflux andgastro-oesophageal reflux-related asthmardquo Journal of InternalMedicine vol 259 no 3 pp 323ndash331 2006

[100] G E Carpagnano M P Foschino Barbaro O Resta et alldquoExhaled markers in the monitoring of airways inflammationand its response to steroidrsquos treatment in mild persistentasthmardquo European Journal of Pharmacology vol 519 no 1-2 pp175ndash181 2005

[101] C M H H T Robroeks K D G van de Kant Q Jobsis et alldquoExhaled nitric oxide and biomarkers in exhaled breath con-densate indicate the presence severity and control of childhoodasthmardquoClinical amp Experimental Allergy vol 37 no 9 pp 1303ndash1311 2007

[102] T-F Leung G W K Wong F W S Ko et al ldquoAnalysis ofgrowth factors and inflammatory cytokines in exhaled breathcondensate from asthmatic childrenrdquo International Archives ofAllergy and Immunology vol 137 no 1 pp 66ndash72 2005

[103] M Profita S La Grutta E Carpagnano et al ldquoNoninvasivemethods for the detection of upper and lower airway inflam-mation in atopic childrenrdquo The Journal of Allergy and ClinicalImmunology vol 118 no 5 pp 1068ndash1074 2006

[104] G E Carpagnano M P F Barbaro M Cagnazzo et al ldquoUse ofexhaled breath condensate in the study of airway inflammationafter hypertonic saline solution challengerdquo Chest vol 128 no 5pp 3159ndash3166 2005

[105] K Matsunaga S Yanagisawa T Ichikawa et al ldquoAirwaycytokine expression measured by means of protein array inexhaled breath condensate correlation with physiologic prop-erties in asthmatic patientsrdquoThe Journal of Allergy and ClinicalImmunology vol 118 no 1 pp 84ndash90 2006

[106] K F Chung and P J Barnes ldquoCytokines in asthmardquoThorax vol54 no 9 pp 825ndash857 1999

[107] Z Zietkowski R Skiepko M M Tomasiak and A Bodzenta-Lukaszyk ldquoEndothelin-1 in exhaled breath condensate of stableand unstable asthma patientsrdquo Respiratory Medicine vol 102no 3 pp 470ndash474 2008


[108] Z Zietkowski M M Tomasiak R Skiepko and A Bodzenta-Lukaszyk ldquoRANTES in exhaled breath condensate of stable andunstable asthma patientsrdquo Respiratory Medicine vol 102 no 8pp 1198ndash1202 2008

[109] K Bloemen R van den Heuvel E Govarts et al ldquoA newapproach to study exhaled proteins as potential biomarkers forasthmardquoClinical amp Experimental Allergy vol 41 no 3 pp 346ndash356 2011

[110] M I Bartoli F Novelli F Costa et al ldquoMalondialdehyde inexhaled breath condensate as a marker of oxidative stress indifferent pulmonary diseasesrdquo Mediators of Inflammation vol2011 Article ID 891752 7 pages 2011

[111] S Carraro G Giordano G Piacentini et al ldquoAsymmetricdimethylarginine in exhaled breath condensate and serum ofchildren with asthmardquo Chest vol 144 no 2 pp 405ndash410 2013

[112] Z Zietkowski M M Tomasiak-Lozowska R Skiepko EZietkowska and A Bodzenta-Lukaszyk ldquoEotaxin-1 in exhaledbreath condensate of stable and unstable asthma patientsrdquoRespiratory Research vol 11 article 110 2010

[113] Z Zietkowski R Skiepko M M Tomasiak-Lozowska andA Bodzenta-Lukaszyk ldquoAirway inflammation and eotaxin inexhaled breath condensate of patients with severe persistentallergic asthma during omalizumab therapyrdquo Advances in Med-ical Sciences vol 56 no 2 pp 318ndash322 2011

[114] Z Zietkowski M M Tomasiak-Lozowska R Skiepko BMroczko M Szmitkowski and A Bodzenta-Lukaszyk ldquoHigh-sensitivity C-reactive protein in the exhaled breath condensateand serum in stable and unstable asthmardquoRespiratoryMedicinevol 103 no 3 pp 379ndash385 2009

[115] E M Klaassen K D van de Kant Q Jobsis et al ldquoIntegra-tive genomic analysis identifies a role for intercellular adhe-sion molecule 1 in childhood asthmardquo Pediatric Allergy andImmunology vol 25 no 2 pp 166ndash172 2014

[116] L LiuWGTeague S Erzurumet al ldquoDeterminants of exhaledbreath condensate pH in a large populationwith asthmardquoChestvol 139 no 2 pp 328ndash336 2011

[117] S Banovic M Navratil Z Vlasic R Z Topic and S DodigldquoCalcium and magnesium in exhaled breath condensate ofchildren with endogenous and exogenous airway acidificationrdquoJournal of Asthma vol 48 no 7 pp 667ndash673 2011

[118] A M Fitzpatrick J T Holbrook C Y Wei M S Brown RA Wise and W G Teague ldquoExhaled breath condensate pHdoes not discriminate asymptomatic gastroesophageal refluxor the response to lansoprazole treatment in children withpoorly controlled asthmardquo The Journal of Allergy and ClinicalImmunology vol 2 no 5 pp 579e7ndash586e7 2014

[119] Y Shimizu K Dobashi J J Zhao et al ldquoProton pumpinhibitor improves breath marker in moderate asthma withgastroesophageal reflux diseaserdquo Respiration vol 74 no 5 pp558ndash564 2007

[120] T Soyer O U Soyer E Birben U Kisa O Kalayci andM Cakmak ldquoPepsin levels and oxidative stress markers inexhaled breath condensate of patients with gastroesophagealreflux diseaserdquo Journal of Pediatric Surgery vol 48 no 11 pp2247ndash2250 2013

[121] A I Papaioannou A Koutsokera K Tanou et al ldquoThe acuteeffect of smoking in healthy and asthmatic smokersrdquo EuropeanJournal of Clinical Investigation vol 40 no 2 pp 103ndash109 2010

[122] A-R Koczulla S Noeske C Herr et al ldquoAcute and chroniceffects of smoking on inflammation markers in exhaled breathcondensate in current smokersrdquo Respiration vol 79 no 1 pp61ndash67 2010

[123] K Tanou A Koutsokera T S Kiropoulos et al ldquoInflammatoryand oxidative stress biomarkers in allergic rhinitis the effect ofsmokingrdquo Clinical amp Experimental Allergy vol 39 no 3 pp345ndash353 2009

[124] B Kane Z Borrill T Southworth A Woodcock and D SinghldquoReduced exhaled breath condensate pH in asthmatic smokersusing inhaled corticosteroidsrdquo Respirology vol 14 no 3 pp419ndash423 2009

[125] L Bentur E Hellou A Goldbart et al ldquoLaboratory and clinicalacute effects of active and passive indoor group water-pipe(Narghile) smokingrdquo Chest vol 145 no 4 pp 803ndash809 2014

[126] M L Nicola H B de Carvalho C T Yoshida et al ldquoYounglsquohealthyrsquo smokers have functional and inflammatory changes inthe nasal and the lower airwaysrdquo Chest vol 145 no 5 pp 998ndash1005 2014

[127] G E Carpagnano S A KharitonovM P Foschino-Barbaro OResta E Gramiccioni and P J Barnes ldquoIncrease inflammatorymarkers in the exhaled breath condensate of cigarette smokersrdquoEuropean Respiratory Journal vol 21 no 4 pp 589ndash593 2003

[128] W Mazur H Stark A Sovijarvi M Myllarniemi and VL Kinnula ldquoComparison of 8-isoprostane and interleukin-8 in induced sputum and exhaled breath condensate fromasymptomatic and symptomatic smokersrdquo Respiration vol 78no 2 pp 209ndash216 2009

[129] J M Diez-Pina M J Fernandez-Acenero M J Llorente-Alonso S Diaz-Lobato S Mayoralas and A Florez ldquoTumornecrosis factor alpha as a marker of systemic and local inflam-mation in lsquohealthyrsquo smokersrdquo International Journal of GeneralMedicine vol 2 pp 9ndash14 2009

[130] D Celik S Doruk H I Koseoglu S Sahin S Celikel and UErkorkmaz ldquoCysteinyl leukotrienes in exhaled breath conden-sate of smoking asthmaticsrdquo Clinical Chemistry and LaboratoryMedicine vol 51 no 5 pp 1069ndash1073 2013

[131] K Kostikas M Minas E Nikolaou et al ldquoSecondhand smokeexposure induces acutely airway acidification and oxidativestressrdquo Respiratory Medicine vol 107 no 2 pp 172ndash179 2013

[132] Z Borrill C Starkey J Vestbo and D Singh ldquoReproducibilityof exhaled breath condensate pH in chronic obstructive pul-monary diseaserdquo European Respiratory Journal vol 25 no 2pp 269ndash274 2005

[133] W MacNee S I Rennard J F Hunt et al ldquoEvaluation ofexhaled breath condensate pH as a biomarker for COPDrdquoRespiratory Medicine vol 105 no 7 pp 1037ndash1045 2011

[134] A I Papaioannou S LoukidesMMinas et al ldquoExhaled breathcondensate pHas a biomarker ofCOPDseverity in ex-smokersrdquoRespiratory Research vol 12 article 67 2011

[135] P N R Dekhuijzen K K H Aben I Dekker et al ldquoIncreasedexhalation of hydrogen peroxide in patients with stable andunstable chronic obstructive pulmonary diseaserdquoTheAmericanJournal of Respiratory and Critical Care Medicine vol 154 no 3pp 813ndash816 1996

[136] K Kostikas G Papatheodorou K Psathakis P Panagou andS Loukides ldquoOxidative stress in expired breath condensate ofpatients with COPDrdquo Chest vol 124 no 4 pp 1373ndash1380 2003

[137] D Nowak M Kasielski A Antczak T Pietras and PBialasiewicz ldquoIncreased content of thiobarbituric acid-reactivesubstances and hydrogen peroxide in the expired breath con-densate of patients with stable chronic obstructive pulmonarydisease no significant effect of cigarette smokingrdquo RespiratoryMedicine vol 93 no 6 pp 389ndash396 1999

[138] M Corradi I Rubinstein R Andreoli et al ldquoAldehydes inexhaled breath condensate of patients with chronic obstructive


pulmonary diseaserdquo The American Journal of Respiratory andCritical Care Medicine vol 167 no 10 pp 1380ndash1386 2003

[139] M Corradi P Pignatti P Manini et al ldquoComparison betweenexhaled and sputum oxidative stress biomarkers in chronicairway inflammationrdquo European Respiratory Journal vol 24 no6 pp 1011ndash1017 2004

[140] B Antus GHarnasi ODrozdovszky and I Barta ldquoMonitoringoxidative stress during chronic obstructive pulmonary diseaseexacerbations using malondialdehyderdquo Respirology vol 19 no1 pp 74ndash79 2014

[141] K Kostikas M Gaga G Papatheodorou T Karamanis DOrphanidou and S Loukides ldquoLeukotriene B

4

in exhaledbreath condensate and sputum supernatant in patients withCOPD and asthmardquo Chest vol 127 no 5 pp 1553ndash1559 2005

[142] J L Izquierdo C Almonacid T Parra and J Perez ldquoSystemicand lung inflammation in 2 phenotypes of chronic obstructivepulmonary diseaserdquo Archivos de Bronconeumologia vol 42 no7 pp 332ndash337 2006

[143] Z L Borrill R C Starkey and S D Singh ldquoVariability ofexhaled breath condensate leukotriene B

4

and 8-isoprostane inCOPD patientsrdquo International Journal of Chronic ObstructivePulmonary Disease vol 2 no 1 pp 71ndash76 2007

[144] M Fumagalli F Ferrari M Luisetti et al ldquoProfiling theproteome of exhaled breath condensate in healthy smokersand COPD patients by LC-MSMSrdquo International Journal ofMolecular Sciences vol 13 no 11 pp 13894ndash13910 2012

[145] G Warwick P S Thomas and D H Yates ldquoNon-invasivebiomarkers in exacerbations of obstructive lung diseaserdquoRespirology vol 18 no 5 pp 874ndash884 2013

[146] A Antczak M Ciebiada T Pietras W J Piotrowski Z Kur-manowska and P Gorski ldquoExhaled eicosanoids and biomarkersof oxidative stress in exacerbation of chronic obstructive pul-monary diseaserdquo Archives of Medical Science vol 8 no 2 pp277ndash285 2012

[147] W A Biernacki S A Kharitonov and P J Barnes ldquoIncreasedleukotriene B4 and 8-isoprostane in exhaled breath condensateof patients with exacerbations of COPDrdquoThorax vol 58 no 4pp 294ndash298 2003

[148] G E Carpagnano O Resta M P Foschino-Barbaro et alldquoExhaled Interleukine-6 and 8-isoprostane in chronic obstruc-tive pulmonary disease effect of carbocysteine lysine saltmonohydrate (SCMC-Lys)rdquo European Journal of Pharmacologyvol 505 no 1ndash3 pp 169ndash175 2004

[149] V Rihak P Zatloukal J Chladkova A Zimulova Z Havlinovaand J Chladek ldquoNitrite in exhaled breath condensate as amarker of nitrossative stress in the airways of patients withasthma COPD and idiopathic pulmonary fibrosisrdquo Journal ofClinical Laboratory Analysis vol 24 no 5 pp 317ndash322 2010

[150] A R Koczulla S Noeske C Herr et al ldquoAlpha-1 antitrypsin iselevated in exhaled breath condensate and serum in exacerbatedCOPD patientsrdquo Respiratory Medicine vol 106 no 1 pp 120ndash126 2012

[151] S Kwiatkowska K Noweta M Zieba D Nowak andP Bialasiewicz ldquoEnhanced exhalation of matrix metallo-proteinase-9 and tissue inhibitor of metalloproteinase-1 inpatients with COPD exacerbation a prospective studyrdquo Respi-ration vol 84 no 3 pp 231ndash241 2012

[152] J L Corhay C Moermans M Henket D N Dang B Duysinxand R Louis ldquoIncreased of exhaled breath condensate neu-trophil chemotaxis in acute exacerbation of COPDrdquo RespiratoryResearch vol 15 no 1 pp 115ndash126 2014

[153] N Kubysheva S Soodaeva L Postnikova V Novikov A Mak-simova and A Chuchalin ldquoAssociations between indicators ofnitrosative stress and levels of soluble HLA-I CD95 moleculesin patients with COPDrdquo Journal of Chronic Obstructive Pul-monary Disease vol 11 no 6 pp 639ndash644 2014

[154] N L Tateosian M J Costa D Guerrieri A Barro J A Mazzeiand H E Chuluyan ldquoInflammatory mediators in exhaledbreath condensate of healthy donors and exacerbated COPDpatientsrdquo Cytokine vol 58 no 3 pp 361ndash367 2012

[155] T Zakharkina A-R Koczulla O Mardanova A Hattesohland R Bals ldquoDetection of microorganisms in exhaled breathcondensate during acute exacerbations of COPDrdquo Respirologyvol 16 no 6 pp 932ndash938 2011

[156] E C Lases V A M Duurkens W B M Gerritsen and F J LM Hass ldquoOxidative stress after lung resection therapyrdquo Chestvol 117 pp 99ndash103 2000

[157] G E Carpagnano O Resta M P Foschino-Barbaro EGramiccioni and F Carpagnano ldquoInterleukin-6 is increased inbreath condensate of patients with non-small cell lung cancerrdquoInternational Journal of BiologicalMarkers vol 17 no 2 pp 141ndash145 2002

[158] G E Carpagnano A Spanevello C Curci et al ldquoIL-2 TNF-120572 and leptin local versus systemic concentrations in NSCLCpatientsrdquo Oncology Research vol 16 no 8 pp 375ndash381 2007

[159] L Brussino B Culla C Bucca et al ldquoInflammatory cytokinesand VEGF measured in exhaled breath condensate are corre-lated with tumor mass in non-small cell lung cancerrdquo Journal ofBreath Research vol 8 no 2 Article ID 027110 2014

[160] G E Carpagnano M P Foschino-Barbaro O Restaa EGramiccioni and F Carpagnano ldquoEndothelin-1 is increasedin the breath condensate of patients with non-small-cell lungcancerrdquo Oncology vol 66 no 3 pp 180ndash184 2004

[161] E Dalaveris T Kerenidi A Katsabeki-Katsafli et al ldquoVEGFTNF-120572 and 8-isoprostane levels in exhaled breath condensateand serum of patients with lung cancerrdquo Lung Cancer vol 64no 2 pp 219ndash225 2009

[162] T Kullmann I Barta E Csiszer B Antus and I HorvathldquoDifferential cytokine pattern in the exhaled breath of patientswith lung cancerrdquo Pathology and Oncology Research vol 14 no4 pp 481ndash483 2008

[163] I Barta T Kullmann E Csiszer and B Antus ldquoAnalysisof cytokine pattern in exhaled breath condensate of patientswith squamous cell lung carcinomardquo International Journal ofBiological Markers vol 25 no 1 pp 52ndash56 2010

[164] W Jungraithmayr C Frings G Zissel A Prasse B Passlickand E Stoelben ldquoInflammatory markers in exhaled breathcondensate following lung resection for bronchial carcinomardquoRespirology vol 13 no 7 pp 1022ndash1027 2008

[165] C Gessner B Rechner S Hammerschmidt et al ldquoAngiogenicmarkers in breath condensate identify non-small cell lungcancerrdquo Lung Cancer vol 68 no 2 pp 177ndash184 2010

[166] G E Carpagnano A Spanevello G P Palladino et alldquoCigarette smoke and increased COX-2 and survivin levels inexhaled breath condensate of lung cancer patients how hot isthe linkrdquo Lung Cancer vol 67 no 1 pp 108ndash113 2010

[167] G E Carpagnano G P Palladino D Martinelli D LacedoniaS Orlando and M P Foschino-Barbaro ldquoExhaled matrixmetalloproteinase-9 in lung cancerrdquo Rejuvenation Research vol15 no 4 pp 359ndash365 2012

[168] Z Cheng A K Chan C R Lewis P S Thomas and M JRaftery ldquoComparative proteomics analysis of exhaled breath


condensate in lung cancer patientsrdquo Journal of Cancer Therapyvol 2 no 1 pp 1ndash8 2011

[169] Y Zou L Wang C Zhao et al ldquoCEA SCC and NSE levels inexhaled breath condensatemdashpossible markers for early detec-tion of lung cancerrdquo Journal of Breath Research vol 7 no 4Article ID 047101 2013

[170] C Gessner H Kuhn K Toepfer S Hammerschmidt J Schauerand H Wirtz ldquoDetection of p53 gene mutations in exhaledbreath condensate of non-small cell lung cancer patientsrdquo LungCancer vol 43 no 2 pp 215ndash222 2004

[171] G E Carpagnano M P Foschino-Barbaro G Mule et al ldquo3pmicrosatellite alterations in exhaled breath condensate frompatients with non-small cell lung cancerrdquo American Journal ofRespiratory and Critical Care Medicine vol 172 no 6 pp 738ndash744 2005

[172] G E Carpagnano M P Foschino-Barbaro A Spanevello et alldquo3p microsatellite signature in exhaled breath condensate andtumor tissue of patients with lung cancerrdquo American Journal ofRespiratory and Critical Care Medicine vol 177 no 3 pp 337ndash341 2008

[173] G E Carpagnano A Spanevello F Carpagnano et al ldquoProg-nostic value of exhaledmicrosatellite alterations at 3p inNSCLCpatientsrdquo Lung Cancer vol 64 no 3 pp 334ndash340 2009

[174] G E Carpagnano G P Palladino C Gramiccioni M PFoschino Barbaro and D Martinelli ldquoExhaled ERCC-1 andERCC-2 microsatellite alterations in NSCLC patientsrdquo LungCancer vol 68 no 2 pp 305ndash307 2010

[175] P Mozzoni I Banda M Goldoni et al ldquoPlasma and EBCmicroRNAs as early biomarkers of non-small-cell lung cancerrdquoBiomarkers vol 18 no 8 pp 679ndash686 2013

[176] J Kordiak J Szemraj K Hamara P Bialasiewicz and DNowak ldquoComplete surgical resection of lung tumor decreasesexhalation of mutated KRAS oncogenerdquo Respiratory Medicinevol 106 no 9 pp 1293ndash1300 2012

[177] W Han T Wang A A Reilly S M Keller and S D SpivackldquoGene promoter methylation assayed in exhaled breath withdifferences in smokers and lung cancer patientsrdquo RespiratoryResearch vol 10 article 86 2009

[178] G E Carpagnano A Koutelou M I Natalicchio et al ldquoHPVin exhaled breath condensate of lung cancer patientsrdquo BritishJournal of Cancer vol 105 no 8 pp 1183ndash1190 2011

[179] G E Carpagnano D Lacedonia G P Palladino et alldquoAspergillus spp colonization in exhaled breath condensateof lung cancer patients from Puglia Region of Italyrdquo BMCPulmonary Medicine vol 14 no 1 article 22 2014

[180] C Gessner S Hammerschmidt H Kuhn et al ldquoExhaled breathcondensate acidification in acute lung injuryrdquo RespiratoryMedicine vol 97 no 11 pp 1188ndash1194 2003

[181] B K Walsh D J Mackey T Pajewski Y Yu B M Gaston andJ F Hunt ldquoExhaled-breath condensate pH can be safely andcontinuously monitored in mechanically ventilated patientsrdquoRespiratory Care vol 51 no 10 pp 1125ndash1131 2006

[182] O Roca S Gomez-Olles M-J Cruz X Munoz M J DGriffiths and J R Masclans ldquoMechanical ventilation induceschanges in exhaled breath condensate of patients without lunginjuryrdquo Respiratory Medicine vol 104 no 6 pp 822ndash828 2010

[183] I Korovesi E Papadomichelakis S E Orfanos et al ldquoExhaledbreath condensate in mechanically ventilated brain-injuredpatients with no lung injury or sepsisrdquo Anesthesiology vol 114no 5 pp 1118ndash1129 2011

[184] P Montushi ldquoIsoprostanes prostanoids and leukotrienes inexhaled breath condensaterdquo in New Prespectives in MonitoringLung Inflammation Analysis of Exhaled Breath Condensate PMontushi Ed pp 53ndash66 CRC Press Boca Raton Fla USA2005

[185] O Roca S Gomez-Olles M-J Cruz X Munoz M J DGriffiths and J R Masclans ldquoEffects of salbutamol on exhaledbreath condensate biomarkers in acute lung injury prospectiveanalysisrdquo Critical Care vol 12 no 3 article R72 2008

[186] L J Nannini R Quintana D H Bagilet et al ldquoExhaled breathcondensate pH in mechanically ventilated patientsrdquo MedicinaIntensiva vol 37 no 9 pp 593ndash599 2013

[187] C T Carpenter P V Price and B W Christman ldquoExhaledbreath condensate isoprostanes are elevated in patients withacute lung injury or ARDSrdquo Chest vol 114 no 6 pp 1653ndash16591998

[188] S R Baldwin RH Simon CMGrum LH Ketai L A Boxerand LJ Devall ldquoOxidant activity in expired breath of patientswith adult respiratory distress syndromerdquoThe Lancet vol 1 no8471 pp 11ndash14 1986

[189] U Sack R Scheibe M Wotzel et al ldquoMultiplex analysis ofcytokines in exhaled breath condensaterdquo Cytometry Part A vol69 no 3 pp 169ndash172 2006

[190] E D Moloney S E Mumby R Gajdocsi et al ldquoExhaled breathcondensate detects markers of pulmonary inflammation aftercardiothoracic surgeryrdquo American Journal of Respiratory andCritical Care Medicine vol 169 no 1 pp 64ndash69 2004

[191] C Gessner S Hammerschmidt H Kuhn et al ldquoExhaled breathcondensate nitrite and its relation to tidal volume in acute lunginjuryrdquo Chest vol 124 no 3 pp 1046ndash1052 2003

[192] C Gessner H Dihazi S Brettschneider et al ldquoPresenceof cytokeratins in exhaled breath condensate of mechanicalventilated patientsrdquo Respiratory Medicine vol 102 no 2 pp299ndash306 2008

[193] G Horonenko J C Hoyt R A Robbins et al ldquoSolubletriggering receptor expressed on myeloid cell-1 is increased inpatients with ventilator-associated pneumonia a preliminaryreportrdquo Chest vol 132 no 1 pp 58ndash63 2007

[194] S Chow P S Thomas M Malouf and D H Yates ldquoExhaledbreath condensate (EBC) biomarkers in pulmonary fibrosisrdquoJournal of Breath Research vol 6 no 1 Article ID 016004 2012

[195] S B Montesi S K Mathai L N Brenner et al ldquoDocosate-traenoyl LPA is elevated in exhaled breath condensate inidiopathic pulmonary fibrosisrdquo BMC Pulmonary Medicine vol14 no 1 article 5 2014

[196] S Tate GMacGregor M Davis J A Innes and A P GreeningldquoAirways in cystic fibrosis are acidified detection by exhaledbreath condensaterdquoThorax vol 57 no 11 pp 926ndash929 2002

[197] B Antus I Barta E Csiszer and K Kelemen ldquoExhaled breathcondensate pH in patients with cystic fibrosisrdquo InflammationResearch vol 61 no 10 pp 1141ndash1147 2012

[198] B Balint S A Kharitonov T Hanazawa et al ldquoIncreasednitrotyrosine in exhaled breath condensate in cystic fibrosisrdquoEuropean Respiratory Journal vol 17 no 6 pp 1201ndash1207 2001

[199] S Celio H Troxler S S Durka et al ldquoFree 3-nitrotyrosinein exhaled breath condensates of children fails as a markerfor oxidative stress in stable cystic fibrosis and asthmardquo NitricOxide vol 15 no 3 pp 226ndash232 2006

[200] F Horak Jr A Moeller F Singer et al ldquoLongitudinal moni-toring of pediatric cystic fibrosis lung disease using nitrite inexhaled breath condensaterdquo Pediatric Pulmonology vol 42 no12 pp 1198ndash1206 2007


[201] C M H H T Robroeks P P R Rosias D van Vliet et alldquoBiomarkers in exhaled breath condensate indicate presenceand severity of cystic fibrosis in childrenrdquo Pediatric Allergy andImmunology vol 19 no 7 pp 652ndash659 2008

[202] S Cunningham J R McColm L P Ho A P Greening andT G Marshall ldquoMeasurement of inflammatory markers in thebreath condensate of children with cystic fibrosisrdquo EuropeanRespiratory Journal vol 15 no 5 pp 955ndash957 2000

[203] G E Carpagnano P J BarnesDMGeddesM EHodson andS A Kharitonov ldquoIncreased leukotriene B

4

and interleukin-6 inexhaled breath condensate in cystic fibrosisrdquo American Journalof Respiratory and Critical Care Medicine vol 167 no 8 pp1109ndash1112 2003

[204] A Bodini C DrsquoOrazio D G Peroni et al ldquoIL-8 and pHvalues in exhaled condensate after antibiotics in cystic fibrosischildrenrdquo International Journal of Immunopathology and Phar-macology vol 20 no 3 pp 467ndash472 2007

[205] C M H H T Robroeks Q Jobsis J G M C Damoiseauxet al ldquoCytokines in exhaled breath condensate of childrenwith asthma and cystic fibrosisrdquo Annals of Allergy Asthma andImmunology vol 96 no 2 pp 349ndash355 2006

[206] G Warwick E Kotlyar S Chow P S Thomas and D H YatesldquoExhaled breath condensate in pulmonary arterial hyperten-sionrdquo Journal of Breath Research vol 6 no 3 Article ID 0360062012

[207] H He Y Tao X Chen et al ldquoHigh levels of interleukin-6 and 8-iso-prostaglandin in the exhaled breath condensate and serumof patients with chronic obstructive pulmonary disease relatedpulmonary hypertensionrdquo Chinese Medical Journal vol 127 no9 pp 1608ndash1612 2014

[208] P Carratu C Scoditti M Maniscalco et al ldquoExhaled andarterial levels of endothelin-1 are increased and correlate withpulmonary systolic pressure in COPD with pulmonary hyper-tensionrdquo BMC Pulmonary Medicine vol 8 article 20 2008

[209] J K Mansoor E S Schelegle C E Davis et al ldquoAnalysis ofvolatile compounds in exhaled breath condensate in patientswith severe pulmonary arterial hypertensionrdquo PLoS ONE vol9 no 4 Article ID e95331 2014

[210] H Ahmadzai B Cameron J Chui A Lloyd D Wakefield andP S Thomas ldquoMeasurement of neopterin TGF-120573

1

and ACEin the exhaled breath condensate of patients with sarcoidosisrdquoJournal of Breath Research vol 7 no 4 Article ID 046003 2013

[211] M A Biltagi M A Maguid M A Ghafar and E Farid ldquoCor-relation of 8-isoprostane interleukin-6 and cardiac functionswith clinical score in childhood obstructive sleep apnoeardquo ActaPaediatrica vol 97 no 10 pp 1397ndash1405 2008

[212] A D Goldbart J Krishna R C Li L D Serpero and DGozal ldquoInflammatory mediators in exhaled breath condensateof children with obstructive sleep apnea syndromerdquo Chest vol130 no 1 pp 143ndash148 2006

[213] G Malakasioti E Alexopoulos C Befani et al ldquoOxidativestress and inflammatory markers in the exhaled breath conden-sate of children with OSArdquo Sleep and Breathing vol 16 no 3pp 703ndash708 2012

[214] V Vlasic J Trifunovic I Cepelak P Nimac R Z Topic and SDodig ldquoUrates in exhaled breath condensate of children withobstructive sleep apneardquo Biochemia Medica vol 21 no 2 pp139ndash144 2011

[215] G E Carpagnano S A Kharitonov O Resta M P Foschino-Barbaro E Gramiccioni and P J Barnes ldquo8-isoprostane inexhaled breath condensate of patients with obstructive sleep

apnea after night and is reduced by continuous positive airwaypressure therapyrdquo Chest vol 124 no 4 pp 1386ndash1392 2003

[216] G E Carpagnano S A Kharitonov O Resta M P Foschino-Barbaro E Gramiccioni and P J Barnes ldquoIncreased 8-isoprostane and interleukin-6 in breath condensate ofobstruc-tive sleep apnea patientsrdquo Chest vol 122 no 4 pp 1162ndash11672002

[217] S Antonopoulou S Loukides G Papatheodorou C Roussosand M Alchanatis ldquoAirway inflammation in obstructive sleepapnea is leptin themissing linkrdquoRespiratoryMedicine vol 102no 10 pp 1399ndash1405 2008

[218] M Westhoff and P Litterst ldquoObstructive sleep apnoea andoxidative stressrdquo Pneumologie vol 66 no 10 pp 610ndash615 2012

[219] G E Carpagnano A Spanevello R Sabato et al ldquoSystemicand airway inflammation in sleep apnea and obesity the roleof ICAM-1 and IL-8rdquo Translational Research vol 155 no 1 pp35ndash43 2010

[220] A Nielepkowicz-Gozdzinska W Fendler E Robak et alldquoExhaled IL-8 in systemic lupus erythematosus with and with-out pulmonary fibrosisrdquo Archivum Immunologiae et TherapiaeExperimentalis vol 62 no 3 pp 231ndash238 2014

[221] A Nielepkowicz-Gozdzinska W Fendler E Robak et alldquoExhaled cytokines in systemic lupus erythematosus with lunginvolvementrdquo Polskie Archiwum Medycyny Wewnetrznej vol123 no 4 pp 141ndash148 2013

[222] G Rolla M Bruno L Bommarito et al ldquoBreath analysis inpatients with end-stage renal disease effect of haemodialysisrdquoEuropean Journal of Clinical Investigation vol 38 no 10 pp728ndash733 2008

[223] J Rysz M Kasielski J Apanasiewicz et al ldquoIncreased hydrogenperoxide in the exhaled breath of uraemic patients unaffected byhaemodialysisrdquoNephrology Dialysis Transplantation vol 19 no1 pp 158ndash163 2004

[224] C R E Esther R C BoucherM R Johnson et al ldquoAirway drugpharmacokinetics via analysis of exhaled breath condensaterdquoPulmonary Pharmacology ampTherapeutics vol 27 no 1 pp 76ndash82 2014

[225] O Beck S Sandqvist P Eriksen J Franck and G PalmskogldquoDetermination of methadone in exhaled breath condensate byliquid chromatography-tandem mass spectrometryrdquo Journal ofAnalytical Toxicology vol 35 no 3 pp 129ndash133 2011

[226] Hippocrates The Corpus The Hippocratic Writings KaplanPublishing New York NY USA 2008

[227] P Montuschi N Mores A Trove C Mondino and P J BarnesldquoThe electronic nose in respiratory medicinerdquo Respiration vol85 no 1 pp 72ndash84 2013

[228] A Koutsokera S Loukides K I Gourgoulianis and KKostikas ldquoBiomarkers in the exhaled breath condensate ofhealthy adults mapping the path towards reference valuesrdquoCurrent Medicinal Chemistry vol 15 no 6 pp 620ndash630 2008


Figure 1 EcoScreen 2 device for exhaled breath condensate (EBC) collection (FILT Lungen-amp Thorax Diagnostik GmbH) EcoScreen 2electrically refrigerates exhaled air conducted through a lamellar PFTE coated aluminum double lumen system EBC is formatted in adisposable polyethylene bag (at approximately minus10∘C) It also allows the fractionated collection of EBC from different areas of the bronchialtree into two disposable polyethylene bags so that the dead space condensate which contains biomarkers of no clinical relevance can bediscarded The device is not portable and weighs 20 kg (published with permission from FILT Lungen-ampThorax Diagnostik GmbH)

gastroesophageal reflux GERD chronic obstructive pul-monary disease smoking COPD lung cancer mechanicalventilation non small lung cancer NSCLC cystic fibro-sis pulmonary arterial hypertension PAH idiopathic pul-monary fibrosis interstitial lung diseases obstructive sleepapnea OSA and drugs

We found 12600 related articles in total in Google Scholar1807 in ScienceDirect and 1081 in PubMedMedline pub-lished from 1980 to October 2014 We used the followingas exclusion criteria (i) studies available only in languagesother than English (ii) studies published only in the formof abstract (iii) studies published in journals without impactfactor and (iv) studies applying EBC-analysis in veterinarymedicine Furthermore we preferably used original investi-gation articles instead of review articles when possible

207 original investigation papers and 21 review articleswhere eligible after applying the abovementioned criteria 47of them refer tomethodological and technical aspects and 181refer to the diagnostic aspects of EBC-analysis

2 Methodology of EBC Collection andAnalysis

21 EBC Collection Devices EBC collection is a simplenoninvasive technique requiring only quiet breathing of thesubject via a system of exhaled air cooling After applying anose-clip the subject breaths quietly for 10minutes through aspecialmouthpiece with a salivary trap and a single-way valveattached diverting the exhaled airflow through a Teflon orpolypropylene tube inside a cooling containerThere exhaledair in the form of droplets is converted to exhaled breathcondensate (EBC) [1 10]

Various house-built devices have been described basedon two simple layouts (a) a Teflon tube dipped in abucket filled with ice and (b) a double glass layer containerwhere exhaled air condensation takes place between the twolayers [1] Commercially available devices are EcoScreen

Turbodeccs Rtube andAnacon glass condenser based on theabovementioned layouts

The EcoScreen device electrically refrigerates exhaledair conducted through a lamellar PFTE coated aluminumdouble lumen system EBC is then formatted in a dispos-able polypropylene collecting cup (at approximately minus10∘C)EcoScreen 1 device has been extensively used in researchprotocols However it is not currently manufactured due toseveral technical drawbacks including lack ofmanual controlof condensing temperature and cleaning requirements ofthe device between consequent trials [11] Therefore theEcoScreen 1 has been replaced by EcoScreen 2 device whichallows the fractionated collection of EBC from different areasof the bronchial tree into two disposable polyethylene bags sothat the dead space condensate which contains biomarkers ofno clinical relevance may be discarded EcoScreen 1 and 2 arenot portable and weight 20 kg (Figure 1)

TurboDECCS consists of a portable TurboUnit and a dis-posable DECCS collection system DECCS is equipped witha mouthpiece a one-way valve a tube and a collection cellinserted in a Peltier-type electrical cooling system (Figure 2)

RTube is also portable and can be used by unsupervisedsubjects at home RTube disposable collection system consistsof a large Tee section (made of polypropylene (PP)) whichseparates saliva from the exhaled breath a one-way valve(made of silicone rubber) and a PP collection tube whichis cooled by a cooling-sleeve placed around (Figure 3)

ANACON condenser has been used by many researchgroups especially in mechanically ventilated patients [1012] It is a condensing device which can be attached to theexpiratory branch of the ventilator circuit Condensationtemperature is constantly monitored within the limits ofminus15∘C to minus5∘C (Figure 4)

22 Sampling

221 Sampling Duration After 10min of quiet breathing 1ndash3mL EBC is collected from adult subjects (1198811015840 = 100 Lmin)


DECCS

DECCS

Turbo








119864of 75 Lmin


119879(tidal






3

To the ventilator


1313

21

13 13

5

9966

4

Patient

Filter191

∘Cminus111

∘C



2O2levels in



119864)






119899119881ALF

119899119881ALF + 119881vapor (1)















2) Therefore













3)












2O2levels were









2O2



4 LTE4







4have also been







2have been


2possibly reflects
















119909(nitrites


119909levels


119909











pH









ILsIL-4


uarr [102]IL-6


uarr [104]IL-10

uarr [70]IL-17

uarr [104]INF-120574





ET-1uarr [106 107] uarruarr [106 107]




CCL11uarr [112]














1values [127 129]


4levels [130] sug-







1decrease has







2O2increase and FEV

1reduction











4may be indicative





pH










2have also


4





1AD)











and H2O2






1-antitrypsin-


4was found









2O2and urine-MDA


2O2and urine-MDA










pHharr [132]



Aldehydes




LTs




Cys-LTs uarr [3]
































2O2(168 plusmn






NO2minusNO3












4were identified





ILsIL-2 uarr [158]


TNF-120572 uarr [158 159 161]IL-1120573 IL-6






GRo-aEotaxin-2




NSEGenomic analysis











1120572












2O2[218]



2O2levels



















uarr [194 195]










1120572

8-isoprostaneIL-6


Sarcoidosis




Pediatric patients

8-IsoprostaneIL-6




Adult patients





pHNitritesNitrates















References














































2















































4



4











2


















































4




4

































































4









1






















DECCS

DECCS

Turbo








119864of 75 Lmin


119879(tidal






3

To the ventilator


1313

21

13 13

5

9966

4

Patient

Filter191

∘Cminus111

∘C



2O2levels in



119864)






119899119881ALF

119899119881ALF + 119881vapor (1)















2) Therefore













3)












2O2levels were









2O2



4 LTE4







4have also been







2have been


2possibly reflects
















119909(nitrites


119909levels


119909











pH









ILsIL-4


uarr [102]IL-6


uarr [104]IL-10

uarr [70]IL-17

uarr [104]INF-120574





ET-1uarr [106 107] uarruarr [106 107]




CCL11uarr [112]














1values [127 129]


4levels [130] sug-







1decrease has







2O2increase and FEV

1reduction











4may be indicative





pH










2have also


4





1AD)











and H2O2






1-antitrypsin-


4was found









2O2and urine-MDA


2O2and urine-MDA










pHharr [132]



Aldehydes




LTs




Cys-LTs uarr [3]
































2O2(168 plusmn






NO2minusNO3












4were identified





ILsIL-2 uarr [158]


TNF-120572 uarr [158 159 161]IL-1120573 IL-6






GRo-aEotaxin-2




NSEGenomic analysis











1120572












2O2[218]



2O2levels



















uarr [194 195]










1120572

8-isoprostaneIL-6


Sarcoidosis




Pediatric patients

8-IsoprostaneIL-6




Adult patients





pHNitritesNitrates















References














































2















































4



4











2


















































4




4

































































4









1






















3

To the ventilator


1313

21

13 13

5

9966

4

Patient

Filter191

∘Cminus111

∘C



2O2levels in



119864)






119899119881ALF

119899119881ALF + 119881vapor (1)















2) Therefore













3)












2O2levels were









2O2



4 LTE4







4have also been







2have been


2possibly reflects
















119909(nitrites


119909levels


119909











pH









ILsIL-4


uarr [102]IL-6


uarr [104]IL-10

uarr [70]IL-17

uarr [104]INF-120574





ET-1uarr [106 107] uarruarr [106 107]




CCL11uarr [112]














1values [127 129]


4levels [130] sug-







1decrease has







2O2increase and FEV

1reduction











4may be indicative





pH










2have also


4





1AD)











and H2O2






1-antitrypsin-


4was found









2O2and urine-MDA


2O2and urine-MDA










pHharr [132]



Aldehydes




LTs




Cys-LTs uarr [3]
































2O2(168 plusmn






NO2minusNO3












4were identified





ILsIL-2 uarr [158]


TNF-120572 uarr [158 159 161]IL-1120573 IL-6






GRo-aEotaxin-2




NSEGenomic analysis











1120572












2O2[218]



2O2levels



















uarr [194 195]










1120572

8-isoprostaneIL-6


Sarcoidosis




Pediatric patients

8-IsoprostaneIL-6




Adult patients





pHNitritesNitrates















References














































2















































4



4











2


















































4




4

































































4









1






























2) Therefore













3)












2O2levels were









2O2



4 LTE4







4have also been







2have been


2possibly reflects
















119909(nitrites


119909levels


119909











pH









ILsIL-4


uarr [102]IL-6


uarr [104]IL-10

uarr [70]IL-17

uarr [104]INF-120574





ET-1uarr [106 107] uarruarr [106 107]




CCL11uarr [112]














1values [127 129]


4levels [130] sug-







1decrease has







2O2increase and FEV

1reduction











4may be indicative





pH










2have also


4





1AD)











and H2O2






1-antitrypsin-


4was found









2O2and urine-MDA


2O2and urine-MDA










pHharr [132]



Aldehydes




LTs




Cys-LTs uarr [3]
































2O2(168 plusmn






NO2minusNO3












4were identified





ILsIL-2 uarr [158]


TNF-120572 uarr [158 159 161]IL-1120573 IL-6






GRo-aEotaxin-2




NSEGenomic analysis











1120572












2O2[218]



2O2levels



















uarr [194 195]










1120572

8-isoprostaneIL-6


Sarcoidosis




Pediatric patients

8-IsoprostaneIL-6




Adult patients





pHNitritesNitrates















References














































2















































4



4











2


















































4




4

































































4









1


























2O2levels were









2O2



4 LTE4







4have also been







2have been


2possibly reflects
















119909(nitrites


119909levels


119909











pH









ILsIL-4


uarr [102]IL-6


uarr [104]IL-10

uarr [70]IL-17

uarr [104]INF-120574





ET-1uarr [106 107] uarruarr [106 107]




CCL11uarr [112]














1values [127 129]


4levels [130] sug-







1decrease has







2O2increase and FEV

1reduction











4may be indicative





pH










2have also


4





1AD)











and H2O2






1-antitrypsin-


4was found









2O2and urine-MDA


2O2and urine-MDA










pHharr [132]



Aldehydes




LTs




Cys-LTs uarr [3]
































2O2(168 plusmn






NO2minusNO3












4were identified





ILsIL-2 uarr [158]


TNF-120572 uarr [158 159 161]IL-1120573 IL-6






GRo-aEotaxin-2




NSEGenomic analysis











1120572












2O2[218]



2O2levels



















uarr [194 195]










1120572

8-isoprostaneIL-6


Sarcoidosis




Pediatric patients

8-IsoprostaneIL-6




Adult patients





pHNitritesNitrates















References














































2















































4



4











2


















































4




4

































































4









1




























119909(nitrites


119909levels


119909











pH









ILsIL-4


uarr [102]IL-6


uarr [104]IL-10

uarr [70]IL-17

uarr [104]INF-120574





ET-1uarr [106 107] uarruarr [106 107]




CCL11uarr [112]














1values [127 129]


4levels [130] sug-







1decrease has







2O2increase and FEV

1reduction











4may be indicative





pH










2have also


4





1AD)











and H2O2






1-antitrypsin-


4was found









2O2and urine-MDA


2O2and urine-MDA










pHharr [132]



Aldehydes




LTs




Cys-LTs uarr [3]
































2O2(168 plusmn






NO2minusNO3












4were identified





ILsIL-2 uarr [158]


TNF-120572 uarr [158 159 161]IL-1120573 IL-6






GRo-aEotaxin-2




NSEGenomic analysis











1120572












2O2[218]



2O2levels



















uarr [194 195]










1120572

8-isoprostaneIL-6


Sarcoidosis




Pediatric patients

8-IsoprostaneIL-6




Adult patients





pHNitritesNitrates















References














































2















































4



4











2


















































4




4

































































4









1
























pH









ILsIL-4


uarr [102]IL-6


uarr [104]IL-10

uarr [70]IL-17

uarr [104]INF-120574





ET-1uarr [106 107] uarruarr [106 107]




CCL11uarr [112]














1values [127 129]


4levels [130] sug-







1decrease has







2O2increase and FEV

1reduction











4may be indicative





pH










2have also


4





1AD)











and H2O2






1-antitrypsin-


4was found









2O2and urine-MDA


2O2and urine-MDA










pHharr [132]



Aldehydes




LTs




Cys-LTs uarr [3]
































2O2(168 plusmn






NO2minusNO3












4were identified





ILsIL-2 uarr [158]


TNF-120572 uarr [158 159 161]IL-1120573 IL-6






GRo-aEotaxin-2




NSEGenomic analysis











1120572












2O2[218]



2O2levels



















uarr [194 195]










1120572

8-isoprostaneIL-6


Sarcoidosis




Pediatric patients

8-IsoprostaneIL-6




Adult patients





pHNitritesNitrates















References














































2















































4



4











2


















































4




4

































































4









1

























1values [127 129]


4levels [130] sug-







1decrease has







2O2increase and FEV

1reduction











4may be indicative





pH










2have also


4





1AD)











and H2O2






1-antitrypsin-


4was found









2O2and urine-MDA


2O2and urine-MDA










pHharr [132]



Aldehydes




LTs




Cys-LTs uarr [3]
































2O2(168 plusmn






NO2minusNO3












4were identified





ILsIL-2 uarr [158]


TNF-120572 uarr [158 159 161]IL-1120573 IL-6






GRo-aEotaxin-2




NSEGenomic analysis











1120572












2O2[218]



2O2levels



















uarr [194 195]










1120572

8-isoprostaneIL-6


Sarcoidosis




Pediatric patients

8-IsoprostaneIL-6




Adult patients





pHNitritesNitrates















References














































2















































4



4











2


















































4




4

































































4









1
























pH










2have also


4





1AD)











and H2O2






1-antitrypsin-


4was found









2O2and urine-MDA


2O2and urine-MDA










pHharr [132]



Aldehydes




LTs




Cys-LTs uarr [3]
































2O2(168 plusmn






NO2minusNO3












4were identified





ILsIL-2 uarr [158]


TNF-120572 uarr [158 159 161]IL-1120573 IL-6






GRo-aEotaxin-2




NSEGenomic analysis











1120572












2O2[218]



2O2levels



















uarr [194 195]










1120572

8-isoprostaneIL-6


Sarcoidosis




Pediatric patients

8-IsoprostaneIL-6




Adult patients





pHNitritesNitrates















References














































2















































4



4











2


















































4




4

































































4









1
























1-antitrypsin-


4was found









2O2and urine-MDA


2O2and urine-MDA










pHharr [132]



Aldehydes




LTs




Cys-LTs uarr [3]
































2O2(168 plusmn






NO2minusNO3












4were identified





ILsIL-2 uarr [158]


TNF-120572 uarr [158 159 161]IL-1120573 IL-6






GRo-aEotaxin-2




NSEGenomic analysis











1120572












2O2[218]



2O2levels



















uarr [194 195]










1120572

8-isoprostaneIL-6


Sarcoidosis




Pediatric patients

8-IsoprostaneIL-6




Adult patients





pHNitritesNitrates















References














































2















































4



4











2


















































4




4

































































4









1
























pHharr [132]



Aldehydes




LTs




Cys-LTs uarr [3]
































2O2(168 plusmn






NO2minusNO3












4were identified





ILsIL-2 uarr [158]


TNF-120572 uarr [158 159 161]IL-1120573 IL-6






GRo-aEotaxin-2




NSEGenomic analysis











1120572












2O2[218]



2O2levels



















uarr [194 195]










1120572

8-isoprostaneIL-6


Sarcoidosis




Pediatric patients

8-IsoprostaneIL-6




Adult patients





pHNitritesNitrates















References














































2















































4



4











2


















































4




4

































































4









1









































2O2(168 plusmn






NO2minusNO3












4were identified





ILsIL-2 uarr [158]


TNF-120572 uarr [158 159 161]IL-1120573 IL-6






GRo-aEotaxin-2




NSEGenomic analysis











1120572












2O2[218]



2O2levels



















uarr [194 195]










1120572

8-isoprostaneIL-6


Sarcoidosis




Pediatric patients

8-IsoprostaneIL-6




Adult patients





pHNitritesNitrates















References














































2















































4



4











2


















































4




4

































































4









1




























2O2(168 plusmn






NO2minusNO3












4were identified





ILsIL-2 uarr [158]


TNF-120572 uarr [158 159 161]IL-1120573 IL-6






GRo-aEotaxin-2




NSEGenomic analysis











1120572












2O2[218]



2O2levels



















uarr [194 195]










1120572

8-isoprostaneIL-6


Sarcoidosis




Pediatric patients

8-IsoprostaneIL-6




Adult patients





pHNitritesNitrates















References














































2















































4



4











2


















































4




4

































































4









1
























ILsIL-2 uarr [158]


TNF-120572 uarr [158 159 161]IL-1120573 IL-6






GRo-aEotaxin-2




NSEGenomic analysis











1120572












2O2[218]



2O2levels



















uarr [194 195]










1120572

8-isoprostaneIL-6


Sarcoidosis




Pediatric patients

8-IsoprostaneIL-6




Adult patients





pHNitritesNitrates















References














































2















































4



4











2


















































4




4

































































4









1
























1120572












2O2[218]



2O2levels



















uarr [194 195]










1120572

8-isoprostaneIL-6


Sarcoidosis




Pediatric patients

8-IsoprostaneIL-6




Adult patients





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