Exhaled nitric oxide and other exhaled biomarkers in bronchial challenge with exercise in asthmatic children: current knowledge

Paediatric Respiratory Reviews xxx (2013) xxx–xxx

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YPRRV-949; No. of Pages 7

Review Article

Exhaled nitric oxide and other exhaled biomarkers in bronchialchallenge with exercise in asthmatic children: current knowledge

Mario Barreto *, Rosanna Zambardi, Maria Pia Villa

Pediatric Unit, Sant’Andrea Hospital, NESMOS Department, Faculty of Medicine and Psychology, Sapienza University of Rome, Rome, Italy

EDUCATIONAL AIMS

� To discuss the potential role of the fractional concentration of exhaled nitric oxide (FENO) in assessing exercise-inducedbronchoconstriction (EIB).� To illustrate changes in FENO and airway-NO exchange dynamics following exercise challenge.� To review other exhaled biomarkers currently being investigated to assess EIB in asthmatic children.

A R T I C L E I N F O

Keywords:

Exercise challenge test

Bronchoconstriction

Exhaled nitric oxide

Biomarkers

Asthma

Atopy

Children.

S U M M A R Y

The fractional concentration of exhaled nitric oxide (FENO), a known marker of atopic-eosinophilic

inflammation, may be used as a surrogate to assess exercise-induced bronchoconstriction (EIB) in

asthmatic children. The predictive value of baseline FENO for EIB appears to be influenced by several

factors, including age, atopy, current therapy with corticosteroids and measurement technique.

Nonetheless, FENO cut-off values appear to be able to rule out EIB. FENO levels decrease during EIB,

apparently through neural mechanisms rather than by decreased airway-epithelial surface. Partition of

FENO into proximal and peripheral contributions of the respiratory tract may improve our understanding

on NO exchange during exercise and help to screen subjects prone to EIB.

Other biomarkers of inflammation and oxidative stress contained in exhaled gases and exhaled breath

condensate (EBC) may shed light on the pathophysiology of EIB. Exhaled breath temperature is a

promising real-time measurement whose routine use for assessing EIB warrants further investigation.

� 2013 Elsevier Ltd. All rights reserved.

Contents lists available at ScienceDirect

Paediatric Respiratory Reviews

INTRODUCTION

The fractional concentration of nitric oxide in exhaled air (FENO)closely reflects atopic-eosinophilic airway inflammation [1–4].FENO measurement is currently being investigated on account of itspotential role as a means of monitoring asthmatic patients inaddition to its lung function testing and clinical roles [5,6]. Givenits non-invasive nature, this marker appears to be particularlysuited to the assessment of asthma in children. FENO may also helpto assess exercise-induced bronchoconstriction (EIB) since theseverity of EIB correlates with airway eosinophilic inflammation inEIB patients [7,8].

* Corresponding author. Clinica Pediatrica, Ospedale Sant’Andrea, Via di

Grottarossa 1035-1039, 00189 Roma (Italia). Tel.: +39/0633775279;

fax: +39/0633775941.

E-mail addresses: [email protected], [email protected]

(M. Barreto).

Please cite this article in press as: Barreto M, et al. Exhaled nitric oexercise in asthmatic children: current knowledge. Paediatr. Respir.

1526-0542/$ – see front matter � 2013 Elsevier Ltd. All rights reserved.

http://dx.doi.org/10.1016/j.prrv.2013.11.006

Exercise is a common trigger of airway narrowing inasthmatic children, typically within the first 5-10 minutes ofexercise cessation [9]. Post-exercise symptoms such as cough,chest pain or dyspnea may be present, though the patient’s andparents’ perception of these symptoms may be in contrast toresults of the exercise challenge test [10,11]. EIB, which occurs in70–90% of asthmatic children, is indicative of poor asthmacontrol, but improves with appropriate asthma therapy [12–15].A post-exercise FEV1 fall of at least 10% from baseline isdiagnostic of EIB [9,16]. The FEV1 fall is more pronounced asasthma severity increases; however, baseline FEV1 does notpredict outcomes of exercise testing [11]. The most likelymechanism of EIB in asthmatic subjects is airway dehydrationand hyperosmolarity from hyperventilation, with the subsequentrelease of inflammatory mediators such as histamine andleukotrienes. Hyperventilation, particularly in healthy subjects(e.g. athletes), can also induce EIB through airway cooling andrewarming, followed by bronchial edema and plasma exudation[17,18].

xide and other exhaled biomarkers in bronchial challenge with Rev. (2013), http://dx.doi.org/10.1016/j.prrv.2013.11.006


mailto:[email protected]

mailto:[email protected]


http://www.sciencedirect.com/science/journal/15260542


M. Barreto et al. / Paediatric Respiratory Reviews xxx (2013) xxx–xxx2

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The main advantage of the exercise challenge test is that,unlike other tests (e.g. methacholine or histamine), it does notstimulate the bronchial receptors directly, but elicits aninflammatory response indirectly, mimicking the natural patho-physiology of asthma [19]. The main disadvantage of this test isthe fact that it needs to be used repeatedly to assess asthmacontrol in children, and hence becomes unpractical, expensiveand time-consuming. The measurement of FENO has beensuggested as a surrogate of EIB because it reflects asthmatic-airway inflammation, it can be measured easily in real-time, andit can be repeated in outpatients at every follow-up examination[20]. Assessing FENO could reduce the need for the exercisechallenge test, thereby avoiding the use of this test in subjects inwhom EIB is unlikely.

This brief review focuses on the relationship between baselineFENO measurements and EIB in asthmatic children. Moreover, wediscuss post-exercise FENO changes, which provide insights intothe airway sources of nitric oxide (NO) during EIB. We alsocomment on the use of other exhaled biomarkers in bronchialchallenge with exercise in asthmatic children, including bio-markers of inflammation, oxidative stress, acidity and tempera-ture in the airways. The few reports available on thesebiomarkers in EIB are discussed in the final part of thismanuscript.

ORIGIN AND SOURCES OF NITRIC OXIDE IN THE AIRWAY

Nitric oxide (NO) is a widely distributed free radical that exertsa number of functions in the human body, including vasodilation,bronchodilation and neurotrasmission, as well as defence againstseveral microorganisms [21,22]. Production of NO in the respira-tory system arises from oxidation of L-arginine to L-citruline viaseveral synthase isoforms, two of which are constitutive, i.e.endothelial (eNOs) and neuronal (nNOs). The activity of nNOS innon-adrenergic non-cholinergic nerves induces bronchial musclerelaxation and prevents bronchial obstruction [23]. Both consti-tutive NOs are calcium- and calmodulin-dependent and can bestimulated by several mediators such as bradykinine, acetylcho-line, histamine, leukotrienes and platelet-activating factor [24].The third NO synthase is inducible (iNOs) and is found in airwayepithelial cells, endothelial and smooth muscle cells, macrophagesand neutrophils; iNOs is upregulated by allergens, infections andtoxins [24].

The expression of iNOs increases during inflammatory pro-cesses and is induced by several interleukins, interferon and tumornecrosis factor [24].

Increased eosinophil activity in the mucosal airway is related tohigh NO concentrations in the airway lumen [3,25,26]. ExcessiveNO production leads to the formation of NO derivatives,particularly peroxynitrite, a potent oxidant that further enhancesairway inflammation [27].

Most of the NO in the airway lumen diffuses through the airwayepithelium, though the oropharynx may also contribute signifi-cantly to NO diffusion; NO concentrations are lower in the alveoli,which display a high NO-reabsorption rate by capillary haemo-globin [5,28]. When NO is released into the airway lumen, diffusiveand convective forces interact to drive the rate of NO reabsorptionby blood vessels (mainly in the distal airways and alveoli) and therate of NO excretion at the mouth opening, where it can bemeasured at a determined expiratory flow, i.e. the so-called FENO.Low expiratory flow rates increase accumulation of NO in theairway lumen, which in turn results in high FENO levels; conversely,high expiratory flow rates dilute NO in the airway lumen with thelow-NO-concentrated alveolar gas, which results in low FENO levels[5,29–31].


FENO MEASUREMENT

For the preceding reasons, FENO should be measured during aconstant expiratory flow; expired gas is analysed in real time,usually by chemiluminescence or electrochemical devices. Therecommended maneuver consists in a single vital capacityexhalation against a resistance of at least 5 cmH2O to ensureclosure of the soft palate, thus avoiding contamination with thehigh NO concentrations coming from the nasal and paranasalcavities [5,31]. Most children over 6 years of age can maintain aconstant expiratory flow of 50 mL/s for at least 3 seconds [32]. Foryounger cooperative children, the target expiratory flow can beobtained with the help of a resistor, which can counteract airflowvariations close to 50 mL/s [20]. Other techniques for measuringFENO, which are used above all on infants and less cooperativepatients, are not suitable for FENO assessment during airwaychallenge with exercise.

Several other factors besides expiratory flow can influence FENO

measurements. These factors include age, atopy (especially duringallergen exposure), current respiratory infections, passive smok-ing, recent food intake and ambient conditions. Thorough reviewson standardized procedures for FENO measurements are available[5].

FENO TO ASSESS BRONCHIAL CHALLENGE WITH EXERCISE

Several studies based on a treadmill or bicycle ergometer haveinvestigated the relationship between baseline FENO and bronchialresponse to exercise in asthmatic children. [11,33–37] Their resultssuggest that increased baseline FENO levels could be used as asurrogate of EIB, [11,33,34,36] as findings from studies onasthmatic adults also indicate [38–42]. Baseline FENO levelscorrelate with the degree of post-exercise fall in FEV1;[11,33,34] consequently, the baseline FENO is higher in subjectswith EIB than in those without [11,33,34,36].

Most studies have used a post-exercise FEV1 fall of either 12%[33,35] or 15% [11,34,36] as a cut-off for defining EIB. Scollo et al.studied 24 asthmatic children between 6 and 13 years of age (20atopic, 17 treated with inhaled corticosteroids, ICs); they alsoincluded an age-matched control group (n=18) for baselinecomparisons alone. [33] Patients with EIB displayed a higherbaseline FENO than patients without EIB or control children, thoughdifferences reached statistical significance in the control groupalone (12.3 � 1.6 vs 9.1� 1.0 vs 6.1 � 0.2, p<0.01 vs control group).The authors reported a correlation between baseline FENO and themaximum post-exercise fall in FEV1 (r=0.61, p<0.01) [33]. Similarfindings were described by Terada et al. in 39 atopic asthmaticchildren (25 treated with ICs) and 6 healthy controls; patients withEIB yielded a significantly higher baseline FENO than controls thoughnot than asthmatic children without EIB [34]. We did not findstatistically significant differences in baseline FENO between patientswith EIB and those without in 46 ICs-naıve (21 atopic) asthmaticchildren in our study either [35]. By contrast, a significantly higherbaseline FENO in subjects with EIB than in those without has beenreported in two other studies; approximately half of the patients inboth those studies were being treated with ICs and over 70% wereatopic [11,36].

The contrasting results in all the afore-mentioned studiessuggest that several factors may affect the relationship betweenbaseline FENO and exercise challenge outcomes. Although therewere differences between the various studies in the subjects’characteristics (atopy and therapy with ICs), in the methods (FENO

technique and expiratory flow) and in the criteria used to defineEIB (percent fall in FEV1), overall the information they yieldsupports the usefulness of FENO measurement in exercise challengetesting (Table 1).



Table 1Studies testing FENO and bronchial challenge with treadmill exercise in asthmatic children.

Authors and FENO technique Subjects FENO baseline, ppb FENO vs FEV1 fall

Terada et al. [34]

On-line

2.0 L/min

Asthmatics (n = 39), 8-18 yr, all atopic, 25 treated with ICs

- EIB (n = 18)

- Non EIB (n = 21)

Controls (n = 6), 9-13 yr

69.3 �9.3*

45.3 �6.8

15.7 �3.6

*p= 0.005 vs control group

r= 0.50

p= 0.012

Scollo et al. [33]

Off-line

180-200 mL/s

Asthmatics (n = 24), 6-13 yr,

20 atopic, 17 treated with ICs

- EIB (n = 10)

- Non EIB (n = 14)

Controls (n = 18), 7-15 yr

12.3 �1.6*

9.1 �1

6.1 �0.2

*p <0.01 vs control group

r = 0.61

p <0.01

Barreto et al. [35]

On-line

50 mL/s

Asthmatic (n = 46), 6-17 yr,

21 atopic, all ICs naıve

- EIB (n = 12)

- Non EIB (n = 34)

38.7 (24.5-61.1)

29.1 (22.0-38.4)

p= n.s.

Buchvald et al. [36]

On-line

50 mL/s


79 atopic, 63 treated with ICs

- EIB (n = 30)

- Non EIB (n = 81)

29,5 (21-25)*

10.2 (8-15)

*p= 0.0001

Lex et al. [11]

On-line

50 mL/s


all atopic, 42 treated with ICs

- EIB (n = 12)

- Non EIB (n = 73)

51.3(31.3-67.3)*

20.2 (10.9-42.3)

* p= 0.0002

r= 0.32

p= 0.003

EIB: exercise-induced bronchoconstriction. ICs: inhaled corticosteroids.

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FENO AS A PREDICTOR OF EIB

The potential role of FENO as a predictor of EIB has beeninvestigated in several studies [11,36,43–45]. In a study conductedon 111 asthmatic schoolchildren (79 atopic, 63 treated with ICs),Buchvald et al. showed that the subjects could be pre-screened intoeither one group with increased FENO, who needed to undergo EIBtesting, or into another group with near-normal FENO, who wereunlikely to suffer from EIB [36]. The authors pointed out that FENO

values under 20 ppb in children not treated with ICs and FENO

values under 12 ppb for children treated with ICs provided highnegative predictive values (NPV, untreated: 88%, treated 96%). Bycontrast, the positive predictive value (PPV) was low in bothgroups (53% in subjects without ICs and 44% in those with ICs) [36].In another study conducted on 85 atopic-asthmatic children (42treated with ICs), Lex et al. found that no patients with baselineFENO of 25 ppb or less (n=42) had EIB, yielding a NPV of 100%,whereas only 12 of the 43 patients with increased FENO had EIB,yielding a PPV of 28% [11]. The authors reported that a positiveanswer to a questionnaire on recent asthma symptoms (2 weeks)in addition to elevated FENO raised the PPV to 40%, while the NPVremained high (97%) [11]. Grzelewski et al. retrospectively studied126 atopic-asthmatic schoolchildren and found that a FENO valuebelow 16 ppb helped to rule out EIB in patients regularly treatedwith ICs (NPV 74.2%), whereas FENO values above this cut-offpredicted EIB with a PPV of 60%; although eosinophil blood cellcounts of >350 cell/mm [3] and allergy to house-dust mites alsoincreased the odds ratio of EIB, the only independent factor thatincreased the odds ratio of EIB four fold in these children was aFENO value above 16 ppb [44].

Results from the three afore-mentioned research groupssuggest that FENO cut-off values are more useful as a means ofexcluding than of confirming EIB; further studies are, however,warranted to lend support to their findings.

Prediction of EIB with baseline FENO has also been studied inpreschool-aged children. Malmberg et al. studied 84 IC-naıvechildren aged 3 to 7 years old with a recent wheeze (within 12


months) and 71 age-matched control subjects without respiratorysymptoms [45]. The authors assessed changes in respiratoryresistance with the impulse oscillometry as well as by means ofskin prick tests. Although both atopic (n=63) and non-atopic(n=21) wheezy children with EIB displayed higher FENO than atopic(n=11) and non-atopic (n=60) control subjects, only atopic wheezychildren exhibited a significant correlation between FENO and theseverity of EIB (r=0.44, p=0.0004). FENO levels of 28 ppb in atopicwheezy children predicted EIB (Rrs5 increased by at least 40% afterexercise) with a PPV of 90%; FENO levels of 24 ppb instead yielded aPPV of 83%; their NPV was low (not provided), possibly owing tothe high prevalence of EIB positive children among atopicwheezers (41 out of 63). The lack of a relationship between FENO

and EIB among non-atopic wheezers allowed the authors toconclude that bronchial responsiveness and airway inflammationmay interact differently in these non-atopic patients [45].

FENO CHANGES DURING EIB

Remarkable variations from baseline FENO have been describedafter airway challenge with exercise in asthmatic children,[34,35,46] though these variations have not been found constantly[33]. Terada et al. showed a decrease in FENO 5 and 10 minutespost-exercise in atopic asthmatic subjects with EIB, especially inIC-naıve subjects, whose FENO 10 minutes post-exercise remainedsignificantly lower than in IC-treated EIB-positive asthmaticpatients. Although post-exercise decreases in both FENO andFEV1 correlated significantly, the change in FENO did not correlatewith the change in minute ventilation, nor did FENO increase afterinduced bronchodilation with an inhaled beta-agonist at the EIBpeak [34]. The authors concluded that the EIB-induced FENO

decrease was not due solely to increased ventilation or to airwayobstruction, suggesting instead that patients with EIB haveimpaired NO production in response to exercise, thereby raisingthe possibility that stable NO production is needed to maintainairway patency during exercise. Avoidance of EIB may requiresynthesis of NO by nonadrenergic-noncholinergic nerves in order




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to maintain smooth-muscle relaxation [47]. In keeping with thefindings by Terada et al., we demonstrated that repeatedmaneuvers of maximal expiratory pressures reduced FENO

regardless of changes in airway patency [48].We recently reported a post-exercise decrease in FENO in 46

steroid-naıve (21 atopic) asthmatic children and adolescents; [35]FENO levels decreased in parallel with the FEV1 decline at 5 minutes(r=0.44, p=0.002). FENO only decreased in the 12 subjects with EIB,remaining unchanged in the 34 subjects without EIB. Sincerespiratory maneuvers can influence FENO, [46,48–50] we excludedthe possibility that changes in FENO depended on spirometry ratherthan on exercise, as demonstrated by the subjects’ FENO measure-ments performed both before and after pre-exercise spirometry.Indeed, pre-exercise FENO changes induced by FVC maneuverswere unrelated to post-exercise changes in FENO and FEV1,suggesting that FVC-induced changes in FENO were unable topredict the occurrence of EIB [35].

AIRWAY EXCHANGE DYNAMICS OF NO DURING EIB

As commented in the initial sections, there is an inverserelationship between FENO and expiratory flow; however, theirproduct (i.e. the NO output) increases with the expiratory flow.Using the NO output from several expiratory flows and a two-compartment model (alveolar and airway regions), it is possible tocalculate flow-independent NO exchange parameters, includingthe NO diffusion across the airways (DawNO), the maximal NO fluxfrom airway NO diffusion (J’awNO), the NO concentration in theairway wall (CawNO) and the alveolar concentration of NO (CANO)[29,51]. These estimates can be used to partition FENO intoproximal and peripheral contributions of the respiratory tree [29].

Linkosalo et al. recently investigated whether J’awNO and CANO

predict EIB in atopic children and adolescents with asthma-likesymptoms (n=30 patients), also including 66 healthy controlsubjects [52]. Both J’awNO and CANO were expressed eitherwithout or with a correction for backward axial NO diffusion(TMAD). [53] Patients with EIB (n=18) had significantly higherJ’awNO, CANO and J’awNO (TMAD) than either patients withoutEIB (n=12) or healthy subjects; no differences in CANO (TMAD)values were found between EIB+ and EIB- atopic patients, whichpoints to falsely increased CANO in patients with increasedbronchial NO flux. Only J’awNO, CANO and J’awNO (TMAD)correlated with the degree of post-exercise drop in FEV1 andmiddle flows. The relatively small number of patients in Linkosaloet al.’s study limited the estimates of sensitivity and specificity ofexhaled NO measurements; nonetheless, their results, which are inkeeping with those of a previous study on adult subjects, suggestthat it may be possible to use J’awNO as a predictive biomarker ofEIB in atopic children [52]. Chinellato et al. reported a significantcorrelation between severity of EIB and both J’awNO and CANO in36 atopic children with intermittent asthma; these NO-estimateswere higher in children sensitized to indoor allergens than in thosesensitized to outdoor allergens alone. On the basis of their results,the authors suggested that inflammation in both the central andperipheral airways is associated with the severity of EIB [54].

Shin et al. estimated flow-independent NO exchange para-meters in 9 mild-intermittent steroid-naıve atopic asthmaticadults with EIB and in 9 healthy controls by means of a decreasing-flow expiratory technique preceded by a 20-second breath hold;[55] FENO values for an expiratory flow of 50 ml/s were alsocalculated. At baseline, subjects with EIB had higher DawNO,J’awNO and FENO as well as lower FEV1% and FEF25-75% than healthycontrols, whereas no differences emerged in CANO and FVC%. Afterexercise, patients with EIB (though not healthy subjects) displayeda significant fall in J’awNO, not associated with changes inany other NO exchange parameters. Following bronchodilator


administration, J’awNO returned to baseline whereas DawNO andCawNO remained unchanged. Changes in NO exchange parametersdid not correlate with spirometric changes, suggesting that highbaseline FENO in subjects with EIB and reduced FENO during EIB areascribable to different mechanisms. Shin et al. hypothesizedseveral anatomical and metabolic determinants of NO exchangeduring EIB, including changes in airway surface, gas trapping,heterogeneous response in airway patency along the airway tree,production rate from NOs isoforms, breakdown of NO derivatives(s-nitrosothiols), presence of eosinophils at sites of inflammationand altered mucus production and hydration [55].

OTHER EXHALED BIOMARKERS DURING EIB ASSESSMENT

Several other non-invasive exhaled biomarkers besides FENO

have been investigated as a means of assessing bronchial responseto exercise. Some of these biomarkers have been measured duringEIB in asthmatic children [35,56–59]. Measurements encompassmolecules involved in inflammatory or oxidative stress either inthe exhaled gas or the exhaled breath condensate (EBC), andphysical measurements such as the exhaled breath temperature(EBT). The technical aspects of these measurements are available inseveral reviews [60–66].

Exhaled gases other than FENO

The levels of carbon monoxide (CO) and ethane, which arebiomarkers of oxidative stress, are reported to be higher inasthmatic than in healthy subjects. [67] CO is produced by thestress-induced protein heme-oxygenase, whereas ethane isreleased from lipid peroxidation on several tissues [68,69]. Studiesin healthy subjects and children with cystic fibrosis have revealeddecreased CO levels but increased CO production (corrected byminute ventilation) shortly after exercise [70,71].

Exhaled concentrations of ethane, and other biomarkers such asacetone, ammonia, and isoprene, have been reported to increasefollowing exercise in healthy adult subjects [72–74]. No studieshave yet measured exhaled gases other than FENO to assess airway-challenge with exercise in asthmatic children; hence, their role onEIB warrants investigation.

Exhaled breath condensate (EBC)

A number of compounds have been measured in EBC to assessthe airway response to exercise, including biomarkers of inflam-mation (e.g. leukotrienes, prostaglandins, cytokines, adenosine)and oxidative stress (e.g. 8-isoprostanes, H2O2).

Leukotrienes are released by mast cells, macrophages andactivated eosinophils in the airways [75]. Carraro et al. investigatedthe relationship between EIB and the baseline concentration ofcysteinyl leukotrienes (Cys-LT) in EBC and FENO in 19 atopic-asthmatic patients and 14 healthy control children [56]. Patientswith EIB (fall in FEV1� 12%, n=11) had significantly higher baselineCys-LT concentrations than either patients without EIB (n=8) orcontrol subjects (42.2 vs 11.7 and 5.8 pg/mL). FENO levels were alsohigher in asthmatic children than in healthy controls. Althoughthere was no correlation between baseline EBC Cys-LT and FENO,each of these biomarkers correlated significantly with the post-exercise FEV1 decrease. The authors of that study suggested thatboth Cys-LT and FENO pathways are involved in the pathogenesis ofEIB [56]. Bikov et al. reported higher baseline EBC Cys-LT levels inatopic-asthmatic patients (n=17) than in healthy adult volunteers(n=6) [76]. EBC Cys-LT levels increased in asthmatic patientswithin 10 minutes of the exercise challenge; this increasecorrelated with the post-exercise fall in FEV1, which lends support




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to the hypothesis that the release of this mediator is involved in thedevelopment of EIB [76].

Isoprostanes are prostaglandin-like compounds that derivefrom peroxidation of arachidonic acid on plasma membranephospholipids [77]. 8-Isoprostane (8-IsoP) concentrations in EBCare high in subjects with asthma [78–80]. We measured 8-IsoPlevels in EBC and FENO in 46 IC-naıve (21 atopic) asthmatic childrenat the baseline and after exercise challenge [35]. The mean baselineconcentrations of 8-IsoP (but not FENO) were significantly higher insubjects with EIB (n=12) than in those without EIB (n=34) [44.9 vs

32.3 pg/mL]. Baseline lung function, 8-IsoP levels and FENO werenot correlated. Baseline 8-IsoP concentrations in EBC (though notbaseline FENO) correlated with a reduction in FEV1 5 minutes afterexercise (r= �0.47; p=0.002). Following exercise, 8-IsoP levels didnot, unlike FENO levels (see section ‘‘FENO changes during EIB’’),change from baseline values. Our data point to a role of oxidativestress in bronchial hyper-reactivity [35].

Hydrogen peroxide (H2O2) is a volatile biomarker of oxidativestress whose levels have been studied in healthy smokers and inseveral respiratory diseases, including asthma [60]. H2O2 levels inEBC increase after cycle-ergometer exercise in young cigarettesmokers, though not in their non-smoker counterparts; [81] theincrease in EBC H2O2 is not followed by an increase in the EBCconcentrations of biological antioxidant potential (BAP), whichpoints to an increased risk of respiratory damage in these subjects[81]. No studies have yet been conducted on asthmatic subjects toassess the airway response to exercise with H2O2 levels in EBC.

Adenosine is a degradation product of adenosine triphosphate(ATP) with bronchoconstrictor effects that are comparable to thoseinduced by exercise [82–84]. Csoma et al. measured adenosinelevels in EBC in two groups of adult subjects, composed of 8 healthynon-atopic subjects and 10 asthmatic patients (7 atopic), atbaseline and after exercise [85]. Adenosine levels in EBC weresimilar in both groups at baseline but increased after exercise inasthmatic patients alone; post-exercise changes in adenosine andthe fall in FEV1 were correlated (r [2] =0.59, p<0.001). The authorshypothesized a contribution of adenosine in EIB [85].

Other compounds that can be measured in EBC are chemokinesand interleukins. The chemokine RANTES (regulated upon activa-tion, expressed and secreted by normal T-cell) is produced bybronchial epithelial cells in response to hyperosmolar stimulationand is a chemoattractant of T lymphocytes, eosinophils andbasophils [86]. Keskin et al. measured the concentrations ofRANTES and interleukin 4 (IL-4) in 56 asthmatic children beforeand 30 minutes after an exercise challenge [57]. No differences inRANTES or IL-4 concentrations were found at the baseline betweensubjects with EIB (n=25, 78% atopic) and those without EIB (n=31,51% atopic). Baseline RANTES levels correlated with baselineFEV1%, total IgE and body mass index (BMI). RANTES levelsincreased after exercise in both subjects with and those withoutEIB, whereas IL-4 levels remained unchanged. None of thesebiomarkers correlated with the post-exercise maximum fall inFEV1. The authors concluded that although RANTES and IL-4 maynot be independent predictors of EIB, increased post-exerciseexhaled RANTES might promote recruitment and activation ofinflammatory cells in the airways of asthmatic children [57].Zietkowski et al. found higher baseline RANTES in the EBC ofatopic-asthmatic adult patients (either with or without EIB) than inhealthy control subjects [87]. Exhaled RANTES increased followingexercise in subjects with EIB alone. The authors reported similarfindings for other compounds in EBC, such as endothelin-1,eotaxin, high sensitive PCR and soluble CD40 ligand; [88–91] thislast compound also distinguished patients with EIB from thesewithout EIB at baseline [91]. The post-exercise increase in most ofthese biomarkers correlated with baseline FENO levels, therebysuggesting common inflammatory pathways in EIB [87–90].


Cyclo-oxygenase (COX) mediators may also play a regulatoryrole in the airways; [92] Pucsok et al. reported a post-exerciseincrease in EBC levels of prostaglandin E2 (PGE2) and thromboxaneB2 (TXB2) in male elite athletes only, [93] which they attributed togender differences in COX metabolism [94,95]. Although changesin PGE2 and TXB2 may be associated with airway adaptation toexercise in healthy subjects, their role in asthma has yet to beelucidated.

EBC acidity (EBC pH) probably reflects airway acidification as aresult of several inflammatory and oxidative processes [60–63].EBC pH decreases in asthmatic patients with severe disease orduring exacerbations [62,96,97]. Increased EBC pH from baselinehas instead been reported in healthy adult subjects after asubmaximal exercise. [98] We also observed an increase in EBC pHafter a treadmill-exercise challenge in asthmatic children whoseFEV1 fell more than 9% from baseline [58]. Greenwald et al.compared the ionic composition of EBC collected before and aftervigorous exercise (track practice) in adolescent athletes; followingexercise, EBCpH and ammonia increased while the propionic acidconcentration fell [99]. Since exercise increases systemic ammoniaand urea, those authors suggested that EBC pH is determined bygas-phase diffusion from epithelial and oral surfaces and, to alesser extent, from the blood/alveolar interface [99].

Exhaled breath temperature (EBT)

Chronic inflammation and increased vascularity of the airwaysare critical features of asthma. EBT levels have been reported tocorrelate with FENO levels [100–103] and bronchial blood flow,[103] and are hence likely to be a biomarker of airwayinflammation and vascular remodelling [102,103]. EBT levels havebeen reported to be higher in asthmatic subjects than in healthysubjects [101–103]. EBT can be measured by means of either asingle breath or tidal breathing techniques [100–104].

Peroni et al. recently found a mean increase in tidal EBT of about0.4 8C from baseline after a 6-minute treadmill-exercise challengein 50 atopic-asthmatic children [59]. The increase in EBT correlatedwith the maximum percentage decrease in FEV1, particularly inthose patients who were not regularly treated with ICs. Thisfinding, combined with the lack of any relationship betweenbaseline EBT and the percent fall in FEV1, further supports thehypothesis that EBT is a composite biomarker that may be used tomonitor various aspects of asthma, including vascular remodelingand airway inflammation [59]. Svensson et al. reported animmediate post-exercise increase in EBT in both asthmatic adultpatients (n=20) and healthy controls (n=21); [105] EBT levelsremained high for at least 60 minutes only in patients whose FEV1

dropped by >10%. The authors hypothesized that a prolongedincrease in EBT may be related to persistent inflammation inpatients with EIB [105].

CONCLUSIONS

Baseline FENO levels are related with EIB in asthmatic children;a low FENO is particularly useful as a means of ruling out EIBwithout exercise testing. However, several factors that influenceFENO should be taken in account to improve its predictive value forEIB. The post-exercise decrease in FENO suggests that neural NOproduction plays an important role in maintaining bronchial tone.Further studies designed to assess the relationship between flow-independent NO exchange parameters and EIB could improve theaccuracy of this non-invasive monitoring technique in asthmaticpatients.

FENO remains the gold standard gas for assessing airwayresponse to exercise, though other exhaled gases could providecomplementary information when monitoring asthmatic patients.




G Model


Studies on EBC biomarkers may shed light on the pathophysiologyof EIB; however, the usefulness of such biomarkers in clinicalpractice remain limited because their levels cannot be assessed inreal time. EBT may represent an alternative real-time measure-ment, though further studies are required to better establish how itcan be used routinely to assess airway challenge with exercise inasthmatic children.

FUTURE RESEARCH DIRECTIONS

� The predictive role of exhaled biomarkers as surrogates of EIB.� The mechanisms of airway inflammation and oxidative stress

during exercise in asthmatic children.� The real-time measurement of exhaled biomarkers involved in

EIB.

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Exhaled nitric oxide and other exhaled biomarkers in bronchial challenge with exercise in asthmatic children: current knowledge