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Exhaled Breath Condensate pH and Childhood Asthma

Unselected Birth Cohort Study

University of Manchester; and North West Lung Centre, Wythenshawe Hospital, Manchester, United Kingdom

Correspondence and requests for reprints should be addressed to Nicolaos Nicolaou, M.D., North West Lung Centre, Wythenshawe Hospital, Manchester M23 9LT, UK. E-mail: nicolaos.nicolaou{at}postgrad.manchester.ac.uk

	ABSTRACT

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Rationale: Exhaled breath condensate pH (EBC-pH) may be useful noninvasive marker for evaluation of patients with asthma.

Objectives: To investigate the relationship between EBC-pH and symptoms suggestive of childhood asthma in an epidemiologic setting and examine its relation to lung function, airway hyperresponsiveness (AHR), and airway inflammation.

Methods: Within the context of a prospective population-based birth cohort, EBC was collected from 630 children at age 8 yr using the RTube (pH measured after deaeration with argon). Lung function was measured by spirometry (FEV₁; n = 521) and plethysmography (sRaw; n = 567), and AHR by methacholine challenge (n = 498). Airway inflammation was assessed using exhaled nitric oxide (eNO; n = 305).

Results: EBC-pH values ranged widely (4.40–8.29), and did not differ between 54 children with parentally reported asthma and 562 nonasthmatic subjects (median [interquartile range]: 7.75 [7.45–7.85] vs. 7.77 [7.59–7.87]; p = 0.35). There was a trend for lower EBC-pH among current wheezers (n = 98; 7.72 [7.50–7.83]) compared with nonwheezers (n = 532; 7.77 [7.60–7.87]; p = 0.07). Wheeze frequency, severity, and use of antiasthma medication were not associated with EBC-pH. There was no consistent association between EBC-pH and lung function, airway reactivity, and airway inflammation (FEV₁, sRaw, PD₂₀ methacholine, or eNO). There was no significant difference in EBC-pH between current wheezers receiving asthma medication who had positive methacholine challenge compared with children without any of these features.

Conclusions: In the epidemiologic setting, EBC-pH does not differ between children with and without parentally reported symptoms suggestive of asthma. We found no consistent association between EBC-pH and lung function, AHR, and airway inflammation in this sample from the general population.

Key Words: airway acidity • asthma • exhaled breath condensate • lung function

Over the last few years, it has become clear that childhood asthma is not a single disease but a spectrum of phenotypes reflecting different degrees of airway inflammation involving several cell types and mediators (1, 2). There is no single test that reliably quantifies airway inflammation, and in daily practice diagnosis and monitoring of asthma in children continues to be largely based on clinical symptoms and signs. Measurements of lung function and airway hyperresponsiveness (AHR) may aid diagnosis and assist in disease management, but these have their limitations (3, 4). The development and application of safe and simple objective methods of assessing airway inflammation may facilitate better understanding of the underlying pathophysiology and natural history of the disease, define asthma phenotypes, and help the treatment decision-making process.

Analysis of exhaled breath condensate (EBC), which contains several volatile and nonvolatile biomarkers of airway lining fluid, may be useful in the evaluation of patients with lung disease (5–9). The measurement of EBC-pH (acidity) has been proposed as a promising noninvasive tool in the assessment of patients with asthma (10–12), but its usefulness as a reliable disease marker has been questioned (13–15). It was found to reflect acute exacerbations in adults, and to normalize with antiinflammatory therapy (11). Children with stable asthma appear to have lower EBC-pH than healthy control subjects (10, 16). Furthermore, relationships among EBC-pH and asthma severity have been observed (e.g., children with moderate and severe asthma tend to have lower EBC-pH when compared with those with mild asthma) (10, 17). However, most studies have been small, performed in selected patient groups (thus not generalizable to the general population), and often have had conflicting results (summarized in Table E1 of the online supplement).

We aimed to assess the relationship between EBC-pH and symptoms suggestive of childhood asthma in an epidemiologic setting within the context of a large, prospective, population-based birth cohort. In addition, we examined this noninvasive biomarker in relation to the objective outcomes of lung function, airway inflammation, and AHR.

	METHODS

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The Manchester Asthma and Allergy Study is an unselected population-based birth cohort study, described in detail elsewhere (18, 19). Participants were recruited prenatally, and attended review clinics at age 8 yr (± 4 wk). The study was approved by the local research ethics committee. Informed consent was obtained from all parents, and children gave their assent.

Outcomes
Symptoms.
A validated questionnaire was interviewer-administered to collect the information on symptoms, physician-diagnosed illnesses, and treatments received (20). We defined "current asthma" as a positive response to the question "Does your child have asthma now?" and "Current wheeze" as a positive response to the question "Has your child had wheezing or whistling in the chest in the last 12 months?"

Allergic sensitization.
Allergic sensitization was ascertained by skin-prick testing (mite, cat, dog, grasses, molds, milk, egg, peanut); sensitization was defined as wheal at least 3 mm greater than the negative control.

EBC collection and pH measurement.
Samples were collected using the RTube (Respiratory Research, Inc., Charlottesville, VA). Children had not eaten or drunk for at least half an hour before collection, and did not wear nose clips during the procedure. The subjects were asked to breathe through the tube's mouthpiece for 8 min and to temporarily discontinue collection if they needed to swallow saliva. The collected sample was separated into five aliquots (four were stored at –80°C, and the fifth was used for immediate pH measurement after deaeration with argon for 10 min). All pH measurements were performed using the same equipment (pH-210 m; Hanna Instruments, Leighton, UK) with a sensitive electrode (Hamilton, Reno, NV). Manufacturers' operation and calibration instructions were strictly followed.

Lung function.
FEV₁ was measured using a wedge bellow spirometer. The test was repeated until three technically acceptable traces were obtained. The highest FEV₁ was recorded.

Specific airway resistance (sRaw) was measured using plethysmography (Master Screen Body 4.3; Viasys, Würzburg, Germany), as previously described (21, 22). Three measurements of sRaw were performed, and each was calculated from the medians of five consecutive technically acceptable loops (21, 23). The mean of these three measurements of effective sRaw was used in the analysis.

Airway reactivity.
The children underwent methacholine challenge using five-breath dosimeter method (24) with a quadrupling dosing schedule (0.0625–16 mg/ml³). The challenge was regarded positive if FEV₁ fell by more than 20%, and the provocative dose of methacholine causing a 20% fall in FEV1 (PD₂₀) was recorded.

Exhaled nitric oxide.
Subjects' exhaled nitric oxide (eNO) was measured using NIOX chemiluminescence analyzer (Aerocrine, Solna, Sweden) according to the European Respiratory Society/American Thoracic Society criteria (25). Subjects were encouraged to maintain the steady flow rate of 50 ml/s with the aid of incentive animation software. NO values were measured at the plateau of the end-expiratory reading and expressed as parts per billion (ppb). The mean of three technically acceptable readings was recorded.

Statistical Analysis
EBC-pH measurements could not be normalized; the data were thus analyzed using nonparametric tests, and results expressed as median and interquartile range (IQR). FEV₁% predicted, sRaw, and eNO values were assigned into quartiles. Differences among groups were tested using a Kruskal-Wallis test, followed, if appropriate, by the Mann-Whitney U test for between-group comparisons (SPSS for Windows, version 11; SPSS, Inc., Chicago, IL).

	RESULTS

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Study Population
Of the 809 children invited within the first 18 mo of the 8-yr follow-up, 736 (91%) attended the review clinic. EBC-pH was measured in 718 subjects; three children refused to complete EBC collection, and 15 were symptomatic at the time of the assessment and did not perform any lung function tests. Eighty-eight children who were prenatally randomized to an environmental control regime (18) were excluded, and a total of 630 children who successfully performed the EBC collection were included in the analysis. Within this group, successful FEV₁ measurement was performed in 521 children, 567 completed sRaw, 498 airway reactivity, and 305 completed the eNO measurement. Atopic status was ascertained in 606 subjects, of whom 177 were sensitized to at least one allergen; 98 children (15.6%) had wheezed in the previous 12 mo.

EBC-pH ranged from 4.40 to 8.29 (n = 630; median [IQR], 7.77 [7.57–7.85]; Figure E1). We found no significant association among EBC-pH, height, weight, and a number of other factors that have previously been associated with childhood asthma in the epidemiologic studies (Table E2).

EBC-pH and Parentally Reported Asthma/Wheeze
EBC-pH was significantly lower in children who ever had asthma (either reported by parents or diagnosed by a physician) when compared with those who had never had asthma (Table 1). Similar differences were observed between children who had parentally reported wheeze and those who have never wheezed. However, EBC-pH did not differ between children with and without parentally reported current asthma, whereas a nonsignificant trend was observed between current wheezers and nonwheezers (p=0.07; Table 1 and Figure 1, Figure E2).

View larger version (37K): [in this window] [in a new window]   Figure 1. Exhaled breath condensate (EBC) pH and parentally reported current asthma (A), and current wheeze (B) among 8-yr-old children. Horizontal lines indicate median values.

EBC-pH in Children with Parentally Reported Current Wheeze
EBC-pH values in this group ranged from 4.60 to 8.09 (n = 98; median [IQR], 7.72 [7.50–7.83]). Skin-prick tests were performed in 93 current wheezers. No significant differences in EBC-pH were found between atopic (n = 62) and nonatopic wheezers (median [IQR], 7.71 [7.51–7.82] vs. 7.74 [7.39–7.84]). Parental atopy, sex, exposure to cigarette smoking, pet ownership, and area of residence had no effect on the EBC-pH measurements in this group (data not shown).

EBC-pH, wheeze frequency, and severity.
The relationship between EBC-pH and a number of variables reflecting the severity of wheeze based on parental report is presented in Table 2. Children with fewer wheezing attacks in the previous year tended to have lower EBC-pH compared with those with more frequent attacks, but the observed differences did not reach statistical significance. There was no consistent pattern in EBC-pH values in relation to the sleep disturbances caused by wheeze. The only significant difference was observed between the children who had never experienced sleep disturbance and those who had sleep disturbance less than one night per week, with no differences between children with no sleep disturbances and those who had them frequently ( 1 night/wk; Table 2). EBC-pH did not differ between current wheezers who experienced a severe wheezing attack limiting their speech and those who did not.

EBC-pH and lung function.
FEV₁ and EBC-pH data were available in 84 current wheezers. No significant difference in EBC-pH was observed among the four quartiles of FEV₁% predicted (Figure 2A). sRaw was measured in 88 current wheezers. EBC-pH was significantly lower among wheezers who had sRaw in the first two quartiles compared with those with sRaw in the third or fourth quartile (i.e., EBC-pH was higher among children with reduced lung function; Figure E3).

View larger version (41K): [in this window] [in a new window]   Figure 2. Children with parentally reported current wheeze. The relationship between EBC pH and (A) lung function (FEV1% predicted), (B) airway hyperreactivity (methacholine challenge test), and (C) airway inflammation (exhaled nitric oxide [eNO]).

EBC-pH and Airway Inflammation (eNO).
eNO was measured in 42 current wheezers. No significant difference in EBC-pH was observed between the four quartiles of eNO (Figure 2C).

In addition, there was no significant difference in EBC-pH between children with symptomatic airway reactivity (current wheeze and positive methacholine challenge) who were currently receiving asthma medication and those without any of these features (median [IQR], 7.80 [7.65–7.88] vs. 7.77 [7.61–7.87]; p = 0.8; Figure 3).

View larger version (22K): [in this window] [in a new window]   Figure 3. EBC pH in participants assigned into groups according to the presence of current wheeze, bronchial hyperreactivity, and the use of antiasthma medication: (1) no wheeze, no airway hyperreactivity (AHR–; negative methacholine challenge), not using antiasthma medication; and (2) current wheezer, AHR positive (AHR+; positive methacholine challenge), using antiasthma medication.

	DISCUSSION

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Limitations of the Study
Our study addresses the question whether EBC-pH measurement is helpful in differentiating between well-defined phenotypes suggestive of asthma commonly used in the epidemiologic studies in the general population. Due to the nature of the study, the asthma phenotype we describe is different than that observed in the emergency room studies, for example. However, it is worth emphasizing that, in most of the prevalence studies on childhood asthma and wheezing, phenotypes are defined in the same way as in the current study. Due to the study design, we could not address the question of the potential value of EBC-pH in asthma monitoring.

The analysis of EBC-pH data before the completion of the 8-yr follow-up is one of the limitations of this study, particularly in terms of the generalizability of the data. However, it is worth emphasizing that the response rate during the first 18 mo of this 8-yr review was 91%, and the analysis of EBC-pH measurement in 630 children is likely to be representative of the whole population.

Food or drink intake before EBC collection may influence the EBC-pH. The cut-off for food/drink intake we used (0.5 h) could potentially have been too short, influencing the outcomes. In a group of 238 children, we performed EBC measurement at least 2 h after the food/drink intake. The results in this group did not materially differ when compared with the whole population, suggesting that food or drink intake did not influence the outcomes (data not shown, available on request).

Another possible limitation of the study is that we measured the EBC-pH after deaeration with argon. Although some authors have questioned this methodology because CO₂ and carbonic acid may be relevant in the airway (13, 16, 26), it has been used in the majority of previous studies (10, 12, 27, 28). Furthermore, as the level of CO₂ in EBC is not informative of the pH of the source fluid, deaeration (or gas standardization) is the preferred method for clinical studies (29).

Comparison with Previous Studies
Only a small number of studies in adult and children have explored the role of EBC-pH in asthma, often with conflicting results (summarized in Table E1). It has to be emphasized that our study is an epidemiologic study, and the outcomes need to be viewed within such context. We studied an unselected, population-based birth cohort, and the results are thus generalizable to the general population. This is markedly different from a number of previous studies, most of which recruited subjects from the tertiary referral centers, resulting in a bias among the cases toward the more severe disease.

Carpagnano and colleagues investigated the EBC-pH in 20 children with stable asthma and 15 healthy control children of a similar age to our population and reported significantly lower values in children with asthma than in the control children (10). These findings are similar to an Italian study in 29 children with atopic asthma and 13 healthy children (28). In another pediatric study, which used nitrogen to deaerate samples, no difference in EBC-pH was observed between 23 children with asthma and 9 control children (16). More recently, MacGregor and colleagues showed no difference in EBC-pH between 74 children with chronic asthma and 47 healthy control subjects (14). Adult studies investigating EBC-pH in asthma have revealed similarly conflicting results (11, 12, 30, 31),

In the current study, the differences in EBC-pH between children who had ever wheezed or ever had asthma (parentally reported or diagnosed by a physician) and those that never had wheeze or asthma were very small (despite being statistically significant). The marked overlap in EBC-pH values among children that ever had, currently had, or never had asthma/wheeze suggests that EBC-pH measurement has limitations in differentiating children with parentally reported current stable asthma from healthy control subjects in this setting. We have not formally pursued the diagnostic test approach for EBC-pH, because we found no significant association between this marker and parentally reported asthma. However, given the nonsignificant findings using the comparison tests and the very small actual differences in EBC-pH levels among groups, it is clear that very poor sensitivity and specificity values would be obtained. However, our results do not exclude the possibility that EBC-pH may be a useful tool for asthma monitoring.

Vaughan and colleagues reported that, in healthy individuals, EBC-pH is slightly alkaline, held in a narrow range, and is a robust and relevant marker of disease (32). Although our findings in more than 500 subjects without asthma/wheeze confirm that, in healthy individuals, EBC-pH is slightly alkaline, they do not support the remainder of the above statement. We have demonstrated that even in healthy children, EBC-pH has a very wide range (4.40–8.29), and furthermore, it does not unequivocally reflect the presence of symptoms. The data we obtained among healthy children are similar to those recently reported by Paget-Brown and coworkers (29).

In contrast to the findings of some other studies (10, 12, 17, 28, 30), our study suggests that EBC-pH does not reflect the underlying severity of wheeze/asthma during the stable phase of the disease in the epidemiologic setting. We explored the wheeze variables that reflect disease severity in terms of frequency of wheezing attacks, sleep disturbance, and the limitation of speech caused by wheeze. Our findings are in line with the report by Rosias and colleagues who demonstrated no relationship between EBC-pH and asthma control questionnaire score (16). In contrast, a recent study from China demonstrated a significant association between EBC-pH and asthma severity (17). In two pediatric studies, EBC-pH was able to differentiate patients with mild from those with moderate/severe asthma (10, 17). In one adult study, EBC-pH did not differentiate patients with stable mild asthma from healthy control subjects, but it was significantly lower in patients with moderate asthma than in those with mild asthma, and significantly lower in steroid-naive asthmatics when compared with those treated with inhaled corticosteroids (ICS) (12).

Recently, another adult study reported the effect of treatment on EBC-pH (30). The levels increased slightly after 2 mo and significantly after 6 mo in a group of patients with asthma receiving ICS, but no change was observed among patients with asthma not treated with ICS or healthy control subjects. Evaluating the role of EBC-pH in monitoring response to asthma therapy was beyond the scope of the current study.

We found no consistent association between EBC-pH and lung function among current wheezers during the stable phase of the disease. Our results are consistent with all previous pediatric studies (10, 14, 16, 17, 28). Interestingly, we found that current wheezers with lower sRaw values had lower EBC-pH when compared with wheezers who had higher sRaw (i.e., poorer lung function). These results are difficult to explain, because one would expect children with poorer lung function to have lower EBC-pH. However, the observed differences were very small.

In our study, 32% of current wheezers who successfully completed the challenge had a positive response to methacholine provocation. A strong trend for higher EBC-pH values was observed among these subjects compared with those who had a negative challenge. However, the difference between the two groups was small. Furthermore, we found no difference in EBC-pH between children with current wheeze and positive methacholine challenge who were currently receiving asthma medication (i.e., the most stringent asthma definition in epidemiologic terms) and those without any of these features.

Hunt and colleagues were the first to report that the EBC-pH is over two log orders lower in patients with acute asthma than in control subjects, and normalizes with corticosteroid therapy (11). Furthermore, they suggested that, at these low pH values, the endogenous airway compound nitrite is converted to NO in quantities sufficient to account for the high concentrations of eNO in patients with asthma. This was supported by the findings of a pediatric study, which showed a negative correlation between EBC-pH and eNO (28). However, Ojoo and colleagues reported that patients with mild stable asthma have raised eNO and EBC nitrite/nitrate, but similar EBC-pH when compared with healthy control subjects, demonstrating dissociation between EBC-pH and eNO (31). In our study, although children in the whole population who had eNO values lower than 6.95 ppb had significantly higher EBC pH values than children with eNO greater than 9.40 ppb, the numeric differences were small, and the overlap considerable. Moreover, no differences in EBC-pH were observed among current wheezers with high or low eNO values, suggesting no association between eNO and EBC-pH in this patient population. The lack of an association between these markers was reported in two further pediatric studies (16, 17).

	CONCLUSIONS

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We have demonstrated that measurement of deargonized EBC-pH using the methodology followed in the current study is not useful in differentiating between children with and without symptoms suggestive of childhood asthma in the epidemiologic setting, and for asthma based on parental reporting. In addition, we found no consistent association between EBC-pH and the objective indices of asthma severity (lung function, airway reactivity, and frequency and severity of symptoms) in this sample of children from the general population.

	Acknowledgments

 
The authors thank Angela Kelsall for her contribution in the lung function laboratory; Mark Craven, M.Sc., and Elizabeth Hadley, Ph.D., for their assistance in the EBC-pH measurements; and Julie Morris, M.Sc., for statistical advice.

	FOOTNOTES

 
Supported by the Moulton Charitable Trust.

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1164/rccm.200601-140OC on May 4, 2006

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of the manuscript.

Received in original form January 31, 2006; accepted in final form April 30, 2006

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