- Email: [email protected]
Long-term repeatability of exhaled breath condensate pH in asthma
ARTICLE IN PRESS Respiratory Medicine (2008) 102, 377–381
Long-term repeatability of exhaled breath condensate pH in asthma Rosalba Accordinoa, Annalisa Visentina, Anna Bordina, Silvia Ferrazzonia, Emanuela Mariana, Federico Rizzatoa, Cristina Canovaa, Roberta Venturinib, Piero Maestrellia, a
Department of Environmental Medicine and Public Health, University of Padova, Padova, Italy Department of Laboratory Medicine, Azienda Ospedaliera di Padova, Padova, Italy
b
Received 20 June 2007; accepted 25 October 2007 Available online 3 December 2007
KEYWORDS Airways biomarkers; Lung function; Respiratory diseases; Seasons
Summary Exhaled breath condensate (EBC) is being used increasingly to sample airway fluid. EBC pH may be a biomarker of airway inflammation in asthma. In this study, we assessed the longterm reproducibility of EBC pH in asthma. We examined 31 asthmatic patients and eight healthy subjects three times over a 1-year period (winter, autumn and summer). EBC pH was measured after argon deaeration. Repeatability of pH measurements was assessed using intraclass correlation coefficients (ICC) and the limits of agreement (LOA) between seasons were calculated according to Bland–Altman method. No significant differences in EBC pH between seasons were detected in healthy subjects and asthmatic patients. EBC pH showed high repeatability either in healthy subjects (ICC ¼ 0.94) or in asthmatics (ICC ¼ 0.97). Variability between seasons was greater in asthmatics than in healthy subjects: winter–autumn LOA 0.68/+0.52 and 0.31/+0.31, autumn–summer LOA 0.75/+0.67 and 0.24/+0.15, winter–summer LOA 0.92/+0.67 and 0.34/+0.23 in asthmatic and healthy subjects, respectively. In a subgroup of 11 asthmatics who remained in stable conditions during the study, no substantially different LOA were observed in EBC pH compared with the whole group of asthmatics. Asthmatic smokers (n ¼ 10) tended to have lower EBC pH (7.5770.46) than asthmatic non-smokers (n ¼ 21) (7.7470.21; p ¼ 0.063) and wider LOA. In conclusion, we demonstrated that EBC pH exhibits good repeatability in long-term assessment. EBC pH in asthmatics tended to fluctuate more than in healthy subjects. However, EBC pH variability in asthma was not influenced by changes in clinical status. Rather, we suggest that cigarette smoke may be implicated in EBC pH variability. & 2007 Elsevier Ltd. All rights reserved.
Corresponding author. Tel.: +39 049 821 2564; fax: +39 049 821 2566.
E-mail address: [email protected] (P. Maestrelli). 0954-6111/$ - see front matter & 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.rmed.2007.10.014
ARTICLE IN PRESS 378
Introduction
R. Accordino et al. ments, Padova, Italy) with a flat membrane electrode (5207; Crison Instruments S.A., Alella, Spain) and an accuracy of 70.01 pH. Amylase was measured in all samples using an enzymatic colorimetric test (IFCC; Roche Diagnostic Modular, lower limit of detection 3 U/L). Samples containing amylase were discarded.
Exhaled breath condensate (EBC) is a biological fluid that is collected by cooling exhaled air during tidal breathing. Collection of EBC is a non-invasive, simple and inexpensive method that can be performed repeatedly without adversely affecting a patient. EBC pH is currently considered a robust variable to determine the degree of acidification of EBC in patients with various inflammatory lung diseases. An abnormally low EBC pH is found during acute exacerbations of asthma, chronic obstructive pulmonary disease (COPD) and cystic fibrosis, and also in stable COPD, bronchiectasis and moderate asthma.1–3 It is still controversial whether EBC pH correlates with other indices of airway inflammation.4,5 However, this simple assay might be a useful surrogate for invasive or more complicated attempts at assessment of airway inflammation in asthma, such as biopsy, bronchoalveolar lavage or induced sputum. Technical confounders that may affect pH measurements have been investigated in order to refine this tool for application in clinical setting.6–8 Short-term repeatability of measuring EBC pH in asthmatic and healthy subjects was evaluated by some studies.5,6,8,9 However, studies on long-term variability of airway pH have not been performed. This information may be relevant for the use of EBC pH as biomarker in longer prospective studies. The aim of this study was to assess the long-term repeatability of EBC pH in asthma. EBC was collected in a standardized manner in healthy subjects and stable asthmatics three times over 1-year period and pH was measured in Argon-deaerated samples.
Long-term repeatability of EBC pH was tested in asthmatic and healthy subjects. Samples of EBC for pH analysis were collected on three occasions over a year period (winter, autumn and summer). On each occasion, EBC was collected for 15 min at a condenser temperature of 55 1C. The level of asthma control was evaluated at each visit with lung function tests (forced expiratory volume in 1 s, FEV1), asthma control questionnaire (ACT),12 regimen of inhaled and/or oral corticosteroids.
Methods
Statistical analysis
Subjects
Comparisons of sampling conditions (different temperatures and duration of collection), among seasons, between asthmatic and healthy subjects, and between smokers and non-smokers were performed by repeated measures analysis of variance. Values of po0.05 were considered significant. Intraclass correlation coefficients (ICC) were determined13 and Bland–Altman analysis14 was performed for pH measured on three occasions over a year to evaluate repeatability. The limits of agreement (LOA) were calculated to estimate the variability expected from individual measurements. Data are presented as mean7SD.
A total of 32 patients with mild to severe asthma, diagnosed according to the Global initiative for Asthma (GINA),10 and 10 healthy volunteers were recruited for the study. Subjects were excluded if they had experienced an exacerbation of asthma or respiratory tract infection within the last 4 weeks. All subjects provided written informed consent and the local ethics committee approved the study.
Collection of EBC and pH measurement EBC was collected with a simple homemade apparatus as described by Goldoni et al.11 Samples were collected during tidal breathing for 15 min. Subjects were instructed to breathe normally through their mouth and to temporarily discontinue collection if they needed to swallow saliva or cough. No food was taken 1 h before collection. Samples were stored in 200 ml aliquots and Argon gas was bubbled in the sample for 3 min to achieve deaeration. In preliminary experiments, we found that pH increases bubbling argon up to 2 min; then, deaeration for 4, 8, and 10 min did not induced further changes in pH. Therefore, we established that bubbling argon for 3 min is sufficient for optimal deaereation of 200 ml EBC samples. Then, pH was measured using a calibrated pH meter (model pH300; Hanna Instru-
Effects of the temperature and the duration of collection on EBC pH The effect of temperature and duration of collection on EBC pH were evaluated in samples of 10 healthy subjects. Consecutive collections at various temperatures (5, 20, and 55 1C) were performed. To evaluate the effect of duration of collection on pH, EBC was collected at 5-min consecutive intervals for a total time of 20 min. All samples were processed for pH assay after deaeration with argon.
Long-term repeatability of EBC pH
Results Subjects Table 1 shows the characteristics of the subjects examined. The two groups of subjects were similar with regard to age and sex. Two healthy subjects were lost to follow-up and one asthmatic patient was excluded due to exacerbation. Thus, 8 healthy subjects and 31 asthmatics completed the study on long-term repeatability. Mean FEV1/FVC of the asthmatic group was 92714%. One patient with severe asthma was on oral corticosteroids.
ARTICLE IN PRESS Exhaled breath condensate in asthma
379
Temperature and duration of collection of exhaled breath condensate Mean values (7SD) of EBC pH collected at condenser temperatures of 5, 20, 55 1C and at collection intervals of 5, 10, 15, and 20 min are presented in Table 2. There were no significant differences identified in the pH of EBC collected at different temperatures in these healthy subjects. Furthermore, the duration of collection did not significantly affect EBC pH values.
Long-term repeatability of EBC pH No significant differences in EBC pH among seasons were detected in healthy subjects (winter 7.8870.08, autumn 7.8970.14, summer 7.8470.10; p ¼ 0.56) and asthmatic patients (winter 7.7570.37, autumn 7.6770.20, summer 7.6370.36; p ¼ 0.34). Asthmatics tended to exhibit lower EBC pH than healthy subjects, but the difference was not significant (p ¼ 0.23). We did not find any relationship of EBC pH with gender, smoked pack-year, or grade of severity of asthma. EBC pH showed high repeatability either in healthy subjects (ICC ¼ 0.94) or in asthmatics (ICC ¼ 0.97). We then asked whether pH variability was related to changes in the level of asthma control assessed by different parameters during the study. Changes in FEV1 by more than 20%, in ACT score by more than 1 point, in dose of daily corticosteroids by 50% or more among the three visits were used to define subgroups of patients with a lower control of asthma. Table 1 Characteristics of healthy subjects and asthmatic patients. Healthy subjects
Asthmatic patients
Subjects (no., sex) Age (mean7SD)
3M, 7F 3571
16M, 16F 3977.6
Severity of asthma (GINA 2006)
–
7 mild (22%) 11 moderate (34%) 14 severe (44%)
Inhaled steroids (no., %) Oral steroids (no., %) Current smokers (no., %) Pack-year (no.)
Table 2
– – 1(10%) –
22 (69%) 1 (3%) 10 (31%) 15713
Table 3 shows ICC in subgroups of asthmatics defined according to these criteria. Patients with higher FEV1 variability tended to have slightly less ICC of EBC pH, while ACT score and dosage of corticosteroids did not affect pH repeatability. Variability between seasons was greater in asthmatics than in healthy subjects: winter–autumn LOA 0.68/+0.52 and 0.31/+0.31, autumn–summer LOA 0.75/+0.67 and 0.24/+0.15, winter–summer LOA 0.92/+0.67 and 0.34/ +0.23 in asthmatic and healthy subjects, respectively (Figure 1a, b). The LOA were calculated in a subgroup of 11 asthmatic patients who were defined in stable conditions along the period of the study, i.e. exhibiting changes in FEV1o20%, in ACT score o2 points, and in daily dose of corticosteroids o50%. Except for autumn–summer LOA (0.28/+0.44), no substantially different LOA were observed in winter–summer (0.86/+0.72) and winter–autumn (0.76/+0.59) EBC pH compared with the whole group of asthmatics. We further investigated whether EBC pH variability was affected by smoking habit. EBC pH tended to be lower in asthmatic smokers (7.5770.46) than in asthmatic non-smokers (7.7470.21), but the difference was not statistically significant (p ¼ 0.063). The LOA were wider in asthmatic smokers than in asthmatic non-smokers (Figure 2a, b).
Discussion In this study, we have demonstrated that measurements of EBC pH exhibit high repeatability in asthmatics and healthy
Table 3 Intraclass correlation coefficients (ICC) of EBC pH in subgroups of asthmatic patients defined by variability of FEV1, ACT score, daily dose of corticosteroids. Parameter
No. of patients
ICC
Change in FEV1a 420% p20%
10 21
0.92 0.98
Change in ACT scorea X2 o2
14 17
0.98 0.95
Change in corticosteroids dosea X50% 16 o50% 15 a
0.96 0.98
Calculated as maximal change among the three visits.
Effects of temperature and duration of collection on EBC pH.
Temperature of collection (1C)
pH (mean7SD)
Duration of collection (min)
pH (mean7SD)
5 20 55
7.7270.38 7.5770.58 7.9170.73
5 10 15 20
ANOVA
p ¼ 0.36
7.3570.88 7.4370.68 7.5270.48 7.5770.49 p ¼ 0.10
ARTICLE IN PRESS 380
R. Accordino et al. Asthmatic subjects 1.5 1
pH
0.5 0 -0.5 -1 -1.5 Summer/Autumn
Summer/Winter
Autumn/Winter
pH
Healthy subjects 1.5 1 0.5 0 -0.5 -1 -1.5 Summer/Autumn
Summer/Winter
Autumn/Winter
Figure 1 Limits of agreement for long-term variability of exhaled breath condensate pH in asthmatic patients (a) and healthy control subjects (b). Asthmatic smokers 1.5 1 pH
0.5 0 -0.5 -1 -1.5 Summer/Autumn
Summer/Winter
Autumn/Winter
Asthmatic Non smokers 1.5 1 pH
0.5 0 -0.5 -1 -1.5 Summer/Autumn
Summer/Winter
Autumn/Winter
Figure 2 Limits of agreement for long-term variability of exhaled breath condensate pH in asthmatic smokers (a) and asthmatic non-smokers (b).
subjects over 1-year interval of time. Greater differences in EBC pH were observed in asthmatics over time suggesting that airway pH fluctuated in this disease. In addition, we confirmed, using a different collection apparatus, that pH in deaerated EBC is not influenced by the temperature of the condenser.8 The trend of increased pH when the duration of collection was increased, although not statistically significant, suggests that the period of condensation should be kept constant to obtain comparable results.
Although the temperature we used to condensate the exhaled breath was lower than in some previous studies, the mean EBC pH values in healthy subjects of current study is consistent with that observed in a large data set collected to obtain normative data for EBC pH.15 Since EBC pH value is dependent on the collection device used, our result not necessarily can be extended to EBC pH collected with commercial devices.16 Asthmatic patients tended to have lower EBC pH than healthy subjects, but the difference was not significant. This is in agreement with the findings of recent studies in adults and children with atopic stable asthma.4,17 A further reason for the lack of significant difference between asthmatic and healthy subjects is that the majority of our asthmatic patients were using inhaled corticosteroids. In fact, the study by Hunt et al.3 found that therapy with glucocorticosteroids increased the EBC pH in asthmatic subjects towards the values measured in normal subjects. Other studies assessed repeatability of EBC pH, but in none a long-term follow-up of asthmatic patients was applied. This is relevant because information of long-term repeatability is necessary to design and interpret prospective studies in asthma when EBC pH is used as a non-invasive marker of airway biochemistry. Vaughan et al.8 reported a good repeatability of deaerated EBC pH using a commercial apparatus. Intra-week coefficient of variation (CV) was 4.5% (range 0.9–20%) in 76 healthy subjects. A similar intra-week CV of pH measured in not deaerated EBC collected with a different device in a group of subjects including 26 healthy controls and 42 asthmatics (CV 5.0%, range 1.8–8.1%) was obtained by Prince et al.9 The coefficients of variation calculated for longer term measurements of EBC pH in our asthmatic (2.4%, range 0.6–10.6%) and healthy subjects (1.2%, range 0.8–1.6%) were less than intra-week CVs previously reported. Kostikas et al.1 assessed the repeatability of EBC pH on 2 consecutive days in normal subjects and patients with asthma and COPD. However, the data were presented as means and the variability between individual samples cannot be estimated. Between day LOA for pH of samples collected maximum 1 week apart reported by Leung et al.5 (0.32/0.31) and by Borrill et al.6 (0.46/0.44) in healthy controls were not narrower than those between seasons in the present study. This suggests that in normal subjects EBC pH is relatively stable and there are not additional sources of variation in the long-term period. Although asthma exhibits more clinical and functional variability than COPD, between-day variability of EBC pH was much higher in COPD6 than in our patients with asthma. The reason by which EBC pH fluctuates more in COPD remains undetermined since the mechanisms of airway pH regulation are poorly understood. It can be hypothesized that oxidative stress and/or bacterial colonization may contribute to variations of pH in COPD.18,1 We examined whether clinical status, functional parameters and drug treatment can explain EBC pH variability. Changes in ACT score, in FEV1 and in the corticosteroids regimen were not associated with higher variability. In addition, the measurements of EBC pH in asthmatics who remain in stable conditions during the study visits were not more repeatable than those in less stable patients. Therefore, the source of pH variability in asthma remains undetermined.
ARTICLE IN PRESS Exhaled breath condensate in asthma It cannot rule out that environmental factors such as inhaled particles and gases, temperature and humidity may influence EBC pH variability. This possibility could explain why less agreement was observed both in stable asthma and healthy controls between EBC pH measurements in winter samples and those performed in the other seasons, than between measurements in autumn and summer. Average concentrations of air-suspended particulate matter (PM10) in the urban area of Padova, where the patients lived, was indeed higher in winter at the time when the patients were examined (84.6729.3 mg/m3) than in summer (43.8712.9 mg/m3, po0.001) or autumn (47.2714.0 mg/m3, po0.001). Similarly, the other pollutants derived from combustion (SO2, NO2, CO) were significantly higher in winter compared with the other seasons (data not shown). Variability of EBC pH due to smoking was not investigated previously in asthma. Vaughan et al.8 stated that being a cigarette smoker did not appear to affect EBC pH in healthy subjects and similar variability was observed in COPD exsmokers and current smokers.6 Since our aim was to investigate whether smoking influences pH variability, not if EBC pH values are different in smoking and non-smoking asthmatics, the study is possibly not powered for this comparison. Indeed, we calculated that 16 subjects for group are necessary to show a difference in EBC pH of 0.3 with a significance of 0.05 and a power of 80% assuming a SD of 0.3, as observed in the present study. Our findings that smoking habit increases EBC pH variability is in line with the hypothesis that environmental factors may affect EBC. In conclusion, we demonstrated that EBC pH exhibits good repeatability even in long-term assessment. EBC pH in asthmatics tended to fluctuate more than in healthy subjects. However, EBC pH variability was not influenced by changes in clinical, functional and therapeutical parameters in asthma. Rather, we suggest that environmental factors may be implicated in EBC pH variability. The significance of EBC pH has been challenged, at least in COPD, by Effros et al.19 Since the present study was not designed to compare controlled and not controlled asthmatics and subjects under exacerbation were not examined, our results leave open the question whether EBC pH is sufficiently sensitive to detect changes in clinical and functional status of asthma milder than those during an exacerbation. Therefore, the clinical utility of longitudinal monitoring of EBC pH remains to be determined.
Conflict of interest statement None of the authors has a financial relationship with a commercial entity that has interest in the subject of this manuscript.
Acknowledgments The authors gratefully acknowledge the supports by the Italian Ministry of University and Research (PRIN 2005), by the University of Padova (grants ex60%), by ARPA Veneto and by Associazione Ricerca Cura Asma, Padova, Italy. The authors also thank Giovanna Fulgeri for secretarial assistance and Luigi Zedda for technical assistance.
381
References 1. Kostikas K, Papatheodorou G, Ganas K, Psathakis K, Panagou P, Loukides S. pH in expired breath condensate of patients with inflammatory airway diseases. Am J Respir Crit Care Med 2002;165:1364–70. 2. Tate S, MacGregor G, Davis M, Innes JA, Greening AP. Airways in cystic fibrosis are acidified: detection by exhaled breath condensate. Thorax 2002;57(11):926–9. 3. Hunt JF, Fang K, Malik R, Snyder A, Malhotra N, Platts-Mills TAE, et al. Endogenous airway acidification implications for asthma pathophysiology. Am J Respir Crit Care Med 2000;161(3): 694–9. 4. Ojoo JC, Mulrennan SA, Kastelik JA, Morice AH, Redington AE. Exhaled breath condensate pH and exhaled nitric oxide in allergic asthma and in cystic fibrosis. Thorax 2005;60(1): 22–6. 5. Leung TF, Li CY, Yung E, Liu EKH, Lam CWK, Wong GWK. Clinical and technical factors affecting pH and other biomarkers in exhaled breath condensate. Pediatr Pulmonol 2006;41(1): 87–94. 6. Borrill Z, Starkey C, Vestbo J, Singh D. Reproducibility of exhaled breath condensate pH in chronic obstructive pulmonary disease. Eur Respir J 2005;25(2):269–74. 7. McCafferty JB, Bradshaw TA, Tate S, Greening AP, Innes JA. Effects of breathing pattern and inspired air conditions on breath condensate volume, pH, nitrite, and protein concentrations. Thorax 2004;59(8):694–8. 8. Vaughan J, Ngamtrakulpanit L, Pajewski TN, Turner R, Nguyen TA, Smith A, et al. Exhaled breath condensate pH is a robust and reproducible assay of airway acidity. Eur Respir J 2003;22(6): 889–94. 9. Prince P, Boulay ME, Boulet LP. A fast, simple, and inexpensive method to collect exhaled breath condensate for pH determination. Ann Allergy Asthma Immunol 2006;97(5):622–7. 10. Workshop report: Global Initiative for Asthma—updated 2006. Available from /http://www.ginasthma.comS. 11. Goldoni M, Catalani S, De Palma G, Manini P, Acampa O, Corradi M, et al. Exhaled breath condensate as a suitable matrix to assess lung dose and effects in workers exposed to cobalt and tungsten. Environ Health Perspect 2004;112(13):1293–8. 12. Nathan RA, Sorkness CA, Kosinski M, Schatz M, Li JT, Marcus P, et al. Development of the asthma control test: a survey for assessing asthma control. J Allergy Clin Immunol 2004;113(1): 59–65. 13. Chinn S. Statistics in respiratory medicine. 2. Repeatability and method comparison. Thorax 1991;46(6):454–6. 14. Bland JM, Altman DG. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1986;1(8476):307–10. 15. Paget-Brown AO, Ngamtrakulpanit L, Smith A, Bunyan D, Hom S, Nguyen A, et al. Normative data for pH of exhaled breath condensate. Chest 2006;129:426–30. 16. Prieto L, Ferrer A, Palop J, Domonech J, Llusar R, Rojas R. Differences in exhaled breath condensate pH measurements between samples obtained with two commercial devices. Respir Med 2007;101:1715–20. 17. Ratnawati MJ, Henry RL, Thomas PS. Exhaled breath condensate nitrite/nitrate and pH in relation to pediatric asthma control and exhaled nitric oxide. Pediatr Pulmonol 2006; 41(10):929–36. 18. Rahman I, MacNee W. Oxidative stress and regulation of glutathione in lung inflammation. Eur Respir J 2000;16:534–54. 19. Effros RM, Casaburi R, Su J, Dunning M, Torday J, Biller J, et al. The effects of volatile salivary acids and bases on exhaled breath condensate pH. Am J Respir Crit Care Med 2006;173: 386–92.
Long-term repeatability of exhaled breath condensate pH in asthma Rosalba Accordinoa, Annalisa Visentina, Anna Bordina, Silvia Ferrazzonia, Emanuela Mariana, Federico Rizzatoa, Cristina Canovaa, Roberta Venturinib, Piero Maestrellia, a
Department of Environmental Medicine and Public Health, University of Padova, Padova, Italy Department of Laboratory Medicine, Azienda Ospedaliera di Padova, Padova, Italy
b
Received 20 June 2007; accepted 25 October 2007 Available online 3 December 2007
KEYWORDS Airways biomarkers; Lung function; Respiratory diseases; Seasons
Summary Exhaled breath condensate (EBC) is being used increasingly to sample airway fluid. EBC pH may be a biomarker of airway inflammation in asthma. In this study, we assessed the longterm reproducibility of EBC pH in asthma. We examined 31 asthmatic patients and eight healthy subjects three times over a 1-year period (winter, autumn and summer). EBC pH was measured after argon deaeration. Repeatability of pH measurements was assessed using intraclass correlation coefficients (ICC) and the limits of agreement (LOA) between seasons were calculated according to Bland–Altman method. No significant differences in EBC pH between seasons were detected in healthy subjects and asthmatic patients. EBC pH showed high repeatability either in healthy subjects (ICC ¼ 0.94) or in asthmatics (ICC ¼ 0.97). Variability between seasons was greater in asthmatics than in healthy subjects: winter–autumn LOA 0.68/+0.52 and 0.31/+0.31, autumn–summer LOA 0.75/+0.67 and 0.24/+0.15, winter–summer LOA 0.92/+0.67 and 0.34/+0.23 in asthmatic and healthy subjects, respectively. In a subgroup of 11 asthmatics who remained in stable conditions during the study, no substantially different LOA were observed in EBC pH compared with the whole group of asthmatics. Asthmatic smokers (n ¼ 10) tended to have lower EBC pH (7.5770.46) than asthmatic non-smokers (n ¼ 21) (7.7470.21; p ¼ 0.063) and wider LOA. In conclusion, we demonstrated that EBC pH exhibits good repeatability in long-term assessment. EBC pH in asthmatics tended to fluctuate more than in healthy subjects. However, EBC pH variability in asthma was not influenced by changes in clinical status. Rather, we suggest that cigarette smoke may be implicated in EBC pH variability. & 2007 Elsevier Ltd. All rights reserved.
Corresponding author. Tel.: +39 049 821 2564; fax: +39 049 821 2566.
E-mail address: [email protected] (P. Maestrelli). 0954-6111/$ - see front matter & 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.rmed.2007.10.014
ARTICLE IN PRESS 378
Introduction
R. Accordino et al. ments, Padova, Italy) with a flat membrane electrode (5207; Crison Instruments S.A., Alella, Spain) and an accuracy of 70.01 pH. Amylase was measured in all samples using an enzymatic colorimetric test (IFCC; Roche Diagnostic Modular, lower limit of detection 3 U/L). Samples containing amylase were discarded.
Exhaled breath condensate (EBC) is a biological fluid that is collected by cooling exhaled air during tidal breathing. Collection of EBC is a non-invasive, simple and inexpensive method that can be performed repeatedly without adversely affecting a patient. EBC pH is currently considered a robust variable to determine the degree of acidification of EBC in patients with various inflammatory lung diseases. An abnormally low EBC pH is found during acute exacerbations of asthma, chronic obstructive pulmonary disease (COPD) and cystic fibrosis, and also in stable COPD, bronchiectasis and moderate asthma.1–3 It is still controversial whether EBC pH correlates with other indices of airway inflammation.4,5 However, this simple assay might be a useful surrogate for invasive or more complicated attempts at assessment of airway inflammation in asthma, such as biopsy, bronchoalveolar lavage or induced sputum. Technical confounders that may affect pH measurements have been investigated in order to refine this tool for application in clinical setting.6–8 Short-term repeatability of measuring EBC pH in asthmatic and healthy subjects was evaluated by some studies.5,6,8,9 However, studies on long-term variability of airway pH have not been performed. This information may be relevant for the use of EBC pH as biomarker in longer prospective studies. The aim of this study was to assess the long-term repeatability of EBC pH in asthma. EBC was collected in a standardized manner in healthy subjects and stable asthmatics three times over 1-year period and pH was measured in Argon-deaerated samples.
Long-term repeatability of EBC pH was tested in asthmatic and healthy subjects. Samples of EBC for pH analysis were collected on three occasions over a year period (winter, autumn and summer). On each occasion, EBC was collected for 15 min at a condenser temperature of 55 1C. The level of asthma control was evaluated at each visit with lung function tests (forced expiratory volume in 1 s, FEV1), asthma control questionnaire (ACT),12 regimen of inhaled and/or oral corticosteroids.
Methods
Statistical analysis
Subjects
Comparisons of sampling conditions (different temperatures and duration of collection), among seasons, between asthmatic and healthy subjects, and between smokers and non-smokers were performed by repeated measures analysis of variance. Values of po0.05 were considered significant. Intraclass correlation coefficients (ICC) were determined13 and Bland–Altman analysis14 was performed for pH measured on three occasions over a year to evaluate repeatability. The limits of agreement (LOA) were calculated to estimate the variability expected from individual measurements. Data are presented as mean7SD.
A total of 32 patients with mild to severe asthma, diagnosed according to the Global initiative for Asthma (GINA),10 and 10 healthy volunteers were recruited for the study. Subjects were excluded if they had experienced an exacerbation of asthma or respiratory tract infection within the last 4 weeks. All subjects provided written informed consent and the local ethics committee approved the study.
Collection of EBC and pH measurement EBC was collected with a simple homemade apparatus as described by Goldoni et al.11 Samples were collected during tidal breathing for 15 min. Subjects were instructed to breathe normally through their mouth and to temporarily discontinue collection if they needed to swallow saliva or cough. No food was taken 1 h before collection. Samples were stored in 200 ml aliquots and Argon gas was bubbled in the sample for 3 min to achieve deaeration. In preliminary experiments, we found that pH increases bubbling argon up to 2 min; then, deaeration for 4, 8, and 10 min did not induced further changes in pH. Therefore, we established that bubbling argon for 3 min is sufficient for optimal deaereation of 200 ml EBC samples. Then, pH was measured using a calibrated pH meter (model pH300; Hanna Instru-
Effects of the temperature and the duration of collection on EBC pH The effect of temperature and duration of collection on EBC pH were evaluated in samples of 10 healthy subjects. Consecutive collections at various temperatures (5, 20, and 55 1C) were performed. To evaluate the effect of duration of collection on pH, EBC was collected at 5-min consecutive intervals for a total time of 20 min. All samples were processed for pH assay after deaeration with argon.
Long-term repeatability of EBC pH
Results Subjects Table 1 shows the characteristics of the subjects examined. The two groups of subjects were similar with regard to age and sex. Two healthy subjects were lost to follow-up and one asthmatic patient was excluded due to exacerbation. Thus, 8 healthy subjects and 31 asthmatics completed the study on long-term repeatability. Mean FEV1/FVC of the asthmatic group was 92714%. One patient with severe asthma was on oral corticosteroids.
ARTICLE IN PRESS Exhaled breath condensate in asthma
379
Temperature and duration of collection of exhaled breath condensate Mean values (7SD) of EBC pH collected at condenser temperatures of 5, 20, 55 1C and at collection intervals of 5, 10, 15, and 20 min are presented in Table 2. There were no significant differences identified in the pH of EBC collected at different temperatures in these healthy subjects. Furthermore, the duration of collection did not significantly affect EBC pH values.
Long-term repeatability of EBC pH No significant differences in EBC pH among seasons were detected in healthy subjects (winter 7.8870.08, autumn 7.8970.14, summer 7.8470.10; p ¼ 0.56) and asthmatic patients (winter 7.7570.37, autumn 7.6770.20, summer 7.6370.36; p ¼ 0.34). Asthmatics tended to exhibit lower EBC pH than healthy subjects, but the difference was not significant (p ¼ 0.23). We did not find any relationship of EBC pH with gender, smoked pack-year, or grade of severity of asthma. EBC pH showed high repeatability either in healthy subjects (ICC ¼ 0.94) or in asthmatics (ICC ¼ 0.97). We then asked whether pH variability was related to changes in the level of asthma control assessed by different parameters during the study. Changes in FEV1 by more than 20%, in ACT score by more than 1 point, in dose of daily corticosteroids by 50% or more among the three visits were used to define subgroups of patients with a lower control of asthma. Table 1 Characteristics of healthy subjects and asthmatic patients. Healthy subjects
Asthmatic patients
Subjects (no., sex) Age (mean7SD)
3M, 7F 3571
16M, 16F 3977.6
Severity of asthma (GINA 2006)
–
7 mild (22%) 11 moderate (34%) 14 severe (44%)
Inhaled steroids (no., %) Oral steroids (no., %) Current smokers (no., %) Pack-year (no.)
Table 2
– – 1(10%) –
22 (69%) 1 (3%) 10 (31%) 15713
Table 3 shows ICC in subgroups of asthmatics defined according to these criteria. Patients with higher FEV1 variability tended to have slightly less ICC of EBC pH, while ACT score and dosage of corticosteroids did not affect pH repeatability. Variability between seasons was greater in asthmatics than in healthy subjects: winter–autumn LOA 0.68/+0.52 and 0.31/+0.31, autumn–summer LOA 0.75/+0.67 and 0.24/+0.15, winter–summer LOA 0.92/+0.67 and 0.34/ +0.23 in asthmatic and healthy subjects, respectively (Figure 1a, b). The LOA were calculated in a subgroup of 11 asthmatic patients who were defined in stable conditions along the period of the study, i.e. exhibiting changes in FEV1o20%, in ACT score o2 points, and in daily dose of corticosteroids o50%. Except for autumn–summer LOA (0.28/+0.44), no substantially different LOA were observed in winter–summer (0.86/+0.72) and winter–autumn (0.76/+0.59) EBC pH compared with the whole group of asthmatics. We further investigated whether EBC pH variability was affected by smoking habit. EBC pH tended to be lower in asthmatic smokers (7.5770.46) than in asthmatic non-smokers (7.7470.21), but the difference was not statistically significant (p ¼ 0.063). The LOA were wider in asthmatic smokers than in asthmatic non-smokers (Figure 2a, b).
Discussion In this study, we have demonstrated that measurements of EBC pH exhibit high repeatability in asthmatics and healthy
Table 3 Intraclass correlation coefficients (ICC) of EBC pH in subgroups of asthmatic patients defined by variability of FEV1, ACT score, daily dose of corticosteroids. Parameter
No. of patients
ICC
Change in FEV1a 420% p20%
10 21
0.92 0.98
Change in ACT scorea X2 o2
14 17
0.98 0.95
Change in corticosteroids dosea X50% 16 o50% 15 a
0.96 0.98
Calculated as maximal change among the three visits.
Effects of temperature and duration of collection on EBC pH.
Temperature of collection (1C)
pH (mean7SD)
Duration of collection (min)
pH (mean7SD)
5 20 55
7.7270.38 7.5770.58 7.9170.73
5 10 15 20
ANOVA
p ¼ 0.36
7.3570.88 7.4370.68 7.5270.48 7.5770.49 p ¼ 0.10
ARTICLE IN PRESS 380
R. Accordino et al. Asthmatic subjects 1.5 1
pH
0.5 0 -0.5 -1 -1.5 Summer/Autumn
Summer/Winter
Autumn/Winter
pH
Healthy subjects 1.5 1 0.5 0 -0.5 -1 -1.5 Summer/Autumn
Summer/Winter
Autumn/Winter
Figure 1 Limits of agreement for long-term variability of exhaled breath condensate pH in asthmatic patients (a) and healthy control subjects (b). Asthmatic smokers 1.5 1 pH
0.5 0 -0.5 -1 -1.5 Summer/Autumn
Summer/Winter
Autumn/Winter
Asthmatic Non smokers 1.5 1 pH
0.5 0 -0.5 -1 -1.5 Summer/Autumn
Summer/Winter
Autumn/Winter
Figure 2 Limits of agreement for long-term variability of exhaled breath condensate pH in asthmatic smokers (a) and asthmatic non-smokers (b).
subjects over 1-year interval of time. Greater differences in EBC pH were observed in asthmatics over time suggesting that airway pH fluctuated in this disease. In addition, we confirmed, using a different collection apparatus, that pH in deaerated EBC is not influenced by the temperature of the condenser.8 The trend of increased pH when the duration of collection was increased, although not statistically significant, suggests that the period of condensation should be kept constant to obtain comparable results.
Although the temperature we used to condensate the exhaled breath was lower than in some previous studies, the mean EBC pH values in healthy subjects of current study is consistent with that observed in a large data set collected to obtain normative data for EBC pH.15 Since EBC pH value is dependent on the collection device used, our result not necessarily can be extended to EBC pH collected with commercial devices.16 Asthmatic patients tended to have lower EBC pH than healthy subjects, but the difference was not significant. This is in agreement with the findings of recent studies in adults and children with atopic stable asthma.4,17 A further reason for the lack of significant difference between asthmatic and healthy subjects is that the majority of our asthmatic patients were using inhaled corticosteroids. In fact, the study by Hunt et al.3 found that therapy with glucocorticosteroids increased the EBC pH in asthmatic subjects towards the values measured in normal subjects. Other studies assessed repeatability of EBC pH, but in none a long-term follow-up of asthmatic patients was applied. This is relevant because information of long-term repeatability is necessary to design and interpret prospective studies in asthma when EBC pH is used as a non-invasive marker of airway biochemistry. Vaughan et al.8 reported a good repeatability of deaerated EBC pH using a commercial apparatus. Intra-week coefficient of variation (CV) was 4.5% (range 0.9–20%) in 76 healthy subjects. A similar intra-week CV of pH measured in not deaerated EBC collected with a different device in a group of subjects including 26 healthy controls and 42 asthmatics (CV 5.0%, range 1.8–8.1%) was obtained by Prince et al.9 The coefficients of variation calculated for longer term measurements of EBC pH in our asthmatic (2.4%, range 0.6–10.6%) and healthy subjects (1.2%, range 0.8–1.6%) were less than intra-week CVs previously reported. Kostikas et al.1 assessed the repeatability of EBC pH on 2 consecutive days in normal subjects and patients with asthma and COPD. However, the data were presented as means and the variability between individual samples cannot be estimated. Between day LOA for pH of samples collected maximum 1 week apart reported by Leung et al.5 (0.32/0.31) and by Borrill et al.6 (0.46/0.44) in healthy controls were not narrower than those between seasons in the present study. This suggests that in normal subjects EBC pH is relatively stable and there are not additional sources of variation in the long-term period. Although asthma exhibits more clinical and functional variability than COPD, between-day variability of EBC pH was much higher in COPD6 than in our patients with asthma. The reason by which EBC pH fluctuates more in COPD remains undetermined since the mechanisms of airway pH regulation are poorly understood. It can be hypothesized that oxidative stress and/or bacterial colonization may contribute to variations of pH in COPD.18,1 We examined whether clinical status, functional parameters and drug treatment can explain EBC pH variability. Changes in ACT score, in FEV1 and in the corticosteroids regimen were not associated with higher variability. In addition, the measurements of EBC pH in asthmatics who remain in stable conditions during the study visits were not more repeatable than those in less stable patients. Therefore, the source of pH variability in asthma remains undetermined.
ARTICLE IN PRESS Exhaled breath condensate in asthma It cannot rule out that environmental factors such as inhaled particles and gases, temperature and humidity may influence EBC pH variability. This possibility could explain why less agreement was observed both in stable asthma and healthy controls between EBC pH measurements in winter samples and those performed in the other seasons, than between measurements in autumn and summer. Average concentrations of air-suspended particulate matter (PM10) in the urban area of Padova, where the patients lived, was indeed higher in winter at the time when the patients were examined (84.6729.3 mg/m3) than in summer (43.8712.9 mg/m3, po0.001) or autumn (47.2714.0 mg/m3, po0.001). Similarly, the other pollutants derived from combustion (SO2, NO2, CO) were significantly higher in winter compared with the other seasons (data not shown). Variability of EBC pH due to smoking was not investigated previously in asthma. Vaughan et al.8 stated that being a cigarette smoker did not appear to affect EBC pH in healthy subjects and similar variability was observed in COPD exsmokers and current smokers.6 Since our aim was to investigate whether smoking influences pH variability, not if EBC pH values are different in smoking and non-smoking asthmatics, the study is possibly not powered for this comparison. Indeed, we calculated that 16 subjects for group are necessary to show a difference in EBC pH of 0.3 with a significance of 0.05 and a power of 80% assuming a SD of 0.3, as observed in the present study. Our findings that smoking habit increases EBC pH variability is in line with the hypothesis that environmental factors may affect EBC. In conclusion, we demonstrated that EBC pH exhibits good repeatability even in long-term assessment. EBC pH in asthmatics tended to fluctuate more than in healthy subjects. However, EBC pH variability was not influenced by changes in clinical, functional and therapeutical parameters in asthma. Rather, we suggest that environmental factors may be implicated in EBC pH variability. The significance of EBC pH has been challenged, at least in COPD, by Effros et al.19 Since the present study was not designed to compare controlled and not controlled asthmatics and subjects under exacerbation were not examined, our results leave open the question whether EBC pH is sufficiently sensitive to detect changes in clinical and functional status of asthma milder than those during an exacerbation. Therefore, the clinical utility of longitudinal monitoring of EBC pH remains to be determined.
Conflict of interest statement None of the authors has a financial relationship with a commercial entity that has interest in the subject of this manuscript.
Acknowledgments The authors gratefully acknowledge the supports by the Italian Ministry of University and Research (PRIN 2005), by the University of Padova (grants ex60%), by ARPA Veneto and by Associazione Ricerca Cura Asma, Padova, Italy. The authors also thank Giovanna Fulgeri for secretarial assistance and Luigi Zedda for technical assistance.
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