Endogenous airway acidification, as assessed by pH in expired breath condensate, has been implicated in asthma pathophysiology. We measured pH in breath condensate of patients with inflammatory airway diseases in stable condition and examined its relationship with the inflammatory process (as assessed by differential cell counts in induced sputum), oxidative stress (as assessed by H2O2 and 8-isoprostane), and nitric oxide metabolism (as assessed by total nitrate/nitrite). We studied 40 patients with bronchial asthma (20 with moderate disease, forced expiratory volume in 1 second 60 % SD predicted), 20 patients with bronchiectasis, 20 patients with chronic obstructive pulmonary disease (COPD), and 10 normal subjects. Mean (95% confidence intervals) pH values were significantly lower in patients with COPD and bronchiectasis compared with patients with asthma and control subjects (7.16, 7.09–7.23 and 7.11, 7.04–7.19 versus 7.43, 7.35–7.52 and 7.57, 7.51–7.64, respectively, p < 0.0001). Patients with moderate asthma had significantly lower values compared with mild and control subjects. In patients with COPD and bronchiectasis, the values of pH were significantly correlated with both sputum neutrophilia and oxidative stress. Respectively, in patients with moderate asthma, a significant correlation was observed between pH and sputum eosinophilia, total nitrate/nitrite, and oxidative stress. The pH of the expired breath condensate might be a simple, noninvasive, inexpensive, and easily repeatable procedure for the evaluation of the inflammatory process in airway diseases.
Airway inflammation has a central role in the development and progression of many lung diseases. Activation of inflammatory cells, such as neutrophils, eosinophils, and macrophages, has been implicated in the pathophysiology of asthma, chronic obstructive pulmonary disease (COPD), and bronchiectasis (1). The measurement of markers in the expired breath condensate has proven to be a useful, noninvasive method for assessing and monitoring airway inflammation (2–5). Expired breath condensate is a simple, noninvasive technique for the monitoring of airway inflammation, which reflects abnormalities in markers obtained bronchoscopically (6), in sputum (7) and in exhaled air (8). Endogenous airway acidification, as assessed by pH in expired breath condensate, has been implicated in asthma pathophysiology (9). In this study, the pH of the expired breath condensate was found to be substantially lower than normal during acute asthmatic exacerbations and to normalize with antiinflammatory therapy and the remission of the exacerbation.
Lowering of the airway pH has been reported to cause bronchoconstriction (10), to impair the ciliary motility (11), to increase the airway mucus viscosity (12), and to induce damage to the airway epithelium (13). These mechanisms are crucial for the development of inflammatory airway diseases. Polymorphonuclear cell metabolism has been implicated in the lowering of pleural fluid pH (14–16). Because pH seems to be neutrophil dependent and related to the inflammatory process, we hypothesized that low pH in patients with inflammatory airway diseases might reflect the airways inflammation and its consequences. The aim of this study was to determine the amounts of pH in the expired breath condensate of patients with asthma, COPD, and bronchiectasis. Furthermore, we investigated the relationship between pH and the inflammatory process (as assessed by the differential cell counts in induced sputum), oxidative stress (as assessed by hydrogen peroxide and 8-isoprostane in breath condensate), nitric oxide (NO) metabolism (as assessed by its stable end products, total nitrite and nitrate [NO2/NO3]), and lung function tests (forced expiratory volume in one second [FEV1]). We report that pH values reflect the underlying airway inflammatory process in the three diseases studied.
General characteristics of patients and control subjects are summarized in Table 1
Control Subjects (n = 10)
COPD (n =20)
Bronchiectasis (n = 20)
(n = 40)||Mild
(n = 20)||Moderate
(n = 20)|
|Age, years||34 (8)||31 (6)||30 (4)||31 (7)||54 (8)||32 (9)|
|FEV1, % predicted||99 (3)||78 (18)||95 (4)||62 (9)||59 (14)||62 (17)|
|H2O2, μM||0.19 (0.06)||0.79 (0.4)||0.59 (0.12)||1.0 (0.4)||0.90 (0.51)||0.95 (0.4)|
|8-Isoprostane, pg/ml||20 (7)||33 (11)||25 (7)||40 (9)||60 (44)||305 (193)|
|Total NO2/NO3, μM||0.3 (0.1)||0.78 (0.6)||0.42 (0.3)||1.13 (0.6)||0.67 (0.20)||0.69 (0.22)|
|Eosinophils, %||0.5 (0.5)||4.7 (4.9)||2.0 (1.9)||7.4 (5.5)||0.6 (1)||0.3 (0.4)|
|Neutrophils||24 (2)||26 (4)||23 (3)||28 (4)||56 (13)||58 (14)|
We used the recommendations for the diagnosis and classification of asthma severity of the NHLBI/WHO Workshop on the Global Strategy for Asthma (17) to classify our patients with asthma into two categories: 20 patients had mild persistent asthma (FEV1 95 % predicted), and 20 had moderate asthma (FEV1 62 % predicted). Ten patients from each group were receiving inhaled corticosteroids (ICSs), and 10 were steroid naive. The inhaled steroid regimen consisted of fluticasone propionate (500 to 1,000 μg daily) or budesonide (400 to 800 μg daily) for a minimum of 3 months before the study. All patients were atopic, as judged by the positive skin prick tests to six common aeroallergens and the elevated amounts of total immunoglobulin (Ig) E. None of the patients was on any other antiinflammatory treatment, including leukotriene antagonists, theophylline, or inhaled or oral mucolytics, or were receiving oxygen therapy. Patients were included in the study only if they were clinically stable and had no evidence of acute exacerbation. All patients were nonsmokers.
All patients had bronchiectasis diagnosed on the basis of clinical and radiologic features and were confirmed by high resolution computed tomography (HRCT) of the thorax. All patients were clinically stable and had no evidence of infection or acute infective exacerbation (lower or upper respiratory tract) for at least 4 weeks before the study. All were nonsmokers and had a negative history of allergy (negative skin prick tests to common aeroallergens). None of our patients had reversibility with inhaled salbutamol of 12% or more of predicted FEV1. All patients had a negative sweat test. Patients with cystic fibrosis, allergic bronchopulmonary aspergillosis, asthma, α1-antitrypsin deficiency, COPD, and atopic diseases were excluded. None of the patients were on inhaled or oral mucolytics, and none were receiving oxygen therapy or long-term oral antibiotics. Seven patients with chronic colonization with Pseudomonas aeruginosa (FEV1 49 , range 33–70% predicted; neutrophils 67 , range 50–90; H2O2 1.21 [0.43], range 0.74–1.86) were separately studied and compared with the remaining 13 who had no evidence of any chronic colonization with bacteria. Chronic colonization with P. aeruginosa was defined as more than three isolations of P. aeruginosa from separate samples over 3 months (18). Ten of the patients received treatment with ICSs (fluticasone propionate, 500 to 1,000 μg daily, or budesonide, 400 to 800 μg daily), and 10 were steroid naive.
Twenty COPD patients (FEV1 54 , range 37–79% predicted) were diagnosed according to the NHLBI/WHO Workshop guidelines (19). Ten of the patients received treatment with inhaled steroids, and 10 were steroid-naive. The inhaled steroid regimen consisted of fluticasone propionate (500 to 1,000 μg daily) or budesonide (400 to 800 μg daily) for a minimum of 3 months before the study. All patients were clinically stable and had no evidence of acute exacerbation for at least 4 weeks before the study. All patients were smokers and had a negative history of allergy (negative skin prick tests to common aeroallergens) and showed no marked spirometric response to inhaled bronchodilators (FEV1 increase of less than 12% or 200 ml 15 minutes after the inhalation of 200 μg of salbutamol). None of the patients were receiving oxygen therapy.
All normal subjects were nonsmokers and had a negative history of allergy (negative skin prick tests to common allergens), normal spirometry (99 , range 95–105), and normal bronchial reactivity with a provocative dose of histamine (PD20) of more than 0.800 mg (1.34 [0.28], range 0.96–1.84). The ethics committee of our hospital approved the study protocol, and all subjects gave informed written consent.
FEV1 was measured with a dry spirometer (Vica-test, Mijnhardt, Holland). The best value of three maneuvers was expressed as a percentage of the predicted value. Airway responsiveness was measured by histamine provocation challenge in normal subjects.
The collection of breath condensate was performed as previously described (20). A heat exchanger unit (RHES; Jaeger, Wuerzburg, Germany) was used to produce cold air of −15° C to −18° C at an airflow of more than 80 L/minute. A double-jacketed glass tube of 45 cm in length (an internal diameter of 4 cm and an external diameter of 7 cm) was specifically adapted to the cold air system, and a Hans-Rudolf two-way unidirectional valve was connected to the proximal end of the tube to separate inspiration from expiration. After rinsing their mouths, subjects were comfortably seated in a chair. They were wearing nose clips and breathed in a relaxed manner (tidal breathing) for 15 minutes. The breath condensate was collected at the distal end of the tube. According to this design, salivary contamination was highly unlikely and was easily observed, as the proximal cold air connection was 25 cm away from the mouthpiece. Approximately 2 ml of breath condensate was collected and divided in two samples in 2 ml sterile plastic tubes. The first sample was used for the measurement of pH, and the second was immediately frozen and stored in −70° C for the measurements of H2O2, 8-isoprostane, and NO2/NO3. In each group, measurements were performed on the same day.
Repeatability of H2O2, 8-isoprostane, and NO2/NO3 measurements and stability of the frozen samples were estimated as previously described (21) in 5 normal subjects, 12 patients with asthma, 6 patients with bronchiectasis, and 6 patients with COPD. All condensate samples were tested for salivary contamination by determination of amylase activity (20). Briefly, amylase activity was performed spectrophotometrically (kinetic method) using a commercial reagent kit (KONE Instruments, Espoo, Finland). In this procedure, a-amylase of the sample and the enzyme a-glycosidase hydrolyzes the substrate p-nitrophenyl-a-d-maltoheptaoside to glucose and p-nitrophenol. The liberation of p-nitrophenol is followed at 405 nm (37° C) for 2 minutes. Two samples were spiked with saliva to ensure that this can be detectable by our method. In all of the samples tested for saliva, no amylase was detected, suggesting no contamination of breath condensate. The samples that were spiked with saliva showed amounts of salivary amylase that were higher than 5,000 IU.
pH was measured as previously described (9) right after the collection of the condensate. Stable pH was achieved in all cases after deaeration of the condensate with an inert gas (argon, 350 ml/minutes for 10 minutes) and was measured using a pH meter (CONSORT P-903).
8-Isoprostane was determined by a competitive enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI), as previously described (24). The detection limit of the assay is 4 pg/ml.
NO2/NO3 measurements were performed as previously described (20). Briefly, NO3 was measured as NO2 after enzymatic conversion by NO3 reductase (25), and the total NO2/NO3 (converted NO3 plus NO2) was measured by using the Griess reaction (26). We decided to measure the end products of the NO metabolism, instead of exhaled NO, to compare compounds generating from the same specimen.
Sputum was induced by the inhalation of an aerosol 3.5% hypertonic saline generated by DeVilbiss ultrasonic nebulizer (Model 2,696, Somerset, PA), with modifications to improve its safety (27). At least 2 ml of sputum was collected into a sterile container. The sample from the first cough was discarded, as it is heavily contaminated with squamous epithelial cells (28). An adequate sample was defined if the number of squamous epithelial cells was less than 30% of the total number of inflammatory cells (29). Cytospine slides were prepared and stained with May-Grunwald-Giemsa. The worker who did the differential cell counts was not aware the clinical and functional status of the patients as well as of the expired breath condensate measurements. Two slides were used for counting, and at least 300 inflammatory cells were counted for each slide. The inflammatory cells in sputum are shown as a percentage of total nonsquamous cells. Sputum measurements were performed on the same day for all the patients.
Data concerning subject characteristics are expressed as means (SD) with ranges. Data concerning the comparisons among the various parameters in the study groups are given as means with a 95% confidence intervals (CIs) for the differences. Data were examined for normal distribution, and when it was not normally distributed, the nonparametric Mann-Whitney test was used for statistical comparisons. For normally distributed data, the paired t test was used for statistical comparisons between two groups. Normality of distribution was tested with Shapiro-Wilk's test. Parameters from patients with asthma, COPD, and bronchiectasis were compared using the one-way analysis of variance with an appropriate post hoc test (Bonferroni) for multiple comparisons. Spearman's correlation coefficient was used to investigate the relationship between the parameters. A p value of less than 0.05 was considered significant.
The measurements of pH in the same subject on two consecutive days were highly repeatable. The respective values were as follows: in patients with asthma, 7.38 (0.19) versus 7.40 (0.21) (p = 0.40); in patients with bronchiectasis, 7.19 (0.08) versus 7.22 (0.1) (p = 0.64); in patients with COPD, 7.24 (0.1) versus 7.22 (0.1) (p = 0.56); and in normal subjects, 7.47 (0.12) versus 7.49 (0.1) (p = 0.42), respectively.
Patients with COPD and bronchiectasis had significantly lower pH values compared with asthma patients and control subjects (7.16, 95% CI, 7.09–7.23 and 7.11, 95% CI, 7.04–7.19 versus 7.43, 95% CI, 7.35–7.52 and 7.57, 95% CI, 7.51–7.64, respectively, p < 0.0001). The pH values in patients with mild asthma (7.6, 95% CI, 7.55–7.65) did not differ significantly from those of control subjects (p = 0.26), whereas patients with moderate asthma had significantly lower pH values (7.27, 95% CI, 7.15–7.39) compared with mild asthma patients (p = 0.0002) and control subjects (p = 0.005) (Figure 1A). Patients with bronchiectasis that were chronically colonized by P. aeruginosa had significantly lower pH values compared with those noncolonized with P. aeruginosa (6.96, 95% CI, 6.88–7.05 versus 7.11, 95% CI, 7.19–7.27, p = 0.005).
Asthma patients on ICSs had significantly higher pH values compared with those who were steroid naive (7.56, 95% CI, 7.47–7.65 versus 7.31, 95% CI, 7.19–7.43, p = 0.0001). This difference was noticed in patients with both mild (7.65, 95% CI, 7.60–7.71 versus 7.54, 95% CI, 7.46–7.64, p < 0.0001) and moderate asthma (7.46, 95% CI, 7.31–7.61 versus 7.08, 95% CI, 7.01–7.15, p < 0.0001). In contrast, there was no significant difference between pH values in patients treated with ICSs and steroid-naive ones, both in bronchiectasis (7.16, 95% CI, 7.02–7.29 versus 7.076, 95% CI, 6.98–7.16, p = 0.1) and COPD (7.17, 95% CI, 7.03–7.30 versus 7.16, 95% CI, 7.06–7.26, p = 0.8) (Figure 1B).
|rs||p Value||rs||p Value||rs||p Value||rs||p Value|
|H2O2||−0.71||< 0.0001*||−0.74||0.0002*||−0.87||< 0.0001*||−0.2||0.57|
|Total NO2/NO3||−0.81||< 0.0001*||0.13||0.57||−0.28||0.2||0.02||0.95|
|Macrophages||0.75||< 0.0001*||0.66||0.001*||0.84||< 0.0001*||0.06||0.85|
|FEV1|| 0.81||< 0.0001*||0.67||0.0009*|| 0.79||< 0.0001*|| 0.3||0.39|
|ICS (+)||ICS (−)||ICS (+)||ICS (−)|
|rs||p Value||rs||p Value||rs||p Value||rs||p Value|
|FEV1|| 0.58||0.006*||0.94||< 0.0001*|| 0.8|| 0.005*|| 0.77||0.008*|
|rs||p value||rs||p value||rs||p value||rs||p value|
|Total NO2/NO3||−0.47||0.02*||−0.76||< 0.0001*||−0.81||< 0.0001*||−0.71||0.0004*|
|FEV1|| 0.82||0.0001*||0.86||< 0.0001*|| 0.74|| 0.0001*|| 0.86||< 0.0001*|
In patients with bronchial asthma, there was a significant negative correlation between pH and markers of oxidative stress (hydrogen peroxide [rs = −0.71, p < 0.0001] and 8-isoprostane [rs = −0.55, p = 0.0002]) as well as with the stable end products of the NO metabolism (total NO2/NO3 [rs = −0.81, p < 0.0001]) (Table 2). Further analysis showed that the correlations between pH and those three markers were significant in patients with moderate asthma (rs = −0.63, p = 0.002 for H2O2, rs = −0.67, p = 0.001 for 8-isoprostane, and rs = −0.76, p < 0.0001 for total NO2/NO3), whereas in patients with mild asthma, we observed only a significant negative correlation between pH and total NO2/NO3 (rs = −0.47, p = 0.02). In patients not treated with ICSs, there was a significant negative correlation between pH and hydrogen peroxide (rs = −0.69, p = 0.0007), 8-isoprostane (rs = −0.58, p = 0.006), and total NO2/NO3 (rs = −0.71, p = 0.0004). Patients on ICS showed no significant correlation between pH and 8-isoprostane (rs = −0.37, p = 0.1) (Table 3). Moreover, a significant negative correlation was observed between pH amounts and eosinophil counts in induced sputum (rs = −0.74, p < 0.0001), and this correlation was not observed in patients with mild asthma (rs = −0.13, p = 0.55), whereas it was present in patients with moderate disease (rs = −0.68, p = 0.0008). In all of the subgroups of patients with asthma, there was a strong positive correlation between pH and FEV1 (percent predicted) (Tables 2 and 4).
Parameters expressing the inflammatory process in each disease (neutrophils for COPD and bronchiectasis and eosinophils for asthma) were significantly correlated with the parameters of oxidative stress, NO metabolism (only in asthma), and airflow limitation (as assessed by FEV1 percent predicted) (data not shown).
In this prospective, cross-sectional study, we have found that endogenous airways acidification, as assessed by pH in expired breath condensate, is mainly related to neutrophilic inflammation and in a lesser extent to the presence of eosinophils. In addition, pH was significantly affected by the degree of oxidative stress and, in patients with asthma, by the end products of NO metabolism.
pH values of expired breath condensate have been found to be identical to those obtained from lower airway secretions (9), thus reflecting the amounts of endogenous acidification in the lower respiratory tract. However, what is not clear is whether the endogenous acidification expresses the presence of inflammation in the airways. Our findings confirmed our initial hypothesis that endogenous airway acidification is strongly related to the underlying inflammatory process in the three diseases studied. However, the mechanism seems to differ between asthma (that is predominantly characterized by eosinophilic inflammation) and COPD or bronchiectasis (that are predominantly characterized by neutrophilic inflammation).
The lower pH values that were observed in bronchiectasis and COPD compared with asthma might be attributed to the neutrophilic inflammation. One plausible explanation for the above observation might be the activity of neutrophil myeloperoxidase. This enzyme catalyzes the reaction between hydrogen peroxide and a chloride to form hypochlorous acid (HOCl) (30). This acid is highly volatile and might be responsible for the acidification of expired breath condensate of patients with stable COPD or bronchiectasis. It has also been shown in an in vivo experiment that leukocyte metabolism in the presence of live bacteria could produce a fall in pleural fluid pH (15). This might also apply to bronchial secretions and interpret the low pH observed in patients with bronchiectasis or COPD that can be chronically colonized by bacteria (31, 32). All of the previously mentioned theory is partially supported by the lower pH values that we observed in patients with bronchiectasis chronically colonized with P. aeruginosa.
Patients with stable bronchial asthma had higher pH values compared with those with COPD and bronchiectasis but lower values compared with the control group. In addition, pH values differ between patients with different clinical severity. It has been shown that a sudden drop in airway pH during asthma exacerbation would cause extensive eosinophilic necrosis with an acute release of inflammatory and bronchoconstricting products. Our results showed that when the inflammatory process of the disease is well controlled, the pH remains within normal limits, whereas in the presence of limited cellular inflammation (as assessed by sputum eosinophils), the pH starts to decrease. This means that pH is probably a consequence of the eosinophilic inflammation and not its cause. The granule matrix of eosinophils contains eosinophil peroxidase, an enzyme that, in the presence of H2O2, is able to oxidize halides to form highly reactive hypohalous acids (33). This might in part explain the lower pH that we observed in stable patients with moderate asthma, characterized by higher eosinophil counts and H2O2 values than those with mild asthma. Furthermore, in the study by Hunt and colleagues (9), an acute fall of the pH values in exacerbated patients with asthma was reported. Those values were significantly lower than the values we have observed in patients with COPD and bronchiectasis. This might be explained by previous studies, which showed a predominance of neutrophils in the sputum of patients with asthmatic exacerbation (34). This leads to the hypothesis that neutrophilic inflammation contributes to endogenous acidification in asthma exacerbations.
Endogenous airway acidification depends on the amount of acid provided to the airways by inflammatory cells and also to the presence of buffers, such as ammonia. Ammonia is produced in airway epithelial cells by glutamine in the presence of glutaminase, and this production is inhibited by cytokines (interferon-γ and tumor necrosis factor-α), whereas corticosteroid administration has the opposite effect (35). This regulatory mechanism has been implicated in the pathophysiology of acute asthma exacerbations (35). Interestingly, there is a gap in pH values at a pH of 7.5, and this is indicative of the presence of a silent buffer with this pKa in the airways. We believe that this buffer might be the reason of the absence of pH values at that amount. Further investigation is required to define the nature of such a buffer.
We observed a significant correlation between 8-isoprostane amounts and pH in patients with asthma, especially in those with moderate disease, whereas no such relationship has been noticed in patients with COPD or bronchiectasis. It has been shown that in patients with asthma, 8-isoprostane represents a marker of disease severity (24), a fact that has not been observed in COPD (36). In addition, there are currently no other data concerning amounts of 8-isoprostane in bronchiectasis. We believe that in bronchiectasis, a disease with an inflammatory process similar to that of COPD, the amounts of 8-isoprostane might not be related to the disease severity. The significant correlation between pH amounts and sputum eosinophils in patients with asthma that receive ICSs might be due to the fact that patients with moderate asthma are included in this group and in such patients sputum eosinophilia might be present despite the treatment with ICSs (37). In steroid-treated patients with asthma, there is a significant correlation between pH and H2O2, whereas there is no relationship between pH and 8-isoprostane. We speculate that this difference implies that H2O2 is a better marker of eosinophilic inflammation (5) than 8-isoprostane, although currently no data exist to support the second part of that hypothesis.
Our results showed a strong correlation between amounts of pH and both oxidative stress markers as well as markers of NO metabolism. The latter relationship has been observed only in asthmatic subjects and is relevant to previous studies reporting that NO2 and NO3 amounts may reflect inflammation in asthma (20, 38). The absence of significant correlations of total NO2/NO3 and pH in patients with COPD and bronchiectasis could be due to the fact that concentrations of NO in such patients are affected by factors such as smoking (39) or mucus hypersecretion (40). Hunt and coworkers (9) have reported that concentrations and bioactivity of many of these inflammatory markers are critically pH dependent, suggesting that low airway pH could contribute substantially to airway inflammation. Furthermore, in lower pH amounts, NO2 would be lost as NO (9). The significant correlations observed in this study might be the result of the initial cellular process, which leads through different ways to oxidative stress, to NO production, and finally to endogenous acidification. This theory is partially supported by previous observations that showed a significant relationship between oxidative stress, exhaled NO, and the respective inflammatory and structural cells in airways (41, 42).
The present cross-sectional study could not demonstrate a causal relationship between treatment with ICS and pH values. Prospective controlled studies are needed for that purpose. However, the available evidence of our study suggests that ICSs are considerably more effective in patients with asthma. This theory is supported by previous observations suggesting that eosinophilic inflammation is more sensitive than neutrophilic inflammation to treatment with ICS (42, 43). We believe that the differences in pH values between steroid-treated and steroid-naive asthma patients might reflect differences in the underlying eosinophilic inflammation of the disease. Moreover, it has been reported that corticosteroids suppress the inflammatory cytokine amounts of patients with asthma, contributing to the increased availability of ammonia in the airways (35). The role of corticosteroids on the availability of buffer in the airways of patients with COPD and bronchiectasis is not clear yet and remains to be clarified. All this information supports our initial belief that pH values are dependent on both the underlying cellular inflammatory process of each disease and the availability of buffer in the airways.
In conclusion, we report that endogenous airway acidification reflects the underlying inflammatory process in various airway diseases in stable condition. This observation suggests that it might be critical to consider endogenous pH when interpreting airways inflammation in lung inflammatory diseases. Because the measurement of pH is an inexpensive, easy-to-perform, and highly reproducible technique, it may prove to be helpful for the understanding of the pathophysiology of inflammatory airway diseases.
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