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Published ahead of print on February 8, 2007, doi:10.1164/rccm.200607-940OC
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American Journal of Respiratory and Critical Care Medicine Vol 175. pp. 905-910, (2007)
© 2007 American Thoracic Society
doi: 10.1164/rccm.200607-940OC

Original Article

An Investigation of Airway Acidification in Asthma Using Induced Sputum

A Study of Feasibility and Correlation

Metka Kodric1, Ajay N. Shah1, Leonardo M. Fabbri2 and Marco Confalonieri1

1 Department of Pulmonology, University Hospital of Trieste, Trieste, Italy; and 2 Department of Respiratory Diseases, University of Modena and Reggio Emilia, Modena, Italy

Correspondence and requests for reprints should be addressed to Metka Kodric, M.D., Department of Pulmonology (SC Pneumologia), University Hospital of Trieste (Azienda Ospedaliero–Universitaria Ospedali Riuniti di Trieste), Strada di Fiume 447, 34100 Trieste, Italy. E-mail: metka.kodric{at}gmail.com


   ABSTRACT
TOP
 ABSTRACT
AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: Acidification of the airways seems to be involved in asthma pathophysiology, but its assessment might be difficult.

Objectives: The aim of our study is to assess the feasibility and validity of airway acidification measurement by induced sputum and its clinical significance in asthma.

Methods: Induced-sputum samples were obtained in 57 outpatients with asthma. The between-sample repeatability after 48 hours was measured in an independent population of 14 patients with asthma. pH was measured using a pH meter. The control of asthma was established by the Asthma Control Questionnaire.

Measurements and Main Results: The pH measurement was feasible in all samples and repeatable both within (intraclass correlation coefficient [ICC], 0.96) and between samples (ICC, 0.621). The mean pH was significantly different between healthy subjects and patients with asthma, including in those with controlled (mean pH: 7.54 in healthy subjects vs. 7.28 in subjects with controlled asthma; p = 0.0105) and uncontrolled disease (mean pH: 7.54 in healthy subjects vs. 7.06 in subjects with uncontrolled disease; p < 0.0001), and between patients with stable asthma and those with poorly controlled asthma (7.28 vs. 7.06, respectively; p = 0.0134). The validity of the method was assessed with the receiver operating characteristic curves and induced-sputum lower pH values (with a cutoff value of 7.3; sensitivity, 72.1%; specificity, 100%).

Conclusions: Patients with asthma show lower pH than healthy subjects. Patients with poorly controlled asthma seem to have the lowest induced-sputum pH, independent of the GINA (Global Initiative for Asthma) severity level. In conclusion, induced sputum is a feasible, repeatable, noninvasive method to measure airway pH. The pH in induced sputum may reflect a different aspect of asthma from sputum eosinophils and be related to different pathophysiologic factors.

Key Words: asthma • airway acidification • induced sputum



   AT A GLANCE COMMENTARY
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Scientific Knowledge on the Subject
The acidification of the airways seems to be involved in asthma pathophysiology. To date, airway pH was assessed mostly by invasive methods or by exhaled breath condensate.

What This Study Adds to the Field
Airway acidification can also be measured by induced sputum and may be clinically relevant.

 
The acidification of the airways seems to be involved in asthma pathophysiology, causing bronchoconstriction, impaired ciliary motility, increased mucus viscosity, and damage to airway epithelium (1–4). To date, airway pH has been assessed either by invasive methods, not suitable in the daily clinical practice, or by exhaled breath condensate (EBC), with possible digestive contamination and with scant content of epithelial lining fluid (5–7). Induced sputum is a simple, noninvasive technique to study the airway inflammation and may contain a huge amount of epithelial lining fluid (8–12).

The aim of our study is to assess whether the airway acidification can be measured by induced sputum and to evaluate its clinical significance in patients with asthma as compared with the level of control of the underlying disease.

Some of the results of this study have been previously reported in the form of abstract (13).


   METHODS
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 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Study Subjects
We studied patients with asthma, according to the definition and classification of severity of National Heart, Lung, and Blood Institute/World Health Organization Global Strategy for Asthma Management and Prevention (14), who were admitted to our respiratory outpatient clinic for a scheduled visit. Patients with asthma with exacerbations or emergency visits within the previous 3 months were excluded. We also excluded subjects who smoked, had colds or other acute illness, or had a chronic disease other than asthma. Healthy control subjects were recruited from hospital staff and medical students. They were nonsmokers, had no history of allergy and negative skin prick test, normal lung function test, and negative methacholine challenge test up to a provocative dose (PD20) of 1,600 µg. All of the patients with asthma and healthy subjects agreed with the purpose of the study and gave their informed consent. This study was approved by the Institutional Human Investigation Ethics Committee of the University Hospital of Trieste.

Study Design
Patients underwent clinical examination, lung function testing (SensorMedics 6002 Autobox DL; SensorMedics Corp., Yorba Linda, CA). Sputum induction was performed within 3 to 5 days; at that time, subjects repeated the spirometry and were also asked to complete the Asthma Control Questionnaire (ACQ), used with the permission of Prof. Elizabeth Juniper (15). We divided patients with asthma into two groups according to the level of control of symptoms, regardless of their current treatment. Poor control was defined on the basis of symptom frequency, sleep disturbance, and use of relievers as assessed by the ACQ with a mean score equal to or greater than 0.5. Healthy subjects also underwent clinical examination, methacholine challenge, and skin prick testing.

For the independent subgroup of patients with asthma, the procedure was repeated after 48 hours to assess the between-sample repeatability.

Induced-Sputum Sampling and Processing
Induced sputum was performed in accordance with the European Respiratory Society task force (16, 17). FEV1 and FVC were measured at baseline and after inhalation of salbutamol (200 µg by metered dose inhalers). After that, subjects were asked to rinse their mouth. Subjects inhaled sterile hypertonic saline (NaCl, 4.5%, prepared by the hospital chemist) nebulized with an ultrasonic device (Syst'Am DP100; System Assistance Medical, Le Ledat, France) at 1 ml/minute for three cycles of 5 minutes each. After each cycle and when needed they were asked to cough on a petri dish.

The collected sputum samples were processed as previously described (16). Cell count was performed on at least two slides for an overall differential count of 800 nucleated nonsquamous cells. Only samples with less than 20% squamous cell contamination, to exclude salivary contamination, and more than 50% viable cells were considered suitable.

pH Measurement
Plugs were selected from fresh induced-sputum samples, without any additive, and carefully aspirated into a 1-ml insulin syringe. A sample in the range of 0.3 to 0.6 ml was sufficient for a microarray evaluation by a blood gas analyzer (Rapidlab 845; Bayer Diagnostic Division, Milan, Italy) Three different samplings were performed in each sample to check for within-sample repeatability and analyzed separately immediately after sputum production. The mean pH of each patient's sputum was calculated.

Statistical Analysis
Analysis was performed using Statview for Windows (version 5.0.1; SAS Institute, Inc., SAS Institute, Cary, NC) and MedCalc (version 7.2.1.0; MedCalc Software, Mariakerke, Belgium). The within- and between-sample repeatability was assessed by intraclass correlation coefficient (ICC) and a standard deviation (SD) of within- and between-sample differences. Repeated measures with an ICC value in excess of 0.6 are believed to be clinically useful. The relation between the SDs and magnitude of the values was estimated by plotting the SDs against their means and assessing the rank correlation coefficient (Kendall's tau). Data showing a normal distribution, as assessed by the Kolmogorov-Smirnov test, are expressed as mean and standard error of the mean and the analysis were performed using analysis of variance with adjustment for multiple comparisons (Bonferroni method); for comparison between two groups, an unpaired t test was applied. Data not following normal distribution are presented as median and interquartile range. Further analysis was performed with the Kruskal-Wallis test with adjustment for multiple comparisons (Dunn method); for comparison between two groups, we performed a Mann-Whitney test. Categorical data were analyzed by the chi-square test. Correlation between variables was evaluated to determine the Spearman rank correlation coefficient (rs). A p value of less than 0.05 was considered significant, except in multiple comparisons.

The validity of the method was assessed with a receiver operating characteristic curves, to determine the ability of the pH to discriminate, first, between healthy subjects and patients with asthma and, second, between patients with controlled asthma and those with poorly controlled asthma.


   RESULTS
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Patient Characteristics
We studied 43 nonsmoking outpatients with asthma and 14 normal subjects; we further studied the between-sample repeatability on an independent group of 14 patients with asthma. We collected data from all patients. All patients provided an adequate sample of sputum without any problem arising during sputum induction and collection. The characteristics of the patients are listed in Table 1. The ACQ score was significantly different between stable and poorly controlled asthma groups (p < 0.0001).


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  		TABLE 1. PATIENT CHARACTERISTICS

 
The level of severity of asthma was significantly higher in the poor control group, as assessed by the prevalence of more severe asthma (p = 0.0206).

Feasibility and Repeatability
The measurement of pH was feasible in all the samples. The amount of each sample allowed us to perform three measurements of pH and an analysis of cellularity. A very small amount (0.3–0.6 ml) was enough to get the proper result from the analyzer. Even with handling denser samples, clogging of the analyzer never occurred. The whole process took from 5 to 10 minutes. Carbon dioxide was not detectable in our samples.

We checked whether the SD was possibly related to the magnitude of the pH measure by plotting the subjects' SDs against their means and assessing the rank correlation coefficient; there did not appear to be a relation (Kendall's tau, –0.096; p = 0.2913). The ICC and SD for the within-sample measures performed on 57 subjects showed high repeatability (ICC = 0.96; SD = 0.063); the ICC for the repeated measures between samples, performed at baseline and after 48 hours, for 14 subjects with asthma was 0.621 with an SD of 0.207.

Validity of the Method
We performed a validation of the methodology to assess the ability of the pH to discriminate between healthy subjects and subjects with asthma. The accuracy of the pH was high as demonstrated by the values of the C statistic, 0.857 (95% confidence interval [CI], 0.739–0.935). For a cutoff pH value of 7.3, the method showed a sensitivity of 72.1% and specificity of 100% in distinguishing patients with asthma from healthy individuals (Figure 1A). We further tried to assess the ability of the pH to discriminate between stable and poorly controlled asthma, and the accuracy was, as demonstrated by the values of the C statistic, 0.703 (95% CI, 0.544–0.832). For a cutoff pH value of 7.22, the method showed a sensitivity of 75% and specificity 63.2% in distinguishing uncontrolled asthma from stable disease (Figure 1B).


Figure 1
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  		Figure 1. Receiver operating characteristic curves for the validity of the technique to discern the presence of asthma (A) and its level of control for a cutoff of Asthma Control Questionnaire mean score >= 0.5 (B).

 
Induced-Sputum Analysis
The pH values were normally distributed, whereas the cell percentages were not. Data are shown in Table 2.


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  		TABLE 2. INDUCED-SPUTUM FEATURES

 
The pH values were significantly different between healthy subjects and patients with stable asthma (7.54 vs. 7.28; p = 0.0105) and between the latter and patients with poorly controlled asthma (7.28 vs. 7.06; p = 0.0134). The percentage of eosinophils was significantly different between patients with poorly controlled asthma and those with stable asthma (11.11 vs. 2%; p < 0.0001) but not between healthy subjects and patients with stable asthma (0 vs. 2%; p = 0.1774). There were no significant differences for the other cellular types between the groups.

The pH did not vary significantly between the GINA (Global Initiative for Asthma) severity groups (p = 0.0513) (Figure 2); the percentage of eosinophils (Figure 3) and neutrophils also did not differ (p = 0.0892 and p = 0.1816, respectively). However, there were significant differences between the asthma severity groups in FEV1 (p < 0.001) and FEV1/FVC (p < 0.001).


Figure 2
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  		Figure 2. pH and severity of asthma. Open squares = patients with poorly controlled asthma; solid circles = patients with stable asthma. GINA = Global Initiative for Asthma.

 

Figure 3
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  		Figure 3. Percentage of eosinophils in induced sputum and severity of asthma. Open squares = patients with poorly controlled asthma; solid circles = patients with stable asthma. GINA = Global Initiative for Asthma.

 
Asthma Control
A significant correlation was observed between the pH values and the ACQ score (rs = –0.518, p = 0.0008); globally, we observed the same for the percentage of eosinophils and the ACQ score (rs = 0.552, p = 0.0004) (Figure 4).


Figure 4
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  		Figure 4. Correlation graphs between levels of control of asthma expressed by the Asthma Control Questionnaire (ACQ) score and pH and percentage of eosinophils. Solid circles = patients with poorly controlled asthma; open circles = patients with stable asthma. Left, rs = –0.518, p = 0.0008; right, rs = 0.552, p = 0.0004.

 
In the poorly controlled group, the ACQ score correlated significantly with pH (rs = –0.531, p = 0.0108) but not with the percentage of eosinophils (rs = 0.291, p = 0.1631). With a greater loss of asthma control (ACQ score >= 0.71), we found out that the values of pH still correlated with the ACQ score (rs = –0.515, p = 0.0213), whereas there was no correlation between the percentage of eosinophils and ACQ score (rs = 0.200, p = 0.3722) (Figure 5). In the controlled asthma group, neither pH nor eosinophils correlated with ACQ score (rs = –0.324, p = 0.1687, and rs = 0.324, p = 0.1687, respectively). In the poorly controlled asthma group, there was no significant difference in pH values between the GINA classes (p = 0.1892).


Figure 5
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  		Figure 5. Correlation graphs between the lower levels of control of asthma expressed by the Asthma Control Questionnaire (ACQ) score (ACQ >= 0.71) and pH and percentage of eosinophils.

 

   DISCUSSION
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We demonstrated that airway pH could be easily and noninvasively assessed in subjects with asthma and healthy subjects by means of a direct measurement in induced sputum. Most previous reports on airway pH in normal and various pathologic settings used mostly EBC; this is the first study using induced sputum (18–20). Induced-sputum pH has a repeatable value within and between samples and is normally distributed in our patient population. We chose to repeat induced sputum after 48 hours because it was recently observed that induction repeated within 24 hours may cause a transient neutrophilic and eosinophilic response (21).

Low induced-sputum pH values (with a cutoff value of 7.3) have a high predictive capability, especially in distinguishing patients with asthma from healthy subjects.

EBC, even if easy to obtain, might not be an ideal tool to assess airway pH because it may present some disadvantages (22). The origin of EBC and its amount of epithelial lining fluid have not been assessed (6, 7). Because the EBC procedure and urea breath test are both performed by exhalation, they might influence each other. Moreover, as recently discussed by Effros and colleagues, the pH of EBC may be influenced by volatile contaminants from the mouth (NH4– and HCO3–). (23) A recent study observed that leukotriene B4 found in EBC may be due to salivary contamination (24).

Major discrepancies between bronchial EBC and bronchial surface pH measurements also suggest that pH values obtained by either method can be misleading (25).

Induced sputum is a validated and standardized noninvasive method to study airway inflammation (11, 26–32, 34). Theoretically, because it has the features of a gel and is thus less affected by external influences, induced sputum should contain a larger amount of purer epithelial lining fluid. Although they have not been directly compared, eosinophil cationic protein and albumin have been found to be more abundant in induced sputum compared with bronchoalveolar lavage fluid (31, 33, 35).

We found airway acidification in subjects with asthma in comparison with healthy control subjects, in accordance with previous studies which compared different respiratory disease (asthma, chronic obstructive pulmonary disease, cystic fibrosis) and different degrees of severity of asthma. Values in normal, healthy subjects in our study are similar to those obtained by analyzing the mucus through tracheostomy access (36), but so far, other types of samplings for pH determination have resulted in slightly different values (1, 6, 7, 36–38). The factors determining airway pH are not completely known, but alterations in pH lead to respiratory symptoms such as cough, wheezing, and dyspnea (1, 18, 19). From a teleologic point of view, an acidic environment may play a protective role against exogenous bacteria by inhibiting their growth through the formation of nitric oxide and the nitrosilation of proteins; gram-negative bacteria and Mycobacterium tuberculosis do not survive in an acidic milieu (39–41). Our knowledge of the effects of airway acidification derives from the effects of aspiration of acidic substances in the atmosphere (acidic fog) or gastroesophageal reflux (1). The hydrogen ions activate the primary capsaicin-sensitive fibers, which, through the release of tachykinins, lead to the following: cough (42–44), bronchial obstruction (45, 46), bronchial hyperreactivity (47), tissue edema (48, 49), increased mucus viscosity (3), reduced ciliary motility, and tissue damage (4). The acid–base homeostasis is a complex mechanism in which the acid production by lamellar bodies (50), ionic pumps (51), and release by degranulation of inflammatory cell vacuoles (52) is counterbalanced by the presence of buffers such as albumin in the epithelial lining fluid, bicarbonates, and ammonia produced through the activation of glutaminase (53, 54). These mechanisms may be altered in pathologic conditions. It has been observed that some patients with asthma have a low concentration of high-molecular-weight proteins or a low buffer capacity with a decreased capacity to protect the H+ ion penetration of surrounding tissues (55). Moreover, it is known that some of the airway-neutralizing mechanisms can be shut off to allow acidification for host defense purposes during inflammation (1). For example, the surest way to inactivate rhinovirus (the common cold virus and the most important trigger of asthma exacerbations) is by means of mild acidification (56). It is known that acidic mucus or mucus with a low protein concentration, as in some patients with asthma, constitutes a risk factor in case of acidic exposures (e.g., air pollution) (57).

Moreover, we distinguished the severity of the disease from its level of control, considering severity as the intrinsic intensity of the disease over a long time and control as the fluctuation of the disease over a short period of some weeks. In both cases, these two aspects cannot be defined by a single parameter (58). Even if our data do not fully support definitive conclusions, we observed the lowest induced-sputum pH in patients with poorly controlled asthma, independent of the GINA severity level. This is in accordance with previous studies (18). In addition, we found that, once lost, the control of asthma, or the degree of poor control, correlates better with pH than does induced-sputum eosinophil count; the latter observation is partially in contrast with previous observations in which the control of asthma was related to the increase of sputum eosinophils (59, 60). We did find an increase in eosinophils in the poorly controlled asthma group, but this was not as strictly related to the degree of poor asthma control as was the pH. The control and severity of asthma presented some overlapping features in their evaluations and, apart from clinical data, we need sensitive and specific biomarkers not affected by the severity of the disease. Even more important, the study of the alterations of pH may lead us to discover new pathophysiologic mechanisms of poorly controlled disease.

Taking into account its simplicity and the good within-sample repeatability of the obtained results, induced sputum may be considered a useful tool to study airway acidification both in clinical practice and in experimental settings. Future studies that use induced sputum as a simple and useful investigative tool may give us more insights into the acidification of the airways and into different aspects of asthma.


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

Originally Published in Press as DOI: 10.1164/rccm.200607-940OC on February 8, 2007

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

Received in original form July 12, 2006; accepted in final form February 5, 2007


   REFERENCES
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 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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