An Investigation of Airway Acidification in AsthmaUsing Induced SputumA Study of Feasibility and Correlation

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

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

Rationale: Acidification of the airways seems to be involved inasthma pathophysiology, but its assessment might be difficult.Objectives: The aim of our study is to assess the feasibility and validityof airway acidification measurement by induced sputum and itsclinical significance in asthma.Methods: Induced-sputum samples were obtained in 57 outpatientswith asthma. The between-sample repeatability after 48 hours wasmeasured in an independent population of 14 patients with asthma.pH was measured using a pH meter. The control of asthma wasestablished by the Asthma Control Questionnaire.Measurements and Main Results: The pH measurement was feasiblein all samples and repeatable both within (intraclass correlationcoefficient [ICC], 0.96) and between samples (ICC, 0.621). The meanpH was significantly different between healthy subjects and patientswith asthma, including in those with controlled (mean pH: 7.54 inhealthy subjects vs. 7.28 in subjects with controlled asthma; p �

0.0105) and uncontrolled disease (mean pH: 7.54 in healthy subjectsvs. 7.06 in subjects with uncontrolled disease; p � 0.0001), andbetween patients with stable asthma and those with poorly con-trolled asthma (7.28 vs. 7.06, respectively; p � 0.0134). The validityof the method was assessed with the receiver operating characteris-tic curves and induced-sputum lower pH values (with a cutoff valueof 7.3; sensitivity, 72.1%; specificity, 100%).Conclusions: Patients with asthma show lower pH than healthy sub-jects. Patients with poorly controlled asthma seem to have thelowest induced-sputum pH, independent of the GINA (Global Initia-tive for Asthma) severity level. In conclusion, induced sputum is afeasible, repeatable, noninvasive method to measure airway pH.The pH in induced sputum may reflect a different aspect of asthmafrom sputum eosinophils and be related to different pathophysio-logic factors.

Keywords: asthma; airway acidification; induced sputum

The acidification of the airways seems to be involved in asthmapathophysiology, causing bronchoconstriction, impaired ciliarymotility, increased mucus viscosity, and damage to airway epithe-lium (1–4). To date, airway pH has been assessed either byinvasive methods, not suitable in the daily clinical practice, orby exhaled breath condensate (EBC), with possible digestivecontamination and with scant content of epithelial lining fluid(5–7). Induced sputum is a simple, noninvasive technique tostudy the airway inflammation and may contain a huge amountof epithelial lining fluid (8–12).

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

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

This article has an online supplement, which is accessible from this issue’s tableof contents at www.atsjournal.org

Am J Respir Crit Care Med Vol 175. pp 905–910, 2007Originally Published in Press as DOI: 10.1164/rccm.200607-940OC on February 8, 2007Internet address: www.atsjournals.org

AT A GLANCE COMMENTARY

Scientific Knowledge on the Subject

The acidification of the airways seems to be involvedin asthma pathophysiology. To date, airway pH was as-sessed mostly by invasive methods or by exhaled breathcondensate.

What This Study Adds to the Field

Airway acidification can also be measured by induced spu-tum and may be clinically relevant.

The aim of our study is to assess whether the airway acidifica-tion can be measured by induced sputum and to evaluate itsclinical significance in patients with asthma as compared withthe level of control of the underlying disease.

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

METHODS

Study Subjects

We studied patients with asthma, according to the definition and classi-fication of severity of National Heart, Lung, and Blood Institute/WorldHealth Organization Global Strategy for Asthma Management andPrevention (14), who were admitted to our respiratory outpatient clinicfor a scheduled visit. Patients with asthma with exacerbations or emer-gency visits within the previous 3 months were excluded. We alsoexcluded subjects who smoked, had colds or other acute illness, or hada chronic disease other than asthma. Healthy control subjects wererecruited from hospital staff and medical students. They were nonsmok-ers, had no history of allergy and negative skin prick test, normallung function test, and negative methacholine challenge test up to aprovocative dose (PD20) of 1,600 �g. All of the patients with asthmaand healthy subjects agreed with the purpose of the study and gavetheir informed consent. This study was approved by the InstitutionalHuman Investigation Ethics Committee of the University Hospital ofTrieste.

Study Design

Patients underwent clinical examination, lung function testing (Sensor-Medics 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 theAsthma Control Questionnaire (ACQ), used with the permission ofProf. Elizabeth Juniper (15). We divided patients with asthma into twogroups according to the level of control of symptoms, regardless of theircurrent treatment. Poor control was defined on the basis of symptomfrequency, sleep disturbance, and use of relievers as assessed by theACQ with a mean score equal to or greater than 0.5. Healthy subjectsalso underwent clinical examination, methacholine challenge, and skinprick testing.

906 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 175 2007

For the independent subgroup of patients with asthma, the proce-dure was repeated after 48 hours to assess the between-samplerepeatability.

Induced-Sputum Sampling and Processing

Induced sputum was performed in accordance with the European Re-spiratory Society task force (16, 17). FEV1 and FVC were measured atbaseline and after inhalation of salbutamol (200 �g by metered doseinhalers). After that, subjects were asked to rinse their mouth. Subjectsinhaled sterile hypertonic saline (NaCl, 4.5%, prepared by the hospitalchemist) nebulized with an ultrasonic device (Syst’Am DP100; SystemAssistance Medical, Le Ledat, France) at 1 ml/minute for three cyclesof 5 minutes each. After each cycle and when needed they were askedto cough on a petri dish.

The collected sputum samples were processed as previously de-scribed (16). Cell count was performed on at least two slides for anoverall differential count of 800 nucleated nonsquamous cells. Onlysamples with less than 20% squamous cell contamination, to excludesalivary contamination, and more than 50% viable cells were consideredsuitable.

pH Measurement

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

Statistical Analysis

Analysis was performed using Statview for Windows (version 5.0.1;SAS Institute, Inc., SAS Institute, Cary, NC) and MedCalc (version7.2.1.0; MedCalc Software, Mariakerke, Belgium). The within- andbetween-sample repeatability was assessed by intraclass correlation co-efficient (ICC) and a standard deviation (SD) of within- and between-sample differences. Repeated measures with an ICC value in excessof 0.6 are believed to be clinically useful. The relation between theSDs and magnitude of the values was estimated by plotting the SDsagainst their means and assessing the rank correlation coefficient(Kendall’s tau). Data showing a normal distribution, as assessed by theKolmogorov-Smirnov test, are expressed as mean and standard error

TABLE 1. PATIENT CHARACTERISTICS

Healthy Subjects Stable Asthma Poor Asthma Control

Number (%) 14 (24.56) 19 (33.33) 24 (42.11)Sex, M/F 5/9 6/13 5/19Age, yr 43.07 (2.71) 44.16 (3.65) 45.71 (3.17)FEV1, % predicted 115.50 (6.63) 101.89 (3.42) 91.54 (5.19)FEV1/FVC 81.36 (1.45) 75.50 (1.64) 69.10 (2.55)ACQ score NA 0.26 (0.04) 1.47 (0.16)�2-agonist use, occasions/d NA 0 (0–0) 0 (0–2)Asthma symptoms

Diurnal symptomsNumber per day NA 0 (0–0) 1.00 (0–2.00)Number per week NA 1.00 (0–2.00) 2.50 (1.00–4.00)

Nocturnal symptoms NA 0 (0–0) 1.00 (0–2.00)Number per weekNumber per month NA 0 (0–1.00) 1.50 (0–3.00)

GINA severity level, nMild intermittent NA 7 3Mild persistent NA 11 10Moderate NA 1 6Severe NA 0 5

Definition of abbreviations: ACQ � Asthma Control Questionnaire; F � female; GINA � Global Initiative for Asthma; M � male;NA � not applicable.

Values of sex, age, FEV1, FEV1/FVC, and ACQ score are expressed as mean (SEM); �2-agonist use and asthma symptoms areexpressed as median (interquartile range).

of the mean and the analysis were performed using analysis of variancewith adjustment for multiple comparisons (Bonferroni method); forcomparison between two groups, an unpaired t test was applied. Datanot following normal distribution are presented as median and inter-quartile range. Further analysis was performed with the Kruskal-Wallistest with adjustment for multiple comparisons (Dunn method); forcomparison between two groups, we performed a Mann-Whitney test.Categorical data were analyzed by the chi-square test. Correlation be-tween variables was evaluated to determine the Spearman rank correla-tion coefficient (rs). A p value of less than 0.05 was considered signifi-cant, except in multiple comparisons.

The validity of the method was assessed with a receiver operatingcharacteristic 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 con-trolled asthma.

RESULTS

Patient Characteristics

We studied 43 nonsmoking outpatients with asthma and 14 nor-mal subjects; we further studied the between-sample repeatabil-ity on an independent group of 14 patients with asthma. Wecollected data from all patients. All patients provided an ade-quate sample of sputum without any problem arising duringsputum induction and collection. The characteristics of the pa-tients are listed in Table 1. The ACQ score was significantlydifferent between stable and poorly controlled asthma groups(p � 0.0001).

The level of severity of asthma was significantly higher in thepoor control group, as assessed by the prevalence of more severeasthma (p � 0.0206).

Feasibility and Repeatability

The measurement of pH was feasible in all the samples. Theamount of each sample allowed us to perform three measure-ments 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 neveroccurred. The whole process took from 5 to 10 minutes. Carbondioxide was not detectable in our samples.

Kodric, Shah, Fabbri, et al.: Induced Sputum and Airway Acidification 907

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

We checked whether the SD was possibly related to the mag-nitude of the pH measure by plotting the subjects’ SDs againsttheir means and assessing the rank correlation coefficient; theredid not appear to be a relation (Kendall’s tau, �0.096; p �0.2913). The ICC and SD for the within-sample measures per-formed on 57 subjects showed high repeatability (ICC � 0.96;SD � 0.063); the ICC for the repeated measures between sam-ples, performed at baseline and after 48 hours, for 14 subjectswith asthma was 0.621 with an SD of 0.207.

TABLE 2. INDUCED-SPUTUM FEATURES

Healthy Subjects Stable Asthma Poor Asthma Control

pH 7.54 (0.03) 7.28 (0.07) 7.06 (0.07)Eosinophils, % 0.00 (0.00–0.00) 2.00 (0.9–4.05) 11.11 (3.19–19.00)Neutrophils, % 29.88 (20.50–37.38) 32.50 (11.75–45.85) 21.20 (9.66–51.07)Macrophages, % 68.63 (60.63–78.00) 61.60 (51.17–82.16) 52.02 (35.58–78.69)Lymphocytes, % 1.42 (0.81–1.50) 0.00 (0.00–0.50) 0.27 (0.00–1.13)

Values of pH are expressed as mean (SEM); percentages of cells are expressed as median (interquartile range).

Validity of the Method

We performed a validation of the methodology to assess theability of the pH to discriminate between healthy subjects andsubjects with asthma. The accuracy of the pH was high as demon-strated by the values of the C statistic, 0.857 (95% confidenceinterval [CI], 0.739–0.935). For a cutoff pH value of 7.3, themethod 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 todiscriminate between stable and poorly controlled asthma, andthe 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, themethod showed a sensitivity of 75% and specificity 63.2% indistinguishing uncontrolled asthma from stable disease (Figure1B).

Induced-Sputum Analysis

The pH values were normally distributed, whereas the cell per-centages were not. Data are shown in Table 2.

The pH values were significantly different between healthysubjects and patients with stable asthma (7.54 vs. 7.28; p �0.0105) and between the latter and patients with poorly con-trolled asthma (7.28 vs. 7.06; p � 0.0134). The percentage ofeosinophils was significantly different between patients withpoorly controlled asthma and those with stable asthma (11.11vs. 2%; p � 0.0001) but not between healthy subjects and patientswith stable asthma (0 vs. 2%; p � 0.1774). There were no signifi-cant differences for the other cellular types between the groups.

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

Asthma Control

A significant correlation was observed between the pH valuesand the ACQ score (rs � �0.518, p � 0.0008); globally, weobserved the same for the percentage of eosinophils and theACQ score (rs � 0.552, p � 0.0004) (Figure 4).

In the poorly controlled group, the ACQ score correlatedsignificantly with pH (rs � �0.531, p � 0.0108) but not with thepercentage of eosinophils (rs � 0.291, p � 0.1631). With a greaterloss of asthma control (ACQ score � 0.71), we found out thatthe values of pH still correlated with the ACQ score (rs ��0.515, p � 0.0213), whereas there was no correlation betweenthe percentage of eosinophils and ACQ score (rs � 0.200, p �0.3722) (Figure 5). In the controlled asthma group, neither pHnor eosinophils correlated with ACQ score (rs � �0.324, p �0.1687, and rs � 0.324, p � 0.1687, respectively). In the poorlycontrolled asthma group, there was no significant difference inpH values between the GINA classes (p � 0.1892).

908 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 175 2007

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

DISCUSSION

We demonstrated that airway pH could be easily and noninva-sively assessed in subjects with asthma and healthy subjects bymeans of a direct measurement in induced sputum. Most previ-ous reports on airway pH in normal and various pathologicsettings used mostly EBC; this is the first study using inducedsputum (18–20). Induced-sputum pH has a repeatable valuewithin and between samples and is normally distributed in ourpatient population. We chose to repeat induced sputum after 48hours because it was recently observed that induction repeatedwithin 24 hours may cause a transient neutrophilic and eosino-philic response (21).

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

EBC, even if easy to obtain, might not be an ideal tool toassess airway pH because it may present some disadvantages(22). The origin of EBC and its amount of epithelial lining fluidhave not been assessed (6, 7). Because the EBC procedure andurea breath test are both performed by exhalation, they mightinfluence each other. Moreover, as recently discussed by Effrosand colleagues, the pH of EBC may be influenced by volatilecontaminants from the mouth (NH4

� and HCO3�). (23) A recent

Figure 4. Correlation graphsbetween levels of control ofasthma expressed by theAsthma Control Question-naire (ACQ) score and pHand percentage of eosino-phils. Solid circles � patientswith poorly controlledasthma; open circles � pa-tients with stable asthma.Left, rs � �0.518, p �

0.0008; right, rs � 0.552,p � 0.0004.

Figure 3. Percentage of eosinophils in induced sputum and severity ofasthma. Open squares � patients with poorly controlled asthma; solidcircles � patients with stable asthma. GINA � Global Initiative forAsthma.

study observed that leukotriene B4 found in EBC may be dueto salivary contamination (24).

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

Induced sputum is a validated and standardized noninvasivemethod to study airway inflammation (11, 26–32, 34). Theoreti-cally, because it has the features of a gel and is thus less affectedby external influences, induced sputum should contain a largeramount of purer epithelial lining fluid. Although they have notbeen directly compared, eosinophil cationic protein and albuminhave been found to be more abundant in induced sputum com-pared with bronchoalveolar lavage fluid (31, 33, 35).

We found airway acidification in subjects with asthma incomparison with healthy control subjects, in accordance withprevious 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 byanalyzing the mucus through tracheostomy access (36), but sofar, other types of samplings for pH determination have resultedin slightly different values (1, 6, 7, 36–38). The factors determin-ing airway pH are not completely known, but alterations in

Kodric, Shah, Fabbri, et al.: Induced Sputum and Airway Acidification 909

Figure 5. Correlation graphsbetween the lower levels ofcontrol of asthma expressedby the Asthma Control Ques-tionnaire (ACQ) score (ACQ� 0.71) and pH and percent-age of eosinophils.

pH lead to respiratory symptoms such as cough, wheezing, anddyspnea (1, 18, 19). From a teleologic point of view, an acidicenvironment may play a protective role against exogenous bacte-ria by inhibiting their growth through the formation of nitricoxide and the nitrosilation of proteins; gram-negative bacteriaand Mycobacterium tuberculosis do not survive in an acidic mi-lieu (39–41). Our knowledge of the effects of airway acidificationderives from the effects of aspiration of acidic substances inthe atmosphere (acidic fog) or gastroesophageal reflux (1). Thehydrogen 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 hyper-reactivity (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 acidproduction by lamellar bodies (50), ionic pumps (51), and releaseby degranulation of inflammatory cell vacuoles (52) is counter-balanced by the presence of buffers such as albumin in theepithelial lining fluid, bicarbonates, and ammonia producedthrough the activation of glutaminase (53, 54). These mecha-nisms may be altered in pathologic conditions. It has been ob-served that some patients with asthma have a low concentrationof high-molecular-weight proteins or a low buffer capacity witha decreased capacity to protect the H� ion penetration of sur-rounding tissues (55). Moreover, it is known that some of theairway-neutralizing mechanisms can be shut off to allow acidifi-cation for host defense purposes during inflammation (1). Forexample, the surest way to inactivate rhinovirus (the commoncold virus and the most important trigger of asthma exacerba-tions) is by means of mild acidification (56). It is known thatacidic mucus or mucus with a low protein concentration, as insome patients with asthma, constitutes a risk factor in case ofacidic exposures (e.g., air pollution) (57).

Moreover, we distinguished the severity of the disease fromits level of control, considering severity as the intrinsic intensityof the disease over a long time and control as the fluctuation ofthe 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, weobserved the lowest induced-sputum pH in patients with poorlycontrolled asthma, independent of the GINA severity level. Thisis in accordance with previous studies (18). In addition, we foundthat, once lost, the control of asthma, or the degree of poorcontrol, correlates better with pH than does induced-sputumeosinophil count; the latter observation is partially in contrastwith previous observations in which the control of asthma wasrelated to the increase of sputum eosinophils (59, 60). We didfind an increase in eosinophils in the poorly controlled asthmagroup, but this was not as strictly related to the degree of poor

asthma control as was the pH. The control and severity of asthmapresented some overlapping features in their evaluations and,apart from clinical data, we need sensitive and specific biomark-ers not affected by the severity of the disease. Even more impor-tant, the study of the alterations of pH may lead us to discovernew pathophysiologic mechanisms of poorly controlled disease.

Taking into account its simplicity and the good within-samplerepeatability of the obtained results, induced sputum may beconsidered a useful tool to study airway acidification both inclinical practice and in experimental settings. Future studies thatuse induced sputum as a simple and useful investigative toolmay give us more insights into the acidification of the airwaysand into different aspects of asthma.

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

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