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Keywords:

  • ammonia;
  • asthma;
  • exhaled breath condensate;
  • nitric oxide;
  • pH

Abstract

  1. Top of page
  2. Abstract
  3. Methods
  4. Study subjects
  5. Exhaled breath condensate collection
  6. Ammonia and pH analysis
  7. Fractional exhaled nitric oxide FENO measurement
  8. Pulmonary function test
  9. Statistical analysis
  10. Results
  11. Ammonia
  12. pH
  13. FENO
  14. Spirometric parameters
  15. Correlations
  16. Discussion
  17. Acknowledgments
  18. References

Background:  The dysregulation of airway pH control may have a role in asthma pathophysiology. The measurement of exhaled breath condensate (EBC) pH and ammonia levels may be used as a noninvasive method to study acid–base status in the airway of asthmatics.

Methods:  Exhaled breath condensate from 29 allergic stable asthmatic children and 13 healthy controls was collected by cooling exhaled air during tidal breathing. Ammonia was measured by high-performance liquid chromatography with fluorescence detection. pH was measured after deaeration of EBC samples by bubbling with argon. The children also underwent FENO measurement.

Results:  Both pH and ammonia values in EBC were significantly lower in the asthmatics than in the control group [pH: ICS-treated (median and interquartile range) 7.70 (7.62–7.74), steroid-naïve 7.53 (7.41–7.68), controls 7.85 (7.80–7.90), P < 0.01 and P < 0.001, respectively; ammonia: ICS-treated 476.17 μM (282.50–594.80), steroid-naïve 253.24 μM (173.43–416.08), controls 788.30 μM (587.29–1310.39), P < 0.05 and P < 0.001, respectively]. Both pH and ammonia values were higher in ICS-treated than in steroid-naïve asthmatic children. There was a significant correlation between EBC pH and ammonia concentrations.

Conclusions:  These data show that EBC pH values of stable asthmatic children are lower compared with those of healthy controls and positively correlated with ammonia concentrations, supporting the hypothesis that airway acidification may have a role in the pathobiology of allergic asthma.

Exhaled breath contains water vapor and micro-droplets, whose composition appears to reflect airway lining fluid (1, 2). Exhaled breath condensate (EBC) collection can be defined as a method to capture exhaled volatile and nonvolatile substances. Through the cooling of exhaled air, both condensed water and micro-droplets are collected, forming EBC, a fluid which is easy to be analyzed.

Exhaled breath condensate contains several compounds which have been used as biomarkers of lung diseases (2). EBC collection is totally noninvasive and therefore particularly easy to perform in children (3). It provides access to volatile and nonvolatile respiratory compounds (2) without the need for invasive diagnostic procedures, such as bronchoalveolar lavage fluid and bronchial biopsies, or for semi-invasive ones, such as sputum induction.

Recently, Hunt et al. (4) showed that the measurement of EBC pH can be used as a marker for acute worsening of childhood asthma, proposing that the simple measurement of EBC pH could be used to study acid–base status in the airway of asthmatic patients. In addition, Kostikas et al. (5) showed that EBC pH values in adults with asthma were lower compared with those in controls and that they were correlated with inflammatory cells in induced sputum. Furthermore, Hunt et al. (6) demonstrated that airway and lung epithelial cells produce ammonia stoichiometrically from glutamine in a reaction catalyzed by glutaminase. The production of ammonia could represent an attempt of epithelial cells to buffer the acidic challenge, maintaining the airway pH homeostasis.

These observations opened a new door on asthma pathophysiology and led to a new interest toward acid–base equilibrium into airways of asthmatics (7, 8). However, there are no data regarding pH values in EBC of clinically stable asthmatic children or about their relationship with EBC ammonia concentrations.

Asthma is characterized by a chronic inflammation of the airways almost invariably associated with tissue damage and healing. These processes are dynamic and may result in remodeling of the airways through mechanisms which are still largely unknown (9). The development and application of noninvasive methods for assessing ongoing biochemical and inflammatory activities in the lung can help us to gain a better understanding of the relationship between the various inflammatory processes occurring in asthma (2).

The aim of the study was to compare pH in EBC of clinically stable asthmatic children with that of healthy controls and to evaluate its relationship with ammonia. In addition, because of the reported inter-relationship between fractional exhaled nitric oxide (FENO) and acid–base equilibria into airways (6), we also measured the levels of FENO in asthmatic and control children.

Asthmatic children.  The study included 29 allergic stable asthmatic children (14 ICS-treated and 15 steroid-naïve) (Table 1). They had mild to moderate persistent asthma clinically and by pulmonary function criteria, accordingly to GINA guidelines (10). Children were recruited among patients attending the Pulmonology/Allergy outpatient's clinic at the Pediatrics Department in Padova. The steroid-naïve asthmatic children had not been treated with inhaled corticosteroids (ICS) for at least 1 month. The ICS-treated asthmatics had been on maintenance therapy with low doses (10) of ICS (budesonide or fluticasone) at a constant dose for at least 2 months. Children who had had acute upper or lower airway infections in the previous 3 weeks or who had a history of poor compliance were excluded from the study.

Table 1.  Subjects’ characteristics, biomarker values in EBC and exhaled NO
 Healthy childrenAsthmatic children ICS-treatedAsthmatic children steroid-naïve
  1. Data are expressed as median (interquartile range).

  2. *P < 0.05 vs healthy children.

  3. **P < 0.01 vs healthy children.

  4. ***P < 0.001 vs healthy children.

  5. P < 0.05 vs steroid-naïve asthmatic children.

Number (males)13 (9)14 (10)15 (9)
Mean age (range)10.2 (7–11)12.4 (7–15)11.3 (6–15)
FEV1 (%pred)98 (87–100)92 (90–95)97 (94–103)
FEF25−75 (%pred)97 (84–109)86 (72–104)94 (82–105)
FENO (ppb)8.75 (6.65–13.10)31.30 (16.83–82.18)***40.00 (29.13–68.73)***
EBC ammonia (μM)788.30 (587.29–1310.39)476.17 (282.50–594.80)*,†253.24 (173.43–416.08)***
EBC pH7.85 (7.80–7.90)7.70 (7.62–7.74)**,†7.53 (7.41–7.68)***

All the children were atopic, sensitized to common allergens (Dermatophagoides pteronissinus and farinae, mixed grass pollen, Parietaria, Artemisia vulgaris, Alternaria, dog and cat), as evaluated by skin prick test and/or RAST. None of the children with grass pollen allergy was studied during the grass pollen season.

Healthy controls.  The study included 13 healthy controls (Table 1) with no history of asthma or atopy. None of the children had had respiratory infections in the previous 4 weeks. None of the control subjects was taking any medication or smoked.

All the children were examined by a physician, and then underwent FENO measurement, spirometry and EBC collection.

The ethics committee of our hospital reviewed and approved the protocol and all parents gave their informed consent.

Exhaled breath condensate collection

  1. Top of page
  2. Abstract
  3. Methods
  4. Study subjects
  5. Exhaled breath condensate collection
  6. Ammonia and pH analysis
  7. Fractional exhaled nitric oxide FENO measurement
  8. Pulmonary function test
  9. Statistical analysis
  10. Results
  11. Ammonia
  12. pH
  13. FENO
  14. Spirometric parameters
  15. Correlations
  16. Discussion
  17. Acknowledgments
  18. References

Exhaled breath condensate samples were collected in a condensing device composed of two glass chambers (Incofar Srl, Modena, Italy) (11, 12). The inner glass chamber was cooled with ice and suspended in a larger glass chamber. The children, with no nose clip, were instructed to breathe tidally through their mouths via a two-way nonrebreathing valve for 15 min. The two-way valve also served as a saliva trap. In addition, the glass condenser was placed at a higher level than child's mouth with a 12 cm banded tube vertically positioned between the mouthpiece and the condensing device. This setting makes unlike the salivary contamination. Children were also asked to swallow their saliva periodically. The temperature of collection was around 0°C. The collected EBC samples were then immediately stored in sterile tubes at −70°C. EBC collection was performed without nose clip, as it was reported that there was no significant difference in ammonia levels between the samples collected with and without nose clip (13).

Ammonia and pH analysis

  1. Top of page
  2. Abstract
  3. Methods
  4. Study subjects
  5. Exhaled breath condensate collection
  6. Ammonia and pH analysis
  7. Fractional exhaled nitric oxide FENO measurement
  8. Pulmonary function test
  9. Statistical analysis
  10. Results
  11. Ammonia
  12. pH
  13. FENO
  14. Spirometric parameters
  15. Correlations
  16. Discussion
  17. Acknowledgments
  18. References

Ammonia was measured by high-performance liquid chromatography with fluorescence detection (HPLC-FLD) using a dansyl chloride precolumn derivatization method (14, 15). Briefly, 200 μl of EBC sample or standard was derivatizated, in 0.7 ml Gilson vials, by adding 200 μl of borate buffer, 0.1 M at pH 9.8, followed by 200 μl of 2 mM dansyl chloride stock solution in acetone. After incubation for 90 min at room temperature, a 20 μl sample was injected into the HPLC. If EBC sample was not sufficient, the derivatization procedure was done with 100 μl.

In aqueous solution there is an equilibrium of two species, H ammonia (NH3) and H ammonium (NHinline image), which are invariably present together in a ratio determined by the pH. At pH 9.8, only 25% is NHinline image.

Chromatography of the NH3 dansyl chloride adduct was achieved with an isocratic elution on a Superchrom LC18 column, 250 × 4.6 mm inside diameter, 5 μm (Varian, Milan, Italy) with a mobile phase composed of acetonitrile/methanol/water (3/7/7), at flow rate of 0.8 ml/min. Fluorimetric detection was performed at 500 nm after excitation at 368 nm (Scanning Fluorescence Detector 474; Waters, Milan, Italy). The retention time was ≈8 min.

The dansyl ammonia derivative was stable in air and ambient light at room temperature for many hours (15). Appropriate water blank was also analyzed and calibration curve for NH3 was made with each batch of samples.

Under the conditions described, method quality parameter for NH3 was: low detection limit >1 μM, recovery >98%, coefficient of variation intra-day <3%, coefficient of variation inter-day <5%, operative linearity range 20–3000 μM. pH was measured with an Hamilton Biotrode electrode for micro-samples (Hamilton, Bonaduz AG, Switzerland) attached to a Crison pH meter (Crison Instruments, S.A., Alella Barcelona, Spain) in naïve EBC samples and after deaeration and decarbonation by bubbling with argon as previously reported (4, 5). Initially, the measures were repeated 5, 10 and 15 min after the deaeration with argon. We observed no difference between the two last measurements; so we assumed that the time necessary to obtain free carbon dioxide samples was 10 min. In order to avoid the contamination with ambient CO2 which may influence pH values, EBC vials were tightly capped during the pH measurements.

Fractional exhaled nitric oxide FENO measurement

  1. Top of page
  2. Abstract
  3. Methods
  4. Study subjects
  5. Exhaled breath condensate collection
  6. Ammonia and pH analysis
  7. Fractional exhaled nitric oxide FENO measurement
  8. Pulmonary function test
  9. Statistical analysis
  10. Results
  11. Ammonia
  12. pH
  13. FENO
  14. Spirometric parameters
  15. Correlations
  16. Discussion
  17. Acknowledgments
  18. References

Fractional exhaled nitric oxide was measured with the NIOX system (Aerocrine, Stockholm, Sweden) using a single-breath online method according to ERS/ATS guidelines for FENO measurement in children (16). Children inhaled NO-free air and exhaled through a dynamic flow restrictor with a target flow of 50 ml/s for at least 6–7 s. Visual incentives provided feedback for flow rate compliance.

Statistical analysis

  1. Top of page
  2. Abstract
  3. Methods
  4. Study subjects
  5. Exhaled breath condensate collection
  6. Ammonia and pH analysis
  7. Fractional exhaled nitric oxide FENO measurement
  8. Pulmonary function test
  9. Statistical analysis
  10. Results
  11. Ammonia
  12. pH
  13. FENO
  14. Spirometric parameters
  15. Correlations
  16. Discussion
  17. Acknowledgments
  18. References

Data are expressed as median and interquartile range (IQR). Differences among groups were analyzed using the nonparametric Kruskal–Wallis test followed, where significant, by the Mann–Whitney U-test for comparisons between groups. Correlations were evaluated by Spearman's rank test. Results were considered significant at a value of P < 0.05.

Ammonia

  1. Top of page
  2. Abstract
  3. Methods
  4. Study subjects
  5. Exhaled breath condensate collection
  6. Ammonia and pH analysis
  7. Fractional exhaled nitric oxide FENO measurement
  8. Pulmonary function test
  9. Statistical analysis
  10. Results
  11. Ammonia
  12. pH
  13. FENO
  14. Spirometric parameters
  15. Correlations
  16. Discussion
  17. Acknowledgments
  18. References

In both the ICS-treated and the steroid-naïve asthmatic children, the EBC concentrations of ammonia were significantly lower than those detected in the EBC of healthy children [ICS-treated (median and IQR) 476.17 μM (282.50–594.80), steroid-naïve 253.24 μM (173.43–416.08), controls 788.30 μM (587.29–1310.39), P < 0.05 and P < 0.001, respectively] (Fig. 1; Table 1). There was also a significant difference in ammonia EBC concentrations between ICS-treated and steroid-naïve asthmatic children (P < 0.05) (Fig. 1).

image

Figure 1. Ammonia levels in exhaled breath condensate (EBC) of asthmatic (black spots) and control (white spots) children. Horizontal bars represent median values.

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pH

  1. Top of page
  2. Abstract
  3. Methods
  4. Study subjects
  5. Exhaled breath condensate collection
  6. Ammonia and pH analysis
  7. Fractional exhaled nitric oxide FENO measurement
  8. Pulmonary function test
  9. Statistical analysis
  10. Results
  11. Ammonia
  12. pH
  13. FENO
  14. Spirometric parameters
  15. Correlations
  16. Discussion
  17. Acknowledgments
  18. References

The median pH measured after argon deaeration was significantly lower in both asthmatic groups compared with that in the control group [ICS-treated asthmatics 7.70 (7.62–7.74), steroid-naïve asthmatics 7.53 (7.41–7.68), healthy controls 7.85 (7.80–7.90), P < 0.01 and P < 0.001, respectively] (Fig. 2; Table 1). EBC pH was lower in steroid-naïve than in ICS-treated asthmatic children (P < 0.05) (Fig. 2).

image

Figure 2. pH levels in exhaled breath condensate (EBC) of asthmatic (black spots) and control children (white spots). Horizontal bars represent median values.

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In a subgroup of 21 children (11 asthmatic and 10 healthy) the baseline pH was measured before and after deaeration of the condensate with argon gas. In both asthmatic and healthy children the pH values were significantly higher after deaeration with argon [asthmatic children: before 6.85 (6.68–7.06), after 7.72 (7.64–7.75), P < 0.001, healthy controls: before 7.05 (6.83–7.31), after 7.85 (7.80–7.90), P < 0.01] (Fig. 3).

image

Figure 3. pH levels in exhaled breath condensate (EBC) in a subgroup of asthmatic and control children. EBC pH was measured in naïve EBC samples (black spots) and in EBC samples after deaeration with argon (white spots) Horizontal bars represent median values.

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Because of technical reasons the pH was not measured in seven of the asthmatic children and in three of the healthy controls.

FENO

  1. Top of page
  2. Abstract
  3. Methods
  4. Study subjects
  5. Exhaled breath condensate collection
  6. Ammonia and pH analysis
  7. Fractional exhaled nitric oxide FENO measurement
  8. Pulmonary function test
  9. Statistical analysis
  10. Results
  11. Ammonia
  12. pH
  13. FENO
  14. Spirometric parameters
  15. Correlations
  16. Discussion
  17. Acknowledgments
  18. References

In both asthmatic groups the FENO values were significantly higher compared with those measured in the healthy children [ICS-treated children, median 31.30 ppb [16.83–82.18]; steroid-naïve children, median 40.0 ppb (29.13–68.73), healthy controls, median 8.75 ppb (6.65–13.10); P < 0.001] (Fig. 4; Table 1). No significant difference in FENO levels was found between the two groups of asthmatic children (P = 0.75).

image

Figure 4. Fractional exhaled nitric oxide (FENO) levels in asthmatic (black spots) and control children (white spots). Horizontal bars represent median values.

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Spirometric parameters

  1. Top of page
  2. Abstract
  3. Methods
  4. Study subjects
  5. Exhaled breath condensate collection
  6. Ammonia and pH analysis
  7. Fractional exhaled nitric oxide FENO measurement
  8. Pulmonary function test
  9. Statistical analysis
  10. Results
  11. Ammonia
  12. pH
  13. FENO
  14. Spirometric parameters
  15. Correlations
  16. Discussion
  17. Acknowledgments
  18. References

There was no significant difference in FEV1 and FEF25−75 between asthmatic and healthy children [ICS-treated children: FEV1 92% pred (90–95), FEF25−75 86% pred (72–104); steroid-naïve asthmatics FEV1 97% pred (94–103) , FEF25−75 94% pred (82–104); healthy controls FEV1 98% pred (87–100), FEF25−75 97% pred (84–109)].

Correlations

  1. Top of page
  2. Abstract
  3. Methods
  4. Study subjects
  5. Exhaled breath condensate collection
  6. Ammonia and pH analysis
  7. Fractional exhaled nitric oxide FENO measurement
  8. Pulmonary function test
  9. Statistical analysis
  10. Results
  11. Ammonia
  12. pH
  13. FENO
  14. Spirometric parameters
  15. Correlations
  16. Discussion
  17. Acknowledgments
  18. References

In the whole group of children there was a significant correlation between EBC ammonia concentrations and pH (r = 0.5, P = 0.003) (Fig. 5), a negative correlation between FENO and EBC pH (r = −0.5, P = 0.005) and a negative correlation between ammonia EBC concentrations and FENO (r = −0.5, P < 0.001) (Fig. 6).

image

Figure 5. Correlation between EBC ammonia concentrations and pH in asthmatic (black spots) and control children (white spots).

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image

Figure 6. Correlation between EBC ammonia concentrations and FENO in asthmatic (black spots) and control children (white spots).

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Discussion

  1. Top of page
  2. Abstract
  3. Methods
  4. Study subjects
  5. Exhaled breath condensate collection
  6. Ammonia and pH analysis
  7. Fractional exhaled nitric oxide FENO measurement
  8. Pulmonary function test
  9. Statistical analysis
  10. Results
  11. Ammonia
  12. pH
  13. FENO
  14. Spirometric parameters
  15. Correlations
  16. Discussion
  17. Acknowledgments
  18. References

The dysregulation of airway pH control is a new aspect of asthma pathophysiology and changes in acid–base equilibrium of airway lining fluid may stimulate neurogenic reflexes leading to cough, bronchospasm and airway reactivity (17).

In this study, we showed that EBC pH values in asthmatic children were significantly lower compared with those in the control group and correlated with EBC ammonia levels. These results confirm and extend the data that Hunt et al. (4) and Kostikas et al. (5) found in adult asthmatic patients.

The measurement of EBC pH is inexpensive and simple to perform, and shows a good reproducibility in both subjects with airway diseases and controls (5, 18). In this study, the measurement of EBC pH was performed after deaerating the EBC samples with argon, a procedure required to obtain stable pH levels. The deaeration changes the equilibrium between CO2 and bicarbonate ion (HCOinline image), therefore the EBC fluid is alkaline and the pH is higher than in the naïve samples (Fig. 3).

The mechanisms leading to airway acidification in asthma are a matter of speculation and interesting hypothesis have been raised. Low EBC pH may be caused by change of buffering systems into airways. Hunt et al. (6) showed that human airway and lung epithelium express glutaminase, an enzyme which produces ammonia from glutamine, buffering endogenous and exogenous acids. The authors demonstrated that the activity of glutaminase is down-regulated by inflammatory cytokines, such as interferon-γ and tumor necrosis factor-α (6), whereas corticosteroid administration, which suppresses inflammatory cytokines (9), up-regulates the glutaminase expression (6). In addition, they observed that during acute asthma exacerbations both EBC ammonia levels and pH values are low, while glutaminase expression may be decreased, suggesting that inhibition of glutaminase may contribute to lower the airway pH in asthmatic subjects. According to this hypothesis, our data show, for the first time in children, that ammonia levels in EBC are lower in asthmatics compared with controls (Fig. 1) and that there is a positive correlation between ammonia concentrations and pH in EBC (Fig. 5). Of course the depletion of ammonia may be necessary but not sufficient to explain the acidification, and further studies are required to investigate the presence and the concentration of other buffers in EBC, such as bicarbonate and citrate. At this regard, further studies are warranted to identify specific acid/base pairs in EBC.

The observed endogenous acidification we found in asthma may also depend on the amount of acid provided to the airways either by low pH lamellar bodies or by acidic vacuoles from inflammatory cells (19). Kostikas et al. (5) showed a negative correlation between EBC pH levels and eosinophil number in induced sputum of asthmatic patients, together with a negative correlation between EBC pH and 8-isoprostane, which is a reliable biomarker of oxidative stress (20).

We found that EBC pH values were higher in asthmatic children under treatment with inhaled steroids than in those who were steroid-naïve (Fig. 2). This is in line with the reported data by Kostikas et al. (5), who observed higher pH levels in EBC of ICS-treated asthmatics compared with steroid-naïve patients. In addition Hunt et al. reported that EBC pH of patients with acute asthma exacerbation significantly increases after systemic corticosteroid therapy. Taken together, these observations indirectly support the role of EBC pH as a noninvasive marker of airway inflammation. Moreover, we did not find a correlation between EBC pH or ammonia and spirometric parameters, suggesting that these inflammatory markers may be more sensitive than spirometry for detecting disease.

We showed that also ammonia concentrations were higher in ICS-treated children (Fig. 1), supporting the hypothesis that corticosteroid therapy may increase glutaminase expression (6).

For the measurement of ammonia in EBC we applied a new method based on liquid chromatography, instead of less-specific spectrophotometric determination reported in previous publications (6, 13). Nevertheless, we found ammonia levels similar to those observed by Hunt et al. (6). Guidelines defining standardized procedures are needed in order to obtain easily comparable data.

Ammonia values in EBC were significantly lower in asthmatic children than in healthy controls. Even if we recognize that EBC ammonia may in part derive from the catabolic degradation of urea in the mouth (13), the relevance of ammonia in asthma pathophysiology is supported by the observation that both EBC ammonia (Fig. 6) and pH are negatively correlated with FENO, a sensitive marker of asthmatic airway inflammation. These correlations let us speculate that airway acid–base equilibrium may influence the NO metabolite concentration. This is consistent with the observation of Hunt et al. (6) who showed that airway acidity favors the protonation of nitrite (NOinline image), which is increased in asthma (21), to form nitrous acid (HNO2) resulting in NO production.

The finding of increased FENO values in both steroid-treated and steroid-naïve asthmatic children is in keeping with some previous studies (22, 23) (Fig. 4). The persistence of increased FENO values in several steroid-treated patients suggests that there is heterogeneity of FENO response to ICS (24) and that NO-related airway inflammation may persist despite treatment with ICS. FENO is considered an attractive biomarker providing immediately available information about the asthmatic state not accessed by clinical characteristics (25) and pulmonary function. However, exhaled NO is expression of just one of multiple pathways of lung nitrogen oxides chemistry and EBC analysis has potential to complement FENO in giving insights into lung redox assessment.

In conclusion, we have shown that EBC pH levels of stable asthmatic children are lower compared with healthy controls and are positively correlated with ammonia levels supporting the hypothesis that airway acidification may have a role in the pathobiology of asthma. The study of airway acid–base equilibrium is a new area of research with potential application for asthma monitoring and development of new therapies.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Methods
  4. Study subjects
  5. Exhaled breath condensate collection
  6. Ammonia and pH analysis
  7. Fractional exhaled nitric oxide FENO measurement
  8. Pulmonary function test
  9. Statistical analysis
  10. Results
  11. Ammonia
  12. pH
  13. FENO
  14. Spirometric parameters
  15. Correlations
  16. Discussion
  17. Acknowledgments
  18. References

This work was supported in part by grant 1R01 HL72323-01 from the National Heart, Blood and Lung Institute (NHLBI), Bethesda, USA. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NHLBI or National Institute of Health.

References

  1. Top of page
  2. Abstract
  3. Methods
  4. Study subjects
  5. Exhaled breath condensate collection
  6. Ammonia and pH analysis
  7. Fractional exhaled nitric oxide FENO measurement
  8. Pulmonary function test
  9. Statistical analysis
  10. Results
  11. Ammonia
  12. pH
  13. FENO
  14. Spirometric parameters
  15. Correlations
  16. Discussion
  17. Acknowledgments
  18. References
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