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Exhaled breath condensate pH standardised for CO₂ partial pressure

¹ Dept of Pathophysiology and, ³ Clinical Laboratory, National Korányi Institute for Tuberculosis and Pulmonology, and ² Institute of Human Physiology and Clinical Experimental Research, Semmelweis University, Budapest, Hungary.

CORRESPONDENCE: I. Horváth, Dept of Human Physiology and Clinical Experimental Research, Semmelweis University, 1082 Budapest Üllüi u. 78/a Hungary. Fax: 36 13343162. E-mail: kiss.horvath{at}t-online.hu

Keywords: Airway biology, airway inflammation, breath test, exhaled biomarkers, exhaled breath condensate

Received: June 27, 2006
Accepted October 23, 2006

	ABSTRACT

TOP ABSTRACT METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Exhaled breath condensate (EBC) pH is considered to reflect the acid-base balance of the airways. Current pH measurements do not take into account the effect of CO₂. The aim of the present study was to determine the effect of condensate CO₂ partial pressure on pH and to provide a more precise mode of EBC pH determination.

Condensate pH and CO₂ partial pressure were measured in parallel from 12 healthy volunteers and 12 asthmatics using a blood gas analyser in neat, argon de-aerated and CO₂-loaded samples. The regression analysis was used to test the relationship between pH and CO₂, and to calculate the pH at a CO₂ level of 5.33 kPa (physiological alveolar CO₂ partial pressure). Reproducibility of different pH readings was compared using the Bland–Altman test.

Condensate CO₂ concentration was variable both in neat and argon de-aerated samples. There was a close negative logarithmic relationship between CO₂ and pH. Calculation of pH at a CO₂ level of 5.33 kPa provided reproducibility approximately six times as good as that of the currently used measurements.

Condensate CO₂ partial pressure influences pH measurements. Determination of pH at a standard CO₂ level provides the most reproducible condensate pH values to date.

Exhaled breath condensate (EBC) analysis is a promising method for the investigation of airway pathology 1. Ease of repeatability and its noninvasive nature make EBC collection attractive to clinicians. However, the measurement of different exhaled biomarkers, such as hydrogen peroxide, nitrogen oxides, cytokines and leukotrienes, yields greatly variable results. The pH is currently considered to be the most robust variable of EBC 2, 3. Measurement of EBC pH has already proven valuable in determining the degree of acidification of EBC in patients with various inflammatory lung diseases 4–7, individuals exposed to hypertonic saline solution inhalation 8 or acute lung injury 9.

It has been acknowledged by the American Thoracic Society (ATS)/European Respiratory Society (ERS) Task Force that the pH of neat EBC samples is unstable 1. Argon de-aeration was suggested as a method of improving the reproducibility of pH readings 1, 2 as, in theory, inert gas removes all volatile components of EBC allowing the measurement of nonvolatile acidity. When assayed continuously by a glass microelectrode, it has been observed that the pH of EBC stabilises after 8–10 min of bubbling with argon 2. It is generally assumed that a stable pH marks the complete removal of CO₂ and other volatile components.

CO₂ is the major volatile component of EBC. In an aqueous environment CO₂ forms H⁺ and HCO₃^- and profoundly affects the pH of dilute solutions, such as EBC. Levels of CO₂ have not yet been systematically tested in EBC. Although argon de-aeration causes a significant decrease in the CO₂ partial pressure of the exhaled breath condensate (P_EBC,CO2), the remaining CO₂ could influence pH results 10. Furthermore, it is not clear whether NH₃, another volatile component of EBC, is important in de-aeration-induced changes in EBC pH 11, 12.

The aim of the present study was to determine the effect of CO₂ on breath condensate pH and to achieve better reproducibility of pH readings by considering P_EBC,CO2 in both healthy subjects and asthmatic patients.

	METHODS

TOP ABSTRACT METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Subjects
A total of 12 healthy nonsmokers without any disease in their medical history (eight females, four males, mean (range) age 41 (21–61) yrs, forced vital capacity (FVC) >90% predicted, forced expiratory volume in one second (FEV₁)>80% pred, FEV₁/FVC >70%) and 12 atopic asthmatic subjects without upper airway diseases in a clinically stable condition (seven females, five males, aged 43 (25–64) yrs, FVC >90% pred, FEV₁ >80% pred, FEV₁/FVC >70%, exhaled nitric oxide fraction <20 ppb), treated with short-acting ß₂-agonists and inhaled corticosteroids (400 µg·day^–1 budesonide), were enrolled in the study. The study was approved by the local ethics committee and participants gave their written informed consent.

EBC collection
EBC was collected for 10 min with a commercially available condenser (EcoScreen; Jaeger, Würzburg, Germany). Nose-clips were not worn. Subjects were asked to inhale through the nose and exhale through the mouth in their normal rhythm of breathing. This sampling method provides a larger sample volume than that using a nose-clip 13. Furthermore, there is no difference in exhaled biomarker concentration between the two types of sampling in subjects without upper airways disease 13, 14.

From healthy subjects, two EBC samples were collected on two consecutive days between 07:00 and 08:00 h. pH and CO₂ from both samples were determined in duplicate as follows: 1) from neat samples within 10 min after sampling; 2) after argon de-aeration for 2.5, 5, 7.5 and 10 min; and 3) after CO₂ loading for 1, 2, 3 and 4 s.

Handling of EBC samples
Neat EBC samples
Neat EBC samples were used for measurement immediately after sampling (all measurements were made within 10 min after sampling).

De-aerated EBC samples
The generally used argon de-aeration method was chosen to obtain data comparable with published results.

Each EBC sample was divided into 250-µL aliquots in eight plastic tubes. Aliquots were simultaneously bubbled with argon (Argon 4.6; Messer Hungarogáz Kft, Budapest, Hungary) using a purpose-made bubbling device with eight arms. The device assured the same argon flow (300 mL·min^–1) in each tube. Samples were de-aerated in duplicate for 2.5, 5, 7.5 and 10 min and aliquots were taken for pH and CO₂ determination after each de-aeration period.

CO₂-loaded EBC samples
CO₂ loading was achieved by bubbling CO₂ gas through the samples (CO₂ 4.5; Messer Hungarogáz Kft, Budapest, Hungary). Since this manoeuvre caused a rapid increase in P_EBC,CO2, very short intervals of bubbling (1 s) were chosen to obtain a stepwise increase in P_EBC,CO2. CO₂ gas was bubbled through the EBC samples four times with a 1-s duration. After each 1-s bubbling period aliquots were taken for pH and CO₂ measurements (i.e. each 1 s CO₂ bubbling was followed by 10–15 s, as samples were taken, when no gas was bubbled through the sample).

pH and CO₂ measurement
EBC samples were immediately transferred into glass capillaries. The closed capillaries were stored for no longer than 1 h at room temperature before measurements. pH and P_EBC,CO2 measurements were performed by means of a blood gas analyser (ABL 520; Radiometer, Copenhagen, Denmark). The reliability of the blood gas analyser in determining EBC pH was tested before the study by comparing it with a glass microelectrode (Radelkis, Budapest, Hungary) and by 10 repeated measurements of a de-aerated EBC sample.

Calculation of EBC pH at predetermined P_EBC,CO2
The pH value at 5.33 kPa P_EBC,CO2 was calculated using data obtained from neat and CO₂-loaded samples by regression analysis.

Comparison of CO₂-normalised EBC pH with other pH readings
The repeatability of the three different types of EBC pH determination (in neat samples, in argon de-aerated samples and the CO₂-normalised EBC pH) and the day-to-day variability of EBC pH in healthy individuals was tested. Furthermore, the EBC pH of healthy subjects was compared with that of stable asthmatic patients by using all three types of EBC pH determination.

The repeatability of EBC pH readings was tested in duplicate EBC samples from healthy participants. From both parts of given EBC samples the following six pH and CO₂ measurements were taken: one from the neat sample; one after 10 min argon de-aeration and four from CO₂-loaded samples following the four 1-s loading periods.

Day-to-day variability of EBC pH was tested for the three pH reading methods in healthy participants.

Comparison of EBC pH of healthy and asthmatic subjects was also performed with all three different pH reading methods.

Ammonia measurement
Ammonia was measured spectrophotometrically in neat EBC samples and after argon de-aeration for 10 min (Diagnostic ammonia assay kit; Randox®, Ardmore, UK) in the same healthy participants.

Statistical analysis
A pH–P_EBC,CO2 plot was created for each sample using the data collected by the blood gas analyser. Logarithmic regression and coefficient of determination (r²) were calculated. The pH and CO₂ values obtained in the neat sample and after CO₂-loading periods were used to calculate pH at a standardised CO₂ level (P_EBC,CO2 of 5.33 kPa).

A Bland–Altman test was performed to compare the repeatability of pH assessment of neat and argon de-aerated samples with CO₂-normalised pH values. Paired t-tests were applied for comparison of ammonia concentrations before and after de-aeration and also for comparison of mean of differences.

	RESULTS

TOP ABSTRACT METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
CO₂ removal (argon de-aeration)
A broad range of CO₂ level variation was seen in neat EBC samples: 4.31–0.67 kPa (mean±SD 2.20±0.65). The corresponding pH values in neat EBC samples were in the range 6.17–7.19 (6.89±0.31).

The time course of CO₂ removal was not predictable. The P_EBC,CO2 could not be reduced to a standard level at a given time point and the reduction of P_EBC,CO2 was not proportional to time. Representative curves of the P_EBC,CO2 reduction time course (fig. 1a) with the corresponding pH increase (fig. 1b) obtained from two parallel aliquots of the same EBC sample are shown in figure 1.

View larger version (7K): [in this window] [in a new window]   Fig. 1— Representative time course of a) CO2 partial pressure of the exhaled breath condensate (PEBC,CO2) and b) pH during parallel argon de-aeration in duplicates of the same exhaled breath condensate (EBC) sample.

CO₂ loading
CO₂ bubbling raised the P_EBC,CO2 very quickly. Each 1-s CO₂ bubbling period caused a 5–10 kPa increase in P_EBC,CO2 and within 10 s, P_EBC,CO2 reached 80–100 kPa and could not be further increased.

pH measurement and calculation
The blood gas analyser provided the same pH values as the glass microelectrode. The mean pH of argon de-aerated EBC samples after 10-min de-aeration was 8.04 (7.91–8.11).

Loading samples with CO₂ revealed a close negative logarithmic correlation between pH and P_EBC,CO2 (r²>0.99, p<0.01; fig. 2). This correlation allows the calculation of EBC pH at any standardised P_EBC,CO2. A value of 5.33 kPa was chosen as it is thought to be identical to the physiological CO₂ level of the alveolar surface lining (ASL) fluid in healthy individuals. The mean EBC pH standardised to 5.33 kPa CO₂ partial pressure was 6.54 (6.06–6.96).

View larger version (17K): [in this window] [in a new window]   Fig. 2— Close negative logarithmic correlation between pH and partial pressure of carbon dioxide in the exhaled breath condensate (PEBC,CO2) upon CO2 load in a) healthy and b) asthmatic subjects. Samples were run in duplicate as indicated by red and blue symbols. Only one regression line is displayed per sample, as the regression lines of the duplicates run almost in parallel. ·····: 5.33 kPa PEBC,CO2. Coefficients of determination were as follows: blue: 0.9982; red: 0.9921; blue: 0.9965; red: 0.9970; blue: 0.9857; red: 0.9926; blue: 0.9993; red: 0.9970; • blue: 0.9958; • red: 0.9995; blue: 0.9991; red: 0.9967; blue: 0.9993; red: 0.9962; blue: 0.9969; red: 0.9955; blue: 0.9956; red: 0.9910; blue: 0.9963; red: 0.9981; x blue: 0.9986; x red: 0.9913; + blue: 0.9999; + red: 0.9996. EBC: exhaled breath condensate.

Repeatability of pH readings
The limits of agreement for parallel samples determined by the Bland–Altman test were 0.27, 0.25 and 0.04 for the argon de-aerated, the neat samples and the CO₂ standardisation, respectively (fig. 3). These results demonstrate that EBC pH standardised to P_EBC,CO2 is approximately six times as precise as pH measurement of either neat or de-aerated samples.

View larger version (5K): [in this window] [in a new window]   Fig. 3— Repeatability of pH measurement. Comparison of two values obtained from the same sample by a) de-aeration, b) neat measurement and c) calculation according to the CO2 loading protocol by the Bland–Altman test. ——: mean; ––––: ±2 SD.

Variability of EBC pH
EBC pH showed daily variability when determined by either method. The mean difference of pH values between de-aerated samples was 0.359, between neat samples 0.376 and between standardised pH values 0.278. Variability of standardised values is demonstrated in figure 4. Standardised pH values showed normal distribution.

View larger version (17K): [in this window] [in a new window]   Fig. 4— Variability of exhaled breath condensate (EBC) pH. pH values standardised to 5.33 kPa partial pressure of carbon dioxide in the EBC (PEBC,CO2) of healthy participants on day 1 () and day 2 ().

Ammonia measurement
There was no significant difference between ammonia concentrations before and after de-aeration by argon for 10 min (86±70 and 82±65 µM·L^–1, respectively). There was no correlation between ammonia levels and pH, either before (r² = 0.09) or after de-aeration (r² = 0.01) or with the calculated values (r² = 0.15).

	DISCUSSION

TOP ABSTRACT METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
EBC analysis is a promising topic of investigation; however, low reproducibility of measurements of different exhaled biomarkers limits its application. pH has been considered to be the most robust parameter of EBC 3. EBC pH is determined by volatile and nonvolatile components 11, 12, 15. Volatile components have been suspected of causing noise in EBC pH measurement. It was assumed that argon bubbling removes the volatile components of EBC almost completely; however, this assumption has never been tested.

Although the general suggestion of the ERS/ATS Task Force Report was to use a nose-clip for EBC sampling, it was acknowledged that samples could be collected without the use of one 1.

By measuring CO₂ partial pressure in EBC the present authors found that CO₂ influences EBC pH to a great extent. However, CO₂ can neither be completely removed from EBC nor can it be decreased to a standard level by argon bubbling even if continued for as long as 20 min.

Quite high levels of P_EBC,CO2 were achieved by repeated CO₂ loading compared with CO₂ within the physiological range and a substantial increase in P_EBC,CO2 was caused by just 1 s of CO₂ load. Lower P_EBC,CO2 levels may be created if the sample is left after one episode of CO₂ loading and aliquots for pH and CO₂ measurements are taken every few minutes (as CO₂ is diffusing out of the EBC). P_EBC,CO2 curves were constructed from eight points in the range 2.5–15 kPa. The same close negative logarithmic correlation was found as in the case of higher CO₂ levels. A repeated CO₂-loading protocol was chosen in the present study because it was less time-consuming. A negative logarithmic correlation was found between pH and P_EBC,CO2. The near-perfect logarithmic correlation between EBC pH and P_EBC,CO2 found in the CO₂-loading protocol is a consequence of the Henderson–Hasselbach equation. Similarly, the very strong logarithmic correlation in the de-aeration protocol means that argon bubbling mainly removes CO₂ and does not influence other components of the condensate. According to the present findings, argon de-aeration does not change the ammonia concentration of EBC.

The close correlation allows the calculation of EBC pH at any standard P_EBC,CO2. Standardisation to 5.33 kPa is justified as it is considered to correspond to the physiological ASL CO₂ level. EBC pH calculation at 5.33 kPa P_EBC,CO2 is six times as reproducible as pH measurements that do not take into account the CO₂ level. The reason for the improved repeatability of the CO₂ standardisation method is that in either de-aerated or neat pH measurements CO₂ level may vary and thus cause a significant change in pH (visually, it means that the standardised pH is read at a fixed point of the CO₂–pH regression line, while the neat and the de-aerated pH value moves along the regression line.)

Repeatability of standardised EBC pH is not increased at the expense of a loss of ability to detect differences between groups, as illustrated in figure 2 where the regression lines run almost in parallel.

Although the present results allow the reliable calculation of EBC pH they do not provide information about the identity of components that determine EBC pH and this could be an area of further investigation.

The possibility that EBC is contaminated with saliva is debatable 1, 11–13. The results of salivary contamination would be that estimated EBC pH does not correspond to ASL pH. Even if this were true it would not weaken the good repeatability of the method itself. Nor does the variability of EBC pH contradict good reproducibility. In fact, reliable pH determination ensures that a change in pH corresponds to real variability instead of the uncertainty of the measurement.

The fact that the current stable asthmatic patients had an EBC pH statistically similar to that of healthy individuals does not exclude the possibility that patients in more severe state of disease or during exacerbations would have a lower EBC pH. Even though the statistical demonstration of EBC acidification in inflammatory airway diseases is interesting from a pathophysiological point of view, it only has clinical importance if a cut-off value between healthy and pathological pH values can be set.

A number of questions remain to be answered, including the reason for the variability of EBC pH and the potential of EBC pH determination in clinical routine.

In summary, by the parallel measurement of pH and CO₂ partial pressure in EBC, CO₂ was found to affect condensate pH to great extent and condensate CO₂ level was unable to be standardised by the currently recommended de-aeration. A near-perfect negative logarithmic correlation was found between pH and CO₂ partial pressure in EBC. This correlation allows the calculation of pH at CO₂ partial pressure of 5.33 kPa. The calculated EBC pH of healthy adults shows variability in the interval of 6–7.

In conclusion, exhaled breath condensate CO₂ partial pressure is an important confounding factor of pH measurements. Determination of exhaled breath condensate pH standardised to CO₂ partial pressure of the exhaled breath condensate provides the most reproducible exhaled breath condensate pH values to date.

	ACKNOWLEDGEMENTS

TOP ABSTRACT METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
The authors would like to thank É. Huszár, laboratory assistants I. Kenéz and J. Csoszor, engineer V. Kuzniarsky and undergraduate students Á. Jendrék and A. Bikov for their important contribution to the study, as well as D. Byrne for English correction.

	REFERENCES

TOP ABSTRACT METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 

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