Science
Partial Oxygen and Carbon Dioxide Pressure
of Exhaled Breath Condensate
Slavica Dodig, PhD,1 Ivana Čepelak, PhD,2 Željka Vlašić, MD,1 Renata Zrinski Topić, PhD,1 Jadranka Živčić, MS,1
Boro Nogalo, MD1
(1Srebrnjak Children’s Hospital, Reference Center for Clinical Pediatric Allergology of the Ministry of Health and Social Welfare,
Zagreb, Croatia, 2Department of Medical Biochemistry and Hematology, School of Pharmacy and Biochemistry, University of Zagreb,
Zagreb, Croatia)
DOI: 10.1309/63E061U2YRV5T2A9
Abstract
Objective: To assess (a) the effect of ambient
and exhaled gases on water gas analysis and
(b) the impact of argonization on the results
of oxygen (Po2) and carbon dioxide (Pco2)
determination in exhaled breath condensate.
Methods: Samples of deionized water exposed
to atmosphere and exhaled air were analyzed
to assess the effect of ambient gases on the
respective analyte results. Exhaled breath
condensate (EBC) samples were argonized by 2
methods. Also, Po2 and Pco2 were determined
in EBC obtained from healthy children.
Results: Deionized water samples exposed
to air showed similar Po2 and Pco2 values in
ambient air. The values of Po2 were higher,
Considering the almost identical battery of biomarkers found
in exhaled breath condensate (EBC), induced sputum, and bronchoalveolar lavage, the latter 2 may likely be substituted with EBC
in the future. The composition of EBC1,2 has been postulated
to correspond to the composition of epithelial lining fluid but at
considerably lower concentrations,3,4 and its analysis appears to
bear a high potential in the detection and monitoring of pathologic changes in the lungs.3,4 Apart from numerous advantages
of the procedure of EBC analysis (a noninvasive, simple, and safe
procedure), a number of problems4,5 remain to be solved: the exact
anatomical site of origin of EBC constituents, the mechanism of
formation, preanalytical factors (eg, methodology of sampling,
storage, repeatability, and reproducibility), determination of low
constituent concentrations, and identification of the most appropriate biomarkers in lung diseases.
The aim of the study was to assess (a) the effect of ambient and exhaled gases on water gas analysis and (b) the impact of
argonization on the results of oxygen (Po2) and carbon dioxide
(Pco2) determination in exhaled breath condensate.
Material and Methods
Deionized water. Deionized water was aliquoted in 6 samples of 2 mL each, and partial oxygen pressure (Po2) and partial
carbon dioxide pressure (Pco2) were determined (experiment 1).
The aliquoted samples were blown through for 5 minutes by 6
adult volunteers (inspiration by nose and maximal expiration by
mouth), then gas analysis was repeated.
Air and exhaled breath. Po2 and Pco2 were determined in
6 air samples and 6 exhaled breath samples (experiment 2) from
healthy adult volunteers (deep inhalation and maximal exhalation).
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and those of Pco2 lower in water after exhaled
air bubbling. The values of Po2 and Pco2 were
significantly higher in EBC samples protected
by an argon layer.
Conclusion: Ambient/exhaled gases influence
gas analysis in water and EBC, respectively.
Additional validation of the method is needed.
Exhaled breath condensate. Exhaled breath condensate
samples were obtained from 14 clinically healthy children aged 7
to 14 years (experiment 3). The study protocol was approved by
the Hospital Ethics Committee. Exhaled breath condensate was
collected according to the American Thoracic Society/European
Respiratory Society Task Force recommendations6 using an EcoScreen condenser (Erich Jaeger GmbH, Hoechberg, Germany).
The subjects were instructed to breathe tidally for 15 minutes
wearing a nose clip. An average of 1.8 (range 0.5 to 3.4) mL EBC
per child was collected. None of the EBC samples showed detectable α-amylase catalytic activity (detection limit 7 U/L). Exhaled
breath condensates were protected from ambient air by an argon
layer (Figure 1), 6 Lmin-1 for 2 minutes, and gases were analyzed
A
B
Figure 1_Sample argonization by protection using an argon layer (A)
and gas standardization using argon bubbling (B).
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Science
(sample A). Then, the samples were argon blown through (350
mL/min-1 for 10 minutes; CO2 elimination [ie, gas standardization]), and gas analysis was repeated (sample B). All EBC samples
were plugged immediately after protection by an argon layer.
Repeatability and internal reproducibility. Seven EBCs protected by argon layer were used for repeatability (within-day precision) and internal reproducibility (day-to-day precision) testing of
Po2 and Pco2 determination (experiment 4). The measurements
were performed 3 and 5 minutes after EBC collection. Exhaled
breath condensate samples were frozen at –20°C as soon as possible after the second measurement for analysis 24 hours later.
Results
Po2 of deionized water dropped and Pco2 rose after exhaled
air had been blown through the water for 5 minutes (experiment
1). The mean Po2 value in deionized water (Figure 2A) was 22.49
kPa, and decreased to 19.51 kPa upon expiration blow-through.
The respective values of Pco2 (Figure 2B) were 0.51 kPa and
1.26 kPa. The results of experiment 2 showed significant differences in Po2 and Pco2 between ambient air and exhaled air. The
mean values of 20.78 kPa O2 and 0.61 kPa CO2 were recorded in
atmosphere air and of 15.77 kPa O2 and 4.15 kPa CO2 in adult
volunteer exhaled air (Table 1). Taking pooled results of the 2
experiments, there was no significant difference in Po2 and Pco2
A
between ambient air (Table 1) and deionized water (Figure 2),
and the exhaled air gas values (Table 1) were similar to those recorded in expiration-blown water for 5 minutes (Figure 2).
The values of Po2 and Pco2 in EBC (experiment 3) showed
significant differences according to the method of sample argonization (Table 2). Both Po2 and Pco2 were statistically significantly higher in samples A (argon protected) than in samples B
(argon blown, standardized). Pco2 and Po2 values measured in
argon-protected EBC samples (Table 2, sample A) were comparable (P>0.05) with Pco2 and Po2 values found in expiration-blown
water (Figure 2). Pco2 values in gas-standardized EBC samples
(Table 2, sample B) were comparable (P<0.05) with atmosphere
(Table 1) and deionized water (Figure 2B) Pco2 values.
Experiment 4 demonstrated significant changes in both Po2
and Pco2 with time (Figure 3). While the Po2 remained stable
during 5 minutes (Figure 3A), the values of Pco2 decreased significantly (Figure 3B). Significant changes of Po2 and Pco2 values
of frozen/thawed EBC samples were observed.
Discussion
Results of our study indicate that the ambient/exhaled gases
influence the gas analysis in water and EBC, respectively.
In the ambient air, Po2 equaled 21 kPa and Pco2 was approximately 0; in the alveoles, Po2 was approximately 14.7 kPa,
whereas Pco2 was about 5.3 kPa.7 In the exhaled air, Po2 equaled
15.9 kPa and Pco2 equaled 4.2 kPa,6 which is consistent with our
findings in both expiration-blown water and exhaled air, respectively. Rozycki and colleagues obtained end-tidal Pco2 values of
5.24 ± 1.57 kPa in neonatal intensive care unit patients.8
The methods of argonization used in the study differ according to the objective to be achieved. Using the procedure of
protection of EBC by argon layer, stability of the analytes influenced by atmosphere might be ensured.9 The present results
indicated the Po2 and Pco2 values in argon-protected EBC
were consistent with the respective values in the expirationblown water. During gas standardization, gases were removed
from the sample and balance in Po2 and Pco2 between the air
and EBC was achieved. Exhaled breath condensate Pco2 leveled
with atmosphere values during gas standardization.
The results on Pco2 and Po2 might suggest that the EBC
sample obtained represented a mixture of expired and inspired
gases, as expected, because EBC sampling was performed during
B
Table 1_Po2 and Pco2 Values in Air and Exhaled Breath
of 6 Adult Volunteers
Figure 2_Values of Po2 (A) and Pco2 (B) in 6 water samples before
and after exhaled air bubbling. Six healthy subjects were included
and each of them was asked to breathe into a water sample.
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LABMEDICINE j Volume 39 Number 9 j September 2008
Po2 (kPa)
Pco2 (kPa)
Sample
Air
Exhaled Air
Air
Exhaled Air
1
2
3
4
5
6
± SD
20.79
20.70
20.82
20.75
20.85
20.74
20.78 ± 0.02
14.87
17.74
16.16
17.09
15.03
13.75
15.77 ± 0.61
0.52
0.58
0.67
0.66
0.59
0.61
0.61 ± 0.06
4.24
2.82
4.62
3.58
4.51
5.10
4.15 ± 0.82
Po2 and Pco2 were determined on an Ecosys II analyzer (Eschweiler GmbH & Co. KG, Kiel,
Germany). The Ecosys II analyzer is a fully automated analytical system based on sensors
for direct measurement of pH, Po2, and Pco2 in the blood/atmosphere/exhaled air.
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Science
A
Table 2_Po2 and Pco2 Values in Exhaled Breath
Condensate of Healthy Children*
Healthy children (n=14)
Sample A
Sample B
Po2
(kPa)
Pco2
(kPa)
16.64
14.60
19.53
4.93
1.25
0.94
1.73
0.79
13.08
11.47
14.30
2.83
0.54
0.46
0.60
0.14
Median**
Q1
Q3
IQR
Median***
Q1
Q3
IQR
* Median, quartile, and interquartile ranges are presented based on nonparametric distribution of data. Sample A, argon-protected EBC; sample B, argon-blown EBC.
** P=0.0001
*** P=0.0008
Q1: first quartile; Q3: third quartile; IQR: interquartile range.
calm breathing when the respiratory volume was small and the dead
space relatively large.10 In this way, the inspired air from the dead
space was mixed with the expired air from the alveoli and bronchi.
The present study revealed a preanalytical factor: the effect of
ambient partial pressures posed a major problem in Po2 and Pco2
determination in EBC. When Po2 and Pco2 can be determined
immediately upon sampling, we consider that EBC protected by
an argon layer should be performed in order to prevent the effect
of ambient air. Further investigations are needed to assess the clinical efficacy of the findings thus obtained. LM
Acknowledgments: The results presented were obtained in
the scope of a scientific project No. 277-2770966-0965, entitled
“Exhaled breath condensate as a source of lung disease biomarkers
in children,” carried out with support from the Ministry of Science, Education, and Sports of the Republic of Croatia. LM
B
Figure 3_Repeatability and internal reproducibility testing of Po2
(A) and Pco2 (B) determination for 7 different argon-protected EBC
samples.
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