Comparison of Biomarkers in Exhaled Breath
Condensate and Bronchoalveolar Lavage
Abigail S. Jackson, Alessandra Sandrini, Charlotte Campbell, Sharron Chow, Paul S. Thomas, and
Deborah H. Yates
Department of Thoracic Medicine, St. Vincent’s Hospital, Darlinghurst; and University of New South Wales, Randwick, Sydney, Australia
Rationale: Exhaled breath condensate (EBC) is increasingly studied
as a noninvasive research method of sampling the lungs, measuring
several biomarkers. The exact site of origin of substances measured
in EBC is unknown, as is the clinical applicability of the technique.
Special techniques might be needed to measure EBC biomarkers.
Objectives: To assess biomarker concentrations in clinical disease
and investigate the site of origin of EBC, we compared EBC and
bronchoalveolar lavage (BAL) biomarkers in 49 patients undergoing
bronchoscopy for clinical indications.
Measurements: We measured exhaled nitric oxide, 8-isoprostane,
hydrogen peroxide, total nitrogen oxides, pH, total protein, and
phospholipid (n  33) and keratin (n  15) to assess alveolar and
mucinous compartments, respectively. EBC was collected over 10
min using a refrigerated condenser according to European Respira-
tory Society/American Thoracic Society recommendations, and BAL
performed immediately thereafter.
Results: 8-Isoprostane, nitrogen oxides, and pH were significantly
higher in EBC than in BAL (3.845 vs. 0.027 ng/ml, 28.4 vs. 3.8 M,
and 7.35 vs. 6.4, respectively; p  0.001). Hydrogen peroxide
showed no difference between EBC and BAL (17.5 vs. 20.6 M,
p  not significant), whereas protein was significantly higher in
BAL (33.8 vs. 183.2 g/ml, p  0.001). Total phospholipid was also
higher in EBC, but keratin showed no difference. No significant
correlationwas found between EBC and BAL for any of the biomark-
ers evaluated either before or after correction for dilution.
Conclusions: In clinical disease, markers of inflammation and oxida-
tive stress are easily measurable in EBC using standard laboratory
techniques and EBC is readily obtained. However, EBC and BAL
markers do not correlate.
Keywords: exhaled breath condensate; bronchoalveolar lavage;
biomarkers; oxidative stress; inflammation
Current methods of assessing and monitoring lung disease do
not directly reflect underlying inflammatory processes. Methods
directly sampling material from the lower respiratory tract in-
clude expectorated and induced sputum analysis and bronchos-
copy with bronchoalveolar lavage (BAL) (1). Of these, BAL is
regarded as the most reliable method for sampling the lining
fluid of the lower respiratory tract. However, it is an invasive
technique requiring sedation and its use in monitoring inflam-
mation is limited in clinical practice.
More recently, novel noninvasive techniques to assess lung
inflammation and oxidative stress have been developed. Mea-
(Received in original form January 24, 2006; accepted in final form November 10, 2006 )
Supported by the Lesley Pockley Clinical Research Trust.
Correspondence and requests for reprints should be addressed to Dr. Deborah
H. Yates, Department of Thoracic Medicine, St. Vincent’s Hospital, Victoria Street,
Darlinghurst, Sydney 2010, Australia. E-mail: deborahy88@hotmail.com
This article has an online supplement, which is accessible from this issue’s table
of contents at www.atsjournals.org
Am J Respir Crit Care Med Vol 175. pp 222–227, 2007
Originally Published in Press as DOI: 10.1164/rccm.200601-107OC on November 16, 2006
Internet address: www.atsjournals.org
AT A GLANCE COMMENTARY
Scientific Knowledge on the Subject
Biomarkers in exhaled breath condensate have been evalu-
ated in several lung diseases in a research setting, but the
clinical applicability of this new tool is unknown.
What This Study Adds to the Field
No correlation was found comparing biomarkers in bron-
choalveolar lavage to biomarkers in exhaled breath conden-
sate in a clinical setting for any biomarker. These findings
demonstrate that exhaled breath condensate sampling can-
not be directly compared with information derived from
bronchoalveolar lavage.
surement of nitric oxide in exhaled air (eNO) has been exten-
sively investigated and is now accepted as reflecting airway in-
flammation (2–5). Another noninvasive method involves collec-
tion of exhaled breath condensate (EBC), with subsequent
analysis of several substances (6, 7). Inflammatory markers as
well as those of oxidative imbalance can be measured in EBC,
although to date only a limited number have been assessed. Infor-
mation regarding EBC is rapidly emerging, and the American
Thoracic Society (ATS) and European Respiratory Society
(ERS) have recently collaborated on publishing recommenda-
tions, which summarize current knowledge, including optimal
collection techniques (8).
The exact origin of EBC is uncertain. Although it is likely
to sample the whole respiratory tract from mouth to alveoli, the
contribution of each compartment to individual EBC markers
has not yet been determined. BAL is generally accepted as
sampling only the smaller airways and alveoli (9). EBC collection
is much less invasive than BAL and therefore has potential as
a substitute for BAL both in research studies and in clinical
practice. To date, there have been no published studies directly
comparing biomarkers in BAL and EBC. Direct comparison of
biomarkers using both techniques would clarify the potential of
EBC for clinical use and investigate the likely origin of EBC.
Current information suggests that levels of the various markers
in EBC will vary according to their individual characteristics
(e.g., solubility and volatility), as well as according to the com-
partment of the lung, and will also be significantly affected by
the equipment used for collection and several other physical
factors. BAL biomarkers may be influenced by dilution with
saline, pH and other changes induced by this, and by instrumen-
tation (which may cause bleeding), and in itself may induce
inflammation.
We aimed to investigate the above and therefore compared
several biomarkers in both EBC and BAL. We studied patients

Jackson, Sandrini, Campbell, et al.: Comparison of EBC and BAL Biomarkers 223
undergoing bronchoscopy for clinical indications. We hypothe-
sized that EBC biomarkers would be measurable in clinical dis-
ease, and that if EBC samples the lower respiratory tract as does
BAL, the concentrations of biomarkers in EBC and BAL would
be significantly correlated after correction for dilution, even if
correlation only occurred with one or two biomarkers. If the
results showed significant differences, this would suggest that
EBCoriginates from a different compartment to BALandwould
help elucidate the sources of likely variability for individual
markers.We studiedmarkers of lung inflammation and oxidative
stress (eNO, 8-isoprostane, hydrogen peroxide, total nitrogen
oxides [NOx], and pH) as well as measured keratin and total
phospholipid (PL) to assess mucinous and alveolar compart-
ments, respectively. We also measured protein concentration as
an estimate of sample dilution.
METHODS
Clinical Methods
Patients referred for bronchoscopy for any clinical indication were
invited to participate in the study, which was approved by St. Vincent’s
Hospital HumanResource Ethics Committee. All patients gave written,
informed consent. Sample size was calculated assuming a power of 0.8
(80%) and a 5% significance level, with standard deviations of different
biomarkers assessed from published literature where available and us-
ing data from our previous work (8, 10, 11). Lung transplant patients
undergoing surveillance bronchoscopies were included in the study but
not on more than one occasion.
All patients had a clinical history and physical examination prior
to bronchoscopy. Appropriate investigations relating to the clinical
presentation together with spirometric and radiologic data were ob-
tained from the records. Subjects underwent eNO measurements fol-
lowed by EBC collection and then BAL.
eNOmeasurements were performed online by means of chemilumi-
nescence using a rapid-response analyzer (LR 2500 [I]; Logan Research,
Rochester, UK) according to ERS and ATS guidelines. EBC was col-
lected using a refrigerating exhaled breath circuit (EcoScreen version
1.1; Jaeger, Wu¨rzburg, Germany) with patients breathing at tidal vol-
ume for 10 min. Samples were immediately stored at –70C for subse-
quent analysis.
BAL was performed according to ERS guidelines (9). The broncho-
scope was wedged in the right middle lobe or lingula and up to 240 ml
of normal saline was instilled in 60-ml aliquots. Between 25 to 100 mm
Hg of suction pressure was applied after each installation. Lavage fluid
was immediately centrifuged at 800 rpm for 10 min; supernatant was
then collected and stored at –70C for subsequent analysis.
Analysis of EBC and BAL Fluid
Reagents were purchased from Sigma-Aldrich (Sydney, Australia) un-
less otherwise indicated. Hydrogen peroxide (H2O2)wasmeasured spec-
tophotometrically by horseradish peroxidise–catalyzed oxidation of tet-
ramethylbenzidine (TMB) (12, 13). Briefly, EBC and BAL fluid was
mixed with 100 l of 33’55-tetramethylbenzidine in 0.42 M potassium
citrate buffer and 52.5 U/ml horseradish peroxidase and incubated for
20 min at room temperature. The reaction was stopped by adding 2 N
sulphuric acid, and the resultant change in absorbance measured specto-
photometrically at 450 nm. H2O2 concentrations were calculated from
serially diluted H2O2 solution. The lower limit of detection was 0.2 M.
8-Isoprostane was measured using a specific enzyme immunoassay
(EIA) kit (Cayman Chemical, Ann Arbor, MI), validated to obtain a
high correlation (0.95) with known amounts of 8-isoprostane and with
a lower detection limit of 5 pg/ml (14). Total NOx were measured after
enzymatic reduction of nitrate using a fluorimetric modification of
the Greiss reaction (15). Samples and standards were treated with
nitrate reductase and incubated for 1 h at 37C. 2,3-Diaminoaphthalene
was added and plates incubated for 10 min in the dark. The reaction was
stopped by adding 2.8 M NaOH. The fluorescent reaction product was
measured immediately in a fluorescent plate reader, excitation 360 nm,
emission 395 nm. Standard curves of nitrite were made in distilled
water. The lower limit of detection was 2 M.
Keratin was measured using an ELISA. Standards (human epider-
mis, Sigma- K0253; Sigma-Aldrich) and samples were applied to an
ELISA, using a primary rabbit polyclonal anti-human keratin antibody
(Biogenesis, Poole, UK) and incubated with a peroxidase-conjugated
donkey anti-rabbit immunoglobulin (Amersham-NA9340; Amersham,
Castle Hill, NSW, Australia). This was followed by the TMB one-
step substrate system (Amersham-US22128), the reaction stopped by
acidification, and the resultant change in absorbance read spectrophoto-
metrically at 450 nm (16). Total PL was measured as previously de-
scribed (17, 18). Briefly, standards (l-phosphatidylcholine) and samples
underwent chloroform extraction in the presence of 3.04% ammonium
thiocyanate and 2.7% ferric chloride hexahydrate. The lipid content of
the chloroform phase was measured by the absorbance at 488 nm.
Total protein concentration was measured using a Quantipro BCA
assay kit (Sigma-Aldrich). Equal amounts of EBC or BAL fluid were
added to a previously prepared working reagent, which was prepared
by mixing 25 parts of a solution of sodium carbonate, tartrate, and
bicarbonate in 0.2 M NaOH, with 25 parts of a 4% bicinchonic acid
solution. This was added to one part 4% copper sulfate pentahydrate
solution. Standard curves were constructed using bovine serum albumin.
The lower limit of detection was 4 g/ml.
pH was measured with a pH sensor probe (pH Boy-P2 ISFET
semiconductor electrode; Shindengen Electric Mfg. Co. Ltd., Fukuya,
Japan). The meter was calibrated daily with pH standards. Measure-
ment range was 2 to 12 ( 0.1 pH).
Statistical Analysis
Data were analyzed using SPSS for MS Windows, version 12.0 (SPSS,
Inc., Chicago, IL). For parametric data, the Student’s unpaired t test
was used to compare groups. For nonparametric variables, the Mann-
Whitney U test was performed (10). Data are expressed as mean 
SEM.
Correlation of inflammatory markers between EBC and BAL
was performed using Pearson’s correlation coefficient test after the
individual concentrations were corrected for protein concentration, and
transformed to the normal distribution using a logarithmic transforma-
tion where required. p values of less than 0.05 were considered
significant.
RESULTS
Forty-nine patients were included in the trial. Twenty-six (53%)
of these were transplant patients, of whom 10 (38%)were under-
going surveillance bronchoscopy. Clinical characteristics are sum-
marized in Table 1.
Significantly higher concentrations in EBC than in BAL
were found for 8-isoprostane (3.845  1.268 ng/ml vs. 0.027 
0.012 ng/ml, respectively; p  0.0001) and NOx (28.4  2.6 M
vs. 3.8  0.7 M, respectively; p  0.0001). At the suggestion
of one of the reviewers of this article, a post hoc experiment was
performed to determine whether the EBC collection apparatus
influenced NOx results. At baseline, normal saline, purified
deionized water and tap water showed no detectable NOx.
Saline and both types of water were then allowed to dwell in
the EcoScreen apparatus collection chamber for 10 min as per
TABLE 1. PATIENT CHARACTERISTICS
Male Female Total
No. of patients 32 (65%) 17 (35%) 49
Age, yr 53.6  2.8 44.8  4.3 50.6  2.4
Transplant/nontransplant, n 17/15 9/8 26/23
Smokers/ex-smokers/never-smokers, n 4/20/8 1/7/9 5/27/17
FEV1, % predicted 66.7  4.8 68.3  4.8 67.3  3.5
FVC, % predicted 77.6  4.3 75.7  5.4 77.0  3.4
eNO, ppb 8.4  0.8 9.1  1.1 8.7  0.6
Definition of abbreviation: eNO  exhaled nitric oxide.
Values unless otherwise stated are mean  SEM.