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 Respiratory 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 correlation was found between EBC and BAL for any of the biomarkers evaluated either before or after correction for dilution. Conclusions: In clinical disease, markers of inflammation and oxidative 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 include expectorated and induced sputum analysis and bronchoscopy 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 inflammation 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:
[email protected] 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 evaluated 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 bronchoalveolar lavage to biomarkers in exhaled breath condensate in a clinical setting for any biomarker. These findings demonstrate that exhaled breath condensate sampling cannot be directly compared with information derived from bronchoalveolar lavage.
surement of nitric oxide in exhaled air (eNO) has been extensively investigated and is now accepted as reflecting airway inflammation (2–5). Another noninvasive method involves collection 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. Information regarding EBC is rapidly emerging, and the American Thoracic Society (ATS) and European Respiratory Society (ERS) have recently collaborated on publishing recommendations, 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 compartment 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 instrumentation (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
undergoing bronchoscopy for clinical indications. We hypothesized that EBC biomarkers would be measurable in clinical disease, 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 EBC originates from a different compartment to BAL and would help elucidate the sources of likely variability for individual markers. We studied markers 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 compartments, 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 Human Resource 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 using 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 obtained from the records. Subjects underwent eNO measurements followed by EBC collection and then BAL. eNO measurements were performed online by means of chemiluminescence using a rapid-response analyzer (LR 2500 [I]; Logan Research, Rochester, UK) according to ERS and ATS guidelines. EBC was collected using a refrigerating exhaled breath circuit (EcoScreen version 1.1; Jaeger, Wu¨rzburg, Germany) with patients breathing at tidal volume for 10 min. Samples were immediately stored at –70⬚C for subsequent analysis. BAL was performed according to ERS guidelines (9). The bronchoscope 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 –70⬚C for subsequent analysis.
Analysis of EBC and BAL Fluid Reagents were purchased from Sigma-Aldrich (Sydney, Australia) unless otherwise indicated. Hydrogen peroxide (H2O2) was measured spectophotometrically by horseradish peroxidise–catalyzed oxidation of tetramethylbenzidine (TMB) (12, 13). Briefly, EBC and BAL fluid was mixed with 100 l of 3⬘3’5⬘5⬘-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 spectophotometrically 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 37⬚C. 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.
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Keratin was measured using an ELISA. Standards (human epidermis, 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 onestep substrate system (Amersham-US22128), the reaction stopped by acidification, and the resultant change in absorbance read spectrophotometrically at 450 nm (16). Total PL was measured as previously described (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. Measurement 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 MannWhitney 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 transformation 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 undergoing surveillance bronchoscopy. Clinical characteristics are summarized 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
No. of patients Age, yr Transplant/nontransplant, n Smokers/ex-smokers/never-smokers, n FEV1, % predicted FVC, % predicted eNO, ppb
Male
Female
Total
32 (65%) 53.6 ⫾ 2.8 17/15 4/20/8 66.7 ⫾ 4.8 77.6 ⫾ 4.3 8.4 ⫾ 0.8
17 (35%) 44.8 ⫾ 4.3 9/8 1/7/9 68.3 ⫾ 4.8 75.7 ⫾ 5.4 9.1 ⫾ 1.1
49 50.6 ⫾ 2.4 26/23 5/27/17 67.3 ⫾ 3.5 77.0 ⫾ 3.4 8.7 ⫾ 0.6
Definition of abbreviation: eNO ⫽ exhaled nitric oxide. Values unless otherwise stated are mean ⫾ SEM.
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the collection process. NOx values were 80.2, 43.4, and 19.4 M, respectively, confirming the influence of the apparatus chamber on NOx levels. We measured total NOx and therefore have no data on levels of different nitrogen oxides in either BAL or EBC. When eNO was correlated with NOx levels, no significant correlation was found for either BAL or EBC (Table 2). pH was higher (7.35 ⫾ 0.04 vs. 6.40 ⫾ 0.05, p ⬍ 0.0001) in EBC than BAL. The pH of the 0.9% saline solution used for BAL was 6.2 prior to instillation. In contrast, protein was significantly higher in BAL than in EBC (33.8 ⫾ 4.8 g/ml vs. 183.2 ⫾ 22.6 g/ml, respectively; p ⬍ 0.0001). Hydrogen peroxide was also higher in BAL than EBC, but the difference was not statistically significant (EBC, 17.5 ⫾ 3.2 M, vs. BAL, 20.6 ⫾ 5.3 M). When analyzed separately, these differences remained significant for both transplant and nontransplant patients. Despite having previously assessed amylase in EBC samples, we also performed a post hoc assay of 19 further subjects using a sensitive amylase assay. This showed measurable amylase contamination in only one subject, indicating that this is unlikely to have been a major confounder in our study. Mean eNO levels were 8.7 ⫾ 0.6 ppb. There was no significant difference in eNO found between males and females, transplant and nontransplant patients, or those who were immunosuppressed. All transplant patients, however, were using oral glucocorticosteroids. A significant difference in eNO was found between those who had ever smoked and lifelong nonsmokers (10.38 ⫾ 1.1 ppb vs. 6.8 ⫾ 0.9 ppb, respectively; p ⫽ 0.018). Mean concentration of 8-isoprostane was higher in transplant patients than in nontransplant patients, whereas NOx were higher in nontransplant subjects. Total PL was measured in 33 subjects. Because PL data did not conform to the normal distribution, they were log transformed for analysis. Mean (SD) PL in EBC was significantly higher than in BAL (geometric mean, 2.47 [0.04] vs. 1.32 [0.21]; p ⬍ 0.001). This difference persisted after correction for protein (EBC log PL/mg protein, 0.19 [0.33], vs. BAL log PL/mg protein, 0.01 [0.00]; p ⬍ 0.001). Keratin was measured in 15 of the same subjects. Mean keratin concentrations were higher in BAL than EBC (428.6 [273] vs. 22.8 [27] ng/ml) but the difference was not statistically significant. Similarly, there was no statistically significant difference between EBC and BAL keratin when corrected for protein. No correlation was demonstrated between the EBC and BAL for any of the substances measured (Table 3), nor when the data were presented as a Bland-Altman plot (not shown). This was
also true when transplant and nontransplant subjects were analyzed separately, whether or not the results were corrected for protein concentration or with a crude adjustment for the lavage volume of saline.
DISCUSSION Collection of EBC is a promising new noninvasive technique for investigating pathologic mechanisms in lung disease. The role of EBC in diagnosis and management of patients is likely to become important in the future. Here, we report the first study that directly compares EBC and BAL in vivo. Our study reports three new findings. First, biomarkers were easily measurable in EBC using commercially available assays, suggesting that EBC sampling may be more useful than predicted in a clinical setting. Second, there was no correlation between identical biomarkers in EBC and BAL. Third, we measured some novel biomarkers to try to clarify the source of origin of EBC. Information about EBC is rapidly evolving. In EBC, the concentration of substances present in the respiratory droplet fraction depends on the characteristics of the individual molecules (water solubility, hydrophilicity, volatility, and electrical charge) as well as their concentration in the lung. The latter may be determined by transportation mechanisms as well as by any underlying disease. Dilutional factors may differ among markers and the physical characteristics of the collection device will have different effects on individual biomarkers. The relative contribution from different parts of the respiratory tract to each biomarker is unknown. EBC samples a larger portion of the respiratory tract than BAL, including the alveolar lining fluid and fluid derived from the larger airways (8). EBC is collected via the mouth, and the final sample may be affected by oral components (e.g., saliva and oral organisms), although the extent to which this occurs is debated. We measured a variety of biomarkers in EBC and BAL, some for the first time, but we found no correlation between any biomarkers either before or after correction for dilution. For most biomarkers, concentrations were higher in EBC than in BAL. We aimed to minimize potential confounding factors, collecting samples after overnight fasting, with EBC sampled immediately prior to BAL. A saliva trap was used and EBC collection performed according to current recommendations (8). Clinical factors (e.g., drug intake, disease) were minimized by direct comparison of samples from the same patient at the same time, and we performed BAL according to published ERS
TABLE 2. CONCENTRATIONS OF BIOMARKERS IN EXHALED BREATH CONDENSATE AND BRONCHOALVEOLAR LAVAGE (UNCORRECTED FOR PROTEIN) All Patients (n ⫽ 49) EBC 8-isoprostane, ng/ml BAL 8-isoprostane, ng/ml EBC hydrogen peroxide, M BAL hydrogen peroxide, M EBC NOx, M BAL NOx, M EBC pH BAL pH EBC protein, g/ml BAL protein, g/ml Exhaled nitric oxide, ppb
3.845 0.027 17.5 20.6 28.4 3.8 7.35 6.4 33.8 183.2 8.7
⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾
1.268* 0.012 3.2 5.3 2.6* 0.7 0.04* 0.05 4.8* 22.6 0.6
Transplant Patients (n ⫽ 26) 6.358 0.037 18.9 14.8 21.1 4.3 7.5 6.4 40.3 144.1 8.4
⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾
2.327* 0.021.4 5.0* 4.4 3.0* 1.1 0.1* 0.1 7.1* 17.2 0.8
Definition of abbreviations: BAL ⫽ bronchoalveolar lavage; EBC ⫽ exhaled breath condensate. All values are mean ⫾ SEM. * Significant difference between the EBC and BAL concentrations, p ⬍ 0.0001. † Significant difference between the EBC and BAL concentrations, p ⬍ 0.003.
Nontransplant Patients (n ⫽ 23) 1.112.6 0.015 15.9 27.1 30.0 3.2 7.2 6.4 26.2 225.9 9.0
⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾
0.311* 0.03. 4.0† 10.1 4.5* 0.7 0.1* 0.1 5.8* 42.2 1.1
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TABLE 3. CORRELATIONS BETWEEN INDIVIDUAL BIOMARKERS IN EXHALED BREATH CONDENSATE AND BRONCHOALVEOLAR LAVAGE AND BETWEEN EXHALED NITRIC OXIDE AND NITROGEN OXIDES All Patients
EBC log 8-isoprostane/BAL log 8-isoprostane EBC log H2O2/BAL log H2O2 EBC log NOx/BAL log NOx EBC pH/BAL pH EBC NOx/eNO BAL NOx/eNO
Correlation Coefficient (r ) Uncorrected for Protein (n ⫽ 49)
p Value
Correlation Coefficient (r ) Corrected for Protein (n ⫽ 49)
p Value
0.08 0.06 0.02 0 0.05 0.06
0.65 0.73 0.92 0.99 0.75 0.7
0.17 0.17 0.26 N/A N/A N/A
0.33 0.33 0.12 N/A N/A N/A
Definition of abbreviation: N/A ⫽ not applicable.
guidelines (9). Contamination with blood can occur during BAL even with minimal trauma, and could affect biomarker levels as well as proteins. To minimize this, all BAL samples were taken prior to invasive procedures and samples were excluded from the study if they were visibly contaminated with blood. Thus, although we cannot exclude differences in blood contamination between samples as a cause of differences between EBC and BAL samples, we do not believe they account for our results. We sampled a range of biomarkers and keratin and PL, using previously reported methods and commercially available assays. We found that almost all biomarkers were present in higher concentrations in EBC than in BAL, whereas protein was higher in BAL. The only biomarker with similar levels in both BAL and EBC was hydrogen peroxide. 8-Isoprostane and NOx were both significantly higher in EBC than BAL. Because EBC is purported to consist primarily of water vapor, with the fraction derived from respiratory droplets comprising a small proportion of the total (8), it has been suggested that biomarkers may be present in such low concentrations in EBC as to be difficult to measure using standard techniques (6, 19). Our study does not support this. We found diverse biomarkers easily measurable in clinical disease in EBC and BAL. We found levels of biomarkers to be comparable or higher than previously reported, and even higher in our lung transplant patients. We believe this reflects the severity of disease in our patients, who underwent bronchoscopy for clinical indications rather than for research purposes. Although we did find differences between transplant patients and other subjects for some markers, this was not the aim of the study, nor was it powered to detect such differences. We would therefore be reluctant to interpret these further, given the number of potential confounding factors. It is possible that ratios or combinations of biomarkers will be found to be useful in future work rather than raw concentrations. To date, only a limited number of markers have been assessed in EBC, and information is limited regarding EBC biomarkers levels in different diseases. Some studies have shown poor reproducibility of biomarkers in EBC, although BAL suffers from the same disadvantage. However, because we merely compared BAL and EBC using identical methods, any sampling error should apply equally to both techniques. EBC biomarker levels are also affected by the collection device used (20, 21), and this may well have affected our results. However, we used the EcoScreen commercial device for sampling EBC, which collects EBC at ⫺20⬚C, which is believed to be beneficial for unstable markers, and a methodology recommended by the ATS/ERS task force (8). Another possible difference between the collection techniques, which might interfere with the assays, would be the presence of a “matrix” effect, but in our study, undiluted samples gave results that were within the dynamic range for the assays. We did not deaerate samples prior to pH because we
measured this immediately and the published literature was divided at the time regarding the necessity of this procedure. Also, there is very little information regarding the effect of deaeration on BAL pH and other biomarkers. From our own measurements and subsequent published information, it has now become clear that samples are best deaerated prior to measuring pH in EBC (22), and this also applies to BAL. Our amylase data suggest that oral contamination is unlikely to have been a significant issue in our study, although this is a relatively crude tool. It would be interesting in the future to perform proteomic analysis on EBC proteins to determine whether these are primarily of oral or lower respiratory tract origin. Our patients had diverse diseases and were treated with a variety of medications, including corticosteroids. Thus, discussion regarding absolute levels of biomarkers in our study is speculative. Nonetheless, our data on some biomarkers are compatible with several previous reports (7, 14, 23, 24). Widely variable levels of hydrogen peroxide have been reported in EBC, possibly because H2O2 is unstable in EBC and/or because the colorimetric assay has poor reproducibility (8, 25). One recent study has shown variable levels of H2O2 correlated with flow rate in EBC, suggesting that it is at least partially produced in conducting airways, despite volatility, solubility, and reactivity remaining an issue (23). We did not confirm this in our study, where levels were, if anything, slightly higher in BAL. We found levels of NOx were higher in EBC than in BAL, but there are several possible reasons for this. NOx levels may be significantly affected by bacterial activity in the mouth (22, 24), and pH differences are also relevant. Differences in proton concentration (measured as pH) alter nitrogen oxide species (26, 27), and in a more acid environment, nitrous acid rapidly evolves to NO, which is lost as a gas (28). Thus, in our study, where the pH of the BAL 0.9% saline fluid was 6.2 at baseline, pH changes would have facilitated reaction of nitrous acid and lower NOx concentrations in BAL than in EBC. This probably accounts for most if not all of the differences in NOx. It would have been of interest to measure individual nitrogen oxides, but we did not have these assays available. Our results confirm that our EBC collection apparatus itself can cause contamination with NOx to confound results, but this may well also apply to BAL procedures. The EBC apparatus is best washed with deionized water and then dried with inert gas (8). We found no correlation between eNO and NOx levels in either EBC and BAL NOx levels, but this can partly be explained by contamination as above. Our data confirm that interpretation of NOx levels in EBC (and BAL) is complex and that EBC NOx are unlikely to be as clinically promising as, for example, eNO. In an attempt to localize the origin of our samples, we measured total PL and keratin in a subset of our subjects. Keratin is expressed in epithelial cells, whereas PL is found primarily in
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surfactant. Thus, we would have expected keratin to be higher in EBC, which samples the total airway from the oropharynx to the alveoli, whereas PL would be expected to be higher in BAL if it mainly samples the alveolar compartment. Contrary to our expectations, keratin concentrations were similar in EBC and BAL, whereas PL levels were significantly higher in EBC. This may suggest that EBC, in fact, samples the alveoli to a greater extent than expected. However, PLs have unusual polar natures with both hydrophilic and hydrophobic moities. Liposomes are spontaneously formed, which may act as a vehicle to allow passage of substances by a fluid mosaic model. These properties may result in concentration of PLs in EBC and may also alter their absorption or adherence to a collection apparatus or into another medium (e.g., mucus), and indeed their solubility in 0.9% saline, such as is used in BAL. It is of interest that 8-isoprostane, which shares some of the same characteristics, was also found in relatively high concentrations in our study. Keratins, in contrast, are relatively insoluble. One major difficulty in interpreting our results is the problem of how to account for differences in dilution between BAL and EBC. BAL primarily samples small airways and alveoli by direct instillation of relatively large volumes of saline and thus on first principles it would be expected to be more diluted than EBC. Despite suggestions that EBC consists primarily of water vapor, and that the fraction derived from respiratory droplets is very small (29), there are few actual data on this area and reports are largely based on theoretical considerations rather than actual measurements. There is much debate as to whether conductivity, electrolytes, or urea may be useful dilutional markers (30), but as yet there is no consensus on this issue. In BAL, several denominators have not been found to be ideal (31). We used protein as an internal dilutional marker because it is practical to assay and has been used in previous studies. Protein may, however, not be an ideal marker because the permeability of protein across the alveolar membrane may vary in different lung diseases. Our choice of dilutional marker is debatable, but there is no current recommended one. In the future, it is possible that the ratio between different substances may prove useful, as has been suggested with BAL (9). EBC has an advantage over BAL in that no external factors are introduced. Thus, dilutional factors should be easier to assess than with BAL, once the physics and normal physiology are better understood. In conclusion, our study has confirmed that EBC biomarkers can be readily measured in patients with clinically active lung disease. Several new markers can be assayed using standard laboratory methods and levels of many biomarkers may in fact be higher in EBC than in BAL. EBC sampling cannot be compared directly with information derived from BAL, and both techniques have significant potential limitations. Although it is tempting to suggest that the discrepancy in our measurements between EBC and BAL is attributable to their sampling different compartments of the lung, so many other factors (volatility, solubility, electric charge, collection technique) are likely to be involved that EBC biomarkers should probably be conceptualized totally differently to BAL, and separately for each individual marker. EBC has an advantage over BAL in that it does not require instrumentation, induce any inflammatory change, or introduce any foreign substances into the lung; furthermore, it can be performed repeatedly in sick patients. Consequently, it is likely that once more is understood about EBC, it will become increasingly useful in a clinical setting. Conflict of Interest Statement : A.S.J. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. A.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. C.C. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. S.C.
does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. P.S.T. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. D.H.Y. has participated as a speaker in scientific meetings organized and financed by various pharmaceutical companies (GlaxoSmithSline, AstraZeneca) but has not participated in any industry-sponsored trials for 6 years and receives no pharmaceutical company funding. She is a chief investigator on a peer-reviewed grant sponsored by Wyeth ($25,000) and awarded in 2004. Acknowledgment : The authors thank the subjects who kindly participated in our study as well as the staff of the Thoracic and Lung Transplant Unit at St. Vincent’s Hospital. They are also grateful for the helpful comments of Benjamin Gaston.
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