Collection and Analysis of Exhaled Breath Condensate in Humans

GÖKHAN M. MUTLU, KEVIN W. GAREY, RICHARD A. ROBBINS, LARRY H. DANZIGER, and ISRAEL RUBINSTEIN

Pulmonary and Critical Care Medicine, Evanston-Northwestern Healthcare and Northwestern University Medical School, Evanston, Illinois; Respiratory and Critical Care Medicine, College of Medicine, Department of Pharmacy Practice, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois; VA Health Care System, West Side Division, Chicago, Illinois; and Research Service, Southern Arizona VA Health Care System, Tucson, Arizona

	INTRODUCTION

TOP INTRODUCTION COLLECTION OF EXHALED BREATH... COMPOUNDS PRESENT IN EXHALED... ADVANTAGES AND LIMITATIONS FUTURE DIRECTIONS CONCLUSIONS REFERENCES

Traditional methods of sampling secretions from the lower respiratory tract include sputum collection, sputum induction, and bronchoscopy with bronchoalveolar lavage (BAL). By far, bronchoscopy with BAL has become the preferred method for sampling the lining fluid of the lower respiratory tract. The invasive nature of bronchoscopy has led to a constant search for less intrusive methods that are easier to implement in ambulant individuals, particularly children. Recently there has been an increasing interest in using exhaled breath as a simple, noninvasive means to sample the lower respiratory tract in humans.

The lining fluid of the lower respiratory tract contains various nonvolatile and over 200 volatile substances (1, 2). Although initial attempts were aimed at identifying volatile substances, particularly nitric oxide (NO), studies are now being conducted to detect nonvolatile macromolecules present in exhaled breath, including proteins, lipids, oxidants, and nucleotides. Analysis of these nonvolatile substances requires cooling of the expired breath, which results in condensation. These macromolecules represent biomarkers of various pathological processes in the lungs.

In this review, we present the current body of knowledge on exhaled breath condensate as published in peer-reviewed journals in the English language. We focus on the use of this modality in determining host inflammatory response to injury in the lung as well as possible future applications, particularly its potential use as a single, noninvasive sampling method for point-of-care real-time analysis.

Keywords: lung; inflammation; cytokines; leukotrienes; hydrogen peroxide

	COLLECTION OF EXHALED BREATH CONDENSATE

TOP INTRODUCTION COLLECTION OF EXHALED BREATH... COMPOUNDS PRESENT IN EXHALED... ADVANTAGES AND LIMITATIONS FUTURE DIRECTIONS CONCLUSIONS REFERENCES

Exhaled breath is saturated with water vapor that can be condensed with cooling. Although most of the condensate consists of water vapor, it also contains aerosol particles from the lower respiratory tract. Although limited, available evidence suggests that nonvolatile substances in the lower respiratory tract can be transported in the form of aerosols in exhaled breath (1). When exhaled breath is collected in a cool-trap at 196° C and freeze-dried, aerosols precipitate and become visible as white particles (1). Comparison of the protein patterns between frozen aerosol particles and saliva samples demonstrates the presence of extra proteins in aerosol particles (not in saliva) suggesting that nonvolatile substances such as proteins from the lower respiratory tract can be transported in the form of aerosols in exhaled air (1). Finding the primary origin (airways versus alveoli) of aerosol particles that carry these proteins presents an important challenge and is yet to be determined. A recent study suggested that the large airways are responsible for most of the exhaled hydrogen peroxide (H₂O₂) based on the flow dependency of H₂O₂ concentration (3). Studies using surfactant-related proteins or specific markers produced by alveolar cells (e.g., placental alkaline phosphatase) or bronchioles are warranted to confirm the cellular source of aerosols and macromolecules (1).

During normal tidal breathing, levels of aerosol particles range between 0.1 and 4 particles/cm³ (4). The mean diameter of particles is less than 0.3 µm (4). The number of aerosols formed in the respiratory tract depends on the current velocity of air and surface tension of the extracellular lining fluid. Higher velocity and lower surface tension favor the production of aerosols. The balance between the low velocity and high surface tension found at the level of alveoli determines the generation of aerosols. In addition, turbulent flow and higher number of edges in the branching points may facilitate aerosol formation.

Breathing air through a cooling system results in condensation, thereby rendering collection of exhaled breath in a liquid form possible. Condensation of exhaled breath can be achieved simply by cooling the tubing through which the patient is exhaling (Figure 1). Successful collection has been reported with a variety of devices with different designs. Most widely used designs include immersion of a Teflon-lined tubing in an ice-filled bucket and a specially designed double-wall glass condenser system. A commercially available condenser (Erich JAEGER GmbH, Hoechberg, Germany) is also available. However, its utility and reliability should first be tested in controlled clinical trials before widespread use is recommended.

View larger version (31K): [in this window] [in a new window]   Figure 1.   Schematic representation of a collection apparatus.

The condensing chambers used to date include glass, polystyrene, or polypropylene containers. The composition of the exhaled breath condensate may be altered by the adhesive properties of the material from which the container is made. When analysis of bioactive lipids is planned, polypropylene containers should be considered for collection to avoid the problem of adsorption that can occur with polystyrene containers. This approach also allows direct organic extraction without dissolution of the collection vehicle.

Cooling of exhaled air can be readily achieved using wet ice as well as with dry ice or liquid nitrogen. Complete obstruction of the collection tube can occur probably due to very low temperatures when liquid nitrogen or dry ice is used to cool the exhaled breath. This complication is a major concern, particularly during collection of exhaled breath from patients receiving mechanical ventilation, because it may lead to significant consequences (i.e., dynamic hyperinflation) if overlooked. Use of wet ice or longer collection tubing may minimize this complication. Nonetheless, dry ice or liquid nitrogen is advised for analysis of substances unstable at 5 to +5° C (e.g., leukotrienes).

The patient is instructed to breathe tidally directly into the tubing or through a mouthpiece connected to the tubing. No nose clips are recommended because their application opens the nasopharyngeal velum, thereby contaminating the exhaled breath with nasal air. Tidal breathing has been used in all but one study that evaluated spontaneously breathing patients. In contrast to others, Ho and coworkers instructed the subjects to inspire to total lung capacity before exhalation (5). The collection system can also be connected to mechanically ventilated patients through the expiratory limb of the ventilatory tubing.

It usually takes 5-10 min in adults and up to 15-20 min in children to obtain 1-3 ml of condensate. The amount of condensate generated per exhalation varies among individuals. Minute ventilation remains the major determinant of the amount of condensate over time, and it does not have any effects on levels of measured substances (6). Although it cannot be generalized to all biomarkers, H₂O₂ concentration in exhaled breath appears to be expiratory flow dependent (3). Humidity of inspired air may be an important determinant of the rate at which exhaled breath condensate is formed. Bronchial circulation is essential in humidification of inspired air (7). Having two intercommunicating venous plexuses, one immediately below the bronchial epithelium and the other deep in the peribronchial connective tissue, the bronchial tree is an ideal system for warming and humidification of air. In animals, bronchial blood flow changes with the humidity and temperature of inspired air (8). Similarly, breathing dry air has been shown to increase bronchial blood flow in humans (9, 10).

As most substances (e.g., nitrite) measured are present not only in the lower airway but throughout the respiratory tract including nasal passages, prevention of exhaled breath from contamination remains an important step in sampling. The use of a two-way nonrebreathing valve that allows exclusive inspiration of ambient air and exhalation into the apparatus prevents potential problems with rebreathing of exhaled samples. Similarly, if level of a given substance (e.g., H₂O₂, nitrite) is high in the saliva, it may contaminate the sample data interpretation. Exclusion of salivary source can be achieved simply with a saliva trap, but rinsing of mouth prior to collection and/ or swallowing accumulated saliva to maintain a dry mouth during collection usually suffice to prevent contamination. Additionally, the undirectional valve of the mouthpiece acts as a saliva trap. Nonetheless, in an attempt to completely exclude contamination, samples can also be tested for salivary amylase. When such measures are undertaken, exhaled aerosol droplets should reflect the composition of extracellular fluid lining the lower respiratory tract.

	COMPOUNDS PRESENT IN EXHALED BREATH CONDENSATE

TOP INTRODUCTION COLLECTION OF EXHALED BREATH... COMPOUNDS PRESENT IN EXHALED... ADVANTAGES AND LIMITATIONS FUTURE DIRECTIONS CONCLUSIONS REFERENCES

Exhaled breath condensate is presently an area of active research. Hence, new macromolecules are being added to the list of substances that can be detected in exhaled breath condensate. Table 1 summarizes the substances detected to date and published in the English literature.

Smokers

Level of H₂O₂, a metabolite of oxidative metabolism and a measure of oxidant activity in tissues, in exhaled breath condensate was measured in cigarette smokers versus healthy control subjects. Cigarette smokers had a 5-fold higher mean expired breath H₂O₂ level than nonsmokers. A gender difference was also detected, as male smokers exhaled more H₂O₂ than female smokers (11). In another study that attempted to correlate exhaled breath H₂O₂ with H₂O₂ generated from the alveolar lining fluid, exhaled H₂O₂ was 5 × 10⁴ times lower than H₂O₂ produced in the alveolar lining fluid (12). This difference was attributed to the presence of antioxidants in the lining fluid of the lower respiratory tract. Removal of H₂O₂ by the antioxidant system, which is also activated by smoking, may explain the lack of correlation between exposure to cigarette smoke and level of H₂O₂ in exhaled breath condensate. To this end, a recent study showed that level of H₂O₂ in exhaled breath condensate of smokers is increased half an hour after combustion of one cigarette (13). Measuring H₂O₂ or other markers of oxidative stress in exhaled breath condensate may be used to identify subjects at a higher risk for developing smoking-related pulmonary diseases by documenting enhanced production of reactive oxygen species.

Asthma

A variety of inflammatory markers present in exhaled breath condensate have been investigated as possible biomarkers of disease activity. The most commonly studied was H₂O₂. Antczak and colleagues found increased levels of H₂O₂ and thiobarbituric acid-reactive (TBAR) substances (a marker of lipid peroxidation/oxidative damage) in patients with asthma compared with healthy volunteers (14). They also documented a high positive correlation between H₂O₂ and TBAR. Importantly, increase in the H₂O₂ levels were associated with a decrease in forced expiratory volume at 1 s (FEV₁) in patients with asthma. Supporting these observations, in a double-blind, placebo-controlled study Antzcak and coworkers demonstrated a significant reduction in exhaled H₂O₂ levels in patients with asthma treated with inhaled corticosteroids compared with placebo (15). As in the previous study, there was a negative correlation between H₂O₂ level and FEV₁ at 29 d of treatment. Interestingly, H₂O₂ levels remained stable for 2 wk after inhaled corticosteroids were discontinued.

As in adults, H₂O₂ levels are increased in exhaled breath condensate of children with stable asthma compared with healthy controls (16). Importantly, H₂O₂ levels correlated with clinical symptoms and were lower in children treated with antiinflammatory medications (17). Therefore, it appears that exhaled breath H₂O₂ may be a simple and useful biomarker of airway inflammation in children and adults with asthma, particularly in monitoring the salutary effects of antiinflammatory drugs.

Other inflammatory mediators derived from oxidant and peroxidase activity have garnered considerable attention as useful biomarkers in patients with asthma. Hanazawa and coworkers compared levels of nitrotyrosine, a stable end-product of the oxidant, peroxynitrite, in exhaled breath condensate of patients with asthma to those in normal nonsmoking controls (18). Patients had mild (corticosteroid naive), moderate (on inhaled corticosteroid), or severe (on oral corticosteroid) asthma. Exhaled nitrotyrosine levels were increased in patients with corticosteroid naive, mild asthma compared with control subjects but not in patients with moderate or severe asthma receiving corticosteroid therapy. In contrast, these patients had stable levels of peroxynitrite but increased levels of cysteinyl-leukotrienes (LTC₄/D₄/E₄) that were unresponsive to corticosteroid therapy. In another study, patients with asthma had threefold higher levels of nitrite, a stable end-product of NO metabolism, relative to healthy control subjects (19). Recently, antioxidant response in asthma was investigated using nitrosothiols, products that are formed as a result of reaction between NO and antioxidant response (e.g., glutathione) (20). Exhaled nitrosothiol levels were elevated in patients with severe asthma compared with normal subjects and patients with mild asthma, suggesting a potential role for classification of asthma.

Isoprostanes, compounds primarily formed as a result of nonenzymatic peroxidation of membrane phospholipids (arachidonic acid) during oxidative stress, have also been investigated as biomarkers of inflammation in exhaled breath condensate of patients with asthma (21, 22). They are relatively stable and specific for lipid peroxidation, which makes them potentially reliable biomarkers for oxidant stress (23). 8-Isoprostane, a member of the F₂ isoprostane class, is the most studied of the isoprostanes. A study performed in patients with mild, moderate, or severe asthma found elevated 8-isoprostane levels compared with normal volunteers (24). Even patients with mild asthma had values twofold greater than the normal group, and 8-isoprostane levels increased with the severity of asthma. However, the difference between mild to moderate asthma was not statistically significant. There was no correlation between 8-isoprostane levels and pulmonary function tests in any group of patients.

It is well established that leukotrienes play an important role in the pathophysiology of asthma, in part, by inducing airway smooth muscle contraction, microvascular leakage, and mucus hypersecretion. A recent study showed increased levels of cysteinyl-leukotrienes and LTB₄ in exhaled breath condensate of patients with asthma as compared with healthy controls (18). Levels of leukotrienes increased with asthma severity. However, there was no correlation between these mediators and FEV₁ in patients with moderate and severe asthma. In another study, LTB₄ levels in exhaled breath condensate increased with the severity of asthma, increasing 9-fold in patients with severe (peak expiratory flow [PEF] < 60% predicted normal values) compared with mild (PEF > 80%) asthma (25). However, there was no correlation between LTB₄ levels and FEV₁.

Hunt and coworkers used exhaled breath condensate to measure the pH of exhaled breath condensate in patients with asthma and controls (26). In the control group, pH values were comparable to those reported in sputum and similar to those in BAL (27, 28). Acute asthma was associated with over two-log orders lower pH compared with control subjects. Importantly, the pH normalized with corticosteroids therapy. These investigators suggested that airway acidification may explain increased levels of exhaled NO and proposed that serial measurements of pH might be helpful in titrating antiinflammatory therapy in patients with asthma. Unfortunately, pH measurements seem to be hampered by poor reproducibility (29).

Despite lack of long-term prospective studies, the outlook for using biomarkers from exhaled breath condensate in asthma is promising. These data suggest that airway inflammation in asthma persists despite corticosteroid therapy. They also underscore the importance of the leukotriene metabolic pathway by demonstrating its lack of suppression by corticosteroids. Persistent elevation of leukotrienes in exhaled breath condensate in corticosteroid-treated patients may be used as an indication to use leukotriene pathway inhibitors. Lack of correlation between FEV₁ and leukotriene levels in these studies was disappointing but does not preclude a potential role for measuring leukotrienes in exhaled breath condensate in the management of asthma.

Collectively, this method may prove a valuable diagnostic tool by providing an objective means to define the severity of asthma and to tailor antiinflammatory medications. If changes in biomarkers precede those of physiological parameters, the utility of these measurements in the management of asthma would be strengthened.

Cystic Fibrosis

Lung disease in cystic fibrosis (CF) is characterized by recurrent bacterial airway infection and exposure to increased oxidative stress. Early in the course of the disease, patients are frequently asymptomatic despite ongoing inflammation (30). Insidious presentation of CF lung disease has made careful monitoring essential in the management of these patients and consequently has led to several studies using exhaled breath condensate.

When adults with clinically stable CF were studied, no difference in exhaled breath H₂O₂ levels was detected between the CF group and normal controls (31). The lack of increase in H₂O₂ levels was attributed to rapid reaction between H₂O₂ and other oxygen-reactive species, impaired diffusion due to large amount of viscous secretions in the airways, and/or increased levels of antioxidants. Conversely, in a study of 16 children with CF experiencing acute infectious pulmonary exacerbations, Jöbsis and colleagues showed that exhaled H₂O₂ levels decreased in exhaled breath condensate as patients improved clinically with antibiotic therapy (32). However, the correlation between H₂O₂ levels and FEV₁ in individual subjects was weak. This study also lacked a control group.

In contrast to H₂O₂, levels of nitrite in exhaled breath condensate are increased in adults with clinically stable CF when compared with controls (5). There was no difference in exhaled NO levels between the two groups. These data are consistent with a study showing increased nitrite levels in sputum of patients with CF (33). A study in children with CF confirmed these findings and showed elevated levels of nitrite that was comparable to those in adults (34). Interestingly, interleukin (IL)-8, a proinflammatory cytokine, was present in only one-third of children with CF. Nitrite may represent a more sensitive marker of NO production in the lower respiratory tract due to its greater stability relative to NO. Evaluation of nitrite levels may thus be useful in assessing subclinical disease activity in patients with CF, thereby initiating appropriate therapy before onset of symptoms.

Exhaled 8-isoprostane levels were increased 3-fold in exhaled breath condensate of patients with clinically stable CF compared with controls and were negatively correlated with FEV₁ (35). Consistent with other studies, a positive correlation was found between 8-isoprostane and exhaled levels of carbon monoxide (CO), which could represent a physiological defense to counteract the oxidative damage evoked by CO (36).

In summary, data from preliminary studies suggest a potential role for exhaled breath condensate to determine biomarkers of oxidative stress in the management of patients with CF. These measurements may enable the clinician to diagnose an exacerbation earlier and intervene before symptoms develop. Exhaled breath condensate may also be used to monitor the efficacy of drugs in these patients.

Bronchiectasis

Bronchiectasis, a suppurative lung disease, is characterized by significant pulmonary oxidant stress that can be measured using exhaled breath H₂O₂. In a study by Loukides and coworkers, patients with bronchiectasis displayed exhaled H₂O₂ levels higher than normal controls, and a negative correlation between the H₂O₂ levels and FEV₁ was documented (37). In contrast to asthma, patients with bronchiectasis receiving corticosteroids therapy had H₂O₂ levels similar to those who did not receive corticosteroids. This was attributed, in part, to the relative ineffectiveness of corticosteroids in downregulating neutrophil activity, a prime source of H₂O₂ in the airways of patients with bronchiectasis.

These findings are consistent with the presence of ongoing airway inflammation in bronchiectasis even when the disease is subclinical. An inverse correlation with physiological parameters may have practical clinical implications such as monitoring disease activity and early detection of heightened inflammation.

Acute Lung Injury/Acute Respiratory Distress Syndrome

Baldwin and coworkers demonstrated increased levels of H₂O₂ in exhaled breath condensate of patients with acute respiratory distress syndrome (ARDS) compared with patients with other respiratory diseases including pneumonia (38). In a subsequent study, no difference was found between patients with ARDS and those with pulmonary infiltrates but without ARDS (6). However, both groups had higher levels of H₂O₂ compared with controls. Patients with sepsis and severe brain injury had the highest level of H₂O₂ in the exhaled breath condensate. Wilson and coworkers confirmed these findings by showing a higher level of exhaled H₂O₂ in patients with ARDS in comparison to control subjects (39). Likewise, patients with ARDS have higher levels of exhaled H₂O₂ relative to other lung diseases (40).

Carpenter and coworkers measured 8-isoprostane levels in exhaled breath condensates of patients with or at risk for acute lung injury (ALI)/ARDS and from healthy intubated individuals (41). More than half of 22 patients with or at risk for ALI/ARDS had levels of 8-isoprostane greater than 2 standard deviations above the mean found in the normal group. There was no correlation between the level of 8-isoprostane and fraction of inspired oxygen in patients with or at risk for ALI/ARDS. Lack of difference in prostaglandin E₂ (PGE₂) levels in exhaled breath condensate between patients with or at risk for ALI/ARDS and healthy individuals suggested that most of the 8-isoprostane was formed through lipid peroxidation.

These findings support the notion about involvement of reactive oxygen species in the pathophysiology of ALI/ARDS. Unfortunately, lack of specificity for ALI/ARDS limits the applicability of these biomarkers in clinical practice. Recent studies in patients with ARDS have shown that injurious ventilatory strategies (high volume and low positive end-expiratory pressure) are associated with an increase in BAL cytokine levels (e.g., IL-6) (42, 43). Repeated measurements of cytokines in exhaled breath condensate may be used as biomarkers to monitor for ventilator-associated lung injury in the management of ARDS.

Chronic Obstructive Pulmonary Disease

An imbalance between oxidative stress and lung antioxidant capacity is important in the development and progression of chronic obstructive pulmonary disease (COPD). Dekhuijzen and coworkers demonstrated increased H₂O₂ in exhaled breath condensate of patients with stable COPD relative to healthy controls with a further increase noted during an acute exacerbation (44). Levels of H₂O₂ also correlated with eosinophil differential counts in induced sputum. Unfortunately, the study failed to find a correlation between H₂O₂ levels and FEV₁ during clinically stable disease and acute exacerbations. In another study, Montuschi and coworkers determined 8-isoprostane in exhaled breath condensate as a biomarker of oxidative stress in patients with COPD and investigated the effects of acute smoking (45). They found that 8-isoprostane levels were similar in COPD ex-smokers and COPD current smokers and were increased 1.8-fold compared with healthy smokers. The latter had 2.2-fold higher 8-isoprostane levels than healthy nonsmokers. The impact of acute smoking on oxidative stress in the lower respiratory tract was suggested by finding a 50% increase in exhaled 8-isoprostane levels 15 min after cigarette smoking. Lastly, patients with COPD have increased levels of nitrite and nitrosothiols in exhaled breath condensate compared with normal subjects and healthy smokers (20).

These findings are consistent with the concept that airway inflammation is present in COPD even when the disease is clinically stable. The oxidative burden is further amplified during exacerbations and following acute smoking. These data suggest that measurement of biomarkers of oxidative stress may be complementary to physiological parameters used to monitor these patients.

Other Pulmonary Disorders

In a small study of 13 patients with a variety of respiratory disorders, analysis of exhaled breath condensate from 3 patients with interstitial lung disease (ILD) detected IL-1, soluble IL-2 receptor, and tumor necrosis factor- (1). Biomarkers of oxidative stress are also detected in exhaled breath condensate of patients with pneumonia, lung cancer, and after thoracotomy (1, 47).

	ADVANTAGES AND LIMITATIONS

TOP INTRODUCTION COLLECTION OF EXHALED BREATH... COMPOUNDS PRESENT IN EXHALED... ADVANTAGES AND LIMITATIONS FUTURE DIRECTIONS CONCLUSIONS REFERENCES

Exhaled breath condensate could provide noninvasive, real-time assessment of pulmonary pathobiology with several advantages (Table 2). It is simple to perform and highly reproducible (26). It is portable and has the potential to be used in an outpatient setting or even at home. Exhaled breath condensate reflects abnormalities noted in traditional sampling methods of the respiratory tract, including BAL and induced sputum (48). It does not disrupt the respiratory mucosa and does not have a dilutional factor as observed in samples obtained via bronchoscopy and BAL. Patients can be studied at any age. Condensate can be obtained from conscious, spontaneously breathing patients and from mechanically ventilated patients (6, 38). Exhaled breath condensate can also provide sequential and longitudinal sampling of the lower respiratory tract without the need for more invasive tests, such as bronchoscopy with BAL and induced sputum.

The use of exhaled breath condensate is promising because it is a simple, noninvasive technique to sample the lining fluid of the lower respiratory tract for various biomarkers. However, it has limitations (Table 2). Among those, lack of a standard method of collection remains the most important one. Exhaled breath condensate may possess a concentration artifact due to evaporation of water from droplets before actual condensation occurs. To verify the accuracy of the collected samples, equipment should be tested using aerosols of solutions with known concentrations of mediators before the actual collection proceeds. Another shortcoming is that the collected fluid is not anatomic-site specific. The condensate consists of particles from fluid lining of the lower respiratory tract and alveoli. The relative contribution of those sites is unknown. Moreover, the exact location where aerosol particles from the lower respiratory tract originate is yet to be determined.

	FUTURE DIRECTIONS

TOP INTRODUCTION COLLECTION OF EXHALED BREATH... COMPOUNDS PRESENT IN EXHALED... ADVANTAGES AND LIMITATIONS FUTURE DIRECTIONS CONCLUSIONS REFERENCES

Collection of exhaled breath condensate is a rapidly expanding area of research. Standardization of the collection procedure must be undertaken to compare data obtained by different research groups. Further characterization of diurnal fluctuations in levels of mediators in normal volunteers and patients is warranted. Correlating patient outcome data with levels of inflammatory mediators would be necessary to identify mediators of distinct disease entities. Likewise, correlating levels obtained from exhaled breath condensate with other established methods of assessing pulmonary inflammation, namely BAL, is necessary to validate this method. The number of mediators detected in exhaled breath condensate is growing rapidly. In addition to mediators, other proteins, lipids, deoxyribonucleic acid (DNA), and ribonucleic acid (RNA) from mammalian cells and microorganisms as well as drugs may be present in exhaled breath rendering exhaled breath condensate a potential area of research for microbiology and gene delivery. For instance, as mycobacteria are present in droplet nuclei, it is reasonable to assume that mycobacterial large proteins and/or RNA could be detected in exhaled breath condensate early in the course of the infection. Obviously, a concerted research initiative is required to advance this evolving field of research. Collection and analysis of exhaled breath condensate could prove a valuable, single, noninvasive tool to evaluate, treat, and follow-up patients with various pulmonary disorders.

	CONCLUSIONS

TOP INTRODUCTION COLLECTION OF EXHALED BREATH... COMPOUNDS PRESENT IN EXHALED... ADVANTAGES AND LIMITATIONS FUTURE DIRECTIONS CONCLUSIONS REFERENCES

Collection and analysis of exhaled breath condensate constitute a rapidly progressing field of research. The collection procedure is simple and noninvasive, and the collected fluid represents aerosolized pulmonary extracellular-lining fluid. The exclusion of salivary contamination is one of the most important concerns of the collection procedure. To date, over a dozen different compounds, the majority of which represent biomarkers of oxidative stress in the lower respiratory tract, have been detected in the exhaled breath condensate from normal volunteers and patients with asthma, COPD, cystic fibrosis, bronchiectasis, ALI/ARDS, and other pulmonary disorders. These biomarkers may be a useful single and noninvasive adjunct in the diagnosis and follow-up of patients with various pulmonary inflammatory conditions at the point of care in real time. Clearly, further studies are indicated to validate, standardize, and better define the clinical utility of this instrument in patients with various pulmonary disorders.

	Footnotes

Correspondence and requests for reprints should be addressed to Dr. Israel Rubinstein, Department of Medicine (M/C 787), University of Illinois at Chicago, 840 S. Wood Street, Chicago, IL 60612-7323. E-mail: IRubinst{at}uic.edu

(Received in original form January 8, 2001 and in revised form May 21, 2001).

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TOP INTRODUCTION COLLECTION OF EXHALED BREATH... COMPOUNDS PRESENT IN EXHALED... ADVANTAGES AND LIMITATIONS FUTURE DIRECTIONS CONCLUSIONS REFERENCES

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