Exhaled breath condensates: a potential novel technique for detecting aspiration

Exhaled Breath Condensates: A Potential Novel Technique for Detecting Aspiration Richard M. Effros, MD, Mark Bosbous, Bradley Foss, Reza Shaker, MD, Julie Biller, MD

There is an urgent need for diagnostic procedures that can detect aspiration of oral and gastrointestinal (GI) secretions into the respiratory tract. Current approaches are limited by poor sensitivity and specificity. These techniques include (1) adding indicators to feedings; (2) recovery of lipid-filled macrophages in respiratory secretions; (3) measurement of changes in the pH of the upper GI and respiratory tracts; (4) endoscopic visualization of reflux events; and (5) measurement of increased glucose concentrations in respiratory secretions. Ideally, specific markers from various sites in the oral and GI tracts might be discovered in respiratory secretions, but conventional bronchoalveolar lavage for sampling respiratory secretions is not practical and involves some risk. Noninvasive measurements of indicators in the exhaled breath condensates could be used to detect aspiration, but a number of theoretical and practical aspects of such studies must be considered before this approach can be applied to the problem of aspiration. Am J Med. 2003;115(3A): 137S–143S. © 2003 by Excerpta Medica, Inc.

From the Pulmonary and Critical Care Division (RME, MB, BF, JB), the Digestive Disease Center (RME, RS), and the Cystic Fibrosis Center (JB), Medical College of Wisconsin, Milwaukee, Wisconsin, USA; and Zablocki VA Hospital (RME, BF, RS). This work was supported by Grant Nos. R01 HL 60057 and P01 DC03191 from the National Institutes of Health. Requests for reprints should be addressed to Richard M. Effros, MD, Department of Medicine, Medical College of Wisconsin, 9200 West Wisconsin Avenue, Milwaukee, Wisconsin, 53226. © 2003 by Excerpta Medica, Inc. All rights reserved.

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spiration of gastric contents into the lungs is among the most serious and most common supraesophageal complications of severe gastroesophageal reflux (GER). Patients with impaired consciousness and swallowing mechanisms are predisposed to this complication, which is often responsible for morbidity and mortality in these individuals, particularly when reflux extends beyond the esophagus. Although reflux of acid into the esophagus can cause bronchospasm, the consequences of acid entering the lungs are much more serious. This has been illustrated in animal studies conducted by Tuchman et al.,1 who showed that instillation of 10 mL of 0.2 N HCl into the esophagus of anesthetized cats increased airway resistance by an average factor of 1.47 from baseline (P ⬍0.05) and affected only 8 of 13 animals studied. In contrast, instillation of just 0.05 mL of 0.2 N HCl into the airways resulted in a 4.65-fold increase in total lung resistance from baseline and affected all of the animals (P ⬍0.005). This response was eliminated by bilateral cervical vagotomy. Using a radioactive indicator, the investigators were able to show that the instilled bolus remained in the trachea, indicating that the afferent limb of the response to acid was from the trachea wall rather than from more distal airways. A wide variety of respiratory disorders have been associated with possible aspiration of oral and gastric contents. These include bronchial asthma, bronchiectasis, cystic fibrosis, aspiration pneumonitis (response to chemical injury), aspiration pneumonia (related primarily to organisms in the mouth), necrotizing pneumonia, abscesses, pulmonary fibrosis, and lipoid pneumonia. Despite the frequency and gravity of aspiration, development of reliable methods of detecting aspiration has proved frustrating. In this article, we describe conventional approaches for detecting aspiration of gastric, oral, or intestinal contents, and propose using exhaled breath condensates (EBC) as a noninvasive method for measuring aspirates.

CONVENTIONAL METHODS FOR DETECTING ASPIRATES One approach for detecting aspirates is the identification of lipid-laden macrophages in respiratory secretions obtained by bronchoalveolar lavage.2–5 The simple observation of lipid-laden macrophages in these specimens lacks sensitivity and specificity, and is seldom used.4,5 0002-9343/03/$22.00 137S doi:10.1016/S0002-9343(03)00212-2

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Aspiration has also been detected by scintigraphic monitoring in patients with both pulmonary disease and GER.6 –10 However, when Ghaed and Stein6 administered a solution at bedtime containing 99mTc-sulfur colloid by nasogastric tube to 10 individuals with asthma and GER, and then scanned their lungs the next morning, the investigators found that this technique lacked sensitivity in their subjects. Although these tests were frequently positive in patients with asthma,6,8,10 positive scans have also been observed in normal subjects.7 Apparently, some nocturnal aspiration is a common event in normal subjects, but it is insufficient to cause significant respiratory disease. One problem with this, as well as with most other approaches for detecting aspiration, is the intermittent nature of aspiration. Indeed, even a single aspiration of a large bolus of material can have serious complications. In addition, the presence of radioactive material in the stomach can be difficult to distinguish from that in the lungs. The use of dyes in enteral feedings to detect aspiration has been popular for some years in the United States. Methylene blue was initially used for this purpose, but this dye can lose color with reduction and it also is expensive. Methylene blue is readily absorbed by the gastrointestinal (GI) tract; thus, the skin as well as the urine and stool can be stained greenish. For this reason, methylene blue has been largely replaced by a commercial food coloring, Blue No. 1 (brilliant blue). Unfortunately, the use of dyes in enteral feedings has been shown to lack both sensitivity and specificity for detecting aspiration.11–14 Furthermore, although Blue No. 1 is not appreciably absorbed from the intestines in normal subjects, it can cross the mucosa of injured bowels. Absorption can result in more serious problems, including hyperthermia and shock, suggesting mitochondrial poisoning.15 With the appearance of these reports of serious complications, it is quite possible that the practice of placing this dye in enteral feedings will lose some of its popularity. Some evidence for aspiration may be identified by dual pH monitoring of the distal and proximal esophagus16 –19; however, most data do not support the theory that proximal GER is specific for asthma. In 1 series, 44% of subjects with asthma had abnormal acid contact times in the distal esophagus, and in 24% acid reached the proximal esophagus.16 Probes placed in the pharynx only identified the presence of acid in 5% of subjects with moderate or severe asthma.17 In the most definitive studies, probes were placed in the trachea by way of a cannula inserted through the cricothyroid membrane, permitting documentation of acidification of the trachea.18 The risks associated with this approach probably outweigh the benefit derived in this fashion. Movement of fluid from the mouth into the airways can be documented by directly visualizing such events by

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fiberoptic endoscopic evaluation of swallowing.20,21 This approach is useful for evaluating swallowing in patients with dysphagia, but it may miss intermittent aspiration. In a now-classic publication, Winterbauer and associates introduced the technique of using glucose oxidase test strips to detect acid reflux in respiratory secretions.22 These investigators found that glucose concentrations in respiratory secretions were very low in normal subjects, generally ⬍5 mg/dL. Enteral feedings generally have very high glucose concentrations (325 mg/dL), and aspiration of these feedings typically increases glucose concentrations in the respiratory secretions. Although this approach continues to enjoy some popularity, questions persist concerning the specificity of the technique.12,13,23–26 Contamination of specimens by blood can raise glucose concentrations, and it appears that injury to the airway and airspace membranes may result in the movement of glucose from the plasma through these membranes into the respiratory secretions, even in the absence of aspiration. Of course, the likelihood of finding increases in glucose concentrations in the secretions will also depend on the presence of high glucose concentrations in the enteral feedings, which decreases when the formula is diluted. It can be concluded that none of the current procedures for detecting aspiration are fully satisfactory. Evidence for aspiration might be secured if oral and/or gastric markers could be discovered in samples of the fluid that lines the airways and/or airspaces. Among patients who cannot produce adequate sputum, information concerning respiratory fluid has been based primarily on bronchoalveolar lavage. Although this approach has proved to be a useful investigational and clinical tool, it is associated with some unavoidable risks and artifacts. Bronchoalveolar lavage is conducted with isotonic saline, which is reabsorbed relatively quickly over the following few hours; however, use of saline makes it virtually impossible to gain any information about the sodium and chloride concentrations present in the small volume of fluid on the lung surfaces before lavage was commenced. Alternative isotonic fluids, such as mannitol, have been used in animal studies,27 but may not be clinically applicable because reabsorption of the instilled fluid would be delayed.

DETECTING ASPIRATES FROM EXHALED BREATH CONDENSATES Measurable concentrations of a variety of nonvolatile solutes have been found in the EBC of normal subjects and in patients with a variety of illnesses.28,29 In this technique, subjects exhale into a cooled condenser, and solutes are measured in the condensate. It has been sug-

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Figure 1. Respiratory droplets are released from the surfaces of the airways/air spaces. Much greater quantities of water are released as vapor (dotted arrows). When the respiratory droplets reach the condenser, they become diluted by large volumes of water vapor that become deposited as large droplets on the walls of the condenser. (Reprinted with permission from Am J Respir Crit Care Med.29)

gested that the condensate approach could provide information about aspirates in the respiratory fluids, thereby avoiding the risk, inconvenience, and unavoidable artifacts associated with bronchoalveolar lavage. Although much has been published concerning exhaled condensates, little attention has been given to their medium, much of which is derived from water vapor, a gas that cannot carry nonvolatile solutes (Figure 1). Therefore, it is impossible to determine the concentrations of solutes on the respiratory surfaces from the condensate unless the factor of dilution of the respiratory droplets by water vapor has been determined. Where no information regarding the factor of dilution has been provided, it is possible to infer that increases in concentrations of inflammatory mediators that have been reported in inflammatory disease could be attributed to an increase in concentrations in respiratory secretions or to increased respiratory droplet formation. It has been argued that if the concentration of mediator A is increased and that of another mediator B is not, then this would suggest a true increase in A. However, it is also possible that concentrations of B have actually decreased, or that it is not possible to measure B with sufficient accuracy to detect parallel increases in B. Changes in the ratios of oxidized to reduced glutathione or NO3⫺ to NO2⫺ could theoretically

provide useful information, but in the absence of an independent measure of dilution, observed changes in the concentrations of most mediators in the condensate cannot be interpreted with confidence. Because the exhaled air is nearly saturated with water that is generated throughout the lungs, the amount of water vapor formed per minute is linked to minute ventilation. In contrast, production of true respiratory droplets may variably depend on local turbulence (audible as rales or rhonchi), which tends to occur in the larger airways, and is consequently less predictable. This variability is presumably responsible for the enormous variation seen in the concentrations of solutes observed between individuals and within individuals from day to day. However, we found that concentrations of Na⫹ were well correlated with those of K⫹ and Cl⫺ (Figure 2). This is consistent with the hypothesis that these variations are caused by changes in the volumetric contribution of respiratory droplets to the much larger volume of water vapor collected in the condensers.30 However, these high correlations occurred because of a few studies in which very high solute concentrations were found in the exhaled condensate. Nevertheless, good correlation has been found between Na⫹ and Cl⫺, even when the concentrations of these ions are confined to those ⬍30 mmol.

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Figure 2. Correlations between ion concentrations in the exhaled breath condensate of 20 normal subjects. The high correlation coefficients are related in part to high values found in several subjects. However, significant correlation was also observed over much smaller ranges of values. (Reprinted with permission from Am J Respir Crit Care Med.29)

These correlations suggest that the variability in observed concentrations of solutes in the condensates is related to differences in the dilution of the respiratory droplets by the water of condensation. In a recent publication,30 we suggested that the factor of dilution (D) of respiratory droplets by water vapor in the collected condensate could be calculated from the equation: D⫽

Volumevapor [Na⫹]plasma ⫹ [K⫹]plasma ⫽ . Volumerespiratory [Na⫹]condensate ⫹ [K⫹]condensate

This equation is based on the assumptions that (1) the airway droplets are derived from the airway surface fluid, (2) the airway surface fluid is isotonic,31–35 and (3) Na⫹ and K⫹ and their anions represent the principal osmotic ingredients of the airway surface lining fluid. X-ray microanalysis of frozen samples of airway fluid obtained from normal individuals and subjects with cystic fibrosis 140S August 18, 2003

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indicated that Na⫹ and Cl⫺ are the principal solutes in the exhaled condensate.36 The value of the factor of dilution averaged 2,500, and suggested that respiratory droplets contributed only 0.2% of the volume of fluid collected in the condenser. This approach provides a method for estimating the concentrations of all solutes, including GI markers, which are present in the respiratory fluids, without the need for performing bronchoalveolar lavage. Detection of oral contaminants, such as amylase, and of digestive enzymes from the stomach (e.g., pepsin) or the small intestine, may prove useful for diagnosing aspiration in patients who have recurrent episodes of reflux. A variety of reference indicators could be used to calculate the actual concentrations of gastric enzymes in the respiratory fluid. However, care must be taken in interpreting these kinds of data. Respiratory droplets may be generated from surfaces that do not have traces of the enzyme. Droplets from these “unsoiled” areas may have

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the same amount of Na⫹ as those from areas containing remnants of the aspirated material but none of the enzymes. This will tend to reduce the calculated concentrations of the enzyme. Furthermore, there is no reason to believe that subjects always produce measurable amounts of respiratory droplets, and Na⫹ concentrations are not infrequently as low as 1 ␮mol/L. This is equivalent to a dilution as great as 100,000 times. When the recovery of respiratory droplets is this low, measurement of any nonvolatile marker may be very problematic. Some of the inflammatory markers found in condensates, such as H2O2, NO2⫺, and NO3⫺, may actually arrive in the condenser as gases. Concentrations of gases in the condensate may vary because of differences in the efficiencies with which they are captured in the condenser fluid. Furthermore, if they are derived from the blood perfusing the lungs, exhaled concentrations will decrease as the ratio of dead space to tidal volume (VD/Vt) of the lungs increases. Rather than calculating the concentration of volatile gases in the exhaled air, it would presumably be more informative to determine the rate at which such gases are exhaled from the lungs. Calculation of the actual concentration of volatile gases in the respiratory fluids is complicated by distribution between the blood, tissue, and gas phases at various sites within the lungs. Because the pH of the gastric contents plays an important role in subsequent development of lung injury, it is tempting to test the pH of the condensate as a surrogate for measuring the pH of the respiratory surfaces. Inflammation of the airways might also promote a decrease in airway pH. Evidence has been obtained that the condensate becomes acidic in patients with severe asthma37 and chronic obstructive pulmonary disease.38 However, there are reasons for believing that these changes in condensate pH are caused by factors other than the pH of the respiratory fluids. These factors include the following: 1. NH4⫹ represents the principal ion found in the collected condensates and frequently exceeds the concentration of other ions (and buffers) by a factor ⬎200.30 It is derived principally from the catalytic degradation of urea in the mouth, and is delivered to the condenser in the exhaled air as NH3 gas. NH3 is trapped as NH4⫹ in the water droplets lining the tubing and condenser. Because the dissociation constant (Ka) of NH4⫹ is 9.25, the delivery of NH3 in the exhaled air alkalinizes the condensate fluid. 2. Individuals with asthma who have a low exhaled condensate pH also have low NH4⫹ concentrations in the condensate. Reduced production and collection of NH4⫹ may be responsible for more acid pH in the condensates. Several factors could be responsible for diminished NH4⫹ concentrations in the condensates: (1) Reductions in Vt should increase trapping of

NH4⫹ in the tubing connecting the mouthpiece and condenser.30 (2) Drying of the mouth secondary to such factors as hyperventilation, catecholamine release, and medications could reduce oral NH4⫹ production. (3) To the extent that exhaled NH3 is derived from the blood, concentrations of NH4⫹ in the condensate will decrease as the ratio of VD to Vt increases. (4) CO2 and HCO3⫺ represent the principal buffer system in the condenser during the collection of condensate. The presence of CO2 in the exhaled air enhances trapping of NH3 in the condensate.30 Reductions in end-tidal PCO2 are typical of patients with asthma who tend to hyperventilate and who have increased VD/Vt ratios. (5) Significant concentrations of lactate are normally present in the exhaled condensate. Low condensate pH in individuals with asthma may therefore reflect reductions in the collection of NH4⫹ buffer rather than increases in acids within the condensates. 3. Superficial analysis would suggest that acidification of the respiratory fluids would slow loss of NH3 from the lungs because more NH4⫹ would be trapped locally in the fluid lining the airways if the pH of this fluid is low. In fact, the volume of respiratory fluid is extremely small compared with the volume of blood and air within the lungs, and equilibration of this compartment with surrounding compartments should be very rapid. Once a steady-state concentration is reached, the parallel movement of NH4⫹ should accelerate the movement of NH3 from the surface of the airways to the air phase, a process referred to as facilitated diffusion.39 4. Because concentrations of buffers are normally very low in the condensate, pH measurements are difficult and can be influenced by such factors as the surfaces of the containers in which they are measured. CO2 is rapidly lost from the fluids, but some HCO3⫺may be present in the condensate at ambient CO2 levels. Attempts to purge all of the CO2 out of the condenser with argon may reduce these levels further, but this maneuver could also release other volatile gases from the condensate. Purging the samples with inert gases does not release NH4⫹ unless pH is increased to very alkaline levels. These considerations make it very unlikely that the pH values of the condensates resemble those in the respiratory fluids. Any decreases in pH that are observed in lung disorders largely reflect the effects of these diseases on the production of NH3 in the mouth and the efficiency with which NH3 is collected in the condenser. It is therefore unlikely that EBC pH will provide a reliable index of the change in lung pH after aspiration of gastric acid.

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SUMMARY A wide variety of respiratory disorders have been associated with possible aspiration of oral and gastric contents. To date, none of the approaches that have been developed for detecting aspiration of gastric, oral, or intestinal contents have proved fully satisfactory. Direct sampling of respiratory fluids by lavage could conceivably allow more precise measurements of products in these fluids, but this approach is not practical or safe, and it is associated with dilutional problems that are difficult to solve. The possibility of using EBC to detect aspiration may provide a noninvasive method for measuring a variety of proteins secreted in the mouth or GI tract. However, before EBC can be used for this purpose, careful consideration must be given to the manner in which these markers of aspiration are generated and then collected by the condenser, and to what extent they are diluted by water vapor that has been deposited in the condensate.

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A Symposium: Exhaled Breath Condensates/Effros et al 31. Verkman AS. Lung disease in cystic fibrosis: is airway surface liquid composition abnormal? Am J Physiol. 2001;281: L306 –L308. 32. Tarran R, Grubb BR, Parsons D, et al. The CF salt controversy: in vivo observations and therapeutic approaches. Mol Cell. 2001;8:149 –158. 33. Knowles MR, Robinson JM, Wood RE, et al. Ion composition of airway surface liquid of patients with cystic fibrosis as compared with normal and disease-control subjects. J Clin Invest. 1997;100:2588 –2595. 34. Matsui H, Grubb BR, Tarran R, et al. Evidence for periciliary liquid layer depletion, not abnormal ion composition, in the pathogenesis of cystic fibrosis airway disease. Cell. 1998; 95:1005–1015.

35. Caldwell RA, Grubb BR, Tarran R, et al. In vivo airway surface liquid Cl⫺ analysis with solid-state electrodes. J Gen Physiol. 2001;119:3–14. 36. Zahm JM, Davidson DJ, Baconnais S, et al. X-ray microanalysis of airway surface liquid collected in cystic fibrosis mice. Am J Physiol. 2001;281:L309 –L313. 37. Hunt J, Fang K, Malik R, et al. Endogenous airway acidification: implications for asthma pathophysiology. Am J Respir Crit Care Med. 2000;161:694 –699. 38. Kostikas K, Papatheodorou G, Ganas K, et al. pH in expired breath condensate of patients with inflammatory airway diseases. Am J Respir Crit Care Med. 2002;165:1364–1370. 39. Gros G, Moll W. Facilitated diffusion of CO2 across albumin solutions. J Gen Physiol. 1974;64:356 –371.

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