"I Don't Know What You Guys Are Measuring But You Sure Are Measuring It!"
A Fair Criticism of Measurements of Exhaled Condensates?

Richard W. Hyde, M.D.

University of Rochester Medical Center, Rochester, New York

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In the 1950s, Robert Forster, Ward Fowler, David Bates, and others in the Department of Physiology at the Graduate School of Medicine at the University of Pennsylvania, modified Marie Krogh's breath-holding estimates of pulmonary carbon monoxide diffusing capacity (DLCO) by adding 10% helium to the inspired mixture. This markedly simplified the breath-holding maneuver and made the measurements of DLCO a simple practical tool to assess pulmonary diffusion. Rapidly, the Penn Group and others embarked on measurements in normal and diseased subjects. One day at Physiology Lunch Club, Forster and his associations enthusiastically discussed their early results. The Chief, Julius Comroe, later the distinguished developer and head of the Cardiovascular Research Institute in San Francisco for many years, listened patiently and then dryly commented: "I don't know what you guys are measuring but you sure are measuring it." Only in subsequent years did investigators do the studies that led to a better understanding of the physiologic limitations of the method and appropriate applications.

This "boom and bust" adventure, later followed by acquisition of the strengths and limitations of a new method, keeps repeating itself for new tests such as closing volumes, gallium scans, angiotensin-converting enzyme levels, bronchoalveolar lavage (BAL), exhaled nitric oxide, and now analyses of exhaled breath condensates. For the past five years, there has been an explosion of over 100 publications of measurements of substances in exhaled condensates in normal subjects; in subjects with a variety of disorders including asthma, chronic bronchitis, cystic fibrosis, and acute lung injury; and in smokers. Substances measured include hydrogen peroxide, leukotrienes, and other mediators, and recently pH. Recently published in the "Pulmonary Perspective" section of the AJRCCM is an excellent and well measured review of the current status of measurements of exhaled condensates (1). The authors point out the rudimentary state of knowledge about the pathophysiologic factors involved in the analysis of exhaled condensates.

Collection of exhaled condensates has huge appeal because the method only requires the noninvasive collection of exhaled gas for 15 to 20 minutes in a cold trap. It is applicable to children as well as patients, even those on ventilators, and provides access to nonvolatile respiratory compounds without requiring fiberoptic bronchoscopy with BAL that may be damaging to the airways and thereby provide distorted data.

However, little attention has been paid to potential pitfalls of the method. For example, do the nonvolatile compounds in condensates come mainly from the mouth, conducting airways, or alveoli? Why is there such a huge variability in concentration of mediators with considerable overlap between diseased and normal subjects?

The report by Effros and coworkers entitled "Dilution of Respiratory Solutes in Exhaled Condensates" on pages 663- 669 of this issue is a welcome innovative approach that begins to figure out "what you guys are measuring" (2). They point out that the collected condensates contain fluid principally from two sources: mostly pure exhaled water vapor that contains no solutes, and traces of nonvolatile solutes that can only be recovered in the expired samples if they are released from airway lining fluid in the form of respiratory tract droplets. They next point out that there is excellent evidence that these droplets come from the lung epithelial lining fluid that coats the airways and have an electrolyte composition similar to plasma (3). During exhalation, the droplets of lining fluid and its electrolytes are markedly diluted by the exhaled water vapor that collects as a liquid in the cold trap. Then, if you measure Na⁺ and K⁺ concentration of the collected specimens and assume that the lining fluid's electrolytes are similar to plasma, you can calculate the degree of dilution of the airway lining fluid by exhaled water vapor. The sum of Na⁺ and K⁺ is appealing as a dilution indicator because these cations are essentially all of the cation content of most biologic fluids. Effros and colleagues reported huge variability in the amount of dilution even in the same subject and the harvested droplets condensate had electrolyte concentrations that varied 200-fold (0.01% to 2% of plasma levels). Using the dilution of the sum of Na⁺ and K⁺ in the collected exhalate will likely permit a semiquantitative analysis of substances such as leukotrienes and H₂O₂ in the epithelial lining fluid. One reassuring check on their hypothesis was that the calculated value of protein in the airway fluid using their method was 7.6 ± 2 g/dl (SEM), a value closely approximating levels in plasma and airway surface liquid (2). It will be of great interest to see if their method of calculating the "true" concentration of mediators in the respiratory tract lining fluid will abolish or markedly reduce the huge variability in currently reported measurements.

Although Effros and coworkers' method for compensating for dilution by water vapor in exhaled condensates is reasonable, there are limitations. Currently, one must collect condensate for an hour to have sufficient sensitivity to accurately measure the very low electrolyte concentrations in the expirate. More sensitive techniques for measuring trace amounts of electrolytes in the condensates will likely be developed to obtain a more practical sampling time of 10 to 15 minutes. Possibly, respiratory maneuvers such as coughing or intermittent hyperventilation with 3% to 5% CO₂ can increase the yield of droplets without altering the concentration of mediators.

Other interesting observations include measurements in three subjects with tracheostomies that showed no NH₄⁺ in the expirate. This means NH₄⁺ and NH₃ in respiratory tract condensates must come from the mouth. Unfortunately, this NH₃ and NH₄⁺ represents the most important buffer in the condensate. As a consequence, what comes from the mouth must be an important determinant of pH of exhaled condensates. This will likely raise havoc with attempts to measure the pH in respiratory tract condensates in individuals with asthma (6).

Effros and coworkers have made an important contribution that will likely permit a semiquantitative analysis of nonvolatile substances in respiratory lining fluid. If Julius Comroe were still with us, he might now comment: "It's about time you guys settle down and figure out what you are measuring."

	References

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1. Mutlu GM, Garey KW, Robbins RA, Danziger LH, Rubenstein I. Pulmonary Perspective: collection and analysis of exhaled breath condensates in humans. Am J Respir Crit Care Med 2001; 164: 731-737 [Free Full Text].

2. Effros RM, Wahlen K, Bosbous M, Castillo D, Foss B, Dunning M, Gare M, Lin W, Sun F. Dilution of respiratory solutes in exhaled condensates. Am J Respir Crit Care Med 2002; 165: 663-669 [Abstract/Free Full Text].

3. Jayaraman S, Song Y, Vetrivel L, Shankar L, Verkman AS. Noninvasive in vivo fluorescence measurement of airway-surface liquid depth, salt concentration, and pH. J Clin Invest 2001; 107: 317-324 [Medline].

4. Effros RM, Feng D, Mason G, Sietsema K, Silverman P, Hukkanen J. Solute concentrations of the epithelial lining fluid of anesthetized rats. J Appl Physiol 1990; 68: 275-281 [Abstract/Free Full Text].

5. Adamson TM, Boyd RDDH, Platt HS, Strang LB. Composition of alveolar liquid in the foetal lamb. J Physiol (Lond) 1969; 204: 159-168 [Abstract/Free Full Text].

6. Hunt JF, Fang K, Malik R, Snyder A, Malhotra N, Platts-Mills TAE, Gaston B. Endogenous airway acidification: implications for asthma pathology. Am J Respir Crit Care Med 2000; 161: 694-699 [Abstract/Free Full Text].