A Simple Method for Estimating Respiratory Solute Dilution in Exhaled Breath Condensates

A Simple Method for Estimating Respiratory Solute Dilution in Exhaled Breath Condensates
December 15, 2003
Effros, Richard M, Biller, Julie, Foss, Bradley, Hoagland, Kelly, Et al
American Journal of Respiratory and Critical Care Medicine,
The broad appeal of exhaled breath condensate studies is readily understandable (1, 2). Until recently, bronchoalveolar lavage has been the only way that fluid lining the airways and airspaces could be sampled in patients who do not produce sputum. Although bronchoalveolar lavage can yield important clinical and investigative information, it involves some risk, including infection, impairment of gas exchange, and problems with sedation. Furthermore, bronchoalveolar lavage is expensive and inconvenient, cannot be readily repeated at frequent intervals, and entails a number of artifacts associated with the instillation of fluid into the airways (3). In contrast, collection of condensates is noninvasive, can be repeated as often as needed, and does not alter the fluids on the lung surfaces.
Unfortunately, the condensate method involves some rather formidable problems of its own. Perhaps the most serious difficulty associated with exhaled condensates is that of extreme and what may be variable dilution of respiratory droplets (4). Most of the water that is exhaled from the lungs is generated in the form of water vapor, a gas that cannot carry nonvolatile solutes. The formation of droplets from exhaled water vapor can be considered as an artifact associated with cooling.
The quantity of water vapor generated each minute by the lungs is determined by the ventilatory rate and the saturation of the exhaled air, which is close to 100%. Full saturation of the exhaled air keeps the airway surfaces moist, thereby enhancing gas exchange. Water exchange is presumably assisted by the presence of a variety of aquaporins on airway surfaces (5). In contrast, little is known about release of droplets from the respiratory surfaces.
We recently drew attention to the need for dilutional reference indicators that would permit calculation of solute concentrations in the respiratory fluid from those in the condensates (4). Although increases in the concentrations of inflammatory mediators that have been described in a variety of lung diseases could reflect increases in the concentrations of these indicators in respiratory fluid, they could also be due to an increase in the volume of respiratory droplets that reaches the condenser and is then dissolved in the water vapor that has collected there.
There is reason for believing that changes in condensate concentrations of inflammatory mediators in lung disease are not simply due to alterations in dilution. Comparable increases in the concentrations of these mediators have been observed when bronchoalveolar lavage samples have been collected in these patients. Furthermore, increases in the concentrations of some markers of inflammation were associated with no change or even decreases in the concentrations of other indicators (6-9). However, measurements of relative concentrations of inflammatory markers may be misleading. An increase in the relative concentration of one mediator to that of a second mediator could be due to an increase in the first or a decrease in the second. Rather than using another inflammatory mediator to determine whether changes in the dilution of respiratory droplets had occurred, it would be preferable to use noninflammatory reference indicators that remain relatively unchanged in the respiratory fluid and, ideally, similar in concentration to those in the plasma.
In a previous study, we measured the concentrations of Na+, K+, and Cl- in condensates and found that these paralleled one another. We suggested that the sum of Na+ and K+ in the plasma could be divided by this sum in the condensate to estimate the dilution of respiratory droplets and solutes by condensed water vapor that accumulated in the condenser. We also attempted to use the conductivity of the condensate to estimate the total concentrations of the respiratory electrolytes in the condensates. This approach was frustrated by the presence of high concentrations of NH^sub 4^^sup +^ in the condensate, much of which represented oral contamination (4, 10, 11).
Our objective in this article is to show that conductivity can be used to estimate airway electrolyte concentrations and the dilution of respiratory droplets by water vapor if most of the NH^sub 4^^sup +^ is first removed by lyophilization. Measurements of conductivity can be performed on small volumes of lyophilized samples without consuming the samples. This technique provides an inexpensive and reliable method of estimating the total concentration of ions in the condensates and the dilution of respiratory droplets by the water vapor. A comparison was between estimates of dilution made from conductivity, the total cations, and the urea concentrations of the condensates.
METHODS
Subjects
Condensates were collected from a group of 18 normal adult subjects with no history of lung disease or recent smoking (average age, 26 ? 6 SD; 7 males and 11 females). Condensates were collected by having subjects exhale through a nonrebreathing valve connected to a 64 ? 22-mm ID ventilator tubing (Corr-a-Flex 2; Hudson RCI, Temecula, CA) into an inexpensive 66-cm commercial Pyrex Allihn condenser, which was cooled with circulating ice water. The condensate dripped into polycarbonate collection tubes. The subjects breathed into the condensers for approximately 1 hour, yielding a total of approximately 10 ml of condensate. Two groups of studies were performed. The effects of lyophilisation on conductivity and NH^sub 4^^sup +^ and electrolyte concentrations were studied in the first group of 11 samples obtained from 11 of the normal subjects. In the second group of 13 sets of samples from 13 normal subjects, the variability of condensate concentrations was studied in two consecutive 30-minute samples. Cations, urea, and conductivity were measured in these samples after lyophilization. Samples were also collected from five subjects with tracheostomies to determine the oral contribution to NH^sub 4^^sup +^ found in the condensates. Six salivary samples and five serum samples were also collected for comparison with condensate concentrations.
Sample Analysis
Condensate samples were stored at -80?C; 5-10 ml samples were lyophilized to dryness at -100?C and less than 1-torr pressure. The samples were reconstituted in 1.7 ml of deionized water. Concentrations of Na+, NH^sub 4^^sup +^, K+, Ca^sup 2+^, and Mg^sup 2+^ were measured in condensate samples before and/or after lyophilization with an ion chromatograph (Metrohm Model 761 compact IC, 0.5-ml sample loop, Metrosep C 2 100 column). Condensate urea concentrations were determined by measuring the concentration of NH^sub 4^^sup +^ released from incubating 1 ml of the lyophilized condensates with purified urease (Sigma, St. Louis, MO). Salivary and scrum urea concentrations were measured spectrophometrically with a Sigma urea kit. Serum rather than plasma was used to avoid interference with lithium and sodium in conventional heparinized tubes. Temperature-corrected conductivities of the condensates were measured in condensate samples before and after lyophilization with a modified Cole Parmer Con 10 conductivity meter (Vernon Hills, IL). The coefficients of variation for the ions and conductivity were less than 2% at 10 ?mol/ L and less than 20% for urea at 0.5 ?mol/L.
Dilution of respiratory droplets was estimated by dividing the total cation concentration in serum (150 mM) by the total concentration of cations estimated from the conductivity (in ?mol/L of NaCl) in the lyophilized samples (see DISCUSSION for justification).
D^sub conductivity^ = [total cations]^sub serum^/conductivity^sub lyophilized condensate^
A one-way analysis of variance with the Student-Newman-Keuls test was used to compare mean concentrations of ions and urea concentrations in the second set of studies (which included 28 samples). Correlation coefficients were also calculated from these data (SigmaStat2; Jandel Scientific, San Rafael, CA). Unless otherwise indicated, means and SEs are indicated in the text and figures.
These studies were approved by the Human Research Review Committees, and consent was obtained from each subject before each study.
RESULTS
As indicated in Figure 1, NH^sub 4^^sup +^ concentrations in the condensate averaged 141 ? 26 (SEM) ?mol/L and represented 93 ? 3% of the total cations in the condensate samples before lyophilization in the first group of condensate samples obtained from 11 of the normal individuals (three males and eight females). Lyophilization removed more than 99% of the NH^sub 4^^sup +^ and reduced both the sum of the total cations and the conductivity of the condensates. NH^sub 4^^sup +^ concentrations were significantly lower in patients with tracheostomies (26 ? 13 ?mol/L, n = 5). This reflects the fact that most of the NH^sub 4^^sup +^ found in the condensates is delivered to the condensates as NH^sub 3^ gas from the mouth