Comment on 'The effect of temperature on exhaled breath condensate collection'

Vyas et al [1] reported that the collection of exhaled breath condensates (EBCs) can be increased 79% by cooling their condenser with dry ice rather than ice water. This was accompanied by what may have been a modest decrease in EBC conductivity and protein concentrations, suggesting that some but not all of the increases in EBC volume were due to the addition of water vapor to the condensate. It is possible that water vapor is trapped more readily than airway droplets at cold temperatures. Alternatively, water vapor in ambient air could have entered through the top of the condenser, which appears to be open to the atmosphere. Despite these decreases in concentration, cold temperatures do appear to increase the rates at which some airway solutes can be collected in the EBC. It should be noted that volatile constituents (e.g., NH4+?and HCO3?), normally account for ~90% of the total conductivity of EBC samples [2?5]. These ions must be removed before measuring conductivity because they are derived primarily from the oral cavity by diffusion. When we originally introduced the conductivity approach [2], the samples were freeze-dried (lyophilized) at ?99% of NH4+?in the samples was removed by lyophilization and that the ratios of plasma conductivities to those of lyophilized EBC concentrations were similar to the ratios for 2 other indicators which are used to estimate the dilution of airway droplets by water vapor, namely urea and total cations [2]. In their current study, Vyas et al [1] chose to remove volatiles from their samples by centrifugal evaporation at room temperature and a pressure of 1.3?kPa. They report conductivity values that are approximately an order of magnitude greater than those found when the samples are lyophilized and what would be expected from studies in which urea or total cations are used [2?9]. Since they failed to indicate how much NH4+?remains in their samples after evaporation or measure dilutional indicators other than conductivity, retention of some oral volatiles may have occurred, complicating interpretation of their data. Dry ice failed to significantly change nitrate/nitrite concentrations and this approach may not be particularly useful for measuring these biomarkers. Interconversion between NO3?, NO2??and their derivatives could greatly complicate interpretation of the effect of low temperatures on NO3??and NO2??concentrations in the EBC. Concentrations of hydrogen peroxide doubled when dry ice rather than ice water was used. This may be due in part to increased solubility of H2O2?in cold water. It is probably more appropriate to measure the rates (VE,x moles min?1) at which each of the volatile solutes (for example, x = NH3, CO2, NO2, H2O2) are exhaled from the lungs than to estimate airway fluid concentrations (moles/liter). If most of the exhaled indicator is in the gas phase, VE,x can be calculated from the product of the expiratory flow rate and the fractional concentration of the indicator in the exhaled gas. Furthermore, it would probably be better if the rates of exhalation of volatile indicators were directly measured in the gas phase (e.g., GC-MS after droplet formation is minimized by warming the exhaled air) rather than in condensates. Most volatile substances (including water) are rapidly lost from alveolar and airway surfaces as gases rather than in airway droplets. Because gases readily diffuse between the blood and airspaces in the lungs, exhaled concentrations may reflect concentrations in the blood and extrapulmonary sites of production and may be influenced by local ventilation to perfusion ratios in the lungs. Many factors can affect the efficiency with which different volatiles are collected in condensates including: [1] the air: EBC distribution coefficients and the volume of water in the condenser during condensation, [2] temperature, flow rate and turbulence of air flow through the condenser, [3] the presence of buffers in the lungs, exhaled air and fluid lining the condenser, which is particularly important for weak acids and bases. EBCs cannot be used to estimate losses of volatile solutes from the lungs unless the rates of efflux out of the condenser in both the air and fluid dripping from the condenser are also known. In contrast to nonvolatiles excretion rates of volatiles should not be significantly affected by dilution of airway droplets by condensed water vapor. We would argue that EBC measurements should be largely confined to non-volatile indicators, using a dilutional indicator to estimate concentrations (moles L?1) in the airway fluid. Although excretion of nonvolatile solutes is largely from the airways and is limited by the miniscule volumes of airway fluid in the EBC, estimates of airway concentrations of nonvolatile markers are relatively immune to the multitude of variables listed above and there is no need to measure losses from the condenser. As illustrated in this study, greater cooling could presumably enhance recovery of nonvolatile EBC indicators. Alternatively, droplet formation might be augmented by intentional coughing, depth or rate of respiration, or chest vibration of one sort or another. Salivary contamination should be routinely monitored with sensitive amylase measurements. Of the greatest importance would be the development of sufficiently sensitive assays of EBC biomarkers and dilution. Acknowledgments This work was supported by the National Institutes of Health (grants HL75405 and HD51857). RC occupies the Grancel/Burns Chair in the Rehabilitative Sciences at LA Biomed. 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