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

Abstract

Exhaled breath condensates have been widely used to detect inflammatory mediators in the fluid that covers airway surfaces of patients with inflammatory lung disorders. This approach is much less invasive than bronchoalveolar lavage, but respiratory droplets are markedly diluted by large and variable amounts of water vapor. We estimated the dilution of respiratory droplets by comparing concentrations of nonvolatile, reference indicators (total nonvolatile cations, urea or conductivity) in 18 normal subjects with normal plasma concentrations by assuming similar concentrations in the respiratory fluid and plasma. The volatile cation, NH4+ (most of which is delivered as NH3 gas from the mouth), represented 93 +/- 3% (SEM) of the condensate cations. More than 99% of the NH4+ was removed by lyophilization, making it possible to use conductivity to estimate total nonvolatile ionic concentrations and facilitating analysis of urea. Conductivity was significantly correlated with electrolyte and urea concentrations. Estimates of dilution based on total cations, conductivity, and urea were not significantly different (cations: 20,472 +/- 2,516; conductivity: 21,019 +/- 2,427; and urea: 18,818 +/- 2,402). These observations suggest that the conductivity of lyophilized samples can be used as an inexpensive, simple, and reliable method for estimating dilution of nonvolatile, hydrophilic mediators in condensates.

Determining Urea Levels in Exhaled Breath Condensate with Minimal Preparation Steps and Classic LC-MS

“…These levels are comparable with those found in other studies of EBC urea in children (21). Assuming 0.2 g/L of urea in the plasma (11,35), and thus the airway fluid, and using the following equation to calculate dilution of the airway fluid in EBC: Dilution urea ¼ Urea airway fluid Urea EBC ;…”

“…A key issue related to the EBC method is the dilution of the lung lining component of EBC, which contains the biomarkers of interest, by condensed water vapor. Exhaled droplets of lung lining fluid are condensed along with water vapor during tidal breathing, and the dilution of the biomarkers from the lung lining fluid by water vapor can vary by orders of magnitude between study subjects (11). An ideal dilution marker would be one that is evenly distributed throughout the body and is not directly related to lung inflammation so that its concentration in the respiratory fluid may be compared with its concentration in the plasma to determine dilution in the respiratory fluid.…”

“…An ideal dilution marker would be one that is evenly distributed throughout the body and is not directly related to lung inflammation so that its concentration in the respiratory fluid may be compared with its concentration in the plasma to determine dilution in the respiratory fluid. Several biomarkers have been proposed to correct dilution effects, including urea (12, 13), tyrosine (14), K þ and Na þ and conductivity (11,15). K þ and Na þ measurements are used as approximation for total cations in the respiratory fluid and conductivity used as an estimate of respiratory electrolytes.…”

“…One complication in using conductivity as a dilution marker is the high levels of NH 4 þ present in the majority of EBC samples; a product of NH 3 gas in the mouth, it makes up 93 + 3% of all cations in the EBC. Using conductivity as a dilution marker, then requires the extra steps of lyophilization and passage through an ion exchange column to remove NH 4 þ from the samples (11). The amino acid, tyrosine, has the advantage of occurring at easily detectable levels in most EBC samples; however, more validation studies are needed.…”

“…Whereas some commercially available chemical, colorimetric assay kits do not appear to be sensitive enough to detect urea in EBC (14), other methods have been more successful when used in conjunction with various forms of pretreatment and sample concentration (9,16,17). Effros et al (11) measured urea concentrations based on the transformation of urea to NH þ 4 following treatment with urease, but this required lyophilization and passage through an ion exchange column to remove pre-existing NH 4þ from the samples before urease treatment. Gas chromatography -mass spectrometry (GC -MS) and liquid chromatography -mass spectrometry (LC -MS) methods have been developed for the detection of urea, many of which involve a derivatization step (e.g., 19 -29).…”