Dilution, Salivary Contamination And Reproducibility In Exhaled Breath Condensate Studies Of Bioactive Purines

Abstract

Corresponding author's email: reffros@labiomed.org: Exhaled breath condensates (EBC) can provide information regarding inflammatory mediators in the fluid lining the airways. RationaleAlthough this noninvasive procedure avoids the risks and costs associated with bronchoalveolar lavage, EBC samples are variably diluted from ~5,000 to 15,000 times by water vapor and they can be contaminated with saliva.These factors were addressed with a pilot study of 10 subjects (5 healthy nonsmokers, 2 healthy smokers, 3 asthmatics) from Methods: whom EBC, saliva, and blood were collected in duplicate on two separate days. EBC was obtained using methods developed for field use, with salivary contamination minimized by insertion of upwardly inclined respiratory tubing between the mouthpiece and condenser. EBC and salivary concentrations of the biomarkers adenosine, adenosine monophosphate (AMP), phenylalanine, and tyrosine plus the dilution marker urea were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and salivary contamination was assessed by comparing salivary and EBC amylase concentrations with a sensitive, fluorometric assay. Airway lining fluid (ALF) concentrations were calculated by multiplying EBC concentrations by the ratios between plasma and EBC concentrations of urea.Salivary contamination of most EBC samples was minimal (1±17% of the respiratory droplet volumes) but exceeded 10% in 5 Results: samples from 3 subjects. EBC biomarkers and urea were readily detected, and although intra-individual variability was relatively high (average coefficients of variation = 0.44 to 0.65), it was reduced by averaging duplicate collections and using plasma:EBC urea ratios to calculate ALF concentrations: Adenosine: 0.41±0.29 μM SD, AMP= 0.43±0.35 μM, phenylalanine= 9.5±5.7 μM and tyrosine = 15.1±10.3 μM. As previously observed, ALF adenosine concentrations in asthmatics exceeded those in normals (p<0.05). Mean concentrations of urea were similar in saliva and plasma, suggesting that salivary samples may be used instead of plasma values, thereby avoiding discomfort of blood collection. Unexpectedly, the calculated concentrations of adenosine, AMP, tyrosine and phenylalanine in the ALF were similar to those measured in the saliva, suggesting that similar mechanisms regulate these parameters.We conclude that EBC can be successfully collected and analyzed using conventional portable equipment, modified to Conclusions: minimize salivary contamination. However, sensitive measures of biomarkers, as well as salivary and dilutional indicators remain essential to accurately determine airway biomarker concentrations.

Exhaled Breath Condensates: Analyzing the Expiratory Plume

“…To ensure that saliva represents less than 10% of ALF volume, less than 10 nl of saliva should be collected in each milliliter of EBC, a daunting requirement. However, this was successfully accomplished in 36 of 40 samples when the mouthpiece was separated from the condenser, as judged by a sufficiently sensitive amylase assay (2).…”