Subjects were excluded if they were experiencing viral respiratory tract (common cold) symptoms. Additional exclusion criteria included physician-diagnosed gastroesophageal reflux disease, use of antacids, histamine type 2 antagonists, or proton pump inhibitors in the past week, or if the subject admitted to any acute or chronic systemic illness. All subjects signed informed consent documents, and the study was approved by the University of Virginia Human Investigation Committee.
Samples of EBC were collected during oral tidal breathing for 7 min using a disposable portable collector (Rtube; Respiratory Research Inc; Charlottesville, VA). Subjects did not wear nose clips and reported that they had ingested no liquids or solids in the previous 1 h. The metal sleeves used to chill the breath condenser surface were stored at approximately — 10°C prior to use, a temperature shown to lead to EBC collection as a liquid, not as a solid, on the condenser surface. The majority of samples were collected under supervised conditions, although samples were also collected unsupervised at home. www.canadian-familypharmacy.com
EBC samples were frozen as soon as possible after collection at – 20°C until assay, which was usually performed within 1 week. Each sample was thawed, removed from the portable collector by plunging, and then aliquoted into several microcentrifuge tubes. Argon gas (350 mL/min) was bubbled for 8 min through a 200-^L aliquot of EBC by means of a glass Pasteur pipette inserted into the sample. EBC was kept in constant motion by this process, maximizing exposure of the fluid to the CO2-free gas. This amount of time was previously shown to be sufficient for complete stabilization of sample pH in previous experiments. pH was measured using an Orion glass combination micro pH electrode attached to a Orion 525-A pH meter (Thermo Electron Corporation; Waltham, MA). This pH measurement system was calibrated at pH 4, pH 7, and pH 10 against standard as well as low-ionic-strength calibration buffers prior to each series of assays.