Ten healthy lifetime non-smokers participated in the study. The exclusion criteria were as follows: 1) a physician diagnosis of cardiovascular or pulmonary disease; 2) the use of cardiovascular or airway medication; 3) a body mass index >30; and 4) a forced expiratory volume in 1 second (FEV1) < 80% of predicted and FEV1-to-forced vital capacity ratio < 0.7. All subjects had been free of an acute respiratory infection for at least 4 weeks before beginning the study, and no subject had an acute respiratory infection during the study. The study was approved by the Western Institutional Review Board and by the Human Subjects Research Office at the University of Miami. A signed informed consent was obtained from the subjects. The study is registered at clinicaltrials.gov: NCT01216748.
Airway blood flow (Q̇aw)
A previously validated soluble inert gas uptake method was used to measure Q̇aw [8,9]. The subjects first inhaled room air to total lung capacity. After exhaling 500 mL, they rapidly re-inhaled the same volume of a pre-mixed gas consisting of 10% dimethylether (DME), balance nitrogen. After a predetermined breathhold time, the subjects then exhaled through a critical flow orifice to standardize the expiratory flow. During the entire maneuver, the instantaneous concentrations of DME and nitrogen were measured at the airway opening with a mass spectrometer (Perkin-Elmer; Pomona, CA). The maneuver was performed with two breathhold times each of 5 and 15 sec in random order. The DME concentration (FDME) at the end of phase 1 of the nitrogen wash-in curve (defining a virtual anatomical dead space, VD) was obtained. The difference in FDME between the two breathhold times (∆FDME) multiplied by VD was used to calculate DME uptake (V̇DME) over the intervening 10 sec. From V̇DME, the mean DME concentration between the two breathholds (FmDME) and the solubility coefficient for DME in blood and tissue (α), was calculated using the Fick principle (Q̇aw = V̇DME/(α•FmDME). Q̇aw was normalized for VD; therefore, VD cancels out and wasn’t measured. Q̇aw was expressed as μl.min-1.mL-1, where μl.min-1 reflects blood flow and mL reflects the virtual anatomical deadspace. At each Q̇aw determination, data from two 5 sec and two 15 sec breathholds were analyzed. A Q̇aw determination took less than 5 min.
Blood pressure and arterial oxygen saturation (SaO2) by pulse-oximetry were monitored at each measurement point. Mean systemic arterial pressure (perfusion pressure for airway blood flow) was calculated as diastolic pressure plus 1/3 pulse pressure.
For spirometry (Forced Expired Volume in one second/FEV1, Forced Vital Capacity/FVC, FEV1/FVC), a Koko spirometer was used (Ferraris Respiratory, Louisville, CO). The tracing with the highest FVC of three forced vital capacity maneuvers was analyzed. Predicted normal values were taken from Crapo et al . The values were expressed in absolute values and percent of predicted.
Exhaled Breath Condensate (EBC) pH was obtained as recommended by an American Thoracic Society/European Respiratory Society task force . The EBC samples were collected with the condenser temperatures close to 0°C. We determined EBC pH immediately following sample collection without argon purging , using a Thermo Orion 3 Star pH Meter and Micro pH Electrode (Thermo Scientific Orion Inc., Carlsbad, CA). During the different breathing maneuvers, EBC samples were collected by directing the subject’s exhaled breath into a pre-cooled (-10°C) tube for 5 min, using the disposable R-tubes® from Respiratory Research System (Charlottesville, VA). Over this period of time, approximately 0.5-1 mL of condensate was collected. For further standardization, the subjects were not allowed to drink or eat for at least one hour before the EBC samples were collected [13,14].
Compressed air was lead through a calibrated airflow regulator (Dakota Instruments, Orangeburg, NY) and an anesthesia bag to a one-way valve at the mouthpiece. During the ventilatory maneuvers, the airflow was adjusted to keep the anesthesia bag from collapsing or overinflating until a steady state was reached . The airflow was read at that point and expressed as l.min-1. The system had a deadspace of 100 mL between the mouthpiece and the valve separating inspiration from expiration. Subjects wore a nose clip for all measurements.
Different respiratory maneuvers were used to change airway pH as reflected by EBC pH.
The same measurements were made in all subjects during quiet breathing, hypercapnic hyperventilation, hypocapnic hyperventilation and eucapnic hyperventilation. To induce hypercapnic hyperventilation, we employed a modification of a previously described procedure . While monitoring SaO2 using pulse oximetry and end-tidal CO2 by mass-spectrometry (Perkin-Elmer, Pomona, CA) on a breath by breath basis, CO2 was bled into the inspired air to achieve an end-tidal pCO2 of at least 55 mmHg, expected to result in a decrease in systemic pH of about 0.1 pH units. For hypocapnic hyperventilation, the subjects were instructed to breathe fast and deep until their end-tidal pCO2 fell to 30 mmHg, corresponding to a systemic pH increase of about 0.1 pH units. For eucapnic hyperventilation, the subjects were instructed to increase their ventilation to the highest level of ventilation recorded in the previous two hyperventilation maneuvers, while CO2 was bled into the inspired air to maintain end-tidal pCO2 at 40 mmHg. This maneuver was used to separate the effect of ventilation from the effect of pH on albuterol responsiveness. The same mouthpiece set-up was used for the measurement of Q̇aw, EBC pH, and ventilation.
The subjects were instructed to abstain from ingesting alcoholic beverages the night before each study day and not to ingest caffeinated drinks for at least 12 hours before the study. The subjects were also instructed not to use phosphodiesterase type 5 inhibitors for 12 hours before coming to the laboratory.
There were 6 visit days. On day 1, informed consent was obtained and the subjects underwent a physical examination to ensure good general health. In females, a urine pregnancy test was performed to rule out current pregnancy. Then, spirometry was performed to ensure normal lung function. For technical reasons, EBC pH, Q̇aw responses to albuterol and the level of ventilation could not be assessed simultaneously during the breathing maneuvers. Therefore, these parameters were measured during different breathing maneuvers on different days in random order (quiet breathing, hypercapnic hyperventilation, hypocapnic hyperventilation and eucapnic hyperventilation.
Exhaled breath condensate collection
For each respiratory maneuver, the subjects breathed at the respective ventilatory level for 2 minutes followed by a 5 minutes EBC collection while maintaining the same breathing pattern.
Determination of ventilation
This was done during the different respiratory maneuvers as described for the EBC collection. Ventilation was measured during the 5 min steady state period.
Q̇aw response to albuterol
This was done during the four breathing protocols as described above. During the 5 min steady state breathing period, Q̇aw was first measured with a short break in the breathing maneuver. After resuming the designated breathing maneuver, the subjects inhaled albuterol (180 μg) delivered by a metered dose inhaler using a holding chamber during a brief interruption of the breathing maneuver. The subjects then continued to perform the prescribed respiratory maneuver for another 5 min. Q̇aw was again measured 15 min after drug administration during quiet breathing. Albuterol responsiveness was expressed as the difference between pre-and post albuterol Q̇aw (ΔQ̇aw).
Values are presented as mean ± standard error (SE). Differences between the groups were analyzed by a non-parametric Kruskal-Wallis ANOVA test followed, when significant, by the Mann-Whitney U test for comparisons between groups. Values were expressed as mean ± SE and a p value less than 0.05 was accepted as a statistically significant difference. All statistics were analyzed with SPSS software (Statistical Product and Services Solutions, version 18.0; SPSS Inc., Chicago, IL).