Exhaled NO was measured by a chemiluminescence analyzer (NIOX; Aerocrine AB; Stockholm, Sweden), which was calibrated according to the instructions of the manufacturer every 14 days. After inhaling NO-free air to total lung capacity, subjects exhaled at a constant flow rate against a resistor to measure the plateau NO concentration. Readings were taken at flow rates of 10, 100, 200, and 260 mL/s by applying resistors of 10, 50, 100, 200, and 300 cm H2O2 mL/s, respectively, to create and maintain the target flow rates. The exhalation times required were 20, 10, 10, 6, and 6 s, respectively, to ensure that the total volume of air exhaled at each flow rate accounted for the exclusion of the dead space.14 The mean amount of exhaled NO at each expiratory flow was calculated by the analyzer during an NO plateau of not less than 3 s with NO variability within 10% of the plateau or ± 1 part per billion (ppb). Jno, Dno, and Calv were calculated according to the following equation:
where the exhaled concentration (Cexh [in ppb]) is a function of the residence time (Tres) of each differential bolus of air in the airway compartment, the volume of the airway compartment (Vair) and the remaining three parameters (Jno, Dno, and Calv).