Methacholine inhalation tests were carried out using a modification of the method described by Chai. At the time of the study, all subjects had been free of acute respiratory tract infections for 4 weeks and were required to stop receiving medications 7 days before the study. In view of the fact that most of the adolescents were sensitized to house dust mites, it was decided to perform the test during the winter season (December to February), when house dust mite numbers are at their lowest and least variable levels in our country. Methacholine (Sigma Chemical; St. Louis, MO) at concentrations of 0.075, 0.15, 0.3, 0.625, 1.25, 2.5,5, 10, and 25 mg/mL was prepared by dilution in buffered saline solution (pH 7.4).
A Rosenthal-French dosimeter (Laboratory for Applied Immunology; Baltimore, MD), triggered by a solenoid valve set to remain open for 0.6 s, was used to deliver the aerosol generated from a DeVilbiss 646 nebulizer (DeVilbiss; Somerset, PA) with pressurized air at 20 pounds per square inch. Each subject inhaled five inspiratory capacity breaths of buffered saline solution, and the study was continued only if the postsaline solution FEV1 was at least 70% of the predicted value. Increasing concentrations of methacholine were then inhaled until the FEV1 fell by > 20% of its postsaline solution value or the highest concentration was reached. The largest value of triplicate FEV1 at 1.5 min after each inhalation was adopted for analysis. The percentage fall of FEV1 from the postsaline solution value was plotted against log concentration of inhaled methacholine. PC20 was calculated by interpolation between two adjacent data points if the FEV1 fell by > 20%. As not all subjects experienced a 20% drop in FEV1 after inhalation up to the highest concentration of methacholine (25 mg/mL), thereby making PC20 calculation impossible, a continuous index of methacholine responsiveness was required for the entire sample. For this purpose, the log of the slope of the percentage decline from baseline FEV1 after the last concentration of methacholine, per unit concentration of methacholine (bronchial responsiveness [BR] index), was calculated for all the subjects, as described by Burrows.
Parents gave written informed consent for their children and themselves to participate in the study. The study protocol was approved by the hospital ethics committee.
Statistical Analysis
Subjects were considered to have BHR if they had a PC20 < 18 mg/mL. Values for PC20 were logarithmically transformed before analysis, and were expressed as a geometric mean and range of1 SD. Other values were presented as mean ± 1 SD. Values or frequencies between the two groups of adolescents or of parents were compared using the Student t test or the x2 test, respectively. In each case, statistical significance was accepted when the two-sided p values were < 0.05.