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Dose-Response Relationship and Reproducibility of the Fall in Exhaled Nitric Oxide After Inhaled Beclomethasone Dipropionate Therapy in Asthma Patients: Materials and Methods

NO Measurement: NO was analyzed using a rapid-response chemiluminescent NO analyzer (280 NOA; Sievers Instruments; Boulder, CO). The NO detection limit is approximately 1 ppb for gas, with a response time of 200 ms for 90% full scale. Data were collected via a computerized data collection program. Calibration was performed at each session. The analyzer was zeroed using 100% nitrogen (Praxair; Mississauga, Ontario, Canada) containing < 1 ppb NO. High-point calibration was performed with a commercial NO gas standard containing 2 ppm (Praxair). The analyzer sample line was connected just distal to the mouthpiece and sampled at a flow rate of 150 mL/min.
Feno Measurement Technique: We used a restricted breath technique, which employed exhalation via a high resistance, and positive mouth pressure to close the velum, thus excluding nasal NO. Subjects inhaled medical-grade compressed air (Praxair) that contained < 2 ppb NO and then exhaled via a high expiratory resistance while targeting a mouth pressure of 20 mm Hg. This produced an expiratory flow rate of 45 mL/s (including analyzer sampling rate). Exhalations were repeated until three plateau Feno values varied by < 5%. The mean of the three replicate Feno values was used in all analyses. canadian family pharmacy

Spirometry and Methacholine Challenge: Spirometry was performed according to American Thoracic Society guidelines using a dry rolling seal spirometer (model 130; P.K. Morgan; Gillingham, Kent, UK) and an XY recorder (model 7045A; Hewlett Packard; Palo Alto, CA). Methacholine challenge was performed with a tidal breathing pattern using a hand-held nebulizer according to American Thoracic Society guidelines.
Statistics: The distributions of Feno and PC20 significantly deviated from normal, so data were transformed to log base 10 for Feno and log base 2 for PC20. A mixed model was used to determine if there were significant differences between any of the end points for all treatments. Post hoc analysis revealed that a compound symmetric covariance structure gave the best fit for FEV1 and an autoregressive structure with order 1 + random subject structure was the best fit for PC20 and FVC. Pairwise comparisons were then made among the four dose levels with results adjusted for multiple comparisons using the Tukey-Kramer method. All statistical tests were two sided and conducted at the 5% significance level.
Study B: Reproducibility of the Fall in Feno
Subjects: We recruited 12 different subjects (five men; age range, 18 to 24 years) with a baseline Feno > 80 ppb with identical exclusion criteria as in study A. The inclusion baseline Feno criteria and dose of medication were determined based on conclusions derived from study A.
Study Design: The design was a single-blinded, prospective study conducted over 8 to 12 weeks in four phases: period 1, placebo metered-dose inhaler (1 week); period 2, iBDP at 200 |j.g/d (2 weeks); period 3, washout of medication using placebo metered-dose inhaler (variable period); and period 4, identical to period 2. Each subject served as his or her own control during periods of placebo and active medications. All subjects underwent history and examination, Feno, spirometry, and PC20 at baseline. Feno was measured two to three times weekly in all periods. PC20 was measured at baseline to confirm the diagnosis of asthma, and was not an outcome measure in this study. Period 3 terminated when two sequential Feno measurements had returned to within 15% of the mean Feno recorded during period 1. If period 3 was > 6 weeks, subjects were withdrawn.
Feno Measurement: This was identical to that described in study A.
Statistical Analysis: Multiple measurements of outcome variables within periods were summarized by taking the average of the visits in the baseline period and the average of the last two visits in subsequent periods, for each subject. The data were summarized using arithmetic means and SEs, or geometric means for those variables with distributions that deviated from a normal distribution after log transformation. A mixed-effects model with a compound symmetric covariance structure was used to determine if there were mean differences for the four treatment periods accounting for repeated measures for subjects across periods. Contrasts within the mixed model were used to test for differences in pairs of means and to determine if the change in outcome from period 1 to period 2 was equal to the change from period 3 to period 4. All tests were two sided and conducted at the 5% level of significance. Analyses were performed using software (SAS version 6.12; SAS Institute; Cary, NC) on a personal computer (Windows NT; Microsoft; Redmond, WA).