This study was a prospective, multicenter study performed at four referral hospitals in Korea: Seoul National University Hospital, Samsung Medical Center, Ajou University Hospital, and Hanyang University Medical Center. Hospital-based recruitment was performed for asthmatic patients and non-asthmatic controls. Asthmatic patients were defined as those who had been diagnosed with asthma by specialist physicians; they had recurrent symptoms of asthma (wheezing and dyspnea) and used anti-asthmatic medication for 6 months before the enrollment. Non-asthmatic controls were voluntarily recruited from hospital visitors; they were included if they had never wheeze or physician diagnosed asthma.
Common inclusion criteria were Korean adults between the age of 18 and 70 years who could understand and perform lung function tests and bronchial challenges. Exclusion criteria were any one of following: history of recent respiratory infection (within the last 4 weeks), history of a recent surgery, history of heart disease that could impose risks during bronchial challenges, history of uncontrolled hypertension, current smokers or ex-smokers with more than 10 pack-years, history of known pulmonary diseases with the exception of asthma, pregnancy or lactation, severe obesity (body mass index \[BMI\] of \>35 kg/m2), history of any health condition considered inappropriate for participation in this study, or a pre-bronchodilator predictive value of forced expiratory volume in 1 second (FEV1) of \<70%.
All participants underwent methacholine bronchial challenge test and mannitol challenge test on different days, separated by least 24 h, as in previous reports.7, 10 All asthmatic patients stopped their anti-asthma medications before the tests according to the predetermined protocols. The protocol was approved by the Institutional Review Board of Seoul National University Hospital. All subjects gave written informed consent.
Methacholine challenge test\> The methacholine challenge test was performed as previously described.11, 12 Pulmonary function testing was carried out using a spirometry system (SensorMedics 2130; SensorMedics, CA, USA). The methacholine challenge test was performed using the Chai method13 with minor modifications. Briefly, methacholine was prepared at the following concentrations, diluted with saline: 0.25, 0.625, 1, 4, 16, and 25 mg/mL. Methacholine was delivered as an aerosol by a Rosenthal-French dosimeter (Laboratory for Applied Immunology, Inc., Baltimore, MD) and a nebulizer. Subjects were instructed to inhale five inspiratory capacity breaths while increasing the methacholine concentration from 0.25 to 25 mg/mL. The methacholine concentration that caused a 20% decrease in the FEV1 from baseline was defined as PC20. AHR was defined as positive at PC20 \< 16 mg/mL.
Mannitol challenge test\> A commercial mannitol (Aridol™; BL\&H Co., Ltd., South Korea) kit was used, and the challenge test was performed according to the manufacturer's protocols.14 The mannitol capsule dose started at 0 mg and increased to a total cumulative dose of 635 mg. Each capsule was placed in the inhalation device, and a hole was made by pressing the device button before inhalation. After a deep breath of mannitol, the FEV1 was measured after 60 seconds. The test was considered positive if the FEV1 value decreased by more than 15% compared with the baseline FEV1. The cumulative mannitol dose that caused a 15% decrease in the FEV1 from baseline was defined as PD15. If the FEV1 did not decline by more than 15%, the dose was increased until a cumulative dose of 635 mg was reached. If the FEV1 did not fall by more than 15% until the last dose, the test was considered negative.
