This study was conducted in the department of Physiology, College of Medicine and King Khalid University Hospital, King Saud University, Riyadh, Saudi Arabia. Written informed consent was obtained from all subjects and the project was approved by the College of Medicine Ethics Review board. We recruited 12 healthy nonsmoker male adults aged 36.6 ± 2.7 (21-50) years. All subjects were free from acute or chronic respiratory infections and allergies. They were included in the study if they were nonsmokers, had no recent or current upper respiratory tract infection, were not on any medications, and had neither atopy nor clinical manifestations of allergic diseases. In addition, all subjects included in the study had normal ventilatory functions on spirometry and serum IgE levels. Patients were excluded from the study if they had physician diagnosis of respiratory disease, symptoms of respiratory disease in the last one year, or were on inhaled medications.
They were called at 8:00 am in the morning and baseline values of FENO were recorded which acted as control values. They had not taken tea or coffee in the morning and had taken similar light breakfast. They were given three traditional cups (about 180 ml) of freshly prepared Arabian Qahwa to drink and then after every 30 minutes, serial levels of FENO were recorded for a period of 4 hours. The ingredients of the Arabian Qahwa prepared were water, ground coffee beans, cardamom (coarsely ground), ginger powder, and saffron.[11
Measurements of fractional exhaled nitric oxide
FENO measurements were performed according to the present recommendations of American Thoracic Society[12
] using a NOX EVA 4000 chemiluminescence analyzer (SERES-FRANCE) with a sensitivity of 1 part per billion (ppb). All subjects were asked to refrain from eating, drinking, and strenuous exercise for 2 hours before FENO measurement. The history of recent meals was also recorded to avoid any alteration in results by nitrate-containing foods. As an additional precaution, all tests were performed at the same time of the day between 08:00 am and 12:00 noon to minimize possible circadian effects. Using online visual monitoring, the subjects were asked to inhale from residual volume to total lung capacity and then performed a slow expiratory vital capacity maneuver with a constant standardized expiratory flow rate of 0.05 l/sec (±10%) resulting in an expiration time of about 20 seconds, into a Teflon cylinder connected to 3-mm Teflon tubing, without clipping the nose.
To exclude nasal NO contamination, an expiratory resistance of 10 to 20 cm H2O was applied. This expiratory resistance was measured by a special pressure sensor (SAMBA 3200 pressure measurement system) connected to restricted breathing configuration set up (Samba Sensors, Vastra Frolunda, Sweden). The subjects inspired from NO-free air and expired in restricted-breath configuration set up. The expiratory flow rate was measured by data acquisition system BIOPAC MP-100 (Biopac Systems Inc, USA). Plateau levels of FENO against time were determined and expressed as ppb.
Mean exhaled NO concentrations were determined between 5 and 15 seconds after start of the expiration. Three successive recordings were made at least at 1 minute intervals and the mean was used in analysis. To ensure standardization and reproducibility, the acceptable variation between the tests was kept less than 10%. NO measurement set up was calibrated before each test using a standard NO calibration gas. Ambient NO levels were recorded and, if >40 ppb, the analyzer was flushed with NO-free gas.
We used SPSS version 19.0 to perform the data analysis. The data were presented as Mean ± SD and as percentage values. We used test of normality to check if FENO data were following normal distribution or not by using Kolmogorov-Smirnov test of normality and observed that our data were not following normal distribution. Therefore, we used non-parametric Friedman test and Wilcoxon Signed Ranks test to compare between FENO data at different time intervals. Friedman test was used to compare between the data at different time intervals and the difference was significant (P<0.0001). Comparison of FENO between different time intervals and baseline was analyzed by Wilcoxon signed-rank test. Significant difference was considered if P<0.025 (two tailed).