- Study subjects
Korea National Health and Nutritional Examination Survey (KNHANES) is a nationally representative cross-sectional survey of non-institutionalized South Korean citizens which is conducted annually by the Korean Center for Disease Control and Prevention (KCDC). Details of the study design and protocol are described in a previous report (14). The survey comprises three main components of a health interview, health examination, and nutritional survey, and the data of socioeconomic status, demographic characteristics, health behaviors, quality of life, biochemical data, and dietary intake are examined. KNHANES is designed as a complex sample survey, and hence, nationally representative data are obtained by stratified multistage clustered probability sampling; complete raw data are freely available to the public on the KCDC website.
In the present study, data of KNHANES Ⅴ conducted from 2010 to 2012 were used, which are the first years with data of all iron indices available, including the serum ferritin level, serum iron level, total iron binding capacity (TIBC), and daily iron intake; the ferritin concentration was examined only for the period of 2007 to 2012. Of 31,596 eligible individuals, 25,534 individuals participated in KNHANES 2010-2012, with response rate of 80.8%. Pulmonary function test (PFT) was performed in 9,281 participants aged over 40 years (4,024 men and 5,257 women). First, 540 men and 3,432 women were identified with both non-smoking and no second-hand smoking. Subsequently, subjects with clinical conditions that may affect serum ferritin level were excluded as follows: Active malignancy (n = 196), treatment for tuberculosis (n = 5), suspicious findings of active tuberculosis or pneumonia on chest radiograph (n = 12), and hepatitis B or C (n = 70), with overlap of subjects in any combination of two categories. In addition, subjects with missing data for variables of interest were excluded. Finally, 342 men and 2,879 women (742 pre-menopausal and 2,137 post-menopausal women) were included in the analysis.
The study protocol was approved by the Institutional Review Board (IRB) of the KCDC (IRB No. 2010-02CON-21-C in 2010, 2011-02CON-06-C in 2011, and 2012-01EXP-01-2C in 2012), and the study was conducted according to the tenets of the Declaration of Helsinki. Moreover, all study procedures were in accordance with STROBE guidelines, and written informed consent was obtained from all individuals before their participation in KNHANES.
- Data collection
Smoking status was categorized into active smokers, passive smokers, and non-smokers based on WHO classification. Non-smokers were defined as subjects who reported smoking <100 cigarettes during their lifetime, and no smoking at the time of survey. Since even self-reported non-smokers may be exposed to second-hand smoking in the workplace or home, the participants with second-hand smoking were confirmed based on their response to the question: Have you ever been exposed to second-hand smoking in work place or at home? Tobacco-naïve population was defined as those who are non-smokers without exposure to second-hand smoking both at work and home.
PFT was performed only in subjects aged ≥40 years by trained medical technicians using Dry rolling seal spirometer (Model 2130, SensorMedics; Yorba Linda, CA, USA). Standardization of spirometry was done based on the criteria of American Thoracic Society and European Respiratory Society (15). Obstructive lung disease (OLD) was defined as the ratio of forced expiratory volume in 1 second (FEV1) divided by forced vital capacity (FVC) of <70% based on the Global Initiative for Chronic Obstructive Lung Disease (GOLD) classification system (16). Restrictive lung disease (RLD) was defined as FEV1/FVC of ≥70% and FVC of <80%.
Information of the subjects’ socio-economic characteristics (age, residential area, education, household income) was collected during face-to-face interviews. Residential area was categorized into two groups (rural or urban); education was categorized into three groups (middle school or lower, high school, and college or more); household income level was categorized into four groups (low, lower middle, upper middle, and high), and calculated by dividing the mean monthly income by the square root of the number of family members. Data of hormonal therapy (HRT) use, including oral HRT or oral contraceptives for at least 1 month was assessed through self-reporting questionnaire.
In all subjects, the laboratory samples were obtained by skilled medical assistants and transported to the Central Laboratory (NEODIN Medical Institute, Seoul, South Korea) that met the quality-control standards of an extramural institute (Hallym University, Seoul, South Korea). Hemoglobin level (g/dL) was measured with XE-2100D (Sysmex; Japan) using cyanide-free method. Serum ferritin level (ng/mL) was measured with 1470 WIZARD gamma-Counter (PerkinElmer; Finland) using immuno-radiometric assay. Serum iron level (µg/dL) and TIBC (µg/dL) were measured with Hitachi Automatic Analyzer 7600 (Hitachi; Japan) using Bathophenanthroline direct method. Iron intake (mg/day) was estimated using 24-hour dietary recall questionnaire validated for good reliability and reproducibility in a previous study (17).
Anthropometric variables such as height and weight were measured by trained medical personnel at the mobile examination center. Body mass index (BMI) was calculated as kg/m2, and was categorized into four groups (underweight, <18.5 kg/m2; normal, 18.5-22.9 kg/m2; pre-obese, 23-24.9 kg/m2; obese, ≥25 kg/m2) based on the Korean Society for the Study of Obesity guideline (18).
- Statistical analysis
Since KNHANES acquired data by multistage, stratified, and probability sampling method to represent the non-institutionalized Korean citizens, all statistical analyses were performed with complex sample analysis method. Moreover, since KCDC assigned sampling weights to account for complex survey design, non-responders, and post-stratification, those sampling weights were applied in all analyses of the current study (14).
The subjects’ general characteristics were compared between men and women using Student’s t-test for continuous variables with normal distribution, chi-square test for categorical variables, and Mann-Whitney test for continuous variables without normal distribution (serum ferritin level and daily iron intake). Dose-dependent relationships between the ferritin level divided into tertiles and spirometry results (predicted FEV1%, predicted FVC%, and FEV1/FVC%) were determined using Jonckheere-Terpstra trend test.
Linear regression analysis was used to evaluate the association between serum ferritin level and lung function. Since daily iron intake, hemoglobin, TIBC, and serum iron level can affect ferritin concentration, and previous studies reported limitations due to insufficient information of the variables (12, 13), those variables were included in the analysis as covariates. In addition, other variables that affect lung function were included (age, height, residential area, and education). Due to left-sided skewed distribution of the serum ferritin, log-transformed value was applied in the linear regression analysis. Multivariate logistic analysis was used to evaluate the odds ratio (OR) for OLD and RLD. Before statistical analysis, multicollinearity test was performed to identify any inter-associations between the variables.
To account for discrepancy between self-reported smoking status and that through biochemical verification, sensitivity analysis was performed on the basis of measurement of the urinary cotinine levels. In the sub-group analysis, cut-off value of 50-ng/mL urinary cotinine was determined for active smokers; hence, urinary cotinine of <50 ng/mL was used to indicate tobacco-naïve population (19).
All tests were two-tailed, and P-value of <0.05 were considered as statistically significant. All statistical analyses were performed using IBM Statistical Package for the Social Sciences (SPSS) for Windows v24.0 (IBM Corp., Armonk, NY, USA).