Subjects of this cohort study were selected from participants of the Tehran Lipid and Glucose Study (TLGS), a population-based prospective study performed to determine the risk factors for non-communicable diseases in a sample of residents from District 13 of Tehran, the capital of Iran (20). The first examination survey was performed from 1999 to 2001 on 15,005 individuals aged ≥3 years, using the multistage stratified cluster random sampling technique, and follow-up examinations were conducted every 3 years; 2002–2005 (survey 2), 2005–2008 (survey 3), 2008–2011(survey 4), and 2012–2015 (survey 5) to identify recently developed diseases.
Of 14712 individuals participating in baseline (surveys 3 and 4) of our study, 9057subjects were randomly selected for dietary assessment based on age- and sex-stratified random sampling; of these, 5531 people who aged≥30 years in baseline and had completed data were included and followed until 2014. Of these participants, we excluded Subjects with under- or over-report of energy intake (<800 or ≥4200 kcal/day) (n=227) and also subjects with history of CVD (n=414) at baseline. Finally, after excluding participants missing any follow up data (n=15), 5102 subjects remained and entered the analysis (Figure 1).
All participants signed a written informed consent form before taking part in this investigation. The study was implemented based on the Declaration of Helsinki and the study protocol was accepted by the ethics committee of the Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran. All methods were performed in line with their relevant guidelines and regulations.
2.2. Dietary intake measurements
Dietary assessment was used by a valid and reliable 168-item semi-quantitative food frequency questionnaire (FFQ); trained interviewers collected information on usual dietary intake, through face-to-face private interviews. The consumption frequency of each food item on a daily, weekly, or monthly basis was converted to daily intakes, and portion sizes were then converted grams using household measures. (20). Dietary intakes of vitamins E, C, A, , and zinc were calculated and considered as grams per week.
Physical activity level was assessed using the Persian-translated modifiable activity questionnaire (MAQ) with high reliability and moderate validity (21). Data on the time and frequency of light, moderate, high, and very hard intensity activities were obtained according to the list of common activities of daily life over the past year and these activity data were transformed into metabolic equivalent-hours/week (Met/h/week) (22).
Blood pressure and anthropometric measurements
Systolic and diastolic blood pressure were measured twice (with a 30 sec interval in between) in a sitting position after 15 min of rest.
The body weight was measured to the nearest 100 g, using a digital scale (Seca 707), while subjects were minimally clothed and barefoot. Height was measured to the 0.5 cm by a tape measure, in standing position without shoes and with shoulders in normal alignment. Waist circumference (WC) was measured with a non-flexible tape meter without any pressure to body surface at the level of the umbilicus and was taken at the end of a normal expiration, over light clothing. Measurements were recorded to the nearest 0.1 cm.
Blood samples were drawn into vacutainer tubes between 7:00 to 9:00 a.m., after a 12–14 hour overnight fast from subjects, who were in sitting position and were centrifuged within 30 to 45 min of collection. All biochemical analyses were performed using a Selectra 2 auto-analyzer at the TLGS research laboratory on the day of blood collection. Fasting blood glucose (FBS) concentration was measured on the day of blood collection by the enzymatic colorimetric method with the glucose oxidase technique. The standard 2-h post-challenge blood glucose test was performed using oral administration of 82.5 g glucose monohydrate solution (equivalent to 75 g anhydrous glucose) for all individuals who were not on glucose-lowering drugs.
HDL-C concentration was assessed after precipitation of the apolipoprotein B-containing lipoproteins with phosphotungstic acid. Total cholesterol (TC) and TG were measured using the enzymatic colorimetric method. For the TC assay, cholesteryl ester hydrolase was used to convert cholesteryl ester to cholesterol, which was then oxidized by cholesterol oxidase to cholesterol-4-en-3-one and H2 O2. For the TG assay, TG was broken down to glycerol and free fatty acids using lipoprotein lipase and glycerol was then phosphorylated to glycerol phosphate by glycerokinase; glycerol phosphate was converted to dihydroxyacetone phosphate and H2 O2 by glycerol phosphate oxidase. The Friedewald equation (LDL-C = TC − HDL-C − TG/5) was used to calculate LDL-C concentrations in samples with TG (23).
Details of CVD outcome data have been described elsewhere (24). Coronary heart disease (CHD) events included cases of definite myocardial infarction (diagnostic electrocardiographic [ECG] results and biomarkers), probable myocardial infarction (positive ECG findings plus cardiac symptoms or signs plus missing biomarkers or positive ECG findings plus equivocal biomarkers), proven CHD by angiography, unstable angina pectoris (new cardiac symptoms or changing symptom patterns and positive ECG findings with normal biomarkers), and CHD death. CVD was defined as stroke (a new neurological deficit that lasted more than 24 h), CHD events, or CVD death (fatal stroke or fatal CHD). CVD risk score was measured according to the sex specific ‘general CVD’ algorithms were derived that age, systolic BP, treatment for hypertension, total cholesterol, HDL-C, type 2 diabetes status, and smoking (25). Hypertension was defined as SBP≥140 or DBP≥90 mmHg, or receiving antihypertensive drug treatment (26).
Statistical analyses were carried out using the Statistical Package for Social Sciences (version 21.0; SPSS). A two-tailed P value <0.05 was used to determine statistical significance. We used a Chi-square test for qualitative variables and the Student’s t test for quantitative variables to compare the characteristics between men and women. In case of non-normal nutritional and biochemical variables (triglyceride concentration), log-transformed values were used for statistical analysis. The hazards ratio (HR) and 95% confidence interval of incident CVD were assessed using multivariable Cox proportional hazard regression models. Person-years of follow-up was calculated for each individual from the date of inclusion to the date of diagnosis of CVD, death, or end of the follow-up, whichever came first. Survival time for censored individuals was calculated as the interval between the first and last observation dates. Event date was considered as the middle-time between the date of follow up visit at which the events were diagnosed for the first time, and the most recent follow up visit preceding the diagnosis. The incidence of CVD during the follow up period were considered as dichotomous variables (yes/no) in the models. Vitamin E, vitamin C, vitamin A and zinc intakes were categorized into quartiles, given the first quartile as the reference. The median of each quartile was used as a continuous variable to assess the overall trends of HRs across quartiles of dietary vitamins A, E, C and zinc intakes in the Cox proportional hazard regression models. The proportional hazard assumption of multivariate Cox models were assessed using Schoenfeld’s global test of residuals.
The confounders were selected based on literature; and each confounder was included in the uni-variable Cox regression model. A two-tailed P value <0.20 was used for determining inclusion in the model. The Cox regression models were adjusted for several potential confounders; the analyses were adjusted for age, sex, CVD risk score(continuous), family history of CVD, physical activity (continuous), total energy intake, fiber (gr/1000 Kcal) and total fat (percentage of energy) intakes; in models for estimating HR.