Biochemical assessment

For biochemical measurements, after 12–14 h of overnight fasting, venous blood samples were collected in vacutainer tubes and centrifuged within 30–45 min of collection for all subjects. The fasting plasma glucose (FPG), high-density lipoprotein-cholesterol (HDL-C), and triglyceride (TG) levels were measured in the TLGS research laboratory on the day of sample collection, using a Selectra 2 autoanalyzer (Vital Scientific, Spankeren, the Netherlands) and commercial kits (Pars Azmoon Inc., Tehran, Iran). FPG level was measured using an enzymatic colorimetric method with the glucose oxidase technique. The inter- and intra-assay coefficients of variation (CV) at baseline and after follow-up were both below 2.3%. TG was also assayed using an enzymatic colorimetric method with glycerol phosphate oxidase. HDL-C was measured after the precipitation of apolipoprotein B-containing lipoproteins with phosphotungstic acid. In all baseline and follow-up assays, intra- and inter-assay CVs were below 2.1% and 3.0% for TG and HDL-C. All samples were analyzed when the internal quality control met the acceptable criteria.