Ldl c

Each sample was collected, processed, stored and transported in the same way across 9 cities. Venous blood samples were drawn from the study subjects after a 12-h fast. Blood samples were kept in the portable blood refrigerator of 4°C and subsequently centrifuged for 20 min in a tabletop refrigerated centrifuge at 2500 rpm. Identical processing procedures were rigorously controlled for at each testing period. Serum samples were frozen and stored at -20°C in the local health-center and were transported via cold chain system to central laboratory in Beijing and stored at -80°C within one month.
Serum total cholesterol (TC) and triglycerides (TG) were measured by the enzymatic colorimetric method (Roche, Basel, Switzerland). Direct methods were applied to assess high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C) (Roche, Basel, Switzerland). A modified hexokinase enzymatic method was used to detect glucose (Glu) (Roche, Basel, Switzerland), and serum insulin was measured by chemiluminescence immunoassay (CLIA) on the ADVIA Centaur immunoassay system. Insulin resistance was estimated according to homeostasis model assessment (HOMA-IR): HOMA-IR = [fasting glucose (mmol/l) × insulin (U/ml)]/22.5 [27 (link)]. To minimize the effects of assay variability, samples from each twin pair were analyzed using the same assay.

Serum samples were separated by centrifugation at 4 °C at 3500 g for 10 min. The routine laboratory parameters were determined from fresh sera with Cobas c501 analyzer (Roche Ltd. Mannheim, Germany). Total cholesterol levels were measured by using enzymatic, colorimetric tests (cholesterol oxidase-p-aminophenazone – GPOD-PAP; Modular P-800 analyzer; Roche/Hitachi). HDL cholesterol and LDL cholesterol levels were determined by a homogenous enzymatic, colorimetric assay (Roche HDL-C plus 3rd generation and Roche LDL-C plus 2nd generation, respectively). Apo A-I, ApoB and Lp(a) examinations were performed by immunoturbidimetric assays (Tina-quant apolipoprotein A-I ver. 2, Tina-quant apolipoprotein B ver. 2 and Tina-quant lipoprotein(a) ver. 2, respectively). The tests were performed according to the recommendation of the manufacturer. Sera were kept frozen at −70 °C for subsequent lipoprotein subfraction analysis and ELISA measurements.

Clinical laboratory test results, including biochemical indexes and blood routine results, were obtained from routine clinical practice. The clinical laboratory examination results included the following: C-reactive protein (CRP, CRP-1003, Shenzhen Lifotronic Technology, Shenzhen, P.R. China), lactate dehydrogenase (LDH, 05169330190, Roche, Switzerland), blood urea nitrogen (BUN, 05171873190, Roche, Switzerland), cholesterol (CHOL, 05168538190, Roche, Switzerland), triglyceride (TG, 0517407190, Roche, Switzerland), high-density lipoprotein cholesterol (HDL-C, 07528582190, Roche, Switzerland), low-density lipoprotein cholesterol (LDL-C, 07005768190, Roche, Switzerland), albumin (ALB, 05166861190, Roche, Switzerland), white blood cell (WBC, SYSMEX, Japan), lymphocyte count (SYSMEX, Japan), neutrophil count (SYSMEX, Japan), and neutrophil-to-lymphocyte ratio (NLR). All parameters were obtained through standard automated laboratory methods with commercially available kits. The systems used in each laboratory were consistent.