Very Low Density Lipoprotein Cholesterol

Spot urine for analysis of albumin and creatinine was collected in plastic containers. Venous blood samples were drawn after an overnight fast (≥8 hours). Participants without known diabetes underwent a standard 75 g OGTT with blood samples drawn at 30 and 120 minutes. Plasma for analysis of glucose was prepared immediately upon collection in fluoride-heparin coated tubes. Samples were placed on ice before centrifugation at 3000 rpm for 10 minutes at 4°C. Plasma for analysis of creatinine, total cholesterol, HDL-cholesterol, and triglycerides was prepared upon collection in lithium-heparin coated tubes by incubating for 0.5-1.5 hours at room temperature with subsequent centrifugation at 3000 rpm for 10 minutes without cooling. Serum for analysis of insulin was prepared by incubating whole blood for 0.5-1.5 hours at room temperature with subsequent centrifugation for 10 minutes at 3000 rpm without cooling. Whole blood for analysis of HbA_1c and DNA was collected in EDTA coated tubes. Additionally, aliquots of plasma (0, 30, 120 min), serum (0, 30, 120) and spot urine were collected for the ADDITION-PRO biobank. Plasma for the biobank was collected in chilled EDTA coated tubes and centrifuged within 30 minutes at 3000 rpm for 10 minutes at 4°C. Biobank samples were subsequently stored at −80°C.
All biochemical measures were analysed at the Clinical Chemistry Department at the Steno Diabetes Center in Gentofte, Denmark. Serum insulin was measured by immunoassay (AutoDELFIA, Perkin Elmer, Massachusetts, United States). Glycated haemoglobin A_1c (HbA_1c) was measured by HPLC (TOSOH G7, Tokyo, Japan). Between 2009 and 2010, plasma glucose, alanine transaminase, alkaline phosphatase, total cholesterol, HDL-cholesterol, triglycerides, plasma creatinine, urine creatinine and urine albumin were measured using the Hitachi 912 system (Roche Diagnostics, Mannheim, Germany). During 2010, the study laboratory gradually implemented the Vitros 5600 Integrated System (Ortho Clinical Diagnostics, Illkirch Cedex, France). There was modest agreement between the Hitachi 912 and Vitros 5600 instruments. Thus, all ‘Vitros’ values were converted to correspond to ‘Hitachi’ values, using regression equations from validation analyses performed by the study laboratory (Table 3).
Albumin creatinine ratio was calculated using the formula: U-albumin mg/l × 8.84)/(U-creatinine(μmol/l)/1000). VLDL cholesterol (VLDL-C) was calculated using the formula: VLDL-C = triglycerides (mmol/l)/2.2). VLDL-C was calculated only for triglyceride values ≤ 5.05 mmol/l. LDL cholesterol was calculated using Friedewald’s equation (LDL-C = TC – VLDL-C – HDL-C mmol/l) [24 (link)]. LDL-cholesterol was calculated only for triglyceride values ≤ 4.55 mmol/l.
Collection of whole saliva was performed at one of the study centres (Steno Diabetes Centre). Participants were asked not to brush teeth on the day of the health assessment. Upon arrival, participants were instructed to chew on paraffin wax for approximately one minute and then to empty their mouth of saliva. The participants were then instructed to chew on the paraffin wax for a further three minutes whilst spitting into a collection container whenever needed. The collected saliva was divided into two cryotubes. One tube was stored immediately at −80°C. RNAlater (Ambion, Austin, TX) was added to the second tube in a 1:3 ratio (saliva:RNAlater) and then refrigerated for approximately 24 hours before being transferred to −80°C storage.

Serum lipids [total cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, very low-density lipoprotein (VLDL) cholesterol, and triglycerides] were quantified using standard enzymatic methods (Roche, Basel, Switzerland). Fasting serum insulin was measured by radio-immuno assay (Diagnostic Products, Cypress, USA). All quantitative parameters were determined by following manufacturer's instructions using a Hitachi 902 auto-analyzer (Roche, Basel, Switzerland).

Eligible participants were randomized in a 2:2:1:1 ratio to receive subcutaneous tafolecimab 150 mg Q2W, tafolecimab 450 mg Q4W, placebo Q2W or placebo Q4W, respectively, in the 12-week double-blind treatment period. Randomization was implemented by an interactive web response system and was stratified by LDL-C levels at screening (≥ or < 4.8 mmol/L), by baseline ezetimibe use (yes/no) and by prior use of PCSK9 inhibitors (yes/no). The participants, investigators and study site personnel involved in treating and assessing participants were masked to treatment allocations.
After the 12-week double-blind treatment period, participants receiving tafolecimab continued to receive open-label tafolecimab with the previous regimens while participants receiving placebo Q2W or Q4W crossed over to receive open-label tafolecimab 150 mg Q2W or 450 mg Q4W, respectively, for 12 weeks, followed by an 8-week safety follow-up.
Fasting LDL-C (OSR6183, Beckman Coulter), HDL cholesterol (HDL-C, OSR6187, Beckman Coulter), total cholesterol (OSR6116, Beckman Coulter) and triglycerides (OSR61118, Beckman Coulter) concentrations were measured by commercial kits on a Beckman Coulter AU600 Chemistry Analyzer. Non-HDL cholesterol (non-HDL-C) concentration was calculated by subtracting HDL-C concentration from total cholesterol concentration. Very low density lipoprotein cholesterol (vLDL-C) concentration was calculated by dividing the triglyceride concentration by 5 (when triglyceride < 4.52 mmol/L) or by subtracting HDL-C and LDL-C concentration from total cholesterol concentration (when triglyceride ≥4.52 mmol/L). Apolipoprotein A1, apolipoprotein B and lipoprotein(a) concentrations were determined using a nephelometric method (OUED, OSAN and OQHL, respectively, Siemens BN ProSpec System). We measured unbound PCSK9 concentrations using an in-house developed enzyme-linked immunosorbent assay. All lipids and PCSK9 were tested in a central laboratory (WuXi AppTec, Shanghai).
DNA extracted from blood samples of all enrolled participants were sequenced by Novogene (Beijing) for variants in all exons of LDLR, APOB, PCSK9 and LDLRAP1 gene. Pathogenicity of variants were annotated using clinical classification of the Leiden Open Variation Database (LOVD) [17 (link)].

The plasma concentration of VLDL-cholesterol (VLDL-c) was calculated according to Friedewald’s equation, expressed below [27 (link)]: $$\mathrm{VLDL-}\mathrm{c}(\mathrm{mg}/\mathrm{dL})=\frac{\mathrm{Triglycerides}}{5}$$

Fasting blood glucose concentrations were measured by ACCU-CHEK^® Performa (Roche, Basel, Switzerland, Germany). Plasma concentrations of cardiometabolic risk factors, including triglycerides (TG), total cholesterol (TC), HDL-cholesterol (HDL-C), and LDL-cholesterol (LDL-C) were measured using a 3100 automatic biochemistry analyzer (Hitachi, Tokyo, Japan), with enzymatic or immunoturbidimetric reagents as per the manufacturer’s protocols. Plasma concentrations of VLDL-cholesterol (VLDL-C) were calculated as VLDL-C = TG/2.2 [27 (link)], and non-HDL-cholesterol (non-HDL-C) was calculated as non-HDL-C = TC − HDL-C [28 (link)]. Plasma PCSK9 concentrations were determined with an ELISA kit (Sino Biological, Beijing, China) according to the manufacturer’s protocol.