Cobas mira plus autoanalyzer

Blood samples were obtained after a 10 h fast. The serum was immediately frozen at −120 °C in liquid nitrogen for transport and stored at −80 °C for final conservation. Total cholesterol and high-density lipoprotein (HDL) cholesterol were analyzed by standardized enzymatic methods (Roche Diagnostic, Basel, Switzerland) adapted to a Cobas Mira Plus autoanalyzer (Hoffmann-La Roche, Basel, Switzerland). Smoking was self-reported by standardized questions.

Complete anamnesis and anthropometric data, including sex, age, clinical history, and medication, were entered into our database after physical examination. Weight and height values were used to determine the BMI. The carotid intima-media thickness (cIMT) of the right and left common carotid arteries was assessed using a MyLab 60-X Vision sonographer (Esaote, Genova, Italy), and the mean cIMT was determined by averaging the readings from the two carotid arteries. cIMT more than 1.5 mm or protrusions into the lumen that were 50% thicker than the cIMT around them were classified as plaques. Blood samples taken from each participant were prepared for storage at −80 °C in our center’s BioBank prior to usage. Standard biochemical parameters including lipids, apolipoproteins, glucose, and high-sensitivity C-reactive protein (hsCRP) were measured utilizing colorimetric, enzymatic, and immunoturbidimetric assays (Spinreact SA, Spain; Horiba SA, Spain), which were adapted for the Cobas Mira Plus Autoanalyzer (Roche Diagnostics, Spain). Plasma levels of insulin were determined with enzyme-linked immunosorbent assays (ELISAs) following the corresponding manufacturer’s instructions (Mercodia AB, Uppsala, Sweden). Insulin resistance was calculated using the homeostasis model assessment of insulin resistance (HOMA-IR): [fasting insulin (µU/mL) × fasting glucose (mg/dL)/405] [23 (link)].

Anamnesis and anthropometric data, including sex, age, clinical history and medication, were recorded and included in the research institute database. BMI was calculated from weight and height measurements (kg/m²). Standard biochemical parameters, including lipids, apolipoproteins, Lp(a), blood glucose and hsCRP, were measured using colorimetric, enzymatic and immunoturbidimetric assays (Spinreact, Girona, Spain; Horiba, Kioto, Japan), which were adapted for automation by clinical chemistry Cobas Mira Plus Autoanalyzer (Roche Diagnostics, Basilea, France).
The standard LDL cholesterol concentration was calculated using the Friedewald formula [62 ], and the remnant cholesterol concentration was calculated by subtracting LDL and HDL cholesterol from total cholesterol.
Circulating PCSK9 was measured by enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA) following the reagent manufacturer’s instructions.

Fasting venous blood samples were collected in EDTA tubes and centrifuged immediately for 15 min at 1500× g and 4 °C to obtain plasma. The samples were then divided into aliquots and stored at −80 °C until analysis.
Standard laboratory methods were used to quantify the total cholesterol, triglycerides and HDL cholesterol contents. LDL cholesterol values were calculated using the Friedewald formula [21 (link)]. Apolipoproteins were quantified using an immunoturbidimetric assay with specific antibodies against apolipoprotein A1 (Apo A1), apolipoprotein B100 (Apo B100) and apolipoprotein CIII (Apo CIII). These analyses were adapted for the Cobas-Mira-Plus autoanalyzer (Roche Diagnosis, Barcelona, Spain).

Animals were fasted for 4h, and a blood sample was collected. Plasma samples were analyzed for triglycerides, total cholesterol, HDL cholesterol (HDLc), glucose (Spinreact; Barcelona, Spain), and non-esterified fatty acids (NEFAs) (Wako; Osaka, Japan) via standardized colorimetric methods adapted to the Cobas Mira Plus Autoanalyzer (Roche Diagnostics, Barcelona, Spain). The low-density lipoprotein cholesterol (LDLc) concentration was calculated using the Friedewäld formula, and the concentration of VLDLc was determined by the formula total cholesterol—(HDLc + LDLc). Plasma levels of insulin, resistin, leptin, and adiponectin were determined by commercial ELISA Kits (Milliplex^®, Millipore; Billerica, MA, USA). The homeostatic model assessment (HOMA) index was calculated as previously described [23 (link),63 (link)].

Total cholesterol, triglyceride, and HDL-cholesterol fractions were determined from fasting blood samples obtained immediately before surgery (prior to anesthesia) using an enzymatic method (Hoffmann-LaRoche, Basel, Switzerland). C-reactive protein (hsCRP) was measured by immunoturbidimetric assay using a Cobas Mira Plus autoanalyzer (Hoffmann-LaRoche, Basel, Switzerland).