Hdl c plus

Lipid profile, C-reactive protein (CRP), total apoJ, Lp-PLA₂ activity, LDL size, and HDL subfraction proportion were determined in plasma obtained in EDTA-containing Vacutainer tubes. The lipid profile included total cholesterol (Roche), triglycerides (Roche), apoB (Roche), and VLDL, LDL, and HDL cholesterol. Cholesterol of lipoprotein fractions was quantified using a direct HDL-cholesterol method (HDL-C plus, Roche) or by ultracentrifugation when the TG concentration was higher than 3 mmol/L, according to the National Cholesterol Education Program [25 (link)]. Non-esterified fatty acids (NEFA) were quantified using a commercial kit (Wako Pure Chemical, Osaka, Japan). All these determinations and CRP (Roche Diagnostics, Basel, Switzerland) were performed in a Cobas 6000/c501 autoanalyzer. ApoJ plasma levels were measured with commercial ELISA kits (Mabtech, Stockholm, Sweden). LDL size and HDL subfraction proportions were evaluated from plasma by non-denaturing polyacrylamide gradient (2.5%–16%) gel electrophoresis, as described previously [26 (link)]. Lp-PLA₂ activity was determined using 2-thio-PAF (Cayman Chemicals, Ann Arbor, MI, USA) as a substrate [27 (link)] according to the manufacturer’s instructions. The distribution of Lp-PLA₂ between lipoprotein fractions was measured by precipitating apoB-containing lipoproteins from plasma with dextran sulfate [28 (link)].

Levels of fasting lipids [total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and triglycerides (TGs)] were measured using in vitro enzyme test kits on a Hitachi system (Cholesterol CHOD-PAD, HDL-C plus, Triglycerides GPO-PAP, and LDL-C plus; Roche Diagnostics GmbH, Mannheim, Germany). The ratios of LDL-C to HDL-C and TGs to HDL-C were calculated as independent markers for cardiovascular risk (27 (link)).
Insulin was measured using an electrochemiluminescence immunoassay (IMMULITE 2000; Siemens Healthcare Diagnostics, NJ, USA). Fasting glucose was measured by enzymatic absorption photometry using a cobas^® 8000 analyzer (via Gluco-Quant; Roche Diagnostics GmbH). Insulin resistance (IR) was defined by using the homeostatic model assessment (HOMA) index, compared with specific pediatric percentiles according to sex and pubertal stage (28 ), and defined by a fasting glucose-to-insulin ratio (FGIR) of less than 7 (29 (link)).
Measurements of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and estradiol (E2) were taken using the ADVIA Centaur^® platform (Siemens Healthcare Diagnostics, NJ, USA) using the chemiluminescent immunometric method.
All biochemical analyses were carried out in the same laboratory.

Blood samples were taken after overnight fasting and immediately transported to the central laboratory of the Gynecologic Obstetrical University Hospital in Poznan for analysis. HbA1c level in whole blood was determined using the turbidimetric inhibition immunoassay (TINIA) (Tina-quant Hemoglobin A1c II test in a Cobas c311 analyser (Roche Diagnostics, Basel, Switzerland)).
The total serum cholesterol, HDL cholesterol and triglyceride (TG) levels were determined with Roche Diagnostics reagents (Cholesterol CHOD-PAP, HDL-C plus, and Triglycerides GPO-PAP, respectively) on a Cobas c501 analyser. The following formula was used to calculate the level of LDL cholesterol: LDL cholesterol = total cholesterol − HDL cholesterol − (TG/5).