2.4. Biochemical Analysis
BLOOD
Blood and urine samples were collected early in the morning after a minimum 8 h fast.The following data were determined: Total cholesterol (T-Chol) was quantitatively determined by the colorimetric cholesterol enzymatic test method (CHOD-PAP). Serum levels of HDL-cholesterol were measured by the enzymatic immunoinhibiting method in an Olympus 5800 (Beckman-Coulter, Brea, CA, USA). LDL-cholesterol was calculated with the Friedewald formula. Serum triglycerides were measured with a colorimetric enzymatic method using glycerol phosphate oxidase p-amino phenazone (GPO-PAP). Dimension Vista (Siemens Healthcare Diagnostics, Munich, Germany) was used to measure apolipoprotein B and C-RP by immunonephelometry and nephelometry, respectively. Fasting serum insulin (FSI) was measured by a chemiluminescence immunoassay in an IMMULITE 2000 Xpi (Siemens, Healthcare Diagnostics, Munich, Germany), with an inter-assay accuracy in concentrations of 11 uIU/mL of 6.3% and for insulin concentration of 21 uIU/mL of 5.91. HOMA-IR was calculated as glucose (mmol/L) × insulin (µUI/mL)/22.7. FSG (glucose oxidase) and HbA1c (%) levels were standardized by the International Federation of Clinical Chemistry and Laboratory Medicine, using ion-exchange high-performance liquid chromatography in gradient, with a Tosoh G8 analyzer (Tosoh Co., Tokyo, Japan). Inter-assay imprecision of HbA1c for levels of 5.1% had standard deviation (SD) of 0.06 and coefficient of variation (CV) of 1.23%; for levels of HbA1c 10.39%, SD was 0.11 and CV was 1.04. An external quality guarantee program of the SEQC (Sociedad Española de Química Clínica) evaluated the quality of the methods monthly.