Advia 1600 1800

Blood samples were obtained from each subject early in the morning, following a 10-hour overnight fast by venipuncture from the antecubital vein. The samples were stored in sterile blood collection tubes in refrigerated conditions (4° to 8°C), and then sent to an analytical laboratory for testing according to standardized procedures; (i) hs-CRP, latex enhanced immunoturbidimetric assay (Siemens ADVIA 1800, Erlangen, Germany); (ii) HDL-Cholesterol, Precipitation of the Apolipoprotein B containing lipoproteins with dextran-magnesium-chloride (Siemens Advia 1600/1800 Erlangen, Germany); (iii) Adiponectin, ELISA (Plate Reader); (iv) Glucose, Hexokinase method (Siemens Advia 1600/1800 Erlangen, Germany);(v) Insulin, Chemiluminescence immunoassay (Siemens ACS Centaur System, Erlangen, Germany). All assays were performed in duplicate according to the manufacturers' instructions. The homeostatic model assessment (HOMA) was calculated as the product of basal glucose and insulin levels divided by 22.5, and was used as a proxy measure of insulin resistance (26) .

Blood samples were collected from the antecubital vein, at least 10 h of fasting conditions, refrigerated (4-8 °C), and sent to laboratory for measure inflammatory biomarkers: CRP by the latex-enhanced turbidimetric assay (Siemens Advia 1600/1800, Erlangen, Germany); IL-6 by the chemiluminescence immunoassay (Immulite 2000, Diagnostic Products Corporation, Los Angeles, CA); complement component 3 (C3) and 4 (C4) by the Immunoturbidimetric assay (Siemens Advia 1600/1800, Erlangen, German).
Inflammatory biomarkers were dichotomized, based on sex-and age-adjusted median values, because they have a very skewed distribution, and no cut-offs values are established for adolescents. Categories considered were higher or lower inflammatory state. Medians of each category (lower/ higher) were defined as follows: 0.11/0.92 mg/L for CRP, 1.90/4.20 ng/L for IL-6, 107.00/127.00 mg/dL for C3 and 17.00/25.55 mg/dL for C4.
We created an overall inflammatory biomarker score considering each biomarker categories, assigning one point to those who were above the sex, age-adjusted median or zero for those who were below, and summing all points assigned. The overall inflammatory biomarker score varies from zero to four inflammatory biomarkers above the median. We also create two categories: 0-1 (49.9%) or 2-4 (50.1%) biomarkers above the median.