Cobase 501

Blood samples were drawn from the antecubital vein at the hours of 6 to 8 AM, after at least 10 hours of fasting and avoidance of alcohol. Two sets of fasting blood samples were collected separately from each subject in sodium fluoride potassium oxalate tubes (for glucose) and lithium heparin vacuum tubes (for lipids).The latter collections were then centrifuged and kept at −80°C until analysis. Fasting glucose was immediately determined by One Touch Ultra 2 (LifeScan, USA). Serum levels of HDL- cholesterol, LDL- cholesterol, and triglycerides were measured by enzymatic assay (CHOD-PAP, Roche Diagnostics GmbH). All analyses were carried out through an automatic biochemical analyzer (COBASE 501, Roche Diagnostics GmbH). Subjects were further divided into two groups, cases with MS and controls without MS, according to the IDF definition. For a subject to be defined as having MS, they must have: Central obesity (waist circumference ≥ 90 cm in males, ≥ 80 cm in females, or BMI is >30 kg/m²) along with any two of the following four factors: triglycerides ≥ 150 mg/dL (1.7 mmol/L); HDL-cholesterol < 40 mg/dl (1.03 mmol/L) in males, < 50 mg/dL(1.29 mmol/L) in females; blood pressure, systolic BP ≥ 130 or diastolic BP ≥ 85 mmHg; fasting glucose ≥5.6 mmol/L (100 mg/dl) [1 (link)].

Total cholesterol (Chol), high-density lipoprotein cholesterol (HDL-C), and low density lipoprotein cholesterol (LDL-C) contents were assessed. As a measurement of liver damage, serum alanine (ALT) activity was also quantified. Enzymatic colorimetric kits (Roche Diagnostics GmbH; Mannheim, Germany) were used to measure these parameters in a Roche HITACHI Cobas e501 instrument (Roche Diagnostics GmbH; Mannheim, Germany) accordingly to manufacturer’s instructions by enzymatic colorimetric assay (Roche Diagnostics GmbH; Mannheim, Germany). ALT/AST activity in cell supernatants upon the treatments described before, were assessed by Alanine Aminotransferase Activity Assay Kit and Aspartate Aminotransferase Activity Assay Kit (Sigma-Aldrich^®, St. Louis, MO, USA). The amount of pyruvate and glutamate quantified with these colorimetric kits are respectively proportional to ALT and AST enzymatic activity in each sample. The assay and the kinetic measurements were performed following manufacturer’s instructions. The absorbance for ALT was measured at 570 nm and for AST at 450 nm using an EnSpire^® Multimode Plate Reader (Perkin Elmer).

Routine laboratory tests and the method to measure blood cell fractions were performed as described previously [19 (link), 20 (link)]. The CRP (reference: 0-0.5 mg/dl) was measured by using turbidimetric inhibition immunoassay with the CRPL3 kit (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer’s manual with the automatic analysis system (Cobas e 501, Roche, Switzerland). In addition, PCT (reference: <0.05 ng/ml) and IL-6 (reference: 0–7 pg/ml) were measured via chemiluminescence with the Elecsys BRAHMS PCT kit and Elecsys IL-6 kit (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer’s manual with the automatic analysis system (Cobas e 601 and Cobas e 801, Roche, Switzerland, respectively). The NLR and LMR were calculated as described previously [21 (link)].