Konelab 20

An automated spectrometric system (Konelab 20 from Thermo Electron Corporation, Cergy-Pontoise, France) was used for biochemical measurements of thawed urine samples, with the manufacturer’s biological chemistry reagents and protocols. The markers measured in urine included amylase, calcium, uric acid, creatinine, glucose, phosphorus, total proteins and urea.

An automated spectrophotometric system (Konelab 20, Thermo Electron Corporation, Cergy-Pontoise, France) was used to measure these parameters, with biological chemistry reagents from the manufacturer or Diagam (Lille, France). Bilirubin was measured with the Thermo Electron Corporation kit. Kits from Diagam were used for measuring total cholesterol, low-density lipoprotein- (LDL-) cholesterol, high-density lipoprotein- (HDL-) cholesterol, triglycerides, alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine, urea, iron, ferritin, transferrin, and ceruloplasmin. Phospholipids B were measured with a kit provided by Diagnostic Partners (Bougival, France).

Fasting plasma concentrations of total cholesterol (CV: 3%), triglycerides (CV: 4%), and FFA (CV: 4%) were measured on an automated analyzer (Konelab 20; Thermo Electron, Waltham, MA). Fasting plasma concentrations of sTNF-R1 (CV: 5%), sTNF-R2 (CV: 4%), leptin (CV: 5%), and adiponectin (CV: 5%) were measured by enzyme-linked immunosorbent assay (ELISA, BioCat, Heidelberg, Germany).

The plasma glucose concentration was measured by using a glucose reagent strip and a glucometer (Glucotouch, Lifescan, Milpitas, CA). Insulin was measured by using an enzyme-linked immunosorbent assay kit purchased from Alpco Diagnostics (Salem, USA). Fasting plasma lipids (ie, triglycerides, total cholesterol and HDL cholesterol) were measured in plasma samples by using an automated system (Konelab 20; ThermoElectron Corporation).

Participants were instructed to come to a fasting state; anthropometric measures and blood samples were collected from all participants by trained nurses. Height (cm), weight (kg), systolic and diastolic blood pressures (SBP and DBP, respectively), waist and hip circumferences (cm) were measured using routine methods by trained nurses [59 (link)]. Body mass index (BMI; kg/m²) and waist–hip ratio (WHR) were calculated. Lipid profile and blood glucose levels were measured using an automated biochemical analyzer (Konelab 20 Thermo-Fischer, Espoo, Finland) using commercially available kits (catalogue nos. 981379, 981812, 981823 and 981301, respectively) [60 (link)]. IL–33 serum levels were measured using available commercial ELISA kit (BioVendor, R&D systems, Brno, Czech Republic) Cat No. RAF064R [61 (link),62 (link)]. According to the manufacturer, intra-assay and inter-assay % CV were less than 4.7% and less than 6.9%, respectively. IL–37 serum levels were measured using Flex MAP 3D System (Luminex Corporation, Austin, TX, USA) using human cytokines Magnetics Bead Panels Cat No. HCYP4MAG-64K Human Cytokine/Chemokine Magnetic Bead Panel IV. Intra-assay and inter-assay % CV were <10 and <15, respectively, according to the manufacturer.

Plasma concentrations of glucose and non-esterified fatty acids (NEFA) were determined using the Infinity Glucose Hexokinase kit (Thermo Electron, Pittsburgh, PA, USA) and the Wako NEFA C kit (Wako Pure Chemical Industries Ltd., Osaka, Japan), respectively. Assays were conducted using a Konelab 20 (Thermo Scientific, Vantaa, Finland). Plasma insulin and leptin concentrations were measured by immunoassay using the ALPCO Insulin (Rat) Ultrasensitive ELISA kit (ALPCO diagnostics, Salem, NH, USA) and the Crystal Chem Rat Leptin ELISA kit (Crystal Chem INC, Downers Grove, IL, USA). All assays were conducted according to manufacturer’s instructions and intra- and inter-assay coefficients of variation were < 10%.