Konelab 20xti analyzer

Venous blood samples were taken in standardized fasting condition (12 h) in the morning (7–9 a.m.). Serum samples were stored frozen at −80 °C until analyzed. Serum triglycerides, total cholesterol and high-density lipoprotein (HDL) were determined by using KONELAB 20XTi analyzer (Thermo Fischer Scientific Inc, Waltham, MA, USA) and described previously [18 (link)]. The intra- and inter-assay correlation coefficients (CVs%) were 3.4% and 2.9% for triglycerides. Serum leptin was assessed using human leptin (ELISA; Diagnostic Systems Laboratories, Inc., Webster, TX, USA). The inter- and intra-assay coefficients of variation (CVs%) were 2.2% and 2.7% for leptin, respectively.

Venous blood samples for biochemical analyses were taken in standardized fasting conditions in the mornings between 7 am and 9 am. Serum samples were stored frozen at -80°C until analyzed. Serum glucose, total cholesterol, HDL, triacylglycerol, alanine amino transferase (S-ALAT); aspartate amino transferase (S-ASAT) and gamma glutamyltransferase (GGT) were analyzed using the KONELAB 20XTi analyzer (Thermo Fischer Scientific inc. Waltham, MA, USA). Insulin was determined by immunofluorescence using the IMMULITE Analyser (Diagnostic Products Corporation, Los Angeles). The homeostasis model assessment of insulin resistance (HOMA-IR) index was calculated as (fasting insulin concentration × fasting glucose concentration)/22.5. The inter- and intra-assay CVs were 2.0% and 3.7% for glucose and 11% and 3.4% for insulin, respectively.

Blood pressure and anthropometrics were measured after overnight fasting. SBP and DBP was measured twice in a sitting position after 10 min rest using Omron M6 Comfort (Omron Healthcare, Kioto, Japan) with a standard size cuff and the mean values of the measurements were used. Waist circumference was measured in light underwear midway between the superior iliac spine and the lower rib margin, and hip circumference at the level of the greater trochanters [21 (link)]. Body mass and height were measured with standard procedures and BMI was computed by dividing the body mass with squared body height. Total body fat mass and percentage, android fat mass, and fat free mass were assessed with dual-energy X-ray absorptiometry (DXA; LUNAR, GE Healthcare, Chicago, IL, USA).
Serum samples collected during menopausal status assignment were also used for outcome variable analysis. Serum glucose, high- (HDL-C), low-density lipoprotein cholesterol (LDL-C), total cholesterol, and triglycerides were measured with KONELAB 20 XTi analyzer (Thermo Fischer Scientific, Vantaa, Finland).
The updated ATP III criteria for MetS risk factors was used [1 (link)]. The defining levels for risk factors were ≥88 cm for waist circumference, ≥130/≥85 mmHg for blood pressure, ≥1.69 mmol/l for serum triglycerides, ≥5.6 mmol/l for blood glucose, and <1.29 mmol/l for HDL-C.

The standard parameters were established as follows: total soluble solids (TSS) was measured by a digital automatic refractometer (Abbemat, Anton Paar^®, St. Albans, Austria). The total saccharide content was enzymatically determined using a Konelab 20XTi analyzer (Thermo Fisher Scientific, Schwerte, Germany) and its proper kits (EnzytecTM fluid, Thermo Fisher Scientific). Total acidity and pH were analyzed by a Schott titrator (Titroline alpha plus, SI-Analytics, Texas City, TX, USA). The content of total phenols was evaluated by the Folin assay (Singleton and Rossi 1965) using an automatic analyzer (Konelab 20XTi, Thermo Fisher Scientific). Amino acids were determined by an Amino Acid Analyzer S433 (Sykam GmbH, Eresing, Germany). All measurements were made in triplicate.

Blood samples were collected in the morning between 07:00 and 09:00 h after an overnight fast at each time point. In girls with regular menses, blood sampling was performed in early follicular phase (between 2 and 5 days after the initiation of the menstrual bleeding) (13 (link)). Serum was extracted by centrifugation and stored immediately at −80°C until analysis. The samples from different time points were analyzed by one technician using the same kits and instrument. Estradiol (E2), testosterone and SHBG were determined by ELISA (NovaTec Immunodiagnostica GmbH, Dietzenbach, Germany). Inter- and intra-assay coefficients of variation were 3.2 and 5.4% for E2, 3.9 and 6.2% for testosterone and 1.1 and 1.1% for SHBG, respectively. Fasting plasma glucose was analyzed using the KONELAB 20XTi analyzer (Thermo Fischer Scientific Inc.) and fasting serum insulin was determined by immunofluorescence using the IMMULITE Analyzer (Diagnostic Products Corporation, Los Angeles, USA). Insulin-like growth factor 1 (IGF-1) was assessed using time-resolved fluoroimmunoassays (IMMULITE, Siemens Healthcare Diagnostics). The homeostatic model assessment of insulin resistance index (HOMA-IR) was calculated as fasting insulin concentration*fasting glucose concentration/22.5 (15 (link)).