Beckman glucose analyzer

Glucose tolerance tests were performed on days 7 and 8 of refeeding, according to the protocol described previously^{7 (link),9 (link)}. Food was removed early in the morning (07:00 h). At 6–7 h later, i.e. in the post-absorptive phase, blood was drained from the tail vein and immediately followed by an intraperitoneal injection of glucose (2 g/kg body weight). At intervals of 30 min for the next 2 h period, blood samples were taken from the tail vein in heparinized tubes and transferred on ice. The blood samples were then centrifuged, and the plasma was frozen and stored at −20 °C for later assays of plasma glucose and insulin. Plasma glucose was determined using a Beckman Glucose Analyzer (Beckman Instruments, Palo Alto, CA, USA), while plasma insulin was assessed using a rat insulin ELISA kit (Crystal Chem, Inc., Downer’s Grove, IL, USA).

All assays were performed using commercially available kits on paired samples. Blood for determination of PAI-1 activity in plasma was collected into 5 mL tubes containing 0.5 mL of sodium citrate. The samples were immediately centrifuged for 10 minutes at 3000 g and 4°C, and then plasma was carefully separated, transferred to small vials, and stored at −80°C until analyzed. The level of PAI-1 activity in plasma was determined by using plasminogen/chromogenic plasmin substrate assay (kit Behring, Germany). The normal range for PAI-1 activity by using this test is 0.3–3.5 U/mL with an interassay CV of 7.7%. Plasma glucose was determined by using glucose oxidase method on Beckman glucose analyzer (Beckman Instruments, Fullerton, USA). Plasma insulin levels were measured by radioimmunoassay with double antibodies kits (INEP, Zemun, Serbia). Insulin resistance was estimated using the homeostasis model assessment (HOMA-IR) and was calculated from fasting plasma insulin and glucose levels according to the formula: (insulin (mU/L) × glucose (mmol/L))/22.5 [21 (link)]. Determination of lipid parameters, total cholesterol (chol), HDL-chol, and triglycerides concentrations were analyzed using commercial enzymatic kit (Boehringer Mannheim GmbH Diagnostics), while LDL-chol levels were calculated by using standard Friedewald formula.

Fasting serum total cholesterol, triglyceride, and high-density lipoprotein (HDL) cholesterol levels were measured using commercially available kits on a Hitachi 7150 Autoanalyzer (Hitachi Ltd., Tokyo, Japan). Low-density lipoprotein (LDL) cholesterol was indirectly estimated in subjects with serum triglyceride concentrations of <400 mg/dl using the Friedewald formula. Fasting glucose levels were measured using the glucose oxidase method with a Beckman Glucose Analyzer (Beckman Instruments, Irvine, CA).