Immulite 2000 insulin kit

Samples were centrifuged at 4000 rpm for 15 minutes using a NF 1200 R centrifuge system and stored at −80°C. At the time of laboratory assessment, the samples were taken out of the freezer and were allowed to thaw to room temperature. Serum insulin levels were detected using Immulite 2000 insulin kit, and brain natriuretic peptide (BNP) levels in heparinised plasma were detected using Immulite 2000 kit for Immulite 2000 XPi system (Siemens). Serum cortisol was detected using the Advia Centaur XP kit for the Advia Centaur XP System (Siemens). Serum creatinine kinase (CK) and creatinine kinase muscle-brain (CK-MB) were detected using Beckman Coulter AU2700 PLUS system, and serum C-reactive protein (CRP) was detected using Beckman Coulter Immage 800 system.

A separate trial was performed to assess plasma glucose and insulin kinetics before (fasting state, time 0) and 30, 50 (corresponding to the start and the end of the haemodynamic measurements, respectively), and 90 min after OGTT. Each participant had an intravenous cannula inserted in a forearm peripheral vein and venous blood (VB) samples collected in pairs: the insulin sample (test tube for serum preparation) immediately after the glucose sample (anticoagulant-coated tube). All test tubes were centrifuged and placed on ice immediately after collection. Serum insulin concentration (c_ins-VB) was determined by the commercially available solid-phase, enzyme-labelled chemiluminescent immunometric assay, using the “Immulite2000 Insulin” kit and Immulite2000-XPI analyser (both Siemens Healthcare Diagnostics Products Ltd., Camberley, UK). For quantitative determination of plasma glucose (c_glc-VB), the enzymatic UV test (hexokinase method) was used (Olympus AU400 system; Mishima Olympus CO., Ltd., Tokyo, Japan; reagent: Beckman Coulter, Inc., Kildare, Ireland).