End point technique

Blood samples were collected in lithium-heparin, EDTA and serum tubes. Lithium-heparin tubes (4.5 mL LH PST^TM II, Becton-Dickinson, NJ, America) were centrifuged at 1300 CRF for 10 min at room temperature (RT), plasma was frozen at −80 °C until it was analyzed for glucose concentrations. Glucose was measured by means of an end-point technique (Siemens, The Netherlands). EDTA tubes (8 mL, Becton-Dickinson, NJ, America) were centrifuged at 1200 G for 15 min at 4 °C, and plasma was frozen at −80 degrees until it was analysed for free fatty acids concentrations. Free fatty acids were assessed using an enzymatic test kit according to the manufacturer’s protocol (InstruChemie, Delfzijl, The Netherlands). Serum tubes (5 mL, Becton-Dickinson, NJ, USA) were set aside for at least 30 min, where after they were centrifuged at 1300 G for 10 min at RT, serum was frozen at −80 degrees until it was analysed for ketone content and cortisol concentration. Beta-hydroxybutyrate (βHB) was determined via a colorimetric enzymatic assay (Sigma-Aldrich; St. Louis, MO, USA). Analysis was performed according to manufacturer’s protocol. Cortisol was measured with immunometric chemiluminescence (sandwich) assay with Immulite XPi (Siemens, Den Haag, The Netherlands).

All blood samples were immediately centrifuged at 1000 g at 4°C for 10 min, after which plasma was stored in −80°C until further analysis. Blood samples were analyzed for cortisol, creatine kinase, glucose, and insulin (Gelderse Vallei hospital, Ede, NL). Cortisol was measured with immunometric chemilumiescence (sandwich) assay with Immulite XPi, (Siemens, the Netherlands). Creatine kinase was measured using Vista device (Siemens, the Netherlands). Free fatty acids were assessed using an enzymatic test kit according to the manufacturer's protocol (InstruChemie, Delfzijl, Netherlands). Glucose was measured using absorption changes caused by the formation of NADH as a measure of glucose concentration and was measured bichromatic (340 and 383 nm) by means of an end point technique (Siemens, the Netherlands). Insulin was measured using an solid-phase enzyme-linked chemilumiescent immunometric assay with Immulite 2000 XPi (Siemens, the Netherlands).