125i radioimmunoassay

Plasma glucose concentrations were measured with the glucose oxidase method using a Biosen C-line plus glucose analyzer (EKF Diagnostics, Barleben/Magedeburg, Germany). Insulin and cortisol were both measured on an IMMULITE 2000 system (Siemens Healthcare Diagnostics B.V., Breda, The Netherlands). Cortisol was measured with a chemiluminescent immunoassay (intra-assay variation: 7–8%; total assay variation: 7–8%; detection limit: 50 nM). Insulin was measured with a chemiluminescent immunometric assay (intra-assay variation: 3–6%; total assay variation: 4%; detection limit: 15 pm). C-peptide levels were measured with a ¹²⁵I radioimmunoassay (Merck Millipore, St. Charles, Mo., USA) (intra-assay variation: 6–9%; total assay variation: 7–11%; detection limit: 50 pM). Glucagon was measured with the ¹²⁵I radioimmunoassay (Merck Millipore) (intra-assay variation: 9–10%; total assay variation: 5–7%; detection limit: 15 ng/l).

Blood samples were centrifuged for 10 min at 3000 rpm at 4 °C, and plasma was separated from the cells. Glucose was determined immediately using the glucose oxidation method with the Biosen glucose analyzer (EKF Diagnostics, Barleben, Germany). Plasma aliquots were temporarily stored at −20 °C. HbA_1c, plasma insulin, and plasma triglyceride levels were measured as described previously (Stenvers et al., 2014 (link)); C-peptide was determined with a ¹²⁵I radioimmunoassay (Linco Research, Inc., St. Charles, MO); and plasma free fatty acid (FFA) levels were determined with an enzymatic calorimetric method (NEFA-HR(2) test kit, Wako Chemical, Neuss, Germany). In patients with type 2 diabetes, 2 additional measurements were performed: Plasma glucagon was determined with a ¹²⁵I radioimmunoassay (Millipore, Billerica, MA), and saliva cortisol was determined by online solid phase extraction LC/MS/MS (Waters Corporation, Milford, MA).