K2 edta plasma vacutainer tubes

Blood samples were collected using an intravenous cannula by a qualified research nurse. Blood samples were either collected in serum vacutainer tubes (BD, USA) for the measurements of glucose, insulin and triglycerides (TG) or collected in K₂ EDTA plasma vacutainer tubes (BD, USA) for the measurements of glucagon and free fatty acids (FFA), concentrations. The serum tubes were centrifuged within approximately 1 hour of collection after clot was formed. The plasma tubes were stored on ice immediately on collection and centrifuged within 30 min of collection. All blood tubes were centrifuged at 1500× g for 10 min at 4°C. Serum and plasma samples were then aliquoted into individual cryotubes and immediately stored at −80°C until analyses. Serum glucose, insulin and TG were measured using the standardized clinical chemistry method of the National University Hospital Referral Laboratories, Singapore, whereby glucose and TG were measured using AU 5800 clinical chemistry analyzer (Beckman Coulter, USA) and insulin was measured using UniCel DxI 800 Access Immunoassay System (Beckman Coulter, USA). Plasma FFA was measured by an in vitro enzymatic colorimetric method (Wako, Germany), while glucagon was measured by ELISAs (Mercodia, Sweden).