Blood sample collection technique, processing and analysis
CLOT, COLD
For blood glucose, 0·5 ml of whole blood (WB) was collected into cold fluoride/citrate tubes, mixed gently, kept on ice and analysed within 2 h using YSI 2300 STAT PLUS (YSI Inc. Yellow Springs).The remaining blood was gently mixed in tubes containing the appropriate preservative, centrifuged (3000 g 4°C for 10 min) and plasma/serum aspirated, aliquoted and frozen at minus 80°C as appropriate for the intended analyses. All samples were kept on ice throughout processing, except for serum samples which were kept at room temperature for the first 15 min to enable the blood to clot. Preservatives used were as follows: active glucagon-like-peptide 1 (GLP-1) required EDTA tubes with added DPP IV inhibitor (10 µl/ml WB), and total peptide YY (PYY) and total ghrelin both required EDTA tubes with added aprotinin (500 Kallikrein Inactivator units/ml WB). Insulin and total glucose-dependent insulinotropic polypeptide (GIP) required serum, and plain serum-separator tubes were used.Analytical techniques were either RIA (PYY, ghrelin, and insulin) or ELISA (active GLP-1 and GIP) as used previously