2.7. Blood Collection and Analysis
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BLOOD, SENSITIVITY
Blood samples were collected into 6 mL EDTA vacutainers via venipuncture. Immediately after collection, a protease inhibitor cocktail containing dipeptidyl peptidase IV inhibitor (10 μL/mL blood; MilliporeSigma Corp., Burlington, MA, USA), sigma protease inhibitor (1 mg/mL blood; SigmaFast, MilliporeSigma Corp.) and pefabloc (1 mg/mL blood; MilliporeSigma Corp.) was added to the sample to prevent degradation of appetite-related hormones. Samples were centrifuged at 2000 rpm for 10 min at 4 °C. Plasma aliquots were stored at −80 °C for later analysis. The concentration of plasma hormones was determined using the Human PYY (Total) kits, High Sensitivity GLP-1 Active Chemiluminescent kits, and Human Ghrelin (Active) ELISA kits (MilliporeSigma Corp., Burlington, MA, USA). Inter- and intra-assay variations were 3.09 ± 3.29% and 2.39 ± 1.14% for PYY, 6.20 ± 6.71% and 6.75 ± 5.46% for GLP-1, and 2.73 ± 3.11% and 2.11 ± 1.50% for acyl-ghrelin. All samples were assayed in duplicate and samples from one participant session were analyzed in the same assay to minimize the effects of inter-assay variation.