2.4. Biomarker Assessment
TG
SETTLE, BLOOD
The measurement of all biomarkers was carried out using capillary blood samples. Plasma glucose concentrations were measured in whole blood at the time intervals specified above, using a HemoCue Glucose 201+ analyzer manufactured by HemoCue AB in Ängelholm, Sweden. Samples for the analysis of serum insulin, free fatty acids (FFA), and TG were obtained using BD Microtainer SST tubes. The serum insulin samples were taken at the same time points as the glucose determinations, except for 15 and 150 min. The concentrations of FFA and TG were measured at 0, 60, 120, 210, 270, and 330 min. The tubes were allowed to settle at room temperature for about 30 min prior to centrifugation for a duration of 5 min. The centrifugation was performed at a speed of 5000 revolutions per minute (rpm) at a temperature of 25 °C, using an Eppendorf centrifuge model 5425. Subsequently, the serum was separated and kept at a temperature of −40 °C until it was subjected to analysis.Blood plasma samples were taken for the analysis of total concentrations of GLP-1, GIP, PYY, and ghrelin at four different time points (0, 60, 210, and 330 min). The samples were collected using BD Microtainer K2E tubes. A mixture of a DPP-4 inhibitor (10 µL/mL blood, Millipore, St. Charles, IL, USA) and aprotinin (50 µL/mL blood, Sigma-Aldrich, St. Louis, MO, USA) was injected into the tubes as an inhibitory cocktail before the blood collections. The tubes were stored on ice prior to and during the collection of samples. Subsequently, the tubes were subjected to centrifugation for a duration of 10 min at a speed of 4200 rpm and a temperature of 4 °C. This centrifugation process was carried out within a time frame of 30 min after the blood was collected. The plasma was thereafter separated, and samples were subjected to freezing at a temperature of −40 °C until the time of analysis.The measurement of insulin concentrations was conducted using a solid phase two-site enzyme immunoassay kit (Insulin ELISA 10-1113-01, Mercordia AB, Uppsala, Sweden). The amounts of FFA were determined using an enzymatic colorimetric approach on a 96 microplate. Specifically, the NEFA-HR (2) ACS-ACOD method provided by FUJIFILM Wako Chemicals Europe GmbH in Germany was used for this analysis. The amounts of serum TG were measured using a multi-sample enzymatic assay known as LabAssay™ Triglyceride 290-63701, which follows the GPO.DAOS technique. This test was conducted by FUJIFILM Wako Chemicals Europe GmbH, Germany. The measurement of total plasma concentrations of GLP-1, PYY, GIP, and ghrelin was conducted using a 10-spot U-plex test kit (Meso Scale Diagnostics LLC, Rockville, MD, USA). In accordance with the suppliers’ description, biotinylated capture antibodies are coupled to U-PLEX Linkers. The U-PLEX Linkers then self-assemble onto unique spots on the U-PLEX plate. After analytes in the sample bind to the capture reagents, detection antibodies conjugated with electrochemiluminescent labels (MSD GOLD SULFO-TAG) bind to the analytes to complete the sandwich immunoassay.