Bio plex diabetes panel

Hormone analysis was performed by Cardinal Bioresearch Pty Ltd. (Brisbane, Australia). Fasting and postprandial total amylin was measured using a Human Metabolic Panel Milliplex kit (Merck Millipore, Darmstadt, Germany). The sensitivity of this assay is 15 pg/mL, and the intra‐ and inter‐assay variation is 9.2% and 13.4%, respectively. Fasting and postprandial total ghrelin, GIP, active GLP‐1, and leptin were measured using a Bio‐Plex diabetes panel (Bio‐Rad, Hercules, California). The sensitivity of the assay is 1.2, 0.8, 5.3, and 3.1 pg/mL for the hormones, as listed previously. The intra‐ and interassay variation is <7% and <15%, respectively. Both assays used are magnetic bead‐based multiplex assays, which allow for the simultaneous detection of multiple biomarkers. For each analyte, all samples were tested in duplicate, using the same kit or panel batch number.

The primary outcome of the RCT was change in fat mass [13 (link)]. The main prespecified exploratory outcome measures of this secondary analysis were changes in adipokine and gut hormone levels. Adipokines and gut hormones were measured as previously described [15 (link)]. All blood tests were drawn in the fasting morning state (8 am to 10 am), including into a BD P800 tube (BD, Franklin Lakes, NJ) for preservation of gut hormones, and were assayed in a single run. Total PP, PYY, and amylin levels were measured using the Millipore Human Metabolic Panel Milliplex kit (Merck, Darmstadt, Germany), with intra-assay coefficient of variation (CVs) of 5.86%, 6.73%, and 2.90% and interassay CVs of 5.25%, 9.60%, and 8.71%, respectively. Leptin, active GLP-1, GIP, and total ghrelin levels were measured using the Bio-plex diabetes panel (Bio-Rad, Hercules, CA) with intra-assay CVs of 5.51%, 7.37%, 3.57%, and 2.47% and interassay CVs of 13.44%, 6.90%, 3.15%, and 5.25%, respectively. Adiponectin was measured using a duo enzyme-linked immunosorbent assay kit (R&D Systems, Minneapolis, MN) with an intra-assay CV of 5.37% and an interassay CV of 7.64%.