P800 blood collection system

The blood samples collected before (d-14) and after intervention (d30) were immediately centrifuged and plasma was transferred and kept at − 20 °C until analysis. Biochemical parameters including alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) concentrations were measured using standard laboratory techniques. Blood for peptide analysis was immediately transferred into specific tube (BD P800 Blood Collection System (BD Biosciences, CA, USA). Plasma obtained for inflammatory markers and peptides were transferred at − 80 °C before analysis. Plasma cytokines (interleukin (IL)-1β, IL-6, IL-8, IL-10, IL-17a, monocyte chemotactic protein-1 (MCP-1), interferon (IFN) γ and tumor necrosis factor (TNF) α) and plasma peptides (pancreatic polypeptide (PP), glucose-dependent insulinotropic peptide (GIP), leptin, ghrelin, insulin) were determined in duplicate by multiplex immunoassays (Millipore, Belgium) and measured using LUMINEX xMAP technology (Biorad, Nazareth, Belgium) following the manufacturer’s instructions.

To measure the concentration of serum diamine oxidase (DAO), the Diamine Oxidase ELISA Kit (KR8500; Immundiagnostik GmbH, Bensheim, Germany) was used, in accordance with the manufacturer’s protocol. Blood samples for GLP-2 analyses were collected with the BD™ P800 Blood Collection System (BD Biosciences, San Jose, CA, USA), which is an evacuated blood collection tube containing a cocktail of protease inhibitors, optimized for the stabilization of serum GLP-2. To measure the concentration of serum GLP-2, Rat GLP-2 ELISA Kit (292-60601; Wako Pure Chemical Industries, Tokyo, Japan) was used, in accordance with the manufacturer’s protocol.