Metabolite and Enzyme Analysis in Mucosa

For the analyses of metabolites and enzyme activities, 30 mg of powdered mucosa tissue was homogenized in 300 µl lysis puffer containing 10 mM HEPES (Thermo Fisher Scientific, Schwerte, Germany), 1% (v/v) Tween20 (Carl Roth, Karlsruhe, Germany), 1 mM EDTA (GE Healthcare, Munich, Germany), 10 mM NaF (Thermo Fisher Scientific), 0.1% (v/v) Triton X-100 (GE Healthcare), 0.5% (v/v) DOC (Sigma-Aldrich), 0.1% (w/v) SDS (USB Corporation, Cleveland, OH, USA) with 0.5 cycles and 80% amplitude (20-times) Ultrasonic Processor UP50H (Hielscher Ultrasound Technology, Teltow, Germany)^{54 (link)}. The homogenized extract was centrifuged at 3000×g for 20 min at 4 °C. The supernatant was used to measure glucose and lactate concentrations and aspartate aminotransferase (AST) and glutamate dehydrogenase (GLDH) activities photometrically (Abx Pentra 400; Horiba, Kyoto, Japan) using kits for glucose (no. A11A01667, Axon Lab, Reichenbach, Germany), lactate (no. A11A01721, Axon Lab), AST activity (no. A11A01629, Axon Lab) and GLDH activity (LT-GD 0010, Labor + Technik Eberhard Lehmann GmbH, Berlin, Germany). Protein concentrations of the extracts were measured using the Bradford kit (Thermo Fisher Scientific). Metabolites and enzyme activities were normalized to the protein concentration of the mucosa extract.