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Sample Analysis

Satiety hormones can be analyzed using commercially available ELISA kits according to the manufacturer's instructions. Porcine GLP-1 can be measured using the human GLP-1 kit from Millipore (EGLP-35K, Billerica, MA, USA), since the GLP-1 hormone gene sequence is highly preserved. GLP-2 can be determined using a competitive human GLP-2 (1-34) ELISA kit from Phoenix Pharmaceuticals Inc. (Belmont, CA, USA) according to the manufacturer's instructions. There is a 92 % homology of GLP-2 protein between humans and pigs. The cross reactivity of this kit with GLP-1 is 6 %.

PYY release can be measured with a commercial available ELISA kit for total PYY (Bachem, Peninsula Laboratories, San Carlos, CA, USA). This kit measures porcine PYY, which is identical to human PYY (Adrian et al. 1987, 11).

CCK concentrations can be determined using a human EURIA-CCK radioimmunoassay (RIA) kit (Euro-diagnostica, Malmö, Sweden) according to the manufacturer's instructions. An identical sequence of CCK-8 has been found form most mammals, among them pigs and human. The kit measures CCK-8 sulfate (CCK 26-33). Tissue concentrations of ECs (AEA and 2-AG) and NAEs (DHEA, EPEA, DLE, OEA, PEA and SEA) can be analyzed according to a method described previously (Balvers et al. 2012). For this analysis 50–100 mg freeze-dried tissue is required to be within the detection limit of LC–MS method. Although the paper describes the extraction of ileal mouse tissue, this method can also be used for porcine pig tissue (unpublished data). Tissue concentrations of NAEs can be analyzed according to the method described previously (Verhoeckx et al. 2011). For this method 50–300 mg intestinal tissue is required.

Monitoring Viability

The viability of the tissue segments is important to analyze, especially when secretagogue effects are studied. It has to be avoided that the release of satiety hormones is secondary to cell lysis or other nonspecific toxic effects. To analyze tissue viability macroscopic and microscopic tissue checks could be done after staining with markers for proliferation and apoptosis (e.g. hematoxylin–eosin staining). Another method to check the viability is measuring leakage of intracellular lactate dehydrogenase (LDH). LDH is stable enzyme common in all cells which can be readily detected when cell membranes are no longer intact for instance due to active proteases or mechanical forces during the preparation of the biopsies. The enzyme activity is determined with respect to the total intracellular lactate dehydrogenase. The total LDH activity of the tissue can be determined by homogenize the tissue in ice cold KRB buffer, using a Potter Elvehjem-type teflon pestle tissue grinder (Braun, Melsungen Germany) for 5 min at 200 rpm. It is also possible to use Triton-x as a positive control. Triton-x dissolves membranes and makes the tissue leaky. Samples are excluded when the LDH leakage is more than 10 % of the total intracellular LDH. The incubation time of the segments should not exceed 1 h, since at longer incubation times, LDH leakage will be >10 % (Voortman et al. 2012).

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