Ek 059 02

Venous blood samples were taken after an overnight fast between 7:00 and 8:00 AM within the first week after admission (T0) and at a second time point during treatment (Tx). Patients were advised not to smoke or exercise and only to have a small amount of water before blood withdrawal. Circulating glucose levels were determined by photometric measurement from blood collection tubes containing sodium fluoride that were kept at room temperature. Blood for measuring peptide concentrations was collected in pre-cooled standard EDTA tubes prepared with aprotinin (1.2 Trypsin Inhibitory Unit per 1 ml blood; ICN Pharmaceuticals, Costa Mesa, CA, United States) for peptidase inhibition. After blood withdrawal the tubes were stored on ice and centrifuged for 10 min at 3000 rpm at 4°C. Plasma was separated and samples were stored at −80°C until further processing. NPY, PYY and PP levels were determined using a commercial enzyme-linked immunosorbent assay (ELISA, catalog # EK-049-03, EK-059-02 and EK-054-02, Phoenix Pharmaceuticals, Inc., Burlingame, CA, United States). According to the manufacturer’s information there is no cross-reactivity (0%) between the respective peptide antigens. All samples were processed in one batch; the intra-assay variability was 8, 5, and 5%, respectively.