260 spectrophotometer

Gastric emptying was determined as in our previous study [33 (link)] in mice deprived of food but not water for 6–7 h during light phase. Mice had no access to food and water after the surgery and were gavaged with 0.3 ml of 1.5% methylcellulose (Sigma-Aldrich, St Louis, MO) and 0.5% phenol red (Sigma-Aldrich) and 20 min later, euthanized by cervical dislocation. The abdominal cavity was opened, gastric pylorus and cardia were clamped, the stomach removed, rinsed, placed into 15 ml of 0.1 N NaOH, homogenized for 30 s (Polytron; Brinkman Instruments, Westbury, NY). The suspension was settled for 1 h at room temperature and then 5 ml of the supernatant added to 0.5 ml of 20% trichloroacetic acid (w/v; Sigma-Aldrich). The samples were centrifuged at 3000 rpm at 4°C for 20 min. Three ml of the supernatant were mixed with 4 ml of 0.5 N NaOH. The absorbency of the sample (1 ml) was read at 560 nm (Shimadzu 260 spectrophotometer). phenol red recovered from animals euthanized immediately after the administration of the methylcellulose/phenol red solution served as a standard (0% emptying). Percentage of emptying in the 20-min period was calculated as % emptying = (1- absorbance of test sample/absorbance of standard) × 100.