4.10. Gastric Emptying and Intestinal Transit

Gastric emptying and intestinal transit were measured on day 7 of the experimental protocol using a modified technique previously described [41]. Mice were deprived of food with free access to water for 18 h. Then, 0.3 mL of phenol red solution (0.75 mg/mL in 5% glucose) was administered by gavage to each mouse. After 20 min, the animals were euthanized, gastrointestinal tissues (stomach and small intestine) were collected, and their lengths were measured. The small intestine was divided into three segments: duodenum (40% of total small intestine length), jejunum (30% of total small intestine length) and ileum (30% of total small intestine length). Each gastrointestinal segment was placed in 15 mL tubes containing 10 mL of 0.1 N NaOH solution. The samples were cut into small pieces, mechanically homogenized and centrifuged (2800 rpm, 4 °C, 10 min). Then, 50 μL of 20% TCA was added to 1 mL of the supernatant to precipitate proteins from the homogenate, and the sample was centrifuged (2800 rpm, 4 °C, 20 min). Finally, 75 μL of each sample was added to 96-well plates, and 100 μL of 0.5 N NaOH was added to each well. The absorbance of the samples was analyzed using an ELISA reader (EPOCH, Winooski, VT, USA) at 570 nm. The fractional dye retention was expressed as a percentage, according to the following equation: Gastric dye retention = amount of phenol red recovered in stomach/total amount of phenol red recovered from two segments (stomach and small intestine). Intestinal transit was calculated for each intestinal segment by dividing the amount of phenol red recovered from a given segment by the amount of phenol red recovered from all three segments and was expressed as a percentage.