Total bile acid test

Blood samples were drawn by cardiac puncture under anesthesia using 2,2,2-tribromoethanol (250 mg/kg intraperitoneal injection; Wako Pure Chemical Industries, Osaka, Japan). Plasma was acquired by centrifugation and stored at −30°C until measurement. The total cholesterol, LDL, HDL, and triglyceride contents were measured with an automated chemistry analyzer. In addition, 50 μl plasma were used for lipoprotein profiling by high-performance liquid chromatography using molecular sieve columns with the LipoSEARCH system (Skylight Biotech Inc., Tokyo, Japan) (23 (link)). For determination of hepatic triglyceride and cholesterol concentrations, total lipids were extracted from the liver by the Folch method (24 (link)), and the hepatic triglyceride and cholesterol contents were measured using the enzyme assay. Total bile acids in the liver were measured with a total bile acid test (Wako Pure Chemical Industries, Osaka, Japan).

Feces were dried with a freeze dryer (FD-1000; Tokyo Rikakikai Co., Ltd., Tokyo, Japan) for 24 h. The trap cooling temperature was −45°C. After drying, weights of freeze-dried feces were measured and the samples were ground in a mortar. Fecal amounts of bile acid were measured by the method described by Kanamoto et al.^{(17 )} A total of 10 mg of feces were suspended in a glass test tube with 2.5 ml of 99.5% ethanol, vortexed for 30 s, incubated for 1 h at 65°C, and centrifuged at 3,000 rpm for 10 min at 4°C. The supernatants were transferred to a glass test tube. The same volume (2.5 ml) as that used in the first extraction of 99.5% ethanol was added to the sediment and the procedure was repeated. The supernatants from both extractions were pooled in the same glass test tube and dried at 65°C gassing with N₂ gas. After drying, 0.5 ml of 90% ethanol was added to the residue and vortexed for 30 s. Total bile acid concentrations were measured with a total bile acid test (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) according to the manufacturer’s instructions.

Here, 0.1 g of homogenized dry fecal matter was added to 4 mL of concentrated sulfuric acid in test tubes for 30 min at room temperature. Diethyl ether was added to reach 25 mL, and mixed. The diethyl ether-containing layer was moved to a flask, and the diethyl ether was evaporated. The fecal lipid in the flask was then weighed. The T-cho concentration in fecal matter was determined in the same way as in the liver T-cho concentration.
Fecal bile acids were measured by the procedure described in the work of Iwami et al. [20 ]; 10 mg of the sample was mixed with 0.2 mL of 90% ethanol during vortex mixing and incubated for 1 h at 65°C. The mixture was subjected to centrifugation at 5,000 rpm for 3 min. The supernatant was transferred to a 1.5 mL tube, and the ethanol was evaporated. Then, 0.2 mL of 90% ethanol was added in the precipitate for vortex mixing. The sample was dissolved in 1 mL of 90% ethanol and measured using test kits (total bile acid test by enzyme colorimetric method, Wako pure Chemical Industries, Osaka, Japan).