Cholesterol e

Total lipid was extracted from intestinal and hepatic tissues essentially as previously described [20] (link). Briefly, 100 mg of tissue was homogenized in 1 ml of isopropanol with tissue disperser followed by vigorous shaking for 45 minutes and then centrifuged at 13,000 RPM for 10 minutes. Supernatant from each sample was equally divided into 2 parts, one was used for triglyceride measurement and the other was used for cholesterol measurement after adding Triton-×100. Intestinal and hepatic triglycerides were measured using an enzymatic kit (L Type Triglyceride M, Wako diagnostics) according to the manufacturer's instructions. Total cholesterol and Free Cholesterol were measured using an enzymatic kit (Cholesterol E and Free Cholesterol, respectively, Wako diagnostics, United states) according to the manufacturer's instructions. The amount of Cholesterol Esters was calculated as the difference between the amount of Free Cholesterol and total cholesterol. To assess the levels of cholesterol and triglycerides in the blood, serum was first collected and then samples from three different animals from the same group were pooled together for further analysis. Measurement of serum cholesterol and trigycerides as well as lipoprotein fractionation in serum samples was achieved by FPLC methods and performed by the Mouse Metabolic Phenotyping Center University of Cincinnati.

Hepatic lipids were extracted and measured as previously described [14 (link)]. Plasma and liver TG and total cholesterol concentrations were measured using Triglyceride-E and Cholesterol-E tests (Wako Pure Chemical Industries, Osaka, Japan), respectively.

Capillary blood from submandibular bleeds was collected in heparinized tubes and centrifuged at 200 x g for 20 minutes at 4°C to separate plasma. Lipid extraction from liver was adapted from the Folch method [57 ]. Approximately 100 mg tissue were homogenized in 3 mL phosphate-buffered saline (PBS). 12 mL 2:1 chloroform: methanol (CHCl₃: MeOH) were added and mixture was vortexed twice for 15 seconds each. After centrifuging at 3000 rpm for 10 minutes, the organic lower layer was transferred to a 20-mL glass scintillation vial. An additional 10 mL 2:1 CHCl₃: MeOH were added to upper layer and vortexing and centrifugation were repeated. Organic lower layer was added to first extraction in scintillation vial. Solvent was dried down under nitrogen (N₂) gas followed by lipid resuspension in 1 mL 15% Triton X-100 in CHCl₃. Solvent was dried down again under N₂ gas and remaining lipid was resuspended in 1mL H₂O. Triglyceride and total cholesterol in plasma and liver extracts were measured with the Infinity Triglycerides (Thermo Scientific) and Cholesterol E (Wako Diagnostics) kits, respectively.