Free cholesterol e

Mice were euthanized by CO₂ inhalation and blood was collected by cardiac puncture. Serum was collected by centrifugation (15 min, 2000 g, 4°C) after allowing blood to clot on ice. Plasma was collected by centrifugation (15 min, 2000 g, 4°C) in EDTA-treated tubes (Sarstedt, Numbrecht, Germany). All serum and plasma samples were snap frozen and stored at −80°C. Kits were used to measure total cholesterol (Infinity Total Cholesterol, Fisher Scientific), free cholesterol (Free Cholesterol E, Wako Diagnostics, Richmond, VA), HDL cholesterol (HDL-C E, Wako Diagnostics), LDL cholesterol (LDL-C Reagent L-type, Wako Diagnostics), NEFAs [HR Series NEFA-HR(2), Wako Diagnostics], and TGs (Infinity Triglycerides, Fisher Scientific) according to the manufacturer's directions. For HFD and LFD male mice, n=5-8; for HFD and LFD female mice, n=8-9; and for CD male mice, n=6-8.

Total cholesterol, free cholesterol, triglycerides and HDL cholesterol in plasma were measured using enzymatic assay kits according to the manufacturer’s protocols (total cholesterol: Infinity Cholesterol, Thermo Fisher Scientific, Ottawa, ON, Canada; free cholesterol: Free Cholesterol E, Wako Diagnostics, Mountain View, CA, USA; triglycerides: l-type triglyceride M, Wako Chemicals, Richmond, VA, USA; HDL cholesterol: HDL-cholesterol E, Wako diagnostics, Mountain View, CA, USA). Non-HDL cholesterol was calculated as the difference between total cholesterol and HDL cholesterol measurements. Cholesteryl ester levels were calculated as the difference between total cholesterol and free cholesterol measurements for each sample. For lipoprotein total cholesterol profiles, plasma was separated by size by gel filtration chromatography using a Tricorn Superose 6 HR 10/300 column on an AKTA fast protein liquid chromatography system (GE Healthcare Life Sciences, Mississauga, ON, Canada), and total cholesterol was measured in each fraction as previously described [58 (link),59 (link)].

Total lipids were extracted from a large portion of the liver comprising all 4 lobes missing a small section of the left lateral lobe that was used for other analyses. The liver was ground with a tissue grinder into a homogenous slurry and ~300 mg of slurry (weighed accurately) was homogenized in 5 mL of chloroform:methanol (2:1, v/v). The samples were incubated overnight on a shaker, centrifuged (10 min, 2000 × g) and the supernatant was collected. Sodium chloride (0.9%, w/v) was added to the supernatant and the organic solvent layer was recovered and evaporated under nitrogen to obtain the weight of total lipids. For determination of TC, free cholesterol and triglyceride concentrations, total lipids were extracted from an ~300 mg section (weighed accurately) of the left lateral lobe as described above. Total lipids were weighed and re-suspended in 1.5 mL of 10% Triton X-100 in isopropanol. TC, free cholesterol and triglycerides were measured using Wako Cholesterol E (999–02601, Wako Chemicals, Richmond, VA, USA), Free Cholesterol E (993–02501, Wako Chemicals) and L-Type Triglyceride M (Wako Chemicals) kits, respectively.