Lipoprotein Fractionation by FPLC

Lipoprotein fractionation was performed as described previously [10 (link),11 (link)]. Briefly, plasma specimens collected from the mice (initially, 8–10 weeks old) after WD feeding with or without ezetimibe (20 μg/g diet) for 3 or 20 weeks were fractionated by size-exclusion chromatography using a fast protein liquid chromatography system equipped with a Superose 6 column (GE Healthcare UK Ltd., Buckinghamshire, UK). Elution was performed in PBS containing 1 mM EDTA and 3 mM sodium azide as the running buffer. After loading 300 μL of serum, the system was run at a constant flow of 200 μL/min, with 500-μL fractions collected. The cholesterol and triglyceride concentrations in each fraction were quantified with the Total Cholesterol E-test Wako and Triglyceride E-test Wako (Wako Pure Chemical Industries, Ltd., Osaka, Japan), respectively.