Enzymatic colorimetric assay kit

Total plasma cholesterol levels were determined using an enzymatic colorimetric assay kit (Wako Pure Chemical Industries, Ltd, 439-17501) (Fig. 2C, Supplemental Fig. 1D,2C,2D).
The lipoprotein cholesterol distribution profile (VLDL, LDL, HDL) was evaluated in pooled plasma samples under fasting conditions after fractionation by fast protein liquid chromatography (FPLC) gel filtration on a single Superose 6 column (Fig. 2D).

Triacylglycerols (TAGs) were extracted from powdered frozen liver (~20 mg) samples via homogenization with a 2:1 chloroform: methanol solution. In brief, after sample homogenization, 0.2 volumes of methanol was added to each sample and samples were kept on ice for 10-min, following which they were centrifuged at 3,500 xg for 10-min. To allow the mixture to separate into two phases, 0.2 volumes of 0.04% (w/v) CaCl 2 was added, followed by centrifugation at 2,400 xg for 20-min. The supernatant was discarded, and the remaining TAG extract was washed twice with 150 μL of pure solvent upper phase. Finally, 50 μL methanol was added to produce a singular phase. The samples were subsequently dried under N 2 gas at 60°C, and the remaining TAGcontaining pellets were dissolved in 50 μL of 3:2 tert-butyl alcohol X-100 methyl alcohol (1:1). The samples were stored overnight at 4°C, and the following day all samples were quantified for TAG content using a commercially available enzymatic colorimetric assay kit (Wako Pure Chemical Industries) as previously described (Al Batran et al. 2020) (link).