Murine liver lipid extraction and quantification

Murine liver samples (about 20 mg) from 271 female and 268 male livers were homogenized and lipids extracted by 10 vol of chloroform:methanol (v:v 2:1) (10). The organic fraction was subsequently analyzed on the untargeted lipidomics platform. One to four livers were included per sex and strain depending on accessibility. In addition, a selection of mouse livers (n = 18) from five different strains of mice (A/J, BALB/cJ, C3H/HeJ, C57BL/6J, and DBA/2J) were chosen for more detailed structural characterization of the endogenous ceramides using a targeted lipidomics platform. Semi-quantification of the lipids was performed by using class-specific exogenous internal standards. The exogenous ceramide, Cer(d18:1/12:0), was used as a class-specific internal standard for ceramides, and was added to the murine and bovine liver homogenates prior to the Folch extraction. The main function of the class-specific internal standards was to normalize the lipid response prior to statistical analysis. The quality control samples were analyzed alongside the murine liver samples (for every tenth sample). The coefficients of variation for Cer(d18:1/16:0), Cer(d18:1/18:0), and Cer(d18:1/20:0) were better than 14, 14, and 10%, respectively. Measurements of total triacylglycerol (TAG) using a colorimetric assay (Sigma-Aldrich, St. Louis, MO) were performed as previously described (11).