Quantitative Hepatic Lipid Profiling

Lipids were extracted from each hepatic construct following exposure to LA and HSA vehicle control using 500 μl of 3:2 hexane:isopropanol overnight. The solvent was removed and evaporated under nitrogen before lipids were converted to FAME and quantified using GC-FID, as previously described [37 (link),38 (link)]. Samples were analyzed by GC-FID on a HP 7890 (Agilent Technologies, Inc., Santa Clara, CA) with a DB-23 column as described above using triheptadecanoin (100μg; NuChek Prep) as an internal standard. Fatty acids were cleaved from complex lipids and converted to methyl esters in duplicate samples when possible, utilizing a modification of the protocol developed by Metcalfe et al [39 (link)] and Sergeant et al [37 (link)] Fatty acids in samples were identified based on retention times of commercially available internal standards. Approximately 12–25 peaks were identified and accounted for >99% of the total fatty acids in the sample. Fatty acid data are presented as the total mass in sample (μg) normalized to protein content (mg).