2.4. Separation and quantitative determination of nonesterified fatty acids

The lipid extracts from livers were mixed with an appropriate amount of undecanoic acid as an internal standard and then separated by silica gel TLC. A part of the nonesterified fatty acids were methyl‐esterified with boron trifluoride–methanol complex (14% in methanol). The reaction solutions were mixed with hexane and saturated saline. The hexane layers were filtered (pore size 0.45 μm, ADVANTEC, Tokyo, Japan) and analyzed by GC‐TOF MS. Deal conditions are shown in Supporting Information.

The concentration of each fatty acid in the lipid extract was determined by comparing the values of peak areas in total ion chromatogram with those in methyl‐esterified fatty acid standards: palmitic, stearic, oleic, and linoleic acids.