Fatty acid analysis

Brain tissue was removed from animals, frozen on dry ice and stored at −80℃ until analysis. Brain tissue was pulverized in liquid nitrogen, and 180 mg of pulverized tissue was mixed with 800 µl of ice-cold phosphate-buffered saline containing 1 mM EDTA and 0.1 mM indomethacin. The tissue was homogenized by vortexing followed by sonication in an ice-water bath (3 min). The homogenate was aliquoted to retain 150 µl for fatty acid analysis and 20 µl for protein analysis by the Bradford assay (Bio-Rad, Hercules, CA).

Folch reagent (chloroform: methanol 2:1, v/v) was used for extraction of brain homogenates, experimental diets, or sera. Fatty acid methyl esters were prepared using METH-PREP II derivatization reagent (Alltech, Deerfield, IL) at room temperature prior to analysis by gas chromatography with mass spectral detection using an Agilent 7890 GC and a 5975C mass-selective detector. Major fatty acids were quantified against standard curves using triheptadecanoin as the internal standard (Nucheck Prep, Elysian, MN) as previously described and expressed as % of total fatty acids.^14–16 All fatty acid assays were performed blinded to treatment identity.