Total VLCFA extraction and derivatization

Sample preparation was performed following published protocols (31). Brain was homogenized at 200 mg tissue/mL water and testes at 100 mg tissue/mL water. Adrenal glands (two per mouse) were homogenized in 1 mL of water per adrenal gland. In brief, 25 μL of homogenized brain, 400 μL of homogenized adrenal glands, or 200 μL of homogenized testes were extracted in the presence of internal standards (d₄C22:0, d₄C24:0, and d₄C26:0) in 2 mL of 2:3 isopropanol/hexane for 1 hour at RT with shaking. For brain, 1.00 μg of d₄C22:0, 2.00 μg of d₄C24:0, and 0.16 μg of d₄C26:0 were added to each sample. For testes, 0.25 μg of d₄C22:0, 0.50 μg of d₄C24:0, and 0.05 μg of d₄C26:0 were added to each sample. For adrenal glands, 0.05 μg of d₄C22:0, 0.15 μg of d₄C24:0, and 0.05 μg of d₄C26:0 were added to each sample. The extracted samples underwent acid hydrolysis, followed by base hydrolysis each for 45 minutes at 100°C. After reacidification, samples were extracted with hexanes and dried under vacuum. Dried samples underwent derivatization with pentafluorobenzyl bromide in the presence of triethylamine for 1 hour at RT, and were further extracted in hexanes and dried under vacuum. The final dried sample was dissolved in 50 µL of hexanes and analyzed by gas chromatography–mass spectrometry (GC-MS).