Adipose Tissue Lipid Metabolite Extraction and Analysis

To extract lipid metabolites from adipose tissues, ~100 mg tissues were mixed with an antioxidant solution (0.2 mg/mL butylated hydroxytoluene and 0.2 mg/mL triphenylphosphine in methanol), 10 µL of deuterated internal standards (500 nM of d₄-6-keto PGF1a, d₄-TXB₂, d₄-PGE₂, d₄-LTB₄, d₁₁-14,15-DHET, d₄-9-HODE, d₈-5-HETE, d₁₁-11,12-EET), and 400 µL extract solution (0.1% acetic acid with 0.2 mg/mL butylated hydroxytoluene in methanol), and then homogenized; the resulting homogenates were kept in −80 °C overnight. After centrifugation of the homogenates, the pellets were washed with methanol (containing 0.1% butylated hydroxytoluene and 0.1% acetic acid) and then combined with the supernatant. The lipid metabolites in the combined solutions were loaded onto pre-washed Waters^® Oasis solid phase extraction (SPE) cartridges, washed with 95:5 v/v water/methanol with 0.1% acetic acid, the analytes were eluted with methanol and ethyl acetate, dried using a centrifugal vacuum evaporator, then reconstituted in methanol for LC-MS/MS analysis. The LC-MS/MS analysis was carried out on an Agilent 1200SL HPLC system (Agilent, Santa Clara, CA) coupled to a 4000 QTRAP MS/MS (AB Sciex, Foster City, CA) as described in our previous report (24 (link)). The lipid mediators whose levels were above the detection limit of LC-MS/MS were reported.