Quantification of Lipid Intermediates in Muscle

For quantification of lipid intermediates in subcellular muscle fractions, lipids were extracted, purified and analyzed from frozen tissue samples, using lipid chromatography mass spectrometry (LC-MS/MS)^{69 (link)}. In brief, 50 mg of tissue was homogenized in 20 mM Tris/HCl, 1 mM EDTA 0.25 mM EGTA, pH 7.4, using a IKA T10 basic Ultra Turrax (IKA; Wilmington, NC, USA) and a tight-fitting glass douncer (Wheaton, Rochdale, UK). An internal standard (d517:0-DAG; Avanti Polar Lipids, Ala, USA) was added and samples were centrifuged for 1 h (100,000 × g, 4 °C). Lipid droplet, cytosol and membrane fractions were collected and lipids of each fraction were extracted according to Folch et al. ⁷⁰ , followed by solid phase extraction (Sep Pak Diol Cartridges; Waters, Milford, MA, USA). The resulting lipid phase was dried under a gentle flow of nitrogen and re-suspended in methanol. Lipid analytes were separated using a Phenomenex Luna Omega column (1.6 µm 100A; Phenomenex, Torrance, CA, USA) on an Infinity 1290 HPLC system (Agilent Technologies, Santa Clara, CA, USA) and analyzed by multiple reaction monitoring on a triplequadrupole mass spectrometer (Agilent 6495; Agilent Technologies), operated in the positive ion mode.