BAT Retinol and Retinyl Ester Quantification

BAT retinol and retinyl ester concentrations were measured using HPLC, according to previously published protocols3 (link)39 (link). In brief, lipids were extracted from tissue homogenates using hexane and analyzed using a Waters modular HPLC system (Waters, Milford, MA). Chromatographic separation of extracted lipids was achieved using a Waters Symmetry C18 column (4.6 × 250 mm), and retinol and retinyl esters were measured at a peak absorbance of 325 nm using a photodiode array (Waters). Quantification of these lipids was calculated based on the recovery of a retinyl acetate internal standard. Tissue concentrations of retinoic acid were measured by LC/MS/MS, using a Xevo TQ MS Acquity UPLC system (Waters), as previously described40 (link). Note, in all cases, when we refer to retinol, retinyl ester and retinoic acid, we are referring to the all-trans-isomers of these compounds. BAT triglyceride content was measured in lipids extracted using a standard Folch solution (2:1 methanol chloroform)41 (link), and measured using a liquid triglycerides reagent (Thermo Fisher Scientific, Waltham, MA).