Quantifying BAT β-Oxidation Activity

The β-oxidation ability of BAT homogenates was examined as previously described [35] (link). Twenty milligrams of BAT were manually homogenized using a Potter-Elvehjem homogenizer in 400 µl of ice-cold buffer A (100 mM KCl, 40 mM Tris-HCl, 10 mM Tris base, 5 mM MgCl₂-6H₂O, 1 mM EDTA, and 1 mM ATP [pH 7.4]). The lysate was centrifuged at 420×g and 4°C for 5 min, and 40 µl of the supernatant was added to 160 µl of the reaction mixture (100 mM sucrose, 10 mM Tris-HCl, 5 mM KH₂PO₄, 1 mM MgCl₂, 2 mM L-carnitine, 0.1 mM malate, 2 mM ATP, 0.05 mM coenzyme A, 1 mM dithiothreitol, 0.2 mM EDTA, and 0.2 µCi ¹⁴C-palmitic acid [Perkin Elmer, CT, USA] complexed to fatty-acid-free albumin at a 3∶1 molar ratio [pH 7.4]). The reactions were performed in an Eppendorf tube and were placed in an incubator at 37°C for 60 min. The mixture was transferred to a new Eppendorf tube containing 200 µl of 6 N HCl. Filter paper containing 20 µl of ethanolamine was previously placed in the tube cap. The sample was further incubated at room temperature for 1 h to collect the ¹⁴CO₂ trapped in the filter paper. The radioactivity in ¹⁴CO₂ was quantified using liquid scintillation counting. The value was normalized with the lysate protein concentration.