Xevo tq ms acquity uplc system

In a subset of 7-10 mice per group, plasma NE was measured as previously described (Jia et al., 2011 (link); Marce et al., 1995 (link); Nirogi et al., 2013 (link)). Briefly, 50 µl of plasma was mixed with isotopical internal standard and protein was subsequently removed by adding 50 µl of 0.4 M perchloric acid. The clear supernatant was mixed with 100 µl of 1 M NaHCO₃ and 200 µl dansyl chloride (1% in acetone) was added to the sample followed by heating at 65°C for 10 min. The sample was chilled on ice and dansylated norepinephrine was extracted with 200 µl ethyl acetate. The organic phase was evaporated under a nitrogen stream and resuspended in acetonitrile for UPLC-MS/MS injection. The assay was carried out on a Waters Xevo TQ MS ACQUITY UPLC system. Dansylated norepinephrine was separated on a Waters Acquity UPLC BEH Phenyl column (3 mm inner diameter×100 mm with 1.7 μm particles) and detected using positive electrospray ionization tandem mass spectrometry (ESI-MS/MS) under optimized multiple reaction monitoring (MRM) mode. The optimized transitions of norepinephrine and internal standard are 869.3→170.3 and 857.7→170.3, respectively.

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).

Hepatic steatosis was assessed using histological and biochemical approaches. Neutral lipid accumulation in the liver was visualized by staining with Oil Red O, following tissue preparation by the Columbia University Medical Centre’s Molecular Pathology core facility. Images of stained livers were collected using an FSX100 microscope (Olympus, Center Valley, PA, USA). To measure hepatic triglycerides (TG), total hepatic lipids were extracted using a Folch solution [22 (link)], and the concentration of TG measured using an Infinity Triglycerides liquid stable reagent (Thermo-Fisher Scientific; Middleton, VA). The measurement of hepatic retinol and retinyl ester levels was performed using standard HPLC methods [23 (link)], with a 4.6 × 260 mm Waters Symmetry C18 column (Waters Corp., Milford, MA, USA). The concentrations of retinol and retinyl ester were calculated using the area under the curve of chromatogram peaks (λ = 325 nm) and corrected to the amount of recovered retinyl acetate internal standard (Sigma-Aldrich, St Louis, MO, USA). A Xevo TQ MS Acquity UPLC system (Waters) was used to measure hepatic RA levels as previously described [24 (link)].