Comprehensive Hepatic Steatosis Analysis

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)].