Infinity cholesterol tr13421

Serum levels of ALT and AST were determined using the MaxDiscovery ALT (3460-08) and AST (5605-01) Assay Kits (Bioo-Scientific, Austin, TX). Plasma or tissue fatty acids, triglyceride and cholesterol were determined using the Infinity Cholesterol (TR13421) and Triglyceride (TR22421) Reagents (Thermo Scientific, Waltham, MA). Serum level of Lcn2 was measured by Lipocalin-2/ab199083 Mouse ELISA Kit (Abcam, Cambridge, MA). Serum level of Saa1 was measured by Saa1/ab157723 Mouse Elisa Kit (Abcam, Cambridge, MA). Serum level of FGF15 was measured by FGF15/LS-F11446 Mouse Elisa Kit (LifeSpan BioSciences, Inc, Seattle, WA). Serum levels of total adiponectin were determined using a commercial ELISA kit from BioVendor (Candler, NC). Serum levels of HMW forms of adiponectin were determined with Mouse Adiponectin Assay Kit from ALPCO # 47-ADPMS-E01 (Salem, NH). Concentrations of total bile acids in serum were determined using a 3α-hydroxysteroid dehydrogenase method (Diazyme). Analyses were performed with a SpectraMax i3x microplate reader (Molecular Devices, Sunnyvale, CA).

Plasma triglycerides and cholesterol concentrations were measured with an enzymatic assay (Konelab i 20XTi, Thermo Electron Corp., Waltham, MA) using commercially available reagents [Infinity cholesterol (TR13421) and triglyceride (TR22421) reagents, Thermo Fisher, Waltham, MA] and standards [Stabio cholesterol standard (SB1012030), Thermo Fisher, Waltham, MA; triglyceride standard (T7531STD), Pointe Scientific, Canton, MI]. Plasma HMW adiponectin was measured using a commercially available kit (EZHWAM-65, Millipore Sigma, Burlington, MA) previously validated for use in lean and obese horses (22 (link)). Average intra-assay variation for plasma adiponectin was 3.14%.

Lipoprotein separation for total cholesterol and triglycerides estimation in plasma samples was performed using fast-protein liquid chromatography (FPLC) as described elsewhere (27 (link)). Briefly, mice were fasted 4 h, and blood was collected in anticoagulant (EDTA)-coated tubes through the submandibular plexus. Samples were centrifuged at 2500 g for 10 min at 4°C, and plasma was collected and the total cholesterol was measured (using an enzymatic kit) to ensure that pooling of samples used plasma with comparable sterols to exclude any outliers with any dramatic differences between individual mice (although no samples needed to be excluded). Column chromatography (10/300GL, Superose 6) was then used for separation of different lipoproteins by loading a minimum of 100 μl of pooled plasma mixed with 100 μl of PBS, from each group, onto the Akta pure FPLC, and a total of 52 fractions each with volume of 500 μl were collected and analyzed for cholesterol content using Infinity cholesterol (TR13421) and triglyceride (TR22421) calorimetric kits procured from ThermoFisher Scientific, Middletown, VA.