Infinity assay reagents

Blood glucose was measured using OneTouch Ultra2 blood glucose meter (Life Scan, Milpitas, CA). Serum β-hydroxybutyrate was determined using the Cayman Chemical β-hydroxybutyrate assay kit (Ann Arbor, MI). Serum triglyceride (TG) and cholesterol were measured using the Infinity Assay Reagents (Thermo Scientific, Waltham, MA). Serum free fatty acids (FFA) were determined with a BioVision FFA Kit (Milpitas, CA). Activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured with Infinity kit (Thermo Scientific). Serum adiponectin and leptin levels were determined using ELISA kits (Millipore, Billerica, MA). Serum ethanol concentration was measured using BioVision ethanol assay kit.

Plasma alanine transaminase (ALT), aspartate transaminase (AST), cholesterol, triglyceride (TRIG), high-density lipoprotein (HDL), low-density lipoprotein (LDL), very low-density lipoprotein (VLDL), and non-HDL cholesterol (nHDLc) levels were determined with lipid panel plus kits on a Piccolo Xpress Chemistry Analyzer (Abbott Laboratories, Chicago, IL, USA). Livers were rinsed in 1× PBS and homogenized as described (Jin et al., 2021 (link)). Hepatic lipids were extracted via a modified Bligh and Dyer method (Bligh and Dyer, 1959 (link)) whereby the livers were bead homogenized in a 50 mM sodium chloride solution and then homogenate extracted with a 2:1 ratio of chloroform to methanol mixture. Lipids extracted in lower chloroform layer were then evaporated to dryness and assayed for TRG or CHOL using Infinity Assay Reagents (Thermo Fisher Scientific, Inc., Middletown, VA, USA). Sample absorbance at 500 nm was calculated from a standard curve constructed from clinical standards from Pointe Scientific (Horiba Medical, Kyoto, Japan) for each analyte that was run simultaneously. Results obtained in mg/dL were mathematically restructured and expressed as mg TRG or CHOL per gram of liver tissue.