Diagnostic kits

At the time of liver collection, liver slices were taken near the porta hepatis, fixed for 24 h in 10% neutral buffered formalin, put into individual cassettes with 70% alcohol, processed and embedded in paraffin, sectioned (4-6 microns), and stained with hematoxylin and eosin for histological evaluation [4 (link),6 (link)]. Serum was obtained after overnight blood coagulation at 4°C, followed by centrifugation at 14,000 rpm for 20 min at 4°C and removal of the serum fraction. Stanbio diagnostic kits (Boerne, TX) were used to determine serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and β-beta-hydroxybutyrate (β-OHB).

Aliquots of liver homogenate (5 mg protein from above) were also used for quantitating lipids including total cholesterol (TC), free cholesterol (C), triacylglycerol (TG), and phospholipid (PL) after solvent-extraction, thin layer chromatography (TLC), and quantitation as in (42 (link)). Alternately lipid classes were determined directly in the homogenate using diagnostic kits available from Wako Chemicals (Richmond, VA). Quantitation did not significantly differ between the two methods. To determine cholesteryl ester concentration (CE), free (non-esterified) cholesterol concentration (C) was subtracted from the total cholesterol. Serum β-hydroxybutyrate (β-Hydroxybutyrate LiquiColor, β-OHB) and high density lipoprotein cholesterol (Direct HDL-Cholesterol, HDL-C) were determined using diagnostic kits from Stanbio Laboratory (Boerne, TX). Serum apolipoprotein B (apoB) and apolipoprotein A-I (apoA1) were quantitated with diagnostic kits from Diazyme Labs (Poway, CA). Serum non-HDL-C was calculated by subtracting serum HDL-C from serum TC.