Tina version 2

Liver lipids were extracted from freshly prepared liver homogenates using methanol and chloroform following the Bligh & Dyer method29 and separated by high‐performance thin‐layer chromatography as described previously.17 Briefly, lipids were stained with a color reagent (5 g of MnCl₂4H₂O, 32 mL of 95%‐97% H₂SO₄ added to 960 mL of CH₃OH/H₂O = 1:1 [vol/vol]) and triglycerides, cholesteryl esters, and free cholesterol were quantified (TINA version 2.09 software, Raytest, Straubenhardt, Germany).
Hepatic total collagen content was determined biochemically after acid hydrolysis of liver biopsies for 20 hours at 95°C. Hydroxyproline was measured in the freshly prepared hydrolysate using a total collagen assay relative to a collagen standard per manufacturer’s instructions (Quickzyme, Leiden, the Netherlands).

Liver lipids were analyzed by high-performance thin-layer chromatography as described previously.^{22 (link)} In brief, lipids were extracted from liver homogenates using methanol and chloroform following the Bligh and Dyer method,^{23 (link)} after which they were separated by high-performance thin-layer chromatography on silica gel plates. Lipid spots were stained with color reagent (5 g of MnCl₂4H₂O, 32 ml of 95–97% H₂SO₄ added to 960 ml of CH₃OH/H₂O=1:1 (v/v)) and triglycerides, cholesteryl esters and free cholesterol were quantified using TINA version 2.09 software (Raytest, Straubenhardt, Germany). Liver lipids were expressed per mg liver protein, which was determined in the same liver homogenates used for the liver lipid analysis, using the Lowry protein assay.^{24 (link)}