4.8. Quantification of Total Lipids, Total Cholesterol, and Triglycerides

Ten sets of tissue samples were used for each genotype and each age group. Total lipids, total cholesterol and triglyceride levels in Pparb/d^fl/fl and FSP1cre-Pparb/d^−/− livers were determined with quantitative enzymatic assays using the Lipid Quantification Kit STA-613 (Cell Biolabs, Inc, San Diego, CA, USA), Total Cholesterol Assay Kit STA-384 (Cell Biolabs, Inc, San Diego, CA, USA) and Serum Triglyceride Quantification Kit STA-396 (Cell Biolabs, Inc, San Diego, CA, USA), respectively. Prior measurement of total lipids, the lipids in the livers were extracted using the Lipid Extraction Kit STA-612 (Cell Biolabs, Inc, San Diego, CA, USA). The procedures were in accordance with the protocols provided by the manufacturer. The enzymatic reactions of the Lipid Quantification Kit and Total Cholesterol Assay Kit generated a dye that absorbs light at 540 nm, whereas enzymatic reactions from the Serum Triglyceride Quantification Kit generated a dye that absorbs light at 570 nm. A standard curve was generated for each individual assay, using the serial dilutions of the standard solution provided in each kit. Using the optical density values at the specific absorbance wavelength against the respective concentrations of standards, the standard curves were generated using Microsoft^® Office Excel 2010 and best fitted to a linear function with an R2 ≥ 0.98. The calculated total lipids level was normalized with the mass of liver tissue, while the calculated triglyceride, total cholesterol, cholesterol ester, and free cholesterol levels were normalized with the total protein determined with the Bradford assay protein assay #5000006 (Bio-Rad Laboratories, Hercules, CA, USA).