2.14. Hydroxyproline Analysis

The total amount of collagen was measured by the hydroxyproline assay. The assay was performed using an ELISA kit (Cell Biolabs) in accordance with the manufacturer’s protocol. Liver tissues (100 mg) were homogenized with 10 mL of 5 N HCl for hydrolysis and incubated overnight at 120 °C. After incubation, the hydrolyzed product was passed through a 0.45 µm PVDF syringe filter (Sigma–Aldrich, St. Louis, MO, USA). It was left to dry under vacuum for 45 min at 70 °C. All samples were analyzed in duplicates, and the mean values were used for analysis. The hydroxyproline level is specified as micrograms per milligrams of protein.