2.3. Skin Hydroxyproline Content Measurement

A hydroxyproline assay kit was purchased from Sigma-Aldrich (MAK008-1KT, St. Louis). Hydroxyproline is a modified amino acid unique to collagen. Hence, fibrosis can be quantitatively estimated by determining the total hydroxyproline content of the tissue. Briefly, skin tissue was first homogenized and hydrolyzed with 12 M hydrochloric acid 120°C for 3 h. The supernatant was then collected to determine the hydroxyproline concentration by the reaction of oxidized hydroxyproline with 4-(Dimethylamino) benzaldehyde. The results were expressed as micrograms of hydroxyproline per gram of skin.