Dermal Hydroxyproline Assay

Skin tissue specimens were hydrolyzed in 12N HCl at 120°C for 24 hours. After evaporation, 200 μL of dextran-charcoal solution (1% charcoal and 0.05% dextran 500T) was mixed with 800 μL of water and centrifuged at 400 × g for 10 minutes. A volume of 100 mL of a 1:2 dilution of the supernatant was mixed with 250 μL of chloramine solution (1.3% chloramine-T, 10% propanol, and 80% citrate-acetate buffer) over 20 minutes at room temperature, followed by the addition of 250 μL of Ehrlich solution and incubation at 60°C for 20 minutes. Absorbance was measured at 550 nm. Standard curves (0 to 1 mg/mL) were generated for each experiment using reagent hydroxyproline as a standard. Results were expressed as micrograms of hydroxyproline per milligram of tissue.⁶