Hydroxyproline assay for collagen content

Hydroxyproline is a major constituent of collagen and is highly expressed in fibrous tissue (Mu et al. 2010). Total hydroxyproline content was determined in the GC muscle using a colorimetric assay kit (BioVision, Milpitas, CA) to evaluate fibrotic changes in the distracted GC muscles, as described previously (Tonogai et al. 2015). Briefly, GC muscles were hydrolyzed in 12 N HCl at 120 °C for 3 h. After hydrolysis, samples were neutralized and treated with chloramine T solution for 5 min at room temperature followed by dimethylamine borane solution for 90 min at 60 °C. Absorbance was measured at 560 nm. Hydroxyproline concentration was determined by comparison with a standard curve prepared from pure hydroxyproline of known concentrations.