Analysis of hydroxyproline in tissue based on alkaline hydrolysis.

Objective Traditional determination of hydroxyproline in tissue by acidic hydrolysis has many shortcomings such as long digestion time,easy volatilization for 6 N HC1and,tedious steps.This study aimed at exploring feasibility and influencing factors of a simplified method based on alkline hydrolysis. Methods Test samples including tissue homogenates or minces were hydroszed with sodium hydroxide and autoclaved at 120℃for 20 min.Chloramine-T was used to oxidize for 10 min at 37℃.The addition of p-dimethylaminobenzaldehyde and incubation of the samples at 80℃ for 10 min resulted in the formation of a chromophore that can be meaeured at 550 nm. We observed critical parameters such as concentration of sodium hydroxide, time of hydrolysis, pH of the reation buffer, and dry and wet weights of tissue, and we made a comparison between the assy methods based on alkaline hydrolysis and acid hydrolysis. Results The optimal condition of the method based on alkaline hydrolysis: 1 yophil ized tissue; 2 N sodium hydroxide (final concentration); 20 min for hydrolysis; pH 6.0-6.5. Conclu-sionsThis assay method reduces meassurement time (whole time is about 2 h), and simplifies experimental procedure. This method is highly sensitive and, reproducible with high consistency and recovery, which can be used in clinical and biomedical research fields.