碱解法测定组织羟脯氨酸的实验研究

Analysis of hydroxyproline in tissue based on alkaline hydrolysis.

目的 在羟脯氨酸的测定方法中,传统的酸解法存在消化时间长,6 mol/L HC1易挥发,步骤繁琐等缺点,故本文目的在于探讨操作简单的碱解法测定羟脯氨酸的可行性及影响因素。方法 将待测组织匀浆或组织碎块按1:4的比例加入2.5 mol/L氢氧化钠,12℃、0.1 kPa消化20 min,然后氯氨T氧化10 min(37℃水浴),与对二甲氨基苯甲醛反应10 min(水浴80℃)生成红色化合物,550nm 处比色。分别观察了氢氧化钠浓度、水解时间、pH值、组织干重及湿重对本实验的影响,并与酸解法作了对比。结果碱解法的适宜条件如下:组织块必须称干重、2 mol/L NaOH(终浓度)、水解时间20 min、pH值6.0-6.5。结论碱解法缩短了测定时间(全部测定用2 h),简化了操作,而且灵敏度高、重复性和一致性较好,回收率较高,可用于临床和生物医学领域。

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.