双试剂速率法测定二胺氧化酶活性的研究

The study of rate method to detect the activity of diamine oxidase with double-reagent

目的建立检测二胺氧化酶(DAO)活性的双试剂连续监测法,并进行方法学评价。方法通过试验来确定反应特异吸收峰、最佳检测时间、合理试剂配比、最佳底物浓度和反应缓冲体系的最佳pH值及浓度等最适反应条件。利用线性范围、精密度、回收试验、干扰试验和试剂及样本稳定性来评价该方法的敏感性、准确度和抗干扰能力。检测150名健康人和30例严重烧伤合并胃肠道黏膜屏障功能障碍或衰竭患者血清DAO活性,确定参考区间和诊断准确度。结果通过试验确定以100 mmol/L三羟甲基氨基甲烷-盐酸(Tris-HCl,pH值8.0)为反应缓冲体系,以3.2 mmol/L 1,4-丁二胺为最佳底物浓度,试剂Ⅰ和试剂Ⅱ按4∶1与样本反应,在340 nm波长下200～300 s期间连续监测还原型辅酶Ⅰ(NADH)吸光度的下降速度计算求得DAO活性。本法线性范围为0～100 U/L,不精密度均<5%,2个水平的回收率分别为95.8%、98.0%,胆红素<150μmol/L、血红蛋白<4 g/L、脂肪乳<4 g/L、NH4+<60μmol/L、单胺氧化酶(MAO)<120 U/L时对本法无明显干扰,试剂置仪器中可稳定2周,正常人血清DAO活性为(3.90±2.96)U/L,按95%可信区间其诊断参考区间为0～9.7 U/L,受试者工作特征(ROC)曲线下面积为0.960,诊断准确度良好。结论建立的DAO活性浓度双试剂连续监测方法敏感性高、重复性好、抗干扰力强、诊断准确度良好,试剂稳定性满足日常工作需要,适用于全自动生化分析仪检测,可做为临床常规检测方法用于肠黏膜损伤的诊断。

Objective To establish a continuous monitoring method to detect the activity of diamine oxidase（DAO） with double-reagent, and to evaluate methodologically. Methods The method was used to identify the optimum reaction conditions about the specific absorption peak, the optimum detection time, a reasonable ratio of reagents, the optimum substrate concentration, the best pH value of reaction buffer system and the concentration of the buffer. The sensitivity, accuracy and anti-interference capability were evaluated by linear range, precision, recovery, interference test and stability of reagents and samples. The reference interval and diagnostic accuracy were identified by detecting the activity of DAO in 150 healthy subjects and 30 patients who were severely burnt and combined with gastrointestinal mucosa barrier dysfunction or failure. Results The final reaction buffer system was based on the 100 mmol/L Tris （ hydroxy- methyl） -aminomethane hydrochloride （Tris-HC1） buffer （ pH 8.0 ）. The optimum substrate was 3.2 mmol/L 1,4- tetranethylenediamine, and the ratio of reagent I and reagent ]I was 4：1 . The activity of DAO was calculated by the continuous monitoring of the lowering speed of nicotinamide adenine dinucleotide （NADH） absorbance under the wavelength of 340 nm during 200-300 s. The linear range was 0-100 U/L, the imprecision was 〈 5% , the recovery rate was 95.8% and 98.0% , the method was not interfered when bilirubin 〈 150 μmol/L, hemoglobin 〈 4 g/L, lipid 〈 4 g/L,NH4^＋ 〈60 p, mol/L and monoamine oxidase（MAO） 〈 120 U/L. The reagents were stable in 2 weeks. The normal activity of serum DAO was （3.90 ± 2.96 ） U/L, and according to 95% confidence interval, the diagnostic reference range was is 0 - 9.7 U/L. The area under receiver operating characteristic（ROC） curve was 0. 960, and the diagnostic accuracy was good. Conclusions The continuous monitoring method to detect the activity of DAO with double-reagent is sensitive and reproducible, and has strong ability of anti-interference and good diagnostic accuracy. The stability of reagent can meet the daily needs of automatic biochemical analyzer for detection. It may become a routine clinical detection method for the diagnosis of intestinal mucosa injury.