尿β-D-半乳糖苷酶活性检测方法的建立及临床意义探讨

Establishment on the determination method of urinary beta-D-galactosidase activity

目的以2-氯-4-硝基苯基-β-D-半乳糖苷(CNP-GAL)为底物,建立测定尿β-D-半乳糖苷酶(GAL)活性的连续监测法,并探讨GAL在肾小管病变早期诊断中的临床意义。方法基于GAL能催化水解底物生成色原2-氯-4-硝基苯酚(CNP)的原理,对最适p H值、底物浓度、反应时间等条件进行研究,建立用于检测尿GAL的连续监测法,并用该法检测125例肾脏病变(30例继发性肾损伤、36例肾小管肾炎、25例肾癌、34例肾囊肿)患者及80名健康体检者(正常对照组)尿GAL活性。结果建立了检测尿GAL活性的连续监测法,采用柠檬酸缓冲液为最佳缓冲体系,酶促反应最适p H值为4.8,最适底物浓度为2.0 mmol/L。该法批内、批间平均变异系数(CV)分别为2.44%、4.47%,线性范围为3.2~25.6 U/L,当GAL<1.6 U/L时不能被检出。继发性肾损伤、肾小管肾炎、肾癌患者的尿GAL活性均明显高于正常对照组(P<0.05),而肾囊肿患者的尿GAL活性无明显增高。结论建立的GAL活性连续监测法敏感性高,结果准确,操作简便,可用于肾小管疾病的早期诊断。

Objective To establish the continuous monitoring method for urinary beta-D-galactosidase (GAL)activity using 2-chloro-4-nitrophenyl-beta-D-galactosidase (CNP-GAL)as substrate,and to investigate the value of GAL in the early diagnosis of renal tubular lesion. Methods Based on the generation of color substance upon the hydrolysis of substrate into 2-chloro-4-nitrophenol(CNP),the optimal pH value,substrate concentration,reaction time and so on were analyzed. The continuous monitoring method for urinary GAL activity was established.Urinary GAL activities in renal tubular lesion patients(30 cases of secondary renal injury,36 cases of tubal nephritis,25 cases of renal carcinoma and 34 cases of renal cyst) and 80 healthy subjects (healthy control group)were determined. Results The continuous monitoring method for urinary GAL activity was established using citric acid buffer solution. The enzyme optimal pH value was 4.8,the optimal substrate concentration was 2.0 mmol/L,and the within-run and between-run coefficients of variation(CV)were 2.44% and 4.47%. The linear range was 3.2-25.6 U/L,which can not be determined at GAL<1.6 U/L. Urinary GAL activities in patients with secondary renal injury,tubal nephritis and renal carcinoma were higher than those in healthy control group(P<0.05),and there was no difference in renal cyst patients. Conclusions The continuous monitoring method for urinary GAL activity has good sensitivity,precision and simplicity,which is suitable for the early diagnosis of renal tubular lesion.