两种血浆壳三糖酶活性测定方法的比较及其临床应用

Comparison and clinical application of two methods for determination of plasma chitotriosidaseactivity

目的比较两种血浆壳三糖酶活性测定方法，并观察其应用于戈谢病和尼曼匹克病的辅助检测及戈谢病治疗监测的效果。方法分别使用4-methylumbelliferyl-β-D-N，N’，N”-triacetyl-chitotrioside（4MU-C3）和4．Methylumbelliferyl4．Deoxy—β—D—chitobiose（4MU-4dC2）作为底物，对45例正常人、31例戈谢病患者及9例尼曼匹克病A／B型患者进行血浆壳三糖酶活性测定，并进行人壳三糖酶基因24个碱基重复序列突变（dutp24）检测。结果（1）对照组使用底物4MU-4dC2测得壳三糖酶活性值为使用底物4MU．C3测定值的3．7倍（Z=-4．703，P〈0．001）；戈谢病未治疗组使用两种底物测得壳三糖酶活性值分别高于对照组794倍和610倍（Z=-3．823，P〈0．001），治疗组治疗药物减量前高于对照组134倍和79倍，减量后高于对照组215倍和118倍（Z=-2．521，P〈0．05）；尼曼匹克病组使用两种底物测得壳三糖酶活性值分别高于对照组8倍和14倍（Z=-1．604，P=0．109），差异无显著意义。（2）85例研究对象中有30例酶活性值极低，经突变检测证实携带dup24纯合突变，为壳三糖酶缺失；野生型及dup24杂合突变型戈谢病患者壳三糖酶活性值显著高于对照组。结论底物4MU-4dC2检测壳三糖酶活性更为敏感，可用于戈谢病患者辅助检测和壳三糖酶非缺失型病例的治疗监测，对尼曼匹克病无辅助检测价值。

Objective Chitotriosidase （CT） is a plasma biomarker for Gaucher disease（ GD）, the enzyme activity is usually markedly elevated in plasma of Gaucher patients, and it was reported that levels of plasma chitotriosidase activity was mildly-moderately increased in patients with Niemann-Pick disease （NPD）. The aim of this study was to compare chitotriosidase activity using 4-methylumbelliferyl-β-D-N, N＇, N” -triacetyl-chitotrioside （4MU-C3） with 4-methylumbelliferyl 4-deoxy-β-D-chitobiose （4MU-4dC2） as substrates, and apply chitotriosidase activity t to help clinical determination of GD and NPD, and to monitor therapy in GD patients. Method Plasma of 45 healthy individuals, 31 patients with GD and 9 patients with NPD type A/B was collected from outpatient clinics of the Department of Pediatric Endocrinologic, Genetic and Metabolic Diseases, Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine. Plasma chitotriosidase activity was measured with the substrates 4MU-C3 and 4MU-4dC2 respectively. Determinations were based on the methods described by Hollak et al and Rodrigues et al. Meanwhile, common mutation dup24 of the human chitotriosidase gene was detected. Result （1） Chitotriosidase activity when measured with 4MU-4dC2 gave higher values than 4MU-C3. In the healthy controls chitotriosidase activity was increased 3.7-fold when the 4MU-dC2 was used as substrate as compared with the 4MU-C3 （ Z = - 4. 703 ,P 〈 0. 001 ）. In the untreated GD patients, the median value was increased 794-fold and 610-fold of the control subjects （ Z = - 3. 823, P 〈 0. 001 ） when the enzyme was measured with two substrates respectively. In the GD patients during therapy, chitotriosidase activity was increased 134-foldand 79-fold, and after changing therapeutic dose chitotriosidase activity was increased 215-fold and 118-fold of the controls（Z = -2. 521 ,P 〈 0.05）. In the NPD patients chitotriosidase activity was increased 8-fold and 14-fold of the controls （ Z = - 1. 604, P = 0. 109）. （2） Consistent with the results of chitotriosidase activity, 30 of 85 （35.3%） individuals were homozygotes of dup24 mutation, which are completely chitotriosidase enzyme deficiency. Among GD patients with wild-type and heterozygotes for the dup24 mutation, chitotriosidase activity highly increased in the plasma compared with the controls. Conclusion The use of 4MU-4dC2 as substrate makes chitotriosidase activity measurement more sensitive. The determination of plasma chitotriosidase activity is a useful tool to assist the clinical identification of Gaucher disease, and to monitor enzyme replacement therapy （ERT） of non-chitotriosidase deficient GD patients. Chitotriosidase activity determination has no value in the clinical identification of NPD.