串联质谱检测干血滤纸片多种溶酶体酶活性方法的建立  

Detecting multiple lysosomal enzymes in dried blood spots by tandem mass spectrometry

作　　者：占霞 顾学范[1] 琳娜 叶军[1] 邱文娟[1] 韩连书[1] 张惠文[1] 梁黎黎[1] 

机构地区：[1]上海交通大学医学院附属新华医院上海市儿科医学研究所小儿内分泌遗传代谢病研究室,200092

出　　处：《中华检验医学杂志》2016年第10期761-765,共5页Chinese Journal of Laboratory Medicine

基　　金：“十二五”国家科技支撑计划项目（2012BA109804）;上海市科委重大课题（11dz1950300）

摘　　要：目的建立串联质谱法测定干血滤纸片中半乳糖神经酰胺酶（GALC）、酸性α－葡萄糖苷酶（GAA）、α－半乳糖苷酶（GLA）、α－L－艾杜糖苷酸酶（IDUA）活性。方法 方法学建立。收集2013年7月、11月在上海市新华医院新生儿筛查中心进行遗传代谢性疾病筛查的2 175份干血滤纸片标本和2012年9月至2014年1月在新华医院儿科就诊的20例溶酶体贮积病（lysosomal storage disorders，LSDs）患儿干血滤纸片样本。干血滤纸片提取液与酶底物及内标孵育后，经液液和固相萃取，氮气吹干，复溶后串联质谱分析酶反应产物和内标，计算酶活性。评价方法的线性、精密度、准确度和最低检测限；检测2 175份新生儿－干血滤纸片样本，0.5～99.5百分位数法确立4种酶活性参考区间；同时检测20例LSDs患儿干血滤纸片样本进行临床验证。结果 酶活性测定批内和批间精密度为1.7%～11.8%，批内和批间准确度为85%～115%，产物和内标实测浓度比值与添加的理论浓度比值线性系数为0.997～0.999。GALC、GAA、GLA和IDUA的最低检测限分别为0.03 μmol/（L·h）、0.09 μmol/（L·h），0.12 μmol/（L·h），0.16 μmol/（L·h）；初步确定新生儿酶活性参考区间分别为GALC[0.51～8.51 μmol/（L·h）]，GAA[1.99～22.22 μmol/（L·h）]，GLA[1.68～41.59 μmol/（L·h）]，IDUA [2.36～19.21 μmol/（L·h）]；20例LSDs患儿酶活性明显低于参考区间，串联质谱法能有效检出Krabbe、Pompe、Fabry、MPSⅠ患儿。结论 成功建立了串联质谱法检测干血滤纸片中GALC、GAA、GLA、IDUA活性。Objective Establish a method for measuring the activities of Galactocerebrosidase （ GALC）, α-Glucosidase （ GAA）, α-Galactosidase （GLA） and α-L-Iduronidase （IDUA） in dried blood spots specimen by tandem mass spectrometry （MS/MS）. Methods A total of 2175 dried blood spot samples forinborn errors of metabolism in neonatalscreening center of Shanghai Xinhua hospital were collected in July and November, 2013. And twenty dried blood spot samples from patients withlysosomalstorage disorders（LSDs） of Shanghai Xin Hua Hospital were collected from September 2012 to January 2014. The extraction of DBS was incubated with enzyme substrates and internal standards. After liquid-liquid and solid- phase extraction, the extraction solution was dried under nitrogen and reconstituted. Then enzyme reaction products and internal standards were analyzed by MS/MS. Linearity, precision, accuracy and the limit of detection were evaluated. 2175 dried blood spot samples were detected to establish the normal reference range for the activities of four enzymes according to 0.5th to 99. 5th percentiles. 20 specimens from patients withLSDs were detected to verify the reference range inclinical judgment. Results The intraassay and interassay precisions ranged from h 7% to lh 8%, and the intraassay and interassay accuracies ranged from 85% to 115%. The linear coefficients for measured concentration of enzyme products/internal standards and theoretical concentration were 0. 997 -0. 999. The limits of detection forGALC, GAA, GLA and GLA were 0.03μmol/（L·h）], 0.09μmol/（L·h）], 0. 12 μmol/（L·h）] and 0. 16μmol/（L·h）] . The normal reference values for GALC, GAA, GLA and GLAwere 0. 51 - 8.51μmol/（L·h）] , 1.99 - 22. 22μmol/（L·h）], 1.68 - 41.59μmol/（L·h）] and 2.36 - 19. 21μmol/（L·h）]. The enzymes of 20 patients with LSDs were remarkably decreased compared to the normal range. The Krabbe, Pompe, Fabry, MPS I patients can be effectively detected by this MS/MS method. Conclusions A MS/M

关 键 词：溶酶体贮积病 串联质谱法 干燥血斑检测 半乳糖神经酰胺酶 d葡糖苷 酶类 Α-半乳糖苷酶 艾杜糖醛酸酶 

分 类 号：R446.1[医药卫生—诊断学] R722.1[医药卫生—临床医学]