Sozmen Eser Yıldırım, Sezer Ebru Demirel
Department of Medical Biochemistry and Metabolism Laboratory, Ege University Faculty of Medicine, Izmir, Turkey.
Methods Mol Biol. 2017;1594:255-264. doi: 10.1007/978-1-4939-6934-0_17.
The lysosomal storage diseases (LDSs) are a heterogeneous group of inherited genetic disorders caused by defects of lysosomal proteins. The accumulation of undigested substrates from different catabolic pathways leads to cellular dysfunction. LSDs generally presents during early childhood and have a devastating impact on the families and on public health. Over the years, approaches for treatment of some LSDs have been developed with different strategies. Increasing availability of treatments of these diseases has accelerated the development of new methods and techniques for rapid diagnosis in patients with clinical indication.The use of dried blood spot (DBS) test has been proposed as a first tier test to identify patients with Gaucher, Pompe, and Fabry diseases. DBS usage is advantageous for the purpose of screening as it is non-invasive, sensitive, has low-cost and fast turnaround time compared to measurements in leucocyte and/or fibroblast culture. This chapter focuses on the activity measurement of three lysosomal enzymes (α-glucosidase, β-glucosidase, and α galactosidase) in DBS samples by using fluorescent substrates and by the LC-MS/MS (liquid chromatography-mass spectrometry) method. All steps of the methods, from preparation of the solutions to calculation of the enzyme activity, will be explained in detail.
溶酶体贮积症（LDSs）是一组由溶酶体蛋白缺陷引起的遗传性基因疾病，具有异质性。来自不同分解代谢途径的未消化底物的积累会导致细胞功能障碍。溶酶体贮积症通常在儿童早期出现，对家庭和公共卫生造成毁灭性影响。多年来，已针对一些溶酶体贮积症开发了不同的治疗方法。这些疾病治疗方法的可及性不断提高，加速了针对具有临床指征患者的快速诊断新方法和新技术的发展。 dried blood spot（DBS）检测已被提议作为用于识别戈谢病、庞贝病和法布里病患者的一线检测方法。DBS检测用于筛查具有优势，因为与白细胞和/或成纤维细胞培养检测相比，它是非侵入性的、灵敏的、成本低且周转时间快。本章重点介绍通过使用荧光底物和液相色谱 - 质谱联用（LC - MS/MS）方法对DBS样本中的三种溶酶体酶（α - 葡萄糖苷酶、β - 葡萄糖苷酶和α - 半乳糖苷酶）进行活性检测。将详细解释该方法的所有步骤，从溶液制备到酶活性计算。
