Tomatsu Shunji, Kubaski Francyne, Sawamoto Kazuki, Mason Robert W, Yasuda Eriko, Shimada Tsutomu, Montaño Adriana M, Yamaguchi Seiji, Suzuki Yasuyuki, Orii Tadao
Nemours/Alfred I. duPont Hospital for Children, Wilmington, DE ; Department of Pediatrics, Gifu University, Gifu, Japan.
Nemours/Alfred I. duPont Hospital for Children, Wilmington, DE ; Department of Biological Sciences, University of Delaware, Newark, DE.
Nihon Masu Sukuriningu Gakkai Shi. 2014;24:19-37.
Mucopolysaccharidoses (MPS) are a group of lysosomal storage disorders caused by the deficiency of lysosomal enzymes. The enzymes are required to break down glycosaminoglycans (GAGs) that help build bone, cartilage, tendons, corneas, skin and connective tissue. In patients with MPS, a missing enzyme leads to the accumulation of GAGs in the cells, blood, connective tissues, and multiple organs. The consequence is permanent, with progressive cellular damage affecting patients' appearance, physical abilities, organ and system function, and skeletal and mental development. The measurement of each specific GAG in a variety of specimens is required to establish the correlation between GAGs and physiological status of patients and/or prognosis and pathogenesis of the disease and to separate the patients with MPS from the healthy controls. We have developed a highly accurate, sensitive, and cost-effective liquid chromatography tandem mass spectrometry (LC-MS/MS) method for measurements of disaccharides derived from four specific GAGs [chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS), and keratan sulfate (KS)]. Disaccharides were produced by specific enzyme digestion of each GAG, and subsequently, quantified by negative ion mode of multiple reaction monitoring. Subclasses of GAGs with the same molecular weights can be separated by liquid chromatography. We have also developed another GAG assay by high-throughput mass spectrometry (HT-MS/MS). The HT-MS/MS consists of an integrated solid phase extraction robot that binds and de-salts samples from assay plates and directly injects them into a MS/MS detector, reducing sample processing time to within ten seconds. HT-MS/MS consequently yields much faster throughput than conventional LC-MS/MS-based methods; however, the HT-MS/MS system does not use a chromatographic step, and therefore, cannot separate GAGs that have the same molecular weights. Both techniques can be applied to the analysis of dried blood spots, blood, and urine specimens. In this review, we describe the assay methods for GAGs and the application to newborn screening and diagnosis of MPS.
黏多糖贮积症（MPS）是一组由溶酶体酶缺乏引起的溶酶体贮积病。这些酶是分解糖胺聚糖（GAGs）所必需的，而GAGs有助于构建骨骼、软骨、肌腱、角膜、皮肤和结缔组织。在MPS患者中，缺失的酶会导致GAGs在细胞、血液、结缔组织和多个器官中蓄积。其后果是永久性的，进行性细胞损伤会影响患者的外貌、身体能力、器官和系统功能以及骨骼和智力发育。需要测量各种标本中每种特定的GAG，以建立GAGs与患者生理状态和/或疾病预后及发病机制之间的相关性，并将MPS患者与健康对照区分开来。我们开发了一种高度准确、灵敏且经济高效的液相色谱串联质谱法（LC-MS/MS）来测量源自四种特定GAGs（硫酸软骨素（CS）、硫酸皮肤素（DS）、硫酸乙酰肝素（HS）和硫酸角质素（KS））的二糖。通过每种GAG的特异性酶消化产生二糖，随后通过多反应监测的负离子模式进行定量。具有相同分子量的GAGs亚类可通过液相色谱分离。我们还开发了另一种通过高通量质谱法（HT-MS/MS）进行的GAG检测方法。HT-MS/MS由一个集成的固相萃取机器人组成，该机器人可结合并对来自检测板的样品进行脱盐处理，然后直接将其注入MS/MS检测器，将样品处理时间缩短至10秒以内。因此，HT-MS/MS的通量比传统的基于LC-MS/MS的方法快得多；然而，HT-MS/MS系统不使用色谱步骤，因此无法分离具有相同分子量的GAGs。这两种技术均可应用于干血斑（DBS）、血液和尿液标本的分析。在本综述中，我们描述了GAGs的检测方法及其在MPS新生儿筛查和诊断中的应用。
