A New Fluorescent Method to Detect Sulfamidase Activity in Blood, Tissue Extracts and Dried Blood Spots

Q3 Medicine

Journal of Inborn Errors of Metabolism and Screening Pub Date : 2021-01-01 DOI:10.1590/2326-4594-JIEMS-2020-0021

Xingxuan He, E. Schuchman, C. Simonaro

{"title":"A New Fluorescent Method to Detect Sulfamidase Activity in Blood, Tissue Extracts and Dried Blood Spots","authors":"Xingxuan He, E. Schuchman, C. Simonaro","doi":"10.1590/2326-4594-JIEMS-2020-0021","DOIUrl":null,"url":null,"abstract":" Abstract Mucopolysaccharidosis type IIIA (MPS IIIA) is a lysosomal storage disorder due to the deficient activity of sulfamidase (SGSH). Traditionally, measurement of this enzymatic activity has been performed using a fluorescently (4-MU) labeled glycoside substrate. While this substrate is inexpensive and readily available, the current method requires a 2-step procedure that is performed over 2 days. Here we report a new and simplified procedure using the 4-MU substrate. Major advantages of this assay method over the existing fluorescent method include a single step vs. 2-step procedure, an incubation time of 1 hour, and high sensitivity. The reaction is also run on UPLC equipment, which is available in most research labs and permits separation of the endogenous, autofluorescent material from the 4-MU signal. This assay method was developed using the MPS IIIA mouse model, and was validated using mouse plasma, liver and brain extracts, and dried blood spots. Human MPS IIIA skin fibroblasts and dried blood spots also were used to validate the","PeriodicalId":56346,"journal":{"name":"Journal of Inborn Errors of Metabolism and Screening","volume":"140 1","pages":""},"PeriodicalIF":0.0000,"publicationDate":"2021-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"2","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of Inborn Errors of Metabolism and Screening","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1590/2326-4594-JIEMS-2020-0021","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"Medicine","Score":null,"Total":0}

引用次数: 2

Abstract

 Abstract Mucopolysaccharidosis type IIIA (MPS IIIA) is a lysosomal storage disorder due to the deficient activity of sulfamidase (SGSH). Traditionally, measurement of this enzymatic activity has been performed using a fluorescently (4-MU) labeled glycoside substrate. While this substrate is inexpensive and readily available, the current method requires a 2-step procedure that is performed over 2 days. Here we report a new and simplified procedure using the 4-MU substrate. Major advantages of this assay method over the existing fluorescent method include a single step vs. 2-step procedure, an incubation time of 1 hour, and high sensitivity. The reaction is also run on UPLC equipment, which is available in most research labs and permits separation of the endogenous, autofluorescent material from the 4-MU signal. This assay method was developed using the MPS IIIA mouse model, and was validated using mouse plasma, liver and brain extracts, and dried blood spots. Human MPS IIIA skin fibroblasts and dried blood spots also were used to validate the

查看原文本刊更多论文

一种检测血液、组织提取物和干血斑中磺胺酶活性的新荧光方法

摘要:粘多糖病IIIA型(MPS IIIA)是一种溶酶体储存障碍，是由于磺胺酶(SGSH)活性不足引起的。传统上，这种酶活性的测量是使用荧光(4-MU)标记的糖苷底物进行的。虽然这种基板价格低廉且容易获得，但目前的方法需要2天以上的2步过程。在这里，我们报告了一个新的和简化的过程，使用4-MU衬底。与现有的荧光检测方法相比，该检测方法的主要优点包括单步检测与两步检测、1小时的孵育时间和高灵敏度。该反应也在UPLC设备上运行，该设备可在大多数研究实验室中使用，并允许从4-MU信号中分离内源性自荧光材料。该方法采用MPS IIIA小鼠模型建立，并采用小鼠血浆、肝、脑提取物和干血斑进行验证。人MPS IIIA皮肤成纤维细胞和干血斑也被用来验证

本文章由计算机程序翻译，如有差异，请以英文原文为准。

求助全文

约1分钟内获得全文求助全文

来源期刊

Journal of Inborn Errors of Metabolism and Screening

CiteScore

0.60

自引率

0.00%

发文量

审稿时长

12 weeks

期刊介绍： The Journal of Inborn Errors of Metabolism and Screening (JIEMS) is an online peer-reviewed open access journal devoted to publishing clinical and experimental research in inherited metabolic disorders and screening, for health professionals and scientists. Original research articles published in JIEMS range from basic findings that have implications for disease pathogenesis and therapy, passing through diagnosis and screening of metabolic diseases and genetic conditions, and therapy development and outcomes as well. Original articles, reviews on specific topics, brief communications and case reports are welcome. JIEMS aims to become a key resource for geneticists, genetic counselors, biochemists, molecular biologists, reproductive medicine researchers, obstetricians/gynecologists, neonatologists, pediatricians, pathologists and other health professionals interested in inborn errors of metabolism and screening.