Fluorimetric assay with a novel substrate for quantification of galactocerebrosidase activity in dried blood spot specimens.
Fluorimetric assay with a novel substrate for quantification of galactocerebrosidase activity in dried blood spot specimens.
复制标题
使用新型底物进行荧光测定，用于定量干血斑标本中的半乳糖脑苷脂酶活性。
DOI：
10.1016/j.plabm.2019.e00141
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发表时间：
2020
影响因子：
1.9
通讯作者：
Pamula,VamseeK
中科院分区：
文献类型：
--
作者：
Ullal,AnirudhJ;Pham,Hong;Singh,Rajendra;Ross,Peter;Graham,CarrieA;Norton,ScottM;Nuffer,MiriamH;Burns,DebbieS;Eckhardt,AllenE;Escolar,Maria;Bali,Deeksha;Pamula,VamseeK
背景半乳糖脑苷酶 (GALC) 酶活性降低是导致克拉伯病的原因，克拉伯病是一种溶酶体贮积症，具有毁灭性的神经退行性后果。已描述了分离血液和皮肤细胞中 GALC 活性的定量荧光测定法；然而，尚未描述过使用干血斑 (DBS) 样本进行此类测定。 方法 使用新型糖苷底物（源自 6-十六酰氨基-4-甲基伞形酮的羧基）的荧光定量测量 GALC 酶活性。 GALC 活性在新生儿 DBS 样本、已知克拉伯病患者样本、能力验证和质量控制样本中得到了证明。结果我们提供了有关新型底物和测定的表征的数据，包括使用荧光分析的 pH 优化和酶动力学。单日和多日精度分析显示严格的分析测量，%CV 范围为 5.2% 至 14.1%。 GALC 酶活性在 0.31 - 12.04 μmol/l/h 范围内呈线性，检测限为 0.066 μmol/l/h。我们的检测结果表明，克拉伯病患者样本中的 GALC 活性与假定的正常新生儿样本中的 GALC 活性存在明显区别。结论 对干血斑标本进行 GALC 酶活性测量的荧光检测是可行的。检测的改进包括新颖的底物设计、增加的底物浓度和去除氯化钠，最大限度地提高了检测的特异性，并最大限度地减少了 β-半乳糖苷酶的干扰。
BackgroundDecreased galactocerebrosidase (GALC) enzyme activity is causative for Krabbe disease, a lysosomal storage disorder with devastating neurodegenerative consequences. Quantitative fluorimetric assays for GALC activity in isolated blood and skin cells have been described; however, no such assay has been described using dried blood spot (DBS) specimens.MethodsGALC enzyme activity was measured quantitatively using fluorescence from a novel glycosidic substrate: carboxy derived from 6-hexadecanoylamino-4-methylumbelliferone. GALC activity was demonstrated on newborn DBS specimens, known Krabbe disease patient specimens, proficiency testing and quality control samples.ResultsWe present data on characterization of the novel substrate and assay, including pH optimization and enzyme kinetics using a fluorimetric profile. Single and multi-day precision analyses revealed tight analytical measurements with %CV ranging from 5.2% to 14.1%. GALC enzyme activity was linear over the range of 0.31 - 12.04 μmol/l/h with a limit of detection of 0.066 μmol/l/h. Our results with this assay show a clear discrimination between GALC activities in samples from Krabbe disease patients versus presumed normal newborn samples.ConclusionsA fluorimetric assay for GALC enzyme activity measurement on dried blood spot specimens is feasible. Improvements to the assay including novel substrate design, increased substrate concentration and removal of sodium chloride maximize the specificity of the assay and minimize interference from β-galactosidase.