■半乳糖脑糖苷酶(GALC)活性降低是Krabbe病的病因,具有毁灭性神经退行性后果的溶酶体贮积症。已经描述了分离的血液和皮肤细胞中GALC活性的定量荧光测定法;然而,没有使用干血斑(DBS)标本描述过这样的测定。
■使用来自新型糖苷底物的荧光定量测量GALC酶活性:衍生自6-十六烷酰基氨基-4-甲基伞形酮的羧基。在新生儿DBS标本上证明了GALC活性,已知的Krabbe病患者标本,能力测试和质量控制样本。
■我们提供了有关新型底物和测定的表征数据,包括使用荧光光谱的pH优化和酶动力学。单日和多日精度分析显示出严格的分析测量值,%CV范围为5.2%至14.1%。GALC酶活性在0.31-12.04μmol/l/h范围内呈线性关系,检出限为0.066μmol/l/h。我们使用此测定法的结果表明,Krabbe病患者样品中的GALC活性与假定的正常新生儿样品之间存在明显的区别。
■对干血斑标本进行GALC酶活性测量的荧光测定法是可行的。对测定的改进,包括新颖的底物设计,增加的底物浓度和氯化钠的去除使测定的特异性最大化,并使β-半乳糖苷酶的干扰最小化。
UNASSIGNED: Decreased 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.
UNASSIGNED: GALC 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.
UNASSIGNED: We 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.
UNASSIGNED: A 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.