Louis A I, Widen K E, Tsay K K, Fluharty A L
University of California, Los Angeles.
Mol Chem Neuropathol. 1991 Apr;14(2):113-30. doi: 10.1007/BF03159931.
N-[7-Nitrobenz-2-oxa-1,3-diazol-4-yl]psychosine sulfate (NBD-PS), a fluorescent analog of cerebroside sulfate (CS), was synthesized and tested as an alternative to the radiolabeled forms of CS used for assaying arylsulfatase A (ASA) in its physiological role as a cerebroside sulfate sulfohydrolase. NBD-PS simulates the natural substrate for ASA. Protocols have been developed for its use in differentiating low enzyme activities in diagnostic samples. Hydrolysis of NBD-PS is specific for ASA and optimal assay parameters were identical to those determined for CS. Differentiations between each of the major phenotypes for ASA activity were possible in the set of samples tested. One particular advantage was the ability to discriminate between individuals exhibiting arylsulfatase A pseudodeficiency and the truly deficient individuals with metachromatic leukodystrophy. Differential diagnosis was possible with fibroblast extracts by an assay that is more sensitive than procedures employing radioisotopes. Reaction products may be analyzed quantitatively by HPLC, or semiquantitatively with TLC. NBD-PS provides a simpler, safer, and more cost-effective means of performing natural substrate enzyme assays for ASA. Phenotyping with the fluorescence assay is an effective alternative to the laborious radioactive CS preparations and tissue culture loading studies that have previously been necessary.
N-[7-硝基苯并-2-恶唑-1,3-二氮杂-4-基]硫酸脑苷脂（NBD-PS）是硫酸脑苷脂（CS）的荧光类似物，已被合成并作为用于测定芳基硫酸酯酶A（ASA）作为硫酸脑苷脂硫酸水解酶的生理作用的放射性标记形式的替代物进行测试。NBD-PS模拟ASA的天然底物。已经开发了用于在诊断样品中区分低酶活性的方案。NBD-PS的水解对ASA具有特异性，最佳测定参数与CS测定确定的参数相同。在所测试的样品组中可以区分ASA活性的每种主要表型。一个特别的优点是能够区分表现出芳基硫酸酯酶A假缺陷的个体和真正患有异染性脑白质营养不良的缺陷个体。通过比使用放射性同位素的方法更灵敏的测定，可以用成纤维细胞提取物进行鉴别诊断。反应产物可以通过HPLC进行定量分析，或者用TLC进行半定量分析。NBD-PS为进行ASA的天然底物酶测定提供了一种更简单、更安全且更具成本效益的方法。荧光测定法进行表型分析是以前必需的费力的放射性CS制剂和组织培养加载研究的有效替代方法。
