Synthesis and characterization of NBD-PS: a fluorescent analog of cerebroside arylsulfatase A deficiency disorders.
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.
University of California, Los Angeles.
A I
AI
Louis
K E
KE
Widen
K K
KK
Tsay
A L
AL
Fluharty
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives*; D000818:Animals; D002553:Cerebroside-Sulfatase / Q000172:deficiency*; D002554:Cerebrosides / Q000138:chemical synthesis; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D005456:Fluorescent Dyes / Q000138:chemical synthesis*; D005838:Genotype; D006801:Humans; D010641:Phenotype; D010718:Phosphatidylserines / Q000138:chemical synthesis*; D051381:Rats; D013050:Spectrometry, Fluorescence; D013462:Sulfur Radioisotopes
D002554:Cerebrosides; D005456:Fluorescent Dyes; C034296:N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylserine; D010718:Phosphatidylserines; D013462:Sulfur Radioisotopes; D002553:Cerebroside-Sulfatase; D009327:4-Chloro-7-nitrobenzofurazan