Synthesis and characterization of NBD-PS: A fluorescent analog of cerebroside sulfate for diagnosis of arylsulfatase a deficiency disorders

Abstract

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.

A New Analytical Bench Assay for the Determination of Arylsulfatase A Activity Toward Galactosyl-3-Sulfate Ceramide: Implication for Metachromatic Leukodystrophy Diagnosis

“…Taking into account the variability of enzyme activity in normal donors and patients, our assay returned activities that were highest for ND-derived cells and lowest for patient’s cells, compared to the range of values previously reported in the literature − or routinely generated through conventional methods in laboratory facilities specialized in prenatal diagnostics. Thus, our assay allows for detection of ARSA activity specifically in cells and tissues from NDs, defining a reference threshold of activity that must be restored in target tissues for a successful therapeutic treatment and clearly identifying samples from MLD patients, in which ARSA activity is undetectable.…”

“…ARSA activity toward the cerebroside sulfate is currently evaluated in conventional assays using a radiolabeled sulfatide or fluorescent-tagged derivatives as substrates. − Notwithstanding being specific, these methods exhibit poor limits of detection and reproducibility and require relatively large quantities of protein as the source of enzyme activity.…”

“…Currently, ARSA activity is measured through a variety of biochemical assays based on radiolabeled sulfatide − or fluorogenic derivatives − or on fluorogenic/colorimetric artificial substrates. − …”