Quantitative assessment of enzymatic reaction byproducts using mass spectrometry or fluorimetry may become a novel diagnostic tool for Pompe disease (PD) and other lysosomal storage diseases (LSDs), according to a study recently published in The FASEB Journal.

Substrates of such enzymes are usually made up of a particle recognized by the enzyme that is attached to another particle (usually a 4-umbelliferone) by a covalent bond, and the enzymatic reaction produces 4-methylumbelliferone as a derivative. Therefore, quantifying this molecule could determine whether the enzyme functions correctly, yielding a screening method for LSDs, as demonstrated by Jayaweera and colleagues.

To evaluate this hypothesis, the investigators obtained samples using dried blood spots (DBS) to analyze the behavior of various substrates of the enzymes involved in LSDs, such as mucopolysaccharidoses (MPS) and neuronal ceroid lipofuscinoses (NCL), after undergoing simultaneous mass spectrometric and fluorimetric tests. The results were later validated with DBS samples from patients previously diagnosed with LSDs.

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Both fluorimetry and multiple reactions monitoring mass spectrometry delivered promising results in single assays.

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“We have developed a high-throughput single and multiplexing assay for rare diseases . . . in DBSs,” the authors wrote.

The researchers performed further triplex and duplex assays by employing the Pechmann condensation technique to produce modified substrates from coumarin, interchanging elements in the 4 and 7 positions of the molecule. Although the experiment included mostly MPS subtypes, these pivotal findings could also represent a significant advance in the clinical setting for patients with PD.

Current medical practice lacks easily accessible diagnostic tools for PD, and few examples of LSDs use fluorimetry or mass spectrometry for diagnosis. Although rare, this disease can produce significant morbidity and decrease the quality of life of affected patients.

The therapeutic approach of enzyme replacement therapy has been proven useful for ameliorating symptoms; nonetheless, initiating treatment requires a timely diagnosis. The pathogenesis of all LSDs, including lysosomal acid lipase deficiency, involves a defective enzyme, and determining the byproducts of its activity through easily obtained DBSs could potentially become a standardized diagnostic approach.


Jayaweera T, Darie Ion L, Petre BA, Darie C. Multiplex mass spectrometry for enzymatic activities screening of lysosomal storage diseases. FASEB J. Published online May 13, 2022. doi:10.1096/fasebj.2022.36.s1.l7862