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Fluorimetric assay with a novel substrate for quantification of galactocerebrosidase activity in dried blood spot specimens.

Publication ,  Journal Article
Ullal, AJ; Pham, H; Singh, R; Ross, P; Graham, CA; Norton, SM; Nuffer, MH; Burns, DS; Eckhardt, AE; Escolar, M; Bali, D; Pamula, VK
Published in: Pract Lab Med
January 2020

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Duke Scholars

Published In

Pract Lab Med

DOI

ISSN

2352-5517

Publication Date

January 2020

Volume

18

Start / End Page

e00141

Location

Netherlands
 

Citation

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Ullal, A. J., Pham, H., Singh, R., Ross, P., Graham, C. A., Norton, S. M., … Pamula, V. K. (2020). Fluorimetric assay with a novel substrate for quantification of galactocerebrosidase activity in dried blood spot specimens. Pract Lab Med, 18, e00141. https://doi.org/10.1016/j.plabm.2019.e00141
Ullal, Anirudh J., Hong Pham, Rajendra Singh, Peter Ross, Carrie A. Graham, Scott M. Norton, Miriam H. Nuffer, et al. “Fluorimetric assay with a novel substrate for quantification of galactocerebrosidase activity in dried blood spot specimens.Pract Lab Med 18 (January 2020): e00141. https://doi.org/10.1016/j.plabm.2019.e00141.
Ullal AJ, Pham H, Singh R, Ross P, Graham CA, Norton SM, et al. Fluorimetric assay with a novel substrate for quantification of galactocerebrosidase activity in dried blood spot specimens. Pract Lab Med. 2020 Jan;18:e00141.
Ullal, Anirudh J., et al. “Fluorimetric assay with a novel substrate for quantification of galactocerebrosidase activity in dried blood spot specimens.Pract Lab Med, vol. 18, Jan. 2020, p. e00141. Pubmed, doi:10.1016/j.plabm.2019.e00141.
Ullal AJ, Pham H, Singh R, Ross P, Graham CA, Norton SM, Nuffer MH, Burns DS, Eckhardt AE, Escolar M, Bali D, Pamula VK. Fluorimetric assay with a novel substrate for quantification of galactocerebrosidase activity in dried blood spot specimens. Pract Lab Med. 2020 Jan;18:e00141.
Journal cover image

Published In

Pract Lab Med

DOI

ISSN

2352-5517

Publication Date

January 2020

Volume

18

Start / End Page

e00141

Location

Netherlands