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

• Published in: Practical laboratory medicine Vol. 18; p. e00141
• Main Authors: Ullal, Anirudh J., Pham, Hong, Singh, Rajendra, Ross, Peter, Graham, Carrie A., Norton, Scott M., Nuffer, Miriam H., Burns, Debbie S., Eckhardt, Allen E., Escolar, Maria, Bali, Deeksha, Pamula, Vamsee K.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.01.2020; Elsevier
• Subjects: Dried blood spot; Fluorescence enzyme assay; Galactocerebrosidase; Krabbe disease; Newborn screening; LoD; PT; QCBP; Dried blood spot; Fluorescence enzyme assay; CHMUG; Galactocerebrosidase; LoQ; CHMU; DBS; Krabbe disease; Newborn screening; NBS; MUG; RFU; CLSI; rhGALC; rhβ-gal; GALC; NEH; HMU; HMUG; QCH; QCM; QCL; LoB; NEH, Non-enzymatic hydrolysis; rhGALC, Recombinant human galactocerebrosidase; HMU, 6-hexadecanoylamino-4-methylumbelliferone; QCH, Quality control high sample; rhβ-gal, Recombinant human β-galactosidase; PT, Proficiency testing; QCBP, Quality control base pool; NBS, Newborn screening; HMUG, 6-hexadecanoylamino-4-methylumbelliferone β-D-galactopyranoside substrate; CHMU, 3-Carboxy 6-hexadecanoylamino-4-methylumbelliferone; CLSI, Clinical and Laboratory Standards Institute; CHMUG, 3-Carboxy 6-hexadecanoylamino-4-methylumbelliferone β-D-galactopyranoside substrate; LoD, Limit of detection; RFU, Relative fluorescence units; DBS, Dried blood spot; GALC, galactocerebrosidase; LoB, Limit of the blank; LoQ, Limit of quantitation; MUG, 4-methylumbelliferyl-β-D-galactopyranoside substrate; QCL, Quality control low sample; QCM, Quality control medium sample
• ISSN: 2352-5517; 2352-5517
• DOI: 10.1016/j.plabm.2019.e00141

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. 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. 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. 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. •First fluorescent assay to measure GALC enzyme activity in dried blood spots.•Linear range covers the activity normally seen in newborn dried blood spots.•Successfully discriminates deficient from normal enzyme activity in DBS specimens.