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

Anirudh J Ullal; Hong Pham; Rajendra Singh; Peter Ross; Carrie A Graham; Scott M Norton; Miriam H Nuffer; Debbie S Burns; Allen E Eckhardt; Maria Escolar; Deeksha Bali; Vamsee K Pamula

doi:10.1016/j.plabm.2019.e00141

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

Pract Lab Med. 2019 Oct 16:18:e00141. doi: 10.1016/j.plabm.2019.e00141. eCollection 2020 Jan.

Authors

Affiliations

¹ Baebies, Inc., PO Box 14403, Durham, NC, 27709, USA.
² Advanced Liquid Logic Inc., PO Box 14025, Durham, NC, 27709, USA.
³ University of Pittsburgh, One Children's Hospital Way, 4401 Penn Ave, Pittsburgh, PA, 15224, USA.
⁴ Duke University, Biochemical Genetics Laboratory, 801 Capitola Dr., Suite 6, Durham, NC, 27713, USA.

Abstract

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.

Keywords: CHMU, 3-Carboxy 6-hexadecanoylamino-4-methylumbelliferone; CHMUG, 3-Carboxy 6-hexadecanoylamino-4-methylumbelliferone β-D-galactopyranoside substrate; CLSI, Clinical and Laboratory Standards Institute; DBS, Dried blood spot; Dried blood spot; Fluorescence enzyme assay; GALC, galactocerebrosidase; Galactocerebrosidase; HMU, 6-hexadecanoylamino-4-methylumbelliferone; HMUG, 6-hexadecanoylamino-4-methylumbelliferone β-D-galactopyranoside substrate; Krabbe disease; LoB, Limit of the blank; LoD, Limit of detection; LoQ, Limit of quantitation; MUG, 4-methylumbelliferyl-β-D-galactopyranoside substrate; NBS, Newborn screening; NEH, Non-enzymatic hydrolysis; Newborn screening; PT, Proficiency testing; QCBP, Quality control base pool; QCH, Quality control high sample; QCL, Quality control low sample; QCM, Quality control medium sample; RFU, Relative fluorescence units; rhGALC, Recombinant human galactocerebrosidase; rhβ-gal, Recombinant human β-galactosidase.

Grants and funding

R44 HD057713/HD/NICHD NIH HHS/United States