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Lymphocyte Galactocerebrosidase Activity by LC-MS/MS for Post-Newborn Screening Evaluation of Krabbe Disease
Hsuan-Chieh Liao1,2, Zdenek Spacil1,3, Farideh Ghomashchi1
1Department of Chemistry, University of Washington, Seattle, WA.
Insights
A new assay accurately measures residual galactosylcerebrosidase (GALC) enzyme activity in lymphocytes. This improved method aids in follow-up analysis for newborns screened positive for Krabbe disease, enabling better family counseling and treatment optimization.
Area of Science:
- Biochemistry
- Genetics
- Pediatrics
Background:
- Krabbe disease is a lysosomal storage disorder caused by galactosylcerebrosidase (GALC) deficiency.
- Current newborn screening for Krabbe disease requires enhanced follow-up methods for accurate diagnosis and treatment planning.
Purpose of the Study:
- To develop and validate a novel assay for quantifying GALC enzymatic activity in lymphocytes.
- To improve the diagnostic accuracy for Krabbe disease in screen-positive newborns.
Main Methods:
- Isolation of T lymphocytes from venous blood using magnetic bead technology.
- Quantification of GALC activity via liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a synthetic substrate analog and an internal standard.
Main Results:
- The new assay demonstrated a 20-fold greater analytical range compared to conventional radiometric methods.
- The LC-MS/MS assay could detect GALC activity as low as 0.3% of normal levels, distinguishing between null and residual activity.
- A strong correlation was observed between residual GALC activity in lymphocytes and Krabbe disease severity.
Conclusions:
- The developed LC-MS/MS assay offers high accuracy in measuring residual GALC activity in leukocytes.
- This assay, combined with other diagnostic tools, can enhance post-newborn screening follow-up strategies for Krabbe disease.
- Improved diagnostic capabilities facilitate better clinical management and family guidance for Krabbe disease.
Background:
Deficiency of the lysosomal enzyme galactosylcerebrosidase (GALC) causes Krabbe disease. Newborn screening for Krabbe disease is ongoing, but improved methods for follow-up analysis of screen-positive babies are needed to better advise families and to optimize treatment. We report a new assay for the enzymatic activity of GALC in lymphocytes.
Methods:
T lymphocytes were isolated from venous blood by magnetic bead technology. The assay used a close structural analog of the natural substrate and LC-MS/MS to quantify the amount of product with the aid of a chemically identical internal standard.
Results:
The analytical range of the assay (ratio of assay response for the QC high standard to that from all non-enzymatic-dependent processes) was 20-fold greater than that for the conventional radiometric GALC assay. The LC-MS/MS could distinguish cells that were null in GALC from those that contained traces of active enzyme (down to 0.3% of normal). There was a good correlation between the level of residual GALC activity in lymphocytes and the severity of Krabbe disease.
Conclusions:
The new assay can measure small amounts of residual GALC activity in leukocytes with high accuracy compared to previous assays and can contribute, along with genotyping, biomarker analysis, and neurological imaging, a better plan for post-newborn screening follow-up for Krabbe disease.
