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Abstract INTRODUCTION:
Metachromatic leukodystrophy (MLD) is a rare, severe, neurodegenerative disorder caused by deficient activity of arylsulfatase A (ARSA), an enzyme responsible for cleaving the sulfate group from sulfatides (3-O-sulfogalactosylceramide) into galactosylceramide (GalCer). In MLD, sulfatide accumulation leads to progressive demyelination. A disease-modifying gene therapy is available for MLD but is currently only approved for the late-infantile and early juvenile forms, highlighting the need for disease stratification. Although genotype–phenotype correlations have been identified, they remain incomplete. Diagnostic biochemical approaches, including ARSA activity measurement and sulfatides quantitation, provide complementary information but remain limited for their prognostic utility. Santhanakumaran et al improved the conventional spectrophotometric ARSA activity assay and demonstrated that residual ARSA activity correlates with disease severity (1). However, spectrophotometric assays have several technical limitations, including poor sensitivity and selectivity due to the use of artificial substrate. We propose that an LC-MS/MS-based ARSA activity assay can overcome these limitations by enabling the use of a natural substrate, thereby providing more accurate and reliable measurements of ARSA activity (2). This will be crucial not only for diagnosis, but also for prognosis and treatment monitoring in MLD.
OBJECTIVES:
The study aims to develop a high-performance LC-MS/MS assay to measure ARSA activity in various biological matrices, including white blood cells (WBCs) and cerebrospinal fluid (CSF) for diagnostic, prognostic, and treatment monitoring purposes; and to conduct a comprehensive validation of the assay to support its implementation in a clinical diagnostic laboratory.
METHODS:
CSF and WBC lysates (isolated from EDTA-treated whole blood) were incubated with a reaction cocktail containing d₃-C18:0-sulfatide as the enzymatic substrate and d7-C18:0-GalCer as the internal standard. D₃-C18:0-sulfatide was selected as the substrate so that the enzymatic product would retain the three deuterium labels, allowing it to be distinguished from endogenous C18:0-GalCer by MS/MS. The reaction was carried out at room temperature with overnight incubation. Following incubation, the mixture was quenched and analyzed by UPLC-MS/MS, where the enzymatic product, d₃-C18:0- GalCer, was chromatographically separated from the undigested substrate, d₃-C18:0-sulfatide. An external calibration curve, combined with isotope-dilution using the internal standard, was used to quantify d₃-C18:0- GalCer generated through the enzymatic reaction. Enzyme activity was calculated by normalizing the amount of enzymatic product to the incubation time and volume (for CSF) or to the WBC cell count (for WBC lysates).
RESULTS:
ARSA activity was readily detectable in 10 µL of normal CSF and approximately 50,000 white blood cells, corresponding to 4.5 to 12.5 µL of whole blood from a healthy individual. The WBC isolation protocol we adopted minimized the loss of ARSA during the procedure, but it also resulted in RBC contamination in the final cell pellet, necessitating normalization of ARSA activity to WBC cell count rather than protein concentration. ARSA activity in RBCs was found to be at least 100-fold lower than in WBCs, supporting this approach. We obtained WBCs from a late-infantile MLD patient during initial evaluation and detected no residual ARSA activity, demonstrating that the assay is highly specific to the enzyme of interest.
The external calibration curve covered a range of 10-20,000 nM d₃-C18:0-GalCer. The accuracy and precision (A&P) and lower limit of quantitation (LLOQ) of the LC-MS/MS platform were assessed with neat d₃-C18:0-GalCer solution. A&P were evaluated at three levels (30, 1,200, 12,000 nM) with bias <10% and imprecision <10%. The LLOQ was determined at 10 nM with the bias <10% and imprecision <12.5%. The linearity and precision of the entire system, including enzymatic reaction and LC-MS/MS measurement, were evaluated using real CSF and WBC lysates. Linearity of ARSA activity in CSF was demonstrated across dilution levels ranging from 6.25% to 100%, with an R² value of 0.9998. For WBC ARSA activity, linearity was demonstrated over a 10% to 100% dilution range, with an R² value of 0.991. Total imprecision for CSF measurements was evaluated using pooled CSF samples mixed with heat-inactivated CSF at 2%, 20%, and 100% activity levels. Similarly, total imprecision for WBC measurements was assessed using pooled WBC lysates mixed with heat-inactivated WBC material at 2%, 10%, and 100% levels. Overall, these results underscore the strong analytical performance of this assay.
CONCLUSION:
Our isotope dilution LC-MS/MS assay for ARSA is highly selective, precise, and sensitive. It shows strong potential for use in presymptomatic diagnosis of MLD and in predicting disease onset.
REFERENCES:
(1) Santhanakumaran, V., Groeschel, S., Harzer, K., Kehrer, C., Elgün, S., Beck-Wödl, S., ... & Laugwitz, L. (2022). Predicting clinical phenotypes of metachromatic leukodystrophy based on the arylsulfatase A activity and the ARSA genotype?–Chances and challenges. Molecular Genetics and Metabolism, 137(3), 273-282.
(2) Hong, X., Kumar, A. B., Daiker, J., Yi, F., Sadilek, M., De Mattia, F., ... & Gelb, M. H. (2020). Leukocyte and dried blood spot arylsulfatase A assay by tandem mass spectrometry. Analytical chemistry, 92(9), 6341-6348.
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= Discovery stage. (53.14%, 2025)
= Translation stage. (22.33%, 2025)
= Clinically available. (24.53%, 2025)
