Sulfatide Analysis by Mass Spectrometry for Screening of Metachromatic Leukodystrophy in Dried Blood and Urine Samples

Abstract

Background: Metachromatic leukodystrophy (MLD) is an autosomal recessive disorder caused by deficiency in arylsulfatase A activity, leading to accumulation of sulfatide substrates. Diagnostic and monitoring procedures include demonstration of reduced arylsulfatase A activity in peripheral blood leukocytes or detection of sulfatides in urine. However, the development of a screening test is challenging because of instability of the enzyme in dried blood spots (DBS), the widespread occurrence of pseudodeficiency alleles, and the lack of available urine samples from newborn screening programs.

Methods: We measured individual sulfatide profiles in DBS and dried urine spots (DUS) from MLD patients with LC-MS/MS to identify markers with the discriminatory power to differentiate affected individuals from controls. We also developed a method for converting all sulfatide molecular species into a single species, allowing quantification in positive-ion mode upon derivatization.

Results: In DBS from MLD patients, we found up to 23.2-fold and 5.1-fold differences in total sulfatide concentrations for early- and late-onset MLD, respectively, compared with controls and pseudodeficiencies. Corresponding DUS revealed up to 164-fold and 78-fold differences for early- and late-onset MLD patient samples compared with controls. The use of sulfatides converted to a single species simplified the analysis and increased detection sensitivity in positive-ion mode, providing a second option for sulfatide analysis.

Conclusions: This study of sulfatides in DBS and DUS suggests the feasibility of the mass spectrometry method for newborn screening of MLD and sets the stage for a larger-scale newborn screening pilot study.

Fig. 1

Concentrations of 19 sulfatides in blood or urine (μg/mL) detected in DBS (A) and DUS (B) from late infantile MLD patients (n = 11, black), juvenile/adult MLD patients (n = 3, medium gray), and controls (n = 50 for DBS and n = 104 for DUS, light gray).

Fig. 2

Mean total sulfatide concentrations in blood (μg/mL) across patient groups (A) and mean concentrations of C18:0 and C24:1 sulfatides (B) in DBS from MLD patients and healthy controls. Error bars show SDs.

Fig. 3

Total sulfatide concentrations in blood (μg/mL) in patients already diagnosed with MLD (n = 14), non-MLD newborns (n = 50), patients with MLD pseudodeficiencies (n = 10), newborn patients who developed infantile MLD (n = 3), and newborn patients who developed juvenile MLD (n = 2).

Scheme 1. Protocol to enhance sensitivity of sulfatide positive-ion detection in DBS

Step I: all sulfatide species are enzymatically converted to a single molecular species (lysosulfatide); step II: the lysosulfatide is reacted with the indicated reagent to improve detection sensitivity in positive-ion mode.