Profiling and semi-quantitation of urine sulfatides by UHPLC-Orbitrap-HRMS

Background: Sulfatides are a class of sphingolipids which are abundant in the myelin sheet and oligodendrocytes, therefore they play a crucial role in the nervous system. Abnormal sulfatide excretion has been linked to several neurodegenerative disorders including metachromatic leukodystrophy (MLD) and multiple sulfatase deficiency (MSD). In MLD and MSD, sulfatide catabolism is impaired due to the reduced lysosomal arylsulfatase A (ARSA) activity resulting in an accumulation of sulfatides, which can be useful in a diagnostic setting. The current study aims to develop a method for semi-quantitation of urine sulfatides as a diagnostic tool for MLD and MSD. Results: We developed a sensitive and accurate method for identifying 48 urinary molecular sulfatide species by UHPLC-Orbitrap-HRMS analysis. Newborns were classified according to their gestational age. The proportion of sulfatides bearing saturated fatty acids attached to d18:1 and d18:0 sulfatide backbone was higher in newborns and increased with prematurity. The 5 most abundant sulfatide species in all samples (controls, MLD and MSD) were C22:0, C24:0, C22:0-OH, C24:0-OH and C24:1-OH fatty acid attached to d18:1 sulfatide backbone. The top discriminant feature between MLD patients and controls was d18:1/C26:1-OH. Total semi-quantitation of 6 sulfatide species (5 most abundant sulfatides + d18:1/C26:1-OH) shows that overall excretion gradually de- creases with age and all MLD patients were successfully discriminated from their age-matched controls. While sulfatide excretion was increased in the severe MSD patients (n = 2), it was normal in the attenuated MSD patients, who had high residual ARSA activity. Significance: This study proves the feasibility of diagnosing MLD and severe MSD based on sulfatide excretion in urine. We established (gestational) age-specific cut-offs of the total sulfatide excretion and composition. Inter- pretation of the composition (e.g. by calculation the ratio(d18:1/C22:0+d18:1/C24:0)/(d18:1/C22:0-OH + d18:1/C24:0-OH)) may reduce false positives, especially when sampling at young age.