Direct tandem mass spectrometric profiling of sulfatides in dry urinary samples for screening of metachromatic leukodystrophy

Abstract

Background Prediagnostic steps in suspected metachromatic leukodystrophy (MLD) rely onclinical chemical methods other than enzyme assays. We report a new diagnostic method which evaluates changes in the spectrum of molecular types of sulfatides (3-O-sulfogalactosyl ceramides) in MLD urine. Methods The procedure allows isolation of urinary sulfatides by solid-phase extraction on DEAE-cellulose membranes, transportation of a dry membrane followed by elution and tandem mass spectrometry (MS/MS) analysis in the clinical laboratory. Major sulfatide isoforms are normalized to the least variable component of the spectrum, which is the indigenous C18:0 isoform. This procedure does not require the use of specific internal standards and minimizes errors caused by sample preparation and measurement. Results Urinary sulfatides were analyzed in a set of 21 samples from patients affected by sulfatidosis. The combined abundance of the five most elevated isoforms, C22:0, C22:0-OH, C24:0, C24:1-OH, and C24:0-OH sulfatides, was found to give the greatest distinction between MLD-affected patients and a control group. Conclusions The method avoids transportation of liquid urine samples and generates stable membrane-bound sulfatide samples that can be stored at ambient temperature. MS/MS sulfatide profiling targeted on the most MLD-representative isoforms is simple with robust results and is suitable for screening.

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

“…In agreement with a previous study (7), the major sulfatide species in DUS were C24:0-OH, C22:0, and C22:0-OH, with MLD-to-control ratios of 120, 102, and 86, respectively. The mean total sulfatide concentration for the sum of 19 detected molecular species in late infantile MLD patients was 4.87 μ g/mL, range 0.42–17.24 μ g/mL.…”

“…In summary, the mean total sulfatide concentrations (sum of all detected species) were 9.5- and 3.3-fold higher in patients with severe and mild forms of MLD, respectively, compared with control samples (0.215 μ g/mL). There was no overlap between groups, and given <20% CVs routinely achieved with single reaction monitoring (SRM) MS/MS technology (7), the difference in sulfatide concentration was sufficient to unambiguously detect MLD patients and stratify them on the basis of severity. Additionally, we found the most discriminatory MLD markers to be C16:1-OH and C16: 0-OH, with 23.2- and 13.1-fold mean increases, respectively, in the infantile form of MLD, and 5.1- and 4.6-fold increases in the juvenile/adult form of MLD.…”

“…Although normalization with the concentration of creatinine is commonly used for soluble analytes, it was proven inefficient in the case of ceramides (12). The isoform profile number has been proposed as a new MLD biomarker (7), which is the combined signal of the major isoforms relative to the intrinsic C18:0 sulfatide reference. The C18:0 isoform was chosen as the least-variable parameter of the total sulfatide profile among controls and MLD patients.…”

“…Newborn screening (NBS) of MLD by direct measurement of arylsulfatase A activity or protein abundance is not likely to be feasible because of a severe pseudodeficiency problem (1) and relative instability of the enzyme in dried blood spots (DBS) (2). Sulfatides, the natural substrates for arylsulfatase A, have been shown to be highly increased in urine from MLD patients compared with healthy individuals (3–7). However, NBS programs typically use DBS, and dried urine samples (DUS) are usually not available.…”

Quantification of sulfatides and lysosulfatides in tissues and body fluids by liquid chromatography-tandem mass spectrometry

“…Several methods have been used to quantify sulfatides, including TLC ( 19 ), HPLC ( 14,20 ), and, more recently, MS ( 4,13,15,(21)(22)(23)(24)(25). Here we describe a LC/MS/MS method to quantify sulfatides and their molecular species over a wide range of concentrations in normal and diseased tissues, plasma, and urine.…”