Abstract
Lysosomal storage disorders (LSDs) are caused by defective enzyme activities in lysosomes, characterized by the accumulation of glycolipids, oligosaccharides, mucopolysaccharides, sphingolipids, and other biological substances. Accumulating evidence has suggested that early detection of individuals with LSDs, followed by the immediate initiation of appropriate therapy during the presymptomatic period, usually results in better therapeutic outcomes. The activities of individual enzymes are measured using fluorescent substrates. However, the simultaneous determination of multiple enzyme activities has been awaited in neonatal screening of LSDs because the prevalence of individual LSDs is rare. In this study, the activities of six enzymes associated with LSDs were examined with 6-plex enzyme assay using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The accumulation of enzyme products was almost linear for 0–20h at 37°C. Dried blood spots (DBSs) provided by the Centers for Disease Control and Prevention (CDC) were used for quality control (QC). The intraday and interday coefficient of variance values were <25%. The enzyme activities of healthy individuals were higher than those of LSD-confirmed individuals. These results suggest that the levels of enzyme activities of six LSDs in a Japanese population were comparable to those of a recent report [Elliott et al. Mol Genet Metab 118 (2016) 304–309], providing additional evidence that the 6-plex LSD enzyme assay is a reproducible analytical procedure for neonatal screening.
Highlights
Lysosomal storage disorders (LSDs) are a group of congenital metabolic disorders caused by the accumulation of glycolipids, oligosaccharides, mucopolysaccharides, sphingolipids, and other biological substances induced by the defective activity of lysosomal enzymes [1,2]
The LSD assay was validated in terms of (1) chromatographic separation of the enzyme reaction products using several commercially available reversed-phase columns, (2) quality control (QC) validation using CDCprovided QC Dried blood spots (DBSs), (3) analysis of the enzyme activity in random neonates and LSD-confirmed individuals in a Japanese population, and
A BEH C18 column was used in a subsequent study of this assay due to its commercial availability, as the enzyme reaction products of GAA, GLA, IDUA, ABG, acid sphingomyelinase (ASM), and GALC were readily detected under the analytical conditions tested (Fig. 1A)
Summary
Lysosomal storage disorders (LSDs) are a group of congenital metabolic disorders caused by the accumulation of glycolipids, oligosaccharides, mucopolysaccharides, sphingolipids, and other biological substances induced by the defective activity of lysosomal enzymes [1,2]. Lysosomal enzyme deficiency can lead to systemic manifestations of LSD symptoms. A prior study demonstrated the efficacy of hematopoietic stem cell transplantation [3]. LSDs, such as Pompe, Fabry, and Gaucher disease and mucopolysaccharidosis (MPS) I, II, and VI [5]. Neonatal screening of six LSDs involving Pompe, Fabry, MPS I, Gaucher, Krabbe, and Niemann–Pick disease type A/B has attracted. Krabbe disease leads to severe neurological manifestations and is linked to a galactosylceramidase (GALC) deficiency [11]. Hematopoietic cell transplant therapy is used to treat Krabbe disease [3]. We examined the ability of LCMS/MS to detect 6-plex LSD enzyme activity and applied the methodology to random Japanese neonates
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