Abstract
AstractLysosomal storage disorders (LSDs) are characterized by the accumulation of lipids, glycolipids, oligosaccharides, mucopolysaccharides, and other biological substances because of the pathogenic deficiency of lysosomal enzymes. Such diseases are rare; thus, a multiplex assay for these disorders is effective for the identification of affected individuals during the presymptomatic period. Previous studies have demonstrated that such assays can be performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with multiple reaction monitoring (MRM) detection. An assay procedure to quantify the activity of 11 enzymes associated with LSDs was provided. First, a validation study was performed using dried blood spot (DBS) samples with 100% and 5% enzyme activity for quality control (QC). Under the assay condition, the analytical range, defined as the ratio of the peak area of the enzyme reaction products from the DBS for QC with 100% enzyme activity to that from the filter paper blank sample, was between 14 for GALN and 4561 for GLA. Based on these results, the distribution of the enzyme activity for the 11 LSD enzymes was further examined. Consistent with the previous data, the enzyme activity exhibited a bell-shaped distribution with a single peak. The averaged enzyme activity for the healthy neonates was as follows: GLA, 3.80 ± 1.6; GAA, 10.6 ± 4.8; IDUA, 6.4 ± 2.3; ABG, 8.6 ± 3.1; ASM, 3.3 ± 1.1; GALC, 2.8 ± 1.3; ID2S, 16.7 ± 6.1; GALN, 1.2 ± 0.5; ARSB, 17.0 ± 8.7; NAGLU, 4.6 ± 1.5; and GUSB, 46.6 ± 19.0 μmol/h/L (mean ± SD, n = 200). In contrast, the enzyme activity in disease-affected individuals was lower than the minimum enzyme activity in healthy neonates. The results demonstrate that the population of disease-affected individuals was distinguished from that of healthy individuals by the use of LC-MS/MS.
Highlights
Lysosomal storage disorders (LSDs) are characterized by the accumulation of biological substances, such as glycolipids, lipids, oligosaccharides, and mucopolysaccharides, in the lysosomes as a result of the pathogenic defects of the lysosomal enzymes [1,2]
During the course of the current study, it was observed that the enzyme activity for the NAGLU was always attenuated when this reaction was performed with the ID2S, ARSB, and GALN assays
It was found subsequently that (Z)-Pugnac, an inhibitor for O-linked N-acetylglucosamine, was included in this batch of ID2S, ARSB, and GALN reagents at the time of production. This implies that the suppression for NAGLU observed above in the preliminary 11-plex assay could be associated with (Z)-Pugnac in assay reagent for ID2S, ARSB, and GALN
Summary
Lysosomal storage disorders (LSDs) are characterized by the accumulation of biological substances, such as glycolipids, lipids, oligosaccharides, and mucopolysaccharides, in the lysosomes as a result of the pathogenic defects of the lysosomal enzymes [1,2]. To accommodate the large number of DBS samples under current NBS platform, the assay was performed using flow-injection analysis under high-throughput assay conditions (8) Based on these innovations, these reagents were used for several pilot studies reported in Austria [11], Taiwan [12], and the United States (U.S.) [10]. Assay reagents for iduronate-2-sulfatase (ID2S) for MPS II, N-acetylgalactosamine-6-sulfatase (ARSB) for MPS IVA, Nacetylgalactosamine-4-sulfatase (GALN) for MPS VI, N-acetyl-α-D-glucosaminidase (NAGLU) for MPS IIIB, and β-glucuronidase (GUSB) for MPS VII [14], as well as lysosomal acid lipase and biotinidase for the associated deficiencies [15,16] were developed in previous studies These enzyme activities can be quantified with a combination of enzymes. The current study has extended that previous study on the multiplex quantification of LSD enzymes involving GAA, GLA, IDUA, ABG, ASM, GALC, NAGLU, and GUSB by using LC-MS/MS
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