Quantification of 11 enzyme activities of lysosomal storage disorders using liquid chromatography-tandem mass spectrometry

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.

UPLC-MS/MS Method for Analysis of Endocannabinoid and Related Lipid Metabolism in Mouse Mucosal Tissue

“…The inclusion of internal standards and non-endogenous substrates in these assays increases the accuracy of the quantitative results when compared to other assays of enzyme activity, including fluorogenic assays. Indeed, the addition of fluorogenic residues on a substrate, including those previously reported for assaying FAAH activity (Ramarao et al, 2005), may alter its enzymatic conversion, decreasing assay sensitivity (Su et al, 2006;Ohira et al, 2018). Furthermore, isomerases are known to change molecular orientation of the substrate without changing its mass (Lambeth and Julian, 2019); however, changes in molecular orientation may lead to changes in solubility and, ultimately, changes in UPLC-MS/MS retention time.…”

“…Coupling a chromatographic step (i.e., liquid chromatography) to MS provides physical and temporal separation of analytes and significantly increases sensitivity. These advantages have motivated work to develop LC-MS/MS based methods for analyzing enzyme activity (Ohira et al, 2018).…”

Production of therapeutic iduronate‐2‐sulfatase enzyme with a novel single‐stranded RNA virus vector

“…IDS activity, as well as activity for other lysosomal enzymes including acid α‐glucosidase (GAA), α‐galactosidase A (GLA), α‐iduronidase (IDUA), acid‐β‐glucocerebrosidase (ABG), acid sphingomyelinase (ASM), and β‐galactocerebrosidase (GALC) in the culture supernatant, was determined as previously described (Ilyinskaya et al., 2018) with a slight modification (Mashima et al., 2018; Ohira et al., 2018). In brief, the sample (5 μl) was incubated with substrate at 37°C for 20 hr (PerkinElmer).…”

Diagnosis and Emerging Treatment Strategies for Mucopolysaccharidosis VII (Sly Syndrome)

“…The enzyme deficiency can be demonstrated in serum, leukocytes, cultured fibroblasts, or dried blood spots (DBS) using fluorimetry with 4-methylumbelliferyl (4-MU) derived substrate, 11 , 17 , 18 and in DBS using liquid chromatography tandem mass spectrometry. 19 , 20 …”