Establishment of glycosaminoglycan assays for mucopolysaccharidoses

Abstract

Mucopolysaccharidoses (MPS) are a group of lysosomal storage disorders caused by deficiency of the lysosomal enzymes essential for catabolism of glycosaminoglycans (GAGs). Accumulation of undegraded GAGs results in dysfunction of multiple organs, resulting in distinct clinical manifestations. A range of methods have been developed to measure specific GAGs in various human samples to investigate diagnosis, prognosis, pathogenesis, GAG interaction with other molecules, and monitoring therapeutic efficacy. We OPEN ACCESSMetabolites 2014, 4 656 established ELISA, liquid chromatography tandem mass spectrometry (LC-MS/MS), and an automated high-throughput mass spectrometry (HT-MS/MS) system (RapidFire) to identify epitopes (ELISA) or disaccharides (MS/MS) derived from different GAGs (dermatan sulfate, heparan sulfate, keratan sulfate, and/or chondroitin sulfate). These methods have a high sensitivity and specificity in GAG analysis, applicable to the analysis of blood, urine, tissues, and cells. ELISA is feasible, sensitive, and reproducible with the standard equipment. HT-MS/MS yields higher throughput than conventional LC-MS/MS-based methods while the HT-MS/MS system does not have a chromatographic step and cannot distinguish GAGs with identical molecular weights, leading to a limitation of measurements for some specific GAGs. Here we review the advantages and disadvantages of these methods for measuring GAG levels in biological specimens. We also describe an unexpected secondary elevation of keratan sulfate in patients with MPS that is an indirect consequence of disruption of catabolism of other GAGs.

Newborn screening for mucopolysaccharidoses: a pilot study of measurement of glycosaminoglycans by tandem mass spectrometry

“…The ratio of di-sulfated KS in total KS was significantly higher in MPS newborn patients compared to general newborns (p = 0.0001) (table 1). We have previously shown a secondary elevation of total KS and increase in ratio of di-sulfated KS in blood from older patients with other MPS types, where 40% for MPS II patients and 54% of MPS IVA had significantly higher levels of ratio di-sulfated KS in total KS (Shimada et al, 2015;Tomatsu et al, 2005;Tomatsu et al, 2014). We did not see a secondary elevation of KS in the MPS II newborn samples, possibly due to low accumulation during early stages of bone development.…”

“…We have previously shown that blood from patients with other forms of MPS (IV, VI, and VII) contain elevated levels of other GAGs, but we do not have access to newborn DBS from such patients at this time and cannot directly determine GAG levels expected in newborn DBS from all MPS types (Tomatsu et al, 2005;Rowan et al, 2013;Shimada et al, 2014b;Tomatsu et al, 2014;Shimada et al, 2015). Nevertheless, based on data from older patients, this newborn screen is likely to capture patients with a severe form of any MPS.…”

“…Thus while we were able to establish stringent cut-off values to distinguish all patient samples in this study from controls, we are not able to predict false negative rates for larger screening purposes. Another limitation is that the running time of the current LC-MS/MS (4 to 5 minutes per sample) is still a challenge for application to mass screening, compared to high-throughput MS/MS (10 to 12 seconds per sample) (Shimada et al, 2014). Although the main drawback of HT-MS/MS is that disaccharides with identical molecular weights cannot be distinguished, this may not be crucial for a method that is proposed as a first-tier screen to identify a high-risk group that may have an MPS.…”

Measurement of Elevated Concentrations of Urine Keratan Sulfate by UPLC-MSMS in Lysosomal Storage Disorders (LSDs): Comparison of Urine Keratan Sulfate Levels in MPS IVA Versus Other LSDs

“…Tomatsu et al (2005) have reported that keratan sulfate is elevated in both plasma and urine samples from patients with all mucopolysaccharidosis disorders (types I, II, III, IV, VI, and VII) compared to agematched controls. It was speculated that elevated heparan sulfate directly inhibits GALNS enzyme activity, resulting in a secondary elevation of keratan sulfate (Rowan et al 2013;Tomatsu et al 2014). This could explain the mild elevation of urine KS that was observed in MPS I and MPS II patients (Fig.…”

Elevation of glycosaminoglycans in the amniotic fluid of a fetus with mucopolysaccharidosis VII

“…We and others have shown that KS is secondarily elevated in plasma (serum) and urine in several types of MPS and LSDs (in addition to MPS IV in which the deficient enzyme is directly involved in the catabolism of KS) . We previously proposed that secondary elevation of KS can be caused by several factors including the release of KS from damaged bone and cartilage . However, newborn DBS from MPS I, II, or III subjects showed no elevation of KS at birth .…”