Determination of monosaccharides and disaccharides in mucopolysaccharidoses patients by electrospray ionisation mass spectrometry

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Abstract

The mucopolysaccharidoses are a group of lysosomal storage disorders characterised by the storage of glycosaminoglycans. With the exception of Hunters syndrome (MPS II), which is X-linked, they are autosomal recessively inherited resulting in a defect in any one of 10 lysosomal enzymes needed to catabolise glycosaminoglycans. The type and size of the glycosaminoglycans stored in lysosomes are determined by the particular enzyme deficiency. These glycosaminoglycan elevations are subsequently observed in tissue, circulation, and urine. A method has been developed for the derivatisation and quantification of sulfated N-acetylhexosamine-containing mono- and disaccharides from patient samples by electrospray ionisation tandem mass spectrometry. Urine from most mucopolysaccharidoses types had significant increases in di- and monosulfated N-acetylhexosamines (GalNAc4,6S, GalNAc6S, GalNAc4S, or GlcNAc6S) and monosulfated N-acetylhexosamine-uronic acid disaccharides (GalNAc6S-UA, GalNAc4S-UA, or GlcNAc6S-UA). Analysis of plasma and dried blood spots on filter paper collected from mucopolysaccharidoses patients showed elevations of total monosulfated N-acetylhexosamines but less than that seen in urine. Urine samples from bone marrow transplant recipients, mucopolysaccharidosis IVA and mucopolysaccharidosis VI patients, showed decreases in HexNAcS, HexNAcS2/GalNAc4,6S, and HexNAcS-UA post-transplant. This decrease correlated with clinical improvement to levels comparable with those identified in patients with less severe phenotypes. These metabolic markers therefore have potential applications in diagnosis, phenotype prediction and monitoring of current and future therapies, particularly for the mucopolysaccharidosis IIID, IVA, VI, and multiple sulfatase deficiency. This paper reports a sensitive and simple method for the measurement of sulfated N-acetylhexosamines and sulfated disaccharides shown to be elevated in some mucopolysaccharidosis and multiple sulfatase deficient patients.

Introduction

The mucopolysaccharidoses (MPS) are characterised by the lysosomal storage of glycosaminoglycans (GAGs) in tissues resulting in elevated concentrations of these compounds in body fluids, urine, plasma, and blood. Lysosomal storage results from an enzyme deficiency in any one of 10 lysosomal enzymes required to catabolise glycosaminoglycans. The lysosomal storage disorder group of genetically inherited metabolic disorders have a collective incidence in Australia of approximately 1 in 7700 [1]. The MPS, a subgroup of lysosomal storage disorders, has an approximate incidence of 1 in 16,000.

Glycosaminoglycans are currently characterised by high resolution electrophoresis and polyacrylamide electrophoresis methods [2], [3]. Methods to ascertain small GAG-derived oligosaccharides have been widely reported using high performance liquid chromatography with various stationary phases and detection systems. These have included porous graphite with pulsed amphometric detection [4], ion-pairing reverse phase with radiolabel detection [5], strong anion exchange incorporating either post column 2-cyanoacetamide derivatisation [6], Δ4,5-unsaturated uronic acid at 232 nm from bacterial lyase digestion [7] or tritium radiolabel detection [8] and gradient gel electrophoresis [9]. Larger GAG oligosaccharides can be analysed by capillary electrophoresis [10], [11], [12] and polyacrylamide gel electrophoresis (PAGE) [3], [7], [13]. PAGE has the ability to analyse most of the glycosaminoglycan oligosaccharides simultaneously. PAGE analysis of GAGs from MPS patients was demonstrated by Byers et al. [3] to be strongly indicative of MPS type. They were able to show elevated amounts of GAG oligosaccharides were present in the urine of MPS patients compared to controls. Sulfated monosaccharides were observed to be elevated in urines from MPS patients [14], [15], [16]. Ion exchange followed by paper chromatography of the urine samples, spiked with radiolabelled monosaccharides, was used to separate sulfated monosaccharides. Hexosamine analysis was then used to determine the amount of N-acetylhexosamine present in acid hydrolysates of individual fractions. These studies where able to quantify the type and amounts of sulfated N-acetylhexosamines present in the urine of MPS patients. Hopwood and Elliott [14], [15], [16] demonstrated that sulfated N-acetylhexosamines present in human urine, were most likely derived from an alternative degradative pathway with β-N-acetylhexosaminidase cleavage of non-reducing end sulfated GlcNAc from keratan sulfate and GalNAc residues from dermatan sulfate and chondroitin sulfate. These sulfated monosaccharides are then stored in lysosomes and secreted in the urine of each sulfatase deficient patient [14], [15], [16]. The analysis of these sulfated monosaccharides suggested that the amount and type of urinary sulfated monosaccharides depend on MPS type and clinical severity. However, these methods were not suitable for a screening protocol enmass nor were they rapid or quantifiable and therefore improved methods were required.

Electrospray tandem mass spectrometry (ESI-MS/MS) has become a common tool to screen for metabolic disorders from dried blood spots [17], [18], [19], [20]. Mass spectrometry has been used to elucidate the structure of oligosaccharides derived from glycosaminoglycans [21], [22], [23], [24], [25], [26] but not to quantify specific sulfated oligosaccharides. Desaire and Leary [27] have however developed a method based on ratios of isomeric product ions (generated by ESI-MS/MS) to provide relative proportions of disaccharides in a mixture without an internal standard but did not provide absolute concentrations. In this paper, an ESI-MS/MS method is described for measuring sulfated mono- and disaccharides in biological fluids using 1-phenyl-3-methyl pyrazolone (PMP) derivatisation and an internal standard, a [2H3]deuterium labelled monosaccharide (GlcNAc6S). We report results and limitations of this technique for the analysis of urine, plasma and blood from MPS I, II, IIIA, IIIB, IIIC, IIID, IVA, VI, and multiple sulfatase deficiency (MS) patients.

Section snippets

Materials

Glucosamine 6-sulfate (GlcN6S), N-acetylglucosamine-6-sulfate (GlcNAc6S), N-acetylgalactosamine-6-sulfate (GalNAc6S), N-acetylgalactosamine-4-sulfate (GalNAc4S), acetic anhydride(d6), and solvents were purchased from Sigma–Aldrich (Castle Hill, NSW, Australia). PMP was purchased from Tokyo Kasei Kogyo (Tokyo, Japan). AG1-X8 (H+ form, 100–200 mesh) and size exclusion gel P2 (fine) were both obtained from Bio-Rad (Richmond, VA, USA). All solvents used for LC-MS were HPLC grade (Unichrom) and

Calibration curves and response ratios

The calibration curves for the four monosaccharides (Fig. 1), using the internal standard GlcNAc6S-d3 at 2 μmol/L, were linear up to 20 μmol/L with R2 values for GalNAc6S=0.997, GalNAc4S=0.993, GlcNAc6S=0.994, and GalNAc4,6S= 0.997. Response ratios were GalNAc6S 0.904, GalNAc4S 1.25, GlcNAc6S 0.938, and GalNAc4,6S 0.173 using the 710/256 MRM or 0.448, using the 710/630 MRM ion pair. Although the 710/630 MRM pair provided a more intense signal than the 710/256 MRM ion pair, the latter was chosen

Discussion

The PMP derivatising method has been used effectively to derivatise oligosaccharides [30], [31]. This method has been incorporated into preparation of sulfated oligosaccharides present in urine, plasma and dried blood spot samples collected from unaffected controls and MPS patients for analysis by ESI-MS. A modified version of this method and the general mechanism is shown in Scheme 1. Ammonia was used to basify the PMP solution instead of the original method which used sodium hydroxide/sodium

Acknowledgements

We are grateful to the MPS patients and their families for the use of their urine and blood samples used in this study. Staff members also kindly donated control urine and blood. Pharming BV (The Netherlands), TLH Research (USA) and the National Health and Medical Research Council (Australia) are gratefully acknowledged for their financial support.

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