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

Molecular Genetics and Metabolism 78 (2003) 193–204 www.elsevier.com/locate/ymgme

Determination of monosaccharides and disaccharides in mucopolysaccharidoses patients by electrospray ionisation mass spectrometry Steven L. Ramsay, Peter J. Meikle, and John J. Hopwood* Lysosomal Diseases Research Unit, Department of Chemical Pathology, Adelaide WomenÕs and ChildrenÕs Hospital, North Adelaide, SA 5006, Australia Received 11 September 2002; received in revised form 6 January 2003; accepted 6 January 2003

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, GalNAc4SUA, 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 Nacetylhexosamines and sulfated disaccharides shown to be elevated in some mucopolysaccharidosis and multiple sulfatase deficient patients. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Mucopolysaccharidoses (MPS); Glycosaminoglycans (GAGs); Electrospray mass spectrometry (ESI-MS); 1-Phenyl-3-methyl pyrazolone (PMP)

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 * Corresponding author. Fax: +618-8161-7100. E-mail address: [email protected] (J.J. Hopwood).

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

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radiolabel detection [5], strong anion exchange incorporating either post column 2-cyanoacetamide derivatisation [6], D4,5-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 [10–12] 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 [14–16]. 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 [14–16] demonstrated that sulfated N-acetylhexosamines present in human urine, were most likely derived from an alternative degradative pathway with b-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 [14–16]. 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 [17–20]. Mass spectrometry has been used to elucidate the structure of oligosaccharides derived from glycosaminoglycans [21–26] 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 [2 H3 ]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.

Experimental Materials Glucosamine 6-sulfate (GlcN6S), N-acetylglucosamine-6-sulfate (GlcNAc6S), N-acetylgalactosamine-6sulfate (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 (Hþ 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 ammonia (28%) and formic acid (95%) were of analytical grade (Univar), both from APS Finechem (Seven Hills, NSW, Australia). Isolute solid phase extraction columns (C18 endcapped, 25 and 100 mg) were obtained from International Sorbent Technology (MidGlamorgan, UK). Samples used in this study were submitted to our department for the diagnosis of lysosomal, peroxisomal and other genetic disorders and were stored at )20 °C. Preparation of deuterated N-acetylglucosamine-6-sulfate (GlcNAc6S-d3 ) GlcNAc6S-d3 was prepared by selective N-acetylation of the GlcN6S. The monosaccharide GlcN6S (25 mg) was dissolved in anhydrous solutions of pyridine (70 lL), dimethyl formamide (700 lL), and methanol (50 lL) by sonication. The solution was stirred on ice and acetic anhydride(d6 ) (2  20 lL) was added at 30min intervals. After one hour the reaction was quenched with a 4% aqueous ammonia solution (500 lL) and then placed under a stream of nitrogen to remove solvents. This step was repeated. The remaining residue was dissolved in water (500 lL) and loaded onto an anion exchange column (AG1-X8, Hþ form, 100–200 mesh, 5 mL bed) and washed with deionised water (4 column volumes). The monosaccharide was eluted with LiCl (2.5 M, pH 4.5 (no buffer)) and the fractions monitored with the bicinchoninic acid microwell assay (BCA, Pierce Chemical, Rockford, IL). The fractions containing GlcNAc6S-d3 were pooled and desalted on a size exclusion column, (50  1 cm) of P2 fine (Bio-Rad) run in water. The desalted fractions were pooled and lyophilised to produce a white solid 5.5 mg (18.7% yield). ESI-MS of this monosaccharide produced an ½M–H ion at m/z 303.2. The purity of the GlcNAc6S-d3 was determined by combining equal amounts of GlcNAc6S (3.02 lg) to GlcNAc6S-d3 (3.05 lg), and the mixture derivatised with 100 lL PMP solution (25 mM) using the

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method below. An ESI-MS scan in negative ion mode of this solution (10 lmol/L) showed two ions only at m/z 630.2 and 633.2. A neutral loss (NL374) ESI-MS/MS experiment was used to assess the purity of the PMP-derivatised GlcNAc6S-d3 . The PMP-derivatised GlcNAc6S-d3 gave an ion intensity 83.95% of the PMPderivatised GlcNAc6S. The impurity was assumed to be residual LiCl from incomplete desalting. The GlcNAc6S-d3 was not purified further and was used as an internal standard.

to remove any unincorporated PMP, and again dried thoroughly. Derivatised oligosaccharides were eluted from each C18 column with 50% (v/v) CH3 CN/0.025% (v/v) formic acid in water (3  200 lL) and dried under a stream of N2 . Each sample was then reconstituted in 200, 500, or 100 lL of 50% (v/v) CH3 CN/0.025% (v/v) formic acid in water for plasma, urine, and blood spot samples, respectively.

Preparation of PMP derivatised GlcNAc6S-d3

Mass spectrometric analysis was performed in negative ion mode using a PE Sciex API 365 triple–quadrupole mass spectrometer with an ionspray source and LC-Tune/Multiview data system (PE Sciex, Concord, Ont., Canada). Samples (20 lL) were injected into the electrospray source with a Gilson 215 autosampler using a solvent consisting of 50% CH3 CN/0.025% formic acid in water, at a flow rate of 30 lL/min. For all analytes nitrogen was used as the collision gas at a pressure 2  105 Torr.

GlcNAc6S-d3 (100 nmol) was dissolved in 200 lL derivatising solution (250 mmol/L PMP, 400 mmol/L NH3 , pH 9.1) and heated at 70 °C for 90 min. The solution was acidified with 200 lL of 800 mmol/L formic acid and then extracted with CHCl3 (3  500 lL) to remove excess PMP. The aqueous phase was lyophilised (3 from water) to remove excess formic acid and NH4 COOH. The PMP-GlcNAc6S-d3 mixture was reconstituted in 1 mL of 50% (v/v) CH3 CN/0.025% (v/v) formic acid in water and stored at )20 °C for up to 6 months. Derivatisation of oligosaccharide standards and biological samples Samples of plasma (20 lL) and urine (1.0 lmol creatinine equivalents) were lyophilised prior to derivatisation. Whole blood samples were dried onto filter paper (S&S 903, Schleicher & Schuell, Dassel, Germany) and 3 mm punches were taken and derivatised directly. To each sample was added derivatising solution (250 mmol/ L PMP, 400 mmol/L NH3 , pH 9.1), 200 lL to plasma, 100 lL to urine, and 50 lL to blood spots. The derivatising solution contained 0.8, 0.4, or 4.0 nmol GlcNAc6S-d3 for plasma, blood spots and urine samples, respectively. The samples were heated in an oven at 70 °C for 90 min. Samples were then acidified with a 2fold molar excess of formic acid (equal volume of 800 mmol/L) and made up to 500 lL with water. Excess PMP was extracted from each sample with 500 lL of CHCl3 and centrifuged (13,000g, 5 min). Solid phase extraction columns (25 mg, C18 for plasma and blood spots; 100 mg, C18 for urine) were primed with successive 1 mL washes of 100% CH3 CN, 50% (v/v) CH3 CN/ 0.025% (v/v) formic acid, and water. The aqueous layer from each CHCl3 extraction (400 lL) was applied to a primed C18 column and allowed to enter the solid phase completely. The column was washed with water (1  500 lL, followed by 2  1000 lL) and dried under vacuum (15 min) on a Supelco, Visiprep24 vacuum manifold (Sigma–Aldrich, St. Louis, USA) or in a lyophiliser (45 min), if in the 96-well format. Each dried C18 column was then washed with CHCl3 (2  1000 lL)

Mass spectrometry

ESI-MS/MS analysis of oligosaccharides Quantification of PMP derivatised oligosaccharides was performed using the multiple-reaction monitoring (MRM) mode. Ion pairs monitored were 633/259 (GlcNAc6S-d3 ), 630/256 (HexNAcS), 710/256 (HexNAcS2 ) and 806/331 (HexNAcS-UA). Each ion pair was monitored for 100 ms using a resolution setting of 1.0 amu at half peak height. For each quantitative measurement, continuous scans were made over the injection period and averaged. Quantification was achieved by relating the peak heights of the PMP-oligosaccharides to the peak height of the PMP-GlcNAc6S-d3 internal standard.

Results Calibration curves and response ratios The calibration curves for the four monosaccharides (Fig. 1), using the internal standard GlcNAc6S-d3 at 2 lmol/L, were linear up to 20 lmol/L with R2 values for GalNAc6S ¼ 0.997, GalNAc4S ¼ 0.993, GlcNAc6S ¼ 0.994, and GalNAc4,6S ¼ 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 as the 256 daughter ion produced from the m/z 710 ion represented the same fragmentation as the internal standards m/z 633 and therefore would better reflect any fluctuations in the instrument.

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Fig. 1. Standard curve of analytes to internal standard GlcNAc6S-d3 . Linear fit and R2 values for the following analytes MRM pairs used (in parentheses) were: GlcNAc6S (630/256), y ¼ 0:904x and R2 ¼ 0:997; GalNAc6S (630/256), y ¼ 0:938x and R2 ¼ 0:994; GalNAc4S (630/256) y ¼ 1:25x and R2 ¼ 0:993; GalNac4,6S (710/630), y ¼ 0:448x and R2 ¼ 0:994; (710/256), y ¼ 0:173x and R2 ¼ 0:997; GalNAcS* (630/256), y ¼ 0:082x; and R2 ¼ 0:998. , GalNAcS is the product formed as a result of in-source desulfation of the 4- or 6-O-sulfate from GalNAc4,6S. Legend: r, GalNAc4S MRM pair 630/256; N, GalNAc6S MRM pair 630/256; +, GlcNAc6S MRM pair 630/256; d, GalNAc4,6S MRM pair 710/630; s, GalNAc4,6S MRM pair 710/256; and , GalNAcS MRM pair 630/256.

Analysis of PMP derivatised oligosaccharides/monosaccharides prepared from MPS patient urine Application of this method to a range of controls and MPS patient urine samples was undertaken for assessment of urinary sulfated monosaccharides and oligosaccharides present. Several types of negative ion ESI-MS/ MS scans were applied to these samples with two of the most facile fragmentations being neutral loss of 254 Da (a possible loss of PMP and SO3 ) and neutral loss of 374 Da (proposed as a loss of CH2 CðPMPÞ2 ). The NL374 scan was quite specific for the PMP-labelled HexNAcS monosaccharides and this was used for the quantification of the sulfated N-acetylhexosamines using MRM mode. The NL254 scan was less sensitive than the NL374 scan but showed the presence of other oligosaccharides (Fig. 2A and B). The ½M–H ions shown in these scans have been tentatively assigned as m/z 523 uronic acid (UA), m/ z 589 phosphohexose (PHex), m/z 630 HexNAcS, m/z 710 HexNAcS2 , and m/z 806 (HexNAcS-UA). Accuracy and precision of the assay Intra-assay (n ¼ 10) and inter-assay (n ¼ 5) coefficients of variations (CVs) were determined for all the analytes monitored in this study using urine samples from a control patient and a MPS VI patient. The results are shown in Table 1. The intra-assay CV results for the MPS VI sample were proportional to the signal

intensity and were satisfactory for all analytes whereas the control samples CV were high for the HexNAcS2 analyte. This was due to extremely low analyte concentrations. The inter-assay CV results showed a similar trend. Semi-quantification of urinary oligosaccharides in MPS patients The elevations in the total HexNAcS, HexNAcS2 , and HexNAcS-UA in urines from MPS patients and controls are summarised in Table 2. The control population samples, when normalised against creatinine, a widely used metabolite to normalise urinary output, show an inverse exponential relationship with age for all analytes (Fig. 3) and presumably therefore urinary GAG content. This has been shown previously in this laboratory in a feline animal model [28,29]. The majority of MPS disorders showed elevations in total HexNAcS above their respective age matched control cohorts, with the largest elevations observed in the MPS IIID, IVA, VI, and MS disorders. Urine from MPS VI patients had increased concentrations of HexNAcS2 /GalNAc4,6S up to 87 lmol/mmol creatinine. Thus the presence of relatively high concentrations of GalNAc4,6S is strongly indicative of MPS VI as a likely diagnosis. The HexNAcS-UA analyte ratios in comparison to the control population were mostly elevated in MPS IVA, VI, and MS urines with the highest

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A

B

Fig. 2. Negative ion ESI-MS/MS neutral loss 254 scan of PMP-derivatised urine from A, a control and B, a severe MPS VI patient showing elevations of HexNAcS (m/z 630), GalNAc4,6S (m/z 710), and the disaccharide HexNAcS-UA/GalNAc4S-UA (m/z 806).

recorded for an MPS VI, a value of 115:3  103 , a 34fold increase above the age matched control value. Correlation between phenotype and analyte concentration The availability of several MPS IVA and VI patient samples allowed comparison and measurement of monoand di-sulfated N-acetylhexosamines between patients with different clinical phenotypes. The HexNAcS

monosaccharide fraction that should predominantly contain GalNAc6S and GalNAc4S for MPS IVA and VI, respectively, showed a broad range in concentration amongst these MPS types. The amounts of HexNAcS were proportional to the reported clinical severity of these patients indicated in Table 2. Clinical severity scores were based on patientsÕ clinical notes and observations. In the absence of a natural history study being available to the investigators or a widely recognised

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Table 1 Intra and inter-assay coefficient of variations of HexNAcS and HexNAcS2 concentrations for a control and an MPS VI urine sample Intra-assay% CV

HexNAcSa

HexNAcS2 a

HexNAcS-UAa

Control (n ¼ 10) Average Standard deviation % CV

0.5390 0.0387 7.2

0.0550 0.0053 9.6

0.0046 0.0007 16.2

MPS VI (n ¼ 10) Average Standard deviation % CV

3.203 0.080 2.5

0.140 0.013 9.5

0.0074 0.0006 7.7

Inter-assay% CV

HexNAcSa

HexNAcS2 a

HexNAcS-UAa

Control (n ¼ 5) Average Standard deviation % CV

0.570 0.058 10.1

0.0420 0.0098 23.3

0.0038 0.0010 25.5

MPS VI (n ¼ 5) Average Standard deviation % CV

3.29 0.26 7.8

0.126 0.021 16.5

0.0078 0.0014 18.3

a

Ratio to final volume concentration of GlcNAc6S-d3 of 8 lmol/L.

scaling index, the scoring scale of 0–5, 0 being attenuated and 5 severe, was implemented. This system is acknowledged by the authors has having shortcomings should this work need be challenged or mirrored elsewhere. Bone marrow transplantation Analysis of the amount of HexNAcS in the urine samples collected from a MPS VI and a MPS IVA patient whom had both undergone bone marrow transplantation (BMT) demonstrated decreased urinary HexNAcS, HexNAcS2 , and HexNAcS-UA with time after transplantation. The MPS IVA patient at 100 months post-BMT showed reductions in total HexNAcS ()78%), HexNAcS2 ()46%), and HexNAcS-UA ()78%). Similarly, urine from the MPS VI patient after 35 months post-BMT, had reduced HexNAcS ()90%), HexNAcS2 ()85%), and HexNAcS-UA ()93%) content. Although, values for these sulfated analytes following BMT were dramatically reduced, they did not reach normal levels and were still comparable to those of patients afflicted with mild MPS phenotypes. Plasma analysis Analysis of the HexNAcS2 and HexNAcS-UA analytes in plasma from MPS patient did not produce significant signals above background. Only HexNAcS monosaccharides were shown to be elevated threshold values and have a similar trend to the urine results by having a broad concentration range within each MPS

type (Table 3). The total amount of HexNAcS monosaccharides present in plasma appeared to correlate with clinical severity. The two highest elevations in HexNAcS were in samples from neonatal MPS VI patients. The amount of HexNAcS in MPS IVA and MPS IIID plasma was also significantly elevated. The other MPS disorders had only slightly elevated HexNAcS amounts above the mean of the controls. Dried blood spot analysis ESI-MS was also applied to the analysis of single 3mm dried blood spots (containing 3.6 lL blood) punched from Guthrie card blood spot samples obtained from neonates, adults and MPS patients. The limit of detection of the API 365 in MRM mode was sufficient only for the detection of the more abundant HexNAcS monosaccharides. The contribution from background interference in negative ion was estimated to be 0.02 lmol/L on average, based on data from the standard alone. The control blood spots provided signal for the MRM 630/256 ion pair only slightly above the tolerable signal/noise threshold of the instrument. Dried blood spots from MPS I, II, IIIA, IIIB, IIID, IVA, and VI patients were analysed with the results shown in Table 4. The average concentration of HexNAcS in adults was 0.19 lmol/L and was slightly lower than what was observed for the neonates 0.39 lmol/L. The MPS I, II, and IIIA blood spot samples did not have elevations significantly above the control adult values. Only the MPS IIID, IVA, and VI samples, when compared to the adult control samples, showed significant increases of HexAcS above the normal range.

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 carbonate. This resulted in minimising sodium adducts that were frequently observed leading to loss of ½M–H signal intensity in the mass spectra observed. Using a volatile ammonia based buffer system has also allowed the preparation of PMPderivatised monosaccharide standards without the need to desalt samples on solid phase extraction C18 cartridges avoiding potential sample loss. The modified method produced no noticeable reduction in yield compared to the original method by Honda et al. [30].

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Table 2 Calculated concentrations and ratios of sulfated oligosaccharides per micromoles of creatinine from MPS and control urines after derivatising with PMP containing the internal standard GlcNAc6S-d3 Sample

Age (years)

Severitya 1 (mild) to 5 (severe)

HexNAcSb (MRM 630/256) lmol/mmol Cr

HNAc4,6S (MRM 710/256) lmol/mmol Crc

HexNAcS-UAd (MRM 806/331) ratio/mol Cr

Controls 0–2 years (n ¼ 7) Average (SD) Range

0.82 ( 0:8) 0.019–2

2.8 ( 0:7) 2.1–4.1

1.3 ( 1:4) 0–3.4

3.4 ( 1:4) 1–5.2

Controls 2–16 years (n ¼ 9) Average (SD) Range

8.9 ( 4:6) 3–16

0.9 ( 0:6) 0.2–1.9

0.0 ( 0) 0–0.1

1.3 ( 1:0) 0.4–2.7

Controls 16+ years (n ¼ 8) Average (SD) Range

35.5 ( 5:3) 27–42

0.7 ( 0:3) 0.4–1.2

0.1 ( 0:1) 0–0.3

0.7 ( 0:2) 0.4–1.0

14.2 4.6 2.5 6.4 2.5 1.8 3.5 2.7 2.9 1.6 1.5 2.8 1.5 0.9 2.6 16.2 18.5 5.3 38.1 5.7 16.3 10.7 24.0

4.2 3.1 1.4 5.1 0.9 0.4 2.1 1.1 1.2 0.0 0.1 0.0 0.1 0.0 0.2 3.4 4.8 1.4 10.0 1.3 3.4 2.5 6.8

9.4 4.5 1.1 5.3 2.2 1.1 8 5.9 3.1 3.5 0.9 2.6 0.8 0.6 1.4 12 10.2 9.6 53.8 5.8 43 11.9 27.4

LSD types 1 MS 2 MPS I 3 MPS I 4 MPS I 5 MPS II 6 MPS II 7 MPS II 8 MPS IIIA 9 MPS IIIA 10 MPS IIIB 11 MPS IIIB 12 MPS IIIB 13 MPS IIIB 14 MPS IIIC 15 MPS IIIC 16 MPS IIID 17 MPS IIID 18 MPS IVA 19 MPS IVA 20 MPS IVA 21 MPS IVA 22 MPS IVA 23 MPS IVA pre BMT 24 MPS IVA + 39 months BMT 25 MPS IVA + 100 months BMT 26 MPS VI 27 MPS VI 28 MPS VI 29 MPS VI 30 MPS VI 31 MPS VI° 32 MPS VI° 33 MPS VI pre BMT 34 MPS VI + 14 months BMT 35 MPS VI + 35 months BMT a

4.1 0.3 2.4 0.8 5.0 4.3 0.7 12.9 2.6 11.8 14.1 18.7 8.7 10.8 12.0 7.5 2.9 19.0 4.0 8.0 9.6 15.2 1.4

4 5 4 5 5 5 2 5 4 2–3 2–3 5 2–3 4 5 5 5 2 4–5 2 4–5 2–3 4–5

4.6

–

6.5

3.5

7.7

12.9

–

5.3

2.8

6

45.4 0.9 7.0 26.0 17.7 15.7 13.8 12.0 13.2

0–1 5 3–4 3 2 2–3 2–3 4 —

9.1 87.4 30.0 22.7 9.9 13.0 8.9 54.8 18.9

3.8 87.0 19.5 12.7 4.2 9.3 6.3 35.8 15.2

14.4 115.3 35.7 34.7 4.5 7.4 7.9 75.2 32.6

16.1

—

5.5

3.8

5.2

A ranking of severity was based on clinical notes and observations on each patient. Concentration lmol/mmol creatinine (Cr). c Calculated after background subtraction of an internal standard containing blank before calculating the concentration lmol/mmol creatinine. d Uncorrected ratio to internal standard/mol creatine. * Siblings. ° Siblings. b

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Fig. 3. Correlation of age versus HexNAcS, HexNAcS2 , and HexNAcS-UA in control urine samples. s, HexNAcS lmol/mmol creatinine; j, HexNAcS2 lmol/mmol creatinine; M, HexNAcS-UA ratio 103 /mmol creatinine

The different response factors of each sulfated monosaccharide made quantification of total HexNAcS in any sample containing a mixture of these analytes formally a semi-quantitative method as a range of concentrations could only be calculated if the various contributions from each monosaccharide were known. Thus the range variation for the total HexNAcS monosaccharides would at most be overestimated by 25% of the calculated value. Additionally, the GalNAc4,6S monosaccharide did not ionise effectively in comparison to the internal standard and tended also to desulfate in the ESI source, typical of highly sulfated oligosaccharides. These limitations had maximum affect in quantifying total HexNAcS in samples that contain high amounts of the HexNAcS2 /GalNAc4,6S monosaccharide shown to occur primarily in samples from MPS VI patients. The labile sulfate ester groups are unavoidably cleaved during the electrospray ionisation process with a subsequent loss of SO3 (80 Da) from the ½M–H ion at m/z 710 of up to 18%. This has the effect of artificially increasing the observed HexNAcS ions at m/z 630 in the source raising the calculated concentration of HexNAcS. Use of liquid chromatography mass spectrometry (LC-MS) of each sample to separate each of the oligosaccharides could be implemented to avoid this dilemma and obtain more accurate quantification but this would increase the analytical time/sample from the current 4 to >30 min. Although this will improve the analytical accuracy of the method it is counter to the original concept of a fast and efficient method for screening neonates and at risk patients. The contribution to total HexNAcS could be corrected for by using equimolar mixtures of GalNAc4,6S and GlcNAc6S-d3 with each sample batch and

subtracting the percentage contribution, but as this was generally less than the inter-assay CV values it was considered unnecessary. The HexNAcS monosaccharides are present in unusually high amounts in MPS urines due to an alternate degradation pathway whereby b-N-acetylhexosaminadase cleaves the non-reducing end sulfated N-acetylhexosamine [14–16]. The MPS disorders MS, IIID, IVA, and VI store GAGs with non-reducing end b-linked sulfated N-acetylated hexosamines. This in turn leads to an elevation in the HexNAcS monosaccharides in the urine of these patients. An elevated amount of sulfated N-acetylated hexosamines in the urine suggests an elevation in larger GAG oligosaccharides is likely in these urine samples. Hopwood and Elliott [16] have shown that urine from MPS VI patients contain mainly GalNAc4S and GalNAc4,6S. Whereas urine from MPS IVA patients contain mainly GalNAc6S. This indicated that 4-sulfatase action precedes 6-sulfatase in the hydrolysis of GalNAc4,6S. With a greater preponderance of HexNAcS2 (GalNAc4,6S) present in the MPS VI urines our results support these findings. Our results are also consistent for the other disorders, MPS I, II, IIIA, IIIB, and IIIC containing a variable mixture of GalNAc6S, GalNAc4S, and GalNAc6S [14–16]. Not surprisingly, the MPS disorders (I, II, IIIA, IIIB, and IIIC) lacking the non-reducing end HexNAcS moiety, do not have these large elevations but still show an excess over control concentrations and hence would only be useful in indicating that a patient may have an MPS disorder. Case 14 (Table 2), an MPS IIIC sample, highlights this point, with all three analytes being in the normal range.

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201

Table 3 The calculated concentration of total HexNAcS monosaccharides from control and MPS plasmas after derivatising with PMP containing the internal standard GlcNAc6S-d3 Plasma samples

Severitya 1 (mild) to 5 (severe)

Age (years)

HexNAcS MRM 630/256 (lmol/mmol)

Controls 0–2 years (n ¼ 16) Average (SD) Range

0.8 0.13–1.8

0.16 ( 0:06) 0.1–0.28

Controls 2–16 years (n ¼ 20) Average (SD) Range

7.6 2.0–16.0

0.11 ( 0:04) 0.09–0.19

40.2 17.0–62.3

0.15 ( 0:05) 0.08–0.35

1.3 0.8 29.0 4.6 2.9 2.0 4.4 3.1 5.3 4.6 9.3 2.7 11.8 4.3 4.0 21.0 20.2 10.8 5.8 0.4 3.3 2.9 4.3 2.3 1.4 1.7 8.0 2.5 3.7 5.6 0.04 5.3 9.6 2.7 0.0 1.4

0.22 0.32 0.17 0.16 0.18 0.16 0.22 0.14 0.21 0.21 0.16 0.14 0.14 0.20 0.17 0.13 0.12 0.14 0.15 0.88 0.67 0.91 1.30 1.89 1.53 1.14 2.59 1.77 1.80 1.54 6.88 0.92 0.75 1.56 4.15 2.79

Controls 16+ year (n ¼ 25) Average (SD) Range LSD types 1 MPS I 2 MPS 1 3 MPS 1 4 MPS 1 5 MPS II 6 MPS II 7 MPS II 8 MPS II 9 MPS IIIA 10 MPS IIIA 11 MPS IIIA 12 MPS IIIA 13 MPS IIIB 14 MPS IIIB 15 MPS IIIA 16 MPS IIIB 17 MPS IIIC 18 MPS IIIC 19 MPS IIIC 20 MPS IIID 21 MPS IIID 22 MPS IIID 23 MPS IVA 24 MPS IVA 25 MPS IVA 26 MPS IVA 27 MPS VI 28 MPS VI 29 MPS VI 30 MPS VI 31 MPS VI 32 MPS VI° 33 MPS VI° 34 MPS VI 35 MPS VI 36 MPS VI

5 5 2 2–3 Unknown 5 4–5 5 Unknown 4–5 4–5 5 2–3 5 4 3–4 3 4 4–5 5 5 5 4 5 4–5 5 4 4 4–5 3–4 5 2 Unknown Unknown 5 4

a

A ranking of severity was based on clinical notes and observations on each patient. Siblings. ° Siblings. *

It remains unclear why these MPS disorders should show an elevation in the sulfated N-acetylated hexosamines. It is possible that the alternative degradation pathway involving b-N-acetylhexosaminadase could be upregulated as a result of GAG storage leading to competition for substrate with other GAG-degrading enzymes in the lysosome that are required to degrade the stored GAG in each MPS-genotype. Alternatively it

could simply be a result of the increased availability of these secondary substrates in the lysosomal organelles. The overall trends in urine elevations and ranges of the combined HexNAcS and HexNAcS2 urine analytes reported here are comparable to that seen by Hopwood and Elliott [14–16] but our results did not give the same fold increases as reported. These differences may be a reflection of the comparisons made by Hopwood and

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Table 4 The calculated concentration of total HexNAcS in blood spots after derivatising with PMP containing the internal standard GlcNAc6S-d3 Dried blood spot samples

Severitya 1 (mild) to 5 (severe)

Neonate controls (n ¼ 24) Average (SD) Range

0 0

Adult controls (n ¼ 34) Average (SD) Range LSD types 1 MPS I 2 MPS I 3 MPS II 4 MPS II 5 MPS IIIA 6 MPS IIID 7 MPS IIID 8 MPS IVA 9 MPS VI 10 MPS VI 11 MPS VI 12 MPS VI a *

Age (year)

5 3 2 4–5 4 5 5 Unknown 4 2–3 5 3–4

HexNAcSMRM 630/256 (lmol/mmol) 0.39 ( 0:15) 0.20–0.83

47 24.7–64.7

0.19 ( 0:04) 0.13–0.26

2.7 27.4 35.8 7.7 19.5 5.0 11.3 8.4 11.8 42.2 0.0 7.0

0.44 0.46 0.19 0.34 0.32 1.41 1.45 1.11 3.14 1.67 2.76 2.02

A ranking of severity was based on clinical notes and observations on each patient. Siblings.

Scheme 1. Derivatisation of GlcNAc6S using 1-phenyl-3-methyl pyrazolone.

Elliott were with urine that was not age matched and contained only a few controls. Additionally, due to the rarity of many of these MPS disorders, only a few samples from each of the MPS categories were available at that time. It is noteworthy to mention the use of creatinine (113.12 Da) to adjust for fluctuations in urine incorporates its own difficulties as creatinine is derived from muscle metabolism. During the first month of life serum creatinine levels fall to below 1 mg/10 L (0.9 mM) [32]. Levels in children then increase with age and are slightly higher in males than females at any age. Consequently, older patients with higher creatinine levels have a lower apparent analyte concentration (or ratio) to creatinine.

This may artificially indicate a less severe phenotype. To overcome this, affected individuals should be compared to age matched control values. The monosaccharide markers HexNAcS, HexNAcS2 , and the disaccharide HexNAcS-UA in urine appear effective in differentiating each of the analysed MPS patients from controls. However, in plasma and dried blood samples, only the amounts of HexNAcS was sufficient for analysis. MPS I, II, IIIA, IIIB, and IIIC, on the whole, showed only slight elevations of all three analytes above the control range and in a screening sense were not distinguishable from each other. Conversely MPS IIID, IVA, VI, and MS, were substantially elevated above control values and could, with a few exceptions, be categorised into sub groups based on the ratios of the analytes monitored. MPS VI was distinguishable from the other MPS due to the relative abundance of the HexNAcS2 and also a high HexNAcS2 /HexNAcS ratio generally above a ratio of 0.6. MPS IVA was distinguishable with elevated HexNAcS and HexNAcS-UA relatively low amounts of HexNAcS2 , yielding a high ratio of HexNAcS-UA/ HexNAcS2 generally greater than 2.7. MPS IIID and MS were distinguishable from the others with small amounts of HexNAcS2 and a ratio for HexNAcS-UA/ HexNAcS2 of less than 2.0. They are not separable from each other using the monitored analytes in this study. Analysis of plasma and blood samples from each MPS showed a similar trend but with reduced elevations of the HexNAcS and HexNAcS2 /GalNAc4,6S analytes. Importantly, only MPS IIID, IVA, and VI displayed elevations of these analytes above normal and therefore

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were distinguishable from control dried blood spots. However as these elevations are not large, an attenuated MPS phenotype from this group would be at risk of not being detected.

Acknowledgments 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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