Quantification of Glycosaminoglycans in Urine by Isotope-Dilution Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry.

Mucopolysaccharidoses (MPSs) are complex lysosomal storage disorders that result in the accumulation of glycosaminoglycans (GAGs) in urine, blood, and tissues. Lysosomal enzymes responsible for GAG degradation are defective in MPSs. GAGs including chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS), and keratan sulfate (KS) are disease-specific biomarkers for MPSs. This article describes a stable isotope dilution-tandem mass spectrometric method for quantifying CS, DS, and HS in urine samples. The GAGs are methanolyzed to uronic or iduronic acid-N-acetylhexosamine or iduronic acid-N-sulfo-glucosamine dimers and mixed with internal standards derived from deuteriomethanolysis of GAG standards. Specific dimers derived from HS, DS, and CS are separated by ultra-performance liquid chromatography (UPLC) and analyzed by electrospray ionization tandem mass spectrometry (MS/MS) using selected reaction monitoring for each targeted GAG product and its corresponding internal standard. This UPLC-MS/MS GAG assay is useful for identifying patients with MPS types I, II, III, VI, and VII. © 2023 Wiley Periodicals LLC. Basic Protocol: Urinary GAG analysis by ESI-MS/MS Support Protocol 1: Prepare calibration samples Support Protocol 2: Preparation of stable isotope-labeled internal standards Support Protocol 3: Preparation of quality controls for GAG analysis in urine Support Protocol 4: Optimization of the methanolysis time Support Protocol 5: Measurement of the concentration of methanolic HCl.

Untargeted LC-HRMS metabolomics reveals candidate biomarkers for mucopolysaccharidoses.

BACKGROUND: Mucopolysaccharidoses (MPSs) are inherited genetic diseases caused by an absence or deficiency of lysosomal enzymes responsible for catabolizing glycosaminoglycans (GAGs). Undiagnosed patients, or those without adequate treatment in early life, can be severely and irreversibly affected by the disease. In this study, we applied liquid chromatography-high resolution mass spectrometry (LC-HRMS)-based untargeted metabolomics to identify potential biomarkers for MPS disorders to better understand how MPS may affect the metabolome of patients. METHODS: Urine samples from 37 MPS patients (types I, II, III, IV, and VI; untreated and treated with enzyme replacement therapy (ERT)) and 38 controls were analyzed by LC-HRMS. Data were processed by an untargeted metabolomics workflow and submitted to multivariate statistical analyses to reveal significant differences between the MPS and control groups. RESULTS: A total of 12 increased metabolites common to all MPS types were identified. Dipeptides, amino acids and derivatives were increased in the MPS group compared to controls. N-acetylgalactosamines 4- or 6-sulfate, important constituents of GAGs, were also elevated in MPS patients, most prominently in those with MPS VI. Notably, treated patients exhibited lower levels of the aforementioned acylaminosugars than untreated patients in all MPS types. CONCLUSIONS: Untargeted metabolomics has enabled the detection of metabolites that could improve our understanding of MPS physiopathology. These potential biomarkers can be utilized in screening methods to support diagnosis and ERT monitoring.

The Effect of Hyperfiltration on Kidney Function in Living Donor Kidney Transplantation: A Prospective Cohort Study.

BACKGROUND: living kidney donation is a safe medical procedure. Kidney function after donation is crucial for donors' health and quality of life. Kidney hyperfiltration is a compensatory mechanism, which will preserve kidney function after unilateral nephrectomy. The number of studies regarding hyperfiltration in living kidney donors is limited. Our study aimed to explain kidney hyperfiltration mechanism and evaluate its effect on the kidney function within 30 days after surgery. METHODS: our study was a prospective cohort study with 46 living-kidney donors participating in the study between April and December 2019. We evaluated main outcomes, the 30-day post-surgery kidney function, which was evaluated by calculating estimated glomerular filtration rate (eGFR) and Urinary Albumin to Creatinine Ratio (ACR). The subjects were categorized into two groups based on their 30-day outcomes, which were the adaptive (eGFR > 60 mL/min/1.73 m2 and/or ACR > 30 mg/g) and maladaptive (eGFR < 60 mL/min/1.73 m2 and/or ACR > 30 mg/g) groups. A series of evaluation including calculating the renal arterial resistive index (RI) and measuring urinary vascular endothelial growth factor (VEGF), neutrophil gelatinase-associated lipocalin (NGAL), and heparan sulfate (HS) levels were performed before surgery and serially until 30 days after surgery. Multivariate analysis with adjustments for confounding factors was done. RESULTS: forty donors were included and mostly were female (67.5%). The average age and body mass index (BMI) were 45.85 (SD 9.74) years old and 24.36 (SD 3.73) kg/m2, respectively. Nineteen donors (47.5%) had maladaptive hyperfiltration outcomes. The hyperfiltration process was demonstrated by significant changes in renal arterial RI, urinary VEGF, NGAL, and HS levels (p<0.005). There was no significant difference regarding RI, urinary VEGF, NGAL, and HS levels between both groups. Several confounding factors (BMI over 25 kg/m2, familial relationship, age over 40 years old, and arterial stiffness) were significantly influenced by kidney hyperfiltration and outcomes (p<0.05). CONCLUSION: the hyperfiltration process does not affect the 30-day post-nephrectomy kidney function of the donors. Several other factors may influence the hyperfiltration process and kidney function. Further study is necessary to evaluate kidney function and its other related variables with a longer period of time study duration.

The North Carolina Experience with Mucopolysaccharidosis Type I Newborn Screening.

OBJECTIVE: To evaluate the performance of a 2-tiered newborn screening method for mucopolysaccharidosis type I (MPS I) in North Carolina. STUDY DESIGN: The screening algorithm included a flow injection analysis-tandem mass spectrometry assay as a first-tier screening method to measure α-L-iduronidase (IDUA) enzyme activity and Sanger sequencing of the IDUA gene on dried blood spots as a second-tier assay. The screening algorithm was revised to incorporate the Collaborative Laboratory Integrated Reports, an analytical interpretive tool, to reduce the false-positive rate. A medical history, physical examination, IDUA activity, and urinary glycosaminoglycan (GAG) analysis were obtained on all screen-positive infants. RESULTS: A total of 62 734 specimens were screened with 54 screen-positive samples using a cut-off of 15% of daily mean IDUA activity. The implementation of Collaborative Laboratory Integrated Reports reduced the number of specimens that screened positive to 19 infants. Of the infants identified as screen-positive, 1 had elevated urinary GAGs and a homozygous pathogenic variant associated with the severe form of MPS I. All other screen-positive infants had normal urinary GAG analysis; 13 newborns had pseudodeficiency alleles, 3 newborns had variants of unknown significance, and 2 had heterozygous pathogenic variants. CONCLUSIONS: An infant with severe MPS I was identified and referred for a hematopoietic stem cell transplant. Newborn IDUA enzyme deficiency is common in North Carolina, but most are due to pseudodeficiency alleles in infants with normal urinary GAG analysis and no evidence of disease. The pilot study confirmed the need for second-tier testing to reduce the follow-up burden.

Urinary sulfated glycosaminoglycan insufficiency and chondroitin sulfate supplement in urolithiasis.

Familial members of urolithiasis have high risk for stone development. We observed the low sulfated glycosaminoglycan (GAG) excretion in urolithiasis patients and their descendants. In this study, we investigated urinary excretion of sulfated GAG, chondroitin sulfate (CS), heparan sulfate (HS) and hyaluronic acid (HA) in urolithiasis and their children, and explored the effect of CS and HA supplement in urolithic hyperoxaluric rats. The 24-hour urines were collected from urolithiasis patients (28) and their children (40), as well as healthy controls (45) and their children (33) to measure urinary sulfated GAG, CS, HS and HA excretion rate. Our result showed that urinary sulfated GAG and CS were diminished in both urolithiasis patients and their children, while decreased HS and increased HA were observed only in urolithiasis patients. Percentage of HS per sulfated GAG increased in both urolithiasis patients and their children. In hyperoxaluric rats induced by ethylene glycol and vitamin D, we found that CS supplement could prevent stone formation, while HA supplement had no effect on stone formation. Our study revealed that decreased urinary GAG and CS excretion are common in familial members of urolithiasis patients, and CS supplement might be beneficial in calcium oxalate urolithiasis prophylaxis for hyperoxaluric patients.

LC-MS/MS method for simultaneous quantification of heparan sulfate and dermatan sulfate in urine by butanolysis derivatization.

Mucopolysaccharidoses are a group of lysosomal storage disorders (LSDs) characterized by the accumulation of glycosaminoglycans (GAGs). Recently, LC-MS/MS has been widely applied in GAGs analysis combined with different sample preparations for cleaving GAGs to disaccharide units. The aim of the present is paper is to present a new method for the simultaneous quantification of urinary dermatan sulfate (DS) and heparan sulfate (HS) by LC-MS/MS, after butanolysis reaction. Chromatographic separation was achieved with a gradient of acetonitrile and water in 0.1% formic acid on a Kinetex Biphenyl analytical column in 21 min. Calibration curves ranging from 0.78 to 50 µg/mL for HS and from 1.56 to 100 µg/mL for DS were prepared and the coefficient of determination (r2) was higher than 0.99 for both analytes. Intra-day and inter-day imprecisions and the bias for both compounds were <10.0%. Up to now, most analytical procedures for quantifying GAGs have not had a high level of reproducibility among laboratories, despite the availability of various techniques. The adoption of a new protocol incorporating the methods outlined in this paper could significantly improve the quality and reproducibility of MS results. A procedure using simple steps for preparing samples and reagents that are easily available on the market could promote the standardization of analytical procedures and increase the use of these measurements in clinical practice.

Prospective Evaluation of Chondroitin Sulfate, Heparan Sulfate and Hyaluronic Acid in Prostate Cancer.

PURPOSE: The present study evaluates chondroitin sulfate (CS) and heparan sulfate (HS) in the urine and hyaluronic acid (HA) in the plasma of patients with prostate cancer before and after treatment compared to a control group. MATERIALS AND METHODS: Plasma samples were used for HA dosage and urine for quantification of CS and HS from forty-four cancer patients and fourteen controls. Clinical, laboratory and radiological information were correlated with glycosaminoglycan quantification by statistical analysis. RESULTS: Serum HA was significantly increased in cancer patients (39.68 ± 30.00 ng/ mL) compared to control group (15.04 ± 7.11 ng/mL; p=0.004) and was further increased in high-risk prostate cancer patients when compared to lower risk patients (p = 0.0214). Also, surgically treated individuals had a significant decrease in seric levels of heparan sulfate after surgical treatment, 31.05 ± 21.01 µg/mL (before surgery) and 23.14 ± 11.1 µg/mL (after surgery; p=0.029). There was no difference in the urinary CS and HS between prostate cancer patients and control group. Urinary CS in cancer patients was 27.32 ± 25.99 µg/mg creatinine while in the men unaffected by cancer it was 31.37 ± 28.37 µg/mg creatinine (p=0.4768). Urinary HS was 39.58 ± 32.81 µg/ mg creatinine and 35.29 ± 28.11 µg/mg creatinine, respectively, in cancer patients and control group (p=0.6252). CONCLUSIONS: Serum HA may be a useful biomarker for the diagnosis and prognosis of prostate cancer. However, urinary CS and HS did not altered in the present evaluation. Further studies are necessary to confirm these preliminary findings.

Prospective evaluation of Chondroitin sulfate, Heparan sulfate and Hyaluronic acid in prostate cancer

ABSTRACT Purpose: The present study evaluates chondroitin sulfate (CS) and heparan sulfate (HS) in the urine and hyaluronic acid (HA) in the plasma of patients with prostate cancer before and after treatment compared to a control group. Materials and Methods: Plasma samples were used for HA dosage and urine for quantification of CS and HS from forty-four cancer patients and fourteen controls. Clinical, laboratory and radiological information were correlated with glycosaminoglycan quantification by statistical analysis. Results: Serum HA was significantly increased in cancer patients (39.68 ± 30.00 ng/ mL) compared to control group (15.04 ± 7.11 ng/mL; p=0.004) and was further increased in high-risk prostate cancer patients when compared to lower risk patients (p = 0.0214). Also, surgically treated individuals had a significant decrease in seric levels of heparan sulfate after surgical treatment, 31.05 ± 21.01 μg/mL (before surgery) and 23.14 ± 11.1 μg/mL (after surgery; p=0.029). There was no difference in the urinary CS and HS between prostate cancer patients and control group. Urinary CS in cancer patients was 27.32 ± 25.99 μg/mg creatinine while in the men unaffected by cancer it was 31.37 ± 28.37 μg/mg creatinine (p=0.4768). Urinary HS was 39.58 ± 32.81 μg/ mg creatinine and 35.29 ± 28.11 μg/mg creatinine, respectively, in cancer patients and control group (p=0.6252). Conclusions: Serum HA may be a useful biomarker for the diagnosis and prognosis of prostate cancer. However, urinary CS and HS did not altered in the present evaluation. Further studies are necessary to confirm these preliminary findings.

The relationships between urinary glycosaminoglycan levels and phenotypes of mucopolysaccharidoses.

BACKGROUND: The aim of this study was to use the liquid chromatography/tandem mass spectrometry (LC-MS/MS) method to quantitate levels of three urinary glycosaminoglycans (GAGs; dermatan sulfate [DS], heparan sulfate [HS], and keratan sulfate [KS]) to help make a correct diagnosis of mucopolysaccharidosis (MPS). METHODS: We analyzed the relationships between phenotypes and levels of urinary GAGs of 79 patients with different types of MPS. RESULTS: The patients with mental retardation (n = 21) had significantly higher levels of HS than those without mental retardation (n = 58; 328.8 vs. 3.2 µg/ml, p < 0.001). The DS levels in the patients with hernia, hepatosplenomegaly, claw hands, coarse face, valvular heart disease, and joint stiffness were higher than those without. Twenty patients received enzyme replacement therapy (ERT) for 1-12.3 years. After ERT, the KS level decreased by 90% in the patients with MPS IVA compared to a 31% decrease in the change of dimethylmethylene blue (DMB) ratio. The DS level decreased by 79% after ERT in the patients with MPS VI compared to a 66% decrease in the change of DMB ratio. CONCLUSIONS: The measurement of GAG fractionation biomarkers using the LC-MS/MS method is a more sensitive and reliable tool than the DMB ratio for MPS high-risk screening, diagnosis, subclass identification, and monitoring the efficacy of ERT.

Glycosaminoglycans analysis in blood and urine of patients with mucopolysaccharidosis.

To explore the correlation between glycosaminoglycan (GAG) levels and mucopolysaccharidosis (MPS) type, we have evaluated the GAG levels in blood of MPS II, III, IVA, and IVB and urine of MPS IVA, IVB, and VI by tandem mass spectrometry. Dermatan sulfate (DS), heparan sulfate (HS), keratan sulfate (KS; mono-sulfated KS, di-sulfated KS), and the ratio of di-sulfated KS in total KS were measured. Patients with untreated MPS II had higher levels of DS and HS in blood while untreated MPS III had higher levels of HS in blood than age-matched controls. Untreated MPS IVA had higher levels of KS in blood and urine than age-matched controls. The ratio of blood di-sulfated KS/total KS in untreated MPS IVA was constant and higher than that in controls for children up to 10 years of age. The ratio of urine di-sulfated KS/total KS in untreated MPS IVA was also higher than that in age-matched controls, but the ratio in untreated MPS IVB was lower than controls. ERT reduced blood DS and HS in MPS II, and urine KS in MPS IVA patients, although GAGs levels remained higher than the observed in age-matched controls. ERT did not change blood KS levels in MPS IVA. MPS VI under ERT still had an elevation of urine DS level compared to age-matched controls. There was a positive correlation between blood and urine KS in untreated MPS IVA patients but not in MPS IVA patients treated with ERT. Blood and urine KS levels were secondarily elevated in MPS II and VI, respectively. Overall, measurement of GAG levels in blood and urine is useful for diagnosis of MPS, while urine KS is not a useful biomarker for monitoring therapeutic efficacy in MPS IVA.

Development and validation of an LC-MS/MS Method for the quantitation of heparan sulfate in human urine.

Heparan sulfate is a linear polysaccharide and serves as an important biomarker to monitor patient response to therapies for MPS III disorder. It is challenging to analyze heparan sulfate intact owing to its complexity and heterogeneity. Therefore, a sensitive, robust and validated LC-MS/MS method is needed to support the clinical studies for the quantitation of heparan sulfate in biofluids under regulated settings. Presented in this work are the results of the development and validation of an LC-MS/MS method for the quantitation of heparan sulfate in human urine using selected high-abundant disaccharides as surrogates. During sample processing, a combination of analytical technologies have been employed, including rapid digestion, filtration, solid-phase extraction and chemical derivatization. The validated method is highly sensitive and is able to analyze heparan sulfate in urine samples from healthy donors. Disaccharide constitution analysis in urine samples from 25 healthy donors was performed using the assay and demonstrated the proof of concept of using selected disaccharides as a surrogate for validation and quantitation.

One-pot analysis of sulfated glycosaminoglycans.

Routine isolation, estimation, and characterization of glycosaminoglycans (GAGs) is quite challenging. This is compounded by the fact that the analysis is technique-intensive and more often there will be a limitation on the quantity of GAGs available for various structural, functional and biological studies. In such a scenario, the sample which can be made available for estimation and elucidation of disaccharide composition and species composition as well remains a challenge. In the present study, we have determined the feasibility where isolated sulfated GAGs (sGAG) that is estimated by metachromasia is recovered for further analysis. sGAG-DMMB complex formed after estimation of sGAG by DMMB dye-binding assay was decomplexed and sGAGs were recovered. Recovered sGAGs were analysed by cellulose acetate membrane electrophoresis and taken up for disaccharide composition analysis by HPLC after fluorescent labelling. Good recovery of sGAGs after metachromasia was observed in all samples of varying levels of purity by this protocol. Further analysis using cellulose acetate membrane electrophoresis showed good separation between species of sGAGs namely chondroitin/dermatan sulfate and heparan sulfate, with comparatively lesser interference from hyaluronic acid, a non-sulfated GAG. Analysis of recovered sGAGs, specifically heparan sulfate by HPLC showed characteristic disaccharide composition akin to that of GAG obtained by the conventional protocol. Thus, in the present paper, we show that sGAG can be recovered in comparatively purer form after routine estimation and can be used for further analysis thus saving up on the precious sample.

Urinary metabolic phenotyping of mucopolysaccharidosis type I combining untargeted and targeted strategies with data modeling.

BACKGROUND: Application of metabolic phenotyping could expand the pathophysiological knowledge of mucopolysaccharidoses (MPS) and may reveal the comprehensive metabolic impairments in MPS. However, few studies applied this approach to MPS. METHODS: We applied targeted and untargeted metabolic profiling in urine samples obtained from a French cohort comprising 19 MPS I and 15 MPS I treated patients along with 66 controls. For that purpose, we used ultra-high-performance liquid chromatography combined with ion mobility and high-resolution mass spectrometry following a protocol designed for large-scale metabolomics studies regarding robustness and reproducibility. Furthermore, 24 amino acids have been quantified using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Keratan sulfate, Heparan sulfate and Dermatan sulfate concentrations have also been measured using an LC-MS/MS method. Univariate and multivariate data analyses have been used to select discriminant metabolites. The mummichog algorithm has been used for pathway analysis. RESULTS: The studied groups yielded distinct biochemical phenotypes using multivariate data analysis. Univariate statistics also revealed metabolites that differentiated the groups. Specifically, metabolites related to the amino acid metabolism. Pathway analysis revealed that several major amino acid pathways were dysregulated in MPS. Comparison of targeted and untargeted metabolomics data with in silico results yielded arginine, proline and glutathione metabolisms being the most affected. CONCLUSION: This study is one of the first metabolic phenotyping studies of MPS I. The findings might help to generate new hypotheses about MPS pathophysiology and to develop further targeted studies of a smaller number of potentially key metabolites.

Minimal shedding of the glycocalyx layer during abdominal hysterectomy.

BACKGROUND: Surgery with and without hypervolaemia may cause shedding (breakdown) of the endothelial glycocalyx layer, but the severity of this problem is unclear. METHODS: In this preliminary report of a larger clinical trial, the plasma and urine concentrations of three biomarkers of glycocalyx shedding (syndecan-1, hyaluronic acid and heparan sulfate) were measured in seven patients before, during, and after open hysterectomy. The fluid therapy consisted of 25 ml/kg (approximately 2 l) of Ringer's lactate, which was infused over 30 min when the surgery started. The resulting plasma volume expansion at the end of the infusion was estimated from the haemodilution. RESULTS: The mean plasma concentration of syndecan-1 was 21.7 ng/ml before surgery and averaged 19.7 ng/ml during and after the surgery. The plasma concentration of hyaluronic acid decreased from 38.0 to 27.7 ng/ml (P < 0.05), while heparan sulfate increased from 3.4 to 5.5 µg/ml (P < 0.05). The urine concentrations of syndecan-1 decreased significantly, while they increased for hyaluronic acid and heparan sulfate. Despite the vigorous fluid load, the urine flow did not exceed 1 ml/min. CONCLUSIONS: No clear evidence was found for shedding of the endothelial glycocalyx layer when 2 l of Ringer's lactate was infused over 30 min during abdominal hysterectomy. Urine analyses yielded patterns of changes that differed from those in plasma. TRIAL REGISTRATION: ISRCTN81005631 . Registered May 17, 2016.

Incomplete biomarker response in mucopolysaccharidosis type I after successful hematopoietic cell transplantation.

BACKGROUND: Residual disease, primarily involving musculoskeletal tissue, is a common problem in patients with neuronopathic mucopolysaccharidosis type I (MPS I, Hurler or severe Hurler-Scheie phenotype) after a successful hematopoietic cell transplantation (HCT). The concentration of the GAG derived biomarkers heparan sulfate (HS) and dermatan sulfate (DS), may reflect residual disease and is used for monitoring biochemical response to therapies. This study investigates the response of HS and DS in blood and urine to HCT in MPS I patients. METHODS: In 143 blood- and urine samples of 17 neuronophatic MPS I patients, collected prior and post successful HCT, the concentration of the disaccharides derived after full enzymatic digestion of HS and DS were analyzed by multiplex liquid chromatography tandem-mass spectrometry (LC-MS/MS). RESULTS: Median follow up after HCT was 2.4years (range 0-11years). HCT led to a rapid decrease of both HS and DS. However, only 38% of the patients reached normal HS levels in blood and even less patients (6%) reached normal DS levels. In none of the patients normalization of HS or DS was observed in urine. CONCLUSIONS: Biomarker response after HCT is incomplete, which may reflect residual disease activity. Novel therapeutic strategies should aim for full metabolic correction to minimize clinical manifestations.

Toward Noninvasive Diagnosis of IgA Nephropathy: A Pilot Urinary Metabolomic and Proteomic Study.

IgA nephropathy is diagnosed by renal biopsy, an invasive procedure with a risk of significant complications. Noninvasive approaches are needed for possible diagnostic purposes and especially for monitoring disease activity or responses to treatment. In this pilot project, we assessed the utility of urine samples as source of biomarkers of IgA nephropathy. We used spot urine specimens from 19 healthy controls, 11 patients with IgA nephropathy, and 8 renal-disease controls collected on day of renal biopsy. Urine samples were analyzed using untargeted metabolomic and targeted proteomic analyses by several experimental techniques: liquid chromatography coupled with mass spectrometry, immunomagnetic isolation of target proteins coupled with quantitation by mass spectrometry, and protein arrays. No single individual biomarker completely differentiated the three groups. Therefore, we tested the utility of several markers combined in a panel. Discriminant analysis revealed that combination of seven markers, three metabolites (dodecanal, 8-hydroxyguanosine, and leukotriene C4), three proteins (α1-antitrypsin, IgA-uromodulin complex, and galactose-deficient IgA1), and heparan sulfate, differentiated patients with IgA nephropathy from patients with other renal diseases and healthy controls. Future studies are needed to validate these preliminary findings and to determine the power of these urinary markers for assessment of responses to therapy.

A Multiplex Assay for the Diagnosis of Mucopolysaccharidoses and Mucolipidoses.

INTRODUCTION: Diagnosis of the mucopolysaccharidoses (MPSs) generally relies on an initial analysis of total glycosaminoglycan (GAG) excretion in urine. Often the dimethylmethylene blue dye-binding (DMB) assay is used, although false-negative results have been reported. We report a multiplexed diagnostic test with a high sensitivity for all MPSs and with the potential to identify patients with I-cell disease (ML II) and mucolipidosis III (ML III). METHODS: Urine samples of 100 treatment naive MPS patients were collected and analyzed by the conventional DMB assay and a multiplex assay based on enzymatic digestion of heparan sulfate (HS), dermatan sulfate (DS) and keratan sulfate (KS) followed by quantification by LC-MS/MS. Specificity was calculated by analyzing urine samples from a cohort of 39 patients suspected for an inborn error of metabolism, including MPSs. RESULTS: The MPS cohort consisted of 18 MPS I, 16 MPS II, 34 MPS III, 10 MPS IVA, 3 MPS IVB, 17 MPS VI and 2 MPS VII patients. All 100 patients were identified by the LC-MS/MS assay with typical patterns of elevation of HS, DS and KS, respectively (sensitivity 100%). DMB analysis of the urine was found to be in the normal range in 10 of the 100 patients (sensitivity 90%). Three out of the 39 patients were identified as false-positive, resulting in a specificity of the LS-MS/MS assay of 92%. For the DMB this was 97%. All three patients with MLII/MLIII had elevated GAGs in the LC-MS/MS assay while the DMB test was normal in 2 of them. CONCLUSION: The multiplex LC-MS/MS assay provides a robust and very sensitive assay for the diagnosis of the complete spectrum of MPSs and has the potential to identify MPS related disorders such as MLII/MLIII. Its performance is superior to that of the conventional DMB assay.

Butanolysis derivatization: improved sensitivity in LC-MS/MS quantitation of heparan sulfate in urine from mucopolysaccharidosis patients.

Heparan sulfate (HS) is a complex oligosaccharide that is a marker of a number of diseases, most notably several of the mucopolysaccharidoses (MPS). It is a very heterogeneous compound and its quantification at physiological concentrations in patient samples is challenging. Here, we demonstrate novel derivatization chemistry for depolymerization/desulfation and alkylation of HS based on butanolysis. The resultant alkylated disaccharides are quantifiable by LC-MS/MS. This new method is at least 70-fold more sensitive than a previously published methanolysis method. Disaccharide yield over time is compared for methanolysis, ethanolysis, and butanolysis. Maximum disaccharide concentration was observed after 2 h with butanolysis and 18 h with ethanolysis whereas a maximum was not reached over the 24 h of the experiment with methanolysis. The sensitivity of the new technique is illustrated by the quantification of HS in 5 µL urine samples from MPS patients and healthy controls. HS was quantifiable in all samples including controls. Disaccharide reaction products were further characterized using exact mass MS/MS.

Mental retardation in mucopolysaccharidoses correlates with high molecular weight urinary heparan sulphate derived glucosamine.

Mucopolysaccharidoses (MPS) are characterized by mental retardation constantly present in the severe forms of Hurler (MPS I), Hunter (MPS II) and Sanfilippo (MPS III) diseases. On the contrary, mental retardation is absent in Morquio (MPS IV) and Maroteaux-Lamy (MPS VI) diseases and absent or only minimal in the attenuated forms of MPS I, II and III. Considering that MPS patients affected by mental disease accumulate heparan sulfate (HS) due to specific enzymatic defects, we hypothesized a possible correlation between urinary HS-derived glucosamine (GlcN) accumulated in tissues and excreted in biological fluids and mental retardation. 83 healthy subjects were found to excrete HS in the form of fragments due to the activity of catabolic enzymes that are absent or impaired in MPS patients. On the contrary, urinary HS in 44 patients was observed to be composed of high molecular weight polymer and fragments of various lengths depending on MPS types. On this basis we correlated mental retardation with GlcN belonging to high and low molecular weight HS. We demonstrate a positive relationship between the accumulation of high molecular weight HS and mental retardation in MPS severe compared to attenuated forms. This is also supported by the consideration that accumulation of other GAGs different from HS, as in MPS IV and MPS VI, and low molecular weight HS fragments do not impact on central nervous system disease.

Plasma and urinary glycosaminoglycans in the course of juvenile idiopathic arthritis.

OBJECTIVES: The aim of the study was to perform analyses of plasma and urinary glycosaminoglycan isolated from juvenile idiopathic arthritis (JIA). METHODS, RESULTS: Chondroitin/dermatan sulfate (CS/DS), heparan sulfate/heparin (HS/H) and hyaluronic acid (HA) were evaluated in samples obtained from JIA patients before and after treatment. Electrophoretic analysis of GAGs identified the presence of CS, DS and HS/H in plasma of healthy subjects and JIA patients. CS were the predominant plasma GAGs constituent in all investigated subject. The plasma CS level in untreated patients was significantly decreased. Therapy resulted in an increase in this glycan level. However, plasma CS concentration still remained higher than in controls. Increased levels of DS and HA in untreated JIA patients were recorded. Anti-inflammatory treatment led to normalization of these parameters concentrations. Plasma and urinary concentrations of HS/H were similar in all groups of individuals. Urinary CS/DS and HA were decreased only in untreated patients. CONCLUSIONS: The data presented indicate that changes in plasma and urinary glycosaminoglycan occur in the course of JIA. There are probably the expression of both local articular cartilage matrix and systemic changes in connective tissue remodeling.