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(1) Background: In this study, we evaluated the modulation of urine glycosaminoglycans (GAGs), which resulted from etanercept (ETA) therapy in patients with juvenile idiopathic arthritis (JIA) in whom methotrexate therapy failed to improve their clinical condition. (2) Methods: The sulfated GAGs (sGAGs, by complexation with blue 1,9-dimethylmethylene), including chondroitin-dermatan sulfate (CS/DS) and heparan sulfate (HS), as well as non-sulfated hyaluronic acid (HA, using the immunoenzymatic method), were determined in the blood of 89 children, i.e., 30 healthy children and 59 patients with JIA both before and during two years of ETA treatment. (3) Results: We confirmed the remodeling of the urinary glycan profile of JIA patients. The decrease in the excretion of sGAGs ( p < 0.05), resulting from a decrease in the concentration of the dominant fraction in the urine, i.e., CS/DS ( p < 0.05), not compensated by an increase in the concentration of HS ( p < 0.000005) and HA ( p < 0.0005) in the urine of patients with the active disease, was found. The applied biological therapy, leading to clinical improvement in patients, at the same time, did not contribute to normalization of the concentration of sGAGs ( p < 0.01) in the urine of patients, as well as CS/DS ( p < 0.05) in the urine of sick girls, while it promoted equalization of HS and HA concentrations. These results indicate an inhibition of the destruction of connective tissue structures but do not indicate their complete regeneration. (4) Conclusions: The metabolisms of glycans during JIA, reflected in their urine profile, depend on the patient's sex and the severity of the inflammatory process. The remodeling pattern of urinary glycans observed in patients with JIA indicates the different roles of individual types of GAGs in the pathogenesis of osteoarticular disorders in sick children. Furthermore, the lack of normalization of urinary GAG levels in treated patients suggests the need for continued therapy and continuous monitoring of its effectiveness, which will contribute to the complete regeneration of the ECM components of the connective tissue and thus protect the patient against possible disability.

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., (© 2023 Wiley Periodicals LLC.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: Musculocontractural Ehlers-Danlos Syndrome (mcEDS) is a rare connective tissue disorder caused by biallelic loss-of-function variants in CHST14 (mcEDS-CHST14) or DSE (mcEDS-DSE), both of which result in defective dermatan sulfate biosynthesis. Forty-one patients with mcEDS-CHST14 and three patients with mcEDS-DSE have been described in the literature., Methods: Clinical, molecular, and glycobiological findings in three additional patients with mcEDS-DSE were investigated., Results: Three patients from two families shared craniofacial characteristics (hypertelorism, blue sclera, midfacial hypoplasia), skeletal features (pectus and spinal deformities, characteristic finger shapes, progressive talipes deformities), skin features (fine or acrogeria-like palmar creases), and ocular refractive errors. Homozygous pathogenic variants in DSE were found: c.960T>A/p.Tyr320* in patient 1 and c.996dupT/p.Val333Cysfs*4 in patients 2 and 3. No dermatan sulfate was detected in the urine sample from patient 1, suggesting a complete depletion of DS., Conclusion: McEDS-DSE is a congenital multisystem disorder with progressive symptoms involving craniofacial, skeletal, cutaneous, and cardiovascular systems, similar to the symptoms of mcEDS-CHST14. However, the burden of symptoms seems lower in patients with mcEDS-DSE., (© 2020 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.)

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., (Copyright © 2019. Published by Elsevier Inc.)

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 (r 2 ) 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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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., (© 2018 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.)

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Purpose: Dermatan sulfate (DS) plays a number of roles in a wide range of biological activities such as cell signaling and tissue morphogenesis through interactions with various extracellular matrix proteins including collagen. Mutations in the carbohydrate sulfotransferase 14 gene (CHST14) encoding CHST14/dermatan 4-O-sulfotransferase-1 (D4ST1), which is responsible for the biosynthesis of DS, cause a recently delineated form of Ehlers-Danlos syndrome (EDS, musculocontractural type 1), an autosomal recessive connective tissue disorder characterized by congenital malformations (specific craniofacial features, and congenital multiple contractures) and progressive fragility-related complications (skin hyperextensibility, bruisability, and fragility with atrophic scars; recurrent dislocations; progressive talipes or spinal deformities; and large subcutaneous hematomas). In an attempt to develop a diagnostic screening method for this type of EDS, the amount of DS in the urine of patients was analyzed., Methods: Urinary DS was quantified by an anion-exchange chromatography after treatment with DS-specific degrading enzyme., Results: DS was not detected in the urine of patients with homo- or compound heterozygous mutations in CHST14. These results suggest that the quantification of DS in urine is applicable to an initial diagnosis of DS-defective EDS., Conclusions: This is the first study to perform a urinary disaccharide compositional analysis of chondroitin sulfate (CS)/DS chains in patients with EDS caused by a CHST14/D4ST1 deficiency, and demonstrated the absence of DS chains. This result suggests systemic DS depletion in this disorder, and also proposes the usefulness of a urinary disaccharide compositional analysis of CS/DS chains as a non-invasive screening method for this disorder., (Copyright © 2017 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

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.

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., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Mucopolysaccharidoses (MPS) are complex storage disorders that result in the accumulation of glycosaminoglycans (GAGs) in urine, blood, brain and other tissues. Symptomatic patients are typically screened for MPS by analysis of GAG in urine. Current screening methods used in clinical laboratories are based on colorimetric assays that lack the sensitivity and specificity to reliably detect mild GAG elevations that occur in some patients with MPS. We have developed a straightforward, reliable method to quantify chondroitin sulfate (CS), dermatan sulfate (DS) and heparan sulfate (HS) in urine by stable isotope dilution tandem mass spectrometry. The GAGs were methanolyzed to uronic acid-N-acetylhexosamine or iduronic acid-N-glucosamine dimers and mixed with stable isotope labeled internal standards derived from deuteriomethanolysis of GAG standards. Specific dimers derived from HS, DS and CS were separated by ultra-performance liquid chromatography and analyzed by electrospray ionization tandem mass spectrometry using selected reaction monitoring for each targeted GAG product and its corresponding internal standard. The method was robust with a mean inaccuracy from 1 to 15%, imprecision below 11%, and a lower limit of quantification of 0.4mg/L for CS, DS and HS. We demonstrate that the method has the required sensitivity and specificity to discriminate patients with MPS III, MPS IVA and MPS VI from those with MPS I or MPS II and can detect mildly elevated GAG species relative to age-specific reference intervals. This assay may also be used for the monitoring of patients following therapeutic intervention. Patients with MPS IVB are, however, not detectable by this method., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Mucopolysaccharidosis type VII (MPS VII, Sly syndrome) is a very rare lysosomal storage disease caused by a deficiency of the enzyme β-glucuronidase (GUS), which is required for the degradation of three glycosaminoglycans (GAGs): dermatan sulfate, heparan sulfate, and chondroitin sulfate. Progressive accumulation of these GAGs in lysosomes leads to increasing dysfunction in numerous tissues and organs. Enzyme replacement therapy (ERT) has been used successfully for other MPS disorders, but there is no approved treatment for MPS VII. Here we describe the first human treatment with recombinant human GUS (rhGUS), an investigational therapy for MPS VII, in a 12-year old boy with advanced stage MPS VII. Despite a tracheostomy, nocturnal continuous positive airway pressure, and oxygen therapy, significant pulmonary restriction and obstruction led to oxygen dependence and end-tidal carbon dioxide (ETCO2) levels in the 60-80mmHg range, eventually approaching respiratory failure (ETCO2 of 100mmHg) and the need for full-time ventilation. Since no additional medical measures could improve his function, we implemented experimental ERT by infusing rhGUS at 2mg/kg over 4h every 2 weeks for 24 weeks. Safety was evaluated by standard assessments and observance for any infusion associated reactions (IARs). Urinary GAG (uGAG) levels, pulmonary function, oxygen dependence, CO2 levels, cardiac valve function, liver and spleen size, and growth velocity were assessed to evaluate response to therapy. rhGUS infusions were well tolerated. No serious adverse events (SAEs) or IARs were observed. After initiation of rhGUS infusions, the patient's uGAG excretion decreased by more than 50%. Liver and spleen size were reduced within 2 weeks of the first infusion and reached normal size by 24 weeks. Pulmonary function appeared to improve during the course of treatment based on reduced changes in ETCO2 after off-ventilator challenges and a reduced oxygen requirement. The patient regained the ability to eat orally, gained weight, and his energy and activity levels increased. Over 24 weeks, treatment with every-other-week infusions of rhGUS was well tolerated with no SAEs, IARs, or hypersensitivity reactions and was associated with measurable improvement in objective clinical measures and quality of life., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Mucopolysaccharidoses are a group of lysosomal storage disorders caused by deficiency of enzymes catalyzing the degradation of glycosaminoglycans. Mucopoly-saccharidosis I can present a wide range of phenotypic characteristics with three major recognized clinical entities: Hurler and Scheie syndromes represent phenotypes at the severe and mild ends of the clinical spectrum, respectively, and the Hurler-Scheie syndrome is intermediate in phenotypic expression. These are caused by the deficiency or absence of alpha-L-iduronidase, essential to the metabolism of both dermatan and heparan sulfate, and it is encoded by the lDUA gene. We report the case of a 34-year-old male patient with enzymatic deficiency of alpha-L-iduronidase, accumulation of its substrate and a previously unreported mutation in the IDUA gene that developed a phenotype of Scheie syndrome.

Objectives: The influence of age and gender factor on the urinary excretion of total glycosaminoglycans (uGAGs) and their particular types: chondroitin/dermatan sulfates (CS/DSs), heparan sulfates (HSs) and hyaluronan (HA) was analyzed in healthy pediatric and adolescent population., Design and Methods: Urine samples were collected from 95 healthy children. Sulfated GAGs excreted in the urine were quantitated using standardized dye-binding method, while the concentrations of HA were determined by immunoassay., Results: Age-dependent decline in total uGAG excretion (r=-0.686; p<0.001), resulting from a decrease in particular GAG fractions i.e. CS/DS (r=-0.757; p<0.001), HS (r=-0.401; p<0.05) and HA (r=-0.638; p<0.001), was found in healthy subjects. The observed differences were not gender specific with the exception of HS, in which excretion declines with age in males (r=-0.501; p<0.05) and does not change in females. Changes in the distribution pattern of uGAG were also found. CS/DS were the predominant uGAG's fraction, representing from 55% to 76% of the total GAGs. Children up to 3 years excreted more GAGs than older subjects and with a higher proportion of CS/DS and less content of HS. Moreover, the relative contribution of HA was increased twofold in adolescents, aged 15-18, as compared to younger subjects. A negative correlation existed between uGAG excretion and body height, except for HS, for which this relationship was found only in males., Conclusions: Changes in urinary distribution pattern of particular GAG types during physiological human growth and development were found. Evaluation of urinary GAG screening procedures during pathological conditions should be based on the GAG/creatinine ratios with age and gender taken into account., (Copyright © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Proteus mirabilis cause urinary tract infections which are recurrent and can lead to formation of urinary calculi. Both bacterial and the host factors are involved in the development of urolithiasis. To determine the impact of glycosaminoglycans (GAGs) in the formation of P. mirabilis-induced urinary stones, we investigated the in vitro crystallization, aggregation and adhesion of crystals in the presence of GAGs naturally appearing in urine. Crystallization experiments were performed in synthetic urine infected with P. mirabilis in the presence of: hyaluronic acid (HA), heparan sulfate (HS), chondroitin sulfate A, B and C (ChSA, ChSB, ChSC). The intensity of crystallization and aggregation were established by counting particles and phase-contrast microscopy. To analyze the adhesion of crystals, we used normal urothelium and (45)Ca isotope-labeled crystals. In the presence of ChSC, both the size of the crystals formed and their number were higher compared with the control. GAGs increased crystals adhesion to the cells, but only for ChSA this effect was significant. Chondroitin sulfates, which accelerate the first stages of infection-induced stones formation, may play an important role in the pathogenesis of infectious urolithiasis., (© 2013 APMIS. Published by John Wiley & Sons Ltd.)

Introduction: Mucopolysaccharidosis type I (MPS I) results in a defective breakdown of the glycosaminoglycans (GAGs) heparan sulfate and dermatan sulfate, which leads to a progressive disease. Enzyme replacement therapy (ERT) results in clearance of these GAGs from a range of tissues and can significantly ameliorate several symptoms. The biochemical efficacy of ERT is generally assessed by the determination of the total urinary excretion of GAGs. However, this has limitations. We studied the concentrations of heparan sulfate and dermatan sulfate derived disaccharides (HS and DS, respectively) in the plasma and urine of seven patients and compared these levels with total urinary GAGs (uGAGs) levels., Methods: Plasma and urine samples were collected at different time points relative to the weekly ERT for three non-consecutive weeks in seven MPS I patients who had been treated with ERT for at least 2.5 years. Heparan and dermatan sulfate in plasma and urine were enzymatically digested into disaccharides, and HS and DS levels were determined by HPLC-MS/MS analysis. uGAGs were measured by the DMB test., Results: The levels of HS and DS were markedly decreased compared with the levels before the initiation of ERT. However, the concentrations of DS in plasma and of both HS and DS in urine remained significantly elevated in all studied patients, while in six patients the level of total uGAGs had normalized. The concentrations of plasma and urinary HS during the weekly ERT followed a U-shaped curve. However, the effect size is small. The concentrations of plasma and urinary DS and uGAGs appeared to be in a steady state., Conclusions: HS and DS are sensitive biomarkers for monitoring the biochemical treatment efficacy of ERT and remain elevated despite long-term treatment. This finding may be related to the labeled dose or antibody status of the patient. The timing of the sample collection is not relevant, at least at the current dose of 100 IU/kg/weekly.

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 unit 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 new GAG assay is useful for identifying patients with MPS types I, II, III, VI, and VII.

Mucopolysaccharidosis type II (MPS II, Hunter syndrome) is an X-linked lysosomal storage disease caused by a deficiency of iduronate-2-sulfatase (IDS). Two affected girls with moderate and severe forms of MPS II with normal karyotypes and increased urinary dermatan sulphate and heparin sulphate excretion and marked deficiencies of IDS activity are reported. Molecular studies showed that case 1 has a heterozygous mutation c.1568A > G (p.Y523C) associated with almost totally skewed inactivation of the normal maternal X chromosome, and case 2 has a heterozygous deletion that includes exons 1-4 of IDS (minimal deletion range c.1-103&#95;184del). The multi-exon deletion correlated with early onset of the disease and severe phenotype with intellectual disability, whereas the missense mutation was associated with moderate developmental delay. Although genotype-phenotype correlation in MPS II is difficult, gene deletions seem to correlate with more severe clinical manifestation of the disease. Enzyme replacement therapy (ERT) in these two females resulted in disease stabilization in both., (Copyright © 2012 Wiley Periodicals, Inc.)

Mucopolysaccharidoses (MPSs) are complex storage disorders caused by specific lysosomal enzyme deficiencies, resulting in the accumulation of glycosaminoglycans (GAGs) in urine, plasma, as well as in various tissues. We devised and validated a straightforward, but accurate and precise tandem mass spectrometry methodology coupled to high performance liquid chromatography (LC-MS/MS) for the quantification of GAGs in urine. The method is applicable to the investigation of patients with MPS I, II, and VI, by quantifying dermatan sulfate (DS) and heparan sulfate (HS) in urine. We analyzed urine samples from 28 MPS patients, aged 1 to 42 years, and 55 control subjects (41 days to 18 years old). Levels of DS and HS in urine from healthy controls of all ages were below the limit of quantification. The levels of DS and HS in urine from 6 treated patients with MPS I were lower than in 6 untreated patients in DS (0.7-45 vs 9.3-177 mg/mmol creat) and HS (0-123 mg/mmol creatinine vs 38-418 mg/mmol creatinine); similar results were obtained for 9 patients with MPS II and 7 patients with MPS VI. Analyses were performed on as little as 250 μL of urine. Methanolysis took 75 min per sample; the total analysis run time for each LC-MS/MS injection was 8 min. Results indicate that the method is applicable to a wide variety of situations in which high accuracy and precision are required, including the evaluation of the effectiveness of existing and emerging treatments., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Mucopolysaccharidosis I (MPS I) is an autosomal recessive disorder caused by deficiency of alpha-L-iduronidase leading to accumulation of its catabolic substrates, dermatan sulfate (DS) and heparan sulfate (HS), in lysosomes. This results in progressive multiorgan dysfunction and death in early childhood. The recent success of enzyme replacement therapy (ERT) for MPS I highlights the need for biomarkers that reflect response to such therapy. To determine which biochemical markers are better, we determined serum and urine DS and HS levels by liquid chromatography tandem mass spectrometry in ERT-treated MPS I patients. The group included one Hurler, 11 Hurler/Scheie, and two Scheie patients. Seven patients were treated from week 1, whereas the other seven were treated from week 26. Serum and urine DS (DeltaDi-4S/6S) and HS (DeltaDiHS-0S, DeltaDiHS-NS) were measured at baseline, week 26, and week 72. Serum DeltaDi-4S/6S, DeltaDiHS-0S, and DeltaDiHS-NS levels decreased by 72%, 56%, and 56%, respectively, from baseline at week 72. Urinary glycosaminoglycan level decreased by 61.2%, whereas urine DeltaDi-4S/6S, DeltaDiHS-0S, and DeltaDiHS-NS decreased by 66.8%, 71.8%, and 71%, respectively. Regardless of age and clinical severity, all patients showed marked decrease of DS and HS in blood and urine samples. We also evaluated serum DS and HS from dried blood-spot samples of three MPS I newborn patients, showing marked elevation of DS and HS levels compared with those in control newborns. In conclusion, blood and urine levels of DS and HS provide an intrinsic monitoring and screening tool for MPS I patients.

Glycosaminoglycans (GAGs) are accumulated in various organs in both mucopolysaccharidoses (MPS) and mucolipidoses II and III (ML II and III). MPS and ML II and III patients can not properly degrade dermatan sulfate (DS) and/or heparan sulfate (HS). HS storage occurs in the brain leading to neurological signs while DS storage involves mainly visceral and skeletal manifestations. Excessive DS and HS released into circulation and thus blood levels of both are elevated, therefore, DS and HS in blood could be critical biomarkers for MPS and ML. Such measurement can provide a potential early screening, assessment of the clinical course and efficacy of therapies. We here assay DS and HS levels in MPS and ML patients using liquid chromatography tandem mass spectrometry (LC/MS/MS). Plasma samples were digested by heparitinase and chondroitinase B to obtain disaccharides of DS and HS, followed by LC/MS/MS analysis. One hundred-twenty samples from patients and 112 control samples were analyzed. We found that all MPS I, II, III and VI patients had a significant elevation of all DS+HS compositions analyzed in plasma, compared with the controls (P<0.0001). Specificity and sensitivity was 100% if the cut off value is 800 ng/ml between control and these types of MPS group. All MPS I, II and III patients also had a significant elevation of plasma HS, compared with the controls (P<0.0001). All MPS VI patients had a significant elevation of plasma DS, compared with the controls (P<0.0001). These findings suggest measurement of DS and/or HS levels by LC/MS/MS is applicable to the screening for MPS I, II, III and VI patients., (Copyright (c) 2009. Published by Elsevier Inc.)

Background: The fact that mucopolysaccharidoses (MPSes) are now treatable, and that the earlier treatment is initiated the better, is an indication for neonatal screening. The most efficient approach seems likely to be a multi-tier procedure in which screening for urinary glycosaminoglycan (GAG) is followed by enzyme determinations in heelprick blood of newborns screening positive. Hitherto the method of choice for the determination of GAG has been the measurement of absorbance by a complex of GAG and 1,9-dimethylmethylene blue (DMB)., Method: We evaluated a DMB method in which absorbance by DMB is measured following its addition to the eluate obtained from paper-borne newborn urine samples and is normalized relative to urinary creatinine. Calibration is performed with chondroitin-6-sulfate (Ch-6-S)., Results: The limits of detection and quantification of GAG were 1.98 and 5.94 mg/dl, respectively. The within-run coefficients of variation (CVs) of the GAG/creatinine ratio for 25, 31, and 70 mg/dl solutions of Ch-6-S in urine were 21.8, 16.4, and 10.5%, respectively, and the corresponding between-run CVs were 25.0, 13.5, and 10.1%. Recovery from the urine spiked with 31 mg Ch-6-S/dl was 94.8%. Accuracy was also acceptable for all other GAGs except hyaluronic acid. For neonatal screening, the diagnostic threshold was tentatively established as 800 mg GAG/g creatinine, the 95th centile of samples from 903 infants aged 3-28 days, but the value of the GAG/creatinine ratio was negatively correlated with age. Application of the new method to samples from older individuals with and without MPS achieved 100% sensitivity and specificity when used with an age-dependent threshold taken from the literature on the original DMB method., Conclusion: If used in the first tier of a multi-tier screening protocol, the proposed method would allow the detection of abnormal levels of all GAGs except hyaluronic acid., ((c) 2010 Wiley-Liss, Inc.)

Introduction and Hypothesis: Urinary glycosaminoglycans (uGAG) have protective effects against urinary tract disorders. Here we investigated whether oral hormonal contraceptives (OC) affect uGAG excretion., Methods: Urine specimens were from young women regularly taking: ethinyl estradiol + drospirenone, n = 9; ethinyl estradiol + cyproterone acetate, n = 9; and ethinyl estradiol + gestodene, n = 7. Controls were from ten women not taking OC. Total uGAG was assayed as hexuronic acid/urinary creatinine. Sulfated uGAG species was determined by electrophoresis., Results: Unlike controls, total uGAG in the two halves of the menstrual cycle was similar in the OC groups. Whole cycle uGAG was higher in the OC groups (p < 0.01), especially for ethinyl estradiol + cyproterone acetate (p < 0.005). The three OC produced decreases of approximately 50% in heparan sulfate (p < 0.02) and dermatan sulfate (p < 0.02), and a approximately 100% increase in chondroitin sulfate (p < 0.004)., Conclusions: uGAG excretion is changed in women taking OC, and this might enhance the protective effects of these molecules against urinary tract disorders.

Multiple Sulfatase Deficiency (MSD) is a rare autosomal recessive disease in which the activities of all sulfatases are reduced; its estimated prevalence is 1:1.4 million births. The disease is caused by mutations in SUMF1, which encodes an enzyme involved in the post-translational modification of sulfatases. The MSD phenotype is a combination of the clinical features found in diseases resulting from a deficiency of the individual sulfatases; i.e., mucopolysaccharidosis II, IIIA, IIID, IVA and VI, metachromatic leukodystrophy, X-linked ichthyosis, and the X-linked recessive form of chondrodysplasia punctata. We describe herein the first case of a Brazilian patient with MSD. The case was initially diagnosed as having mucopolysaccharidosis (MPS), due to skeletal alterations, coarse facial features, and urinary excretion of dermatan sulfate and heparan sulfate. Later, after a detailed biochemical investigation, the diagnosis of MSD was established. The analysis of the SUMF1 showed the patient was a compound heterozygote for two novel mutations (p.R349G and p.F244S). This case illustrates the challenges in the diagnosis of a disease considered rare, such as MSD. We point out that the availability of therapy for certain MPS disorders necessitates correct disease assignment, and the need to exclude the likelihood of MSD.

We compared substrate reduction in patients with lysosomal storage disorder treated with hematopoietic stem cell transplant and found that it was significantly reduced compared with patients treated with pharmacological enzyme replacement therapy. These data might support the wider application of hematopoietic stem cell transplant in the treatment of lysosomal storage disorders.

Hurler Syndrome is corrected by allogeneic BMT by the action of donor enzyme on recipient tissue. In this paper, we describe monitoring of 39 patients transplanted in two centres to determine donor chimerism, enzyme level and residual substrate - expressed as dermatan sulphate to chondroitin sulphate ratio. We show that in fully engrafted recipients, the enzyme level, expressed as mumol/g total protein/h, post-transplant is 24.2 from an unrelated donor and 10.2 from a heterozygote family donor (P<0.0001). There is a tight relationship between mean post-transplant enzyme level and residual substrate - Spearman's rank correlation coefficient (Rho) was -0.76 and -0.80 at 12 and 24 months, respectively (P<0.0001). We propose that these differences affect patient outcome. As unrelated donor transplant outcomes improve and especially given the higher levels of donor cell engraftment following cord transplants, our data might influence donor selection where only heterozygote-matched family members are available.

Within cells, dermatan sulfate (DS) and heparan sulfate (HS) are degraded in two steps. The initial endohydrolysis of these polysaccharides is followed by the sequential action of lysosomal exoenzymes to reduce the resulting oligosaccharides to monosaccharides and inorganic sulfate. Mucopolysaccharidosis (MPS) type II is a lysosomal storage disorder caused by a deficiency of the exoenzyme iduronate-2-sulfatase (I2S). Consequently, partially degraded fragments of DS and HS have been shown to accumulate in the lysosomes of affected cells and are excreted in the urine. Di- to hexadecasaccharides, isolated from the urine of a MPS II patient using anion exchange and gel filtration chromatography, were identified using electrospray ionization-tandem mass spectrometry (ESI-MS/MS). These oligosaccharides were shown to have non-reducing terminal iduronate-2-sulfate residues by digestion with recombinant I2S. A pattern of growing oligosaccharide chains composed of alternating uronic acid and N-acetylhexosamine residues was identified and suggested to originate from DS. A series of oligosaccharides consisting of hexosamine/N-acetylhexosamine alternating with uronic acid residues was also identified and on the basis of the presence of unacetylated hexosamine; these oligosaccharides are proposed to derive from HS. The presence of both odd and even-length oligosaccharides suggests both endo-beta-glucuronidase and endo-N-acetylhexosaminidase activities toward both glycosaminoglycans. Furthermore, the putative HS oligosaccharide structures identified indicate that heparanase activities are directed toward regions of both low and high sulfation, while the N-acetylhexosaminidase activity acted only in regions of low sulfation in this polysaccharide.

The aim of the present study was to characterize the urinary excretion of glycosaminoglycans in kittens and adult healthy cats, as well as in cats with a low urinary tract disease, the feline urologic syndrome (FUS). The main urinary glycosaminoglycan in cats was found to be chondroitin sulfate, with smaller amounts of dermatan sulfate and heparan sulfate. There was no difference in the urinary glycosaminoglycan concentration with sex, but a marked decrease occurred with age, due to chondroitin sulfate. Trace amounts of keratan sulfate were also detected in the urine of kittens, but not of healthy adult cats. Dermatan sulfate and heparan sulfate were the only glycosaminoglycans found in the urinary tract and kidney, and chondroitin sulfate was the only glycosaminoglycan found in the plasma. These data suggest that the main urinary glycosaminoglycan chondroitin sulfate is of systemic origin and filtered in the kidney, while the minor components dermatan sulfate and heparan sulfate may come from the urinary tract. The urinary glycosaminoglycan concentration was greatly decreased in animals with FUS, as compared to normal adults. We hypothesize that these low glycosaminoglycan levels reflect a damage to the bladder surface, resulting in absorption and/or degradation of the endogenous urinary glycosaminoglycans.

Background: Substantial amounts of glycosaminoglycans (GAGs) are present in the urine of healthy individuals, but the concentration in the serum is very low. This finding suggests that urinary GAGs come from the glomerulus and may reflect the turnover of GAGs in the glomerulus., Hypothesis: However, little is known about the physiologic regulation of the urinary GAGs in humans, and so investigations are needed to evaluate the effects of age and sex on urinary GAGs in normal individuals., Methods: Eighty-seven healthy subjects were included in this study. Urinary GAGs were isolated and quantified at the nanogram level by combined azure A-silver staining in agarose gels., Results: The level of urinary GAGs peaked at 10-19 years in both sexes. The proportion of chondroitin sulfate decreased with age, but the proportion of heparan sulfate increased with age., Conclusion: The total amount of GAGs and the proportions of chondroitin sulfate, heparan sulfate, and dermatan sulfate appear to change with age. Therefore, investigations in which urinary GAG is used as a parameter of glomerular GAG turnover should ensure that control groups are precisely matched for age. Changes in the proportions of each GAG may be more informative than their absolute levels., (Copyright 2003 S. Karger AG, Basel)

Mucopolysaccharidosis (MPS) type I (alpha-iduronidase deficiency) is characterized by storage and massive urinary excretion of dermatan sulfate and heparan sulfate; it may be distinguished into three different subtypes based on age at onset and severity of the clinical symptoms. We report on progressive white matter involvement documented by serial MR imaging in a patient with the MPS type I, severe skeletal involvement and preserved mental capabilities (intermediate phenotype or Hurler/Scheie syndrome).The natural history of white matter abnormalities in patients with MPS is still unclear; based on the present study, it appears that degenerative changes of the white matter mimicking a leukodystrophy may mark the course of MPS type I. We also suggest that the degree of MR changes in patients with MPS does not always reflect their neurological impairment.

A new, simple, and rapid pretreatment method for the determination of chondroitin sulfate, dermatan sulfate, and hyaluronan from urine and blood plasma samples has been developed. Plasma proteins were first converted into small peptides by digestion using a nonspecific protease, actinase E, and the resulting small peptides were removed by centrifugal filtration. The retained, residual crude glycosaminoglycans, including chondroitin/dermatan sulfates and hyaluronan, were converted into unsaturated disaccharides through the action of chondroitin sulfate lyses. Next, these disaccharides were recovered and purified using centrifugal filtration together with DeltaDi-UA2S, added as an internal standard. The filtered disaccharide mixture was analyzed by HPLC with fluorometric postcolumn derivatization using 2-cyanoacetamide as a fluorogenic reagent. This method was applied to a pharmacokinetic study of chondroitin sulfate administered intravenously to mice. The half-life of the administered chondroitin sulfates, having molecular masses from 6 to 50 kDa, varied depending on their molecular sizes. This new method should be useful for studies on the metabolic fate of exogenously administered glycosaminoglycans in small experimental animals., ((C)2002 Elsevier Science (USA).)

Hurler disease resulting from a deficiency in alpha-L-iduronidase, which causes an accumulation of dermatan sulfate and heparan sulfate glycosaminoglycans, is characterized by connective tissue and skeletal deformations, cardiomyopathy, cardiac valve defects, and progressive coronary artery stenosis. In this report, we present evidence that accumulation of dermatan sulfate but not heparan sulfate moieties is linked to impaired elastic fiber assembly that, in turn, contributes substantially to the development of the clinical phenotype in Hurler disease. Our data suggest that dermatan sulfate-bearing moieties bind to and cause functional inactivation of the 67-kd elastin-binding protein, a molecular chaperone for tropoelastin, which normally facilitates its secretion and assembly into elastic fibers. We demonstrate that, in contrast to normal skin fibroblasts and cells from Sanfilippo disease, which accumulate heparan sulfate, Hurler fibroblasts show reduced expression of elastin-binding protein and do not assemble elastic fibers, despite an adequate synthesis of tropoelastin and sufficient production of a microfibrillar scaffold of elastic fibers. Because cultured Hurler fibroblasts proliferate more quickly than their normal counterparts and the addition of exogenous insoluble elastin reduces their proliferation, we suggest that cell contacts with insoluble elastin play an important role in controlling their proliferation.

Diabetes mellitus was induced in one group of rats by a single injection of streptozotocin. The glycemia, the body weight, and the blood systolic pressure were measured every week, and the 24 h urine volume and urinary excretions of creatinine, albumin and glycosaminoglycans were measured every 2 weeks. At the end of the experiment (12 weeks) the weight and the glycosaminoglycan composition of the kidneys were determined. All the diabetic animals were hyperglycemic, hypertense, and did not gain weight during all the experimental period. Albuminuria appeared from the second week on. Rat urine was shown to contain heparan sulfate, chondroitin sulfate, and dermatan sulfate, and the glycosaminoglycan excretion decreased in all diabetic animals. The onset of the change in glyco-samino-glycan excretion rate was a very early event, appearing in the second week after diabetes induction. The main glycosaminoglycan found in normal rat kidney was heparan sulfate and, in contrast to the urine, the total kidney glycosaminoglycans increased in diabetic kidney, due to chondroitin sulfate and dermatan sulfate accumulation. The heparan sulfate concentration (per tissue dry weight) did not change. Our results suggest that quantification of urinary glycosaminoglycans may be a useful tool for the early diagnosis of diabetic nephropathy.

Background and Purpose: The effect of glycosaminoglycans (GAGs) in urinary crystal inhibition has been shown in vitro, but their inhibitor role in vivo has not been precisely determined in stone-forming patients. The aim of this study was to compare the levels of total GAGs and their components in primary stone-forming patients and a healthy control group and to investigate the impact of shockwave lithotripsy (SWL)., Patients and Methods: Thirty-eight patients with primary kidney stones and 31 healthy controls were included in this prospective study. Total urinary GAG concentrations were determined by the dimethylene blue assay (DMB), and GAG fractions (chondroitin sulfate, heparan sulfate, and dermatan sulfate) were studied by cellulose acetate electrophoresis. Analysis was repeated after SWL in the stone patients., Results: Chondroitin sulfate was the major component secreted in the urine of the control subjects. Heparan sulfate was the major component in the urine of the stone patients with less chondroitin sulfate and dermatan sulfate (48%, 35%, 16.5%, respectively). Our study showed a significant increase in total urinary GAGs (4.75 v. 7.43 microg/mg of creatinine; P<0.0001) after SWL. Dermatan sulfate was the main component in this group (P<0.0001). The total urinary GAG concentrations remained high for at least 2 days after SWL., Conclusion: The elevation in total GAGs after SWL indicates the presence of tissue injury, which also renders dermatan sulfate the principal excreted component. Studies with longer follow-up periods are needed to determine whether these changes in the excretion of GAG components persist.

We present a case of Hunter syndrome diagnosed because of skin eruption. A 4-year-old Japanese boy presented with a 3-4-months history of papular lesions on the back and extremities. His growth and development were almost normal. His face was not of coarse appearance. He had multiple, whitish to skin-coloured, papules and nodules symmetrically distributed on the scapular regions and the extensor aspects of the upper arms and thighs. There was no family history of similar symptoms. Skin biopsy showed the deposition of a considerable amount of mucin in the dermis. Although physical examinations failed to detect any other signs of Hunter syndrome, X-rays showed the characteristic features of mucopolysaccharidosis: deformities of the vertebral bone, ribs, and pelvis. Mucopolysaccharide analysis of the urine revealed a marked increase in dermatan sulphate and heparan sulphate. The activity of iduronate sulphatase in the lymphocytes was deficient, which was diagnostic for Hunter syndrome. We emphasize that the skin eruption can be the earliest sign of Hunter syndrome, particularly in the mild form presenting with normal development and growth.

1. Orbital accumulation of hydrophilic, interstitial glycosaminoglycans (GAGs) and subsequent expansion of retrobulbar tissue lead to the clinical manifestation of exophthalmos in patients with Graves' eye disease. 2. A highly specific method to determine the concentration and biochemical composition of different GAGs was developed in order to obtain a sensitive test system for the activity of the disease. By means of this method, GAG excretion in 24 h urine collections of 56 patients and 21 controls was analysed by precipitation with cetylpyridinium chloride and potassium acetate in ethanol, followed by sequential enzymic hydrolysis with chondroitin AC lyase, chondroitin ABC lyase and hyaluronate lyase, with HPLC analysis of the resulting alpha, beta-unsaturated disaccharides by anion-exchange chromatography. 3. Concentrations of GAG, chondroitin sulphate A (CA), dermatan sulphate (DS) and hyaluronic acid (HA) were determined in patients and controls, with high recovery rates [72.2 +/- 5.3%, mean +/- SEM; detection limit, 4.2 micrograms/l (0.01 mumol/l)], revealing marked differences in urinary concentrations of total GAG and HA, as well as an elevation of CA in patients compared with controls. 4. Method sensitivity was 0.86 for patients with active Graves' eye disease, and 0.87 for patients with untreated ophthalmopathy, whereas specificity was 1.0 for patients with inactive disease. Patients with increased GAG concentration responded well to steroids and/or orbital irradiation (before therapy: GAG, 111.49 +/- 40.32; CA, 59.58 +/- 21.34; DS, 25.05 +/- 8.12; HA, 26.88 +/- 11.63 mg/24 h; during therapy: GAG, 54.22 +/- 10.94; CA, 20.52 +/- 4.58; DS, 17.65 +/- 3.46; HA, 16.05 +/- 3.69 mg/24 h), whereas GAG excretion increased markedly 2-3 months after stopping prednisone therapy in patients with still active eye disease (GAG, 109.9 +/- 10.51; CA, 63.8 +/- 7.34; DS, 24.1 +/- 5.07; HA, 22.0 +/- 6.28 mg/24 h). 5. This sensitive method determines the nature of renally excreted GAGs, reflecting the aberrant synthesis pattern of fibroblasts in patients with Graves' disease.

Five patients presenting Hunter's syndrome were biochemically studied. Quantification of urinary glycosaminoglycans (GAGs), electrophoretic characterization and correlation with enzymatic activity in leucocytes were carried out. In all cases, urinary GAGs/creatinine ratio was increased. Electrophoresis revealed the presence of heparan sulfate (HS) and dermatan sulfate (DS) in four cases (80%), but in the remaining patient, only DS was present. In all patients, deficient enzymatic activity was demonstrated. These results show evidences of biochemical differences in this syndrome.

Enzymatic and chemical analyses of the structures of heparan sulfates excreted in the urine by patients with Sanfilippo's and Hunter's syndromes revealed that their nonreducing ends differ from each other and reflect the enzyme deficiency of the syndromes. The heparan sulfates from the different syndromes were treated with heparitinase II, crude enzyme extracts from Flavobacterium heparinum, and nitrous acid degradation. The heparan sulfates from patients with Sanfilippo A (deficient in heparan N-sulfatase) and Sanfilippo B (deficient in alpha-N-acetylglucosaminidase) were degraded with heparitinase II producing, besides unsaturated disaccharides, substantial amounts of glucosamine N-sulfate and N-acetylglucosamine, respectively. The heparan sulfate from patients with Hunter's syndrome (deficient in iduronate sulfatase) were degraded by heparitinase II or crude enzyme extracts to several products, including two saturated disaccharides containing a sulfated uronic acid at their nonreducing ends. The heparan sulfate from patients with Sanfilippo's C syndrome (deficient in acetyl Co-A: alpha-glucosaminide acetyltransferase) produced, by action of heparitinase II, among other products, two sulfated trisaccharides containing glucosamine with a nonsubstituted amino group. In addition to providing a new tool for the differential diagnosis of the mucopolysaccharidoses, these results bring new insights into the specificity of the heparitinases from Flavobacterium heparinum.

The glycosaminoglycans of the tumor mass and from the urine of patients with a nephroblastoma of embryonic origin (Wilms' tumor) and hypernephroma were analyzed. The urine of patients with Wilms' tumors prior to treatment, and two patients with metastasis contained high levels of hyaluronic acid (2-5 mg/l of urine) when compared to patients after surgery or chemotherapy where the content of hyaluronic acid was less than 0.1 mg/l. Urine of patients with hypernephroma and normal individuals contained even smaller amounts of hyaluronic acid. Normal kidneys contain mainly dermatan sulfate and heparan sulfate, while the hypernephroma and Wilms' tumor contain substantial amounts of chondroitin sulfate. The amount of glycosaminoglycans isolated from Wilms' tumor and hypernephroma were 10 times and 3 times, respectively, greater than normal kidneys. The amounts of hyaluronic acid in Wilms' tumor varied from 56 to 73% whereas normal kidneys contained about 13%. Chondroitin sulfate was also increased in Wilms' tumor and hypernephroma. It corresponded to 11% and 42%, respectively, of the total glycosaminoglycans. These and other findings indicate that the glycosaminoglycans of Wilms' tumors resemble those present during embryonic development of normal tissues whereas those in hypernephroma are typical of other carcinomas of different origins.

Two thousand urine samples (from patients presenting with clinical features suggestive of a mucopolysaccharidosis, MPS) were analysed by a procedure that included a quantitative measurement of glycosaminoglycan (GAG) hexuronic acids (harmine reagent), a qualitative GAG analysis (cellulose acetate electrophoresis) and a study of urinary oligosaccharide patterns. One hundred and seventy MPS and 29 oligosaccharidosis-affected patients were found, but 23 MPS patients among the 170 would have been missed by use of a quantitative procedure only. Fourteen of these (mainly MPS IV A) were detected on the basis of abnormal electrophoresis and the 9 others on the basis of abnormal urinary oligosaccharide patterns (MPS IV B patients). Our results emphasize that normal quantitative GAG excretion alone cannot rule out a diagnosis of MPS; qualitative analysis is also required, as well as oligosaccharide screening.

Dermatan sulfate excreted in normal human urine was isolated and characterized by TLC and cellulose acetate strip electrophoresis after cetylpyridinium chloride precipitation and pronase digestion. In these separation methods, dermatan sulfate and chondroitin sulfate were extracted and then monitored by sensitive HPLC methods with post column fluorometric derivatization coupled with chondroitinase ABC, ACII and B digestion. From the results, we demonstrated that human urinary dermatan sulfate contains iduronic acid as its major uronic acid (80-90% of total uronic acid), and is composed mainly of repeated mono-sulfated disaccharide units [Di-4S (structure shown in Fig. 1), 89%] and small numbers of di-sulfated disaccharide units (Di-diSB, 7% and Di-diSE, 1%).

A 37-year-old man with coarse facies, stiff joints, corneal clouding, and normal intelligence sought medical attention. The diagnosis of a systemic mucopolysaccharidosis (MPS) type I-S (Scheie's syndrome) was confirmed by the presence of lysosomal alpha-L-iduronidase deficiency and excessive urinary dermatan and heparan sulfate excretion. The corneal button after perforating keratoplasty of the right eye demonstrated mucopolysaccharides consisting of numerous vacuoles containing fibrillogranular and partly membranebound material in epithelial cells, histiocytes, keratocytes, and extracellular matrix. Endothelial cells were distinctly free of storage material. The epithelial basement membrane showed frequent breaks, whereas Bowman's layer was only slightly attenuated. Irregular collagen fibrils and fibrous long-spacing collagen were noted near degenerating distended keratocytes. The Descemet's membrane was normal. The literature of six reported histopathological examinations of the cornea in Scheie's syndrome is reviewed. Detection of fibrous long-spacing collagen seems to be a typical abnormality of the cornea in MPS I-S.

A mildly affected Maroteaux-Lamy patient is described. Electrophoretic separation of acid mucopolysaccharides (MPS) in the urine showed an increased excretion of dermatan sulphate in spite of a normal total excretion of MPS.

A method for the determination of hyaluronic acid (HA), chondroitin sulphate (CS) and dermatan sulphate (DS) was developed. HA, CS and DS were converted to the corresponding unsaturated disaccharides by digestion with chondroitinase ABC and/or chondroitinase AC-II and determined by high-performance liquid chromatography with fluorimetric detection using 2-cyanoacetamide as a post-column derivatization reagent. The calibration graphs for the unsaturated disaccharides were linear over the range 2 ng - 2 micrograms for each unsaturated disaccharide. This method was applied to the analysis of normal human urine.

A sensitive and specific, monoclonal antibody-based immunoquantification assay has facilitated determination of the N-acetylgalactosamine-4-sulfatase (4-sulfatase) protein content in cultured fibroblasts from normal controls and mucopolysaccharidosis type VI (MPS VI) patients. The assay enabled the quantification of 4-sulfatase protein by using a panel of seven monoclonal antibodies and has shown that fibroblasts from 16 MPS VI patients contained less than or equal to 5% of the level determined for normal controls. Fibroblasts from the most severely affected patients contained the lowest levels of 4-sulfatase protein, usually with few epitopes detected, while fibroblasts from mildly affected patients had higher levels of 4-sulfatase protein, with all seven epitopes detected. The pattern of epitope expression is proposed to reflect the conformational changes in the 4-sulfatase protein that arise from different mutations in the 4-sulfatase gene. Immunoquantification in combination with a specific and highly sensitive 4-sulfated trisaccharide-based assay of enzyme activity in these MPS VI patient fibroblasts enabled the determination of residual 4-sulfatase catalytic efficiency (kcat/Km). The capacity of fibroblasts to degrade substrate (catalytic capacity) was calculated as the product of 4-sulfatase catalytic efficiency and the content of 4-sulfatase in fibroblasts. One patient, 2357, with no clinical signs of MPS VI but with reduced 4-sulfatase activity and protein (both 5% of normal) and dermatansulfaturia, had 5% of normal catalytic capacity. The other 15 MPS VI patient fibroblasts had 0%-1.4% of the catalytic capacity of fibroblasts from normal controls and were representative of the spectrum of MPS VI clinical phenotypes, from severe to mild.(ABSTRACT TRUNCATED AT 250 WORDS)

An abnormal urinary excretion of sulphated glycosaminoglycans in a patient with GM-2 gangliosidosis (Tay-Sachs disease) is described. Besides the accumulation of GM-2 ganglioside in liver and lack of hexosaminidase A, the patient shows an abnormal urinary excretion of an iduronic acid-rich low molecular weight heparan sulphate. Also, no dermatan sulphate could be detected in the urine, whereas this compound was the main sulphated glycosaminoglycan in the liver of the patient. Heparan sulphate was the main glycosaminoglycan of normal liver. The total amount of sulphated glycosaminoglycans in the urine and liver of the patient did not differ significantly from the amounts found in the liver and urine of normal subjects. Several plasma glycosidases have been assayed and the activities did not differ significantly from the values obtained for the plasma of normal subjects.