heparitin-sulfate and Mucopolysaccharidosis-II

Mucopolysaccharidosis type I (MPS I) is a rare monogenic disease in which glycosaminoglycans' abnormal metabolism leads to the storage of heparan sulfate and dermatan sulfate in various tissues. It causes its damage and impairment. Patients with the severe form of MPS I usually do not live up to the age of ten. Currently, the therapy is based on multidisciplinary care and enzyme replacement therapy or hematopoietic stem cell transplantation. Applying gene therapy might benefit the MPS I patients because it overcomes the typical limitations of standard treatments. Nanoparticles, including nanoemulsions, are used more and more in medicine to deliver a particular drug to the target cells. It allows for creating a specific, efficient therapy method in MPS I and other lysosomal storage disorders. This article briefly presents the basics of nanoemulsions and discusses the current state of knowledge about their usage in mucopolysaccharidosis type I.

Topics: Enzyme Replacement Therapy; Genetic Therapy; Glycosaminoglycans; Heparitin Sulfate; Humans; Mucopolysaccharidosis I; Mucopolysaccharidosis II

Mucopolysaccharidosis type II (MPS II; Hunter syndrome; OMIM 309900) is a rare, multisystemic, progressive lysosomal storage disease caused by deficient activity of the iduronate-2-sulfatase (I2S) enzyme. Accumulation of the glycosaminoglycans dermatan sulfate and heparan sulfate results in a broad range of disease manifestations that are highly variable in presentation and severity; notably, approximately two-thirds of individuals are affected by progressive central nervous system involvement. Historically, management of this disease was palliative; however, during the 1990s, I2S was purified to homogeneity for the first time, leading to cloning of the corresponding gene and offering a means of addressing the underlying cause of MPS II using enzyme replacement therapy (ERT). Recombinant I2S (idursulfase) was produced for ERT using a human cell line and was shown to be indistinguishable from endogenous I2S. Preclinical studies utilizing the intravenous route of administration provided valuable insights that informed the design of the subsequent clinical studies. The pivotal Phase II/III clinical trial of intravenous idursulfase (Elaprase

Topics: Blood-Brain Barrier; Dermatan Sulfate; Enzyme Replacement Therapy; Heparitin Sulfate; Humans; Iduronate Sulfatase; Mucopolysaccharidosis II

Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disorder characterized by an accumulation of glycosaminoglycans (GAGs), including heparan sulfate, in the body. Major manifestations involve the central nerve system (CNS), skeletal deformation, and visceral manifestations. About 30% of MPS II is linked with an attenuated type of disease subtype with visceral involvement. In contrast, 70% of MPS II is associated with a severe type of disease subtype with CNS manifestations that are caused by the human iduronate-2-sulfatase (IDS)-Pro86Leu (P86L) mutation, a common missense mutation in MPS II. In this study, we reported a novel Ids-P88L MPS II mouse model, an analogous mutation to human IDS-P86L. In this mouse model, a significant impairment of IDS enzyme activity in the blood with a short lifespan was observed. Consistently, the IDS enzyme activity of the body, as assessed in the liver, kidney, spleen, lung, and heart, was significantly impaired. Conversely, the level of GAG was elevated in the body. A putative biomarker with unestablished nature termed UA-HNAc(1S) (late retention time), one of two UA-HNAc(1S) species with late retention time on reversed-phase separation,is a recently reported MPS II-specific biomarker derived from heparan sulfate with uncharacterized mechanism. Thus, we asked whether this biomarker might be elevated in our mouse model. We found a significant accumulation of this biomarker in the liver, suggesting that hepatic formation could be predominant. Finally, to examine whether gene therapy could enhance IDS enzyme activity in this model, the efficacy of the nuclease-mediated genome correction system was tested. We found a marginal elevation of IDS enzyme activity in the treated group, raising the possibility that the effect of gene correction could be assessed in this mouse model. In conclusion, we established a novel Ids-P88L MPS II mouse model that consistently recapitulates the previously reported phenotype in several mouse models.

Topics: Animals; Biomarkers; Disease Models, Animal; Heparitin Sulfate; Humans; Iduronate Sulfatase; Iduronic Acid; Mice; Mucopolysaccharidosis II; Mutation

Mucopolysaccharidosis type II (MPS II) is a neurometabolic disorder, due to the deficit of the lysosomal hydrolase iduronate 2-sulfatase (IDS). This leads to a severe clinical condition caused by a multi-organ accumulation of the glycosaminoglycans (GAGs/GAG) heparan- and dermatan-sulfate, whose elevated levels can be detected in body fluids. Since 2006, enzyme replacement therapy (ERT) has been clinically applied, showing efficacy in some peripheral districts. In addition to clinical monitoring, GAG dosage has been commonly used to evaluate ERT efficacy. However, a strict long-term monitoring of GAG content and composition in body fluids has been rarely performed. Here, we report the characterization of plasma and urine GAGs in Ids knock-out (Ids-ko) compared to wild-type (WT) mice, and their changes along a 24-week follow-up, with and without ERT. The concentration of heparan-sulfate (HS), chondroitin-sulfate (CS), and dermatan-sulfate (DS), and of the non-sulfated hyaluronic acid (HA), together with their differentially sulfated species, was quantified by capillary electrophoresis with laser-induced fluorescence. In untreated Ids-ko mice, HS and CS + DS were noticeably increased at all time points, while during ERT follow-up, a substantial decrease was evidenced for HS and, to a minor extent, for CS + DS. Moreover, several structural parameters were altered in untreated ko mice and reduced after ERT, however without reaching physiological values. Among these, disaccharide B and HS 2s disaccharide showed to be the most interesting candidates as biomarkers for MPS II. GAG chemical signature here defined provides potential biomarkers useful for an early diagnosis of MPS II, a more accurate follow-up of ERT, and efficacy evaluations of newly proposed therapies. KEY MESSAGES : Plasmatic and urinary GAGs are useful markers for MPS II early diagnosis and prognosis. CE-LIF allows GAG structural analysis and the quantification of 17 different disaccharides. Most GAG species increase and many structural features are altered in MPS II mouse model. GAG alterations tend to restore to wild-type levels following ERT administration. CS+DS/HS ratio, % 2,4dis CS+DS, and % HS 2s are potential markers for MPS II pathology and ERT efficacy.

Topics: Animals; Biomarkers; Body Fluids; Dermatan Sulfate; Disaccharides; Disease Models, Animal; Enzyme Replacement Therapy; Glycosaminoglycans; Heparitin Sulfate; Mice; Mice, Knockout; Mucopolysaccharidosis II

Mucopolysaccharidosis II (MPS II), a lysosomal storage disease caused by mutations in iduronate-2-sulfatase (IDS), is characterized by a wide variety of somatic and neurologic symptoms. The currently approved intravenous enzyme replacement therapy with recombinant IDS (idursulfase) is ineffective for CNS manifestations due to its inability to cross the blood-brain barrier (BBB). Here, we demonstrate that the clearance of heparan sulfate (HS) deposited in the brain by a BBB-penetrable antibody-enzyme fusion protein prevents neurodegeneration and neurocognitive dysfunctions in MPS II mice. The fusion protein pabinafusp alfa was chronically administered intravenously to MPS II mice. The drug reduced HS and attenuated histopathological changes in the brain, as well as in peripheral tissues. The loss of spatial learning abilities was completely suppressed by pabinafusp alfa, but not by idursulfase, indicating an association between HS deposition in the brain, neurodegeneration, and CNS manifestations in these mice. Furthermore, HS concentrations in the brain and reduction thereof by pabinafusp alpha correlated with those in the cerebrospinal fluid (CSF). Thus, repeated intravenous administration of pabinafusp alfa to MPS II mice decreased HS deposition in the brain, leading to prevention of neurodegeneration and maintenance of neurocognitive function, which may be predicted from HS concentrations in CSF.

Topics: Administration, Intravenous; Animals; Antibodies; Blood-Brain Barrier; Brain; Disease Models, Animal; Glycoproteins; Heparitin Sulfate; Humans; Iduronate Sulfatase; Immunoglobulin G; Mice; Mucopolysaccharidosis II; Neurocognitive Disorders; Receptors, Transferrin; Recombinant Fusion Proteins; Recombinant Proteins; Spatial Learning

A lysosomal storage disease, identified as a mucopolysaccharidosis (MPS), was diagnosed in a free-living Kaka (Nestor meridionalis), an endemic New Zealand parrot, which exhibited weakness, incoordination, and seizures. Histopathology showed typical colloid-like cytoplasmic inclusions in Purkinje cells and many other neurons throughout the brain. Electron microscopy revealed that storage bodies contained a variety of linear, curved, or circular membranous profiles and electron-dense bodies. Because the bird came from a small isolated population of Kaka in the northern South Island, a genetic cause was deemed likely. Tandem mass spectrometry revealed increased levels of heparan sulfate-derived disaccharides in the brain and liver compared with tissues from controls. Enzymatic assays documented low levels of iduronate-2-sulfatase activity, which causes a lysosomal storage disorder called MPS type II or Hunter syndrome. A captive breeding program is currently in progress, and the possibility of detecting carriers of this disorder warrants further investigation.

Topics: Animals; Heparitin Sulfate; Mucopolysaccharidosis II; New Zealand; Parrots; Tandem Mass Spectrometry

Mucopolysaccharidosis type II is a lysosomal storage disorder caused by a deficiency of iduronate-2-sulfatase (IDS) and characterized by the accumulation of the primary storage substrate, glycosaminoglycans (GAGs). Understanding central nervous system (CNS) pathophysiology in neuronopathic MPS II (nMPS II) has been hindered by the lack of CNS biomarkers. Characterization of fluid biomarkers has been largely focused on evaluating GAGs in cerebrospinal fluid (CSF) and the periphery; however, GAG levels alone do not accurately reflect the broad cellular dysfunction in the brains of MPS II patients. We utilized a preclinical mouse model of MPS II, treated with a brain penetrant form of IDS (ETV:IDS) to establish the relationship between markers of primary storage and downstream pathway biomarkers in the brain and CSF. We extended the characterization of pathway and neurodegeneration biomarkers to nMPS II patient samples. In addition to the accumulation of CSF GAGs, nMPS II patients show elevated levels of lysosomal lipids, neurofilament light chain, and other biomarkers of neuronal damage and degeneration. Furthermore, we find that these biomarkers of downstream pathology are tightly correlated with heparan sulfate. Exploration of the responsiveness of not only CSF GAGs but also pathway and disease-relevant biomarkers during drug development will be crucial for monitoring disease progression, and the development of effective therapies for nMPS II.

Topics: Adolescent; Animals; Biomarkers; Brain; Child; Child, Preschool; Dermatan Sulfate; Enzyme Replacement Therapy; Female; Gangliosides; Glycosaminoglycans; Hematopoietic Stem Cell Transplantation; Heparitin Sulfate; Humans; Iduronate Sulfatase; Infant; Inflammation; Lipid Metabolism; Lysosomes; Male; Mass Spectrometry; Mice; Mice, Knockout; Mucopolysaccharidosis II; Neurofilament Proteins; Recombinant Proteins

We recently developed a blood-brain barrier (BBB)-penetrating enzyme transport vehicle (ETV) fused to the lysosomal enzyme iduronate 2-sulfatase (ETV:IDS) and demonstrated its ability to reduce glycosaminoglycan (GAG) accumulation in the brains of a mouse model of mucopolysaccharidosis (MPS) II. To accurately quantify GAGs, we developed a plate-based high-throughput enzymatic digestion assay coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to simultaneously measure heparan sulfate and dermatan sulfate derived disaccharides in tissue, cerebrospinal fluid (CSF) and individual cell populations isolated from mouse brain. The method offers ultra-high sensitivity enabling quantitation of specific GAG species in as low as 100,000 isolated neurons and a low volume of CSF. With an LOD at 3 ng/mL and LLOQs at 5-10 ng/mL, this method is at least five times more sensitive than previously reported approaches. Our analysis demonstrated that the accumulation of CSF and brain GAGs are in good correlation, supporting the potential use of CSF GAGs as a surrogate biomarker for brain GAGs. The bioanalytical method was qualified through the generation of standard curves in matrix for preclinical studies of CSF, demonstrating the feasibility of this assay for evaluating therapeutic effects of ETV:IDS in future studies and applications in a wide variety of MPS disorders.

Topics: Animals; Biomarkers; Blood-Brain Barrier; Brain; Chromatography, Liquid; Dermatan Sulfate; Disaccharides; Disease Models, Animal; Glycosaminoglycans; Heparitin Sulfate; Humans; Iduronate Sulfatase; Mice; Mucopolysaccharidosis II; Tandem Mass Spectrometry

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.

Topics: Adolescent; Adult; Biomarkers; Child; Child, Preschool; Dermatan Sulfate; Female; Glycosaminoglycans; Heparitin Sulfate; Humans; Keratan Sulfate; Male; Mucopolysaccharidoses; Mucopolysaccharidosis II; Mucopolysaccharidosis III; Mucopolysaccharidosis IV; Mucopolysaccharidosis VI; Tandem Mass Spectrometry; Young Adult

Mucopolysaccharidosis II (MPS II) is caused by a deficiency of iduronate-2-sulfatase that results in accumulation of glycosaminoglycans (GAG), including heparan sulfate (HS), which is considered to contribute to neuropathology. We examined the efficacy of intracerebroventricular (ICV) enzyme replacement therapy (ERT) of idursulfase-beta (IDS-β) and evaluated the usefulness of HS as a biomarker for neuropathology in MPS II mice. We first examined the efficacy of three different doses (3, 10, and 30 μg) of single ICV injections of IDS-β in MPS II mice. After the single-injection study, its long-term efficacy was elucidated with 30 μg of IDS-β ICV injections repeated every 4 weeks for 24 weeks. The efficacy was assessed by the HS content in the cerebrospinal fluid (CSF) and the brain of the animals along with histologic examinations and behavioral tests. In the single-injection study, the 30 μg of IDS-β ICV injection showed significant reductions of HS content in brain and CSF that were maintained for 28 days. Furthermore, HS content in CSF was significantly correlated with HS content in brain. In the long-term repeated-injection study, the HS content in the brain and CSF was also significantly reduced and correlated. The histologic examinations showed a reduction in lysosomal storage. A significant improvement in memory/learning function was observed in open-field and fear-conditioning tests. ICV ERT with 30 μg of IDS-β produced significant improvements in biochemical, histological, and functional parameters in MPS II mice. Furthermore, we demonstrate for the first time that the HS in the CSF had significant positive correlation with brain tissue HS and GAG levels, suggesting HS in CSF as a useful clinical biomarker for neuropathology.

Topics: Animals; Biomarkers; Blood-Brain Barrier; Disease Models, Animal; Enzyme Replacement Therapy; Heparitin Sulfate; Iduronate Sulfatase; Infusions, Intraventricular; Maze Learning; Mice; Mice, Inbred C57BL; Mice, Knockout; Mucopolysaccharidosis II

Mucopolysaccharidosis type II (MPS II or Hunter syndrome) is a lysosomal storage disorder caused by a deficiency of iduronate-2-sulfatase (IDS), an enzyme that catabolizes glycosaminoglycans (GAGs) including heparan sulfate (HS) and dermatan sulfate (DS). GAG accumulation leads to severe neurological and somatic impairments. At present, the most common treatment for MPS II is intravenous enzyme replacement therapy; however, the inability of recombinant IDS to cross the blood-brain barrier (BBB) restricts therapeutic efficacy for neurological manifestations. We recently developed a BBB-penetrating IDS fusion protein, JR-141, and demonstrated its ability to reduce GAG accumulation in the brain of human transferrin receptor knock-in and Ids knock-out mice (TFRC-KI/Ids-KO), an animal model of MPS II, following intravenous administration. Given the impossibility of measuring GAG accumulation in the brains of human patients with MPS II, we hypothesized that GAG content in the cerebrospinal fluid (CSF) might serve as an indicator of brain GAG burden. To test this hypothesis, we optimized a high-sensitivity method for quantifying HS and DS in low-volume samples by combining acidic methanolysis and liquid chromatography-tandem mass spectrometry (LC/MS/MS). We employed this method to quantify HS and DS in samples from TFRC-KI/Ids-KO mice and revealed that HS but not DS accumulated in the central nerve system (CNS). Moreover, concentrations of HS in CSF correlated with those in brain. Finally, intravenous treatment with JR-141 reduced levels of HS in the CSF and brain in TFRC-KI/Ids-KO mice. These results suggest that CSF HS content may be a useful biomarker for evaluating the brain GAG accumulation and the therapeutic efficacy of drugs in patients with MPS II.

Topics: Animals; Biomarkers; Blood-Brain Barrier; Brain; Chromatography, Liquid; Dermatan Sulfate; Disease Models, Animal; Heparitin Sulfate; Humans; Iduronate Sulfatase; Mice; Mice, Knockout; Mucopolysaccharidosis II; Nervous System Diseases; Receptors, Transferrin; Tandem Mass Spectrometry

Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disease caused by the deficient activity of iduronate 2-sulfatase (IDS), which is involved in the lysosomal catabolism of the glycosaminoglycans (GAGs) dermatan and heparan sulfate. Such a deficiency leads to the accumulation of undegraded GAGs in some organs. Although enzyme replacement therapy is available as a treatment of MPS II, there are some limitations, such as the requirement of weekly administration for whole life. To avoid such limitations, hematopoietic cell transplantation (HSCT) is a possible alternative. In fact, some report suggested positive effects of HSCT for MPS II. However, HSCT has also some limitations. Strong conditioning regimens can cause severe side effects. For overcome this obstacle, we studied the efficacy of ACK2, an antibody that blocks KIT, followed by low-dose irradiation as a preconditioning regimen for HSCT using a murine model of MPS II. This protocol achieves 58.7±4.92% donor chimerism at 16weeks after transplantation in the peripheral blood of recipient mice. GAG levels were significantly reduced in liver, spleen, heart and intestine. These results indicated that ACK2-based preconditioning might be one of the choices for MPS II patients who receive HSCT.

Topics: Animals; Antibodies, Anti-Idiotypic; Bone Marrow Transplantation; Dermatan Sulfate; Disease Models, Animal; Glycoproteins; Heparitin Sulfate; Humans; Lysosomes; Mice; Mice, Knockout; Mucopolysaccharidosis II; Proto-Oncogene Proteins c-kit

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.

Topics: Adolescent; Adult; Aged; Child; Child, Preschool; Chondroitin Sulfates; Chromatography, Liquid; Dermatan Sulfate; Glycosaminoglycans; Heparitin Sulfate; Humans; Infant; Middle Aged; Mucopolysaccharidoses; Mucopolysaccharidosis II; Mucopolysaccharidosis III; Mucopolysaccharidosis IV; Mucopolysaccharidosis VI; Radioisotope Dilution Technique; Reference Values; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry; Young Adult

Mucopolysaccharidosis type II (MPS II) is an X-linked lysosomal storage disorder caused by a deficiency of iduronate 2-sulfatase (IDS). Progressive, intralysosomal accumulation of the glycosaminoglycans (GAGs) dermatan and heparan sulfate in almost all tissues leads to multi-organ involvement in affected males but to virtual absence of symptoms in heterozygote female carriers due to preferential inactivation of the mutant allele. Diagnosis of MPS II in males is based on IDS analysis in leukocytes, fibroblasts, plasma, or dried blood spots (DBS), whereas IDS activities may be within the normal range in heterozygote females. The advent of fluorometric and mass spectrometry methods for enzyme analysis in DBS has simplified the diagnostic approach for MPS II males. Molecular analysis of the IDS gene confirms the diagnosis of MPS II in males and is the only diagnostic test to confirm carrier status in females. This unit provides detailed analytical protocols for measurement of IDS activity in DBS and plasma using a fluorometric assay.

Topics: Dermatan Sulfate; Dried Blood Spot Testing; Female; Fluorometry; Glycoproteins; Heparitin Sulfate; Humans; Lysosomes; Male; Mucopolysaccharidosis II

Mucopolysaccharidoses (MPSs) are a group of lysosomal storage disorders (LSDs) caused by a defect in the degradation of glycosaminoglycans (GAGs). The accumulation of GAGs in MPS patients results in extensive, severe and progressive disease. Disease modifying therapy is available for three of the MPSs and is being developed for the other types. Early initiation of treatment, before the onset of irreversible tissue damage, clearly provides a favorable disease outcome. However, early diagnosis is difficult due to the rarity of these disorders in combination with the wide variety of clinical symptoms. Newborn screening (NBS) is probably the optimal approach, and several screening techniques for different MPSs have been studied. Here we describe a relatively simple and sensitive method to measure levels of dermatan and heparan sulfate derived disaccharides in dried blood spots (DBS) with HPLC-MS/MS, and show that this reliably separates MPS I, II and MPS III newborns from controls and heterozygotes.. Newborn DBS of 11 MPS I, 1 MPS II, and 6 MPS III patients, with phenotypes ranging from severe to relatively attenuated, were collected and levels of dermatan and heparan sulfate derived disaccharides in these DBS were compared with levels in DBS of newborn MPS I and MPS III heterozygotes and controls.. The levels of dermatan and heparan sulfate derived disaccharides were clearly elevated in all newborn DBS of MPS I, II and III patients when compared to controls. In contrast, DBS of MPS I and III heterozygotes showed similar disaccharide levels when compared to control DBS.. Our study demonstrates that measurement of heparan and dermatan sulfate derived disaccharides in DBS may be suitable for NBS for MPS I, II and MPS III. We hypothesize that this same approach will also detect MPS VI, and VII patients, as heparan sulfate and/or dermatan sulfate is also the primary storage products in these disorders.

Topics: Biomarkers; Child; Child, Preschool; Dermatan Sulfate; Disaccharides; Heparitin Sulfate; Humans; Infant; Infant, Newborn; Mucopolysaccharidoses; Mucopolysaccharidosis I; Mucopolysaccharidosis II; Mucopolysaccharidosis III; Neonatal Screening; Reproducibility of Results; Tandem Mass Spectrometry

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.

Topics: Adolescent; Adult; Biomarkers; Case-Control Studies; Child; Child, Preschool; Chromatography, Liquid; Creatinine; Dermatan Sulfate; Enzyme Replacement Therapy; Female; Glycosaminoglycans; Heparitin Sulfate; Humans; Infant; Male; Mucopolysaccharidosis I; Mucopolysaccharidosis II; Mucopolysaccharidosis VI; Reference Values; Tandem Mass Spectrometry; Young Adult

The reaction of heparan sulfate (HS) and dermatan sulfate (DS) oligosaccharides with 1-phenyl-3-methyl-5-pyrazolone (PMP) yields hydrophobic derivatives that are amenable to separation by reversed-phase high-performance liquid chromatography (RP-HPLC) and analysis by electrospray ionization-tandem mass spectrometry (ESI-MS/MS). We describe here the development of an RP-HPLC-ESI-MS/MS assay for the measurement of di- to pentasaccharides derived from HS and DS in the urine of mucopolysaccharidosis (MPS) type II patients, as PMP derivatives. HPLC separation was performed on a 3-mum Alltima C18-LL column (50 x 2.1mm) using a gradient elution of up to 25% acetonitrile over 17 min, and an API-4000 mass spectrometer equipped with a turbo-ion-spray source was used in the negative ion multiple reaction monitoring mode for PMP-oligosaccharide determination. Using this method, we found that the derivatization kinetics of the oligosaccharides was influenced by the type of residue present at the reducing end (i.e., N-acetylglucosamine, N-acetylgalactosamine, or uronic acid). The elevation of each of the measured oligosaccharides in MPS II urine enabled complete discrimination of a cohort of MPS II patient urines from unaffected controls. This assay is rapid and reproducible and may be useful for the diagnosis of MPS II, and also for monitoring of disease progression and efficacy of therapy.

Topics: Adolescent; Adult; Child; Child, Preschool; Chromatography, High Pressure Liquid; Dermatan Sulfate; Heparitin Sulfate; Humans; Mucopolysaccharidosis II; Oligosaccharides; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry; Young Adult

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.

Topics: Carbohydrate Conformation; Carbohydrate Sequence; Chromatography, Liquid; Dermatan Sulfate; Female; Glycoproteins; Heparitin Sulfate; Humans; Male; Molecular Sequence Data; Mucopolysaccharidosis II; Oligosaccharides; Spectrometry, Mass, Electrospray Ionization; Uronic Acids

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.

Topics: Child, Preschool; Dermatan Sulfate; Facies; Heparitin Sulfate; Humans; Iduronate Sulfatase; Male; Mucopolysaccharidosis II; Pelvic Bones; Radiography; Ribs; Skin Diseases; Spine

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.

Topics: Child; Child, Preschool; Dermatan Sulfate; Electrophoresis, Cellulose Acetate; Genetic Carrier Screening; Genetic Testing; Glycosaminoglycans; Heparitin Sulfate; Humans; Iduronate Sulfatase; Leukocytes; Male; Mucopolysaccharidosis II; Phenotype; Sensitivity and Specificity

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.

Topics: Biomarkers; Carbohydrate Sequence; Chondroitin Sulfates; Dermatan Sulfate; Diagnosis, Differential; Electrophoresis, Agar Gel; Heparitin Sulfate; Humans; Molecular Sequence Data; Mucopolysaccharidosis II; Mucopolysaccharidosis III; Polysaccharide-Lyases; Reference Values; Substrate Specificity

This male infant was first brought to attention in the neonatal period because he presented clinical and radiological evidence of multiple bone deformities. He was readmitted at 21/2 months for hydrocephaly, hepatosplenomegaly and poor somatic and psychomotor development. In addition, coarse facies, corneal opacities and stiff joints were noticed. Bone X-ray anomalies and vacuolized lymphocytes supported the clinical presumption of lysosomal storage disorder. The diagnosis of multiple sulphatase deficiency rests on the presence of MPS and sulphatides in the urine, the finding of a mixed storage process in conjunctival biopsy and the demonstration of deficiencies in arylsulphatases A, B, C, iduronate sulphatase and heparan sulphatase in serum, leukocytes and cultured fibroblasts.

Topics: Abnormalities, Multiple; Arylsulfatases; Bone and Bones; Cerebroside-Sulfatase; Chondro-4-Sulfatase; Heparitin Sulfate; Humans; Infant; Male; Mucopolysaccharidosis II; Steryl-Sulfatase; Sulfatases

Glycosaminoglycan isolated from the urine of a patient with the Hunter syndrome was composed of heparan sulfate (59.9%), dermatan sulfate (30.6%) and chondroitin sulfate (9.5%), and was heterogeneous in molecular weight (1,500-10,000) and in sulfate content (0.35-2.05 moles/mole of hexosamine). About 60% of dermatan sulfate and 10% of heparan sulfate had molecular weight of 7,000 to 10,000, while about 10% of the former and 60% of the latter had those of 1,500 to 3,500. Sulfate contents of dermatan sulfate and heparan sulfate were inversely related to their molecular weights. Higher total- and N-sulfate contents were measured in smaller molecular-weight heparan sulfate, and higher acetyl content was in larger molecular-weight heparan sulfate. On the basis of the chemical properties of dermatan sulfate and heparan sulfate isolated in this experiment, their catabolic processes in the Hunter syndrome were discussed.

Topics: Amino Acids; Child, Preschool; Chondroitin; Dermatan Sulfate; Electrophoresis; Glycosaminoglycans; Heparitin Sulfate; Humans; Male; Molecular Weight; Mucopolysaccharidosis II

Some structural features of heparitin sulfate excreted by patients with Hunter syndrome are described. It is shown, with the aid of heparitinases and heparinase from Flavobacterium heparinum, that the Hunter heparitin sulfate is a very complex structure composed of nine different disaccharide units containing regions akin to normal heparitin sulfate and regions akin the heparin. Two-thirds of the iduronic acid residues of Hunter heparitin sulfate are devoid of sulfate, contrasting with heparin in which most of the iduronic acid residues are sulfated. The isolation and characterization of the non-reducing ends of heparin and of the heparitin sulfates is also described. Based on these results the specificity of the heparinase and heparitinases as well as the biosynthesis of iduronic acid-containing heparin-like compounds is discussed.

Topics: Chemical Phenomena; Chemistry; Disaccharides; Glycosaminoglycans; Heparin; Heparitin Sulfate; Humans; Iduronic Acid; Mucopolysaccharidosis II; Sulfates

Multidisciplinary studies were conducted on the brain and other tissues of patients who died with the antemortem diagnosis of mucopolysaccharidosis (MPS) of one of the following types; type V, Scheie disease (MPS-V); type I, Hurler disease (MPS-I): and type II, Hunter disease (MPS-II). The principal new finding in the brain of the patient with MPS-V is the presence of lesions in the periadventitial mesenchymal tissue of the white matter, similar to those of MPS-I, while the nerve cells in MPS-V are histologically normal, in contradistinction to MPS-I, in which the neuronal abnormality is severe. Electron microscopical studies of the brain in MPS-I demonstrated numerous complex membranous inclusions in the neurons, whereas the neurons in MPS-V contained only a small number of lipofuscin-like inclusions and typical lipofuscin granules. There was a threefold increase of glycosaminoglycans (GAG) in the brain of MPS-I, but only a slight increase in the MPS-V; GAG in the liver and spleen of all patients was noticeably increased. alpha-L-iduronidase activity was not detectable in the brain and liver of patients with MPS-I and MPS-V, thus suggesting a similar enzymatic defect.

Topics: Adult; Brain; Cerebral Cortex; Child; Dermatan Sulfate; Diagnosis, Differential; Female; Glycosaminoglycans; Heparitin Sulfate; Humans; Iduronidase; Liver; Male; Mucopolysaccharidoses; Mucopolysaccharidosis I; Mucopolysaccharidosis II

Glycosaminoglycans were isolated from the urines of 46 patients with mucopolysaccharidosis; 11 with Type I (Hurler), 8 with Type II (Hunter), 16 with Type III (Sanfilippo A and B), 9 with Type V (Scheie), one with Type VI (Marateaux-Lamy), and one unclassified. All 46 patients excreted in their urine excessive amounts of dermatan sulfate, heparan sulfate or both. In addition, patients of certain types excreted excessive amounts of chondroitin sulfates A and/or C. There is a trend in each type of the disease towards the same carbazole/orcinol ratio, glucosamine/galactosamine ratio and glycosaminoglycan composition. Molecular weight distribution of the urinary glycosaminoglycans by gel filtration from Sephadex G-200 is characteristic for each different type of mucopolysaccharidosis and is distinguished from normal controls and patients without mucopolysaccharidosis. Preparation of elution diagrams from Sephadex G-200 allows an estimation of the composition of the glycosamino-glycans. Practically all heparan sulfate and a sizable part of dermatan sulfate from the urinary glycosaminoglycans of all these patients have been highly degraded. In all the patients in which the specific enzyme defect was demonstrated, the assignment of the type of mucopolysaccharidosis, on the basis of the elution diagrams, was correct. Patients with mucopolysaccharidosis Type V displayed two conspicuously different types of elution patterns, suggesting heterogeneity. Indeed, only a portion of these patients showed alpha-L- iduronidase deficiency. Carriers had normal urinary glycosaminoglycan output and composition and exhibited normal elution diagrams.

Topics: Adolescent; Adult; Bone and Bones; Carbohydrate Metabolism, Inborn Errors; Child; Child, Preschool; Chondroitin; Dermatan Sulfate; Female; Glycosaminoglycans; Heparitin Sulfate; Humans; Hypertrichosis; Infant; Intellectual Disability; Joint Diseases; Male; Mucopolysaccharidoses; Mucopolysaccharidosis I; Mucopolysaccharidosis II; Retinitis Pigmentosa

Heparan sulfates were isolated from the urine of normal individuals and patients with genetic mucopolysaccharidoses after exhaustive digestion with chondroitinase ABC. Electrophoresis of these preparations on cellulose acetate membrane revealed one spot corresponding in mobility to reference heparan sulphate in barium acetate buffer, while electrophoresis in 0.1 M HCl resulted in two distinct spots for each case; one corresponded in migration rate to reference heparan sulfate, and the other was faster in mobility than reference heparan sulfate but slightly retarded when compared with reference heparin. On thin-layer gel filtration on Sephadex G-200 (superfine) heparan sulfate from normal urine was polydispersed in character and its molecular size was larger than those of other preparations. Heparan sulfates from Hunter's and Sanfilippo's urine were monodispersed and small in molecular size. The molecular size of heparan sulfate from Sanfilippo's urine was the smallest of all. Heparin sulfate from Hurler's urine appeared to be composed of two populations; one corresponded in molecular size to heparan sulfate from normal urine, and the other corresponded to that of Hunter's urine.

Topics: Adolescent; Child; Child, Preschool; Chromatography, Gel; Chromatography, Thin Layer; Electrophoresis, Cellulose Acetate; Galactosamine; Glucosamine; Glycosaminoglycans; Heparitin Sulfate; Humans; Male; Molecular Weight; Mucopolysaccharidoses; Mucopolysaccharidosis I; Mucopolysaccharidosis II; Mucopolysaccharidosis III