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1.
Mol Genet Metab ; 121(3): 259-270, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28532689

RESUMO

Gaucher disease is a lysosomal storage disorder caused by a deficiency in glucocerebrosidase activity that leads to accumulation of glucosylceramide and glucosylsphingosine. Membrane raft microdomains are discrete, highly organized microdomains with a unique lipid composition that provide the necessary environment for specific protein-lipid and protein-protein interactions to take place. In this study we purified detergent resistant membranes (DRM; membrane rafts) from the occipital cortex and spleen from sheep affected with acute neuronopathic Gaucher disease and wild-type controls. We observed significant increases in the concentrations of glucosylceramide, hexosylsphingosine, BMP and gangliosides and decreases in the percentage of cholesterol and phosphatidylcholine leading to an altered DRM composition. Altered sphingolipid/cholesterol homeostasis would dramatically disrupt DRM architecture making them less ordered and more fluid. In addition, significant changes in the length and degree of lipid saturation within the DRM microdomains in the Gaucher brain were also observed. As these DRM microdomains are involved in many cellular events, an imbalance or disruption of the cell membrane homeostasis may impair normal cell function. This disruption of membrane raft microdomains and imbalance within the environment of cellular membranes of neuronal cells may be a key factor in initiating a cascade process leading to neurodegeneration.


Assuntos
Doença de Gaucher/metabolismo , Lipídeos/química , Microdomínios da Membrana/química , Baço/química , Animais , Encéfalo/patologia , Química Encefálica , Colesterol/análise , Galactosiltransferases/análise , Gangliosídeos/análise , Glucosilceramidas/análise , Fosfatidilcolinas/análise , Ovinos , Baço/patologia
2.
J Neurochem ; 137(3): 409-22, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26762778

RESUMO

Repeated replacement of sulphamidase via cerebrospinal fluid injection is an effective treatment for pathological changes in the brain in mice and dogs with the lysosomal storage disorder, mucopolysaccharidosis type IIIA (MPS IIIA). Investigational trials of this approach are underway in children with this condition, however, infusions require attendance at a specialist medical facility. We sought to comprehensively evaluate the effectiveness of sustained-release (osmotic pump-delivered) enzyme replacement therapy in murine MPS IIIA as this method, if applied to humans, would require only subcutaneous administration of enzyme once the pump was installed. Six-week-old MPS IIIA and unaffected mice were implanted with subcutaneous mini-osmotic pumps connected to an infusion cannula directed at the right lateral ventricle. Either recombinant human sulphamidase or vehicle were infused over the course of 7 weeks, with pumps replaced part-way through the experimental period. We observed near-normalisation of primarily stored substrate (heparan sulphate) in both hemispheres of the MPS IIIA brain and cervical spinal cord, as determined using tandem mass spectrometry. Immunohistochemistry indicated a reduction in secondarily stored GM 3 ganglioside and neuroinflammatory markers. A bias towards the infusion side was seen in some, but not all outcomes. The recombinant enzyme appears stable under pump-like conditions for at least 1 month. Given that infusion pumps are in clinical use in other nervous system disorders, e.g. for treatment of spasticity or brain tumours, this treatment method warrants consideration for testing in large animal models of MPS IIIA and other lysosomal storage disorders that affect the brain. Clinical trials of repeated injection of replacement enzyme into CSF are underway in patients with the inherited neurodegenerative disorder mucopolysaccharidosis type IIIA. In this pre-clinical study, we examined an alternative approach - slow, continual infusion of enzyme using pumps. We observed significant reductions in substrate accumulation and other disease-based lesions in treated mouse brain. Thus, the strategy warrants consideration for testing in large animal models of MPS IIIA and also in other neurodegenerative lysosomal storage disorders.


Assuntos
Encéfalo/patologia , Terapia de Reposição de Enzimas/métodos , Hidrolases/uso terapêutico , Mucopolissacaridose III/tratamento farmacológico , Mucopolissacaridose III/patologia , Animais , Biomarcadores/metabolismo , Química Encefálica , Gliose/tratamento farmacológico , Gliose/patologia , Heparitina Sulfato/metabolismo , Humanos , Hidrolases/administração & dosagem , Bombas de Infusão Implantáveis , Ventrículos Laterais , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/uso terapêutico , Medula Espinal/metabolismo
3.
Neurobiol Dis ; 91: 143-54, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-26976737

RESUMO

Gaucher disease arises from mutations in the ß-glucocerebrosidase gene which encodes an enzyme required for the lysosomal catabolism of glucosylceramide. We have identified a naturally occurring mutation in the ß-glucocerebrosidase gene in sheep that leads to Gaucher disease with acute neurological symptoms. Here we have examined the clinical phenotype at birth and subsequently quantified lipids in Gaucher lamb brain, in order to characterise the disorder. Enzyme activity assessments showed that a reduction in ß-glucocerebrosidase activity to 1-5% of wild-type occurs consistently across newborn Gaucher lamb brain regions. We analyzed glucosylceramide, glucosylsphingosine, bis(monoacylglycero)phosphate and ganglioside profiles in brain, liver, and spleen, and observed 30- to 130-fold higher glucosylceramide, and 500- to 2000-fold higher glucosylsphingosine concentrations in Gaucher diseased lambs compared to wild-type. Significant increases of bis(monoacylglycero)phosphate and gangliosides [GM1, GM2, GM3] concentrations were also detected in the brain. As these glycosphingolipids are involved in many cellular events, an imbalance or disruption of the cell membrane lipid homeostasis would be expected to impair normal neuronal function. To our knowledge, this is the first detailed analysis of glycosphingolipids in various brain regions in a large animal model of neuronal disease, which permits the mechanistic investigation of lipid deregulation and their contribution to neurodegenerative process.


Assuntos
Doença de Gaucher/metabolismo , Glucosilceramidas/metabolismo , Glicoesfingolipídeos/metabolismo , Lisossomos/metabolismo , Doença Aguda , Animais , Encéfalo/metabolismo , Modelos Animais de Doenças , Neurônios/metabolismo , Fenótipo , Ovinos , Baço/metabolismo
4.
J Inherit Metab Dis ; 38(2): 341-50, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25421091

RESUMO

Intracerebrospinal fluid (CSF) infusion of replacement enzyme is under evaluation for amelioration of disease-related symptoms and biomarker changes in patients with the lysosomal storage disorder mucopolysaccharidosis type IIIA (MPS IIIA; www.clinicaltrials.gov ; NCT#01155778; #01299727). Determining the optimal dose/dose-frequency is important, given the invasive method for chronically supplying recombinant protein to the brain, the main site of symptom generation. To examine these variables, we utilised MPS IIIA Huntaway dogs, providing recombinant human sulphamidase (rhSGSH) to young pre-symptomatic dogs from an age when MPS IIIA dog brain exhibits significant accumulation of primary (heparan sulphate) and secondary (glycolipid) substrates. Enzyme was infused into CSF via the cisterna magna at one of two doses (3 mg or 15 mg/infusion), with the higher dose supplied at two different intervals; fortnightly or monthly. Euthanasia was carried out 24 h after the final injection. Dose- and frequency-dependent reductions in heparan sulphate were observed in CSF and deeper layers of cerebral cortex. When we examined the amount of immunostaining of the general endo/lysosomal marker, LIMP-2, or quantified activated microglia, the higher fortnightly dose resulted in superior outcomes in affected dogs. Secondary lesions such as accumulation of GM3 ganglioside and development of GAD-reactive axonal spheroids were treated to a similar degree by both rhSGSH doses and dose frequencies. Our findings indicate that the lower fortnightly dose is sub-optimal for ameliorating existing and preventing further development of disease-related pathology in young MPS IIIA dog brain; however, increasing the dose fivefold but halving the frequency of administration enabled near normalisation of disease-related biomarkers.


Assuntos
Encéfalo/efeitos dos fármacos , Terapia de Reposição de Enzimas , Hidrolases/administração & dosagem , Mucopolissacaridose III/tratamento farmacológico , Animais , Biomarcadores/metabolismo , Encéfalo/enzimologia , Encéfalo/patologia , Modelos Animais de Doenças , Cães , Esquema de Medicação , Cálculos da Dosagem de Medicamento , Glicolipídeos/metabolismo , Heparitina Sulfato/metabolismo , Infusões Intraventriculares , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Mucopolissacaridose III/enzimologia , Mucopolissacaridose III/genética , Mucopolissacaridose III/patologia , Proteínas Recombinantes/administração & dosagem , Fatores de Tempo
5.
Eur J Neurosci ; 29(6): 1197-214, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19302155

RESUMO

Mucopolysaccharidosis type IIIA is a neurodegenerative lysosomal storage disorder characterized by progressive loss of learned skills, sleep disturbance and behavioural problems. Absent or greatly reduced activity of sulphamidase, a lysosomal protein, results in intracellular accumulation of heparan sulphate. Subsequent neuroinflammation and neurodegeneration typify this and many other lysosomal storage disorders. We propose that intra-cerebrospinal fluid protein delivery represents a potential therapeutic avenue for treatment of this and other neurodegenerative conditions; however, technical restraints restrict examination of its use prior to adulthood in mice. We have used a naturally-occurring Mucopolysaccharidosis type IIIA mouse model to determine the effectiveness of combining intravenous protein replacement (1 mg/kg) from birth to 6 weeks of age with intra-cerebrospinal fluid sulphamidase delivery (100 microg, fortnightly from 6 weeks) on behaviour, the level of heparan sulphate-oligosaccharide storage and other neuropathology. Mice receiving combination treatment exhibited similar clinical improvement and reduction in heparan sulphate storage to those only receiving intra-cerebrospinal fluid enzyme. Reductions in micro- and astrogliosis and delayed development of ubiquitin-positive lesions were seen in both groups. A third group of intravenous-only treated mice did not exhibit clinical or neuropathological improvements. Intra-cerebrospinal fluid injection of sulphamidase effectively, but dose-dependently, treats neurological pathology in Mucopolysaccharidosis type IIIA, even when treatment begins in mice with established disease.


Assuntos
Hidrolases/administração & dosagem , Doenças por Armazenamento dos Lisossomos/tratamento farmacológico , Doenças por Armazenamento dos Lisossomos/fisiopatologia , Proteínas/administração & dosagem , Análise de Variância , Animais , Anticorpos/administração & dosagem , Peso Corporal/efeitos dos fármacos , Peso Corporal/genética , Encéfalo/enzimologia , Cromatografia Líquida de Alta Pressão/métodos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Vias de Administração de Medicamentos , Comportamento Exploratório/efeitos dos fármacos , Comportamento Exploratório/fisiologia , Heparitina Sulfato/administração & dosagem , Hidrolases/imunologia , Doenças por Armazenamento dos Lisossomos/genética , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucopolissacaridose III/genética , Mucopolissacaridose III/metabolismo , Necrose/tratamento farmacológico , Necrose/etiologia , Proteínas/imunologia , Espectrometria de Massas em Tandem/métodos , Fatores de Tempo
6.
Blood Cells Mol Dis ; 40(3): 420-7, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18054258

RESUMO

Enzyme replacement therapy has been in clinical practice for the non-neuronopathic form of Gaucher disease for 15 years. However, the wide phenotypic variability in this disorder poses challenges to clinicians to assess patient severity and disease progression in order to effectively manage patients. Once therapy is initiated, methods to monitor the complex biochemical changes associated with the disease, and the response of these changes to therapy, are required in order to tailor therapy regimens to individual patients. We have evaluated the suitability of plasma sphingolipids and phospholipids as biochemical markers of disease burden and the efficacy of therapy to reduce that burden. Over 60 lipid species were measured using electrospray ionization-tandem mass spectrometry in plasma from controls and Gaucher patients, pre- and post-therapy. Glucosylceramide, molecular species of phosphatidylglycerol and G(M3) ganglioside were elevated in Gaucher disease, whereas species of ceramide, dihexosylceramide and sphingomyelin were decreased. Multivariate analysis enabled us to calculate the combined response of these lipids to therapy in Gaucher patients and correlate them with patient severity. Plasma lipids are proposed to be useful biomarkers for Gaucher disease.


Assuntos
Doença de Gaucher/sangue , Doença de Gaucher/tratamento farmacológico , Hexosaminidases/sangue , Lipídeos/sangue , Biomarcadores/sangue , Doença de Gaucher/enzimologia , Humanos , Espectrometria de Massas por Ionização por Electrospray
7.
Mol Genet Metab ; 93(4): 437-43, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18162427

RESUMO

Gaucher disease (GD) is an inborn error of glycosphingolipid metabolism resulting from a deficiency of the lysosomal enzyme beta-glucosidase leading to the accumulation of glucosylceramide (GC) in lysosomes of affected cells. In order to determine the effect of GC accumulation on intracellular lipid content in fibroblasts from patients with GD, we measured individual species of ceramide, di- and trihexosylceramide, sphingomyelin, phosphatidylcholine, phosphatidylinositol and phosphatidylglycerol using electrospray ionisation-tandem mass spectrometry. The different subspecies of each lipid class correlated with each other and were summed to give total lipid concentrations. In addition to GC, we also noted secondary elevations in other lipids, especially in type 2 GD. Sub-cellular fractionation showed that GC was not confined to the lysosome but increased throughout the cell. The sequelae of extra-lysosomal accumulation may have implications in the pathogenic mechanisms of GD by interaction with biochemical and metabolic pathways located outside the lysosome. The elevation of ceramide in confluent type 2 GD fibroblasts redistributed from its primary site of accumulation in the lysosome to the endosomal region at four-weeks post-confluence. The accumulation of lipids in the endosome and lysosome suggests both impaired trafficking of lipids and reduced capacity of the lysosome to degrade lipids.


Assuntos
Fibroblastos/metabolismo , Doença de Gaucher/fisiopatologia , Glucosilceramidas/metabolismo , Lisossomos/metabolismo , Fracionamento Celular , Células Cultivadas , Fibroblastos/ultraestrutura , Doença de Gaucher/patologia , Humanos , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem
8.
JIMD Rep ; 29: 59-68, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26620043

RESUMO

Mucopolysaccharidosis IIIA (MPS IIIA) is a neurodegenerative lysosomal storage disorder characterised by progressive loss of learned skills, sleep disturbance and behavioural problems. Reduced activity of lysosomal sulfamidase results in accumulation of heparan sulfate and secondary storage of glycolipids in the brain. Intra-cisternal sulfamidase infusions reduce disease-related neuropathology; however, repeated injections may subject patients to the risk of infection and tissue damage so alternative approaches are required. We undertook a proof-of-principle study comparing the ability of slow/continual or repeat/bolus infusion to ameliorate neuropathology in MPS IIIA mouse brain. Six-week-old MPS IIIA mice were implanted with subcutaneously located mini-osmotic pumps filled with recombinant human sulfamidase (rhSGSH) or vehicle, connected to lateral ventricle-directed cannulae. Pumps were replaced at 8 weeks of age. Additional MPS IIIA mice received intra-cisternal bolus infusions of the same amount of rhSGSH (or vehicle), at 6 and 8 weeks of age. Unaffected mice received vehicle via each strategy. All mice were euthanised at 10 weeks of age and the brain was harvested to assess the effect of treatment on neuropathology. Mice receiving pump-delivered rhSGSH exhibited highly significant reductions in lysosomal storage markers (lysosomal integral membrane protein-2, GM3 ganglioside and filipin-positive lipids) and neuroinflammation (isolectin B4-positive microglia, glial fibrillary acidic protein-positive astroglia). MPS IIIA mice receiving rhSGSH via bolus infusion displayed reductions in these markers, but the effectiveness of the strategy was inferior to that seen with slow/pump-based delivery. Continual low-dose infusion may therefore be a more effective strategy for enzyme delivery in MPS IIIA.

9.
Exp Neurol ; 263: 79-90, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25246230

RESUMO

Injection of lysosomal enzyme into cisternal or ventricular cerebrospinal fluid (CSF) has been carried out in 11 lysosomal storage disorder models, with each study demonstrating reductions in primary substrate and secondary neuropathological changes, and several reports of improved neurological function. Whilst acute studies in mucopolysaccharidosis (MPS) type II mice revealed that intrathecally-delivered enzyme (into thoraco-lumbar CSF) accesses the brain, the impact of longer-term treatment of affected subjects via this route is unknown. This approach is presently being utilized to treat children with MPS types I, II and III. Our aim was to determine the efficacy of repeated intrathecal injection of recombinant human sulfamidase (rhSGSH) on pathological changes in the MPS IIIA dog brain. The outcomes were compared with those in dogs treated via intra-cisternal or ventricular routes. Control dogs received buffer or no treatment. Significant reductions in primary/secondary substrate levels in brain were observed in dogs treated via all routes, although the extent of the reduction differed regionally. Treatment via all CSF access points resulted in large reductions in microgliosis in superficial cerebral cortex, but only ventricular injection enabled amelioration in deep cerebral cortex. Formation of glutamic acid decarboxylase-positive axonal spheroids in deep cerebellar nuclei was prevented by treatment delivered via any route. Anti-rhSGSH antibodies in the sera of some dogs did not reduce therapeutic efficacy. Our data indicates the capacity of intra-spinal CSF-injected rhSGSH to circulate within CSF-filled spaces, penetrate into brain and mediate a significant reduction in substrate accumulation and secondary pathology in the MPS IIIA dog brain.


Assuntos
Hidrolases/administração & dosagem , Mucopolissacaridoses/patologia , Animais , Cromatografia Líquida , Modelos Animais de Doenças , Cães , Ensaio de Imunoadsorção Enzimática , Heparitina Sulfato/análise , Humanos , Imuno-Histoquímica , Injeções Espinhais , Espectrometria de Massas , Proteínas Recombinantes/administração & dosagem
10.
Gene ; 491(1): 53-7, 2012 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-21963444

RESUMO

Mucopolysaccharidosis type IIIA (MPS-IIIA) is a severe neurodegenerative lysosomal storage disorder caused by a deficiency of N-sulfoglucosamine sulfohydrolase (SGSH) activity with subsequent accumulation of partially-degraded heparan sulfate and other glycolipids. In this study, we have evaluated a gene therapy approach using a helper-dependent canine adenovirus vector that expresses human SGSH as a means of delivering sustained transgene expression to the brain. Initial testing in a mixed neural cell culture model demonstrated that the vector could significantly increase SGSH activity in transduced cells, resulting in near-normalization of heparan sulfate-derived fragments. While administration of vector by direct injection into the brain of adult MPS-IIIA mice enabled transgene expression for at least 8.5 months post-treatment, it was only in discrete areas of brain. Heparan sulfate storage was reduced in some regions following treatment, however there was no improvement in secondary neuropathological changes. These data demonstrate that helper-dependent canine adenovirus vectors are capable of neural transduction and mediate long-term transgene expression, but increased SGSH expression throughout the brain is likely to be required in order to effectively treat all aspects of the MPS-IIIA phenotype.


Assuntos
Adenovirus Caninos/genética , Hidrolases/genética , Mucopolissacaridose III/terapia , Animais , Encéfalo/metabolismo , Células Cultivadas , Técnicas de Transferência de Genes , Terapia Genética , Vetores Genéticos , Vírus Auxiliares/genética , Transgenes
11.
Exp Neurol ; 230(1): 123-30, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21515264

RESUMO

Mucopolysaccharidosis type IIIA (MPS IIIA) is a neurodegenerative lysosomal storage disorder that results from a deficiency of sulfamidase (N-sulfoglucosamine sulfohydrolase), with consequential accumulation of its substrate, partially degraded heparan sulfate. Conventional doses (e.g. 1mg/kg) of intravenously delivered recombinant human sulfamidase (rhSGSH) do not improve neuropathology in MPS IIIA mice due to an inability to traverse the blood-brain barrier; however high-dose treatment or administration of enzyme that has been chemically modified to remove mannose-6-phosphate glycans has been shown to reduce neuropathology in related animal models. We have combined these approaches to evaluate the ability of 1, 5, 10 or 20mg/kg of similarly chemically modified or unmodified rhSGSH to reduce neuropathology following repeated intravenous delivery to adult MPS IIIA mice. rhSGSH was detected in brain homogenates from mice treated with all doses of modified rhSGSH and those receiving the two higher doses of unmodified rhSGSH, albeit at significantly lower levels. Immunohistochemically, rhSGSH visualized in the brain was localized to the endothelium, meninges and choroid plexus, with no convincing punctate intra-neuronal staining seen. This presumably underlies the failure of the treatment to reduce the relative level of a heparan sulfate-derived oligosaccharide (GlcNS-UA), or secondarily stored substrates that accumulate in MPS IIIA brain cells. However, modification of rhSGSH significantly increased its effectiveness in degrading GlcNS-UA in non-CNS tissues, potentially as a result of its reduced plasma clearance. If this observation is generally applicable, chemical modification may permit the use of significantly lower doses of lysosomal enzymes in patients currently receiving intravenous enzyme replacement therapy.


Assuntos
Hidrolases/uso terapêutico , Animais , Encéfalo/patologia , Plexo Corióideo/patologia , Cromatografia Líquida de Alta Pressão/métodos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Esquema de Medicação , Endotélio/patologia , Ensaio de Imunoadsorção Enzimática , Humanos , Hidrolases/sangue , Hidrolases/química , Manosefosfatos/sangue , Meninges/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mucopolissacaridose III/sangue , Mucopolissacaridose III/tratamento farmacológico , Mucopolissacaridose III/patologia , Oligorribonucleotídeos/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Fatores de Tempo
12.
Exp Neurol ; 225(2): 445-54, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20673764

RESUMO

Mucopolysaccharidosis type IIIA (MPS IIIA) is a neurodegenerative metabolic disorder caused by mutations in the N-sulfoglucosamine sulfohydrolase gene with resultant accumulation of partially degraded heparan sulfate (HS). Whilst allogeneic bone marrow transplantation (BMT) is indicated for several lysosomal storage disorders featuring neurodegeneration, its use in MPS III is highly controversial. Published evidence suggests that BMT does not improve cognitive function in MPS III patients. Despite this, patients continue to be transplanted in some centers. We therefore sought to determine the clinical effectiveness of BMT in a murine model of MPS IIIA. Pre-symptomatic young adult mice pre-conditioned with total body irradiation generated complete and stable donor-type chimerism. Whilst HS-derived disaccharides were reduced by up to 27% in the brain parenchyma, this was insufficient to decrease secondary cholesterol and GM3 ganglioside storage or permit clinical improvement. These results suggest that BMT is ineffective in its unmodified form and should not be considered as a treatment for MPS IIIA children.


Assuntos
Encéfalo/cirurgia , Mucopolissacaridose III/terapia , Transplante de Células-Tronco , Análise de Variância , Animais , Encéfalo/fisiopatologia , Células Cultivadas , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Marcha , Masculino , Camundongos , Camundongos Transgênicos , Atividade Motora , Mucopolissacaridose III/fisiopatologia , Resultado do Tratamento
13.
Clin Chem ; 51(4): 688-94, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15695328

RESUMO

BACKGROUND: Fabry disease is an X-linked lysosomal storage disorder resulting from a deficiency of the lysosomal hydrolase, alpha-galactosidase, for which enzyme replacement therapy is now available. In this study, we aimed to identify Fabry heterozygotes not only for genetic counseling of families but because it is becoming increasingly obvious that many heterozygous (carrier) females are symptomatic and should be considered for treatment. METHODS: We measured 29 individual lipid species, including ceramide, glucosylceramide, lactosylceramide, and ceramide trihexoside, in urine samples from Fabry hemizygotes and heterozygotes and from control individuals by electrospray ionization tandem mass spectrometry. Individual analyte species and analyte ratios were analyzed for their ability to differentiate the control and patient groups. RESULTS: The Fabry hemizygotes had increased concentrations of the substrate for the deficient enzyme, ceramide trihexoside, as well as lactosylceramide and ceramide, along with decreased concentrations of both glucosylceramide and sphingomyelin. Ratios of these analytes improved differentiation between the control and Fabry groups, with the Fabry heterozygotes generally falling between the Fabry hemizygotes and the control group. CONCLUSIONS: These lipid profiles hold particular promise for the identification of Fabry individuals, may aid in the prediction of phenotype, and are potentially useful for the monitoring of therapy in patients receiving enzyme replacement.


Assuntos
Doença de Fabry/genética , Lipídeos/urina , Doença de Fabry/urina , Heterozigoto , Homozigoto , Humanos , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização por Electrospray
14.
Pediatr Res ; 56(5): 733-8, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15347771

RESUMO

Unprecedented demands are now placed on clinicians for early diagnosis as we enter into an era of advancing treatment opportunities for the mucopolysaccharidoses (MPS). Biochemical monitoring of any therapeutic avenue will also be prerequisite. To this end, we aimed to identify a range of urinary oligosaccharides that could be used to identify and characterize patients with MPS. We analyzed 94 urine samples from 68 patients with MPS and 26 control individuals for oligosaccharides derived from glycosaminoglycans using electrospray ionization-tandem mass spectrometry. The oligosaccharide profile for each patient group was compared with that of the control group. The Mann-Whitney U test was used to measure the difference between each patient group and the controls for each analyte. Urine samples from patients before and at successive times after bone marrow transplantation were also evaluated. A number of oligosaccharides were identified in the urine of each MPS subtype, and for each of these, specific oligosaccharide profiles were formulated. These profiles enabled the identification of all 68 patients and their subtypes with the exception of MPS IIIB and IIIC. Selected oligosaccharides were used to assess three individuals after a bone marrow transplant, and, in each case, a substantial reduction in the level of diagnostic oligosaccharides, posttransplantation, was observed. The identification and measurement of glycosaminoglycan-derived oligosaccharides in urine provides a sensitive and specific screen for the early identification of individuals with MPS. The resulting oligosaccharide profiles not only characterize subtype but also provide a disease-specific fingerprint for the biochemical monitoring of current and proposed therapies.


Assuntos
Mucopolissacaridoses/diagnóstico , Mucopolissacaridoses/urina , Oligossacarídeos/urina , Biomarcadores , Sequência de Carboidratos , Humanos , Dados de Sequência Molecular , Espectrometria de Massas por Ionização por Electrospray
15.
Clin Chem ; 48(1): 131-9, 2002 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11751548

RESUMO

BACKGROUND: The development of therapies for lysosomal storage disorders has created a need for biochemical markers to monitor the efficacy of therapy and methods to quantify these markers in biologic samples. In Pompe disease, the concentration of a tetrasaccharide, consisting of four glucose residues, is reputedly increased in urine and plasma, but faster and more sensitive methods are required for the analysis of this, and other oligosaccharides, from biologic fluids. METHODS: We optimized the derivatization of storage oligosaccharides with 1-phenyl-3-methyl-5-pyrazolone for the measurement, by electrospray ionization tandem mass spectrometry, of oligosaccharide concentrations in urine (n = 6), plasma (n = 11), and dried-blood spots (n = 17) from Pompe-affected individuals. Age-matched control samples of urine (n = 10), plasma (n = 28), and blood spots (n = 369) were also analyzed. RESULTS: The mean tetrasaccharide concentration was increased in urine from infantile-onset (0.69-12 mmol/mol of creatinine) and adult-onset (0.22-3.0 mmol/mol of creatinine) Pompe individuals compared with age-matched controls. In plasma samples, an increased tetrasaccharide concentration was observed in some infantile patients (up to 22 micromol/L) compared with age-matched controls (mean, 2.2 micromol/L). The method developed was sensitive enough to determine oligosaccharide concentrations in a single 3-mm blood spot, but no differences were observed between blood spots from control and Pompe-affected individuals. CONCLUSIONS: Measurements of oligosaccharide concentrations in urine by this new method have potential application for the diagnosis and monitoring of patients with Pompe disease. Plasma analysis may have limited application for infantile patients, but analysis of blood spots does not discriminate between controls and affected individuals.


Assuntos
Doença de Depósito de Glicogênio Tipo II/sangue , Doença de Depósito de Glicogênio Tipo II/urina , Oligossacarídeos/sangue , Oligossacarídeos/urina , Adolescente , Adulto , Antipirina/análogos & derivados , Criança , Pré-Escolar , Edaravone , Humanos , Indicadores e Reagentes , Lactente , Recém-Nascido , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização por Electrospray
16.
Pediatrics ; 114(4): 909-16, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15466084

RESUMO

OBJECTIVE: To evaluate the use of protein markers using immune-quantification assays and of metabolite markers using tandem mass spectrometry for the identification, at birth, of individuals who have a lysosomal storage disorder. METHODS: A retrospective analysis was conducted of Guthrie cards that were collected from newborns in Denmark during the period 1982-1997. Patients whose lysosomal storage disorder (LSD; 47 representing 12 disorders) was diagnosed in Denmark during the period 1982-1997 were selected, and their Guthrie cards were retrieved from storage. Control cards (227) were retrieved from the same period. Additional control cards (273) were collected from the South Australian Screening Centre (Australia). RESULTS: From 2 protein and 94 metabolite markers, 15 were selected and evaluated for their use in the identification of LSDs. Glycosphingolipid and oligosaccharide markers showed 100% sensitivity and specificity for the identification of Fabry disease, alpha-mannosidosis, mucopolysaccharidosis (MPS) IVA, MPS IIIA, Tay-Sachs disease, and I-cell disease. Lower sensitivities were observed for Gaucher disease and sialidosis. No useful markers were identified for Krabbe disease, MPS II, Pompe disease, and Sandhoff disease. The protein markers LAMP-1 and saposin C were not able to differentiate individuals who had an LSD from the control population. CONCLUSIONS: Newborn screening for selected LSDs is possible with current technology. However, additional development is required to provide a broad coverage of disorders in a single, viable program.


Assuntos
Biomarcadores/sangue , Doenças por Armazenamento dos Lisossomos/diagnóstico , Triagem Neonatal/métodos , Anticorpos Monoclonais , Antígenos CD/sangue , Antígenos CD/imunologia , Glicoesfingolipídeos/sangue , Humanos , Recém-Nascido , Proteínas de Membrana Lisossomal , Espectrometria de Massas/métodos , Oligossacarídeos/sangue , Estudos Retrospectivos , Saposinas/sangue , Saposinas/imunologia , Sensibilidade e Especificidade
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