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1.
Clin Biochem ; 50(7-8): 431-435, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28088454

RESUMO

BACKGROUND: The goal of this study was to assess the biochemical parameters of the enzymes α-l-iduronidase (IDUA) and arylsulfatase B (ASB), which are deficient in mucopolysaccharidosis (MPS) I and VI, respectively, in dried blood spot (DBS) samples impregnated on filter paper. METHODS AND RESULTS: The optimal pH, Km, and Vmax of IDUA and ASB in DBS are hereby presented. After these analyses, the reference values for the activities of these enzymes in DBS with cutoff of 3.65nmol/h/mL for IDUA and 6.80nmol/h/mL for ASB were established. The research also showed that the stability (21days) of the IDUA activity is lower than ASB, which maintained its enzymatic activity stable up until 60days of analysis, after impregnating the filter paper with blood. CONCLUSION: Currently, DBS ensures important advantages in handling storage and transportation of samples with respect to neonatal screening programs. This study contributes to characterizing and differentiating the biochemistry of deficient enzymes in MPSs I and VI of DBS samples.


Assuntos
Teste em Amostras de Sangue Seco/métodos , Iduronidase/sangue , Mucopolissacaridose IV/sangue , Mucopolissacaridose I/sangue , N-Acetilgalactosamina-4-Sulfatase/sangue , Teste em Amostras de Sangue Seco/instrumentação , Feminino , Humanos , Masculino
2.
Braz. j. pharm. sci ; 52(4): 693-697, Oct.-Dec. 2016. tab, graf
Artigo em Inglês | LILACS | ID: biblio-951881

RESUMO

ABSTRACT Mucopolysaccharidoses (MPS) are a group of inherited metabolic disorders caused by deficiency of enzymes that degrade glycosaminoglycans (GAGs). Urinary excretion of GAGs is a common feature of MPS, and is considered their major biomarker. We aimed to adapt the GAG electrophoresis method to a commercial agarose gel which would be able to separate urinary GAGs in a simpler way with good sensitivity and reproducibility. Urine samples from patients previously diagnosed with MPS I, IV, and VI were used as electrophoretic standards. Samples from patients on enzyme replacement therapy (ERT) were also assessed. Commercial agarose gel electrophoresis was effective, showing proper definition and separation of GAG bands. Detection sensitivity exceeded 0.1 µg and band reproducibility were consistent. GAG bands quantified in urine samples from patients on ERT correlated very strongly (correlation coefficient = 0.98) with total GAG concentrations. This application of gel electrophoresis demonstrates the possibility of monitoring patients with MPS treated with ERT by analyzing separately the GAGs excreted in urine. We suggest this process should be applied to MPS screening as well as to follow-up of patients on treatment.


Assuntos
Humanos , Masculino , Feminino , Pré-Escolar , Mucopolissacaridoses/diagnóstico , Eletroforese em Gel de Ágar , Glicosaminoglicanos/uso terapêutico , Urina , Eletroforese/métodos
3.
Clin Chim Acta ; 445: 60-4, 2015 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-25804996

RESUMO

BACKGROUND: Due to the importance and the difficulty still present in determining the biochemical diagnosis of Fabry disease (FD), the aim of this study was to establish and compare the biochemical and kinetic properties of alpha-galactosidase A (GLA) in dried blood spots (DBS), plasma and leukocyte samples of FD patients and healthy subjects to evaluate the possible use of these parameters as an auxiliary tool in the diagnosis of this disease. METHODS: GLA activity in DBS, plasma and leukocyte samples from Fabry disease patients and healthy subjects was compared and characterized in terms of optimal pH, Km and Vmax and heat stability. RESULTS: A difference was observed between the Km and Vmax of FD patients and healthy controls using DBS, plasma and leukocyte samples. In leukocytes, pre-incubation at 50°C for 60 min was effective to differentiate FD patients from healthy controls. CONCLUSION: These results can be used as an auxiliary method to the FD diagnosis, especially in cases of patients whose GLA activity is within normal range.


Assuntos
Doença de Fabry/sangue , Doença de Fabry/diagnóstico , Leucócitos Mononucleares/enzimologia , alfa-Galactosidase/metabolismo , Estudos de Casos e Controles , Teste em Amostras de Sangue Seco , Estabilidade Enzimática , Doença de Fabry/patologia , Feminino , Temperatura Alta , Humanos , Cinética , Leucócitos Mononucleares/patologia , Masculino
4.
Clin Chim Acta ; 433: 49-53, 2014 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-24613516

RESUMO

BACKGROUND: Mucopolysaccharidoses (MPSs) are a group of lysosomal storage diseases caused by the deficiency/absence of enzymes which catalyze the degradation of glycosaminoglycans (GAGs). The use of biological samples dried on filter paper has been increasing because it makes it easy to ship them to reference laboratories. Urinary GAGs are the main biomarkers of MPS and, thus, we studied the correlations of determinations to GAGs and creatinine, as well as compared the GAGs' profile on electrophoresis, between urine and dried urine in filter paper (DUFP) samples. We also assessed the GAG stability over time under different storage temperatures. METHODS: We quantified the GAG concentration in both sample types and compared the results by Pearson correlation. RESULTS: The results were very similar, with r=0.97 for creatinine and with r=0.94 and r=0.98 for GAGs for controls and patients, respectively, with similar electrophoretic profiles. The GAG stability in DUFP was up to 30days at -20, 4, and 25°C and up to 21days at 37°C. CONCLUSION: Our proposal assessed urinary GAGs in DUFP and concluded that these samples can be used in the investigation of MPS, replacing urine samples in neonatal screening and monitoring of therapies, due to ease of transportation and storage.


Assuntos
Filtração , Glicosaminoglicanos/urina , Papel , Manejo de Espécimes/métodos , Temperatura , Creatinina/urina , Humanos , Fatores de Tempo
5.
Neurol Res ; 35(1): 59-64, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23317800

RESUMO

OBJECTIVE: The present study was undertaken to evaluate whether resveratrol (RSV) modulates membrane lipid composition, as well as on ganglioside profile in ischemia/reperfusion injury. METHODS: Global cerebral ischemia was induced by four-vessel occlusion for 10 minutes. RSV (30 mg/kg) or vehicle was intraperitoneally administered to rats 7 days prior to ischemia. Brain structures were homogenized with chloroform/methanol for ganglioside, phospholipids, and cholesterol levels. RESULTS: RSV significantly prevented the reduction in the total content of gangliosides, phospholipids, and cholesterol in hippocampi and cerebral cortex induced by global cerebral ischemia. Although ischemia/reperfusion decreased ganglioside content, the ganglioside profiles were apparently not modified. CONCLUSIONS: Our experiments suggest that lipid metabolism is important for development of ischemic damage and indicate that RSV treatment 7 days prior to ischemia may prevent membrane lipid loss.


Assuntos
Antioxidantes/uso terapêutico , Isquemia Encefálica/complicações , Isquemia Encefálica/tratamento farmacológico , Transtornos do Metabolismo dos Lipídeos/etiologia , Transtornos do Metabolismo dos Lipídeos/prevenção & controle , Estilbenos/uso terapêutico , Animais , Colesterol/metabolismo , Cromatografia em Camada Fina , Modelos Animais de Doenças , Gangliosidoses/metabolismo , Masculino , Fosfolipídeos/metabolismo , Ratos , Ratos Wistar , Resveratrol
6.
Gene ; 508(2): 197-8, 2012 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-22884741

RESUMO

This study investigates the miniaturization of the screening technique using dried blood spots on filter paper (DBS) to measure GBA and CT activities, and GBA and ß-galactosidase activities in leukocytes. 274 DBS from individuals with suspected GD were screened for 1.5 years. Of these, we confirmed the diagnosis in 13.5%. The miniaturization of the DBS and leukocyte techniques afforded to reduce costs and sample size appropriate for a reliable diagnosis.


Assuntos
Bioensaio , Doença de Gaucher/sangue , Doença de Gaucher/diagnóstico , Hexosaminidases/sangue , Leucócitos/metabolismo , Programas de Rastreamento , beta-Galactosidase/sangue , Coleta de Amostras Sanguíneas , Brasil , Estudos de Casos e Controles , Humanos
7.
Neurochem Int ; 59(5): 648-55, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21723896

RESUMO

The effect of Aß25-35 peptide, in its fibrillar and non-fibrillar forms, on ganglioside expression in organotypic hippocampal slice cultures was investigated. Gangliosides were endogenously labeled with D-[1-C(14)] galactose and results showed that Aß25-35 affected ganglioside expression, depending on the peptide aggregation state, that is, fibrillar Aß25-35 caused an increase in GM3 labeling and a reduction in GD1b labeling, whereas the non-fibrillar form was able to enhance GM1 expression. Interestingly, GM1 exhibited a neuroprotective effect in this organotypic model, since pre-treatment of the hippocampal slices with GM1 10 µM was able to prevent the toxicity triggered by the fibrillar Aß25-35, when measured by propidium iodide uptake protocol. With the purpose of further investigating a possible mechanism of action, we analyzed the effect of GM1 treatment (1, 6, 12 and 24h) upon the Aß-induced alterations on GSK3ß dephosphorylation/activation state. Results demonstrated an important effect after 24-h incubation, with GM1 preventing the Aß-induced dephosphorylation (activation) of GSK3ß, a signaling pathway involved in apoptosis triggering and neuronal death in models of Alzheimer's disease. Taken together, present results provide a new and important support for ganglioside participation in development of Alzheimer's disease experimental models and suggest a protective role for GM1 in Aß-induced toxicity. This may be useful for designing new therapeutic strategies for Alzheimer's treatment.


Assuntos
Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/toxicidade , Gangliosídeo G(M1)/farmacologia , Gangliosídeos/biossíntese , Fármacos Neuroprotetores , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/toxicidade , Animais , Western Blotting , Morte Celular/efeitos dos fármacos , Cromatografia em Camada Fina , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Masculino , Neurofibrilas/efeitos dos fármacos , Neurofibrilas/metabolismo , Técnicas de Cultura de Órgãos , Fosforilação , Ratos , Ratos Wistar
8.
Braz. j. pharm. sci ; 46(4): 643-649, Oct.-Dec. 2010. ilus, graf, tab
Artigo em Inglês | LILACS | ID: lil-622863

RESUMO

Gaucher disease is a sphingolipidosis that leads to an accumulation of glucosylceramide. The objective of this study was to develop a methodology, based on the extraction, purification and quantification of glucosylceramide from blood plasma, for use in clinical research laboratories. Comparison of the glucosylceramide content in plasma from Gaucher disease patients, submitted to enzyme replacement therapy or otherwise, against that from normal individuals was also carried out. The glucosylceramide, separated from other glycosphingolipids by high performance thin layer chromatography (HPTLC) was chemically developed (CuSO4 / H3PO4) and the respective band confirmed by immunostaining (human anti-glucosylceramide antibody / peroxidase-conjugated secondary antibody). Chromatogram quantification by densitometry demonstrated that the glucosylceramide content in Gaucher disease patients was seventeen times higher than that in normal individuals, and seven times higher than that in patients on enzyme replacement therapy. The results obtained indicate that the methodology established can be used in complementary diagnosis and for treatment monitoring of Gaucher disease patients.


A doença de Gaucher é uma esfingolipidose caracterizada pelo acúmulo de glicosilceramida. O objetivo deste estudo foi desenvolver metodologia baseada na extração, purificação e quantificação da glicosilceramida plasmática a qual possa ser usada em laboratórios de pesquisa clínica. Após o desenvolvimento desta metodologia, foi proposto, também, comparar o conteúdo de glicosilceramida presente no plasma de pacientes com doença de Gaucher, submetidos ou não a tratamento, com aquele de indivíduos normais. A glicosilceramida, separada de outros glicoesfingolipídios por cromatografia de camada delgada de alto desempenho (HPTLC), foi revelada quimicamente (CuSO4/H3PO4) e a respectiva banda foi confirmada por imunorrevelação (anticorpo anti-glicosilceramida humana/anticorpo secundário conjudado à peroxidase). A quantificação do cromatograma por densitometria demonstrou que o conteúdo de glicosilceramida nos pacientes com doença de Gaucher era 17 vezes maior que aquele de indivíduos normais e 7 vezes maior que aquele dos pacientes com doença de Gaucher submetidos a tratamento com terapia de reposição enzimática. Os resultados obtidos demonstram que a metodologia estabelecida pode ser usada como diagnóstico complementar e como monitoração do tratamento de pacientes com doença de Gaucher.


Assuntos
Humanos , Doença de Gaucher/sangue , Glucosilceramidas/química , Glucosilceramidas/sangue , Técnicas e Procedimentos Diagnósticos/estatística & dados numéricos , Análise de Variância , Análise Química do Sangue/métodos , Cromatografia Líquida de Alta Pressão/métodos
9.
Mol Cell Biochem ; 303(1-2): 121-30, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17440688

RESUMO

GRX cell line represents hepatic stellate cell and can be transformed from an actively proliferation myofibroblast phenotype into a quiescent fat-storing lipocyte phenotype. Both express the same gangliosides (GM3, GM2, GM1 and GD1a), which are resolved as doublets on HPTLC. Upper/lower band ratio is increased in lipocyte-like cells and the upper band is composed by ceramides with long-chain fatty acids. This study evaluated the contribution of de novo synthesis, sphingosine and Golgi recycling pathways on ganglioside biosynthesis, in both phenotypes. Cells were preincubated with 5 mM beta-chloroalanine (SPT: serine palmitoyltransferase inhibitor) or with 25 muM fumonisin B1 (ceramide synthase inhibitor) and then radiolabeled with [U-(14)C]galactose in the continued presence of inhibitors. Gangliosides were extracted, purified and analyzed by HPTLC. In myofibroblast-like cells, simple gangliosides use the de novo pathway while complex gangliosides are mainly synthesized by recycling pathways. In lipocyte-like cells, de novo pathway has a lesser contribution and this is in agreement with the lower activity of the committed enzyme of sphingolipid synthesis (SPT) detected in this phenotype. SPT mRNA has an identical expression in both phenotypes. It was also observed that gangliosides doublets from myofibroblast-like cells have the same distribution between triton soluble and insoluble fractions (upper band > lower band) while the gangliosides doublets from lipocyte-like cells show an inversion in the insoluble fraction (lower band > upper band) in comparison to soluble fraction. These results indicate that myofibroblast- and lipocyte-like cells have important differences between the glycosphingolipid biosynthetic pathways, which could contribute with the respective glycosphingolipid-enriched membrane microdomain's composition.


Assuntos
Adipócitos/metabolismo , Adipócitos/fisiologia , Vias Biossintéticas , Fibroblastos/metabolismo , Gangliosídeos/metabolismo , Hepatócitos/citologia , Mioblastos/fisiologia , Adipócitos/citologia , Animais , Linhagem Celular , Fibroblastos/citologia , Hepatócitos/metabolismo , Camundongos , Mioblastos/citologia , Fenótipo , Serina/metabolismo , Serina C-Palmitoiltransferase/metabolismo , Esfingolipídeos/química , Esfingolipídeos/metabolismo , Esfingomielinas/química , Esfingomielinas/metabolismo
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