Detecção de glicosilceramida em plasma de pacientes com doença de Gaucher

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Diagnóstico laboratorial da Doença de Gaucher no Brasil

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Estudo das propriedades cinéticas da enzima quitotriosidase humana e dos parâmetros pró-inflamatórios interleucina 6 e fator de necrose tumoral alfa em plasma de pacientes com as doenças de Gaucher tipo I, Krabbe e gangliosidose GM1 e de heterozigotos para a doença de Gaucher tipo I : comparação com indivíduos normais

Um aumento acentuado da atividade da quitotriosidase (QT) tem sido encontrado em plasma de pacientes afetados por várias esfingolipidoses, incluindo a doença de Gaucher tipo I (DG), a doença de Krabbe (DK) e a gangliosidose GM1 (GM1). Na DG, causada pela deficiência da enzima glicocerebrosidase, a QT é considerada um marcador auxiliar no diagnóstico desta enfermidade. Já nas DK e GM1 causadas, respectivamente pelas deficiências na atividade das enzimas galactosilceramidase e b-galactosidase, há um aumento moderado da atividade da QT. O objetivo inicial do presente trabalho foi o de caracterizar as propriedades cinéticas da enzima quitotriosidase em plasma de pacientes com as DG tipo I, DK, GM1 e heterozigotos para a DG tipo I (HG), bem como comparar essas propriedades entre os vários grupos e com as de indivíduos normais. Verificamos inicialmente que nos pacientes com DG tipo I a atividade plasmática da QT apresentou-se muito acima da normalidade (em média 600 vezes), o mesmo ocorrendo em indivíduos com DK (em média 12 vezes) e com GM1 (em média 15 vezes). No que se refere às propriedades cinéticas, observamos que o pH ótimo da QT dos pacientes com DK e GM1 foi superior ao dos controles, enquanto a QT de homozigotos (DG) e heterozigotos para DG (HG) teve um pH ótimo inferior ao dos controles. Além disso, o maior Km observado para a QT foi observado em pacientes com GM1, seguido daquele com DG, que foram muito superiores aos Km de todos os outros grupos (DK, HG e controles), refletindo uma menor afinidade da enzima pelo substrato nos pacientes com GM1 e DG Além disso, o Km dos pacientes com DK e HG foram, respectivamente, superior e inferior ao dos controles. Já no que se refere à velocidade máxima (Vmax) observou-se um aumento acentuado desse parâmetro no grupo DG tipo I, seguindo-se do GM1 e DK, relativamente aos grupos controle e HG. Esses achados foram semelhantes aos encontrados para a atividade da QT nos grupos estudados. Finalmente verificamos que a QT de indivíduos normais é muito mais termoestável relativamente à QT dos pacientes com DG tipo I, DK e GM1, bem como ao grupo HG. É possível que as variações das propriedades cinéticas da QT entre os vários grupos analisados em relação aos controles e também aos HG possam potencialmente representar marcadores bioquímicos simples e rápidos na diferenciação de indivíduos homozigotos para algumas dessas doenças lisossomais, bem como de HG com indivíduos normais. Estes resultados indicam, portanto, que indivíduos normais podem ser diferenciados dos HG através do pH ótimo, Km e termoestabilidade da QT O próximo objetivo de nossa investigação foi comparar as propriedades cinéticas da quitotriosidase em pacientes com a DG tipo I não tratados e em tratamento com terapia de reposição enzimática. Verificamos que a atividade da enzima apresentou um decréscimo ao longo do tratamento e que o Km se elevou significativamente em relação àquele do início do tratamento, enquanto as outras propriedades (Vmax, pH ótimo e termoestabilidade) não foram alteradas. Embora não se tenha uma explicação no momento para o aumento do Km com o tratamento, podemos supor que isoenzimas da QT com uma afinidade menor ao substrato (maior Km) pudessem ser mais expressas no sangue dos pacientes tratados. Também medimos e comparamos os níveis dos fatores pró-inflamatórios interleucina 6 (IL-6) e fator de necrose tumoral alfa (TNF- ) no plasma de pacientes com DG tipo I, DK, GM1, HG e de indivíduos normais. Observamos que tanto a IL-6 quanto o TNF- apresentaram concentrações plasmáticas aumentadas nos pacientes com DG tipo I, DK e GM1, bem como nos HG relativamente aos controles. O aumento verificado nas concentrações plasmáticas de TNF- nos pacientes afetados por DG tipo I, GM1 e DK foi aproximadamente de 3 a 4 vezes superior ao dos controles Já as concentrações plasmáticas de IL-6 nesses mesmos pacientes foram superiores a dos controles na ordem de magnitude de 3 (DG tipo I e HG), 5 (GM1) e 15 (DK) vezes, indicando que esse parâmetro é mais sensível que o TNF- na diferenciação entre os indivíduos normais e os com as doenças lisossomais estudadas. Esses achados indicam uma ativação do sistema imunológico a nível sistêmico nos pacientes com as doenças lisossomais acima descritas, bem como nos HG. Além disso, observamos que os níveis plasmáticos de IL-6 e TNF- não estavam correlacionados com a atividade e as propriedades bioquímicas da QT, bem como com as atividades da -glicosidase, galactocerebrosidase e -galactosidase, indicando que o processo inflamatório sistêmico detectado no plasma dos pacientes com DG tipo I, DK e GM1 por níveis elevados de IL-6 e TNF- não está associado à atividade ou às propriedades cinéticas da QT e com a atividade das enzimas deficientes nestas doenças. Também investigamos associações entre o sexo, algumas manifestações clínicas comuns encontradas nos pacientes com DK, GM1 e entre a atividade e as propriedades cinéticas da QT com os níveis das citocinas IL-6 e TNF- em plasma desses indivíduos. Verificamos que o sexo não alterou significativamente nenhum desses parâmetros. Já nos pacientes com GM1 a presença de hepatomegalia associou-se a uma elevação da atividade e da Vmax da QT, indicando que esse sinal clínico possa estar relacionado a uma ativação do sistema imunológico. Ainda nos pacientes com GM1, foi verificado que a presença de convulsões nos mesmos associou-se a uma menor Vmax da QT e a níveis baixos de TNF- . Nenhuma outra associação significante nesses parâmetros pode ser verificada nos pacientes com DK e GM1.Sphingolipidoses are lysossomal storage disorders characterized by the accumulation of sphingolipids in one or more tissues of the affected individuals as a result of a primary deficiency in activity of lysosomal enzymes or their activating proteins involved in routes of degradation. A high elevation of chitotriosidase enzyme has been found in plasma of patients affected by several sphingolipidoses, such as Gaucher Disease type 1 (GD), Krabbe Disease (KD) and GM1 Gangliosidosis. In GD, caused by deficiency of glucerebrosidase enzyme, chitotriosidase (CT) activity is very high in plasma of affected patients, been considered a useful marker in the diagnosis of this disease. On the other hand, in DK and GM1, due respectively to a deficiency in the galactocerebrosidase enzyme and -galactosidase enzyme, there is a moderate increase of CT activity. The initial objective of this study was to characterize the kinetic properties of chitotriosidase enzyme in plasma of patients with GD type I, KD, GM1 and heterozygotes for GD type I (HG) and compare these properties among the various groups and with those of normal subjects. We noticed initially that in patients with GD, CT activity was much higher than in normal individuals (600 times on average), the same occurred in DK patients (increased 12 times on average) and GM1 patients (increased 15 times on average). Concerning the kinetic properties, we observed that the optimum pH of CT in patients with KD and GM1 was higher than in controls, whereas homozygotes and heterozygotes for GD the CT had an optimum pH lower than that of controls Moreover, the highest Km of CT was observed in GM1 patients, followed that with DG, which were much higher than the Km of all other groups (DK, HG and controls), reflecting a lower affinity of the enzyme by the substrate in patients with GM1 and DG. Furthermore, the Km of patients with KD and HG were respectively higher and lower than of controls. Besides, regarding to the Vmax, there was an increase of this parameter in GD type I, followed by GM1 and KD, relatively to controls and HG. These findings were similar to those found for CT activity in the groups studied. Finally, we noted that CT activity of normal individuals was much more stable to heating, compared to CT of patients with GD, KD and GM1, and HG group. It is possible that the changes in the kinetic properties of CT between different groups in relation to controls and also to HG could potentially represent rapid and simple biochemical markers in the differentiation of individuals homozygous for some of these lysosomal diseases, as well as HG from normal subjects. These results indicate therefore that normal individuals can be distinguished from HG through optimum pH , Km and thermostability of the CT The next objective of our research was to compare the kinetic properties of CT in patients with GD type I under enzyme replacement therapy (imiglucerase) from those withouth treatment. We found that the enzyme activity was decreased and the Km increased significantly during treatment, while the other properties (Vmax, optimum pH and thermostability) were not altered by treatment. Although at the moment we do not have an explanation for an increase of CT with treatment, we may presume that CT isoenzymes with a lower affinity to the substrate (increased Km) are more expressed in blood of treated patients. We also measured and compared the levels of the pro-inflammatory interleukin-6 and tumor necrosis factor alpha (TNF- ) in plasma of patients with DG type I, DK, GM1, HG and normal individuals. We observed that both IL-6 as TNF- concentrations were significantly increased in plasma from patients with type I DG, DK and GM1, as well as in HG compared with controls. The increase observed in plasma concentrations of TNF- in patients affected by type I DG, GM1 and DK was approximately 3 to 4 times higher than that of controls. Furthermore, the plasma concentrations of IL-6 in these patients were higher than controls in the order of magnitude of 3 (type I DG and HG) , 5 (GM1) and 15 (GM1) times, indicating that this parameter is more sensitive than TNF- in the differentiation between normal subjects and patients with the lysosomal diseases studied. These findings indicate an activation of the immune system at the systemic level in patients with the lysosomal diseases above described, as well as in HG We also observed that IL-6 and TNF- were not correlated with the activity and biochemical properties of CT, as well as with the activities of -glicosidase, galactocerebrosidase and -galactosidase, indicating that the systemic inflammatory process found in plasma of patients with GD type I, KD and GM1 detected by high serum levels of IL-6 and TNF- is not associated with the activity and the kinetic properties of QT and with the activity of the enzymes deficient in these diseases. We also investigated associations between sex, some clinical manifestations found in patients with KD, GM1 and between the activity and the kinetic properties of CT and the levels of citokines IL-6 and TNF- in plasma of these individuals. We verified that the sex did not significantly alter any of these parameters. Furthermore, in GM1 patients, the presence of hepatomegaly was associated with an elevation of QT activity and Vmax, indicating that this clinical sign may be related to an activation of the immune system. Still in GM1 patients, we observed that the presence of seizures was associated with a decreased CT Vmax and TNF- . No other significant alteration in these parameters could be verified in patients with DK and GM1

Disturbance of mitochondrial functions associated with permeability transition pore opening induced by cis-5-tetradecenoic and myristic acids in liver of adolescent rats

Patients affected by very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency commonly present liver dysfunction whose pathogenesis is poorly known. We demonstrate here that major metabolites accumulating in this disorder, namely cis-5-tetradecenoic acid (Cis-5) and myristic acid (Myr), markedly impair mitochondrial respiration, decreasing ATP production in liver mitochondrial preparations from adolescent rats. Other parameters of mitochondrial homeostasis such as membrane potential (Delta Psi m) and Ca2+ retention capacity were strongly compromised by these fatty acids, involving induction of mitochondrial permeability transition. The present data indicate that disruption of mitochondrial bioenergetics and Ca2+ homeostasis may contribute to the liver dysfunction of VLCAD deficient patients50113CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DO RIO GRANDE DO SUL - FAPERGS425914/2016‐017/17728-82266‐2551/14‐

Disturbance of bioenergetics and calcium homeostasis provoked by metabolites accumulating in propionic acidemia in heart mitochondria of developing rats

Propionic acidemia is caused by lack of propionyl-CoA carboxylase activity. It is biochemically characterized by accumulation of propionic (PA) and 3-hydroxypropionic (3OHPA) acids and clinically by severe encephalopathy and cardiomyopathy. High urinary excretion of maleic acid (MA) and 2-methylcitric acid (2MCA) is also found in the affected patients. Considering that the underlying mechanisms of cardiac disease in propionic acidemia are practically unknown, we investigated the effects of PA, 3OHPA, MA and 2MCA (0.05–5 mM) on important mitochondrial functions in isolated rat heart mitochondria, as well as in crude heart homogenates and cultured cardiomyocytes. MA markedly inhibited state 3 (ADP-stimulated), state 4 (non-phosphorylating) and uncoupled (CCCP-stimulated) respiration in mitochondria supported by pyruvate plus malate or α-ketoglutarate associated with reduced ATP production, whereas PA and 3OHPA provoked less intense inhibitory effects and 2MCA no alterations at all. MA-induced impaired respiration was attenuated by coenzyme A supplementation. In addition, MA significantly inhibited α-ketoglutarate dehydrogenase activity. Similar data were obtained in heart crude homogenates and permeabilized cardiomyocytes. MA, and PA to a lesser degree, also decreased mitochondrial membrane potential (ΔΨm), NAD(P)H content and Ca2+ retention capacity, and caused swelling in Ca2+-loaded mitochondria. Noteworthy, ΔΨm collapse and mitochondrial swelling were fully prevented or attenuated by cyclosporin A and ADP, indicating the involvement of mitochondrial permeability transition. It is therefore proposed that disturbance of mitochondrial energy and calcium homeostasis caused by MA, as well as by PA and 3OHPA to a lesser extent, may be involved in the cardiomyopathy commonly affecting propionic acidemic patients18665CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DO RIO GRANDE DO SUL - FAPERGS425914/2016-017/17728-817/2551-0000/800-

Detecção de heterozigotos para mucopolissacaridose do tipo I através das características bioquímicas da enzima alfa-iduronidase

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Mechanistic Bases of Neurotoxicity Provoked by Fatty Acids Accumulating in MCAD and LCHAD Deficiencies

Fatty acid oxidation defects (FAODs) are inherited metabolic disorders caused by deficiency of specific enzyme activities or transport proteins involved in the mitochondrial catabolism of fatty acids. Medium-chain fatty acyl-CoA dehydrogenase (MCAD) and long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiencies are relatively common FAOD biochemically characterized by tissue accumulation of medium-chain fatty acids and long-chain 3-hydroxy fatty acids and their carnitine derivatives, respectively. Patients with MCAD deficiency usually have episodic encephalopathic crises and liver biochemical alterations especially during crises of metabolic decompensation, whereas patients with LCHAD deficiency present severe hepatopathy, cardiomyopathy, and acute and/or progressive encephalopathy. Although neurological symptoms are common features, the underlying mechanisms responsible for the brain damage in these disorders are still under debate. In this context, energy deficiency due to defective fatty acid catabolism and hypoglycemia/hypoketonemia has been postulated to contribute to the pathophysiology of MCAD and LCHAD deficiencies. However, since energetic substrate supplementation is not able to reverse or prevent symptomatology in some patients, it is presumed that other pathogenetic mechanisms are implicated. Since worsening of clinical symptoms during crises is accompanied by significant increases in the concentrations of the accumulating fatty acids, it is conceivable that these compounds may be potentially neurotoxic. We will briefly summarize the current knowledge obtained from patients with these disorders, as well as from animal studies demonstrating deleterious effects of the major fatty acids accumulating in MCAD and LCHAD deficiencies, indicating that disruption of mitochondrial energy, redox, and calcium homeostasis is involved in the pathophysiology of the cerebral damage in these diseases. It is presumed that these findings based on the mechanistic toxic effects of fatty acids may offer new therapeutic perspectives for patients affected by these disorders