Free carnitine and branched chain amino acids are not good biomarkers in Huntington's disease / Carnitina livre e aminoácidos de cadeia ramificada não são bons biomarcadores na doença de Huntington

ABSTRACT Background: Huntington's disease (HD), caused by an expanded CAG repeat at HTT, has no treatment, and biomarkers are needed for future clinical trials. Objective: The objective of this study was to verify if free carnitine and branched chain amino acids levels behave as potential biomarkers in HD. Methods: Symptomatic and asymptomatic HD carriers and controls were recruited. Age, sex, body mass index (BMI), age of onset, disease duration, UHDRS scores, and expanded CAG tract were obtained; valine, leucine, isoleucine, and free carnitine were measured. Baseline and longitudinal analysis were performed. Results: Seventy-four symptomatic carriers, 20 asymptomatic carriers, and 22 non-carriers were included. At baseline, valine levels were reduced in symptomatic and asymptomatic HD carriers when compared to non-carriers. No difference in free carnitine or isoleucine+leucine levels were observed between groups. BMI of symptomatic individuals was lower than those of non-carriers. Valine levels correlated with BMI. Follow-up evaluation was performed in 43 symptomatic individuals. UHDRS total motor score increased 4.8 points/year on average. No significant reductions in BMI or valine were observed, whereas free carnitine and isoleucine+leucine levels increased. Conclusions: Although valine levels were lower in HD carriers and were related to BMI losses observed in pre-symptomatic individuals, none of these metabolites seem to be biomarkers for HD.

RESUMO Introdução: A doença de Huntington (DH), causada por uma repetição CAG expandida no HTT, não possui tratamento e biomarcadores são necessários para futuros ensaios clínicos. Objetivo: Nosso objetivo foi verificar se os níveis de carnitina livre e aminoácidos de cadeia ramificada se comportam como potenciais biomarcadores na DH. Métodos: Portadores sintomáticos e assintomáticos e controles foram recrutados. Idade, sexo, índice de massa corporal (IMC), idade de início, duração da doença, escores UHDRS e trato CAG expandido foram obtidos; valina, leucina, isoleucina e carnitina livre foram medidas. Foram realizadas análises basal e longitudinal. Resultados: Setenta e quatro portadores sintomáticos, 20 portadores assintomáticos e 22 não portadores foram incluídos. No início do estudo, os níveis de valina estavam reduzidos em portadores de DH sintomáticos e assintomáticos quando comparados aos não portadores. Não foram observadas diferenças nos níveis de carnitina livre ou isoleucina + leucina entre os grupos. O IMC dos indivíduos sintomáticos foi menor que o dos não portadores. Níveis de valina correlacionaram-se com o IMC. Avaliação de acompanhamento foi realizada em 43 indivíduos sintomáticos. A pontuação do escore motor total da UHDRS aumentou 4,8 pontos/ano em média. Não foram observadas reduções significativas no IMC ou na valina, enquanto os níveis de carnitina livre e isoleucina+leucina aumentaram. Conclusões: Embora os níveis de valina tenham sido menores nos portadores de DH e estivessem relacionados às perdas de IMC observadas em indivíduos pré-sintomáticos, nenhum desses metabólitos parece ser biomarcador para a DH.

Monitoring of Phenylalanine Levels in Patients with Phenylketonuria Using Dried Blood Spots: a Comparison of Two Methods

Abstract Phenylketonuria (PKU) is caused by deficient activity of phenylalanine hydroxylase (PAH), responsible for the conversion of phenylalanine (Phe) to tyrosine (Tyr). Monitoring of patients with PKU requires the measurement of Phe in plasma using high-performance liquid chromatography (HPLC) or in dried blood spots (DBS) using different techniques to adjust treatment strategy. The objective of this study was to evaluate Phe levels in DBS measured by two different methods and compare them with Phe levels measured in plasma by HPLC. We analyzed 89 blood samples from 47 PKU patients by two different methods: fluorometric method developed in-house (method A) and the commercially available PerkinElmer® Neonatal Phenylalanine Kit (method B) and in plasma by HPLC. The mean Phe levels by method A, method B, and HPLC were 430.4±39.9μmol/L, 439.3±35.4μmol/L, and 442.2±41.6μmol/L, respectively. The correlation values between HPLC and methods A and B were 0.990 and 0.974, respectively (p < 0.001 for both). Our data suggest that methods A and B are useful alternatives for monitoring Phe levels in patients with PKU, with method A being in closer agreement with the reference standard (HPLC).

DNA damage in homocystinuria: 8-oxo-, 8-dihydro-2'-deoxyguanosine levels in cystathionine-ß-synthase deficient patients and the in vitro protective effect of N-acetyl­L­cysteine

Introduction: Homocysteine (Hcy) tissue accumulation occurs in a metabolic disease characterized biochemically by cystathionine ß-synthase (CBS) deficiency and clinically by mental retardation, vascular problems, and skeletal abnormalities. Previous studies indicate the occurrence of DNA damage secondary to hyperhomocysteinemia and it was observed that DNA damage occurs in leukocytes from CBS-deficient patients. This study aimed to investigate whether an oxidative mechanism could be involved in DNA damage previously found and investigated the in vitro effect of N-acety-L-cysteine (NAC) on DNA damage caused by high Hcy levels. Methods: We evaluated a biomarker of oxidative DNA damage in the urine of CBS­deficient patients, as well as the in vitro effect of NAC on DNA damage caused by high levels of Hcy. Moreover, a biomarker of lipid oxidative damage was also measured in urine of CBS deficient patients. Results: There was an increase in parameters of DNA (8-oxo-7,8-dihydro-2'- deoxyguanosine) and lipid (15-F2t-isoprostanes levels) oxidative damage in CBS-deficient patients when compared to controls. In addition, a significant positive correlation was found between 15-F2t-isoprostanes levels and total Hcy concentrations. Besides, an in vitro protective effect of NAC at concentrations of 1 and 5 mM was observed on DNA damage caused by Hcy 50 µM and 200 µM. Additionally, we showed a decrease in sulfhydryl content in plasma from CBS-deficient patients when compared to controls. Discussion: These results demonstrated that DNA damage occurs by an oxidative mechanism in CBS deficiency together with lipid oxidative damage, highlighting the NAC beneficial action upon DNA oxidative process, contributing with a new treatment perspective of the patients affected by classic homocystinuria.

In vitro effect of N-acetyl-L-cysteine on glutathione and sulfhydryl levels in X-linked adrenoleukodystrophy patients

Introduction: Recent evidence shows that oxidative stress seems to be related with the pathophysiology of X-linked adrenoleukodystrophy (X-ALD), a neurodegenerative disorder. Methods: In the present study, the in vitro effect of N-acetyl-L-cysteine (NAC) on glutathione (GSH) and sulfhydryl levels in X-ALD patients was evaluated. Results: A significant reduction of GSH and sulfhydryl content was observed in X-ALD patients compared to the control group. Furthermore, 5 mM of NAC, in vitro, led to an increase in GSH content and sulfhydryl groups in these patients. Conclusion: These data probably indicate that an adjuvant therapy with the antioxidant NAC could improve the oxidative imbalance in X-ALD patients(AU)

Validation of a Multiplex Tandem Mass Spectrometry Method for the Detection of Selected Lysosomal Storage Diseases in Dried Blood Spots

Abstract Background: Interest in screening methods for lysosomal storage diseases (LSDs) has increased in recent years, since early diagnosis and treatment are essential to prevent or attenuate the onset of symptoms and the complications of these diseases. In the current work, we evaluated the performance of tandem mass spectrometry (MS/MS) for the detection of some LSDs, aiming the future use of this methodology for the screening of these disorders. Methods: Standard curves and quality control dried blood spots were assayed to evaluate the precision, linearity, and accuracy. A total of 150 controls were grouped according to age and subjected to measurement of lysosomal enzymes deficient in Niemann-Pick A/B, Krabbe, Gaucher, Fabry, Pompe, and Mucopolysaccharidosis type I diseases. Samples from 59 affected patients with a diagnosis of LSDs previously confirmed by fluorimetric methods were analyzed. Results: Data from standard calibration demonstrated good linearity and accuracy and the intra- and interassay precisions varied from 1.17% to 11.60% and 5.39% to 31.24%, respectively. Except for galactocerebrosidase and ?-l-iduronidase, enzyme activities were significantly higher in newborns compared to children and adult controls. Affected patients presented enzymatic activities significantly lower compared to all control participants. Conclusion: Our results show that MS/MS is a promising methodology, suitable for the screening of LSDs, but accurate diagnoses will depend on its correlation with other biochemical and/or molecular analyses.

Neurological outcomes after hematopoietic stem cell transplantation for cerebral X-linked adrenoleukodystrophy, late onset metachromatic leukodystrophy and Hurler syndrome / Desfechos neurológicos após transplante de células tronco hematopoiéticas na adrenoleucodistrofia ligada ao X, forma cerebral, na leucodistrofia metacromática de início tardio e na síndrome de Hurler

ABSTRACT Hematopoietic stem cell transplantation (HSCT) is the only available treatment for the neurological involvement of disorders such as late-onset metachromatic leukodystrophy (MLD), mucopolysaccharidosis type I-Hurler (MPS-IH), and X-linked cerebral adrenoleukodystrophy (CALD). Objective To describe survival and neurological outcomes after HSCT for these disorders. Methods Seven CALD, 2 MLD and 2 MPS-IH patients underwent HSCT between 2007 and 2014. Neurological examinations, magnetic resonance imaging, molecular and biochemical studies were obtained at baseline and repeated when appropriated. Results Favorable outcomes were obtained with 4/5 related and 3/6 unrelated donors. Two patients died from procedure-related complications. Nine transplanted patients were alive after a median of 3.7 years: neurological stabilization was obtained in 5/6 CALD, 1/2 MLD, and one MPS-IH patient. Brain lesions of the MPS-IH patient were reduced four years after HSCT. Conclusion Good outcomes were obtained when HSCT was performed before adulthood, early in the clinical course, and/or from a related donor.

RESUMO O transplante de células tronco hematopoiéticas (TCTH) é o único tratamento disponível para o envolvimento neurológico de doenças como a leucodistrofia metacromática (MLD), a mucopolissacaridose tipo I-Hurler (MPS-IH) e a adrenoleucodistrofia (CALD). Objetivos Descrever a sobrevida e os desfechos neurológicos após o TCTH nessas doenças. Métodos Sete pacientes CALD, 2 MLD e 2 MPS-IH realizaram TCTH entre 2007 e 2014. Avaliações neurológicas, ressonância nuclear magnética e estudos bioquímicos e moleculares foram feitos no baseline e repetidos quando apropriado. Resultados Desfechos favoráveis foram obtidos em 4/5 TCTH de doadores relacionados e em 3/6 não relacionados. Dois pacientes faleceram de complicações do procedimento. Nove transplantados sobreviveram após uma mediana de 3,7 anos: estabilização neurológica foi obtida em 5/6 CALD, ½ MLD e em um caso MPS-IH. As lesões encefálicas de um caso MPS-IH reduziram-se quatro anos após o TCTH. Conclusão Bons desfechos foram obtidos quando o TCTH foi feito antes da vida adulta, cedo no curso clínico e/ou a partir de um doador relacionado.

Acidúrias D-2-hidroxiglutárica e L-2-hidroxiglutárica: uma revisão da literatura / D-2-hydroxyglutaric and L-2-hydroxyglutaric acidurias:a literature review

As acidúrias D-2-hidroxiglutárica (D-2-HGA) e L-2-hidroxiglutárica (L-2-HGA) são raras doenças neurometabólicas que constituem um grupo de erros inatos do metabolismo. Essas doenças são causadas pela deficiência das atividades enzimáticas da D-2-hidroxiglutarato desidrogenase na D-2-HGA do tipo I ou isocitrato desidrogenase na D-2-HGA do tipo II, e da L-2-hidroxiglutarato desidrogenase na L-2-HGA. Os principais achados clínicos nos pacientes caracterizam-se por sintomas neurológicos, como convulsões, coma e atrofia cerebral. Também ocorrem lesões cerebrais nos gânglios da base (D-2-HGA, L-2-HGA) e cerebelo (L-2-HGA). Bioquimicamente, essas acidúrias caracterizam-se por acúmulo em tecidos e elevada excreção urinária dos ácidos D-2-hidroxiglutárico (na D-2-HGA) e L-2-hidroxiglutárico (na L-2-HGA). Ainda, uma terceira variante bioquímica da acidúria, a D,L-2-hidroxiglutárica (D,L-2-HGA), é caracterizada por excreção aumentada de ambos enantiômeros do ácido 2-hidroxiglutárico. Em modelo animal, estudos de toxicidade dos ácidos D e L-2-hidroxiglutárico mostraram injúria cerebral, mas não foi elucidado o mecanismo exato causador do dano. Além disso, altos níveis dos ácidos D e L-2-hidroxiglutárico foram encontrados em tumores cerebrais. No entanto, a relação entre a acidúria e o câncer ainda precisa ser esclarecida. Tendo em vista a gravidade da doença, este trabalho teve como objetivo fazer uma revisão bibliográfica acerca do tema, enfatizando as consequências do metabolismo, principalmente para o tecido cerebral, bem como apontar possíveis abordagens terapêuticas.

The D-2-hydroxyglutaric (D-2-HGA) and L-2-hydroxyglutaric acidurias (L-2-HGA) are rare neurometabolic diseases that form a group of inborn errors of metabolism. They are caused by a deficiency on the enzyme activities of D-2-hydroxyglutarate dehydrogenase in D-2-HGA type I or isocitrate dehydrogenase in D-2-HGA type II, and L-2-hydroxyglutarate dehydrogenase in L-2-HGA. The main clinical findings in affected patients are related to neurological symptoms, such as convulsions, coma and brain atrophy. Brain injuries also occur in the basal ganglia (D-2-HGA, L-2-HGA) and cerebellum (L-2-HGA). These acidurias are biochemically characterized by the accumulation in tissues and increased urinary excretion of D-2-hydroxyglutaric acid (in D-2-HGA) and L-2-hydroxyglutaric acid (in L-2-HGA). Still, a third biochemical variant of aciduria, called D,L-2-hydroxyglutaric (D,L-2-HGA), is characterized by increased excretion of both enantiomers of 2-hydroxyglutaric acid. In an animal model, toxicity studies on D- and L-2-hydroxyglutaric acids showed brain injury, but the exact mechanism of brain damage was not elucidated. Furthermore, high levels of D- and L-2-hydroxyglutaric acids were found in brain tumors. However, the relationship between cancer and aciduria still needs to be clarified. In view of the severity of the disease, this study aimed to do a literature review on the topic, emphasizing metabolic consequences, particularly for the brain tissue, as well as to identify possible therapeutic approaches.

Encefalopatia diabética e depressão: dano oxidativo no cérebro / Diabetic encephalopathy and depression: brain oxidative damage

O diabetes é um distúrbio complexo e heterogêneo caracterizado por hiperglicemia resultante de defeitos na secreção e ação da insulina. Tem sido reconhecido que, além do comprometimento de órgãos como rins, olhos, fígado e coração, o sistema nervoso central é suscetível aos efeitos deletérios da hiperglicemia em longo prazo. A encefalopatia diabética representa uma das complicações do diabetes, na qual os danos são caracterizados por alterações do funcionamento cognitivo, modificações estruturais e neurofisiológicas no cérebro. Existe uma associação bem reconhecida entre a depressão e o diabetes, uma vez que a prevalência de depressão é maior na população diabética comparada com a população geral. Porém, os mecanismos atribuídos a essa relação ainda estão em fase de investigação. O estresse oxidativo desempenha um papel importante nas complicações do diabetes e pode ser um mecanismo biológico envolvido na relação entre a depressão e o diabetes, relacionado à encefalopatia diabética. Neste artigo de revisão, apresentamos uma visão geral dos principais conceitos relacionados ao assunto, bem como dos dados clínicos e experimentais que suportam a relação entre o dano oxidativo no cérebro e a depressão relacionada com encefalopatia diabética...

Diabetes is a complex and heterogeneous disorder characterized by hyperglycemia resulting from defects in the secretion and action of insulin. It has been recognized that, in addition to the involvement of organs such as kidney, eye, liver, and heart, the central nervous system is susceptible to the deleterious effects of hyperglycemia in the long term. Diabetic encephalopathy is one of the complications of diabetes, in which the damage is characterized by changes in cognitive functioning, structural and neurophysiologic changes in the brain. There is a well-known association between depression and diabetes, since the prevalence of depression is higher in the diabetic population compared to the general population. However, the mechanisms assigned to this relationship are still under investigation. Oxidative stress plays an important role in the complications of diabetes and can be a biological mechanism involved in the relation between depression and diabetes related to diabetic encephalopathy. This review article is an overview of key concepts related to the subject, as well as of the clinical and experimental data supporting the relationship between oxidative damage in the brain and depression related to diabetic encephalopathy...

Homocysteine and other markers of cardiovascular risk during a manic episode in patients with bipolar disorder

Objective: To evaluate serum levels of different biomarkers associated with cardiovascular disease in patients with bipolar disorder (BD). Patients were prospectively evaluated in two separate instances: during acute mania and after remission of manic symptoms. All measurements were compared with those of healthy controls. Methods: The study included 30 patients with BD and 30 healthy controls, matched for gender and age. Biochemical parameters evaluated included homocysteine (Hcy), folic acid, vitamin B12, ferritin, creatine kinase (CK) and C-reactive protein (CRP). Results: Hcy levels were significantly higher in the BD patients, both during mania and after achieving euthymia. When Hcy was adjusted for body mass index, there was no significant difference between patients and controls. Ferritin was the only marker that showed a significant decrease during mania when compared to both euthymic patients and controls. There were no significant differences for folate, vitamin B12, CK and CRP. Conclusions: These findings do not show an association between alterations of markers of cardiovascular risk during manic episodes. Further studies are necessary to determine factors and mechanisms associated with cardiovascular risk in patients with BD. .