RESUMO
Gyrate atrophy of the choroid and retina (GACR) is caused by pathogenic biallelic variants in the gene encoding ornithine-δ-aminotransferase (OAT), and is characterized by progressive vision loss leading to blindness. OAT is a pyridoxal-5'-phosphate (PLP) dependent enzyme that is mainly involved in ornithine catabolism, and patients with a deficiency develop profound hyperornithinemia. Therapy is aimed at lowering ornithine levels through dietary arginine restriction and, in some cases, through enhancement of OAT activity via supraphysiological dosages of pyridoxine. In this study, we aimed to extend diagnostic practices in GACR by extensively characterizing the consequences of pathogenic variants on the enzymatic function of OAT, both at the level of the enzyme itself as well as the flux through the ornithine degradative pathway. In addition, we developed an in vitro pyridoxine responsiveness assay. We identified 14 different pathogenic variants, of which one variant was present in all patients of Dutch ancestry (p.(Gly353Asp)). In most patients the enzymatic activity of OAT as well as the rate of [14C]-ornithine flux was below the limit of quantification (LOQ). Apart from our positive control, only one patient cell line showed responsiveness to pyridoxine in vitro, which is in line with the reported in vivo pyridoxine responsiveness in this patient. None of the patients harboring the p.(Gly353Asp) substitution were responsive to pyridoxine in vivo or in vitro. In silico analysis and small-scale expression experiments showed that this variant causes a folding defect, leading to increased aggregation properties that could not be rescued by PLP. Using these results, we developed a diagnostic pipeline for new patients suspected of having GACR. Adding OAT enzymatic analyses and in vitro pyridoxine responsiveness to diagnostic practices will not only increase knowledge on the consequences of pathogenic variants in OAT, but will also enable expectation management for therapeutic modalities, thus eventually improving clinical care.
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A deficiency of 3-hydroxyisobutyric acid dehydrogenase (HIBADH) has been recently identified as a cause of primary 3-hydroxyisobutyric aciduria in two siblings; the only previously recognized primary cause had been a deficiency of methylmalonic semialdehyde dehydrogenase, the enzyme that is immediately downstream of HIBADH in the valine catabolic pathway and is encoded by the ALDH6A1 gene. Here we report on three additional patients from two unrelated families who present with marked and persistent elevations of urine L-3-hydroxyisobutyric acid (L-3HIBA) and a range of clinical findings. Molecular genetic analyses revealed novel, homozygous variants in the HIBADH gene that are private within each family. Evidence for pathogenicity of the identified variants is presented, including enzymatic deficiency of HIBADH in patient fibroblasts. This report describes new variants in HIBADH as an underlying cause of primary 3-hydroxyisobutyric aciduria and expands the clinical spectrum of this recently identified inborn error of valine metabolism. Additionally, we describe a quantitative method for the measurement of D- and L-3HIBA in plasma and urine and present the results of a valine restriction therapy in one of the patients.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos , Espectrometria de Massas em Tandem , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Cromatografia Líquida , Humanos , Hidroxibutiratos/urina , Oxirredutases , ValinaRESUMO
Mitochondrial trifunctional protein (MTP) is involved in long-chain fatty acid ß-oxidation (lcFAO). Deficiency of one or more of the enzyme activities as catalyzed by MTP causes generalized MTP deficiency (MTPD), long-chain hydroxyacyl-CoA dehydrogenase deficiency (LCHADD), or long-chain ketoacyl-CoA thiolase deficiency (LCKATD). When genetic variants result in thermo-sensitive enzymes, increased body temperature (e.g. fever) can reduce enzyme activity and be a risk factor for clinical decompensation. This is the first description of five patients with a thermo-sensitive MTP deficiency. Clinical and genetic information was obtained from clinical files. Measurement of LCHAD and LCKAT activities, lcFAO-flux studies and palmitate loading tests were performed in skin fibroblasts cultured at 37°C and 40°C. In all patients (four MTPD, one LCKATD), disease manifested during childhood (manifestation age: 2-10 years) with myopathic symptoms triggered by fever or exercise. In four patients, signs of retinopathy or neuropathy were present. Plasma long-chain acylcarnitines were normal or slightly increased. HADHB variants were identified (at age: 6-18 years) by whole exome sequencing or gene panel analyses. At 37°C, LCHAD and LCKAT activities were mildly impaired and lcFAO-fluxes were normal. Remarkably, enzyme activities and lcFAO-fluxes were markedly diminished at 40°C. Preventive (dietary) measures improved symptoms for most. In conclusion, all patients with thermo-sensitive MTP deficiency had a long diagnostic trajectory and both genetic and enzymatic testing were required for diagnosis. The frequent absence of characteristic acylcarnitine abnormalities poses a risk for a diagnostic delay. Given the positive treatment effects, upfront genetic screening may be beneficial to enhance early recognition.
Assuntos
Erros Inatos do Metabolismo Lipídico , Miopatias Mitocondriais , Doenças Musculares , 3-Hidroxiacil-CoA Desidrogenases , Adolescente , Cardiomiopatias , Criança , Pré-Escolar , Coenzima A , Diagnóstico Tardio , Ácidos Graxos/metabolismo , Humanos , Erros Inatos do Metabolismo Lipídico/diagnóstico , Erros Inatos do Metabolismo Lipídico/genética , Erros Inatos do Metabolismo Lipídico/metabolismo , Miopatias Mitocondriais/diagnóstico , Miopatias Mitocondriais/genética , Proteína Mitocondrial Trifuncional/deficiência , Doenças Musculares/diagnóstico , Doenças Musculares/genética , Doenças do Sistema Nervoso , RabdomióliseRESUMO
PURPOSE: In this study we investigate the disease etiology in 12 patients with de novo variants in FAR1 all resulting in an amino acid change at position 480 (p.Arg480Cys/His/Leu). METHODS: Following next-generation sequencing and clinical phenotyping, functional characterization was performed in patients' fibroblasts using FAR1 enzyme analysis, FAR1 immunoblotting/immunofluorescence, and lipidomics. RESULTS: All patients had spastic paraparesis and bilateral congenital/juvenile cataracts, in most combined with speech and gross motor developmental delay and truncal hypotonia. FAR1 deficiency caused by biallelic variants results in defective ether lipid synthesis and plasmalogen deficiency. In contrast, patients' fibroblasts with the de novo FAR1 variants showed elevated plasmalogen levels. Further functional studies in fibroblasts showed that these variants cause a disruption of the plasmalogen-dependent feedback regulation of FAR1 protein levels leading to uncontrolled ether lipid production. CONCLUSION: Heterozygous de novo variants affecting the Arg480 residue of FAR1 lead to an autosomal dominant disorder with a different disease mechanism than that of recessive FAR1 deficiency and a diametrically opposed biochemical phenotype. Our findings show that for patients with spastic paraparesis and bilateral cataracts, FAR1 should be considered as a candidate gene and added to gene panels for hereditary spastic paraplegia, cerebral palsy, and juvenile cataracts.
Assuntos
Aldeído Oxirredutases/genética , Éteres , Lipídeos , Paraplegia Espástica Hereditária/genética , Humanos , FenótipoRESUMO
BACKGROUND: The high variability in clinical outcome of patients with Classical Galactosemia (CG) is poorly understood and underlines the importance of prognostic biomarkers, which are currently lacking. The aim of this study was to investigate if residual galactose metabolism capacity is associated with clinical and biochemical outcomes in CG patients with varying geno- and phenotypes. METHODS: Galactose Metabolite Profiling (GMP) was used to determine residual galactose metabolism in fibroblasts of CG patients. The association between the galactose index (GI) defined as the ratio of the measured metabolites [U13C]Gal-1-P/ [13C6]UDP-galactose, and both intellectual and neurological outcome and galactose-1-phosphate (Gal-1-P) levels was investigated. RESULTS: GMP was performed in fibroblasts of 28 patients and 3 control subjects. The GI of the classical phenotype patients (nâ¯=â¯22) was significantly higher than the GI of four variant patients detected by newborn screening (NBS) (pâ¯=â¯.002), two homozygous p.Ser135Leu patients (pâ¯=â¯.022) and three controls (pâ¯=â¯.006). In the classical phenotype patients, 13/18 (72%) had a poor intellectual outcome (IQâ¯<â¯85) and 6/12 (50%) had a movement disorder. All the NBS detected variant patients (nâ¯=â¯4) had a normal intellectual outcome (IQâ¯≥â¯85) and none of them has a movement disorder. In the classical phenotype patients, there was no significant difference in GI between patients with a poor and normal clinical outcome. The NBS detected variant patients had significantly lower GI levels and thus higher residual galactose metabolism than patients with classical phenotypes. There was a clear correlation between Gal-1-P levels in erythrocytes and the GI (pâ¯=â¯.001). CONCLUSIONS: The GI was able to distinguish CG patients with varying geno- and phenotypes and correlated with Gal-1-P. The data of the NBS detected variant patients demonstrated that a higher residual galactose metabolism may result in a more favourable clinical outcome. Further research is needed to enable individual prognostication and treatment in all CG patients.
Assuntos
Fibroblastos/metabolismo , Galactose/metabolismo , Galactosemias/diagnóstico , Galactosemias/metabolismo , Estudos de Coortes , Feminino , Galactosemias/genética , Galactosemias/fisiopatologia , Galactosefosfatos/metabolismo , Genótipo , Homozigoto , Humanos , Recém-Nascido , Deficiência Intelectual/diagnóstico , Masculino , Transtornos dos Movimentos/diagnóstico , Triagem Neonatal , FenótipoRESUMO
CTP:phosphoethanolamine cytidylyltransferase (ET), encoded by PCYT2, is the rate-limiting enzyme for phosphatidylethanolamine synthesis via the CDP-ethanolamine pathway. Phosphatidylethanolamine is one of the most abundant membrane lipids and is particularly enriched in the brain. We identified five individuals with biallelic PCYT2 variants clinically characterized by global developmental delay with regression, spastic para- or tetraparesis, epilepsy and progressive cerebral and cerebellar atrophy. Using patient fibroblasts we demonstrated that these variants are hypomorphic, result in altered but residual ET protein levels and concomitant reduced enzyme activity without affecting mRNA levels. The significantly better survival of hypomorphic CRISPR-Cas9 generated pcyt2 zebrafish knockout compared to a complete knockout, in conjunction with previously described data on the Pcyt2 mouse model, indicates that complete loss of ET function may be incompatible with life in vertebrates. Lipidomic analysis revealed profound lipid abnormalities in patient fibroblasts impacting both neutral etherlipid and etherphospholipid metabolism. Plasma lipidomics studies also identified changes in etherlipids that have the potential to be used as biomarkers for ET deficiency. In conclusion, our data establish PCYT2 as a disease gene for a new complex hereditary spastic paraplegia and confirm that etherlipid homeostasis is important for the development and function of the brain.
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Fosfatidiletanolaminas/biossíntese , RNA Nucleotidiltransferases/genética , Paraplegia Espástica Hereditária/genética , Adolescente , Alelos , Animais , Atrofia , Encéfalo/patologia , Criança , Pré-Escolar , Deficiências do Desenvolvimento/genética , Epilepsia/genética , Feminino , Técnicas de Inativação de Genes , Variação Genética , Humanos , Lipidômica , Masculino , Camundongos , RNA Nucleotidiltransferases/deficiência , Adulto Jovem , Peixe-ZebraRESUMO
Primary carnitine deficiency is caused by a defect in the active cellular uptake of carnitine by Na+ -dependent organic cation transporter novel 2 (OCTN2). Genetic diagnostic yield for this metabolic disorder has been relatively low, suggesting that disease-causing variants are missed. We Sanger sequenced the 5' untranslated region (UTR) of SLC22A5 in individuals with possible primary carnitine deficiency in whom no or only one mutant allele had been found. We identified a novel 5'-UTR c.-149G>A variant which we characterized by expression studies with reporter constructs in HeLa cells and by carnitine-transport measurements in fibroblasts using a newly developed sensitive assay based on tandem mass spectrometry. This variant, which we identified in 57 of 236 individuals of our cohort, introduces a functional upstream out-of-frame translation initiation codon. We show that the codon suppresses translation from the wild-type ATG of SLC22A5, resulting in reduced OCTN2 protein levels and concomitantly lower transport activity. With an allele frequency of 24.2% the c.-149G>A variant is the most frequent cause of primary carnitine deficiency in our cohort and may explain other reported cases with an incomplete genetic diagnosis. Individuals carrying this variant should be clinically re-evaluated and monitored to determine if this variant has clinical consequences.
Assuntos
Regiões 5' não Traduzidas , Cardiomiopatias/genética , Carnitina/deficiência , Códon de Iniciação , Predisposição Genética para Doença , Hiperamonemia/genética , Doenças Musculares/genética , Mutação , Membro 5 da Família 22 de Carreadores de Soluto/genética , Alelos , Sequência de Aminoácidos , Sequência de Bases , Transporte Biológico , Cardiomiopatias/diagnóstico , Cardiomiopatias/metabolismo , Carnitina/genética , Carnitina/metabolismo , Linhagem Celular , Frequência do Gene , Genes Reporter , Estudos de Associação Genética , Humanos , Hiperamonemia/diagnóstico , Hiperamonemia/metabolismo , Doenças Musculares/diagnóstico , Doenças Musculares/metabolismo , Membro 5 da Família 22 de Carreadores de Soluto/metabolismoRESUMO
Multiple acyl-CoA dehydrogenase deficiency (MADD) is an ultra-rare inborn error of mitochondrial fatty acid oxidation (FAO) and amino acid metabolism. Individual phenotypes and treatment response can vary markedly. We aimed to identify markers that predict MADD phenotypes. We performed a retrospective nationwide cohort study; then developed an MADD-disease severity scoring system (MADD-DS3) based on signs and symptoms with weighed expert opinions; and finally correlated phenotypes and MADD-DS3 scores to FAO flux (oleate and myristate oxidation rates) and acylcarnitine profiles after palmitate loading in fibroblasts. Eighteen patients, diagnosed between 1989 and 2014, were identified. The MADD-DS3 entails enumeration of eight domain scores, which are calculated by averaging the relevant symptom scores. Lifetime MADD-DS3 scores of patients in our cohort ranged from 0 to 29. FAO flux and [U-13 C]C2-, C5-, and [U-13 C]C16-acylcarnitines were identified as key variables that discriminated neonatal from later onset patients (all P < .05) and strongly correlated to MADD-DS3 scores (oleate: r = -.86; myristate: r = -.91; [U-13 C]C2-acylcarnitine: r = -.96; C5-acylcarnitine: r = .97; [U-13 C]C16-acylcarnitine: r = .98, all P < .01). Functional studies in fibroblasts were found to differentiate between neonatal and later onset MADD-patients and were correlated to MADD-DS3 scores. Our data may improve early prediction of disease severity in order to start (preventive) and follow-up treatment appropriately. This is especially relevant in view of the inclusion of MADD in population newborn screening programs.
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Carnitina/análogos & derivados , Ácidos Graxos/sangue , Deficiência Múltipla de Acil Coenzima A Desidrogenase/fisiopatologia , Índice de Gravidade de Doença , Carnitina/sangue , Feminino , Humanos , Recém-Nascido , Masculino , Deficiência Múltipla de Acil Coenzima A Desidrogenase/sangue , Estudos RetrospectivosRESUMO
In recent years the short-chain fatty acid, 4-phenylbutyrate (PB), has emerged as a promising drug for various clinical conditions. In fact, PB has been Food and Drug Administration-approved for urea cycle disorders since 1996. PB is more potent and less toxic than its metabolite, phenylacetate (PA), and is not just a pro-drug for PA, as was initially assumed. The metabolic pathway of PB, however, has remained unclear. Therefore, we set out to identify the enzymes involved in the ß-oxidation of PB. We used cells deficient in specific steps of fatty acid ß-oxidation and ultra-HPLC to measure which enzymes were able to convert PB or its downstream products. We show that the first step in PB oxidation is catalyzed solely by the enzyme, medium-chain acyl-CoA dehydrogenase. The second (hydration) step can be catalyzed by all three mitochondrial enoyl-CoA hydratase enzymes, i.e., short-chain enoyl-CoA hydratase, long-chain enoyl-CoA hydratase, and 3-methylglutaconyl-CoA hydratase. Enzymes involved in the third step include both short- and long-chain 3-hydroxyacyl-CoA dehydrogenase. The oxidation of PB is completed by only one enzyme, i.e., long-chain 3-ketoacyl-CoA thiolase. Taken together, the enzymatic characteristics of the PB degradative pathway may lead to better dose finding and limiting the toxicity of this drug.
Assuntos
Ensaios Enzimáticos , Enzimas/metabolismo , Fenilbutiratos/metabolismo , OxirreduçãoRESUMO
Ketoacidosis is a potentially lethal condition caused by the imbalance between hepatic production and extrahepatic utilization of ketone bodies. We performed exome sequencing in a patient with recurrent, severe ketoacidosis and identified a homozygous frameshift mutation in the gene encoding monocarboxylate transporter 1 (SLC16A1, also called MCT1). Genetic analysis in 96 patients suspected of having ketolytic defects yielded seven additional inactivating mutations in MCT1, both homozygous and heterozygous. Mutational status was found to be correlated with ketoacidosis severity, MCT1 protein levels, and transport capacity. Thus, MCT1 deficiency is a novel cause of profound ketoacidosis; the present work suggests that MCT1-mediated ketone-body transport is needed to maintain acid-base balance.
Assuntos
Corpos Cetônicos/metabolismo , Cetose/genética , Transportadores de Ácidos Monocarboxílicos/deficiência , Transportadores de Ácidos Monocarboxílicos/genética , Mutação , Simportadores/deficiência , Simportadores/genética , Transporte Biológico , Criança , Pré-Escolar , Mutação da Fase de Leitura , Genótipo , Humanos , Lactente , Cetonas/metabolismo , Transportadores de Ácidos Monocarboxílicos/fisiologia , Polimorfismo de Nucleotídeo Único , Simportadores/fisiologiaRESUMO
BACKGROUND: N-acetylglutamate synthase (NAGS) plays a key role in the removal of ammonia via the urea cycle by catalyzing the synthesis of N-acetylglutamate (NAG), the obligatory cofactor in the carbamyl phosphate synthetase 1 reaction. Enzymatic analysis of NAGS in liver homogenates has remained insensitive and inaccurate, which prompted the development of a novel method. METHODS: UPLC-MS/MS was used in conjunction with stable isotope (N-acetylglutamic-2,3,3,4,4-d5 acid) dilution for the quantitative detection of NAG produced by the NAGS enzyme. The assay conditions were optimized using purified human NAGS and the optimized enzyme conditions were used to measure the activity in mouse liver homogenates. RESULTS: A low signal-to-noise ratio in liver tissue samples was observed due to non-enzymatic formation of N-acetylglutamate and low specific activity, which interfered with quantitative analysis. Quenching of acetyl-CoA immediately after the incubation circumvented this analytical difficulty and allowed accurate and sensitive determination of mammalian NAGS activity. The specificity of the assay was validated by demonstrating a complete deficiency of NAGS in liver homogenates from Nags -/- mice. CONCLUSION: The novel NAGS enzyme assay reported herein can be used for the diagnosis of inherited NAGS deficiency and may also be of value in the study of secondary hyperammonemia present in various inborn errors of metabolism as well as drug treatment.
Assuntos
Aminoácido N-Acetiltransferase/genética , Carbamoil-Fosfato Sintase (Amônia)/genética , Hiperamonemia/diagnóstico , Distúrbios Congênitos do Ciclo da Ureia/diagnóstico , Acetilcoenzima A/metabolismo , Aminoácido N-Acetiltransferase/metabolismo , Animais , Carbamoil-Fosfato Sintase (Amônia)/deficiência , Humanos , Hiperamonemia/genética , Hiperamonemia/metabolismo , Hiperamonemia/fisiopatologia , Fígado/enzimologia , Camundongos , Camundongos Knockout , Espectrometria de Massas em Tandem , Distúrbios Congênitos do Ciclo da Ureia/genética , Distúrbios Congênitos do Ciclo da Ureia/metabolismo , Distúrbios Congênitos do Ciclo da Ureia/fisiopatologiaRESUMO
The importance of mitochondrial fatty acid ß-oxidation (FAO) as a glucose-sparing process is illustrated by patients with inherited defects in FAO, who may present with life-threatening fasting-induced hypoketotic hypoglycemia. It is unknown why peripheral glucose demand outpaces hepatic gluconeogenesis in these patients. In this study, we have systematically addressed the fasting response in long-chain acyl-CoA dehydrogenase-deficient (LCAD KO) mice. We demonstrate that the fasting-induced hypoglycemia in LCAD KO mice was initiated by an increased glucose requirement in peripheral tissues, leading to rapid hepatic glycogen depletion. Gluconeogenesis did not compensate for the increased glucose demand, which was not due to insufficient hepatic glucogenic capacity but rather caused by a shortage in the supply of glucogenic precursors. This shortage in supply was explained by a suppressed glucose-alanine cycle, decreased branched-chain amino acid metabolism and ultimately impaired protein mobilization. We conclude that during fasting, FAO not only serves to spare glucose but is also indispensable for amino acid metabolism, which is essential for the maintenance of adequate glucose production.
Assuntos
Gluconeogênese/genética , Glucose/metabolismo , Hipoglicemia/metabolismo , Oxirredução , Acil-CoA Desidrogenase de Cadeia Longa/deficiência , Acil-CoA Desidrogenase de Cadeia Longa/genética , Acil-CoA Desidrogenase de Cadeia Longa/metabolismo , Aminoácidos/metabolismo , Animais , Modelos Animais de Doenças , Ácidos Graxos/metabolismo , Humanos , Hipoglicemia/genética , Hipoglicemia/patologia , Erros Inatos do Metabolismo Lipídico/metabolismo , Glicogênio Hepático/genética , Glicogênio Hepático/metabolismo , Camundongos , Camundongos Knockout , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Hepáticas/patologiaRESUMO
PURPOSE: Very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency (VLCADD) is an inherited disorder of mitochondrial long-chain fatty acid ß-oxidation (LC-FAO) and is included in many newborn screening (NBS) programs worldwide. Patients may present with hypoketotic hypoglycemia, cardiomyopathy, and/or myopathy, but clinical severity varies widely and the clinical outcome is unpredictable. We investigated predictive markers that may determine clinical severity. METHODS: We developed a clinical severity score (CSS), which was determined for 13 Dutch patients with VLCADD, all of whom were diagnosed before the introduction of VLCADD in NBS to prevent bias from early diagnosis. In cultured skin fibroblasts from these patients, we measured LC-FAO flux (the rate of oleate oxidation), VLCAD activity, and acylcarnitine profiles following palmitate loading. RESULTS: The strongest correlation (r = 0.93; P < 0.0001) was observed between LC-FAO flux and the CSS. VLCAD activity measurement and the C14/C16-to-acylcarnitine ratio correlated much less. A median LC-FAO flux of 6% of control values (range 5.6-6.8%) was associated with cardiomyopathy (P < 0.01), and 32.4% (range 5.6-50.5%) was associated with myopathy (P < 0.05). CONCLUSION: Our results demonstrate a very strong correlation between LC-FAO flux in fibroblasts and the clinical severity of VLCADD. LC-FAO flux measurements may thus predict whether patients are likely to develop symptoms.
Assuntos
3-Hidroxiacil-CoA Desidrogenases/metabolismo , Acetil-CoA C-Aciltransferase/metabolismo , Acil-CoA Desidrogenase de Cadeia Longa/deficiência , Biomarcadores , Isomerases de Ligação Dupla Carbono-Carbono/metabolismo , Enoil-CoA Hidratase/metabolismo , Erros Inatos do Metabolismo Lipídico/metabolismo , Erros Inatos do Metabolismo Lipídico/fisiopatologia , Doenças Mitocondriais/metabolismo , Doenças Mitocondriais/fisiopatologia , Doenças Musculares/metabolismo , Doenças Musculares/fisiopatologia , Racemases e Epimerases/metabolismo , Índice de Gravidade de Doença , Acil-CoA Desidrogenase de Cadeia Longa/metabolismo , Síndrome Congênita de Insuficiência da Medula Óssea , Dinamarca , Humanos , Recém-Nascido , Erros Inatos do Metabolismo Lipídico/diagnóstico , Doenças Mitocondriais/diagnóstico , Doenças Musculares/diagnóstico , Triagem Neonatal , OxirreduçãoRESUMO
Oxidation of unsaturated fatty acids requires the action of auxiliary enzymes, such as Δ(3),Δ(2)-enoyl-CoA isomerases. Here we describe a detailed biochemical, molecular, histological, and evolutionary characterization of Eci3, the fourth member of the mammalian enoyl-CoA isomerase family. Eci3 specifically evolved in rodents after gene duplication of Eci2. Eci3 is with 79% identity homologous to Eci2 and contains a peroxisomal targeting signal type 1. Subcellular fractionation of mouse kidney and immunofluorescence studies revealed a specific peroxisomal localization for Eci3. Expression studies showed that mouse Eci3 is almost exclusively expressed in kidney. By using immunohistochemistry, we found that Eci3 is not only expressed in cells of the proximal tubule, but also in a subset of cells in the tubulointerstitium and the glomerulus. In vitro, Eci3 catalyzed the isomerization of trans-3-nonenoyl-CoA to trans-2-nonenoyl-CoA equally efficient as Eci2, suggesting a role in oxidation of unsaturated fatty acids. However, in contrast to Eci2, in silico gene coexpression and enrichment analysis for Eci3 in kidney did not yield carboxylic acid metabolism, but diverse biological functions, such as ion transport (P=7.1E-3) and tissue morphogenesis (P=1.0E-3). Thus, Eci3 picked up a novel and unexpected role in kidney function during rodent evolution.
Assuntos
Dodecenoil-CoA Isomerase/metabolismo , Rim/enzimologia , Animais , Sequência de Bases , Primers do DNA , Imunofluorescência , Humanos , CamundongosRESUMO
OBJECTIVE: To investigate whether statin therapy affects coenzyme Q10 (CoQ10) status in children with heterozygous familial hypercholesterolemia (FH). STUDY DESIGN: Samples were obtained at baseline (treatment naïve) and after dose titration with rosuvastatin, aiming for a low-density lipoprotein cholesterol level of 110 mg/dL. Twenty-nine patients were treated with 5, 10, or 20 mg of rosuvastatin for a mean period of 29 weeks. RESULTS: We found a significant (32%) decrease in peripheral blood mononuclear cell (PBMC) CoQ10 level (P = .02), but no change in PBMC adenosine triphosphate synthesis (P = .60). Uncorrected plasma CoQ10 values were decreased significantly, by 45% (P < .01). In contrast, ratios of plasma CoQ10/total cholesterol and CoQ10/low-density lipoprotein cholesterol remained equal during treatment. CONCLUSIONS: In children with FH, rosuvastatin causes a significant decrease in cellular PBMC CoQ10 status but does not affect mitochondrial adenosine triphosphate synthesis in children with FH. Further studies should address whether (rare) side effects of statin therapy could be explained by a deterioration in CoQ10 status.
Assuntos
Trifosfato de Adenosina/biossíntese , Fluorbenzenos/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Hiperlipoproteinemia Tipo II/tratamento farmacológico , Pirimidinas/farmacologia , Sulfonamidas/farmacologia , Ubiquinona/análogos & derivados , Adolescente , Criança , Colesterol/sangue , Relação Dose-Resposta a Droga , Humanos , Leucócitos Mononucleares/metabolismo , Mitocôndrias/metabolismo , Países Baixos , Rosuvastatina Cálcica , Ubiquinona/sangue , Ubiquinona/efeitos dos fármacosRESUMO
Many biological systems including the oxidative catabolic pathway for branched-chain amino acids (BCAAs) are affected in vivo by valproate therapy. In this study, we investigated the potential effect of valproic acid (VPA) and some of its metabolites on the metabolism of BCAAs. In vitro studies were performed using isovaleryl-CoA dehydrogenase (IVD), isobutyryl-CoA dehydrogenase (IBD), and short branched-chain acyl-CoA dehydrogenase (SBCAD), enzymes involved in the degradation pathway of leucine, valine, and isoleucine. The enzymatic activities of the three purified human enzymes were measured using optimized high-performance liquid chromatography procedures, and the respective kinetic parameters were determined in the absence and presence of VPA and the corresponding CoA and dephosphoCoA conjugates. Valproyl-CoA and valproyl-dephosphoCoA inhibited IVD activity significantly by a purely competitive mechanism with K(i) values of 74 ± 4 and 170 ± 12 µM, respectively. IBD activity was not affected by any of the tested VPA esters. However, valproyl-CoA did inhibit SBCAD activity by a purely competitive mechanism with a K(i) of 249 ± 29 µM. In addition, valproyl-dephosphoCoA inhibited SBCAD activity via a distinct mechanism (K(i) = 511 ± 96 µM) that appeared to be of the mixed type. Furthermore, we show that both SBCAD and IVD are active, using valproyl-CoA as a substrate. The catalytic efficiency of SBCAD turned out to be much higher than that of IVD, demonstrating that SBCAD is the most probable candidate for the first dehydrogenation step of VPA ß-oxidation. Our data explain some of the effects of valproate on the branched-chain amino acid metabolism and shed new light on the biotransformation pathway of valproate.
Assuntos
Aminoácidos de Cadeia Ramificada/metabolismo , Butiril-CoA Desidrogenase/metabolismo , Isovaleril-CoA Desidrogenase/metabolismo , Ácido Valproico/metabolismo , Cromatografia Líquida de Alta Pressão , Oxirredução , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em TandemRESUMO
BACKGROUND: Variations in the gene ACADS, encoding the mitochondrial protein short-chain acyl CoA-dehydrogenase (SCAD), have been observed in individuals with clinical symptoms. The phenotype of SCAD deficiency (SCADD) is very heterogeneous, ranging from asymptomatic to severe, without a clear genotype-phenotype correlation, which suggests a multifactorial disorder. The pathophysiological relevance of the genetic variations in the SCAD gene is therefore disputed, and has not yet been elucidated, which is an important step in the investigation of SCADD etiology. AIM: To determine whether the disease-associated misfolding variant of SCAD protein, p.Arg107Cys, disturbs mitochondrial function. METHODS: We have developed a cell model system, stably expressing either the SCAD wild-type protein or the misfolding SCAD variant protein, p.Arg107Cys (c.319 C > T). The model system was used for investigation of SCAD with respect to expression, degree of misfolding, and enzymatic SCAD activity. Furthermore, cell proliferation and expression of selected stress response genes were investigated as well as proteomic analysis of mitochondria-enriched extracts in order to study the consequences of p.Arg107Cys protein expression using a global approach. CONCLUSIONS: We found that expression of the p.Arg107Cys variant SCAD protein gives rise to inactive misfolded protein species, eliciting a mild toxic response manifested though a decreased proliferation rate and oxidative stress, as shown by an increased demand for the mitochondrial antioxidant SOD2. In addition, we found markers of apoptotic activity in the p.Arg107Cys expressing cells, which points to a possible pathophysiological role of this variant protein.
Assuntos
Butiril-CoA Desidrogenase/metabolismo , Doenças Mitocondriais/metabolismo , Proteínas Mitocondriais/química , Animais , Antioxidantes/química , Butiril-CoA Desidrogenase/química , Butiril-CoA Desidrogenase/toxicidade , Proliferação de Células , Variação Genética , Genótipo , Humanos , Camundongos , Estresse Oxidativo , Fenótipo , Desnaturação Proteica , Dobramento de Proteína , Proteômica/métodosRESUMO
Several mouse models for mitochondrial fatty acid beta-oxidation (FAO) defects have been developed. So far, these models have contributed little to our current understanding of the pathophysiology. The objective of this study was to explore differences between murine and human FAO. Using a combination of analytical, biochemical and molecular methods, we compared fibroblasts of long chain acyl-CoA dehydrogenase knockout (LCAD(-/-)), very long chain acyl-CoA dehydrogenase knockout (VLCAD(-/-)) and wild type mice with fibroblasts of VLCAD-deficient patients and human controls. We show that in mice, LCAD and VLCAD have overlapping and distinct roles in FAO. The absence of VLCAD is apparently fully compensated, whereas LCAD deficiency is not. LCAD plays an essential role in the oxidation of unsaturated fatty acids such as oleic acid, but seems redundant in the oxidation of saturated fatty acids. In strong contrast, LCAD is neither detectable at the mRNA level nor at the protein level in men, making VLCAD indispensable in FAO. Our findings open new avenues to employ the existing mouse models to study the pathophysiology of human FAO defects.
Assuntos
Ácidos Graxos/metabolismo , Mitocôndrias/metabolismo , Acil-CoA Desidrogenase de Cadeia Longa/deficiência , Animais , Carnitina/análogos & derivados , Carnitina/química , Carnitina/metabolismo , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Fibroblastos/enzimologia , Humanos , Camundongos , Oxirredução , Espectrometria de Massas em TandemRESUMO
Oxidation of fatty acids in mitochondria is a key physiological process in higher eukaryotes including humans. The importance of the mitochondrial beta-oxidation system in humans is exemplified by the existence of a group of genetic diseases in man caused by an impairment in the mitochondrial oxidation of fatty acids. Identification of patients with a defect in mitochondrial beta-oxidation has long remained notoriously difficult, but the introduction of tandem-mass spectrometry in laboratories for genetic metabolic diseases has revolutionalized the field by allowing the rapid and sensitive analysis of acylcarnitines. Equally important is that much progress has been made with respect to the development of specific enzyme assays to identify the enzyme defect in patients subsequently followed by genetic analysis. In this review, we will describe the current state of knowledge in the field of fatty acid oxidation enzymology and its application to the follow-up analysis of positive neonatal screening results.