RESUMO
Hereditary tyrosinemia type 1 (HT1) is a severe human autosomal recessive disorder caused by the deficiency of fumarylacetoacetate hydroxylase (FAH), an enzyme catalyzing the last step in the tyrosine degradation pathway. Lack of FAH causes accumulation of toxic metabolites (fumarylacetoacetate and succinylacetone) in blood and tissues, ultimately resulting in severe liver and kidney damage with onset that ranges from infancy to adolescence. This tissue damage is lethal but can be controlled by administration of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC), which inhibits tyrosine catabolism upstream of the generation of fumarylacetoacetate and succinylacetone. Notably, in animals lacking FAH, transient withdrawal of NTBC can be used to induce liver damage and a concomitant regenerative response that stimulates the growth of healthy hepatocytes. Among other things, this model has raised tremendous interest for the in vivo expansion of human primary hepatocytes inside these animals and for exploring experimental gene therapy and cell-based therapies. Here, we report the generation of FAH knock-out rabbits via pronuclear stage embryo microinjection of transcription activator-like effector nucleases. FAH-/- rabbits exhibit phenotypic features of HT1 including liver and kidney abnormalities but additionally develop frequent ocular manifestations likely caused by local accumulation of tyrosine upon NTBC administration. We also show that allogeneic transplantation of wild-type rabbit primary hepatocytes into FAH-/- rabbits enables highly efficient liver repopulation and prevents liver insufficiency and death. Because of significant advantages over rodents and their ease of breeding, maintenance, and manipulation compared with larger animals including pigs, FAH-/- rabbits are an attractive alternative for modeling the consequences of HT1.
Assuntos
Hidrolases/genética , Tirosinemias/genética , Animais , Modelos Animais de Doenças , Feminino , Técnicas de Inativação de Genes , Hepatócitos/transplante , Humanos , Hidrolases/metabolismo , Rim/metabolismo , Rim/patologia , Fígado/metabolismo , Fígado/patologia , Falência Hepática/etiologia , Falência Hepática/metabolismo , Falência Hepática/patologia , Falência Hepática/terapia , Masculino , Coelhos , Tirosinemias/complicações , Tirosinemias/metabolismo , Tirosinemias/patologiaRESUMO
Aldosterone is a mineralocorticoid steroid hormone, the measurement of which in the clinical laboratory is principally performed for the investigation of primary hyperaldosteronism. Primary hyperaldosteronism is a specifically treatable and potentially curable form of hypertension, which typically presents as drug-resistant hypertension and, in up to 37% of cases, hypokalemia. Accurate measurement of aldosterone concentration is essential for correct diagnosis. The serum concentrations of aldosterone are in the picomolar range and therefore sensitive aldosterone assays are required. With the advancement in instrumentation of LC-MS/MS, the picomolar range of aldosterone can be easily measured by the newer models, but for those with a less sensitive instrument, special technique for sample preparation to enhance assay sensitivity is required. This work described the use of charge-tagging for the picomolar measurement of serum aldosterone in a less sensitive LC-MS/MS instrument. The assay was linear up to 3000 pmol/L with lower limit of quantitation at 80 pmol/L. The mean relative recovery was 96.5% with a range of 89.3-101.6% for aqueous calibrators and the mean relative recovery was 94.8% with a range of 87.5-101.4% for serum calibrators. Intra-assay CVs range from 8.2% to 11.3%, and inter-assay CVs ranged from 8.5% to 13.5% at concentration range from 229 to 1720 pmol/L. The LC-MS/MS method compared well (y = 1.04x + 8.97) with the in-use radioimmunoassay method. There was no significant difference found (p = 0.7135) between results determined by LC-MS/MS and radioimmunoassay method.
Assuntos
Aldosterona/sangue , Hiperaldosteronismo/sangue , Espectrometria de Massas em Tandem/métodos , Adulto , Idoso , Calibragem , Cromatografia Líquida/métodos , Feminino , Humanos , Hiperaldosteronismo/diagnóstico , Limite de Detecção , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Adulto JovemRESUMO
Influenza-associated encephalopathy (IAE) is a potentially fatal neurological complication of influenza infection usually in the presence of high and persistent fever. Thermolabile carnitine palmitoyltransferase II enzyme (CPT-II) predisposes IAE, so far only described in Japanese. As the genetic origins of Japanese and Chinese are alike, similar genetic risk factors in CPT-II are expected. We report the first two unrelated Chinese patients of thermolabile CPT-II variants that underlain the persistent high fever-triggered viral infection-associated encephalopathy, multi-organ failure and death. Elevated (C16:0+C18:1)/C2 acylcarnitines ratio and the CPT2 susceptibility variant allele [p.Phe352Cys; p.Val368Ile] were detected. The asymptomatic family members of one patient also had abnormal long-chain acylcarnitines. In our experience of biochemical genetics, the elevated (C16:0+C18:1)/C2 acylcarnitines ratio is unusual and specific for thermolabile CPT-II variants. Allele frequency of [p.Phe352Cys; p.Val368Ile] among Hong Kong Chinese was 0.104, similar to Japanese data, and [p.Phe352Cys] has not been reported in Caucasians. This may explain the Asian-specific phenomenon of thermolabile CPT-II-associated IAE. We successfully demonstrated the thermolabile CPT-II variants in patients with viral infection-associated encephalopathy in another Asian population outside Japanese. The condition is likely under-recognized. With our first cases, it is envisaged that more cases will be diagnosed in subsequent years. The exact pathogenic mechanism of how other factors interplay with thermolabile CPT-II variants and high fever leading to IAE, is yet to be elucidated. Fasting and decreased intake during illness may aggravate the disease. Further studies including high risk and neonatal screening are warranted to investigate its expressivity, penetrance and temperature-dependent behaviors in thermolabile CPT-II carriers. This may lead to discovery of the therapeutic golden window by aggressive antipyretics and L-carnitine administration in avoiding the high mortality and morbidity of IAE.
Assuntos
Carnitina O-Palmitoiltransferase/metabolismo , Encefalite Viral/enzimologia , Influenza Humana/complicações , Substituição de Aminoácidos , Sequência de Bases , Carnitina/análogos & derivados , Carnitina/metabolismo , Carnitina O-Palmitoiltransferase/genética , Pré-Escolar , Análise Mutacional de DNA , Encefalite Viral/complicações , Encefalite Viral/genética , Estabilidade Enzimática , Saúde da Família , Evolução Fatal , Feminino , Predisposição Genética para Doença/genética , Genótipo , Humanos , Masculino , Dados de Sequência Molecular , Mutação , Linhagem , Fatores de Risco , TemperaturaRESUMO
Metformin is a widely used antidiabetic agent that is generally considered safe. However, metformin-associated lactic acidosis (MALA), though not common, occurs from time to time and results in significantly high mortality. A series of 23 MALA cases in a local major hospital in Hong Kong is reported in this article to demonstrate the epidemiological data, risk factors, clinical features as well as the clinical outcomes for better understanding of this disease entity. It is the first MALA case series in which plasma metformin levels were assessed. However, the results show that plasma metformin levels in MALA bear no diagnostic and prognostic values. Risk factors of mortality were identified as shock and high plasma lactate levels. The majority of patients were found to have significantly raised creatinine versus a normal baseline value before the acute illness. Concomitant illnesses taking place alongside MALA were common. With a high utility rate of renal replacement therapy (82.6%) in the study group, the mortality rate was 30.4%.
Assuntos
Acidose Láctica/induzido quimicamente , Diabetes Mellitus Tipo 2/complicações , Hipoglicemiantes/efeitos adversos , Metformina/efeitos adversos , Acidose Láctica/sangue , Acidose Láctica/epidemiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Amilases/sangue , Povo Asiático , China/epidemiologia , Creatinina/sangue , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/epidemiologia , Diabetes Mellitus Tipo 2/terapia , Overdose de Drogas , Feminino , Cardiopatias/epidemiologia , Humanos , Hipertensão/epidemiologia , Hipoglicemiantes/sangue , Nefropatias/epidemiologia , Masculino , Metformina/sangue , Pessoa de Meia-Idade , Terapia de Substituição Renal , Fatores de Risco , Acidente Vascular Cerebral/epidemiologiaRESUMO
Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was applied to the direct analysis of melamine cyanurate (MC). The three commonly used MALDI matrixes, namely, alpha-cyano-4-hydroxycinnamic acid (CHCA), sinapinic acid (SA), and 2,5-dihydroxybenzoic acid (DHB), were able to desorb/ionize melamine from MC upon N(2) laser irradiation, with CHCA showing the highest detection sensitivity in the positive mode. Only DHB and SA were able to desorb/ionize cyanuric acid from MC in the negative mode but with remarkably lower sensitivity. The method is able to detect melamine unambiguously from a small amount of MC (down to 12.5 microg) spiked into urine and was successfully applied for the rapid and sensitive detection of melamine in urine stones/residues of the samples collected from patients clinically confirmed of having kidney stones associated with the consumption of melamine-tainted food products. The urine matrix resulted in interfering ion peaks and suppressed the ion intensity of melamine, while a cleanup process consisting of simply washing with water eliminated such interference and enhanced the ion intensity. The merit of the method is simplicity in sample preparation. The analytical time of the method for high-throughput analysis from the time of sample treatment to analysis is less than 7 minutes per sample, with sensitive detection of the presence of melamine in the urine stones/residues of the patient samples.
Assuntos
Triazinas/urina , Criança , Contaminação de Alimentos , Humanos , Cálculos Renais/etiologia , Cálculos Renais/urina , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Triazinas/análise , Triazinas/químicaRESUMO
Cough mixture abuse has been reported to cause severe folate deficiency and neurological defects. We carried out a prospective case-controlled survey to confirm this association and define the incidence and severity of the problem. A total of 57 cough mixture abusers and 47 other substance abusers (controls) were studied. When compared with controls, cough mixture abusers had a high incidence of low folate levels that could only be detected by screening.
Assuntos
Antitussígenos/efeitos adversos , Codeína/efeitos adversos , Dextrometorfano/efeitos adversos , Deficiência de Ácido Fólico/induzido quimicamente , Transtornos Relacionados ao Uso de Substâncias/complicações , Adulto , Estudos de Casos e Controles , Coleta de Dados , Cárie Dentária/induzido quimicamente , Índices de Eritrócitos , Feminino , Deficiência de Ácido Fólico/sangue , Deficiência de Ácido Fólico/epidemiologia , Hemoglobinas/análise , Hong Kong/epidemiologia , Humanos , Incidência , Masculino , Programas de Rastreamento , Doenças do Sistema Nervoso/induzido quimicamente , Contagem de Plaquetas , Estudos Prospectivos , Transtornos Relacionados ao Uso de Substâncias/sangue , Transtornos Relacionados ao Uso de Substâncias/epidemiologia , Deficiência de Vitamina B 12/sangue , Deficiência de Vitamina B 12/induzido quimicamente , Deficiência de Vitamina B 12/epidemiologiaRESUMO
The conventional method for the determination of mercury in clinical samples is cold vapor atomic absorption spectrometry. Sample digestion or pretreatment require large sample volume and long sample preparation time. The inductively coupled plasma mass spectrometry (ICP-MS) method developed in this study requires only 100 microL of sample with practically no preparation, except for dilution with diluent. Significant savings in sample volumes, reagents, technician time, and analysis time are realized. Among different types of diluents, the one containing acid, tert-butanol, and potassium dichromate gave the best results to remove the mercury memory effect. The interassay precisions for whole blood and urine were < 5% and < 8%, respectively, and the intra-assay precisions were < 3% and < 7%, respectively. The lower limits of detection were 0.13, 0.17, and 0.26 microg/L for aqueous standard, urine, and whole blood, respectively. The developed ICP-MS method correlated well with the atomic absorption method and can offer an alternative to the atomic absorption method for mercury analysis with less sample volume requirement as well as shorter analysis time.
Assuntos
Poluentes Ambientais/sangue , Poluentes Ambientais/urina , Mercúrio/sangue , Mercúrio/urina , Humanos , Ácido Clorídrico , Indicadores e Reagentes , Espectrometria de Massas , Dicromato de Potássio , terc-Butil ÁlcoolAssuntos
Nefropatias/genética , Síndrome Metabólica/genética , Síndrome Metabólica/metabolismo , Transportadores de Ânions Orgânicos/genética , Proteínas de Transporte de Cátions Orgânicos/genética , Ácido Úrico/metabolismo , Adulto , Idoso , Creatinina/sangue , Análise Mutacional de DNA , Diabetes Mellitus Tipo 2/genética , Família , Feminino , Humanos , Nefropatias/metabolismo , Testes de Função Renal , Masculino , Mutação/genéticaRESUMO
BACKGROUND: Carnitine palmitoyltransferase II (CPT II) deficiency is one of the most common disorders of oxidative fatty acid metabolism. In this disorder, long-chain acylcarnitines cannot be converted to acyl CoA and there is impairment of ß-oxidation of fatty acids. RESULTS: In the 3 distinct clinical subtypes of CPT II deficiency, adult onset myopathic form shows mild clinical manifestations, characterized by recurrent rhabdomyolysis after intense physical stress. In this study, we report a case with adult myopathic form of CPT II deficiency presenting recurrent exercise-induced myoglobinuria. CONCLUSION: The acylcarnitine profile showed characteristic CPTII deficiency profile and sequencing of the CPT2 gene showed 2 novel missense mutations p. H369Q and p G497S.