RESUMEN
BACKGROUND: Mitochondrial neurogastrointestinal encephalopathy (MNGIE), due to mutations in TYMP, often presents with gastrointestinal symptoms. Two sisters, initially managed for Crohn's disease based upon clinical, imaging and pathological findings, were later found to have MNGIE. The cases provide novel clinicopathological insight, for two further reasons: both sisters remain ambulant and in employment in their late 20s and 30s; diagnosis in one sister was made after a suspected azathioprine-precipitated acute illness. CASE PRESENTATION: A 25-year-old female presented with diarrhoea, vomiting, abdominal pain, and bloating. Faecal calprotectin, colonic biopsies and magnetic resonance enterography were consistent with a diagnosis of Crohn's disease. Azathioprine initiation preceded admission with a sore throat, headache, myalgia, and pyrexia. Withdrawal led to rapid clinical improvement. MRI brain revealed persistent, extensive white matter changes. Elevated plasma and urine thymidine and deoxyuridine, and genetic testing for TYMP variants, confirmed MNGIE. Testing of the patient's sister, also diagnosed with Crohn's disease, revealed identical variants. In this context, retrospective review of colonic biopsies identified histological findings suggestive of MNGIE. CONCLUSIONS: Azathioprine interference in nucleic acid metabolism may interact with the mitochondrial DNA depletion of MNGIE. Nucleotide supplementation, proposed for treatment by manipulating mitochondrial nucleoside pools, may require caution. The late onset and mild phenotype observed confirms presentation can occur later in life, and may reflect residual thymidine phosphorylase activity. Clinicians should consider measuring plasma thymidine levels in suspected Crohn's disease to rule out MNGIE, particularly if white matter abnormalities are identified on neuroimaging.
Asunto(s)
Enfermedad de Crohn/diagnóstico , Enfermedades Gastrointestinales/diagnóstico , Enfermedades Gastrointestinales/patología , Encefalomiopatías Mitocondriales/diagnóstico , Encefalomiopatías Mitocondriales/patología , Adulto , Edad de Inicio , Azatioprina/efectos adversos , Desoxiuridina/sangre , Desoxiuridina/orina , Diagnóstico Diferencial , Femenino , Humanos , Fenotipo , Mutación Puntual , Estudios Retrospectivos , Timidina/sangre , Timidina/orina , Timidina Fosforilasa/genética , Sustancia Blanca/patologíaRESUMEN
Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare, autosomal-recessive mitochondrial disorder caused by TYMP mutations presenting with a multisystemic, often lethal syndrome of progressive leukoencephalopathy, ophthalmoparesis, demyelinating neuropathy, cachexia and gastrointestinal dysmotility. Hemodialysis (HMD) has been suggested as a treatment to reduce accumulation of thymidine and deoxyuridine. However, all studies so far have failed to measure the toxic metabolites in cerebrospinal fluid (CSF), which is the crucial compartment for CNS damage.Our study is the first prospective, longitudinal investigation, exploiting detailed serial testing of predefined clinical and molecular outcome parameters (including serial CSF assessments) in a 29-year-old MNGIE patient undergoing 1 year of extensive HMD. We demonstrate that HMD only transiently restores increased serum and urine levels of thymidine and deoxyuridine, but fails to reduce CSF levels of the toxic metabolites and is ineffective to influence neurological function. These findings have direct important implications for clinical practice: They prevent a burdensome, long-term invasive, but ultimately probably ineffective procedure in future MNGIE patients.
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Enfermedades del Sistema Nervioso Central/etiología , Desoxiuridina/sangre , Seudoobstrucción Intestinal/terapia , Encefalomiopatías Mitocondriales/terapia , Diálisis Renal , Timidina/sangre , Adulto , Enfermedades del Sistema Nervioso Central/patología , Enfermedades del Sistema Nervioso Central/terapia , Desoxiuridina/orina , Humanos , Seudoobstrucción Intestinal/genética , Seudoobstrucción Intestinal/patología , Masculino , Encefalomiopatías Mitocondriales/genética , Encefalomiopatías Mitocondriales/patología , Distrofia Muscular Oculofaríngea , Mutación , Oftalmoplejía/congénito , Enfermedades Raras , Timidina/orina , Timidina FosforilasaRESUMEN
The objectives of this study were to evaluate the safety, tolerability and pharmacokinetics (PK) of BMS-986001 as a single oral dose in healthy male subjects. Sixty-four healthy male subjects were randomized to receive a single dose of BMS-986001 or placebo in this single-blind, placebo-controlled, sequential ascending-dose study. There were eight treatment groups (10, 30, 100, 300, 600, and 900 mg fed; and 100 and 300 mg fasted) of eight subjects each (BMS-986001 n = 6/placebo n = 2). BMS-986001 was well tolerated, with no serious adverse events (AEs), deaths, or discontinuations due to AEs reported. AEs were experienced by 14.6% of subjects receiving BMS-986001; however, these did not appear to be dose related and were not considered to be related to study drug. BMS-986001 was rapidly absorbed and exhibited a linear dose-exposure relationship across the dose range studied. PK appeared similar whether administered with or without food. Administration of BMS-986001 as a single dose was generally safe and well tolerated. A linear dose-exposure relationship was seen across all doses studied, with no apparent food effect. Further clinical development is warranted.
Asunto(s)
Fármacos Anti-VIH/farmacocinética , Interacciones Alimento-Droga , Inhibidores de la Transcriptasa Inversa/farmacocinética , Timidina/análogos & derivados , Adulto , Fármacos Anti-VIH/efectos adversos , Fármacos Anti-VIH/sangre , Fármacos Anti-VIH/orina , Relación Dosis-Respuesta a Droga , Ayuno/metabolismo , Voluntarios Sanos , Humanos , Masculino , Persona de Mediana Edad , Inhibidores de la Transcriptasa Inversa/efectos adversos , Inhibidores de la Transcriptasa Inversa/sangre , Inhibidores de la Transcriptasa Inversa/orina , Método Simple Ciego , Timidina/efectos adversos , Timidina/sangre , Timidina/farmacocinética , Timidina/orina , Adulto JovenRESUMEN
Manufacturers have developed prototype cigarettes yielding reduced levels of some tobacco smoke toxicants, when tested using laboratory machine smoking under standardised conditions. For the scientific assessment of modified risk tobacco products, tests that offer objective, reproducible data, which can be obtained in a much shorter time than the requirements of conventional epidemiology are needed. In this review, we consider whether biomarkers of biological effect related to oxidative stress can be used in this role. Based on published data, urinary 8-oxo-7,8-dihydro-2-deoxyguanosine, thymidine glycol, F2-isoprostanes, serum dehydroascorbic acid to ascorbic acid ratio and carotenoid concentrations show promise, while 4-hydroxynonenal requires further qualification.
Asunto(s)
Desoxiguanosina/análogos & derivados , F2-Isoprostanos/orina , Neoplasias Pulmonares/diagnóstico , Timidina/análogos & derivados , Productos de Tabaco/efectos adversos , 8-Hidroxi-2'-Desoxicoguanosina , Aldehídos/orina , Ácido Ascórbico/sangre , Biomarcadores/sangre , Biomarcadores/orina , Carotenoides/sangre , Ácido Deshidroascórbico/sangre , Desoxiguanosina/orina , Humanos , Neoplasias Pulmonares/sangre , Neoplasias Pulmonares/etiología , Neoplasias Pulmonares/orina , Estrés Oxidativo , Riesgo , Fumar/efectos adversos , Timidina/orina , Productos de Tabaco/análisis , Contaminación por Humo de TabacoRESUMEN
We describe detailed methods to measure thymidine (dThd) and deoxyuridine (dUrd) concentrations and thymidine phosphorylase (TP) activity in biological samples. These protocols allow the detection of TP dysfunction in patients with mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). Since the identification of mutations in TYMP, the gene encoding TP, as the cause of MNGIE (Nishino et al. Science 283:689-692, 1999), the assessment of TP dysfunction has become the best screening method to rule out or confirm MNGIE in patients. TYMP sequencing, to find the causative mutations, is only needed when TP dysfunction is detected. dThd and dUrd are measured by resolving these compounds with high-performance liquid chromatography (HPLC) followed by the spectrophotometric monitoring of the eluate absorbance at 267 nm (HPLC-UV). TP activity can be measured by an endpoint determination of the thymine formed after 1 h incubation of the buffy coat homogenate in the presence of a large excess of its substrate dThd, either spectrophotometrically or by HPLC-UV.
Asunto(s)
Desoxiuridina/sangre , Pruebas de Enzimas/métodos , Timidina Fosforilasa/sangre , Timidina Fosforilasa/metabolismo , Timidina/sangre , Métodos Analíticos de la Preparación de la Muestra , Desoxiuridina/orina , Humanos , Seudoobstrucción Intestinal/sangre , Seudoobstrucción Intestinal/enzimología , Seudoobstrucción Intestinal/orina , Encefalomiopatías Mitocondriales/sangre , Encefalomiopatías Mitocondriales/enzimología , Encefalomiopatías Mitocondriales/orina , Distrofia Muscular Oculofaríngea , Oftalmoplejía/congénito , Timidina/orinaRESUMEN
Blueberries are rich in antioxidants known as anthocyanins, which may exhibit significant health benefits. Strenous exercise is known to acutely generate oxidative stress and an inflammatory state, and serves as an on-demand model to test antioxidant and anti-inflammatory compounds. The purpose of this study was to examine whether 250 g of blueberries per day for 6 weeks and 375 g given 1 h prior to 2.5 h of running at â¼72% maximal oxygen consumption counters oxidative stress, inflammation, and immune changes. Twenty-five well-trained subjects were recruited and randomized into blueberry (BB) (N = 13) or control (CON) (N = 12) groups. Blood, muscle, and urine samples were obtained pre-exercise and immediately postexercise, and blood and urine 1 h postexercise. Blood was examined for F2-isoprostanes for oxidative stress, cortisol, cytokines, homocysteine, leukocytes, T-cell function, natural killer (NK), and lymphocyte cell counts for inflammation and immune system activation, and ferric reducing ability of plasma for antioxidant capacity. Muscle biopsies were examined for glycogen and NFkB expression to evaluate stress and inflammation. Urine was tested for modification of DNA (8-OHDG) and RNA (5-OHMU) as markers of nucleic acid oxidation. A 2 (treatment) × 3 (time) repeated measures ANOVA was used for statistical analysis. Increases in F2-isoprostanes and 5-OHMU were significantly less in BB and plasma IL-10 and NK cell counts were significantly greater in BB vs. CON. Changes in all other markers did not differ. This study indicates that daily blueberry consumption for 6 weeks increases NK cell counts, and acute ingestion reduces oxidative stress and increases anti-inflammatory cytokines.
Asunto(s)
Antioxidantes/uso terapéutico , Arándanos Azules (Planta) , Ejercicio Físico , Frutas , Células Asesinas Naturales/inmunología , Miositis/prevención & control , Estrés Oxidativo , Adolescente , Adulto , Antiinflamatorios no Esteroideos/uso terapéutico , Antioxidantes/análisis , Atletas , Biomarcadores/sangre , Biomarcadores/orina , Arándanos Azules (Planta)/química , F2-Isoprostanos/sangre , Frutas/química , Humanos , Interleucina-10/sangre , Recuento de Linfocitos , Miositis/sangre , Miositis/inmunología , Miositis/orina , Resistencia Física , Carrera , Timidina/análogos & derivados , Timidina/orina , Adulto JovenRESUMEN
Mitochondrial neurogastrointestinal encephalomyopathy is an autosomal recessive disorder in which a nuclear mutation of the thymidine phosphorylase gene leads to mitochondrial genomic dysfunction. Herein, we report a 29-year-old Iranian man with abdominal pain, diarrhea, hearing loss, ophthalmoplegia, sensorimotor axonal neuropathy, and elevated muscle enzymes. Magnetic resonance imaging showed leukoencephalopathic changes. Metabolite analysis revealed a very high thymidine concentration in the patient's urine consistent with the diagnosis of mitochondrial neurogastrointestinal encephalomyopathy.
Asunto(s)
Enfermedades Gastrointestinales/diagnóstico , Encefalomiopatías Mitocondriales/diagnóstico , Mutación , Timidina Fosforilasa/genética , Adulto , Enfermedades Gastrointestinales/genética , Humanos , Imagen por Resonancia Magnética , Masculino , Encefalomiopatías Mitocondriales/genética , Timidina/orinaRESUMEN
BACKGROUND: The objective of this study was to obtain baseline data on biomarkers of exposure (BoE) and biomarkers of potential harm (BoPH) in smokers, former smokers and never-smokers. METHODS: This was a cross-sectional study of 80 healthy male and female volunteers over 21 years old, self-selected for smoking status. Subjects were prescreened by medical staff at an independent clinical research unit, within 1 week prior to a single overnight residential visit and sample collection. RESULTS: All BoE were able to differentiate between the two smoking groups and smokers from all nonsmokers. There was a strong correlation between cigarettes smoked per day and total urinary nicotine equivalents (TNE; r=0.85). TNE correlated better with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol levels than cigarettes smoked per day (r=0.75 and r=0.56, respectively). Of the BoPH included in this study, seven (11-dehydro-thromboxane B2, 2, 3-dinorthrom-boxane B2, 8-epi prostaglandin F(2alpha), 8-hydroxy-2 deoxyguanosine, cis-thymidine glycol, low-density lipoprotein cholesterol and IgG) were significantly different between the group who smoked more cigarettes per day and never-smokers. These differences became more apparent and extended to the group who smoked 10 or less cigarettes per day, when total urinary recovery values were corrected for creatinine clearance. CONCLUSIONS: While BoE clearly differentiate between groups based on self-declared smoking status, most BoPH examined could not do so in a consistent manner. The dynamics of BoPH levels are not well understood. Future studies of BoPH should eliminate potential confounding factors and increase the number of subjects to allow the investigation of genetic polymorphism in metabolic pathways.
Asunto(s)
Biomarcadores/sangre , Biomarcadores/orina , Humo/efectos adversos , Cese del Hábito de Fumar , Fumar/sangre , Fumar/orina , 8-Hidroxi-2'-Desoxicoguanosina , Adulto , Anciano , LDL-Colesterol/sangre , LDL-Colesterol/orina , Estudios Transversales , Desoxiguanosina/análogos & derivados , Desoxiguanosina/sangre , Desoxiguanosina/orina , Dinoprost/análogos & derivados , Dinoprost/sangre , Dinoprost/orina , Femenino , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/orina , Masculino , Persona de Mediana Edad , Nicotina/orina , Nitrosaminas/sangre , Nitrosaminas/orina , Piridinas/sangre , Piridinas/orina , Reproducibilidad de los Resultados , Tromboxano B2/análogos & derivados , Tromboxano B2/sangre , Tromboxano B2/orina , Timidina/análogos & derivados , Timidina/sangre , Timidina/orinaRESUMEN
Gamma-radiation exposure of humans is a major public health concern as the threat of terrorism and potential hostile use of radiological devices increases worldwide. We report here the effects of sublethal gamma-radiation exposure on the mouse urinary metabolome determined using ultra-performance liquid chromatography-coupled time-of-flight mass spectrometry-based metabolomics. Five urinary biomarkers of sublethal radiation exposure that were statistically significantly elevated during the first 24 h after exposure to doses ranging from 1 to 3 Gy were unequivocally identified by tandem mass spectrometry. These are deaminated purine and pyrimidine derivatives, namely, thymidine, 2'-deoxyuridine, 2'-deoxyxanthosine, xanthine and xanthosine. Furthermore, the aminopyrimidine 2'-deoxycytidine appeared to display reduced urinary excretion at 2 and 3 Gy. The elevated biomarkers displayed a time-dependent excretion, peaking in urine at 8-12 h but returning to baseline by 36 h after exposure. It is proposed that 2'-deoxyuridine and 2'-deoxyxanthosine arise as a result of gamma irradiation by nitrosative deamination of 2'-deoxycytidine and 2'-deoxyguanosine, respectively, and that this further leads to increased synthesis of thymidine, xanthine and xanthosine. The urinary excretion of deaminated purines and pyrimidines, at the expense of aminopurines and aminopyrimidines, appears to form the core of the urinary radiation metabolomic signature of mice exposed to sublethal doses of ionizing radiation.
Asunto(s)
Rayos gamma/efectos adversos , Purinas/orina , Pirimidinas/orina , Análisis de Varianza , Animales , Biomarcadores/orina , Desaminación , Desoxirribonucleósidos/orina , Desoxiuridina/orina , Relación Dosis-Respuesta en la Radiación , Masculino , Ratones , Ratones Endogámicos C57BL , Análisis Multivariante , Purinas/metabolismo , Pirimidinas/metabolismo , Ribonucleósidos/orina , Espectrometría de Masas en Tándem , Timidina/orina , Factores de Tiempo , Xantina/orina , XantinasRESUMEN
OBJECTIVE: Evaluation of altered urinary nucleosides before and after tumor removal of breast cancer (BCa). DESIGN AND METHODS: Targeted metabolite profiling of 14 urinary nucleosides was conducted with both pre- and post-operative female patients with BCa (n=150, age: 46.6+/-7.7 years), and female controls (n=150, age: 46.8+/-7.7 years) by liquid chromatography-tandem mass spectrometry coupled to on-line extraction. RESULTS: Levels of modified nucleosides (5-hydroxymethyl-2'-deoxyuridine, P<0.001; 8-hydroxy-2'-deoxyguanosine, P<0.001; 1-methyladenosine, P<0.02; N(2),N(2)-dimethylguanosine, P<0.001) were significantly higher in pre-operative patients than in both normal controls and post-operative patients. CONCLUSIONS: This approach could be used to further understand the pathogenesis of BCa as well as to evaluate the effects of medical treatment.
Asunto(s)
Neoplasias de la Mama , Mastectomía , Nucleósidos/orina , 8-Hidroxi-2'-Desoxicoguanosina , Adenosina/análogos & derivados , Adenosina/orina , Adulto , Neoplasias de la Mama/cirugía , Neoplasias de la Mama/orina , Cromatografía Liquida , Desoxiguanosina/análogos & derivados , Desoxiguanosina/orina , Femenino , Guanosina/análogos & derivados , Guanosina/orina , Humanos , Persona de Mediana Edad , Estrés Oxidativo/fisiología , Periodo Posoperatorio , Especies Reactivas de Oxígeno/metabolismo , Espectrometría de Masas en Tándem , Timidina/análogos & derivados , Timidina/orinaRESUMEN
Modified nucleosides are formed by DNA repair as a result of oxidative DNA damage and post-transcriptionally modified tRNA in cells. In the present study, profiling of 14 nucleoside was investigated in rat urine to evaluate bisphenol A (BPA)-induced oxidative stress after intraperitoneally injecting rats with 0, 10 or 50 mg kg(-1) per day of BPA for four consecutive days. The urinary concentrations of individual nucleosides were measured by liquid chromatography-tandem mass spectrometry combined with column switching on-line extraction. Increased levels of 5-hydroxymethyl-2'-deoxyuridine (P < 0.01 on first, P < 0.005 on second, P < 0.001 on third and P < 0.01 on fourth day) and 8-hydroxy-2'-deoxyguanosine (P < 0.005 on second, P < 0.001 on third and P < 0.001 on fourth day) were found. Also, the patterns of urinary nucleosides in three dosage groups (control, BPA1, BPA2) were significantly different. Statistical significance was observed between BPA1 (5-hydroxymethyl-2'-deoxyuridine, P < 0.05; 8-hydroxy-2'-deoxyguanosine, P < 0.005) and BPA2 (5-hydroxymethyl-2'-deoxyurindine, P < 0.005; 8-hydroxy-2'-deoxyguanosine, P < 0.001) during the treatment period. Supervised analysis with partial least-squares-discrimination analysis led to discrimination between the three dosage groups. Quantitative alterations showed the metabolic trajectories responsible for physiological responses. The described methods could be used to evaluate and monitor BPA-induced oxidative stress early after exposure.
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Estrógenos no Esteroides/farmacología , Espectrometría de Masas/métodos , Metabolómica , Estrés Oxidativo/efectos de los fármacos , Fenoles/farmacología , 8-Hidroxi-2'-Desoxicoguanosina , Animales , Compuestos de Bencidrilo , Cromatografía Liquida/métodos , Desoxiguanosina/análogos & derivados , Desoxiguanosina/orina , Relación Dosis-Respuesta a Droga , Femenino , Nucleósidos/orina , Ratas , Ratas Sprague-Dawley , Timidina/análogos & derivados , Timidina/orina , Factores de Tiempo , Urinálisis/métodosAsunto(s)
Transfusión de Eritrocitos/métodos , Enfermedades Gastrointestinales/tratamiento farmacológico , Hepatopatías/tratamiento farmacológico , Enfermedades Mitocondriales/tratamiento farmacológico , Enfermedades del Sistema Nervioso Periférico/tratamiento farmacológico , Timidina Fosforilasa/uso terapéutico , Adulto , Desoxiuridina/orina , Sistemas de Liberación de Medicamentos , Eritrocitos/enzimología , Resultado Fatal , Femenino , Enfermedades Gastrointestinales/enzimología , Enfermedades Gastrointestinales/fisiopatología , Predisposición Genética a la Enfermedad/genética , Humanos , Hepatopatías/enzimología , Hepatopatías/fisiopatología , Enfermedades Mitocondriales/enzimología , Enfermedades Mitocondriales/fisiopatología , Mutación/genética , Enfermedades del Sistema Nervioso Periférico/enzimología , Enfermedades del Sistema Nervioso Periférico/fisiopatología , Neumonía/complicaciones , Síndrome , Timidina/orina , Timidina Fosforilasa/genética , Insuficiencia del TratamientoRESUMEN
Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare autosomal recessive disorder in which a nuclear mutation of the thymidine phosphorylase (TP) gene causes mitochondrial genomic dysfunction. Patients suffer from gastrointestinal dysmotility, cachexia, ptosis, external ophthalmoparesis, myopathy and polyneuropathy. Magnetic resonance imaging (MRI) shows leukoencephalopathy. We describe clinical, genetic and neuroradiological features of three brothers affected with MNGIE. Clinical examination, laboratory analyses, MRI and magnetic resonance spectroscopy (MRS) of the brain, and genetic analysis have been performed in all six members of the family with the three patients with MNGIE. Two of them are monozygous twins. They all suffered from gastrointestinal dysmotility, cachexia, ophthalmoplegia, muscular atrophies, and polyneuropathy. Urinary thymidine was elevated in the patients related to the severity of clinical disease, and urinary thymidine (normally not detectable) was also found in a heterozygous carrier. Brain MRI showed leukoencephalopathy in all patients; however, their cognitive functioning was normal. Brain MRS demonstrated reduced N-acetylaspartate and choline in severely affected areas. MRI of heterozygous carriers was normal. A new mutation (T92N) in the TP gene was identified. Urinary thymidine is for the first time reported to be detectable in a heterozygous carrier. MRS findings indicate loss of neurons, axons, and glial cells in patients with MNGIE, but not in heterozygous carriers.
Asunto(s)
Cuerpo Estriado/diagnóstico por imagen , Encefalomiopatías Mitocondriales , Hermanos , Sustancia Negra/diagnóstico por imagen , Adulto , Cuerpo Estriado/patología , Enfermedades en Gemelos , Exones , Humanos , Imagen por Resonancia Magnética/métodos , Espectroscopía de Resonancia Magnética/métodos , Masculino , Encefalomiopatías Mitocondriales/diagnóstico por imagen , Encefalomiopatías Mitocondriales/genética , Encefalomiopatías Mitocondriales/fisiopatología , Encefalomiopatías Mitocondriales/orina , Mutación , Conducción Nerviosa/fisiología , Cintigrafía , Análisis de Secuencia de ADN/métodos , Sustancia Negra/patología , Timidina/orina , Timidina Fosforilasa/genéticaRESUMEN
Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder characterized by severe gastrointestinal dysmotility, cachexia, ptosis, ophthalmoparesis, peripheral neuropathy and leukoencephalopathy. The disease is due to a thymidine phosphorylase defect. This enzyme catalyses the phosphorolysis of thymidine to thymine and deoxyribose 1-phosphate. For this reason, increased levels of thymidine in plasma and urine are found in MNGIE patients. Haemodialysis can reduce circulating plasma thymidine levels and can be beneficial in some MNGIE patients. We developed a fast analytical method based on HPLC-ESI-MS/MS capable of identifying pyrimidine nucleotides (thymine, cytosine, uracil) and nucleosides (thymidine, citidine, uridine) in plasma and urine after direct dilution of the samples without pre-treatment. In the patient studied, we observed a significant reduction of plasmatic and urinary thymidine levels during and after dialysis. However, we noted a progressive reduction of the initial thymidine level after some dialytic trials. This method will be useful not only for thymidine level follow-up during dialysis in MNGIE patients but also for the improvement of the diagnosis or diagnostic suspect in other pyrimidine defects such as dihydropyrimidine dehydrogenase deficiency, dihydropyrimidinase deficiency and ureidopropionase deficiency.
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Cromatografía Líquida de Alta Presión/métodos , Encefalomiopatías Mitocondriales/sangre , Encefalomiopatías Mitocondriales/orina , Espectrometría de Masa por Ionización de Electrospray/métodos , Timidina , Adulto , Resultado Fatal , Femenino , Humanos , Encefalomiopatías Mitocondriales/terapia , Diálisis Renal , Timidina/análisis , Timidina/sangre , Timidina/orina , Timidina Fosforilasa/deficienciaRESUMEN
The mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) syndrome is characterized by the association of gastrointestinal and neurological symptoms. It is a rare autosomal recessive mitochondrial disorder with multiple mitochondrial DNA deletions and/or depletion. It is caused by thymidine phosphorylase (TP) gene mutations resulting in a complete abolition of TP activity. We tested 31 unrelated patients presenting either with a complete MNGIE syndrome (8 patients), a severe intestinal pseudo-obstruction (10 patients), and multiple deletions and/or depletion of mitochondrial DNA (13 patients). All the tested patients presenting with a complete MNGIE had increased thymidine levels in plasma and urine, and no TP activity. The group with pseudo-obstruction syndrome had normal or partial reduction of TP activity. We found pathogenic mutations on TP gene only in the MNGIE syndrome group: all the MNGIE patients were compound heterozygous or homozygous for mutations in the TP gene. Eight of these mutations are yet unreported, confirming the lack of genotype/phenotype correlation in this syndrome. Enzymatic activity and thymidine level are thus rapid diagnosis tests to detect MNGIE affected patients prior to genetic testing for patients with gastrointestinal symptoms.
Asunto(s)
Encefalomiopatías Mitocondriales/genética , Mutación , Timidina Fosforilasa/genética , Adulto , Niño , ADN Mitocondrial/genética , Humanos , Seudoobstrucción Intestinal/genética , Eliminación de Secuencia , Síndrome , Timidina/sangre , Timidina/orina , Timidina Fosforilasa/metabolismoRESUMEN
To assess novel liquid chromatography/mass spectrometric methods for measuring oxidative damage to nucleic acids and lipids, we compared urinary excretion of 8-hydroxy-2'-deoxyguanosine (8-OHdG), 5-hydroxymethyl-2'-deoxyuridine (5-OHmU), and 8-hydroxyguanosine (8-OxoG), and an isoprostane, 8-iso-prostaglandin F(2)alpha (IsopF(2)alpha) in 234 healthy men (n = 113) and women (n = 121), 80 current smokers, 96 never-smokers), and 58 ex-smokers (no tobacco use for 3 years). The 8-OHdG and 8-OxoG did not differ significantly by group; 5-OHmU was higher in smokers, compared with ex- (p <.003) and never- (p <.0001) smokers and in ex- vs. never-smokers (p =.014) at, respectively, 13.5 +/- 0.7, 11.3 +/- 1.0, and 8.7 +/- 0.3 microg/g creatinine. IsopF(2)alpha was higher in smokers, compared with ex- (p =.007) and never-smokers (p <.0001) and in ex- vs. never- smokers (p =.002) at, respectively, 1.1 +/- 0.10; 0.74 +/- 0.07, and 0.51 +/- 0.04 microg/g creatinine. There were significant correlations among all three nucleic acid adducts and between IsopF(2)alpha and both 5-OHmU and 8-OHdG. Many smokers and ex-smokers had high levels of either 5-OHmU excretion or IsopF(2)alpha excretion, but not both. We conclude that 5-OHmU and IsopF(2)alpha are more discriminating of oxidative stress from tobacco smoke than the other two compounds measured. Whether characteristic patterns of excretion of these indicators forecast differential disease risk should be explored in future research.
Asunto(s)
Aductos de ADN/metabolismo , Desoxiguanosina/análogos & derivados , Desoxiguanosina/orina , Dinoprost/orina , Guanosina/análogos & derivados , Guanosina/orina , Fumar/metabolismo , Timidina/análogos & derivados , Timidina/orina , 8-Hidroxi-2'-Desoxicoguanosina , Adulto , Distribución por Edad , Anciano , Anciano de 80 o más Años , Cromatografía Liquida , Dinoprost/análogos & derivados , Femenino , Humanos , Masculino , Espectrometría de Masas , Persona de Mediana Edad , Oxidación-Reducción , Estrés Oxidativo , Distribución por Sexo , Fumar/efectos adversosRESUMEN
Reactive oxygen species are generated during ischemia-reperfusion tissue injury. Oxidation of thymidine by hydroxyl radicals (HO*) causes formation of 5,6-dihydroxy-5,6-dihydrothymidine (thymidine glycol). Thymidine glycol excreted in urine can be used as a biomarker of oxidative DNA damage. The aim of this study was to investigate the oxidative DNA damage in patients showing immediate allograft function after kidney transplantation, and to find out whether this damage correlates with glomerular and tubular lesions. Time dependent changes in urinary excretion rates of thymidine glycol, but also of total protein, albumin, low molecular weight (alpha1-microglobulin, beta2-microglobulin) and high molecular weight proteins (transferrin, IgG, alpha2-macroglobulin) were analyzed quantitatively and by polyacrylamide-gel electrophoresis in six patients. Urinary thymidine glycol was determined by a fluorimetric assay in combination with affinity chromatography and HPLC. After kidney transplantation the urinary excretion rate of thymidine glycol increased gradually reaching a maximum within the first 48 hours (16.56+/-11.3 nmol/m mol creatinine, ref. 4.3+/-0.97). Severe proteinuria with an excretion rate of up to 7.2 g/mmol creatinine was observed and declined within the first 24 hours of allograft function (0.35+/-0.26 g/mmol creatinine). The gel-electrophoretic pattern showed a nonselective glomerular and tubular proteinuria. The initial nonselective glomerular proteinuria disappeared within 48 hours, changing to a mild selective glomerular proteinuria. In this period (12-48 hours) higher levels of thymidine glycol excretion were observed, when compared to the initial posttransplant phase (13.66+/-9.76 vs. 4.31+/-3.61 nmol/mmol creatinine; p<0.05). An increased excretion of thymidine glycol is seen after kidney transplantation and is explained by the ischemia-reperfusion induced oxidative DNA damage in the kidney. In the second phase higher levels of excretion were observed parallel to the change from a nonselective to a selective glomerular and tubular proteinuria. An explanation may be sought in the repair process of DNA in the glomerular and tubular epithelial cells, appearing simultaneously with functional recovery.
Asunto(s)
Rechazo de Injerto/diagnóstico , Trasplante de Riñón/fisiología , Estrés Oxidativo , Timidina/análogos & derivados , Adulto , Biomarcadores/orina , Cromatografía Líquida de Alta Presión , Estudios de Seguimiento , Supervivencia de Injerto , Humanos , Fallo Renal Crónico/cirugía , Pruebas de Función Renal , Trasplante de Riñón/efectos adversos , Trasplante de Riñón/métodos , Masculino , Persona de Mediana Edad , Periodo Posoperatorio , Probabilidad , Daño por Reperfusión/etiología , Daño por Reperfusión/orina , Sensibilidad y Especificidad , Estadísticas no Paramétricas , Timidina/orinaRESUMEN
Reactive oxygen species are generated during ischaemia-reperfusion of tissue. Oxidation of thymidine by hydroxyl radicals (HO) leads to the formation of 5,6-dihydroxy-5,6-dihydrothymidine (thymidine glycol). Thymidine glycol is excreted in urine and can be used as biomarker of oxidative DNA damage. Time dependent changes in urinary excretion rates of thymidine glycol were determined in six patients after kidney transplantation and in six healthy controls. A new analytical method was developed involving affinity chromatography and subsequent reverse-phase high-performance liquid chromatography (RP-HPLC) with a post-column chemical reaction detector and endpoint fluorescence detection. The detection limit of this fluorimetric assay was 1.6 ng thymidine glycol per ml urine, which corresponds to about half of the physiological excretion level in healthy control persons. After kidney transplantation the urinary excretion rate of thymidine glycol increased gradually reaching a maximum around 48 h. The excretion rate remained elevated until the end of the observation period of 10 days. Severe proteinuria with an excretion rate of up to 7.2 g of total protein per mmol creatinine was also observed immediately after transplantation and declined within the first 24 h of allograft function (0.35+/-0.26 g/mmol creatinine). The protein excretion pattern, based on separation of urinary proteins on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), as well as excretion of individual biomarker proteins, indicated nonselective glomerular and tubular damage. The increased excretion of thymidine glycol after kidney transplantation may be explained by ischaemia-reperfusion induced oxidative DNA damage of the transplanted kidney.
Asunto(s)
Daño del ADN , Trasplante de Riñón/efectos adversos , Especies Reactivas de Oxígeno/fisiología , Timidina/análogos & derivados , Adulto , Biomarcadores , Cromatografía de Afinidad , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Glomérulos Renales/fisiología , Túbulos Renales/fisiología , Masculino , Persona de Mediana Edad , Proteinuria/orina , Daño por Reperfusión/metabolismo , Daño por Reperfusión/orina , Timidina/orinaRESUMEN
We have measured the levels of thymine glycol (TG) and thymidine glycol (dTG) in human urine, using an HPLC method. The results show that all 30 specimens examined (including 10 non-neoplastic and 20 neoplastic human urines) contained significant amounts of TG and dTG, average levels (mean +/- SEM) were 0.376 +/- 0.026 and 0.138 +/- 0.009 nmol/kg body weight/day respectively. The average levels of TG and dTG were 0.435 +/- 0.038 and 0.164 +/- 0.017 nmol/kg body weight/day in 10 healthy human urine specimens and 0.347 +/- 0.035 and 0.125 +/- 0.010 nmol/kg body weight/day in 20 neoplastic human urine specimens respectively. No significant differences were found between female and male as well as between the non-neoplastic and neoplastic human urine specimens. There were also wide interindividual variations, which were not age-dependent.