RESUMO
Cancer is associated with strong changes in lipid metabolism. For instance, normal cells take up fatty acids (FAs) from the circulation, while tumour cells generate their own and become dependent on de novo FA synthesis, which could provide a vulnerability to target tumour cells. Betulinic acid (BetA) is a natural compound that selectively kills tumour cells through an ill-defined mechanism that is independent of BAX and BAK, but depends on mitochondrial permeability transition-pore opening. Here we unravel this pathway and show that BetA inhibits the activity of steroyl-CoA-desaturase (SCD-1). This enzyme is overexpressed in tumour cells and critically important for cells that utilize de novo FA synthesis as it converts newly synthesized saturated FAs to unsaturated FAs. Intriguingly, we find that inhibition of SCD-1 by BetA or, alternatively, with a specific SCD-1 inhibitor directly and rapidly impacts on the saturation level of cardiolipin (CL), a mitochondrial lipid that has important structural and metabolic functions and at the same time regulates mitochondria-dependent cell death. As a result of the enhanced CL saturation mitochondria of cancer cells, but not normal cells that do not depend on de novo FA synthesis, undergo ultrastructural changes, release cytochrome c and quickly induce cell death. Importantly, addition of unsaturated FAs circumvented the need for SCD-1 activity and thereby prevented BetA-induced CL saturation and subsequent cytotoxicity, supporting the importance of this novel pathway in the cytotoxicity induced by BetA.
Assuntos
Cardiolipinas/metabolismo , Mitocôndrias/efeitos dos fármacos , Triterpenos/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Morte Celular/efeitos dos fármacos , Linhagem Celular/efeitos dos fármacos , Linhagem Celular/metabolismo , Citocromos c/metabolismo , Ácidos Graxos/metabolismo , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Triterpenos Pentacíclicos , Estearoil-CoA Dessaturase/metabolismo , Ácido BetulínicoRESUMO
BACKGROUND: Sanfilippo disease (Mucopolysaccharidosis III) is a neurodegenerative lysosomal disorder characterized by accumulation of the glycosaminoglycan heparan sulfate (HS). MPS III has a large phenotypic variability and early assessment of disease severity is difficult. We investigated the correlation between disease severity and the plasma concentration of HS (pHS, defined by the sum of the heparan sulfate derived disaccharides obtained after enzymatic digestion) and urinary total GAGs level (uGAGs, measured by the dimethylene blue test) in a cross-sectional cohort of 44 MPS III patients. METHODS: Disease severity was established on the basis of the age of complete loss of independent walking and of full loss of speech in all patients. Hazard ratios (HR) were obtained with cox-regression analysis. In order to allow prediction of a severe phenotype based on a cut-off value for pHS, patients were divided in two groups (severely affected and less severely affected) based on predictive mutations or on the age of full loss of speech. Receiver operator characteristics (ROC) were obtained for pHS. RESULTS: pHS and uGAGs were independently and linearly associated with an increased risk of speech loss with a HR of 1.8 (95 % CI 1.3-2.7) per 500 ng/ml increase of HS in plasma (p = 0.002), and a HR of 2.7 (95 % CI 1.6-4.4) per 10 mg/mmol creatinine increase of uGAGs (p < 0.001). pHS and uGAGS were less strongly associated with loss of walking. The area under the ROC curve for pHS was 0.85, indicating good discrimination. CONCLUSION: pHS and uGAGs may be useful biomarkers for prediction of severity in MPS III.
Assuntos
Dissacarídeos/sangue , Glicosaminoglicanos/urina , Heparitina Sulfato/sangue , Mucopolissacaridose III/sangue , Mucopolissacaridose III/urina , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mucopolissacaridose III/patologia , Adulto JovemRESUMO
AIM: To assess the effect of three times daily mealtime inhaled insulin therapy compared with once daily basal insulin glargine therapy on 72-h glucose profiles, glucose variability and oxidative stress in type 2 diabetes patients. METHODS: In an inpatient crossover study, 40 subjects with type 2 diabetes were randomized to receive 9 days of inhaled insulin three times daily before meals or 9 days of glargine administered in the morning before breakfast in a randomized order. During the last 72 h in each phase, glucose was measured with continuous glucose monitoring. Activation of oxidative stress was measured by determining the 15(S)-8-iso-PGF(2alpha)-secretion in 24-h urine samples. RESULTS: Inhaled insulin improved overall and postprandial glucose control significantly better than insulin glargine (p < 0.0001). There was a trend towards a greater reduction in glucose variability (8-9%) in the inhaled group [p = 0.1430 and p = 0.3298 for mean amplitude of glycaemic excursions (MAGEs) and mean of daily differences respectively]. Oxidative stress, estimated by determining the urinary isoprostane excretion (15(S)-8-iso-PGF(2alpha)), was equally reduced from baseline by both treatments. No correlation was found between glucose variability and oxidative stress in both groups. CONCLUSIONS: This study showed a mealtime insulin approach to improve glycaemic control more than a basal insulin approach. These findings indicate also that lowering glucose using insulin treatment lowers oxidative stress over time, at least for the study period of 9 days, in type 2 diabetes patients. Contrary to earlier data, we found no correlation between glucose variability (MAGE) and oxidative stress (15(S)-8-iso-PGF(2alpha)) in this study.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Hipoglicemiantes/administração & dosagem , Insulina/análogos & derivados , Insulina/administração & dosagem , Estresse Oxidativo/efeitos dos fármacos , Administração por Inalação , Idoso , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Estudos Cross-Over , Diabetes Mellitus Tipo 2/sangue , Esquema de Medicação , Feminino , Humanos , Insulina Glargina , Insulina de Ação Prolongada , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Resultado do TratamentoRESUMO
In this study, we measured the activity of dihydropyrimidine dehydrogenase (DPD), dihydropyrimidinase (DHP) and beta-ureidopropionase (beta-UP), using radiolabeled substrates, in 16 different tissues obtained at autopsy from a single patient. The activity of DPD could be detected in all tissues examined, with the highest activity being present in spleen and liver. Surprisingly, the highest activity of DHP was present in kidney followed by that of liver. Furthermore, a low DHP activity could also be detected in 8 other tissues. The highest activity of beta-UP was detected in liver and kidney. However, low UP activities were also present in 8 other tissues. Our results demonstrated that the entire pyrimidine catabolic pathway was predominantly confined to the liver and kidney. However, significant residual activities of DPD, DHP and beta-UP were also present in a variety of other tissues, especially in bronchus.
Assuntos
Regulação Enzimológica da Expressão Gênica , Pirimidinas/química , Amidoidrolases/biossíntese , Di-Hidrouracila Desidrogenase (NADP)/biossíntese , Humanos , Fígado/enzimologia , Masculino , Pessoa de Meia-Idade , Pirimidinas/metabolismo , Baço/enzimologia , Distribuição TecidualRESUMO
In this article, we describe a fast and specific method to measure 5FU with HPLC tandem-mass spectrometry. Reversed-phase HPLC was combined with electrospray ionization tandem mass spectrometry and detection was performed by multiple-reaction monitoring. Stable-isotope-labeled 5FU (1,3-15N2-5FU) was used as an internal standard. 5FU was measured within a single analytical run of 16 min with a lower limit of detection of 0.05 microM. The intra-assay variation and inter-assay variation of plasma with added 5FU (1 microM, 10 microM, 100 microM) was less then 6%. Recoveries of the added 5FU in plasma were > 97%. Analysis of the 5FU levels in plasma samples from patients with the HPLC tandem mass spectrometry method and a HPLC-UV method yielded comparable results (r2 = 0.98). Thus, HPLC with electrospray ionization tandem mass spectrometry allows the rapid analysis of 5FU levels in plasma and could, therefore, be used for therapeutic drug monitoring.
Assuntos
Antimetabólitos Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Fluoruracila/farmacocinética , Espectrometria de Massas por Ionização por Electrospray/métodos , Área Sob a Curva , Cromatografia Líquida de Alta Pressão/métodos , Di-Hidrouracila Desidrogenase (NADP)/metabolismo , Monitoramento de Medicamentos/métodos , Humanos , Fatores de TempoRESUMO
The pyrimidine bases uracil and thymine are degraded via the consecutive action of three enzymes to beta-alanine and beta-aminoisobutyric acid, respectively. To date, a number of patients have been described with a deficiency of dihydropyrimidine dehydrogenase and dihydropyrimidinase, the first two enzymes of the pyrimidine degradation pathway. In this study, we demonstrate that the first patient presenting with N-carbamyl-beta-amino aciduria, due to a deficiency of beta-ureidopropionase, was easily diagnosed at the metabolite level using HPLC-tandem mass spectrometry. Urinary analysis showed strongly elevated levels of N-carbamyl-beta-alanine and N-carbamyl-beta-aminoisobutyric acid, with normal or moderately increased levels of the pyrimidine bases and the dihydropyrimidines, respectively. The deficiency of beta-ureidopropionase was confirmed by measuring all three enzymes of the pyrimidine degradation pathway. No activity of beta-ureidopropionase could be detected in a liver biopsy of the patient, while a normal activity of dihydropyrimidine dehydrogenase and dihydropyrimidinase was present. Thus, HPLC-tandem mass specrometry proved to be a powerful tool for the initial diagnosis of patients with deficiency of beta-ureidopropionase.
Assuntos
Amidoidrolases/deficiência , Cromatografia Líquida de Alta Pressão , Fígado/enzimologia , Espectrometria de Massas , Amidoidrolases/análise , Ácidos Aminoisobutíricos/análise , Di-Hidrouracila Desidrogenase (NADP) , Feminino , Humanos , Lactente , Oxirredutases/análise , Pirimidinas/metabolismo , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
BACKGROUND: Urinary concentrations of thymine, uracil, and their degradation products are useful indicators of deficiencies of enzymes of the pyrimidine degradation pathway. We describe a rapid, specific method to measure these concentrations to detect inborn errors of pyrimidine metabolism. METHODS: We used urine or urine-soaked filter-paper strips as samples and measured thymine, uracil, and their degradation products dihydrothymine, dihydrouracil, N:-carbamyl-ss-aminoisobutyric acid, and N:-carbamyl-ss-alanine. Reversed-phase HPLC was combined with electrospray ionization tandem mass spectrometry, and detection was performed by multiple-reaction monitoring. Stable-isotope-labeled reference compounds were used as internal standards. RESULTS: All pyrimidine degradation products could be measured in one analytical run of 15 min. Detection limits were 0.4-4 micromol/L. The intraassay imprecision (CV) of urine samples with added compounds was 1.3-12% for liquid urines and 1. 0-10% for filter-paper extracts of the urines. The interassay imprecision (CV) was 3-11% (100-200 micromol/L). Recoveries were 89-99% at 100-200 micromol/L and 95-106% at 1 mmol/L in liquid urines, and 93-103% at 100-200 micromol/L and 100-106% at 1 mmol/L in filter-paper samples. Correct identifications of deficiencies of the pyrimidine-degrading enzymes were readily made with urine samples from patients with known defects. CONCLUSIONS: HPLC with electrospray ionization tandem mass spectrometry allows rapid testing for disorders of the pyrimidine degradation pathway, and filter-paper samples allow easy collection, transport, and storage of urine samples.
Assuntos
Pirimidinas/urina , Cromatografia Líquida de Alta Pressão , Humanos , Papel , Pirimidinas/metabolismo , Reprodutibilidade dos Testes , Manejo de Espécimes , Espectrometria de Massas por Ionização por Electrospray , Timina/metabolismo , Timina/urina , Uracila/metabolismo , Uracila/urinaRESUMO
BACKGROUND: Dihydropyrimidine dehydrogenase (DPD) catalyzes the degradation of thymine, uracil, and the chemotherapeutic drug 5-fluorouracil. To identify patients suffering from complete or partial DPD deficiency and to identify pitfalls that can preclude the proper diagnosis of patients with partial DPD deficiency, a sensitive and accurate assay is necessary. METHODS: The activity of DPD was measured using [4-(14)C]thymine followed by separation of substrate and products with reversed-phase HPLC with on-line detection of the radioactivity. RESULTS: Complete baseline separation of radiolabeled thymine and all degradation products was achieved within 15 min. The detection limit for dihydrothymine was 0. 4 pmol. In lymphocytes, the DPD activity deviated from linearity at low protein concentrations (<0.2 g/L). Profoundly decreased activity of DPD was detected in the peripheral blood mononuclear cells (PBM cells) of two tumor patients when measured at low protein concentrations. Low DPD activity comparable to that observed in obligate heterozygotes was initially detected in PBM cells, containing substantial amounts of myeloid cells, from a patient suffering from 5-fluorouracil toxicity. However, after the patient experienced full clinical recovery, normal DPD activity was observed in the PBM cells. No significant differences in DPD activity were observed between exponentially growing fibroblasts and those at confluence. The range of DPD activities of obligate heterozygotes overlaps the range of DPD activities of controls. CONCLUSIONS: The low activity of DPD measured in PBM cells containing myeloid cells or that measured at a low protein concentration in the assay mixture is not indicative of heterozygosity for a mutant DPD allele. Although fibroblasts are suitable to establish a complete deficiency of DPD, unambiguous detection of heterozygotes is not possible.
Assuntos
Oxirredutases/metabolismo , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Di-Hidrouracila Desidrogenase (NADP) , Fibroblastos/enzimologia , Heterozigoto , Humanos , Leucócitos Mononucleares/enzimologia , Oxirredutases/genética , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of 5-fluorouracil (5FU), and it is suggested that patients with a partial deficiency of this enzyme are at risk for developing a severe 5FU-associated toxicity. To evaluate the importance of this specific type of inborn error of pyrimidine metabolism in the etiology of 5FU toxicity, an analysis of the DPD activity, the DPD gene, and the clinical presentation of patients suffering from severe toxicity after the administration of 5FU was performed. Our study demonstrated that in 59% of the cases, a decreased DPD activity could be detected in peripheral blood mononuclear cells. It was observed that 55% of patients with a decreased DPD activity suffered from grade IV neutropenia compared with 13% of patients with a normal DPD activity (P = 0.01). Furthermore, the onset of toxicity occurred, on average, twice as fast in patients with low DPD activity as compared with patients with a normal DPD activity (10.0 +/- 7.6 versus 19.1 +/- 15.3 days; P < 0.05). Analysis of the DPD gene of 14 patients with a reduced DPD activity revealed the presence of mutations in 11 of 14 patients, with the splice site mutation IVS14+1G-->A being the most abundant one (6 of 14 patients; 43%). Two novel missense mutations 496A-->G (M166V) and 2846A-->T (D949V) were detected in exon 6 and exon 22, respectively. Our results demonstrated that at least 57% (8 of 14) of the patients with a reduced DPD activity have a molecular basis for their deficient phenotype.
Assuntos
Antimetabólitos Antineoplásicos/toxicidade , Antimetabólitos Antineoplásicos/uso terapêutico , Fluoruracila/toxicidade , Fluoruracila/uso terapêutico , Mutação , Neoplasias/tratamento farmacológico , Neoplasias/genética , Oxirredutases/deficiência , Oxirredutases/genética , Adulto , Idoso , Alelos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Di-Hidrouracila Desidrogenase (NADP) , Éxons , Feminino , Genótipo , Granulócitos/enzimologia , Humanos , Íntrons , Leucócitos Mononucleares/enzimologia , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Oxirredutases/metabolismo , Fenótipo , Reação em Cadeia da Polimerase , Polimorfismo Genético , Neoplasias Retais/tratamento farmacológico , Neoplasias Retais/genéticaRESUMO
A full-length cDNA clone encoding human beta-ureidopropionase was isolated. A 1152-nucleotide open reading frame which corresponds to a protein of 384 amino acids with a calculated molecular weight of 43¿ omitted¿158 Da, surrounded by a 5'-untranslated region of 61 nucleotides and a 3'-untranslated region of 277 nucleotides was identified. The protein showed 91% similarity with the translation product of the rat beta-ureidopropionase cDNA. Expression of the human cDNA in an Escherichia coli and eukaryotic COS-7 expression system revealed a very high beta-ureidopropionase enzymatic activity, thus confirming the identity of the cDNA. Since human EST libraries from brain, liver, kidney and heart contained partial beta-ureidopropionase cDNAs, the enzyme seems to be expressed in these tissues, in agreement with the expression profile of this enzyme in rat. Using the human cDNA as a probe a genomic P1 clone could be isolated containing the complete human beta-ureidopropionase gene. The gene consist of 11 exons spanning approximately 20 kB of genomic DNA. Fluorescence in situ hydridization localized the human beta-ureidopropionase gene to 22q11.2.
Assuntos
Amidoidrolases/genética , Cromossomos Humanos Par 22 , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Mapeamento Cromossômico , Clonagem Molecular , DNA Complementar/genética , Escherichia coli , Expressão Gênica , Humanos , Dados de Sequência Molecular , Ratos , Alinhamento de SequênciaRESUMO
A radiochemical assay was developed to measure the activity of dihydropyrimidinase (DHP) in human liver homogenates. The method is based on the separation of radiolabeled dihydrouracil from N-carbamyl-beta-alanine by HPLC with on-line detection of radioactivity combined with detection of 14CO2 by liquid scintillation counting. The assay was linear with time and protein concentration. The minimum amount of radiolabeled products which could be determined proved to be 12 pmol using a purified stock solution of [2-(14)C]-5,6-dihydrouracil. This highly sensitive assay is especially suitable to identify patients with a dihydropyrimidinase deficiency.
Assuntos
Amidoidrolases/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Radioisótopos de Carbono , Humanos , Cinética , Fígado/enzimologia , Reprodutibilidade dos Testes , Contagem de Cintilação , Sensibilidade e EspecificidadeRESUMO
A radiochemical assay was developed to measure the activity of beta-ureidopropionase in human liver homogenates which is based on the detection of the reaction product (14)CO(2) by liquid scintillation counting. Radiolabeled N-carbamyl-beta-alanine was prepared within 15 min by a simple hydrolysis of [2-(14)C]5, 6-dihydrouracil under alkaline conditions at 37 degrees C. The enzymatic reaction proved to be linear with time up to at least 3.5 h and protein concentrations up to at least 1 mg/ml. Human beta-ureidopropionase obeyed Michaelis-Menten kinetics with an apparent Km for N-carbamyl-beta-alanine of 15.5 +/- 1.9 microM. The assay proved to be very accurate and sensitive with an intraassay coefficient of variation of 2% and a detection limit of 28 pmol for the product CO(2).
Assuntos
Amidoidrolases/análise , Fígado/enzimologia , Uracila/análogos & derivados , beta-Alanina/análogos & derivados , Amidoidrolases/metabolismo , Dióxido de Carbono/análise , Radioisótopos de Carbono , Humanos , Hidrólise , Cinética , Técnica de Diluição de Radioisótopos , Reprodutibilidade dos Testes , Contagem de Cintilação/métodos , Sensibilidade e Especificidade , Uracila/química , beta-Alanina/síntese química , beta-Alanina/metabolismoRESUMO
Dihydropyrimidine dehydrogenase (DPD) deficiency is an autosomal recessive disease characterised by thymine-uraciluria in homozygous deficient patients and has been associated with a variable clinical phenotype. In order to understand the genetic and phenotypic basis for DPD deficiency, we have reviewed 17 families presenting 22 patients with complete deficiency of DPD. In this group of patients, 7 different mutations have been identified, including 2 deletions [295-298delTCAT, 1897delC], 1 splice-site mutation [IVS14+1G>A)] and 4 missense mutations (85T>C, 703C>T, 2658G>A, 2983G>T). Analysis of the prevalence of the various mutations among DPD patients has shown that the G-->A point mutation in the invariant splice donor site is by far the most common (52%), whereas the other six mutations are less frequently observed. A large phenotypic variability has been observed, with convulsive disorders, motor retardation and mental retardation being the most abundant manifestations. A clear correlation between the genotype and phenotype has not been established. An altered beta-alanine, uracil and thymine homeostasis might underlie the various clinical abnormalities encountered in patients with DPD deficiency.
Assuntos
Oxirredutases/deficiência , Oxirredutases/genética , Animais , Di-Hidrouracila Desidrogenase (NADP) , Genótipo , Humanos , Oxirredutases/química , FenótipoRESUMO
Dihydropyrimidine dehydrogenase (DPD) is responsible for the breakdown of the widely used antineoplastic agent 5-fluorouracil (5FU), thereby limiting the efficacy of the therapy. To identify patients suffering from a complete or partial DPD deficiency, the activity of DPD is usually determined in peripheral blood mononuclear cells (PBM cells). In this study, we demonstrated that the highest activity of DPD was found in monocytes followed by that of lymphocytes, granulocytes and platelets, whereas no significant activity of DPD could be detected in erythrocytes. The activity of DPD in PBM cells proved to be intermediate compared with the DPD activity observed in monocytes and lymphocytes. The mean percentage of monocytes in the PBM cells obtained from cancer patients proved to be significantly higher than that observed in PBM cells obtained from healthy volunteers. Moreover, a profound positive correlation was observed between the DPD activity of PBM cells and the percentage of monocytes, thus introducing a large inter- and intrapatient variability in the activity of DPD and hindering the detection of patients with a partial DPD deficiency.