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1.
Anal Chim Acta ; 1081: 103-111, 2019 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-31446947

RESUMO

Both DNA cytosine methylation (5-methyl-2'-deoxycytidine, m5dC) and RNA cytosine methylation (5-methylcytidine, m5rC) are important epigenetic marks that play regulatory roles in diverse biological processes. m5dC and m5rC can be further oxidized by the ten-eleven translocation (TET) proteins to form 5-hydroxymethyl-2'-deoxycytidine (hm5dC) and 5-hydroxymethylcytidine (hm5rC), respectively. 2'-O-methyl-5-hydroxymethylcytidine (hm5rCm) was recently also identified as a second oxidative metabolite of m5rC in RNA. Previous studies showed that the dysregulation of cytidine modifications in both DNA and RNA are closely related to a variety of human diseases. These cytidine modifications are generally excreted from cell into urine. If these cytidine modifications exhibit specific features related to certain diseases, determination of the cytidine modifications in urine could be utilized as non-invasive diagnostic of diseases. Here, we established a solid-phase extraction in combination with liquid chromatography-mass spectrometry (LC-MS/MS) analysis for simultaneous detection of these cytidine modifications in human urine samples. The developed method enabled the distinct detection of these cytidine modifications. We reported, for the first time, the presence of hm5rCm in human urine. Furthermore, we found that compared to the healthy controls, the contents of hm5dC, hm5rC, and hm5rCm showed significant increases in urine samples of cancer patients, including lymphoma patients, gastric cancer patients, and esophageal cancer patients. This study indicates that the urinary hydroxylmethylation modifications of hm5dC, hm5rC, and hm5rCm may serve as potential indicator of cancers.


Assuntos
Cromatografia Líquida/métodos , Citidina/análogos & derivados , Citidina/urina , Extração em Fase Sólida/métodos , Espectrometria de Massas em Tandem/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , DNA/química , Metilação de DNA , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , RNA/química
2.
Anal Chim Acta ; 1034: 110-118, 2018 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-30193624

RESUMO

5-Methyl-2'-deoxycytidine (5-mdC), 5-hydroxymethyl-2'-deoxycytidine (5-hmdC), 5-methylcytidine (5-mrC) and 5-hydroxymethylcytidine (5-hmrC) are epigenetic marks of DNA and RNA, and aberrant levels of these modified nucleosides were found to be associated with various cancers. Urine is a preferred source of biological fluid for biomarker discovery because the sample collection process is not invasive to patients. Herein, we developed a novel malic acid-enhanced hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method for sensitive and simultaneous quantification of the modified cytosine nucleosides in human urine. Malic acid markedly increased the detection sensitivities of all four cytosine nucleosides, with the limits of detection (LODs) for 5-mdC, 5-hmdC, 5-mrC and 5-hmrC being 0.025, 0.025, 0.025 and 0.050 fmol, respectively. By using this method, we demonstrated, for the first time, the presence of 5-hmrC in human urine, and we successfully quantified 5-mdC, 5-hmdC, 5-mrC and 5-hmrC in urine samples collected from 90 patients with colorectal cancer (CRC) and 90 healthy controls. We found that the levels of 5-mdC, 5-hmdC, 5-mrC and 5-hmrC in urine were all substantially decreased in CRC patients, suggesting that these modified nucleosides might have great potential to be noninvasive biomarkers for early detection and prognosis of CRC. Together, we established a novel and sensitive method for detecting 5-methylated and 5-hydroxymethylated cytosine nucleosides in human urine and the results from this study may stimulate future investigations about the regulatory roles of these cytosine derivatives in the initiation and development of CRC.


Assuntos
Citidina/análogos & derivados , Desoxicitidina/análogos & derivados , Malatos/química , Cromatografia Líquida , Citidina/química , Citidina/urina , Desoxicitidina/química , Desoxicitidina/urina , Humanos , Interações Hidrofóbicas e Hidrofílicas , Conformação de Ácido Nucleico , Espectrometria de Massas em Tandem
3.
Mikrochim Acta ; 185(3): 195, 2018 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-29594694

RESUMO

Cytidine is regarded as an early marker of colon cancer. The authors describe a surface enhanced Raman scattering (SERS) technique to detect trace levels of cytidine in urine. The Raman band at 784 cm-1 can be acquired best. Compared to earlier methods, an improvement in detection sensitivity by a factor of 6.2 × 105 is achieved by using a magnetically induced method in which cytidine is captured in the vicinity of the SERS hot spots of the type Fe3O4/Au/Ag. Cytidine can be quantified at 1 nM levels by this method which is simple and reliable. Graphical Abstract Clusters consisting of magnetite (Fe3O4) nanoparticles, gold nanoparticles and silver nanoparticles were prepared and used in a SERS based method for detection of cytidine in urine by using magnetic improvement. The lowest detectable concentration of cytidine are at the nM level.


Assuntos
Neoplasias do Colo , Citidina/análise , Ouro/química , Limite de Detecção , Nanopartículas de Magnetita/química , Prata/química , Análise Espectral Raman/métodos , Biomarcadores Tumorais/análise , Citidina/urina , Humanos , Fenômenos Magnéticos
4.
Clin Chim Acta ; 428: 57-62, 2014 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-24177150

RESUMO

BACKGROUND: Gastric cancer is a major public health concern as the fourth most common cancer, and it is of particular relevance as the second most common cause of cancer death worldwide. We caparisoned the urinary nucleoside concentrations between the gastric patients and healthy volunteers that try to evaluate the diagnostic value in the gastric cancer. METHOD: Urinary nucleosides from 49 gastric patients and 40 healthy volunteers were evaluated by high-performance liquid chromatography/electrospray ionization-tandem mass spectrometry (HPLC/ESI-MS/MS) under optimized conditions as determined in our previous study. RESULTS: The mean concentrations of 5 urinary nucleosides, cytidine, 3-methylcytidine (m3C), 1-methyladenosine (m1A), adenosine, and inosine, were found to be elevated in cancer patients, but only cytidine showed a significant elevation. Moreover, cytidine concentrations were significantly elevated by an average of 1.42-fold in patients with late stage (S3+4) disease. Combining the determined concentrations of preoperative serum alpha-fetoprotein (AFP, cutoff of 20 µg/l) or carbohydrate antigen 19-9 (CA19-9, cutoff of 37 U/ml) with the mean urinary cytidine concentration was shown to improve the diagnostic ratio (sensitivity) for gastric cancer from 16.3% (8/49 patients) to 38.8% (8+11/49 patients) or from 28.6% (14/49 patients) to 51.0% (14+11/49 patients), respectively. CONCLUSIONS: Urinary cytidine may be an important adjunct biomarker for gastric cancer.


Assuntos
Biomarcadores Tumorais/urina , Citidina/urina , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/urina , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Taiwan
5.
Biomarkers ; 13(4): 435-49, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18484357

RESUMO

Modified nucleosides are formed post-transcriptionally in RNA. In cancer disease, the cell turnover and thus RNA metabolism is increased, yielding higher concentrations of excreted modified nucleosides. In the presented study, urinary ribonucleosides were used to differentiate between breast cancer patients and healthy volunteers. The nucleosides were extracted from urine samples using affinity chromatography and subsequently analyzed via liquid chromatography ion trap mass spectrometry (LC-IT-MS). The peak areas were related to the internal standard isoguanosine and to the urinary creatinine level. For bioinformatic pattern recognition we used the support vector machine. We examined 113 urine samples from breast cancer patients (stage Tis-T4) and 99 control samples from healthy volunteers. We achieved a sensitivity of 87.67% and a specificity of 89.90% when including 31 nucleosides. The medical metabonomics concept based on the urinary nucleoside profile reveals a significantly improved classification compared with currently applied breast cancer biomarkers such as CA15-3.


Assuntos
Neoplasias da Mama/diagnóstico , Espectrometria de Massas/métodos , Nucleosídeos/urina , Adenosina/urina , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Algoritmos , Inteligência Artificial , Biomarcadores/urina , Neoplasias da Mama/urina , Citidina/urina , Reações Falso-Negativas , Reações Falso-Positivas , Feminino , Guanosina/urina , Humanos , Inosina/urina , Pessoa de Meia-Idade , Análise de Componente Principal , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Uridina/urina
6.
J Pharm Biomed Anal ; 31(1): 47-55, 2003 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-12560048

RESUMO

A liquid chromatographic-electrospray ionization tandem mass spectrometric (LC-ESI/MS/MS) method was developed for the quantitative analysis of a novel anticancer drug, 3'-C-ethynylcytidine (I) in human plasma and urine. I and its stable isotope-labeled internal standard (II) were extracted from human plasma and urine samples using a polymer-based cation-exchange cartridge, and LC-ESI/MS/MS analysis was performed by monitoring the positive fragment ions of I and II. The linear ranges are 1-500 ng/ml in plasma and 10-5000 ng/ml in urine. The limits of quantitation for I were 1 ng/ml in plasma and 10 ng/ml in urine. The relative errors (RE) for I ranged from -8.4 to 3.0% in plasma and from 0.8 to 4.4% in urine. The relative standard deviations (RSD) for I ranged from 1.2 to 8.9% in plasma and from 0.7 to 2.8% in urine. This validated analytical method is demonstrated to be useful for the analysis of I in human plasma and urine in clinical studies.


Assuntos
Antineoplásicos/análise , Citidina/análogos & derivados , Citidina/análise , Antineoplásicos/sangue , Antineoplásicos/urina , Calibragem , Cromatografia Líquida , Citidina/sangue , Citidina/urina , Humanos , Indicadores e Reagentes , Neoplasias/sangue , Neoplasias/urina , Controle de Qualidade , Padrões de Referência , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização por Electrospray
7.
Br J Rheumatol ; 36(9): 990-5, 1997 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-9376997

RESUMO

The levels of the five methylated nucleosides pseudouridine (psi-Urd), 1-methyladenosine (1-MeAdo), 4 acetylcytidine (4-AcCyd), 1 methylinosine (1-Melno) and 7 methylguanosine (7-MeGuo) resulting from RNA degradation were examined in the urine of rheumatoid arthritis (RA) patients. Of these five, 1-MeAdo and psi-Urd were correlated with the active phase of the disease, while two others (4-AcCyd and 1-Melno), which require further evaluation, appeared to be linked to the prognosis of the disease. As RNA turnover is closely associated with cell proliferation, including that of lymphocytes in RA, there may be a hitherto unsuspected benefit in measuring 1-MeAdo and psi-Urd as biochemical markers of RA disease activity.


Assuntos
Artrite Reumatoide/urina , Nucleosídeos/urina , Adenosina/análogos & derivados , Adenosina/urina , Idoso , Biomarcadores , Citidina/análogos & derivados , Citidina/urina , Feminino , Seguimentos , Guanosina/análogos & derivados , Guanosina/urina , Humanos , Inosina/análogos & derivados , Inosina/urina , Masculino , Pessoa de Meia-Idade , Pseudouridina/urina , Capuzes de RNA/urina , RNA Ribossômico/metabolismo , RNA de Transferência/metabolismo
8.
J Chromatogr B Biomed Sci Appl ; 692(1): 169-79, 1997 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-9187397

RESUMO

Cyclopentenyl cytosine (CPE-C) is a synthetic carbocyclic nucleoside that possesses diverse antitumor and antiviral activity. CPE-C has been studied extensively at the preclinical level and has been evaluated in a Phase I clinical trial involving patients with solid tumors. A narrow-bore, reversed-phase HPLC method that has been developed for the sensitive measurement of CPE-C in plasma and urine in order to carry out these studies is described. Covalent solid-phase extraction based on an immobilized phenylboronic acid ligand is employed to isolate both CPE-C and endogenous ribonucleosides from the biological matrix selectively and efficiently. This is followed by isocratic elution of the extract with pH 5.0, 0.1 M ammonium formate buffer at 0.150 ml/min on a tandem, switchable, C18 narrow-bore (2.1 mm I.D.) column system in which the precolumn is automatically backflushed to eliminate late-eluting components. UV detection at 278 nm provides a limit of quantitation of 0.1 microM for CPE-C in rat and human plasma with a precision better than 4% for the range 1-20 microM in rat plasma. Application of this assay to the determination of the bolus dose plasma kinetics and disposition of 2 mg/kg CPE-C in rats is illustrated. This method is amenable to partial automation and is well-suited for the analysis of clinical samples.


Assuntos
Antineoplásicos/sangue , Antineoplásicos/urina , Citidina/análogos & derivados , Animais , Antineoplásicos/farmacocinética , Proteínas Sanguíneas/metabolismo , Cromatografia Líquida de Alta Pressão , Citidina/sangue , Citidina/farmacocinética , Citidina/urina , Cães , Estabilidade de Medicamentos , Feminino , Humanos , Concentração de Íons de Hidrogênio , Hidrólise , Masculino , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Espectrofotometria Ultravioleta
9.
Neuropediatrics ; 24(1): 15-8, 1993 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-7682674

RESUMO

Three children with the late onset form of argininosuccinic aciduria are presented. The first two are sisters. The clinical features are characterized by mild retardation and ataxia, complicated by episodes of hyperammonemia. All patients showed elevated concentrations of argininosuccinic acid and its anhydrides in all body fluids, most pronounced in cerebrospinal fluid (CSF). Moreover, in Cases 1 and 2, we found elevated concentrations of pseudouridine and uridine limited to CSF, which was not reported before. In Case 3, with some residual activity of argininosuccinate lyase (ASL), we found normal values of these compounds. In urine we found elevated concentrations of uracil in Cases 1 and 2, and orotic acid in Case 2. Plasma showed an elevated concentration of orotic acid in all three patients, uracil was elevated in Case 2, cytidine was elevated in Cases 2 and 3. The results are being discussed and indicate that CSF values of pyrimidines reveal new biochemical abnormalities of brain tissue in urea cycle disorders.


Assuntos
Ácido Argininossuccínico/metabolismo , Doenças Metabólicas/enzimologia , Pirimidinas/metabolismo , Argininossuccinato Liase/análise , Argininossuccinato Liase/líquido cefalorraquidiano , Argininossuccinato Liase/metabolismo , Ácido Argininossuccínico/análise , Ácido Argininossuccínico/líquido cefalorraquidiano , Encéfalo/anormalidades , Encéfalo/metabolismo , Química Encefálica , Pré-Escolar , Citidina/sangue , Citidina/líquido cefalorraquidiano , Citidina/urina , Deficiências do Desenvolvimento/etiologia , Deficiências do Desenvolvimento/metabolismo , Feminino , Humanos , Lactente , Masculino , Doenças Metabólicas/complicações , Ácido Orótico/sangue , Ácido Orótico/líquido cefalorraquidiano , Ácido Orótico/urina , Pirimidinas/análise , Pirimidinas/líquido cefalorraquidiano , Uracila/sangue , Uracila/líquido cefalorraquidiano , Uracila/urina
10.
Invest New Drugs ; 9(1): 9-17, 1991 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-2026489

RESUMO

A hybrid compartmental-physiological model for cyclopentenyl cytosine (CPE-C) is designed on the basis of early limited rodent pharmacokinetic data. Application of model independent pharmacokinetics and biochemical knowledge was first used to conceptualize such a model. The approach was to scale the physiological parameters of the model (compartmental clearances) and keep constant the anatomic parameters of the model (compartment volumes). Scaling of physiological mechanisms was based on body weight/surface area ratios. Using these principles, simulations with the model can reasonably anticipate the in vivo behavior of (CPE-C) in several species (mouse, rat, dog). The model is useful in understanding species differences in pharmacokinetic behavior of CPE-C.


Assuntos
Antineoplásicos/farmacocinética , Citidina/análogos & derivados , Animais , Antineoplásicos/sangue , Antineoplásicos/urina , Simulação por Computador , Citidina/sangue , Citidina/farmacocinética , Citidina/urina , Cães , Esquema de Medicação , Injeções Intravenosas , Camundongos , Modelos Biológicos , Ratos , Especificidade da Espécie
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