RESUMO
Renal clinical chemistry only detects kidney dysfunction after considerable damage has occurred and is imperfect in predicting long term outcomes. Consequently, more sensitive markers of early damage and better predictors of progression are being urgently sought, to better support clinical decisions and support shorter clinical trials. Transglutaminase 2 (TG2) is strongly implicated in the fibrotic remodeling that drives chronic kidney disease (CKD). We hypothesized that urinary TG2 and its ε-(γ-glutamyl)-lysine crosslink product could be useful biomarkers of kidney fibrosis and progression. Animal models: a rat 4-month 5/6th subtotal nephrectomy model of CKD and a rat 8-month streptozotocin model of diabetic kidney disease had 24-hour collection of urine, made using a metabolic cage, at regular periods throughout disease development. Patients: Urine samples from patients with CKD (n = 290) and healthy volunteers (n = 33) were collected prospectively, and progression tracked for 3 years. An estimated glomerular filtration rate (eGFR) loss of 2-5 mL/min/year was considered progressive, with rapid progression defined as > 5 mL/min/year. Assays: TG2 was measured in human and rat urine samples by enzyme-linked immunosorbent assay (ELISA) and ε-(γ-glutamyl)-lysine by exhaustive proteolytic digestion and amino acid analysis. Urinary TG2 and ε-(γ-glutamyl)-lysine increased with the development of fibrosis in both animal model systems. Urinary TG2 was 41-fold higher in patients with CKD than HVs, with levels elevated 17-fold by CKD stage 2. The urinary TG2:creatinine ratio (UTCR) was 9 ng/mmol in HV compared with 114 ng/mmol in non-progressive CKD, 1244 ng/mmol in progressive CKD and 1898 ng/mmol in rapidly progressive CKD. Both urinary TG2 and ε-(γ-glutamyl)-lysine were significantly associated with speed of progression in univariate logistic regression models. In a multivariate model adjusted for urinary TG2, ε-(γ-glutamyl)-lysine, age, sex, urinary albumin:creatinine ratio (UACR), urinary protein:creatinine ratio (UPCR), and CKD stage, only TG2 remained statistically significant. Receiver operating characteristic (ROC) curve analysis determined an 86.4% accuracy of prediction of progression for UTCR compared with 73.5% for UACR. Urinary TG2 and ε-(γ-glutamyl)-lysine are increased in CKD. In this pilot investigation, UTCR was a better predictor of progression in patients with CKD than UACR. Larger studies are now warranted to fully evaluate UTCR value in predicting patient outcomes.
Assuntos
Biomarcadores/urina , Nefropatias Diabéticas/metabolismo , Nefrectomia/efeitos adversos , Proteína 2 Glutamina gama-Glutamiltransferase/urina , Insuficiência Renal Crônica/metabolismo , Estreptozocina/efeitos adversos , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Estudos de Casos e Controles , Nefropatias Diabéticas/induzido quimicamente , Nefropatias Diabéticas/urina , Dipeptídeos/urina , Modelos Animais de Doenças , Progressão da Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Ratos , Análise de Regressão , Insuficiência Renal Crônica/etiologia , Insuficiência Renal Crônica/urinaRESUMO
In recent years, radiolabeled tracers targeting prostate-specific membrane antigen (PSMA) have had a tremendous impact on prostate cancer management. Here, we report on the formation of radioactive impurities formed during the clinical production of 177Lu-labeled PSMA-617. We provide compelling evidence that these impurities are the result of a spontaneous, thermally mediated condensation reaction of the Glu-CO-Lys moiety resulting in the formation of three different five-membered ring systems. Density functional theory (DFT) calculations show that the condensation and cyclization of the Glu-CO-Lys moiety is thermodynamically spontaneous. In cell experiments, no affinity of these cyclized compounds toward PSMA was observed. HPLC analyses of urine samples from patient studies showed rapid renal excretion of these radioactive cyclized species. Radiolabeling conditions were identified that significantly reduced the formation of cyclized side products yielding 177Lu-labeled PSMA-617 in high radiochemical yield and purity in concordance with current good manufacturing practice (cGMP) requirements.
Assuntos
Dipeptídeos/química , Compostos Heterocíclicos com 1 Anel/química , Compostos Radiofarmacêuticos/síntese química , Motivos de Aminoácidos , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Ciclização , Teoria da Densidade Funcional , Dipeptídeos/metabolismo , Dipeptídeos/urina , Compostos Heterocíclicos com 1 Anel/metabolismo , Compostos Heterocíclicos com 1 Anel/urina , Humanos , Lutécio/química , Espectroscopia de Ressonância Magnética , Antígeno Prostático Específico , Radioisótopos/química , Compostos Radiofarmacêuticos/metabolismo , Compostos Radiofarmacêuticos/urina , Espectrometria de Massas por Ionização por Electrospray , TermodinâmicaRESUMO
Analytical methods to determine the potential misuse of the ghrelin mimetics capromorelin (CP-424,391), macimorelin (macrilen, EP-01572) and tabimorelin (NN703) in sports were developed. Therefore, different extraction strategies, i.e. solid-phase extraction, protein precipitation, as well as a "dilute-and-inject" approach, from urine and EDTA-plasma were assessed and comprehensive in vitro/in vivo experiments were conducted, enabling the identification of reliable target analytes by means of high resolution mass spectrometry. The drugs' biotransformation led to the preliminary identification of 51 metabolites of capromorelin, 12 metabolites of macimorelin and 13 metabolites of tabimorelin. Seven major metabolites detected in rat urine samples collected post-administration of 0.5-1.0 mg of a single oral dose underwent in-depth characterization, facilitating their implementation into future confirmatory test methods. In particular, two macimorelin metabolites exhibiting considerable abundances in post-administration rat urine samples were detected, which might contribute to an improved sensitivity, specificity, and detection window in case of human sports drug testing programs. Further, the intact drugs were implemented into World Anti-Doping Agency-compliant initial testing (limits of detection 0.02-0.60 ng/ml) and confirmation procedures (limits of identification 0.18-0.89 ng/ml) for human urine and blood matrices. The obtained results allow extension of the test spectrum of doping agents in multitarget screening assays for growth hormone-releasing factors from human urine.
Assuntos
Dipeptídeos , Dopagem Esportivo , Indóis , Piperidinas , Pirazóis , Triptofano/análogos & derivados , Animais , Biomarcadores/metabolismo , Biomarcadores/urina , Cromatografia Líquida/métodos , Dipeptídeos/metabolismo , Dipeptídeos/urina , Feminino , Grelina , Humanos , Indóis/metabolismo , Indóis/urina , Limite de Detecção , Masculino , Piperidinas/metabolismo , Piperidinas/urina , Pirazóis/metabolismo , Pirazóis/urina , Ratos , Reprodutibilidade dos Testes , Extração em Fase Sólida , Espectrometria de Massas em Tandem/métodos , Triptofano/metabolismo , Triptofano/urinaRESUMO
OBJECTIVE: Gout is the most common inflammatory arthritis and the worldwide incidence is increasing. By revealing the metabolic alterations in serum and urine of gout patients, the first aim of our study was to discover novel molecular biomarkers allowing for early diagnosis. We also aimed to investigate the underlying pathogenic pathways. METHODS: Serum and urine samples from gout patients (n = 30) and age-matched healthy controls (n = 30) were analysed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) to screen the differential metabolites and construct a diagnostic model. Next, the model was verified and optimized in the second validation cohort (n = 100). The pathways were illustrated to understand the underlying pathogenesis of gout. RESULTS: In general, serum metabolomics demonstrated a clearer distinction than urine metabolomics. In the discovery cohort, 40 differential serum metabolites were identified that could distinguish gout patients from healthy controls. Among them, eight serum metabolites were verified in the validation cohort. Through regression analysis, the final model consisted of three serum metabolites-pyroglutamic acid, 2-methylbutyryl carnitine and Phe-Phe-that presented optimal diagnostic power. The three proposed metabolites produced an area under the curve of 0.956 (95% CI 0.911, 1.000). Additionally, the proposed metabolic pathways were primarily involved in purine metabolism, branched-chain amino acids (BCAAs) metabolism, the tricarboxylic acid cycle, synthesis and degradation of ketone bodies, bile secretion and arachidonic acid metabolism. CONCLUSION: The metabolomics signatures could serve as an efficient tool for early diagnosis and provide novel insights into the pathogenesis of gout.
Assuntos
Carnitina/análogos & derivados , Dipeptídeos , Gota/sangue , Gota/urina , Metaboloma , Ácido Pirrolidonocarboxílico , Área Sob a Curva , Biomarcadores/sangue , Biomarcadores/urina , Carnitina/sangue , Carnitina/urina , Estudos de Casos e Controles , Cromatografia Líquida de Alta Pressão/métodos , Intervalos de Confiança , Creatinina/sangue , Dipeptídeos/sangue , Dipeptídeos/urina , Humanos , Masculino , Análise Multivariada , Ácido Pirrolidonocarboxílico/sangue , Ácido Pirrolidonocarboxílico/urina , Análise de Regressão , Ácido Úrico/sangueRESUMO
In this study, the levels of concentration of homocysteine thiolactone (HTL), cysteine (Cys), and cysteinylglycine (CysGly) in the urine of autistic and non-autistic children were investigated and compared. HTL has never been analyzed in autistic children. The levels of low molecular weight sulfur compounds in the urine of both groups were determined by validated methods based on high-performance liquid chromatography with spectrofluorometric and diode-array detectors. The statistical data show a significant difference between the examined groups. Children with autism were characterized by a significantly higher level of HTL (p = 5.86 × 10-8), Cys (p = 1.49 × 10-10) and CysGly (p = 1.06 × 10-8) in urine compared with the control group. A difference in the p-value of <0.05 is statistically significant. Higher levels of HTL, Cys, and CysGly in the urine of 41 children with autism, aged 3 to 17, were observed. The obtained results may indicate disturbances in the metabolism of methionine, Cys, and glutathione in some autistic patients. These preliminary results suggest that further research with more rigorous designs and a large number of subjects is needed.
Assuntos
Transtorno Autístico/urina , Cisteína/urina , Homocisteína/análogos & derivados , Compostos de Enxofre/urina , Adolescente , Transtorno Autístico/patologia , Criança , Pré-Escolar , Dipeptídeos/urina , Feminino , Homocisteína/urina , Humanos , Masculino , Peso MolecularRESUMO
Biomonitoring of methylene diphenyl diisocyanate (MDI) in urine may be useful in industrial hygiene and exposure surveillance approaches toward disease (occupational asthma) prevention and in understanding pathways by which the internalized chemical is excreted. We explored possible urine biomarkers of MDI exposure in mice after respiratory tract exposure to MDI, as glutathione (GSH) reaction products (MDI-GSH), and after skin exposure to MDI dissolved in acetone. LC-MS analyses of urine identified a unique m/ z 543.29 [M + H]+ ion from MDI-exposed mice but not from controls. The m/ z 543.29 [M + H]+ ion was detectable within 24 h of a single MDI skin exposure and following multiple respiratory tract exposures to MDI-GSH reaction products. The m/ z 543.29 [M + H]+ ion possessed properties of dilysine-MDI, including (a) an isotope distribution pattern for a molecule with the chemical formula C27H38N6O6, (b) the expected collision-induced dissociation (CID) fragmentation pattern upon MS/MS, and (c) a retention time in reversed-phase LC-MS identical to that of synthetic dilysine-MDI. Further MDI-specific Western blot studies suggested albumin (which contains multiple dilysine sites susceptible to MDI carbamylation) as a possible source for dilysine-MDI and the presence of MDI-conjugated albumin in urine up to 6 days after respiratory tract exposure. Two additional [M + H]+ ions ( m/ z 558.17 and 863.23) were found exclusively in urine of mice exposed to MDI-GSH via the respiratory tract and possessed characteristics of previously described cyclized MDI-GSH and oxidized glutathione (GSSG)-MDI conjugates, respectively. Together the data identify urinary biomarkers of MDI exposure in mice and possible guidance for future translational investigation.
Assuntos
Dipeptídeos/urina , Isocianatos/urina , Pele/química , Animais , Biomarcadores/urina , Cromatografia Líquida , Dipeptídeos/administração & dosagem , Dipeptídeos/química , Glutationa/química , Glutationa/urina , Isocianatos/administração & dosagem , Isocianatos/química , Camundongos , Estrutura Molecular , Espectrometria de Massas em TandemRESUMO
Ethylene oxide (EO), a genotoxic industrial chemical and sterilant, forms covalent adducts with DNA and also with nucleophilic amino acids in proteins. The adduct with N-terminal valine in globin [N-(2-hydroxyethyl)valine (HEV)] has been used in biomonitoring of cumulative exposures to EO. Here we studied in rats the fate of EO-adducted N-termini of globin after life termination of the erythrocytes. Rat erythrocytes were incubated with EO to produce the HEV levels in globin at 0.4-13.2 µmol/g as determined after acidic hydrolysis. Alternative hydrolysis of the isolated globin with enzyme pronase afforded N-(2-hydroxyethyl)-L-valyl-L-leucine (HEVL) and N-(2-hydroxyethyl)-L-valyl-L-histidine (HEVH), the EO-adducted N-terminal dipeptides of rat globin α- and ß-chains, respectively. The ratio of HEVL/HEVH (1:3) reflected higher reactivity of EO with the ß-chain. The EO-modified erythrocytes were then given intravenously to the recipient rats. HEVL and HEVH were found to be the ultimate cleavage products excreted in the rat urine. Finally, rats were dosed intraperitoneally with EO, 50 mg/kg. Herein, the initial level of globin-bound HEVL (11.7 ± 1.3 nmol/g) decreased almost linearly over 60 days corresponding to the life span of rat erythrocytes. Daily urinary excretion of HEVL was almost constant for 30-40 days, decreasing faster in the subsequent phase of elimination. Recoveries of the total urinary HEVL from its globin-bound form were 84 ± 6% and 101 ± 17% after administrations of EO and the EO-modified erythrocytes, respectively. In conclusion, urinary HEVL appears to be a promising novel non-invasive biomarker of human exposures to EO.
Assuntos
Dipeptídeos/urina , Óxido de Etileno/toxicidade , Substâncias Perigosas/toxicidade , Animais , Biomarcadores/urina , Dipeptídeos/metabolismo , Monitoramento Ambiental , Eritrócitos , Globinas/metabolismo , Hidrólise , Leucina , Ratos , Valina/químicaAssuntos
Aberrações Cromossômicas , Análise Mutacional de DNA , Dipeptidases/genética , Dermatoses da Perna/genética , Deficiência de Prolidase/genética , Úlcera Cutânea/genética , Adolescente , Adulto , Criança , Cromossomos Humanos Par 19/genética , Terapia Combinada , Dipeptídeos/urina , Fácies , Feminino , Seguimentos , Humanos , Dermatoses da Perna/diagnóstico , Dermatoses da Perna/patologia , Dermatoses da Perna/terapia , Cooperação do Paciente , Deficiência de Prolidase/diagnóstico , Deficiência de Prolidase/patologia , Deficiência de Prolidase/terapia , Recidiva , Análise de Sequência de DNA , Pele/patologia , Úlcera Cutânea/diagnóstico , Úlcera Cutânea/patologia , Úlcera Cutânea/terapia , Adulto JovemRESUMO
Unhydrolyzed prolyl hydroxyproline (Pro-Hyp) and total 4-hydroxyproline (Hyp) in urine have been suggested as disease biomarkers for bone turnover and osteoporosis. Here, a rapid method was developed to accurately and selectively determine free prolyl compounds in unhydrolyzed urine samples. Urine samples were treated with o-phthalaldehyde to block primary amines followed by selective fluorogenic derivatization of secondary amines using 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) at room temperature. The derivatized mixture was then directly analyzed and quantitated on a flow-gated capillary electrophoresis system. Six prolyl compounds: Pro-Hyp, Pro-Pro, Pro-Gly, Pro-Leu, Hyp, and Pro in unhydrolyzed urine samples were separated in 30 s, which was > 60-fold faster than the reported HPLC method, using the separation buffer (pH 9.2) composed of tetraborate, cholate, and deoxycholate at 40 mM each. The limits of detection were ~ 20 nM for the dipeptides and ~ 60 nM for Hyp and Pro. The levels of these prolyl compounds in fresh urine samples were determined by using the one-point standard addition method with nipecotic acid as the internal standard. The present protocol was significantly simplified compared with reported techniques, which could improve accuracy and analytical speed. This method is potentially useful in the determination of prolyl dipeptides and Hyp in biological fluids. Graphical abstract Rapid quantitative analysis of prolyl dipeptides in urine using flow-gated capillary electrophoresis coupled with laser-induced fluorescence detection.
Assuntos
Dipeptídeos/química , Dipeptídeos/urina , Eletroforese Capilar/métodos , Hidroxiprolina/química , Hidroxiprolina/urina , Adulto , Humanos , Masculino , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Background: Meat and fish intakes have been associated with various chronic diseases. The use of specific biomarkers may help to assess meat and fish intake and improve subject classification according to the amount and type of meat or fish consumed.Objective: A metabolomic approach was applied to search for biomarkers of meat and fish intake in a dietary intervention study and in free-living subjects from the European Prospective Investigation into Cancer and Nutrition (EPIC) study.Design: In the dietary intervention study, 4 groups of 10 subjects consumed increasing quantities of chicken, red meat, processed meat, and fish over 3 successive weeks. Twenty-four-hour urine samples were collected during each period and analyzed by high-resolution liquid chromatography-mass spectrometry. Signals characteristic of meat or fish intake were replicated in 50 EPIC subjects for whom a 24-h urine sample and 24-h dietary recall were available and who were selected for their exclusive intake or no intake of any of the 4 same foods.Results: A total of 249 mass spectrometric features showed a positive dose-dependent response to meat or fish intake in the intervention study. Eighteen of these features best predicted intake of the 4 food groups in the EPIC urine samples on the basis of partial receiver operator curve analyses with permutation testing (areas under the curve ranging between 0.61 and 1.0). Of these signals, 8 metabolites were identified. Anserine was found to be specific for chicken intake, whereas trimethylamine-N-oxide showed good specificity for fish. Carnosine and 3 acylcarnitines (acetylcarnitine, propionylcarnitine, and 2-methylbutyrylcarnitine) appeared to be more generic indicators of meat and meat and fish intake, respectively.Conclusion: The meat and fish biomarkers identified in this work may be used to study associations between meat and fish intake and disease risk in epidemiologic studies. This trial was registered at clinicaltrials.gov as NCT01684917.
Assuntos
Dieta , Comportamento Alimentar , Peixes , Carne , Metaboloma , Avaliação Nutricional , Adulto , Idoso , Aminas/urina , Animais , Área Sob a Curva , Biomarcadores/urina , Galinhas , Dipeptídeos/urina , Feminino , Humanos , Masculino , Metabolômica/métodos , Pessoa de Meia-Idade , Estudos Prospectivos , Curva ROC , Alimentos MarinhosRESUMO
Wheat bran, and especially wheat aleurone fraction, are concentrated sources of a wide range of components which may contribute to the health benefits associated with higher consumption of whole-grain foods. This study used NMR metabolomics to evaluate urine samples from baseline at one and two hours postprandially, following the consumption of minimally processed bran, aleurone or control by 14 participants (7 Females; 7 Males) in a randomized crossover trial. The methodology discriminated between the urinary responses of control, and bran and aleurone, but not between the two fractions. Compared to control, consumption of aleurone or bran led to significantly and substantially higher urinary concentrations of lactate, alanine, N-acetylaspartate acid and N-acetylaspartylglutamate and significantly and substantially lower urinary betaine concentrations at one and two hours postprandially. There were sex related differences in urinary metabolite profiles with generally higher hippurate and citrate and lower betaine in females compared to males. Overall, this postprandial study suggests that acute consumption of bran or aleurone is associated with a number of physiological effects that may impact on energy metabolism and which are consistent with longer term human and animal metabolomic studies that used whole-grain wheat diets or wheat fractions.
Assuntos
Dieta , Fibras na Dieta/metabolismo , Período Pós-Prandial , Sementes/química , Triticum/química , Grãos Integrais/metabolismo , Adulto , Alanina/urina , Ácido Aspártico/análogos & derivados , Ácido Aspártico/urina , Betaína/urina , Ácido Cítrico/urina , Dipeptídeos/urina , Feminino , Manipulação de Alimentos , Hipuratos/urina , Humanos , Ácido Láctico/urina , Espectroscopia de Ressonância Magnética , Masculino , Metabolômica/métodos , Fatores Sexuais , Adulto JovemRESUMO
1. The safety, tolerability, pharmacokinetics, pharmacodynamics, and food effect of LB30870, a new selective thrombin inhibitor, were studied in 16 healthy men. 2. A double-blind, placebo-controlled single ascending dose study was done at oral doses of 5, 15, 30, 60, 120, and 240 mg under fasting conditions. An open, randomized, balanced cross-over food effect study was done at 60 mg dose. Plasma and urinary concentrations were measured up to 48 h post-dose. Coagulation and thrombin activity markers were measured at selected time points. 3. Cmax of LB30870 was at 1.3-3.0 h post-dose with a mean apparent terminal half-life (t1/2) of 2.8-4.1 h. AUC after doses above 15 mg appeared greater than dose-proportional. In fed state, AUC showed 80% reduction relative to fasting condition. 4. At doses 60 and 120 mg, peak activated partial thromboplastin time (aPTT) increased by 1.5- and 2-fold, respectively, from baseline. The aPTT and international normalized ratio (INR) were concentration-dependent, with less within-individual variability than ecarin clotting time (ECT), prothrombin time (PT), or thrombin time (TT). 5. Single oral doses of LB30870 up to 240 mg were well tolerated. The food effect must be overcome if LB30870 is to be used as an oral anti-coagulant.
Assuntos
Amidinas/administração & dosagem , Amidinas/farmacocinética , Anticoagulantes/farmacocinética , Antitrombinas/farmacocinética , Coagulação Sanguínea/efeitos dos fármacos , Dipeptídeos/administração & dosagem , Dipeptídeos/farmacocinética , Interações Alimento-Droga/fisiologia , Administração Oral , Adulto , Amidinas/sangue , Amidinas/urina , Anticoagulantes/sangue , Anticoagulantes/urina , Antitrombinas/sangue , Antitrombinas/urina , Biomarcadores Farmacológicos/sangue , Biomarcadores Farmacológicos/urina , Estudos Cross-Over , Dipeptídeos/sangue , Dipeptídeos/urina , Relação Dose-Resposta a Droga , Método Duplo-Cego , Fluoracetatos , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Leucine is an essential branched-chain amino acid that acts as a substrate for protein synthesis and as a signaling molecule. Leucine not incorporated into muscle protein is ultimately oxidized through intermediates such as ß-hydroxy-ß-methylbutyrate (HMB) which itself is reported to enhance muscle mass and function in rats and humans. HMB has been reported in the plasma following oral leucine administration in sheep and pigs but not in Sprague-Dawley rats, the standard preclinical model. Therefore, we conducted radiolabeled absorption, distribution, metabolism and excretion (ADME) studies in rats using a low (3 mg/kg) or high dose (1,000 mg/kg) of (14)C-leucine. Blood, tissue, and urine samples were analyzed for (14)C-leucine and its metabolites by HPLC-MS. Our results show for the first time that (14)C-HMB appears in plasma and urine of rats following an oral dose of (14)C-leucine. (14)C-leucine appears in plasma as (14)C-α-ketoisocaproic acid (KIC) with a slower time course than (14)C-HMB, a putative product of KIC. Further, two novel metabolites of leucine were detected in urine, N-acetyl leucine and glycyl leucine. Mass balance studies demonstrate that excretory routes accounted for no more than 0.9 % of the radiolabel and approximately 61 % of the dose was recovered in the carcass. Approximately 65 % of the dose was recovered in total, suggesting that approximately one-third of the leucine dose is oxidized to CO2. In conclusion, this study demonstrates endogenous production of HMB from leucine in adult rats, a standard preclinical model used to guide design of clinical trials in nutrition.
Assuntos
Dipeptídeos/urina , Cetoácidos/sangue , Leucina/análogos & derivados , Leucina/farmacocinética , Valeratos/sangue , Animais , Transporte Biológico , Radioisótopos de Carbono , Cromatografia Líquida de Alta Pressão , Dipeptídeos/sangue , Absorção Intestinal/fisiologia , Cetoácidos/urina , Leucina/sangue , Leucina/urina , Masculino , Espectrometria de Massas , Oxirredução , Ratos , Ratos Sprague-Dawley , Valeratos/urinaRESUMO
BACKGROUND: Tissue transglutaminase (TG2), a cross-linking enzyme, modulates deposition of extracellular matrix protein in renal fibrosis. This study aimed to examine TG2 and its cross-link product ε(γ-glutamyl)-lysine in the Fisher-Lewis rat renal transplantation (RTx) model of chronic allograft nephropathy (CAN). MATERIALS AND METHODS: Left renal grafts from male Fisher and Lewis were transplanted into Lewis rats, generating allografts and isografts, respectively. Blood pressure, renal function, and proteinuria were monitored for up to 52 weeks. At termination, CAN was assessed in the renal tissue by light and electron microscopy, TG2 and ε(γ-glutamyl)-lysine by immunofluorescence, and the urinary ε(γ-glutamyl)-lysine by high performance liquid chromatography. RESULTS: Compared to the isograft, the allografts were hypertensive, proteinuric, and uraemic and developed CAN. Extracellular TG2 (glomerulus: 64.55 ± 17.61 versus 2.11 ± 0.17, P < 0.001; interstitium: 13.72 ± 1.62 versus 3.19 ± 0.44, P < 0.001), ε(γ-glutamyl)-lysine (glomerulus: 21.74 ± 2.71 versus 1.98 ± 0.37, P < 0.01; interstitium: 37.96 ± 17.06 versus 0.42 ± 0.11, P < 0.05), TG2 enzyme activity (1.09 ± 0.13 versus 0.41 ± 0.03 nmol/h/mg protein, P < 0.05), TG2 mRNA (20-fold rise), and urinary ε(γ-glutamyl)-lysine (534.2 ± 198.4 nmol/24 h versus 57.2 ± 4.1 nmol/24 h, P < 0.05) levels were significantly elevated in the allografts and showed a positive linear correlation with tubulointerstitial fibrosis. CONCLUSION: CAN was associated with upregulation of renal TG2 pathway, which has a potential for pharmacological intervention. The elevated urinary ε(γ-glutamyl)-lysine, measured for the first time in RTx, is a potential biomarker of CAN.
Assuntos
Dipeptídeos/metabolismo , Proteínas de Ligação ao GTP/genética , Nefropatias/enzimologia , Nefropatias/etiologia , Transplante de Rim/efeitos adversos , Transglutaminases/genética , Regulação para Cima , Aloenxertos , Animais , Doença Crônica , Reagentes de Ligações Cruzadas/metabolismo , Dipeptídeos/urina , Modelos Animais de Doenças , Imunofluorescência , Proteínas de Ligação ao GTP/metabolismo , Nefropatias/sangue , Nefropatias/fisiopatologia , Glomérulos Renais/patologia , Glomérulos Renais/ultraestrutura , Masculino , Proteína 2 Glutamina gama-Glutamiltransferase , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Endogâmicos F344 , Ratos Endogâmicos Lew , Transglutaminases/metabolismoRESUMO
Biologically active low-molecular-mass thiols, mainly including glutathione (GSH), cysteine (Cys), homocysteine (Hcy), and cysteinylglycine (Cys-Gly), are important physiological components in biological fluids, and their analytical methods have gained continuous attention over recent years. We developed and validated a novel HPLC method for the quantification of GSH, Cys, Hcy, and Cys-Gly in human plasma, urine, and saliva using 4-chloro-3,5-dinitrobenzotrifluoride as the derivatization reagent. Analyses were linear from 0.15 to 500 µM with the coefficient regression range of 0.9987-0.9994. Detection limits ranged from 0.04 to 0.08 µM (S/N=3). The developed method was applied to quantification of four thiols in human biological fluids collected from five donors with the concentration range of 2.50-124.25 µM, 0-72.81 µM, and 0-4.25 µM for plasma, urine, and saliva, respectively. The present method seemed to be an attractive choice for the determination of thiols in plasma, urine, and saliva.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Cisteína/análise , Dipeptídeos/análise , Glutationa/análise , Homocisteína/análise , Saliva/química , Adulto , Cromatografia Líquida de Alta Pressão/instrumentação , Cisteína/sangue , Cisteína/urina , Dipeptídeos/sangue , Dipeptídeos/urina , Glutationa/sangue , Glutationa/urina , Homocisteína/sangue , Homocisteína/urina , Humanos , Masculino , Adulto JovemRESUMO
A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed which, with sample preparation using a commercially available kit, allows rapid quantitation of 39 chloroformate-derivatised amino acids (AAs), polyamines (PAs) and dipeptides (DPs) in complex biological matrices. Lower limits of quantitation (LOQ) were 20-150nM for putrescine, spermine, spermidine, cadaverine, agmatine, and below 5µM for all analytes. Responses were linear for all analytes between 0.5 and 50µM. Quantitative measurements of all 39 metabolites were achieved within a 15min runtime. The method was evaluated with a Pseudomonas aeruginosa cell extract study (n=24) and a larger human urine study (n=308). Batch effects were observed in the urine study and an investigation of instrument and sample stability showed a wave-like pattern in the MS responses. Both the run order and inter-batch variation were successfully corrected by normalising to pooled urine quality control data. Thus, this method should be suitable for diverse biological matrices and for large as well as small sample sets.
Assuntos
Aminoácidos/química , Cromatografia Líquida/métodos , Dipeptídeos/química , Poliaminas/química , Pseudomonas aeruginosa/química , Espectrometria de Massas em Tandem/métodos , Aminoácidos/urina , Dipeptídeos/urina , Formiatos/química , Humanos , Poliaminas/urina , Pseudomonas aeruginosa/metabolismoRESUMO
A method for determination of proline-hydroxyproline dipeptide (PHP) was developed using high-performance anion-exchange chromatography-pulsed amperometric detection (HPAEC-PAD). This method resulted in good separation of proline (Pro), hydroxyproline (Hyp), and PHP within 20min using a mobile phase of 1.2mM Ba(OH)2+1.5mM Ba(OAc)2. The linear dynamic ranges and their detection limits (S/N=3) were 1-100 (r(2)=0.9990-0.9999) and 0.05-0.3µM, respectively. Mean recoveries were 91.6-121.3% and 92.2-110.3% for intra- and inter-day assays, respectively. Our HPAEC-PAD method showed clear differences in the corrected PHP levels measured in urine samples from two groups of rats, sham-operated and ovariectomized, without the need for prior acid hydrolysis or sample derivatization.
Assuntos
Cromatografia por Troca Iônica/métodos , Dipeptídeos/urina , Técnicas Eletroquímicas/métodos , Animais , Ânions/química , Compostos de Bário/química , Dipeptídeos/química , Feminino , Limite de Detecção , Ovariectomia , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , TemperaturaRESUMO
PURPOSE: MTC-220, a conjugate of paclitaxel and muramyl dipeptide analogue, was reported to exhibit anti-tumor ability and anti-metastatic effect. The aim of present study was to investigate the elimination of MTC-220 and the related mechanisms in rats. METHODS: The excretion of MTC-220 and its metabolites in bile and urine were determined in rats after intravenous administration at 4 mg/kg. Caco-2 cell monolayer, in situ liver perfusion model and in vivo pharmacokinetics with selected inhibitors in rats were used to confirm the involvement of hepatic transporters in the elimination of MTC-220. The metabolic stability of MTC-220 was assessed by the incubation with rat liver microsomes and plasma. RESULTS: Approximately 72 % of MTC-220 was excreted into bile and less than 0.02 % into urine after administration in rats. The Caco-2 cell monolayer was impermeable to MTC-220. In in situ liver perfusion model, the hepatic extraction ratio of MTC-220 was reduced to 40 % of control in the presence of rifampicin, an Oatps inhibitor, and the cumulative biliary excretion rates of MTC-220 were reduced to 52.9, 71.5 and 62.9 % of control when concomitant perfusion with probenecid, novobiocin and verapamil, the inhibitors of Mrp2, Bcrp and P-gp, respectively. Co-administration of rifampicin, probenecid, novobiocin and verapamil with MTC-220 increased the AUC0-t and decreased the CL of MTC-220 in certain extents in rats. MTC-220 remained metabolically intact in rat liver microsomes, but less stable in plasma incubation. CONCLUSIONS: In summary, the elimination of MTC-220 was mainly through the biliary excretion in unchanged form in rats. Liver transporters including Oatps, Mrp2, Bcrp and P-gp might be all involved in the hepatic elimination of MTC-220. MTC-220 exhibited the high metabolic stability in liver microsomes, but less stable in plasma. The esterases might involve in the metabolism of MTC-220 in plasma.
Assuntos
Acetilmuramil-Alanil-Isoglutamina/análogos & derivados , Acetilmuramil-Alanil-Isoglutamina/farmacocinética , Antineoplásicos/farmacocinética , Dipeptídeos/farmacocinética , Paclitaxel/análogos & derivados , Paclitaxel/farmacocinética , Acetilmuramil-Alanil-Isoglutamina/sangue , Acetilmuramil-Alanil-Isoglutamina/urina , Animais , Antineoplásicos/sangue , Antineoplásicos/urina , Bile/química , Transporte Biológico , Células CACO-2 , Permeabilidade da Membrana Celular/efeitos dos fármacos , Dipeptídeos/sangue , Dipeptídeos/química , Dipeptídeos/urina , Interações Medicamentosas , Humanos , Masculino , Taxa de Depuração Metabólica , Microssomos Hepáticos/metabolismo , Paclitaxel/sangue , Paclitaxel/química , Paclitaxel/urina , Ratos , Ratos Sprague-DawleyRESUMO
Benzene is known to produce hematotoxicity in occupational exposure workers. This study examined the utility of metabonomic biomarkers to ascertain subacute toxicity produced by benzene in male C3H/He mice. A 30-d intermittent collection of urine was obtained from mice in this experiment. The relative organ weights, blood parameters, and bone marrow smears were examined to identify specific changes of benzene-induced toxicity. In addition, an integrated analytical approach based on liquid chromatography coupled with mass spectrometry (LC-MS) was developed to map metabolic responses in urine. Five endogenous metabolites, hypoxanthine, spermidine, 4-aminohippuric acid, indolelactic acid, and glutamylphenylalanine, were identified as potential biomarkers of benzene-induced toxicity, indicating that pathways of purine, spermidine, fatty acid, tryptophan, and peptides metabolism might be disturbed in benzene-exposed mice. Our findings showed that the use of urine metabonomics was a more sensitive tool to detect benzene-induced toxicity compared to body weight or blood parameter changes.
Assuntos
Benzeno/toxicidade , Carcinógenos Ambientais/toxicidade , Metabolômica , Solventes/toxicidade , Animais , Biomarcadores/urina , Dipeptídeos/urina , Relação Dose-Resposta a Droga , Hipoxantina/urina , Indóis/urina , Injeções Subcutâneas , Cinética , Masculino , Programas de Rastreamento/métodos , Metabolômica/métodos , Camundongos , Camundongos Endogâmicos C3H , Análise de Componente Principal , Distribuição Aleatória , Espermidina/urina , Ácido p-Aminoipúrico/urinaRESUMO
BACKGROUND AND OBJECTIVES: The pharmacokinetics of some medications may be affected by differences in race and ethnicity, which can lead to suboptimal outcomes. The present study was conducted to assess the single- and multiple-dose pharmacokinetics of saxagliptin in healthy Chinese subjects living in China. METHODS: This was an open-label, 9-day study conducted at the Drug Clinical Trial Center, Peking University Third Hospital, Beijing, China. Sixteen healthy Chinese subjects of both sexes between 21 and 33 years of age were administered saxagliptin 5 mg orally on day 1, then once daily on days 3-7. Pharmacokinetic variables for saxagliptin (primary outcome) and its active metabolite, 5-hydroxy saxagliptin (secondary outcome), after single and multiple oral doses of saxagliptin were assessed. Safety was also assessed. RESULTS: Saxagliptin was absorbed rapidly (median time to reach maximum concentration [t(max)]: 0.5 and 1 hour on days 1 and 7, respectively), and its pharmacologically active metabolite, 5-hydroxy saxagliptin, appeared in plasma (median t(max): 1.0 and 1.5 hours, respectively). Plasma exposure to 5-hydroxy saxagliptin was approximately 2- to 3-fold higher than exposure to saxagliptin. Plasma concentration-time profiles for saxagliptin and 5-hydroxy saxagliptin were similar on days 1 and 7, with no evidence of drug accumulation on repeated dosing. The elimination half-lives (t(½)) for saxagliptin and 5-hydroxy saxagliptin were approximately 3 and 4 hours, respectively, with renal excretion as the primary route of elimination. After single and multiple dosing, 54.48% and 52.60%, respectively, of the administered saxagliptin dose was recovered in urine as unchanged drug or 5-hydroxy saxagliptin. Saxagliptin was generally well tolerated. Six (37.5%) subjects experienced an adverse event (AE). All AEs were mild in intensity and judged by the investigator as not related to the study medication. There were no deaths, serious AEs, discontinuations due to AEs, or other clinically significant AEs during this study. CONCLUSION: Saxagliptin 5 mg (single dose and once-daily doses for 5 days) was generally well tolerated; the pharmacokinetics of saxagliptin and 5-hydroxy saxagliptin in healthy Chinese subjects were consistent with previous assessments in the saxagliptin clinical development program. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT00770302.