RESUMEN
BACKGROUND: Iodinated contrast (IC) is a leading cause of hospital-based acute kidney injury (AKI). Contrast-induced acute kidney injury (CI-AKI) is a decline in renal function due to iodinated contrast administration and occurs more frequently in individuals with increasingly common risk factors, such as diabetes mellitus (DM). Physical training (PT) can have renoprotective effects on CI-AKI in diabetic nephropathy. The aim of this study was to evaluate the injury in kidneys of diabetic rats submitted to treatment with IC, evaluating the impact of PT on hemodynamics and renal function in addition to oxidative profile in diabetic rats submitted to IC-AKI. MATERIALS AND METHODS: Adult male Wistar rats are randomized into four groups: citrate (n = 7): control group, citrate buffer (streptozotocin-STZ vehicle), intravenous tail (iv), single dose; DM (n = 7): STZ, 60 mg/kg, iv, single dose; DM+IC (n = 7): DM rats treated with IC (sodium meglumine ioxithalamate, 6 mL/kg, intraperitoneal (ip), single dose); DM+IC+PT (n = 7): DM rats treated with IC as mentioned and submitted to physical training. Renal function parameters (inulin clearance, neutrophil gelatinase-associated lipocalin (NGAL), serum creatinine, and urinary albumin), hemodynamics (renal blood flow and renal vascular resistance), and oxidative profile (urinary peroxides, urinary TBARS, urinary nitric oxide, and renal tissue thiols) were evaluated. RESULTS: It was possible to observe a decrease in inulin clearance, renal blood flow, and thiols in renal tissue accompanied by an increase in urinary flow, serum creatinine, urinary albumin, renal vascular resistance, urinary peroxides, urinary nitrate, and TBARS in the DM group compared to the citrate group. The DM+IC group showed a reduction in inulin clearance, and the renal dysfunction was also seen by the increased NGAL. Renal hemodynamics and oxidative profile compared were also worsened in the DM group. PT improved renal function by increasing renal blood flow and thiol levels in renal tissue and reduced renal vascular resistance, metabolites of reactive oxygen, nitrogen species, and lipid peroxidation in the DM+IC+PT group compared to DM+IC. CONCLUSIONS: Our results confirmed that DM induction increases renal vulnerability to the toxicity of IC and an association between DM with IC predisposes to severe AKI with reduced renal function alongside with renal hemodynamic alterations and oxidative mechanism of injury. The PT showed a renoprotective effect in DM animals subjected to damage with IC by modulating renal hemodynamics and oxidative profile, confirming a potential to modify the risk of CI-AKI when diabetes mellitus is present.
Asunto(s)
Lesión Renal Aguda/inducido químicamente , Lesión Renal Aguda/patología , Medios de Contraste/efectos adversos , Diabetes Mellitus Experimental/complicaciones , Condicionamiento Físico Animal , Lesión Renal Aguda/fisiopatología , Lesión Renal Aguda/orina , Animales , Diabetes Mellitus Experimental/orina , Hemodinámica , Riñón/fisiopatología , Pruebas de Función Renal , Masculino , Nitratos/orina , Oxidación-Reducción , Peróxidos/orina , Ratas Wistar , Factores de Riesgo , Compuestos de Sulfhidrilo/orina , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismoRESUMEN
Homocysteine and related thiols (cysteine, cysteinylglycine, and glutathione) in the urine of a cystathionine ß-synthase (CBS)-deficient mouse model were quantified using hydrophilic interaction chromatography with fluorescence detection. Urine samples were incubated with tris(2-carboxyethyl) phosphine to reduce disulfide bonds into thiols. After deproteinization, thiols were fluorescently derivatized with ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F). Homocysteine, cysteine, cysteinylglycine, and glutathione in mouse urine were analyzed using an amide-type column with a mobile phase of acetonitrile/120 mM ammonium formate buffer (pH 3.0) (81:19). The developed method was well-validated. Thiol concentrations in the urine of CBS-wild type (-WT), -heterozygous (-Hetero), and -knockout (-KO) mice were quantified using the developed method. As expected, total homocysteine concentration in CBS-KO mice was significantly higher than that in CBS-WT and CBS-Hetero mice. The developed method shows promise for diagnoses in preclinical and clinical studies.
Asunto(s)
Cromatografía , Cistationina betasintasa/deficiencia , Homocistinuria/etiología , Homocistinuria/orina , Compuestos de Sulfhidrilo/orina , Animales , Biomarcadores , Cromatografía/métodos , Cromatografía/normas , Cromatografía Líquida de Alta Presión/métodos , Cromatografía Líquida de Alta Presión/normas , Modelos Animales de Enfermedad , Ratones , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Compuestos de Sulfhidrilo/aislamiento & purificaciónRESUMEN
Quick and effective detection of biothiols in biological fluids has gained increasing attention due to its vital biological functions. In this paper, a novel reversible fluorescence chemosensor (L-Cu2+) based on a benzocoumarin-Cu2+ ensemble has been developed for the detection of biothiols (Cys, Hcy and GSH) in human urine. The chemosensing ensemble (L-Cu2+) contains a 2:1 stoichiometry structure between fluorescent ligand L and paramagnetic Cu2+. L was found to exclusively bond with Cu2+ ions accompanied with a dramatic fluorescence quenching maximum at 443 nm and an increase of an absorbance band centered at 378 nm. Then, the in situ generated fluorescence sluggish ensemble, L-Cu2+, was successfully used as a chemosensor for the detection of biothiols with a fluorescence "OFF-ON" response modality. Upon the addition of biothiols, the decomplexation of L-Cu2+ led to the liberation of the fluorescent ligand, L, resulting in the recovery of fluorescence and absorbance spectra. Studies revealed that L-Cu2+ possesses simple synthesis, excellent stability, high sensitivity, reliability at a broad pH range and desired renewability (at least 5 times). The practical application of L-Cu2+ was then demonstrated by the detection of biothiols in human urine sample.
Asunto(s)
Técnicas Biosensibles/métodos , Cobre/química , Fluorescencia , Compuestos de Sulfhidrilo/orina , HumanosRESUMEN
Mercaptopyruvate sulfurtransferase (Mpst) and its homolog thiosulfate sulfurtransferase (Tst = rhodanese) detoxify cyanide to thiocyanate. Mpst is attracting attention as one of the four endogenous hydrogen sulfide (H2S)/reactive sulfur species (RSS)-producing enzymes, along with cystathionine ß-synthase (Cbs), cystathionine γ-lyase (Cth), and cysteinyl-tRNA synthetase 2 (Cars2). MPST deficiency was found in 1960s among rare hereditary mercaptolactate-cysteine disulfiduria patients. Mpst-knockout (KO) mice with enhanced liver Tst expression were recently generated as its model; however, the physiological roles/significances of Mpst remain largely unknown. Here we generated three independent germ lines of Mpst-KO mice by CRISPR/Cas9 technology, all of which maintained normal hepatic Tst expression/activity. Mpst/Cth-double knockout (DKO) mice were generated via crossbreeding with our previously generated Cth-KO mice. Mpst-KO mice were born at the expected frequency and developed normally like Cth-KO mice, but displayed increased urinary 3-mercaptolactate excretion and enhanced passive systemic anaphylactic responses when compared to wild-type or Cth-KO mice. Mpst/Cth-DKO mice were also born at the expected frequency and developed normally, but excreted slightly more 3-mercaptolactate in urine compared to Mpst-KO or Cth-KO mice. Our Mpst-KO, Cth-KO, and Mpst/Cth-DKO mice, unlike semi-lethal Cbs-KO mice and lethal Cars2-KO mice, are useful tools for analyzing the unknown physiological roles of endogenous H2S/RSS production.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos/etiología , Errores Innatos del Metabolismo de los Aminoácidos/metabolismo , Compuestos de Sulfhidrilo/orina , Sulfurtransferasas/deficiencia , Alelos , Errores Innatos del Metabolismo de los Aminoácidos/orina , Animales , Biomarcadores , Modelos Animales de Enfermedad , Marcación de Gen , Genotipo , Hígado/metabolismo , Ratones , Ratones Noqueados , MutaciónRESUMEN
Biothiols play important roles in regulating redox balance in biological systems, but their discrimination is challengeable. In this work, a colorimetric nanosensing array for biothiols was established, which was composed of gold nanorods (AuNRs) and metal ions (Hg2+, Pb2+, Cu2+, Ag+). By employing label-free AuNRs as the colorimetric probe, and the color and spectral changes of AuNRs as the output signal, principal component analysis (PCA) was applied to processing the signal and generating a clustering map. Due to the different binding affinity between biothiols and metal ions, AuNRs exhibited a unique pattern to form a fingerprint-like colorimetric array, which was able to discriminate five biothiols by the naked eyes. This strategy combines PCA and sensor array to achieve rapid and accurate discrimination and detection of biothiols. In addition, the method shows the great potential in analysis of biothiols in human urine samples.
Asunto(s)
Colorimetría/métodos , Oro/química , Nanotubos/química , Compuestos de Sulfhidrilo/orina , Acetilcisteína/orina , Cisteamina/orina , Cisteína/orina , Glutatión/orina , Homocisteína/orina , Humanos , Análisis de Componente PrincipalRESUMEN
PURPOSE: Surgical stone treatment induces oxidative stress in kidney tissue. We hypothesized that tubeless percutaneous nephrolithotomy (tPCNL) may induce less oxidative stress than classic percutaneous nephrolithotomy (cPCNL) with nephrostomy tube. METHODS: Seventy-two consecutive patients with kidney stones qualified for PCNL were enrolled in the study. Patients were assigned to one of two groups (first group 33 patients-cPCNL and second group 39 patients-tPCNL). Four urine samples were collected in four consecutive days, starting the day before operation. Four oxidative stress markers were analyzed in each sample: catalase (CAT), protein sulfhydryl group (SH), total antioxidant capacity (TAC) and superoxide dismutase (SOD). RESULTS: Baseline mean levels of CAT (IU/l), SH (µmol/l), TAC (mmol/l) and SOD (NU/ml) were 19.4 versus 11.7; 18 versus 58.7; 2.02 versus 1.99; 20.5 versus 22.6 in cPCNL and tPCNL group, respectively. On day two, the levels were 89 versus 104.9; 334.7 versus 518.9; 1.87 versus 1.79; 33.7 versus 41.4, respectively. On the third day, the levels were: 67.4 versus 28.3; 206.8 versus 306.9; 2.01 versus 2.06; 38.2 versus 36.6, respectively. On the fourth day, the concentrations were 47.4 versus 18.5; 129.3 versus 208.7; 2 versus 2.06; 35 versus 45.2, respectively. Significant differences were observed only for CAT and TAC concentrations in days 3 (p = 0.04 and 0.04) and 4 (p = 0.02 and < 0.001) in favor of tPCNL. CONCLUSIONS: CAT, SH and SOD significantly rise after operation. TAC represents the inversion of other parameters. CAT is significantly lower, and TAC is significantly higher in tPCNL postoperatively favoring this method.
Asunto(s)
Cálculos Renales/cirugía , Cálculos Renales/orina , Nefrostomía Percutánea/métodos , Estrés Oxidativo , Antioxidantes/metabolismo , Catalasa/orina , Femenino , Humanos , Masculino , Persona de Mediana Edad , Nefrostomía Percutánea/instrumentación , Periodo Posoperatorio , Periodo Preoperatorio , Compuestos de Sulfhidrilo/orina , Superóxido Dismutasa/orinaRESUMEN
Several diseases are associated with perturbations in redox signaling and aberrant hydrogen sulfide metabolism, and numerous analytical methods exist for the measurement of the sulfur-containing species affected. However, uncertainty remains about their concentrations and speciation in cells/biofluids, perhaps in part due to differences in sample processing and detection principles. Using ultrahigh-performance liquid chromatography in combination with electrospray-ionization tandem mass spectrometry we here outline a specific and sensitive platform for the simultaneous measurement of 12 analytes, including total and free thiols, their disulfides and sulfide in complex biological matrices such as blood, saliva and urine. Total assay run time is <â¯10â¯min, enabling high-throughput analysis. Enhanced sensitivity and avoidance of artifactual thiol oxidation is achieved by taking advantage of the rapid reaction of sulfhydryl groups with N-ethylmaleimide. We optimized the analytical procedure for detection and separation conditions, linearity and precision including three stable isotope labelled standards. Its versatility for future more comprehensive coverage of the thiol redox metabolome was demonstrated by implementing additional analytes such as methanethiol, N-acetylcysteine, and coenzyme A. Apparent plasma sulfide concentrations were found to vary substantially with sample pretreatment and nature of the alkylating agent. In addition to protein binding in the form of mixed disulfides (S-thiolation) a significant fraction of aminothiols and sulfide appears to be also non-covalently associated with proteins. Methodological accuracy was tested by comparing the plasma redox status of 10 healthy human volunteers to a well-established protocol optimized for reduced/oxidized glutathione. In a proof-of-principle study a deeper analysis of the thiol redox metabolome including free reduced/oxidized as well as bound thiols and sulfide was performed. Additional determination of acid-labile sulfide/thiols was demonstrated in human blood cells, urine and saliva. Using this simplified mass spectrometry-based workflow the thiol redox metabolome can be determined in samples from clinical and translational studies, providing a novel prognostic/diagnostic platform for patient stratification, drug monitoring, and identification of new therapeutic approaches in redox diseases.
Asunto(s)
Disulfuros/aislamiento & purificación , Metaboloma , Estrés Oxidativo , Compuestos de Sulfhidrilo/aislamiento & purificación , Cromatografía Liquida , Disulfuros/sangre , Disulfuros/orina , Glutatión/sangre , Glutatión/aislamiento & purificación , Glutatión/orina , Humanos , Espectrometría de Masas , Oxidación-Reducción , Compuestos de Sulfhidrilo/sangre , Compuestos de Sulfhidrilo/orinaRESUMEN
Ratiometric fluorescent probes could eliminate the influence from experimental factors and improve the detection accuracy. In this article, a ratiometric nanoprobe was constructed based on silver nanoclusters (AgNCs) with nitrogen-doped carbon dots (NCDs) and used for the detection of biothiols. The fluorescence peak of AgNCs was observed at 650nm with excitation wavelength at 370nm. In order to construct the ratiometric fluorescent probe, NCDs with the excitation and emission wavelengths at 370nm and 450nm were selected. After adding AgNCs, the fluorescence of NCDs was quenched. The mechanism of the fluorescence quenching was studied by fluorescence, UV-Vis absorption and the fluorescence lifetime spectra. The results indicated that the quenching could be ascribed to the inner filter effect (IFE). With the addition of biothiols, the fluorescence of AgNCs at 650nm decreased due to the breakdown of AgNCs, and the fluorescence of NCDs at 450nm recovered accordingly. Thus, the relationship between the ratio of the fluorescence intensities (I450/I650) and biothiol concentration was used to establish the determination method for biothiols. Cysteine (Cys) was taken as the model of biothiols, and the working curve for Cys was I450/I650=0.60CCys-1.86 (CCys: µmol/L) with the detection limit of 0.14µmol/L (S/N=3). Then, the method was used for the detection of Cys in human urine and serum samples with satisfactory accuracy and recovery ratios. Furthermore, the probe could be applied for the visual semi-quantitative determination of Cys by naked eyes.
Asunto(s)
Técnicas Biosensibles/métodos , Carbono/química , Colorantes Fluorescentes/química , Nanopartículas del Metal/química , Plata/química , Compuestos de Sulfhidrilo/sangre , Compuestos de Sulfhidrilo/orina , HumanosRESUMEN
Detection of polar organic compounds (POCs) using gas chromatography (GC) is not straightforward due to high polarity, hydrophilicity, and low volatility of POCs. In this study, we report a tandem microwave-assisted derivatization method combined with salting-out assisted liquid-liquid microextraction (SALLME) to modify successively the polar groups of POCs in protic and aprotic solvents. Biothiols (cysteine and homocysteine) served as a proof of concept for this method because they possess three polar groups (thiol, amine, and carboxyl); the derivatizing reagent was 3,4,5-trifluorobenzyl bromide (Br-TFB) for alkylation. The solubility of the POCs in the protic or aprotic reaction medium affected the number of TFB molecules attached. Using the tandem derivatization with Br-TFB, the thiol and amine groups of biothiols were alkylated in the protic system, and the carboxylic groups of biothiols were alkylated in the aprotic system. The developed method was then successfully applied to measure biothiols in human urine. Because of the complex urine matrix and the lack of urine samples without endogenous biothiols, the standard addition method was utilized to avoid the matrix effect, check the recovery, and calculate the initial biothiol content in the urine. Regarding the linearity of the standard addition curves, the coefficient of determination was >0.996, and the linear regression showed satisfactory reproducibility with a relative standard deviation <3.9% for the slope and <8.8% for the intercept. The levels of cysteine and homocysteine in healthy human urine ranged from 28.8 to 111µmolL-1 and from 1.28 to 3.73µmolL-1, respectively. The proposed method effectively increased the sensitivity of GC-MS assays of water-soluble compounds in human urine.
Asunto(s)
Cromatografía de Gases y Espectrometría de Masas , Microextracción en Fase Líquida , Compuestos de Sulfhidrilo/orina , Urinálisis/métodos , Cisteína/orina , Humanos , Límite de Detección , Modelos Lineales , Microextracción en Fase Líquida/instrumentación , Microondas , Reproducibilidad de los Resultados , SolventesRESUMEN
This work describes a new approach for the determination of free biothiols in biological fluids that exploits some of the basic principles of early photographic chemistry - that was based on silver-halide recording materials - and uses broadly-available imaging devices (i.e. flatbed scanners) as detectors. Specifically, the proposed approach relies on the ability of biothiols to bind to silver ions and dissociate the silver halide crystals thus changing the photosensitivity of silver halide crystal suspension. The changes induced by biothiols on the light intensity transmitted through the silver halide suspension, after photochemical reduction, were measured with a simplified photometric approach that employs a flatbed scanner operating in transmittance mode. The overall analytical procedure for the determination of biothiols was easily executable, fast and could be applied with inexpensive and commercially available materials and reagents. What is more, physiologically relevant biothiol levels could be inspected even by the unattended eye. The developed assay was successfully applied to the determination of biothiols in urine and blood plasma samples with detection limits as low as 10µM, satisfactory recoveries (92-97%), good reproducibility (6.7-8.8%) and high selectivity against other major components of biological fluids. The utility of the method to the determination of reduced/oxidized thiol ratio's as well as its application under natural light illumination, without external energy sources, was also demonstrated and is discussed with regard to point-of need applications in facility-limited settings.
Asunto(s)
Colorimetría/economía , Colorimetría/instrumentación , Equipos y Suministros Eléctricos , Halógenos/química , Procesos Fotoquímicos , Plata/química , Compuestos de Sulfhidrilo/análisis , Costos y Análisis de Costo , Humanos , Oxidación-Reducción , Compuestos de Sulfhidrilo/sangre , Compuestos de Sulfhidrilo/química , Compuestos de Sulfhidrilo/orinaRESUMEN
The determination of thiol based biological molecules and drugs, such as cysteine (Cys) (I), α-lipoic acid (II), and sodium 2-sulfanylethane sulphonate (Mesna (III)) in human plasma are becoming progressively more important due to the growing body of knowledge about their essential role in numerous biological pathways. Herein we demonstrate a sensitive colorimetric sensor for the determination of medicinally important thiol drugs based on aggregation of the citrate capped silver nanoparticles (Ag NPs). This approach exploited the high affinity of thiols towards the Ag NPs surface which could tempt replacement of the citrate shell by the thiolate shell of target molecules, resulting in aggregation of the NPs through intermolecular electrostatic interaction or hydrogen-bonding. Because of aggregation, the plasmon band at around 400nm decreases gradually, along with the appearance of a new band connoting a red shift. The calibration curves are derived from the intensity ratios of A530/A400, which display a linear relation in the range of 1µM-150µM, 5µM-200µM and 10µM-130µM, respectively. The obtained detection limits (3σ) were found to be 1.5µM, 5.6µM and 10.2µM for compound I-III, respectively. The proposed method has been successfully applied for the detection of thiol compounds in real samples.
Asunto(s)
Nanopartículas del Metal/química , Plata/química , Espectrofotometría/métodos , Compuestos de Sulfhidrilo/química , Calibración , Humanos , Concentración de Iones de Hidrógeno , Nanopartículas del Metal/ultraestructura , Concentración Osmolar , Espectroscopía Infrarroja por Transformada de Fourier , Compuestos de Sulfhidrilo/orina , Resonancia por Plasmón de Superficie , Agua/químicaRESUMEN
Four HD urinary metabolites including hydrolysis metabolite thiodiglycol (TDG), glutathione-derived metabolite 1,1'-sulfonylbis[2-S-(N-acetylcysteinyl)ethane] (SBSNAE), as well as the ß-lyase metabolites 1,1'-sulfonylbis[2-(methylsulfinyl)ethane] (SBMSE) and 1-methylsulfinyl-2-[2-(methylthio) ethylsulfonyl]ethane (MSMTESE) are considered as important biomarkers for short-term retrospective detection of HD exposure. In this study, a single method for simultaneous quantification of the four HD metabolites in urine samples was developed using ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The four urinary metabolites were simultaneously extracted from urinary samples using a solid phase extraction (SPE) method with high extraction recoveries for all four metabolites varied in the range of 71.1-103% followed by UHPLC-MS/MS analysis. The SPE is simple and high effective only requiring 0.1mL of urinary samples and 0.5h time consuming. The problem of previous co-elution of TDG and SBSNAE in UHPLC was well solved, and complete separation of TDG, SBSNAE, SBMSE and MSMTESE from SPE-processed urine matrix was obtained to increase specificity and sensitivity. A full method validation was performed for each analyte in urine matrix. The linear range of calibration curves for the four analytes were respectively from 0.50-500ngmL-1 for TDG and SBSNAE, 0.05-500ngmL-1 for SBMSE and MSMTESE with coefficient of determination value (R2) ≥0.990. The limit of detection was 0.25ngmL-1 for TDG and SBSNAE, 0.01ngmL-1 for SBMSE and MSMTESE spiked in normal urine. The intra/inter-day precision for each analyte at three QC levels had relative standard deviation (%RSD) of ≤10.3%, and the intra/inter-day accuracy ranged between 88.0-108%. This developed method allows for simultaneous and trace measurement of four HD urinary metabolites within one single determination with the lowest usage amount of urine samples over all previous methods This study provides a useful tool for early diagnosis and monitoring of HD poisoning for medical treatment with high confidence, avoiding the need for application of several analysis methods.
Asunto(s)
Sustancias para la Guerra Química/metabolismo , Gas Mostaza/metabolismo , Acetatos/química , Biomarcadores/orina , Sustancias para la Guerra Química/análisis , Sustancias para la Guerra Química/aislamiento & purificación , Cromatografía Líquida de Alta Presión , Humanos , Gas Mostaza/análisis , Gas Mostaza/aislamiento & purificación , Reproducibilidad de los Resultados , Extracción en Fase Sólida , Compuestos de Sulfhidrilo/aislamiento & purificación , Compuestos de Sulfhidrilo/orina , Espectrometría de Masas en TándemRESUMEN
The biological importance of aminothiols is well recognized with the concentration of these compounds within biological fluids such as plasma and urine functioning as valuable biomarkers in a number of clinical circumstances and a wide variety of diseases. Herein, for the first time, chromatographic coupled chemiluminescent assay was used for simultaneous determination of aminothiols in human urine. The method exploits nucleophilic nature of aminothiols to form adducts in the existence of quinones. The released adducts retain the redox-cycling capability of parent quinones and able to liberate reactive oxygen species (ROS) when come in contact with dithiothreitol (DTT). Strong glow is released upon reaction of ROS with luminol. The method succeeded to determine aminothiols in human urine after solid phase extraction achieving good linearity and high sensitivity shown by low limit of detection (LOD) ranged from 3.8 to 16 (fmol per injection).
Asunto(s)
Mediciones Luminiscentes/métodos , Compuestos de Sulfhidrilo/química , Compuestos de Sulfhidrilo/orina , Urinálisis/métodos , Humanos , Oxidación-Reducción , Extracción en Fase Sólida , Compuestos de Sulfhidrilo/aislamiento & purificaciónRESUMEN
A simple and rapid HPLC method using 2-chloro-1-methyllepidinium tetrafluoroborate (CMLT) as a derivatization reagent was developed for simultaneous determination of homocysteine (Hcy), glutathione (GSH), γ-glutamylcysteine (γ-GluCys), cysteinylglycine (CysGly), N-acetylcysteine (NACys) and cysteine (Cys) in human saliva, plasma and urine. Separation of the analytes was achieved in just 7min using an HPLC, followed by UV detection at 355nm. Chromatographic separation was accomplished on Aeris PEPTIDE XB-C18 (150mm×4.6mm, 3.6µm) column from Phenomenex with a gradient elution: 0-4.0min, 7-30% B; 4.0-5.5min, 30-7% B; 5.5-7.5min, 7% B; (A: B, v/v); (A) 0.5% CH3COOH and (B) EtOH. Mobile phase was delivered at a flow rate 1.0mLmin(-1). Linearity in detector response for total thiols was observed over the range of 0.1-20µmolL(-1) for Hcy, GSH and γ-GluCys, 0.25-50µmolL(-1) for NACys and CysGly and 5-300 for Cys. The LOQ values for Hcy, GSH, γ-GluCys, NACys, CysGly and Cys were 0.05, 0.05, 0.10, 0.06, 0.12 and 0.08µmolL(-1), respectively. The method was successfully implemented to analysis of the samples donated by 15 apparently healthy volunteers and 10 patients.
Asunto(s)
Análisis Químico de la Sangre/métodos , Saliva/química , Compuestos de Sulfhidrilo/análisis , Urinálisis/métodos , Adulto , Calibración , Cromatografía Líquida de Alta Presión , Femenino , Humanos , Límite de Detección , Masculino , Persona de Mediana Edad , Espectrofotometría Ultravioleta , Compuestos de Sulfhidrilo/sangre , Compuestos de Sulfhidrilo/orina , Adulto JovenRESUMEN
A rapid and robust CE method using a long wavelength fluorescent reagent 1,7-dimethyl-3,5-distyryl-8-phenyl-(2-maleimide)difluoroboradiaza-s-indacene as the labeling reagent has been developed for the simultaneous determination of thiols, including glutathione, cysteine, homocysteine, N-acetylcysteine, cysteinylglycine, and penicillamine. The derivatization reaction was carried out in 14 mmol/L pH 8.5 borate buffer at 30°C for 6 min and the labeled thiols derivatives were separated with the running buffer containing 30 mmol/L pH 7.4 phosphate, 30% v/v acetonitrile and 8 mmol/L SDS within 12 min. Detection limits ranged from 0.4 to 2.4 nmol/L. To demonstrate the capability of this method, it was applied to the analysis of thiols in human urine with recoveries of 92.4-105.6%. The derivatization reaction was much faster at milder conditions, and the analysis was rapider. Moreover, with excitation wavelength at long wavelength region, background interference from samples was reduced effectively. The present method seems to be a potential choice for quantifying thiols in human urine.
Asunto(s)
Electroforesis Capilar/métodos , Colorantes Fluorescentes/química , Compuestos de Sulfhidrilo/orina , Humanos , Límite de Detección , Modelos LinealesRESUMEN
Precursor ion scan and multiple reaction monitoring scan (MRM) are two typical scan modes in mass spectrometry analysis. Here, we developed a strategy by combining stable isotope labeling (IL) with liquid chromatography-mass spectrometry (LC-MS) under double precursor ion scan (DPI) and MRM for analysis of thiols in 5 types of human cancer urine. Firstly, the IL-LC-DPI-MS method was applied for non-targeted profiling of thiols from cancer samples. Compared to traditional full scan mode, the DPI method significantly improved identification selectivity and accuracy. 103 thiol candidates were discovered in all cancers and 6 thiols were identified by their standards. It is worth noting that pantetheine, for the first time, was identified in human urine. Secondly, the IL-LC-MRM-MS method was developed for relative quantification of thiols in cancers compared to healthy controls. All the MRM transitions of light and heavy labeled thiols were acquired from urines by using DPI method. Compared to DPI method, the sensitivity of MRM improved by 2.1-11.3 folds. In addition, the concentration of homocysteine, γ-glutamylcysteine and pantetheine enhanced more than two folds in cancer patients compared to healthy controls. Taken together, the method demonstrated to be a promising strategy for identification and comprehensive quantification of thiols in human urines.
Asunto(s)
Espectrometría de Masas/métodos , Neoplasias/orina , Compuestos de Sulfhidrilo/orina , Anciano , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Here we developed a novel strategy of isotope labeling in combination with high-performance liquid chromatography-double precursor ion scan mass spectrometry (IL-LC-DPIS-MS) analysis for nontargeted profiling of thiol-containing compounds. In this strategy, we synthesized a pair of isotope labeling reagents (ω-bromoacetonylquinolinium bromide, BQB; ω-bromoacetonylquinolinium-d7 bromide, BQB-d7) that contain a reactive group, an isotopically labeled moiety, and an ionizable group to selectively label thiol-containing compounds. The BQB and BQB-d7 labeled compounds can generate two characteristic product ions m/z 218 and 225, which contain an isotope tag and therefore were used for double precursor ion scans in mass spectrometry analysis. The peak pairs with characteristic mass differences can be readily extracted from the two precursor ion scan (PIS) spectra and assigned as potential thiol-containing candidates, which facilitates the identification of analytes. BQB and BQB-d7 labeled thiol-containing compounds can be clearly distinguished by generating two individual ion chromatograms. Thus, thiol-containing compounds from two samples labeled with different isotope reagents are ionized at the same time but recorded separately by mass spectrometry, offering good identification and accurate quantification by eliminating the MS response fluctuation and mutual interference from the two labeled samples. Using the IL-LC-DPIS-MS strategy, we profiled the thiol-containing compounds in beer and human urine, and 21 and 103 thiol candidates were discovered in beer and human urine, respectively. In addition, 9 and 17 thiol candidates in beer and human urine were successfully identified by further comparison with thiol standards or tandem mass spectrometry analysis. Taken together, the IL-LC-DPIS-MS method is demonstrated to be a promising strategy in the profiling of compounds with identical groups in metabolomics study.
Asunto(s)
Cerveza/análisis , Hidrocarburos Bromados/química , Metabolómica/métodos , Compuestos de Quinolinio/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Compuestos de Sulfhidrilo/orina , Cromatografía Liquida , Humanos , Marcaje Isotópico , Metabolómica/instrumentación , Espectrometría de Masa por Ionización de Electrospray/instrumentación , Espectrometría de Masas en TándemRESUMEN
Autism is a complex neurodevelopmental disorder usually presents in early childhood and thought to be influenced by genetic and environmental factors. Individuals with autism vary widely in abilities, intelligence, and behaviors. It is common for children with autism to exhibit eating disorders and some have preferences for soft and sweetened food making them susceptible to caries. Furthermore, a wide spectrum of medical and behavioral symptoms exhibited by children with autism makes routine dental care very difficult. Intellectual disability is evident in approximately 70% of individuals with autism and most psychiatric disorders, including autism, are associated with increased oxidative stress. 29 subjects diagnosed with autism, in the age group of 6 to 12 years, were a part of the study. Furturemore, 24 normal healthy siblings of same age group were taken as the control group. The present study aimed to evaluate oxidative stress biomarkers such as urinary total antioxidant concentration (TAC), catalase activity (CAT) and total thiol molecules (TTM). The results showed the autism group have significantly higher CAT activity and concomitant lower TAC and TTM concentration in comparison with control group. The results are discussed in relation to an increased vulnerability to oxidative damage, which may contribute to the development and clinical manifestation of symptoms of autism.
Asunto(s)
Antioxidantes/metabolismo , Trastorno Autístico/orina , Catalasa/metabolismo , Compuestos de Sulfhidrilo/orina , Trastorno Autístico/enzimología , Biomarcadores/orina , Estudios de Casos y Controles , Niño , Femenino , Humanos , Irán , Masculino , Estrés Oxidativo , HermanosRESUMEN
A highly sensitive method for the determination of sulfur mustard (SM) metabolites thiodiglycol (TDG) and thiodiglycol sulfoxide (TDGO) in urine was established and validated using isotope-dilution negative-ion chemical ionization (NICI) gas chromatography-mass spectrometry (GC-MS). TDGO in the samples was reduced with TiCl3, and then determined together with TDG as a single analyte. The sample preparation procedures, including two solid-phase-extraction (SPE) clean-up steps, were optimized to improve the sensitivity of the method. The limits of detection (LOD) for both TDG and TDG plus TDGO (TDG + TDGO) were 0.1 ng mL(-1), and the limits of quantitation (LOQ) for both were 0.3 ng mL(-1). The method was used in a rabbit cutaneous SM exposure model. Domestic rabbits were exposed to neat liquid SM at three dosage levels (0.02, 0.05, and 0.15 LD50), and the urinary excretion of four species of hydrolysis metabolites, namely free TDG, free plus conjugated TDG (total TDG), free TDG + TDGO, and free plus conjugated TDG + TDGO (total TDG + TDGO), was evaluated to investigate the metabolic processes. The total urinary excretion profiles of the metabolites, including the peak time, time window, and dose-response and time-response relationships, were clarified. The results revealed that the concentrations of TDG and TDG + TDGO in the urine increased quickly and then decreased rapidly in the first two days after SM exposure. The cumulative amount of total TDG + TDGO excreted in urine during the first five days accounted for 0.5-1% of the applied dose of SM. It is also concluded that TDG and TDGO in urine existed mainly in free form, the levels of glucuronide and of sulfate conjugates of TDG or TDGO were very low, and most hydrolysis metabolites were present in the oxidized form (TDGO). The study indicates that the abnormal increase of TDG and TDGO excretion levels can be used as a diagnostic indicator and establishes a reference time-window for retrospective analysis and sampling after SM exposure.
Asunto(s)
Sustancias para la Guerra Química/toxicidad , Fármacos Dermatológicos/toxicidad , Gas Mostaza/toxicidad , Compuestos de Sulfhidrilo/orina , Sulfóxidos/orina , Administración Cutánea , Animales , Biotransformación , Sustancias para la Guerra Química/metabolismo , Fármacos Dermatológicos/metabolismo , Relación Dosis-Respuesta a Droga , Cromatografía de Gases y Espectrometría de Masas/métodos , Técnicas de Dilución del Indicador , Masculino , Gas Mostaza/metabolismo , Oxidación-Reducción , Conejos , Piel/irrigación sanguínea , Piel/efectos de los fármacos , Piel/metabolismo , Extracción en Fase Sólida , Titanio/químicaRESUMEN
PURPOSE: Oxidative stress can cause tissue damage in many diseases. Oxidative status depends on the balance between total oxygen radical absorbance capacity and antioxidants. Neurogenic bladder (NB) is a special state where oxidative status can influence urinary tract function. We decided to measure antioxidant (thiol) status in patients with NB and assess the effect of NB on the urinary antioxidant status and to correlate it with urodynamic findings. MATERIALS AND METHODS: The investigation was conducted on two groups. The first group, constituted of 41 children with NB. The second group, consisted of 20 healthy children with no abnormality in urinary and nervous systems. The antioxidant status was assessed based on the enzyme-linked immunosorbent assay of thiols. RESULTS: The median value of urinary protein thiol level was significantly lower in NB patients than in reference group [median 48 (0.0-633.33) and 221.55 (0.17-1293] µmoL/g protein, respectively (P < .01). We found out the statistically significant differences in urinary thiol level between patients with and without overactivity (P = .017) and between catheterized and noncatheterized patients (P = .048). CONCLUSION: This study demonstrates that antioxidant status in patients with NB decreased and the level of thiol status depends on the grade of bladder overactivity. Oxidative stress may be involved in the pathophysiology of bladder dysfunction related to neurogenic damage.