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We examined standard clinical and laboratory biochemical parameters, as well as the levels of aminothiols in the blood and urine (homocysteine (Hcy), cysteine (Cys), S-adenosylmethionine (SAM), and S-adenosylhomocysteine (SAH)) via capillary electrophoresis in patients with CKD at stages II-V. Patient outcomes were assessed after five years. To complete forecasting, correlation and ROC analysis were performed. It was found that the levels of Cys and Hcy in blood plasma were earlier markers of CKD starting from stage II, while the levels of SAM and SAM/SAH in urine made it possible to differentiate between CKD at stages II and III. Blood plasma Hcy and urinary SAM and SAM/SAH correlated with mortality, but plasma Hcy concentrations were more significant. Thus, plasma Hcy, urine SAM, and SAM/SAH can be considered to be potential diagnostic and prognostic markers in patients with CKD.
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The very high intensity of exercise accompanied by mental stress triggers adaptive mechanisms associated with adrenocortical steroidogenesis. However, the association between adrenocortical steroidogenesis and the high intensity of exercise in elite athletes is poorly studied. A significant obstacle to solving this complex task is the wide range (4-5 orders) of steroid concentrations in serum and limitations related to the amount of biological samples taken from professional athletes. To solve this task, we have developed and validated a non-trivial approach for targeted serum metabolic profiling based on the use of LC-MS/MS with dual-polarity electrospray ionization. The developed method based on the proposed approach allows for the quantitative determination of 14 stress resistance biomarkers in elite athletes using a small amount of specimen within 8.5 min.
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Platinum nanoparticles (nPts) have neuroprotective/antioxidant properties, but the mechanisms of their action in cerebrovascular disease remain unclear. We investigated the brain bioavailability of nPts and their effects on brain damage, cerebral blood flow (CBF), and development of brain and systemic oxidative stress (OS) in a model of cerebral ischemia (hemorrhage + temporary bilateral common carotid artery occlusion, tBCAO) in rats. The nPts (0.04 g/L, 3 ± 1 nm diameter) were administered to rats (N = 19) intraperitoneally at the start of blood reperfusion. Measurement of CBF via laser Doppler flowmetry revealed that the nPts caused a rapid attenuation of postischemic hypoperfusion. The nPts attenuated the apoptosis of hippocampal neurons, the decrease in reduced aminothiols level in plasma, and the glutathione redox status in the brain, which were induced by tBCAO. The content of Pt in the brain was extremely low (≤1 ng/g). Thus, nPts, despite the extremely low brain bioavailability, can attenuate the development of brain OS, CBF dysregulation, and neuronal apoptosis. This may indicate that the neuroprotective effects of nPts are due to indirect mechanisms rather than direct activity in the brain tissue. Research on such mechanisms may offer a promising trend in the treatment of acute disorders of CBF.
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OBJECTIVE: Acute brain ischemia is accompanied by a disruption of low-molecular-weight aminothiols (LMWTs) homeostasis, such as homocysteine (Hcy), cysteine (Cys), and glutathione (GSH). We investigated the redox balance of LMWTs in blood plasma and its influence on ischemic stroke severity and the functional outcome in patients with an acute period. PATIENTS AND METHODS: A total of 177 patients were examined. Total and reduced forms of LMWTs were determined in the first 10-24â h. Stroke severity and functional state were estimated using the National Institutes of Health Stroke Scale (NIHSS) and the modified Rankin Scale (mRs) at admission and after 21 days. RESULTS: Patients with high levels of total Hcy (> 19â µM) showed significantly reduced redox statuses of all LMWTs. Patients with low total GSH levels (≤ 1.07â µM) were at an increased risk of higher stroke severity (NIHSS > 10) compared to patients with a total GSH level > 2.64â µM (age/gender-adjusted odds ratio: 4.69, 95% CI: 1.43-15.4). DISCUSSION: (1) low total GSH level can be considered as a novel risk marker for the severity of acute stroke in conditions of low redox status of LMWTs and (2) high Hcy levels associated with low redox status of LMWTs.
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Isquemia Encefálica , AVC Isquêmico , Acidente Vascular Cerebral , Glutationa , Humanos , OxirreduçãoRESUMO
OBJECTIVE: To determine whether urine S-adenosylmethionine (SAM) might be an indicator of chronic kidney disease (CKD). METHODS: We investigated urine levels of SAM and related metabolites (S-adenosylhomocysteine and homocysteine cysteine) in 62 patients (average age, 65.9 years) with CKD (stages II-V). RESULTS: Patients with stages III-V CKD stages have significantly decreased urine levels and SAM/S-adenosylhomocysteine ratio and also cysteine/homocysteine ratio in blood plasma (P <.05), compared with patients with stage II CKD. Urine SAM levels allowed us to distinguish patients with mildly decreased kidney function from those with moderate to severe renal impairment (AUC, 0.791; sensitivity, 85%; specificity, 78.6%). CONCLUSIONS: Our study results demonstrate that urine SAM is a potent biomarker for monitoring renal function decline at early CKD stages. Urine SAM testing confers an additional advantage to healthcare professionals in that it is noninvasive.
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Homocisteína/sangue , Insuficiência Renal Crônica/urina , S-Adenosil-Homocisteína/urina , S-Adenosilmetionina/urina , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Taxa de Filtração Glomerular , Humanos , Masculino , Pessoa de Meia-Idade , Insuficiência Renal Crônica/sangue , Insuficiência Renal Crônica/fisiopatologiaRESUMO
Experiments from flight- and ground-based model systems suggest that unexpected alterations of the human lymphoblastoid cell line Jurkat, as well as effects on cell growth, metabolism, and apoptosis, can occur in altered gravity conditions. Using a desktop random positioning machine (RPM), we investigated the effects of simulated microgravity on Jurkat cells and their multidrug-resistant subline, Jurkat/A4 cells. The viability of Jurkat/A4 cells decreased after simulated microgravity in contrast with the Jurkat cells. At the same time, the viability between the experimental Jurkat cells and control Jurkat cells was not significantly different. Of note, Jurkat cells appeared as less susceptible to apoptosis than their multidrug-resistant clone Jurkat/A4 cells, whereas cell-cycle analysis showed that the percentage of Jurkat/A4 cells in the S-phase was increased after 72 and 96 h of RPM-simulated microgravity relative to their static counterparts. The differences in Jurkat cells at all phases between static and simulated microgravity were not significant. The surface expression of the intercellular adhesion molecule 3 (ICAM-3)-also known as cluster of differentiation (CD)50-protein was changed for Jurkat/A4 cells following exposure to the RPM. Changes in cell morphology were observed in the Jurkat/A4 cells after 96 h of RPM-simulated microgravity. Thus, we concluded that Jurkat/A4 cells are more sensitive to RPM-simulated microgravity as compared with the parental Jurkat cell line. We also suggest that intercellular adhesion molecule 3 may be an important adhesion molecule involved in the induction of leukocyte apoptosis. The Jurkat/A4 cells with an acquired multidrug resistance phenotype could be a useful model for studying the effects of simulated microgravity and testing anticancer drugs.
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Antígenos CD/metabolismo , Moléculas de Adesão Celular/metabolismo , Resistência a Múltiplos Medicamentos , Células Jurkat/citologia , Simulação de Ausência de Peso/instrumentação , Apoptose , Ciclo Celular , Proliferação de Células , Sobrevivência Celular , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Humanos , Células Jurkat/metabolismoRESUMO
A new approach for direct determination of S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), and methylthioadenosine (MTA) in urine was developed based on MEKC by using SDS modified with isobutanol in the presence of PEG-300. Analytes were first extracted with grafted phenylborononic acid. Using a 50 µm internal diameter silica capillary of 32 cm total length filled with 0.05 M SDS, 0.05 M H3 PO4 , 5% (v/v) isobutanol, and 10% (v/v) PEG-300, LOQ of 0.15 µM for SAM and SAH, and 0.2 µM for MTA was reached. Accuracy was 92% for MTA, 109% for SAH, and 105% for SAM, intra- and interday imprecision were <2.5 and ≤3%, respectively. The total time of analysis for one sample was 10 min. Analysis of 30 urine samples from healthy volunteers showed that the median SAM and SAH levels were 12.1 and 0.73 µM, respectively. MTA levels, which were determined in urine for the first time (according to our data), were 0.43 µM, and these values correlated well with the SAM level (r = 0.748, p < 0.01).
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Adenosina/análogos & derivados , Cromatografia Capilar Eletrocinética Micelar/métodos , S-Adenosil-Homocisteína/urina , S-Adenosilmetionina/urina , Tionucleosídeos/urina , Adenosina/urina , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Limite de Detecção , Modelos Lineares , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Adulto JovemRESUMO
OBJECTIVE: To evaluate the association of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) in urine with chronic kidney disease (CKD). METHODS: Case-control study including 50 patients with CKD and 20 healthy volunteers. RESULTS: SAM level and SAM/SAH ratio in urine were significantly lower in patients than in control individuals (P <.001 and P = .01, respectively). The estimated glomerular filtration rate was associated with the SAM level (P = .04) and the SAM/SAH ratio in urine (P = .01). CONCLUSION: CKD is associated not only with the decline in the SAM level but also with the decrease in the SAM/SAH ratio in urine. Thus, use of the urinary SAM/SAH ratio as a noninvasive diagnostic indicator of renal function seems promising.
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Insuficiência Renal Crônica/urina , S-Adenosil-Homocisteína/urina , S-Adenosilmetionina/urina , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/urina , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Insuficiência Renal Crônica/diagnósticoRESUMO
A validated approach to determine various methionine cycle metabolites (S-adenosylmethionine, S-adenosylhomocysteine, and methylthioadenosine) in human blood plasma is offered. The approach is based on solid-phase extraction (with grafted phenylboronic acid) and derivatization with chloroacetaldehyde followed by ultra-performance liquid chromatography with fluorescence detection. We used a 100â¯×â¯2.1â¯mmâ¯×â¯1.8⯵m C18 column for the selective separation of analytes. Chromatographic separation was achieved with gradient elution of acetonitrile (flow rate 0.2â¯mL/min) from 2 to 20%. The eluent was initially composed of 10â¯mM KH2PO4 with 10â¯mM acetic acid and 25⯵M heptafluorobutyric acid. The total analysis time was 11â¯min. Validation of the method included detection of the limit of detection (2â¯nM), limit of quantification (5â¯nM), accuracy (97.2-101%), and intra- and interday precision (2.2-9.0%). Analysis of plasma samples from healthy volunteers revealed that the average levels of S-adenosylmethionine, S-adenosylhomocysteine, and methylthioadenosine were 93.6, 20.9 and 14.8â¯nM, respectively.
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Cromatografia Líquida de Alta Pressão/métodos , S-Adenosil-Homocisteína/sangue , S-Adenosilmetionina/sangue , Cromatografia Líquida de Alta Pressão/instrumentação , Fluorescência , Humanos , Plasma/química , S-Adenosil-Homocisteína/isolamento & purificação , S-Adenosilmetionina/isolamento & purificação , Extração em Fase SólidaRESUMO
OBJECTIVES: To determine the disruption of low-molecular-weight aminothiols (LMWTs: cysteine, cysteinylglycine, homocysteine, and glutathione) homeostasis in blood plasma during the acute and early subacute stages after ischemic stroke. PATIENTS AND METHODS: We admitted 41 patients with primary large-artery atherosclerosis and cardioembolic stroke in the carotid arteries within the first 6-24â¯h from the moment of neurologic symptoms development. We included 31 patients with chronic cerebral ischemia in the control group. Total LMWT levels and their reduced forms were measured in blood plasma on the 1st, 3rd, 7th, and 15th days after stroke. RESULTS: Our study demonstrated a decrease of cysteine and cysteinylglycine reduced forms and an increase of total glutathione and cysteine levels. There were no differences in LMWT levels among stroke subtypes (large-artery atherosclerosis and cardioembolic stroke). The decrease (or increase) in GSH and Hcy redox status on the 3rd day after stroke was associated with severe neurological deficit. Total Hcy (1st day), Cys (3rd day) and CG(7th day) levels were associated with the size of cerebral infarction area. Logistic regression analysis indicated that reduced homocysteine, total cysteinylglycine levels, and cysteine redox status at admission were predictive factors for ischemic stroke occurrence with a probability of 86.2% (pâ¯<â¯0.001). CONCLUSIONS: LMWTs may indicate the severity of neurological deficit and the size of the cerebral infarct, and their complex determination can be of diagnostic importance both at an early stage of ischemic stroke development and during its monitoring.
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Isquemia Encefálica/sangue , Dissulfetos/sangue , Homeostase/fisiologia , Acidente Vascular Cerebral/sangue , Compostos de Sulfidrila/sangue , Feminino , Glutationa/sangue , Humanos , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo/fisiologia , Fatores de RiscoRESUMO
Cerebral ischemia has previously been shown to cause a systemic decrease in levels of the reduced forms of low-molecular-weight aminothiols [cysteine (Cys), homocysteine (Hcy), and glutathione (GSH)] in blood plasma. In this study, we examined the effect of beta-adrenergic receptor (ß-AR) antagonists metoprolol (Met) and nebivolol (Neb) on the redox status of these aminothiols during acute cerebral ischemia in rats. We used a model of global cerebral ischemia (bilateral occlusion of common carotid arteries with hypotension lasting for 10 minutes). The antagonists were injected 1 hour before surgery. Total and reduced Cys, Hcy, and GSH levels were measured 40 minutes after the start of reperfusion. Neb (0.4 and 4 mg/kg) and Met (8 and 40 mg/kg) treatment increased the levels of reduced aminothiols and the global methylation index in the hippocampus. The treatments also prevented any decrease in reduced aminothiol levels in blood plasma during ischemia. Although both of these drugs eliminated delayed postischemic hypoperfusion, only Neb reduced neuronal damage in the hippocampus. The results indicate an essential role of ß1-AR blockage in the maintenance of redox homeostasis of aminothiols in the plasma and brain during acute cerebral ischemia.
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Antagonistas de Receptores Adrenérgicos beta 1/farmacologia , Isquemia Encefálica/tratamento farmacológico , Cisteína/sangue , Glutationa/sangue , Hipocampo/efeitos dos fármacos , Homocisteína/sangue , Metoprolol/farmacologia , Nebivolol/farmacologia , Fármacos Neuroprotetores/farmacologia , Doença Aguda , Animais , Isquemia Encefálica/sangue , Isquemia Encefálica/patologia , Isquemia Encefálica/fisiopatologia , Circulação Cerebrovascular/efeitos dos fármacos , Modelos Animais de Doenças , Hipocampo/metabolismo , Hipocampo/patologia , Masculino , Peso Molecular , Oxirredução , RatosRESUMO
A sensitive capillary electrophoresis method was developed for the determination of aminothiol (cysteine, homocysteine, and glutathione) total levels in human blood plasma. Analytes were derivatized with Ellman's reagent (5,5'-dithiobis(2-nitrobenzoic acid)) after reduction with dithiothreitol. Liquid-liquid extraction was applied to purify the samples and concentrate the analytes. Total analysis time was 7.5 min using a silica capillary (50 µm i.d.; effective separation length 23.5 cm). Electrophoretic separation was performed using 50 mM citric acid with 20 mM triethanolamine (pH 3) containing 2% Ficoll 400. Detection limit was 0.8 µM for glutathione and 0.3 µM for both cysteine and homocysteine. Accuracy was 94 - 107%, repeatability and reproducibility were ca. 2.7 - 3.5 and 2.5 - 6.5%, respectively.
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Análise Química do Sangue/métodos , Compostos de Sulfidrila/sangue , Compostos de Sulfidrila/química , Adulto , Eletroforese Capilar , Feminino , Humanos , Extração Líquido-Líquido , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Compostos de Sulfidrila/isolamento & purificaçãoRESUMO
The purpose of the present study was to investigate the use of laser Doppler flowmetry (LDF) signals coupled with spectral wavelet analysis to detect endothelial link dysfunction in the autoregulation of cerebral blood flow in the setting of hyperhomocysteinaemia (HHcy). Fifty-one rats were assigned to three groups (intact, control, and HHcy) according to the results of biochemical assays of homocysteine level in blood plasma. LDF signals on the rat brain were recorded by LAKK-02 device to measure the microcirculatory blood flow. The laser operating wavelength and output power density were1064 nm and 0.051 W/mm2, respectively. A Morlet mother wavelet transform was applied to the measured 8-min LDF signals, and periodic oscillations with five frequency intervals were identified (0.01-0.04 Hz, 0.04-0.15 Hz, 0.15-0.4 Hz, 0.4-2 Hz, and 2-5 Hz) corresponding to endothelial, neurogenic, myogenic, respiratory, and cardiac origins, respectively. In initial state, the amplitude of the oscillations decreased by 38% (P < 0.05) in the endothelial range in HHcy rats than in control rats. Cerebral autoregulation was challenged by hemorrhagic hypotension. The lower limit of autoregulation raised in a rat model of chronic HHcy (71.5 ± 0.7 mmHg in HHcy vs. 62.3 ± 0.5 mmHg in control). The data obtained indicate that the laser Doppler method and wavelet analysis may be successfully applied to detect the dysfunction of the endothelial link in cerebral vessel tone and to reveal the pathological shift of lower limit of autoregulation.
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Circulação Cerebrovascular/fisiologia , Homeostase , Hiper-Homocisteinemia/fisiopatologia , Análise de Ondaletas , Animais , Encéfalo/irrigação sanguínea , Encéfalo/fisiologia , Doença Crônica , Hemodinâmica , Hemorragia/fisiopatologia , Humanos , Fluxometria por Laser-Doppler , Masculino , Ratos , Pele/irrigação sanguíneaRESUMO
A rapid and selective method has been developed for highly sensitive determination of total cysteine and homocysteine levels in human blood plasma and urine by capillary electrophoresis (CE) coupled with liquid-liquid extraction. Analytes were first derivatized with 1,1'-thiocarbonyldiimidazole and then samples were purified by chloroform-ACN extraction. Electrophoretic separation was performed using 0.1 M phosphate with 30 mM triethanolamine, pH 2, containing 25 µM CTAB, 2.5 µM SDS, and 2.5% polyethylene glycol 600. Samples were injected into the capillary (with total length 32 cm and 50 µm id) at 2250 mbar*s and subsequent injection was performed for 30 s with 0.5 M KÐÐ. The total analysis time was less than 9 min, accuracy was 98%, and precision was <2.6%. The LOD was 0.2 µM for homocysteine and 0.5 µM for cysteine. The use of liquid-liquid extraction allowed the precision and sensitivity of the CE method to be significantly increased. The validated method was applied to determine total cysteine and homocysteine content in human blood plasma and urine samples obtained from healthy volunteers and patients with kidney disorders.
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Cisteína/sangue , Cisteína/urina , Eletroforese Capilar/métodos , Homocisteína/sangue , Homocisteína/urina , Extração Líquido-Líquido/métodos , Acetonitrilas/química , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Clorofórmio/química , Feminino , Humanos , Limite de Detecção , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Adulto JovemRESUMO
OBJECTIVE: Recent studies have shown that cerebral ischaemia causes not only local, but also systemic oxidative stress. This leads to oxidation of thiol-containing compounds, including low-molecular-weight thiols (cysteine, glutathione, homocysteine and others). Therefore, the aim of this work was to verify the hypothesis that the thiol/disulphide homeostasis of low-molecular-weight thiols is disturbed in the early stages of cerebral ischaemia. METHODS: Two experimental rat models of ischaemia were used: a global model of vascular ischaemia (clamping the common carotid arteriesâ +â haemorrhage) and focal ischaemia (middle cerebral artery occlusion). The total levels of thiols and their reduced forms were measured before surgery and after 40 minutes of reperfusion (global) or 3â hours (focal) ischaemia. RESULTS: The global ischaemia model caused a marked (2.5-4 times, Pâ <â 0.01) decrease in the plasma thiol/disulphide redox state, and focal ischaemia caused an even larger decrease (30-80 times, Pâ <â 0.001). DISCUSSION: These results suggest that plasma low-molecular-weight thiols are actively involved in oxidation reactions at early stages of cerebral ischaemia; therefore, their reduced forms or redox state may serve as a sensitive indicator of acute cerebrovascular insufficiency.
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Biomarcadores/sangue , Isquemia Encefálica/sangue , Dissulfetos/sangue , Compostos de Sulfidrila/sangue , Animais , Glutationa/sangue , Homeostase/fisiologia , Masculino , Oxirredução , Estresse Oxidativo/fisiologia , RatosRESUMO
An approach that allows direct analysis of the ratio of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) by using CE is presented. The analytes were extracted on phenylboronic acid phase and eluted with 100 mmol/L HCl. CE separation of the analytes took place in the transient isotachophoresis mode with addition of NaCl and meglumine to the samples. The sensitivity (S/N = 3) and quantification limit (S/N = 10) of the method were 0.07 and 0.2 µmol/L, respectively, using a silica capillary with 50 µm internal diameter and 30.5 cm total length. The BGE was 0.02 mol/L Tris with 1 mol/L HCOOH (pH 2.2), and the separation voltage was 15-17 kV. Accuracy of SAM and SAH analysis in urine was 96 and 105%, respectively; interday precision for the SAM/SAH ratio was within 6%. The theoretical plate number exceeded a million. Total analysis time was 8.5 min.
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Ácidos Borônicos/química , Eletroforese Capilar/métodos , S-Adenosil-Homocisteína/urina , S-Adenosilmetionina/urina , Extração em Fase Sólida/métodos , Adulto , Idoso , Feminino , Humanos , Modelos Lineares , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Adulto JovemRESUMO
A simple and rapid approach is described for the determination of total plasma cysteine and homocysteine using capillary electrophoresis. Human plasma samples were reduced with dithiothreitol and then processed with 1,1'-thiocarbonyldiimidazole in acetonitrile. After centrifugation, the sample supernatant was injected directly into a capillary by applying negative voltage and analytes were stacked after alkaline post-injection. Using a 50µm i.d. silica capillary of 35cm total length, filled with 0.1M triethanolamine, 0.15M formic acid, and 50µM hexadecyltrimethylammonium bromide (pH 3.9), we reached a limit of quantification of 2.5µM for homocysteine. Accuracy was 94.7-105.1%, intra- and inter-day imprecisions were <2.5 and <3%, respectively. The total analysis time was 6min. Furthermore, liquid-phase extraction with isopropanol led to a fourfold increase in sensitivity.
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Cisteína/sangue , Eletroforese Capilar/métodos , Homocisteína/sangue , Imidazóis/química , Centrifugação , Cromatografia Líquida de Alta Pressão , Humanos , Espectrofotometria UltravioletaRESUMO
RATIONALE: The presence in a urinary matrix of a large number of endogenous steroids and corticosteroids with similar structures can hamper the detection of specific exogenous steroids using liquid chromatography/mass spectrometry (LC/MS) with reversed-phase columns. Therefore, the development of LC/MS methods using alternative columns is of great interest. Porous graphitized carbon is a unique stationary phase for high-performance liquid chromatography (HPLC), with properties differing from traditional silica-based and polymeric stationary phases. METHODS: The new method involves enzymatic hydrolysis, liquid-liquid extraction, and determination by high-temperature HPLC/Orbitrap mass spectrometry (HTLC/Orbitrap MS) with atmospheric pressure photoionization (APPI). To achieve APPI of doping substances, the mobile phase consisted of 0.1% CF3COOH (A) and a mixture of acetonitrile/2-propanol (25:75 v/v), containing 0.1% CF3COOH (B), which was used as an effective proton source. RESULTS: A screening method for the detection of 57 exogenous steroids has been developed. The method was validated by spiking 10 different blank urine samples at different concentration levels. Validation parameters included limit of detection (LOD), selectivity, ion suppression, extraction recovery, and repeatability. All studied compounds had an LOD lower than the minimum required performance level. Of the 57 steroids studied, 55 showed recovery better than 70%. For all of the analytes, the relative retention times proved to be stable between days, with relative standard deviations (RSDs) smaller than 0.3%. In addition, the interday RSDs of the peak area ratios ranged between 0.7% and 14.5%. CONCLUSIONS: The proposed method matches the basic requirements of all methods used to analyze drugs or metabolites in an antidoping laboratory, i.e., sensitivity, selectivity, and specificity. The acquisition of full-scan mass spectra with accurate masses can be a valuable tool in the retrospective evaluation of analyzed samples for anabolic steroids recently added to the prohibited list.
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Anabolizantes/urina , Espectrometria de Massas/métodos , Cromatografia Líquida de Alta Pressão/métodos , Dopagem Esportivo , Grafite/química , Temperatura Alta , Humanos , Masculino , PorosidadeRESUMO
This work proposes an approach to the direct analysis of S-adenosylhomocysteine (SAH) and the methylation index in blood using CE with UV detection (CE-UV). After application of meglumine postinjection, we achieved SAH in-capillary preconcentration in the HClO4 extracts of erythrocytes, which improved the detection limit (S/N = 3) of SAH up to 3 fmol or 180 nmol/L at the injection volume of 50 nL, taking into account the sample dilution rate. CE-UV was carried out in 30 mM glycine and 45 mmol/L HCl (pH ~1.8) at 17 kV in a capillary 48 cm in length and 50 µm id. Accuracy of the technique was 101% and reproducibility was about 12%.
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Eletroforese Capilar/métodos , S-Adenosil-Homocisteína/sangue , Adolescente , Adulto , Eritrócitos/química , Humanos , Limite de Detecção , Meglumina , Metilação , Percloratos/química , Reprodutibilidade dos Testes , S-Adenosil-Homocisteína/química , Adulto JovemRESUMO
Peroxisome proliferator-activated receptor-δ (PPARδ) agonists are the drug candidates with potential performance-enhancing properties, and therefore their illegitimate use in sports should be controlled. To simulate the metabolism of PPARδ agonist GW0742, in vitro reactions were performed which demonstrated that the main metabolic pathway is oxidation of the acyclic divalent sulfur to give the respective sulfoxide and sulfone. After being characterized by liquid chromatography-mass spectrometry (LC-MS), these metabolites were evaluated in urine samples collected after a controlled excretion study. For comparative purposes, GW1516 excretion study was also performed. It has been shown that GW1516 and GW0742 are best monitored as the sulfone metabolites which are detectable in urine using LC-MS/MS based procedure up to 40 and 20 days after a single oral dose of 15 mg each, respectively. The unmetabolized compounds are measurable only for a short period of time and at low ng/ml level. The sulfoxide-to-sulfone ratio for both GW1516 and GW0742 changed irregularly in the range of 1:3 to 1:15 depending on time elapsed after administration with a tendency of increasing the ratio with time. The other important finding was that the abundance of GW0742 and its metabolites in urine is about ten times lower than in case of GW1516.