RESUMEN
We developed a method of sensitive capillary electrophoresis using UV detection for the determination of certain free aminothiols (reduced cysteinylglycine (rCysGly), cysteine (rCys), glutathione (rGln), and cystine (CysS) in human blood plasma. The reduced thiols were derivatized with N-ethylmaleimide. The plasma was purified from proteins via ultrafiltration. Electrophoretic separation was performed using 115 mM Na phosphate with 7.5% (v/v) polyethylene glycol 600, pH 2.3. The in-capillary concentration of the analytes was achieved with a pH gradient created via the preinjection of triethanolamine and postinjection of phosphoric acid. The separation was carried out using a silica capillary (50 µm i.d.; total/effective separation length 42/35 cm) at a 25 kV voltage. The total analysis/regeneration time was 18 min. The quantification limits varied from 1.3 µM (rCysGly) to 5.4 µM (CysS). The accuracy was 95%-99%, and the repeatability and reproducibility were approximately 1.8%-3.8% and 1.9%-5.0%, respectively. An analysis of plasma samples from healthy volunteers (N = 41) showed that the mean levels of rCysGly, rCys, rGln, and CysS were 1.64, 10.6, 2.58, and 46.2 µM, respectively.
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Cistina , Compuestos de Sulfhidrilo , Humanos , Reproducibilidad de los Resultados , Electroforesis Capilar/métodos , Aminas , Plasma , Concentración de Iones de HidrógenoRESUMEN
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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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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Coronary artery disease (CAD) and the coronary artery bypass graft (CABG) are associated with a decreased blood glutathione (bGSH) level. Since GSH metabolism is closely related to other aminothiols (homocysteine and cysteine) and glucose, the aim of this study was to reveal the associations of bGSH with glucose and plasma aminothiols in CAD patients (N = 35) before CABG and in the early postoperative period. Forty-three volunteers with no history of cardiovascular disease formed the control group. bGSH and its redox status were significantly lower in CAD patients at admission. CABG had no significant effect on these parameters, with the exception of an increase in the bGSH/hemoglobin ratio. At admission, CAD patients were characterized by negative associations of homocysteine and cysteine with bGSH. All these associations disappeared after CABG. An association was found between an increase in oxidized GSH in the blood in the postoperative period and fasting glucose levels. Thus, CAD is associated with the depletion of the intracellular pool and the redox status of bGSH, in which hyperhomocysteinemia and a decrease in the bioavailability of the extracellular pool of cysteine play a role. The present study indicates that CABG causes disruptions in aminothiol metabolism and induces the synthesis of bGSH. Moreover, glucose becomes an important factor in the dysregulation of GSH metabolism in CABG.
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A new approach has been developed for the direct determination of reduced (glutathione [GSH]) and oxidized (glutathione disulfide [GSSG]) GSH in whole blood by means of capillary electrophoresis. Its features include GSH-stabilizing sample preparation, the use of an internal standard, and pH-mediated stacking. Blood stabilized with acid citrate and K3 EDTA was treated with acetonitrile with N-ethylmaleimide, and then the analytes were extracted with diethyl ether. The total analysis time was 8 min using a 50-µm (i.d.) by 32.5-cm (eff. length) silica capillary. The background electrolyte was 0.075-M citrate Na pH 5.8 with 200-µM cetyltrimethylammonium bromide and 5-µM sodium dodecyl sulfate, and the separation voltage was -14 kV. The quantification limit (S/N = 15) of the method was 1.5 µM for GSSG. The accuracy levels of GSH and GSSG analysis were 104% and 103%, respectively, and between-run precision levels were 2.6% and 3.2%, respectively. Analysis of blood samples from healthy volunteers (N = 24) showed that the levels of GSH and GSSG and the GSH/GSSG ratio in the whole blood were 1.05 ± 0.14 mM, 3.9 ± 1.25 µM, and 256 ± 94, respectively. Thus, the presented approach can be used in clinical and laboratory practice.
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Éter , Glutatión , Acetonitrilos , Cetrimonio , Citratos , Ácido Edético , Electroforesis Capilar/métodos , Etilmaleimida , Glutatión/análisis , Disulfuro de Glutatión/análisis , Humanos , Concentración de Iones de Hidrógeno , Dióxido de Silicio , Dodecil Sulfato de SodioRESUMEN
OBJECTIVE: S-Adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) are indicators of global transmethylation and may play an important role as markers of severity of COVID-19. METHODS: The levels of plasma SAM and SAH were determined in patients admitted with COVID-19 (n = 56, mean age = 61). Lung injury was identified by computed tomography (CT) in accordance with the CT0-4 classification. RESULTS: SAM was found to be a potential marker of lung damage risk in COVID-19 patients (SAM > 80 nM; CT3,4 vs. CT 0-2: relative ratio (RR) was 3.0; p = 0.0029). SAM/SAH > 6.0 was also found to be a marker of lung injury (CT2-4 vs. CT0,1: RR = 3.47, p = 0.0004). There was a negative association between SAM and glutathione level (ρ = -0.343, p = 0.011). Interleukin-6 (IL-6) levels were associated with SAM (ρ = 0.44, p = 0.01) and SAH (ρ = 0.534, p = 0.001) levels. CONCLUSIONS: A high SAM level and high methylation index are associated with the risk of lung injury in patients with COVID-19. The association of SAM with IL-6 and glutathione indicates an important role of transmethylation in the development of cytokine imbalance and oxidative stress in patients with COVID-19.
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COVID-19/complicaciones , Lesión Pulmonar/sangre , S-Adenosilhomocisteína/sangre , S-Adenosilmetionina/sangre , SARS-CoV-2 , Adulto , Anciano , Anciano de 80 o más Años , Aterosclerosis/epidemiología , Biomarcadores , COVID-19/epidemiología , Comorbilidad , Diabetes Mellitus/epidemiología , Femenino , Glutatión/sangre , Humanos , Hipertensión/epidemiología , Interleucina-6/sangre , Lesión Pulmonar/diagnóstico por imagen , Lesión Pulmonar/etiología , Masculino , Metilación , Persona de Mediana Edad , Personal Militar , Riesgo , Tomografía Computarizada por Rayos X , Adulto JovenRESUMEN
OBJECTIVE: Aminothiols (glutathione (GSH), cysteinylglycine (CG)) may play an important role in the pathogenesis of coronavirus disease 2019 (COVID-19), but the possible association of these indicators with the severity of COVID-19 has not yet been investigated. METHODS: The total content (t) and reduced forms (r) of aminothiols were determined in patients with COVID-19 (n = 59) on admission. Lung injury was characterized by computed tomography (CT) findings in accordance with the CT0-4 classification. RESULTS: Low tGSH level was associated with the risk of severe COVID-19 (tGSH ≤ 1.5 µM, mild vs. moderate/severe: risk ratio (RR) = 3.09, p = 0.007) and degree of lung damage (tGSH ≤ 1.8 µM, CT < 2 vs. CT ≥ 2: RR = 2.14, p = 0.0094). The rGSH level showed a negative association with D-dimer levels (ρ = -0.599, p = 0.014). Low rCG level was also associated with the risk of lung damage (rCG ≤ 1.3 µM, CT < 2 vs. CT ≥ 2: RR = 2.28, p = 0.001). Levels of rCG (ρ = -0.339, p = 0.012) and especially tCG (ρ = -0.551, p = 0.004) were negatively associated with platelet count. In addition, a significant relationship was found between the advanced oxidation protein product level and tGSH in patients with moderate or severe but not in patients with mild COVID-19. CONCLUSION: Thus, tGSH and rCG can be seen as potential markers for the risk of severe COVID-19. GSH appears to be an important factor to oxidative damage prevention as infection progresses. This suggests the potential clinical efficacy of correcting glutathione metabolism as an adjunct therapy for COVID-19.
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COVID-19/diagnóstico , Dipéptidos/sangre , Glutatión/sangre , Productos Avanzados de Oxidación de Proteínas/sangre , Anciano , Aminoácidos Sulfúricos/sangre , Biomarcadores/sangre , COVID-19/sangre , COVID-19/patología , Dipéptidos/metabolismo , Femenino , Glutatión/metabolismo , Humanos , Pulmón/diagnóstico por imagen , Pulmón/patología , Masculino , Persona de Mediana Edad , Oxidación-Reducción , SARS-CoV-2/aislamiento & purificación , Índice de Severidad de la EnfermedadRESUMEN
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 , Accidente Cerebrovascular Isquémico , Accidente Cerebrovascular , Glutatión , Humanos , Oxidación-ReducciónRESUMEN
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/sangre , Insuficiencia Renal Crónica/orina , S-Adenosilhomocisteína/orina , S-Adenosilmetionina/orina , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Tasa de Filtración Glomerular , Humanos , Masculino , Persona de Mediana Edad , Insuficiencia Renal Crónica/sangre , Insuficiencia Renal Crónica/fisiopatologíaRESUMEN
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 , Cromatografía Capilar Electrocinética Micelar/métodos , S-Adenosilhomocisteína/orina , S-Adenosilmetionina/orina , Tionucleósidos/orina , Adenosina/orina , Adolescente , Adulto , Niño , Preescolar , Femenino , Humanos , Límite de Detección , Modelos Lineales , Masculino , Persona de Mediana Edad , Reproducibilidad de los Resultados , Adulto JovenRESUMEN
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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Insuficiencia Renal Crónica/orina , S-Adenosilhomocisteína/orina , S-Adenosilmetionina/orina , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/orina , Femenino , Humanos , Masculino , Persona de Mediana Edad , Insuficiencia Renal Crónica/diagnósticoRESUMEN
In clinical practice, the metabolic syndrome (MetS) is often associated with chronic obstructive pulmonary disease (COPD). Although gender differences in MetS are well documented, little is known about sex-specific differences in the pathogenesis of COPD, especially when combined with MetS. Consequently, it is not clear whether the same treatment regime has comparable efficacy in men and women diagnosed with MetS and COPD. In the present study, using sodium glutamate, lipopolysaccharide, and cigarette smoke extract, we simulated lipid metabolism disorders, obesity, hyperglycemia, and pulmonary emphysema (comorbidity) in male and female C57BL/6 mice. We assessed the gender-specific impact of lipid metabolism disorders and pulmonary emphysema on angiogenic precursor cells (endothelial progenitor cells (EPC), pericytes, vascular smooth muscle cells, cells of the lumen of the nascent vessel), as well as the biological effects of pegylated glucagon-like peptide 1 (pegGLP-1) in this experimental paradigm. Simulation of MetS/COPD comorbidity caused an accumulation of EPC (CD45-CD31+CD34+), pericytes, and vascular smooth muscle cells in the lungs of female mice. In contrast, the number of cells involved in the angiogenesis decreased in the lungs of male animals. PegGLP-1 had a positive effect on lipids and area under the curve (AUC), obesity, and prevented the development of pulmonary emphysema. The severity of these effects was stronger in males than in females. Furthermore, PegGLP-1 stimulated regeneration of pulmonary endothelium. At the same time, PegGLP-1 administration caused a mobilization of EPC (CD45-CD31+CD34+) into the bloodstream in females and migration of precursors of angiogenesis and vascular smooth muscle cells to the lungs in male animals. Gender differences in stimulatory action of pegGLP-1 on CD31+ endothelial lung cells in vitro were not observed. Based on these findings, we postulated that the cellular mechanism of in vivo regeneration of lung epithelium was at least partly gender-specific. Thus, we concluded that a pegGLP-1-based treatment regime for metabolic disorder and COPD should be further developed primarily for male patients.
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Células Progenitoras Endoteliales/efectos de los fármacos , Péptido 1 Similar al Glucagón/administración & dosificación , Síndrome Metabólico/tratamiento farmacológico , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Enfisema Pulmonar/tratamiento farmacológico , Animales , Fumar Cigarrillos/efectos adversos , Modelos Animales de Enfermedad , Células Progenitoras Endoteliales/citología , Femenino , Péptido 1 Similar al Glucagón/farmacología , Humanos , Lipopolisacáridos/efectos adversos , Masculino , Síndrome Metabólico/inducido químicamente , Ratones Endogámicos C57BL , Enfermedad Pulmonar Obstructiva Crónica/inducido químicamente , Enfisema Pulmonar/inducido químicamente , Caracteres Sexuales , Glutamato de Sodio/efectos adversos , Resultado del TratamientoRESUMEN
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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Cromatografía Líquida de Alta Presión/métodos , S-Adenosilhomocisteína/sangre , S-Adenosilmetionina/sangre , Cromatografía Líquida de Alta Presión/instrumentación , Fluorescencia , Humanos , Plasma/química , S-Adenosilhomocisteína/aislamiento & purificación , S-Adenosilmetionina/aislamiento & purificación , Extracción en Fase SólidaRESUMEN
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/sangre , Disulfuros/sangre , Homeostasis/fisiología , Accidente Cerebrovascular/sangre , Compuestos de Sulfhidrilo/sangre , Femenino , Glutatión/sangre , Humanos , Masculino , Persona de Mediana Edad , Estrés Oxidativo/fisiología , Factores de RiesgoRESUMEN
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/farmacología , Isquemia Encefálica/tratamiento farmacológico , Cisteína/sangre , Glutatión/sangre , Hipocampo/efectos de los fármacos , Homocisteína/sangre , Metoprolol/farmacología , Nebivolol/farmacología , Fármacos Neuroprotectores/farmacología , Enfermedad Aguda , Animales , Isquemia Encefálica/sangre , Isquemia Encefálica/patología , Isquemia Encefálica/fisiopatología , Circulación Cerebrovascular/efectos de los fármacos , Modelos Animales de Enfermedad , Hipocampo/metabolismo , Hipocampo/patología , Masculino , Peso Molecular , Oxidación-Reducción , RatasRESUMEN
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/sangre , Cisteína/orina , Electroforesis Capilar/métodos , Homocisteína/sangre , Homocisteína/orina , Extracción Líquido-Líquido/métodos , Acetonitrilos/química , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Cloroformo/química , Femenino , Humanos , Límite de Detección , Persona de Mediana Edad , Sensibilidad y Especificidad , Adulto JovenRESUMEN
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/sangre , Isquemia Encefálica/sangre , Disulfuros/sangre , Compuestos de Sulfhidrilo/sangre , Animales , Glutatión/sangre , Homeostasis/fisiología , Masculino , Oxidación-Reducción , Estrés Oxidativo/fisiología , RatasRESUMEN
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 Borónicos/química , Electroforesis Capilar/métodos , S-Adenosilhomocisteína/orina , S-Adenosilmetionina/orina , Extracción en Fase Sólida/métodos , Adulto , Anciano , Femenino , Humanos , Modelos Lineales , Masculino , Persona de Mediana Edad , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Adulto JovenRESUMEN
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.