1,721,020 research outputs found
Micro-method for the determination of glutathione in human blood
No full textA new procedure is described for the visible-range spectrophotometric analysis of glutathione (GSH) in microvolumes of blood (as low as 0.5 mu L) collected by fingerstick. Samples are diluted 1 to 300 (v/v) in a stabilizing solution, followed by determination of haemoglobin concentration and by acid deproteination. GSH is then measured in the clear supernatant by colorimetry using DTNB, i.e., 5,5'-dithio-bis(2-nitrobenzoic acid), in aqueous solution at pH 7.8. The DTNB reagent is prepared and kept at pH 6.2 until just prior its addition, thus avoiding spontaneous decomposition of the reagent. The assay is rapid, easy to adapt to large-scale studies and it avoids artefactual oxidation of GSH, a common methodological shortcoming. The method is precise with 1.7 to 3.4% intra-day relative standard deviation (RSD) and 2.2 to 4.2% inter-day RSD, and accurate with -1.4% to 2.3% intra-day relative error (RE) and -2.8% to 1.6% inter-day RE. GSH is recovered by 97.5 to 100% at all tested concentrations. The new colorimetric micro-method was validated by a reliable previously reported HPLC method. The procedure is suitable for minimally invasive investigation of oxidative stress in peripheral blood
Glutathione S-transferase P influences the Nrf2-dependent response of cellular thiols to seleno-compounds
No full textRecent findings suggest a functional interaction of the drug resistance enzyme glutathione S-transferase P (GSTP) with the transcription factor Nrf2, a master regulator of the adaptive stress response to cellular electrophiles. The effect of this interaction on the metabolism and redox of cellular thiols was investigated in this study during the exposure to alkylating Se-compounds in murine embryonic fibroblasts (MEFs). GSTP1-1 gene ablation was confirmed to upregulate Nrf2 activity and to increase Cys uptake and the de novo biosynthesis of reduced glutathione (GSH) that was readily released in the extracellular medium together with other cellular thiols. This latter response was associated with a higher expression of the membrane transporter MRP1 and was markedly stimulated by the treatment with alkylating Se-compounds together with protein S-glutathionylation that was observed to be under the influence of GSTP expression. The response of cellular thiols to Se-compounds was not altere..
The pro-oxidant role of protein SH groups of haemoglobin in rat erythrocytes exposed to menadione
No full textMenadione is selectively toxic to erythrocytes. Although GSH is considered a primary target of menadione, intraerythrocyte thiolic alterations consequent to menadione exposure are only partially known. In this study alterations of GSH and protein thiols (PSH) and their relationship with methemoglobin formation were investigated in human and rat red blood cells (RBC) exposed to menadione. In both erythrocyte types, menadione caused a marked increase in methemoglobin associated with GSH depletion and increased oxygen consumption. However, in human RBC, GSH formed a conjugate with menadione, whereas, in rat RBC it was converted to GSSG, concomitantly with a loss of protein thiols (corresponding to menadione arylation), and an increase in glutathione-protein mixed disulfides (GS-SP). Such differences were related to the presence of highly reactive cysteines, which characterize rat hemoglobin (cys beta125). In spite of the greater thiol oxidation in rat than in human RBC, methemoglobin formation and the rate of oxygen consumption elicited by menadione in both species were rather similar. Moreover, in repeated experiments under N2 or CO-blocked heme, it was found that menadione conjugation (arylation) in both species was riot dependent oil the presence of oxygen or the status of heme. Therefore, we assumed that GSH (human RBC) and protein (rat RBC) arylation was equally responsible for increased oxygen consumption and Hb oxidation. Moreover, thiol oxidation of rat RBC was strictly related to methemoglobin formation. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved
Immediate stabilization of human blood for delayed quantification of endogenous thiols and disulfides
No full textEndogenous thiols undergo rapid and reversible oxidation to disulfides when exposed to oxidants and are, therefore, suitable biomarkers of oxidative stress. However, accurate analysis of thiols in blood is frequently compromised by their artifactual oxidation during sample manipulation, which spuriously elevates the disulfide levels. Here, we describe a validated pre-analytical procedure that prevents both artifactual oxidation of thiols during sample manipulation and their oxidative decay for months in biosamples that are stored at -80 degrees C. Addition of N-ethylmaleimide to blood samples from healthy donors was used to stabilize whole blood, red blood cells, platelets and plasma disulfides, whereas addition of citrate buffer followed by dilution of plasma with H2O was used to stabilize plasma thiols. The concentrations of thiols and disulfides were stable in all biosamples for at least 6 months when analyzed by UV/Vis HPLC at regular intervals. Only 3 ml of blood were needed to perform the analyses of thiols and disulfides in the different blood fractions. This pre-analytical procedure is reliable for use in both animal and human prospective studies. Its ease of implementation makes the method suitable for application to multicenter studies where blood samples are collected by different sites and personnel and are shipped to specific specialized laboratories. (C) 2016 Elsevier B.V. All rights reserved
Studies on the biological effects of ozone: 9. Effects of ozone on human platelets
No full textDuring the course of ozonated autohaemotherapy (O3-AHT) using heparin as an anticoagulant, it was occasionally observed that a few clots were retained in the filter during blood reinfusion. This observation prompted an investigation on the effect of ozone (O3) on human platelets. We have now shown, both by biochemical and morphological criteria, that heparin in the presence of O3 can promote platelet aggregation. In contrast, after Ca2+ chelation with citrate, platelet aggregation is much reduced. The potential role of the transient formation of hydrogen peroxide (H2O2) in the presence of Ca2+ with the possible expression of adhesion molecules is briefly discussed
Responses of thiols to an oxidant challenge: differences between blood and tissues in the rat
No full textTreatment of rats with diamide (100 mg/kg ip) altered the thiol components of the blood to a very different extent than in tissues (liver, kidney, lung, spleen, heart and testis). A total consumption (10 min) and regeneration (120 min) of blood glutathione (GSH), matched by a parallel increase and decrease in glutathione-protein mixed disulfides (GS-SP) was observed. In contrast, no modification of non-protein SH groups (NPSH) and protein SH groups (PSH), GS-SP and malondialdehyde (MDA) was observed in liver, kidney, lung, testis spleen and heart within same time range. In particular, only glutathione disulfide (GSSG) levels and some activities of antioxidant enzymes were modified to a small extent and in an opposite direction in some organs. For example, GSSG, and glucose-6-phosphate dehydrogenase (G-6-PDH) and catalase (CAT) activities appeared up-regulated in one tissue and down-regulated in another. The least modified organ was the liver, whereas lung and spleen were the most affected (lung, GSSG, significantly increased whereas G-6-PDH, glutaredoxin (GRX), GPX, superoxide dimutase (SOD) levels were significantly lowered; spleen, GSSG and the activity of glutathione reductase (GR), G-6-PDH and glutathione transferase (GST) were significantly decreased). The different responses of erythrocytes and organs to diamide were explained by the high affinity of hemoglobin and by the relatively high potential of thiol regeneration in organs. The rapid reversibility of the process of protein S-thiolation in blood and the small effects in organs leads us to propose the existence af an inter-organ cooperation in the rat that regulates protein S-thiolation in blood. Plasma thiols may well play a role in this process. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved
The role of cysteine in the regulation of blood glutathione-protein mixed disulfides in rats treated with diamide
No full textThe kinetics of GSH, GSSG, and thiol-protein mixed disulfides (RS-SP) of GSH (GS-SP) and cysteine (CYS-SP) were studied in rat blood and liver in the time range 0-120 min after treatment with 100 and 200 mg/kg ip of diamide. Total consumption (10 min) and regeneration (120 min) of blood GSH, matched by parallel increases and decreases in RS-SP, were observed. GSSG did not change appreciably. No dose-effect relationship was obtained with either treatment. On the contrary, in vitro treatment of blood with 0.75 mM diamide provoked the same trends of GSH and RS-SP as in vivo (e.g., reversible modifications), whereas treatment with 1.5 mM caused drops and rises in GSH and RS-SP, respectively, without any subsequent return to control values. The presence of a hematic factor responsible for RS-SP regulation is hypothesized in the in vivo experiment. Successive experiments involving in vitro pretreatment with 2 mM diamide and treatment with 0.5 mM of various thiols indicated that cysteine (CYS), but not GSH or N-acetylcysteine, rapidly restored erthyrocyte GSH and RS-SP to their basal levels. No evident sign of hemolysis was observed in these experiments. These results indicate that CYS is a diffusible thiol important for RS-SP regulation. Analysis of whole blood of rats treated with 100 mg/kg ip diamide and the presence of two reversible peaks (about 10 times the corresponding control level) of CYS-SP and free CYS confirmed the plausible role of CYS in maintaining the reversibility of the process. Preliminary results in liver of rats treated with 100 mg/kg diamide indicated that CYS may act by metabolic cooperation between organs. We suggest that CYS may have a role in the regulation of the intracellular redox state of rat erythrocytes during oxidative stress
Minor thiols cysteine and cysteinylglycine regulate the competition between glutathione and protein SH groups in human platelets subjected to oxidative stress
No full textChanges in the concentrations of protein-mixed disulfides (XS-SP) of glutathione (GSH), cysteine (CSH), and cysteinylglycine (CGSH) were studied in human platelets treated with diamide and t-BOOH in timecourse experiments (time range, 1-30 min) in order to understand the contribution of minor thiols CSH and CGSH to the regulation of glutathione-protein mixed disulfides (GS-SP). Diamide was much more potent than t-BOOH in altering the platelet thiol composition of XS-SP (threshold dose: diamide, 0.03 mM; t-BOOH, 0.5 mM) and caused reversible XS-SP peaks whose magnitude was related to the concentration of free thiols in untreated cells. Thus maximum levels of GS-SP (8 min after 0.4 mM diamide) were about 16-fold higher than those of controls (untreated platelets, GS-SP = 0.374 nmol/109 platelets), whereas those of CS-SP and CGS-SP were only 4-fold increased (untreated platelets, CS-SP = 0.112 nmol/109 platelets; CGS-SP = 0.024 nmol/109 platelets). The greater effects of diamide with respect to t-BOOH were explained on the basis of the activities of fast reactive protein SH groups for diamide and glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G-6-PDH) for t-BOOH. The addition of cysteine (0.3 mM, at 4 min) after treatment of platelets with 0.4 mM diamide increased the rate of reversal of GS-SP peaks to normal values, but also caused a relevant change in CGS-SP with respect to that of platelets treated with diamide alone. An increased γ-glutamyltranspeptidase activity was found in platelets treated with diamide. Moreover, untreated platelets were found to release and hydrolyze GSH to CGSH and CSH. Ratios of thiols/disulfides (XSH/XSSX) and activities of GR and G-6PDH were also related to a high reducing potential exerted by GSH but not by minor thiols. The lower mass and charge of minor thiols is a likely requisite of the regulation of GS-SP levels in platelets. (C) 2000 Academic Press
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