1,721,080 research outputs found
S-Nitrosothiols in blood: does photosensitivity explain a 4-order-of-magnitude concentration range?
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The time course of mixed disulfide formation between GSH and proteins in rat blood after oxidative stress with tert-butyl hydroperoxide
No full textVariations in time of GSH, GSSG and glutathione-protein mixed disulfides (GSSP) were studied in rat blood in vitro experiments of oxidative stress with tert-butyl hydroperoxide (t-BOOH, dose range 0.3-2 mM; time range 15 sec-60 min). The aim was to elucidate the potential for GSSG reduction of protein-bound SH groups (PSH). GSSP was estimated by two methods, indirectly from GSHt (GSH + 2 GSSG) variations and directly from precipitated and washed proteins. After treatment with t-BOOH, GSH and GSSG concentrations showed an immediate (15-30 sec) drop and a peak respectively and returned to control levels (time zero values) between 30 and 60 min. A t-BOOH dose-dependent minimum of GSHt and a corresponding GSSP maximum were obtained within 1-6 min and subsequently returned to control values. Basal GSH, GSSG and GSSP levels were similar in aged and fresh blood. In contrast, after treatment with 1 mM t-BOOH substantial differences in kinetic patterns were observed: for intance GSSP concentrations were higher in aged than in fresh blood with no return to the initial values. The pretreatment of aged blood with 10 mM glucose decreased GSSP formation and produced a reversible pattern similar to that observed in fresh blood. The role of glucose in regulating GSSP generation is discussed. © 1994
Different mechanisms of formation of glutathione-protein mixed disulfides of diamide and tert-butyl hydroperoxide in rat blood
No full textThe mechanisms of glutathione-protein mixed disulfide (GSSP) formation caused by diamide and tert-butyl hydroperoxide were studied in rat blood after in vitro treatment in the 0.3–4 mM dose range. tert-Butyl hydroperoxide formed GSSP, via GSSG, according to the reaction, GSSG + PSH → GSSP + GSH, whereas diamide reacted first with protein SH groups, giving PS-diamide adducts and then, after reaction with GSH, GSSP. Moreover, after diamide treatment, GSSP patterns were characterized by a much slower or irreversible dose-related return to basal levels in comparison with those observed with tert-butyl hydroperoxide, always reversible. Experiments with purified hemoglobin revealed the existence of a large fraction of protein SH groups which formed GSSP and had a higher reactivity than GSH. Experiments on glucose consumption and role of various erythrocyte enzymes, carried out to explain the inertness of GSSP to reduction after treatment of blood with diamide, were substantially negative. Other tests carried out to confirm the efficiency of the enzymatic machinery of blood samples successively treated with diamide and tert-butyl hydroperoxide, indicated that GSSP preformed by diamide was difficult to reduce, whereas those generated by tert-butyl hydroperoxide were reversible as normal. Our results suggest that a fraction of GSSP generated by diamide is different and less susceptible to reduction than that obtained with tert-butyl hydroperoxide
HPLC determination of novel dithiolethione containing drugs and its application for in vivo studies in rats
No full textA panel of new drugs obtained by grafting a sulfurated moiety, i.e. 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione (ADTOH) onto existing drugs have been synthesized and their in vivo action is under preclinical evaluation. In the present paper we describe rapid HPLC methods to detect ADTOH derivatives of valproic acid (ACS2), sildenafil (ACS6), aspirin (ACS14) and diclofenac (ACS15) in plasma. These methods allow the simultaneous detection of the potential drugs and of ADTOH moiety. In the case of ACS14 the de-acetylated metabolite (ACS21) can also be concomitantly measured. The chromatographic separation was performed on a C18 column, applying a mobile phase consisting of a mixture of trifluoroacetic acid and acetonitrile. ADTOH, ACS6, ACS14, ACS21 were separated isocratically whereas ACS2 and ACS15 were separated applying gradient elution. The methods are precise and accurate, with a low quantification limit of 200 nM for ACS2, ACS15 and ACS21 or 100 nM for ADTOH, ACS6 and ACS14. The mean absolute recovery for all tested molecules was always found to be close to 100%. The methods are shown to be selective and linear in the range 0.2-50 μM and thus appear suitable for pharmacokinetic studies with ADTOH containing compounds, as indicated by exemplificative experiments performed with intravenous administration of the drugs to rats
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
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
Protein S-glutathionylation: a regulatory device from bacteria to humans
No full textS-Glutathionylation is the specific post-translational modification of protein cysteine residues by the addition of the tripeptide glutathione, the most abundant and important low-molecular-mass thiol within most cell types. Protein S-glutathionylation is promoted by oxidative or nitrosative stress but also occurs in unstressed cells. It can serve to regulate a variety of cellular processes by modulating protein function and to prevent irreversible oxidation of protein thiols. Recent findings support an essential role for S-glutathionylation in the control of cell-signalling pathways associated with viral infections and with tumour necrosis factor-(-induced apoptosis. Glyceraldehyde-3-phosphate dehydrogenase has recently been implicated in the regulation of endothelin-1 synthesis by a novel, S-glutathionylation-based mechanism involving messenger RNA stability. Moreover, recent studies have identified S-glutathionylation as a redox signalling mechanism in plants
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
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