1,721,068 research outputs found
Interactive alkaptonuria database: investigating clinical data to improve patient care in a rare disease
No full textAlkaptonuria (AKU) is an ultrarare autosomal recessive disorder (MIM 203500) that is caused byby a complex set of mutations in homogentisate 1,2-dioxygenasegene and consequent accumulation of homogentisic acid (HGA), causing a significant protein oxidation. A secondary form of amyloidosis was identified in AKU and related to high circulating serum amyloid A (SAA) levels, which are linked with inflammation and oxidative stress and might contribute to disease progression and patients' poor quality of life. Recently, we reported that inflammatory markers (SAA and chitotriosidase) and oxidative stress markers (protein thiolation index) might be disease activity markers in AKU. Thanks to an international network, we collected genotypic, phenotypic, and clinical data from more than 200 patients with AKU. These data are currently stored in our AKU database, named ApreciseKUre. In this work, we developed an algorithm able to make predictions about the oxidative status trend of each patient with AKU based on 55 predictors, namely circulating HGA, body mass index, total cholesterol, SAA, and chitotriosidase. Our general aim is to integrate the data of apparently heterogeneous patients with AKUAKU by using specific bioinformatics tools, in order to identify pivotal mechanisms involved in AKU for a preventive, predictive, and personalized medicine approach to AKU.-Cicaloni, V., Spiga, O., Dimitri, G. M., Maiocchi, R., Millucci, L., Giustarini, D., Bernardini, G., Bernini, A., Marzocchi, B., Braconi, D., Santucci, A. Interactive alkaptonuria database: investigating clinical data to improve patient care in a rare disease
Anethole dithiolethione lowers the homocysteine and raises the glutathione levels in solid tissues and plasma of rats: a novel non-vitamin homocysteine-lowring agent..
No full textHigh homocysteine (Hcys) levels are suspected to contribute to the pathogenesis of cardiovascular disease and of other chronic conditions. Failure of B vitamins to reduce the incidence of cardiovascular events while lowering the Hcys levels, has prompted the search for alternative treatments. We tested the ability of anethole dithiolethione (ADT) to lower the Hcys levels in rats and we explored possible underlying mechanisms. Parenteral administration of 10mg/kg ADT to normal rats for 3 days lowered the Hcys levels between 51.4% and 31.5% in kidneys, liver, testis and plasma. Concomitantly, glutathione (GSH) increased between 112% and 28% in kidneys, brain, liver and plasma whereas protein thiolation index decreased 30%. In hyperhomocysteinemic rats, the plasma Hcys levels dropped 70% following a single ip injection of 10mg/kg ADT, while they decreased 55% following oral administration of 2mg/kg/day ADT for one week. Significant additive effects occurred when sub-therapeutic doses of ADT and folic acid were used in combination. To test the possible mechanism(s) of these actions, we perfused isolated rat livers and kidneys with albumin-bound Hcys, the prevalent form of plasma Hcys, and physiological thiols and disulfides at different ratios. In both organ preparations, the elimination rate of albumin-bound Hcys was progressively faster as the amount of reduced thiols was increased in the perfusate. These findings indicate that ADT shifts the redox ratio of GSH and other thiols with their oxidized forms toward the reduced forms, thus favoring the dissociation of albumin-bound Hcys and its transfer to renal and hepatic cells for further processing
An improved HPLC mesurement for GSH and GSSG in human blood
No full textThe pathophysiological sequelae of oxidative/nitrosative stress are notoriously difficult to quantify. Despite these impediments, the medical significance of oxidative/nitrosative stress has become increasingly recognized to the point that it is now considered to be a component of virtually every disease. The level of oxidative stress can be quantified in blood by the measurement of the increase in glutathione disulfide (GSSG) and the decrease in the GSH/GSSG ratio, which has been shown to be altered in a variety of human diseases such as lung inflammation, amyotrophic lateral sclerosis, chronic renal failure, malignant disorders, and diabetes. Among the proposed methods for GSH/GSSG detection, the amino group derivatization with 2,4- dinitrofluorobenzene followed by HPLC separation has the advantage of allowing evaluation of both parameters within a single run contemporaneously. However, it has been shown that the application of this method on blood samples is not reproducible. In this report, we offer an explanation for these experimental limits and suggest some modifications that allow the application of this method to blood samples. The modified method has a low detection limit (0.5 μM, i.e., 1.4 pmoles) and a high reproducibility with a within-run imprecision of less than 2%. It could have a wide application as it is simple, virtually artifact-free, and not time-consuming, especially for large-scale screening studies
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
How to Increase Cellular Glutathione
Full text linkGlutathione (GSH) has special antioxidant properties due to its high intracellular concentration, ubiquity, and high reactivity towards electrophiles of the sulfhydryl group of its cysteine moiety. In most diseases where oxidative stress is thought to play a pathogenic role, GSH concentration is significantly reduced, making cells more susceptible to oxidative damage. Therefore, there is a growing interest in determining the best method(s) to increase cellular glutathione for both disease prevention and treatment. This review summarizes the major strategies for successfully increasing cellular GSH stores. These include GSH itself, its derivatives, NRf-2 activators, cysteine prodrugs, foods, and special diets. The possible mechanisms by which these molecules can act as GSH boosters, their related pharmacokinetic issues, and their advantages and disadvantages are discussed
Red blood cells as a physiological source of glutathione for extracellular fluids
No full textPlasma low molecular mass thiols are represented by glutathione, cysteine, cysteinylglycine and homocysteine. The physiological mechanisms responsible for maintaining the homeostasis of these compounds in the intracellular and extracellular spaces have not been fully clarified. Erythrocytes possess the enzymatic machinery to synthesize glutathione and an efflux of glutathione disulfide and glutathione conjugates from erythrocytes under various conditions occurs. In this study, the property of red blood cells (RBCs) to export low molecular mass thiols has been assessed. Plasma concentration of low molecular mass thiols has been measured in healthy volunteers by HPLC and a significant correlation with RBC number has been observed for glutathione and cysteinylglycine. A sustained export of reduced glutathione has been observed (about 21 nmol/h/ml RBCs) together with a lower, though significant, efflux of both cysteine and homocysteine. These results suggest that erythrocytes can contribute significantly to the extracellular pool of glutathione (GSH), thus cooperating with liver and other tissues to the dynamics of inter-organ GSH metabolism
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 altered by the transient (SiRNA-induced) or stable inactivation of NRF2 in GSTP competent or hGSTP1 transfected cells, while defects of GSH biosynthesis, efflux, and redox were observed after NRF2 silencing in GSTP−/− MEFs. In conclusion, GSTP is confirmed to functionally interact with Nrf2 and to have a prominent position in the pecking order of factors that control both the Nrf2-dependent and independent response of cellular thiols to alkylating agents
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 0.5μL) 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
Measurement of S-glutathionylated proteins by HPLC
Full text linkS-glutathionylated proteins (GSSP), i.e., protein-mixed disulfides with glutathione (GSH), are considered a suitable biomarker of oxidative stress. In fact, they occur within cells at low level and their concentration increases markedly under pro-oxidant conditions. Plasma is something different, since it is physiologically rich in S-thiolated proteins (RSSP), i.e., protein-mixed disulfides with various types of low molecular mass thiols (LMM-SH). However, albumin, which is largely the most abundant plasma protein, possesses a cysteine residue at position 34 that is mostly reduced (about 60%) under physiological conditions, but easily involved in the formation of additional RSSP in the presence of oxidants. The quantification of GSSP requires special attention to sample handling, since their level can be overestimated as a result of artefactual oxidation of GSH. We have developed the present protocol to avoid this methodological problem. Samples should be treated as soon as possible after their collection with the alkylating agent N-ethylmaleimide that masks –SH groups and prevents their oxidation. The GSH released from mixed disulfides by reduction with dithiothreitol is then labeled with the fluorescent probe monobromobimane and quantified by HPLC. The method can be applied to many different biological samples, comprising blood components, red blood cell plasma membrane, cultured cells, and solid organs from animal models
The age-dependent decline of the extracellular thiol-disulfide balance and its role in SARS-CoV-2 infection
Full text linkSARS–CoV-2 (COVID-19) infection can cause a severe respiratory distress syndrome. The risk of severe manifestations and mortality characteristically increase in the elderly and in the presence of non-COVID-19 comorbidity. We and others previously demonstrated that the low molecular weight (LMW) and protein thiol/disulfide ratio declines in human plasma with age and such decline is even more rapid in the case of inflammatory and premature aging diseases, which are also associated with the most severe complications of COVID-19 infection. The same decline with age of the LMW thiol/disulfide ratio observed in plasma appears to occur in the extracellular fluids of the respiratory tract and in association with many pulmonary diseases that characteristically reduce the concentrations and adaptive stress response of the lung glutathione. Early evidence in literature suggests that the thiol to disulfide balance of critical Cys residues of the COVID-19 spike protein and the ACE-2 receptor may influence the risk of infection and the severity of the disease, with a more oxidizing environment producing the worst prognosis. With this hypothesis paper we propose that the age-dependent decline of LMW thiol/disulfide ratio of the extracellular fluids, could play a role in promoting the physical (protein-protein) interaction of CoV-2 and the host cell in the airways. Therefore, this redox-dependent interaction is expected to affect the risk of severe infection in an age-dependent manner. The hypothesis can be verified in experimental models of in vitro CoV-2 infection and at the clinical level in that LMW thiols and protein thiolation can now be investigated with standardized, reliable and versatile laboratory protocols. Presenting the verification strategy of our hypothesis, we also discuss available nutritional and ancillary pharmacological strategies to intervene on the thiol/disulfide ratio of extracellular fluids of subjects at risk of infection and COVID-19 patients
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