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Biomedical subjects

Isabella Dalle-Donne

Publications and source records attributed to Isabella Dalle-Donne.

At least 19 recordsLinked to original sources
PubMed2007

Intervention strategies to inhibit protein carbonylation by lipoxidation-derived reactive carbonyls.

Protein carbonylation induced by reactive carbonyl species (RCS) generated by peroxidation of polyunsaturated fatty acids plays a significant role in the etiology and/or progression of several human diseases, such as cardiovascular (e.g., atherosclerosis, long-term complications of diabetes) and neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson's disease, and cerebral ischemia). Most of the biological effects of intermediate RCS, mainly alpha,beta-unsaturated aldehydes, di-aldehydes, and keto-aldehydes, are due to their capacity to react with the nucleophilic sites of proteins, forming advanced lipoxidation end-products (ALEs). Because of the emerging deleterious role of RCS/protein adducts in several human diseases, different potential therapeutic strategies have been developed in the last few years. This review sheds focus on fundamental studies on lipid-derived RCS generation, their biological effects, and their reactivity with proteins, with particular emphasis to 4-hydroxy-trans-2-nonenal (HNE)-, acrolein (ACR)-, malondialdehyde (MDA)-, and glyoxal (GO)-modified proteins. It also discusses the recently developed pharmacological approaches for the management of chronic diseases in which oxidative stress and RCS formation are massively involved. Inhibition of ALE formation, based on carbonyl-sequestering agents, seems to be the most promising pharmacological tool and is reviewed in detail.

Aldehydes↗
PubMed2006

Membrane skeletal protein S-glutathionylation and hemolysis in human red blood cells.

In this work, protein-glutathione mixed disulfide formation in human red blood cells (RBCs) was evaluated in vitro by using the thiol-specific reagent diamide. We investigated what mechanism could lead to S-glutathionylation of membrane skeletal proteins, what are the main target proteins, and the correlation between protein S-glutathionylation and RBC hemolysis. Diamide caused a decrease in the reduced form of glutathione (GSH), which was accompanied by an increase in the basal level of glutathione disulfide (GSSG) and in S-glutathionylation of protein 4.2 and spectrin. The increase in membrane skeletal protein S-glutathionylation was correlated with a lower susceptibility of RBCs to osmotic hemolysis, suggesting that S-glutathionylation of protein 4.2 and spectrin could contribute to regulate RBC membrane stability.

Diamide↗
PubMed2006

Detection of S-nitrosothiols in biological fluids: a comparison among the most widely applied methodologies.

Many different methodologies have been applied for the detection of S-nitrosothiols (RSNOs) in human biological fluids. One unsatisfactory outcome of the last 14 years of research focused on this issue is that a general consensus on reference values for physiological RSNO concentration in human blood is still missing. Consequently, both RSNO physiological function and their role in disease have not yet been clarified. Here, a summary of the values measured for RSNOs in erythrocytes, plasma, and other biological fluids is provided, together with a critical review of the most widely used analytical methods. Furthermore, some possible methodological drawbacks, responsible for the highlighted discrepancies, are evidenced.

Animals↗
PubMed2006

Protein S-glutathionylation and platelet anti-aggregating activity of disulfiram.

Blood platelets are central to haemostasis, and reactions in platelets involving sulfhydryl groups play important roles in platelet function. Reduced glutathione (GSH) plays an important role in platelet aggregation and glutathione-depleting chemicals inhibit platelet aggregation. The lipophilic drug disulfiram, because of its affinity for sulfhydryl groups, is a highly thiol-reacting agent. As a consequence, GSH and sulfhydryl groups of protein cysteines in human platelets, in analogy to other components of human blood, are a potential target of disulfiram. In the present study, we have shown that exposure of human platelets to disulfiram causes the depletion of platelet GSH and augmentation of mixed disulfides between GSH and protein sulfhydryl groups to form protein-glutathione mixed disulfides (S-glutathionylated proteins). The depletion of platelet GSH and the increase in S-glutathionylated proteins occurred at concentrations of disulfiram that inhibited platelet aggregation, suggesting that protein S-glutathionylation is involved in the inhibition of platelet aggregation caused by disulfiram.

Blood Platelets↗
PubMed2006

Oxidized forms of glutathione in peripheral blood as biomarkers of oxidative stress.

BACKGROUND: Reduced glutathione (GSH) and its redox forms, glutathione disulfide (GSSG) and glutathionylated proteins (PSSG), are biomarkers of oxidative stress, but methodologic artifacts can interfere with their measurement. We evaluated the importance of correct sample handling during the preanalytical phase for GSH, GSSG, and PSSG measurement. METHODS: We used human blood for in vitro experiments with oxidants [tert-butylhydroperoxide (t-BOOH), diamide, and menadione]. For in vivo experiments, we used rats in which we cannulated the jugular and femoral veins for both oxidant administration and blood collection. We measured GSH, GSSG, and PSSG with HPLC with or without sample pretreatment with N-ethylmaleimide (NEM) to prevent artifacts. We also measured malondialdehyde (MDA) with HPLC, and protein carbonyls (PCO) with spectrophotometric procedures. RESULTS: When methodologic artifacts were prevented by pretreatment with NEM, GSSG results increased up to 3-fold over the basal concentrations, even in the presence of 5 micromol/L t-BOOH or diamide and 20 micromol/L menadione. PSSG increased by approximately 50% at 20 micromol/L t-BOOH or diamide and at 50 micromol/L menadione. PCO and MDA remained unchanged. In vivo oxidation treatments elicited immediate and significant increases in GSSG and PSSG over basal values (up to 200-fold), whereas PCO and MDA showed only slight variation 120 or 180 min after treatment. CONCLUSIONS: With the use of artifact-free measurement methods, GSH, GSSG, and PSSG are potentially powerful and reliable biomarkers of oxidative stress status and can be used to evaluate whether, and to what extent, oxidative stress may be involved in various diseases.

Animals↗
PubMed2006

Metabolism of oxidants by blood from different mouse strains.

Haemoglobins bearing reactive sulfhydryl groups have been shown to be able to interplay with glutathione in some detoxification processes. Blood from different mouse strains commonly used as experimental animal models, i.e., C57, DBA and ICR, was treated with oxidants with the aim of evaluating: (i) the involvement of protein SH groups in oxido-reductive reactions that are commonly carried out by glutathione and (ii) the impact of this phenomenon on blood-mediated metabolism of thiol reactants. All the main forms of glutathione (reduced, disulfide, and mixed disulfide with haemoglobin) were measured after oxidant treatment. Significant differences were found among the studied strains: DBA mice formed preferably mixed disulfides instead of glutathione disulfide, whereas the opposite behaviour was shown by C57 mice. Unexpectedly, the ICR strain resulted to be composed of three different subgroups (ICRa, ICRb, and ICRc), with the ICRa behaving similarly to the DBA strain, ICRc to the C57 strain, and ICRc showing an intermediate behaviour. These results are due to the different number of haemoglobin SH groups in the studied mouse strains. In particular, additional fast-reacting SH groups were found in haemoglobin from DBA, ICRa, and ICRb mice, but not in the C57 and ICRc strain. These differences were also reflected in the susceptibility of haemoglobin to dimerize and in its ability to react with S-nitrosocysteine. Because of the widely different reactivity of haemoglobin cysteinyl residues, the mouse strains examined are an interesting but complicated model in which to study the pharmacological and toxicological action of some drugs.

Animals↗
PubMed2006

Biomarkers of oxidative damage in human disease.

Oxidative/nitrosative stress, a pervasive condition of increased amounts of reactive oxygen/nitrogen species, is now recognized to be a prominent feature of many acute and chronic diseases and even of the normal aging process. However, definitive evidence for this association has often been lacking because of recognized shortcomings with biomarkers and/or methods available to assess oxidative stress status in humans. Emphasis is now being placed on biomarkers of oxidative stress, which are objectively measured and evaluated as indicators of normal biological processes, pathogenic processes, or pharmacologic responses to therapeutic intervention. To be a predictor of disease, a biomarker must be validated. Validation criteria include intrinsic qualities such as specificity, sensitivity, degree of inter- and intraindividual variability, and knowledge of the confounding and modifying factors. In addition, characteristics of the sampling and analytical procedures are of relevance, including constraints and noninvasiveness of sampling, stability of potential biomarkers, and the simplicity, sensitivity, specificity, and speed of the analytical method. Here we discuss some of the more commonly used biomarkers of oxidative/nitrosative damage and include selected examples of human studies.

Biomarkers↗
PubMed2006

Age-related influence on thiol, disulfide, and protein-mixed disulfide levels in human plasma.

In this study, plasma levels of both low-molecular-mass sulfhydryls/disulfides and mixed disulfides with proteins in 41 healthy humans aged 21-92 years were measured, with the aim of assessing whether there is a shift of the thiol/disulfide balance during aging and verifying some of the possible effects of the thiol imbalance. Our data suggest that aging is strictly correlated to a decrease in plasma glutathione and cysteinylglycine with the concomitant increase of most oxidized forms of thiols and a parallel increase in total cysteine and total homocysteine, probably due to an augmented efflux of these amino acids from various organs. The occurrence of two distinct regulatory systems for plasmatic pools of glutathione/cysteinylglycine on the one hand and cysteine/homocysteine on the other hand is hypothesized.

Adult↗
PubMed2005

S-glutathionylation in human platelets by a thiol-disulfide exchange-independent mechanism.

Protein-glutathione mixed disulfide formation was investigated in vitro by exposure of human platelets to the thiol-specific oxidant azodicarboxylic acid-bis-dimethylamide (diamide). We found that diamide causes a decrease in the reduced form of glutathione (GSH), paralleled by an increase in protein-GSH mixed disulfides (S-glutathionylated proteins), which was not accompanied by any significant increase in the basal level of glutathione disulfide (GSSG). The increase in the appearance of S-glutathionylated proteins was inversely correlated with ADP-induced platelet aggregation. Platelet cytoskeleton was analyzed by SDS-PAGE followed by Western immunoblotting with anti-GSH antibody. The main S-glutathionylated cytoskeletal protein proved to be actin, which accounts for 35% of the platelet total protein content. Our results suggest that neither GSSG formation nor a consequent thiol-disulfide exchange mechanism is involved in actin S-glutathionylation of human platelets exposed to diamide. Instead, a mechanism involving the initial oxidative activation of actin thiol groups, which then react with GSH to the protein-GSH mixed disulfides, makes it likely that platelet actin is S-glutathionylated without any significant increase in the GSSG content.

Actins↗
PubMed2005

Covalent modification of actin by 4-hydroxy-trans-2-nonenal (HNE): LC-ESI-MS/MS evidence for Cys374 Michael adduction.

We demonstrate for the first time, by a combined mass spectrometric and computational approach, that G- and F-actin can be covalently modified by the lipid-derived aldehyde, 4-hydroxy-trans-2-nonenal, providing information on the molecular mass of modified protein and the mechanism and site of adduction.ESI-MS analysis of actin treated with different molar ratios of HNE (1 : 1 to 1 : 20) showed the formation of a protein derivative in which there was an increase of 156 Da (42028 Da) over native actin (41872 Da), consistent with the adduction of one HNE residue through Michael addition. To identify the site of HNE adduction, G- and F-actin were stabilized by NaBH(4) reduction and digested with trypsin. LC-ESI-MS/MS analysis in data-dependent scan mode of the resulting peptides unequivocally indicated that Cys374 is the site of HNE adduction. Computational studies showed that the reactivity of Cys374 residue is due to a significant accessible surface and substantial thiol acidity due to the particular microenvironment surrounding Cys374.

Actins↗
PubMed2004

Nitric oxide, S-nitrosothiols and hemoglobin: is methodology the key?

Two main hypotheses describe the role of hemoglobin in the regulation of nitric oxide (NO) bioavailability. It has been suggested that hemoglobin interacts with circulating NO, forming Fe-nitrosyl hemoglobin and then S-nitrosothiols, which deliver NO extracellularly by an allosterically regulated mechanism. Alternatively, the existence of diffusional barriers that protect NO from hemoglobin-mediated degradation has been proposed. The reliability of each model in vivo is supported by the detection of physiological hematic levels of S-nitrosohemoglobin. However, the measured concentrations of S-nitrosohemoglobin are largely divergent between the two models. Moreover, recent reports suggest that circulating levels of S-nitrosohemoglobin in human blood could be significantly lower than assessed previously. We suggest that solving the methodological controversies that make the field of NO research a 'minefield', even for skilled analysts, is fundamental to understanding the role of S-nitrosothiols in the vasculature.

Hemoglobins↗
PubMed2004

Interference of plasmatic reduced glutathione and hemolysis on glutathione disulfide levels in human blood.

The blood reduced glutathione (GSH)/GSH disulfide (GSSG) ratio is an index of the oxidant/antioxidant balance of the whole body. Nevertheless, data indicating GSH and GSSG physiological levels are still widely divergent, especially those on GSSG, probably due to its low concentration. Standardization in methodological protocols and sample manipulation could help to minimize these discrepancies. Therefore, we have investigated how plasma reduced GSH, which is rapidly oxidized after blood withdrawal, could alter the blood GSSG measurement if the sample is not suitably processed. We have observed that an increase in plasma GSH concentration, due to red blood cell hemolysis, is responsible for a significant overestimation of blood GSSG level. Our results show that, before performing blood GSSG determination, thiols have to be rapidly blocked, to avoid possible pitfalls in GSSG measurement, in particular when hemolysis is present.

Animals↗
PubMed2004

Adaptation of the Griess reaction for detection of nitrite in human plasma.

The determination of nitrite in human plasma or serum has been most frequently used as a marker of nitric oxide (NO) production. In addition, it has recently been suggested that nitrite could act as a vasodilating agent at physiological concentrations by NO delivery. Therefore, nitrite determination in biological fluids is becoming increasingly important. The most frequently used method to measure nitrite is based on the spectrophotometric analysis of the azo dye obtained after reaction with the Griess reagent. This method has some limitations regarding detection limit and sensitivity, thus resulting unsuitable for nitrite detection in plasma. We have identified some drawbacks and modified the original procedure to overcome these problems. By the use of the newly developed method, we measured 221+/-72 nM nitrite in human plasma from healthy donors.

Health↗
PubMed2003

An improved HPLC measurement for GSH and GSSG in human blood.

The 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 microM, 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.

Chromatography, High Pressure Liquid↗
PubMed2003

Actin S-glutathionylation: evidence against a thiol-disulphide exchange mechanism.

Many proteins, including actin, are targets for S-glutathionylation, the reversible formation of mixed disulphides between protein cysteinyl thiol groups and glutathione (GSH) that can be induced in cells by oxidative stress. Proposed mechanisms of protein S-glutathionylation follow mainly two distinct pathways. One route involves the initial oxidative modification of a reduced protein thiol to an activated protein, which may then react with GSH to the mixed disulphide. The second route involves the oxidative modification of GSH to an activated form such as glutathione disulphide (GSSG), which may then react with a reduced protein thiol, yielding the corresponding protein mixed disulphide. We show here that physiological levels of GSSG induce a little extent of actin S-glutathionylation. Instead, actin with the exposed cysteine thiol activated by diamide or 5,5'-dithiobis(2-nitrobenzoic acid) reacts with physiological levels of GSH, incorporating about 0.7 mol GSH/mol protein. Differently, an extremely high concentration of GSSG induces an increased level of S-glutathionylation that causes a 50% inhibition in actin polymerization not reversed by dithiotreitol. In mammalian cells, GSH is present in millimolar concentrations and is in about 100-fold excess over GSSG. The high concentration of GSSG required for obtaining a significant actin S-glutathionylation as well as attendant irreversible changes in protein functions make unlikely that actin may be S-glutathionylated by a thiol-disulphide exchange mechanism within the cell.

Actins↗
PubMed2003

Protein carbonyl groups as biomarkers of oxidative stress.

Oxidative stress, an imbalance toward the pro-oxidant side of the pro-oxidant/antioxidant homeostasis, occurs in several human diseases. Among these diseases are those in which high levels of protein carbonyl (CO) groups have been observed, including Alzheimer's disease (AD), rheumatoid arthritis, diabetes, sepsis, chronic renal failure, and respiratory distress syndrome. What relationships might be among high level of protein CO groups, oxidative stress, and diseases remain uncertain.The usage of protein CO groups as biomarkers of oxidative stress has some advantages in comparison with the measurement of other oxidation products because of the relative early formation and the relative stability of carbonylated proteins. Most of the assays for detection of protein CO groups involve derivatisation of the carbonyl group with 2,4-dinitrophenylhydrazine (DNPH), which leads to formation of a stable dinitrophenyl (DNP) hydrazone product. This then can be detected by various means, such as spectrophotometric assay, enzyme-linked immunosorbent assay (ELISA), and one-dimensional or two-dimensional electrophoresis followed by Western blot immunoassay. At present, the measurement of protein CO groups after their derivatisation with DNPH is the most widely utilized measure of protein oxidation.

Animals↗