GSSG) And Reduced Glutathione Research Articles

Evaluation of oxidative stress biomarkers for differentiating bacterial and viral infections: a comparative study of glutathione disulfide (GSSG) and reduced glutathione (GSH)

Background and aims. This study evaluates the potential of oxidative stress biomarkers, specifically glutathione disulfide (GSSG) and reduced glutathione (GSH), for differentiating bacterial and viral infections. Oxidative stress plays a crucial role in the immune response, and glutathione is a key regulator of cellular redox balance. The aim was to assess whether differences in GSH and GSSG levels could be used as diagnostic markers for infection type. Methods. A chemiluminescence-based method evaluated GSH and GSSG as potential biomarkers for distinguishing between bacterial and viral infections. The GSH and GSSG concentrations were analyzed across bacterial, viral, and control groups. Results. Our data revealed significant differences in the GSH/GSSG ratio between the analyzed groups, with bacterial infections showing higher oxidative stress markers compared to viral infections. A combined analysis of GSH and GSSG concentrations, visualized through heatmaps and ROC curves, improved diagnostic accuracy, with clustering patterns distinguishing infection types. Conclusions. These findings suggest that the GSH/GSSG ratio could be used as a biomarker in distinguishing between bacterial and viral infections, offering potential clinical applications for more accurate diagnosis. Further research is required to validate these results in larger cohorts and to explore the underlying mechanisms of oxidative stress in pathogen-specific immune responses.

Effects of increased nitrate intake from beetroot juice on blood markers of oxidative stress and inflammation in older adults with hypertension

BackgroundVascular oxidative stress and low-grade inflammation are important in the pathology of cardiovascular disorders, including hypertension. Cell culture and animal studies suggest that inorganic dietary nitrate may attenuate oxidative stress and inflammation through nitric oxide (NO), and there is a need to investigate whether this translates to humans. AimIn this randomised, placebo-controlled crossover study, by measuring a combination of multiple blood biomarkers, we evaluated whether previously reported benefits of dietary nitrate translate to a reduced oxidative stress and an improved inflammation status in 15 men and women (age range: 56–71 years) with treated hypertension. MethodsWe investigated the effects of a single ∼400 mg-dose of nitrate at 3 h post-ingestion (3H POST) and the daily consumption of 2 × ∼400 mg of nitrate over 4 weeks (4WK POST), through nitrate-rich versus nitrate-depleted (placebo) beetroot juice. Measurements included plasma nitrate and nitrite (NOx), oxidised low-density lipoprotein (oxLDL), F2-isoprostanes, protein carbonyls, oxidised (GSSG) and reduced glutathione (GSH); and serum high-sensitive C-reactive protein (hsCRP), chemokines, cytokines, and adhesion molecules. Flow cytometry was used to assess the relative proportion of blood monocyte subsets. ResultsAt 4WK POST nitrate intervention, the oxLDL/NOx ratio decreased (mainly due to increases in plasma nitrate and nitrite) and the GSH/GSSG ratio (a sensitive biomarker for alterations in the redox status) increased, compared with placebo (for both ratios P < 0.01). The relative proportion of classical (CD14+CD16−) monocytes decreased at 4WK POST for placebo compared to nitrate intervention (P < 0.05). Other oxidative stress and inflammatory markers were not altered by increased nitrate intake relative to placebo. ConclusionsThe data from this study point toward a subtle alteration in the redox balance toward a less pro-oxidative profile by a regular intake of inorganic nitrate from plant foods. Clinical trial registry numberNCT04584372 (ClinicialTrials.gov).

Alcohol and its metabolites dysregulate cellular bioenergetics and induce oxidative and endoplasmic reticulum stress in primary human bronchial epithelial cells.

Chronic alcohol consumption/misuse is a significant risk factor for pneumonia and lung infection leading to the development of chronic pulmonary disorders such as chronic obstructive pulmonary disease (COPD) and lung fibrosis. In this study, we sought to delineate the mechanism of alcohol-associated lung disease. We did so by measuring invitro mitochondrial, endoplasmic reticulum (ER) oxidative stress in human bronchial epithelial cells (hBECs) treated with ethanol and its oxidative (acetaldehyde) and nonoxidative (fatty acid ethyl esters or FAEEs) metabolites. Primary hBECs from a normal subject were treated with relevant concentrations of ethanol and its metabolites and incubated at 37°C for 24 h. Viability and cytotoxicity were determined using cell viability and lactate dehydrogenase (LDH) assay kits, respectively. Oxidized glutathione (GSSG) and reduced glutathione (GSH) were measured by colorimetric reaction, and 4-hydroxynenonal (4HNE) by immunohistochemistry. Endoplasmic reticulum stress and dysregulated cellular bioenergetics were determined by western blot analysis. Mitochondrial stress and real-time ATP production rates were determined using a Seahorse Extracellular Flux analyzer. Amelioration of ethanol-induced oxidative/ER stress and mitochondrial energetics was determined using an AMPKα agonist. Human bronchial epithelial cells treated with ethanol, acetaldehyde, and FAEEs showed a concentration-dependent increase in the secretion of LDH, oxidative/ER stress, deactivation of AMPKα phosphorylation and mitochondrial stress (decreased spare respiratory capacity) with concomitant decreases in mitochondrial and glycolytic ATP production rates. FAEEs caused greater cytotoxicity, ER stress, and dysregulated cellular bioenergetics than those ethanol and its oxidative metabolite. AMPKα agonist-pretreated cells significantly ameliorated ethanol-induced oxidative/ER stress, deactivation of AMPKα, and dysregulated cellular bioenergetics. Findings of this study suggest that ethanol and its metabolites contribute to cytotoxicity, oxidative/ER stress, and dysregulation of cellular bioenergetics in hBECs. The attenuation of ethanol-induced ER/oxidative stress and mitochondrial respiration by an AMPKα agonist may reflect a potential for it to be developed as a therapeutic agent for chronic alcohol-associated lung disease.

Components of the Glutathione Cycle as Markers of Biological Age: An Approach to Clinical Application in Aging.

The oxidative-inflammatory theory of aging states that aging is the result of the establishment of a chronic oxidative-inflammatory stress situation in which the immune system is implicated. Among the redox parameters, those involved in the glutathione cycle have been suggested as essential in aging. Thus, the first objective of this study was to determine if several components of the glutathione cycle (glutathione reductase (GR) and glutathione peroxidase (GPx) activities, and concentrations of oxidized glutathione (GSSG) and reduced glutathione (GSH)) in leukocytes) are associated with the biological age (ImmunolAge) estimated using the Immunity Clock in 190 men and women. The second objective was to identify the best blood fraction (whole blood, blood cells, erythrocytes, or plasma) to quantify these components and correlate them with the estimated ImmunolAge. The results show that the oxidative state of peripheral leukocytes correlates with their functionality, supporting the idea that this is the basis of immunosenescence. In blood, the correlations are more significant in the fraction of blood cells with respect to ImmunolAge (positive correlations with GSSG concentration and the GSSG/GSH ratio, and negative correlations with GPx and GR activities). Therefore, blood cells are proposed as the most effective sample to estimate the biological age of individuals in clinical settings.

Facile and Green Fabrication of Highly Competent Surface-Modified Chlorogenic Acid Silver Nanoparticles: Characterization and Antioxidant and Cancer Chemopreventive Potential.

The eco-friendly, cost-effective, and green fabrication of nanoparticles is considered a promising area of nanotechnology. Here, we report on the green synthesis and characterization of bovine serum albumin (BSA)-decorated chlorogenic acid silver nanoparticles (AgNPs-CGA-BSA) and the studies undertaken to verify their plausible antioxidant and antineoplastic effects. High-resolution transmission electron microscopy (HR-TEM), dynamic light scattering, X-ray diffraction, and Fourier transform infrared analyses depict an average mean particle size of ∼96 nm, spherical morphology, and nanocrystalline structure of AgNPs-CGA-BSA. DPPH scavenging and inhibition of lipid peroxidation signify the noticeable in vitro antioxidant potential of the nanoparticles. The in vitro experimental results demonstrate that AgNPs-CGA-BSA shows significant cytotoxicity to Dalton's lymphoma ascites (DLA) cells and generates an enhanced intracellular reactive oxygen species and oxidized glutathione (GSSG) and reduced glutathione (GSH) in DLA cells. Furthermore, mechanism investigation divulges the pivotal role of the downregulated expression of superoxide dismutase (SOD) and catalase (CAT), and these ultimately lead to apoptotic chromatin condensation in AgNPs-CGA-BSA-treated DLA cells. In addition, in vivo experiments reveal an excellent decrease in tumor cell count, an increase in serum GSH and CAT, SOD, and glutathione peroxidase activities, and a decrease in the malondialdehyde (MDA) level in DLA-bearing mice after AgNPs-CGA-BSA treatment. These findings suggest that the newly synthesized biogenic green silver nanoparticles have remarkable in vitro antioxidant and antineoplastic efficacy that triggers cytotoxicity, oxidative stress, and chromatin condensation in DLA cells and in vivo anticancer efficacy that enhances the host antioxidant status, and these might open a new path in T-cell lymphoma therapy.

Oxidative stress and metabolic parameters in hypertensive patients with/without diabetes mellitus in cardiovascular risk evaluation

Abstract Introduction: Hypertension and diabetes mellitus affect a large number of patients and can significantly influence their life expectancy. Changes in metabolic and oxidative stress parameters are common in these pathologies, contributing to associated complications. The aim of the study was assessment of relationship between laboratory parameters and their role in evaluation of cardiovascular risk, and possible gender-related differences in the protective factors. Material and methods: Blood samples were collected from hypertensive patients with/without diabetes mellitus admitted to the Cardiovascular Rehabilitation Clinic in Tîrgu Mureș and controls without these pathologies. Biochemical analyses were performed on Konelab analyzer (glycemia, lipid profile, kidney function tests, zinc, hsCRP). Oxidative stress markers, such as serum malondialdehyde (MDA), oxidized (GSSG) and reduced glutathione (GSH) were evaluated using an HPLC-UV/VIS technique at GEP UMPhST. Statistical analysis was performed by GraphPad InStat3. Results: Mean age of hypertensive patients (n=131) was 69.44 ± 9.02 years, 45.8% males, 31.3% being diabetics. 74.1% of the studied patients had zinc deficiency, 19.8% presented slightly elevated hsCRP. The control group included 24 nonhypertensive/nondiabetic patients of similar age. Average GSH was significantly lower (p=0.0002) in hypertensive patients, 1.89 ± 0.82 µg/ml, compared to the control group (3.23 ± 0.49 µg/ml), and no correlation could be observed between GSH and MDA values. GSH concentration was significantly higher in males (p=0.0395) and HDL-cholesterol significantly higher in females (p=0.0132). A negative correlation was observed between serum triglyceride and HDL-cholesterol concentration. Conclusions: Gender differences are present in the level of protective factors against cardiovascular diseases, while oxidative stress is intensified in hypertensive/diabetic patients.

Stability of glutathione added as a supplement to parenteral nutrition.

Most very premature newborns (<32 weeks of gestation) receive parenteral nutrition (PN) that is inherently contaminated with peroxides. Oxidative stress induced by PN is associated with bronchopulmonary dysplasia, a main pathological complication in these infants who have weak antioxidant capacity to detoxify peroxides because of their glutathione deficiency. In animals, glutathione supplementation of PN prevented oxidative stress and alveolar loss (the main characteristic of bronchopulmonary dysplasia). Of its two forms-oxidized glutathione (GSSG) and reduced glutathione (GSH)-GSSG was used because of its better stability. However, a 30% loss of GSSG in PN is observed. The potentially high therapeutic benefits of GSSG supplementation on the health of very premature infants make the study of its stability highly important. GSSG was incubated in combination with the following components of PN: dextrose, multivitamins, Primene, and Travasol, and with cysteine, cystine, and peroxides, for 24 h. Total glutathione in these solutions was measured 0-24 h after the addition of GSSG. The combination of cysteine and multivitamins caused the maximum loss of glutathione. The stability of GSSG was not affected by multivitamins. The cysteine was responsible for ∼20% of the loss of GSSG; in the presence of multivitamins, the loss reached >70%. Removing the cysteine prevented the degradation of glutathione. GSSG reacts with cysteine to form cysteine-glutathione mixed disulfide, another suitable glutathione substrate for preterm neonates. The study confirms that GSSG added to PN can potentially provide a precursor to de novo synthesis of glutathione in vivo.

Stiffness Induces NAFLD‐like Metabolic Dysfunction in Primary Hepatocytes

BackgroundNon‐alcoholic fatty liver disease (NAFLD), the liver manifestation of the metabolic syndrome linked to obesity and insulin resistance, affects 25% of the general population both in western and developing countries. The development and progression of NAFLD is mediated by increase in lipid accumulation and triglycerides levels, excessive oxidative stress and inflammation that in turn exacerbates the diseased condition and impairs metabolic parameters. NAFLD progression is also characterized by liver stiffness due to extensive remodeling of the extracellular matrix. The role of matrix stiffness as related to subtle but pivotal changes in hepatocytes physiology and metabolic dysfunction is under explored. The overall goal of our study is the development and implementation of a platform that enables the convergence of engineered cell microenvironments with the phenotypic and functional analysis of hepatocytes. Using our innovative biomimetic liver fibrosis model that allows modulation of substrate stiffness, we investigated the role of liver matrix stiffness in modulating hepatocytes metabolic function in fibrotic‐like microenvironment.Materials and MethodsPrimary hepatocytes were cultured on our novel polymer film coated polydimethylsiloxane (PDMS) gels with 2 kPa, 9 kPa 25 kPa and 55 kPa elastic modulus mimicking healthy, early fibrotic, fibrotic and extremely fibrotic substrates. A wide range of functional/metabolic analysis were performed to determine: lipogenic and oxidation genes, glutathione expression, dynamics by Western Blot; Respirometry by Seahorse XFe24 Analyzer.ResultsWe demonstrated that stiffness impedes hepatic urea and albumin production, expression of drug transporter gene and epithelial cell phenotype marker, hepatocyte nuclear factor 4 alpha (HNF4a). We also demonstrated that hepatocytes cultured on NAFLD‐like stiffness showed an induction of lipogenic genes along with lowered‐oxidation genes expression, mitochondrial respiration, and glycolytic capacity, 2) increased ROS production, and 3) disruption of the mitochondrial fusion process and dynamics. We observed a significant increase in oxidized glutathione (GSSG) and reduced glutathione (GSH) in hepatocytes cultured on NAFLD‐like stiffness compared to healthy liver stiffness. Similar effect was observed in hepatocytes isolated from NAFLD rat models indicating correlation to physiological conditions. These data suggest a plausible mechanism that increased stiffness modulates hepatocyte function causing liver functional failure.ConclusionsTogether, all these data demonstrates the plausible role of stiffness in regulating hepatocytes function and contribute to metabolic dysregulation in NAFLD. Understanding the impact of stiffness on hepatocytes biology will provide significantly more nuanced data to aid drug development for NAFLD.Support or Funding InformationNIH R01AA027189‐01A1; Nebraska Research Initiative; NIH 1P20GM113126Project SummaryFigure 1

The evaluation of glutathione concentration in whole blood of Holstein dairy calves

The aim of this study was to evaluate the concentrations of total (T-GSH), oxidized (GSSG) and reduced glutathione (GSH) in blood of clinically healthy calves, to assess the effect of sex and age together with the type of feeding on its concentrations, and to assess the relationships between glutathione and metabolic indices. A total of 117 Holstein calves at the age of 1 day to 6 months were divided according to age and together with the type of feeding into 4 groups: colostrum and transition milk (n = 20), native milk (n = 39), milk replacer (n = 29) and total mixed ration (n = 29). Blood serum was used for the assessment of the metabolic profile of calves. The concentrations of individual forms of glutathione (mean ± SEM) were as follows: T-GSH: 789.1 ± 19.4 µmol/l, GSSG: 54.2 ± 4.3 µmol/l, GSH: 681.6 ± 15.3 µmol/l, and GSH/GSSG: 41.1 ± 5.9, with the coefficients of variability of 26.6%, 85.3%, 24.3% and 157.4%, respectively. Age and the type of feeding affected GSSG (P≤ 0.01) and GSH/GSSG (P≤ 0.01). The relationships between glutathione and biochemical parameters were mostly weak. T-GSH correlated with creatinine (P≤ 0.05) and chlorides (P≤ 0.05); GSSG correlated with albumin (P≤ 0.01), creatinine (P≤ 0.01), cholesterol (P≤ 0.05), aspartate aminotransferase (P≤ 0.01), alanine aminotransferase (P≤ 0.01), gamma-glutamyltransferase (P≤ 0.05) and calcium (P≤ 0.05); GSH/GSSG ratio correlated with creatinine (P≤ 0.01), cholesterol (P≤ 0.05), aspartate aminotransferase (P≤ 0.05), gamma-glutamyltransferase (P≤ 0.01) and calcium (P≤ 0.05). The evaluation of individual forms of glutathione helps in estimation of redox status in healthy animals and their monitoring can detect the stress extent.

Oxidative stress assessment by glutathione peroxidase activity and glutathione levels in response to selenium supplementation in patients with Mucopolysaccharidosis I, II and VI.

We assessed levels of plasma selenium (Se), selenoproteins and their change after Se supplementation in patients with mucopolysaccharidosis (MPS) types I, II and VI. This was done in a retrospective study of the medical records of 30 patients with MPS I (n=13), MPS II (n=9) and MPS VI (n=8) who were being treated with enzyme replacement therapy. As part of routine nutritional monitoring, Se levels were measured, revealing that 28 patients (93.3%) had values below the normal range. Therefore, they received supplementation for 12 months, and Se was measured after 6 and 12 months. Glutathione peroxidase (GPx) activity, total glutathione (GSHt), oxidized glutathione (GSSG) and reduced glutathione (GSH) were measured at baseline and 6 months after Se supplementation. The mean GSHt at baseline was 7.90 ± 2.36 μmol/g Hb, and after Se supplementation it was 5.76 ± 1.13 μmol/g Hb; GSH/GSSG was 2.3 ± 1.16 at baseline and 0.58 ± 0.38 after supplementation. GPx activity was 16.46 ± 3.31 U/g Hb at baseline and 4.53 ± 4.92 U/g Hb after Se supplementation. The difference was shown to be statistically significant by paired t-test. In conclusion, our study demonstrated that oxidative stress parameters were altered by Se supplementation in patients with MPS I, II and VI who were previously deficient in Se.

Liquiritigenin and liquiritin alleviated MCT-induced HSOS by activating Nrf2 antioxidative defense system

Hepatic sinusoidal obstruction syndrome (HSOS) is a serious and life-threatening liver disease. Liquiritigenin (LG) and liquiritin (LQ) are natural flavonoids distributed in Glycyrrhizae Radix et Rhizoma (Gan-cao). This study aims to investigate the protective effect and mechanism of LG and LQ against monocrotaline (MCT)-induced HSOS. Results of serum alanine/aspartate aminotransferases (ALT/AST) activities, liver histological evaluation and scanning electron microscope observation, and hepatic metalloproteinase-9 (MMP-9) expression demonstrated that LG and LQ both alleviated HSOS induced by MCT in rats. Results of hepatic reactive oxygen species (ROS), malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), oxidized glutathione (GSSG) and reduced glutathione (GSH) contents, glutathione reductase (GR) and superoxide dismutase (SOD) activities showed that LG and LQ attenuated MCT-induced liver oxidative stress injury. Furthermore, LG and LQ were found to promote Nrf2 nuclear translocation and lead to the increased expression of Nrf2 downstream antioxidative genes. Molecule docking analysis indicated the potential interaction of LG and LQ with Nrf2 binding site in the kelch-like ECH-associated protein-1 (Keap1) protein. Finally, Nrf2 knock-out mice were used. The results showed that LG and LQ both alleviated MCT-induced HSOS in wild-type mice, but such protection was totally diminished in Nrf2 knock-out mice. In conclusion, our study revealed that LG and LQ alleviated MCT-induced HSOS by inducing the activation of hepatic Nrf2 antioxidative defense system.

Abstract P216: Elevated Blood Glutathione Does Not Reduce Arterial Stiffness or Central Blood Pressure in Healthy Males and Females

Intro: Glutathione is endogenous within human plasma, erythrocyte lysate and is also bound to the protein within plasma. Glutathione mediates redox chemistry and prevents oxidative damage within and around cellular components via reduction of reactive species (e.g. reactive oxygen, nitrogen, or sulfur species). Polyphenols and antioxidants have been shown to improve NO bioavailability which may reduce long term incidence of endothelial dysfunction. Less is known about whether changes in antioxidant capacity augments the risk of developing hypertension. Hypothesis: We hypothesized that acute glutathione supplementation would decrease arterial stiffness and reduce both brachial (bBP) and central blood pressure (cBP) in healthy male and female volunteers. Methods: Six males and six females (25 ± 3 and 22 ± 1 years, respectively) participated in a randomized, double blind, placebo controlled, crossover protocol. On two visits separated by 1 week, following a 12-hour fast, participants consumed either a placebo or glutathione (negligible and 200 mg, respectively) supplement via 90 second sublingual absorption which was then swallowed. Concentrations of oxidized (GSSG) and reduced glutathione (GSH) were spectrophotometrically measured in plasma (protein-bound) and erythrocyte lysate using a kinetic, enzymatic assay. Arterial stiffness was measured via pulse wave velocity (PWV) using applanation tonometry, and cBP was determined non-invasively using pulse wave analysis. All data were recorded before supplementation (baseline) and at 10, 30, 60 and 120 minutes post-consumption. Results: Linear mixed effect models revealed a significant (p&lt;0.01) increase in total glutathione (GSH+GSSG) in the supplement group compared to placebo across all post-supplementation time points with the greatest increase occurring at 120 minutes (mean 99.0; 95%CI: 7.9,190.1). At 120 minutes post-consumption, no difference was present between glutathione and placebo groups for PWV (5.86 ± 1.19 and 6.08 ± 1.25 m/s, respectively; p=0.43), resting heart rate (52.95 ± 3.55 and 55.83 ± 6.36, respectively; p=0.16), systolic bBP (123.05 ± 12.75 and 123.13 ± 14.52 mmHg; p=0.22), diastolic bBP (71.81 ± 7.87 and 74.21 ± 6.53; p=0.48), systolic cBP (108.05 ± 10.45 and 108.68 ± 11.14 mmHg, respectively; p=0.11) and diastolic cBP (72.03 ± 7.82 and 74.94 ± 6.42 mmHg, respectively; p=0.46). Conclusion: Young healthy males and females experienced an increase in circulating humoral antioxidants in response to glutathione supplementation. However, supplementation had minimal effects on resting hemodynamics. Future research should examine glutathione supplementation’s effect in participants with decreased antioxidant capacity and increased oxidative stress including patients with known disease such as hypertension or peripheral artery disease.

Simultaneous Electrochemical Speciation of Oxidized and Reduced Glutathione. Redox Profiling of Oxidative Stress in Biological Fluids with a Modified Carbon Electrode.

The simultaneous electrochemical quantification of oxidized (GSSG) and reduced glutathione (GSH), biomarkers of oxidative stress, is demonstrated in biological fluids. The detection was accomplished by the development of a modified carbon electrode and was applied to the analysis of biological fluids of model organisms under oxidative stress caused by lead intoxication. Nanocomposite molecular material based on cobalt phthalocyanine (CoPc) and multiwalled carbon nanotubes functionalized with carboxyl groups (MWCNTf) was developed to modify glassy carbon electrodes (GCE) for the detection of reduced and oxidized glutathione. The morphology of the nanocomposite film was characterized by scanning electron microscopy (SEM) and profilometry. The electrochemical behavior of the modified electrode was assessed by cyclic voltammetry (CV) to determine the surface coverage (Γ) by CoPc. The electrocatalytic behavior of the modified electrode toward reduced (GSH) and oxidized (GSSG) forms of glutathione was assessed by CV studies at physiological pH. The obtained results show that the combined use of CoPc and MWCNTf results in an electrocatalytic activity for GSH oxidation and GSSG reduction, enabling the simultaneous detection of both species. Differential pulse voltammetry reveals detection limits of 100 μM for GSH and 8.3 μM for GSSG, respectively. The potential interference from ascorbic acid, cysteine, glutamic acid, and glucose was also studied, and the obtained results show limited effects from these species. Finally, the hybrid electrode was used for the determination of GSH and GSSG in rat urine and plasma samples, intoxicated or not by lead. Both glutathione forms were detected in these complex biological matrixes without any pretreatment. Our results portray the role of GSH and GSSG as markers of oxidative stress in live organisms under lead intoxication.

Effect of time-dependent cryotherapy on redox balance of quadriceps injuries

Muscle trauma represents a high number of injuries in professional sport and recreation and may occur through several mechanisms. This study aims at analyzing time-dependent effects of cryotherapy on the redox balance in lesioned quadriceps muscles in F1 mice. Twenty male F1 mice were divided into five groups: (a) animals were not subjected to muscle lesioning or treatment (CTR); (b) quadriceps muscle was lesioned without treatment (L); (c) quadriceps muscle was lesioned and treated with cryotherapy for 5 min (LC5); (d) quadriceps muscle was lesioned and treated with cryotherapy for 20 min (LC20); and quadriceps muscle was lesioned and treated with cryotherapy for 40 min (LC40). The mice were euthanized; the quadriceps muscles were collected and subjected to analyses for levels of protein, hydroperoxides, nitrite, catalase (CAT) activity, oxidized glutathione (GSSG) and reduced glutathione (GSH). Protein levels were reduced in L (−39%; p < 0.05), LC5 (−54%; p < 0.05), LC20 (−40%; p < 0.05) and LC40 (−50%; p < 0.05) compared to CTR. There was an increase in lipid peroxidation in L (158%; p < 0.05), LC5 (300%; p < 0.01), LC20 (292%; p < 0.01) and LC40 (362%; p < 0.01) compared to CTR. We observed a significant increase in CAT activity in L (164%; p < 0.05) and LC5 (193%; p < 0.01) compared to CTR; a significant reduction in GSH in L (−60%; p < 0.05) and LC20 (−61%; p < 0.05) compared to CTR; and a significant increase in GSSG in LC5 (171%; p < 0.05) compared to CTR. In addition, GSH/GSSG was reduced in L (−89%; p < 0.01), LC5 (−95%; p < 0.01), LC20 (−59%; p < 0.05), and LC40 (−82%; p < 0.01) compared to CTR. This study showed that the cryotherapy does not improve the oxidative stress in lesioned muscles.

Sequential radial and circumferential strain and oxidative stress assessment in dogs with tachycardia-induced cardiac dysfunction.

Quantitative analysis of left ventricular (LV) deformation based on two-dimensional speckle tracking echocardiography (2D STE) has increasingly been used to assess segmental and global function because conventional echocardiography is influenced by tethering effects of adjacent myocardium and cardiac translational motion. (1) 2D STE can be useful to detect subtle regional changes in the LV contractility during development of tachycardia-induced cardiomyopathy (TIC) to heart failure in awake dog; (2) oxidized glutathione (GSSG) and reduced glutathione (GSH) levels are associated with the development of its respective segmental wall motion abnormalities (WMA). 6 healthy canine female Beagles were examined using 2D STE myocardial strain analysis before and for 8 weeks on week basis of rapid ventricular pacing (RVP) and at the end of study each myocardial segment were evaluated for oxidative status (GSH:GSSG ratio). 2D STE showed an initial peak of reduced global radial strain at 2 weeks of RVP in all three cardiac levels analyzed in which the affected segments showed a decreased transmural fiber shortening from anteroseptal to inferior segments distributed in helical pattern suggesting impaired contractility from part of left band to apical loop of spiral muscle band while global circumferential strain showed to be reduced since the fifth week of RVP particularly in the base and midventricular levels of ascending segment in clockwise direction from lateral to inferior segments. The most affected segments are localized in the lateral to posterior wall, however, segmental oxidative stress analysis did not show correlation with WMA detected by strain. 2D STE strain demonstrated to be a reliable tool for evaluation of LV myocardial deformation in TIC canine model showing an earlier significant WMA using radial strain and later using circumferential strain which may be of importance for improvement of diagnosis and therapy in naturally occurring canine cardiomyopathy and for earlier detection of WMA after suboptimal pacemaker lead placement.

Structural elucidation and biological studies of a novel exopolysaccaride from Klebsiella pneumoniae PB12

An exopolysaccharide (KNPS) of an average molecular weight ∼1.8×105Da was isolated from the culture medium of Klebsiella pneumoniae PB12. Structural characterization of KNPS was carried out using sugar and methylation analysis, Smith degradation and 1D/2D NMR experiments. Sugar analysis showed that the KNPS composed of arabinose, galactose, 3-O-methyl-galctose and glucose in a molar ratio of nearly 4:3:1:1. The proposed repeating unit of the KNPS has a backbone chain consisting of two (1→6)-galactopyranosyl residues, two (1→5)-arabinofuranosyl residues, one (1→6)-glucopyranosyl residue and one (1→3)-arabinopyranosyl residue, out of which one (1→6)-galactopyranosyl residue was branched at O-2 position with a (1→2)-linked-galactopyranosyl residue terminated with non reducing arabinofuranosyl residue and one (1→5)-arabinofuranosyl residue branched at O-3 position with non reducing end 3-O-Me-galactopyranosyl residue. KNPS was found non-toxic toward human lymphocyte up to the dosage of 100μg/ml. KNPS enhanced malondialdehyde (MDA), reactive oxygen species (ROS), and have the potential to alter the ratio of oxidized glutathione (GSSG) and reduced glutathione (GSH) levels in the cellular system.

Glutathione redox system, immune status, antioxidant enzymes and metabolism of purine nucleotides in hypothyroidism

The immune status, components of the glutathione redox system, the activity of antioxidant enzymes and metabolism of purine nucleotides have been investigated in animals with experimental hypothyroidism. On day 8 after an increase in the number of leukocytes, lymphocytes, T-helpers and T-suppressors as well as increased number of B-lymphocytes was found in blood of thyroidectomized rats. This was accompanied by decreased activity of adenosine deaminase (AD), AMP-deaminase (AMPD), and 5'-nucleotidase (5'N) in blood, but the ratio of enzyme activity AD/AMPD increased. These changes in the activity of enzymes, involved in purine catabolism can be regarded as increased functional relationships between T and B lymphocytes in hypothyroidism. The functional changes of immune system cells were accompanied by increased activity of glutathione peroxidase (GPx), a decrease in the activity of superoxide dismutase (SOD), glutathione reductase (GR) and the ratio GH/GPx. Thyroidectomized rats had increased amounts of total, oxidized (GSSG) and reduced glutathione (GSH), but the ratio GSH/GSSG decerased as compared with control animals. In the liver, hypothyroidism resulted in activation of SOD, GPx, decreased activity of GR and decreased ratio GR/GPx. At the same time, the levels of total, oxidized, and reduced glutathione increased, but the ratio GSH/GSSG as well as activities of enzymes involved in purine nucleotide metabolism ratio (and their ratio 5'N/AD + AMPD) decreased. All these data suggest a functional relationship of the glutathione redox system not only with antioxidant enzymes, but also activity of enzymes involved purine nucleotide metabolism and immune status.

Oxidative stress biomarkers as indicator of chronic inflammatory joint diseases stage

ObjectiveTo evaluate the participation of oxidative stress (OS) on chronic inflammatory joint disease (CIJD), as well as its possible use as a diagnostic biomarker. Patients and methodsThe study population comprised 29 patients with CIJD: 18 with rheumatoid arthritis (RA: 13 active/5 inactive); 11 with ankylosing spondylitis (AS: 7 active/4 inactive) and 13 healthy subjects. Activity of the disease was assessed by: RA patients, Disease Activity Score (DAS 28) and AS patients by means of Bath Ankylosing Spondylitis Disease Activity Index (BASDAI). Oxidative stress biomarkers were determined in plasma using spectrophotometrical techniques. The statistical analysis was carried out using the SSPS statistical package. ResultsActive CIJD showed a high oxidative stress characterized by increases in oxidative damage markers and a reduction in antioxidative systems, together with a higher myeloperoxidase (MPO) concentration. Inactive CIJD only showed changes in oxidized glutathione (GSSG) and reduced glutathione (GSH)/GSSG ratio levels, without changes in oxidative damage parameters or in antioxidative systems. ConclusionsOur data revealed that: i) CIJD presents with a high oxidative stress; ii) inactive CIJD shows a production of reactive species without triggering oxidative damage and maintaining red-ox homeostasis, and iii) the combination of oxidative stress biomarkers may be used as markers of active-inactive stages of CIJD.

Biomarcadores de estrés oxidativo como indicadores de actividad en la enfermedad articular inflamatoria crónica

Objective To evaluate the participation of oxidative stress (OS) on chronic inflammatory joint disease (CIJD), as well as its possible use as a diagnostic biomarker. Patients and methods The study population comprised 29 patients with CIJD: 18 with rheumatoid arthritis (RA: 13 active/5 inactive); 11 with ankylosing spondylitis (AS: 7 active/4 inactive) and 13 healthy subjects. Activity of the disease was assessed by: RA patients, Disease Activity Score (DAS 28) and AS patients by means of Bath Ankylosing Spondylitis Disease Activity Index (BASDAI). Oxidative stress biomarkers were determined in plasma using spectrophotometrical techniques. The statistical analysis was carried out using the SSPS statistical package. Results Active CIJD showed a high oxidative stress characterized by increases in oxidative damage markers and a reduction in antioxidative systems, together with a higher myeloperoxidase (MPO) concentration. Inactive CIJD only showed changes in oxidized glutathione (GSSG) and reduced glutathione (GSH)/GSSG ratio levels, without changes in oxidative damage parameters or in antioxidative systems. Conclusions Our data revealed that: i) CIJD presents with a high oxidative stress; ii) inactive CIJD shows a production of reactive species without triggering oxidative damage and maintaining red-ox homeostasis, and iii) the combination of oxidative stress biomarkers may be used as markers of active-inactive stages of CIJD.

Rat testicular mitochondrial antioxidant defence system and its modulation by aging

Accumulation of oxidative damage caused by reactive oxygen species (ROS) underlies fundamental changes found during aging. In the present study, age related effect on testicular mitochondrial oxidant generation and antioxidant defence profile was investigated in Wistar rats at 3 months (young adults), 12 months (old adults) and 24 months (senescent animals) of age. Mitochondrial oxidative stress parameters viz., lipid peroxidation (LPx), protein carbonylation, hydrogen peroxide (H2O2) generation and activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR), levels of total, oxidized (GSSG) and reduced glutathione (GSH) were studied to find out their roles in maintenance of mitochondrial glutathione redox pool as a function of age. Increased levels of LPx, H2O2 and decreased GSH content accompanied by a decline in activities of SOD, GPx and GR with advancing age suggest that antioxidant defense profile of testicular mitochondria exhibit age related alterations which might play a critical role in regulating physiological functions of the testis such as steroidogenesis and spermatogenesis.