Involvement of mammalian mitochondrial glycerophosphate dehydrogenase (mGPDH, EC 1.1.99.5) in reactive oxygen species (ROS) generation was studied in brown adipose tissue mitochondria by different spectroscopic techniques. Spectrofluorometry using ROS-sensitive probes CM-H2DCFDA and Amplex Red was used to determine the glycerophosphate- or succinate-dependent ROS production in mitochondria supplemented with respiratory chain inhibitors antimycin A and myxothiazol. In case of glycerophosphate oxidation, most of the ROS originated directly from mGPDH and coenzyme Q while complex III was a typical site of ROS production in succinate oxidation. Glycerophosphate-dependent ROS production monitored by KCN-insensitive oxygen consumption was highly activated by one-electron acceptor ferricyanide, whereas succinate-dependent ROS production was unaffected. In addition, superoxide anion radical was detected as a mGPDH-related primary ROS species by fluorescent probe dihydroethidium, as well as by electron paramagnetic resonance (EPR) spectroscopy with DMPO spin trap. Altogether, the data obtained demonstrate pronounced differences in the mechanism of ROS production originating from oxidation of glycerophosphate and succinate indicating that electron transfer from mGPDH to coenzyme Q is highly prone to electron leak and superoxide generation.
- MeSH
- Antimycin A analogs & derivatives pharmacology MeSH
- Cell Respiration MeSH
- Electron Spin Resonance Spectroscopy MeSH
- Ethidium analogs & derivatives chemistry MeSH
- Ferricyanides pharmacology MeSH
- Financing, Organized MeSH
- Glycerolphosphate Dehydrogenase metabolism MeSH
- Glycerophosphates metabolism MeSH
- Adipose Tissue, Brown enzymology drug effects ultrastructure MeSH
- Cricetinae MeSH
- Mitochondria enzymology metabolism drug effects MeSH
- Reactive Oxygen Species analysis metabolism MeSH
- Electron Transport Complex III metabolism MeSH
- Oxygen Consumption MeSH
- Electron Transport MeSH
- Ubiquinone metabolism MeSH
- Animals MeSH
- Check Tag
- Cricetinae MeSH
- Male MeSH
- Animals MeSH
Diagnostics of hypoxic-ischemic encephalopathy (HIE) that is based mainly on clinical and neurosonographic criteria should be added with the research of biochemical parameters of the generation of reactive oxygen species (ROS) and ROS-scavenger enzymes activity in umbilical blood which can be useful. It is revealed, that the level of ROS generation at women from the group of risk (with an anemia and FPI) before giving a birth and in umbilical blood has an identical direction of changes. In chronic hypoxia caused by an anemia of mothers, the increase of catalase activity and decrease of SOD activity are detected both in blood of mother, and umbilical blood. Chronic hypoxia at FPI is accompanied by the oppression of activity both SOD and catalase. According to the condition of mother it is possible to predict the current of early neonatal period at newborn, according to the condition of SOD activity and catalase in umbilical blood, and also it is possible to judge about the intensity of protective systems of the organism on activation of ROS generation that accompanies hypoxia/re-oxygenation and to determine the approaches to the directed antioxidant therapies.
- MeSH
- Blood Chemical Analysis MeSH
- Biomarkers blood MeSH
- Fetal Blood cytology MeSH
- Fetal Hypoxia diagnosis blood physiopathology MeSH
- Pregnancy Complications MeSH
- Humans MeSH
- Malondialdehyde blood MeSH
- Hypoxia-Ischemia, Brain * diagnosis blood physiopathology MeSH
- Infant, Newborn MeSH
- Oxidative Stress MeSH
- Reactive Oxygen Species MeSH
- Risk Factors MeSH
- Pregnancy MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Infant, Newborn MeSH
- Pregnancy MeSH
- Female MeSH
Enhanced production of superoxide radicals by nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase in the brain and/or kidney of salt hypertensive Dahl rats has been proposed to participate in the pathogenesis of this form of experimental hypertension. Most information was obtained in young Dahl salt-sensitive (DS) rats subjected to high salt intake prior to sexual maturation. Therefore, the aim of our study was to investigate whether salt hypertension induced in adult DS rats is also accompanied with a more pronounced oxidative stress in the brain or kidney as compared to Dahl salt-resistant (DR) controls. NADPH oxidase activity as well as the content of thiobarbituric acid-reactive substances (TBARS) and conjugated dienes (oxidative index), which indicate a degree of lipid peroxidation, were evaluated in two brain regions (containing either hypothalamic paraventricular nucleus or rostral ventrolateral medulla) as well as in renal medulla and cortex. High salt intake induced hypertension in DS rats but did not modify blood pressure in DR rats. DS and DR rats did not differ in NADPH oxidase-dependent production of ROS, TBARS content or oxidative index in either part of the brain. In addition, high-salt diet did not change significantly any of these brain parameters. In contrast, the enhanced NADPH oxidase-mediated ROS production (without significant signs of increased lipid peroxidation) was detected in the renal medulla of salt hypertensive DS rats. Our findings suggest that there are no signs of enhanced oxidative stress in the brain of adult Dahl rats with salt hypertension induced in adulthood.
- MeSH
- Hypertension chemically induced metabolism MeSH
- Blood Pressure drug effects MeSH
- Rats MeSH
- Sodium Chloride, Dietary * MeSH
- Kidney drug effects metabolism MeSH
- Brain metabolism MeSH
- Organ Specificity drug effects MeSH
- Oxidative Stress drug effects MeSH
- Rats, Inbred Dahl MeSH
- Reactive Oxygen Species metabolism MeSH
- Tissue Distribution MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Male MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Cíl: Cílem práce bylo zjistit výskyt jednotlivých kardiovaskulárních projevů systémového lupus erythematodes (SLE), popsat jejich typ, stupeň závažnosti, zjistit vztah k jednotlivým charakteristickám SLE a posoudit možnosti časné detkce těchto komplikací. Metodika: Bylo vyšetřeno 29 nemocných se SLE a 15 zdravých kontrol. Pacienti absolvovali podrobné echokardiografické vyšetření včetně pozátěžového po absolvovaném testu šestiminutové chůze a 24hodinové monitorování dle Holtera. Byla hodnocena základní demografická data, typ imunosupresivní léčby a délka její aplikace, dávka kortikosteroidů, kterou nemocní užívali, přítomnost autoprotilátek (anti ds DNA, anti Ro, La, Sm, ACLA), hemokoagulační parametry, spektrum lipidů, přítomnost orgánových projevů, plicní funkce a byla měřena aktivita nemoci dle skóre SLEDAI. Byl zjišťován vztah mezi jednotlivými nalezenými kardiologickými patologiemi a parametry SLE. Výsledky: Výskyt kardiovaskulárních projevů v našem souboru byl celkově relativně nízký. Byl zjištěn rozdíl při vyšetření v klidové a pozátěžové echokardiografii, po zátěži testem šestiminutové chůze došlo k odkrytí některých srdečních patologií. Sledované echokardiografické parametry nevykázaly při vyšetření v klidovém stavu žádné významné nálezy. Zvýšené hodnoty indexu Tei významně odlišovaly kontrolní skupinu od nemocných se SLE a na hranici statistické významnosti byly jeho rozdíly u nemocných s nízkou a vyšší aktivitou SLE. Klidové echokardiografické hodnoty trikuspidálního Pg max a TAPSE u nemocných se SLE nebyly odlišné od skupiny zdravých kontrol ani mezi pacienty s odlišnou aktivitou SLE. Biochemická a hemokoagulační vyšetření prokázala statisticky významné rozdíly mezi zdravými kontrolami a SLE pouze ve výši D-dimérů, jejichž hodnota je významně zvýšena u nemocných se SLE a narůstá s aktivitou. Hodnota DLCO naopak významně klesala v závislosti na nárůstu aktivity SLE. Význam hodnocení poměru indexu FVC/DLCO pro diagnostiku plicní hypertenze jsme nepotvrdili. Významně snížené hodnoty DLCO byly spojeny s nárůstem Pg max v zátěžovém testu. DLCO mělo větší výtěžnost než poměr FVC/DLCO. Přínosná pro diagnostiku časných forem plicní hypertenze byla pozátěžová echokardiografie hlavně v hodnocení parametru trikuspidálního Pg max, který po zátěží vzrůstal až k hodnotám na hranici prokazatelné plicní hypertenze. Pozátěžový nárůst hodnot indexu Tei nekoreloval se vzestupem hodnot Pg max u nemocných s rizikem plicní hypertenze. Perikardiální výpotek byl diagnostikován u 17,3 % nemocných. Závěr: Pozátěžová echokardiografie prokázala přínos při detekci možné počínající plicní hypertenze. Nemocní se SLE s vyšším rizikem vzniku kardiovaskulárních komplikací by měli být pravidelně echokardiograficky sledováni a možností zpřesnění detekce srdečních patologií je pozátěžová echokardiografie. Dalším dostupným a přínosným vyšetřením je posouzení změn DLCO.
Objective: the aim of the study was to determine particular cardiovascular manifestations in systemic lupus erythematosus (SLE), to describe their type and severity, find out a relationship with particular SLE characteristics and suggest possibilities for early detection of those complications. Methods: Twenty-nine patients with SLE and 15 healthy controls were investigated. Patients had extensive echocardiographic evaluation including exercise stress testing evaluation after 6 minutes of walk as well as Holter monitoring. Basic demographic data, type of immunosuppressive treatment and time-course of its administration, the dose of glucocorticoids, presence of autoantibodies (anti-dsDNA, anti-Ro, La, Sm, aCL), haemostatic parameters, lipid spectrum, presence of organ manifestations, pulmonary functions as well as activity of the disease according the SLEDAI score were evaluated. The association between particular cardiovascular pathologies and SLE parameters was studied. Results: Presence of cardiovascular manifestations in our group was relatively small. The difference between standard and exercise stress testing echocardiography was found. After the 6 minute-walk test, several heart pathologies appeared. No significant changes were found during echocardiographic examination. Higher levels of Tei index significantly differentiated between control individuals and patients with SLE. Moderate difference, however not statistically significant, was also observed between SLE patients with high and low disease activity. PG max on tricuspidal valve according to echocardiography and systolic excursion (TAPSE) were not different from those in healthy controls. Those values did not differ also between patients with different disease activity of SLE. Biochemistry and haemostatic parameters revealed statistically significant differences between healthy controls and SLE patients for D-dimers that are significantly increased in SLE patients and correlate with the disease activity. The value of DLCO, on the contrary, decreases with the disease activity of SLE patients. The significance of evaluating the ratio of FVC/DLCO index for the diagnosis of pulmonary hypertension was not confirmed. Significantly lower values of DLCO were associated with increased PG max in the exercise stress testing. DLCO was of greater importance than the FVC/DLCO index. Exercise stress testing echocardiography was beneficial for the diagnosis of early forms of pulmonary hypertension particularly in the evaluation of PG max on tricuspidal valve that increased after the exercise up-to the borderline levels of detectable pulmonary hypertension. Increase of Tei after the exercise stress testing did not correlate with the increase of PG max in patients with the risk of pulmonary hypertension. Pericardial effusion was diagnosed in 17.3 % of patients. Conclusion: Exercise stress testing echocardiography demonstrated benefit for the detection of potential onset of pulmonary hypertension. Patients with SLE and higher risk of cardiovascular manifestation should be regularly monitored by echocardiography. More specific examination for cardiovascular pathologies is exercise stress testing echocardiography. Another reasonable and beneficial examination is DLCO.
Polycyclic aromatic hydrocarbons (PAHs) associated with particulate matter (PM) may induce oxidative damage via reactive oxygen species (ROS) generation. However, the kinetics of ROS production and the link with antioxidant response induction has not been well studied. To elucidate the differences in oxidative potential of individual PAH compounds and extractable organic matter (EOM) from PM containing various PAH mixtures, we studied ROS formation and antioxidant response [total antioxidant capacity (TAC) and expression of HMOX1 and TXNRD1] in human alveolar basal epithelial cells (A549 cells) and human embryonic lung fibroblasts (HEL12469 cells). We treated the cells with three concentrations of model PAHs (benzo[a]pyrene, B[a]P; 3-nitrobenzanthrone, 3-NBA) and EOM from PM <2.5 μm (PM2.5). ROS levels were evaluated at 8 time intervals (30 min-24 h). In both cell lines, B[a]P treatment was associated with a time-dependent decrease of ROS levels. This trend was more pronounced in HEL12469 cells and was accompanied by increased TAC. A similar response was observed upon 3-NBA treatment in HEL12469 cells. In A549 cells, however, this compound significantly increased superoxide levels. This response was accompanied by the decrease of TAC as well as HMOX1 and TXNRD1 expression. In both cell lines, a short-time exposure to EOMs tended to increase ROS levels, while a marked decrease was observed after longer treatment periods. This was accompanied by the induction of HMOX1 and TXNRD1 expression in HEL12469 cells and increased TAC in A549 cells. In summary, our data indicate that in the studied cell lines B[a]P and EOMs caused a time-dependent decrease of intracellular ROS levels, probably due to the activation of the antioxidant response. This response was not detected in A549 cells following 3-NBA treatment, which acted as a strong superoxide inducer. Pro-oxidant properties of EOMs are limited to short-time exposure periods.
- MeSH
- A549 Cells MeSH
- Fibroblasts drug effects metabolism pathology MeSH
- Kinetics MeSH
- Cells, Cultured MeSH
- Humans MeSH
- Organic Chemicals adverse effects MeSH
- Particulate Matter adverse effects MeSH
- Lung drug effects metabolism pathology MeSH
- Polycyclic Aromatic Hydrocarbons adverse effects MeSH
- Reactive Oxygen Species metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Jedním z problémů při kloubních náhradách jsou komplikace spojené s pooperačními infekcemi. Infekce způsobené bakteri-emi, které tvoří biofilmy na površích, se nazývají infekce související s biofilmem (biofilm related infections, BRI). V důsledku následné biologické odezvy organismu dochází k silným patofyziologickým změnám vmikroklimatu kolem takto postiženého povrchu (pokles pH, tvorba různých reaktivních forem kyslíku (ROS), vyčerpání iontů železa a zvýšení koncentrace vápenatých iontů). Vytvořili jsme robustní selektivní polymerní potenciometrický senzor ROS a pH senzor pro detekci změn způsobených sterilním zánětem a bakteriálními a plísňovými infekcemi. Senzor ROS se skládá z vodivé polymerní vrstvy na bázi polythiofenu se zabudovaným komplexem porfyrin-kov, který potenciometricky deteguje přítomnost ROS, jak bylo demonstrováno na peroxi-du vodíku. Tento senzor je kovalentně potažen vrstvou odolnou biopasivaci (non-biofouling layer, NBL) tvořenou poly(2-methyl--2-oxazolin)em, která funguje jako biokompatibilizátor.Bylo prokázáno, že potenciometrický senzor vykazuje rychlou odezvu naperoxid vodíku, nezaznamenává interferenci s hovězím sérovým albuminem jako modelovým sérovým proteinem a je schopenplně reverzibilně detegovat ROS s lineární odezvou v širokém rozsahu biologicky relevantních koncentrací (od 0,05μM do10μM). Polymerní pH senzor na bázi polyanilinu a poly(2-methyl-2-oxazolinu) na nosiči z titanové slitiny byl vyvinut pro po-tenciometrickou detekci změn pHv okolí implantátu, aby bylo možné včas detegovat výše uvedené záněty. Vyvinuté elektrody měří změnu pH v rozsahu pH5 až 8, tedy vrozmezí relevantním pro jednotlivé infekce baktériemi a kvasinkami.
One of the problems occurring after the joint replace-ment is connected with the post-surgery infections which are caused by bacteria that form biofilms on surfaces and are referred to as biofilm-related infections (BRI). It is also worth noting that due to the bioresponse, strong path-ophysiologicalchangesinthemicroclimateofanaffectedsurface occur (decrease in pH, formation of various reac-tive oxygen species (ROS), depletion of Fe ions, and in-crease in the concentration of Ca ions). In this work we have prepared a robust selective potentiometric sensorof ROS and pH sensor for the detection of pH changes caused by sterile inflammation and bacterial and fungal infections. The ROS sensor consists of a conductive poly-mer layer based on polythiophene with an incorporated porphyrin-metal complex that potentiometrically detects the presence of ROS (H2O2and ClO–ions). This sensor is connected by the covalent bonds with a non-biofouling layer of poly(2-methyl-2-oxazoline), which works as abio-compa-tibilizer. It was shown that the potentiometric sen-sor shows a rapid response to hydrogen peroxide, does not record any interference with bovine serum albumin as amodel serum protein, and is able to fully reversibly de-tect ROS with a linear response within a wide range of biological relevant concentrations (from 0.05 μM to 10μM). The sensing electrode based on polyaniline and poly(2-methyl-2-oxazoline) on a titanium alloy support was developed for the potentiometric detection of peri-implant pH changes to enable early detection of the afore-mentioned pathologies. The developed electrodes show the changing of pH in the range between 5 and 8 for the individual pathogenic bacteria or pathogenic yeast, with aNernstian slope of −59.6/pH.
- Keywords
- potenciometrický senzor,
- MeSH
- Biofilms MeSH
- Joints surgery microbiology MeSH
- Hydrogen-Ion Concentration MeSH
- Humans MeSH
- Potentiometry * methods instrumentation MeSH
- Joint Prosthesis microbiology MeSH
- Reactive Oxygen Species analysis MeSH
- Research MeSH
- Inflammation diagnosis etiology prevention & control MeSH
- Check Tag
- Humans MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
OBJECTIVES: Glutathione belongs to the family of small-molecular weight antioxidants like ascorbic acid, cysteine, α-tocopherol, uric acid, etc. These molecules play important role in the neutralization of free radicals and reactive oxygen species (ROS). Oxidative stress may lead to ageing and the development of large scale of pathological states of organism. This low molecular weight antioxidant´s level can alter under pathological conditions from reduced (GSH, thiols) to oxidized (oxidized glutathione -GSSG, disulfides) form. A GSSG-to-GSH ratio is indicative marker of oxidative stress. There is a large scale of methods how to determine this biomarker. The trend of the analysis is to minimalize the instrument equipment, sample application volume and analysis cost. DESIGN: Reduced glutathione (GSH) solutions were prepared in water in the concentration 0-16 mmol/L. Other small-molecular weight antioxidants like 0.25 mmol/L ascorbic acid, 0.15 mmol/L TROLOX and 0.02 mmol/L N-acetyl-cysteine (NAcCys) were studied as possible interferents. The samples were mixed with 5,5´-dithiobis-(2-nitrobenzoic) acid (DTNB) resulting in yellow colored drops forming. Coloration was assayed using camera integrated in a smartphone and color channels analysis. The total volume of 10 µl of sample was applied for one analysis. The smartphone-based data were compared with the reference Ellman assay. RESULTS: The calibration of glutathione was evaluated. The blue channel intensity data were decreasing according to the increasing glutathione concentration. Red and green channel intensities were stagnating during the whole concentration scale of glutathione. Limits of detection were 0.4 mmol/l for glutathione. Addition of 0.25 mmol/L of ascorbic acid, 0.15 mmol/L of TROLOX and 0.02mmol/L of N-acetylcysteine to GSH in final concentration 0-16 mmol/L had minimal influence on the assay. The results from smartphone-based analysis correlate with the standard Ellman method. The detection limit for GSH was 0.03 mmol/L. CONCLUSION: The smartphone-based assay seems to be promising because of simplicity, reliability, robustness and low cost. In spite of the fact that there is a large scale for approaches for the glutathione determination, the main advantage of our colorimetric method is portability and easibility to perform the assay in the field and publically availability of smartphones for home applications.
- MeSH
- Antioxidants analysis MeSH
- Biomarkers MeSH
- Biological Assay methods MeSH
- Smartphone utilization MeSH
- Glutathione analysis MeSH
- Colorimetry MeSH
- Dithionitrobenzoic Acid MeSH
- Humans MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
