Complexity of free radical metabolism in human erythrocytes

The auto-oxidation of oxyhaemoglobin to methaemoglobin generating superoxide anion radical (O2 .-) represents the main source of free radicals in the erythrocytes. Hydrogen peroxide is produced by O2 .- dismutation or originates from the circulation. Human erythrocytes are also exposed to the prooxidative actions of nitric oxide (NO) from circulation. Free radicals that may induce reactions with direct dangerous consequences to erythrocytes are also preceded by the reaction of O2 .- and NO producing per oxynitrite. In physiological settings, erythrocytes show a self-sustaining activity of antioxidative defense (AD) enzymes, such as: superoxide dismutase (SOD, EC 1.11.16), catalase (CAT, EC 1.11.1.6), glutathione peroxidase (GSHPx, EC 1.11.1.9) and glutathione reductase (GR, EC 1.6.4.2), as well as low molecular weight antioxidants: glutathione and vitamins E and C. Their coordinate actions protect the erythrocyte's biomacromolecules from free radical-mediated damage. Since there is no de novo synthesis of AD enzymes in mature erythrocytes, their defense capacity is limited. Free radicals influence antioxidative enzymes capacities and relative share of particular components in the whole anti oxidative system. Therefore, by measuring changes in the activity of individual AD components, as well as their inter relations by statistical canonical discriminate methods, valuable data about the complexity, overall relations and coordinated actions in the AD system in erythrocytes and its relevance for systemic effects can be acquired.Produkcija slobodnih radikala u eritrocitima uglavnom se odnosi na nastajanje superoksid anjon radikala (O2 .-) putem autooksidacije oksihemoglobina u methemoglobin. Ljudski eritrociti izloženi su prooksidacionom delovanju vodonik-peroksida nastalog dismutacijom O2 .- ili iz cirkulacije, kao i azot oksidu (NO) iz cirkulacije. Od di rektnih reakcija slobodnih radikala, reakcija O2 .- i NO uz nastajanje peroksinitrita je reakcija sa primarno štetnim posledicama po eritrocite. U eritrocitima se nalaze enzimi zaštite od oksidacionih oštećenja, kao što su superoksid dismutaza (SOD, EC 1.15.1.1), katalaza (CAT, EC 1.11.1.6), glutation peroksidaza (GSHPx, EC 1.11.1.9) i glutation reduktaza (GR, EC 1.6.4.2) kao i komponente male molekulske mase (glutation, vitamini E i C). Njihovim sadejstvom se kanališu reakcije slobodnih radikala tako da direktna oštećenja biomakromolekula budu što manja. Međutim, kako nema de novo sinteze enzima u maturiranim eritrocitima, kapacitet ovih sistema je ograničen, jer slobodnoradikalske vrste i direktno inhibiraju neke od enzima. Promene na enzimima i njihova inhibicija slobodnim radikalima utiču na kapacitet zaštite od oksidacionih oštećenja i relativni udeo pojedinih komponenti u ukupnom antioksidativnom potencijalu. To se može pratiti i preko promena aktivnosti pojedinačnih komponenti, ali i međusobnih odnosa između komponenti antioksidativne odbrane diskriminacionim statističkim metodama, koje ukazuju na sveukupnost i kompleksnost odnosa antioksidativnih komponenti u eritrocitima i njihov sistemski značaj.nul

Effects of hemazin SC 500 (terbuthylazine) on antioxidative enzymes in human erythrocytes in vitro

The aim of this work was to investigate the effect of the commercial formulation hemazin SC 500, an herbicide containing terbuthylazine as the active compound, on the isoenzyme patterns and activities of Cu-Zn superoxide dismutase (SOD1) and catalase (CAT), as well as on the glutathione S-transferase (GST) activity, in human erythrocytes in vitro. The human erythrocytes were treated with hemazin SC 500 over a broad range of terbuthylazine concentrations (37 nmol L-1– –37 mol L-1) for 1 and 3 h at a temperature of 37°C. Native electrophoresis of the control and treated samples revealed two SOD1 and one CAT isoform. Treatment did not affect the SOD1 and CAT isoenzyme profile, but induced a change in their activities. Terbuthylazine at lower concentration induced a significant increase of the total SOD1 activity and decreased the GST activity in samples incubated for 1 and 3 h. On the other hand, the highest increase in the CAT activity was observed for the sample treated for 1 h with a higher concentration of terbuthylazine. Hemazin SC 500 containing terbuthylazine induces changes in the erythrocyte antioxidative system whereby the response of individual enzymatic antioxidants depends on the concentration of the pesticide and the incubation time

Complexity of free radical metabolism in human erythrocytes

The auto-oxidation of oxyhaemoglobin to methaemoglobin generating superoxide anion radical (O2 .-) represents the main source of free radicals in the erythrocytes. Hydrogen peroxide is produced by O2 .- dismutation or originates from the circulation. Human erythrocytes are also exposed to the prooxidative actions of nitric oxide (NO) from circulation. Free radicals that may induce reactions with direct dangerous consequences to erythrocytes are also preceded by the reaction of O2 .- and NO producing per oxynitrite. In physiological settings, erythrocytes show a self-sustaining activity of antioxidative defense (AD) enzymes, such as: superoxide dismutase (SOD, EC 1.11.16), catalase (CAT, EC 1.11.1.6), glutathione peroxidase (GSHPx, EC 1.11.1.9) and glutathione reductase (GR, EC 1.6.4.2), as well as low molecular weight antioxidants: glutathione and vitamins E and C. Their coordinate actions protect the erythrocyte's biomacromolecules from free radical-mediated damage. Since there is no de novo synthesis of AD enzymes in mature erythrocytes, their defense capacity is limited. Free radicals influence antioxidative enzymes capacities and relative share of particular components in the whole anti oxidative system. Therefore, by measuring changes in the activity of individual AD components, as well as their inter relations by statistical canonical discriminate methods, valuable data about the complexity, overall relations and coordinated actions in the AD system in erythrocytes and its relevance for systemic effects can be acquired.Produkcija slobodnih radikala u eritrocitima uglavnom se odnosi na nastajanje superoksid anjon radikala (O2 .-) putem autooksidacije oksihemoglobina u methemoglobin. Ljudski eritrociti izloženi su prooksidacionom delovanju vodonik-peroksida nastalog dismutacijom O2 .- ili iz cirkulacije, kao i azot oksidu (NO) iz cirkulacije. Od di rektnih reakcija slobodnih radikala, reakcija O2 .- i NO uz nastajanje peroksinitrita je reakcija sa primarno štetnim posledicama po eritrocite. U eritrocitima se nalaze enzimi zaštite od oksidacionih oštećenja, kao što su superoksid dismutaza (SOD, EC 1.15.1.1), katalaza (CAT, EC 1.11.1.6), glutation peroksidaza (GSHPx, EC 1.11.1.9) i glutation reduktaza (GR, EC 1.6.4.2) kao i komponente male molekulske mase (glutation, vitamini E i C). Njihovim sadejstvom se kanališu reakcije slobodnih radikala tako da direktna oštećenja biomakromolekula budu što manja. Međutim, kako nema de novo sinteze enzima u maturiranim eritrocitima, kapacitet ovih sistema je ograničen, jer slobodnoradikalske vrste i direktno inhibiraju neke od enzima. Promene na enzimima i njihova inhibicija slobodnim radikalima utiču na kapacitet zaštite od oksidacionih oštećenja i relativni udeo pojedinih komponenti u ukupnom antioksidativnom potencijalu. To se može pratiti i preko promena aktivnosti pojedinačnih komponenti, ali i međusobnih odnosa između komponenti antioksidativne odbrane diskriminacionim statističkim metodama, koje ukazuju na sveukupnost i kompleksnost odnosa antioksidativnih komponenti u eritrocitima i njihov sistemski značaj.nul

Predloženi mehanizam uticaja oligosaharida dobijenih iz pektina na intestinalnu mikrobiotu

We prepared pectin-derived oligosaccharides (apple and citrus) and polygalacturonic acid-derived oligosaccharides, using alkaline hydrolysis by hydrogen peroxide, and analyzed them by Fourier Transform Infrared spectrometry. Furthermore, we analyzed the effects of pectin-derived oligosacharides on hydroxyl radical generating Fenton reaction using electron paramagnetic resonance spin-trapping spectroscopy, and the effects on the growth of Escherichia coli and Staphylococcus aureus in the presence of dietary relevant HO.-generating system (iron + ascorbate).Alkalnom hidrolizom sa vodonik-peroksidom smo dobili oligosaharide iz pektina (jabuka i citrusi) i poligalakturonske oligosaharide, koje smo analizirali infracrvenom spektrometrijom sa Furijeovom transformacijom. Pored toga, elektron paramagnetnom rezonantnom spin-traping spektorskopijom smo analizirali efekat oligosaharida iz pektina na hidroksil-radikal (HO.)-generisanu Fentonovu reakciju i na rast Escherichia coli i Staphylococcus aureus u prisustvu sistema koji generiše HO.- (gvožđe + askorbat)

Comparation of the muscle of the antioxidant defence enzymes in pigs and bulls

Anti-oxidant defence (AD) system prevent oxidative cell damage through the coordinated expression of antioxidant defence enzymes. Main AD enzymes are: Mn SOD –mitochondrial manganese containing superoxide dismutase and CuZn-superoxide dismutase (SOD, EC 1.15.1.1), catalyses the dismutation of superoxide (O2.-) to hydrogen peroxide (H2O2) which is then independently converted to water by catalase (CAT, EC 1.11.1.6) or by selenium-dependent glutathione peroxidase (GSH-Px, EC 1.11.1.9) (Chance at al., 1979). Glutathione reductase (GR, EC 1.6.4.2) catalyses the reduction of oxidised GSH back into GSH, the latter being the co-substrate of GSH-Px (Gul et al., 2000). An imbalance between oxidative stress and the cell’s anti-oxidant defence system may have adverse effects on cell membranes through the indiscriminate oxidation of susceptible molecules such as polyunsaturated fatty acids (PUFAs), the main substrates for lipid peroxidation (Crastes de Paulet, 1987). Some investigators (de Haan et al., 1995; Percy et al., 1990) have suggested that the alteration in the SOD/GSHPx + CAT ratio correlate well with increases in lipid damage. Animals (pigs and bulls) have different lipid metabolism, different plasma lipid profiles and different erythrocyte anti-oxidant defence compositions, but have similar content of cholesterol in meat (Nikolić et al., 2006; Turubatović et al., 2006). Since mitochondrias are both the main source and the main target for ROS in skeletal muscle, the comparative study on specific mitochondrial antioxidative defence systems (e.g. mitochondrial superoxide dismutase) and citosolyc antioxidative defence enzymes in pigs and bulls is of particular interest. Therefore, the aim of the task was the comparative study on specific mitochondrial antioxidative defence systems (mitochondrial SOD) and citosolyc antioxidative defence enzymes (CAT, GSH-Px and GR) in the selected identical groups of beef and pork muscles ( thick flank, loin and neck).Poster: [https://cer.ihtm.bg.ac.rs/handle/123456789/5000

Diethyldithiocarbamate potentiates the effects of protamine sulphate in the isolated rat uterus

Protamine sulphate causes potassium ion channel-mediated relaxation of spontaneous and calcium ion-induced contractions of the isolated rat uterus. Diethyldithiocarbamate (DDC) potentiated the effect of protamine sulphate. A mechanism for DDC's action was postulated on the basis of its interactions with divalent iron ions and Cui Zn-SOD. DDC chelates divalent iron ions creating DDC-iron (Fe-DDC) complexes. Fe-DDC forms stable NO-Fe-DDC2 complexes by NO scavenging and de-nitrosylation processes, which in combination with DDC (5 mM) provoke inhibition of Cui Zn-SOD resulting in specific oxidative conditions culminating in potassium ion channel opening, membrane hyperpolarisation, inhibition of calcium ion influx and subsequent muscle relaxation. As Fe-DDC and NO-Fe-DDC2 complexes exclude divalent iron ions from participating in the hydroxy radical generating Fenton reaction, DDC can also prevent iron-related pathophysiological manifestations. Such permissive roles of DDC open the possibility for application of its pharmacological form (disulfiram) to a wider spectrum of pathophysiological conditions related to smooth muscles

Different roles of radical scavengers - ascorbate and urate in the cerebrospinal fluid of amyotrophic lateral sclerosis patients

Ferrous iron, released from iron deposits in the motor cortex and other brain regions of amyotrophic lateral sclerosis (ALS) patients, participates in the Fenton reaction in cerebrospinal fluid (CSF) alongside H2O2, which is continuously released by neuronal cells. In vivo, the production of notoriously reactive hydroxyl radicals via this reaction could lead to the progression of the disease. Herein, we have examined the effect of ascorbate and uric acid on the production of hydroxyl radicals in CSF from both sporadic ALS patients and control subjects. Electron paramagnetic resonance spectroscopy identified ascorbyl radicals in CSF from ALS patients whereas it was undetectable in control CSF. The addition of H2O2 to the CSF from ALS patients provoked further formation of ascorbyl radicals and the formation of hydroxyl radicals ex vivo. The hydroxyl addition of uric acid to CSF from ALS patients diminished the production of hydroxyl radicals. In conclusion, there are clear differences between the roles of the two examined radical scavengers in the CSF of ALS patients indicating that the use of ascorbate could have unfavourable effects in ALS patients

The effects of human wild-type and FALS mutant L144P SOD1 on non-vascular smooth muscle contractions

Background: Mutated copper, zinc-containing superoxide dismutase (SOD1) may self-aggregate, an event that could also be an initial cause of motor neuron malfunction leading to disease onset. The effects of human mutated SOD1 protein from the blood of familial amyotrophic lateral sclerosis (FALS) patients bearing Leu144Phe (L144F) mutation were compared to wild-type (WT) human SOD1 derived from healthy examinees, for enzymatic activity and the effects on isometric contractions of non-vascular smooth muscle. Methods: We isolated WT and L144F SOD1 enzymes from eight patients with FALS, L144F mutation in exon 5 and eight healthy controls. We then investigated SOD1 activities in the obtained samples by the adrenaline method and profiled them electrophoretically. Finally, we applied WT and L144F SOD1 on the isolated rat uterus. Results: L144F SOD1 showed lower superoxide-dismutating activity compared to WT human SOD1. We found that, in contrast to WT human SOD1, mutated L144F does not induce smooth muscle relaxation. Conclusions: Our data suggest that the lack of relaxation of muscle tonus in the presence of mutated SOD1 may have pathogenic feedback effects in FALS.Uvod: Mutirana bakar, cink superoksid-dizmutaza (SOD1) može da pravi agregate, sto predstavlja početni uzrok oštećenja motornog neurona može da izazove nastanak bolesti. U ovom radu su pokazani efekti humane bakar, cink super-oksid dizmutaze iz krvi pacijenata obolelih od familijarne amiotrofične lateralne skleroze (FALS) sa Leu144Phe (L144F) mutacijom i normalne (wild-type - WT) humane SOD1, iz krvi zdravih kontrola, na glatkom mišiću. Metode: Izolovali smo WT i L144F SOD1 enzime kod osam odabranih FALS pacijenata sa L144F mutacijom na egzonu 5 i pet zdravih kontrola. Dalje smo ispitivali aktivnost SOD1 u dobijenim uzorcima adrenalinskom metodom i elektro-foretski ih profilisali. Konačno, izolovanu WT i L144F SOD1 aplicirali smo na izolovani uterus pacova. Rezultati: Aktivnost L144F SOD1 je statistički značajno manja (p<0,05) u poređenju sa aktivnosti WT SOD1 zdravih kontrola. L144F ne izaziva relaksaciju glatkog mišića, kao sto je to slučaj sa WT SOD1. Zaključak: Naši rezultati pokazuju da izostanak relaksacije mišićnog tonusa u prisustvu mutirane SOD1 može imati štetni povratni efekat kod FALS pacijenata.Projekat ministarstva br. 173014 i br. 17508

The effects of human wild-type and FALS mutant L144P SOD1 on non-vascular smooth muscle contractions

Uvod: Mutirana bakar, cink superoksid-dizmutaza (SOD1) može da pravi agregate, sto predstavlja početni uzrok oštećenja motornog neurona može da izazove nastanak bolesti. U ovom radu su pokazani efekti humane bakar, cink super-oksid dizmutaze iz krvi pacijenata obolelih od familijarne amiotrofične lateralne skleroze (FALS) sa Leu144Phe (L144F) mutacijom i normalne (wild-type - WT) humane SOD1, iz krvi zdravih kontrola, na glatkom mišiću. Metode: Izolovali smo WT i L144F SOD1 enzime kod osam odabranih FALS pacijenata sa L144F mutacijom na egzonu 5 i pet zdravih kontrola. Dalje smo ispitivali aktivnost SOD1 u dobijenim uzorcima adrenalinskom metodom i elektro-foretski ih profilisali. Konačno, izolovanu WT i L144F SOD1 aplicirali smo na izolovani uterus pacova. Rezultati: Aktivnost L144F SOD1 je statistički značajno manja (p lt 0,05) u poređenju sa aktivnosti WT SOD1 zdravih kontrola. L144F ne izaziva relaksaciju glatkog mišića, kao sto je to slučaj sa WT SOD1. Zaključak: Naši rezultati pokazuju da izostanak relaksacije mišićnog tonusa u prisustvu mutirane SOD1 može imati štetni povratni efekat kod FALS pacijenata.Background: Mutated copper, zinc-containing superoxide dismutase (SOD1) may self-aggregate, an event that could also be an initial cause of motor neuron malfunction leading to disease onset. The effects of human mutated SOD1 protein from the blood of familial amyotrophic lateral sclerosis (FALS) patients bearing Leu144Phe (L144F) mutation were compared to wild-type (WT) human SOD1 derived from healthy examinees, for enzymatic activity and the effects on isometric contractions of non-vascular smooth muscle. Methods: We isolated WT and L144F SOD1 enzymes from eight patients with FALS, L144F mutation in exon 5 and eight healthy controls. We then investigated SOD1 activities in the obtained samples by the adrenaline method and profiled them electrophoretically. Finally, we applied WT and L144F SOD1 on the isolated rat uterus. Results: L144F SOD1 showed lower superoxide-dismutating activity compared to WT human SOD1. We found that, in contrast to WT human SOD1, mutated L144F does not induce smooth muscle relaxation. Conclusions: Our data suggest that the lack of relaxation of muscle tonus in the presence of mutated SOD1 may have pathogenic feedback effects in FALS

The effects of wild-type and mutant sod1 on smooth muscle contraction

In this work we compared the mutated liver copper zinc-containing superoxide dismutase (SOD1) protein G93A of the transgenic rat model of familial amyotrophic lateral sclerosis (FALS), to wild-type (WT) rat SOD1. We examined their enzymatic activities and effects on isometric contractions of uteri of healthy virgin rats. G93A SOD1 showed a slightly higher activity than WT SOD1 and, in contrast to WT SOD1, G93A SOD1 did not induce smooth muscle relaxation. This result indicates that effects on smooth muscles are not related to SOD1 enzyme activity and suggest that heterodimers of G93A SOD1 form an ion-conducting pore that diminishes the relaxatory effects of SOD1. We propose that this type of pathogenic feedback affects neurons in FALS