Duško Blagojević

Relevance of the capacity of phosphorylated fructose to scavenge the hydroxyl radical

The hydroxyl radical (*OH) has detrimental biological activity due to its very high reactivity. Our experiments were designed to determine the effects of equimolar concentrations of glucose, fructose and mannitol and three phosphorylated forms of fructose (fructose-1-phosphate (F1P); fructose-6-phosphate (F6P); and fructose-1,6-bis(phosphate) (F16BP)) on *OH radical production via the Fenton reaction. EPR spectroscopy using spin-trap DEPMPO was applied to detect radical production. We found that the percentage inhibition of *OH radical formation decreased in the order F16BP>F1P>F6P>fructose>mannitol=glucose. As ketoses can sequester redox-active iron thus preventing the Fenton reaction, the Haber-Weiss-like system was also employed to generate *OH, so that the effect of iron sequestration could be distinguished from direct *OH radical scavenging. In the latter system, the rank order of *OH scavenging activity was F16BP>F1P>F6P>fructose=mannitol=glucose. Our results clearly demonstrate that intracellular phosphorylated forms of fructose have more scavenging properties than fructose or glucose, leading us to conclude that the acute administration of fructose could overcome the bodys reaction to exogenous antioxidants during appropriate therapy in certain pathophysiological conditions related to oxidative stress, such as sepsis, neurodegenerative diseases, atherosclerosis, malignancy, and some complications of pregnancy.

Hydrogen peroxide affects contractile activity and anti‐oxidant enzymes in rat uterus

Background and purpose:  The effects of hydrogen peroxide (H2O2) on uterine smooth muscle are not well studied. We have investigated the effect and the mechanism of action of exogenous hydrogen peroxide on rat uteri contractile activity [spontaneous and calcium ion (Ca2+)‐induced] and the effect of such treatment on anti‐oxidative enzyme activities.

Effects of zinc on the mineralization of bone nodules from human osteoblast-like cells

Zinc is an important mineral that is required for normal bone development. However, the direct effects of zinc on the mineralization of bone cells of human origin are not clear. The objective of this study was to determine the effects of zinc on the differentiation of SaOS-2 human osteoblast-like cells and the formation of mineralized bone nodules. Cells were cultured for 8 d and then transferred to zinc-free medium and treated with varying concentrations (0–50 μM) of zinc. Alkaline phosphatase (ALP) activity was used as a measure of osteoblast differentiation, and bone nodules were detected by von Kossa staining. After 4, 6, and 8 d of treatment, zinc increased ALP activity at 1 and 10 μM, but decreased activity at 50 μM. After 9 d of treatment, zinc increased both the number and area of mineralized bone nodules at low concentrations (1 and 10 μM), but decreased both at higher concentrations (25 and 50 μM). These findings demonstrate that zinc has biphasic effects on the differentiation and mineralization of human osteoblast-like cells.

Alterations in anti-oxidative defence enzymes in erythrocytes from sporadic amyotrophic lateral sclerosis (SALS) and familial ALS patients.

Abstract Background: Overproduction of nitric oxide (NO) and hydrogen peroxide (H2O2) may be an important factor in the pathogenesis of amyotrophic lateral sclerosis (ALS). Owing to their ability to permeate through biological membranes, excess NO and H2O2 may be present in the media surrounding motor neurones. Anti-oxidative defence enzymes (ADEs) in erythrocytes are capable of detoxifying reactive oxygen species (produced endogenously or exogenously), but may also be structurally modified and inactivated by reactive oxygen and nitrogen species. Both balanced and coordinated ADE activities are of utmost importance for their correct physiological function. Methods: We determined activity of the following ADEs: copper-zinc superoxide dismutase (CuZn SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and glutathione reductase (GR) in erythrocytes from sporadic ALS patients [SALS (−/+)], familial ALS patients with the Leu144Phe mutation in the SOD1 gene [FALS (+/+)], asymptomatic carriers with the Leu144Phe mutation in the SOD1 gene (+/−), and control subjects (−/−). We also examined the in vitro effect of diethyldithiocarbamate (DDC) on CuZn SOD activity in erythrocytes from FALS patients, SALS patients and control subjects. Results: The influence of the Leu144Phe mutation and/or disease was apparent for ADE activities measured in all three patient groups. The SOD1 gene mutation decreased CuZn SOD and GSH-Px activity (two-way ANOVA, significant mutation effect). We noted that the disease also contributed to decreased CuZn SOD activity in SALS patients in comparison with the control group (two-way ANOVA, mutation and disease effect). The disease also influenced CAT and GR activity. CAT activity was decreased in both SALS and FALS patients. In all three patient groups, GR activity was higher than in the control group. Finally, DDC inhibited CuZn SOD activity in erythrocytes from control subjects, FALS (Leu144Phe) patients and SALS patients; however, its effect was more pronounced and significant in FALS patients. Conclusions: Changes in erythrocyte ADE activities suggest that oxidative stress, involved in the motor neurone pathogenesis of SALS and FALS, also has systemic effects. Differences in ADE systems between the study groups revealed the presence of different types of oxidative pressure, indicating the potential additional benefit of individually designed anti-oxidant cocktail therapies.

Complexity of free radical Metabolism in human Erythrocytes

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 erythro-cytes. 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 peroxynitrite. In physiological settings, erythrocytes show a self-sustaining activity of antioxidative defence (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 erythrocytes bio-macromolecules from free radical-mediated damage. Since there is no de novo synthesis of AD enzymes in mature erythrocytes, their defence capacity is limited. Free radicals influence antioxidative enzymes capacities and relative share of particular components in the whole antioxidative system. Therefore, by measuring changes in the activity of individual AD components, as well as their interrelations by statistical canonical discriminant 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. Kompleksnost Metabolizma Slobodnih Radikala u humanim Eritrocitima 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 direktnih 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.

Citrus flavanones naringenin and hesperetin improve antioxidant status and membrane lipid compositions in the liver of old-aged Wistar rats

This study aimed to investigate effects of citrus flavanones naringenin (NAR) and hesperetin (HES) on liver antioxidant status and membrane phospholipid composition in 24-month-old rats. NAR and HES (15mg/kg) were administrated orally to male Wistar rats, once per day, for 4weeks. Control group received either vehicle (sunflower oil) or remained intact. The results showed decreased (p<0.05) activity of antioxidant enzymes (AOE), specifically catalase (CAT), superoxide dismutase (SOD) 1 and glutathione reductase (GR) in the liver of intact control old-aged rats in comparison to young intact controls. Flavanone administration to old-aged males increased (p<0.05) examined AOE activities in comparison to vehicle-administered animals. Namely, NAR was more potent in comparison to HES regarding the increase (p<0.05) in activities of examined antioxidant enzymes (SOD 1 and 2, glutathione peroxidase-GPx and GR) and the liver glutathione (GSH), while HES elevated (p<0.05) only activity of CAT and GR. Both flavanones significantly decreased (p<0.05) TBARS and improved (p<0.05) membrane phospholipid composition in favor of n-3 PUFA and n-6/n-3 PUFA ratio. Both flavanones did not affect liver histology and reduced (p<0.05) alanine aminotransferase and aspartate aminotransferase levels in serum. The results of this study indicate beneficial potential of citrus flavanones in the old-aged rat liver.

ACTIVITY OF SUPEROXIDE DISMUTASE AND CATALASE IN THE BEAN WEEVIL (ACANTHOSCELIDES OBTECTUS) SELECTED FOR POSTPONED SENESCENCE

Relationship of superoxide dismutase and catalase activities and aging were tested using bean weevil lines selected for postponed senescence. The beetles of different age (young and old) and mating status (virgin and mated) from the extended longevity lines were compared with their counterparts derived from the short-lived lines for activities of SOD and catalase. The old beetles from the long-lived lines had statistically significant higher activity of SOD than their controls. Although we did not find a significant effect of catalase on longevity, beetles originating from both types of lines exhibited an increased catalase activity during mating processes. In addition, we did observe an increased activity of catalase in one-day-old beetles of the short-lived lines relative to the same-aged individuals of the long-lived lines.

Effect of atypical antipsychotics on antioxidant enzyme activities in human erythrocytes (in vitro study)

This study was set out to examine the impact of atypical antipsychotic drugs: aripiprazole, clozapine, ziprasidone, olanzapine, quetiapine, sertindole and amisulpride on the activity of antioxidant defence enzymes in human erythrocytes in vitro.

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

Abstract 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.

Correlation analysis confirms differences in antioxidant defence in the blood of types I and II schizophrenic male patients treated with anti-psychotic medication

The activities of antioxidant defence enzymes were determined in erythrocytes isolated from types I and II schizophrenic male patients and from healthy controls. Significant differences in superoxide dismutase (SOD) activity (type I: 3284+/-577; type II: 2959+/-697 compared with controls: 3778+/-577; analysis of variance (ANOVA) P<0.001), catalase (CAT) activity (type I: 17.8+/-1.8 compared to type II: 19.2+/-1.5 and both compared with controls: 19.2+/-1.5; ANOVA P<0.05), glutathione peroxidase (GSH-Px) activity (controls: 17.8+/-2.3; type I: 13.9+/-2.9 and type II: 11.6+/-1.9; ANOVA P<0.001) as well as in glutathione reductase (GR) activity (controls: 5,0+/-0.8; type I: 4.3+/-0.9 and type II: 4.5+/-0.8; ANOVA P<0.01) were apparent. Correlation analysis of antioxidant defence enzymes showed significant negative correlation between GSH-Px and CAT activities (P<0.01) in type I patients. In type II patients, GSH-Px activity was significantly positively correlated with GR (P<0.01). Canonical discriminant analysis separated type I and type II patients from controls (and among each other) with a high degree of certainty according to the overall group composition of antioxidant defence enzymes. Our results indicate differences in the composition of antioxidant defence between controls and anti-psychotic treated type I and type II patients with a possible negative feedback influence on the pathological process, which could provide a rationale for applying antioxidants during schizophrenic therapy.