H. S. Ozturk

Impaired antioxidant defense system in the kidney tissues from rabbits treated with cyclosporine. Protective effects of vitamins E and C.

Enzymatic antioxidant defense system and antioxidant defense potential (AOP) were studied in kidney tissue from rabbits treated with cyclosporine (CsA, 25 mg/kg/day), antioxidant vitamins (E, 100 mg/kg/day plus C, 200 mg/kg/day), and CsA plus antioxidant vitamins, and in kidney tissue from control animals. Although no change was observed in superoxide dismutase (SOD) activity, glutathione peroxidase (GSH-Px) and catalase (CAT) activities were found decreased in kidney tissue exposed to CsA for 10 days compared with control tissue. The level of thiobarbituric acid-reagent substances (TBARS) was higher and antioxidant defense potential (AOP) lower in the CsA-treated group compared with the other groups. Histopathological examination reveals important subcellular damage in the renal tissue from the animals treated with CsA. Antioxidant vitamin therapy caused full improvement in the enzyme activities, TBARS levels and AOP, but the subcellular damage was partly ameliorated in the CsA plus vitamin group. Results suggest that CsA impairs the antioxidant defense system and reduces the antioxidant defense potential in the renal tissue. Antioxidant vitamin treatment protects the tissue in part against toxic effects of the drug.

Antioxidant interferences in superoxide dismutase activity methods using superoxide radical as substrate.

Sir, In several methods for the measurement of the superoxide dismutase (SOD; E.C 1.15.1.1) activity, superoxide radical (O2• -) produced from various sources such as the xanthin/xanthine oxidase system, pyrogallol oxidation etc. has been used as the substrate (1–4). In general, SOD activity is measured on the basis of competition between O2• dismutation by this enzyme or by an oxidising substance such as nitroblue tetrazolium salt (NBT), nicotinamide adenine dinucleotide (NAD) etc. Accordingly, when SOD activity is high, reduction rate of the oxidising substance is low and vice versa. In these methods, there is however interference from non-enzymatic antioxidants, which mainly results from cellular components with antioxidant capacity. Some of these substances, such as hemoglobin, transferrin, albumin, ceruloplasmin and haptoglobin, exert their antioxidant activities by binding metal ions which play a part in free radical production. Others, such as uric acid and vitamins A, C and E, mainly act as chainbreaking antioxidants. Due to this fact, it is not possible to carry out a valid measurement of activity without removing these non-enzymatic antioxidant substances from the medium. Although some researchers use chloroform/ethanol extraction to eliminate these substances, this procedure cannot remove all of them; hydrophilic antioxidants remain in the alcohol/water phase. Therefore, in the present methods, total (enzymatic plus non-enzymatic) superoxide radical scavenger activity (TSSA) instead of SOD activity is measured. We have used human erythrocytes and, rabbit liver and lung to assess this interference. First, we measured SOD activites in erythrocyte hemolyzates and in lung and liver by using the method described previously (5, 6). Second, activity measurements were made in trichloroacetic acid (TCA)-treated fractions, which were prepared by treating part of the sample with 20 % (w/v) TCA solution and centrifuging at 5000 xg for 30 min. We observed that TCA-treatment causes no meaningful changes in the pH of the final assay mixture (pH = 10.5 in untreated assay and 10.2 in TCAtreated assay). Activity assays were performed in the supernatant fraction. One unit of SOD activity was defined as the amount of protein causing 50% inhibition in NBT reduction rate. Results are given in Table 1. As shown in the table, a significant amount of SOD activity still exists in the supernatant fluids after TCAtreatment, which have removed all enzyme. There must therefore be other, non-enzymatic, factors which remove O2• from the reaction medium. Therefore, activity values measured by the present methods should be regarded as TSSA rather than enzyme activity and SOD activity should be calculated from the equation below.

Effects of smoking on plasma and erythrocyte antioxidant defense systems.

In this study, activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) enzymes were measured in the erythrocytes, and levels of thiobarbituric acid-reactive substances (TBARS) and antioxidant potential (AOP) values were measured in both erythrocyte and plasma samples from smokerS and nonsmokers. No significant differences were observed in erythrocyte parameters, serum triglycerides, and total cholesterol. AOP was significantly lower and TBARS level higher in the plasma samples from smokers compared with those of nonsmokers. Results suggest that smoking causes no impairment in the enzymatic antioxidant defense system and does not lead to oxidant stress in the erythrocytes, possibly because these cells have potent antioxidant defense capacity.

Oxidative stress in patients with chronic renal failure: effects of hemodialysis.

Objective: To investigate blood oxidative status of patients with chronic renal failure (CRF) and possible effects of hemodialysis on the development of oxidative stress in blood. Materials and Methods: The levels of malondialdehyde (MDA) and oxidation resistance (OR) values were measured in blood plasma, erythrocyte hemolysate and erythrocyte membrane fractions of 33 patients with CRF and of 12 healthy controls. Of the 33 patients, 17 subjects were under hemodialysis treatment. Results: MDA levels were found to be increased in all blood fractions of the patients. OR values were unchanged in erythrocyte hemolysates but decreased in plasma and erythrocyte membrane fractions of the CRF patients. Moreover, erythrocyte MDA levels were determined to be higher in hemodialyzed patients compared with both controls and non-hemodialyzed patients. OR values were lower in all blood fractions of the hemodialyzed patients relative to controls and non-hemodialyzed patients. Conclusion: Results suggest that there is a significant oxidative stress (expressed as peroxidation) in blood samples from patients with CRF, which is further exacerbated by hemodialysis.

Impaired antioxidant defence in guinea pig heart tissues treated with halothane

PurposeTo investigate the effects of halothane and halothane plus vitamin E treatment on myocardial free radical metabolism in guinea pigs.MethodsFour groups of seven animals were studied; control, halothane, halothane plus vitamin E and vitamin E groups. In the halothane group, halothane 1.5% in oxygen was given for 90 min over three days. In the halothane plus vitamin E group, 300 rng · kg−1 · day−1 vitamin Eim was started three days before the first halothane treatment and continued for three days. Following sacrifice, the hearts were assayed for superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) and malondialdehyde (MDA) level was determined. Electron spin resonance (ESR) analysis and electron microscopy (EM) were also performed.ResultsIn the halothane group, SOD activities and MDA concentrations were increased compared with control and GSH-Px and CAT activities were decreased. In the halothane plus vitamin E group, there were no differences in enzyme activity compared with halothane alone but the MDA level was decreased. In the vitamin E group, enzyme activities were increased compared with control. Mainly the CF3CHCl radical was identified by ESR analysis in heart tissues exposed to halothane and the concentration of this radical was reduced by vitamin E. Electron microscopy showed cytoplasmic vacuolisation and dilation in sarcoplasmic reticulum in the heart tissues exposed to halothane: both were prevented by vitamin E.ConclusionAlthough halothane causes impairment in enzymatic antioxidant defence potential, due to lowered GSH-Px and CAT activity, and accelerates peroxidative reactions in the tissues affected, no subcellular damage occurred. Vitamin E may protect tissues against free radical attack by scavenging toxic free radicals formed in heart tissue during halothane anaesthesia.RésuméObjectifÉtudier les effets de l’halothane et de l’association halothane-vitamine E sur la production myocardique de radicaux libres.MéthodesL’étude portait sur quatre groupes de sept animaux: contrôle, halothane, halothane+vitamine E, et vitamine E. Le groupe halothane a reçu de l’halothane 1,5% en oxygène pendant 90 min pour 3 jours. Le groupe halothane+vitamine E a reçu une doseim de 300 mg · kg−1 · j−1 de vitamine E pendant trois jours avant un premier traitement a l’halothane. Une fois l’animal sacrifié, la superoxyde dismutase (SOD), la glutathion peroxydase (GSH-Px) et catalase (CAT), et la malondialdéhyde (MDA) ont été titrées dans le tissu cardiaque. La résonance paramagnétique électronique (RPÉ) et la microscopie électronique ont complété ces analyses.RésultatsDans le groupe halothane, l’activité de la SOD et la concentration de MDA augmentaient comparativement au contrôle et l’activité de la GSH-Px et de la CAT diminuait. Dans le groupe halothane+vitamine E, l’activité enzymatique ne changeait pas comparativement à l’halothane seul mais le niveau de MDA diminuait. Dans le groupe vitamine E, l’activité enzymatique augmentait comparativement au contrôle. Le radical CF3CHCl était principalement identifié par l’analyse RPÉ dans le tissu cardiaque exposé à l’halothane alors que la vitamine E diminuait la concentration de ce radical. La microscopie électronique révélait une vacuolisation et une dilatation cytoplasmiques du réticulum sarcoplasmique du tissu cardiaque exposé à l’halothane; la vitamine E prévenait ces effets.ConclusionMalgré l’altération par l’halothane de la capacité de protection enzymatique contre l’oxydation, due à la baisse de l’activité de la GSH-Px et de la CAT et l’accélération des réactions peroxydatives dans les tissus affectés, il n’y a pas eu de dommages infracellulaires. La vitamine E protège les tissus contre l’agression des radicaux libres en épurant les radicaux toxiques libérés dans le tissu cardiaque pendant l’anesthésie à l’halothane.

The effects of gentamicin and vitamin E on enzymatic antioxidant defence in guinea-pig lung.

Objective: To study the possible effects of gentamicin on the enzymic free‐radical defence system in the lung.

The effects of fasting on blood antioxidant potential and malondialdehyde levels.

Sir, Blood anitoxidant potential (AOP) and malondialdehyde level (MDA) have been measured to obtain more information into oxidant and antioxidant status of the body (1, 2). However, there is no information regarding whether it is necessary to obtain blood samples after overnight fasting or not. This study was performed to elucidate this. We measured AOP and MDA levels in plasma and erythrocytes after 16 hours of fasting in order to establish possible effects of fasting on these parameters. For this purpose, blood samples were obtained from 11 healthy volunteers four times a day. The first samples were taken just before breakfast, the second 1 hour later and others 8 and 16 hours after breakfast. AOP and MDA levels of plasma and erythrocyte samples were measured as previously described, respectively (3, 4). To measure AOP, in reaction medium enriched with fish oil, samples were exposed to superoxide radicals (O2) produced by xanthine/xanthine oxidase system for 1 hour. Then thiobarbituric acid reactive substances (TBARS) levels (nmol/ml) were measured before and after O2 radical attack. The difference between the values is inversely proportional to AOP of the samples (l/nmol/ml · h) (3). The MDA method was based on the absorbance measurement of thiobarbituric acid-malondialdehyde complexone formed by heating in boiling water for 30 min (4). Although this method is not very specific since thiobarbituric acid reacts with some other unconjugated substances as well, it is commonly used as a lipid peroxidation index. Results (mean ± SD values) for AOP and MDA are given in Table 1. As seen from the table, no statistically meaningful differences exist between first and following AOP and MDA values except AOP in the erythrocytes. Erythrocyte AOP increases just after breakfast and then decreases to steady state level. It seems that some antioxidant substances in the food such as antioxidant vitamins (vitamins E and C), flavonoids and flavones etc. make a significant contribution to cellular AOP for a certain period. Possibly, antioxidant substances entering into erythrocytes just after food ingestion are consumed to prevent free radical-mediated peroxidation reactions, which can occur during glycolysis and/or hexosemonophosphate pathway etc. Plasma AOP does not change, possibly due to the fact that antioxidant substances derived from food rapidly enter into the tissues and cells including erythrocytes. The results of the present study suggest that it is necessary to use fasting blood samples for AOP measurement in erythrocytes but fasting is not necessary for the MDA measurement in either plasma or erythrocytes.