A novel analytical method to evaluate directly catalase activity of microorganisms and mammalian cells by ESR oximetry

Abstract

Electron spin resonance (ESR) oximetry technique was applied for analysis of catalase activity in the present study. Catalase activity was evaluated by measuring oxygen from the reaction between hydrogen peroxide (H₂O₂) and catalase-positive cells. It was demonstrated that the ESR spectra of spin-label probes, 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPOL), 4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxy (4-oxo-TEMPO) and 4-maleimido-2,2,6,6-tetramethyl-1-piperidinyloxy (4-maleimido-TEMPO) in the presence of H₂O₂ were broadened with the concentrations of catalase. It was possible to make a calibration curve for catalase activity by peak widths of the spectra of each spin-label probe, which are broadened dependently on catalase concentrations. The broadened ESR spectra were also observed when the catalase-positive micro-organisms or the mammalian cells originally from circulating monocytes/macrophages were mixed with TEMPOL and H₂O₂. Meanwhile, catalase-negative micro-organisms caused no broadening change of ESR spectra. The present study indicates that it is possible to evaluate directly the catalase activity of various micro-organisms and mammalian cells by using an ESR oximetry technique.     

A possible mechanism for the action of some myxoviruses

Summary The redox properties of some myxoviruses [Fowl plaque virus strain Rostock (FPV), New Castle Disease virus strain Italy (NDV), B/Hong Kong, A/Port Chalmers, A/Victoria, A/Scotland, and A/Fort Dix (FD)] have been investigated by means of electron spin resonance (ESR) and electron microscopic studies as well as by the determination of the hemagglutination (HA) titer (antigen efficiency). The results have shown that viruses decrease the spin concentration of Cu²⁺ by acting as a reducing species (electron donor) which will result in the inactivation (oxidation) of the virus. Addition of an oxidizing substance, such as H₂O₂, to a virus suspension also leads to an oxidation of the viruses and, thus, to their inability to reduce Cu²⁺. This result is confirmed by the decrease of the HA titer of viruses with increasing Cu²⁺ concentrations. H₂O₂ could not be applied for the HA titer test since it interacts with the erythrocytes of the chicken blood used for this determination. Therefore, another oxidizing substance (oxidized glutathione, GSS) was selected which exhibited a slightly less pronounced effect than Cu²⁺. Since reduced glutathione (GSH) exerts a similar but less pronounced effect than GSS, it might be concluded that viruses have a redox system of their own and act as reducing or oxidizing substance depending on the biological receptor system. Electron microscopic studies confirm this hypothesis. As can be seen by the electron micrographs, increasing concentrations of either Cu²⁺, GSS, H₂O₂, KMnO₄, or GSH will, finally, result in a complete destruction of the virus. Because of structural similarities it might be assumed that other types of viruses behave very similarly.     

Lipid peroxidation induced by phenylbutazone radicals

Lipid peroxidation was investigated to evaluate the deleterious effect on tissues by phenylbutazone (PB). PB induced lipid peroxidation of microsomes in the presence of horseradish peroxidase and hydrogen peroxide (HRP-H2O2). The lipid peroxidation was completely inhibited by catalase but not by superoxide dismutase. Mannitol and dimethylsulfoxide had no effect. These results indicated no paticipation of superoxide and hydroxyl radical in the lipid peroxidation. Reduced glutathione (GSH) efficiently inhibited the lipid peroxidation. PB radicals emitted electron spin resonance (ESR) signals during the reaction of PB with HRP-H2O2. Microsomes and arachidonic acid strongly diminished the ESR signals, indicating that PB radicals directly react with unsaturated lipids of microsomes to cause thiobarbituric acid reactive substances. GSH sharply diminished the ESR signals of PB radicals, suggesting that GSH scavenges PB radicals to inhibit lipid peroxidation. Also, 2-methyl-2-nitrosopropan strongly inhibited lipid peroxidation. R-Phycoerythrin, a peroxyl radical detector substance, was decomposed by PB with HRP-H2O2. These results suggest that lipid peroxidation of microsomes is induced by PB radicals or peroxyl radicals, or both.     

Enzyme defense against reactive oxygen derivatives. II. Erythrocytes and tumor cells

Summary The enzymatic destruction of oxidizing products produced during metabolic reduction of oxygen in the cell (such as singlet oxygen, H₂O₂ and OH radical) involves the concerted action of superoxide dismutase-which removes O ₂ ^- and yields H₂O₂-and H₂O₂ removing enzymes such as catalase and glutathione peroxidase. A difference in distribution or ratio of these enzymes in various tissues may result in a different reactivity of oxygen radicals.It was found that in red blood cells superoxide dismutase and catalase are extracted in the same fraction as hemoglobin, while glutathione peroxidase appears to be loosely bound to the cellular structure. This suggests that in red blood cells catalase acts in series with superoxide dismutase against bursts of oxygen radicals formed from oxyhemoglobin, while glutathione & peroxidase may protect the cell membrane against low concentrations of H₂O₂. On the other hand, catalase activity is absent in various types of ascites tumor cells, while glutathione peroxidase and superoxide dismutase are found in the cytoplasm. However, the peroxidase/dismutase ratio is lower than in liver cells, and this may provide an explanation for the higher susceptibility of tumor cells to treatments likely to involve oxygen radicals.     

Developmental patterns in the antioxidant defenses of the housefly, Musca domestica

The activities of superoxide dismutase (SOD), catalase, glutathione peroxidase, glutathione S-transferases, GSSG reductase, thiol transferases, gamma glutamylcysteine synthetase, and glucose-6-phosphate dehydrogenase, and the concentrations of H2O2 and reduced and oxidized glutathione were determined in the various developmental stages of houseflies. Housefly development was correlated with a progressive increase of cellular oxidizing equivalents and a loss of cellular reducing capacity. The loss of reducing equivalents appeared to result from a decrease in the activity of enzymes involved in glutathione and NADPH synthesis and a concomitant increase in glutathione-oxidizing enzymes. Relatively little change was observed in SOD activity during housefly development; however, the electrophoretic pattern of MnSOD varied in a manner specific to developmental stage. A striking increase in H2O2 concentration occurred prior to pupation possibly due to changes in substrate catabolism. These results support the hypothesis that the cellular environment becomes progressively more oxidizing during development.     

In vitro interaction of mutagenic chromium (VI) with red blood cells

The interaction of mutagenic Cr(VI) with red blood cells has been studied by ESR spectroscopy. Signals of two Cr(V) species are observed almost immediately after contacting red cells with chromate(VI) aqueous solution at pH 7.4. The signal at go = 1.985, which decays within one hour, is attributed to a Cr(V) complex formed by glutathione due its reducing and chelating ability. The other signal at go = 1.979, which is distinctly more persistent, may indicate that some immobilization of the formed Cr(V) ions takes place on the macromolecular cell components, e.g. glycoproteins.     

Short-term gemfibrozil treatment reverses lipid profile and peroxidation but does not alter blood glucose and tissue antioxidant enzymes in chronically diabetic rats

In this study, we investigated the efficiency of short-term treatment with gemfibrozil in the reversal of diabetes-induced changes on carbohydrate and lipid metabolism, and antioxidant status of aorta. Diabetes was induced by a single injection of streptozotocin (45 mg/kg, i.p.). After 12 weeks of induction of diabetes, the control and diabetic rats were orally gavaged daily with a dosing vehicle alone or with 100 mg/kg of gemfibrozil for 2 weeks. At 14 weeks, there was a significant increase in blood glucose, plasma cholesterol and triglyceride levels of untreated-diabetic animals. Diabetes was associated with a significant increase in thiobarbituric acid reactive substances (TBARS) in both plasma and aortic homogenates, indicating increased lipid peroxidation. Diabetes caused an increase in vascular antioxidant enzyme activity, catalase, indicating existence of excess hydrogen peroxide (H₂O₂). However, superoxide dismutase (SOD) and glutathione peroxidase (GSHPx) activities in aortas did not significantly change in untreated-diabetic rats. In diabetic plus gemfibrozil group both plasma lipids and lipid peroxides showed a significant recovery. Gemfibrozil treatment had no effect on blood glucose, plasma insulin and vessel antioxidant enzyme activity of diabetic animals. Our findings suggest that the beneficial effect of short-term gemfibrozil treatment in reducing lipid peroxidation in diabetic animals does not depend on a change of glucose metabolism and antioxidant status of aorta, but this may be attributed to its decreasing effect on circulating lipids. The ability of short-term gemfibrozil treatment to recovery of metabolism and peroxidation of lipids may be an effective strategy to minimize increased oxidative stress in diabetic plasma and vasculature.     

Study on the antioxidant status of vitiligo patients of different age groups in Baroda

One of the major hypotheses in the pathogenesis of vitiligo is the oxidative stress hypothesis. Pollution plays a major role in the production of free radicals. Gujarat, a highly industrialized state in India has a high prevalence of vitiligo patients. No previous studies were done on the age-dependent antioxidant status of vitiligo patients in Baroda city, Gujarat. Blood samples were collected from vitiligo patients of different age groups (5-15, 16-25, 26-35, 36-45 yr) and from age matched healthy volunteers. Antioxidant enzymes in blood such as catalase, superoxide dismutase, glutathione peroxidase and non-enzymatic antioxidants such as reduced glutathione and plasma vitamin E were estimated. Lipid peroxidation levels in erythrocytes and the reducing equivalent system, i.e. glucose-6-phosphate dehydrogenase were also measured. Significant increase in superoxide dismutase activity and lipid peroxidation levels in erythrocytes was observed in all age groups of vitiligo patients as compared with age-matched healthy controls, wherein an increase of 55% (P < 0.02) was observed in superoxide dismutase activity and lipid peroxidation levels in 36-45 yr age group. Whole blood glutathione levels, erythrocyte glutathione peroxidase and glucose-6-phosphate dehydrogenase activity were decreased significantly, whereas erythrocyte catalase activity and plasma vitamin E levels were not different in vitiligo patients as compared with age-matched healthy controls. No specific age group showed a significant difference. This is the first report on the age-dependent antioxidant status of vitiligo patients in Baroda. The disease affects individuals of any age group as shown in this study and systemic oxidative stress might precipitate the pathogenesis of vitiligo in susceptible patients.     

Computer modeling of ESR spectra of the plasma in patients with Down's syndrome

ESR technique at 77 degrees K was used for studying the blood plasma ESR signals of patients with Down syndrome and of healthy people. It was observed that the first exhibited a tendency towards a decrease of ESR signal with g = 4.3 and increase of the ratio of ceruplasmin (g = 2.05) and transferrin (g = 4.3) ESR signal amplitude. A computer simulation has been carried out by means of special mathematical program of experimental ESR spectra of the blood plasma.     

Protective effect of green tea against lipid peroxidation in the rat liver, blood serum and the brain

This paper reports data on the effect of green tea on the lipid peroxidation products formation and parameters of antioxidative system of the liver, blood serum and central nervous tissue of healthy young rats drinking green tea for five weeks. The rats were permitted free access to solubilized extract of green tea. Bioactive ingredients of green tea extract caused in the liver an increase in the activity of glutathione peroxidase and glutathione reductase and in the content of reduced glutathione as well as marked decrease in lipid hydroperoxides (LOOH), 4-hydroksynonenal (4-HNE) and malondialdehyde (MDA). The concentration of vitamin A increased by about 40%. Minor changes in the measured parameters were observed in the blood serum. GSH content increased slightly, whereas the index of the total antioxidant status increased significantly. In contrast, the lipid peroxidation products, particularly MDA was significantly diminished. In the central nervous tissue the activity of superoxide dismutase and glutathione peroxidase decreased while the activity od glutathione reductase and catalase increased after drinking green tea. Moreover the level of LOOH, 4-HNE and MDA significantly decreased. The use of green tea extract appeared to be beneficial to rats in reducing lipid peroxidation products. These results support and substantiate traditional consumption of green tea as protection against lipid peroxidation in the liver, blood serum, and central nervous tissue.     

The tryptic digestion of spin-labelled heavy meromyosin

The S₁ thiol groups of heavy meromyosin (HMM) have been selectively spin labeled with a paramagnetic analog of iodoacetamide (10) and the effects of tryptic digestion on the ESR spectrum and ATPase activity have been studied. The loss of ATPase activity on tryptic digestion occurs at the same rate with spin-labeled or unlabeled HMM suggesting that spin labeling produces no major change in the conformation of HMM. ESR spectra indicate that spin labels bound to S₁ groups of HMM are strongly immobilized; spectra of subfragment-1 isolated from tryptic digests of spin-labeled HMM are the same as those of labeled HMM. ESR spectra of S₁-spin-labeled peptides produced by tryptic digestion of HMM indicate essentially no immobilization of labels, the spectra being similar to that of a solution of free labels. The ESR spectrum of an unfractionated digest of HMM exhibits a peak attributable to strongly immobilized labels on HMM and subfragment-1 and a peak attributable to weakly immobilized labels bound to peptides. The rate at which spin-labeled peptides are released on tryptic digestion can be measured on the unfractionated digest by the decrease in the ESR peak corresponding to HMM and subfragment-1. The appearance of peptides containing spin-labeled S₁ groups parallels the loss of ATPase activity. No evidence has been found for the existence of an enzymatically active subfragment-1 lacking S₁ thiol groups.     

Effect of some reducing agents on water stress-induced oxidative and deteriorative processes ofVigna seedlings

Decreasing substrate osmotic potential produced in seedlings ofVigna catjang Endl. (cv. Pusa Barsati) proportional decrease in relative water content and leaf water potential, increase in respiration rate, proline content, H₂O₂ content, and the activities of indole acetic acid oxidase, ascorbic acid oxidase, peroxidase and glycolate oxidase but decrease in catalase activity and glycolate content. Pretreatment with reducing agents like L-cysteine or reduced glutathione (10?3 M) caused lower decrease in the relative water content, leaf water potential and glycolate content and reduced the rise of respiration rate, proline content and H₂O₂ content and also the activities of aforementioned oxidative enzymes, except catalase activity which was increased. Such treatments also maintained the chlorophyll and protein levels and decreased the tissue permeability. It was concluded that the treatment ofVigna seedlings with reducing agents reduced the deteriorative changes and oxidative processes which are characteristic of water stressed tissue.     

Capacity of ascorbyl palmitate to produce the ascorbyl radical in vitro: an electron spin resonance investigation

This study aims to compare the electron spin resonance (ESR) spectra emitted by human blood loaded with either ascorbyl-6-palmitate (AP), a lipid-soluble derivative of ascorbic acid (AA), or with AA. Whole blood of a healthy male individual was equilibrated with equimolar concentrations of AP and AA of 200, 400, and 800 micromol/l. The intensity of the ESR signal, expressed as the peak-to-peak amplitude, reflects the amount of unpaired spins that are created due to the reducing action of AA and is proportional, in relative terms, to the amount of the ascorbyl radical formed. We found that the blood with AP emitted an ESR signal whose singlet shape, width, and location precisely correlate with the known characteristics of the ascorbyl radical in vitro. The signal magnitude increased linearly with increasing concentrations of AP and was similar to that of AA. We conclude that AP is biologically active, as it generates the ascorbyl radical, an action that also underlies the scavenging process by ascorbic acid. To this end, ascorbyl-6-palmitate might have potential advantages, due to its ability to penetrate biomembranes and to act at the lipid-related molecular target sites.     

Partial characterization of a glutathione oxidase present in rat kidney plasma membrane fraction.

The previously reported glutathione oxidizing activity of isolated renal cells was recovered in the plasma membrane fraction of a rat kidney homogenate. Glutathione disulfide and hydrogen peroxide were formed during the reaction which was dependent on the access to molecular oxygen and inhibited by KCN and EDTA, but not by NaN₃. The EDTA-inhibited activity could be restored by addition of CuSO₄, but not FeCl₃, to the plasma membrane fraction after dialysis. The results strongly suggest that a Cu-protein present in the plasma membrane is responsible for the glutathione oxidase activity of renal cells.     

Effects of supplementation with acai (Euterpe oleracea Mart.) berry-based juice blend on the blood antioxidant defence capacity and lipid profile in junior hurdlers. A pilot study

The purpose of this pilot study was to examine whether regular consumption of an acai berry-based juice blend would affect sprint performance and improve blood antioxidant status and lipid profile in junior athletes. Seven junior hurdlers (17.5±1.2 years) taking part in a pre-season conditioning camp were supplemented once a day, for six weeks, with 100 ml of the juice blend. At the start and the end of the camp the athletes performed a 300-m sprint running test on an outdoor track. Blood samples were taken before and immediately after the test and after 1 h of recovery. Blood antioxidant status was evaluated based on activities of antioxidant enzymes (superoxide dismutase [SOD], catalase [CAT], glutathione peroxidase [GSH-Px], glutathione reductase [GR]), concentrations of non-enzymatic antioxidants (reduced glutathione [GSH], uric acid), total plasma polyphenols, ferric reducing ability of plasma (FRAP), thiobarbituric acid reactive substances (TBARS) and activities of creatine kinase (CK) and lactate dehydrogenase (LDH) as muscle damage markers. In order to evaluate potential health benefits of the acai berry, the post-treatment changes in lipid profile parameters (triglycerides, cholesterol and its fractions) were analysed. Six weeks’ consumption of acai berry-based juice blend had no effect on sprint performance, but it led to a marked increase in the total antioxidant capacity of plasma, attenuation of the exercise-induced muscle damage, and a substantial improvement of serum lipid profile. These findings strongly support the view of the health benefits of supplementation with the acai berry-based juice blend, mainly attributed to its high total polyphenol content and the related high in vivo antioxidant and hypocholesterolaemic activities of this supplement.     

Effect of hydrogen peroxide on nitrate reductase activity in detached oat leaves in darkness

Nitrate reductase (NR, EC 1.6.6.1) is sensitive to O₂ concentration, and therefore it was of interest to study the action of H₂O₂, a normal substance in plant metabolism, on NR activity in segments of 7-, 14- and 17-day-old leaves of oat (Avena sativa L. ev. Suregrain). After 4 h of treatment in the dark, H₂O₂ decreased NR activity as measured with the in vivo assay. The effect was stronger in 14- and 17- than in 7-day-old leaves. Vacuum infiltration of cysteine did not prevent this decrease. When NR was determined with the in vitro assay, H₂O₂ did not seem to affect the activity after the 4 h treatment. but NR decreased when crude extracts prepared from untreated 14-day-old leaves were incubated directly with H₂O₂. This effect was prevented by addition of cysteine, ascorbate or reduced glutathione to the extracts. In order to study the possibility that low activity of the system for defense against oxidations could account for the age-dependent response of NR to H₂O₂ in the in vivo test, activities of catalase, ascorbate peroxidase and glutathione reductase were measured during leaf development and after a 4-h treatment with H₂O₂ in the dark. No clear correlation was found between the activities of those enzymes and changes in in vivo NR activity caused by H₂O₂. The results suggest that H₂O₂ might affect NR both directly by oxidizing SH-groups and indirectly by decreasing reductant availability as a result of NADH oxidation. The age-dependent response of NR to H₂O₂ treatment could also be explained in terms of decreased NADH availability in the tissues due to decreased NADH synthesis and/or increased degradation.     

Exposure Time Related Oxidative Action of Hyperbaric Oxygen in Rat Brain

Hyperbaric oxygen (HBO) is known to cause oxidative stress in several organs and tissues. Due to its high rate of blood flow and oxygen consumption, the brain is one of the most sensitive organs to this effect. The present study was performed to elucidate the relation of HBO exposure time to its oxidative effects in rats’ brain cortex tissue. For this purpose, 49 rats were randomly divided into five groups. Except the control group, study groups were subjected to three atmospheres HBO for 30, 60, 90, and 120 min. Their cerebral cortex layer was taken immediately after exposure and used for analysis. Thiobarbituric acid reactive substances (TBARS), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and nitrate–nitrite (NO_X) levels were determined. TBARS and SOD levels were found to increase in a time-dependent manner. GSH-Px activity reflected an inconsistent course. NO_X levels were found to be increased only in the 120 min exposed group. The results of this study suggests that HBO induced oxidative effects are strongly related with exposure time.     

Protective effect of gallic acid isolated from Peltiphyllum peltatum against sodium fluoride-induced oxidative stress in rat’s kidney

In the present study, the nephroprotective effect of gallic acid isolated from Peltiphyllum peltatum was examined in sodium fluoride (NaF) treated rats. Nephrotoxicity was induced by 1-week intoxication of NaF at 600 ppm through drinking water. The levels of thiobarbituric acid reactive substances, reduced glutathione as well as activities of superoxide dismutase and catalase in renal tissues homogenates were determined. The serum biochemical markers of renal injuries including creatinine, serum urea, blood urea nitrogen, uric acid levels as well as the levels of phosphate and calcium were also assessed. Intoxication with NaF caused a significant increase in the levels of thiobarbituric acid reactive substances (46 % versus to control) and reduced the glutathione concentration (47 %) and the activities of superoxide dismutase (46 %) and catalase (41 %) in renal tissues homogenates. NaF intoxication also induced significant alterations in the kidney biochemical markers increasing the levels of urea, uric acid, blood urea nitrogen, creatinine, and phosphate and decreasing the levels of calcium. Daily administration of gallic acid (20 mg/kg) for 1 week before NaF intoxication brought the antioxidant–oxidant balance similar to the NaF-untreated group. Silymarin, used a standard antioxidant agent, also showed a nephroprotective activity. We concluded that NaF caused nephrotoxicity and oxidative stress in renal tissues and daily administration of gallic acid for 1 week prior to intoxication inhibited toxicity and oxidative stress.     

Coimmobilization of Superoxide Dismutase,Catalase, and Peroxidase

Various orders of sequential coimmobilization of superoxide dismutase (SOD), catalase, and horseradish peroxidase (HRP) were tested in order to prepare a multienzyme antioxidant complex of these enzymes. Simultaneous coimmobilization of catalase with a preliminarily cross-linked complex between SOD and HRP was found to be the optimum procedure. The catalytic enzyme activity and working stability of catalase was tested kinetically in the multienzyme complexes prepared by different methods. The effects of ascorbic acid, glutathione, and ethanol on the kinetic parameters of catalase were studied. A possible scheme of H₂O₂ degradation catalyzed by coimmobilized SOD, catalase, and HRP in the presence of reducing substrates is suggested.     

Biochemical evaluation of lipid and oxidative stress status in relation to high fat-high antioxidant diets

Free radicals are produced through biological processes and environmental interactions. They are metabolised by the enzymatic and non-enzymatic antioxidants present in the tissues. In this study, a 90 days long feeding of high fat diet to rats, resulted in significantly elevating the lipid and oxidative stress levels of the rat liver and blood as became evident from the changes in the levels of lipids, thiobarbituric acid reactive substances(TBARS), reduced glutathione (GSH), and three hepatic antioxidant enzymes; glutathione peroxidase (EC 1.11.1.9), catalase (EC 1.11.1.6) and superoxide dismuatase (EC 1.15.1.1). However, a concomitant feeding of high antioxidant combination, as high fat high antioxidant diets, reduced the lipid levels and diminished the oxidative stress. The results suggest that apart from reducing lipid levels, dietary antioxidants also support endogenous antioxidants in their oxidative stress reducing endeavours.