Determination Of Superoxide Dismutase Activity Research Articles

Superoxide Dismutase (SOD) Activity in Cryopreserved Semen of Itik Pinas-Khaki (Anas platyrhynchos L.)

Cryopreservation induces oxidative stress on sperm due to an increase in the number of reactive oxygen species (ROS), thereby resulting in decreased sperm quality. ROS's destructive potential is normally counteracted in sperm by their innate antioxidant system consisting of enzymes, which include superoxide dismutase (SOD). This study aimed to assess the quality of semen from Itik Pinas-Khaki (IP-Khaki) drakes that were cryopreserved with either 4.5% DMSO or 7.0% glycerol as cryoprotectant through evaluation of total sperm motility (%) and determination of SOD activity (U/mL). Here, semen samples were collected from 12 sexually mature IP-Khaki drakes, an improved egg-type breed of Philippine mallard duck, and processed using modified reported cryopreservation procedure for ducks. Results showed that post-thawing total sperm motility averages of 12.04±5.61% using 4.5% DMSO and 13.99±5.28% using 7.0% glycerol were comparable. Moreover, similar SOD activity levels of 0.39±0.18 U/mL with 4.5% DMSO and 0.33±0.21 U/mL with 7.0% glycerol in 2.00 x 108 IP-Khaki sperm cells were also observed. The observed very low intracellular SOD activity indicates severe damage to sperm cells due to cryopreservation, which resulted in a comparably low total sperm motility with either of the cryoprotectants. Thus, the cryopreservation protocol used is not the optimum for IP-Khaki semen based on the observed considerable decline in sperm motility and very low SOD activity after cryopreservation.

Melatonin and garlic cytoprotective-ameliorative effects on dibutyl phthalate intoxication on sperm DNA and testicular biomakers of rabbits

The study investigated the cytoprotective and ameliorative effects of melatonin and Allium sativum (garlic) on dibutyl phthalate (DBP)-induced oxidative stress, its impact on sperm DNA integrity and testicular oxidative stress biomarkers. Forty two rabbit bucks were randomly divided into 7 groups of 6 bucks each labeled as A, B, C, D, E, F and G: The treatment were as follows: A (served as negative control, received olive oil for 16 weeks); B (served as positive control, exposed to DBP for 16 weeks, no treatment); C (given melatonin for 8 weeks, thereafter DBP for 8 weeks); D (administered garlic for 8 weeks, thereafter DBP for 8 weeks); E (exposed to DBP for 8 weeks, thereafter melatonin for 8 week); F (exposed to DBP for 8 weeks, thereafter garlic for 8 weeks); and G (exposed to DBP for 8 weeks, thereafter melatonin + garlic for 8 weeks). Ejaculated semen was collected on the last day (112th) using artificialv vagina for rabbit and pooled for each group was used for sperm DNA fragmentation index (SDFI) determination, rabbits were sacrificed and the testes harvested for determination of superoxide dismutase activity, reduced glutathione and malondialdehyde concentration. Results showed a significant increase (P = 0.0018) in the mean SDFI in group B (78.20 ± 4.72), compared to other groups. A significant increase (P ≤ 0.0001) in superoxide dismutase activity, increase reduced glutathione concentration and decrease malondialdehyde concentrations in the treatment groups compared to the DBP exposed group without treatment (group B) were observed. Melatonin and garlic demonstrated cytoprotective and ameliorative effects against DBP-induced oxidative stress in rabbit bucks.
 Keywords: Dibutyl phthalate, Garlic, Melatonin, Sperm DNA, Testicular biomarkers

Superdioxide dismutase (SOD) and metallothionein (MT) Tubifex tubifex at the acute mercury exposure

Oxidative stress is a condition of over production of free radicals that can cause cell damage, pathological conditions and cell death. This study aims was to determined the activity of superoxide dismutase (SOD) and metallothionein (MT) levels in Tubifex tubifex worms exposed to HgCl2 in various concentration levels. The concentration of HgCl2 used was based on LC50-48 h values in acute toxicity test (15,85 μg / L, 25,12 μg / L; 38,81 μg / L; 63,09 μg / L; 99,98 μg / L ). The method used was experiment. Determination of SOD activity done using spectrophotometry and MT measurement using ELISA method. The results was showed that HgCl2 exposure to Tubifex tubifex caused increased levels of superoxide dismutase enzyme and metallothionein levels. SOD and MT levels were directly proportional to the concentration of HgCl2, the higher the concentration, SOD and MT levels will increase accordingly.

Standardization and regulation of the rate of the superoxide-generating reaction of adrenaline autoxidation used for evaluation of pro/antioxidant properties of various materials

The superoxide-generating reaction of adrenaline autoxidation is widely used for determination of superoxide dismutase activity and pro/antioxidant properties of various materials. There are two variants of the spectrophotometric registration of the products of this reaction. The first one is based on registration of adrenochrome (a product of adrenaline autoxidation) at 347 nm; the second approach employs nitro blue tetrazolium (NBT) and registration of diformazan (a product of NBT reduction) at 560 nm. In the present work, recommendations for the standardization of the reaction rate in both variants have been given. The main approach consists in the use of a pharmaceutical form of 0.1% adrenaline hydrochloride solution. Although each of two adrenaline preparations available in the Russian market has some individual features in kinetic behavior of adrenaline autoxidation, they are applicable for the superoxide generating system. Performing measurements at 560 nm, the reaction rate can be regulated by lowering concentration of added adrenaline, whereas during spectrophotometric registration at 347 nm, this is not applicable. These features of the adrenaline autoxidation reaction may be attributed to the multistage process of adrenaline conversion to adrenochrome and also to coupled electron transfer from adrenaline and intermediate products of its oxidation to oxygen, carbon dioxide, and carbonate bicarbonate ions. This results in formation of corresponding radicals detectable by adding NBT.

Determination of Superoxide Dismutase Activities in Different Cyanobacteria for Scavenging of Reactive Oxygen Species

There is considerable interest in cyanobacteria as sources of antioxidant pigments, antiinflammatory substances, enzymes such as superoxide dismutase (SOD) and vitamins. Antioxidant enzymes have significant role in the removal of reactive oxygen species produced during visible and ultraviolet irradiance stress in cyanobacteria. One of the most ubiquitous protective enzyme is superoxide dismutase (SOD). Most of cyanobacteria are known to have both FeSOD and MnSOD which are associated with photosystem I and II. These enzymes are responsible for scavenging superoxide radicals.Spirulina platensis and Synechococcus nidulans are widely used cyanobacteria as food supplement and cosmetic due to their bioactive compounds. Although the chemical composition of S.platensis has been investigated by many researchers, research on the relationship between protein content and comparison of SOD activity with other cyanobacteria is limited.Among several SOD producing cyanobacteria, the most promising organisms were investigate in terms of SOD activities in this study. The aim of the present work was to investigate and compare the SOD activities of different cyanobacteria including S. platensis, Pseudanabeana sp., S. nidulans and associate with protein contents. SOD activities of these cyanobacteria were determined by xanthine/xanthineoxidase method.Modified Lowry method was used for protein determination. The analysis results showed that the maximum specific SOD activity obtained was 50.4 U/mg from S.nidulans and the minimum specific SOD activity was 18.4 U/mg from Pseudanabeana sp. Total protein amount of S. nidulans, S. platensis, Pseudanabeana sp. was determined as 0.25 mg/ml, 0.27 mg/ml and 0.43 mg/ml respectively. As a result, Synechococcus nidulans was more efficient at scavenging peroxide radicals than Pseudanabeana sp.

Use of nitro blue tetrazolium in the reaction of adrenaline autooxidation for the determination of superoxide dismutase activity

Addition of nitro blue tetrazolium (NBT) into the reaction of adrenaline autooxidation allows to identify directly superoxide anion formation (02.-) in this superoxide-generating system. The kinetics of formation of adrenochrome and 02.- were compared under the same conditions. Three possible approaches to the use the adrenaline autooxidation reaction for determining the activity of superoxide dismutase (SOD) and revealing the antioxidant properties of various compounds are discussed. Two of these approaches have been described previously: the spectrophotometric method of the registration of adrenochrome, the final product of adrenaline autoxidation, at 347 nm (Sirota, 1999) and the polarographic method, based on determination of oxygen consumption for 02.- formation (Sirota, 2011). Here, a novel approach to this problem is presented; it consists in the spectrophotometric determination of 02.- using NBT. The application of this approach enables one to lower the pH of carbonate buffer from 10.5 to 9.7 and to decrease (4-fold) the amount of added adrenaline, i.e., to generate milder and more effective conditions for revealing and studying the antioxidant properties of examined materials.

Use of nitro blue tetrazolium in the reaction of adrenaline autooxidation for the determination of superoxide dismutase activity

Use of nitro blue tetrazolium in the reaction of adrenaline autooxidation for the determination of superoxide dismutase activity

A new approach to studying the autoxidation of adrenaline: possibility of the determination of superoxide dismutase activity and the antioxidant properties of various preparations by polarography

The reaction of adrenaline autoxidation in an alkaline buffer with the formation of superoxide radicals and the product of its oxidation, adrenochrome, which models the quinoid pathway of adrenaline conversion in the body, is accompanied by oxygen consumption. This reaction is applicable for polarographic determination of the activity of superoxide dismutase and the antioxidant properties of biological and chemical compounds, it is based on evaluation of the latent period and the rate of oxygen consumption, which are measured in the presence of the compounds examined. It was assumed that the neuro- and cardiotoxicity of quinone products of adrenaline oxidation is related not only to their "own" properties and reactive oxygen species formed but also the hypoxia of those regions of the cell and tissue where the quinoid oxidation of adrenaline occurs.

A novel approach to study the reaction of adrenaline autooxidation: A possibility for polarographic determination of superoxide dismutase activity and antioxidant properties of various preparations

The reaction of adrenaline autooxidation in an alkaline carbonate buffer followed by formation of superoxide radicals and the product of its oxidation, adrenochrome, which models the quinone pathway of adrenaline metabolism in the body, is accompanied by oxygen consumption. A study of this process by the polarographic method enabled one to apply this reaction to determine the activity of superoxide dismutase and antioxidant properties of biological and chemical compounds; it is based on evaluation of a latent period and the rate of oxygen consumption, which are measured in the presence of examined compounds. It was suggested that known neuroand cardiotoxicity of the quinone products of adrenaline oxidation may be associated not only to their intrinsic properties and reactive oxygen species formed but also local hypoxia of those regions of the cell and tissue where the quinone oxidation of adrenaline occurs.

Determination of superoxide dismutase and SOD-mimetic activities by a chemical system: Co2/H2O2/lucigenin

The bright chemiluminescence has been observed in the system: Co(2+)/hydrogen peroxide/lucigenin. The chemiluminescence intensity was directly proportional to either cobalt, hydrogen peroxide, or lucigenin concentrations. A procedure of determination of superoxide dismutase (SOD) activity by the chemiluminescence method in the cobalt-hydrogen peroxide-lucigenin system at pH 8.5 is suggested. A linear dependence was established between a relative chemiluminescence intensity and SOD concentration in the range of SOD concentrations between 0 and 4.5 nM, c (1/2) = 0.8 nM. The determination of SOD activity was performed in several tissue samples (rat plasma, erythrocyte hemolysate, and liver mitochondria). A technique of tissue sample preparation with the use of thermal inactivation of interfering proteins at 60 °C was used. The method was successfully applied for comparison of the efficiency of SOD mimetics.

Aromatization and antioxidant capacity in the testis of seasonally breeding bank voles: Effects of LH, PRL and IGF-I

Aromatization and antioxidant strategies in the male gonads are important processes, which are involved in control of normal fertility. The objective of this study was to show whether luteinizing hormone (LH), prolactin (PRL), and insulin-like growth factor-I (IGF-I) as well as the length of photoperiod are able to exert an effect on aromatase expression, steroid hormone levels, and antioxidant concentrations in testes of bank voles, seasonally breeding rodents. Mature bank voles that were kept under short light cycles or long light cycles served as the animal model. Testicular sections were used for immunohistochemical visualization of aromatase expression, whereas testicular homogenates were used for radioimmunological measurement of steroids, biochemical determination of superoxide dismutase activity, total antioxidant status (TAS) and protein content. In the testes of hormone-treated voles a stronger immunostaining for aromatase was concurrent with the increase in testosterone and estradiol levels, and total antioxidant status compared with the controls. In contrast, there was a decrease in superoxide dismutase (SOD) activity. The strongest effect on aromatase immunoexpression and steroid hormone levels was detected after combined doses of LH and IGF-I, indicating a stimulatory role of these hormones on estrogen synthesis in the bank vole. An increase in total antioxidant status in testes of hormone-treated bank voles suggests the presence of testicular defense, whereas a decrease in superoxide dismutase activity indicates a protective role of administered hormones against toxic oxygen radicals. The present study also demonstrates a significant, photoperiod-dependent relationship between aromatization and antioxidant capacity in the testis of the bank vole.

Study of 2-(2-Pyridyl)benzothiazoline as a Novel Fluorescent Probe for the Identification of Superoxide Anion Radicals and the Determination of Superoxide Dismutase Activity in Scallion Genus Foods

This paper presents a novel spectrofluorometric method using the novel fluorescent probe 2-(2-pyridyl)benzothiazoline for the determination of superoxide dismutase (SOD) activity. The fluorescent probe was synthesized in house and fully characterized by elemental analysis and by IR and (1)H NMR spectra. It could specially identify and trap superoxide anion radicals (O2(.-)), and then was oxidized by O2(.-) to form a strong fluorescence product. On the basis of this reaction, the spectrofluorometric method was proposed and successfully used to determine SOD activity. The proposed method has a better selectivity in the determination of reactive oxygen species, because the probe can be oxidized to afford a highly fluorescent product only by O2(.-) excluding hydrogen peroxide and hydroxyl radical. As a kind of simple, rapid, precise, and sensitive technique, it could avoid the errors caused by detection time and was applied to the measurement of SOD activity in scallion genus foods with satisfactory results.

BIOCHEMICAL AND PHYSIOLOGICAL EFFECTS OF MELATONIN, VITAMIN E + SELENIUM ON HEART, LUNG AND KIDNEY ANTIOXIDANT ENZYMES OF MALE AGING RATS

Free radicals are one of the potential major causes of age related destruction. They are defined as any atom or molecule that possesse one or more unpaired electron. Antioxidants such as vitamin E+ Selenium and melatonin are substances that prevent deterioration damage caused by free radicals. They have been used in prophylaxis and treatment of various diseases affect elderly persons include cancer, cardiac diseases multiple sclerosis and arthritis. In this study 48 male albino rats were kept at constant environmental and nutritional conditions, then classified into 4 equal groups. Free radicals are one of the potential major causes of age related destruction. They are defined as any atom or molecule that possesse one or more unpaired electron. Antioxidants such as vitamin E+ Selenium and melatonin are substances that prevent deterioration damage caused by free radicals. They have been used in prophylaxis and treatment of various diseases affect elderly persons include cancer, cardiac diseases multiple sclerosis and arthritis. In this study 48 male albino rats were kept at constant environmental and nutritional conditions, then classified into 4 equal groups. Group I used as control young (2-4 months old) not supplied by any additives, group II control aging (12-27 months old) not supplied by any additives, group III aged male rats administered by melatonin at a dose 0.27 mg/kg body weight daily for three months orally, and group IV aged rats injected quickly by I.M route with vitamin E+ Selenium 20 mg/kg body weight and 0.15 mg/k. gm. b. w, respectively for three months. After 12 weeks from the onset of administration the rats were killed by decapitation.The heart, lung and kidney were rapidly removed, washed with saline and processed directly for determination of superoxide dismutase activity (SOD), Glutathione peroxidase (GSHPX), catalase activity and Reduced glutathione (GSH). The obtained data revealed significant decrease in heart, lung and kidney (SOD) and (GSHPX) activities of aged male rates. A high significant increase in heart and lung (SOD), kidney (GSHPX) activities as well as significant increase in (GSHPX) of heart and lung in melatonin administered group. Moreover significant increase in heart and lung (SOD) and heart, kidney (GSHPX) of aged male rats treated with vitamin E+ selenium. After administration of melatonin, heart and lung catalase activity increased significantly while (GSH) concentration showed significant decrease in kidney of aged rates, significant increase in heart and kidney of melatoin administrated group, significant increase in heart of vit. E+ selenium injected group.

Flow injection-chemiluminescent assay for the determination of superoxide dismutase activity

Superoxide dismutase (SOD) activity was measured using a flow injection-chemiluminescent assay (FI-CLA) based on xanthine–xanthine oxidase dependent superoxide (O2.–) formation. The mobile phase consists of 50 mM potassium phosphate buffer (pH 7.4) containing lucigenin (5 µM) and xanthine (0.3 mM). Under optimum conditions, bovine albumin did not affect chemiluminescence at concentrations of 1–1000 µg mL–1and KCN inhibited 100% of the Cu, Zn-SOD activity using 5 µL of a 0.23 mM concentration. The analysis of one sample was done in less than 30 s with a relative standard deviation of ±3.1%. SOD activity in the biological samples was correlated to the amount of exogenously applied SOD. The FI-CLA method reported here appears to be one of the faster and more useful tools used to assay SOD activity.Key words: superoxide dismutase, flow injection-chemiluminescent assay, superoxide, xanthine oxidase, lucigenin.

Improvement of a direct spectrophotometric assay for routine determination of superoxide dismutase activity

The growing interest in measuring superoxide dismutase (EC 1.15.1.1) in many diseases calls for useful routine assays. For this purpose, the direct spectrophotometric method of Marklund (J Biol Chem 1976;251:7504-7) was improved to offer an alternative to the imprecise, indirect assays currently used. The decay of O2.- (from KO2) at pH 9.5 was monitored as the decrease in delta A (delta A = A250nm-A360nm). Superoxide dismutase was determined from the pseudo-first-order rate constant of O2.- dismutation. The precision of the assay was improved by increasing the concentration of O2.- and expanding the interval for measurements of O2.- concentrations to 4-16 mumol/L. Other assay characteristics, including temperature, were also optimized. In hemolysate the assay had a within-day CV of 5.5-13% and a between-day CV of 4%. Mn-superoxide dismutase and some superoxide dismutase mimics are inhibited at alkaline pH. Therefore, the method is primarily recommended for Cu,Zn-superoxide dismutase.

Determination of Superoxide Dismutase Activity by the Polarographic Method of Catalytic Currents in the Cerebrospinal Fluid of Aging Brain and Neurologic Degenerative Diseases

The activity of the superoxide dismutase was measured by the polarographic method of catalytic currents in the cerebrospinal fluid of patients with age-related neurologic degenerative diseases, namely, amyotrophic lateral sclerosis and Alzheimer's disease, and of a reference group of normal subjects. The superoxide dismutase activity was found to increase with age in reference subjects (r = 0.81) while no significant correlation was found in amyotrophic lateral sclerosis and Alzheimer's disease patients. The activity mean values were significantly lower (P less than 0.01) in patients with neurologic degenerative diseases than in the reference subjects. The changes of superoxide dismutase activity in the aging brain and in age-related neurologic degenerative diseases are discussed.

Amperometric method for the determination of superoxide dismutase activity at physiological pH

An amperometric method for the determination of superoxide dismutase activity is reported. Superoxide is produced by the enzymatic reaction of xanthine oxidase and detected at a poised potential of 0.000 V vs. SCE using a glassy carbon electrode. Limits of detection are 8 × 10 −11 M at pH 7.0 and 2 × 10 −12 M at pH 9.0.

Determination of superoxide dismutase activity with an electrochemical oxygen probe

A flow system is described for measurements of superoxide dismutase activities over wide concentration ranges by varying the substrate, pH and flow conditions. Pyrogallol and 6-hydroxydopamine were the best substrates found; the limits of detection were 1.5×10 −9 M superoxide dismutase at pH 9.5 and 2×10 −10 M at pH 7.4, respectively. Epinephrine was less suitable; catechol was not useful. Epinephrine provided good sensitivity at pH 10 when a residence time of 8 min in the system was allowed, but the measurements were then less reproducible than with pyrogallol or 6-hydroxydopamine.

A spectrophotometric assay for superoxide dismutase activities in crude tissue fractions.

A sensitive and reliable assay method was developed to characterize crude cell homogenates and subcellular fractions with regard to their superoxide dismutase (SOD) activities. The determination of SOD activities was based on the well-known spectrophotometric assay introduced by McCord & Fridovich [(1969) J. Biol. Chem. 244, 6049-6055], with partially succinylated (3-carboxypropionylated) rather than native ferricytochrome c as indicating scavenger. Partial succinylation of cytochrome c resulted in minimization of interference associated with the interaction of cytochrome c with mitochondrial cytochrome c oxidase or cytochrome c reductases. The further increase in specificity, with regard to exclusion of cytochrome c oxidase interference, gained as a consequence of the high pH of 10 enabled the analysis of samples as rich in cytochrome c oxidase activity as the mitochondrial fraction in the presence or absence of membrane-disrupting detergents. Linear relationships for the dependence of the SOD activities with protein concentration were obtained with rat liver homogenate, mitochondrial and microsomal fractions, indicating negligible interference. Furthermore, by choosing a high pH for the assay medium, a 4-fold increase in sensitivity compared with the classical SOD assay, carried out at pH 7.8, was gained as well as a more precise resolution of Cu/Zn-SOD and Mn-SOD by 2 mM-KCN in samples with a high ratio of Mn-SOD to Cu/Zn-SOD, such as mitochondria. The complete trapping of the O2.- radicals, which was more feasible at pH 10 than at pH 7.8, enabled the application of a simple equation derived for the calculation of appropriately defined units of SOD activity from a single experiment.

A sensitive spectrophotometric method for the determination of superoxide dismutase activity in tissue extracts

Superoxide dismutase (EC 1.15.1.1) has been assayed by a spectrophotometric method based on the inhibition of a superoxide-driven NADH oxidation. The assay consists of a purely chemical reaction sequence which involves EDTA, Mn(II), mercaptoethanol, and molecular oxygen, requiring neither auxiliary enzymes nor sophisticated equipment. The method is very flexible and rapid and is applicable with high sensitivity to the determination of both pure and crude superoxide dismutase preparations. The decrease of the rate of NADH oxidation is a function of enzyme concentration, and saturation levels are attainable. Fifty percent inhibition, corresponding to one unit of the enzyme, is produced by approximately 15 ng of pure superoxide dismutase. Experiments on rat liver cytosol have shown the specificity of the method for superoxide dismutase. Moreover, common cellular components do not interfere with the measurement, except for hemoglobin when present at relatively high concentrations. The assay is performed at physiological pH and is unaffected by catalase.