A method to reduce interference by sucrose in the detection of thiobarbituric acid-reactive substances

A thiobarbituric acid (TBA) reaction for measuring lipid peroxidation products was evaluated for interference by several ingredients commonly used in solutions to prepare or analyze tissue homogenates or subcellular organelles. These included sucrose (up to 100 mm final concentration in the assay medium), Tris-maleate (up to 40 mm), imidazole (up to 20 mm), inorganic phosphate (up to 10 mm), and 4-morpholinepropanesulfonic acid (up to 20 mm). When the samples were heated at 95°C as recommended in some procedures, only sucrose significantly affected color development. Sucrose concentrations as low as 10 mm significantly increased absorbance at 532 nm of aqueous tetramethoxypropane (TMP) standards, and so the assay could not be applied reliably to tissue samples prepared in sucrose. Sucrose interference was only partially reduced by subsequent organic extraction (n-butanol plus pyridine), with measured absorbances remaining significantly greater (50–100%) than sucrose-free controls at sucrose concentrations of 20 mm or more. Modifying the assay to include sucrose in blanks and TMP standards failed to adequately correct for interference when the absorbance of unextracted (aqueous) solutions was measured. Further modification by adding sucrose to blanks and TMP standards, followed by butanolpyridine extraction, gave standard curves that were linear, through the origin, and had slopes equivalent to those of sucrose-free standards. This modification enabled almost complete recovery (average 2% error) of known amounts of TMP added to aliquots of tissue homogenates containing amounts of sucrose that otherwise significantly interfered. Also, with the modified method the content of TBA-reactive substances in tissues homogenized in sucrose was found to be not significantly different from that measured in tissues homogenized in a noninterfering substance, KCl.     

Free radical scavenging activity of the Japanese herbal medicine Toki-Shakuyaku-San (TJ-23) and its effect on superoxide dismutase activity,lipid peroxides,glutamate, and monoamine metabolites in aged rat brain

The free radical scavenging activity of the Japanese herbal medicine, Toki-Shakuyaku-San (TJ-23; TSUMURA & Co., Tokyo, Japan), was examined using electron spin resonance (ESR) spectrometry. TJ-23 scavenged 1,1-diphenyl-2-picrylhydrazyl radicals (DPPH), superoxide (O₂ ⁻), and hydroxyl radicals (·OH) dose-dependently. It also diminished carbon centered radicals (·C) generated by oxidative stress and inhibited thiobarbituric acid-reactive substances (TBARS) formation in mouse cortex homogenate. In addition, the effect of TJ-23 on the concentration of neurotransmitters and TBARS formation, and superoxide dismutase (SOD) activity in the cortex, hippocampus and striatum of the aged rat brain was studied. The concentrations of the metabolites of monoamines, glutamate and glutamine were decreased by 4 weeks of oral administration of TJ-23. The SOD activity of mitochondrial fraction was increased and TBARS formation was significantly suppressed. These results suggest that TJ-23 has an antioxidant action and would have a prophylactic effect against free radical-mediated neurological diseases associated with aging.     

Ontogeny of human fetal catalase superoxide dismutase and lipid peroxidation: A comparative study

A comparative study on the activity profile of catalase and superoxide dismutase, the two scavenging enzymes, as well as the developmental profile of lipid peroxidation in the human fetal brain, liver and kidney have been done for gestation periods ranging from 12 weeks to 28 weeks and beyond. The activity of the scavenging enzymes increase gradually in_all the tissues with the advancement of pregnancy. Brain is an exception in case of catalase where the activity remains more or less same throughout the developmental period except in the case of fetuses, 28 weeks and above where significant decrease in the catalase activity is observed. A high level of lipid peroxidation is observed during early stages of development which declines thereafter.     

Generation of superoxide radicals in alkaline solutions of hydrogen peroxide and the effect of superoxide dismutase on this system

Superoxide radicals in high concentrations were generated from alkaline H₂O₂ without using catalysts or irradiation. The dependence of the intensity and parameters of the superoxide radical EPR spectrum on pH, temperature, viscosity and H₂O₂ concentration were studied. The observed changes are explained on the base of matrix effects. The addition of superoxide dismutase to alkaline H₂O₂ led initially to a drop in the EPR spectrum intensity, followed by an increase in the concentration of superoxide radicals.     

Manganese superoxide dismutase in rat liver peroxisomes: Biochemical and immunochemical evidence

By using highly purified peroxisomes from rat liver, we have shown that peroxisomes contain manganese superoxide dismutase (MnSOD) activity and a 23 kDa protein immunoreactive with antibodies against purified mitochondrial MnSOD. Immunocytochemical studies have also revealed immunoreaction (immunogold) with MnSOD antibodies in mitochondria and peroxisomes. Studies of the intraperoxisomal localization of MnSOD have shown that in peroxisomes MnSOD is a component of the peroxisomal limiting membranes and dense core. Furthermore, the MnSOD level in peroxisomes was modulated by oxidative stress conditions such as ischemia-reperfusion or the treatment with ciprofibrate, a peroxisomal proliferator. These findings suggest that MnSOD in peroxisomes may play an important role in the dismutation of superoxide generated on the peroxisomal membrane for keeping the delicate balance of the redox state.     

A ready-to-use fluorimetric biosensor for superoxide radical using superoxide dismutase and peroxidase immobilized in sol-gel glasses

In this work, a highly sensitive fluorescent biosensor for quantitative superoxide radical detection, based on the coupled reaction superoxide dismutase-peroxidase enzymes and the use of the probe Amplex red, is described. Superoxide anion radical was produced via oxidation of xanthine by xanthine oxidase. Dismutation of superoxide was catalyzed by superoxide dismutase, generating hydrogen peroxide, which reacted stoichiometrically with the nonfluorescent Amplex red, in the presence of peroxidase, yielding the red-fluorescent oxidation product resorufin. The coupled superoxide dismutase-peroxidase system was immobilized in a single sol-gel matrix. The enzymatic activity of the encapsulated superoxide dismutase-peroxidase system was nearly identical to that of one of the soluble enzymes, indicating that sol-gel encapsulation preserved the hierarchy of the enzyme's activity. Specificity and reusability of the encapsulated system for up to four cycles were also demonstrated. The fluorescent biosensor was able to detect concentrations of superoxide as low as 20 nM in phospholipid model membranes composed of saturated or unsaturated phospholipids. These facts make this biosensor a simple, reliable, and highly sensitive method with a potential use in biological systems, food, and drinks.     

The stimulatory effect of negative air ions and hydrogen peroxide on the activity of superoxide dismutase

The activity of erythrocyte cytosolic superoxide dismutase from rat, bovine, man and duck was considerably increased when measured after preparation or incubation in media pretreated with negative air ions (mostly superoxide) from electroeffluvial ion generator. 0.5–1.0 μM H₂O₂ was found in incubation medium after treatment with air ions. The stimulatory effect of air ions on superoxide dismutase activity was mimicked by addition of 0.5–6 μM H₂O₂. The primary physicochemical mechanism of beneficial biological action of negative air ions is suggested to be related to the stimulation of superoxide dismutase activity by micromolar concentrations of H₂O₂.     

Effect of superoxide dismutase,allopurinol and glucocorticoids on liver and lung metallothionein induction by endotoxin in the rat

Liver and lung metallothionein (MT) levels were increased by endotoxin. The administration of superoxide dismutase (SOD) or allopurinol (ALLO) before (30–60 min) or after (24–32 h) the endotoxin treatment either increased or did not affect the effect of endotoxin on MT levels, depending on the particular treatment and tissue. SOD and ALLO also increased liver and lung MT levels in control rats. In contrast, liver MT levels tended to be decreased by the glucocorticoid prednisolone (PRED) when administered before the endotoxin and were significantly decreased when it was administered after endotoxin. The effect of PRED on lung MT levels was completely different, since it decreased the effect of endotoxin when injected before the lipopolysaccharide, but increased it when injected after the endotoxin. Liver lipid peroxidation, as measured by thiobarbituric acid reactants (TBARs), increased after endotoxin in the liver but not in the lung, an effect even potentiated in some cases by the antioxidants studied. As expected, tissue MT and TBARs could not be correlated.     

Assay of superoxide dismutase activity in animal tissues

Convenient assays for superoxide dismutase have necessarily been of the indirect type. It was observed that among the different methods used for the assay of superoxide dismutase in rat liver homogenate, namely the xanthine-xanthine oxidase ferricytochromec, xanthine-xanthine oxidase nitroblue tetrazolium, and pyrogallol autoxidation methods, a modified pyrogallol autoxidation method appeared to be simple, rapid and reproducible. The xanthine-xanthine oxidase ferricytochromec method was applicable only to dialysed crude tissue homogenates. The xanthine-xanthine oxidase nitroblue tetrazolium method, either with sodium carbonate solution, pH 10.2, or potassium phosphate buffer, pH 7·8, was not applicable to rat liver homogenate even after extensive dialysis. Using the modified pyrogallol autoxidation method, data have been obtained for superoxide dismutase activity in different tissues of rat. The effect of age, including neonatal and postnatal development on the activity, as well as activity in normal and cancerous human tissues were also studied. The pyrogallol method has also been used for the assay of iron-containing superoxide dismutase inEscherichia coli and for the identification of superoxide dismutase on polyacrylamide gels after electrophoresis.     

Effects of catechin and epicatechin on superoxide dismutase and glutathione peroxidase activity,in vivo

Abstract

Objectives

The objective of this study was to investigate the effects of catechin and epicatechin on the activity of the endogenous antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx) (as well as the total antioxidant capacity (TAC)) of rats after intra-peritoneal (i.p.) administration.

Methods

Twenty-four Wistar rats were randomly divided into two groups: the experimental group which was administered daily with a 1:1 mixture of epicatechin and catechin at a concentration of 23 mg/kg body weight for 10 days and the control group which was injected daily with an equal amount of saline. Blood and urine samples were collected before and after the administration period, as well as 10 days after (follow-up).

Results

Intra-peritoneal administration of catechins led to a potent decrease in GPx levels and a significant increase in SOD levels. TAC was significantly increased in plasma and urine. Malonaldehyde levels in urine remained stable. In the animals treated with catechins, SOD activity showed a moderate negative correlation with GPx activity.

Discussion

Boosting the activity of the antioxidant enzymes could be a potential adjuvant approach for the treatment of the oxidative stress-related diseases.     

Effect of temperature and htpR on the biosynthesis of superoxide dismutase in Escherichia coli

The synthesis of Mn- and FeSODs in response to temperature changes was examined in strains of Escherichia coli with different mutations in sod and htpR genes. Growth at or shift to elevated temperatures induced FeSOD but not MnSOD. The induction of FeSOD by heat was inhibited by chloramphenicol and was independent of the heat shock (htpR-controlled) regulon. FeSOD was more stable at 42 degrees C than was MnSOD.     

Thiobarbituric acid-reactive substances relate to arterial stiffness and blood pressure in 6 to 8-year-old boys stratified by maternal risk

Early cardiovascular disease (CVD) onset can be inflicted by familial cardiovascular and lifestyle risk factors. We aimed to compare phenotypic characteristics and explore associations between oxidative stress and vascular function in boys stratified by maternal cardiovascular and lifestyle risk. We included 40 Black and 41 White boys (ages 6–8 years), along with the biological mother of each child. The study population was divided into two groups (nonmaternal risk vs. maternal risk) according to maternal risk predetermined by their selfreported cardiovascular and lifestyle risk factors. Pulse wave velocity (PWV) was measured at various sites and blood pressures were recorded. Urine samples were collected for analyses of thiobarbituric acid-reactive substances (TBARS), 8-hydroxy-2-deoxy guanosine (8-OHdG), albumin, and creatinine. Higher levels of urinary albumin to creatinine ratio (uACR) were found in the maternal risk group compared to the nonmaternal risk group (p?=?.038). Multiple regression analysis in the maternal risk group revealed diastolic blood pressure (R²?=?0.159; β?=?0.293; p?=?.050), carotid femoral PWV (R²?=?0.158; β?=?0.297; p?=?.038) and carotid dorsalis pedis PWV (adj R²?=?0.322; β?=?0.505; p?R²?=?0.161; β?=??0.261; p?=?.046) with TBARS was observed. Also, in the maternal risk group, independent associations of DBP (R²?=?0.273; β?=?0.289; p?=?.040) and uACR (R²?=?0.283; β?=?0.268; p?=?.027) with 8-OHdG were indicated. In boys, as young as 6 years of age, oxidative stress related to arterial stiffness and diastolic blood pressure was observed. This association was only evident in boys with linked maternal lifestyle and cardiovascular risk factors, suggesting potential family-related early onset of cardiovascular risk.     

Effect of sulphite on superoxide dismutase activity in the mycorrhizal fungus Rhizopogon roseolus

 Sulphite at a concentration of 1 mM did not strongly affect the growth of mycelium. Higher concentrations of 5–20 mM almost completely inhibited the growth of mycelium and superoxide dismutase (SOD) [EC.1.15.1.1.] activity. The activity of this enzyme was not detectable on polyacrylamide gels. The lack of induction of SOD and the resulting oxidative stress may in part be responsible for the growth inhibition caused by high concentrations of sulphite. Accepted: 20 July 1995     

Isolation and characterization of superoxide dismutase: A personal history and tribute to Joe McCord and Irwin Fridovich

The discovery of superoxide dismutase twenty years ago gave new meaning to work on erythrocuprein. This tribute to the achievement of Joe McCord and Irwin Fridovich is an account of experience of superoxide dismutase from old obscure copper protein of red blood cells to new exciting enzyme of oxygen free-radical metabolism, and an affirmation of the superoxide theory of oxygen toxicity.     

人SOD1基因在大鼠血管平滑肌细胞的表达及对抗氧自由基损伤的作用

本实验构建含人铜锌超氧化物歧化酶（ｈＳＯＤ１）基因的逆转录病毒载体,将其导入离体培养的鼠血管平滑肌细胞,观察ｈＳＯＤ１基因表达及其抗氧自由基损害作用,结果表明：（１）载体构建策略和方法正确,ｈＳＯＤ１基因可在靶细胞中高效稳定表达;（２）转化ｈＳＯＤ１的ＶＳＭＣｓ可对抗大剂量氧自由基对细胞的直接损伤作用;（３）小剂量氧自由基刺激ＶＳＭＣｓ增殖,而转化ｈＳＯＤ１的ＶＳＭＣｓ增殖反应受到抑制,本研究结果