A comparison of chemical systems for luminometric determination of antioxidant capacity towards individual reactive oxygen species.

The aim of the study was to investigate the reactive oxygen species (ROS) production in the hypoxanthine-xanthinoxidase (HX-XO), hydrogen peroxide-ferrous sulphate (H2O2-FeSO4) and hydrogen peroxide (H2O2) systems by using various concentrations of ROS scavengers, such as superoxide dismutase (SOD), dimethylthiourea (DMTU) or catalase (CAT). Luminol (0.8 mmol/L) was dissolved in a borate buffer, pH 9.0, and was used as a luminophor in the chemiluminescence (CL) measurements. In the HX-XO system SOD, CAT and DMTU deepened the CL signal, whereas in the H2O2-FeSO4 system, only CAT and DMTU deepened the CL signal, and in the H2O2 system SOD and CAT increased and DMTU deepened the CL signal. Electron spin resonance (ESR) measurements were performed only in the H2O2-FeSO4 system. 5,5-dimethyl-pyrroline-N-oxide (DMPO) was used as a spin trap. According to typical ESR spectra, .OH was produced in this chemical system. It can be concluded that the chemical systems do not produce single reactive oxygen species but a mixture of them.     

A rapid TLC autographic method for the detection of xanthine oxidase inhibitors and superoxide scavengers

A new bioautographic assay suitable for the localization of xanthine oxidase inhibitors and superoxide radical scavengers present in a complex matrix is described. Enzyme activity is detected by reaction of superoxide radicals with nitroblue tetrazolium to form a blue formazan salt. Both activities can be differentiated using a non-enzymatic version of the autographic assay wherein superoxide is chemically generated.     

Toxic oxygen species and protective systems of the chloroplast

Salin, M. L. 1988. Toxic oxygen species and protective systems of the chloroplast. -Physiol. Plant. 72: 681–689.
As a consequence of living in an environment enriched in oxygen, which they themselves at least partially generate, photosynthetic organisms are exposed to large fluxes of oxyradicals and reactive oxygen species. Among these are superoxide, hydrogen peroxide, hydroxyl radical and singlet oxygen. These highly reactive intermediates pose the threat of toxicity unless neutralized by scavenger substrates or enzymes. The production of oxyradicals and intermediates by chloroplasts as well as the means of protection are discussed in this review.     

The effect of exogenous spermidine on superoxide dismutase activity,H<Subscript>2</Subscript>O<Subscript>2</Subscript> and superoxide radical level in barley leaves under water deficit conditions

Barley (Hordeum vulgare) seedlings were treated with spermidine prior to water deficit to determine whether this polyamine is able to affect the activity of superoxide dismutase -SOD (EC 1.15.1.1) responsible for hydrogen peroxide and superoxide radical level. Short-term dehydration (24h) resulted in decrease of the SOD specific activity and a distinct increase in the superoxide anion and hydrogen peroxide contents. Polyamine treatment caused a substantial reduction in the contents of these two stress-raised reactive oxygen species and thereby lowered the oxidative stress in plant cells. Antioxidant system as an important component of the water-stress-protective mechanism can be changed by polyamines, able to moderate the radical scavenging system and to lessen in this way the oxidative stress.     

Nox4 overexpression activates reactive oxygen species and p38 MAPK in human endothelial cells

Nicotine adenine dinucleotide phosphate (NADPH) oxidase (Nox) complexes are the main sources of reactive oxygen species (ROS) formation in the vessel wall. We have used DNA microarray, real-time PCR and Western blot to demonstrate that the subunit Nox4 is the major Nox isoform in primary human endothelial cells; we also found high levels of NADPH oxidase subunit p22^phox expression. Nox4 was localized by laser scanning confocal microscopy within the cytoplasm of endothelial cells. Endothelial Nox4 overexpression enhanced superoxide anion formation and phosphorylation of p38 MAPK. Nox4 down-regulation by shRNA has in contrast to TGF-β no effect on p38 MAPK phosphorylation. We conclude that Nox4 is the major Nox isoform in human endothelial cells, and forms an active complex with p22^phox. The Nox4-containing complex mediates formation of reactive oxygen species and p38 MAPK activation. This is a novel mechanism of redox-sensitive signaling in human endothelial cells.     

Determination of sulpiride in pharmaceutical preparations and biological fluids using a Cr (III) enhanced chemiluminescence method

A highly sensitive and simple method for identifying sulpiride in pharmaceutical formulations and biological fluids is presented. The method is based on increased chemiluminescence (CL) intensity of a luminol–H₂O₂ system in response to the addition of Cr (III) under alkaline conditions. The CL intensity of the luminol–H₂O₂–Cr (III) system was greatly enhanced by the addition of sulpiride and the CL intensity was proportional to the concentration of sulpiride in a sample solution. Various parameters affecting the CL intensity were systematically investigated and optimized for determination of the sulpiride in a sample. Under the optimum conditions, the CL intensity was proportional to the concentration of sulpiride in the range of 0.068–4.0 µg/mL, with a good correlation coefficient of 0.997. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 8.50 × 10^‐6 µg/mL and 2.83 × 10^‐5 µg/mL, respectively. The method presented here produced good reproducibility with a relative standard deviation (RSD) of 2.70% (n = 7). The effects of common excipients and metal ions were studied for their interference effect. The method was validated statistically through recovery studies and successfully applied for the determination of sulpiride in pure form, pharmaceutical preparations and spiked human plasma samples. The percentage recoveries were found to range from 99.10 to 100.05% for pure form, 98.12 to 100.18% for pharmaceutical preparations and 97.9 to 101.4% for spiked human plasma. Copyright © 2012 John Wiley & Sons, Ltd.     

The superoxide anion and singlet molecular oxygen: their role in the microbicidal activity of the polymorphonuclear leukocyte

Superoxide dismutase was found to partially inhibit both chemiluminescence and nitroblue tetrazolium (NBT) reduction from intact human polymorphonuclear leukocytes. This capacity to reduce NBT was lost when the polymorphonuclear leukocytes were sonicated, but could be regained if exogenous NADPH (or NADH) was added to the system. Superoxide dismutase was found to inhibit this NADPH- and NADH-dependent NBT reduction. A mechanism is proposed that relates superoxide anion generation to the univalent reduction of O₂ by the activated NADPH (and NADH) oxidase. The relationship of superoxide anion production to NBT reduction, singlet molecular oxygen generation, and chemiluminescence is discussed.     

Free Radicals and the Ischemia-Evoked Extracellular Accumulation of Amino Acids in Rat Cerebral Cortex

The effects of free radical generating systems on basal and ischemia/reperfusion-evoked release of amino acids into cortical superfusates was examined in the rat using the cortical cup technique. Xanthine oxidase plus xanthine significantly enhanced GABA levels 358 fold over controls during 20 min of four vessel occlusion. Glutamate and phosphoethanolamine release following reperfusion were also elevated. Prostaglandin synthase plus arachidonic acid significantly enhanced the ischemia-evoked release of all amino acids (aspartate 360 fold; glutamate 433 fold; glycine 6 fold; GABA 689 fold; phosphoethanolamine 69 fold) and increased the pre-ischemic levels of glutamate, glycine and phosphoethanolamine. Administration of H₂O₂ plus ferrous sulfate significantly elevated both pre-ischemic amino acid release and ischemia-evoked release. A role for free radical generating systems in the development of ischemic injury is supported by the ability of superoxide dismutase plus catalase to reduce ischemia-evoked amino acid efflux into cortical superfusates. Thus, the species of free radical produced, as well as the amount generated, may alter the pattern of amino acid release under both ischemic and non-ischemic conditions.     

Effect of zinc nutritional status on activities of superoxide radical and hydrogen peroxide scavenging enzymes in bean leaves

The effect of varied zinc (Zn) supply on the activities of superoxide dismutase (SOD), ascorbate (AsA) peroxidase, glutathione (GSSG) reductase, catalase and guaiacol peroxidase was studied in leaves of bean (Phaseolus vulgaris) plants grown for 15 days in nutrient solution. Zinc deficiency severely decreased plant growth and the leaf concentrations of soluble protein and chlorophyll. Resupply of Zn to deficient plants for up to 72h restored protein concentrations more rapidly than chlorophyll and plant growth. With the exception of guaiacol peroxidase, the activities of all enzymes were significantly decreased by Zn deficiency, in particular GSSG reductase and SOD. Within 72h of resupplying Zn to deficient plants, the enzyme activities reached the level of the Zn sufficient plants. The results indicate severe impairment in the ability of Zn-deficient leaves to enzymically scavenge O₂ ^- and H₂O₂. Consequences and reasons of this impairment are discussed in terms of photooxidation of chloroplast pigments and inhibition of the biosynthesis of the related scavenger enzyme proteins.     

Involvement of hydroxyl radical in NAD(P)H oxidation and associated oxygen reduction by the granule fraction of human blood polymorphonuclears.

The cyanide-insensitive NAD(P)H oxidase activities have been measured in particulate fractions isolated from resting or zymosan-stimulated polymorphonuclear leukocytes. The particulate fraction was primarily composed of granules. The activities were measured both in the presence and absence of Mn⁺⁺. It was found, in all experiments, that hydroxyl radical scavengers such as Tris, benzoate or mannitol, were powerful inhibitors of the NAD(P)H oxidase activities. This was taken as evidence for the involvement of hydroxyl radical as an intermediate in the aerobic oxidation of both NADH and NADPH. Possibles sources of hydroxyl radical are suggested, but none of them is demonstrated.     

Protein-mediated hydroxyl radical generation--the primary event in NADH oxidation and oxygen reduction by the granule rich fraction of human resting leukocytes.

The granule rich-fraction isolated from human resting polymorphonuclear leukocytes is capable of CN-insensitive NADH oxidation and O₂-uptake, accompanied by production of superoxide anion, hydroxyl radicals and H₂O₂. We showed that H₂O₂ initiates and maintains NADH oxidation and O₂-uptake but is also necessary for the formation of superoxide anion and hydroxyl radicals. It acts as a primary substrate for CN-insensitive protein-mediated formation of hydroxyl radicals, which in turn produce superoxide anions, probably through univalent oxidation of NADH as an intermediary.     

Purification and properties of cytochrome P-450 from untreated monkey liver microsomes

Untreated monkey liver cytochrome P-450 (monkey P-450) has been purified to a specific content of 14.9 n mole/mg protein. The purified preparation was apparently homogeneous and the minimum molecular weight was estimated to be 50,000 by SDS-PAGE. Absolute spectrum of the oxidized form showed peaks at 565, 535 and 417 nm. The monkey P-450 was active in the mixed function oxidation of benzphetamine, aminopyrine, ethylmorphine, aniline and 7-ethoxycoumarin in the presence of rat liver NADPH-cytochrome P-450 reductase and DLPC. Anti monkey P-450 IgG could not inhibit rat P-450s (PB P-450, MC P-448(1) and MC P-448(2] catalyzed 7-ethoxycoumarin O-deethylation activities.     

A simple spectrophotometric method for the measurement of ribonuclease activity in biological fluids

We have developed a rapid and sensitive method for detecting ribonuclease (RNAase). The method makes use of a RNa-Pyronine Y complex which has a different absorption spectrium from that of Pyronine Y alone. When the RNA is hydrolyzed by RNAase, the spectrum of the complex changes to that of unbound Pyronine Y. The resultant decrease in absorbance at 572 nm is linear for final RNAase concentrations ranging from 2 to 45 ng/ml. Optimal assay conditions were 11.5 μg/ml Pyronine Y, 0.56 mg/ml RNA, 80 μmol/ml Tris-HCl buffer, pH 7.8 and 2–45 ng/ml RNAase. The effect of complex concentration, PH, molarity and temperature upon the rate of the reaction were determined.The assay is applicable to crude cell-free extracts.     

A chemiluminescent method for the measurement of pregnanetriol-3α-glucuronide in human diluted urine

Pregnanetriol-3α-glucuronide (PTG) is the majority urinary metabolite of 17-hydroxyprogesterone (17OHP) and it typically increases in the commonest form of congenital adrenal hyperplasia (CAH), due to 21 hydroxylase deficiency. We developed a simple chemiluminescent immunoassay for the direct measurement of PTG in diluted urine in order to avoid the preliminary hydrolysis and extraction steps that are usually employed in gas–liquid chromatographic methods. The immunogenic complex PTG-bovine-serum-albumin was used to induce the formation of specific antibodies in New Zealand rabbits. In addition, PTG was conjugated to aminoethylethylisoluminol and the resulting tracer was characterized by mass spectrometry and used to monitor the immunological reaction. The characteristics of the antibody were determined with regard to specificity and sensitivity. The precision of the assay method was also established. PTG excretion was studied before and after the ACTH stimulation test (1 mg synthetic ACTH i.m.) in 11 normal women and in one subject affected by CAH due to 21-hydroxylase deficiency. PTG levels well correlated with 17OHP plasma concentrations both under basal and stimulated conditions, in normal women as well as in the patient affected by CAH.     

Chilling-enhanced photooxidation: The production,action and study of reactive oxygen species produced during chilling in the light

Chilling-enhanced photooxidation is the light- and oxygen-dependent bleaching of photosynthetic pigments that occurs upon the exposure of chilling-sensitive plants to temperatures below approximately 10 °C. The oxidants responsible for the bleaching are the reactive oxygen species (ROS) singlet oxygen (¹O₂), superoxide anion radical (O ₂ ,hydrogen peroxide (H₂O₂), the hydroxyl radical (OH·), and the monodehydroascorbate radical (MDA) which are generated by a leakage of absorbed light energy from the photosynthetic electron transport chain. Cold temperatures slow the energy-consuming Calvin-Benson Cycle enzymes more than the energy-transducing light reactions, thus causing leakage of energy to oxygen. ROS and MDA are removed, in part, by the action of antioxidant enzymes of the Halliwell/Foyer/Asada Cycle. Chloroplasts also contain high levels of both lipid- and water-soluble antioxidants that act alone or in concert with the HFA Cycle enzymes to scavenge ROS. The ability of chilling-resistant plants to maintain active HFA Cycle enzymes and adequate levels of antioxidants in the cold and light contributes to their ability to resist chilling-enhanced photooxidation. The absence of this ability in chilling-sensitive species makes them susceptible to chilling-enhanced photooxidation. Chloroplasts may reduce the generation of ROS by dissipating the absorbed energy through a number of quenching mechanisms involving zeaxanthin formation, state changes and the increased usage of reducing equivalents by other anabolic pathways found in the stroma. During chilling in the light, ROS produced in chilling-sensitive plants lower the redox potential of the chloroplast stroma to such a degree that reductively-activated regulatory enzymes of the Calvin Cycle, sedohepulose 1,7 bisphosphatase (EC 3.1.3.37) and fructose 1,6 bisphosphatase (EC 3.1.3.11), are oxidatively inhibited. This inhibition is reversible in vitro with a DTT treatment indicating that the enzymes themselves are not permanently damaged. The inhibition of SBPase and FBPase may fully explain the inhibition in whole leaf gas exchange seen upon the rewarming of chilling-sensitive plants chilled in the light. Methods for the study of ROS in chilling-enhanced photooxidation and challenges for the future are discussed.Abbreviations ASP ascorbate-specific peroxidase - -TH reduced -tocopherol - DTT dithiothreitol - FBP fructose 1,6 bisphosphate - FBPase fructose 1,6 bisphosphatase (EC 3.1.3.11) - HFA Cycle the Halliwell/Foyer/Asada Cycle responsible for the enzymatic removal of ROS in the chloroplast stroma - MDA monodehydroascorbate radical - MDAR monodehydroascorbate reductase - ROS reactive oxygen species - SBP sedohepulose 1,7 bisphosphate - SBPase sedohepulose 1,7 bisphosphatase (EC 3.1.3.37) - SOD superoxide dismutase     

A spectrophotometric method for the determination of free fatty acid in serum using acyl-coenzyme A synthetase and acyl-coenzyme A oxidase

A mixture of Ti(IV) and 4-(2-pyridylazo)resorcinol was found to be useful in the spectrophotometric determination of trace amounts of hydrogen peroxide. The absorbance at 508 nm was proportional to the concentration of hydrogen peroxide added. The reagent was successfully applied to the assay of free fatty acid in serum through the combined use of acyl-CoA synthetase and acyl-CoA oxidase. The latter enzyme produces H₂O₂. As a result, hydrogen peroxide was produced through the enzymatic oxidation of free fatty acid. It was possible to determine free fatty acid in 50 μl of serum at concentrations ranging from 0.02 to 1.5 mm. The coefficient of variation was less than 3% at concentrations ranging from 0.1 to 1.5 mm. In the present method, there is the advantage of minimal influence from reducible substances as well as greater simplicity and accuracy.