Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P47989 (xanthine oxidase)
 8,633 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

XTT (3'-{1-[(phenylamino)-carbonyl]-3, 4-tetrazolium}bis(4-methoxy-6-nitro)benzenesulfonic acid hydrate) was reduced to a water-soluble product with an absorbance maximum at about 470 nm by superoxide anion generated by xanthine-xanthine oxidase (XO). The rate of XTT reduction was linearly related to XO activity and the reduction was inhibited by superoxide dismutase (SOD). A perfect inhibition of the reduction of XTT by SOD was achieved, suggesting that XTT does not interact with XO. The present XTT-based assay had a higher sensitivity than a conventional nitroblue tetrazolium-based assay by a factor of 2.5 at pH 10.2. This method was applicable to the SOD assay in the pH range 8.0-10.2.
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PMID:Spectrophotometric assay for superoxide dismutase based on tetrazolium salt 3'--1--(phenylamino)-carbonyl--3, 4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfonic acid hydrate reduction by xanthine-xanthine oxidase. 929 17

The tetrazolium dyes MTS and XTT were reduced to their soluble formazans by superoxide radical anions (O2-) produced by the oxidation of xanthine by xanthine oxidase under standard conditions. These reactions were compared to the well-known reductions of NBT and cytochrome c by the xanthine/xanthine oxidase system. Reduction of the dyes was completely inhibited by superoxide dismutase (SOD). Rate constants for the reaction of MTS and XTT with O2- were estimated at 1.3 +/- .1 x 10(5) M-1S-1 and 8.6 x 10(4) M-1S-1 respectively. The stable MTS and XTT formazans have high extinction coefficients in the visible range which enable sensitive detection and quantification of superoxide radicals, avoiding some of the problems inherent in assays based on production of the insoluble NBT formazan. MTS and XTT have considerable potential both for the quantitative assay of radical production in living tissues and for the assay of superoxide dismutase activity in tissue extracts. Implications for the interpretation of cell culture growth assays which employ these dyes are discussed.
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PMID:The tetrazolium dyes MTS and XTT provide new quantitative assays for superoxide and superoxide dismutase. 935 Apr 32

Two commonly used assays for superoxide dismutase (SOD) activity have been compared, one using cytochrome c and the other using XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) as the indicating scavenger of superoxide. The use of cyanide to selectively suppress Cu,Zn-SOD and thus to allow assay of both Cu,Zn-SOD and Mn-SOD in mixtures of the two was also explored, as was the influence of pH. The XTT assay became more sensitive at elevated pH, because the rate of the superoxide/XTT reaction declines with increasing pH. This was clearly seen with the Cu,Zn-SOD but barely with Mn-SOD because the former retains full activity from pH 5 to 10 while the latter does not. Cyanide reacted with cytochrome c, but not XTT, in a concentration- and time-dependent manner and thus diminished its reducibility by superoxide. Cytochromes endogenous to tissue fractions were reduced by the xanthine oxidase reaction and this caused a decrease in absorbance 470 nm which interfered with the XTT assay. The alkalinizing effect of cyanide salts and the problems encountered in neutralizing cyanide stock solutions are discussed.
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PMID:Assay of superoxide dismutase: cautions relevant to the use of cytochrome c, a sulfonated tetrazolium, and cyanide. 1170 Sep 91

The sulfonated tetrazolium 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2-tetrazolium 5-carboxanilide (XTT) is advantageous in that it yields a water-soluble formazan, unlike most other available tetrazoliums. XTT is reducible by superoxide, as are other tetrazoliums, but is not directly reduced by xanthine oxidase plus xanthine or by glucose oxidase plus glucose. This led to the suggestion that XTT reduction might serve as a reliable index of intracellular O(2)(-) production. We now show that soluble extracts of Escherichia coli contain two NADPH:XTT reductases that act aerobically or anaerobically. That being the case, XTT reduction is not a reliable measure of intracellular O(2)(-).
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PMID:Is reduction of the sulfonated tetrazolium 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2-tetrazolium 5-carboxanilide a reliable measure of intracellular superoxide production? 1242 37

Kinobeon A was originally isolated from cultured cells of safflower (Carthamus tinctorius L., Compositae). It had never previously been directly isolated from safflower or other plants, animals or microorganisms. In this report, we demonstrate the anti-oxidative effects of kinobeon A and compare the results with those two known natural antioxidants, lignan (nordihydroguaiaretic acid) and quercetin. The NADPH-induced microsomal lipid peroxidation system was employed to assess anti-oxidative effects of kinobeon A. Addition of kinobeon A to the system significantly decreased the formation of thiobarbituric acid reactive substances (TBARS) in a dose-dependent manner with effects similar to those of lignan and quercetin. Formation of TBARS was completely inhibited at 10 microM of kinobeon A. Employing the xanthine/xanthine oxidase/nitroblue tetrazolium system and the KO2/XTT system, the superoxide anion scavenging activity of kinobeon A was greater than that of lignan or quercetin. IC50 values calculated for kinobeon A in these two systems were 1 microM and 0.8 microM, respectively. Kinobeon A exerted cytoprotective effects following oxidative treatments with hydrogen peroxide, cumene hydroperoxide, menadione and xanthine oxidase (XOD). Addition of kinobeon A to the systems markedly enhanced survival ratios of Madin-Darby bovine kidney cells, while their survival significantly decreased with the oxidative treatment alone. Kinobeon A exhibited stronger effect on the cell viability than lignan or quercetin when menadion or XOD were used as inducing reagents of oxidative stress. The present study demonstrates for the first time that kinobeon A prevents oxidative stresses and could be a useful cytoprotective reagent.
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PMID:A novel and potent biological antioxidant, Kinobeon A, from cell culture of safflower. 1457 15

Two highly sensitive spectrophotometric methods are developed and described for the measurement of superoxide ion radical derived from KO2 as well as O2*- generated either from the xanthine-xanthine oxidase reaction or by the addition of nicotinamide adenine dinucleotide (NADH) to skeletal muscle sarcoplasmic reticulum (SR) vesicles. These methods allow quantification of superoxide ion concentration by monitoring its reaction with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), either by recording absorbance of the final reaction product at a wavelength of 470 nm or by measuring its fluorescence emission intensity at 550 nm using an excitation wavelength of 470 nm. The extinction coefficient of the active product was determined to be 4000 M(-1) cm(-1). A lower limit second-order bimolecular rate constant of 1.5+/-0.3x10(5) M(-1) s(-1) was estimated from kinetic stopped-flow analysis for the reaction between NBD-Cl and KO2. A plot of absorbance versus concentration of superoxide was linear over the range 2 to 200 microM KO2, whereas higher sensitivities were obtained from fluorometric measurements down into sub-micromolar concentrations with a limit of detection of 100 nM KO2. This new spectrophotometric assay showed higher specificity when compared with some other commonly used methods for detection of superoxide (e.g., nitroblue tetrazolium). Results presented showed good experimental agreement with rates obtained for the measurement of superoxide ion when compared with other well-known probes such as acetylated ferri cytochrome c and 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT). A detailed discussion of the advantages and limitations of this new superoxide ion probe is presented.
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PMID:Spectrophotometric and fluorometric assay of superoxide ion using 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole. 1579 75

The chemopreventive chalcone xanthohumol (Xh) has been reported to decrease xanthine oxidase (XOD) catalysed formation of formazan from nitroblue tetrazolium (NBT) and is discussed as a potent scavenger of superoxide. Re-evaluation of the scavenging capacity indicated that Xh disturbed detection of superoxide with NBT, in case of an insufficient NBT/Xh ratio. Xh lacked superoxide scavenging activity in contrast to the Xh-derivative 3'-hydroxy-Xh with catechol substructure, used as positive control. This was shown by the use of sufficient concentration of NBT and other detectors such as hydroxylamine, XTT, cytochrome c and hydroethidine. HPLC analysis of reaction products in a xanthine/XOD/peroxidase system demonstrated beside enhanced inhibition of NBT-formazan by Xh that NBT even prevented oxidation of Xh. p-coumaric acid or ferulic acid could replace Xh in that system, indicating that superoxide detection using NBT is likely jeopardized by interference of phenoxyl-radicals. Furthermore, this study provides evidence that Xh can moderately generate superoxide via auto-oxidation.
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PMID:Re-evaluation of superoxide scavenging capacity of xanthohumol. 2103 56

In vitro cell-based assays are an essential and universally used step in elucidation of biological processes as well as in drug development. However, results obtained depend on the validity of protocols used. This statement certainly pertains to in vitro assays of oxidative stress. The holy grail of in vitro models is reliability and predictability of outcomes that relate to a single variable like addition of hydrogen peroxide or xanthine oxidase. Without such validated outcomes, comparison of results among different laboratories is not possible. Achieving this goal requires a thorough understanding of the complex interplay between the cells, their environment, and the experimental assays. Furthermore, as this knowledge is attained, it must be disseminated and used to update and standardize existing protocols. Here, we confirm and extend the effect of pyruvate and cell density on in vitro oxidative stress assays. Cell viability was assessed using a colorimetric assay measuring the reduction of a tetrazolium salt (XTT) into a colored formazan dye. Extracellular hydrogen peroxide concentrations were measured using the foxp3 assay. We confirmed a previously reported finding that pyruvate, a common ingredient in cell culture media, acts as an extracellular scavenger of reactive oxygen species. We also demonstrated that cell density directly correlates with resistance to oxidative stress in tissue culture. It is theorized that the protective effect due to cell density predominantly relates to intracellular factors such as reduced glutathione and extracellular factors such as catalase.
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PMID:High cell density attenuates reactive oxygen species: implications for in vitro assays. 2210 55