Nitroblue Tetrazolium Method Research Articles

Effectiveness of domestic antibacterial products in decontaminating food contact surfaces

Four commercially available antibacterial products (two wipes and two sprays) were tested under laboratory conditions on a range of food contact surfaces (wood, glass, plastic, Microban ® incorporated plastic). The products’ effectiveness at preventing cross-contamination of Escherichia coli and Staphylococcus aureus and the influence of surface type and drying time were assessed. Survival of the bacterial culture (approximately 400 colonies per 8 cm 2) on the above preparation surfaces was determined using an in situ nitroblue tetrazolium (NBT) method. In the absence of any antibacterial products, both bacteria survived up to 120 min on all test surfaces with glass and plastic showing no reduction in bacterial number. The order of survival is: glass>plastic⪢Microban ® incorporated plastic>wood (<8%). The length of drying time did not affect the survival of either bacterium on glass and plastic surfaces. On wood and Microban ® incorporated plastic, E. coli appeared to be more sensitive to drying time than S. aureus. Only plastic appeared to affect the effectiveness of the antibacterial products, where the reduction in bacterial number was significantly lower than the other test surfaces ( p < 0.05 ). The overall results suggest the antibacterial products are effective in disinfecting food preparing surfaces, provided products instructions are carefully followed.

Iodinated radiographic contrast media possess antioxidant properties in vitro

To study potential properties of iodinated radiographic contrast media (IRCM) for intravascular use in in vitro free radical generating reactions. Superoxide (*O2-) and hydroxyl (*OH) radicals were generated in xanthine oxidase and Fenton reactions. *O2- was assayed by the nitroblue tetrazolium (NBT) method, whereas *OH was assayed by an aromatic hydroxylation (2-hydroxybenzoic acid) method. Total antioxidant status (TAS) of test substances was determined by a colorimetric assay. Finally, acetyl-cholinesterase (AChE) activity was measured in the absence and presence of IRCM. High concentrations (>50 mM) of IRCM inhibited *O2- production, ionic more than non-ionic IRCM. Medium concentrations (25-50 mM) of IRCM reduced *OH production, and both types of IRCM were equally potent. Low concentrations (<25 mM) of non-ionic IRCM displayed higher antioxidant capacity than their ionic counterparts when tested in the TAS assay. Visipaque 320 (iodixanol) was found to have the highest TAS value, followed by Omnipaque 350 (iohexol), Hexabrix 320 (ioxaglate), and Urografin 370 (diatrizoate). IRCM have in vitro antioxidant properties in concentrations relevant for their clinical application. These properties may therefore be of potential importance when evaluating IRCM effects in vivo, particularly those concerning cardiovascular and renal function.

Synthesis and biological evaluation of polyhydroxycurcuminoids

A series of curcumin analogs ( 1– 3, 5a– 5t) was synthesized through the condensation of appropriately protected hydroxybenzaldehydes with acetylacetone, followed by deprotection. The antioxidant activity of these analogs was determined by superoxide free radical nitroblue tetrazolium and DPPH free radical scavenging methods and the polyhydroxycurcuminoids ( 5l– 5s) displayed excellent antioxidant activity. These analogs showed cytotoxicity to lymphocytes and promising tumor-reducing activity on Dalton’s lymphoma ascites tumor cells.

MRNA Study on Cu/Zn Superoxide Dismutase Induction by Hemodialysis Treatment

Background/Aims: There is little or no controversy about the increased oxidative stress of hemodialysis (HD) patients. Several reports show that the activity of superoxide dismutase (SOD), one of the major endogenous antioxidant enzymes, in plasma is elevated among HD patients. It is still unclear, however, whether this elevation is due to the promotion of SOD production or a decrease in renal excretion of SOD. This study was designed to investigate the cause of the SOD activation in HD patients, and we examined the expression of SOD mRNA levels in leukocytes of patients with chronic renal failure. Methods: The total plasma SOD activity was determined by the nitroblue tetrazolium method, plasma SOD contents by ELISA, and SOD mRNA levels in leukocytes by RT-PCR. Results: Our results demonstrated that contents and mRNA levels of Cu/Zn SOD in HD patients are 4.4 times and 2.0 times, respectively, as large as those in healthy controls. Furthermore, in contrast to nondialyzed chronic renal failure patients, we observed higher concentrations of Cu/Zn SOD in plasma and a more enhanced mRNA expression of Cu/Zn SOD in leukocytes of HD patients. Conclusion: Increased Cu/Zn SOD mRNA reflects enhanced antioxidant capacity of leukocytes and can be a promising oxidative stress marker in HD patients.

Crystal Structure and Superoxide Dismutase Activity of [Cu(ethylenediamine)2Cl][PF6

The preparation, spectroscopic properties, and crystal structure of chlorobis(ethylenediamine)copper(II) hexafluorophosphate [Cu(en)2Cl][PF6], (en=ethylendiamine) are reported. The complex crystallizes in the monoclinic system, space group P21/c, with cell constants a=6.1488(9) A, b=12.696(2) A, c=17.7424(17) A, β=97.265(12)°, and Z=4. The copper(II) ion is coordinated to two bidentate en molecules, to one chlorine ion, and to a more distant fluorine atom of the PF6 group, leaving the copper ion in a distorted octahedral coordination geometry. The superoxide dismutase mimetic activity of the complex was investigated using the indirect xanthine-xanthine oxidase- nitroblue tetrazolium method and compared to that of the native enzyme.

Comparative Intralobular Distribution of Low Km Aldehyde Dehydrogenase Activities in Rat and Guinea Pig Livers

To determine species differences in the hepatic lobular distribution of low Km aldehyde dehydrogenase (ALDH, ALDH 1+ALDH 2) activity (substrate, 50 μM acetaldehyde), the activity in Wistar rats and Hartley guinea pigs was examined histochemically by the nitroblue tetrazolium method. In the rat liver, low Km ALDH activity was observed throughout the lobules, with enhanced centrilobular staining. Interestingly, the guinea pig liver exhibited a U-shaped lobular distribution, with even localization in both the centrilobular and periportal zones, but with little activity in the midlobular region. These findings indicate that the distribution pattern of low Km ALDH activity in the hepatic lobule of guinea pigs differs strikingly from that of rats, hamsters and mice. This study shows that rodents, which are widely utilized as experimental animals, differ as much in the hepatic lobular distribution of low Km ALDH activity as in that of ADH activity, indicating that the species most suitable to the purpose of ethanol studies should be selected according to the profiles of the liver ADH and low Km ALDH activities.

Antioxidative Properties of Extracts from Ancient Rice Brans

In this study, the superoxide dismutase (SOD)-like activity and the ROS (superoxide anion, hydroxyl radical, singlet oxygen, t-butylperoxyl radical) scavenging activities were evaluated for the extracts from 2 varieties (black and red rices) of ancient rice brans (8 types) by the nitro-blue tetrazolium (NBT) and ESR-spin trapping methods. All the extracts from ancient rice brans (black and red rice) had SOD-like activity, which was stronger than those from present-day rice brans (Koshihikari, Akitakomachi, Haenuki). It was proven by NBT method that the activities of ancient rice brans were significantly stronger than those of L-ascorbic acid, however, L-ascorbic acid exhibited higher scavenging activity as evaluated by ESR spin-trap method. The ancient rice brans have remarkably strong ROS scavenging activities compared with those of the present-day rice brans (Koshihikari, etc.). The ROS scavenging activities and SOD-like activities of the extracts varied depending on the rice species and the planting region. In addition, the extracts from ancient rice brans inhibited the Maillard reaction, which is known to be involved in physiological aging processes. Thus, the present results suggest the utility of the extracts from ancient rice brans as antioxidative materials.

Age-related changes in the glycation of human aortic elastin

Non-enzymatic glycation of proteins is a consequence of hyperglycemia in diabetes and correlates with aging. The aim of the study was to investigate age-related changes in the glycation of human aortic elastin in healthy subjects by two approaches: (1) assessment by fluorescence method of formed in vivo advanced glycation end products (AGEs) of elastins, purified from human aortas, obtained from different age groups; (2) in vitro glycation of elastins from different age groups and investigation of their capacity to form early (by colorimetric nitroblue tetrazolium method) and AGEs (fluorescence method). Human insoluble elastins were prepared from macro- and microscopic unaltered regions of thoracic aortas, obtained from 68 accident victims, distributed in 15 age-groups, using the method of Starcher and Galione. Soluble α-elastins were obtained by the method of Partridge et al. The direct assessment of Maillard reaction related fluorescence in the age groups showed increase of the fluorescence with age. The ‘young’ elastin had the highest capacity to form both fructosamine and AGEs under glycation in vitro. The glycation of ‘old’ elastin did not increase markedly during the incubation. These results are consistent with the interpretation that because of its long biological half-life, elastin is susceptible to the slow process of glycation and the following modifications would contribute to the age-related changes of connective tissue.

Microscopic detection of reactive oxygen species generation in the compatible and incompatible interactions of Alternaria alternata Japanese pear pathotype and host plants

Reactive oxygen species (ROS) generation was examined in the interaction of Alternaria alternata Japanese pear pathotype and host plants using three methods: nitro blue tetrazolium (NBT) method for microscopic detection of O2 −, diaminobenzidine (DAB) methods for microscopic detection of H2O2, and cerium chloride methods for ultrastructural detection of H2O2. ROS generation was detected by NBT and DAB methods at appressoria on leaves of susceptible cultivars and heat-shocked leaves of resistant cultivars but not in leaves of resistant cultivars. Ultrastructural detection by the cerium chloride method identified ROS generation at cell walls of appressoria and penetration pegs in susceptible, resistant leaves and heat-shocked leaves. These differences in the ultrastructural and microscopic data in resistant areas were due to the restriction of ROS generation in limited areas, the side facing the plant surface, of appressoria and penetration pegs. Therefore, ROS generation was apparently induced regardless of the resistance or susceptibility of the cultivar with the difference being in the volumes generated. After evaluating the pathological role of ROS generation in fungal structures, such generation was found to be associated with early penetration of cell walls in pear plants. Additionally, ROS generation in plants was also found in degrading pectin layers near infected hyphae and in plasma membrane modification sites in susceptible leaves but not in resistant leaves. ROS generation in susceptible leaves might be accompanied with plasma membrane damage, although the role of ROS generation in the pectin layers is not clear. ROS generation in both fungal and plant cells during their interaction was likely associated with the expression of susceptibility.

Crystal structure of the first SH-containing tetrahedral cobalt(II) complex, Co(quinoline)2(SH)2]. Superoxide dismutase activity

The preparation, spectroscopic properties and crystal structure of the bis-quinoline–bis-mercaptocobalt(II) complex [Co(quinoline)2(SH)2] are reported. The complex is tetrahedral with nitrogen donor atoms from two quinoline ligands and sulphur donor atoms from two mercapto groups. The superoxide dismutase mimetic activity of the complex was investigated using the indirect xanthine–xanthine oxidase–nitroblue tetrazolium method and compared to that of the native enzyme.

Protective effect of ascorbic acid in experimental gastric cancer: reduction of oxidative stress.

Oxidative stress participates in the cell carcinogenesis by inducing DNA mutations. Our aim was to assess whether ascorbic acid, an antioxidant, could have a role in preventing ROS (Reactive Oxygen Species) generation in experimental gastric carcinoma in a rat model. Experimental gastric cancer was induced in twelve Wistar male rats (weighting 250-350 g) by profound duodeno-gastric reflux throught split gastrojenunostomy. The rats were allocated to the following groups: Group I (n=6) was the control; Group II (n=6) which was mantained with daily intake of tape water with Vitamin C (30 mg/Kg). After 6 or 12 months, samples of gastric tumor or non tumor mucosa were taken from the anastomosis of both groups. Oxidative stress was measured by superoxide quantification through lucigenin-amplified chemiluminescence base and by staining with Nitrobluetetrazolium. The histopathologic confirmation of adenocarcinoma was made by eosin-hemathoxilin method. The intestinal type of gastric adenocarcinoma was microscopically identified in all animals of group I whereas only 3 rats of group II showed an adenocarcinoma without macroscopic evidence of them. The cancers were located in the anastomosis in all cases. Basal luminescence from tumor gastric tissue generated 38.4+/-6.8 count per minute/mg/X10(6) (mean+/-SD) and 14.9+/-4.0 count per minute/mg/X10(6), respectively, in group I and II animals (P<0.05). The Nitrobluetetrazolium method showed intense staining in tumor tissues but not in non neoplasic mucosa. Experimental gastric tumors seem to produce more reactive oxygen species than non neoplasic gastric tissue. The reduction of oxidative stress and gastric tumor incidence in rats were induced by the intake of ascorbic acid. Therefore, it may have a role in the prevention of gastric carcinoma.

Erythrocyte membrane and cytoskeletal protein glycation and oxidation in short-term diabetic rabbits.

The objective of this study was to elucidate the glycation and oxidation processes in plasma and erythrocyte membrane proteins as well as the major erythrocyte cytoskeletal protein, spectrin, using a short-term experimental rabbit diabetes model. Diabetes was induced with a single-dose alloxan injection. Spectrin was purified from erythrocyte ghosts with selective solubilization followed by gel filtration chromatography techniques, and tested for purity using sodium dodecyl sulfate-poly-acrylamide gel electrophoresis. Glycation in plasma proteins was measured as fructosamine using the nitroblue tetrazolium method, and in erythrocyte membrane and purified spectrin, as ketoamine equivalents, by the hydrazine/phenylhydrazine method. Protein oxidation in plasma, erythrocyte membrane proteins, and purified spectrin was evaluated in terms of sulfhydryl oxidation, based on cis-dichlorodiammine platinum (II) binding. Carbonyl formation was also measured in plasma and membrane proteins. Sulfhydryl oxidation, carbonyl groups and glycated protein levels showed statistically significant differences between the diabetic and control groups for both the plasma and the erythrocyte membrane proteins. The cis-dichlorodiammine platinum (II) binding was significantly different in diabetic rabbit erythrocyte spectrin, while glycation was not significantly different for this protein. Our data clearly demonstrate that both protein glycation and oxidation are biochemical alterations occurring in diabetes, even of short duration.

Effect of Microenvironment Created by Surfactant on Superoxide Dismutase Activity

The activity of SOD (superoxide dismutase) catalyzing dismutation of superoxide radical anion O2 − was studied by a modified NBT (nitroblue tetrazolium) method in aqueous solution, micellar solutions and lamellar liquid crystals (LLC) consisting of the surfactants CTAB and TX-100. The experiments showed that the inhibition by SOD of NBT reduction in organized assemblies of surfactants was weaker than in aqueous solution. The order of inhibition was as follows: in phosphate buffer (pH = 7.8) > in TX-100 micelle > in CTAB micelles > in TX-100 LLC > in CTAB LLC. The results were explained by decrease of α-helical content in SOD and difficulty in the transformation of SOD configurations in organized assemblies of surfactant, as well as by catalytic effects of micelles on NBT reduction.

Clinical value of fructosamine measurements in non-healthy dogs.

Ninety-three unhealthy dogs (including some with diabetes mellitus or insulinoma) of different ages, sex and breeds were divided into 10 groups according to their pathology. Serum fructosamine concentrations were determined using a commercial colorimetric nitroblue tetrazolium method. Diabetic dogs had the highest fructosamine concentrations (454.85 +/- 149.34 micromol/L). Dogs with insulinoma had significantly lower fructosamine concentrations (202.80 +/- 31.22 micromol/L), similar to those with leishmaniosis (202.83 +/- 99.83 micromol/L). Fructosamine concentrations in non-healthy dogs, except those with diabetes mellitus, insulinoma or leishmaniosis, were within the reference limits previously reported.

Cytokine, elastase and oxygen radical release by Fusobacterium nucleatum-activated leukocytes: a possible pathogenic factor in periodontitis.

Periodontitis is characterised by tissue destruction caused by reactive oxygen species (ROS) and proteolytic enzymes, which are released by the interaction between bacteria and phagocytes. We estimated the ability of Fusobacterium species to induce release of tissue destructive and proinflammatory mediators from in vitro activated peripheral leukocytes. ROS was measured with the nitroblue tetrazolium (NBT) method, elastase with a specific chromogenic substrate and cytokines, including interleukin 1beta (IL-1beta), tumour necrosis factor alpha (TNF-alpha), and interleukin 8 (IL-8) with a sandwich ELISA method. Various clinical isolates of unopsonized Fusobacterium species stimulated the neutrophils to an increased NBT- reduction. IL-1beta, TNFalpha, IL-8 and elastase were released in significantly higher levels from neutrophils stimulated by Fusobacterium species. In conclusion, unopsonized Fusobacterium species can induce increased production of oxygen radicals, cytokines and elastase from leukocytes activated in vitro.

Intralobular distribution of class I alcohol dehydrogenase and aldehyde dehydrogenase 2 activities in the hamster liver.

The hepatic lobular localization of class I alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) 2 activities was examined histochemically using livers of hamsters with high ethanol preferences. The activity of class I ADH detected by the nitro blue tetrazolium method using 5 mM ethanol as a substrate was extremely high and was almost homogeneously distributed throughout the lobule. The ALDH 2 activity (substrate, 8 microM acetaldehyde) was localized to the centrilobular zone, whereas low Km ALDH (ALDH 1 + ALDH 2) activity (substrate, 50 microM acetaldehyde) showed a gradient distribution in the lobule with high centrilobular to moderate periportal activity, suggesting that the ALDH 1 activity was distributed throughout the lobule.

Some Properties of the H2O2/O2− Generating System from the Lignifying Xylem of Zinnia elegans

The properties of the enzymatic system responsible for generating H2O2/O2- in the lignifying xylem of Zinnia elegans were studied using the starch/KI method for monitoring H2O2 production and the nitroblue tetrazolium method for monitoring superoxide anion production. The results showed that H2O2/O2- production by lignifying xylem tissues was insensitive to inhibitors of peroxidase and poly(di)amine oxidases. However, H2O2/O2 production in the xylem of Z. elegans was sensitive to the inhibitors of phagocytic plasma membrane NADPH oxidase, pyridine, imidazole, quinacrine and diphenylene iodonium. The sensitivity of H2O2/O2- production to the respective inhibitors of calmodulin (R-24571), phospholipase C (neomycin sulfate), and protein kinase (staurosporine), and its reversion by an inhibitor of protein phosphatases (cantharidin); pointed to the analogies existing between the mechanism of H2O2/O2- production in the lignifying xylem of Z. elegans and the oxidative burst observed during the hypersensitive plant cell response. These results suggest the existence of a metabolic cascade involving calmodulin, IP3 and protein phosphorylation in the activation of the enzymatic system responsible for H2O2/O2- production in the lignifying xylem of Z. elegans.

The generation of H 2 O 2 in the xylem of Zinnia elegans is mediated by an NADPH-oxidase-like enzyme

The nature of the enzymatic system responsible for the generation of H2O2 in the lignifying xylem of Zinnia elegans (L.) was studied using the starch/KI method for monitoring H2O2 production and the nitroblue tetrazolium method for monitoring superoxide production. The results showed that lignifying xylem tissues are able to accumulate H2O2 and to sustain H2O2 production. Hydrogen peroxide production in the xylem of Z. elegans was sensitive to pyridine, imidazole, quinacrine and diphenylene iodonium, which are inhibitors of phagocytic plasma-membrane NADPH oxidase. The sensitivity of H2O2 production to the inhibitor of phospholipase C, neomycin, and to the inhibitor of protein kinase, staurosporine, and its reversion by the inhibitor of protein phosphatases, cantharidin, pointed to the analogies existing between the mechanism of H2O2 production in lignifying xylem and the oxidative burst observed during the hypersensitive plant cell response. A further support for the participation of an NADPH-oxidase-like activity in H2O2 production in lignifying xylem was obtained from the observation that areas of H2O2 production were superimposed on areas producing superoxide anion, the suspected product of NADPH oxidase, although attempts to demonstrate the existence of superoxide dismutase activity in intercellular washing fluid from Z. elegans were unsuccessful. Even so, the levels of NADPH-oxidase-like activity in microsomal fractions, and of peroxidase in intercellular washing fluids, are consistent with a role for NADPH oxidase in the delivery of H2O2 which may be further used by xylem peroxidases for the synthesis of lignins. This hypothesis was further confirmed through a direct histochemical probe based on the H2O2-dependent oxidation of tetramethylbenzidine by xylem cell wall peroxidases. These results are the first evidence for the existence of an NADPH oxidase responsible for supplying H2O2 to peroxidase in the lignifying xylem of Z. elegans.

漢方製剤のスーパーオキシド消去作用について

The superoxide anion scavenging activity of traditional Chinese medicines (Kampo) was estimated by the nitrate and nitrobluetetrazolium methods. No.15 and 34 revealed a strong superoxide anion scavenging activity and No.1, 7, 19, 25 and 45 revealed a relatively strong superoxide anion scavenging activity by nitrate method. On the other hand the nitrobluetetrazolium method revealed a relatively strong activity in No.1, 15, 25, 45 and 96. In particular, both methods denoted a relatively strong superoxide anion scavenging activity in No.1, 15, 25 and 45. These results suggest that the superoxide anion scavenging activity of each component accumulatively contribute to those Kampo medicines and might play an anti-inflamatory effect and “Ku-Oketsu” (improved blood circulation) effect in these medicines.

Histochemical Localization of Alcohol Dehydrogenase Activities in the Livers of Mice, Rats, Hamsters, and Guinea-Pigs.

The hepatic lobular localization of alcohol dehydrogenase (ADH) activity was examined histochemically in mice, rats, hamsters and guinea-pigs with the nitro blue tetrazolium method. In the mouse liver, the ADH activity was localized mainly in the centrilobular zone. In the rat and hamster livers, it was observed throughout the lobule, with increased centrilobular staining. Interestingly, in the guinea-pig liver, it was found to be evenly localized in both the centrilobular and periportal zones, with little or no midlobular staining. These findings show that the distribution pattern of ADH activity in the hepatic lobule of the guinea-pigs differs strikingly from that of the mice, rats and hamsters.