Nitroblue Tetrazolium Reduction Method Research Articles

Activation of human neutrophils by dotriacontane from Tynanthus micranthus Corr. Méllo (Bignoniaceae) for the production of superoxide anions

The species Tynanthus micranthus Corr. Méllo, is popularly known as "Cipó Cravo" or "Craveiro", and is usually used as a stimulant and aphrodisiac, and found in the north of the State of Paraná - Brazil. The isolate the alkane dotriacontane from T. micranthus and to verify its action on the activation of human neutrophils to produce superoxide anions by the nitroblue tetrazolium reduction method (NBT), in comparison with the activation obtained by commercial dotriacontane. The ethanolic extract dried leaves was prepared by modified Soxhlet apparatus. Then the ethanolic extract was fractionated with n-hexane to obtain the hexane fraction which was submitted to the chromatographic column to obtain the compound dotriacontane. The substance was identified by 1H and 13C NMR spectroscopy. Isolated and commercial dotriacontane were evaluated by the NBT assay for verification of the activation of human neutrophils. The isolated and commercial dotriacontane activated neutrophils (10.0 μg/mL) when compared to phorbol myristate acetate (PMA) (10.0 μg/mL). In the quantitative test commercial dotriacontane induced significant activation when compared to PMA (10.0 μg/mL). The substance dotriacontane, first time identified in the genus Tynanthus. The dotriacontane was shown to be important to confirm the activation of human neutrophils to produce superoxide anions.

Two new flavonol glycosides and biological activities of Diplotaxis harra (Forssk.) Boiss.

Two new flavonol glycosides, isorhamnetin 3-O-β-glucopyranoside-4′-O-β-xylopyranoside (1) and kaempferol 3-O-β-glucopyranoside -4′-O-β-xylopyranoside (2), were isolated from the defatted aqueous methanol extract of the whole plant Diplotaxis harra along with 12 known flavonols (3–14). They were characterised by chemical and spectral methods. The 70% aqueous methanol, chloroform and defatted aqueous methanol plant extracts exhibited significant antioxidant effects (nitroblue tetrazolium reduction method). Their cytotoxic activity was carried out against 11 tumour cell lines (sulphorhodamine B assay). The three extracts expressed the greatest antiproliferative activity against colon 38, P388 and MKN-28 with GI50 (0.45, 0.4, 0.07 μg/mL) and against P388 [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay] with IC50 (0.26, 0.24, 0.25 μg/mL), respectively. The chloroform extract showed the highest activity as eukaryotic DNA topoisomerase II inhibitors of P388 with IC50 0.24 μg/mL. Antiviral screening of the extracts and the pure compounds against foot-and-mouth disease virus types A and O revealed a prominent inhibition of its cytopathic effect.

Vinpocetine and α‐tocopherol prevent the increase in DA and oxidative stress induced by 3‐NPA in striatum isolated nerve endings

Vinpocetine is a neuroprotective drug that exerts beneficial effects on neurological symptoms and cerebrovascular disease. 3-nitropropionic acid (3-NPA) is a toxin that irreversibly inhibits succinate dehydrogenase, the mitochondrial enzyme that acts in the electron transport chain at complex II. In previous studies in striatum-isolated nerve endings (synaptosomes), we found that vinpocetine decreased dopamine (DA) at expense of its main metabolite 3,4-dihydroxyphenylacetic acid (DOPAC), and that 3-NPA increased DA, reactive oxygen species (ROS), DA-quinone products formation, and decreased DOPAC. Therefore, in this study, the possible effect of vinpocetine on 3-NPA-induced increase in DA, ROS, lipid peroxidation, and DA-quinone products formation in striatum synaptosomes were investigated, and compared with the effects of the antioxidant α-tocopherol. Results show that the increase in DA induced by 3-NPA was inhibited by both 25μM vinpocetine and 50μM α-tocopherol. Vinpocetine, as α-tocopherol, also inhibited 3-NPA-induced increase in ROS (as judged by DCF fluorescence), lipid peroxidation (as judged by TBA-RS formation), and DA-quinone products formation (as judged by the nitroblue tetrazolium reduction method). As in addition to the inhibition of complex II exerted by 3-NPA, 3-NPA increases DA-oxidation products that in turn can inhibit other sites of the respiratory chain, the drop in DA produced by vinpocetine and α-tocopherol may importantly contribute to their protective action from oxidative damage, particularly in DA-rich structures.

Effect of Lifestyle and Health Behavior on Neutrophil Function -Assessed by Nitroblue Tetrazolium Reduction Method Using Neutrophils in Elderly in Tianjin, China-

In this study, we investigated the relationships of neutrophil functions with lifestyle factors (namely, subjective stress, exercise habits, smoking habits, alcohol-drinking habits, and self-perceived status health) and health behavior in the Chinese urban elderly. We performed a health survey of the elderly aged 65 years or older living in Tianjin. The subjects were 42 males (69.1 ± 4.1 years old) and 41 females (69.1 ± 4.1 years old). Investigations of subjective stress, exercise habits, smoking habits, alcohol-drinking habits, and self-perceived health status were performed. The phagocytosis and superoxide productivity of neutrophils were measured by the nitroblue tetrazolium (NBT) reduction method. In addition, leukocyte count and serum total protein (TP) level were examined. The investigations revealed the associations of health behavior (p<0.05) and self-perceived health (p<0.10) with the balance between phagocytosis and subsequent superoxide production. The present study revealed that there were correlations of neutrophil functions with lifestyle factors (subjective stress, exercise habits, smoking habits, alcohol-drinking habits, and self-perceived status health) and health behavior in the Chinese urban elderly.

247 ADENOSINE TRIPHOSPHATE CONTENT AND SUPEROXIDE DISMUTASE ACTIVITY IN SINGLE OOCYTES BEFORE AND AFTER IN VITRO MATURATION

The developmental competence of in vitro-produced embryos is strictly related to oocyte quality. Analyses of energy and redox status parameters are emerging technologies useful for further oocyte quality characterisation. Mitochondrial (mt) activity is a necessary feature involved in cytoplasmic maturation, and the primary function of mitochondria is adenosine triphosphate (ATP) production. Mitochondria distribution pattern and ATP content are important parameters in the evaluation of oocyte metabolic activity, particularly activities driving microtubules dynamics leading to chromosomes segregation. Superoxide dismutase (SOD), a first-line antioxidant enzyme, has also been hypothesised as being associated to oocyte quality. The aim of the present study was to analyse ATP content and SOD activity in single equine oocytes examined before and after in vitro maturation. Cumulus–oocyte complexes surrounded by a compact cumulus oophorus were recovered from the ovaries of slaughtered mares and analysed before or after in vitro maturation (Ambruosi et al. 2009 Theriogenology 71, 1093–1104). After cumulus cell removal, all oocytes underwent evaluation of signs of meiotic maturation, and only those oocytes showing cumulus expansion, regular ooplasmic size (&gt;160 μm in diameter) and morphology, and 1st polar body extrusion were selected for analysis. Adenosine triphosphate intracellular levels were analysed by luciferin-luciferase bioluminescent reaction (ATPlite, PerkinElmer, Monza, Italy). Quantification of SOD activity was performed by spectrophotometrical assay with WST1 and by polyacrylamide native gel and nitro blue tetrazolium reduction method. Intracellular ATP levels were influenced by meiotic stage in that oocytes at the germinal vesicle stage (GV, n = 15) showed 1.25 ± 0.8 pmol cell–1, whereas metaphase II (MII) oocytes (n = 15) showed significantly higher levels (2.29 ± 1.69 pmol cell–1; P &lt; 0.05). This is in line with our previous observations on mt distribution pattern analysed by Mitotracker Orange CMTM Ros staining and confocal microscopy (Ambruosi et al. 2009). In vitro-matured MII oocytes showed significantly higher rates of perinuclear mt distribution pattern, indicating mt aggregation around meiotic metaphase spindle, compared with GV oocytes (3/12, 25% v. 0/13, 0% in GV oocytes; P &lt; 0.05). Superoxide dismutase spectrophotometrical activity was 0.72 ± 0.55 U mg–1 prot in GV oocytes (n = 4) and 2.33 ± 0.33 U mg–1 prot in MII oocytes (n = 2; P &lt; 0.001). In native gel SOD activity was 16 285.05 arbitrary densitometric units (ADU) in a GV oocyte and 22 501.35 ADU in a MII oocyte. To our knowledge, this is the first study reporting intracellular SOD activity in single oocytes in mammals. Moreover, this is the first study reporting ATP content in single equine oocytes. Observed quantitative differences seem to be related to meiotic stage. Financial support was provided by MIUR PRIN 2007 Project Quota di Ateneo University of Bari, Italy, Resp. Sci. Pro M. E. DellAquila (prot. 2007S75KSE_003).

Respiratory Burst Assay of Head Kidney Macrophages of Ayu, Plecoglossus altivelis, Stimulated with Glugea plecoglossi (Protozoa: Microspora) Spores

The respiratory burst assay was conducted using the nitroblue tetrazolium reduction method and horseradish peroxidase method to investigate the events when ayu head kidney macrophages phagocytize fresh and formalin-killed Glugea plecoglossi spores. The production of O2 against G. plecoglossi spores was negligible compared to zymosan (P < 0.01). Zymosan-induced O2 production was markedly inhibited by adding G. plecoglossi spores simultaneously (P < 0.01). This phenomenon was dose dependent, and killed spores were less inhibitory than fresh spores. The production of H2O2 was drastically increased when G. plecoglossi spores were added (P < 0.01), and most spores were phagocytized. From these results, it is suggested that G. plecoglossi spores modulate the host's phagocytic response to establish infection.

Participation of Reactive Oxygen Species in Phototoxicity Induced by Quinolone Antibacterial Agents

To elucidate the mechanism of phototoxicity induced as a side effect by some of the new quinolone antibiotics, we studied sparfloxacin (SPFX), lomefloxacin, enoxacin, ofloxacin, and ciprofloxacin. We first examined the photosensitized formation of reactive oxygen species such as singlet oxygen (1O2) and superoxide anion (O−2) mediated by the new quinolones. Although a large number of studies have been reported, there is no direct evidence that these drugs generate reactive oxygen species. We employed a near-infrared emission spectrometer to detect1O2-specific emission (1268 nm), and the nitroblue tetrazolium reduction method to detect O−2. All the quinolones investigated in this study were found to produce1O2. Four drugs, but not SPFX, produced O−2. We also examined photodynamic DNA strand-breaking activity as a possible mechanism to explain the participation of reactive oxygen species in the phototoxicity of the drugs. All the drugs exhibited photodynamic DNA strand-breaking activity. The inhibitory effect of scavengers of reactive oxygen species indicated that the main active species was1O2. The DNA strand-breaking activity was correlated not with the1O2-forming ability, but with the affinity of the drugs for DNA. This result may be due to the short lifetime of1O2. These data suggested that the phototoxicity of the new quinolones was related to DNA damage caused by reactive oxygen species, especially1O2.

Studies on the mechanism of mutagenicity and genotoxicity induced by dihydralazine.

Dihydralazine was found to be mutagenic towards S. typhimurium TA1537, TA97, TA1538 and TA98 and genotoxic towards E. coli PQ37. Using the nitro blue tetrazolium reduction method we have found that dihydralazine can generate active oxygen species. The possible role of active oxygen species in mutagenicity (Ames test) and genotoxicity (SOS Chromotest) of dihydralazine was studied by testing the influence of the different active oxygen species scavengers on these two processes. Of the active oxygen scavengers tested, only superoxide dismutase suppressed partially the mutagenic and genotoxic activity of dihydralazine. This result seems to indicate that superoxide anion play a role in these two biological events.

Radical trapping by PBN during reperfusion in rabbit gastric mucosa.

Oxygen free radicals have been considered as a cause of ischemia-reperfusion injury in several organs, but this injury in a stomach, containing acid, may progress to severe damage. Thus, we examined the effect of ischemia-reperfusion and milk-intake on rabbit gastric mucosa. The gastric mucosal blood flow was increased after milk-intake, and gastric rupture was detected. Superoxide dismutase activity measured by an improved nitroblue tetrazolium reduction method and thiobarbituric acid reactive substances in serum is increased during ischemia-reperfusion and milk-intake. By using alpha-phenyl N-tert-butyl nitrone (PBN) as a spin trap and electron paramagnetic resonance (EPR), we detected lipidic radicals from tissue samples in chloroform-methanol solvent only during reperfusion and milk-intake period; no signal was detected before. The EPR signal of spin adducts obtained in the sample after ischemia-reperfusion and milk-intake would be a mixture of peroxyl and alkoxyl radicals from the analysis of coupling constants.

Fructosamine. A new parameter for diagnosis and metabolic control in diabetic dogs and cats.

Fructosamines are glycated serum proteins that, depending on their life span, reflect glycemic control over the previous 2 to 3 weeks. The nitroblue tetrazolium reduction method adapted to auto analysis appeared to be a practical means to assay fructosamine quickly, economically, and accurately. The upper limit of the reference range is 374 mumol/L in dogs (95% percentile) and 340 mumol/L in cats (95% percentile). Newly diagnosed diabetic dogs and cats that had not undergone previous insulin therapy had significantly higher fructosamine concentrations than nondiabetic animals. In diabetic dogs that were receiving insulin therapy, the fructosamine test reflected the glycemic state far more accurately than did individual blood glucose measurements. Animals with satisfactory metabolic control revealed fructosamine concentrations within the reference range, whereas fructosamine concentrations above 400 mumol/L indicated insufficient metabolic control. On the basis of fructosamine concentrations, cats with a transitory hyperglycemia and cats with diabetes mellitus were differentiated. The fructosamine test is a valuable parameter for the diagnosis and metabolic control of diabetes mellitus in dogs and cats.

Evaluation of the fructosamine test in obesity: consequences for the assessment of past glycemic control in diabetes

The effect of obesity in diabetic and nondiabetic states on serum fructosamine levels, as measured by the nitro blue tetrazolium reduction method, was investigated. In 26 nondiabetic obese subjects, the mean (SD) fructosamine (1.78 +/- 0.16 mmol/L) and protein corrected fructosamine concentrations (25.7 +/- 2.5 mumol/g) were significantly lower than in nondiabetic lean control subjects (2.06 +/- 0.18 mmol/L and 30.5 +/- 2.5 mumol/g, respectively; p less than 0.01). Hemoglobin A1C, blood glucose and serum protein concentrations were normal in obese subjects. Interference from hypertriglyceridemia, hemolysis, or drugs was excluded. In diabetic subjects, fructosamine correlated with hemoglobin A1C, but the least-squares regression lines were different in 16 nonobese and in 19 obese patients, so that for the same hemoglobin A1C value, fructosamine level was 16% lower in obese compared to nonobese diabetic subjects. In vitro studies showed a significant decrease in 14C-glucose incorporation in serum proteins of obese nondiabetic subjects compared to control subjects. Similarly, the rate of formation of fructosamine in sera of obese nondiabetic subjects incubated with 12 mmol/L and 30 mmol/L glucose concentrations was slower than in sera of control subjects. In conclusion, fructosamine is underestimated in obesity, both in diabetic and nondiabetic patients, and its validity as an index of glycemic control may be impaired in obese subjects. This decrease is due to an alteration in the glycation process itself.

Fructosamine: structure, analysis, and clinical usefulness.

Glucose molecules are joined to protein molecules to form stable ketoamines, or fructosamines, through glycation, a nonenzymatic mechanism involving a labile Schiff base intermediate and the Amadori rearrangement. The amount of fructosamine in serum is increased in diabetes mellitus owing to the abnormally high concentration of sugar in blood. The concentration of fructosamine in serum thus reflects the degree of glycemic control attained by the diabetic patient and is useful in monitoring the effectiveness of therapy in diabetes over a period of several weeks, in a manner analogous to the determination of glycated hemoglobin. Of the analytical approaches used to measure fructosamine, affinity chromatography with m-aminophenylboronic acid and the nitroblue tetrazolium reduction method appear to be the most practical means for clinical chemists to assay fructosamine quickly, economically, and accurately. Fructosamine values can readily distinguish normal individuals and diabetic patients in good glycemic control from diabetics in poor control. Unlike glycated hemoglobin, which reflects the average blood sugar concentration over the past six to eight weeks, fructosamine reflects the average blood sugar concentration over the past two to three weeks. Thus a clinical advantage is that fructosamine responds more quickly to changes in therapy, thereby allowing for improved glycemic control. Used in conjunction with determinations of blood sugar and (or) of glycated hemoglobin, or by itself, the fructosamine assay can provide clinically useful information for the detection and control of diabetes.

Naphthalene oxygenase fromCorynebacterium renale: Characterisation and mechanism of oxygenation

The formation ofcis-l,2,-dihydroxy-l,2,-dihydronaphthalene from naphthalene by naphthalene oxygenase, purified fromCorynebacterium renale ATCC 15075, was demonstrated to involve oxidation of a mol NADH and consumption of one mol oxygen. The enzyme contains one g-atom Fe2+ and one FAD. Catalase inhibited product formation and H2O2 could substitute for NADH in the reaction. Superoxide dismutase inhibited enzyme activity when either NADH or H2O2 was present; the generation of superoxide anion on addition of NADH to the enzyme, in the absence of naphthalene, was detected by the nitro blue tetrazolium reduction method. Hydroxyl radical scavengers, ethanol, mannitol and sodium benzoate, inhibited product formation when either NADH or H2O2 was present. Electron spin resonance studies, under aerobic conditions, indicated that iron of the enzyme underwent valence changes during the course of the reaction