1-methoxyphenazine Methosulfate Research Articles

MTT FORMAZAN REPLACED WST-8 AS A BETTER SIMPLE SCREENING METHOD FOR DETECTION OF GLUCOSE-6-PHOSPHATE DEHYDROGENASE DEFICIENCY

We have previously developed the WST-8 method as a simple and rapid screening test for detection of glucose-6-phosphate dehydrogenase (G6PD) deficiency accomplished by the naked eye. However, it was little difficult to distinguish between faint orange colors developed by heterozygous females and pink colors of normal hemolyzed blood, since both have similar tones. To solve this problem, we established a new and simple screening method that utilizes another formazan substrate, MTT (3-(4,5-dimethyl-2- thiazolyl)-2,5-diphenyl-2H tetrazolium bromide) in combination with a hydrogen carrier, 1-methoxy phenazine methosulfate. MTT formazan exhibits a purple color, thus allowing for the ability to easily distinguish the pink colors of hemolyzed blood. However, MTT has been reported to react with hemoglobin non-specifically and to interfere with the interpretation of the color reaction. In our examinations by mixing MTT with hemolyzed blood, we found that the non-specific reaction was very slow, and that the addition of a small amount of blood (5~10 μl) into a reaction mixture (800 μl) did not interfere with the reaction of G6PD activity. In this new MTT method, a strong purple color was generated in normal blood samples at 20~30 min after incubation, which could be distinguished by the naked eye from G6PD-deficient blood samples with less than 50% residual activity. In addition, quantitative measurement using a spectrophotometer was also possible despite the fact that MTT formazan is water-insoluble.

Improved Electrochemical Analysis of Neuropathy Target Esterase Activity by a Tyrosinase Carbon Paste Electrode Modified by 1-Methoxyphenazine Methosulfate

A graphite-paste tyrosinase biosensor was improved by adding 1-methoxyphenazine methosulfate as a mediator. Mediator modification enhanced sensitivity to phenol 4-fold and long-term stability 3-fold. Phenol could be detected at 25 nM (S/N = 2) using an Ag/AgCl reference electrode. The biosensor was used to measure the activity of a toxicologically significant enzyme, neuropathy target esterase (NTE), which yields phenol by hydrolysis of the substrate, phenyl valerate. Using the new biosensor, blood and brain NTE inhibition by organophosphorus (OP) compounds with different neuropathic potencies were well correlated (r = 0.990, n = 7), supporting the use of blood NTE as a biochemical marker of exposure to neuropathic OP compounds.

Measurement of cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction activity and lactate dehydrogenase release using MTT.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay and lactate dehydrogenase (LDH) release assay have been widely used for evaluating cell viability in culture. MTT reduction assay measures the redox activity of living cells, while LDH assay measures the activity of LDH released into the medium from dead cells. In this paper, we introduce a quick and simple method of measuring cellular MTT reduction and LDH release with the same dye, MTT. The substrate mixture for measuring LDH activity contained lactate, beta-nicotinamide adenine dinucleotide, 1-methoxyphenazine methosulfate, MTT and Triton X-100. When the medium containing LDH was mixed with the substrates, MTT was converted into MTT formazan in proportion to LDH activity. This method was successfully applied for evaluating t-butyl hydroperoxide toxicity in cultured rat cortical astrocytes and glutamate toxicity in cultured rat hippocampal neurons. Our method is economical and convenient especially for measuring cellular MTT reduction and LDH release in the same culture.

Superoxide produced by activated neutrophils efficiently reduces the tetrazolium salt, WST-1 to produce a soluble formazan: a simple colorimetric assay for measuring respiratory burst activation and for screening anti-inflammatory agents

Activation of the respiratory burst of granulocytes and macrophages by invading microorganisms is a key first line cellular defence against infection. Failure to generate this response leads to persistent life-threatening infection unless appropriate antibiotic treatment is given. The respiratory burst of neutrophils is usually measured spectrophotometrically by following ferricytochrome c reduction, and histologically by using the tetrazolium salt, nitroblue tetrazolium, which is reduced intracellularly to an insoluble formazan. In both assays, reduction is mediated by superoxide generated via NADPH oxidase. Because ferricytochrome c has a high molecular mass and high background absorbance at 550 nm, the assay lacks sensitivity and is not ideally suited to microplate measurement. We have circumvented these limitations by using the cell-impermeable, sulfonated tetrazolium salt, WST-1, which exhibits very low background absorbance and is efficiently reduced by superoxide to a stable water-soluble formazan with high molar absorptivity. This has permitted adaptation of the WST-1 assay to microplate format while retaining sensitivity. Reduction of WST-1 by activated human peripheral blood neutrophils correlated closely with ferricytochrome c reduction across a range of PMA concentrations and with time of activation by PMA and fMLP. Reduction of WST-1 was inhibited by 98% by superoxide dismutase (20 μg/ml) and by 88% by the NADPH oxidase inhibitor, diphenyleneiodinium (10 μM) but was resistant to catalase, azide and the NADH oxidase inhibitor, resiniferatoxin. WST-1 and ferricytochrome c reduction were also compared using xanthine/xanthine oxidase to generate superoxide. Under optimised assay conditions, both WST-1 and ferricytochrome c reduction were directly proportional to added xanthine. WST-1 generated approximately 2-fold greater increase in absorbance than ferricytochrome c at their respective wavelengths, and this translated into increased assay sensitivity. Addition of the intermediate electron acceptor, 1-methoxy phenazine methosulfate, increased the background of the neutrophil assay but did not affect the overall magnitude of the response. We have used the WST-1 assay to assess human neutrophil dysfunction and to compare anti-inflammatory activity.

Quantitative in situ analysis of xanthine oxidoreductase activity in rat liver.

The tetrazolium salt method previously developed for the detection of xanthine oxidoreductase activity in unfixed cryostat sections has been validated for quantitative purposes. The specificity of the enzyme reaction was studied by incubating unfixed cryostat sections of rat liver in test medium containing the substrate hypoxanthine, in control medium that lacked the substrate, and in medium containing substrate and allopurinol, a specific inhibitor of xanthine oxidoreductase activity. The specific reaction rate was determined cytophotometrically by subtracting the amount of final reaction product generated in the control reaction from that formed in the test reaction. Highest specific enzyme activity in rat liver was found when the incubation medium contained 18% (w/v) polyvinyl alcohol, 100 mM phosphate buffer, pH 7.8, 0.45 mM 1-methoxyphenazine methosulfate, 5 mM tetranitro BT, and 0.5 mM hypoxanthine. Enzyme activity was present in liver parenchymal cells and in sinusoidal cells (endothelial and Kupffer cells) and was completely inhibited by allopurinol. A linear relationship was observed between the specific amount of final reaction product generated at 37 degrees C and incubation time at least up to 21 min, as well as section thickness up to 12 microns. Xanthine oxidoreductase activity, expressed as mumoles substrate converted per cm3 tissue/min, was 1.61 +/- 0.34 in pericentral areas and 1.24 +/- 0.16 in periportal areas. These values are similar to biochemical data reported in the literature. In conclusion, the tetrazolium method to detect xanthine oxidoreductase activity in unfixed cryostat sections of rat liver gives a reliable reflection of in situ activity.

Homogeneous distribution of phosphofructokinase in the rat liver acinus: A quantitative histochemical study

A quantitative histochemical method was developed for the demonstration in rat liver of the activity of phosphofructokinase, one of the enzymes assumed to be rate-limiting for glycolysis. The procedure was based on the reduction of a tetrazolium salt as final electron acceptor and a multistep reaction using the exogenous or endogenous auxiliary enzymes aldolase, triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase. The highest activity was found in unfixed cryostat sections of rat liver when the incubation medium contained 17% (wt/vol) polyvinyl alcohol, 100 mmol/L Tris-maleate buffer (pH 8.4), 20 mmol/L fructose-6-phosphate, 2 mmol/L ATP, 2 mmol/L MgCl2, 5.9 mmol/L NAD+, 0.47 mmol/L 1-methoxyphenazine methosulfate, 5 mmol/L sodium azide and 5 mmol/L Nitro BT. The addition of auxiliary enzymes was not necessary to demonstrate maximum activity in rat liver. The specificity of the reaction was proven by the absence of any specific (test minus control) reaction when the incubation was performed in the presence of 25 mmol/L phosphoenolpyruvate, a competitive inhibitor of phosphofructokinase. Cytophotometric analysis revealed that linear relationships exist between the amount of specific reaction product formed and incubation time and the section thickness. The Km values for fructose-6-phosphate and the Vmax values were not significantly different in periportal and pericentral areas of livers from either normally fed or 24-hr-fasted rats. The homogeneous distribution of phosphofructokinase activity in the liver acinus is in line with biochemical findings using hepatocytes isolated from the two different areas showing that these cells contained similar amounts of enzyme activity. It indicates that either phosphofructokinase is not a rate-limiting enzyme of glycolysis or the capacity for glycolysis is not different in periportal and pericentral areas of the rat liver acinus. (HEPATOLOGY 1991;14:634–639.)

Homogeneous distribution of phosphofructokinase in the rat liver acinus: A quantitative histochemical study

A quantitative histochemical method was developed for the demonstration in rat liver of the activity of phosphofructokinase, one of the enzymes assumed to be rate-limiting for glycolysis. The procedure was based on the reduction of a tetrazolium salt as final electron acceptor and a multistep reaction using the exogenous or endogenous auxiliary enzymes aldolase, triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase. The highest activity was found in unfixed cryostat sections of rat liver when the incubation medium contained 17% (wt/vol) polyvinyl alcohol, 100 mmol/L Tris-maleate buffer (pH 8.4), 20 mmol/L fructose-6-phosphate, 2 mmol/L ATP, 2 mmol/L MgCl2, 5.9 mmol/L NAD+, 0.47 mmol/L 1-methoxyphenazine methosulfate, 5 mmol/L sodium azide and 5 mmol/L Nitro BT. The addition of auxiliary enzymes was not necessary to demonstrate maximum activity in rat liver. The specificity of the reaction was proven by the absence of any specific (test minus control) reaction when the incubation was performed in the presence of 25 mmol/L phosphoenolpyruvate, a competitive inhibitor of phosphofructokinase. Cytophotometric analysis revealed that linear relationships exist between the amount of specific reaction product formed and incubation time and the section thickness. The Km values for fructose-6-phosphate and the Vmax values were not significantly different in periportal and pericentral areas of livers from either normally fed or 24-hr-fasted rats. The homogeneous distribution of phosphofructokinase activity in the liver acinus is in line with biochemical findings using hepatocytes isolated from the two different areas showing that these cells contained similar amounts of enzyme activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of xanthine oxidoreductase activity using the tissue protectant polyvinyl alcohol and final electron acceptor Tetranitro BT.

We have detected xanthine oxidoreductase activity in unfixed cryostat sections of rat and chicken liver, rat duodenum, and bovine mammary gland using the tissue protectant polyvinyl alcohol, the electron carrier 1-methoxyphenazine methosulfate, the final electron acceptor Tetranitro BT, and hypoxanthine as a substrate. Enzyme activity was localized in rat duodenum at lateral membranes and brush borders of enterocytes and in goblet cells and mucus. Hepatocytes in pericentral areas and especially sinusoidal cells showed high activity in rat liver. Xanthine oxidoreductase was also detected in epithelial cells and milk lipid globules of lactating bovine mammary gland, which is known to contain large quantities of the oxidase form of the enzyme. Chicken liver, which contains an inconvertible dehydrogenase form, also showed high activity in sinusoidal cells. Therefore, we conclude that the tetrazolium reaction demonstrates both the dehydrogenase and the oxidase form of xanthine oxidoreductase. Control activity, in the absence of hypoxanthine or in the presence of the competitive inhibitor allopurinol, was low in all tissues studied. Addition of O2 or NAD to the incubation medium did not change the specific reaction in bovine mammary gland or chicken liver, implying that the dehydrogenase and the oxidase form are not dependent on their natural electron acceptors in this tetrazolium salt reaction. We conclude that the present light microscopic method gives specific and precise localization of xanthine oxidoreductase activity in situ.

Indoxyl-tetranitro blue tetrazolium method for detection of alkaline phosphatase in immunohistochemistry.

A sensitive method for detection of alkaline phosphatase in immunohistochemistry, using lymphoid cells, has been optimized. The conditions for staining are 0.23 mM 5-bromo-4-chloro-indoxyl phosphate, 0.55 mM tetranitro blue tetrazolium, 2.0 mM levamisole, 5.0 mM sodium azide, 10.0 mM magnesium chloride, and 0.15 mM 1-methoxyphenazine methosulfate dissolved in 100 mM Tris-HCl buffer, pH 9.5.

Fluorometric cyclic assays for pyridine nucleotides reduced or oxidized in enzyme-coupled assays

Sensitive fluorometric cyclic assays have been developed for pyridine nucleotides that are reduced or oxidized in enzyme-coupled assays. The cyclic assays are based on the regeneration of substrate being measured in order to achieve a measurable sustained rate of reaction which otherwise is not possible if the concentration of substrate being measured is so low that it would run out in less than a second. The use of resazurin reduction as an indicator reaction increases the sensitivity and hence the amount of catalyst needed to obtain high sensitivity is less. The resazurin reduction also introduces irreversibility in the cycling assay. The use of photostable 1-methoxyphenazine methosulfate as an electron mediator between resazurin and reduced pyridine nucleotides is introduced, which allows direct monitoring of the increase in fluorescence due to reduction of resazurin to highly fluorescent resorufin. Glucose-6-phosphate dehydrogenase and alcohol dehydrogenase induce specificity in the cycling assays for triphosphopyridine nucleotide and diphosphopyridine nucleotide, respectively. The assays are capable of detecting pyridine nucleotides as low as 100 fmol without difficulty and can be modified further to detect a much smaller amount if desired. As analyte models, glucose-6-phosphate dehydrogenase in erythrocytes and ATP in pure solution were determined. The cycling method used is capable of detecting 20 nU of glucose-6-phosphate dehydrogenase, which is roughly the amount of enzyme in 50 erythrocytes. It was also found that cycling is capable of determining 100 fmol of ATP.

The application of glucose oxidase-labeled antibodies for the detection of proteins on nitrocellulose

We have evaluated the sensitivity of immunostaining with glucose oxidase for the detection of monomeric human serum albumin (HSA) and monomeric human immunoglobulin G (IgG). A modification of a histochemical procedure was utilized by replacing phenazine methosulfate (PMS) with 1-methoxyphenazine methosulfate (mPMS) and by replacing Tris-HCl with Tris-citrate to improve the solubility of the tetrazolium compounds tested. mPMS is less sensitive to light, and may be stored for long period in solution; it is now used routinely by histochemists in place of PMS in dehydrogenase cytochemistry. pH values of 6.3–8.3 were tested, with the reaction at pH 8.3 providing a slight increase in sensitivity. The reaction rate increased markedly as the pH became more alkaline. The minimum quantity of HSA detected was 3 ng applied directly to nitrocellulose and 10 ng when blotted. Human IgG was routinely detected at 250 pg and occasionally at 100 pg when dotted on the nitrocellulose.