Neotetrazolium Chloride Research Articles

Diffusion during dehydrogenase reactions: the effects of intermediate electron acceptors.

A hamster cheek pouch model has been used to study the diffusion of reactants from the epithelium into adjacent muscle and connective tissue during the histochemical demonstration of glucose-6-phosphate dehydrogenase activity. The effects of the addition of intermediate electron acceptors to the incubation medium varied considerably from one acceptor to another, but were independent of the grade of polyvinyl alcohol incorporated into the medium. Menadione was the least effective intermediate both in transferring reducing equivalents from the primary dehydrogenase to Neotetrazolium chloride and in preventing diffusion. Phenazine methosulphate, Methylene Blue and Thionin were more efficient intermediates. Nevertheless, considerable diffusion occurred in the presence of Phenazine methosulphate, although very little diffusion was detectable with either of the thiazine dyes. It is suggested that these differences are related to different modes and sites of action of the carriers.

A modified tetrazolium reaction for identifying malignant cells from gastric and colonic cancer.

When non-malignant cells were reacted for glucose-6-phosphate dehydrogenase activity, with neotetrazolium chloride as the indicator of the activity, oxygen competed with the neotetrazolium and nullified the reaction. In contrast, about 30% of the activity was retained in malignant cells, in sections and in smears, from cancer of the stomach or colon. This could provide the basis of a qualitative (black-or-white) functional test for distinguishing malignant cells in these conditions.

The sensitivity to light of solutions of phenazine methosulphate: a quantitative cytochemical study.

The ability of phenazine methosulphate to transfer electrons from reduced coenzymes to a tetrazolium salt, neotetrazolium chloride, after exposure to light for various periods of time has been studied. Enzymes assayed for this purpose were: glucose-6-phosphate dehydrogenase been studied. Enzymes assayed for this purpose were: glucose-6-phosphate dehydrogenase (NADP+-dependent); lactate dehydrogenase (NAD+-dependent) and succinate dehydrogenase (flavoprotein-dependent). Enzyme activity was measured in sections of rodent liver by scanning and integrating microdensitometry. Phenazine methosulphate in solution was found to be sufficiently stable in light for up to two hours for reproducible quantitative measurements of cytochemical dehydrogenase activity to be obtained over this period.

A quantitative cytochemical method for the measurement of beta-hydroxyacyl CoA dehydrogenase activity in rat heart muscle.

Although cytochemical methods exist for measuring dehydrogenases that act on substrates involved in the production of chemical energy from sugars, virtually no methods exist for measuring the dehydrogenases that act on fatty acids. Yet the oxidation of fatty acids accounts for over 60% of the oxidative activity of cardiac muscle. Consequently a new quantitative cytochemical method, based on a new substrate (DL-S-beta-hydroxybutyryl-N-acetyl cysteamine), has been developed for measuring the activity of hydroxy-acyl coenzyme A dehydrogenase, which is the penultimate step of the beta-oxidation of fatty acids to acetyl-coenzyme A that is used in the Krebs' cycle. Menadione or phenazine methosulphate is used as the intermediate hydrogen-acceptor, with neotetrazolium chloride as the final acceptor. The medium contains nitroprusside, ostensibly to react with any cysteamine liberated by hydrolysis of the substrate. As a control, cysteamine is substituted for the substrate. The concentrations of reactants have been optimized for cardiac muscle; the reaction is linear with thickness of the sections and with time of reaction from 15 to 60 min.

Investigations of the extraction equilibrium of ternary ion-association complexes of thallium(III) with ditetrazolium salts, neo-tetrazolium chloride and nitrobluetetrazolium chloride

The extraction equilibrium of ternary ion-association complexes formed between the tetrachloro complexes of thallium(III) and ditetrazolium salts, neotetrazolium chloride (NTC)[2,2′,5,5′-tetraphenyl-3-3′-(p-biphenylene)ditetrazolium chloride] and nitrobluetetrazolium chloride (NBT)[3,3′-dianizole-4,4′-bis-2-(4-nitrophenyl-5-phenyltetrazolium chloride], has been studied. As a result, the extraction constants, the distribution constants, the association constants, the distribution coefficients of thallium(III) and the extraction indicators R, have been calculated. The results have been evaluated statistically. The relative standard deviations Sr have been calculated at a statistical certainty of 95%. The ratio ditetrazolium salts/TlCl4−=1∶2 has been confirmed once again.

Pigment distribution and nitrogen fixation inAnabaena azollae

The symbiotic heterocystous cyanobacteriumAnabaena azollae present in the leaf cavities of the water fernAzolla spp. was studied. The cyanobacteria extracted from the leaf cavities showed differences in pigment composition in three species ofAzolla, i.e A.pinnata var.pinnata, A.caroliniana and A.filiculoides, as observed by pigment absorption and epifluorescence tests. These differences suggest that of these species the cyanobiont ofA. pinnata is the most actively nitrogenfixing form. This has been confirmed by nitrogen fixation (acetylene reduction) tests. Heterocysts of the symbiont ofA. pinnata were characterized by high chlorophylla and low phycocyanin content, a low fluorescence yield of chlorophyll in the heterocysts compared to vegetative cells and a gradient of phycocyanin concentration in the vegetative cells adjacent to heterocysts. This indicates that only photosystem I is present in the heterocyst. In the two otherAzolla species quantitative shifts in the pigment composition occurred suggesting a lower nitrogen fixation activity. In the cyanobiontAnabaena azollae the heterocyst frequency could reach a value of 44–45%. It is argued that there are two generations of heterocysts in a matureAzolla plant, which are concomitant with two peaks of nitrogen fixation activity correlated with leaf age,i.e. leaf number along the main axis of the plant. At both peaks of maximal N2-ase activity, only 20–25% of the heterocysts present are metabolically active as demonstrated by the reduction of Neotetrazolium chloride (NTC) in the heterocysts and darkening of nuclear emulsions by silver salt reduction. Vegetative cells of the cyanobiont reduce Neotetrazolium chloride (NTC) to formazan more rapidly than has been observed in the free-living heterocystous cyanobacteriumAnabaena cylindrica tested in parallel experiments. This feature may be due to a more permeable cell wall of the vegetative cells of the cyanobiont compared to the free-living form, since the vegetative cells of the symbiont play a role in cross-feeding of the host (Azolla). Evidence is obtained that only the heterocysts of the cyanobiont ofAzolla are involved in the nitrogen fixation process as in free-living heterocystous cyanobacterium species. This situation is different from other cyanobacterial symbioses such as inGunnera, Blasia andAnthoceros, where physiological modifications are reported in the symbiosis with another photosynthetic partner such as the absence of O2 evolution and the absence of photo-fixation of CO2 in the cyanobionts. Pigment composition and N2-ase activity in the symbiotic cyanobacteria of three Azolla species have indicated the superiority of theA. pinnata symbiont.A. pinnata var.pinnata is a semidomesticated form used in S.E. Asia for agricultural purposes (irrigated rice culture) to increase soil fertility. It is suggested that by selection (domestication) more efficient strains (clones) can be obtained, and further that with more advanced techniques such as gene mutation and genetic manipulation even more efficient and for agriculture more beneficial clones can be obtained.

Evaluation of Inhibitors for Rapid Enumeration of Psychrotrophic Bacteria

Two inhibitors of the 17 tested inhibited growth of gram positive bacteria without causing inhibition of gram negative bacteria. These were crystal violet at 2 mg/1 and neotetrazolium chloride at 2 mg/1. In addition, basic fuchsin at 6 mg/1 produced only marginal reduction in counts of gram negative bacteria. These inhibitors might find further use in developing a test for psychrotrophic bacteria in the presence of non-psychrotrophic bacteria.

Neotetrazolium Staining of Three Avian Mycoplasma Serotypes for the Microagglutination Test

Mycoplasma meleagridis, M. gallisepticum, and M. synoviae antigens were stained with neotetrazolium chloride for use in the microagglutination test. Only the stained antigens of M. gallisepticum were acceptable in color intensity and test readability.

Oxygen and the production of formazan from neotetrazolium chloride.

Electrons, generated from dehydrogenase reactions, are transferred to oxygen in preference to neotetrazolium chloride. In model systems in solution the presence of a small amount of oxygen drastically reduces the rate of formazan production. The rate of reaction in tissue sections has been followed using scanning and integrating microdensitometry. As in solution, electrons are transferred preferentially to oxygen. However, oxygen seems unable to diffuse through the incubation medium and thus the supply of oxygen at the site of the enzyme activity becomes exhausted; the time taken to use up the oxygen will depend on the rate of the enzyme activity. It is only then that electrons are passed to the tetrazolium salt and formazan is precipitated.

The quantification of ormazans in tissue sections by microdensitometry. III. The effect of objective power and scanning spot size.

In this article, it is reported that the formazans derived from neotetrazolium chloride (NT) and 2-(2-benzothiazolyl)-3-(4-phthalhydrazidyl)-5-styryl-tetrazolium chloride (BPST) can be measured by microdensitometry of tissue sections with a wide range of scanning spot sizes, without any significant effect on the recorded extinctions. The spot sizes tested ranged from 0.20 to 16 mum. Thus when large fields have to be measured, it is possible to use low-power objectives and still obtain valid results.

The quantification of formazans in tissue sections by microdensitometry. I. The use of neotetrazolium chloride.

This article describes the use of a microdensitometer for the measurement of formazan deposits in tissue sections. Some examples are given to illustrate the various applications of this technique in the assessment of glucose-6-phosphate dehydrogenase activity. These are (I) the separate measurement of the red half-formazan intermediate and purple diformazan of neotetrazolium, and the effect of incubation time on their production, (2) the measurement of activities in different regions of the liver lobule, and the selective effect of phenobarbitone, and (3) the measurement of enzyme activity in individual cartilage cells in normal and osteoarthrosis-prone animals. All activities can be expressed in absolute units as nmol hydrogen/mm3/hr, and thus compared with standard biochemical data. The activities obtained all fall within the range of published values for biochemical systems.

Acceleration of the acrosome reaction and activation of guinea pigs spermatozoa by detergents and other reagents.

In vivo, guinea pig spermatozoa do not begin to undergo the acrosome reaction and to penetrate eggs until 4 to 10 h after they are deposited in the female genital tract. When spermatozoa are incubated in a modified Krebs-Ringer’s solution containing 0.003 percent Hyamine, the majority of them exhibit both the acrosome reaction and activation within 10-15 mm and become capable of penetrating eggs. The acrosome reaction and activation induced by Hyamine are reversibly blocked when calcium ions are omitted from the medium. Other reagents capable of inducing an accelerated acrosome reaction and/or activation include: Triton, Brig, saponin, nonanol, nystatin, mersalyl acid, dibucane, procaine, neotetrazolium chloride, nitro blue tetrazolium, chloroquine, calcium ionophore, unheated sera of the rabbit and guinea pig, rabbit complement and pronase. The study shows that guinea pig spermatozoa are potentially capable of an immediate acrosome reaction and activation upon leaving the epididymis.

Spectrum of function of neutrophils from carriers of sex-linked chronic granulomatous disease.

Bactericidal and metabolic activities were compared for polymorphonuclear leukocytes from normal controls, patients with sex-linked chronic granulomatous disease, five obligate carriers, and six potential carriers of CGD. Bacteria surviving at one hour were quantitated in a standardized assay which employed 1.25 Staphylococcus aureus per neutrophil. Heat-killed bacteria or a chemical agent, phorbol myristate acetate, were used to stimulate increases in utilization of oxygen, oxidation of [1--14C] glucose, and reduction of neotetrazolium chloride by PMN. The results demonstrate that PMN from the individual obligate carriers of CGD have a broad spectrum of functional capabilities. Neutrophils from one obligate carrier performed in the above in vitro tests and others on a par with normal control cells, whereas the PMN of others displayed deficiencies nearly as profound as those of the affected CGD patients. The observations parallel the broad range of phenotypic expression observed in heterozygotic carriers of other sex-linked recessive disorders as a result of random inactivation of the X chromosome. Although predictable from the current concept of random X inactivation, the spectrum has not been previously demonstrated for carriers of sex-linked recessive CGD and thus has important implications for the detection and counseling of carriers of CGD.

Spectrophotometric determination of dipyrone with neotetrazolium chloride.

Spectrophotometric determination of dipyrone with neotetrazolium chloride.

Liver metabolic reactions: Tertiary amine N-dealkylation, tertiary amine N-oxidation, N-oxide reduction, and N-oxide N-dealkylation : I. Tricyclic tertiary amine drugs

N-oxide reduction and N-oxide demethylation, shown to occur in homogenates of various rat tissues with imipramine- N-oxide, were also observed with the N-oxides of chlorpromazine and amitriptyline. With imipramine- N-oxide the rates of N-oxide reduction and N-oxide demethylation were higher in liver homogenates of pigs and horses than of rats. The subcellular sites of the two reactions could not be clearly localized in rat liver fractions, however, microsomes, mitochondria, and probably nuclei, alone and in combination with cytosol or NADPH, displayed negligible activity. Next to the total homogenate the crude nuclear fraction was the most active. N-oxide reduction and N-oxide demethylation are clearly differentiated from tertiary amine N-demethylation and N-oxidation by oxygen, nitrogen, heat pretreatment, ferricytochrome c, and neotetrazolium chloride. The microsomal reactions, tertiary amine demethylation and N-oxidation, differ in that the former but not the latter reaction is inhibited by CO, SKF 525-A, and excess substrate. N-oxide reduction and N-oxide demethylation showed identical responses to the procedures mentioned, except for a higher degree of heat stability of the latter reaction. Experiments with labeled imipramine or imipramine- N-oxide and unlabeled pools of one of the compounds unequivocally confirmed the presence or absence of the following metabolic reactions in rat liver preparations: Tertiary amine demethylation and N-oxidation occur in 9000 g supernatant fraction or microsomes + NADPH but not in homogenate without added NADPH; N-oxide reduction and N-oxide demethylation occur in the latter system but not in the former. All four reactions are simultaneously operative in homogenate fortified by an NADPH-generating system, i.e., the combination of the above systems. The kinetics of the four individual reactions have been estimated by incubating labeled imipramine and a pool of unlabeled imipramine- N-oxide in rat liver homogenate with an NADPH-generating system and determination of amounts, absolute and specific radioactivities of these compounds as well as desmethylimipramine as a function of time. In the steady state the reaction constants could be arranged in the following order: N-oxide reduction ⪢ N-oxide demethylation ⩾ tertiary amine demethylation > tertiary amine- N-oxidation. The results demonstrate that the N-oxide can be ruled out as an intermediate in the oxidative N-demethylation of imipramine in liver microsomal systems and is negligible even in NADPH-fortified liver homogenate. The oxidative N-dealkylation of this tertiary amine is thus likely to represent a C-oxygenation proceeding independently from an N-oxygenation.

Changes in the liver mitochondrial oxidation of succinate during cold-exposure.

1. Exposure of rats to low environmental temperature resulted in increased activities of several hepatic oxidative-enzyme systems. 2. Simultaneous with increase in liver ubiquinone in cold-exposed rats, the ubiquinone-dependent succinate-neotetrazolium chloride reductase activity also increased. Such an increase could also be obtained by enriching liver with ubiquinone by feeding with an exogenous source. 3. Succinate-neotetrazolium chloride reductase activity could be increased by preincubation of mitochondria with succinate and the mechanism of this activation appears to be different from that obtained on addition of ubiquinone. 4. Succinate-neotetrazolium chloride reductase activity was found to be more labile than succinate dehydrogenase on freezing and thawing and storage, and the presence of succinate gave protection against this loss in hepatic mitochondria obtained from both normal and cold-exposed animals.

Colorimetric estimation of succinic dehydrogenase activity by neotetrazolium chloride as a tumor sensitivity test to chemotherapeutic agents.

In the search for tumor sensitivity test to chemotherapeutic agents succinic dehydrogenas e activity by neotetrazolium chloride was measured colorimetrically. The rate of formazan production was proportional to the amount of Ehrlich ascites tumor cells, accelerated by the addition of ascorbic acid, and inhibited by the addition of iodoacetic acid. The inhibitory rate of formazan production of tumor cells treated with chemotherapeuti c agents was well correlated with the survival time of mice. Of the chemotherapeutic agents, mitomycin C was the most effective, either singly or in combination with other drugs. Considering the mechanisms of action of drug and of the host's defense, an in. vivo tumor system was studied as a screening test for the chemotherapeutic agents.

The use of a recording microdensitometer for the quantitative measurement of enzyme activities inside tissue sections.

1. A microdensitometric technique is described which permits enzyme activities to be measured inside tissue sections. 2. The end products of neotetrazolium chloride reduction, nitroblue tetrazolium chloride reduction, and leucyl 2 naphthylamine hydrolysis coupled to the reaction with tetra-azotised o-dianisidine, have so far been calibrated. 3. Activities can be expressed in absolute terms.

The incorporation of p-hydroxybenzoate and isopentenyl pyrophosphate into polyprenylphenol precursors of ubiquinone by broken cell preparations of Rhodospirillum rubrum

Broken cell preparations of Rhodospirillum rubrum have been obtained by subjecting the cells to the combined action of lysozyme and osmotic shock. These lysed preparations incorporate radioactivity from p-hydroxybenzoate-U- 14C and acetate-2- 14 C into 2-polyprenylphenols and ubiquinones. They also incorporate methionine(methyl- 14C) and S-adenosylmethionine-(methyl- 14C) into ubiquinones and 2-decaprenyl-6-methoxyphenol. Relatively cell-free extracts were also obtained by sonicating Rhodospirillum rubrum cells. These extracts were efficient in incorporating p-hydroxybenzoate-U- 14C into 2-polyprenylphenols. Although ubiquinone synthesis was occasionally observed in both preparations it could be accounted for by the presence of small numbers of unbroken cells in the extract which were detected by means of viable cell counts. These cells were not sufficient to account for the vast majority of 2-polyprenylphenol synthesis and were virtually absent from sonicates capable of synthesizing substantial amounts of 2-polyprenylphenol after centrifugation for 1 hr at 48,000g. The incorporation of p-hydroxybenzoate-U- 14C into 2-polyprenylphenol by both types of extract is greatly stimulated by the addition of a source of side-chain precursor in the form of a crude extract of Micrococcus lysodeikticus cells and isopentenyl pyrophosphate. Cysteine and ascorbic acid stimulate this conversion of p-hydroxybenzoate to 2-polyprenylphenol. Attempts to stimulate ubiquinone synthesis in the broken cell preparations by the use of various redox reagents (dichlorobenzenoneindophenol, phenazine methosulfate, methylene blue, neotetrazolium chloride, triphenyltetrazolium bromide, cytochrome c) either in catalytic quantities or as terminal electron acceptors, failed to restore ubiquinone synthesis; most of the reagents proved to be extremely inhibitory to 2-polyprenylphenol synthesis. The R. rubrum extracts, especially when supplemented with Micrococcus extract and isopentenyl pyrophosphate, incorporate radioactivity from p-hydroxybenzoate-U- 14 C into another intermediate in ubiquinone synthesis. p-Hydroxybenzoate-7- 14 C is also incorporated into this compound, but not methionine-(methyl- 14C). The sonicated preparations of R. rubrum are capable of converting this compound to 2-polyprenylphenol. On this basis a reasonable structure to assign to this compound is 3-polyprenyl-4-hydroxybenzoate. A bacterium, found as a contaminant in some cultures of R. rubrum, has been isolated and shown to incorporate phydroxybenzoate-U- 14 C into ubiquinone. The organism, when incubated anaerobically with phydroxybenzoate-U- 14 C (or p-hydroxybenzoate-7- 14C) in the presence of potassium ferricyanide, accumulated 3-polyprenyl-4-hydroxybenzoate. When the cells were washed and the p-hydroxybenzoate-U- 14C was diluted with unlabeled p-hydroxybenzoate, subsequent aerobic incubation of the cells converted almost all the 3-polyprenyl-4-hydroxybenzoate to ubiquinone. The distribution of prenylogues of 2-polyprenylphenol in R. rubrum extracts supplemented with MLE and IPP is the same as that observed in whole cells.

The purification and some properties of neotetrazolium chloride and its chief monotetrazolium salt contaminant

A simple method which does not involve chromatography is described for the purification of neotetrazolium chloride (NT). Commercial samples of NT were shown to contain variable amounts of a major monotetrazolium salt contaminant, which was isolated and identified as 2-(4-biphenyl-)-3,5-diphenyltetrazolium chloride (BDTC). A qualitative method for checking the purity of NT and BDTC by thin-layer chromatography is described; with this technique the presence of traces of other tetrazolium salt contaminants was detected. The preparation of pure samples of formazan corresponding to NT and BDTC is described; molar extinction coefficients are given with sufficient other information to enable estimation of the amounts of NT and BDTC in mixtures of the two to be made.