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PubMed1999

Enzymatic cycling assay for D-carnitine determination.

An enzymatic method for d-carnitine determination using the enzyme d-carnitine dehydrogenase is described. The assay is based on the amplified signal produced during NAD(+) cycling in the presence of a tetrazolium salt and using phenazine methosulfate as electron carrier. Optimum assay conditions were studied with two tetrazolium salt pairs: 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT)/MTT-formazan and 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride (INT)/INT-formazan. The first pair (MTT) showed higher sensitivity. The calibration curve was linear from 0.1 to 5 mM d-carnitine, with a quantification limit of 0.1 mM and a relative standard deviation of 1.51%. The procedure is simple, rapid, accurate, and easily automated. It was satisfactorily applied to following d-carnitine levels during the microbial transformation of d-carnitine into l-carnitine and to determining the d-carnitine content of pharmaceutical preparations.

Alcohol Oxidoreductases↗

PubMed1980

Structure-staining relationships in histochemistry and biological staining. I. Theoretical background and a general account of correlation of histochemical staining with the chemical structure of the reagents used.

A description is given of the choice, rationale and calculation of several numerical coefficients for describing the chemical structures of staining reagents such as dyestuffs, enzyme substrates, diazonium and tetrazolium salts. Using such coefficients, structure-staining correlations may be made for many staining systems. Examples include the structural correlations of the quantitative affinity measurements of the staining of chromatin with basic dyes, the tetrazolium salts for use as visualizing agents in enzyme histochemistry, and the use of bis-azo compounds as acid or basic dyes, or as fat stains. The value of such structure-staining correlation models for assisting the rational selection and design of new histochemical reagents, and for understanding old reagents and procedures, is discussed.

Chemical Phenomena↗

PubMed1988

Use of the MTT assay for rapid determination of chemosensitivity of human leukemic blast cells.

A microcytotoxicity assay employing a tetrazolium salt has been adapted for testing the response of human leukemic blast cells to a variety of chemotherapeutic agents. After exposure to various concentrations of drugs, the viability of fresh leukemic blast cells was measured using a tetrazolium salt, MTT, which is converted to blue formazan crystals by living cells. The amount of formazan produced was quantitated using a microtitre plate spectrophotometer. In the present study, optimal conditions for chemosensitivity testing of human leukemia samples were determined, and the relative chemosensitivity of five patient samples was tested.

Antineoplastic Agents↗

PubMed1987

[Presence of dehydrogenases of the pentose phosphate pathway in boar spermatozoa and testis].

There is insufficient evidence of direct oxidation of glucose through the pentose phosphate cycle in mammalian spermatozoa. We separated the key enzymes (G6P-DH, 6PG-DH) in the pentose pathway using 2',5'ADP-Sepharose 4B. The purpose of this study is to establish the intracellular location of these two enzymes in boar spermatozoa and testis by histochemical techniques. Histochemical localization of the two pentose phosphate pathway dehydrogenases was achieved by the use of the tetrazolium salt nitro blue tetrazolium (Nitro BT). Both G6P-DH and 6PG-DH was present in the mid pieces of boar spermatozoa and in the Leydig cells and seminiferous tubules of testis. The present findings indicate that the pentose pathway could be involved in the energy production of spermatozoa and that these enzymes may be important in supplying NADPH for steroid synthesis in the testis.

Animals↗

PubMed1958

The cytochemical localization of oxidative enzymes. I. Diphosphopyridine nucleotide diaphorase and triphosphopyridine nucleotide diaphorase.

Cytochemical methods involving metal chelation of the formazan of an N-thiazol-2-yl tetrazolium salt are described for the localization of diphosphopyridine nucleotide diaphorase (DPND) and triphosphopyridine nucleotide diaphorase (TPND) in mitochondria. These methods utilize the reduced coenzymes DPNH or TPNH as substrate. The reaction involves a direct transfer of electrons from reduced coenzyme to the respective diaphorase which in turn transfers the electrons to tetrazolium salt, reducing it to the insoluble formazan. Competition for electrons by preferential acceptors in the respiratory chain was prevented by various inhibitors. In the presence of respiratory inhibitors the rate of tetrazolium reduction was markedly increased. The greatest reduction was observed when amytal was used. Sites of diaphorase activity appeared as deposits of blue-black metal formazan chelate measuring 0.2 to 0.3 micro in diameter. Small mitochondria contained 2 deposits, while larger ones contained up to 6. Considerable differences were observed in the rate of tetrazolium reduction and cellular localization of diaphorase activity when DPNH was used as substrate as compared to TPNH. In each instance DPNH was oxidized more rapidly by tissues than TPNH. These findings support the concept that the oxidation of coenzymes I and II is mediated through separate diaphorases.

Coenzymes↗

PubMed2004

Metabolism of benzoquinone by yeast cells and oxidative characteristics of corresponding hydroquinone: application to highly sensitive measurement of yeast cell density by using benzoquinone and a chemiluminescent probe.

The metabolic efficiency of seven derivatives of 1,4-benzoquinone (BQ) by yeast cells and the oxidative characteristics of the corresponding hydroquinones (HQs) were studied by electrochemical, spectrophotometric and chemiluminescent methods. The spectrophotometric method was based on the reduction of a tetrazolium salt to formazan dye during the autoxidation of HQs generated by yeast cells under alkaline conditions. The amounts of HQs detected directly by the electrochemical method did not agree with those calculated from the formazan dye obtained by the spectrophotometric method. A tetrazolium salt was reduced to a formazan dye by both the superoxide anion radical (O2-*) generated during the autoxidation of 2,3,5,6-tetramethyl-1,4-HQ and by HQ itself. Little formazan dye was formed, and hydrogen peroxide (H2O2) was then finally produced during the autoxidation of 1,4-HQ or 2-methyl-1,4-HQ. Formazan dye and H2O2 were generated at a certain ratio during the autoxidation of derivatives of dimethyl-1,4-HQ or 2,3,5-trimethyl-1,4-HQ. The analytical method based on chemiluminescence with lucigenin and 2,3,5,6-tetramethyl-1,4-BQ was applied to highly sensitive measurement of the yeast cell density. A linear relationship between the chemiluminescence intensity and viable cell density was obtained in the range of 1.2 x 10(3) - 4.8 x 10(4) cells/ml. The detection limit was 4.8 x 10(2) cells/ml.

Benzoquinones↗

PubMed2005

Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction.

Tetrazolium salts have become some of the most widely used tools in cell biology for measuring the metabolic activity of cells ranging from mammalian to microbial origin. With mammalian cells, fractionation studies indicate that the reduced pyridine nucleotide cofactor, NADH, is responsible for most MTT reduction and this is supported by studies with whole cells. MTT reduction is associated not only with mitochondria, but also with the cytoplasm and with non-mitochondrial membranes including the endosome/lysosome compartment and the plasma membrane. The net positive charge on tetrazolium salts like MTT and NBT appears to be the predominant factor involved in their cellular uptake via the plasma membrane potential. However, second generation tetrazolium dyes that form water-soluble formazans and require an intermediate electron acceptor for reduction (XTT, WST-1 and to some extent, MTS), are characterised by a net negative charge and are therefore largely cell-impermeable. Considerable evidence indicates that their reduction occurs at the cell surface, or at the level of the plasma membrane via trans-plasma membrane electron transport. The implications of these new findings are discussed in terms of the use of tetrazolium dyes as indicators of cell metabolism and their applications in cell biology.

Animals↗

PubMed1974

Improved isolation and differentiation of enterococci in cheese.

Further documentation of an enterococcus selective differential (ESD) medium was obtained in isolations from eight different cheeses. An improved differentiation of tetrazolium salt (2, 3, 5-triphenyl tetrazolium hydrochloride [TTC])-reducing strains of Streptococcus faecalis from TTC-nonreducing or TTC-faintly-reducing Streptococcus faecium was attained. The sensitivity of the medium was evaluated in comparison with that of KF streptococcal, Pfizer selective enterococcus (PSE), the medium of Reinbold, Swern, and Hussong (RSH), and the medium of Saraswat, Clark, and Reinbold (SCR). Selective counts, rate of colony formation, and ease of isolation and differentiation of colonies were examined. The specificity of the medium was also investigated. ESD supported the fastest rate of growth and the maximum size of colonies; counts in this medium were in most cases possible with 17 h of incubation, whereas the other media required 24 to 48 h. A presumptive identification of 1,077 isolates by four biochemical tests disclosed that SCR, RSH, and ESD selected high, comparable percentages of strains that approximated most closely the typical description of enterococci (66, 60.1, and 58%, respectively). Low percentages (21.1 and 30.7%) were yielded by KF and PSE. The utility of ESD for a rapid, presumptive identification of enterococci was confirmed by serological and biochemical testing of color TTC-differentiated colonies isolated from 18 cheeses.

Cell Count↗

PubMed1978

[Tetrazolium methods for the histochemical investigation of hydrolases (author's transl)].

Using freeze-dried or sections from fresh-frozen or aldehyde-fixed material nitro BT (NBT), tetranito BT (TNBT), distyryl nitro BT (DS-NBT), thiocarbamyl nitro BT (TC-NBT) or benzothiazolylstyrylphthalhydrazidyl tetrazolium chloride (BSPT) were tested as auxiliary reagents for the localization of glycosidases, phosphatases and non-specific esterases with indoxyl substrates in rat tissues. By means of NBT or TNBT as a tetrazolium salt acid beta-D-galactosidase, beta-D-N-acetylglucosaminidase, acid phosphatase, neuraminidase and non-specific esterase can only be localized at the cellular level; a more precise localization is possible for lactase-beta-D-glucosidase in the intestinal brush border, and the best results are obtained in the demonstration of alkaline phosphatase; among all methods described previously the tetrazolium procedure with TNBT is the method of choice for the light microscopic localization of this enzyme. Reverse data are observed with BSPT as a tetrazolium salt; then, all acid and neutral hydrolases can be exactly localized in lysosomes, secretion granules, cytoplasm and/or microvilli of many cells and tissues provided BSPT-formazan is stabilized by osmification. Furthermore, this procedure enables the reliable ultracytochemical demonstration of these enzymes. However, in the case of alkaline phosphatase only sites with high enzyme activity reveal a positive reaction. -DS- and TC-NBT are inferior to NBT, TNBT or BSPT.

Acetylglucosaminidase↗

PubMed1996

Cytochemical colocalization and quantitation of phenotypic and genotypic characteristics in individual bacterial cells.

The widely accepted view that most bacterial species have yet to be cultivated in vitro has gained support from recent ribosomal DNA-based environmental studies. To enable elucidation of the phenotypes of organisms recognized solely by molecular genetic techniques, we developed and evaluated cytochemical methods which colocalize phenotypic properties with in situ rRNA probe hybridization signals. Application of these methods to artificial mixtures of Pseudomonas putida and Escherichia coli or Vibrio vulnificus showed that biochemical properties, such as the cytochrome oxidase reaction and specific substrate-enhanced tetrazolium salt reduction, can be assigned to cells identified by signals from determinative fluorescent rRNA probe binding. By doing the reactions directly on the stage of an inverted microscope and monitoring reaction product formation with a charge-coupled device video camera, it was possible to determine the kinetics of oxidizable substrate utilization in single cells. Analysis of digitized images permitted quantitative study of the relationship between rRNA signal strength and the rate of tetrazolium salt reduction. The approach used in this study opens up new opportunities to investigate the biochemistry, physiology, and behavior of both culturable and nonculturable bacteria in their natural environments.

Bacteria↗

PubMed1989

Enzyme cytochemistry of unfixed leukocytes and bone marrow cells using polyvinyl alcohol for the diagnosis of leukemia.

Cytochemical methods for the demonstration of enzyme activities in blood and bone marrow cells were systematically improved by the addition of an inert polymer, polyvinyl alcohol (PVA), to the incubation medium and by using optimized reaction media. The methods investigated were tetrazolium salt methods for lactate, glucose-6-phosphate, succinate and glutamate dehydrogenase, the indoxyl-tetrazolium salt method for alkaline phosphatase, the diaminobenzidine method for peroxidase, and diazonium salt methods for chloroacetate esterase, beta-glucosaminidase, beta-glucuronidase, acid phosphatase, and dipeptidylpeptidase II and IV. PVA in the media preserved the morphology of cells very well and prevented leakage of large molecules such as enzymes from the cells. Therefore, fixation or long periods of air-drying prior to incubation leading to substantial loss of enzyme activity could be avoided. A brief period of drying (2 min at 37 degrees C) of the cell preparations just before the incubation was sufficient for making the cells permeable. Localization of enzyme activities was very precise and precipitation of the final reaction product was confined to sites which are known to contain the enzyme under study (granules, mitochondria, lysosomes). These advantages advocate the use of PVA in haematological enzyme cytochemistry and especially for diagnosis of leukemia.

Bone Marrow↗

PubMed2000

Endothelial and smooth muscle changes of the thoracic and abdominal aorta with various types of cryopreservation.

BACKGROUND: There is a need for cryopreserved arterial allografts in vascular surgery. Vitrification was examined as an alternative to slow cooling. Function of endothelial and adjacent cells was evaluated in the thoracic and abdominal regions of the porcine aorta. MATERIAL AND METHODS: Aortas from young pigs were harvested and cryopreserved with several treatments: slow cooling at 1 degrees C/min with 10% dimethyl sulfoxide (DMSO) and vitrification with a vitrification solution (VS), performed either in the presence (medium) or in the absence (air) of the cryoprotective medium surrounding the sample. Tetrazolium salt reduction and oxygen consumption were used to assess the mitochondrial activity of the endothelial cells and smooth muscle cells from the aorta. RESULTS: Fresh aorta showed an increased function from below the aortic arch to the iliac branch. Exposure to cryoprotective solutions reduced significantly the oxygen consumption of the abdominal sections. Samples from the thoracic region had a better response to cryopreservation than those from the abdominal region. There was a significant reduction in tetrazolium salt reduction and oxygen consumption when the samples were cryopreserved immersed in the medium. Significant flaking of the endothelium was observed after vitrification and contributes generally to lower the function. CONCLUSION: The thoracic region of the aortic wall was more tolerant to the preservation injury, and freezing with DMSO in air offered a better choice of preservation.

Animals↗

PubMed1989

[Fluorescent formazans in flow cytometry. Studies of their oxygen sensitivity].

In order to supply the tetrazolium salt procedure to flow cytometry we have synthesized monotetrazolium salts which are converted into fluorescent formazans on reduction. In this preliminary study we investigated the "oxygen sensitivity" of four of our compounds and compared them to the commercially available nitrotetrazole blue (NBT). The amount of formazan produced by Ehrlich ascites tumour cells via the succinic dehydrogenase reaction during oxic and anoxic incubations was determined by elution technique. Under oxic conditions no NBT formazan but much cyano-di-chlorophenyl formazan (==CCPC) was generated. The yields of this formazan differed only slightly between oxic and anoxic incubations. Twice as much of the amount of the other three formazans cyano-ditolyl- (==CTC, Stellmach 1984), cyanodiphenyl- (==CPC), and cyano-dianisyl tetrazolium chloride (==CAC), were produced in nitrogen atmosphere as in oxygen. The amount of all formazans investigated increased both in oxic and in anoxic conditions if cyanide was present. All cyano-aryl formazans have the maximum of absorbance between 430 and 470 nm and fluoresce in the spectral range above 580 nm with CTC formazan being the brightest. The tetrazolium salts, i.e. in unreduced state, did not fluoresce in the visible part of the spectrum. The formazans could be applied to flow cytometry. After staining the nuclear DNA with the fluorochrome DAPI, we obtained detailed two-parametric distributions correlating the amount of formazan per cell with the DNA content. The results are a step to establish a method for automated identification of malignant cells.

Animals↗

PubMed1986

Resolution of alkaline phosphatase isoenzymes in serum by isoelectric focusing in immobilized pH gradients.

This new method for fractionation of serum alkaline phosphatase isoenzymes is based on isoelectric focusing on a mixed-type polyacrylamide support containing an immobilized pH gradient with a superimposed carrier-ampholyte gradient. All known forms of alkaline phosphatase are separated in an Immobiline pH 3.5-6.0 gradient, the sample being applied into pockets cast on a pH 8.0 plateau. Sharp zymogram bands are obtained by substituting alkaline-stable 5-bromo-4-chloro-3-indoxyl phosphate and tetrazolium salts for the standard 1- and 2-naphthyl phosphate-diazonium salt combinations. After hydrolysis of the phosphate group by the alkaline phosphatase the indoxyl moieties reduce tetrazolium salts to nearly insoluble and nondiffusible formazan precipitates. Normal sera show an array of about 10 isobands isoelectric between pH 3.9 and pH 4.79. In Paget's disease, two sharp isobands with pls of 4.97 and 5.09 are seen. Placental alkaline phosphatase overlaps with the higher pl bands of normal serum; however, upon heat destruction of the latter, it shows four sharp bands with the following pl's: 4.59, 4.62, 4.67 and 4.73.

Acrylamides↗

PubMed2001

Pterin interactions with distinct reductase activities of NO synthase.

Besides oxidizing L-arginine, neuronal NO synthase (NOS) NADPH-dependently reduces various electron acceptors, including cytochrome c and tetrazolium salts. The latter NADPH diaphorase reaction is used as a NOS-specific histochemical stain. Both reductase activities have been utilized to analyse electron transfer mechanisms within NOS. Basal L-arginine turnover by homodimeric NOS is enhanced by exogenous tetrahydrobiopterin, and the intra-subunit electron flow may include intermediate trihydrobiopterin. In the present work we have investigated the possible role of the tetrahydrobiopterin binding site of NOS in its reductase activities by examining the effects of anti-pterin type (PHS) NOS inhibitors. Although the type I anti-pterin, PHS-32, which does not affect basal dimeric NOS activity, also had no effect on either reductase activity, the type II anti-pterin, PHS-72, which inhibits basal NOS activity, inhibited both reductase activities and the NADPH diaphorase histochemical stain. Pterin-free NOS monomers catalysed both cytochrome c and tetrazolium salt reduction. Our data suggest that both NOS reductase activities are independent of tetrahydrobiopterin. However, occupation of an exosite near the pterin site in NOS by type II anti-pterins may interfere with the electron flow within the active centre, suggesting that steric perturbation of the pterin binding pocket or reductase interaction contribute to the mechanism of inhibition by this class of NOS inhibitors.

Biopterins↗

PubMedDate not supplied

Adhesion of different Candida spp. to plastic: XTT formazan determinations.

Adhesion of synchronized yeast-phase Candida cells to tissue culture plastic was investigated using the tetrazolium salt, XTT. The procedure permits the direct enumeration of adherent yeasts following the metabolic conversion of the XTT tetrazolium salt, to its reduced formazan form, by mitochondrial dehydrogenases. Using this procedure, the formation of XTT formazan by Candida cells was typically related to the inoculum size. The adhesion of Candida yeast-phase cells from different Candida spp. to plastic was of the following order: C. krusei (n = 5) > C. albicans (n = 10) > C. glabrata (n = 6). Furthermore, preliminary experiments with several other species indicated that C. tropicalis (n = 2) may adhere as well as C. albicans and that one strain each of C. guilliermondii and C. parapsilosis appear to adhere to plastic in a similar fashion to C. glabrata. The data indicate the utility of the XTT tetrazolium based assay in enumerating the adhesion of different Candida spp. to plastic.

Adhesiveness↗

PubMed1989

Source of superoxide anion radical in aerobic mixtures consisting of NAD[P]H, 5-methylphenazinium methyl sulfate and nitroblue tetrazolium chloride.

The source of superoxide anion radical (O2-.) in aerobic mixtures consisting of NAD[P]H, 5-methylphenazinium methyl sulfate (or its 1-methoxy derivative) and tetrazolium salt was investigated using superoxide dismutase (SOD), Mn(II), ferricytochrome-C, and epinephrine as probes. NAD[P]H + phenazine + O2 was found to reduce nitroblue tetrazolium, iodonitrotetrazolium, and thiazolyl blue in a manner sensitive to agents that dismutase O2-., viz., SOD and Mn(II). It also mediated the reduction of ferricytochrome-C, and augmented the autooxidation of epinephrine to the adrenochrome, without a tetrazolium salt present in the medium. The autooxidation of epinephrine, but not the reduction of ferricytochrome-C, was found to be sensitive to SOD. Nitroblue tetrazolium, either singly or in combination with SOD, did not stimulate the reduction of ferricytochrome-C. The oxidation of NADH, mediated by a catalytically low concentration of phenazine(+O2), was augmented two-fold by SOD. These observations are consistent with, and lend support to, a scheme of redox events (Scheme-3) wherein it is proposed that the source of O2-. in the NAD[P]H + phenazine + tetrazolium(+O2) system is the reduced phenazine, that the tetrazoinyl radical (a one-electron reduction product of tetrazolium) may not reduce O2 to O2-., that the redox reaction between semiquinone radicals of phenazine and O2 is reversible, and that the disproportionation of semiquinone radicals constitutes an important rate-limiting reaction in the expression of phenazine redox couple.

Aerobiosis↗

PubMed2003

5-Cyano-2,3-ditolyl tetrazolium chloride (CTC) reduction in a mesophilic anaerobic digester: measuring redox behavior, differentiating abiotic reduction, and comparing FISH response as an activity indicator.

The tetrazolium salt 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) has been widely applied to assess microbiological activity in environmental samples. CTC reduction has previously been quantified in a variety of anaerobic systems (i.e., fermentative, nitrate reducing, sulfate reducing) using direct microscopy, solvent extraction, and flow cytometry. In this work, extracellular CTC reduction was observed and distinguished from its intercellular counterparts by the amorphous character and near uniform fluorescence of the resulting formazan precipitates (CTF). Fluorescence yielded by non-cellular-associated formazan precipitates bleached much more rapidly than CTF formed within cells under identical UV exposure (<2 min). Dehydrogenase activity assays and fluorescent in situ hybridization (FISH) were simultaneously carried out in microcosms containing active anaerobic digester biomass, propylene glycol, and settled sewage centrate for direct comparison. In substrate limited microcosms, quantitative FISH measurements remained well above their detection limit indicating sustained intercellular ribosomal RNA concentrations over a 5-day period, while dehydrogenase assays (CTC) decreased to background levels within 14 h of substrate limitation. Results from this work suggest that CTC reduction in cell-free samples may impede accurate enzyme activity measurements, particularly when quantification involves solvent extraction, flow cytometry, or software-aided counting. In addition, activity assessment in anaerobic digesters using FISH and CTC reduction assays may be comparable until substrate becomes limited.

Bacteria, Anaerobic↗