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At least 289 records · Page 16Linked to original sources
PubMed1975

Effect of chronic ethanol ingestion on fatty acid oxidation by hepatic mitochondria.

To study possible factors in the pathogenesis of the ethanol-induced fatty liver, we investigated the effect of chronic ethanol consumption on the metabolism of fatty acids by isolated hepatic mitochondria. Chronic ethanol consumption resulted in decreased fatty acid oxidation, as evidenced by a reduction in oxygen uptake and CO2 production associated with the oxidation of fatty acids. The State 3 rate of oxygen uptake was depressed to a greater extent than the State 4 or the uncoupler-stimulated rate; the respiratory control ratio was also decreased. Therefore, one site of action of chronic ethanol feeding is on oxidative phosphorylation. The reduction in fatty acid oxidation, in general, is not due to an effect on the activation or translocation of fatty acids into the mitochondria. There was no effect by ethanol feeding on the activity of palmitoyl coenzyme A synthetase, whereas carnitine palmitoyltransferase activity was increased. The use of an artificial system (formazan production) to study beta oxidation in the absence of the electron transport chain is described. In the presence of fluorocitrate, which inhibits citric acid cycle activity, ketogenesis and formazan production were increased by chronic ethanol consumption. Thus beta oxidation to the level of acetyl-CoA is not impaired by chronic ethanol consumption. Total oxidation of fatty acids to CO2 is depressed by chronic ethanol intoxication because of effects on oxidative phosphorylation or the citric acid cycle (or both). Neither nutritional deficiency, cofactor depletion, nor the presence of ethanol in vitro explains these effects. Several of the effects of chronic ethanol consumption on fatty acid oxidation are mimicked by acetaldehyde and acetate, products of ethanol oxidation. Chronic ethanol consumption leads to persistent impairment of mitochondrial oxidation of fatty acids to CO2. However, oxidation of fatty acids to acetyl-CoA is not decreased by chronic ethanol consumption.

Alcoholic Intoxication↗
PubMed1992

Polymicrobial sepsis disrupts normal neutrophil extracellular matrix protein interactions.

The purpose of this study was to examine how intra-abdominal sepsis and extracellular matrix proteins (fibronectin, laminin) affect adherent polymorphonuclear leukocyte (PMN) function. Two groups of swine were studied: Group I (n = 5) underwent sham laparotomy; Group II (n = 8) underwent cecal ligation and incision. PMN adherent to either fibronectin (F) or laminin (L) had increased candicidal activity over buffer (B) by Group I but not by post-operative day 8 Group II PMN. (Percent specific release 51Cr-Group I--35.00, 68.25, 64.75% for B, F, and L; P less than 0.001 comparing B vs. F or L; Group II--14.25, 12.50, 12.75% for B, F, and L; P = NS comparing B vs. F or L.) To determine the mechanism for this finding, PMN priming was then assessed by evaluating both PMN adherence to extracellular matrix proteins and the cell surface expression of CR1/CR3 by using sheep RBC opsonized with C3b or C3bi. PMN activation was assayed by using MTT-Formazan, myeloperoxidase, and hypochlorous acid (HOCl) production. Fibronectin and laminin increased PMN adherence and CR1/CR3 expression over buffer by Group I and Group II animals. Fibronectin and laminin increased MTT-Formazan, myeloperoxidase, and HOCl production over buffer by Group I PMN but not POD 8 Group II PMN. These results suggest that untreated intra-abdominal sepsis partially abrogates the effect of extracellular matrix proteins on PMN function; in particular, the activation but not priming of adherent PMN by extracellular matrix proteins is reduced in this clinical situation.

Animals↗
PubMed1992

Validation of the MTT dye assay for enumeration of cells in proliferative and antiproliferative assays.

Increasing use is being made of colorimetric assays to quantitate viable cells, e.g., the cellular reduction of the tetrazolium salt, MTT, to formazan by mitochondrial succinate dehydrogenase. We validated this assay for cell proliferation in mixed lymphocyte cultures as compared with 3H-thymidine uptake, and for inhibition of cell proliferation induced by interferon with results compared by direct cell counting. We also found that cells do not, as previously assumed, require functional mitochondria: there were no differences in formazan production by normal cells or respiratory defective cells in which mitochondria had been poisoned by the nucleic acid toxin, ethidium bromide. The high reproducibility of MTT metabolism by lymphoblasts and various cultured cell lines establishes the reliability and versatility of this method for quantitating cell numbers.

Animals↗
PubMed1989

[Determination of the activity of superoxide dismutase in terms of rea substrate conversion].

On the basis of photochemical generation of O2.- and the tetrazolium/formazan system as indicating reaction an assay for SOD was developed which allows the determination of initial rate velocities of the enzyme. This was achieved by a special apparatus yielding the continuous registration of product formation simultaneously with the photochemical generation of O2.-. The investigation of the indicating reaction revealed that the reduction of nitro blue tetrazolium chloride to formazan is stoichiometrical with respect to riboflavin/methionine generated O2.- above pH 9. On this basis, an activity unit of SOD can be defined as real substrate consumption. One unit of the enzyme causes a decrease in the formation of formazan of 1 mumol/min under defined conditions.

Formazans↗
PubMed1988

Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines.

We have previously described the application of an automated microculture tetrazolium assay (MTA) involving dimethyl sulfoxide solubilization of cellular-generated 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-formazan to the in vitro assessment of drug effects on cell growth (M.C. Alley et al., Proc. Am. Assoc. Cancer Res., 27:389, 1986; M.C. Alley et al., Cancer Res. 48:589-601, 1988). There are several inherent disadvantages of this assay, including the safety hazard of personnel exposure to large quantities of dimethyl sulfoxide, the deleterious effects of this solvent on laboratory equipment, and the inefficient metabolism of MTT by some human cell lines. Recognition of these limitations prompted development of possible alternative MTAs utilizing a different tetrazolium reagent, 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl] -2H- tetrazolium hydroxide (XTT), which is metabolically reduced in viable cells to a water-soluble formazan product. This reagent allows direct absorbance readings, therefore eliminating a solubilization step and shortening the microculture growth assay procedure. Most human tumor cell lines examined metabolized XTT less efficiently than MTT; however, the addition of phenazine methosulfate (PMS) markedly enhanced cellular reduction of XTT. In the presence of PMS, the XTT reagent yielded usable absorbance values for growth and drug sensitivity evaluations with a variety of cell lines. Depending on the metabolic reductive capacity of a given cell line, the optimal conditions for a 4-h XTT incubation assay were 50 micrograms of XTT and 0.15 to 0.4 microgram of PMS per well. Drug profiles obtained with representative human tumor cell lines for several standard compounds utilizing the XTT-PMS methodology were similar to the profiles obtained with MTT. Addition of PMS appeared to have little effect on the metabolism of MTT. The new XTT reagent thus provides for a simplified, in vitro cell growth assay with possible applicability to a variety of problems in cellular pharmacology and biology. However, the MTA using the XTT reagent still shares many of the limitations and potential pitfalls of MTT or other tetrazolium-based assays.

Azo Compounds↗
PubMed1985

[Demonstration of succinate dehydrogenase activity in ascites tumor cells using fluorescent tetrazolium salts].

In Ascites tumor cells a fluorescent tetrazolium salt, new synthesized (Stellmach 1984) was tested on its suitability for the histochemical enzyme demonstration. Optimal incubation conditions for the demonstration of the succinate dehydrogenase were found out. The red coloured and red fluorescent formazane formed by enzymatic reduction can be localized as well under the light microscope as under the fluorescent microscope. A quantification of the formed formazane as a measure for the enzyme activity was possible by measuring the absorption and fluorescent intensity.

Animals↗
PubMed1983

[Histochemical detection of tissue changes using tetrazolium salts of differing nitrosensitivity].

By histochemical detection of succinic dehydrogenase in the myocardium of rats, it was shown that cell alterations induced by isoprenaline can be demonstrated in different qualities by the use of the atypical formazans of suitable nitro-monotetrazolium salts. The chemical structure of these nitro-monotetrazolium salts defines their different trends ("nitro-sensitivity") to react in their atypically-coloured formazans under histochemical conditions. A range of nitro-monotetrazolium salts with graded nitro-sensitivities is stated for improved histochemical detection of cell and tissue injuries.

Animals↗
PubMed1984

[Value of histochemical tetrazolium technics].

A short review is given about the value of the tetrazolium technique in histochemistry including some new aspects of the tetrazolium reduction. The different factors and properties determining the localization, pattern and colours of the formazans in the tissue slide are discussed. The current knowledge of the atypically coloured formazans caused by the nitro groups is presented.

Formazans↗
PubMed1976

[Histochemical evidence of the aminotransferases. V. Quantitative histochemical investigations of the aminotransferases in organs of the rat (author's transl)].

The quantitative histochemical method was employed the activity of some aminotransferases in the rat organs. Determination was based on extraction of the enzymatic reaction product formazan with alkaline dimethylsulfoxid. Evaluation of the enzymatic activity was given by the formazan quantity related to 1 mug tissue protein. The following transaminases have been studied: aspartate-, alanine-, tyrosine-, phenylalanine-, tryptophane-, glycine-, and serine aminotransferases, as the leucine aminotransferase with substrate leucine, isoleucine and valine. Difference was observed as with respect to one and the same aminotransferase in the examined organs, as well, with respect to the different aminotransferases in one and the same organ.

Animals↗
PubMed1995

[Usefulness of the microcolorimetric method of XTT reduction for evaluation of intracellular killing of pathogens].

The aim of this study was to evaluate a XTT assay in testing the killing activity of mouse phagocytes in vitro. Live microorganisms converted XTT to water soluble orange formazan in the presence of CQ. Absorption of formazan measured at 492 nm was directly related to the number of viable cells. The percentage of staphylococci killed by granulocytes and macrophages was 10-40% (1 h- and 2 h-respectively), in the cultures containing 10 bacteria/phagocyte. Killing of Candida albicans (1-3 blastospors/phagocyte) was seen after 2 h of incubation. The percentage of Listeria and Mycobacterium killed by phagocytes depended on pathogenicity of the tested strains. The bactericidal activity of phagocytes estimated in the XTT assay and by the CFU method was quite similar.

Animals↗
PubMed1998

Microtiter plate assay for superoxide dismutase using MTT reduction by superoxide.

A simple microtiter plate based colorimetric assay for superoxide dismutase is described. The method, involves generation of superoxide by pyrogallol autoxidation and the inhibition of superoxide dependent reduction of the tetrazolium dye MTT [3-(4,5-dimethyl-thiazol-2-yl) 2,5-diphenyl tetrazolium bromide] to its formazan, measured at 570 nm. The reaction was terminated by the addition of dimethyl sulfoxide (DMSO) which also helps to solubilize the formazan formed and the colour evolved was stable for many hours. The method was compared with other known methods to measure the activity of purified erythrocyte Cu,ZnSOD and superoxide dismutase activity from various rat tissues. This procedure involves inexpensive reagents, allows a rapid and sensitive measurement of SOD activity and the microtiter plate assay is suitable for use with large number of samples.

Animals↗
PubMed1989

Glucose-6-phosphate dehydrogenase activity in the olfactory system of the young rat: an enzyme histochemical study using computerized image analysis.

An understanding of olfactory system glucose metabolism is necessary for the interpretation of radiolabeled 2-deoxyglucose studies of odor processing since the relationship between glucose uptake and neural activity is based on assumptions regarding cellular glucose utilization. As part of an ongoing study examining divergent pathways of glucose metabolism in the olfactory system, the relative activity of glucose-6-phosphate dehydrogenase, the rate-limiting enzyme of the hexose monophosphate shunt, was examined among cells of the rat olfactory bulb and anterior olfactory nucleus, by using enzyme histochemistry on fresh frozen tissue. Optical density measurement of formazan reaction product in stained tissue were quantified by computerized image analysis. To aid in the identification of histochemically stained neurons, alternate sections were Nissl-stained. The highest olfactory bulb dehydrogenase levels were found in the olfactory nerve and glomerular layers. Individual mitral and tufted cells also showed high dehydrogenase activity. In most stained neurons, formazan reaction product filled the cytoplasm and sometimes extended into the proximal part of dendrites and axons. The external plexiform and granule cell layers had low enzyme activity. High activity also was seen in pyramidal cells of pars dorsalis and pars lateralis of the anterior olfactory nucleus, one of the first, and most rostral of the olfactory bulb projection sites. High glucose-6-phosphate dehydrogenase activity in the olfactory system indicates that a significant amount of glucose can be channeled through the hexose monophosphate shunt in these neurons, with a concomitant production of NADPH. This may reflect high activity of cellular detoxification enzymes that rely on NADPH for reducing power. Such detoxification processes may be engaged in response to the potential entry and transsynaptic movement of airborne chemicals into the brain via the olfactory system.

Animals↗
PubMed1985

Differentiation of HL-60 promyelocytic leukemia cells monitored by flow cytometric measurement of nitro blue tetrazolium (NBT) reduction.

Reduction of nitro blue tetrazolium (NBT) to insoluble blue formazan granules occurs during the stimulus-induced respiratory burst of mature granulocytes and is routinely used as an indicator of the extent of granulocytic differentiation of HL-60 acute promyelocytic leukemia cells. In the present study, the differentiation of HL-60 leukemia cells induced by dimethylsulfoxide (DMSO) or retinoic acid was monitored by flow cytometric (FCM) measurement of forward and 90 degree light scatter of NBT treated cells. Two-parameter correlated analysis permitted a distinction between cells with increased forward and decreased 90 degree light scatter (NBT-), and cells with decreased forward and increased 90 degree light scatter (NBT+). Fixation of NBT treated cells with 1% paraformaldehyde facilitated flow cytometric analysis, and allowed differences in NBT reduction to be quantitated. DMSO-induced cells expressed an all-or-none reduction of NBT to formazan, compared with retinoic acid treated cells that exhibited a graded response. Three parameter flow cytometric analysis of HL-60 leukemia cells stained with propidium iodide in combination with NBT allowed the determination of the cell cycle distribution of NBT-treated cells.

Cell Differentiation↗
PubMed1992

NADH-dependent dehydrogenase activity estimation by flow cytometric analysis of 3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction.

MTT reduction is usually analysed by colorimetric assay to study mitochondrial dehydrogenase activity as a test of cytotoxicity. This enzymatic reaction produces dark-blue granules of formazan, which increase cell refringency. In this work, we define the conditions for MTT use in quantitative flow cytometric analysis. MTT reduction provides a non-fluorescent dye usable by this technique to study an intracellular NADH-dependent dehydrogenase activity in vital cells. We observe that formazan production increases asymptotically with cell concentration and that this temperature-dependent Michaelis enzymatic reduction is produced essentially by mitochondrial dehydrogenases. In isolated mitochondria from rat hepatocytes and in whole L1210 murine leukemia cells, the Michaelis constants (KM) observed in the presence of respiratory substrates were, respectively, 10 microM and 500 microM. The inhibition of mitochondrial protein synthesis by chloramphenicol, which induces a rise of MTT reduction due to the correlative stimulation of glycolysis (Pasteur effect), is a limit of the MTT assay as a cytotoxicity test.

Animals↗
PubMed1986

Heterogeneity in chronic granulomatous disease detected with an improved nitroblue tetrazolium slide test.

The microscopic nitroblue tetrazolium (NBT) slide test, used to score the ability of individual phagocytic leukocytes to produce superoxide, was improved according to the following procedure. Purified granulocyte suspensions are incubated with NBT and fixed in suspension, thereafter centrifuged on microscope slides and stained with nuclear fast red. This method precludes stimulation and selection of cells by adherence and washing. The number of formazan grains per cell can be judged in a semiquantitative way. In parallel incubations, the cells are stained with May-Grünwald/Giemsa, allowing identification of formazan-positive and -negative cells. The test discriminates well between cells from normal individuals, cells from patients with chronic granulomatous disease (CGD), and cells from heterozygotes for the X-linked form of CGD. Several patients and heterozygotes with an autosomal or variant form of CGD were detected with decreased NBT-reducing activity in their neutrophils and/or eosinophils. The relation between NBT-reductase activity of the phagocytes and the clinical situation is discussed.

Cytochrome b Group↗
PubMed1993

Rapid colorimetric bioassay for screening of Fusarium mycotoxins.

The cytotoxicity of Fusarium mycotoxins was evaluated using a trichothecene sensitive cell line (BHK-21, baby hamster kidney cells) in combination with the MTT-cleavage test as an end-point measurement. Cells tended to be more sensitive to the type A trichothecenes with midpoint cytotoxicity values ranging from 1.6 ng/ml for T-2 toxin to 60 ng/ml for scirpentriol. The cytotoxicity value for deoxynivalenol (type B) was 112 ng/ml. The inherent disadvantage of the MTT-assay (formation of insoluble formazan) was overcome by using the analog MTS and measuring the water-soluble formazan directly in the culture media. The MTS-midpoint cytotoxicity values for T-2 toxin and deoxynivalenol (2.1 and 141 ng/ml, respectively), although slightly higher, showed a good correspondence to the MTT-test. Both the MTT- and MTS-cleavage tests are useful for evaluating the cytotoxicity of Fusarium mycotoxins. The replacement of MTT by MTS substantially reduced the number of sample processing steps and the length of time required to complete the cytotoxicity assay.

Animals↗
PubMed1976

Exeriences with isoprenaline induced myocardial necrosis in the rat.

An attempt has been made to quantify myocardial lesions produced in the rat by isoprenaline for use as a model to assess possible incremental effects of environmental and dietary factors. This was initially made difficult by variation in the cardiotoxicity of different samples of isoprenaline. Investigation of these samples failed to reveal the basis for the differences. Active preparations have, however, produced profound changes both clinically and pathologically. The earliest light-microscopic changes both clinically and pathologically. The earliest light-microscopic change was loss of fuchsinophilia of fibres in sections stained by the picro-Mallory technique. By 24 hr obvious necrosis, fragmentation and lysis of the fibres had occurred. Treatment of frozen sections to demonstrate succinate dehydrogenase showed early changes in the character of formazan, suggesting the possibility of a transient alteration in the hydrogen transport system. By 48 hr, this is reversed except in those fibres undergoing necrosis where there is a complete loss of formazan. This contrast in staining between normal and necrotic fibres constitutes the basis for quantification which has been carried out by point counting. The results show some differences in the amount of myocardial necrosis between different batches of animals but relatively small differences within individual batches, suggesting that the introduction of additional variants into the system should be capable of producing clear cut results.

Animals↗
PubMed2000

Quantitative alpha-ketoglutarate dehydrogenase activity staining in brain sections and in cultured cells.

The activity of a key mitochondrial enzyme, the alpha-ketoglutarate dehydrogenase complex (KGDHC), declines in the brains of patients with neurodegenerative diseases such as Alzheimer's disease, as well as in thiamine-deficient (TD) animals. The decreased activity often occurs without a reduction in enzyme protein, which negates the use of immunocytochemistry to study cellular or regional changes in enzyme activity within the brain. To overcome this limitation, an activity staining method using nitroblue tetrazolium was developed. The histochemical activity staining was standardized in cultured cells. The assay was linear with time and was highly specific for KGDHC. The dark-blue reaction product (formazan) formed a pattern that was consistent with mitochondrial localization. Treatment of the cultured cells with both reversible and irreversible inhibitors decreased formazan production, whereas conventional enzyme assays on cell lysates only revealed loss of KGDHC activity with irreversible inhibitors. The activity staining was also linear with time and highly specific for KGDHC activity in mouse brain sections. Staining occurred throughout the brain, and discrete neuronal populations exhibited particularly intense staining. The pattern of staining differed markedly from the distribution of KGDHC protein by immunocytochemistry. Generalized decreases in the intensity of activity staining that occurred in the TD brains compared to controls were comparable with the loss of KGDHC activity by conventional enzyme assay. Thus, the present study introduces a new histochemical method to measure KGDHC activity at the cellular and regional level, which will be useful to determine changes of in situ enzyme activity.

Animals↗