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PubMed1989

Metabolic changes in undifferentiated and differentiated human colon adenocarcinoma cells studied by multinuclear magnetic resonance spectroscopy.

Aspects of energetic and intermediary metabolism were studied in a colon adenocarcinoma cell line (HT29) by multinuclear magnetic resonance spectroscopy. Experiments were carried out on the HT29-D4 clone, which was isolated by limit dilution techniques. This clone, usually undifferentiated (D4-UD), can be maintained in a differentiated state (D4-D) in a glucose-free medium. Metabolic data were obtained by NMR analysis of perchloric acid extracts from D4-UD and D4-D cells. Phosphorus-31 and proton NMR spectra showed the presence of a large amount of choline and phosphorylcholine in the differentiated state (400% and 200%, respectively, of the levels found in D4-UD cells). Other differences appeared in the content of phosphocreatine (absent in D4-D cells) and myoinositol (absent in D4-UD cells). Carbon-13 spectra were recorded from perchloric acid extracts of cells incubated with [1-13C]-labeled glucose or [2-13C]-labeled acetate. The data indicated that both types of cells metabolize glucose through the glycolytic pathway to give lactate, but only D4-D cells were able to store glucose as glycogen at a very high level. A mathematical analysis of fluxes through the tricarboxylic acid (TCA) cycle was developed on the basis of models derived from previous 14C tracer studies. The model was based on the steady-state labeling of glutamate carbons by the 13C isotope and gave the fraction of labeled acetyl-Coa entering the TCA cycle, and the activity y of anaplerotic reactions relative to the flux through the citrate synthetase reaction. The data indicated that y greater than 0.3 in all cases. Only 15% and 30% of labeled acetyl CoA entered the TCA cycle in D4-UD and D4-D cells, respectively, under labeled glucose incubation: these values were significantly different upon labeled acetate feeding, reaching 55% for D4-UD cells and 85% for D4-D cells. The main result of this study is that the process of differentiation of HT29 cells is correlated with a large increase in the activity of oxidative metabolism.

Acetates↗

PubMed1985

Serum and plasma beta-carotene levels measured with an improved method of high-performance liquid chromatography.

An isocratic high-performance liquid chromatographic method specifically developed to allow simple and rapid determination of beta-carotene concentrations in serum and plasma is reported. Using a method modified from a previously published technique, serum and plasma proteins are denatured by exposure to perchloric acid, and beta-carotene is subsequently extracted into an organic matrix consisting of ethyl acetate-tetrahydrofuran (1:1); no evaporation step is required. Separation is achieved using isocratic elution from a reversed-phase C18 column with UV detection at 436 nm. Recovery of beta-carotene from water and plasma was greater than 98.1%; beta-carotene was stable in the extraction matrix for at least 4 h. Three anticoagulants (oxalate, citrate, and EDTA) caused losses of beta-carotene; perchloric acid and tetrahydrofuran could also destroy beta-carotene under certain conditions. Each run required less than 15 min; within-day coefficient of variation for identical samples averaged 2.3%, between-day coefficient of variation was 4.4% and sensitivity was better than 10 ng/ml. Stability of beta-carotene in plasma was also examined. This method permits a simple, rapid, sensitive, precise, and accurate determination of beta-carotene using 0.5 ml of serum or heparinized plasma.

Butylated Hydroxytoluene↗

PubMed1977

[Proteolytic breakdown of human inter-alpha-trypsin inhibitor by plasmin (author's transl)].

Inter-alpha-trypsin inhibitor was isolated from human plasma and submitted to proteolytic degradation by plasmin. A split product of low molecular weight (18 000 daltons) is obtained by gel filtration or solubilisation in perchloric acid. This fragment reacts with an anti-inter-alpha-trypsin inhibitor immune serum and migrates as beta1 globulins. Its specific activity against trypsin (after absorption of residual plasmin on sepharose lysine) was estimated to be 900 mU1/mg. Thus one molecule of fragment can inhibit one molecule of trypsin. As well with native protein as with its fragment, complexes formed with trypsin can be dissociated by urea or sodium dodecyl sulfate. This fragment is similar to the small molecular weight inhibitors obtained directly by solubilisation in perchloric acid from serum, urine and bronchial secretions.

Antigens↗

PubMed1992

Beta-adrenergic stimulation of C6 glioma cells: effects of cAMP overproduction on cellular metabolites. A multinuclear NMR study.

We used 31P-NMR spectroscopy to investigate the response of living C6 glioma cells to stimulation by a beta-adrenergic agonist, isoproterenol. In the presence of 3-isobutyl-1-methylxanthine, stimulation induced an accumulation of cAMP, making possible the NMR detection of the second messenger in living cells grown on microcarrier beads and perfused in the NMR tube. The cAMP signal rose to a maximum level within 20-25 min of stimulation; thereafter it decreased to the detection threshold within 60 min. At the same time, 40% increases of phosphomonoester and diphosphodiester signals were observed, whereas no significant change in phosphocreatine and nucleotide signals was detected. The kinetics of changes of the cellular content in phosphorylated metabolites were analyzed after recording 31P-NMR spectra of cell perchloric acid extracts as a function of time of stimulation. cAMP accumulation in stimulated cells was evidenced by a near linear increase of its NMR signal as a function of incubation time (from 0 to 60 min). Concomitantly with the production of cAMP, the data showed 30% decreases of phosphocreatine and ATP levels within 60 min of stimulation, and an unexpected redistribution of pyrimidine and purine nucleoside triphosphates. At the same time, levels of phosphomonoesters (phosphorylcholine and phosphorylethanolamine) and phosphodiesters (glycerophosphorylcholine and glycerophosphorylethanolamine) rose (50% increase). 13C-NMR spectra of cell perchloric acid extracts prepared after isoproterenol stimulation of cells incubated in the presence of [1-13C]glucose indicated a higher glucose content in stimulated cells, whereas the resonance of ribose C1 was diminished. Moreover, the resonances of C1 of ethanolamine and choline (and their derivatives) were increased in spectra of stimulated cells, whereas that of C3 of serine was decreased. In addition, the 13C-NMR data indicated that neither the pattern of glutamate carbon enrichment nor the glutamate/glutamine ratio was modified in stimulated cells. On the other hand, the heteronuclear coupling pattern of the lactate (methyl group) resonance in 1H-NMR spectra of cell incubation media indicated that no change occurred in the carbon flux through the pentose-phosphate shunt under stimulation. The results of this multinuclear NMR approach are discussed in terms of metabolic responses of C6 cells to beta-adrenergic stimulation and cAMP overproduction.

Adenosine Triphosphate↗

PubMed1971

Studies of colonic carcinoma antigens.

Extracts of human colonic adenocarcinomata were made with perchloric acid and shown to contain antigens which were not present in pooled samples of normal colonic mucosa or in healthy colonic mucosa from patients providing the tumours. Antisera prepared against the tumour extracts gave two precipitin lines on immunodiffusion, and antiserum prepared in Montreal according to the methods devised by Gold and Freedman (1965a and b) also gave two lines of precipitation when tested against some of these extracts; one of these precipitin lines gave a reaction of identity with that given by the antiserum produced here. The results suggest that perchloric acid may extract two antigens from colonic carcinomata.

Adenocarcinoma↗

PubMed1991

A malachite green colorimetric assay for protein phosphatase activity.

A simple and sensitive colorimetric assay for protein phosphatase activity based on the determination of released Pi by an improved malachite green procedure (A. A. Baykov, O. A. Evtushenko, and S.M. Avaeva, 1988, Anal. Biochem. 171, 266-270) is described. Proteins must be removed or stabilized prior to Pi determination with 0.25 N sulfuric acid or 3% (w/w) perchloric acid. Alternatively, to avoid possible acid hydrolysis of phosphate groups from organic compounds during deproteinization, the protein present in the phosphatase assay mixture can be stabilized with sodium dodecyl sulfate. In this case, the excess detergent is subsequently removed by precipitation with KCl because it colors with the malachite green reagent. The above procedure was applied to the determination of phosphorylase phosphatase activity in bovine brain extracts and the results are comparable to those obtained with the radioisotopic phosphatase assay.

Animals↗

PubMed1995

In vivo and in vitro studies of cyclophosphamide chemotherapy in a mouse mammary carcinoma by 31P NMR spectroscopy.

The effect of cyclophosphamide on the metabolic profile of a mammary carcinoma implanted on the foot of mouse was studied by 31P NMR spectroscopy both in vivo and in perchloric acid extracts. The ratio nucleotide triphosphate:P(i) was significantly elevated in cyclophosphamide treated tumours relative to untreated tumours after 96 h in vivo (p < 0.05). Phosphocreatine:P(i) was similarly elevated from 48 to 168 h (p < 0.01). Resolution of the phosphomonoester peak into two distinct resonances allowed us to estimate the ratio of PME' to phosphocholine (PC), where PME' is a composite peak consisting, in part, of phosphoethanolamine (PE). PME':PC was found to be significantly higher in treated animals relative to control animals in vivo (p < 0.01 from 48 to 168 h). Perchloric acid extract spectra suggest that the increase in PME':PC was in part due to a decrease in PC concentration and also due to an increase in a previously unidentified resonance which was coresonant with PE. Extract data show that there was a significant increase in the concentration of the phosphodiesters, glycerophosphocholine (p < 0.01) and glycerophosphoethanolamine (p < 0.05) in treated relative to control tumours. The changes in the phosphomonoester resonances are qualitatively similar to previously described changes following radiation and suggest that they may be a marker of cell kill or lack of cell growth after antineoplastic therapy.

Adenocarcinoma↗

PubMed1982

[Polyphosphate and ATP content of Propionibacterium shermanii cells in nitrogen starvation].

When Propionibacterium shermanii was cultivated in a medium with glucose under the conditions of nitrogen deficiency, the content of high molecular weight polyphosphates (salt-soluble fraction, alkali-soluble fraction, and fraction extracted with hot perchloric acid) decreased. In contrast, it rose in a medium with lactate within two and three days of starvation. The culture growth stopped after a day under these conditions, the energy substrate was assimilated during the entire process of starvation, and the content of ATP continuously decreased. When ammonium sulfate was added after two and three days of starvation, the intracellular content of polyphosphates (in all of the three fractions) increased in the medium with glucose. If ammonium sulfate was added after three days of starvation in the medium with lactate, the content of polyphosphates rose in the fraction extracted with hot perchloric acid while the content of salt-soluble and alkali-soluble polyphosphates decreased. The culture started to grow again and the content of ATP in its cells rose abruptly when ammonium sulfate was added under these conditions. If the culture was grown in the medium with glucose and ammonium sulfate, the content of acid-insoluble polyphosphates increased twofold during the first day and dropped to the initial level by the third day of growth.

Adenosine Triphosphate↗

PubMed1966

Histochemical observations on Mycoplasma after staining with acridine orange.

Mycoplasma colonies and Mycoplasma cells in preparations from infected milk and lymph nodes were observed for their fluorescent qualities after treatment with acridine orange. Mycoplasma colonies fluoresced brilliant red or red-orange. When treated after exposure to ribonuclease, the colonies fluoresced lime-green. There was no fluorescence when both ribonucleic acid and deoxyribonucleic acid were destroyed by perchloric acid. Detection of Mycoplasma in smears of mastitic milk or smears of infected lymph nodes was not definitive because of the large amount of nonspecific ribonucleic acid-rich material present during inflammatory reactions.

Acridines↗

PubMed1988

Determination of cefetamet and its orally active ester, cefetamet pivoxyl, in biological fluids by high-performance liquid chromatography.

Two different, simple and rapid high-performance liquid chromatographic methods with ultraviolet detection, using a common sample work-up procedure, were developed for the determination of cefetamet, an in vitro active cephalosporin, and its orally absorbed pivaloyloxymethyl ester, cefetamet pivoxyl. After protein precipitation with perchloric acid, plasma samples were analysed on C18 reversed-phase columns with 4 mM perchloric acid-acetonitrile (83:17, v/v) and 0.1 M phosphate buffer (pH 6.5)-acetonitrile (60:40, v/v) as mobile phases for the determination of cefetamet and cefetamet pivoxyl, respectively. Urine samples were diluted with water and analysed in the same manner, using 4 mM perchloric acid-acetonitrile (85:15, v/v). The limits of quantification were 0.2, 0.5 and 20 micrograms/ml for the determination of cefetamet and cefetamet pivoxyl in plasma and cefetamet in urine, respectively. The intra-assay precision was less than or equal to 1.5% for cefetamet and less than or equal to 2.3% for cefetamet pivoxyl. The inter-assay precision for cefetamet was less than or equal to 2.4%. Cefetamet was stable in human plasma when stored at -20 degrees C for three months or at 22 degrees C for 24 h. For the determination of cefetamet pivoxyl, which was extremely unstable in plasma (greater than 70% degradation in 1 h), samples were drawn into vacutainers containing citric acid and immediately added to sodium fluoride. The method for cefetamet was successfully applied to several thousand plasma and urine samples from humans, dogs and rats. No unchanged drug could be detected in human or dog plasma after the administration of cefetamet pivoxyl.

Ceftizoxime↗

PubMed1999

Application of experimental design for the characterisation of a novel elution system for high-capacity anion chromatography with suppressed conductivity detection.

A novel elution system for the application of high-capacity anion exchangers with suppressed conductivity detection in ion chromatography is presented. The ternary elution system is based on perchloric acid, sodium hydroxide and sodium carbonate. The novel elution system was applied to a self-made high-capacity anion-exchange column (Q = 453 mu equiv. Cl-). A central composite design with 20 experiments was used to investigate the influence of the eluent compounds, which varied from 0.2 to 1.0 mM HClO4, 20 to 100 mM NaOH and 0 to 20 mM Na2CO3, on the retention factor k' of seven common anions. A quadratic model including interactions was postulated. The model equations were used to estimate retention factors at known eluent compositions. No significant differences of calculated and experimental retention factors were found. Further statistical analysis was done by analysis of variance. The results showed that the three eluent compounds have completely different effects on the retention behaviour of the anions investigated. The elution of soft and weakly hydrated anions like bromide and nitrate is strongly influenced by the content of perchloric acid, whereas strongly hydrated anions like fluoride and sulphate are mainly affected by the hydroxide or carbonate content of the eluent. The results can qualitatively be explained comparing the relation of ionic radii to charge for eluent and analyte anions. As a consequence, the retention of the anions investigated can easily be manipulated by varying the contents of the eluent compounds. The usefulness of the novel elution system and high-capacity anion chromatography is demonstrated by the determination of trace anions in phosphate and fluoride matrices.

Anion Exchange Resins↗

PubMed1985

31P NMR studies of Clostridium thermocellum. Mechanism of end product inhibition by ethanol.

31P NMR studies of intact cells and perchloric acid extracts are used to investigate the effect of ethanol on the bioenergetics and glycolysis of Clostridium thermocellum, an anaerobic bacterium potentially useful for the single step conversion of biomass to ethanol. Whole cells suspended in phosphate buffer and given a carbon source (cellobiose) at 60 degrees C rapidly establish a pH gradient across the membrane that can be monitored by the chemical shifts of inorganic phosphate in the exterior buffer and in the cytoplasm. Peak intensities can be related to phosphate active transport rates. Wild type bacteria and cells grown in inhibiting concentrations of ethanol establish similar pH gradients, but with slower kinetics and slower phosphate transport rates for the cells adapted to growth in ethanol. Direct addition of ethanol does not affect the rate of pH gradient formation or phosphate transport. Thus, while ethanol does not directly affect processes for energy conservation carried out by the membrane, adaptation to ethanol does alter membrane functions such as phosphate transport. 31P NMR spectra of perchloric acid extracts show that when wild type cells are adapted to grow in inhibiting concentrations of ethanol and then energized with cellobiose, sugar phosphate content is increased and the steady state distribution of glycolytic intermediates is altered. Nucleotide triphosphate/nucleotide diphosphate ratios are unaltered in these cells. These results strongly indicate that in C. thermocellum growth inhibition by ethanol is related to a blockage in glycolysis.

Anaerobiosis↗

PubMed2005

Estimation of fluoride distribution in the mandible and teeth of the red deer (Cervus elaphus L.) from industrially polluted areas in Poland.

MINI SUMMARY: Animals from areas contaminated by industrial emissions containing fluoride may accumulate it predominantly in hard tissues. Therefore, an analysis was performed for the determination of fluoride content in mandibles and teeth of deer living at large in different areas in Western Pomerania (Poland). Samples of hard tissues obtained with a dental drill, were dissolved in perchloric acid and analyzed for fluoride content with an ion-selective electrode. Using the Generalized Regression Neural Network (GRNN), it was possible to arrange definite variables according to their order of importance concerning the fluoride mean content in investigated bone material. Parametric analysis revealed in which sites of mandibles and teeth the fluoride accumulation was the highest. In our calculation, the traditional statistic as well as artificial neural network was applied. OBJECTIVE: One of the interesting properties of fluoride is its balancing accumulation in the hard and soft tissues. In this paper, we decided estimate how the changeable doses of fluoride in natural environment influence on fluoride distribution and accumulation in the examined sites of mandible and teeth. MATERIAL AND METHOD: The material consist of 103 mandibles of red deer (Cervus elaphus L.) from five forestry districts of Western Pomerania in Poland, which are located near or far away from two major industrial plants: Police Chemical Work and the Dolna Odra Coal Power Plant. Samples (10 mg of powdered bone or tooth) were dissolved in perchloric acid and the fluoride content was determined with an ion-selective electrode. Comparisons were done with the Statistica 5.5 and Statistica Neural Networks. RESULTS: Animals living in Western Pomerania (Poland) have high fluoride contents in their bones. Fluoride accumulation in mandibles and teeth is irregular. The higher content of fluoride show incisal area, but coronoid process of the mandible was not sensitive to different exposure of environmental fluoride at all. Parametric analysis revealed that the accumulation pattern of fluorine was different in the two groups of deer. CONCLUSIONS: Many biological and environmental factors may influence on incessant fluoride accumulation in osseous tissue of ruminants living at large. The use of artificial neural networks enables a more accurate insight into the process of fluorine accumulation in the mandible and teeth and helps in the ranking of factors influencing this process.

Animals↗

PubMed1999

Perchloric acid-soluble proteins from goat liver inhibit chemical carcinogenesis of Syrian hamster cheek-pouch carcinoma.

Chemically induced Syrian hamster cheek-pouch squamous cell carcinoma is very similar to the corresponding human tumour. This paper describes a blind study in which inhibition of dimethylbenzanthracene-induced cheek-pouch tumours by a goat liver extract denominated UK101 was investigated. Less than 40% of animals treated with UK101 developed tumours compared with 100% of the controls. Intermediate results (80%) were noted in a positive control group treated with Calmette-Guerin bacillus. Immunocytochemical testing of cheek-pouch mucosa by Mib5 showed significantly less proliferating cells in UK101 animals than in the controls. The effect of UK101 was completely reversed when dexamethasone was added in a third control group. A significant difference in complement-mediated cytotoxicity was noted in the sera of UK101-tested and control animals. These findings suggest that an immune mechanism is responsible for the inhibition of hamster cheek-pouch carcinoma by UK101.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1985

[Immune response in rabbits to tumoral tissue extracts of human colon].

Carcinoembryonic antigen (CEA) was purified after perchloric acid extraction of glycoprotein from human normal and tumoral colon. Antibodies against those extracts as well as purified CEA were raised in rabbits. Sera obtained from animals immunized with tumoral extract in protracted schemes showed disperse behavior on polyacrylamide gel electrophoresis (PAGE) for proteins in the area with gamma, mobility which were markedly increased. In addition, a large increase of proteins with a mobility comparable to alpha 2 proteins was observed. The latter increase was not present in sera of rabbits immunized with normal colon extracts. Low titer antisera were obtained after immunization with either antigen as revealed by immunodiffusion. The relatively highest titers were seen against the glycoprotein fraction of tumor extracts which had previously been enriched by isoelectric focusing. Immunoelectrophoresis of tumor extracts against homologous antisera revealed bands with beta mobility due to the presence of CEA antibodies only in the sera of animals injected with tumor extracts. Antibody titers assayed by radioimmunoassay (RIA) revealed that the highest titers were obtained from tumor glycoprotein extracts was used as an immunogen. Measurements of CEA concentrations in serum of patients with low and high levels of CEA showed non significant differences by statistical analysis, when antisera obtained from different bleedings of the same animals were used. It is concluded that in order to establish a CEA RIA for clinical purposes it is necessary to purify up to the isoelectric focusing step the material to be labelled as tracer or used as standard. Antibody reagent may be obtained by immunizing schemes or protracted schemes using crude perchloric acid extracts of colon tumor.

Adult↗

PubMed1998

Acid nuclear extracts as mediators of gene transfer and expression.

In an attempt to demonstrate transfection-active DNA packaging proteins in the cell nucleus, we prepared acid nuclear extracts with perchloric acid and subsequent protein fractions by stepwise acetone precipitation. The original extract and these fractions containing different compositions of nuclear proteins were used as DNA packaging agents. After the formation of complexes between these protein fractions and reporter genes, the addition of these complexes to the cells resulted in high transfection rates. Gel electrophoresis shows that the most active fractions contain histone H1 and HMG17. HMG1 exhibits a smaller activity. This result was confirmed by positive transfection experiments with commercial histone H1. Our results show that the transfection activity of acid nuclear protein fractions and histone H1 is dependent on the presence of calcium.

3T3 Cells↗

PubMed1987

Comparison between acute and chronic effects of ammonia on branched-chain amino acid oxidation and incorporation into protein in primary cultures of astrocytes and of neurons.

A comparison was made of acute and chronic effects of ammonia on production of 14CO2 from the [U-14C] labeled branched-chain amino acids (BCAA) leucine, isoleucine, and valine as well as from [1-14C] leucine, and on the incorporation of radioactivity from these amino acids into a perchloric-acid-precipitable protein fraction in astrocytes and neurons in primary cultures. Acute exposure of astrocytes to 3mM ammonium chloride suppressed 14CO2 production from [U-14C] BCAA and especially from [1-14C] leucine. This inhibitory effect was abolished or even reversed [( U-14C] leucine) after chronic exposure to ammonia. Analogously, incorporation of radioactivity into the protein fraction was inhibited after acute exposure but not after chronic exposure of astrocytes to ammonia. The total protein content per culture was increased after chronic exposure. In neurons, production of 14CO2 and incorporation of 14C into proteins were less affected than in astrocytes. These results are discussed in relation to the ability of the two cell types to synthesize glutamine.

Amino Acids, Branched-Chain↗

PubMed1996

Method for determination of free intracellular and extracellular methylglyoxal in animal cells grown in culture.

Methylglyoxal is present at low levels in most cells as a by-product of glycolysis and a product of lipid and amino acid catabolism. The most widely accepted method for measurement of methylglyoxal involves the derivatization of methylglyoxal with 1,2-diaminobenzene derivatives, such as o-phenylenediamine, followed by quantification of the resulting quinoxaline with high-performance liquid chromatography (HPLC). Here we describe the modification of this procedure for the measurement of free intra- and extracellular methylglyoxal in animal cells grown in culture. Cell harvest and sample volume measurement techniques were developed. Solid-phase extraction prior to methylglyoxal derivatization reduced interferences unique to cell culture, such as the phenol red indicator dye used in most cell culture media, and extended the useful life of the HPLC column. In addition, this extraction step significantly lessened the interference represented by oxidative degradation of nucleic acids to methylglyoxal by perchloric acid under assay conditions. The concentration of free intracellular methylglyoxal in Chinese hamster ovary (CHO) cells grown in culture ranged from 0.7 +/- 0.3 microM (mean +/- 2 standard deviations; n = 4) to 1.2 +/- 0.3 microM (mean +/- 2 standard deviations; n = 7). The concentration of free extracellular methylglyoxal in the growth medium was 0.07 +/- 0.02 microM (mean +/- 2 standard deviations; n = 4), severalfold less than that found inside the cell. A possible explanation for the difference between measured free intracellular and extracellular methylglyoxal levels is that the assay for free intracellular methylglyoxal also measures some reversibly bound methylglyoxal.

Animals↗