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Learning-induced change in neural activity during acquisition and consolidation of a passive avoidance response in the rat.

Time-dependent alterations in neural activity have been established during the acquisition and consolidation of a stepdown passive avoidance paradigm. Change in neural activity was established by administering a glucose analogue, [3H]2-deoxyglucose, 50min prior to sacrifice and estimating perchloric acid soluble counts in nine hand dissected brain regions. Change in [3H]2-deoxyglucose uptake was closely paralleled in both trained and yoked animals for up to 40min following task acquisition however the striatum was the only area to exhibit a task-specific increase in [3H]2-deoxyglucose uptake at 20-30min after training. Longterm changes in neural activity were also apparent as the amygdala and brainstem showed increased [3H]2-deoxyglucose uptake at the 24 h time point. No further paradigm-specific changes were apparent at 48 h. These findings are concluded to suggest that the striatum is involved in the early events of acquiring a passive avoidance response and the amygdala and brainstem during the later events.

Amygdala↗

Changes in organic solutes, volume, energy state, and metabolism associated with osmotic stress in a glial cell line: a multinuclear NMR study.

Diffusion-weighted in vivo 1H-NMR spectroscopy of F98 glioma cells embedded in basement membrane gel threads showed that the initial cell swelling to about 180% of the original volume induced under hypotonic stress was followed by a regulatory volume decrease to nearly 100% of the control volume in Dulbecco's modified Eagle's medium (DMEM) but only to 130% in Krebs-Henseleit buffer (KHB, containing only glucose as a substrate) after 7 h. The initial cell shrinkage to approx. 70% induced by the hypertonic stress was compensated by a regulatory volume increase which after 7 h reached almost 100% of the control value in KHB and 75% in DMEM. 1H-, 13C- and 31P-NMR spectroscopy of perchloric acid extracts showed that these volume regulatory processes were accompanied by pronounced changes in the content of organic osmolytes. Adaptation of intra- to extracellular osmolarity was preferentially mediated by a decrease in the cytosolic taurine level under hypotonic stress and by an intracellular accumulation of amino acids under hypertonic stress. If these solutes were not available in sufficient quantities (as in KHB), the osmolarity of the cytosol was increasingly modified by biosynthesis of products and intermediates of essential metabolic pathways, such as alanine, glutamate and glycerophosphocholine in addition to ethanolamine. The cellular nucleoside triphosphate level measured by in vivo 31P-NMR spectroscopy indicated that the energy state of the cells was more easily sustained under hypotonic than hypertonic conditions.

Animals↗

Rapid determination of tyramine in fish feed and slaughter offal by HPLC using coulometric detection.

Fish feed and slaughter offal products may contain decomposition compounds such as biogenic amines. Owing to their harmful effects on animals fed with such products, there is a need for determining the amine content. In the work a simple and fast HPLC method, based on coulometric detection (EC) measuring only tyramine, was developed for routine quality screening. The samples were extracted with 0.4 mol/L perchloric acid and analysed directly by HPLC using a mobile phase of 0.2 mol/L potassium dihydrogen phosphate in water at pH 3. Tyramine was detected using a coulometric detector consisting of two analytical cells, the first one at 0.4 V and the second at 0.7 V. The calibration was linear over the range 4.52 to 452 ng/ml. The minimum detectable quantity was 10 pg/20 microliters. The reproducibility and the recoveries were high. Comparison of the tyramine content measured by EC or derivatization followed by ultraviolet detection showed that both methods gave similar results. HPLC using EC is a fast and sensitive method for analysing tyramine reliably in fish feed and slaughter offal samples without any time-consuming derivatization steps.

Animal Feed↗

[Determination of biogenic amines by RP-HPLC for monitoring the microbial spoilage of poultry].

A simple high-performance liquid chromatographic (HPLC) analysis is described for the determination of biogenic amines in broiler carcasses. The clean-up procedure consists of an extraction with 0.6 M-perchloric acid, formation of dansyl derivatives, separation by a RP-18 column and UV detection at 254 nm. Within 7 min eight amines could be separated. The quantitative determination of spermidine and spermine requires an additional ion-exchange clean-up with Amberlite CG 50 after the extraction. This procedure gives recoveries of 82%-96% with detection limits of 0.2-0.5 microgram/g of broiler skin. Putrescine and cadaverine are good indicators for the onset of spoilage of poultry carcasses, since both amines could be detected at total colony counts of 10(5) cfu/cm2 and their concentration increases rapidly with advancing decomposition.

Animals↗

Determination of the formaldehyde content in fishery products.

The influence of external factors, such as storage temperature and time on the content of free formaldehyde (FA) in fishery products is described. On the basis of the examination of several methods for the determination of free and bound FA, the following procedures are recommended: (1) for measuring free FA, the samples are extracted using 6% perchloric acid at room temperature and the FA content of the extracts is measured by the formation of 3,5-diacetyl-1,4-dihydrolutidine; (2) bound, acid-labile FA is released by steam distillation using 1% sulphuric acid, giving a pH value of about 1. The FA content of the distillates may be determined either by chromotropic acid assay or by the method described for the extracts.

Amino Acids↗

Malondialdehyde is a biochemical marker of peroxidative damage in the isolated reperfused rat heart.

Concentration of MDA in isolated control, ischemic, and reperfused rat hearts was determined by using a new sensitive and reproducible HPLC method on the perchloric acid extract of the freeze-clamped tissues. By means of this HPLC assay for the direct measurements of MDA, concentrations of adenine nucleotide derivatives were also obtained in the same chromatographic run. Under the present experimental conditions, no detectable amount of MDA could be observed in control hearts while ischemic hearts showed 0.009 mumoles/g d.w. of MDA (s.d. = 0.001), this value representing the sensitivity limit of the method employed. On the contrary, reperfused hearts showed 0.118 mumoles/d d.w. of MDA (s.d. = 0.036), thereby indicating that this compound originates from an oxygen free radical-mediated breakdown of phospholipids and demonstrating the existence of quantifiable molecular damage occurring upon reperfusion. On the whole, our data demonstrate that MDA, if properly assayed, is a reliable index of peroxidative injury to biological systems.

Animals↗

Small intestinal mucosa changes, including epithelial cell proliferative activity, of children receiving total parenteral nutrition (TPN).

We examined the small intestinal histology disaccharidase activities as well as the incorporation of [3H]thymidine into DNA of biopsies maintained in organ culture from seven children (ages 9 months to 5 years) receiving total parenteral nutrition (TPN). Three children suffered from inflammatory bowel disease and received TPN for one month (short term). Four required long-term TPN (> 9 months) for short-bowel syndrome. DNA was extracted from the samples following serial precipitation with perchloric acid. Results were compared to those from 22 age-matched children investigated for abdominal pain or chronic diarrhea. Short-term TPN resulted in slightly lower lactase, sucrase, and palatinase activities that were not statistically different from controls. Long-term TPN resulted in focal mild villus atrophy and a decrease in disaccharidase activity in two patients. Biopsies from long-term TPN patients incorporated less thymidine compared to those of controls (P < 0.001) when data was expressed per total biopsy (3.6 +/- 1.1 vs. 8.4 +/- 1.1 fmol) or per milligram of tissue (1.0 +/- 0.12 vs 2.7 +/- 0.7 fmol). The above data are in general agreement with the hypoplastic effect of TPN in animals. However, in children, much longer periods of TPN are required to realize the changes.

Adolescent↗

The use of Cerenkov radiation in the measurement of 115mCd in blood and other tissues.

A new method for the activity measurement of 115mCd in biological samples was proposed. After tissues mineralization with 60% perchloric acid and 30% hydrogen peroxide (I) or with 0.5 M ethanolic sodium hydroxide (II) the solutions were measured in automatic liquid scintillation counter SL-30 by CERENKOV technique. The channels ratio method was successfully applied for standardization. The recoveries of 115mCd varied in the ranges: 93.6-102% and 82.7-99.3% for method I and II, respectively.

Animals↗

Reactivity of an anti-(human gastric carcinoma) monoclonal antibody with core-related peptides of gastrointestinal mucin.

A murine anti-(human gastric carcinoma) monoclonal antibody, GL-013 (IgG1), which reacts with a high-molecular-mass glycoprotein from colorectal tumour tissue [Yang and Price (1989) Anticancer Res 9: 1707], was examined for reactivity against a panel of purified and partially purified antigens associated with tumours of the gastrointestinal tract. These included carcinoembryonic antigen (CEA), normal cross-reacting antigen, Y-hapten glycoproteins, and perchloric acid extracts and glycolipid preparations from colorectal tumours. While the GL-013 antibody failed to bind to these antigens, it was found to react strongly with synthetic peptides with sequences based upon that reported for the protein core of a human gastrointestinal mucin [Barnd et al. (1989) Proc Natl Acad Sci USA 86: 7159; Gum et al. (1989) J Biol Chem 264: 6480]. In control tests, a series of other anti-(colorectal tumour) antibodies (IgG1 and IgG3), with broad reactivity towards gastrointestinal carcinomas, as well as an anti-CEA antibody, (IgG1) failed to react with the synthetic peptides. It is concluded that the anti-(gastric carcinoma) monoclonal antibody GL-013 binds to a threonine-rich peptide epitope expressed within the protein core of gastrointestinal mucins.

Adenocarcinoma↗

A comparison of an enzymatic and a gas-chromatographic method for measuring the acetate concentration in the blood plasma of cattle.

The concentration of acetate in blood plasma was measured by both a gas-liquid chromatographic method and an enzymatic method using acetyl-CoA synthetase. When acetate was measured enzymatically without previous protein precipitation, the apparent concentration was lower than when the concentration measured by either a gas-chromatographic method or by the enzymatic method after protein precipitation with perchloric acid and neutralization.

Acetate-CoA Ligase↗

Heterogeneity of calcium compartmentation: electron probe analysis of renal tubules.

The objective of this study has been to determine the intracellular localization of calcium in cryofixed, cryosectioned suspensions of kidney proximal tubules using quantitative electron probe X-ray microanalysis. Two populations of cells have been identified: 1) "Viable" cells, representing the majority of cells probed, are defined by their relatively normal K/Na concentration ratio of approximately 4:1. Their measured Ca content is 4.1 +/- 1.4 (SEM) mmol/kg dry wt in the cytoplasm and 3.1 +/- 1.1 mmol/kg dry wt in the mitochondria, or an average cell calcium content of approximately 3.8 mmol/kg dry wt. 2) "Nonviable" cells, defined by the presence of dense inclusions in their mitochondria and a K/Na concentration ratio of approximately 1. The Ca content is 15 +/- 2 mmol/kg dry wt in the cytoplasm and 685 +/- 139 mmol/kg dry wt in the mitochondria of such cells. Assuming 25 to 30% of the cell volume is mitochondrial, the overall calcium content of such nonviable cells is approximately 210 mmol/kg dry wt. The presence of these inclusions in 4 to 5% of the cells would account for the average total Ca content measured in perchloric acid extracts of isolated proximal tubule suspensions (approximately equal to 18 nmol/mg protein or 12.6 mmol/kg dry wt). Whole kidney tissues display a large variability in total Ca content (4.5 to 18 nmol/mg protein, or 3.4 to 13.5 mmol/kg dry wt), which could be accounted for by inclusions in 0 to 4% of the cells. The electron probe X-ray microanalysis (EPXMA) data conclusively demonstrate that the in situ mitochondrial Ca content of viable cells from the kidney proximal tubule is low and support the idea that mitochondrial Ca may regulate dehydrogenase activity but probably does not normally control cytosolic free Ca.

Animals↗

High pressure liquid chromatographic analysis of the serum concentration of cefuroxime after an intravenous bolus injection of cefuroxime in patients with a coronary artery bypass grafting.

A simple reversed-phase high pressure liquid chromatographic method was developed for the determination of cefuroxime in the serum of patients undergoing coronary artery bypass grafting. The serum was cleaned up with a 3.3% solution of perchloric acid in water. Cefalexine was used as an internal standard. Detection was made by a UV multi-wavelength detector. The optimum wavelength for cefuroxime is 275 nm. The absolute recovery of this method was 90.9%; the limit of quantification was 0.7 mg/l. This analytical method was used in a study to investigate the cefuroxime serum concentration--time curves in 26 patients undergoing coronary artery bypass grafting. It was found that one single dose is sufficient to obtain effective serum concentrations.

Adult↗

A macromolecular inhibitor of in vitro calcification of tendon matrix.

Bovine and human tendon tissue do not induce calcification in vitro. However, extraction of those tissues with 3% Na2HPO4 converts them to calcifiable matrices. The supernatant fraction derived from the extraction contains a nondialyzable, perchloric acid soluble component that inhibits calcification of the extracted matrix. This inhibitory substance is characterized by a molecular weight in the range of 85,000-100,000. Exposure to pronase or hyaluronidase did not alter the inhibitory potency but did render the inhibitor dialyzable. Commercial sources of hyaluronic acid, chondrotitin-6-sulfate, chrondroitin-4-sulfate, dermatan sulfate, heparin and lysozyme did not inhibit calcification of the extracted matrix. Phosvitin, a phosphoglycoprotein is a potent inhibitor. Although phosvitin and the tendon extract also inhibit calcification of previously calcified matrix, they have no detectable effect on the rate of decalcification. We conclude that the mechanism of inhibition is characterized by a degree of specificity and that phosvitin and a macromolecular component of tendon tissue blocks conversion of an intermediate matrix-bound CaP complex to crystalline apatite. It seems reasonable that the tendon inhibitor could function in situ and possibly in vivo to control calcification of tendon tissue.

Animals↗

Plasma indomethacin assay by reversed-phase ion pair high pressure liquid chromatography.

A new rapid, selective and sensitive high pressure liquid chromatographic (HPLC) assay for indomethacin in plasma is described. The method involves precipitation of proteins with perchloric acid, followed by dichloromethane extraction using flurbiprofen as an internal standard. The organic solvent was evaporated and the residue dissolved in a water-methanol (2 + 3) phosphate buffer mixture with an apparent pH of 6.8. Aliquots of 100 microliters were injected automatically into the chromatograph. The separation of indomethacin was achieved on a reversed phase (C18, 10 micron) column with a mobile phase consisting of 65% (vol/vol) methanol in water solution of apparent pH 6.8 containing tetrabutylammonium hydrogensulfate as an ion pairing agent. Quantitation of indomethacin was performed by UV detection at 235 nm. At a 2.0 mg X l-1 concentration of indomethacin in plasma the analytical recovery was 82.9 +/- 3.4% (n = 7), the intra-day variability (CV) was 3.6% (n = 7) and the inter-day variability (CV) was 11.0% (n = 7). The calibration curve was linear (typical r-values greater than 0.990) in the range of plasma concentrations as usually found during indomethacin therapy (up to 6 mg X l-1). The limit of sensitivity is 0.025 mg X l-1. The metabolites O-desmethylindomethacin and O-desmethyldeschlorobenzoylindomethacin did not interfere with the method. The capacity of an analyst using this method with automated injection and peak integration is about forty samples in duplicate per day. The applicability of the method for pharmacokinetic studies is demonstrated.

Adult↗

Monitoring hybridoma metabolism in continuous suspension culture at the intracellular level. I. Steady-state responses to different glutamine feed concentrations.

A model mouse hybridoma cell line was grown in continuous culture experiments in a serum-free low-protein lipid-free medium. The steady-state responses of cell numbers, extra- and intracellular metabolite concentrations, substrate and (by) product consumption/production rates, and yield coefficients were investigated as a function of step changes in the glutamine concentration of the feed medium. In addition to the commonly performed analysis of metabolites in culture supernatants, we prepared perchloric acid extracts of cells and determined the amount and the composition of intracellular amino acids and organic acids. Significant differences were found with respect to intracellular metabolite pools for cells growing at nearly identical specific growth rates. To our knowledge this is the first time that data on the intracellular concentrations (pools) of amino acids and Krebs cycle intermediates are reported in the literature that were obtained under carefully defined culture conditions such as those attained in continuous culture experiments.

Amino Acids↗

Regional and developmental variations in metabolite concentration in the rat brain and eye: a study using 1H NMR spectroscopy and high performance liquid chromatography.

Regional and developmental changes in metabolite concentrations were measured by 1H NMR spectroscopy and HPLC of perchloric acid extracts from rat brain and eye. The highest concentrations of N-acetylaspartate were found in grey matter as opposed to white matter with concentration increasing with age from neonate to adult, while the related compound N-acetylaspartylglutamate was highest in adult optic nerve. Creatine and choline-containing compounds were present in all regions throughout development, with higher levels of creatine found in grey matter compared to other regions. Choline-containing compounds were present at the highest concentrations in the eye at all ages examined, and tended to decrease in concentration to minimum values in adulthood in all regions. The presence of hypotaurine in corpus callosum and optic nerve was consistent with the metabolic profiles of O-2A progenitor cells and oligodendrocytes, which are cells composing these tissues. The neurotransmitters glutamate and GABA reached their highest concentrations in the olfactory bulb (higher than in adult cortex).

Aging↗

A 2-phase liquid scintillation assay for glycolipid synthetases.

Glycolipid synthetases can be assayed conveniently by incubating the lipid substrate with the radiosugar-labeled nucleotide in a small plastic scintillation vial. At the end of the incubation period, water and perchloric acid are added, then n-butanol, then a toluene-based scintillation cocktail. The radioactive lipid partitions into the scintillation fluid, leaving excess sugar nucleotide in the aqueous phase. Only a small fraction of the total radioactivity in the aqueous layer is detectable. This method is illustrated for ceramide:UDP-glucose glucosyltransferase. The approach should be applicable to other lipid synthetases that can be assayed with radioactive hydrophilic substrate.

Animals↗

Clinical significance of the preoperative plasma carcinoembryonic antigen (CEA) level in patients with carcinoma of the large bowel.

Preoperative levels of perchloric acid extractable plasma CEA were measured in 911 patients with complaints of the digestive system. A final diagnosis of benign disease was made for 579 patients; 332 patients were found to have cancer. Data for the preoperative CEA values were examined for clinical significance as an aide to diagnosis, preoperative disease staging, and prognosis. The results of our analysis support the conclusions of many investigators that the CEA assay is not a clinically useful diagnostic test, but it shows limited value in preoperative staging and a somewhat stronger correlation with prognosis.

Actuarial Analysis↗