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Quantitative analysis of glycogen repletion by nuclear magnetic resonance spectroscopy in the conscious rat.

In order to directly determine the amount of label exchange that occurs in the tricarboxylic cycle from labeled alanine and lactate after the ingestion of a glucose load [1-13C]glucose was administered by continuous intraduodenal infusion to awake catheterized rats to achieve steady state jugular venous glycemia (160 mg/dl) for 180 min. Liver was freeze-clamped at 90 and 180 min, and perchloric acid extracts of the liver were subjected to 13C and 1H nuclear magnetic resonance analysis. Dilution in the oxaloacetate pool was determined by comparing the intrahepatic 13C enrichments of C2, C3 positions of glutamate with the C2, C3 positions of alanine and lactate. In addition steady state flux equations were derived for calculation of relative fluxes through pyruvate dehydrogenase/TCA cycle flux and pyruvate kinase flux/total pyruvate utilization. After glucose ingestion in a 24-h fasted rat direct conversion of glucose was responsible for 34% of glycogen. The intrahepatic dilution factor for labeled pyruvate in the oxaloacetate pool was 2.4. Using this factor, alanine and lactate contributed approximately 55% to glycogen formation. Pyruvate dehydrogenase flux ranged between 24 and 35% of total acetyl-coenzyme A (CoA) production and pyruvate kinase flux relative to total pyruvate utilization was approximately 40%.

Amino Acids↗

An improved quantitative assay of glycogen in erythrocytes.

BACKGROUND: Current methods for the quantitative determination of glycogen in erythrocytes are unsatisfactory. METHODS: Erythrocytes were deproteinized with perchloric acid either after haemolysing by freezing and thawing twice or without freezing and thawing, and the glycogen content was determined by an enzymatic method. RESULTS: Freezing and thawing resulted in a significantly higher glycogen content, and the recovery of added glycogen using this method was nearly 100%. The mean erythrocyte glycogen content in healthy volunteers (n = 17) was 69.5 microg/g haemoglobin, a value much higher than the previously reported values in healthy subjects. CONCLUSION: Freezing and thawing improves the assay of erythrocyte glycogen.

Erythrocytes↗

Evaluation of a commercial enzyme radioimmunoassay kit for serum carcinoembryonic antigen.

An enzyme immunoassay kit for carcinoembryonic antigen (CEA) that uses plastic beads coated with guinea-pig anti-CEA as first antibody and goat anti-CEA conjugated with horseradish peroxidase as second antibody has been evaluated. The method does not involve perchloric acid extraction and therefore avoids a dialysis procedure. It is accurate, sensitive, and inexpensive to operate, and provides values comparable to those obtained with the Roche-CEA Z-Gel radioimmunoassay.

Carcinoembryonic Antigen↗

A direct assay for pyridoxal 5'-phosphate using pig heart apo-aspartate transaminase.

The development of a simple enzymatic method for the determination of blood and plasma pyridoxal 5'-phosphate (PLP) using pig heart apo-aspartate transaminase (apo-AST) is described. The technique requires three steps: sample extraction using perchloric acid, a binding step in which PLP in the sample extract is attached to the apo-AST and the enzymatic assay of the reconstituted holo-AST. PLP extracts were analysed with and without a known concentration of added PLP to correct for variation in recovery between different specimens. Procedures are outlined for manual and automatic analysis of the PLP extracts. Using the KONE Clinical Analyser after the extraction step, it is possible to measure enzyme activity in 100 specimens (i.e. 400 tubes) in a 5 h period. Results are shown from 185 healthy women aged 20-45 years, in which plasma PLP concentrations ranged from 5 to 165 nmol/L, and 142 men and 56 women in government service aged 17-64 years, whose plasma PLP ranged from 8 to 169 nmol/L. Values less than 20 nmol/L are believed to indicate vitamin B6 deficiency and the method is able to measure 5 nmol/L.

Adolescent↗

An improved method for measurement of common tricyclic antidepressant drugs and their metabolites using normal phase HPLC.

A sensitive HPLC method that allows the simultaneous determination of imipramine, amitriptyline and their desmethyl metabolites, using a 10 cm straight silica cartridge column with a mobile phase consisting of 0.004% perchloric acid in methanol is presented. Detection of the eluent is at 210 nm. The drugs are back-extracted from serum, giving both a good recovery and sensitivity of at least 5 micrograms/L. This method has proved to be extremely robust and reliable, with coefficients of variation around 3% being reported.

Antidepressive Agents, Tricyclic↗

A simple method for determination of plasma thiocyanate.

We describe a method for the determination of thiocyanate in plasma from blood collected under field conditions in heparinised glass capillary tubes. After deproteinisation of plasma with perchloric acid, thiocyanate is directly determined colorimetrically by the König reaction with sodium hypochlorite as the chlorinating reagent and barbituric acid as the coupling agent. This simple method cannot be applied to urine as the latter contains interfering compounds.

Humans↗

Fluoride in dental plaque and its effects.

Total plaque fluoride is in the range 5-10 mg/kg (ppm) on a wet-weight basis. The variability of literature data on plaque fluoride is partly ascribed to analytical problems, many assays being close to or below the concentration detection limit of the fluoride electrode. A change in classification of plaque fluoride compartments is necessary, since recent work indicates that there are two pools of plaque F: less than 5% of the total F is in plaque fluid as the free ion, and the large remaining portion of total plaque F is designated as bound F, with the total F being greater than 95% extractable by cold 0.5 mol/L perchloric acid. Sources of plaque fluoride include the diet, saliva, and crevicular fluid; enamel is unlikely to be a regular source for plaque F unless it is either coated daily with labile fluoride compounds, such as calcium fluoride, or released by demineralization. The location and nature of plaque bound F are not established, but the present evidence is consistent with an intracellular location. Bound F may be released by acids produced in plaque during sugar fermentation, but it is unlikely to reach ion concentrations high enough for sufficient time periods to exert significant inhibition of plaque acidogenesis. Epidemiological evidence showing correlations between pooled plaque F concentrations and caries prevalence in the plaque donors does not exclude the possibility of coincidental effects of water F on both caries and plaque F concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Dental Plaque↗

Carcinoembryonic antigen and cancer: a comparative study.

The sera of 212 patients with malignant and non-malignant diseases have been radioimmunoassayed for the presence of the carcinoembryonic antigen (CEA) using 3 different kits produced of Hoffman La Roche, Switzerland (RCK), BY Sorin-IRE, Italy and Belgium (SCK), and by the Istituto Sieroterapico Milanese, Italy (ICK). In the presence of endodermically-derived system carcinomas, the RCK gave more positive results (72.6%) than did the SCK (63.1%) or ICK (56.2%). With regard to other carcinomas, ICK (50.0%) and SCK (47.1%) gave better results than did RCK (30.6%). The results are discussed in terms of clinical usefulness of the CEA assay and as regards reproducibility, procedural advantages, and economical cost of each kit. It is concluded that the CEA assay cannot be used for the diagnosis of gastrointestinal cancers, although it is useful as a measure of "cancerosity" for prognostic purposes. In this sense the double antibody method employed by SCK and ICK is clinically more advantageous than is the perchloric acid extraction-zirconyl phosphate gel precipitation method of RCK.

Carcinoembryonic Antigen↗

Anionic glycoproteins and their hexosamine content in neoplastic patients: relationships with the clinical stage and the course of the disease.

In 40 healthy subjects, in 47 non-cancer patients, and in 142 cancer patients, perchloric acid soluble glycoproteins (PASG) and hexosamines were determined to investigate their tumor specificity and correlation with the tumor mass. Cancer patients were divided into three subgroups: CI, no evidence of cancer (after radical surgery); CII, locoregional disease; CIII, widespread metastatic disease. There was no statistically significant difference in PASG among normals, non-cancer and CI patients; hexosamines in non-cancer and in CI patients were higher (P less than 0.002) than in normals; both PASG and hexosamines were significantly higher in CII and CIII patients than in normals (P less than 0.001). In the CI group, 62% of patients who relapsed within 10 months after surgery had high hexosamine values, whereas 69% of patients who did not relapse showed normal levels (P less than 0.05). PASG and hexosamines significantly increased with cancer progression and decreased when objective response to treatment was achieved. They are not tumor specific, but seem to be related to the tumor burden; hexosamines seem to have some prognostic value.

Adult↗

Tumor-associated antigens in female genital tract cancers.

The immunologic reactivity of glycoprotein antigens extractable from individual, histologically different ovarian and uterine cancers was studied taking into account their relationship with carcinoembryonic antigen (CEA), nonspecific cross-reacting antigen (NCA), alpha-feto-protein (AFP), and alpha-1-antichymotrypsin. All studies were performed using specific immune sera against perchloric acid (PCA) extracts of ovarian mucinous cystadenocarcinoma (anti-PCA-CaOm) and cervical squamous cell carcinoma (anti-PCA-CaCx), and antisera against the reference antigens mentioned above. A considerable antigenic heterogeneity and the existence of several immunologically related antigenic systems were found: 1) CEA-like antigens; 2) NCA-type antigens; 3) an antigen different from CEA and NCA present in ovarian mucinous adenocarcinomas and often cross-reacting, but not identical with respective antigens of uterine body and cervical carcinomas; 4) an antigen reacting with anti-alpha-1-anti-chymotrypsin serum; and 5) an antigen reacting with anti-AFP serum.

Adenocarcinoma↗

Determination of serum diclofenac by high-performance liquid chromatography by electromechanical detection.

Diclofenac was isolated from plasma by double extraction with organic solvent. Chromatography was performed on Lichrosorb NH2 column with acetonitrile and perchloric acid (0.0025 mol/l; 35:65, v/v) as the mobile phase (flow rate 0.9 ml/min). The electrochemical detector was operated at +900 mV and sensitivity of 5-10 nA. The sensitivity of the method was 10 micrograms/l and the coefficients of variation below 10% in the entire range of the concentration in a dose interval.

Chromatography, High Pressure Liquid↗

Immunofluorescence of histone H1 in stimulated lymphocytes measured by flow cytophotometry.

The modification of histones or their redistribution during the transition from actively transcribing chromatin to the heterochromatic chromosomes seems to play a major in regulation of gene expression. The purpose of this study was to monitor the change in immunofluorescence of histone HI during phytohemagglutinin stimulation in peripheral lymphocytes. The histone antigens were prepared from pig thymus, proven to be pure by gel electrophoresis and repeatedly injected as RNA-complexes into rabbits. The antihistone HI antiserum titer was 1:4000, and there was no cross-reactivity with other histone fractions as shown by microcomplement fixation tests. Affinity chromatography purified antibody after being labeled with fluorescein isothiocyanate was able to differentially stain HeLa cells as controls and those, where histone HI had been extracted by perchloric acid treatment. The measurements were done on a Los Alamos Scientific Laboratories-flow cytophotometer cell sorter. Staining peripheral lymphocytes resulted in a bimodal distribution. The increase in number of cells with high fluorescence intensity had its maximum about 20 hr before the maximum proliferative activity of the lymphocytes as measured by number of cells in S phase with the DNA-stain mithramycin.

Cell Cycle↗

Whole body autoradiographic distribution of exogenously administered renin in mice.

The distribution of exogenously administered renin was investigated using whole body autoradiography. Purified renin from mouse submaxillary gland (SR) was labeled with radioactive iodine (125I). This labeled renin (125I-SR) and Na125I were administered into the tail vein of male ddY mice, in doses of 10.2 and 16.4 mu Ci/30 g body weight, respectively. Mice were killed by an overdose of ether, and autoradiography was performed on whole body sections. To separate free 125I liberated from 125I-SR, sections were treated with perchloric acid. A major accumulation of 125I-SR, acid-insoluble, was evident in the renal cortex, whereas the hepatic accumulation of 125I-SR was minor. Radioactivity in the thyroid and submaxillary glands, in the stomach, and in urine was also apparent, but disappeared after acid treatment, except in the thyroid glands. Radioactivity in the brain, intestinal content, spleen, and adrenal glands was nil. These autoradiograms provide the first evidence that exogenously administered renin is mainly distributed in the renal cortex.

Animals↗

Whole-body autoradiographic distribution of exogenously administered renal renin in rats.

We studied, by whole-body autoradiography, the distribution of exogenously administered renal renin in rat. Rat renal renin was completely purified and labeled with 125I ([125I]-renin) and was then injected into the tail veins of conscious rats at a dose of 30 microCi, 430 ng. After various intervals, rats were killed by an overdose of ether, the whole body rapidly frozen in acetone-dry ice, and autoradiography performed on sagittal whole-body sections. To remove breakdown products ([125I]-tyrosine and free 125I) from [125I]-renin, sections were treated with perchloric acid solution. The main accumulation of [125I]-renin acid-insoluble radioactivity was observed in liver and renal cortex. The accumulation in these organs was already evident 2 min after the injection, reached a maximum level by 15 min, then gradually decreased. A small amount of [125I]-renin was also evident in spleen, bone marrow, and adrenal gland. Thirty min after the injection, radioactivity began to appear in the thyroid gland, stomach, and small intestine, but disappeared with acid treatment, except in the thyroid. Radioactivity was negligible in other organs including brain, submaxillary gland, lung, heart, and testis. These autoradiographs clearly demonstrate that exogenously administered renal renin is distributed mainly in the liver and renal cortex.

Animals↗

Detection of the inhibitory neurotransmitter GABA in macrophages by magnetic resonance spectroscopy.

Macrophages are key components of the inflammatory response to tissue injury, but their activities can exacerbate neuropathology. High-resolution magnetic resonance spectroscopy was used to identify metabolite levels in perchloric acid extracts of cultured cells of the RAW 264.7 murine macrophage line under resting and lipopolysaccharide-activated conditions. Over 25 metabolites were identified including gamma-aminobutyric acid (GABA), an inhibitory neurotransmitter not previously reported to be present in macrophages. The presence of GABA was also demonstrated in extracts of human peripheral blood monocyte-derived macrophages. This finding suggests that there may be communication between damaged central nervous system (CNS) tissue and recruited macrophages and resident microglia, which could help orchestrate the immune response. On activation, lactate, glutamine, glutamate, and taurine levels were elevated significantly, and GABA and alanine were reduced significantly. Strong resonances from glutathione, evident in the macrophage two-dimensional 1H spectrum, suggest that this may have potential as a noninvasive marker of macrophages recruited to the CNS, as it is only present at low levels in normal brain. Alternatively, a specific combination of spectroscopic changes, such as lactate, alanine, glutathione, and polyamines, may prove to be the most accurate means of detecting macrophage recruitment to the CNS.

Amino Acids↗

32P-labeling patterns in rat pancreatic islets: tissue source of the radiophosphate released after glucose stimulation.

Immediately after stimulation with glucose in vitro, isolated rat pancreatic islets prelabeled with [32P]orthophosphate release a pulse of [32P]orthophosphate into the media (the "phosphate flush"). Islets have been rapidly frozen before, during, and after this pulse to assess the concurrent changes in the distribution of tissue radioactivity. Under the present experimental conditions, approximately 90% of the islet radioactivity was soluble in perchloric acid (PCA-soluble) immediately before stimulation and slightly more than half of that was present as [32P]orthophosphate. The tissue pool of [32P]orthophosphate declined 55% and 62% after 7 and 14 min of stimulation which, respectively, incorporated the peak and the end of the heightened efflux of radioactivity. The net decrementa in tissue orthophosphate could account for all of the radioactivity which was released during the "phosphate flush." During the 14-min period of stimulation, labeled ATP and GTP (which had accounted for 13% and 4% of total PCA-soluble radioactivity before stimulation) increased 51% and 35%, respectively, and labeled ADP and AMP (which had accounted for 5.4% and 1.5% of PCA-soluble counts) fell 36% and 77%, respectively. Certain other PCA-soluble components, such as phosphorylcholine and phosphorylethanolamine, and total PCA-insoluble radioactivity were not demonstrably altered. The findings indicate that the "phosphate flush" originates from a labile pool of tissue orthophosphate. It remains to be established whether the simultaneous changes in the turnover of selected nucleotides are coupled to the translocation of orthophosphate or are mediated separately.

Animals↗

Characterization of prolactin-releasing factor in the rat posterior pituitary.

We previously reported that the rat posterior pituitary contains a potent PRL-releasing factor (PRF) which is distinct from oxytocin (OT), TRH, and angiotensin II (AII). The objectives of this study were 1) to examine whether posterior pituitary extracts stimulate PRL release in the presence of dopamine (DA), 2) to determine the chemical nature of PRF, and 3) to estimate its mol wt. Perifused anterior pituitary cells were used to assess PRF activity. Posterior pituitaries and medial basal hypothalamus (MBH) fragments were extracted with perchloric acid and lyophilized. Subsequent to various treatments, samples were reconstituted in the perifusion medium and introduced to the cells in short pulses. Fractions were collected and analyzed for hormone content by RIA. During a constant infusion of DA (50 nM), PRL secretion was inhibited by 75%, yet the posterior pituitary extract retained its ability to rapidly stimulate PRL release. Studies using proteolytic enzymes showed that posterior pituitary PRF was resistant to inactivation by trypsin, whereas the PRF activity of AII was abolished. Both chymotrypsin and proline-specific endopeptidase significantly reduced the PRF activity in the posterior pituitary. The PRL-releasing activity of TRH was not affected by chymotrypsin. Immunoreactive vasoactive intestinal polypeptide was undetectable in posterior pituitary extracts. Oxidation of posterior pituitary extracts with performic acid caused only a modest reduction of their PRF activity, while the ability of OT to stimulate PRL release as well as immunoreactive OT was abolished. Studies using ultrafiltration membranes showed that the PRF activity in the posterior pituitary was less than 5,000 mol wt. Furthermore, posterior pituitary PRF partitioned in nearly equal amounts across 1K membranes, as did AII and OT. In contrast, about 80% of the PRF activity in the MBH and all of the synthetic TRH passed through the 1K membranes. We conclude that 1) posterior pituitary PRF can stimulate PRL secretion from perifused anterior pituitary cells in the presence of physiological concentrations of DA; 2) PRF is a small peptide(s) of less than 5,000, and perhaps closer to 1,000, mol wt; 3) PRF is resistant to inactivation by trypsin and to oxidation by performic acid, but is hydrolyzed by both chymotrypsin and proline-specific endopeptidase; and 4) these data further distinguish posterior pituitary PRF from known PRL secretagogues.

Animals↗