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PubMed1975

Migration inhibition with various cell fractions in human colorectal cancer.

The unseparated leucocytes, separated mononuclear cells and granulocytes of six control subjects and nine patients with colorectal cancer have been studied by a direct cell migration inhibition technique. A migratory index was calculated from the migration in the presence and absence of a perchloric acid extract of large bowel tumours. In 10% homologous AB serum, no significant migration inhibition occurred with any of the cells from control subjects. Five of the nine cancer patients showed significant inhibition with their unseparated leucocytes, seven of seven with their mononuclear cells, and none of nine with their granulocytes. In 10% autologous serum, some controls exhibited migration inhibition with their unseparated leucocytes and their granulocyte fraction, but not with the mononuclear cell fraction. Migration inhibition was also now apparent in the granulocyte fraction of the cancer patients. It is concluded that, with a soluble tumour antigen preparation, a mononuclear cell population increases the sensitivity of the direct migration inhibition test and that autologous serum may interfere directly with the migration of granulocytes, by an action not dependent upon the release of inhibitory factors from sensitized lymphocytes. This could explain some of the inconsistencies of the assay when using an unseparated leucocyte population.

ABO Blood-Group System↗
PubMed1961

The determination of magnesium in biological materials by flame photometry.

A method is described for the measurement of magnesium in biological materials and has been applied to the Unicam flame spectrophotometer SP 900. Samples were diluted in 10 mM strontium ethylenedinitrolo tetraacetate (EDTASr), 10 mM potassium carbonate, giving final magnesium concentrations of about 0.1 to 0.2 mM. The EDTASr almost eliminated the depression of magnesium emission by interfering anions. The potassium carbonate slightly depressed both magnesium emission and flame background, which were then virtually unaffected by further addition of cations. Spectral interference by sodium was controlled by including 14 mM sodium chloride in the plasma standards and making small corrections (usually under 2%) on the basis of the plasma sodium level. The urine standards contained no sodium, but a small correction (usually about 2%) was made on the basis of the urine sodium level. There was insufficient sodium in faeces to cause spectral interference. Using the diluent described, added magnesium was satisfactorily recovered from plasma deproteinized with perchloric acid, urine, and faeces. When different volumes of identical samples of plasma and urine were analysed, the magnesium found was proportional to the volume of sample diluted. The plasma magnesium levels reported for a small group of healthy people were similar to those found by previous workers.

Carbonates↗
PubMed1973

Carcinoembryonic antigen in serum in diseases of the liver and pancreas.

Carcinoembryonic antigen (CEA) was measured in whole serum and in serum extracted with perchloric acid by microradioimmunoassay in patients with benign and malignant diseases of the liver and pancreas. The level of detectability was 5 ng per ml. This level or greater was present in the serum of 50% of patients with chronic diffuse liver disease, 64% with pancreatitis, 94% with cancer of the digestive system, and 3% of controls. The incidence of levels of CEA of 5 ng/ml or more differed for various categories of chronic liver disease: from 22% in active chronic hepatitis, 46% in primary biliary cirrhosis, 63% in hepatoma, 78% in cryptogenic cirrhosis, and 88% in alcoholic cirrhosis; levels of CEA correlated with degrees of impairment of liver function as judged by bromsulphalein retention and serum levels of alkaline phosphatase and transaminase. In pancreatitis, 64% of cases had levels of CEA ranging from 5 to 20 ng/ml and in cancer of the pancreas 94% had levels above 5 ng/ml and 50% above 20 ng/ml.

Adolescent↗
PubMed1981

Regulation of the dephosphorylation of glycogen phosphorylase a and synthase b by glucose and caffeine in isolated hepatocytes.

Synergistic regulation of glycogen phosphorylase a by the competitive inhibitors glucose and caffeine in vitro indicates a possible physiological role for the negative effector site which binds caffeine (nucleoside site). In intact viable hepatocytes glucose promotes the phosphorylase a to be conversion by phosphorylase phosphatase. This conversion is considered to be a necessary prelude to the activation of glycogen synthase by phosphatase and of importance in hepatic regulation of glucose homeostasis. The effects of glucose and(or) caffeine on the conversion of phosphorylase a to b and synthase b to a were studied. Assays of phosphorylase a were used which limited synergistic inhibition (in the assay) by these ligands. Such an approach is necessary to achieve an accurate measure of phosphatase activity in the viable hepatocyte when the combination of ligands is used. The data indicate that in the presence of caffeine and glucose together, the rate of loss of phosphorylase a is significantly increased (1.7-fold) over that in the presence of glucose alone. Phosphorylase phosphatase is activated. The sequential activation of glycogen synthase was also accelerated in the presence of both ligands. The results are consistent with an in vivo function for the nucleoside site, similar to that of glucose. A controlling role or phosphorylase in the regulation of glycogen metabolism by glucose is supported. Although the existence and nature of an intracellular effector is as yet unknown, crystallographic analyses of phosphorylase a crystals soaked in perchloric acid extracts of liver demonstrate that the negative effector site binds a natural metabolite.

Animals↗
PubMed1982

Glycosylation of plasma proteins with [U-14C]glucose in vivo.

Within 1-12 h after subcutaneous injection of [U-14C]glucose, significant amounts of 14C attached by stable (probably covalent) bonds were found in plasma proteins of rats. More than 30% of 14C of [U-14C]glucose injected per 1 g body weight was incorporated into proteins contained in 1 mL of plasma. Less than 5% of 14C of the [14C]glucose-labelled plasma proteins was soluble in cold dilute perchloric acid, whereas more than 80% of 14C in the [14C]glucose-labelled plasma proteins was soluble in 50% saturated solutions of ammonium sulfate. From [U-14C]glucose-labelled plasma proteins, approximately 50% of the incorporated 14C was recovered in carbohydrate moieties (sialic acid, 8-12%; mannose and galactose, 15-31%; hexosamines, 8-14%) and the rest of the 14C (42-64%) was recovered from protein residue. Gel-filtration and electrophoresis profiles of distribution of 14C in [U-14C]leucine-labelled plasma proteins were very similar to those of [U-14C]glucose-labelled plasma proteins; a relatively high specific radioactivity was observed in fractions having an apparent molecular weight of 105 X 10(3) or its multiples (220 X 10(3) and 520 X 10(3)). However, about 99% of the incorporated 14C was recovered from the protein residue of [14C]leucine-labelled plasma proteins, whereas only traces of 14C were found in the carbohydrate moieties. [U-14C]Alanine is also a relatively poor precursor of 14C for incorporation into carbohydrate moieties of plasma proteins as compared with [U-14C]glucose.

Animals↗
PubMed1993

Intracellular pH determination by 13C-NMR spectroscopy.

A noninvasive method for the determination of pH by the 13C-nuclear magnetic resonance (NMR) chemical shift of the C-3 carbon of sn-glycerol 3-phosphate is described. Nonlinear least-squares analysis of the chemical shift variation of the C-3 resonance of sn-glycerol 3-phosphate with pH at 37 degrees C in solutions and in perchloric acid extracts of tissue yielded a pKa of 6.2, making it a very sensitive indicator of pH in the approximate range of 5-7. Intracellular pH determined by the present 13C-NMR method correlated well with simultaneous measurements of pH by 31P-NMR spectroscopy over a wide range during normal perfusion and ischemic conditions in intact rat hearts. These findings indicate that this approach is particularly suited for quantification of intracellular pH over the physiological range in intact tissues and that observed in ischemic myocardium.

Animals↗
PubMed1989

Quantitation of metabolites of isolated chicken enterocytes using NMR spectroscopy.

Isolated enterocytes prepared from chicken small intestine were studied by proton nuclear magnetic resonance (NMR). Perchloric acid extracts of cells were prepared from duodenum, jejunum, and ileum, and concentrations of 19 different metabolites were determined after the virtually complete assignment of all peaks in the aliphatic region of the spectrum. High concentrations of serine ethanolamine phosphodiester (SEP), creatine, taurine, and acidic amino acids were found in all segment extracts. Relatively high concentrations of SEP (approximately 12 mM) and acidic amino acids (approximately 3.5 mM) were found in the ileum, whereas creatine (3 mM) and lactate (6.5 mM) were found at higher concentrations in jejunum. Taurine (approximately 8 mM), choline (0.5 mM), and betaine (approximately 0.5 mM) were evenly distributed throughout the segments. Fasting (40 h) led to substantial increases in the concentrations of pyruvate, succinate, SEP, and taurine, while neutral hydrophobic amino acid concentrations fell appreciably. The significance of the findings is discussed, and the possible contributions of SEP, taurine, and choline to membrane lipid homeostasis are considered.

Animals↗
PubMed1993

Cerebral metabolic and histological effects of thioacetamide-induced liver failure.

Acute liver failure was induced in rats by successive administrations of thioacetamide over 3 days. At progressing stages of hepatic encephalopathy (HE), brains were fixed with microwave irradiation for analysis of metabolite levels or with formaldehyde for histopathological analysis. Metabolite levels were determined using 1H-nuclear magnetic resonance spectroscopy of perchloric acid extracts of the frontal cortex, parietal or occipital cortex, hippocampus, striatum, brain stem, and cerebellum. After thioacetamide treatment, thioacetamide and its metabolites were detected in the brains at levels that did not correlate with the stage of HE. No changes were observed in the levels of N-acetylaspartate, alanine, gamma-aminobutyric acid, aspartate, or inositol in any brain region after thioacetamide treatment. HE was accompanied by elevated glutamine, glucose, and lactate throughout the brain. At all stages of HE, taurine was decreased in the neocortex and hippocampus, and glutamate and choline compounds were decreased in the frontal cortex. None of the metabolite changes showed progression with the stage of HE. Progressing HE was accompanied by increasing neuronal injury in layer III of the neocortex, in the Purkinje cells of the cerebellum, and in the hippocampus, particularly in the CA4 sector. The similarity of this distribution of injury to that associated with excitotoxic injury suggests that metabolic abnormalities after acute hepatic failure may give rise to adverse effects at excitatory (glutamatergic) neuronal receptors, leading to neuronal injury and clinical symptoms of progressing encephalopathy in this model. However, neuronal injury and the presence of thioacetamide and its metabolites in the brain raise questions about the validity of thioacetamide-induced liver failure as a model for clinical HE.

Animals↗
PubMed1980

Cardiac physiologic-metabolic changes after chronic low-level heavy metal feeding.

Female Long-Evans hooded rats received 0 or 5 parts per million Cd, Pb, or Cd + Pb in the drinking water from weaning to 20 mo. Growth, measured as body weight, and organ weights were comparable among heavy metal and control animals. Indirect blood pressure responses measured trimonthly are discussed. Analysis of His bundle electrograms (HBE) and electrocardiograms recorded in sodium pentobarbital-anesthetized animals at 20 mo demonstrated that Cd selectively depressed conduction system excitability proximal to the common His bundle (atrioventricular node region), whereas Pb and Pb + Cd impaired conductivity distal to the common His bundle (His-Purkinje system). Perchloric acid extracts of liquid N2-freeze-clamped isolated perfused hearts, derived from a subpopulation of control and Cd-fed rats in which HBE analyses were not performed, were analyzed by phosphorus-31 nuclear magnetic resonance (31P-NMR) spectroscopic techniques to quantitate [31P]phosphate metabolite profiles. Cardiac active tension and atrioventricular node excitability were depressed in the Cd group. High-energy phosphate and glycerol 3-phosphorylcholine concentrations were depressed. Results demonstrate potentially significant pathophysiological changes within cardiovascular tissues that develop in the absence of overt heavy metal toxicity manifestations.

Animals↗
PubMed1983

Tissue adenosine content in active soleus and gracilis muscles of cats.

Vascularly isolated cat soleus and gracilis muscles were stimulated to contract isometrically and were then frozen in situ. Adenosine, inosine, and hypoxanthine (nucleosides), and lactate were measured in neutralized, perchloric acid extracts of muscle. During contraction, nucleoside content increased in soleus muscle but changed little in gracilis muscle. However, adenosine content did not correlate with vascular conductance or oxygen consumption in either soleus or gracilis muscle. Adenosine content did correlate with lactate content in soleus but not gracilis muscle. The activity of AMP deaminase was highest in cat gracilis muscle and lowest in dog cardiac muscle. The activity of 5'-nucleotidase was lowest in cat gracilis muscle and highest in dog cardiac muscle. Cat soleus and dog gracilis muscles had intermediate activities of both enzymes. The findings of the present study do not support a role for adenosine in mediating prolonged active hyperemia in fast-twitch gracilis muscle of cats and cast doubt on such a role in slow-twitch soleus muscle of cats. Differences in the activities of AMP deaminase and 5'-nucleotidase provide a qualitative, biochemical explanation for apparent differences in net adenosine production among muscles composed of different fiber types and between skeletal and cardiac muscle.

5'-Nucleotidase↗
PubMed1983

Physiological and metabolic characterization of a cardiomyopathy induced by chronic copper deficiency.

Male weanling rats were made copper deficient with a purified diet containing all known essential dietary nutrients except copper. Copper deficiency was verified by indirect (anemia, growth retardation, hypercholesterolemia, gross pathology, and abnormal electrocardiograms) and direct (tissue copper analysis) criteria. His bundle electrographic and electrocardiographic changes detected in the copper-deficient group consisted most notably of depressed His-Purkinje system conductivity and S-T segment depression. Phosphorus-31 nuclear magnetic resonance spectroscopic analysis of cardiac, renal, and hepatic tissue perchloric acid extracts revealed significant metabolic changes associated with the dietary copper deficiency, including a generalized marked decrease in ATP and phosphocreatine levels and a corresponding increase in inorganic orthophosphate and ADP levels in the various tissues. Tissue-specific changes consisting of elevated ribose 5-phosphate (heart), phosphocholine (heart), and inosine monophosphate (kidney) and decreased glycerol 3-phosphorylethanolamine (liver) and glycerol 3-phosphorylcholine (liver) levels were detected in copper-deficient rats. Microscopic examination of heart tissue from copper-deficient rats revealed extensive disruption of mitochondrial fine structure, including fragmentation of cristae and inner and outer mitochondrial membranes, which resulted in pronounced vacuolization throughout the tissue. Although the physiological and metabolic disturbances manifested in hearts from copper-deficient animals generally mimic myocardial responses to chronic ischemia, the observed changes are interpreted in a broader context to represent the appearance of a copper-dependent cardiomyopathy.

Animals↗
PubMed1991

Sensitivity of myocardial fluorodeoxyglucose lumped constant to glucose and insulin.

The lumped constant (LC) that relates the steady-state phosphorylation rate of 2-[18F]-fluoro-2-deoxy-D-glucose (2-FDG) to that of glucose was determined in an isolated working rat heart model by direct assay of phosphorylation product formation. Five conditions were tested: 5 and 30 mM glucose without insulin, and 2, 3.5, and 5 mM glucose + 10 mU/ml insulin, all at high external work load. Hearts were continuously perfused with 2-FDG and tritiated glucose without recirculation. The steady-state production of tritiated water was used to monitor the glucose phosphorylation rate. Perfused hearts were freeze-clamped and extracted in perchloric acid, and 2-FDG-6-phosphate was separated from 2-FDG with a formate column. The accumulation of 2-FDG phosphorylation products in tissue was also determined from the slopes of the total tissue radioactivity time courses measured by external gamma-ray detection. Without insulin, the LC value decreased 18% as perfusate glucose concentration was increased sixfold (0.94 +/- 0.06 at 5 mM vs. 0.77 +/- 0.17 at 30 mM). With insulin, the LC rose from 0.33 +/- 0.03 at 5 mM to 1.19 +/- 0.05 at 2 mM glucose concentration. The trends can be interpreted in terms of the concept of control strength; the LC value rises as glycolysis becomes rate limited by transport into cells. This potential variability of the LC must be addressed in the quantitative interpretation of myocardial deoxyglucose studies.

Animals↗
PubMed1983

Aldosterone response in the turtle bladder is associated with an increase in ATP.

Urinary bladders from freshwater turtles, mounted as sacs, were stripped of their serosa and submucosa. This did not alter conductance. They were maintained in open circuit except for brief observation of short-circuit current (SCC) every 15 min. Potential difference (PD) averaged 68 +/- 14 mV and SCC 485 +/- 100 microA. Acetazolamide 10(-3) M increased SCC by 46 +/- 27 microA. Aldosterone 10(-7) M following acetazolamide resulted in a rise in SCC that began at about 75 min and reached a plateau between 3 and 5 h. SCC rose 127 +/- 15% compared with control bladder halves. ATP measured in perchloric acid extracts 5 h after addition of aldosterone increased by 33% (P less than 0.01) and (ATP)/(ADP) X (Pi) by 81% (P less than 0.01). These results support the view that the stimulatory effects of aldosterone on active sodium transport involve an increase in ATP and (ATP)/(ADP) X (Pi).

Acetazolamide↗
PubMed1988

Accumulation of major organic osmolytes in rat renal inner medulla in dehydration.

Osmotically active organic solutes, osmolytes, exist at high concentrations in the renal inner medulla; however, their modulation during antidiuresis remains largely undefined. Renal osmolyte levels were measured by nuclear magnetic resonance spectroscopy and biochemical assays in perchloric acid extracts from normal and dehydrated (3 days) rats. Dehydration increased urine osmolality from 1,503 to 3,748 mosmol/kg and inner medullary urea content from 2,036 to 4,405 nmol/mg protein. In addition, inner medullary trimethylamines [glycerophosphorylcholine (GPC) and betaine] and polyhydric alcohols (inositol and sorbitol) significantly increased by 95 and 78%, respectively. Ninhydrin-positive substances (amino acids), although abundant, were unchanged. Renal cortex also contained GPC, betaine, and inositol but only inositol increased with dehydration. Analysis of correlations among inner medullary osmolytes showed that only GPC was consistently elevated by dehydration and was not directly correlated with the other osmolytes. In contrast, betaine and inositol contents were linearly related to each other and both tended to rise only when sorbitol content was unchanged. In conclusion, the major osmolytes in the rat renal inner medulla can increase during antidiuresis but they are regulated in a complex manner.

Animals↗
PubMed1986

Effects of delayed freezing on content of phosphagens in human skeletal muscle biopsy samples.

The concentrations of ATP, phosphocreatine (PCr), creatine, and lactate were determined in muscle biopsy samples frozen immediately or after a delay of 1-6 min. During the delay the samples were exposed to normal air or a gas mixture of 6.5% CO2-93.5% O2. The ATP content was unchanged, but PCr increased significantly from 72 mmol after rapid freezing to 85 mmol X kg dry muscle-1 during the 1st min in air. The lactate concentration increased (2.8 to 5.2 mmol X kg-1). If muscles were made anoxic by circulatory occlusion for 4-6 min before sampling, no increase in PCr was observed. Direct homogenization of fresh tissue in perchloric acid gave the same ATP, PCr, and lactate contents as frozen samples. It is concluded that the ATP and PCr contents in muscle are unaffected by freezing but that the biopsy procedure activates the energy utilization processes resulting in PCr decrease. It is suggested that the muscle PCr content after a 1-min delay in tissue freezing corresponds to the level in resting fresh muscle.

Adenosine Triphosphate↗
PubMed1999

Hyperoxia-induced changes in antioxidant capacity and the effect of dietary antioxidants.

We investigated, by measuring oxygen radical absorbance capacity (ORAC), whether hyperoxia causes alterations in antioxidant status and whether these alterations could be modulated by dietary antioxidants. Rats were fed for 8 wk a control diet or a control diet supplemented with vitamin E (500 IU/kg) or with aqueous extracts (ORAC: 1.36 mmol Trolox equivalents/kg) from blueberries or spinach and then were exposed to air or >99% O2 for 48 h. Although the constituents of the extracts were not extensively characterized, HPLC indicated that blueberry extract was particularly rich in anthocyanins, and the spinach extract did not contain any anthocyanins. The ORAC was determined in samples without proteins [serum treated with perchloric acid (PCA); ORACPCA] and with proteins (ORACtot). Hyperoxia induced a decrease in serum protein concentration, an increase in serum ORACPCA, decreases in lung ORACPCA and ORACtot, and an equilibration of proteins and ORACPCA between serum and pleural effusion. These alterations suggested a redistribution of antioxidants between tissues and an increase in capillary permeability during hyperoxia. Only the blueberry extract was effective in alleviating the hyperoxia-induced redistribution of antioxidants between tissues.

Animals↗
PubMed1996

Adaptation of cellular metabolism to anisosmotic conditions in a glial cell line, as assessed by 13C-NMR spectroscopy.

13C-NMR spectroscopy of perchloric acid and lipid extracts of F98 glioma cells showed that volume-regulatory processes under anisosmotic conditions were accompanied by marked alterations in cellular metabolism. Production of alanine, glutamate, and glycine from [U-13C]-glucose is decreased under hypotonic stress and is oppositely increased under hypertonic stress. In contrast, degradation of these molecules is raised under hypotonic conditions and reduced under hypertonic conditions. Furthermore, phospholipid synthesis is decreased under hypertonic stress and increased under hypotonic stress. Obviously, glial metabolism is directed under hypertonic conditions to maintain a high level of small, osmotically active molecules, whereas under hypotonic conditions molecular fragments are increasingly incorporated into the phospholipids and so do not contribute to the osmotic pressure. The latter is evoked by the activation of membrane synthesis process to compensate for stretching and/or damaging of the membranes due to cell swelling.

Adaptation, Physiological↗
PubMed1989

Ontogeny of prolactin releasing and inhibiting activities in the posterior pituitary of male rats.

Plasma PRL levels in male rats are highest during the peripubertal period. We previously reported that the posterior pituitary (PP) contains a potent PRL-releasing factor (PRF), a trypsin-insensitive small peptide which is distinct from known PRL secretagogues. The objectives were to determine the ontogeny of PRF activity in the PP as well as age-related alterations in anterior pituitary responsiveness to PRF. We also explored if the PP contains a nondopaminergic PRL-inhibiting factor (PIF). PRF/PIF activities were assessed by the ability of PP extracts to alter PRL release from cultured anterior pituitary cells. The PP were extracted with perchloric acid and lyophilized, thus eliminating endogenous dopamine. PRF activity in PP extracts from 10- and 20 day-old (d) rats was very low, increased gradually in 30d and 40d rats, and remained unchanged in adult (90d) rats. In a second experiment, age-related changes in anterior pituitary responsiveness to PP extracts from adult rats and to TRH were determined. The responsiveness of anterior pituitary cells from 10d rats to PRF was low, increased dramatically in cells from 20d rats, and was reduced in cells from 30d and adult rats. The responsiveness to TRH was highest in cells from 10d rats. In a third experiment, anterior pituitary responsiveness to age-matched PP extracts was assessed. Only PIF activity was observed when PP extracts from 10d rats were incubated with anterior pituitary cells from 10d rats. In contrast, PP extracts from 20d, 30d and adult rats exhibited only PRF activity when incubated with age-matched cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗