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PubMed1994

Metabolic studies and neurotoxicity in tumors and brain of mice after hypoxic cell sensitizers.

PURPOSE: The effects of the radiosensitizers RK-28 and RP-170, both 2-nitroimidazole nucleoside analogues, and KU-2285, a fluorinated 2-nitroimidazole, as well as etanidazole (ETA) on glucose metabolism in mouse tumors and brain were studied to assess their degree of neurotoxicity. METHODS AND MATERIALS: Adult male C57Bl mice received differing doses of the above sensitizers IP. Blood, brain, and tumor samples were removed at various times and the levels of glycolytic metabolites determined. Glucose uptake and phosphorylation in brain was measured by the 2-deoxyglucose method of Sokoloff et al. (6). RESULTS: RP-170 showed neither signs of toxicity nor significant alterations in glucose metabolism in brain or tumor at doses up to 4 g/kg b.w. up to 4 h. By contrast, RK-28 was extremely neurotoxic at a dose of 1 g/kg b.w. with a high degree of lethality, resulting in a highly significant increase in the brain glucose level from 0.38 mumol/g to 2.20 mumol/g (p < 0.001) 2 h after administration, whereas that in the tumor was decreased. KU-2285 and ETA were significantly (p < 0.01) less toxic than RK-28 at this dose, as reflected in a lower increase in the brain glucose level (0.60 mumol/g), although KU-2285 approached that of RK-28 (1.43 mumol/g; p < 0.01) after 2 h following a dose of 2 g/kg b.w. However, in contrast to the other sensitizers, KU-2285 concomitantly also resulted in a highly significant continuous increase (p < 0.01) in tumor glucose levels. Labeled 3H-2-deoxyglucose studies showed that RP-170 neither markedly affected the uptake of total radioactivity into the brain nor its degree of phosphorylation whereas, KU-2285 (2 g/kg) and RK-28 (1 g/kg) decreased uptake by approximately 50% and phosphorylation approximately 3 and 4-fold, respectively. At doses of 1 g/kg, ETA and KU-2285 showed no significant changes in these parameters. This indicates a decreased level of neurotoxicity. CONCLUSION: Since the adult brain relies solely on glucose metabolism for its energy supply, interference to this pathway may be instrumental in the development of neurotoxicity, thus, underlining the need for such metabolic studies to assess the level of toxicity by radiosensitizers.

Adenosine Triphosphate↗
PubMed2004

Glucose production, gluconeogenesis, and hepatic tricarboxylic acid cycle fluxes measured by nuclear magnetic resonance analysis of a single glucose derivative.

A triple-tracer method was developed to provide absolute fluxes contributing to endogenous glucose production and hepatic tricarboxylic acid (TCA) cycle fluxes in 24-h-fasted rats by (2)H and (13)C nuclear magnetic resonance (NMR) analysis of a single glucose derivative. A primed, intravenous [3,4-(13)C(2)]glucose infusion was used to measure endogenous glucose production; intraperitoneal (2)H(2)O (to enrich total body water) was used to quantify sources of glucose (TCA cycle, glycerol, and glycogen), and intraperitoneal [U-(13)C(3)] propionate was used to quantify hepatic anaplerosis, pyruvate cycling, and TCA cycle flux. Plasma glucose was converted to monoacetone glucose (MAG), and a single (2)H and (13)C NMR spectrum of MAG provided the following metabolic data (all in units of micromol/kg/min; n = 6): endogenous glucose production (40.4+/-2.9), gluconeogenesis from glycerol (11.5+/-3.5), gluconeogenesis from the TCA cycle (67.3+/-5.6), glycogenolysis (1.0+/-0.8), pyruvate cycling (154.4+/-43.4), PEPCK flux (221.7+/-47.6), and TCA cycle flux (49.1+/-16.8). In a separate group of rats, glucose production was not different in the absence of (2)H(2)O and [U-(13)C]propionate, demonstrating that these tracers do not alter the measurement of glucose turnover.

Acetonitriles↗
PubMed1975

The action pattern of Penicillium lilacinum dextranase.

The product distributions resulting from the action of Penicillium lilacinum dextranase on end-labelled oligosaccharides of the isomaltose series have been determined. The initial rates of formation of labelled products were measured for isomaltotriose up to isomalto-octaose, and the molar proportions and radioactivity of the final products from isomaltotriose up to isomaltohexaose were determined. D-Glucose was released only from isomaltotriose and isomaltotetraose, by hydrolysis of the first linkage from the reducing end (linkage 1); the terminal bonds of higher members of the series were not attacked. All oligosaccharides except isomaltotriose were hydrolyzed at more than one linkage. The main points of attack on isomaltotetraose up to isomalto-octaose were at linkage 2, and at the third linkage from the non-reducing end; these two positions coincide for isomaltopentaose. The degradation of isomaltotriose up to isomalto-octaose was entirely hydrolytic. The enzyme also catalyzed an extremely slow, concentration-dependent degradation of isomaltose, and this may have occurred via a condensation to isomaltotetraose, followed by hydrolysis of linkage 1 to give D-glucose and isomaltotriose.

Dextranase↗
PubMed1999

Application of enzyme field-effect transistors for determination of glucose concentrations in blood serum.

Glucose-sensitive enzyme field effect transistors (ENFETs) modified by an additional Nafion membrane have been developed and used for diluted blood samples analysis. The ENFET was used in the linear portion of the calibration curve up to 1.5 mM glucose in a model solution, which corresponds with up to 60 mM glucose in the undiluted samples (dilution 1:40). The high linearity of the Grans curve (factor of linearity is 1.03) obtained by the method of standard additions indicates the high precision of analysis. Glucose concentrations in different blood serum samples determined by ENFETs were compared with those measured by the commercial analyzer 'Eksan-G' and colorimetric method ('Diagluc' enzymatic kit), and good correlation between these methods was revealed. The high reproducibility and operational stability of the biosensor developed were demonstrated.

Animals↗
PubMed1974

Effects of antimycin and 2-deoxyglucose on adenine nucleotides in human platelets. Role of metabolic adenosine triphosphate in primary aggregation, secondary aggregation and shape change of platetets.

1. Human platelet-rich plasma prelabelled with [(3)H]adenine was incubated at 37 degrees C with antimycin A and 2-deoxy-d-glucose. Variations in the amounts of ATP, ADP and P(i), and in the radioactivity of ATP, ADP, AMP, IMP, hypoxanthine+inosine and adenine were determined during incubation. Adrenaline- and ADP-induced platelet aggregation and the ADP-induced shape change of the platelets were determined concurrently. 2. 2-Deoxyglucose caused conversion of [(3)H]ATP to [(3)H]hypoxanthine+inosine. The rate of this conversion increased with increasing 2-deoxyglucose concentration and was markedly stimulated by addition of antimycin, which had no effect alone. At maximal ATP-hypoxanthine conversion rates, the IMP radioactivity remained at values tenfold higher than control, whereas [(3)H]ADP and [(3)H]AMP radioactivity gave variations typical for product/substrates in consecutive reactions. The specific radioactivityof ethanol-soluble platelet ATP decreased during incubation to less than one-tenth of its original value. The amounts and radioactivity of ethanol-insoluble ADP did not vary during incubation with the metabolic inhibitors. 3. The rate of ADP- and adrenaline-induced primary aggregation decreased as the amount of radioactive ATP declined, and complete inhibition of aggregation was obtained at a certain ATP concentration (metabolic ATP threshold). This threshold decreased with increasing concentration of inducer ADP. 4. Secondary platelet aggregation (release reaction) had a metabolic ATP threshold markedly higher than that of primary aggregation. 5. Shape change was gradually inhibited as the ATP radioactivity decreased, and had a metabolic ATP threshold distinctly lower than that of primary aggregation, and which decreased with increasing concentration of ADP. 6. A small but distinct fraction of [(3)H]ATP disappeared rapidly during the combined shape change-aggregation process induced by ADP in platelets incubated with metabolic inhibitors, whereas no ATP disappearance occurred during aggregation in their absence.

Adenine↗
PubMed1985

Protein-bound glycogen is linked to tyrosine residues.

Tyrosine-glycogen obtained from retina proteoglycogen by exhaustive proteolytic digestion was radiolabelled with 125I. The 125I-labelled tyrosine-glycogen was degraded by amylolytic digestion to a very small radioactive product, which was identified as iodotyrosine by h.p.l.c. The amylolytic mixture used released glucose and maltose that were alpha-linked to the phenolic hydroxy group of p-nitrophenol. No free iodotyrosine was found before or after the intact [125I]iodotyrosine-glycogen was subjected to two cycles of the Edman degradation procedure. The linkage between protein and glycogen was alkali-stable. Therefore it is concluded that the protein-bound glycogen was O-glycosidically linked to the phenolic hydroxy group of tyrosine. The amino acid has not been heretofore found to be involved in the linkage of carbohydrates to proteins.

Amylases↗
PubMed1990

Kinetic and inhibitor profiles of soluble and particulate inositol 1,4-5-trisphosphate 5-phosphatase from GH3 and IMR-32 cells.

A simple procedure for assay of Ins(1,4,5)P3 5-phosphatase is described. The reaction products [( 3H]Ins(1,4)P2, [3H]InsP and myo-[3H]inositol) are completely separated from one another, with quantitative yield, on Amprep SAX (100 mg) minicolumns. [3H]Ins(1,4,5)P3 [and [3H]Ins(1,3,4,5)P4] are adsorbed to the columns but not released to any appreciable extent by the elution conditions used. In GH3 cells, the stepwise dephosphorylation of [3H]Ins(1,4,5)P3 to myo-[3H]inositol was demonstrated, and was inhibited by 2.3-bisphosphoglycerate. The Km of the soluble form of the enzyme was lower in GH3 cells (8-13 microM) than in IMR-32 cells (26-32 microM) or in rat cerebral-cortical samples (22 microM. The Km of the particulate form of the enzyme was similar in all three preparations (10-16 microM). The pH profiles of the two soluble 5-phosphatases differed, with a wider pH optimum for the GH3-cell activity than for the IMR-32-cell activity. The soluble and particulate GH3 enzymes were more sensitive than the corresponding IMR-32 enzymes to inhibition by p-hydroxymercuribenzoate, whereas there were no differences in their sensitivities to glucose 6-phosphate, 2,3-bisphosphoglycerate, fructose 1.6- and 2.6-bisphosphate and non-radioactive Ins(1,3,4,5)P4. Dialysis of the soluble fractions and washing of the particulate fractions did not affect the inhibitor sensitivities, except for the soluble IMR-32 fraction and p-hydroxymercuribenzoate. The Km value of the soluble GH3 5-phosphatase activity was lower, and the inhibition by Ins(1,3,4,5)P4 greater, after adsorption to and elution from phosphocellulose. It is concluded that there are qualitative differences in the properties of the soluble 5-phosphatase activity from GH3 and IMR-32 cells.

2,3-Diphosphoglycerate↗
PubMed2003

Increased intestinal permeability as a cause of fluctuating postprandial blood glucose levels in Type 1 diabetic patients.

BACKGROUND: In diabetic patients, postprandial glucose levels, which have a major impact on metabolic control, are determined by the rate of nutrient delivery into the intestine, absorption of nutrients from the small intestine, and the metabolism of the absorbed nutrients by the liver. The present study addresses whether Type 1 diabetic patients have increased intestinal permeability and intestinal permeability predicts postprandial glucose variability. MATERIAL AND METHODS: Thirty Type 1 diabetic patients together with 15 sex- and age-matched healthy controls were enrolled in the study. After an overnight fasting all patients and controls received 100 micro Ci 51Cr of EDTA as a radioactive tracer and the percentage of the isotope excreted in a 24-h urinary specimen was the permeability measure. Instant blood glucose was measured just before the test, and the patients performed and recorded self-monitoring of fasting and 2nd-hour postprandial blood glucose levels during the following week. RESULTS: We found that intestinal permeability is increased in Type 1 diabetic patients compared with age- and sex-matched healthy controls. Increased intestinal permeability is related at least in part to the instant blood glucose level and the presence of diabetic autonomic neuropathy. CONCLUSION: Increased intestinal permeability leads to higher variation in postprandial blood glucose levels, thereby worsening metabolic control.

Blood Glucose↗
PubMed1992

Effects of phenobarbital on cerebral blood flow in the newborn piglet.

To determine the neonatal cerebrovascular effect of a therapeutic dose and a high dose of phenobarbital (Pb), the effect of Pb on cerebral blood flow (CBF) and total brain oxygen consumption (CMRO2) was studied in three groups of awake newborn piglets (aged 1-3.5 days). Group I (control n = 9) received normal saline solution, group II (n = 9) received a therapeutic dose of Pb (15 mg/kg i.v.) and group III (n = 9) received a high Pb dose (45 mg/kg i.v.). Four CBF measurements per piglet using radioactive microspheres (141Ce, 85Cr, 95Nb, 46Sc), arterial blood gases, O2 content, hematocrit and plasma glucose were obtained at 0, 15, 30, 60 min after saline or Pb injections. In all groups, pH, PaO2, PaCO2, blood pressure, heart rate, temperature and plasma glucose remained unchanged except a 14% decrease (p < 0.01) in blood pressure and an increase (p < 0.05) in PaCO2, 60 min after drug injection in groups II and III. Total CBF in group II decreased by 14% (p < 0.05) 15 min after drug injection and was significantly lower (p < 0.05) than control (group I) but returned to baseline after 30 min. High Pb dose progressively lowered CBF by 11% 15 min after drug injection and produced a significant decrease by 20% (p < 0.01) 30 min after drug injection with return to baseline after 60 min. Similar effects were noted in different brain regions (cerebrum and thalamus). CMRO2 remained unchanged in the control group; however, it was decreased by 35% (< 0.01 p > 0.05) 15 min after drug injection and returned to baseline after 60 min. In group III, high Pb dose lowered CMRO2 by 31% 30 min (p = 0.02) after drug injection. Data indicate that Pb exerts a minimal but transient dose-dependent effect on CBF and CMRO2.

Animals↗
PubMedDate not supplied

Simultaneous in vivo measurement of lumped constant and rate constants in experimental cerebral ischemia using F-18 FDG.

Lumped and transfer rate constants in ischemic brain tissue must be measured to estimate accurately cerebral glucose utilization by the deoxyglucose method. We studied the bilateral middle cerebral artery occlusion model in 17 cats, 5 with a 1-hour occlusion, 5 with a 4-hour occlusion, and 7 with a sham operation. The time course of cerebral tissue radioactivity (Ci*(t)) was monitored by external coincidence counting during a programmed infusion of [18F]-2-deoxy-2-fluoro-d-glucose (18F-2-FDG). Arterial plasma concentration (Cp*(t)) of the tracer was kept constant during the first 45 minutes. Rate constants were estimated from Ci*(t) and Cp*(t) by a nonlinear least-squares fitting routine. The lumped constant was estimated from the fit of the ratio of extraction fractions of glucose and 18F-2-FDG by nonweighted, nonlinear least-squares fitting. In the 4-hour-occlusion model, the transfer constant k1* was 23% lower, k3* 39% lower, and the lumped constant 78% higher than in the sham-operated animals. In the 1-hour-occlusion model, k3* was 26% lower than in the sham-operated animals but the lumped constant was not significantly different. The rate of glucose utilization was significantly different in the 4-hour-occlusion model compared to the sham-operated animals (48% decrease, p less than 0.05) but was not significantly different in the 1-hour-occlusion model.

Animals↗
PubMed2003

Defective RNA ribose synthesis in fibroblasts from patients with thiamine-responsive megaloblastic anemia (TRMA).

Fibroblasts from patients with thiamine-responsive megaloblastic anemia (TRMA) syndrome with diabetes and deafness undergo apoptotic cell death in the absence of supplemental thiamine in their cultures. The basis of megaloblastosis in these patients has not been determined. Here we use the stable [1,2-13C2]glucose isotope-based dynamic metabolic profiling technique to demonstrate that defective high-affinity thiamine transport primarily affects the synthesis of nucleic acid ribose via the nonoxidative branch of the pentose cycle. RNA ribose isolated from TRMA fibroblasts in thiamine-depleted cultures shows a time-dependent decrease in the fraction of ribose derived via transketolase, a thiamine-dependent enzyme in the pentose cycle. The fractional rate of de novo ribose synthesis from glucose is decreased several fold 2 to 4 days after removal of thiamine from the culture medium. No such metabolic changes are observed in wild-type fibroblasts or in TRMA mutant cells in thiamine-containing medium. Fluxes through glycolysis are similar in TRMA versus control fibroblasts in the pentose and TCA cycles. We conclude that reduced nucleic acid production through impaired transketolase catalysis is the underlying biochemical disturbance that likely induces cell cycle arrest or apoptosis in bone marrow cells and leads to the TRMA syndrome in patients with defective high-affinity thiamine transport.

Anemia, Megaloblastic↗
PubMed1979

[Use of maltose and a mixture of maltose, fructose and xylitol in parenteral feeding].

Maltose or maltose in combination with fructose and xylitol was administered intravenously to eight healthy male subjects. Constant maltose levels could not be attained in the blood at an infusion rate of 0.125 g maltose/kg body-weight and hour. Maximal concentrations of maltose were found at the end of the infusion period. 8.6 +/- 1.2% of the administered radioactivity was excreted into urine within 8 hours. Regarding the enzymatically determined maltose and glucose, the maltose balance was more favorable with the loss of only 3--3.5% carbohydrates into urine. The highest oxidation rate of the administered maltose was 1.5 g maltose/human volunteer and hour. During the experimental period of 8 hours 7.4 g maltose, corresponding to 37% of the applied dosis of the disaccharid, has been oxidized to and excreted as 14CO2. Xylitol and fructose did not effect utilisation and balance of maltose. Only the urinary excretion of glucose was higher when the combined solution was applied. At a limited infusion rate (0.125 g maltose/kg body-weight and hour) maltose or the combined solution maltose--xylitol--fructose may be recommended for parenteral nutrition.

Adult↗
PubMedDate not supplied

[Carbohydrate-dependent lipidogenesis. Review of the literature and experimental study].

The literature on glycide-induced lipidogenesis is reviewed. This is an important question also because of its relation to the pathogenesis of pathological conditions such as ketoacidosis, obesity and hypertriglyceridaemia. Doubt is expressed concerning the interpretation of some experimental procedures used in determining the extent of lipidogenesis in man. The questions raised are fully discussed. Their solution is sought via an experiment on the rat involving the comparison of tissue and plasma lipid metabolites values following infusion of a labelled glucose bolus. Little correlation was noted, low plasma levels being found with high tissue radioactivity. It is emphasized that it is very difficult to ideate a valid experimental approach in order to investigate the extent of lipidogenesis in man in vivo: in this respect, the long term risk arising from the use of tracers with a very long half-life (e.g. 14C) in man must be carefully valuated.

Adipose Tissue↗
PubMed1977

Use of 3H and 14C double-labeled glucose to assess in vivo pathways of amino acid biosynthesis in Escherichia coli.

3H and 14C tracing data concerning amino acid biosynthetic pathways in Escherichia coli K12 are presented. Thirteen acidic and neutral amino acids were isolated from protein hydrolysates of wild type E. coli K12 grown aerobically or anaerobically in the presence of [U-14C]glucose together with [1-3H]glucose, [3-3H]glucose, [4-3H]glucose, or [6-3H]glucose. The observed 3H/14C counts of the amino acids were compared with the ratios expected on the basis of the input substrate specific activities and present understanding of biosynthetic pathways. For nine amino acids, serine, valine, leucine, threonine, isoleucine, glycine, glutamate, proline, and phenylalanine, the agreement between anticipated and observed specific activities was satisfactory. For the remaining four, methionine, alanine, aspartate, and (in cells labeled with [3-3H]glucose) tyrosine, the anticipated and observed specific activities differed markedly. For alanine, aspartate, and tyrosine, the differences are probably due to exchange of tritium in the course of biosynthesis; for methionine, it may be that there is a principle source of the methyl group other than carbon 3 of serine.

Alanine↗
PubMed1978

Evidence for the existence of a countercurrent exchanger in the small intestine in man.

The vasculature in the human villus forms vascular loops by the supplying arterial vessel and the draining capillaries and/or veins. This study reports two experimental observations that strongly suggest that these vascular loops function as countercurrent exchangers. (1) The elimination of intraarterially injected 85Kr from the human small bowel exhibits an initial very rapid component of the type earlier reported in the feline gut. This component in all probability reflects the extravascular "shunting" in the exchanger of the injected radioactive tracer. (2) When exposing the intestinal mucosa to an isotonic electrolyte solution containing glucose, an osmolality gradient from the tip to the base of the human villi was demonstrated, the tips having an osmolality of around 700 milliosmoles per kg H2O. This hyperosmolality is created by the exchanger acting as a countercurrent multiplier.

Colon↗
PubMed1984

[Use of several precursors for in vivo lipid synthesis in different animal organs and tissues in the neonatal period].

The radioactivity of total lipids and their classes synthesized from the 14C-labeled palmitate, glucose, lactate, pyruvate, oxybutyrate and alanine in the liver, lungs, skeletal muscle, subcutaneous adipose tissue and jejunal mucosa of one- and five-day-old piglets was investigated. The lipid synthesis from the majority of the precursors under study in animal organs and tissues decreases in the following order: lungs greater than liver greater than jejunal mucosa greater than adipose tissue greater than skeletal muscle. Incorporation of the label from [1-14C]palmitate and [3-14C]oxybutyrate into the lipids occurs more intensively than that from other labeled compounds. In piglets of one to five days of age, the expenditure of [1-14C]palmitate and [1-14C] glucose for lipid synthesis in most organs and tissues decreases, whereas that of [1-14C]alanine and [1-14C]pyruvate increases. The label from [1-14C]palmitate and [1-14C]glucose is mainly localized in the liver, lungs, muscular tissue and jejunal mucosa phospholipids, while that in the adipose tissue--in acylglycerols. The amount of phospholipids in most organs and tissues of one-day-old animals is greater, while that of acylglycerols smaller in comparison with the five-day-old ones. There is a correlation between the synthesis of individual lipid classes and the degree of functional maturity of organs and tissues of young animals.

Acetates↗
PubMed1982

Exogenous amino acids as fuel in shock.

It has been suggested that in shock branched-chain amino acids are preferentially oxidized resulting in continued proteolysis and stimulated gluconeogenesis. To determine if exogenous amino acids could be used as fuel in shock, dogs rendered hypotensive by controlled cardiac tamponade and normotensive controls were infused with amino acid mixtures and individual amino acids. When Nephramine, a mixture rich in branched-chain amino acids, was infused, plasma alpha-amino nitrogen levels rose but urea output did not increase in either the control state or in shock, suggesting that these amino acids were not rapidly deaminated to serve as fuels. Travasol, which in addition contained large amounts of alanine and glycine, tripled urea output in the controls and doubled it in shock. The limit of urea production was reached in both groups at 35 mumoles urea/minute/kg. In the Travasol-infused animals plasma alpha-amino nitrogen levels were maintained in normotension but rose sharply in shock. When glycine alone was infused into five dogs in shock urea production rate was 30.6 + 2.1 mumoles/minute/kg; with alanine the same value was 22.5 + 2.2 mumoles/minute/kg. In both cases plasma alpha-amino nitrogen levels were high, suggesting that transport of these amino acids into the cell was slow in shock. In four dogs in shock glycine-14C was added to the glycine infusate as a tracer. At radioactive equilibrium 28% of the label infused appeared in CO2; another 22% appeared in glucose. It is concluded that of all the amino acids tested only glycine and alanine are deaminated rapidly enough to serve as exogenous fuels in shock.

Alanine↗