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PubMed1990

Elimination of 14C-glycated albumin from serum of rabbit.

Glycated albumin was prepared by incubation of serum from rabbits with randomly labelled 14C-glucose. The isolated glycated albumin fraction was reinfused to the same animal. 14C-labelled glucose was given to alloxan-treated rabbits. The disappearance of the radioactivity showed a rapid initial phase and a slow elimination phase, which could be acceptably described by first-order kinetics. The estimated half-life of glycated albumin was about 6 days, which is about 70% of that generally stated for albumin in the rabbit. If these findings were transferred to human conditions, the half-life of fructosamine would be in the range of 13-14 days.

Animals↗
PubMed1986

Characterization of the fatty acid synthetase system of Curtobacterium pusillum.

Curtobacterium pusillum contains 11-cyclohexylundecanoic acid as a major component of cellular fatty acids. A trace amount of 13-cyclohexyltridecanoic acid is also present. Fatty acids other than omega-cyclohexyl fatty acids present are 13-methyltetradecanoic, 12-methyltetradecanoic, n-pentadecanoic, 14-methylpentadecanoic, 13-methylpentadecanoic, n-hexadecanoic, 15-methylhexadecanoic, 14-methylhexadecanoic, and n-heptadecanoic acids. The fatty acid synthetase system of this bacterium was studied. Various 14C-labeled precursors were added to the growth medium and the incorporation of radioactivity into cellular fatty acids was analyzed. Sodium [14C]acetate and [14C]glucose were incorporated into almost all species of cellular fatty acids, the incorporation into 11-cyclohexylundecanoic acid being predominant. [14C]Isoleucine was incorporated into 12-methyltetradecanoic and 14-methylhexadecanoic acids: [14C]leucine into 13-methyltetradecanoic and 15-methylhexadecanoic acids; and [14C]valine into 14-methylpentadecanoic acid. [14C]-Shikimic acid was incorporated almost exclusively into omega-cyclohexyl fatty acids. The fatty acid synthetase activity of the crude enzyme preparation of C. pusillum was reconstituted on the addition of acyl carrier protein. This synthetase system required NADPH and preferentially utilized cyclohexanecarbonyl-CoA as a primer. The system was also able to use branched- and straight-chain acyl-CoAs with 4 to 6 carbon atoms effectively as primers but was unable to use acetyl-CoA. However, if acetyl acyl carrier protein was used as the priming substrate, the system produced straight-chain fatty acids. The results imply that the specificity of the initial acyl-CoA:acyl carrier protein acyltransferase dictates the structure of fatty acids synthesized and that the enzymes catalyzing the subsequent chain-elongation reactions do not have the same specificity restriction.

Acetyltransferases↗
PubMed2005

Functional importance of the family 1 glucosyltransferase UGT72B1 in the metabolism of xenobiotics in Arabidopsis thaliana.

The Arabidopsis type 1 UDP-glucose-dependent glucosyltransferase UGT72B1 is highly active in conjugating the persistent pollutants 3,4-dichloroaniline (DCA) and 2,4,5-trichlorophenol (TCP). To determine its importance in detoxifying xenobiotics in planta, mutant plants where the respective gene has been disrupted by T-DNA insertion have been characterized. Extracts from the knockout ugt72B1 plants showed radically reduced conjugating activity towards DCA and TCP and the absence of immunodetectable UGT72B1 protein. In contrast, activities towards phenolic natural products were unaffected. When aseptic root cultures were fed [14C]-DCA, compared with wild types, the ugt72B1 plants showed a reduced rate of uptake of the xenobiotic and very little metabolism to soluble DCA-glucose or associated polar conjugates. Instead, the knockouts accumulated non-extractable radioactive residues, most probably associated with lignification. When the feeding studies were carried out with [14C]-TCP, rates and routes of metabolism were identical in the wild type and knockouts, with TCP-glucoside a major product in both cases. Similar differential effects on the metabolism of DCA and TCP were obtained in whole plant studies with wild type and ugt72B1 mutants, demonstrating that while UGT72B1 had a central role in metabolizing chloroanilines in Arabidopsis, additional UGTs could compensate for the conjugation of TCP in the knockout. TCP was equally toxic to wild type and ugt72B1 plants, while surprisingly, the knockouts were less sensitive to DCA. From this it was concluded that the glucosylation of DCA may not be as effective in xenobiotic detoxification as bound-residue formation.

Aniline Compounds↗
PubMed1972

Automatic radiometric measurement of antibiotic effect on bacterial growth.

A new method for rapid, automatic radiometric measurement of antibiotic effects on bacterial growth was developed and compared with a conventional broth dilutior technique. The radiometric method measures the amount of radioactive CO(2) generated by the bacterial metabolism of (14)C-glucose in the presence of antibiotics. Antibiotic effect on bacterial growth was standardized by measuring the evolution of (14)CO(2) 3 hr after inoculation. This measurement was found to be quantitatively related to increasing concentration of antibiotic provided the organism was susceptible to the antibiotic tested. In 50 of 179 experiments (28%), each testing one organism against serial concentrations of an antibiotic, the concentration of antibiotic producing a 50% reduction of (14)CO(2) within 3 hr after inoculation in comparison with a control culture was the same as the minimal inhibitory concentration (MIC) determined by the broth dilution technique. In 129 experiments (72%), the antibiotic concentrations inhibiting (14)CO(2) release to 50% of the control level were less than the MIC values. Results of the radiometric method were related to those of the broth dilution method by constant factors characteristic of the organism and antibiotic tested. Our results indicate that the radiometric method provides a reproducible, quantitative, rapid, and sensitive measurement of the inhibitory effects of antibiotics on bacterial growth. The constant relationship between the results of the radiometric and conventional technique should facilitate the adaptation of the automated method to clinical testing of antibiotic susceptibility.

Anti-Bacterial Agents↗
PubMed1982

Effect of insulin on fat and protein deposition in diabetic lean and obese rats.

Five-week-old male obese and lean Zucker rats were made comparably diabetic by intracardiac injections of alloxan (65-72 mg/kg body wt). Lean rats were then given daily injections of protamine zinc insulin at 3 doses: 0.25, 1.25, and 4.0 U.100 g body wt-1.day-1 for 3 wk. Obese rats received identical amounts as corresponding lean controls independent of body weights. The drop of blood glucose concentration after injections of regular insulin and the percentage fall in radioactive plasma insulin after injections of 125I-insulin were comparable in lean and obese rats. Weight gain, fat gain, and protein gain over 21 days increased with increasing amounts of insulin administered. However, at the same dose of insulin, although weight gain was comparable, fat gain was higher and protein gain was lower in obese rats when compared to lean controls. These results suggest that the enhanced lipid deposition of the obese Zucker rat is not totally dependent on insulin levels, but is exaggerated by hyperinsulinemia.

Animals↗
PubMed1984

High accumulation of 2,3,4,7,8-pentachlorodibenzofuran to hepatic microsomes of rats.

The tissue and subcellular distributions of 14C-2,3,4,7,8-pentachlorodibenzofuran (PenCDF), one of the most important causal agents of yusho, were studied using rats. More than 60% of the radioactivity given orally was accumulated in the liver after 5 d and this high percentage persisted over a period of 3 weeks. Subcellular fractionation of the liver homogenate showed unusual separation by PenCDF-pretreatment, but the distribution of radioactivity was just parallel to those of cytochrome P-450 content and glucose-6-phosphatase (EC3.1.3.9) activity. Gas chromatographic analysis provided evidence that the extracts from the liver and its subcellular fractionations contained only unchanged PenCDF. Those results strongly suggest that PenCDF has some affinity to endoplasmic reticulum of rat liver.

Animals↗
PubMed1996

Rapid gastric emptying of a solid pancake meal in type II diabetic patients.

OBJECTIVE: To estimate the rate of gastric emptying of a solid pancake carbohydrate meal in recently diagnosed asymptomatic type II diabetic patients compared with nondiabetic control subjects. RESEARCH DESIGN AND METHODS: Gastric emptying studies using radiolabeled meals were performed on eight recently diagnosed asymptomatic diabetic patients and on eight sex-, BMI- and age-matched nondiabetic control subjects. Although a liquid protein drink was administered along with the pancake meal, the radioactivity was adherent to only the pancake portion of the meal. Plasma glucose and serum insulin levels were measured in fasting and postprandial blood samples collected at 15-min intervals up to 120 min after ingestion of the mixed nutrient meal. RESULTS: The average gastric half-emptying time (time it takes for one-half of the meal to empty) was significantly more rapid for the diabetic patients (45.3 +/- 4.8 min) when compared with the nondiabetic control subjects (60.4 +/- 5.1 min; P = 0.05). The serum insulin concentrations were not statistically different between the two groups. Plasma glucose values were significantly higher in the diabetic patients compared with the nondiabetic control subjects. CONCLUSIONS: Type II diabetic patients with no clinical evidence of neuronal dysfunction have a significantly more rapid rate of gastric emptying of a solid high-carbohydrate meal when compared with nondiabetic control subjects.

Adult↗
PubMed1983

[Biosynthesis of amino acids from glucose in the central nervous system in the Parkinson syndrome].

The incorporation of labelled carbon from glucose U-14C into CSF amino acids was investigated in three patients with Parkinson's disease and in three control persons with comparable age and physical stature. Comparing the specific radioactivities of serum and CSF one can postulate that the labelled amino acids found in the CSF are synthesized mainly by brain tissue. The resorption of glucose into the CNS and therefore the synthesis of amino acids from glucose was more rapid in controls; labelled alanine and glutamine appeared later in the CSF of the patients. As expected, in the controls the specific radioactivity of glutamic acid was found to be higher than that of glutamine, in patients the labelling of glutamine was higher as was that of serine, glycine, aspartic acid and asparagine. From our knowledge concerning the compartmentation of the metabolism of glutamate, we assume that in Parkinsonism the metabolic activity of neurons is reduced but that of astroglia is enhanced.

Aged↗
PubMed1976

Evoked release from guinea pig cerebral cortex slices of endogenous 14C-labelled amino acids, labelled via D-[U-14C]glucose.

Release of endogenous amino acids labelled via D-[U-14C]glucose was compared with that of several exogenous labelled amino acids using slices of guinea pig cerebral cortex. Electrical field stimulation evoked a selective release of endogenous [14C]glutamate, [14C]aspartate, and gamma-amino[14C]butyrate (14C-labelled GABA). The selectivity of release correlated well with 14C incorporation into endogenous amino acids. Calculations of the fraction of the tissue radioactivity released indicated that the selectivity was not an artifact due to differential incorporation. Because glucose in mammalian brain is metabolized almost entirely by the so-called 'large compartment', it is tentatively concluded that the releasable 'transmitter pool' of glutamate, aspartate, and GABA is located in this 'large compartment'.

Amino Acids↗
PubMed1982

Elongation of teichuronic acid chains by a wall-membrane preparation from Micrococcus luteus.

A wall-plus-membrane preparation from Micrococcus luteus catalyzes the incorporation of [14C]glucose from UDP-[14C]glucose, into two fractions of teichuronic acid, which is the cell wall polysaccharide consisting of alternating residues of glucose and N-acetylmannosaminuronic acid (ManNAcUA). Membrane-associated teichuronic acid was extracted from the wall-membrane fraction of reaction mixtures by sodium dodecyl sulfate. The synthesis of membrane-associated teichuronic acid required UDP-glucose, UDP-ManNAcUA, and UDP-N-acetylglucosamine and was inhibited by tunicamycin. Glucose incorporated into wall-bound teichuronic acid remained in wall fragments after extraction with sodium dodecyl sulfate, and its incorporation required UDP-glucose and UDP-ManNAcUA (but not UDP-N-acetylglucosamine) and was insensitive to tunicamycin. Radioactive material incorporated into wall-bound teichuronic acid could be released by treatment with mild acid or by digestion with lysozyme, indicating that the wall-bound teichuronic acid was covalently linked to peptidoglycan. There were about 600 pmol of wall-bound teichuronic acid acceptor sites for glucose per mg of protein as measured in incorporation reaction mixtures lacking UDP-ManNAcUA. In the presence of both UDP-glucose and UDP-ManNAcUA, elongation of teichuronic acid acceptor sites occurred, with the addition of six to eight disaccharide units to each acceptor site.

Adenosine Triphosphate↗
PubMed1979

Carboxylation and decarboxylation reactions. Anaplerotic flux and removal of citrate cycle intermediates in skeletal muscle.

A system for in situ perfusion of rat hindquarters using a fluorocarbon for oxygen and CO2 exchange, and a polyol to provide oncotic pressure is described. Perfusion with glucose plus insulin resulted in no significant change in the tissue level of citrate cycle intermediates, phosphocreatine, ATP, ADP, AMP, and glycogen. Glucose was consumed at a linear rate, and lactate, pyruvate, alanine, glutamine, glutamate, and citrate were released into the perfusing medium. Inclusion of pyruvate resulted in elevation of citrate cycle intermediates and alanine, whereas acetate elevated the level of cycle intermediates without significant effect on tissue alanine or its release. Radioactivity from NaH[14C]O3 was incorporated into citrate cycle intermediates, glutamate, aspartate, and lactate by glucose-perfused hindquarters, the extent of which was markedly elevated as the tissue pyruvate was increased. When pyruvate was in the physiological range, acetate caused elevation in incorporation of CO2 into these metabolites, increased the concentration of citrate, and doubled the concentration of acetyl-CoA. Thirty-five to forty-four per cent of 14C incorporated into citrate was retained after enzymic degradation to 2-oxoglutarate. Perfusion with [2-14C-]propionate led to elevation in the level of citrate cycle intermediates, and radioactivity was incorporated into the latter, as well as glutamate, aspartate, lactate, pyruvate, alanine, and CO2. Two independent calculations estimated the rate of flux of 4-carbon cycle intermediates to 3-carbon metabolites of about 68 mumol/h (approximately 38 nmol/min/g of tissue), a rate in excess of those reported for alanine release from human or rat muscle during starvation. Arsenite blocked carbohydrate flux through the citrate cycle and effected accumulation of lactate, pyruvate, alanine, and 2-oxoglutarate. Flux from 4- to 3-carbon acids was diminished by arsenite, apparently as a result of lowered substrate concentration for decarboxylation. 3-Mercaptopicolinic acid, an inhibitor of phosphoenolpyruvate carboxykinase, was without effect on the parameters studied, suggesting that this enzyme is not involved in the decarboxylation reaction. It is concluded that (a) a constant level of citrate cycle intermediates is maintained in part by continuous flux of carbon into and out of the cycle by carboxylation and decarboxylation reactions; (b) the carbon skeleton of alanine released from skeletal muscle is derived in part from other amino acids which are catabolized to cycle intermediates; and (c) the subsequent removal of these intermediates is probably mediated by malic enzyme(s) (EC 1.1.1.40, or 1.1.1.36, or both.

Animals↗
PubMed1990

Underestimation of glucose turnover determined using [6-3H]glucose tracer in non-steady state. The role of a tritiated tracer impurity.

The use of tritiated glucose tracers may result in underestimation of glucose turnover during hyperinsulinaemic clamps giving paradoxical negative endogenous glucose production rates. While mathematical modelling errors in the analysis of tracer data are major determinants of this underestimate in the non-steady state, the relative importance of tracer contamination under these conditions remains in doubt. We have used high performance liquid chromatography to assess the possible contribution to this problem of a labelled tracer impurity found in [6-3H]glucose. In conventional 4 h hyperinsulinaemic clamps performed in six normal subjects, labelled impurity increased as a percentage of the neutral plasma radioactivity fraction from 5.3 +/- 0.9% after a 2 h equilibration period (0 min) to 13.5 +/- 2.2% at 120 min and 15.4 +/- 2.4% at 240 min, as plasma glucose specific activities fell following the infusion of insulin. Negative endogenous glucose production rates were observed both at 90-120 min (-8.8 +/- 1.6 mumol.kg-1min-1) and at 210-240 min (-8.5 +/- 1.4 mumol.kg-1min-1) implying a persistent underestimate in isotopically determined glucose appearance rate. Using chromatography data to correct for impurity increased glucose appearance rates by 7.9 +/- 2.1% at 120 min and 11.0 +/- 2.5% at 240 min. Purified tracer was then used for a further six clamps. When the conventional protocol was used with unlabelled glucose infusion an obvious negative error persisted only at 90-120 min. In contrast, labelled infusions gave exclusively positive values for endogenous glucose production.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1987

Effects of early chronic phenobarbital treatment on the maturation of energy metabolism in the developing rat brain. I. Incorporation of glucose carbon into amino acids.

The influence of phenobarbital (PhB) on the utilization of glucose by the cerebral cortex and the cerebellum was studied in rats during postnatal development. The animals were treated from day 2 to day 35 after birth by a daily injection of 50 mg/kg PhB or by saline. The rats were studied at 5 postnatal stages: 7, 10, 14, 21 and 35 days. PhB treatment induced a 10% decrease in body and brain weight over the whole period studied and a transient significant decrease in circulating thyroxin levels at 14 days after birth. Amino acid levels in the cerebral cortex and particularly in the cerebellum were not greatly affected by the pharmacological treatment. The conversion of [2-14C]glucose into amino acids was significantly decreased in both cerebral structures between day 7 and day 14 after birth. The distribution of radioactivity between amino acids was not affected in the cerebral cortex but was significantly changed by PhB treatment in the cerebellum. Specific radioactivity values of amino acids were lower in PhB- than in saline-treated animals in both studied structures. The results of the present study show that glucose utilization is reduced in the brain of PhB-treated animals as compared to the controls and that the cerebellum seems to be more affected than the cerebral cortex.

Aging↗
PubMed1987

Fluorodeoxyglucose rate constants, lumped constant, and glucose metabolic rate in rabbit heart.

The isolated arterially perfused rabbit interventricular septum was used to measure myocardial metabolic rate for glucose (MMRGlc) and rate constants and lumped constant (LC) for the glucose analogue [18F]fluorodeoxyglucose (FDG) using a tracer kinetic model. FDG was delivered by constant infusion during coincidence counting of tissue 18F radioactivity. The MMRGlc was measured by the Fick method. Control septa were paced at 72 beats/min and perfused at 1.5 ml/min with oxygenated perfusate containing 5.6 mM glucose and 5 mU/ml insulin. The following conditions were tested: 3.0 and 4.5 ml/min; insulin increased to 25 mU/ml; insulin omitted; 2.8 mM and 11.2 mM glucose; 144 beats/min and 96 paired stimuli/min; and anoxia. Under all conditions studied the phosphorylation (hexokinase) reaction was rate limiting relative to transport. Compared with control conditions, the phosphorylation rate constant was significantly increased with 2.8 mM glucose as well as in anoxia. With 4.5 ml/min and 11.2 mM glucose, conditions that should increase glucose flux into tissue without increasing demand, the phosphorylation rate constant decreased significantly. With 11.2 mM glucose, 96 paired stimuli/min, and anoxia without insulin, a significant increase in the hydrolysis rate of FDG 6-phosphate was observed and suggests that hydrolysis is also an important mechanism for regulating the MMRGlc. Increased transport rate constants were observed with increased flow rates, 96 paired stimuli/min, and anoxia at 96 beats/min. The LC was not significantly different from control in 11 of 14 conditions studied. Therefore, under most conditions, an average LC can be used to calculate MMRGlc estimates.

Animals↗
PubMed1998

Glucose transporter protein-independent tumor cell accumulation of fluorine-18-AFDG, a lipophilic fluorine-18-FDG analog.

UNLABELLED: Fluorine-18-fluorodeoxyglucose (FDG) is used clinically for tumor diagnosis, but its mechanism of accumulation in tumor cells is complicated because two factors, glucose transporter protein (GLUT) and hexokinase, govern [18F]FDG uptake directly. We selected a lipophilic [18F]FDG analog, 1,3,4,6-tetra-acetyl-2-[18F]-2-deoxy-D-glucose ([18F]AFDG), to regulate the effects of hexokinase and evaluated its characteristics in an in vitro cell culture system. METHODS: Fluorine-18-AFDG was synthesized by the method used to produce [18F]FDG, as an intermediate of [18F]FDG. Fluorine-18-AFDG uptake study was performed with LS180 tumor cells, and its metabolites were also investigated by thin-layer chromatography. To evaluate the relationship between [18F]AFDG and GLUT, we also examined [18F]AFDG uptake in the presence of cytochalasin B or with increased medium glucose concentration. The effects of lowered temperature (4 degrees C) on [18F]AFDG uptake were also investigated. RESULTS: Fluorine-18-AFDG (lipophilicity: octanol/water = 3.5) uptake was 3.3-fold higher than that of [18F]FDG. Metabolic analysis showed that [18F]AFDG was extremely stable in the incubation medium but was quickly hydrolyzed and metabolized to 2-fluoro-[18F]-2-deoxy-D-glucose-6-phosphate ([18F]FDG-6P) in tumor cells. Fluorine-18-FDG-6P accounted for approximately 45% of the total radioactivity after a 60-min incubation of [18F]AFDG. Incubation with 50 microM cytochalasin B did not affect [18F]AFDG uptake. In medium with double the control glucose level, [18F]FDG uptake was decreased by about 50%, but [18F]AFDG uptake was not affected. Fluorine-18-AFDG uptake and [18F]FDG-6P production did not show saturation and increased linearly with addition of a 10-fold higher concentration of [18F]AFDG. Lowered incubation temperature caused decreased [18F]AFDG uptake due to reduced [18F]FDG-6P production. CONCLUSION: Fluorine-18-AFDG rapidly penetrated the cell membrane as a result of its high lipophilicity and was metabolized to [18F]FDG-6P within cells. Fluorine-18-AFDG was thus characterized as "GLUT-independent [18F]FDG."

Cytochalasin B↗
PubMed1997

Positron emission tomography for quantitative determination of glucose metabolism in normal and ischemic brains in rats: an insoluble problem by the Harderian glands.

To examine the reliability of quantitative positron emission tomography studies in the rat (Rat-PET), we assessed the influence of radioactivity accumulated in the Harderian glands on PET CMRglc determination. We measured CMRglc by PET and ex vivo dissection methods by using 2-[18F]fluoro-2-deoxy-D-glucose in rats with and without focal brain ischemia. The CMRglc values obtained by PET, after correcting with recovery coefficients, were higher than those measured by the ex vivo method at rostral slices, and reduction of the CMRglc in the ischemic brain was not demonstrated by PET in the frontal cortex. The radioactivity accumulated in the Harderian glands prevents the quantitative determination of CMRglc using Rat-PET.

Animals↗