Ammonia detoxification by accelerated oxidation of branched chain amino acids in brains of acute hepatic failure rats

BCAA aminotransferase and BCKA dehydrogenase activities are increased in the mitochondrial fractions from the brains of hepatic failure rats treated with two-thirds removal of CCl4-injured liver. Cerebral leucine decarboxylation was accelerated, and it well correlated with arterial blood ammonia levels. Elevation of brain ammonia content following an intraperitoneal injection of ammonium acetate to hepatic failure rats could be prevented by intravenous infusion of BCAA. Significantly increased brain glutamic acid, glutamine, and alanine contents were noted. These results suggested that accelerated brain BCAA catabolism in acute hepatic failure rats reduce the neurotoxicity of ammonia by promoting the synthesis of glutamic acid and glutamine from BCAA.     

The effects of L-2,4-diaminobutyric acid on the uptake of gamma-aminobutyric acid by a synaptosomal fraction from rat brain

l-2,4-diaminobutyric acid was studied as an inhibitor of gamma-aminobutyric acid uptake by a synaptosomal fraction isolated from rat brain. Competitive inhibition was observed during short-term exposure of the synaptosomal fraction to the inhibitor but noncompetitive inhibition was observed following prolonged exposure. Studies on the mode of action of l-2,4-diaminobutyric acid showed that the synaptosomal fraction was capable of accumulating this compound and that both the uptake and the effectiveness of the inhibitor were sodium-dependent and temperature-sensitive. In addition, the degree of inhibition of gamma-aminobutyric acid uptake was related to the amount of l-2,4-diaminobutyric acid accumulated. It is suggested that the observed noncompetitive inhibition of gamma-aminobutyric acid uptake by l-2,4-diaminobutyric acid is a result of the accumulation of the inhibitor which exerts its effect from within the synaptosomes. Raising the external concentration of gamma-aminobutyric acid to saturating levels did not completely inhibit the accumulation of l-2,4-diaminobutyric acid. Thus, the transport of l-2,4-diaminobutyric acid appears to be mediated, at least in part, by a carrier which is not involved in the transport of gamma-amiuobutyric acid.     

Superoxide Production and Antioxidant Enzymes in Ammonia Intoxication in Rats

Injection of large doses of ammonium salts lead to the rapid death of animals. However, the molecular mechanisms involved in ammonia toxicity remain to be clarified. We have tested the effect of injecting 7 mmol/kg of ammonium acetate on the production of superoxide and on the activities of some antioxidant enzymes in rat liver, brain, erythrocytes and plasma. Glutathione peroxidase, superoxide dismutase and catalase activities were decreased in liver and brain (both in cytoso-lic and mitochondrial fractions) and also in blood red cells, while glutathione reductase activity remained unchanged. Superoxide production in submitochon-drial particles from liver and brain was increased by more than 100% in both tissues. Both diminished activity of antioxidant enzymes and increased superoxide radical production could lead to oxidative stress and cell damage, which could be involved in the mechanism of acute ammonia toxicity.     

Stimulation of uric acid production and depression of increase in plasma glutamine concentration by insulin in chickens infused with ammonia

1. A single intraportal injection of insulin remarkably stimulated uric acid production in the chicken infused with ammonium acetate and significantly depressed the increase in plasma glutamine concentration by the infusion of ammonia (P less than 0.01 at 20 min, P less than 0.05 at 40 min after the start of infusion). 2. The increases in ammonia concentration in the blood and kidney during the infusion of ammonia were not affected by the insulin pretreatment. 3. The depressive effect of insulin on the increase in plasma glutamine concentration by ammonia may be due to the acceleration by insulin of incorporation of glutamine into biosynthetic pathway of uric acid in the chicken.     

Severity of Hyperammonemic Encephalopathy Correlates with Brain Ammonia Level and Saturation of Glutamine Synthetase In Vivo

Abstract: Correlation among in vivo glutamine synthetase (GS) activity, brain ammonia and glutamine concentrations, and severity of encephalopathy was examined in hyperammonemic rats to obtain quantitative information on the capacity of GS to control these metabolites implicated in the etiology of hepatic encephalopathy. Awake rats were observed for neurobehavioral impairments after ammonium acetate infusion to attain a steady-state blood ammonia concentration of 0.9 (group A) or 1.3 µmol/g (group B). As encephalopathy progressed from grade III to IV, brain ammonia concentration increased from 1.9 to 3.3 µmol/g and then decreased to 1.3 µmol/g on recovery to grade III. In contrast, brain glutamine concentration was 26, 23, and 21 µmol/g, respectively. NH₄⁺-infused rats pretreated with l -methionine dl -sulfoximine reached grade IV when brain ammonia and glutamine concentrations were 3.0 and 5.5 µmol/g, respectively; severity of encephalopathy correlates with brain ammonia, but not glutamine. In vivo GS activity, measured by NMR, was 6.8 ± 0.7 µmol/h/g for group A and 6.2 ± 0.6 µmol/h/g for group B. Hence, the in vivo activity, shown previously to increase with blood ammonia over a range of 0.4–0.64 µmol/g, approaches saturation at blood ammonia >0.9 µmol/g. This is likely to be the major cause of the observed accumulation of brain ammonia and the onset of grade IV encephalopathy.     

Brain Ion and Amino Acid Contents During Edema Development in Hepatic Encephalopathy

Abstract: Brain edema in hepatic encephalopathy has been associated with circulating ammonia that is metabolized to glutamine. We measured alterations in blood chemistry and brain regional specific gravity and ion and amino acid contents in models of simple hyperammonemia and liver failure induced by daily administrations of ammonium acetate (AAc) or thioacetamide (TAA), respectively. Serum and brain ammonia increased to similar levels (200 and 170% of control, respectively) in both experimental groups. Serum transaminase activities increased 10-fold in animals injected with TAA but were unchanged in animals given AAc injections. In both experimental groups glutamine was elevated in cerebral white matter, cerebral gray matter, and basal ganglia, whereas brain tissue specific gravity decreased in all brain regions, indicating edema formation. In the AAc group, we observed a decrease in glutamate and taurine contents concomitant with the development of brain edema. In these animals, cerebral gray matter specific gravity and taurine contents returned to control levels 24 h after the third AAc injection. TAA-injected animals demonstrated similar decreases in brain tissue specific gravity, whereas glutamine, glutamate, and taurine contents were all elevated. During hepatic encephalopathy, ammonia-induced changes in brain amino acid content may contribute to brain edema development.     

Metabolic adaptations to nitrogen excess in late gestation in rat.

Pregnant rats of 19th and 21st days were given an acute nitrogen overload produced by an infusion of either 0.2 M ammonium acetate or 0.2 M glutamine. Metabolic adaptations to nitrogen excess were studied measuring--in fetomaternal unit--non-protein nitrogen content and the activities of enzymes related with ammonia metabolism. Maternal and fetal plasma urea levels were increased by ammonium acetate treatment. Glutamine overload increased more the amino acid content in the mothers than in conceptus. As response to ammonium acetate treatment, glutamate dehydrogenase activity in liver was more sensitive in pregnant than in nonpregnant rats, suggesting more nitrogen incorporation into amino acids in pregnancy. Regarding glutamine synthetase activity, both treatments had an opposite effect except in kidney. The adenylate deaminase activity of pregnant rats was inhibited similarly to nonpregnant rats by nitrogen overloads, but stronger after glutamine infusion. Placenta and fetal metabolism were adjusted, as the dams, to lack of ammonia production by nitrogen overloads and to glutamine synthesis by ammonium acetate infusion.     

Manganese and ammonia interactions in the brain of cirrhotic rats: effects on brain ammonia metabolism

Hepatic encephalopathy is a major complication of cirrhosis. Ammonia and manganese have been associated with hepatic encephalopathy underlying mechanisms. Motor impairment and brain edema are common signs of hepatic encephalopathy. In the present study a model of liver damage in rats was combined with ammonia and manganese exposure to evaluate the role of these substances separately and their interactions on brain glutamine, water content and motor coordination. Additionally, we explored brain levels of each substance -Mn and ammonia- in the presence or absence of the other. Liver damage was induced by bile duct ligation. Rats were exposed to MnCl₂ in drinking water (1 mg Mn/ml) and to ammonia in chow pellets containing 20% ammonium acetate (w/w). As expected, manganese and ammonia levels increased in the brain of cirrhotic rats exposed to these substances; in these animals, glutamine brain levels also increased and positively correlated with tissue water content in cortex. A three way-ANOVA showed that manganese favored ammonia and glutamine accumulation in brain, and possibly their subsequent deleterious effects, as evidenced by the fact that manganese and ammonia accumulation in the brain of cirrhotic rats severely affected motor function. These results suggest that even when controlling ammonia levels in cirrhotic patients, reduction of manganese intake is also a potential strategy to be considered in clinical practice.     

Effect of ammonium acetate-induced hyperammonemia on metabolism of guanidino compounds.

Guanidino compounds are synthesized from arginine in various tissues such as liver, kidney, brain, and skeletal muscle. Guanidino compounds such as arginine and creatine play an important role in nitrogen metabolism, whereas other guanidino compounds such as guanidinosuccinic acid and alpha-N-acetylarginine are known toxins. In order to understand the changes in the metabolism of guanidino compounds during ammonia toxicity, we investigated the effect of hyperammonemia induced by an ammonium acetate injection on the levels of guanidino compounds in plasma, liver, kidney, and brain of rats. Control animals were injected with an equal volume of saline. Blood and tissues were removed 1 h following ammonium acetate or saline injection and guanidino compounds were analyzed by high-performance liquid chromatography. Plasma and kidney levels of guanidinosuccinic acid were significantly elevated in rats challenged with ammonium acetate. Brain alpha-N-acetylarginine levels were also significantly higher in rats injected with ammonium acetate as compared to those in controls. Our results suggest that guanidinosuccinic acid and alpha-N-acetylarginine may play an important role in hyperammonemia.     

Regional Differences in the Capacity for Ammonia Removal by Brain Following Portocaval Anastomosis

Portocaval anastomosis (PCA) in the rat leads, within 4 weeks, to severe liver atrophy, sustained hyperammonemia, and increased brain ammonia. Because brain is not equipped with an effective urea cycle, removal of ammonia involves glutamine synthesis and PCA results in significantly increased brain glutamine. Glutamine synthetase activities, however, are decreased by 15% in cerebral cortex and are unchanged in brainstem of shunted rats. Administration of ammonium acetate to rats following PCA results in severe encephalopathy (loss of righting reflex and, ultimately, coma). Glutamine concentrations in brainstem of comatose rats are increased a further two-fold, whereas those of cerebral cortex are unchanged. Consequently, ammonia levels in cerebral cortex reach disproportionately high levels (of the order of 5 mM). These findings suggest a limitation in the capacity of cerebral cortex to remove additional blood-borne ammonia by glutamine formation following PCA. Such mechanisms may explain the hypersensitivity of rats with PCA and of patients with portal-systemic shunting to small increases of blood ammonia. Disproportionately high levels of brain ammonia in certain regions, such as cerebral cortex, may then result in alterations of inhibitory neurotransmission and, ultimately, loss of cellular (astrocytic) integrity.     

Tricarboxylic acid-cycle metabolism in brain. Effect of fluoroacetate and fluorocitrate on the labelling of glutamate aspartate, glutamine and γ-amino butyrate

1. The effect of fluoroacetate and fluorocitrate on the compartmentation of the glutamate-glutamine system was studied in brain slices with l-[U-(14)C]glutamate, l-[U-(14)C]aspartate, [1-(14)C]acetate and gamma-amino[1-(14)C]butyrate as precursors and in homogenates of brain tissue with [1-(14)C]acetate. The effect of fluoroacetate was also studied in vivo in mouse brain with [1-(14)C]acetate as precursor. 2. Fluoroacetate and fluorocitrate inhibit the labelling of glutamine from all precursors but affect the labelling of glutamate to a much lesser extent. This effect is not due to inhibition of glutamine synthetase. It is interpreted as being due to selective inhibition of the metabolism of a small pool of glutamate that preferentially labels glutamine.     

Effects of Acute Hyperammonemia on Cerebral Amino Acid Metabolism and pHi In Vivo, Measured by 1H and 31P Nuclear Magnetic Resonance

The effects of an acute intravenous infusion of ammonium acetate on rat cerebral glutamate and glutamine concentrations, energy metabolism, and intracellular pH were measured in vivo with 1H and 31P nuclear magnetic resonance (NMR). The level of blood ammonia maintained by the infusion protocol used in this study (approximately 500 microM, arterial blood) did not cause significant changes in arterial PCO2, PO2, or pH. Cerebral glutamate levels fell to at least 80% of the preinfusion value, whereas glutamine concentrations increased 170% relative to the preinfusion controls. The fall in brain glutamate concentrations followed a time course similar to that of the rise of brain glutamine. There were no detectable changes in the content of phosphocreatine (PCr) or nucleoside triphosphates (NTP), within the brain regions contributing to the sensitive volume of the surface coil, during the ammonia infusion. Intracellular pH, estimated from the chemical shift of the inorganic phosphate resonance relative to the resonance of PCr in the 31P spectrum, was also unchanged during the period of hyperammonemia. 1H spectra, specifically edited to allow quantitation of the brain lactate content, indicated that lactate rose steadily during the ammonia infusion. Detectable increases in brain lactate levels were observed approximately 10 min after the start of the ammonia infusion and by 50 min of infusion had more than doubled. Spectra acquired from rats that received a control infusion of sodium acetate were not different from the spectra acquired prior to the infusion of either ammonium or sodium acetate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of ammonia on monoamine oxidase and enzymes of GABA metabolism in mouse brain.

Acute and chronic ammonia toxicity was produced in the mice by intraperitoneal injection of ammonium chloride (200 mg/kg) and by exposure of mice to ammonia vapours (5% v/v) continuously for 2 days and 5 days respectively. The ammonia content was elevated in the cerebellum, cerebral cortex and brain stem and in liver. In acute ammonia intoxication there was a decrease in the monoamine oxidase (MAO) activity in all the three regions of brain. In chronic ammonia toxicity (2 days of exposure) a significant increase in the activity of MAO was observed in the cerebral cortex while in cerebellum and brain stem there was a significant decrease. In cerebral cortex and cerebellum there was a rise in the activity of MAO as a result of exposure to ammonia vapours for 5 days. A significant decrease was observed in the activity of glutamate decarboxylase (GAD) in all the three regions of the brain both in acute and chronic ammonia toxicity (2 days). There was a decrease in the activity of this enzyme only in the cerebral cortex in the animals exposed to ammonia for 5 days. The activity of GABA-aminotransferase (GABA-T) showed a significant rise in cerebellum and a fall in the brain stem in acute ammonia toxicity. In chronic ammonia toxicity GABA-T showed a rise in all the three regions of brain. Chronic ammonia toxicity produced a significant decrease in the content of glutamate in all the three regions without a significant change in the content of aspartate. GABA and glutamine. The content of alanine increased in all the three regions of brain under these experimental conditions. The ratio of glutamate + aspartate/GABA and glutamate/glutamine showed a decrease in all the three regions as a result of ammonia toxicity.     

Guanosine Exerts Neuroprotective Effect in an Experimental Model of Acute Ammonia Intoxication

The nucleoside guanosine (GUO) increases glutamate uptake by astrocytes and acts as antioxidant, thereby providing neuroprotection against glutamatergic excitotoxicity, as we have recently demonstrated in an animal model of chronic hepatic encephalopathy. Here, we investigated the neuroprotective effect of GUO in an acute ammonia intoxication model. Adult male Wistar rats received an intraperitoneal (i.p.) injection of vehicle or GUO 60 mg/kg, followed 20 min later by an i.p. injection of vehicle or 550 mg/kg of ammonium acetate. Afterwards, animals were observed for 45 min, being evaluated as normal, coma (i.e., absence of corneal reflex), or death status. In a second cohort of rats, video-electroencephalogram (EEG) recordings were performed. In a third cohort of rats, the following were measured: (i) plasma levels of glucose, transaminases, and urea; (ii) cerebrospinal fluid (CSF) levels of ammonia, glutamine, glutamate, and alanine; (iii) glutamate uptake in brain slices; and (iv) brain redox status and glutamine synthetase activity in cerebral cortex. GUO drastically reduced the lethality rate and the duration of coma. Animals treated with GUO had improved EEG traces, decreased CSF levels of glutamate and alanine, lowered oxidative stress in the cerebral cortex, and increased glutamate uptake by astrocytes in brain slices compared with animals that received vehicle prior to ammonium acetate administration. This study provides new evidence on mechanisms of guanine-derived purines in their potential modulation of glutamatergic system, contributing to GUO neuroprotective effects in a rodent model of by acute ammonia intoxication.     

Cerebral Cortex Ammonia and Glutamine Metabolism During Liver Insufficiency-Induced Hyperammonemia in the Rat

Hyperammonemia has been suggested to induce enhanced cerebral cortex ammonia uptake, subsequent glutamine synthesis and accumulation, and finally net glutamine release into the blood stream, but this has never been confirmed in liver insufficiency models. Therefore, cerebral cortex ammonia- and glutamine-related metabolism was studied during liver insufficiency-induced hyperammonemia by measuring plasma flow and venous-arterial concentration differences of ammonia and amino acids across the cerebral cortex (enabling estimation of net metabolite exchange), 1 day after portacaval shunting and 2, 4, and 6 h after hepatic artery ligation (or in controls). The intra-organ effects were investigated by measuring cerebral cortex tissue ammonia and amino acids 6 h after liver ischemia induction or in controls. Arterial ammonia and glutamine increased in portacaval-shunted rats versus controls, and further increased during liver ischemia. Cerebral cortex net ammonia uptake, observed in portacaval-shunted rats, increased progressively during liver ischemia, but net glutamine release was only observed after 6 h of liver ischemia. Cerebral cortex tissue glutamine, gamma-aminobutyric acid, most other amino acids, and ammonia levels were increased during liver ischemia. Glutamate was equally decreased in portacaval-shunted and liver-ischemia rats. The observed net cerebral cortex ammonia uptake, cerebral cortex tissue ammonia and glutamine accumulation, and finally glutamine release into the blood suggest that the rat cerebral cortex initially contributes to net ammonia removal from the blood during liver insufficiency-induced hyperammonemia by augmenting tissue glutamine and ammonia pools, and later by net glutamine release into the blood. The changes in cerebral cortex glutamate and gamma-aminobutyric acid could be related to altered ammonia metabolism.     

Compartmentation of citric acid cycle metabolism in brain: labelling of glutamate, glutamine, aspartate and gaba by several radioactive tracer metabolites

—(1) Compartmentation of the metabolism of amino acids in brain has been studied in slices of cerebral cortex incubated with sodium [1-¹⁴C]acetate, sodium [1-¹⁴C]-bicarbonate, [1-¹⁴C]GABA or l-[1-¹⁴C]glutamate and in samples of brain after injection in vivo of [1-¹⁴C]- or [³H]acetate. (2) The method of treatment of the slices (a) maintained in ice-cold medium prior to incubation; (b) preincubation at 37°C and transfer to fresh medium affected the metabolism of the added, labelled substrate, particularly its labelling of glutamine. (3) The specific activity of glutamine labelled from the above metabolites was greater than that of glutamic acid in experiments of 10–30 minutes duration, whether or not subjected to pretreatment in the cold. (4) Incubation in medium containing 27 mm-K⁺ was associated with a decrease in the relative specific activity (RSA) of glutamine, except for the increase when l-[1-¹⁴C]glutamate was the precursor. (5) The data have been discussed in terms of metabolic compartmentation and their consistency with the concept of the presence in brain of more than one citric acid cycle, one containing the relatively smaller pools of intermediates and associated with synthetic processes; the other containing the relatively larger pools of intermediates and functioning as a homeostatic buffer for energy metabolism.     

The effect of sub-lethal ammonia exposure on fed and unfed rainbow trout: the role of glutamine in regulation of ammonia

Many species of fishes have evolved mechanisms for coping with ammonia caused by either high ammonia environments or an inability to excrete nitrogenous wastes. Rainbow trout (Oncorhynchus mykiss), have not been known to have such a mechanism. The present study investigated whether rainbow trout can use amino acid synthesis and storage to cope with ammonia. Experiments were performed on fed and unfed rainbow trout under both control and elevated ammonia conditions (0 and 10 mgN/l (total ammonia nitrogen), pH 7.2). The results indicate that both feeding and ammonia exposure increased plasma ammonia significantly 6 h postprandial and post ammonia exposure. After 48 h the fed/ammonia exposed fish had plasma ammonia levels that were not significantly different than the fed/control fish. Plasma ammonia was reduced by more than 50%, attributable to ammonia being converted to glutamine in brain, liver and muscle tissue. Feeding alone also increased glutamine levels in brain tissue. Activity of glutamine synthetase in brain and liver was increased corresponding to an increase in glutamine concentrations when fish were exposed to ammonia. This is the first report showing that rainbow trout can detoxify endogenous and exogenous ammonia.     

Growth behavior of Chinese hamster ovary cells in a compact loop bioreactor. 2. Effects of medium components and waste products.

Effects of biochemical factors, i.e., medium components and metabolic byproducts, on growth of Chinese hamster ovary (CHO) cells were investigated. Glucose and ammonia were found to inhibit the growth. Kinetic analysis gave the inhibition constants, 0.14 g l-1 for ammonia and 5.0 g l-1 for glucose. Since glutamine was unstable and was a main source of ammonia, precise studies on glutamine degradation and ammonia formation process were done. By evaluating the spontaneous reactions, net glutamine utilization and net ammonia production by the cells could be estimated. It became evident that asparagine could support the growth of CHO cells as a stable substitute for glutamine. Then, a glucose fed-batch culture was grown on a glutamine free and asparagine supplemented medium. Because of (1) low glucose concentration, but (2) no glucose limitation and (3) low ammonia accumulation, the maximum total cell concentration reached 3.4 x 10(6) ml-1, which was 1.8 times greater than that in the control experiment (initial 1.15 g l-1 glucose and 0.29 g l-1 glutamine, and no glucose feed).     

The Release and Neosynthesis of Glutamic Acid Are Increased in Experimental Models of Hepatic Encephalopathy

The effects of ammonium ions on the release of glutamic acid from the rat cerebral cortex were measured in vivo using cortical cups and a multiple ion detection technique. The neosynthesis of this amino acid from glucose was also studied in two experimental models of hepatic encephalopathy: (1) rats receiving large amounts of ammonium acetate (i.p.) and (2) rats with a surgically constructed portocaval anastomosis. Intraperitoneal administration of 8 mmol/kg of ammonium acetate increased the cortical release of glutamic acid from 9.1 +/- 0.8 to 19 +/- 2 (nmol X cm-2 X min-1). Moreover, 20 min after ammonium acetate administration the rate of incorporation of 13C2, originating from [13C]glucose, into glutamic acid increased by 65%. In several brain areas of rats bearing a portocaval anastomosis and fed ad libitum for 4 weeks, the content of glutamic acid slightly increased and the rate of formation of [13C2]glutamate from [13C]glucose approximately doubled. These results indicate that ammonium ions increase the release and the formation of glutamic acid in the brain. The resulting increased concentration of this amino acid in the extracellular spaces may be one of the mechanisms of ammonia toxicity in vivo.     

Compartmentation of citric acid cycle metabolism in brain: effect of aminooxyacetic acid, ouabain and Ca2+ on the labelling of glutamate, glutamine, aspartate and gaba by [1-14C]acetate, [U-14C]glutamate and [U-14C]asparate

—(1) The effects of aminooxyacetic acid, ouabain and Ca²⁺ on the compartmentation of amino acid metabolism have been studied in slices of brain incubated with sodium-[1-¹⁴C]acetate, l-[U-¹⁴C]glutamate and l-[U-¹⁴C]aspartate as tracer metabolites. (2) Aminooxyacetic acid (10^-3 m) inhibited the labelling of aspartate from [¹⁴C]acetate and [¹⁴C]glutamate, as well as the incorporation of label from [¹⁴C]aspartate into glutamate and glutamine. It also inhibited the labelling of GABA from all three radioactive precursors, as would be anticipated if there was inhibition of several transaminases as well as glutamate decarboxylase. The RSA of glutamine labelled from [1-¹⁴C]acetate was increased. This finding indicated that the glutamate pool which is utilized for glutamine formation is associated with glutamate dehydrogenase, and this enzyme appears to be related to the ‘synthetic tricarboxylic acid cycle’. AOAA exerted its major inhibitory effects on the citric acid‘energy cycle’with which transaminases are associated. (3) Ouabain (10^-5 m) inhibited the labelling of glutamine to a much greater extent than the labelling of glutamate from [1-¹⁴C]acetate. It also caused leakage of amino acids from the tissue into the medium. Its effect on the glutamate–glutamine system was interpreted to be a selective inhibition of the 'synthetic’citric acid cycle. (4) The omission of Ca²⁺ from the incubation medium was associated with formation of glutamine with RSA less than 1·0 when labelled from [U-¹⁴C]glutamate, [U-¹⁴C]aspartate and lower than normal when labelled from [1-¹⁴C]acetate.