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.     

Early changes in the nucleoside diphosphate kinase activity of the rat brain and liver during whole body gamma irradiation in an absolutely lethal dose

Nucleoside diphosphatkinase activity in mitochondria and post-mitochondrial supernatant fraction from brain and liver tissues was shown to decrease sharply during the first 60 min after total-body gamma-irradiation of rats with an absolutely lethal dose (30 Gy). A significant increase in the enzyme activity was registered 3 and 24 h following irradiation. Changes in nucleoside diphosphatkinase activity were more pronounced in mitochondria (particularly in liver mitochondria) than in the supernatant fraction.     

EFFECTS OF ACUTE HYPERTHERMIA ON POLYRIBOSOMES, IN VIVO PROTEIN SYNTHESIS AND ORNITHINE DECARBOXYLASE ACTIVITY IN THE NEONATAL RAT BRAIN

—Acute hyperthermia produces in situ disaggregation of brain polyribosomes in infant rats, as determined by electron microscopy. Protein synthesis is inhibited in infant, but not weanling, rat brain by 45 min of hyperthermia; this inhibition is reversed during a 2 h recovery period at normothermic conditions. Hepatic protein synthesis was inhibited less than that of brain. Acute hyperthermia also leads to a profound loss of ornithine decarboxylase activity in brain; during recovery the activity of this enzyme overshoots to values greater than those of normothermic control rats. This increase is blocked by cycloheximide administration. In testis, a tissue with high ornithine decarboxylase activity, enzyme activity was not affected by hyperthermia and recovery, indicating tissue specificity for these effects.     

Irradiation increases superoxide dismutase in rat intestinal smooth muscle

We investigated whether X-irradiation could induce the enzyme superoxide dismutase (SOD) in intestinal muscle. Groups of rats received abdominal irradiation and the time course and dose response for SOD activity determined. Jejunal smooth muscle homogenates were analyzed for the activities of copper/zinc (CuZn) and manganese (Mn) SOD activity and for a mitochondrial marker enzyme, citrate synthase. A progressive rise in Mn SOD activity occurred at 20, 46, and 72 h after 1500 R. No significant changes in Cu-Zn SOD activity occurred at any time after 1500 R. At 20 h after 250 R of X-irradiation, Mn SOD activity increased but no further increase occurred at higher irradiation exposures. At the same time, CuZn SOD activity at 20 h after irradiation was greater than controls only at an exposure of 1000 R (p less than 0.05). Using Western blotting, we were able to clearly demonstrate an increase in immunoreactive Mn SOD protein in muscle samples 20 h after 1500 R. The rise in Mn SOD is not simply due to increase in mitochondrial numbers or increase in all mitochondrial enzyme activities because activity of the mitochondrial marker enzyme citrate synthase was decreased after X-irradiation. Transmission electron microscopic studies demonstrated damage to mitochondria after a dose of 3000 R. The data yield evidence that free radicals play a role in irradiation-induced intestinal smooth muscle injury.     

Nucleoside mono- and nucleoside diphosphate kinase activities in rat liver and brain in radiation sickness

Activity of nucleoside di- and nucleoside triphosphates metabolism enzymes in tissues of rats gamma-irradiated by a dose of 30 Gy was studied 0.5, 1, 3, 6 and 24 hours after the radiation effect. It is shown that the nucleoside monophosphate kinase activity of the liver and brain is enhanced almost at all stages of the studies and the nucleoside diphosphate kinase activity is inhibited. A significant but reversible decrease of the nucleoside monophosphate kinase activity is observed in the liver 3 h later. By an end of the first day after irradiation the nucleoside mono- and nucleoside diphosphate kinase activities increase significantly both in the liver and brain.     

Effect of Hyperammonemia and Methionine Sulfoximine on the Kinetic Parameters of Blood-Brain Transport of Leucine and Phenylalanine

The activity of the blood-brain neutral amino acid transport system is increased in rats infused with ammonium salts or rendered hyperammonemic by a portacaval anastomosis. This effect may be due to a direct action of ammonia or to some metabolic consequence of high ammonia levels, such as increased brain glutamine synthesis. To test these possibilities we evaluated the kinetic parameters of blood-brain transport of leucine and phenylalanine in control rats, in rats after continuous 24 h infusion of ammonium salts (NH4+ = 2.5 mmol X kg-1 X h-1), and in rats treated with methionine sulfoximine, an inhibitor of glutamine synthetase, before infusion of ammonium salts. In ammonia-infused rats without methionine sulfoximine treatment, the KD and Vmax of phenylalanine transport were increased, respectively, about 170% and 80% compared to controls, whereas the Km and Vmax of leucine transport were increased, respectively, about 100% and 200%. Electron microscopy demonstrated marked swelling of astrocytic processes around brain capillaries of ammonia-infused rats; however, capillary permeability to horseradish peroxidase apparently was not increased by ammonia infusion. Administration of methionine sulfoximine before ammonia infusion inhibited glutamine synthesis and prevented the changes in transport of leucine and phenylalanine, but apparently did not reverse the perivascular swelling. These results suggest that the ammonia-induced increase in the activity of transport of large neutral amino acids across the blood-brain barrier requires glutamine synthesis in brain, and is not a direct effect of ammonia.     

Activity of renal enzymes producing ammonia from glutamine in the absence of phosphate: 2. Effect of phosphate dependent glutaminase inhibition by heating

It is known that heating at 50 degrees C for 10 minutes inhibits phosphate dependent glutaminase (PDG) activity of renal cortex, without any effect on gamma-glutamyl transpeptidase (gamma GT) and its phosphate independent glutaminase (PIG) activity. The effect of heating on PIG and total gamma GT activities was evaluated in renal cortex homogenates of rats both in normal acid-base equilibrium and in chronic metabolic acidosis (CMA). Homogenates were incubated in a medium containing glutamine 2 mM, no phosphate, at pH 7,40. PIG activity was measured as glutamate production and total gamma GT activity as ammonia production. In normal rats PIG activity was unchanged after heating, whereas a significant decrease of total gamma GT activity was observed (p less than 0,01). CMA caused an increase in both PIG and total gamma GT activity (p less than 0,01) and these increased to a further extent after heating. In both normal and acidotic rats the glutamate production/ammonia production ratio rose to about 1. In conclusion: a) in the experimental setting used for this study PDG activity does not intervene in glutamate and ammonia production from glutamine; b) heating causes an inhibition of gamma GT activities, other than PIG, both in normal and in acidotic rats; c) in CMA heating increases PIG activity of gamma GT.     

Regional differences in antioxidative response of rat brain after cranial irradiation

In order to examine if differences in activity and inducibility of antioxidative enzymes in rat cerebral cortex and hippocampus are underlying their different sensitivity to radiation, we exposed four-day-old female Wistar rats to cranial radiation of 3 Gy of gamma-rays. After isolation of hippocampus and cortex 1 h or 24 h following exposure, activities of copper-zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD) and catalase (CAT) were measured and compared to unirradiated controls. MnSOD protein levels were determined by SDS-PAGE electrophoresis and Western blot analysis. Our results showed that CuZnSOD activity in hippocampus and cortex was significantly decreased 1 h and 24 h after irradiation with 3 Gy of gamma-rays. MnSOD activity in both brain regions was also decreased 1 h after irradiation. 24 h following exposure, manganese SOD activity in hippocampus almost achieved control values, while in cortex it significantly exceeded the activity of the relevant controls. CAT activity in hippocampus and cortex remained stable 1 h, as well as 24 h after irradiation with 3 Gy of gamma-rays. MnSOD protein level in hippocampus and cortex decreased 1 h after irradiation with 3 Gy of gamma-rays. 24 h after exposure, MnSOD protein level in cortex was similar to control values, while in hippocampus it was still significantly decreased. We have concluded that regional differences in MnSOD radioinducibility are regulated at the level of protein synthesis, and that they represent one of the main reasons for region-specific radiosensitivity of the brain.     

The effect of low doses of external gamma irradiation on the chromatin structure and activity of the histone-specific proteinases of rat brain cells]

Irradiation of rats with doses of 0.5 to 2 Gy was shown to cause dose-dependent changes in the sensitivity of brain cell chromatin to the effect of DNAase I that were manifested by the increased level of DNA hydrolysis and a high content of the chromatin soluble fractions. The chromatin structure was only partially restored 24 h after irradiation. Changes in the chromatin structure were accompanied by the increase in the histone-specific proteinase activity.     

Sympathetic hyperfunction causes increased sensitivity of the autonomic nervous system to whole-body X irradiation

Although the etiology of radiation sickness is still unknown, disturbance of the autonomic nervous system is suggested to be a factor. This study was designed to compare the radiosensitivity of spontaneously hypertensive rats possessing sympathetic hyperfunction and control Wistar-Kyoto rats, and to analyze the effects of radiation on the autonomic nervous system in both strains. After a 7.5-Gy dose of whole-body X irradiation, the blood pressure decreased significantly at 8 h and 2 days in the spontaneously hypertensive rats, but not in the Wistar-Kyoto rats. Epinephrine levels in the adrenal gland of spontaneously hypertensive rats decreased at 4, 8 and 24 h, unlike the Wistar-Kyoto rats. Radiation evoked a stronger increase in norepinephrine in the jejunum and colon of spontaneously hypertensive rats than in Wistar-Kyoto rats. Acetylcholine levels in the jejunum of spontaneously hypertensive rats decreased, in contrast to the increase in Wistar-Kyoto rats within 24 h after irradiation. The survival rate of spontaneously hypertensive rats was lower than that of Wistar-Kyoto rats and weight loss, appetite loss and morphological changes in the jejunum were greater in spontaneously hypertensive rats than in Wistar-Kyoto rats after irradiation. These results indicated that X irradiation caused greater activities in autonomic nervous function and severe radiation injury in spontaneously hypertensive rats. Sympathetic hyperfunction may be associated with a higher sensitivity to radiation, including radiation injury and radiation sickness.     

Two-phase response of rat pineal melatonin to lethal whole-body irradiation with gamma rays.

Male Wistar rats adapted to artificial light:dark (LD) regimen 12:12 h were whole-body irradiated with a single dose of 9.6 Gy of gamma rays and sham/irradiated in the night in darkness. The rats were examined 60 min, 1, 3 and 5 days after exposure between 22:00 and 01:30 h in the darkness. The results obtained indicate a two-phase reaction of pineal melatonin after the lethal irradiation of rats: the decline of melatonin concentration early after the exposure (at 60 min) with unchanged serotonin N-acetyltransferase (NAT) activity followed by an increase of melatonin synthesis, accompanied by an increase of pineal and serum melatonin on day 5 after the exposure. NAT activity was increased on day 3 after the exposure. Serum corticosterone concentrations in irradiated rats were increased 60 min and 3 days after exposure. With respect to the antioxidant, immunomodulating and stress-diminishing properties of melatonin, we consider the increase in melatonin synthesis during later periods after irradiation as part of adaptation of the organism to overcome radiation stress.     

Changes in expression of transferrin,insulin-like growth factor 1, and interleukin 4 receptors after irradiation of cells of primary malignant brain tumor cell lines

Various immunotoxins have been developed for the treatment of cancer. The toxin is internalized by target cells through cell-surface receptors, and it is essential for these receptors to be expressed for the immunotoxin to have specific anti-tumor activity. Radiation therapy is one of the main treatment modalities for primary malignant brain tumors. The purpose of this study was to determine whether radiation influences the expression of cell-surface receptors. Cells of one human medulloblastoma (Daoy) and two glioblastoma (U373-MG and T98-G) cell lines were tested by exposing the cells to a single dose of 5 Gy gamma rays. Expression of transferrin receptors, type-1 insulin-like growth factor receptors (IGF1R), and interleukin 4 receptors (IL4R) was measured by flow cytometry analysis on unirradiated cells and on cells 3 to 120 h after irradiation. In Daoy cells, the absolute expression index of transferrin receptors increased during the 24 h after irradiation with the greatest change of 26% above control at 9 h. The absolute expression index of IGF1R increased 26.5% above control at 12 h. The absolute expression index of IL4R decreased 9 h after irradiation. In U373-MG cells the absolute expression index of transferrin receptors increased during the 24 h after irradiation, and the greatest increase was 45% above control at 9 h. The absolute expression index of IGF1R increased during the 12 h after irradiation with a maximum increase of 33% above control at 6 h. The absolute expression index of IL4R decreased with time after irradiation. In T98-G cells, the absolute expression index of both transferrin receptors and IL4R decreased after irradiation. The results suggest that the expression of growth factor receptors on brain tumor cells may be influenced by radiation. The effect of ionizing radiation on receptor expression should be considered when administration of targeted toxin is combined with radiation. Similar studies with other growth factor receptors used in targeted toxin therapy are recommended.     

The metabolic fate of 13N-labeled ammonia in rat brain.

13N-labeled ammonia was used to study the cerebral uptake and metabolism of ammonia in conscious rats. After infusion of physiological concentrations of [13N]ammonia for 10 min via one internal carotid artery, the relative specific activities of glutamate, glutamine (alpha-amino), and glutamine (amide) in brain were approximately 1:5:400, respectively. The data are consistent with the concept that ammonia, entering the brain from the blood, is metabolized in a small pool of glutamate that is both rapidly turning over and distinct from a larger tissue glutamate pool (Berl, S., Takagaki, G., Clarke, D.D., and Waelsch, H. (1962) J. Biol. Chem. 237, 2562-2569). Analysis of 13N-metabolites, after infusion of [13N]ammonia into one lateral cerebral ventricle, indicated that ammonia entering the brain from the cerebrospinal fluid is also metabolized in a small glutamate pool. Pretreatment of rats with methionine sulfoximine led to a decrease in the label present in brain glutamine (amide) following carotid artery infusion of [13N]ammonia. On the other hand, 13N activity in brain glutamate was greater than that in the alpha-amino group of glutamine, i.e. following methionine sulfoximine treatment the expected precursor-product relationship was observed, indicating that the two pools of glutamate in the brain were no longer metabolically distinct. The amount of label recovered in the right cerebral hemisphere, 5 s after a rapid bolus injection of [13N]ammonia via the right common carotid artery, was found to be independent of ammonia concentration within the bolus over a 1000-fold range. This finding indicates that ammonia enters the brain from the blood largely by diffusion. In normal rats that were killed by a freeze-blowing technique 5 s after injection of an [13N]ammonia bolus, approximately 60% of the label recovered in brain had already been incorporated into glutamine, indicating that the t1/2 for conversion of ammonia to glutamine in the small pool is in the range of 1 to 3 s or less. The data emphasize the importance of the small pool glutamine synthetase as a metabolic trap for the detoxification of blood-borne and endogenously produced brain ammonia. The possibility that the astrocytes represent the anatomical site of the small pool is considered.     

Investigations into mechanisms responsible for seizures induced by chlorinated hydrocarbon insecticides: the role of brain ammonia and glutamine in convulsions in the rat and cockerel

Abstract— Seizures were produced in adult female rats and in cockerels (Gallus domesticusL.) after the intracarotid administration of four structurally-unrelated chlorinated hydrocarbon insecticides. The temporal relationship of the course of the convulsions to levels in whole brain of ammonia and glutamine was examined. High levels of brain ammonia occurred in rats and cockerels poisoned acutely with lindane, dieldrin, heptachlor and DDT. The intensity of convulsions and other neurological signs coincided with and were directly correlated with the increases in brain ammonia, whereas interictal periods were associated with decreased levels of ammonia. In both species the order of drug toxicity was lindane > dieldrin > heptachlor > DDT. Significantly elevated levels of brain glutamine were induced in the cockerels after administration of all four insecticides. In contrast, increased levels of glutamine in rats were found only in those intoxicated with lindane and dieldrin. Although the ammonia-binding mechanism (conversion of free ammonia into glutamine) was saturated throughout the experiments in both the rats and cockerels, the latter were more resistant to ammonia toxicity by virtue of being able to convert free ammonia into glutamine more efficiently than rats. These data, together with data reported by others, lead to the conclusion that the four structurally-unrelated chlorinated hydrocarbon insecticides probably induced convulsions in both species by a common mechanism, involving interference with the production and/or utilization of ammonia.     

A single transient episode of hyperammonemia induces long-lasting alterations in protein kinase A

Hepatic encephalopathy in patients with liver disease is associated with poor prognosis. This could be due to the induction by the transient episode of hepatic encephalopathy of long-lasting alterations making patients more susceptible. We show that a single transient episode of hyperammonemia induces long-lasting alterations in signal transduction. The content of the regulatory subunit of the protein kinase dependent on cAMP (PKA-RI) is increased in erythrocytes from cirrhotic patients. This increase is reproduced in rats with portacaval anastomosis and in rats with hyperammonemia without liver failure, suggesting that hyperammonemia is responsible for increased PKA-RI in patients. We analyzed whether there is a correlation between ammonia levels and PKA-RI content in patients. All cirrhotic patients had increased content of PKA-RI. Some of them showed normal ammonia levels but had suffered previous hyperammonemia episodes. This suggested that a single transient episode of hyperammonemia could induce the long-lasting increase in PKA-RI. To assess this, we injected normal rats with ammonia and blood was taken at different times. Ammonia returned to basal levels at 2 h. However, PKA-RI was significantly increased in blood cells from rats injected with ammonia 3 wk after injection. In conclusion, it is shown that a single transient episode of hyperammonemia induces long-lasting alterations in signal transduction both in blood and brain. These alterations may contribute to the poor prognosis of patients suffering hepatic encephalopathy.     

血糖水平对缺氧缺血新生大鼠体重和脑重的影响观察

目的　观察不同血糖水平及缺氧缺血 (hypoxicischemia ,HI)前后血糖对缺氧缺血新生大鼠体重和脑重的影响。方法　通过制备缺氧缺血新生大鼠合并高低血糖模型 ,分别在脑HI后 2、2 4、48、72h和 7d共 5个时段 ,分别在断头前测体重和断头后取脑称脑重 ,并辅以免疫组化分析HI后 2 4h时段各组脑内葡萄糖转运蛋白 1(GLUT1)及葡萄糖转运蛋白 3 (GLUT3 )合成量的变化。结果　 2 4h时段HI组、HI前低血糖组、HI后低血糖组体重呈显著意义 ,低于正常组 ;2 4h时段缺氧缺血前后低血糖脑重均呈显著意义 ,低于其他各组 ,7d时段I前重高血糖组脑重高于HI组及HI前低血糖组。HI前重高血糖组HI后 2 4h在皮质部位GLUT1的合成量显著高于其他各组。HI前低血糖组HI后 2 4h在皮质部位GLUT3的合成量显著低于其他各组。结论　HI前低血糖对新生大鼠体重、脑重增长不利 ,HI前重高血糖有可能缓解HI引起的体重、脑重的减轻而显示一定的保护作用     

The `neurotoxicity'' of l-2,4-diaminobutyric acid

The neurolathyrogen l-2,4-diaminobutyric acid is concentrated by liver, and liver damage can yield neurotoxicity; thus the neurotoxicity caused by this compound may be due to liver damage followed by secondary brain damage. 1. The intraperitoneal administration of toxic doses of l-2,4-diaminobutyric acid to rats resulted in hyperirritability, tremors and convulsions in 12-20hr. and increased the concentration of ammonia of blood and brain slightly and the concentration of glutamine of brain two- to three-fold. By contrast, toxic doses of l-homoarginine, l-lysine, l-leucine and ammonium acetate caused dyspnoea, extreme prostration, and in some cases coma in 15-30min., and increased the concentration of ammonia of blood significantly and the concentration of glutamine of brain slightly. These results indicate that l-2,4-diaminobutyric acid caused a chronic ammonia toxicity, whereas the other amino acids and ammonium acetate resulted in an acute ammonia toxicity. 2. Liver slices from l-2,4-diaminobutyric acid-treated animals and normal liver slices preincubated with l-2,4-diaminobutyric acid utilized ammonia and formed urea at a lower rate than control slices from normal rats. 3. l-2,4-Diaminobutyric acid inhibited competitively ornithine carbamoyltransferase of rat liver homogenates, thus demonstrating that this reaction is a primary site of toxicity for this neurolathyrogen. Although we were unable to show marked elevations of blood ammonia concentration after treatment with l-2,4-diaminobutyric acid, these results are interpreted to mean that ammonia utilization (urea synthesis) in liver is inhibited by l-2,4-diaminobutyric acid and that at least part of the neurotoxicity is due to a prolonged slight increase in body ammonia concentration.     

ACETYL-COENZYME A: 1,4-DIAMINOBUTANE N-ACETYLTRANSFERASE: ACTIVITY IN RAT BRAIN DURING DEVELOPMENT, IN EXPERIMENTAL BRAIN TUMOURS AND IN BRAINS OF FISH OF DIFFERENT METABOLIC ACTIVITY

—Acetyl-CoA: 1,4-diaminobutane N-acetyltransferase catalyses the first step of putrescine catabolism in mammalian brain. It may be important in putrescine degradation of other tissues as well. Its specific activity is higher in homogenates of immature than of mature rat brains. A steady decline of putrescine acetylase activity is observed from birth until approx adult levels are reached at day 30. Microsomes and purified nuclei from brains of 2-day-old rats show considerably higher putrescine acetylase activities than the corresponding subcellular organelles from adult brains. Increased putrescine acetylase activities were found in nitroso-ethylurea-induced gliomas, together with a dramatic increase of putrescine concentration. High tissue concentrations of putrescine are, however, not necessarily correlated with enhanced putrescine acetylase activities. In trout brains a linear increase of acetyl-CoA: 1,4-diaminobutane N-acetyltransferase activity was observed together with a decrease of putrescine concentration after adaptation of the animals to increased water temperature.     

ELEVATED CONCENTRATIONS OF HISTAMINE IN THE RAT BRAIN

—Whole brain concentrations of histamine (Hm) in the rat were measured with a sensitive and specific single-isotope enzymatic microassay and found to be elevated about 2-fold in rats killed by focused microwave irradiation compared with rats killed by decapitation. However, regardless of the method of killing, whole brain Hm concentrations were elevated about 4-fold when the brains were homogenized in 12.5 vol vs 2.5 vol of water or phosphate buffer. Homogenization of brain regions in 12.5 vol of water resulted in Hm concentrations that were about 10 times greater than previously reported values (except in the hypothalamus). Since (1) the‘extra’Hm could be extracted from pellets of low volume homogenates, and (2) synthesis of Hm is not likely to occur through an increase in the volume of water or buffer used for homogenization, we suggest that the elevated concentrations of Hm seen after high volume homogenization of brain tissue are due to a more complete extraction of Hm from brain tissue, and not to an artifactual production of this amine from its precursor (histidine) or some other compound(s).     

Tissue ammonia and amino acids in rats at various oxygen pressures

The amino acid and ammonia profiles in various tissues of the rat exposed to different pressures of pure oxygen have been studied. Well-defined changes in behavioral activity accompanied a profile of increasing pressure, culminating in convulsive activity in each group of exposed animals. After an initial depression of ammonia, in all tissues studied at 0.68 atm oxygen ammonia increased significantly at higher oxygen pressures. A rise in tissue ammonia took place in the absence of undue muscular activity on the part of the exposed animals. A significant increase in ammonia occurred first in brain and liver at 3.40 atm. Ammonia concentration was high in all tissues after convulsions occurred at 4.08 atm. Between 0.68 and 2.72 atm oxygen, tissue ammonia concentration was generally low and brain glutamate and gamma-aminobutyric acid were high. At pressures higher than 2.72 atm oxygen, tissue glutamate declined and glutamine increased. Alanine became significantly elevated in serum and muscle at high oxygen pressure, and aspartate was depressed in heart, liver, and muscle. These pressure-course experiments on ammonia accumulation in tissue confirm previous serial time course observations that ammonia accumulates in the brain and several tissues of the rat even in the absence of undue muscular activity during high-pressure oxygen exposure and is a significant factor in inducing convulsions.