Thomas E. Duffy

Effect of acute ammonia intoxication on cerebral metabolism in rats with portacaval shunts.

Rats were made chronically hyperammonemic by portal-systemic shunting and, 8 wk later, were subjected to acute ammonia intoxication by the intraperitoneal injection of 5.2 mmol/kg of ammonium acetate. In free-ranging animals, ammonia treatment induced a brief period of precoma (10-15 min) that progressed into deep, anesthetic coma lasting for several hours and was associated with a high mortality. In paralyzed, artificially ventilated animals that were lightly anesthetized with nitrous oxide, acute ammonia intoxication caused major disturbances of cerebral carbohydrate, amino acid, and energy metabolism that correlated in time with the change in functional state. At 10 min after injection (precoma), the concentrations of most glycolytic intermediates were increased, as was the lactate/pyruvate ratio. Citrate declined, despite a twofold rise in pyruvate, suggesting that the conversion of pyruvate to citrate had been impaired. Concentrations of phosphocreatine, and of the putative neurotransmitters, glutamate and aspartate, declined during precoma, but the concentrations of the adenine nucleotides in the cerebral hemispheres, cerebellum, and brain stem remained within normal limits. At 60 min after injection (coma), ATP declined in all regions of brain; the reduction in total high-energy phosphates was most notable in the brain stem. The findings indicate that cerebral dysfunction in chronic, relapsing ammonia intoxication is not due to primary energy failure. Rather, it is suggested that ammonia-induced depletion of glutamic and aspartic acids, and inhibition of the malate-asparate hydrogen shuttle are the dominant neurochemical lesions.

Regional Energy Balance in Rat Brain After Transient Forebrain Ischemia

Abstract: Phosphocreatine, ATP, and glucose were severely depleted, and the lactate levels were increased in the paramedian neocortex, dorsal‐lateral striatum, and CA1 zone of hippocampus of rats exposed to 30 min of forebrain ischemia. Upon recirculation of the brain, phosphocreatine, ATP, and lactate concentrations recovered to control values in the paramedian neocortex and CA1 zone of hippocampus and to near‐control values in the striatum. The phosphocreatine and ATP concentrations then fell and the lactate levels rose in the striatum after 6–24 h, and in the CA1 zone of hippocampus after 24–72 h. The initial recovery and subsequent delayed changes in the phosphocreatine, ATP, and lactate concentrations in the striatum and hippocampus coincided with the onset and progression of morphological injury in these brain regions. The results suggest that cells in these regions regain normal or near‐normal mitochondrial function and are viable, in terms of energy production, for many hours before unknown mechanisms cause irreversible neuronal injury.

The dynamics of ammonia metabolism in man. Effects of liver disease and hyperammonemia.

The cyclotron-produced radionuclide, 13N, was used to label ammonia and to study its metabolism in a group of 5 normal subjects and 17 patients with liver disease, including 5 with portacaval shunts and 11 with encephalopathy. Arterial ammonia levels were 52-264 micron. The rate of ammonia clearance from the vascular compartment (metabolism) was a linear function of its arterial concentration: mumol/min = 4.71 [NH3]a + 3.76, r = +0.85, P less than 0.005. Quantitative body scans showed that 7.4 +/- 0.3% of the isotope was metabolized by the brain. The brain ammonia utilization rate, calculated from brain and blood activities, was a function of the arterial ammonia concentration: mumol/min per whole brain = 0.375 [NH3]a - 3.6, r = +0.93, P less than 0.005. Assuming that cerebral blood flow and brain weights were normal, 47 +/- 3% of the ammonia was extracted from arterial blood during a single pass through the normal brains. Ammonia uptake was greatest in gray matter. The ammonia utilization reaction(s) appears to take place in a compartment, perhaps in astrocytes, that includes less than 20% of all brain ammonia. In the 11 nonencephalopathic subjects the [NH3]a was 100 +/- 8 micron and the brain ammonia utilization rate was 32 +/- 3 mumol/min per whole brain; in the 11 encephalopathic subjects these were respectively elevated to 149 +/- 18 micron (P less than 0.01), and 53 +/- 7 mumol/min per whole brain (P less than 0.01). In normal subjects, approximately equal to 50% of the arterial ammonia was metabolized by skeletal muscle. In patients with portal-systemic shunting, muscle may become the most important organ for ammonia detoxification. Muscle atrophy may thereby contribute to the development of hyperammonemic encephalopathy with an associated increase in the brain ammonia utilization rate.

CARBOHYDRATE AND ENERGY METABOLISM IN PERINATAL RAT BRAIN: RELATION TO SURVIVAL IN ANOXIA

The ability of rats of different ages to survive exposure to anoxia was correlated with rates of high energy phosphate consumption (metabolic rates) of the fore‐brain. Fetal rats at term, delivered by hysterotomy following maternal decapitation, survived in nitrogen at 37°C twice as long as 1‐day‐old neo‐nates, 5 times longer than 7‐day‐old rats, and 45 times longer than adults. During ischemia induced by decapitation, the cerebral concentrations of the labile energy reserves (ATP, ADP, P‐creatine, glucose and glycogen) and of lactate were determined in fetuses, 1‐ and 7‐day post‐natal animals. From the changes, the cerebral energy use rates were calculated to be 1·57 mmol/kg/min in fetuses, 1·33 mmol/kg/min in 1‐day‐olds and 2·58 mmol/kg/min in 7‐day‐olds. Maximal rates of lactate accumulation during ischemia, as a measure of glycolytic capacity, were comparable in fetuses and neonates, but were about twice as great in 7‐day‐old rats. It is concluded that in post‐natal animals survival in anoxia and cerebral energy consumption are inversely, and nearly quantitatively, related. However, the reduced cerebral energy requirement cannot entirely account for the greater anoxic resistance of fetuses.

Cerebral energy metabolism during experimental status epilepticus.

—The concentration of ATP, ADP, AMP, phosphocreatine and of 5 intermediates of carbohydrate metabolism were determined in rodent brain after single and repeated seizures induced by either electroshock (ES), flurothyl or pentylenetetrazol (PTZ). In paralysed‐ventilated rats, one ES produced a 4–5 fold increase in cortical glycolytic flux (estimated from changes in glucose and lactate), and associated increases in pyruvate and in the lactate/pyruvate ratio. Total high energy phosphates declined during the seizure; a decrease was also calculated in cortical tissue pH and in the cytoplasmic [NAD+]/[NADH] ratio. Similar changes in brain were observed in ventilated mice after ES, but in paralysed animals, no decrease in high energy phosphates occurred during the first seizure. More vigorous and prolonged chemically‐induced seizures in both rats and mice elicited a decrease in the cerebral energy reserves with a rise in lactate and in the lactate/pyruvate ratio. At all times during the seizures the cerebral venous blood had a higher oxygen tension than that of control animals (rats) or was visibly reddened (mice), implying that oxygen availability to brain exceeded metabolic demands. It is proposed that the development of‘non‐hypoxic’cerebral lactacidosis during seizures is part of the overall metabolic response of the brain to an abrupt increase in energy consumption. The response constitutes a homeostatic influence which promotes cerebral vasodilatation, thereby increasing blood flow and the delivery of substrates.

Brain dysfunction in mild to moderate hypoxia

Hypoxia is commonly invoked to explain alterations in mental function, particularly in patients with cardiac pulmonary failure. The effects of acute graded hypoxia or higher integrative functions are well documented experimentally in man. Hypoxia in experimental animal models demonstrates that the pathophysiology is complex. In mild to moderate hypoxia, in contrast to severe hypoxia and to ischemia, the supply of energy for the brain is not impaired; cerebral levels of adenosine triphosphate (ATP) and adenylate energy charge are normal. In contrast, the turnover of several neurotransmitters is altered by mild hypoxia. For example, acetylcholine synthesis is reduced proportionally to the reduction in carbohydrate oxidation. This relationship holds in vitro and with several in vivo models of hypoxia. Pharmacologic and physiologic studies in man and experimental animals are consistent with acetylcholine having an important role in mediating the cerebral effects of mild hypoxia. These observations raise the possibility that treatments directed to cholinergic or other central neurotransmitter systems may benefit patients with cerebral syndromes secondary to chronic hypoxia.

Glutathione and Ascorbate During Ischemia and Postischemic Reperfusion in Rat Brain

Thirty minutes of total cerebral ischemia (decapitation) decreased total glutathione (GSH + GSSG) by 7% but had no detectable effect on the concentration of oxidized glutathione (GSSG), reduced ascorbate, or total ascorbate. In a model of reversible, bilateral hemispheric ischemia (four‐vessel occlusion) no changes in glutathione or ascorbate were detected after 30 min of ischemia. During 24 h of reperfusion following such an insult no detectable change in total ascorbate, reduced ascorbate, or oxidized glutathione was noted; however, total brain glutathione declined by 25%. The findings are discussed in relation to the hypothesis that the deleterious effects of ischemia are due to an increase in free radical production which in turn leads to increased lipid peroxidation.

CEREBRAL ENERGY METABOLISM DURING TRANSIENT ISCHEMIA AND RECOVERY IN THE GERBIL

Energy metabolism was studied in the cerebral cortex of gerbils during and following ischemia induced by 1 h of unilateral carotid artery occlusion. An aneurysm clip was applied to the right common carotid artery of 50‐70 g gerbils under brief halothane anesthesia, and the clip was removed 1 h later. Clinical state (gait, responsiveness, seizures) was evaluated during carotid occlusion, and 40% of the animals showed clinical evidence of stroke. Cortical energy stores (2 ATP + ADP + P‐creatine) were more than half depleted in the ipsilateral cortex of clinically‐affected gerbils, and glucose fell by 75%; lactate rose over 7‐fold in the same specimens. After release of the carotid clip, clinical state improved, and biochemical abnormalities partially resolved. However, even after 24 h, the concentration of ATP and the total pool of adenine nucleotides remained subnormal. Metabolic activity in the ischemic cortex, assessed as the utilization of high‐energy phosphates following decapitation, was normal after 1 h of recovery and decreased (‐50%) after 24 h but was increased by more than 50% after 4 h. Cerebral glucose utilization, evaluated from autoradiographs prepared after intravenous administration of 2‐[1‐14C]deoxyglucose, was also increased in the cortex, hippocampus, and thalamus after 4 h of recovery. This post‐ischemic hypermetabolism in tissue damaged by ischemia may identify a critical period for cell repair, when therapy could be decisive.

Impaired Synthesis of Acetylcholine by Mild Hypoxic Hypoxia or Nitrous Oxide

The effect of mild hypoxic hypoxia on brain metabolism and acetylcholine synthesis was studied in awake, restrained rats. Since many studies of hypoxia are done with animals anesthetized with nitrous oxide (N2O), the effects of N2O were evaluated. N2O (70%) increased the cerebral cortical blood flow by 33% and the cortical metabolic rate of oxygen by 26%. In addition, the synthesis of acetylcholine in N2O‐anesthetized animals, measured with [U‐14C]glucose and [1‐2H2,2‐2H2]choline, decreased by 45 and 53%, respectively. Consequently, mild hypoxia was studied in unanesthetized rats. Control rats breathing 30% O2 (partial pressure of oxygen, Pao2= 120 mm Hg) were compared with rats exposed to 15% O2 (Pao2= 57 mm Hg) or 10% O2 (Pao2= 42 mm Hg). The synthesis of acetylcholine, measured with [U‐14C]glucose, was decreased by 35 and 54% with 15% O2 and 10% O2 respectively; acetylcholine synthesis, measured with [1‐2H2,2‐2H2]choline, was decreased by 50 and 68% with 15% O2 and 10% O2 respectively. Animals breathing either 15% or 10% O2 had normal cerebral metabolic rates of oxygen but had increased brain lactates and increased cortical blood flows compared with animals breathing 30% O2. These results show that even mild hypoxic hypoxia impairs acetylcholine synthesis, which in turn may account for the early symptoms of brain dysfunction associated with hypoxia.

Use of β‐Methylene‐D,L‐Aspartate to Assess the Role of Aspartate Aminotransferase in Cerebral Oxidative Metabolism

Abstract: Several inhibitors of aspartate aminotransferase, a key enzyme of the malate‐aspartate shuttle, were investigated for their effects on cerebral oxidative metabolism in vitro. β‐Methylene‐D,L‐aspartate (2 mM), aminooxyacetate (0.1 mM), and D,L‐vinylglycine (20 mM) all significantly reduced the activity of aspartate aminotransferase and the rate of oxygen consumption of rat cerebral cortex slices respiring on glucose. In the presence of β‐methyleneaspartate, a one‐to‐one correlation was found between the degree of inhibition of tissue respiration and the degree of inhibition of transaminase activity. Slices of rat liver incubated in the presence of glucose and β‐methyleneaspartate showed a similar oneto‐one relationship between inhibition of oxygen cornsumption and inhibition of aspartate aminotransferase activity, whereas with rat kidney cortex slices, the inhibition of aspartate aminotransferase activity was greater than the inhibition of oxygen consumption. Structural analogs of β‐methyleneaspartate (D,L‐β‐methyl‐D,L‐aspartate, ‐γ‐methyl‐D,L‐glutamate, and α‐methyl‐D,L‐didehydroglutamate) that did not inhibit the activity of aspartate aminotransferase similarly did not inhibit the rate of oxygen consumption by cerebral cortex slices. In the presence of β‐methyleneaspartate, pyruvate oxidation by cerebral cortex slices was inhibited to almost the same extent as was glucose oxidation, and the oxidation of succinate was decreased by approximately 20%. The artificial electron acceptor phenazine methosulfate (0.1 mM) only partially overcame the β‐methyleneaspartate‐mediated inhibition of respiration with glucose as substrate. The content of ATP and phosphocreatine declined steadily in slices incubated with glucose and β‐methyleneaspartate. At 1 h the concentration of lactate and the lactate/ pyruvate ratio, an indicator of the cytoplasmic redox state, increased threefold, whereas the concentrations of malate, citrate, and aspartate decreased. The findings are interpreted in the context of the hypothesis that enzymes common to the malate‐aspartate shuttle and the tricarboxylic acid cycle are physically complexed in brain, so that inhibition of aspartate aminotransferase, a component of the complex, impedes the flow of carbon through both metabolic pathways. The operation of the malateaspartate shuttle may provide a link between cerebral glycolysis (a continued need for NAD+) and the tricarboxylic acid cycle (supply of oxaloacetate) that is vulnerable to several metabolic disturbances that impair brain function.