Uptake of Amino Acids by Brain Microvessels Isolated from Rats after Portacaval Anastomosis

Abstract: The uptake of amino acids by microvessels isolated from brains of rats was studied. Previous studies have demonstrated alterations in blood-brain amino acid transport after portacaval shunt in rats. In order to elucidate whether such changes in the blood-brain barrier were located in the microvessels, brain microvessels were isolated from both rats with portacaval shunt and controls. Brain microvessels from rats 2 weeks after shunt operations took up significantly greater amounts of ¹⁴C-labeled neutral amino acids, but not of glutamic acid. lysine, or α-methylaminoisobutyric acid than microvessels from sham-operated controls. Measurement of uptake kinetics showed a higher V _max for phenylalanine and leucine uptake and a lower V _max for lysine uptake in microvessels from shunted rats compared with control, whereas the respective K _m's of uptake were similar in both preparations. The results suggest that changes in brain microvessel transport activity account for altered brain neutral amino acid concentrations after portacaval shunt and that such changes can be studied in vitro in isolated microvessels.     

Effects of Dietary Amino Acids on Brain Amino Acids and Transmitter Amines in Rats with a Portacaval Shunt

The etiologic relationship between disturbances in metabolism of amino acids and amines and hepatic coma was investigated by examining the effects of diets containing various mixtures of amino acids on brain amine metabolism in rats with a portacaval shunt, using a method for simultaneous analysis of amino acids and amines. Rats with a portacaval shunt were fed on four different amino acid compositions with increased amounts of various amino acids suspected to be etiologically related to hepatic coma, such as methionine, phenylalanine, tyrosine, and tryptophan. The animals were killed 4 weeks after operation. During the experimental period, these animals did not become comatose, but exhibited various behavioral abnormalities. Marked increase in the plasma and brain levels of the augmented amino acids, especially methionine and tyrosine, were observed in rats with a portacaval shunt. Brain noradrenaline, dopamine, and serotonin levels were significantly decreased when the brain tyrosine level was increased. These results indicate that in rats with a portacaval shunt the dietary levels of amino acids greatly influence the brain levels of both amino acids and transmitter amines.     

Failure of Glucose and Branched-Chain Amino Acids to Normalize Brain Glucose Use in Portacaval Shunted Rats

Several abnormalities in brain and plasma amino acid concentrations caused by portacaval shunting in rats return toward normal after 4 days of intravenous infusion with either glucose or glucose with branched-chain amino acids. To assess the effect of such treatment on brain energy metabolism, regional brain glucose use was measured using [14C]glucose and autoradiography, 5 weeks after portacaval shunting. In one experiment intravenous glucose or glucose with branched-chain amino acids was given for 4 days. In a separate experiment the treatment was given orally for 2 weeks, and in addition to glucose use, brain monoamines and amino acids were measured. No other food was provided; the rats had free access to water. Normally fed shunted rats and sham-operated rats served as controls. Both types of oral treatment lowered the high concentrations of tyrosine, phenylalanine, and glutamine in plasma and brain. Glucose without amino acids normalized brain tryptophan. Levels of brain norepinephrine, 5-hydroxytryptamine (serotonin), and 5-hydroxyindoleacetic acid were significantly raised after shunting. Treatment had no effect on norepinephrine but the glucose diet brought the indoles into the normal range. In contrast, neither intravenous nor oral treatment affected brain glucose use, which remained depressed by 25-30% in all brain areas examined.     

Brain Tryptophan Hydroxylation in the Portacaval Shunted Rat: A Hypothesis for the Regulation of Serotonin Turnover In Vivo

Regional and whole-brain tryptophan-hydroxylating activity and serotonin turnover were investigated in portacaval shunted (PCS) rats using an in vivo decarboxylase inhibition assay. To saturate tryptophan hydroxylation with amino acid substrate, rats were administered a high dose of tryptophan 1 h prior to analysis of brain tryptophan, 5-hydroxytryptophan, serotonin, and 5-hydroxyindoleacetic acid. The analysis revealed, as expected, higher brain concentrations of tryptophan and 5-hydroxyindoles and increased serotonin synthesis rate in PCS rats as compared with shamoperated controls. Saturating levels of brain tryptophan were achieved in both PCS and sham animals after exogenous tryptophan administration. The tryptophan load resulted in increased brain serotonin turnover in all regions and in whole brain compared with rats that did not receive a tryptophan load. Tryptophan-loaded PCS rats showed increased brain serotonin turnover compared with tryptophan-loaded sham rats. Regionally, this supranormal tryptophan-hydroxylating activity was most pronounced in the mesencephalon-pons followed by the cortex. It is concluded that, at least in the PCS rat, brain tryptophan hydroxylation is an inducible process. Since it is known that brain tissue from PCS rats undergoes a redox shift toward a reduced state and that the essential cofactor tetrahydrobiopterin is active in tryptophan hydroxylation only when present in its reduced form, it is hypothesized that this is the reason for the supranormal tryptophan-hydroxylating activity displayed by the PCS rats. The hypothesis further suggests that alterations in tetrahydrobiopterin availability may serve as a mechanism by which brain tryptophan hydroxylation, and therefore serotonin turnover, can be regulated with high sensitivity in vivo.     

Metabolic Abnormalities and Grade of Encephalopathy in Acute Hepatic Failure

Abstract: Acute hepatic failure is associated with many biochemical abnormalities in plasma and brain. Changes that correlate well with the degree of behavioral impairment may be important factors in the development of encephalopathy. We measured the concentrations of intermediary metabolites, ammonia, and amino acids in brain and plasma and the rate of whole-brain glucose utilization in rats with an acutely devascularized liver. In all rats an estimate of the grade of encephalopathy (reflected by behavioral impairment) was made. Rats underwent portacaval shunting and hepatic artery ligation (or sham operation) and were kept normoglycemic and normothermic thereafter. We sampled blood and whole brain (by near-instantaneous freeze-blowing) 2, 4, or 6 h later. There were no alterations in levels of high-energy phosphate metabolites in the brain or in metabolites associated with the glycolytic pathway and Krebs cycle, except lactate and pyruvate. Brain glucose use was decreased similarly at all times after surgery. Levels of ammonia and many amino acids were increased in brain and plasma; brain aspartate, glutamate, and arginine levels were decreased. The increases in content of plasma ammonia and brain glutamine, proline, alanine, and aromatic amino acids and the decreases in brain aspartate and glutamate were most strongly correlated with behavioral impairment.     

Long-Term Effects of Portacaval Anastomosis on the 5-Hydroxytryptamine, Histamine, and Catecholamine Neurotransmitter Systems in Rat Brain

Abstract: Portacaval anastomosis (PCA) in the rat is used as a model for portal systemic encephalopathy. Changes in the serotonergic, histaminergic, and catecholaminergic neurotransmitter systems are often found shortly after PCA. We have examined the long-term effects of PCA on the aminergic systems in brains of male Wistar rats, which 8 months previously had been subjected to PCA. Precursors, amines, and metabolites were assayed by HPLC. Eight months after PCA, the catecholamine levels were unchanged in all brain regions. In contrast, tryptophan was evenly increased throughout the brain. The accumulation of 5-hydroxytryptophan after decarboxylase inhibition (NSD-1015; 100 mg/kg i.p.) and the endogenous levels of 5-hydroxyindoleacetic acid were significantly higher in PCA rats, particularly in the hypothalamus and midbrain, whereas 5-hydroxytryptamine concentrations were unchanged. Histamine levels were elevated throughout the brain with the greatest increase found in the hypothalamus and in the striatum. tele -Methylhistamine levels were significantly elevated in cortex and hypothalamus. We conclude that 8 months after PCA, catecholaminergic systems had reestablished their homeostasis, whereas serotonergic and histaminergic systems still show profound disturbances in their function. With histamine, this is reflected as an increase in the amounts of both transmitter and metabolite; serotonergic neurons respond by increasing only the level of the metabolite.     

Variables Influencing the Effect of a Meal on Brain Tryptophan

Previous work from our laboratory points to plasma free tryptophan being a useful predictor of brain tryptophan concentration in many circumstances. Other work, in particular various studies on the acute effects of food intake, has emphasized the roles of plasma total tryptophan and of plasma large neutral amino acids that compete with tryptophan for transport to the brain. We have now studied associations between the above variables under different dietary conditions. Rats were allowed to feed for restricted periods during a 12-h light-12-h dark cycle. In the first study, rats were given access to a carbohydrate diet for 2 h midway through the light cycle and following an 18-h fast. The resultant rise of brain tryptophan was explicable largely by the associated fall in large neutral amino acids. In a second study, rats were adapted to a regimen whereby they were allowed access to the standard laboratory diet for 4 h during the dark cycle for 3 weeks. A postprandial decrease in brain tryptophan was associated with a fall in free tryptophan and of its ratio to competing amino acids. The brain change could be attributed neither to changes in plasma total tryptophan (which increased) nor to changes of its ratio to the competers (which remained unchanged). Results as a whole are thus consistent with changes of plasma free tryptophan and large neutral amino acid concentrations affecting brain tryptophan concentration under different dietary circumstances. It is suggested that these influences serve to maintain brain tryptophan when dietary supplies are defective.     

Brain Uptake of L-Tryptophan and Diazepam: The Role of Plasma Protein Binding

The brain uptake index (BUI) of L-tryptophan and diazepam into the right and left hemispheres and the cerebellum has been measured after a bolus injection into the carotid artery of the anaesthetised rat. The effect of a range of albumin concentrations (38 microM to 1.4 mM; 0.25-9 g/100 ml) on the viscosity and osmotic pressure of the bolus was studied as a preliminary to the brain uptake experiments. Dextran (Mr 60,000-90,000) was included in the injection to ensure constant viscosity and osmotic pressure. An increase in albumin concentration up to 2 g/100 ml substantially reduced the BUI of L-tryptophan, but a further increase in albumin concentration up to 9 g/100 ml resulted in only a slow fall in the BUI of L-tryptophan which was not proportional to the larger fall in the concentration of unbound L-tryptophan. Furthermore, the use of norharmane as an inhibitor of L-tryptophan binding did not reveal a simple relationship between its unbound concentration and BUI. A decrease in the unbound concentration of diazepam also reduced its BUI, but again there was no straightforward relationship between this and unbound diazepam concentration. The differences observed in the BUI of inulin from solutions of either dextran or albumin indicate not only that each macromolecule may exert particular effects on the BUI, perhaps by an influence on cerebral blood flow, but also show how difficult it is to devise solutions for injection which differ in respect of only one variable, that of the unbound ligand concentration.     

Dialytrode Technology and Local Profiles of Amino Acids in the Awake Cat Brain

Methods to investigate the in vivo effects and release of neuroactive substances include cortical cups, push-pull cannulae, chemitrodes, and dialytrodes. Critical evaluation of these procedures is necessary in order to interpret related results and to select the most suitable devices for further studies. Recent improvements in the dialytrode include structural modifications and the use of a small, permeable membrane constructed of thin polyester. The dialytrode system and its diffusion rates have been characterized with in vitro studies. In vivo long-term experiments in awake cats have been conducted to test injection rates, diffusion of [14C]urea, temporal variability, pressure factors, and other experimental variables. Using dialytrodes we have measured the normal profile of amino acids present in different cerebral structures and their possible correlations.     

Effect of Jejunoileal Bypass on Plasma and Brain Amino Acids in the Rat

Abstract: Liver failure and coma are serious complications of Jejunoileal bypass (JIB) in man. Rats underwent either a 90–95% JIB or a sham operation. Six weeks later all animals were sacrificed, and plasma and brain amino acids were determined. In the plasma of rats with JIB compared with sham operation, the concentrations of valine, leucine, isoleucine, lysine, tryptophan, and tyrosine were significantly lower, while in the brain, phenylalanine, tyrosine, histidine, and glutamine were significantly higher. These changes in the brain are similar to those resulting from portalsystemic shunting in the rat.     

Tryptophan Overload in the Pregnant Rat: Effect on Brain Amino Acid Levels and In Vitro Protein Synthesis

The concentration of most amino acids was higher in the brains of 19- and 21-day rat fetuses than in their respective mothers. After an intraperitoneal load of tryptophan to the mother, the intracerebral concentration of several amino acids (including leucine) decreased not only in the mothers, but also in their fetuses. The in vitro incorporation of pHJleucine into proteins in brain postmitochondrial supernatant fractions was enhanced in both the mothers and fetuses after tryptophan administration, but this effect disappeared when protein synthesis was calculated by using specific activities corrected for the amount of unlabeled leucine in the preparation. By this criterion, protein synthesis activity appeared similar in the brains of 19- and 21-day pregnant rats but was higher in their fetuses, especially in the 21-day subjects. Thus, protein synthesis in the brain was not altered by marked changes in the amino acid pool and more profound and prolonged metabolic disturbances must occur to cause permanent damage in the developing brain.     

Effect of Ammonia on Brain Serotonin Metabolism in Relation to Function in the Portacaval Shunted Rat

Four weeks following portacaval anastomosis (PCA) in the rat, severe liver atrophy, sustained hyperammonemia, and increased plasma and brain tryptophan are observed. Administration of ammonium acetate (NH4Ac) to rats with PCA precipitates severe signs of hepatic encephalopathy (HE) (loss of righting reflex progressing to loss of consciousness and ultimately deep coma). To evaluate the relationship between the deterioration of neurological status in HE and serotonin (5-HT) metabolism, the levels of 5-HT, its precursor 5-hydroxytryptophan, and its major metabolite 5-hydroxy-indole-3-acetic acid (5-HIAA) were measured by HPLC with ion-pairing and electrochemical detection in three well-defined areas of the cerebral cortex: anterior cingulate, piriform and entorhinal, and frontoparietal; as well as in the caudate-putamen, the raphe nuclei, and the locus ceruleus in rats with PCA at different stages of HE, before and after injection of NH4Ac, as well as in sham-operated controls. The results demonstrate increased 5-HIAA/5-HT ratios after PCA and NH4Ac loading, suggesting increased 5-HT turnover in the brains of these animals. However, these changes do not appear to be related to the precipitation of coma as no significant difference in 5-HT turnover was observed between precoma and coma stages of HE. Increased 5-HT turnover in brain of shunted rats may be related to early symptoms of HE such as altered sleep patterns and disorders of motor coordination.     

Amino Acids in Plasma and CSF and Monoamine Metabolites in CSF: Interrelationship in Healthy Subjects

Plasma and cerebrospinal fluid (CSF) concentrations of amino acids were measured in 65 healthy volunteers (50 men and 15 women). The CSF levels of the monoamine metabolites homovanillic acid (HVA), 3-methoxy-4-hydroxyphenylethylene glycol (MOPEG), and 5-hydroxyindoleacetic acid (5-HIAA) were also determined. Sex differences were observed in both plasma and CSF amino acid levels as well as in the relationship between these concentrations. No significant correlations were observed between the CSF levels of HVA and 5-HIAA, and the concentrations of their precursor amino acids in either plasma or CSF. The MOPEG level in CSF correlated positively with the plasma concentrations of several amino acids.     

Portacaval Anastomosis Results in Altered Neuron-Astrocytic Metabolic Trafficking of Amino Acids: Evidence from 13C-NMR Studies

Abstract: ¹³C-NMR spectroscopy was used to evaluate the dynamic consequences of portacaval anastomosis on neuronal and astrocytic metabolism and metabolic trafficking between neurons and astrocytes. Glutamate is predominantly labeled from [1-¹³C]glucose, whereas [2-¹³C]acetate is more efficient in labeling glutamine, in accordance with its primary metabolism in astrocytes. Alanine and succinate labeling was only observed with [1-¹³C]glucose as precursor. Brain [1-¹³C]glucose metabolism in portacaval-shunted rats was similar to that in sham-operated controls with the exception of labeled glutamine and succinate formation, which was increased in shunted rats. The ¹³C enrichment was, however, decreased owing to an increase in total glutamine and succinate. Using [2-¹³C]acetate, on the other hand, flux of astrocytic label to neurons was severely decreased because label incorporation into glutamate, aspartate, and GABA was decreased following portacaval shunting. The latter amino acids are predominantly localized in neurons. These findings demonstrate that metabolic trafficking of amino acids from astrocytes to neurons is impaired in portacaval-shunted rats.     

Mass Fragmentographic Method for the Determination of Trace Amounts of Putative Amino Acid Neurotransmitters and Related Compounds from Brain Perfusates Collected In Vivo

Abstract: A mass fragmentographic method for the determination of trace amounts of amino acid neurotransmitter candidates from brain perfusates is described. The analytical procedure includes the measurements of glycine, β-alanine, γ-aminobutyric acid, proline, aspartic acid, and glutamic acid; αalanine, leucine, and sarcosine, undergoing gas chromatographic coelution, are detected simultaneously. Amino acids extracted from dried perfusate residues are converted to the corresponding N -pentafluoropropionyl hexafluoroisopropyl esters by a single-step procedure. Gas chromatographic separation of the amino acid derivatives is achieved on a packed glass column filled with trifluoropropylsilicone as stationary phase. The limit of detection for the different derivatives (signal-to-noise, 3:1) ranges from 50 femtomol to 1 picomol. Deuterium-labeled amino acid analogues are used as internal standards for quantitative measurements. The mass spectral characteristics of the derivatives are compared and discussed. The technique has been applied to the assay of amino acids released in vivo within the pigeon optic tectum, demonstrating the capabilities of the present analytical approach.     

Amino Acid Analysis Demonstrates that Increased Plasma Free Tryptophan Causes the Increase of Brain Tryptophan During Exercise in the Rat

Rats were trained to run on a horizontal treadmill for 2 h at 20 m/min. This activity considerably increased plasma free tryptophan (TRP) (+70%) but did not alter plasma total TRP levels and had little or no effect on plasma concentrations of the other large neutral amino acids (LNAAs) that compete with TRP for entry into the brain. Brain TRP levels increased by 80%. The only other brain LNAA to be affected by exercise was threonine, which rose moderately. The results indicate that increased plasma free TRP was specifically responsible for the increase of brain TRP after 2 h of exercise. Brain lysine was also increased whereas glycine, alanine, and gamma-aminobutyric acid were decreased. The differences between the present findings and those previously obtained following 2 h immobilization stress are discussed.     

Diversity of Metabolic Patterns in Human Brain Tumors: Enzymes of Energy Metabolism and Related Metabolites and Cofactors

Biopsies from 15 human gliomas, five meningiomas, four Schwannomas, one medulloblastoma, and four normal brain areas were analyzed for 12 enzymes of energy metabolism and 12 related metabolites and cofactors. Samples, 0.01-0.25 microgram dry weight, were dissected from freeze-dried microtome sections to permit all the assays on a given specimen to be made, as far as possible, on nonnecrotic pure tumor tissue from the same region. Great diversity was found with regard to both enzyme activities and metabolite levels among individual tumors, but the following generalities can be made. Activities of hexokinase, phosphorylase, phosphofructokinase, glycerophosphate dehydrogenase, citrate synthase, and malate dehydrogenase levels were usually lower than in brain; glycogen synthase and glucose-6-phosphate dehydrogenase were usually higher; and the averages for pyruvate kinase, lactate dehydrogenase, 6-phosphogluconate dehydrogenase, and beta-hydroxyacyl coenzyme A dehydrogenase were not greatly different from brain. Levels of eight of the 12 enzymes were distinctly lower among the Schwannomas than in the other two groups. Average levels of glucose-6-phosphate, lactate, pyruvate, and uridine diphosphoglucose were more than twice those of brain; 6-phosphogluconate and citrate were about 70% higher than in brain; glucose, glycogen, glycerol-1-phosphate, and malate averages ranged from 104% to 127% of brain; and fructose-1,6-bisphosphate and glucose-1,6-bisphosphate levels were on the average 50% and 70% those of brain, respectively.     

Effect of Altered Blood Plasma Osmolalities on Regional Brain Amino Acid Concentrations and Focal Seizure Susceptibility in the Rat

Blood plasma hypo- or hyperosmolality alters significantly the concentration of some amino acids in brain tissues of the medial septum and hippocampus of adult Sprague-Dawley rats. With some notable exceptions, brain amino acid concentrations decreased under hypoosmotic conditions and increased under hyperosmotic conditions. Osmotic changes and brain amino acid changes appear to be related to each other in an almost linear fashion. A comparison of rats and toads indicates that the patterns of changes in brain amino acid concentrations in response to a hypoosmotic plasma osmolality were almost identical for both species. Changes achievable under hyperosmotic conditions were considerably greater in toads. When rats with kindled epileptogenic foci were made hypoosmotic by water-loading, seizure thresholds decreased dramatically. Our data suggest a possible relationship between the hypoosmotically induced biochemical changes in brain tissues (especially some amino acid neurotransmitters and neurotransmitter precursors) and the hypoosmotically induced increase in seizure susceptibility.     

Developmental Changes in the Neutral α-Amino Acid Transport Systems of Rat Brain Over the First Three Weeks After Birth

Abstract: Transport of seven different amino acids into brain slices increased as donor rats aged from 1 to 6 days. Uptakes of 2-aminoisobutyric acid, 2-(methyl-amino)isobutyric acid, and L-alanine then decreased by day 14, while uptakes of other amino acids continued to increase or remained fairly constant. Neutral α-amino acid transport systems were characterized by measuring inhibition of uptakes and kinetics for representative amino acids at different ages. Results indicate that 2-ami-noisobutyrate and 2-(methylamino)isobutyrate used only one (and the same) system in brain slices from 6-day-old rats, with characteristics of system A (the major sodium-dependent system in most mammalian cells). They used at least two systems at ages 1, 14, and 23 days, but, of these, only at 1 day did they use the same systems in the same proportions. Alanine and leucine used more than one system at all four ages, and somewhat different combinations than used by each other or by 2-aminoisobu-tyrate or 2-(methylamino)isobutyrate. Their transport characteristics showed they used mostly system ASC (a sodium-dependent system distinguished from A) and/or system L (sodium-independent). We conclude that system A increases as the brain ages from 1 to 6 days and declines thereafter. System L probably increases with aging from 1 to 23 days.     

A Comparison of the Distribution of Neurotransmitters in Brain Regions of the Rat and Guinea-Pig Using a Chemiluminescent Method and HPLC with Electrochemical Detection

Six brain areas of rats and guinea-pigs, killed by microwave irradiation, were used for the concomitant measurement of the levels and regional distribution of cholinergic, biogenic amine, and amino acid neurotransmitters and metabolites. Acetylcholine (ACh) and choline (Ch) were quantified by chemiluminescence; noradrenaline (NA), dopamine (DA), 5-hydroxytryptamine (5-HT), and their metabolites by HPLC with electrochemical detection (HPLC-EC); and six putative amino acid neurotransmitters by HPLC-EC following derivatisation. The levels and regional distribution of these transmitters and their metabolites in the rat were similar to those reported in previous studies, except that biogenic amine transmitter levels were higher and metabolite concentrations were lower. The guinea-pig showed a similar regional distribution, but the absolute levels of ACh were lower in striatum and higher in hippocampus, midbrain-hypothalamus, and medulla-pons. In all areas, the levels of Ch were higher and those of NA, 5-HT, and taurine were lower than in the rat. The most marked differences between the rat and guinea-pig were in the relative proportion of DA metabolites and 5-HT turnover, as estimated by metabolite/transmitter ratios. This study can be used as a basis for a comprehensive understanding of the central effects of drugs on the major neurotransmitter systems.