Reevaluation of Taurine Levels and Distribution of Cysteic Acid Decarboxylase in Developing Human Fetal Brain Regions

The possibilities of interference by glycerophosphoryl ethanolamine (GPE) in the estimation of taurine levels in cerebral cortex, midbrain, cerebellum, medullapons, and spinal cord of developing human fetal brain regions were eliminated by hydrolyzing tissue extracts with 6 M HCl. Cysteic acid thus produced was separated from taurine by ion-exchange chromatography using Biorad-AG resin. Fluorescamine was used as fluorogen. Data reveal that the estimation of taurine in human fetal brain regions is affected if GPE is present as a contaminant in the assay system. Cysteic acid decarboxylase activity was measured using cysteic acid as the substrate. Higher enzymic activity was recorded with increased fetal body weight, but the reverse was true for taurine level.     

Determination of amino acids in different regions of the rat brain. Application to the acute effects of tetrahydrocannabinol (THC) and trimethyltin (TMT)

A modified HPLC method is described for the determination of amino acids [aspartic acid, glutamic acid, glutamine, glycine, taurine, and gamma-aminobutyric acid (GABA)] in brain tissue utilizing precolumn derivatization with o-phthalaldehyde (OPA)-tert-butyl-thiol and electrochemical detection. A simple extraction procedure was employed and DL-homoserine used as internal standard. A neurotoxin previously shown to affect brain amino acids (trimethyltin, TMT) and a psychoactive compound hypothesized to act on these neurochemicals (delta-9-tetrahydrocannabinol, THC) were administered to adult male rats and amino acids were measured. Results revealed a gradient of distribution of most amino acids, with lowest levels posteriorly in the brain stem and increasing to the highest values in anterior cortical regions. TMT increased glutamine significantly in all brain regions examined, but increased glycine and decreased taurine only in the frontal cortex and hippocampus. No significant changes in any amino acid were found in hippocampus after THC treatment. The results establish the validity and usefulness of this HPLC method for detecting neurotoxicity-related changes in brain amino acid metabolism.     

Regional Amino Acid Distribution in Relation to Function in Insulin Hypoglycaemia

The amino acids glutamate, aspartate, gamma-aminobutyric acid (GABA), and glutamine were measured as their dansyl derivatives in whole brain and specific brain regions by a sensitive double-labelling technique at various times during the development of hypoglycaemic encephalopathy. Hypoglycaemia was induced by administration of insulin (100 i.u./kg) to 24-h fasted rats. No significant changes in glutamate, GABA, or glutamine were detected in whole brain at any time up to and including the onset of hypoglycaemic convulsions. In cerebral cortex, however, GABA levels were reduced to 65% or normal prior to the appearance of neurological symptoms of hypoglycaemia. Onset of symptoms (severe catalepsy and loss of righting reflex, but before the onset of convulsions) was accompanied by marked decreases of glutamate and glutamine in striatum and hippocampus. These regions, in addition to cerebral cortex, show the greatest vulnerability to hypoglycaemic insult, according to previous anatomical studies. Aspartate levels were significantly increased (p less than 0.01) in the cerebral cortex of convulsing animals, confirming a previous report. No changes were detectable in any of the amino acids studied in medulla-pons at any time during the progression of hypoglycaemia. Cerebral cortex and striatum showed a selective net loss of amino acids (2.2 and 3.5 mumol/g. respectively) prior to the onset of insulin-hypoglycaemic convulsions.     

High-Affinity Transport of γ-Aminobutyric Acid, Glycine, Taurine, L-Aspartic Acid, and L-Glutamic Acid in Synaptosomal (P2) Tissue: A Kinetic and Substrate Specificity Analysis

In a cortical P2 fraction, [14C]gamma-aminobutyric acid ([14C]GABA), [14C]glycine, [14C]taurine, and [14C]glutamic and [14C]aspartic acids are transported by four separate high-affinity transport systems with L-glutamic acid and L-aspartic acid transported by a common system. GABA transport in cortical synaptosomal tissue occurs by one high-affinity system, with no second, low-affinity, transport system detectable. Only one high-affinity system is observed for the transport of aspartic/glutamic acids; as with GABA transport, no low-affinity transport is detectable. In the uptake of taurine and glycine (cerebral cortex and pons-medulla-spinal cord) both high- and low-affinity transport processes could be detected. The high-affinity GABA and high-affinity taurine transport classes exhibit some overlap, with the GABA transport system being more specific and having a much higher Vmax value. High-affinity GABA transport exhibits no overlap with either the high-affinity glycine or the high-affinity aspartic/glutamic acid transport class, and in fact they demonstrate somewhat negative correlations in inhibition profiles. The inhibition profiles of high-affinity cortical glycine transport and those of high-affinity cortical taurine and aspartic/glutamic acid transport also show no significant positive relationship. The inhibition profiles of high-affinity glycine transport in the cerebral cortex and in the pons-medulla-spinal cord show a significant positive correlation with each other; however, high-affinity glycine uptake in the pons-medulla-spinal cord is more specific than that in the cerebral cortex. The inhibition profile of high-affinity taurine transport exhibits a nonsignificant negative correlation with that of the aspartic/glutamic acid transport class.     

FREE AMINO ACIDS AND RELATED COMPOUNDS IN BIOPSIES OF HUMAN BRAIN

Abstract— Contents (μmol/g wet wt.) of 35 free amino acids and related compounds were measured in biopsies of human brain from ten patients. Brain specimens were frozen in liquid nitrogen within 10 sec of their removal at neurosurgery; thus, the values found should approximate those which occur in living brain.
Levels in free pools of biopsied cerebral cortex of most of the amino acids that are constituents of proteins were only 20-50 per cent of those found in autopsied cortex. The content of cystine and ethanolamine was much lower in biopsied than in autopsied cortex. Concentrations of GABA in biopsied cortex were only 20 per cent as high as those found in autopsied cortex, and levels of γ-aminobutyryl dipeptides were also significantly lower in biopsied cortex. Amounts of cystathionine in biopsied cortex varied markedly, but averaged much higher than in autopsied cortex; a single biopsy specimen of cerebellar grey matter had a cystathionine content 36-fold greater than the mean found in autopsied cerebellum.
Appreciable variability in contents among cortical biopsies was found for glycerophosphoethanolamine, phosphoethanolamine, ethanolamine, taurine, aspartic acid, glutamic acid, glutamine, and GABA, as well as for cystathionine. Whether this variability occurred between different subjects, or between different cortical areas, was not clear, although the former possibility was suggested by findings in multiple cortical biopsies from one patient.     

Effects of dorsal root section and occlusion of dorsal spinal artery on the neurotransmitter candidates in rat spinal cord

In order to obtain further evidence of putative neurotransmitters in primary sensory neurons and interneurons in the dorsal spinal cord, we have studied the effects of unilateral section of dorsal roots and unilateral occlusion of the dorsal spinal artery on cholinergic enzyme activity and on selected amino acid levels in the spinal cord. One week after sectioning dorsal roots from caudal cervical (C₇) to cranial thoracic (T₂) levels, the specific activity of choline acetyltransferase (ChAT) was significantly decreased and acetylcholinesterase (AChE) showed a tendency to decrease in the dorsal quadrant on the operated side of the spinal cord. Dorsal root sectioning had little effect on the levels of free glutamic acid or other amino acids in the dorsal spinal cord. These results suggest that primary sensory neurons may include some cholinergic axons, and that levels of putative amino acid transmitters are not regulated by materials supplied by axonal transport from the dorsal root ganglia. By contrast, one week following unilateral occlusion of the dorsal spinal artery, the activities of ChAT and AChE were unchanged in the operated quadrant of the spinal cord, while decreases of Asp, Glu, and GABA, and an increase in Tau were detected. These findings are consistent with the proposals that such amino acids, but not ACh, may function as neurotransmitter candidates in interneurons of the dorsal spinal cord.Abbreviation used ACh acetylcholine - AChE acetylcholinesterase - Asp aspartic acid - ChAT choline acetyltransferase - GABA -aminobutyric acid - Glu glutamic acid - Gly glycine - SP substance P - Tau taurine     

Caffeine-induced changes in the composition of the free amino acid pool of the cerebral cortex

The free amino acid content in the cerebral cortex of rats administered caffeine orally, and with automutilation behavior similar to that observed in the Lesch-Nyhan syndrome, have been measured. Amino acids significantly elevated were taurine, histidine and aspartic acid, whereas tyrosine showed a significant reduction. There was no change in the concentration of -aminobutyric acid and glutamic acid. It has been conjectured that changes in amino acids levels in the cortex might be responsible for the pharmacological action of caffeine and for the progressive behavior abnormalities observed in these rats. Interestingly these results are similar to these found recently in experimental uremia.     

Pyruvate Dehydrogenase Activity in Regions of the Rat Brain During Postnatal Development

Abstract: Activity of the pyruvate dehydrogenase complex (PDHC) was measured in seven brain regions of themale rat at various times during the postnatal period usingan arylamine acetyltransferase coupled assay. Three daysafter birth, PDHC activity was found to be < 15% ofadult values in all brain regions with the exception of hypothalamus and medulla-pons (30% of adult values ineach case). Activity of the enzyme complex in these latterregions attained adult levels by 21 days postnatally, some 5-15 days ahead of that found in cerebral cortex, striatum, hippocampus, and cerebellum. Such differences in PDHC maturation reflect the greater degree of earlymaturity of the phylogenetically older brain structures. Cerebellar PDHC developed more slowly than in otherbrain regions to attain only 40% of adult levels by thetime of weaning. The pattern of maturation of cerebellarPDHC is paralleled by increased incorporation of glucoseinto cerebral amino acids and by the pattern of develop-ment of parallel fiber synaptogenesis. These findings sug-gest that PDHC may play a key role in the regional de-velopment of metabolic compartmentation and the asso-ciated maturation of cerebral function in the rat.     

Excess of taurine supplementation in rat: effects on GABA-related amino acids in developing nervous tissues.

The effects of taurine supplementation on GABA-related amino acid homeostasis in developing nervous tissues of suckling rats were studied. In the first two weeks of postnatal growth, cerebral cortex and cerebellum appear more accessible to taurine supplementation in comparison to retina; in addition, different changes in excitatory/inhibitory amino acids were observed. After the 5th day of life, in the retina and cerebellum of taurine-supplemented pups a decrease in GABA levels was found; in contrast, in cerebral cortex GABA content significantly increased throughout 20 days of postnatal growth. In all nervous tissues studied (except for cerebellum) glutamine concentration increased at the 5th day; then in cerebellum and in retina, but not in cerebral cortex, a significant decrease until the 20th day occurred. Furthermore, in cerebellum and retina taurine supplementation decreased glutamate levels, in comparison to controls, at the 10th and until the 20th day of postnatal life, respectively, whereas in cerebral cortex an increase in glutamate level was observed only at the 5th day. In conclusion, taurine supplementation, in excess to the usual amount from the mother's milk, affected the glutamate compartments in various cell types. The changes in GABA-related amino acid concentrations in cerebral cortex, cerebellum, and retina may depend on the different pattern of the metabolic processes at different maturative stages.     

Lithium: Effect on [3H]spiperone binding,ionic content,and amino acid levels in the brain of rats

After prolonged treatment of rats with lithium (pellets, 0.21% lithium carbonate, or 0.5 mg/ml lithium chloride in drinking water) for three months, the level of lithium in plasma was 0.87 meq/liter; in several brain regions, between 1.06–1.39 eq/g wet weight. The content of sodium and potassium in the plasma was normal. The level of potassium in the brain regions tested increased by 13–30% and that of sodium by about 10%. Glycine levels increased significantly in all the regions (cerebral cortex, midbrain, cerebellum, and spinal cord). In the cerebellum GABA was also increased, while glutamine was decreased. In midbrain, apart from increases in glycine levels, alamine, valine, GABA and lysine were also increased. In the spinal cord, glutamic acid was also increased. Changes were largely in the putative neurotransmitters. Long-term treatment with lithium also influenced the high-affinity binding of [³H]spiperone in the cerebral cortex and corpus striatum. Two specific binding sites were found in both brain regions; the main change was the reduction in the lower affinity binding site (B _max2).     

Topical insulin and accumulation of excitotoxic and other amino acids in ischemic rat cerebral cortex

Insulin plays a neuroprotectant role in the brain and spinal cord during ischemia. However, studies have shown insulin to increase the sensitivity of cultured cortical cells to glutamate toxicity. The present study looked at the relationship between topically administered insulin (1 mIU insulin/ml and 100 mIU insulin/ml) during a four-vessel model of global ischemia and the accumulation of amino acids, especially glutamate, from the ischemic rat cerebral cortex. The lower dose of insulin was found to attenuate the release of excitotoxic and other amino acids from the cortex in ischemia/reperfusion. This may occur because insulin increases glucose availability to glial cells resulting in maintenance of glycolysis and ionic pumps that can reduce glutamate release and maintain uptake during ischemia/reperfusion. The higher dose of insulin, which significantly increased the amount of aspartate, glutamate, taurine, and GABA during reperfusion, may act to stimulate the amount of glycogen stored in astrocytes, reducing the availability of glucose for metabolic purposes.     

Maternal Undernutrition During Lactation: Effect on Amino Acids in Brain Regions of Offspring

Sprague-Dawley dams were fed either a protein-calorie deficient or control diet from day 5 to day 21 after parturition. The concentrations of seven amino acids (aspartate, glutamate, gamma-aminobutyric acid, glycine, glutamine, serine, and taurine) were determined in brain regions from 17-day-old undernourished offspring and from 35-day-old rehabilitated rats. The brain regions examined were the cortex, cerebellum, corpus striatum, hippocampus, hypothalamus, brainstem, and midbrain. At 17 days of age, taurine was the amino acid with the highest concentration, whereas at 35 days glutamate had the highest concentration. This change was due to the fact that the concentration of taurine decreased significantly in all brain regions between 17 and 35 days, whereas the concentration of glutamate remained high or increased somewhat in all brain regions except the hypothalamus and brainstem. When the age-matched offspring of control and undernourished rats were compared, several interesting and significant differences were found. The concentrations of glutamate and aspartate were significantly lower (decreased 16-34%) in the cerebellum, brainstem, cortex, and midbrain in 17-day-old undernourished rats. The aspartate level was also significantly decreased in the corpus striatum and hypothalamus in 17-day-old offspring. However, the deficiencies of aspartate and glutamate were transient and reversible. In contrast, the concentration of taurine was increased in the hypothalamus (31%) and hippocampus (12-33%) at both 17 and 35 days of age and in the midbrain (17%) at 17 days. Other transient abnormalities in amino acid levels were found in undernourished offspring. The results of these experiments suggest that undernutrition during lactation causes delayed CNS development, which is manifested in altered concentrations of the neurotransmitters aspartate, glutamate, and taurine.     

Amino acid changes in regions of the CNS in relation to function in experimental portal-systemic encephalopathy

Sustained hyperammonemia resulting from portocaval anastomosis (PCA) in the rat, is accompanied by neurological symptoms and reversible morphological changes in brain, the nature and distribution of which suggest selective vulnerability of certain brain structures. the present study was initiated to investigate the effects of increasing CNS ammonia on the distribution of amino acids in regions of the rat brain in relation to the degree of neurological impairment in PCA rats. Four weeks following PCA, rats were administered ammonium acetate (5.2 mmol/kg, i.p.) to precipitate neurological symptoms of encephalopathy which included diminished locomotor activity, loss of hindlimb extension and righting reflexes and ultimately coma. At various stages during the development of encephalopathy, rats were sacrificed and the amino acids glutamine, glutamate and aspartate measured simultaneously, using a sensitive double-isotope dansyl microassay. Homogenates of the following regions of the CNS were assayed: cerebral cortex, hippocampus, striatum, midbrain, hypothalamus, cerebellum, medulla-pons, spinal cord (gray matter) and spinal cord (white matter). Sustained hyperammonemia associated with PCA alone resulted in a non-uniform 2–4 fold increase of glutamine in all regions of the CNS. Glutamate, on the other hand, was selectively increased in striatum and cerebellum, two regions of brain shown to exhibit early morphologically-characterised astrocytic abnormalities in rats with PCA. Onset of severe neurological dysfunction was accompanied by significantly decreased glutamine and glutamate in striatum and cerebellum. Thus, sustained hyperammonemia in association with portocaval shunting results in region-selective effects with respect to glutamine-glutamate metabolism in the CNS.     

Regional changes in amino acid content in developing rat brain

Abstract— The content of several amino acids was measured in five discrete regions of the central nervous system of the developing rat, using a [³H]dansylation assay procedure. Both regional differences in amino acid content and regional differences in rate of change of amino acids during maturation were found. Particularly prominent were the maturational changes and high adult contents of glutamic acid in the cerebral cortex, GABA in the hypothalamus, and aspartic acid in the medulla.     

Valproate doubles the anoxic survival time of normal developing mice: possible relevance to valproate-induced decreases in cerebral levels of glutamate and aspartate, and increases in taurine

We have previously reported that chronic administration of valproate in developing mice decreased brain aspartic and glutamic acid levels and increased the brain taurine content. The direction of the valproate-induced changes in the cerebral levels of these neurotransmitter amino acids - excitatory in the case of aspartate and glutamate, inhibitory in the case of taurine - appeared relevant to the mechanism of its anticonvulsant action. Since the neuropathology of hypoxia-ischemia also appears to be mediated by release of glutamate/aspartate at the synapse, the valproate-induced reduction of the levels of these neuroexcitatory/neurotoxic amino acids suggested that valproate might increase the tolerance of young mice to anoxia. A doubling of the length of survival of the intact animal in an atmosphere of pure nitrogen gas and a three-fold increase in the duration of respiratory activity (gasping) of the isolated head after chronic administration of valproate support the speculation.     

Effect of unilateral motor cortex ablation on activity of choline acetyltransferase and levels of amino acid transmitter candidates in the spinal cord of adult monkeys

Evidence thatl-glutamate is a neurotransmitter of corticofugal fibers was sought by measuring changes in several biochemical markers of neurotransmitter function in discrete regions of spinal cord after ablation of sensorimotor cortex in monkeys. One and five weeks after unilateral cortical ablation, samples from six areas of spinal cord (ventral, lateral and dorsal regions of the left and right sides) were analysed for choline acetyltransferase (ChAT) activity and contents of amino acid transmitter candidates-glutamic acid (Glu), aspartic acid (Asp), glycine (Gly), taurine (Tau) and -aminobutyric acid (GABA). During one to five weeks after unilateral cortical ablation of the monkey, prolonged hemiplegia in the contralateral side was observed. Histological examination of the spinal cord 5 weeks after unilateral (left) cortical ablation showed no apparent change in either control (ipsilateral, left) or affected (contralateral, right) sides of the cord as examined by the Klüver-Barrera method. The ChAT activity as a cholinergic marker was scarcely changed in any region of either left (control) or right (affected) side of the spinal cord at one and five weeks after unilateral (left side) ablation of the motor cortex. Amino acid levels in each region of the spinal cord were not significantly changed one week after unilateral ablation of the motor cortex. However, a significant decrease of Glu content was observed in the lateral column of the affected (right) side compared to the control (left) side of cervical and lumbar cord five weeks after cortical ablation of the left motor area. No concomitant alterations of other amino acids were detected. These data strongly suggest thatl-Glu is a neurotransmitter for corticofugal pyramidal tract fibers to anterior horn secondary neurons related to motor control activity in monkey spinal cord.     

POSTNATAL CHANGES IN THE LEVELS OF GLYCINE AND THE ACTIVITIES OF SERINE HYDROXYMETHYLTRANSFERASE AND GLYCINE: 2-OXOGLUTARATE AMINOTRANSFERASE IN THE RAT CENTRAL NERVOUS SYSTEM

Abstract— Of the amino acids found in the CNS of 10-day-old rats the concentration of glycine alone was significantly higher in the spinal cord than in all other regions. Spinal levels of glycine, cystathionine, isoleucine and lysine from 1- and 10-day-old rats did not differ significantly from adult values, whereas the levels of most other amino acids, including GABA, glutamate, glutamine and taurine, were higher in the young animals than in the adults. Aspartate was the only amino acid found in lower concentration in the spinal cord of young animals than in adult animals. These and other observations support the conclusion that glycine is used as an inhibitory transmitter in rat spinal cord early in postnatal life. There was a general decrease in the activity of serine hydroxymethyltransferase and a slight increase in the activity of glycine:2-oxoglutarate aminotransferase in the CNS during development. The activity of neither enzyme correlated on a regional basis with the glycine content. The high level of hydroxymethyltransferase activity in the cerebellum of 10-day-old rats suggests that the activity of this enzyme reflects cell growth rate.     

Effect of transient neonatal hypothyroidism on the free aspartic acid, glutamic acid, and GABA content of different central auditory regions in the rat

The aspartic acid, glutamic acid, and gamma-aminobutyric acid (GABA) contents were determined in four central auditory system regions in rats with transient neonatal hypothyroidism compared with control ones: the ventral and dorsal parts of the cochlear nucleus, the central nucleus of the inferior colliculus, the auditory cortex, and in an extra-auditory structure, the substantia nigra pars reticulata. The animals were sacrificed at 50 days of age, brain tissue samples were taken out by microdissection, and the free amino acids were extracted. The amino acid content was assessed by double-isotope labelling following two-dimensional thin-layer chromatography separation. GABA content was significantly decreased in both cochlear nucleus regions and glutamic acid was elevated in the inferior colliculus. Neonatal hypothyroidism had no significant effect on the aspartic acid levels in the regions studied. The results suggest an effect of neonatal hypothyroidism on regional contents of free amino acids known as candidate neurotransmitters in the auditory system.     

FREE AMINO ACIDS AND RELATED COMPOUNDS IN FIVE REGIONS OF BIOPSIED CAT BRAIN

Abstract— Contents (μmol/g wet wt.) of 34 free amino acids and related compounds were measured in grey matter from three areas of cerebral cortex, from the cerebellum, and from the caudate nucleus in unanaesthetized cats with classical cerveau isolé preparations. Brain specimens were frozen in liquid nitrogen within 10 s of removal; thus, the values found were expected to approximate those which occur in living cat brain. Levels of most of the compounds measured were lower than those previously reported for the cat. In the case of GABA, alanine, and ethanolamine, the lower values found seemed attributable to the rapid freezing of brain tissue, and may more closely approximate levels occurring in living cat brain. On the other hand, the relatively low levels of aspartic and glutamic acids found may have resulted from use of the cerveau isolé preparation. Little difference in levels of amino compounds was found among the three cerebral cortical areas examined. However, there were significant differences in the contents of a number of amino acids between cerebral cortex and the cerebellum or caudate nucleus. These differences resembled those previously observed in autopsied human brain. The content of GABA was two-fold higher in biopsied cat cortex than in biopsied human cortex, whereas the content of cystathionine was only 10 per cent of that in human cortex. Homocarnosine and α-(γ-aminobutyryl)-lysine, two GABA-containing dipeptides found in relatively large amounts in human brain, were not detectable in cat brain. Living cat brain contained two amino acids not previously reported for this species:putreanine and ɛ- N -methyllysine.     

Amino Acid Changes in a Genetic Strain of Epileptic Beagle Dogs

A neurochemical evaluation of beagle dogs with naturally occurring spontaneous generalized convulsive seizures was performed. Amino acid profiles of serum, cerebrospinal fluid (CSF), and biopsied cerebral cortex from epileptic dogs were compared with those from seizure-free siblings. No differences in absolute levels were noted. However, when levels were normalized as a percent of total free amino acids, seizures was performed. Amino acid profiles of serum, cerebrospinal fluid (CEF), and biopsied cerebral cortex from epileptic dogs were compared with those seizure-free siblings. No differences also the two groups differed in certain respects. Ten significant correlations between amino acid pairs appeared in epileptic dogs, but only one was seen in seizure-free animals. Seven of these ten correlations involved glutamate or taurine. It was noted that the highly correlated amino acids (taurine, glutamate, glycine, glutamine, alanine) all utilize sodium-dependent membrane transport processes. The sum of glutamate, aspartate, and glycine levels (competing sodium-dependent high-affinity systems) was significantly lower in epileptic beagles. Since this difference was noted in serum but not CSF or brain, it may indicate a diminished capacity of sodium-dependent high-affinity renal transport for acidic and certain small neutral amino acids.