STUDIES OF THE INTER-RELATIONSHIP BETWEEN CEREBROSPINAL FLUID AND PLASMA AMINO ACID CONCENTRATIONS IN NORMAL INDIVIDUALS

Abstract— —The concentration of free amino acids has been determined in lumbar CSF in 37 fasting normal subjects. The values obtained have been compared with the concentration of the same amino acids measured in venous plasma collected simultaneously and with ventricular CSF amino acid concentrations. Twenty-three amino acids have been identified and quantitated in CSF and plasma. Trace quantities of eight other amino acids have been also detected.
The concentration of 13 amino acids in CSF has been shown to be directly related to the plasma concentration. No such relationship was noted for the other 7 amino acids. Significant variations in the concentration of individual amino acids relating to both age and sex have been noted. A large number of unidentified ninhydrin positive compounds have been found in CSF. Preliminary studies have identified one of these as ɛ-aminobutyric acid (GABA).     

THE FREE AMINO ACIDS OF HUMAN SPINAL FLUID DETERMINED BY ION EXCHANGE CHROMATOGRAPHY

(1) The free amino acids in human CSF from eighteen subjects have been determined. The analyses were performed on 0-75 ml of CSF by an ion exchange chromatographic method which is capable of detection to the 10^?10 mole level. (2) The amino acids always found in readily detectable amounts were: taurine, threonine, serine, glutamine, glutamic acid, citrulline, glycine, alanine, α-NH₂-n- butyric acid, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, ethanolamine, ornithine, lysine, histidine and arginine. Urea was present. Aspartic acid and cystine, though always present, occurred in small or trace amounts. Proline was found in four cases and tryptophan in thirteen cases. In addition, twelve unknown peaks were nearly always evident in every chromatogram. (3) Filtrates 10 times more concentrated than those used regularly were prepared from pooled CSF and analysed. These analyses clearly confirmed the presence of those amino acids which were normally in very low concentration and they also served to distinguish the twelve unknown compounds from confusion with baseline artifacts. (4) The distribution of free amino acids in CSF was different from their distribution in blood plasma. (5) Despite a variety of neurological conditions and a wide age span few marked deviations were found in any of the amino acid concentrations.     

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.     

Pharmacokinetics and cerebral distribution of glycine administered to rats

High doses of glycine have been reported to improve negative schizophrenic symptoms, suggesting that ingested glycine activates glutamatergic transmission via N-methyl-d-aspartate (NMDA) receptors. However, the pharmacokinetics of administered glycine in the brain has not been evaluated. In the present study, the time- and dose-dependent distributions of administered glycine were investigated from a pharmacokinetic viewpoint. Whole-body autoradiography of radiolabeled glycine was performed, and time–concentration curves for glycine and serine in plasma, cerebrospinal fluid (CSF), and brain tissues were obtained. Furthermore, pharmacokinetic parameters were calculated. For a more detailed analysis, the amount of glycine uptake in the brain was evaluated using the brain uptake index method. Radiolabeled glycine was distributed among periventricular organs in the brain. Oral administration of 2?g/kg of glycine significantly elevated the CSF glycine concentration above the ED₅₀ value for NMDA receptors. The glycine levels in CSF were 100 times lower than those in plasma. Glycine levels were elevated in brain tissue, but with a slower time-course than in CSF. Serine, a major metabolite of glycine, was elevated in plasma, CSF, and brain tissue. Glycine uptake in brain tissue increased in a dose-dependent manner. Time–concentration curves revealed that glycine was most likely transported via the blood–CSF barrier and activated NMDA receptors adjacent to the ventricles. The pharmacokinetic analysis and the brain uptake index for glycine suggested that glycine was transported into brain tissue by passive diffusion. These results provide further insight into the potential therapeutic applications of glycine.     

BIOCHEMTCAL CHANGES IN THE BRAIN OF EXPERIMENTAL ANIMALS IN RESPONSE TO ELEVATED PLASMA HOMOCYSTINE AND METHIONINE

The effects of high plasma concentrations of homocystine and methionine on the free amino acids of brain have been examined. Incorporation of the label from [³⁵S]methionine into the free amino acid pools of rabbit brain was enhanced in response to high plasma homocystine or high plasma homocystine and mcthionine. Under comparable conditions a marked decrease in the incorporation of the label from [¹⁴C]glycine into the free pool was observed. The corresponding incorporation of ³⁵S and ¹⁴C into brain proteins parallelled the results obtained with incorporation into the free pools of amino acids. Amino acid analyses of the free amino acid pools of rabbit brain revealed a general decrease in the concentration of amino acids in response to high plasma homocystine or high plasma homocystine and methionine. Inhibition of protein synthesis which occurs under the above experimental conditions is a general phenomenon. myelin and other brain fractions being equally affected. The decrease in concentration of brain amino acids also results in a diminution in concentration of the neurotransmitters GABA, dopamine and noradrenaline. The possible relationship of the observed changes to homocystinuria is discussed.     

THE CONCENTRATIONS OF FREE AMINO ACIDS AND OTHER ELECTROLYTES IN CEREBROSPINAL FLUID,IN VIVO DIALYSATE OF BRAIN,AND BLOOD PLASMA OF THE DOG*

The concentrations of free amino acids in plasma, CSF and in vivo dialysates of peripheral blood (neck sac fluid) and central nervous tissue (brain sac fluid) from each of five dogs (neck sac fluid from four of five dogs) were determined by ion-exchange chromatography. Dialysates were obtained by implanting small dialysis sacs filled with a dextran-saline solution into the subcutaneous tissue of the neck or the parenchyma of the brain at least 10 weeks before sample collection. The mean plasma concentration of most amino acids was within the range of values reported in the literature for human or dog plasma. The concentrations of most amino acids were higher in the neck sac fluid than in plasma; this discrepancy, however, was, for the most part, small and could most likely be accounted for by falling plasma free amino acid levels prior to sample taking. Previous conclusions that the CSF concentrations of most amino acids are lower than plasma concentrations are confirmed, although the present work indicates that there may be considerable individual variation in the CSF/plasma distribution ratio with respect to most amino acids. In the brain sac fluid the concentration of nearly every amino acid was consistently higher than that in CSF and lower than that in the neck sac fluid. The potassium concentration in the brain sac fluid was significantly higher than, and the total osmolality significantly lower than, those in the neck sac fluid. On the assumption that the brain sac fluid represents a dialysate of the brain extracellular fluid, these results contradict recent findings (Bito and Davson , 1965; 1966) indicating that the potassium concentration of the cortex extracellular fluid is lower than that of ventricular or cisterna magna CSF and certainly lower than that of plasma. Because of this and on the basis of consideration of the reaction of the brain to a foreign body, the possibility that the implanted brain sac lay on the‘blood side’of the bloodbrain barrier was suggested. Some implications of this possibility are discussed.     

Glycine Binding to Rat Cortex and Spinal Cord: Binding Characteristics and Pharmacology Reveal Distinct Populations of Sites

Glycine is the principal inhibitory neurotransmitter in posterior regions of the brain. In addition, glycine serves as an allosteric regulator of excitatory neurotransmission mediated by the N-methyl-D-aspartate (NMDA) acidic amino acid receptor subtype. The studies presented here characterize [3H]glycine binding to washed membranes prepared from rat spinal cord and cortex, areas enriched in glycine inhibitory and NMDA receptors, respectively, in an attempt to define the glycine recognition sites on the two classes of receptors. Specific binding for [3H]glycine was seen in both cortex and spinal cord. Saturation analyses in cortex were best fitted by a two-site model with respective equilibrium dissociation constants (KD values) of 0.24 and 5.6 microM and respective maximal binding constants (Bmax values) of 3.4 and 26.7 pmol/mg of protein. Similar analyses in spinal cord were best fitted by a one-site model with a KD of 5.8 microM and Bmax of 20.2 pmol/mg of protein. Na+ had no effect on [3H]glycine binding to cortical membranes but increased the binding to spinal cord membranes by greater than 15-fold. This Na+-dependent binding may reflect glycine binding to the recognition site of the high-affinity, Na+-dependent glycine uptake system. Several short-chain, neutral amino acids displaced [3H]glycine binding from both cortical and spinal cord membranes. The most potent displacers of [3H]glycine binding to cortical membranes were D-serine and D-alanine, followed by the L-isomers of serine and alanine and beta-alanine. In contrast, D-serine and D-alanine were similar in potency to L-serine in spinal cord membranes. Compounds active at receptors for the acidic amino acids had disparate effects on the binding of [3H]glycine. At 10 microM, NMDA resulted in a 25% increase, whereas D- and L-2-amino-5-phosphonovaleric acid at 100 microM resulted in a 30% decrease, in [3H]glycine binding to cortical membranes. Kynurenic acid was the most potent of the acidic amino acid-related compounds at displacing [3H]glycine binding. In cortical membranes, kynurenic acid displacement was resolved into a high- and a low-affinity component; the high-affinity component displaced the high-affinity component of [3H]glycine binding.(ABSTRACT TRUNCATED AT 400 WORDS)     

Spinal and cranial contributions to total cerebrospinal fluid transport

In this study, we quantified cerebrospinal fluid (CSF) transport from the cranial and spinal subarachnoid spaces separately in sheep and determined the relative proportion of total CSF drainage that occurred from both CSF compartments. Cranial and spinal CSF systems were separated by placement of an extradural ligature over the spinal cord between C(1) and C(2). In one approach, two different radiolabeled human serum albumins (HSA) were introduced into the appropriate CSF compartment by a perfusion system (method 1) or as a bolus injection (method 2). Plasma tracer recoveries in conjunction with a mass balance equation were used to estimate CSF transport. In method 3, catheters connected to reservoirs filled with artificial CSF were introduced into the cranial and spinal CSF compartments. Incremental CSF pressures were established in each CSF system, and the corresponding steady-state flow rates were measured. Total CSF drainage ranged from 0.51 to 0.75 ml. h(-1). cmH(2)O(-1). Expressed as a percentage of the total CSF transport, the ratios of cranial-to-spinal clearance estimated from methods 1, 2, and 3 were 75:25, 88:12, and 75:25, respectively. Primarily on the basis of the data derived from methods 1 and 3, we conclude that the spinal subarachnoid compartment has an important role in CSF clearance and is responsible for approximately one-fourth of total CSF transport.     

HOMOVANILLIC ACID AND 3-METHOXY-4- HYDROXYPHENYLETHYLENEGLYCOL PRODUCTION BY THE MONKEY SPINAL CORD

The release of homovanillic acid (HVA) and 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) into CSF by the monkey spinal cord was investigated with spinal subarachnoid perfusion of 20 rhesus monkeys. The preperfusion concentration of HVA in lumbar CSF was 365 ng/ml and in cisternal CSF was 365 ng/ml, while the concentrations of MHPG were 28.3 and 40.4 ng/ml respectively. HVA originating from the spinal cord appeared in the perfusate at a rate of 2.4 and MHPG at 1.4 ng/min. Treatment with probenecid either intraperitoneally or intrathecally did not alter the rate of release into CSF of these metabolites by the spinal cord but did significantly increase the rate of appearance in the cisterna magna of HVA originating from the brain. MHPG and HVA in lumbar CSF are therefore derived in part from spinal cord metabolism.     

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.     

Spinal subarachnoid perfusion in the rat: glycine transport from spinal fluid

Abstract— A method was developed for perfusion of the spinal subarachnoid space in the rat. Bidirectional steady-state fluxes of [¹⁴C]glycine between spinal fluid and plasma were measured. [¹⁴C]glycine clearance from spinal fluid was 5-fold greater than its clearance from plasma. Glycine was transported out of spinal fluid by a saturable process, and the rate of transport was unaffected by the other depressant amino acids, GABA, β-alanine, and taurine. Perfused [¹⁴C]glycine and [³H]GABA distributed in an intracellular compartment in spinal cord. The preparation should be useful for study of the release of these inhibitory amino acids from the intact spinal cord.     

THE FREE AMINO ACIDS IN HUMAN CEREBROSPINAL FLUID

—(a) The free amino acids in human cerebrospinal fluid from 78 subjects have been determined by means of ion exchange chromatography. Analyses were performed on 2 to 3 ml of CSF. (b) Normal values are given for a control group of 13 subjects. (c) The presence of homocarnosine in human CSF is ascertained. The concentration of this dipeptide is very high in CSF samples from phenylketonuric patients. (d) The CSF amino acid pattern in 20 patients with a metabolic disease is discussed. (e) The CSF amino acid pattern of 38 patients suffering from a variety of neurological conditions are reported.     

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     

The specific radioactivity of the tissue free amino acid pool as a basis for measuring the rate of protein synthesis in the rat in vivo

1. Rats were infused in vivo with [U-(14)C]glycine for periods of 2-6h, during which time the specific radioactivity of the free glycine in plasma and tissue approached a constant value. 2. Free serine also became labelled. The ratio of specific radioactivity of serine to that of glycine in the protein of liver, kidney, brain, jejunum, heart, diaphragm and gastrocnemius muscle was closer to the ratio in the free amino acid pool of the tissue than that of the plasma. 3. The kinetics of incorporation of [(14)C]glycine and [(14)C]serine into the protein of gastrocnemius muscle further suggested that the plasma free amino acids were not the immediate precursors of protein. 4. Infusion of rats with [U-(14)C]serine resulted in labelling of free glycine. The ratio of specific radioactivity of glycine to serine in the protein of liver, kidney, brain, jejunum and heart again suggested incorporation from a pool similar to the free amino acid pool of the tissue. 5. Rates of tissue protein synthesis calculated from the incorporation into protein of both radioactive glycine and serine, either infused or derived, were very similar when the precursor specific radioactivity was taken to be that in the total free amino acids of the tissue. Except for gastrocnemius muscle and diaphragm during the infusion of radioactive serine, the rates of tissue protein synthesis calculated from the specific radioactivity of the free glycine and serine in plasma differed markedly.     

Effect of tetanus toxin on the content of glycine, gamma-aminobutyric acid, glutamate, glutamine and aspartate in the rat spinal cord

Tetanus toxin injected intramuscularly induced no significant changes in the levels of glycine, GABA, glutamate, glutamine or aspartate in extracts of spinal cord from rats killed at timed intervals during the development of local and generalized tetanus. The amino acid contents in the hemisegment (longitudinal one-half) of the spinal cord (L₂-L₆) on the injected side (left gastrocnemius muscle) did not differ significantly from the contents in the hemisegment of the spinal cord on the non-injected side. Nor were there any consistent changes in the contents of the amino acids in either hemisegment of the spinal cord as the tetanic symptoms became progressively more severe. Hence, the amino acid pool in the spinal cord was relatively stable despite the metabolic changes known to occur in tetanus. Our observations are consistent with the view of Johnston , De Groat and CURTIS (1969) who suggested that if glycine were indeed a spinal inhibitory neurotransmitter released by interneurons affected by tetanus toxin, the toxin should interfere with the release of the amino acid rather than deplete the transmitter stores.     

THE INTRASPINAL DISTRIBUTION OF SOME DEPRESSANT AMINO ACIDS

Abstract— —Six known depressant amino acids were found in extracts of feline spinal grey matter: α-alanine, cystathionine, GABA, glycine, serine and taurine.
Of these, glycine has the distribution within the nervous system anticipated of a sub-stance whose main function is as a spinal inhibitory transmitter at strychnine-sensitive synapses, GABA being a likely transmitter candidate at any strychnine-insensitive spinal inhibitory synapses.
The intraspinal distributions of α-alanine, cystathionine, serine and taurine reveal little to indicate that a synaptic role is a major function of these amino acids.     

Transport of GABA at the Blood-CSF Interface

Abstract: The entry of GABA into cerebrospinal fluid (CSF) was studied in dogs anesthetized with pentobarbital and relaxed with suxamethonium. GABA was administered intravenously as a priming dose and subsequent maintenance infusion to compensate for the rapid elimination of the amino acid. Steady state concentrations of GABA in CSF were reached between 10 and 60 min after injection, the rate of entry tending to decrease with increasing plasma levels. During steady state conditions CSF concentrations showed great interin-dividual differences and varied between 0.03 and 5.1% of those in plasma. Probenecid and sodium valproate considerably enhanced the CSF/plasma concentration ratio of GABA. When GABA was directly injected into the liquor space, probenecid slowed down the elimination of GABA from CSF. The results suggest a transport of GABA into and out of CSF, the outward transport being inhibited by probenecid and sodium valproate.     

Spinal cord ischemia-induced elevation of amino acids: Extracellular measurement with microdialysis

Excitatory amino acids have been implicated in the production of calcium mediated neuronal death following central nervous system ischemia. We have used microdialysis to investigate changes in the extracellular concentrations of amino acids in the spinal cord after aortic occlusion in the rabbit. Glutamate, aspartate, glutamine, asparagine, glycine, taurine, valine, and leucine were measured in the micordialysis perfusate by high pressure liquid chromatography. The concentrations of glutamate, glycine, and taurine were significantly higher during ischemia and reperfusion than controls. Delayed elevations in the concentrations of asparagine and valine were also detected. The elevation of glutamate is consistent with the hypothesis that excitotoxins may mediate neuronal damage in the ischemic spinal cord. Increased extracellular concentrations of asparagine and valine may reflect preferential use of amino acids for energy metabolism under ischemic conditions. The significance of increased concentrations of inhibitory amino acid neurotransmitters is unclear.     

Elevation of cerebral levels of nonessential amino acids in vivo by administration of large doses

Taurine, aspartic acid, glutamic acid, glycine, and GABA were administered either intragastrically or in liquid diets to mice and rats. This resulted in a great increase in the plasma concentration of the administered amino acid, with plasma levels remaining elevated for several days.The prolonged increase in plasma levels resulted in significant increases in brain levels. Under these experimental conditions, taurine, aspartic acid, and glutamic acid were increased 30–60%; glycine and GABA 100%. During these experiments, plasma levels of taurine, aspartate, and glutamate were below brain levels; those of glycine and GABA were above.The findings show that even slowly penetrating amino acid levels can be increased in brain after parenteral administration of large doses.     

Hydrodynamic modeling of cerebrospinal fluid motion within the spinal cavity

The fluid that resides within cranial and spinal cavities, cerebrospinal fluid (CSF), moves in a pulsatile fashion to and from the cranial cavity. This motion can be measured hy magnetic resonance imaging (MRI) and may he of clinical importance in the diagnosis of several brain and spinal cord disorders such as hydrocephalus, Chiari malformation, and syringomyelia. In the present work, a geometric and hydrodynamic characterization of an anatomically relevant spinal canal model is presented. We found that inertial effects dominate the flow field under normal physiological flow rates. Along the length of the spinal canal, hydraulic diameter was found to vary significantly from 5 to 15 mm. The instantaneous Reynolds number at peak flow rate ranged from 150 to 450, and the Womersle number ranged from 5 to 17. Pulsatile flow calculations are presented for an idealized geometric representation of the spinal cavity. A linearized Navier-Stokes model of the pulsatile CSF flow was constructed based on MRI flow rate measurements taken on a healthy volunteer. The numerical model was employed to investigate effects of cross-sectional geometry and spinal cord motion on unsteady velocity, shear stress, and pressure gradientfields. The velocity field was shown to be blunt, due to the inertial character of the flow, with velocity peaks located near the boundaries of the spinal canal rather than at the midpoint between boundaries. The pressure gradient waveform was found to be almost exclusively dependent on the flow waveform and cross-sectional area. Characterization of the CSF dynamics in normal and diseased states may be important in understanding the pathophysiology of CSF related disorders. Flow models coupled with MRI flow measurements mnay become a noninvasive tool to explain the abnormal dynamics of CSF in related brain disorders as well as to determine concentration and local distribution of drugs delivered into the CSF space.