FREE AMINO ACID LEVELS IN THE CEREBROSPINAL FLUID OF NORMAL HUMANS AND THEIR VARIATION IN CASES OF EPILEPSY AND SPIELMEYER-VOGT-BATTEN DISEASE

Abstract— The concentrations of free amino acids in the plasma and cerebrospinal fluid from control subjects and from patients suffering from epilepsy and Spielmeyer-Vogt-Batten disease were determined using an automatic amino acid analyser. It was found that the plasma/cerebrospinal fluid ratio of amino acid concentrations showed the variation in the amounts of free amino acids in epilepsy more clearly than the cerebrospinal fluid levels alone.     

Chiral separation of amino acids in biological fluids by micellar electrokinetic chromatography with laser-induced fluorescence detection

A method is presented for the chiral analysis of amino acids in biological fluids using micellar electrokinetic chromatography (MEKC) and laser-induced fluorescence (LIF). The amino acids are derivatized with the chiral reagent (+/−)-1-(9-anthryl)-2-propyl chloroformate (APOC) and separated using a mixed micellar separation system. No tedious pre-purification of samples is required. The excellent separation efficiency and good detection capabilities of the MEKC-LIF system are exemplified in the analysis of urine and cerebrospinal fluid. This is the first time MEKC has been reported for chiral analysis of amino acids in biological fluids. The amino acids -alanine, -glutamine, and -aspartic acid have been observed in cerebrospinal fluid, and -alanine and -glutamic acid in urine. To the best of our knowledge no measurements of either -alanine in cerebrospinal fluid or -glutamic acid in urine have been presented in the literature before.     

Direct Colorimetric Method for the Determination of Free Ammonia in Blood

In an attempt to develope a rapid and simple method for the colorimetric determination of free ammonia in blood, the conditions for the indophenol reaction, the direct application of it for the determination of blood ammonia, and the interferences of a number of substances for the color development, have been investigated. This color reaction was sensitive enough for the direct method, but must be done under the limiting conditions, for the interfereing substances are contained in blood. Interferences of many amino acids for the color development were observed, but the effects differed among different amino acids, and many of substances other than amino acids showed very little or no intereference. Under the conditions applied, neither the amino acids nor other substances in blood interfered the method.     

Determination of pyrrolidone carboxylate and gamma-glutamyl amino acids by gas chromatography.

Gas chromatographic methods for the quantitation of pyrrolidone carboxylate and γ-glutamyl amino acids are described. These intermediates of the γ-glutamyl cycle were separated by ion exchange chromatography and converted to their N-acyl-ester derivatives in a reaction with a mixture of 2,2,3,3,3-pentafluoro-1-propanol and pentafluoropropionic anhydride. The derivatives have excellent electron capture properties thus making possible their determination even in small amounts of material of biological origin. The method was applied for the determination of concentrations of pyrrolidone carboxylate in human urine and cerebrospinal fluid, and in the brain, liver, and kidney of the mouse. It was also used to demonstrate the formation in mouse tissues of several γ-glutamyl derivatives of amino acids after administration of the corresponding free amino acid.     

Indolic Substances in Plasma, Cerebrospinal Fluid, and Frontal Cortex of Human Subjects Infused with Saline or Tryptophan

Psychiatric patients undergoing the psychosurgical operation of stereotactic subcaudate tractotomy were infused intravenously with either saline or L-tryptophan (15 mg/kg/h). Plasma, lumbar cerebrospinal fluid (CSF), ventricular CSF and a specimen of frontal cortex were collected. The relationships of plasma concentrations of substances claimed to influence brain tryptophan concentration (total tryptophan, free tryptophan, large neutral amino acids) with the concentration of tryptophan in the cortex and CSF were investigated. Tryptophan infusion resulted in plasma tryptophan values comparable to those found after oral doses used in treating depression or insomnia, and about sixfold increases of tryptophan in the cerebral cortex. Increased brain 5-hydroxytryptamine synthesis was indicated by significant rises of CSF 5-hydroxyindoleacetic acid. The concentration of plasma free tryptophan was a better predictor than plasma total tryptophan of cortex tryptophan concentration. As all correlation coefficients of plasma versus brain or plasma versus ventricular CSF tryptophan concentrations were decreased when allowance was made for differences of concentration of large neutral amino acids, the results suggest that the role of these substances within their physiological range as inhibitors of tryptophan transport to the brain may previously have been overemphasised.     

Motor activity and the content of neuroactive amino acids in brain tissues

The influence of forced motor activity (swimming) on quantitative shifts in neuroactive amino acids (GABA, glutamic and aspartic acids and glycine) was studied in brain tissues of rats and cerebrospinal fluid of cats in health and brain circulation disturbances. The data obtained point to the elevation of the content of amino acids in the brain and appearance of GABA in the cerebrospinal fluid during brain ischemia.     

Analysis of aspartate and glutamate in human cerebrospinal fluid by high-performance liquid chromatography with automated precolumn derivatization

A method is described for the analysis of the neuroexcitatory amino acids, aspartate and glutamate, in human cerebrospinal fluid (CSF) by reverse-phase, high-performance liquid chromatography. Fluorescent isoindole derivatives of the amino acids were prepared by reacting the amino acids with ortho-phthalaldehyde in an automated, precolumn procedure. Chromatographic conditions were developed that resolve the isoindole derivatives of aspartate and glutamate from those of at least 10 unidentified components of CSF. Amino acids were reliably quantified in 5-microliter samples of CSF, and deproteinization of the specimens was not required. Furthermore, it was found that deproteinization by precipitation with strong acid can lead to artifactually high measurements of glutamate. The concentrations of free aspartate and glutamate in lumbar CSF from 15 neurologically normal children were 0.30 +/- 0.11 and 0.48 +/- 0.26 microM (mean +/- SD), respectively. The value for glutamate is considerably lower than has been reported in any previous study of human CSF.     

Metabolic factors affecting the reproductive axis in male sheep

Changes in food intake affect the reproductive axis in both sexes, and the nutritional signals involved and the sites that receive those signals are now beginning to be unravelled. Our studies have focussed on the mature male sheep, a model in which high food intake stimulates GnRH-LH pulse frequency for only 10-20 days but continues to promote testicular growth over several months. Different signals and different target organs seem to be responsible for these short- and long-term responses. Short-term dietary treatments lead to changes in blood concentrations of glucose, fatty acids, insulin and leptin, and concentrations of glucose, insulin, leptin and some amino acids in cerebrospinal fluid. It seems unlikely that amino acids affect GnRH-LH secretion directly in sheep. Intracerebroventricular infusions of insulin specifically increase LH pulse frequency, but intravenous, intra-abomasal or intracerebroventricular infusions of glucose have no effect, despite their effects on cerebrospinal fluid insulin concentrations. The addition of fatty acids to the diet also increases LH pulse frequency, but does not affect the concentrations of insulin or leptin in the cerebrospinal fluid. It appears that acute responses to changes in nutrition involve a range of alternative pathways, possibly including interactions among insulin, leptin and energy substrates. Effects of long-term dietary treatments on testicular size are only partly dependent on the GnRH-LH system (that is, on brain control) and so must also depend on other, as yet unknown, pathways. Concepts of 'metabolic sensing and integration' are being developed from the basis of existing knowledge of the central control of appetite and reproduction.     

A rapid high-performance liquid chromatographic procedure for the simultaneous determination of methionine, ethionine, S-adenosylmethionine, S-adenosylethionine, and the natural polyamines in rat tissues

A method for the analysis of S-adenosyl-L-methionine (SAM) and S-adenosyl-L-ethionine (SAE) and their major metabolites by high-performance liquid chromatography is described. The procedure allows the simultaneous analysis of the natural polyamines, putrescine, spermidine, and spermine, and some of the major amino acids, methionine, tyrosine, and tryptophan. The uv absorbance at 254 nm is used for the determination of the SAM and SAE analogs, whereas the polyamines and amino acids are analyzed by fluorescence detection after postcolumn derivatization with o-phthalaldehyde. The method allows SAM and polyamine determinations by direct injection of the tissue extracts without prepurification. The procedure is applied to study the effects of DL-ethionine treatment on the SAM, SAE, methionine, and polyamine levels in various tissues of rats.     

Extra- and intracellular amino acids in the hippocampus during development of hepatic encephalopathy

Fulminant hepatic failure was induced in rabbits by intravenous administration of galactosamine hydrochloride. The animals were sacrified after 45 h and the hippocampus analyzed for free amino acids. In addition, free amino acids were measured in plasma and in the extracellular fluid of the hippocampus 20, 30 and 45 h after galactosamine injection. The extracellular fluid compartment was analyzed by slow perfusion of a thin dialysis tube which was implanted in the hippocampus one day prior to galactosamine administration. The amino acid concentration in the extracellular fluid agreed fairly well with that of the cerebrospinal fluid in the control situation. During development of hepatic failure, the plasma concentrations of all amino acids increased. The changes in extracellular amino acids were smaller, except for phosphoethanolamine and glutamate. The concentration ratio intra/extracellular amino acids decreased in the hippocampus for amino acids with a normally high concentration gradient.     

A micromethod for the analysis of free amino acids by gas chromatography and its application to biological systems.

A gas chromatographic procedure was developed for determination of minute amounts of free amino acids in natural waters and laboratory models simulating biological systems. Sample pretreatment included removal of interfering organic substances by chloroform extraction and isolation of amino acids by cation exchange. Amino acids were converted to their N-heptafluorobutyryl isobutyl ester derivatives in glass capillary tubes, permitting considerable concentration of the sample prior to gc injection. The derivatives of 19 amino acids were successfully separated on either a glass column packed with a mixture of OV-101 and OV-17 on Chromosorb W, a glass capillary column coated with OV-101, or a support-coated capillary column supported with SE-30. One to five nanograms of individual amino acids were detected using flame ionization detector. The detection limit was reduced more than 100-fold using the electron capture detector and more than 1000-fold by mass fragmentography. The procedure allowed determination of less than 1 ppb of individual amino acids in lake and river water samples and was used to estimate the exeretion of free amino acids from microbial populations.     

Glucose and amino acid metabolism in chick telencephalon slices: Changes with incubation conditions and animals' development

Glucose and amino acid metabolism in 1- and 30-day-old chick telencephalon slices was studied in two incubation media in the presence or in the absence of a continuous oxygenation. Medium 1 has a composition and a tonicity similar to cerebrospinal fluid, medium 2 is hypertonic and does not contain any K⁺ ions. The incorporation of glucose carbon into amino acids and the distribution of radioactivity between the different amino acids are close to the ones observed in the chick brain in vivo only when the slices are incubated in medium 1, with oxygen at 30 days and without oxygen for the 1-day-old chick. It also appears that if oxygenation is necessary for incubation of mature brain tissue in vitro, the absence of the medium oxygenation is more suitable for the study of glucose metabolism in 1-day-old chick brain slices.     

Sensitive enzyme-immunostaining and densitometric determination of ganglio-series gangliosides on thin-layer plate: pmol detection of gangliosides in cerebrospinal fluid

An immunochemical method has been developed for sensitive detection and determination of ganglio-series gangliosides using thin-layer chromatography/enzyme-immunostaining. After chromatography of gangliosides, the plate was treated with Arthrobacter ureafaciens sialidase to remove all sialic acids from ganglio-series gangliosides. For the complete hydrolysis of gangliosides, sodium taurodeoxycholate was found to be required. The resulting asialo-glycolipids, GA2 and GA1 were reacted first with affinity-purified anti-GA2 and anti-GA1, respectively, and second with horseradish peroxidase-conjugated anti-rabbit IgG. Being highly sensitive and reproducible, it allows the characterization of gangliosides in cerebrospinal fluid which cannot be detected by classical methods.     

Rapid and sensitive step gradient assays of glutamate, glycine, taurine and γ-aminobutyric acid by high-performance liquid chromatography–fluorescence detection with o-phthalaldehyde–mercaptoethanol derivatization with an emphasis on microdialysis samples

We developed a rapid step-gradient HPLC method for determination of glutamate, glycine and taurine, and a separate method for determination of γ-aminobutyric acid (GABA) in striatal microdialysates. The amino acids were pre-column derivatized with o-phthalaldehyde–2-mercaptoethanol by using an automated refrigerated autoinjector. Separation of the amino acids was established with a non-porous ODS-II HPLC column, late-eluting substances were washed out with a one-step low-pressure gradient. Concentrations of the amino acids were determined with a fixed-wavelength fluorescence detector. The detection limit for GABA was 80 fmol in a 15 μl sample, detection limits for glutamate, glycine and taurine were not determined because their concentrations in striatal perfusates were far above their detection limits. Total analysis time was less than 12 min, including the wash-out step. The methods described are relatively simple, sensitive, inexpensive, and fast enough to keep up with the microdialysis sampling.     

Little change in cerebrospinal fluid amino acids in subtypes of multiple sclerosis compared with acute polyradiculoneuropathy.

Levels of free amino acids were determined in randomised, blinded samples of cerebrospinal fluid (CSF) from patients with relapsing-remitting or chronic progressive multiple sclerosis (MS), all in the active phase of disease. The levels were compared with amino acid amounts in patients with an acute polyradiculoneuropathy (Guillain-Barré syndrome (GBS)) and a control population of patients with no known neurological disease or deficit. The data did not indicate any significant changes in amino acid levels between MS subgroups. The only significant differences between MS patients and controls were a modest reduction in glutamate and a slight increase in taurine, but the changes were so small that the biological relevance is dubious. These results contrasted with the marked increases for many amino acids in CSF from patients with acute polyradiculoneuropathy compared with controls. The amino acid profile in cerebrospinal fluid (CSF) does not appear to provide evidence of differential pathology in multiple sclerosis (MS). The increase in hydrophobic amino acids and lysine in CSF from patients with acute polyradiculoneuropathy is consistent with transudation over the blood-CSF barrier following an infection. The increases in glutamine and alanine may reflect increased nitrogen removal from brain.     

Behavioral study of chemoreception in the sea starMarthasterias glacialis: Structure-activity relationships of lactic acid,amino acids,and acetylcholine

Behavioral responses of Marthasterias glacialis to low molecular compounds were studied under laboratory conditions. Feeding postures, stomach eversions and locomotion of initially inactive animals can be released with very dilute solutions of lactic acid, neutral 2 and 3 carbon amino acids, L isomers of 4 to 6 carbon neutral amino acids, L-arginine, acetylcholine iodide, and several of their analogues. Hunger was induced by temporary withdrawal of food. Responsiveness to feeding stimuli was controlled with L-cysteine and L-leucine. The lowest behavioral thresholds for the most effective feeding stimuli were 3 X 10(-11) mol/l for both enantiomers of lactic acid, 10(-8) mol/l for L-proline and both enantiomers of cysteine and 10(-7) mol/l for acetylcholine iodide and some of the effective neutral amino acids. The behavioral threshold values for chemical stimuli differed by a factor between 30 and 100 in different sea stars. The test concentration was 3 X 10(-7) mol/l, the level at which L-cysteine elicited a complete feeding response from all the animals. Structure-activity comparison of substances less effective than the control stimulus was thus possible. The behavioral threshold of fully effective substances was determined later. The independence of receptor mechanisms for different substances can be inferred as: L-cysteine controlled responsiveness is not always accompanied by responsiveness to neutral amino acids. Autotomized marthasterias arms crawled after stimulation with lactic acid, cysteine, and acetylcholine iodide but did not respond to the feeding stimuli betaine and L-proline. An animal became inactive if electric shocks were paired with L-proline or L-cysteine emanating from an 'electric' food model.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative studies on partial microsequence-analysis of Bence-Jones proteins

1. Sequence analyses of Bence-Jones proteins up to 15 amino acids from the N-terminus provide decision of subgroups. 2. Investigation of primary structure of 3 Bence-Jones proteins, monomer and dimer of a kappa type, is limited at position 9-10 using DABITC reagent, whereas DABITC/PITC double coupling method allows sequencing up to 20-21 amino acids. 3. Sequencing of tryptic peptides allows only determination of 6-9 amino acids. 4. Microsequencing of tryptic peptide T13a shows the allotypic variant, inv b+, of Bence-Jones proteins TRA and GAN.     

The interaction of amino acids with o-phthaldialdehyde: A kinetic study and spectrophotometric assay of the reaction product

A detailed study has been made of the kinetics of interaction between amino acids and esters of amino acids and o-phthaldialdehyde in the presence of mercaptoethanol. The reaction products have been characterized. A spectrophotometric method for quantitative analysis of all amino acids, except proline and hydroxyproline, has been developed. The possibility of determination of amino acid esters in mixtures containing free amino acids has been demonstrated. It is noted that determination of glycine and histidine with the help of o-phthaldialdehyde has certain specificities associated with faster, compared to other amino acids, degradation of their derivatives. Optimal conditions for quantitative analysis of amino acids in solutions of higher than 10^?5m concentration are recommended. The reproducibility of the determination was ±2%.     

Plasma and Cerebrospinal Fluid Amino Acids in Epileptic Patients

Altered plasma and cerebrospinal fluid amino acid levels may be associated with human epilepsy. We studied three groups of patients, those with a generalized epileptic syndrome, juvenile myoclonic epilepsy, patients with refractory localization-related epilepsies, and patients with acute seizures (within 24 h). Plasma levels of amino acids were studied in all patient groups, as were those in the cerebrospinal fluid (CSF) of patients with acute seizures. After acute seizures, the amino acid changes in the CSF were limited to a reduction in the level of taurine, whereas the levels of most amino acids in plasma were decreased. On the other hand, levels of the excitatory amino acids glutamate and aspartate were increased. The most notable finding in the juvenile myoclonic epilepsy patients was an increase in glutamate level in the plasma. Our study supports the conception of an altered metabolism of glutamate in generalized epilepsies.     

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.