Theodore A. Hare

Low cerebrospinal fluid γ‐aminobutyric acid content in seizure patients

γ-Aminobutyric acid (GABA) has been implicated in the neurochemistry of epilepsy. Lumbar cerebrospinal fluid (CSF) GABA concentrations determined using an ion-exchange fluorometric assay reflect brain GABA content. The mean lumbar CSF GABA concentration among 21 medicated patients with intractable seizures was significantly lower (p < 0.001) than that of 20 urimedicated normal volunteers. Patients with generalized tonic-clonk (grand mal) and complex partial (psychomotor) seizures had significantly lower (p < 0.05) CSF GABA concentrations than those with simple partial (focal sensory/motor) seizures. Although lumbar CSF GABA levels in our seizure patients did not significantly correlate with serum concentrations of phenytoin, phenobarbital, or primidone, additional study of medication-free epileptic patients may be required to evaluate the possibility of anticonvulsant-drug-induced CSF GABA alterations.

Free and conjugated amino acids in human CSF: Influence of age and sex

An extended baseline characterization of amino acids (AAs) and related amino compounds in CSF is reported. Thirty-one amino compounds were measured in deproteinized CSF before and after acid hydrolysis using a triple-column HPLC/fluorometric analyzer. CSF specimens were collected under strictly controlled conditions from neurologically normal myelogram patients and carefully pooled with regard to subject age and sex. Consideration was given to factors which may produce artifactual alterations in AA levels during CSF collection, storage and handling. Conjugated AAs were determined as the difference between levels of free AAs (measured in CSF prior to hydrolysis) and total AAs (measured in hydrolyzed CSF) and are taken as an index of total CSF peptide AAs. Results documented conjugated forms of all non-acid-labile CSF AAs except citrulline and ethanolamine. In general, ratios of conjugated to free AAs were relatively low, however for the neurotransmitter AAs aspartate, glutamate, glycine and GABA as well as for beta-alanine hydrolysis produced marked increases indicating that these compounds are present predominantly in bound form in CSF. Results also revealed the significant influence of both age and sex on levels of a number of CSF free and conjugated AAs. Compared to younger individuals (those less than 40 years of age), older individuals exhibited significantly higher levels of free aspartate, glycine, alpha-aminobutyric acid, valine, isoleucine, leucine, phenylalanine and 3-methylhistidine as well as significantly lower levels of free phosphoethanolamine, serine, GABA, homocarnosine, conjugated GABA and conjugated beta-alanine. Additionally, significantly higher levels of free tyrosine, ethanolamine, arginine and conjugated aspartate were documented in males compared to females.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid and sensitive ion-exchange fluorometric measurement of γ-aminobutyric acid in physiological fluids

Abstract Improvements of the ion-exchange/fluorometric method for measuring γ-aminobutyric acid (GABA) have resulted in an automatic three-column analyzer which produces an analysis every 15 min using 200-μl samples to a sensitivity of 1 pmol. This high-performance liquid chromatographic procedure utilizes three stainless-steel microbore columns containing cation-exchange resin. GABA is eluted using a lithium citrate buffer and then detected in the flow stream after reaction with the fluorogenic reagent orthophthalaldehyde. Each column utilizes a timer-controlled, pneumatically actuated two-way 10-port valve containing a 200-μl loop for sample injection and a 1-ml loop for regeneration. Similar 4-port valves sequentially direct the output from one of the columns to the reaction manifold. Measurement of GABA levels in sequential aliquets of the first 20 ml of human lumbar cerebrospinal fluid revealed an increasing gradient of GABA level in cerebrospinal fluid from six patients and no gradient in a seventh patient, the mean rate of increase being 2% per milliliter.

Rat plasma levels of amino acids and related compounds during stress

Forty-one amino acids and related compounds were measured (using an HPLC physiological amino acid analysis procedure fully validated for plasma studies) in rat plasma obtained through an indwelling jugular catheter before, during and following a 30 min period of immobilization. Taurine, phosphoethanolamine, aspartic acid, glutamic acid, alanine, cystine, tyrosine, beta-alanine and ethanolamine were increased during the period of stress; whereas, valine, tryptophan and arginine were decreased. Most of these alterations were restored toward normal during the 30 min of rest following the stress period. However, cystine, ethanolamine and beta-alanine remained significantly elevated, and valine, tryptophan and arginine remained significantly reduced. Serine, isoleucine, leucine and glutamine were not significantly altered during the stress period, but became significantly reduced during the 30 min following the stress period. While the patterns of amino acid alterations were generally consistent from animal to animal, the magnitude of the responses were variable with some rats demonstrating much larger responses than others. These results may implicate amino acids as important markers for stress related pathologies. The individual differences noticed may explain why some individuals show more stress effects than others.

Measurement of GABA in human cerebrospinal fluid.

Abstract An automatic method is described utilizing ion-exchange column chromatography and fluorescence detection after reaction with orthophthalaldehyde to determine levels of GABA in human CSF. The standard curve was linear over the range tested (50–800 picomoles). The method is rapid enough (2 hrs) for clinical application and provides resolution comparable to that of usual amino acid analyses of physiological fluids. The level of GABA was found to be 270 ± 260 (SD) picomoles/ml in CSF from 25 patients with a variety of neurological disorders.

GABA levels in cerebrospinal fluid of patients with Huntington's chorea: A preliminary report

Abstract GABA levels were measured in CSF from seven patients with Huntingtons chorea and nine control patients with a variety of other neurological disorders. The values for the patients with Huntingtons chorea averaged (± SD) 110 ± 29 pmoles/ml; whereas those for the control population averaged 534 ± 266 pmoles/ml. The difference between the values for the two populations was highly significant ( P ≤ 0.001).

Effect of 1-methyl-4-phemyl-1,2,3,6-tetrahydropyridine (MPTP) on levels of glutathione in the extrapyramidal system of the mouse

Treatment of mice with the proximate neurotoxin MPTP depletes striatal dopamine levels. Depletion of striatal dopamine and metabolites in MPTP-treated mice is accompanied by depletion of glutathione (GSH) in the substantia nigra (SN). Striatal GSH and nigral amino acid levels were not significantly affected by MPTP. Results suggest that GSH depletion in SN may represent an index of regional vulnerability to metabolic oxidative stress and also of selective susceptibility to the toxic effects of MPTP.

Gamma-Aminobutyric Acid in Peripheral Tissue, with Emphasis on the Endocrine Pancreas: Presence in Two Species and Reduction by Streptozotocin

A screen of selected peripheral organs of the rat found that γ-aminobutyric acid (GABA) is generally present outside the central nervous system, and, of those organs examined, GABA was present at the highest concentration in the pancreas (∼40 pmol/mg wet wt). Furthermore, this putative inhibitory neurotransmitter was found to be present at even higher levels in islets of Langerhans tissue isolated from rat pancreas (190 pmol/mg). Administration of streptozotocin, a selective β-cell toxin, decreased pancreatic GABA levels significantly, but had no or only small effects on the GABA content of other organs. Normal teleost (catfish) Brockmann body contains about the same level of GABA as normal rat islet tissue.

In vivo modulation of excitatory amino acid receptors: microdialysis studies on N-methyl-d-aspartate-evoked striatal dopamine release and effects of antagonists

Striatal dopamine (DA) release was measured following intrastriatal (i.s.) administration of N-methyl-D-aspartate (NMDA) to unanesthetized, freely-moving rats. One hour after insertion of a removable microdialysis probe and perfusion with normal Ringers solution, a modified Ringers solution containing 100 mM potassium (high-K+ Ringers) was used to standardize the preparation. DA release following i.s. administration of NMDA (12.5 mM in normal Ringers) was dose-dependent. When NMDA (12.5 mM) was administered in high-K+ Ringers, DA release was greatly potentiated. Administration of the competitive NMDA receptor antagonist aminophosphonovalerate (APV) in normal Ringers prior to treatment with NMDA in high-K+ Ringers resulted in a significant reduction of DA release compared to control animals. In contrast, administration of APV priot to treatment with NMDA in normal Ringers resulted in a significantly increased release of DA compared to controls. Administration of the non-competitive NMDA antagonist, dextromethorphan (DXT) prior to treatment with NMDA in normal Ringers or NMDA in high-K+ Ringers caused significant reductions of DA release compared to controls. Intrastriatal DXT also caused dose-dependent inhibition of high-K+ Ringers-induced DA release. Similarly, administration of the non-specific calcium channel blocker, cadmium, prior to treatment with NMDA resulted in a significant decrease when compared to control values. Results of this study indicate that dose-dependent NMDA-induced striatal DA release is greatly potentiated by potassium suggesting that under physiological conditions in vivo, striatal NMDA receptors are mostly inactivated.(ABSTRACT TRUNCATED AT 250 WORDS)

Verification and quantification of GABA in human cerebrospinal fluid

MEASCREMEW of putative neurotransmitters and their metabolites in human cerebrospinal fluid (CSF) has provided new insights into the neurochemical characteristics of CNS disorders (KLAWANS. 1971; CURZON et 01.. 1972; WOOD et a/., 1977a. 19776). Since y-aminobutyric acid (GABA) is felt to be, quantitatively, one of the more important neurotransmitters in the mammalian CNS ( B ~ M & IVERSEN. 1971) and abnormalities in GABAergic transmission have been implicated in epilepsy (VAN GELDER er al.. 1972), Huntingtons disease (PERRY ~t a/.. 1973; BIRD & IVERSEN, 1974; ENNA et a/., 1976). cerebrovascular disease (ACHAR et al., 1976). meningitis (BURVAKOVA et a/., 1975) and psychiatric illness (ROBERTS, 1972; ROBERTS er a/.. 1976; LANGER et a/., 1975). Methods to accurately determine the quantity of this amino acid in human CSF may aid in the diagnosis and treatment of some CNS disease states. Recently, several reports have appeared on attempts to measure CSF GABA using a variety of techniques but, since some investigators have been unable to detect measurable quantities of GABA in this fluid, the issue of CSF GABA remains in doubt (GLAESER & HARE. 1975; ACHAR et a/., 1976: PERRY & HANSEN. 1976; GROSSMAN e l a/.. 1976; ENNA et a/.. 1977b; Htilzlxca et a/., 1977). In the present study, GABA levels were estimated on the same human CSF samples using different analytical techniques in an attempt to demonstrate conclusively. and to quantitate accurately, GABA in CSF.