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Mice which had been housed in groups were introduced into the home cages of isolated mice and the aggressive defensive behaviour assessed. The grouped mice were chronically treated with testosterone, cyproterone acetate or arachis oil. The concentration of GABA was determined in 10 regions following the last behavioural assessment. The grouped mice which were treated with testosterone or with the anti-androgen, cyproterone acetate, were subjected to fewer aggressive attacks by the isolated mice thatn the grouped controls. The concentration of GABA in particular brain areas appears to depend on the degree of aggressive behaviour exhibited, the type of environment into which the mouse is introduced, the hormone treatment, and the housing conditions.

gamma-Acetylenic gamma-aminobutyric acid (gamma-acetylenic GABA) produces several-fold sustained elevations of brain GABA concentrations when administered intraperitoneally to mice. It protects mice against seizures induced by audiogenic stimuli, electroshock, thiosemicarbazide, isoniazid and strychnine. The duration and degree of audiogenic seizure protection appears to correlate with elevations in whole brain GABA levels. gamma-Acetylenic GABA does not protect against seizures induced by pentylenetetrazol or picrotoxin even at doses that increase brain GABA concentrations approximately 6-fold. This differential antiseizure activity suggests that the GABA system may play a role in some, but not all experimentally produced seizures.

Amino acid analysis of the acid hydrolyzate of polymyxin T1 revealed the amino acid composition. Isolation of the constituent amino acids and measurement of their optical activities clarified their chiralities. These were 2,4-diaminobutyric acid (6L), Thr(1L), Leu(2L) and Phe(1D). The constituent fatty acid was identified as anteisononanoic acid by gas chromatography and mass spectrometry. Deacylation with polymyxin acylase afforded deacyl polymyxin T. Successive EDMAN degradation on deacyl polymyxin T revealed most of its amino acid sequence. The chemical cleavage reaction for fragmentation of threonyl peptide amino acid sequence. The chemical cleavage reaction for fragmentation of threonyl peptide on penta(DNP)-polymyxin T1 cleaved it at the C-terminal side of the Thr residue to afford a DNP-octapeptide, whose sequence was clarified by EDMAN degradation. Thus, the structure of polymyxin T1 was determined.

The ability to withstand a fasting stress as related to glucose homeostasis was studied in malnourished rats. Young male Wistar rats were fed a low-protein (D), or a low-protein, low-energy diet (M) containing, respectively, 4% protein with 70% carbohydrate (D) and 4% protein and 45% carbohydrate (M). They were compared with rats fed a control diet with 18% protein and 70% carbohydrate (C). All diets contained 8% fat. After consuming the respective diets for 4 weeks, the rats were either killed or fasted for 24 or 48 hours. Hypoglycemia developed in the malnourished rats upon fasting, but not in those receiving a complete diet. Malnourished rats had a higher blood glycine level after 4 weeks of dietary treatment. There was a marked decrease of circulating alanine and glutamate-glutamine after 48 hours of fasting in all rats. There were no differences in lysine, histidine, glycine and serine-valine. Protein malnutrition produced an increase in brain lysine, gamma-aminobutyric acid, glycine and alanine, and of glycine and alanine in liver. In contrast, D and M rats showed a depletion in muscle lysine, alanine, serine-valine and glutamate-glutamine. The fasting stress produced no changes in brain or muscle glycogen. However, liver glycogen of rats fed the D and M diets was below that of rats fed the C diet for 4 weeks. In addition, after 48 hours of fasting the C rats mobilized more exhaustively their glycogen stores that the M rats. These data suggest that in chronic malnutrition, rats may maintain their glucose homeostasis by mobilization of muscular gluconeogenic amino acids through proteolysis or de novo synthesis. Rats which were fed protein and protein-energy-deficient diets lacked adequate liver glycogen stores and their overall gluconeogenic capacity was exceeded during fasting with resultant hypoglycemia.

The content of dicarboxylic amino acids lowers with age in all the structures of cerebral cortex and cerebellum under study when calculating per I g of fresh tissue and 100 mg of protein. The greatest decrease in the amount of glutaminic and aspartic acids is found in the parietal region at the senile age. The content of GABA in all the brain structures of three-year dogs is significantly higher than in the one-year dogs and at the senile age it is somewhat lower as compared to the three-year animals when calculating per 1 g of fresh tissue, the degree of the decrease in all the studied regions being practically the same.

Amino acids have been separated by gas-liquid chromatography as their N-heptafluorobutyryl isobutyl esters. Complete resolution of derivatives of all the common amino acids has been achieved using a high-performance support-coated open-tubular capillary column. The analysis time was 30 min. Modifications to the derivatization procedure of MacKenzie and Tenaschuk have been introduced. Acylation by heating at 150 degrees was shown to be destructive; 110 degrees has been selected for routine preparation. To obtain a volatile histidine derivative it has been found necessary to add an antioxidant and to heat samples with ethoxyformic anhydride prior to injection. Amino acid analysis of beta-lactoglobulin after 6 N HCl digestion yielded results in good agreement with those obtained by the conventional ion-exchange method. The method has also been successfully applied to estimation of the different caseins in whole casein and in purified fractions by amino acid analysis of residues liberated by carboxy-peptidase digestion.

Baclofen and the internal standard are isolated from biological samples by adsorption on charcoal. After elution, the compounds are converted into butyl esters and purified by base-specific solvent extraction. The butyl esters are then N-acylated with heptafluorobutyrylimidazole, and the neutral derivatives are isolated by solvent extraction and subjected to gas-liquid chromarography, with use of an electron-capture detector. Baclofen can be determined in concentrations down to about 30 ng per sample.

A procedure is available for the production of DL-[carboxy-14C]canaline from [14C]cyanide by reaction of ethyl N-hydroxyacetimidate and acrolein to form ethyl N-[3-oxopropoxy]acetimidate. The reaction product is converted to the nitrile and then to the hydantoin derivative of DL-canaline; alkaline hydrolysis produces the free amino acid (2-amino-4-aminooxypropionic acid). This procedure can be extended to the production of DL-[carboxy-14C]canavanine by guanidination of C-1-labeled DL-canaline with O-methylisourea. A markedly improved colorimetric assay for canaline has been achieved by a procedure involving carbamylation of canaline with cyanate to form O-ureidohomoserine (2-amino-4-ureidooxybutyric acid). Colorimetric analysis of the latter amino acid markedly enhances the sensitivity, reproducibility, and accuracy of the analysis of L-canaline from biological materials.

The quantitative gas--liquid chromatographic analysis of non-protein amino acids, in the presence of protein amino acids, is described. The amino acids were determined as their N-trifluoroacetyl n-butyl esters on an ethylene glycol adipate column. The relative molar responses of 38 amino acids are reported.

The level of y-aminobutyric acid (GABA) was determined in the brain of rats 1 hr. after i.p. injection of chlorpromazine, prochlorperazine, diazepam, trimipramine, methamphetamine and nikethamide. Diazepam increased, and, trimipramine and amphetamine decreased the brain GABA level over wide dose ranges. Low doses of chlorpromazine and prochlorperazine increased but high doses of the drugs reduced the GABA level. Low doses of nikethamide reduced whereas high doses increased the level of GABA. The effects of the drugs have been discussed in relation to the brain GABA level.

A method for the simultaneous quantitation of glutamic acid and gamma-aminobutyric acid (GABA) in tissue by mass fragmentography has been developed. The amino and carboxylic groups of the two amino acids were in a convenient one-step reaction derivatized with pentafluoropropionic anhydride and hexafluoroisopropanol. Deuterium-labeled glutamic acid and GABA and a homologue of GABA have been used as internal standards. The usefulness of the technique has been demonstrated by measurements in parts of rat cerebellum and in the superior cervical ganglion.

A procedure for the identification and quantification of picomole quantities of gamma-aminobutyric acid in tissue samples is given. This procedure combines the chemical specificity of dinitrophenylation with that of gas chromatography mass spectrometry to eliminate the interferences encountered with other direct derivatization procedures. Only a limited number of dinitrophenyl amino acid ethyl esters and some fatty ethyl esters are detected in the solution used for analysis. Identification is based on retention time and on the relative abundances of the three major ion fragments of the gamma-aminobutyric acid derivative. Quantitation is accomplished using isotope dilution techniques with [2H2]gamma-aminobutyric acid as an internal standard. The procedure has been successfully applied to samples of human cerebrospinal fluid and to extracts of ganglia from the mollusc, Aplysia californica.

GAD activity and GABA, the product of GAD action on L-glutamate, are both prominent in mature human renal cortex. GAD activity is low in fetal kidney but rises several fold preterm to establish the characteristic post-term specific activity. The ontogeny of the initial step in the GABA pathway parallels the need for kidney to accommodate acid-base regulation after birth. PLP coenzyme is required for GAD holoenzyme integrity. Fetal renal GAD was frequently undersaturated with PLP in our series of observations, raising the suggestion that maternal vitamin B6 nutrition is not always adequate.

Cysteinesulfinate, hypotaurine and taurine, which are key metabolites of cysteine, can be separated from each other and other closely eluting amino acids in biological samples by reversed-phase high-performance liquid chromatography on a Waters Nova-Pak C18 column. Samples were derivatized with o-phthalaldehyde-2-mercaptoethanol prior to injection. The elution system consisted of 100 mM potassium phosphate buffer, pH 7.0, with 3% (v/v) tetrahydrofuran with an initial isocratic phase at 1.2% acetonitrile and a gradient from 1.2 to 12.8% acetonitrile. This method is suitable for measurement of the production of metabolites from cysteine by isolated cells and for analysis of plasma and tissue extracts. Low levels of hypotaurine in rat tissues were easily measured with this method and are reported here for the first time.

The distributions of gamma-aminobutyric acid (GABA), glycine, glutamate and aspartate were measured in cochlear nuclei of two rats by quantitative histochemical mapping procedures. The levels and distributions in the two rats were comparable, and resembled those previously reported for cat cochlear nucleus. The results are consistent with a concept that these putative transmitter amino acids have similar levels and distributions in the cochlear nucleus among mammals.

A sensitive and specific method for the determination of gamma aminobutyric acid in small (30 mg) brain samples is described. It is based on preparation of the dansyl-butyrolactam (DBL), subsequent separation by thin layer chromatography and measurement of fluorescence after elution. Recovery is checked by routinely adding 14C labelled GABA.

The effect of factors inducing somatic differentiation on cellular GABA level has been investigated. C6 glial cells from a rat astrocytoma present a significantly higher GABA level than the other glial or neuronal cells studied. A significant decrease in GABA levels in most cases parallels the somatic differentiation induced either by withdrawal of fetal serum, or by addition of dibutyryl cyclic AMP to the culture medium.