Jean K. Tews

FREE AMINO ACIDS AND RELATED COMPOUNDS IN DOG BRAIN: POST-MORTEM AND ANOXIC CHANGES, EFFECTS OF AMMONIUM CHLORIDE INFUSION, AND LEVELS DURING SEIZURES INDUCED BY PICROTOXIN AND BY PENTYLENETETRAZOL*

PREVIOUS studies in this laboratory have been concerned with chemical changes in the brain associated with convulsive activity (STONE et al., 1960) as revealed by analysis of brain tissue after in vivo fixation with liquid air. The development by MOORE and STEIN (1954) of ion exchange procedures for the separation of amino acids has made it feasible to extend the study of cerebral constituents during convulsions to include many free amino acids and related substances. These compounds are of particular interest in view of recent investigations by GEIGER and his coworkers (1960 a, 6) indicating that protein turnover in the brain is accelerated during convulsive activity induced by pentylenetetrazol. Accordingly, the cerebral levels of a large number of nitrogenous constituents have been determined in control experiments and during seizures induced by picrotoxin or by pentylenetetrazol. In an attempt to classify different types of seizures on the basis of specific chemical patterns (STONE et al., 1960), the seizures induced by these two agents have been tentatively classed together as representing a type in which distinctive metabolic changes have not yet been observed. As an aid in the interpretation of any changes which might be observed during seizures or other conditions, it was considered essential to study also post-mortem changes and the effects of anoxia and of ammonium chloride infusion.

CHEMICAL CHANGES IN THE BRAIN DURING INSULIN HYPOGLYCAEMIA AND RECOVERY

EARLY studies of insulin shock, particularly those by HIMWICH and his collaborators, showed that profound hypoglycaemia is accompanied by a decreased cerebral uptake of glucose and of oxygen from the blood stream. HIMWICH (1951) reviewed these studies and cited much evidence indicating that the behavioural and electrographic manifestations of hypoglycaemia are related to the reduced supply of glucose to the brain and the consequent decrease in available oxidative energy. More recently, GEIGER (1958) and his coworkers found that the perfused brain of the cat is able to function for more than 1 hr without exogenous glucose if a high perfusion rate is maintained ; this finding suggests that the brain can utilize endogenous non-carbohydrate substrates and that hypoglycaemic symptoms are due in part to the accumulation of toxic breakdown products of these substrates. ABOOD and GEIGER (1955) found decreases in the protein, lipid and nucleic acid contents of the perfused cat brain deprived of glucose and an associated accumulation in the perfusing blood of small quantities of nitrogenous substances, especially GABA, glutathione, glutamate and creatine. On the other hand, SAMSON, DAHL, DAHL and HIMWICH (1959) found that insulin shock did not significantly decrease the cerebral proteins in cats, and DAWSON (1950) made the same observation in rats. A number of other cerebral constituents are known to undergo quantitative changes during hypoglycaemia. These include glycogen, acid-soluble phosphates, and certain of the free amino acids and related compounds. Some of these substances represent possible sources of limited amounts of energy, either as oxidative substrates or as phosphorylated intermediates, while others may occur as breakdown products of proteins, lipids, or other substrates. Pertinent reports are cited in subsequent sections of this paper; in general, these chemical changes have not been studied in temporal relation to the development of electrographic abnormality. We have attempted to follow simultaneously the progression of the electrographic and a number of chemical changes in insulin shock, and their reversal on termination of the hypoglycaemia. E X P E R I M E N T A L The experiments were done on adult male dogs weighing 8-23 kg. Food was withheld for a period of 18-24 hr, after which the cranium was exposed and opened and most of the calvarium was removed, the dura mater remaining intact. Preparation was made for electrographic recording from three cortical areas and for freezing the brain in situ with liquid air. Blood pressure was measured from a cannulated femoral artery in the final stages of the experiment. The details of these procedures have been described (TEWS, CARTER, ROA and STONE, 1963).

A neurochemical study of thiosemicarbazide seizures and their inhibition by amino-oxyacetic acid

Abstract Effects of intravenous thiosemicarbazide were studied in dogs by recording cortical electrical activity and by analyzing cerebral tissue frozen in situ. Free amino acids and related substances were measured. Generalized seizures were accompanied by a decrease in γ-aminobutyric acid and increases in alanine, ammonia, lactic acid, and tyrosine. In another group of dogs, intravenous amino-oxyacetic acid induced a decrease in brain aspartic acid and increases in alanine, γ-aminobutyric acid, ammonia, glutamine, lactic acid, lysine, and tyrosine. The changes in aspartate, alanine, glutamine, and lactate may be secondary to the high brain ammonia, which in turn can be attributed at least in part to ammonia appearing in the blood. These chemical changes were usually accompanied by varying degrees of electrographic depression, but occasionally by signs of cerebral excitation and in one instance by a generalized seizure (the excitatory effects being more prominent at higher dose levels). Amino-oxyacetic acid had an inhibitory effect on thiosemicarbazide seizures. The inhibition was overcome by increasing the dose of thiosemicarbazide, although γ-aminobutyric acid in the brain remained above normal. The combined effects of the two drugs on the chemical pattern were essentially the same as the effect of amino-oxyacetic acid alone. Amino-oxyacetic acid raised the convulsive threshold to pentylenetetrazol but showed little antagonism to picrotoxin. The inhibitory effects of amino-oxyacetic acid may be due to the increase in brain of γ-aminobutyric acid. The excitatory effects of hydrazides and of amino-oxyacetic acid, which are carbonyl-trapping agents, may involve the same (unknown) basic mechanism, with a decrease in y-aminobutyric acid potentiating excitation by hydrazides and an increase opposing excitation by amino-oxyacetic acid.

CEREBRAL AMINO ACIDS AND LIPIDS IN DRUG-INDUCED STATUS EPILEPTICUS*

—Dogs were given repeated doses of pentylenetetrazol to maintain a condition of status epilepticus for periods of 30–40 min. Analyses of cerebral tissue frozen in situ showed significant increases in alanine, arginine, γ‐aminobutyric acid, glycine, histidine, leucine, lysine, phenylalanine, serine, tyrosine, and valine. Decreases occurred in the glutamic and aspartic acid levels. Other measured amino compounds were unchanged. Ammonia was increased, but not more than occurs in seizures of brief duration. A large decrease was noted in the ganglioside fraction, and a decrease also in a fraction containing the lecithins and sphingomyelins. The cephalin, cerebroside–sulphatide, and cholesterol fractions were not affected.

Transport of threonine and tryptophan by rat brain slices: relation to other amino acids at concentrations found in plasma.

Threonine content of brain decreases in young rats fed a threonine‐limiting, low protein diet containing a supplement of small neutral amino acids (serine, glycine and alanine), which are competitors of threonine transport in other systems (Tewset al., 1977). Threonine transport by brain slices was inhibited more by a complex amino acid mixture resembling plasma from rats fed the small neutral amino acid supplement than by mixtures resembling plasma from control rats or from rats fed a supplement of large neutral amino acids. Greater inhibition was seen with mixtures containing only the small neutral amino acids than with mixtures containing only large neutral amino acids. On an equimolar basis, serine and alanine were the most inhibitory; large neutrals were moderately so; and glycine and lysine were without effect. Threonine transport was also strongly inhibited by α‐amino‐n‐butyric acid and homoserine, less so by α‐aminoisobutyric acid, and not at all by GABA.

Free amino acids and related compounds in brain and other tissues: effects of convulsant drugs.

Publisher Summary This chapter summarizes a series of studies on free amino acids and related compounds in the brain. Cerebral tissue was analyzed by ion-exchange chromatography and other methods, and the technique of freezing the brain in situ with liquid air was used to avoid post-mortem increases that otherwise occur in alanine, ammonia, γ-aminobutyric acid and lactic acid. Dogs were used for most of the experiments, but supplemental studies on the effects of fluoro compounds in mice are included in the chapter. Anoxia induced by the administration of 4.5% oxygen in nitrogen for 12–13 min brought about significant increase in alanine, y-aminobutyric acid, glutamic acid, lactic acid, leucine, and tyrosine and decrease in aspartic acid and a fraction consisting of methionine and cystathionine. Ammonia was not significantly increased. Infusion of buffered ammonium chloride for a 10 min or 45 min period generally caused some degree of depression of cortical electrical activity, although in one animal a seizure developed. Amino-oxyacetic acid, which is known to antagonize the convulsant actions of thiosemicarbazide and methionine sulfoximine, was found to induce increase in brain γ-aminobutyrate, alanine, ammonia, glutamine, lactate, lysine, and tyrosine and a decrease in aspartate. Methionine sulfoximine was found to have extensive effects on the nitrogenous metabolism of the brain, causing increases in alanine, ammonia, lysine, phosphoethanolamine, and serine and decrease in aspartate, glutamate, glutamine, leucine, and the methionine and cystathionine fraction.

Transport of nonmetabolizable amino acids in rat liver slices

Abstract Transport of α-amino [ i -14C]isobutyric acid (AIB) by rat liver slices against a concentration gradient has been demonstrated; uptake was improved by including a preincubation step. Similarities to other systems included linearity of uptake over an extended period of time, as well as indications of saturability of the system with increasing concentrations of substrate. The transport of AIB was inhibited by anoxia and by 2,4-dinitrophenol, while glucose was without effect; inhibition also occurred in the presence of ouabain. No evidence for the active transport of AIB was seen when Na+ was totally replaced in the medium. The removal of extracellular K+ or Ca2+ markedly decreased the transport of AIB, although some uptake against a concentration gradient still occurred. The replacement of Mg2+ had little or no effect on the gradients achieved. Uptake was lower in a Krebs-Ringer phosphate than in a Krebs-Ringer bicarbonate buffer. The Na+ and K+ concentrations of slices incubated in the presence of AIB were similar to those of fresh liver. Cycloleucine was also transported against a concentration gradient but to a lesser extent than AIB.

Metabolism and transport of γ-carboxyglutamic acid

Abstract γ-Carboxyglutamic acid residues have been shown to be present in prothrombin, the other vitamin K-dependent clotting factors, and more recently in bone and kidney proteins. This amino acid is formed by a posttranslational vitamin K-dependent carboxylation of glutamyl residues in polypeptide precursors of these proteins. It has now been demonstrated that this amino acid, either in the free or peptide-bound form, it not metabolically degraded by the rat, but is quantitatively excreted in the urine. In nephrectomized rats, the tissue concentration of intravenously administered γ-carboxyglutamic acid is increased, but there is still no evidence of any oxidative metabolism of this amino acid. This amino acid is transported by kidney slices against a concentration gradient, but does not accumulate in liver, intestinal or brain tissues. Prelimininary data suggest that γ-carboxyglutamic acid may be concentrated by a carrier system from the utilized by other amino acids.

The effect of a nutritional pyridoxine deficiency on free amino acids and related substances in mouse brain.

IT IS well known that seizures frequently result from pyridoxine insufficiency. The convulsions often may be induced by feeding a diet deficient in pyridoxine or by administering any of a number of antagonists (see TOWER, 1956; WILLIAMS and BAIN, 1961 ; HOLTZ and PALM, 1964; for extensive reviews). Vitamin B, in the phosphorylated form is intimately involved in amino acid metabolism (MEISTER, 1965). Although attention has been devoted to examining changes in free amino acid levels in brains of animals treated with pyridoxine antagonists (e.g. KILLAM and BAIN, 1957; KAMRIN and KAMRIN, 1961; ROA, TEWS and STONE, 1964), these effects are not necessarily the same as those which might be seen in a simple nutritional deficiency. Therefore, it was thought desirable to determine levels of amino acids in brains of animals made either mildly or severely deficient by feeding a diet lacking the vitamin and to note whether or not reversal of the neurochemical effects occurred upon restoration of pyridoxine to the diet. M E T H O D S Female, weanling, albino mice (Rolfsmeyer Farms, Madison) were divided into three groups. Group I received a commercial semi-synthetic diet (Nutritional Biochemicals Corporation, Cleveland, Ohio) containing no added vitamin Be; Group LI was fed ad libitum the same diet but with pyridoxine-HCl present (22 mg/kg diet); Group 111 also received the complete ration, the amount given being restricted to that consumed by the deficient mice of Group I. The animals were weighed weekly, and food consumption measurements were made at 2 day intervals. At the end of a 4-week period of deficiency the complete diet was restored to the remaining depleted animals, and each was given an intraperitoneal injection of 50 pg of pyridoxine-HC1 in 0.1 ml saline (MIRONE and JACKSON, 1959). The injection was repeated 2 days later. Randomly chosen animals from each group were killed by dropping into liquid nitrogen after 2,4, or 6 weeks on the diet, the last period being 2 weeks after resumption of the complete diet in Group I. The brains (not including cerebella) were removed and kept frozen in nitrogen until extraction procedures could be carried out. Levels of free amino acids and related compounds were determined on picric acid extracts of seven to ten pooled brains according to the ion exchange procedure of MOORE and STEIN (1954), slightly modified. Ammonia, glutamine, and lactate levels were measured in trichloroacetic acid extracts from three pooled brains. The analytical details were described previously (TEWS, CARTER, ROA and STONE, 1963). Statistical evaluation of results was based on the t-test method for small samples (MAINLAND, 1938).

Some Characteristics of Threonine Transport Across the Blood-Brain Barrier of the Rat

Abstract: Threonine entry into brain is altered by diet‐induced changes in concentrations of plasma amino acids, especially the small neutrals. To study this finding further, we compared effects of various amino acids (large and small neutrals, analogues, and transport models) on transport of threonine and phenylalanine across the blood‐brain barrier. Threonine transport was saturable and was usually depressed more by natural large than small neutrals. Norvaline and 2‐amino‐n‐butyrate (AABA) were stronger competitors than norleucine. 2‐Aminobicyclo[2.2.1]heptane‐2‐carboxylate (BCH), a model in other preparations for the large neutral (L) system, and cysteine, a proposed model for the ASC system only in certain preparations, reduced threonine transport; 2–(methylamino)isobutyrate (MeAIB; a model for the A system for small neutrals) did not. Phenylalanine transport was most depressed by cold phenylalanine and other large neutrals; threonine and other small neutrals had little effect. Norleucine, but not AABA, was a strong competitor; BCH was more competitive than cysteine or MeAIB. Absence of sodium did not affect phenylalanine transport, but decreased threonine uptake by 25% (p < 0.001). Our results with natural, analogue, and model amino acids, and especially with sodium, suggest that threonine, but not phenylalanine, may enter the brain partly by the sodium‐dependent ASC system.