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The metabolism of radioactive glutamic acid in normal and epileptic cat brain Steiner, K. David 1962

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THE METABOLISM IN NORMAL OF RADIOACTIVE GLUTAMIC ACID AND EPILEPTIC CAT BRAIN by K.. DAVID STEINER B i S c , University of Br i t ish Columbia, I960 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science in the Kinsmen Laboratory of Neurological Research Department of Psychiatry We accept this thesis as conforming to the requi red standard THE UNIVERSITY OF BRITISH COLUMBIA October, 1962 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis f o r scholarly purposes may be granted by the Head of my Department or by his representatives. It i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of / /O The University of B r i t i s h Columbia, Vancouver 8, Canada. Date Q(j. Of ) I Ii ABSTRACT The indirect in vivo inhibit ion of the enzyme glutamic acid decarboxy-lase (GAD) by either vitamin B-6 deficiency or the administration of vitamin B-6 antimetabolites decreases the conversion of glutamic acid to gamma aminobutyric acid (GABA) in brain. This decrease is a concomitant to the occurrence of seizures resembling grand mal. The in vivo reactivation of GAD by vitamin B-6 administration and/or by the topical application of GABA to the brain surface reduces the intensity of the convulsions. Because of th is , i t has been suggested that a decrease in the conversion of glutamic acid to GABA in brain may be a factor in the precipitation and maintenance of epi lept ic seizures. In order to investigate this suggestion, the in vivo metabolism of C-14 labelled glutamic acid to GABA and other amino acids was quantitatively determined in various brain areas of several normal cats; a cat with a e p i l -eptogenic lesion in the lef t motor cortex produced by alumina cream; and one cat in status epi lept icus. Throughout most non-epileptogenic brain areas there were similar rates of conversion of glutamic acid to GABA, aspartic acid and glutamine. Notable exceptions to this consistency were found repeatedly in the quadri-geminal plate, thalamus and putamen-globus pallidus where there was a higher conversion of glutamic acid to GABA. No apparent ^consistency in the degree of conversion of glutamic acid to the other amino acids could be discerned throughout the brain of the cat in status epi lept icus. i i i In the epileptogenic lesion there was a decrease in the conversion of glutamic acid to GABA, which is compatible with the suggestion that a reduction in GABA levels increases the degree of brain exc i tab i l i t y . INDEX fv Page INTRODUCTION . . . . 1 Part I. LITERATURE SURVEY . . 4 A . Biochemical Considerat ions . . . . . . . . . . . 4 1. The Glutamic A c i d Shunt . . . . 4 2 . The Role of I n t r a c e l l u l a r pH in the Regulat ion of GABA Levels in the CNS . . 8 3. Cerebral Compartments in A c i d Metabolism 10 B. Phys io log ica l and Chemical Roles o f GABA . . 14 1. General Phys io log i ca l E f f e c t s of GABA . . 14 2 . GABA as a Neurotransmitter 17 3. GABA as a Precursor f o r other CNS Metabol i tes 20 C. Funct ional Considerat ions . . . . . . . . . . 25 1. Ammoniagenic Coma 25 2 . Amino A c i d L e v e l s , Vitamin B-6 and Seizures . 28 Part II. DEVELOPMENT OF METHOD A . Intravenous In ject ion of Control Animals . . 36 B. Preparat ion of T issue Ext racts . . . . . . . . 36 C. Chromatographic Separat ion of Amino Ac ids . . 37 D. Radioautography . . . . . . 37 E. A Note on L iqu id S c i n t i l l a t i o n Counting . . . . 37 F. Assay f o r Label led Amino Ac ids Separated.by;•. Paper Chromatography . . . . . . 39 1. Experiments Designed to Test C r i t e r i a . . 40 2 . Loss of Cpm During Spot Locat ion . . . . 40 3. Locat ion of Amino Ac ids by F luorescence 41 4. Proof of L i n e a r i t y 43 5. E f f e c t of Paper Immersion in the S c i n t -i l l a t i o n S o l u t i o n on Counting E f f i c i e n c y 45 6. R e p r o d u c i b i l i t y of Counts on Dupl icate Chromatograms 45 G. Procedure f o r Radioact ive A n a l y s i s 46 H. Measurement of Amino A c i d Pools 47 I. Animal Preparat ions . . . . . . . . 49 1. Cat with Ep i lep togen ic Focus 49 V INDEX, c o n t ' d . Page 2 . Preparat ion of Cat f o r Study of the Tune Changes of the Metabolism of C-\k Glutamic Ac id in the Cortex 49 J . Post Mortem Changes in L a b e l l i n g Patterns . . 50 Part III . RESULTS AND DISCUSSION 51 A . Q u a l i t a t i v e Metabolism of Glutamic A c i d . . 51 B. Quant i ta t i ve D i s t r i b u t i o n of L a b e l l i n g . . 5*+ C. S i z e of Amino A c i d Pools in Brain . . . . 5*t D. Changes in the D i s t r i b u t i o n of L a b e l l i n g i n T i me . . 58 E . Deviat ions from Cons is tent L a b e l l i n g Patterns 6h F. L a b e l l i n g Patterns in the Focal Lesion . . 65 G. L a b e l l i n g Patterns Observed in a Cat in Status E p i l e p t i c u s . . 66 SUMMARY 71 APPENDIX . . . . 72 A . Introduction:; . . . . 72 B. Def in i t ions 72 C. Determination of Counting E f f i c i e n c y of C-]k Labe l led Toluene Insoluble Mater ia l on Paper . . . . 72 D. E f f i c i e n c y - V o l t a g e Curves . . . . . . . . 7** E. Background . . 71* BIBLIOGRAPHY . . 77 vi INDEX TO TABLES AND FIGURES Page Table 1. E f f e c t o f Chromatography on the Recovery of R a d i o a c t i v i t y .. .. .. 40 Graph A . E f f e c t of Ninhydrin on the E f f i c i e n c y of L iqu id S c i n t i l l a t i o n Counting . . .. .. 42 Table 2. E f f e c t o f Heating on the Recovery of R a d i o a c t i v i t y in Amino Ac ids on F i l t e r Paper . . .. . . . . .. 43 Graph B. L iqu id S c i n t i l l a t i o n Counting of C-14 Labe l led Glutamic A c i d on Paper a f t e r Chromatographic Separat ion . . 44 Table 3* E f f e c t o f Paper Immersion in L iqu id S c i n t i l l a t i o n S o l u t i o n on Counting E f f i c i e n c y 46 Table 4. R e p r o d u c i b i l i t y of Cpm in Amino Ac ids Separated by Paper Chromatography 47 Graph C. E f f e c t o f Heat on Amino A c i d Recovery . . * 48 Diagram I. Major Pathways of Glutamic A c i d Metabolism in Brain . . 52 Diagram II . Chromatogram of Components in Aqueous Brain Ex t rac t . . 53 Table 5* Comparisons o f the D i s t r i b u t i o n of L a b e l l i n g in Amino Ac ids in D i f f e r e n t Brain Areas . . .. 55 Table 6. Concentrat ions of Amino Ac ids in D i f f e r e n t Bra in Areas . . • 56 Table 7. S p e c i f i c A c t i v i t i e s o f Amino Ac ids in D i f f e r e n t Bra in Areas 57 Table 8. Average Brain Values of Amino A c i d Pools 58 Table 9 . Longi tudinal Time Study of Glutamic A c i d Metabolism in Cat Cortex . . .. 60 Graph D. Changes in L a b e l l i n g Patterns in Time in Cat Cortex . . 61 Table 10. Comparison of Longitudinal and Cross Sec t iona l Time Studies 62 Table 11. D i s t r i b u t i o n of R a d i o a c t i v i t y in Blood Glutamic A c i d and Glutamine 62 v i i INDEX TO TABLES AND FIGURES, c o n t ' d . Page Table 1 2 . Estimated Cont r ibut ion of Label led Glutamic A c i d and Glutamine in Blood, to the R a d i o a c t i v i t y in Brain T i ssue . . . . . . . . . . 63 Table 1 3 . D i s t r i b u t i o n of R a d i o a c t i v i t y in Amino Ac ids Throughout the Brain o f a Cat in Status E p i l e p t i c u s 67 Table \k. Changes in L a b e l l i n g Patterns in T i ssue L e f t a t Room Temperature f o r Two Hours 70 Graph E. E f f i c i e n c y Curves f o r D i f f e r e n t C-14 Counting Systems . . 75 Graph F. Background Curve of Paper Blank . . . . 76 INTRODUCTION Much biochemical and physiological evidence has been assembled in recent years which associates an interference in brain glutamic acid metabolism with both pharmacologically-induced and naturally occurring convulsive states. Tower (98) proposed that a biochemical lesion in the metabolism of glutamic acid to GABA was responsible for seizures induced by vitamin B-6 deficiency. Killam and Bain (17). Roberts (8), and Purpura (93) observed that seizures could be induced by the Vitamin B-6 antimetabolites thiosemicarbazide (TSC) (8, 17) and methoxypyridoxine (M0-B6) (93), with a subsequent decrease in the cerebral levels of gamma aminobutyric acid (GABA). These workers suggested that the enzyme glutamic acid decarb-oxylase (GAD) which converts glutamic acid to GABA was indirectly inhibited by these antimetabolites resulting in a decrease in the rate of GABA formation. Gammon (9*0 and Dasgupta et a K (90) have successfully protected mice (3k) and cats (90) from M0-B6 and TSC induced seizures with GABA, while Woodsbury and Espin (105) have demonstrated that the level of cerebral GABA and the cerebral glutamine/glutamic acid ratios are inversel proportional to the levels of brain excitation in pharmacologically and e lec t r ica l ly induced convulsions. These observations are consistent with Curt is ' findings (37, 38, 39) that GABA depressed the act iv i ty of cat motoneurones, with Kuffler and Edwards' findings (28) that low concentta-tion of GABA inhibited or blocked discharges from a stimulated crayfish receptor neurone, and with Hayashi's findings (106) that GABA inhibited both e lec t r ica l ly and chemically induced convulsions. 2. Tower (99) has shown that the In v i t r o metabolism of glutamic a c i d and GABA was a l t e r e d in bra in t i s s u e taken from experimental animals with se izures induced by th iosemicarbazide (TSC), megamid, methionine su l fox imine , and in excised human c o r t i c a l e p i l e p t i c f o c i . The patterns of metabol ic change in these t i ssues were s i m i l a r to each other but d i f f e r e n t from contro l bra in t i s s u e . The a d d i t i o n of GABA to the incuba-t ion medium tended to s i g n i f i c a n t l y co r rec t the a l t e r e d metabol ic patterns in the a c t i v e t i s s u e . In c o r t i c a l foca l les ions produced by ethyl c h l o r i d e spray , Berl et a K (102) found that glutamic a c i d decreased in the l e s i o n s i t e , when compared with c o n t r a l a t e r a l contro l a r e a s . GABA, on the other hand, d id not change s i g n i f i c a n t l y , but i t d id suppress paroxysmal discharges from the les ion (103) a f t e r intravenous i n j e c t i o n . It has been suggested ( 6 ) that the normal func t ion ing of neurones in the CNS is dependent upon the balance between those mechanisms which produce neuronal e x c i t a t i o n and those mechanisms which produce neuronal i n h i b i t i o n . The evidence given would i n d i c a t e that GABA c o u l d , in p a r t , be responsib le f o r neuronal i n h i b i t i o n , and a subsequent decrease of i t in bra in could be a f a c t o r in e p i l e p s y . C u r t i s (37, 38, 39) has found that glutamic a c i d was exc i ta to ry to cat motoneurons, whi le GABA ( i t s decarboxy lat ion product) was i n h i b i t o r y . This observat ion suggests that the mechanisms of neuronal e x c i t a t i o n and i n h i b i t i o n could be a r e f l e c t i o n of the r e l a t i v e concentrat ion of glutamic ac id and GABA in neurones. These r e l a t i v e concentrat ions are c o n t r o l l e d in part by the i n t e g r i t y of the enzyme GAD, which,.when blocked (8, 17), does produce convuls ions with a f a l l in cerebral GABA l e v e l s . 3. The purpose of this thesis is to measure the metabolism of glutamic acid through the glutamic acid shunt in chronic focal i r r i ta t ive lesions produced in cats by alumina cream (107). Methods (8, 2k) using injections of labelled C-14 glutamic acid were previously shown to be ef fect ive, in the study of glutamic acid metabolism in brain. These techniques were modified in order to f i t this particular problem. Berl and Waelsch (108) designed an excellent method to separate and measure glutamic acid and i ts metabolites in brain t issue. This method, although very precise, is cumbersome and time consuming. Since an assay of thirty key anatomical areas was anticipated in this study, the use of Berl and Waelsch1s method was impractical. Therefore, a more rapid method for the measurement of labelled products of glutamic acid was devised. The development and testing of this method forms the f i r s t portion of the experimental work. The second portion is concerned with applying this technique to the analysis of both normal and epileptogenic brain areas with the view of determining i f any qualitative or quantitative differences exist in the metabolism of glutamic acid in excitable t issue. 4. PART I. LITERATURE SURVEY A . Biochemical Cons ide ra t ions . 1. The Glutamic Ac id Shunt. The development of paper chromatography o f f e r e d s c i e n t i s t s a technique by which they could rap id ly determine the d i s t r i b u t i o n of d i f f e r e n t c lasses of metabol i tes in var ious t i ssues and organs . Using th i s technique to survey substances in aqueous bra in e x t r a c t s , Roberts (1) observed large amounts of an unknown ninhydr in p o s i t i v e spot on h i s chromatograms. This mater ia l was i s o l a t e d and t e n t a t i v e l y i d e n t i f i e d as gamma aminobutyric ac id (GABA) by Roberts (2) and by Awapara (3) in independent s t u d i e s . P o s i t i v e i d e n t i f i c a t i o n was made a short time l a t e r by Udenfriend (4) us ing the isotope d e r i v a t i v e method. The discovery of th i s prev ious ly unknown biochemical compound st imulated Roberts to make extensive measurements (5) o f GABA in the t i s s u e ex t rac ts of var ious species of mammals, b i r d s , r e p t i l e s , amphibians and f i s h . From a synthesis of h is observat ions , Roberts drew the fo l l ow ing conc lus ions : (a) In a l l the species measured GABA occurs in detectab le amounts, only in the CNS, mainly in grey matter (6), and (b) the quant i ty of ' f r e e ' o r e a s i l y ex t rac tab le GABA is s p e c i f i c f o r any given s p e c i e s . By experimenting with various enzyme systems in v i t r o , Roberts (7) proved that the enzymatic decarboxy lat ion of glutamic a c i d was one poss -i b l e pathway which could form GABA. As y e t , no other GABA forming pa th -way has been detected . 5. The speci f ic enzyme for this step is glutamic acid decarboxylase (GAD). This enzyme requires pyridoxal phosphate (Pyr-P) as a coenzyme, and, l ike GABA, has only been found in detectable amounts in the CNS. In vi tro evidence of GAD act iv i ty was obtained in mice (8) when C-)k GABA was isolated from brain extracts after an intracerebral injection of C-14 ac id . Studies done by Albers (9) on GABA catabolism strongly suggested that gamma aminobutyric-alphaketoglutate transaminase (GABA-T) catalyzes the conversion of the carbon skeleton of GABA to succinic semi aldehyde (SSA). Apparently alphaketoglutarate is the specif ic receiver of the amino group from GABA. GABA-T, l ike GAD, is a Pyr-P-dependent enzyme, but the degree of a f f in i t y of the enzyme-coenzyme complex is greater for GABA-T than for GAD. Unlike GAD, GABA-T has been isolated in l iver (1), a tissue lying outside the CNS. It has been conjectured that i ts only function in this organ is to destroy traces of GABA circulat ing in the blood. SSA is a very labi le constituent of brain. There Is evidence that this metabolite is oxidized both enzymatically (11) and non-enzymatically (12) to succinic ac id . Albers (9) found the oxidative enzyme to be very active in mito-chondria. Although this enzyme displays many of the properties of a typical aldehyde dehydrogenase, i t exhibits a relative spec i f ic i ty for semi aldehydes. GABA l ies in the center of a pathway which offers an alternate metabolic route from alphaketoglutarate to succinate. This route, called the glutamic acid shunt (GAS) by-passes the succinyl CoA step in the TCA cycle. One noteworthy characterist ic of this shunt is itssproperty to regenerate one precursor molecule (glutamic acid) f o r every molecule of product (GABA) converted to s u c c i n i c semialdehyde. This property gives the shunt a ' b u i l t i n 1 s e l f - r e g u l a t i n g system which makes i t p o s s i b l e f o r the ra t e of GABA degradation to c o n t r o l the ra t e of GABA formation. The c r i t i c a l f a c t o r o f t h i s p o s i t i v e feed-back system i s the maintenance of an adequate l e v e l of a l p h a k e t o g l u t a r a t e to r e c e i v e the amine group from GABA. According to A l b e r s (9) the nature of t h i s i n t r i n s i c regulatory system could be one explanation f o r the d i f f i c u l t y i n producing large changes i n cere b r a l GABA l e v e l s , although a rapid turnover ra t e f o r these l e v e l s has been reported (8). In p r e l i m i n a r y i n v i t r o s t u d i e s , McKhann (13) has determined that approximately 40% of the a l p h a k e t o g l u t a r a t e i s metabolized v i a the glutamic a c i d shunt. Microtechniques have r e c e n t l y been devised by A l b e r s (9, 14) which enabled him to measure a c c u r a t e l y the s p e c i f i c GAD and GABA-T a c t i v i t i e s i n very small t i s s u e segments. U t i l i z i n g these techniques, A l b e r s has made great progress i n mapping the l e v e l s of these enzymes i n various areas of monkey b r a i n and s p i n a l cord. G e n e r a l l y , h i s data shows that both enzymes, l i k e GABA, are found predominantly i n grey matter. The a c t i v i t i e s o f these two enzymes vary from area to a r e a , i n some areas the a c t i v i t y of the decarboxylase enzyme i s greater than the t r a n s -aminase, but, i n general, the transaminase-decarboxylase a c t i v i t y r a t i o (T/A r a t i o ) i s greater than one. A l b e r s b e l i e v e s that the higher transaminase a c t i v i t y r e l a t i v e to decarboxylase a c t i v i t y could be a means by which the accumulation of i n t r a c e l l u l a r GABA would be impeded i f the intracel lu lar glutamic acid were suddenly increased. He reasons that i f the T/A ratio were greater than one, and i f the substrate were freely accessible to the enzymes, then GABA would be transaminated to SSA more rapidly than i t would be formed from glutamic ac id . Under these condi-tions, i t would be impossible to increase the GABA levels by increasing the glutamic acid levels . Following the same line of reasoning, i t was postulated that in the areas where the T/A ratio are the highest, there should be a cor-respondingly low level of GABA. Albers (14) found the highest T/A ratios in the midbrain structures. If his postulate were true, GABA should be low in these areas. Cursory observations made on cats (15) and humans (16) show that GABA reaches i ts highest levels in these areas. If the T/A ratios in cats and humans are found to be similar to Albers measurements on monkeys, i t would completely invalidate his conten-t ion. No simultaneous measurements of the GABA levels and the T/A ratios in any experimental animal have been reported so far in the l i terature . A short note on a possible physiological role of GABA is necessary at this point in order to give some background to the concepts which wi l l be examined in the next portion of this review. This background material wi l l be discussed in a more comprehensive manner in further sections. In i t ia l l y , because of i ts property to mimic superf ic ia l ly the process of neuronal inhibi t ion, GABA had been suggested as a possible candidate for the chemical transmission of inhibitory impulses in the CNS. In the l ight of recent data, this role has been modified. At the present time, GABA is thought to function as a 'physiological damper,' which acts on the neuronal membrane in such a manner as to keep the level 8. of excitation below a certain l imi t . Seizure susceptibi l i ty has been linked to the fa i lure of this mechanism to operate ef fect ive ly . If this is true, one manner by which this mechanism could be impaired is to lower the amount of GABA in the c e l l , thus effect ively lowering the neuronal threshold to excitatory impulses. The partial or total selective inhibit ion of GAD by chemical means has resulted in a decreased GABA level which has been correlated with the occurrence of seizures ( 1 7 ) . The state which appears to be physiologically opposite to seizures is lethargy and coma. This latter state has been reported (8) to occur when GABA levels were raised by the selective competitive inhibit ion of GABA-T. 2 . The Role of Intracellular pH in the Regulation of GABA Levels in  the CNS. The data on the pH optimums (6) of GAD and GABA-T suggest that the relative act iv i t ies of these enzymes can be greatly altered by s l ight changes in the intracel lular pH. GAD reaches i ts optimum act iv i ty at acid pH, while GABA-T reaches i ts optimum act iv i ty at basic pH. The intersection of the pH act iv i ty curves (6) of GAD and GABA-T occurs at pH 7 . 5 . which is close to the estimated intracel lu lar pH of 7 . 1 . At this pH both enzymes are operating at about one-half their maximum act iv i ty . If one assumes that the enzymes behave s imi lar ly in vivo as they do in v i t ro , then intracel lular acidosis would be expected to favor GAD and an increase in GABA, while intracel lu lar alkalosis would favor GABA-T and a decrease in GABA. The intracel lular pH of neurones is controlled part ia l ly by the CO^-bi carbonate buffer system (6). Carbonic anhydrase (6), an enzyme which accelerates the interconversion of C0 2 and bicarbonate, is a c r i t i c a l factor in the maintenance of this buffer system. It has been shown by Caldwell (18) that an increase of the pC02 outside the neurone tends to decrease intracel lu lar pH. Carbonic anhydrase appears (6) to modulate the resulting pH fluctuations which arise from the fluctuations of the intra- and extracellular C0 2 levels . Any inhibit ion of carbonic anhydrase would thus tend to lead to intracel lu lar acidosis and an 'a p r i o r i ' increase in GABA levels. The anticonvulsant properties of diamox and methazolamide have been attributed to their direct inhibit ion of carbonic anhydrase (6). Simi lar ly , the anticonvulsant properties of C0 2 can also be explained by Roberts' model, since this would tend to have the same effect ( int ra -ce l lu lar pH reduction) as the inhibit ion of carbonic anhydrase. The epileptogenic effects of hyperventilation f i t very well into this scheme since this process results in a gradual depletion of C0 2 levels from the blood which would tend to raise the intracel lu lar pH, thus depleting GABA. It has been reported (19) that diamox has blocked the paroxysmal discharges in a provoked attack of seizures induced by hyper-vent i lat ion. The indirect evidence just presented firmly implies that a l ink exists between the anticonvulsant effects of C0 2 acetazolamide and the GABA pathway. Presumably a great deal of modification of this model wi l l take place before a f inal understanding of the mechanisms involved is obtained. 10. 3. Cerebral Compartments in Glutamic Acid Metabolism. Waelsch and his coworkers have been concerned for many years with the metabolism of nitrogenous compounds in the brain. During in i t i a l investigations (20) Schwerin et a l . found that glutamine could readily pass into the brain from circulat ing blood. Glutamic acid, on the other hand, showed no signif icant increase even when the blood levels were increased f i f t y - f o l d . Therefore, there appears to be a selective barrier which tends to keep cerebral glutamic acid levels constant by not allowing this metabolite to pass either in or out of the brain. After an injection of labelled glutamic ac id , a measurable amount of radioactivity appeared in the brain (21). Waelsch (21) believes that these data are indicative of a process of rapid exchange between the un-label led brain glutamic acid and the labelled plasma glutamic ac id . The rapidity of this exchange offers an experimental situation in which the metabolic fate of a labelled dose of glutamic acid could be studied in brain. Experiments (22) performed on mice after the intravenous injection of C-14 glutamic acid showed that within minutes after the dose was given signif icant counts could be^detected in brain glutamine, glutathione (GSH) and GABA. The specif ic act iv i ty (dpm ' ) of the plasma glutamine was (mi cromol e) surprisingly high; higher in fact than the speci f ic act iv i ty of this metabolite in any organ investigated (brain, l i ver , muscle, kidney, lung, and red blood c e l l s ) . Two possible routes by which the radioactivity from the C-14 label -led glutamic acid could enter the brain are (a) by direct exchange between plasma and brain c e l l s , or (b) by being i n i t i a l l y converted to glutamine 11. outside the CNS and then taken up by brain in this form. The high speci f ic act iv i ty of plasma glutamine implies that the second route is taken, although, as wil l be shown, this is not the case. In order to determine the or igin of the high speci f ic act iv i ty of the plasma glutamine, C-14 glutamic acid was injected intravenously into rats with part of their blood circulat ion obstructed. The organs with the restricted blood flow had, as expected, a much lower glutamine speci f ic act iv i ty than the organs with their c i rculat ion intact. But the speci f ic act iv i ty of the plasma glutamine was s t i l l higher than any of the other organs measured including red blood c e l l s . These results, according to Waelsch and his coworkers (22, 23) suggest that the plasma glutamine is derived from some source of glutamic acid with higher than average speci f ic ac t iv i ty . Lajtha et a l . (22) believes that glutamine is synthesized within certain ce l lu lar loci and is then returned to the plasma without prior complete mixing with the total glutamine within the c e l l . In order to explore more fu l ly the metabolic conversions of glutamic acid within the brain, an intracisternal injection of C-\k glutamic acid was given to experimental animals. Even in the shortest time interval (15 seconds) between injection and brain tissue freezing, a large proportion of the injected glutamic acid had already been con-verted to glutamine. In i t i a l l y , the radioactivity of the brain glutamic acid was higher than glutamine but there was a progressive decrease in time of the d.p.m.'s in glutamic ac id , coupled with an increase of counts in glutamine. At a time interval of one minute after the injection the d.p.m.'s of the amide had surpassed the d.p.m.'s of the ac id . 12. When the total radioactivity in glutamic acid and glutamine was observed, i t was found that this remained relatively constant for the f i r s t f ive minutes after injection, after which i t gradually decreased. From this data i t appears that a steady state of C-14 glutamic acid to C-14 glutamine conversion is reached before 15 seconds after inject ion. This steady state lasts for at least 5 minutes, then f a l l s , presumably because the metabolic demand for these labelled compounds exceeds their supply. Measurements of the changes of the speci f ic act i v i t ies of these metabolites showed a parallel ism with the before-mentioned changes of their respective radioactive C-l4 content in time. Spec i f i ca l l y , there appeared i n i t i a l l y to be a very high speci f ic act iv i ty of glutamic acid which gradually f e l l with a reciprocal increase in the glutamine speci f ic ac t i v i t y . On the basis of these results, Waelsch et a l . (22) have postulated that a greater part of the injected C-14 glutamic acid passes from plasma into brain ce l ls by a process of rapid exchange. This is then converted to glutamine without prior equil ibration with the total intracel lu lar free glutamic acid. The model proposed is a small metabolically active glutamic acid pool which is forming glutamine. The radioactive glutamic acid which enters the brain mostly exchanges with the glutamic acid in this pool. The process of tissue homogenization obliterates the high speci f ic act iv i ty of this pool by di lut ing i t with the rest of the free glutamic acid in the eel 1. This model shows expl ic i t l y how glutamine, one of the products of glutamic acid metabolism, could progressively increase in speci f ic act iv i ty at the expense of a decreasing speci f ic act iv i ty of glutamic ac id . 13. The speci f ic act iv i ty of glutamine at its maximum was f ive times higher (Zk) than the specif ic act iv i ty of glutamic ac id . Therefore, according to Waelsch1s concept, the glutamic acid compartment which forms glutamine must have at least f ive times the speci f ic act iv i ty as the total free glutamic acid which had been extracted from brain t issue. Looking at i t another way; the active glutamic acid compartment which forms glutamine cannot be larger than 1/5th or 20% of the total free glutamic acid pool. Since i t is possible that part of the C - l4 glutamine also exists in an intracel lu lar compartment and its speci f ic act iv i ty can also be diluted by tissue homogenization, then the size of the glutamic acid compartment could actually be smaller than 20% of the free glutamic acid pool. The general principle which is an expl ic i t part of Waelsch's theory i s : If a labelled metabolic compound has a higher speci f ic act iv i ty than its precursor, then i t must be formed from a precursor sub-pool which has a specif ic act iv i ty which is higher than the average speci f ic act iv i ty of the cel lular-precursor pool. On the other hand, i f the speci f ic act iv i ty of the product were less than or equal to the speci f ic act iv i ty of its precursor then i t could be formed either from the total precursor pool, or from a small precursor sub-pool of higher than average speci f ic act iv i ty , after which the product can be diluted by unlabel led product from the cel l during homogenization. This latter manipulation would effect ively di lute the specif ic act iv i ty of the product so that i ts previously high speci f ic act iv i ty could not be distinguished. The average specif ic act i v i t ies of GABA and GSH, relative to glutamic acid, were calculated (Zk) to be 0.30 and one respectively. \k. In some of the experiments performed (23) the speci f ic act iv i ty of GSH did exceed the speci f ic act iv i ty of glutamic ac id . This was never observed with GABA. Waelsch1s principle implies that because GSH does at times exceed glutamic acid in speci f ic ac t iv i ty , i t is partly derived from a glutamic acid compartment of higher than average speci f ic ac t iv i ty . Since the speci f ic act iv i ty of GABA never exceeds that of glut-amic acid i t is probably derived from the total ce l lu lar glutamic acid pool, but a possib i l i ty s t i l l exists that GABA is also derived from a small glutamic acid compartment. B. Physiological and Chemical Roles of GABA. 1. General Physiological Effects of GABA. The assay of GABA, with both vertebrate and invertebrate test systems, employing microelectrodes, has shown that this amino acid is associated with the process of neuronal inhibi t ion. Inhibition was defined by Jasper (25) as a 'process which in a l l nerve ce l ls counteracts the depolarizing action of excitatory processes to maintain polarization of a cel l at an equilibrium level near or at i ts resting value. ' At the present time, i t is thought that post-synaptic membranes are generally unexcited by in vivo generated e lectr ical impulses and that both excitatory and inhibitory effects are transmitted by chemical mediators (6). These agents are thought to act on a specialized s i te or receptor situated in the post-synaptic membrane. One of the most thoroughly studied test systems has been the crustacean stretch receptor organ. This organ as-described by Edwards (26) consists of a sensory neurone whose endings are embedded in a '5-receptor muscle strand. A motor nerve supplies the muscle strand and an inhibitory axon forms a synapse with the dendrites of the sensory neurone. The sensory neurone is activated by muscular contraction, e l i c i ted by motor nerve impulses and/or by passive stretch of the muscle. The activation of the sensory neurone can be suppressed by stimulating the inhibitory axon, even in the presence of stretch. When the abdominal organs are stretched the receptor organs are deformed. This deformation sets up a generator potential in the nerve. This potential persists for the duration of stretch, its magnitude depends on the amount of stretch deformation. This magnitude, in turn, controls the frequency of sensory discharge from the neurone. There are two types of receptor organs, one which responds to moderate stretch with a sustained discharge, the other responds to extreme stretch with a short period of discharge. The former, the slow-adapting stretch receptor neurone, is used mostly in bioassay procedures. An analysis of the inhibitory mechanism of the crayfish stretch receptor neurone (CSR) employing intracel lu lar microelectrodes (27), indicates that there are e lectr ica l changes in the post-synaptic membrane of the sensory cel l associated with a stimulation of the inhibitory nerve. This observation suggests the possible l iberation of an inhibitory agent from pre-synaptic s i tes . Kuffler and Edwards (28) found that a low concentration of GABA had effects in the crayfish preparation which were remarkably similar to those which occur when the inhibitory fibers are stimulated. The action of GABA blocks or decreases the frequency of discharge of the neurone with a concomitant s tab i l i z ing effect on the neuronal resting potent ial . 16. The action of GABA is not specif ic for stretch receptors inner-vated by inhibitory f ibers . One stretch receptor in the lobster possesses no physiologically or histological ly demonstrable inhibitory f ibers , yet is inhibited by GABA (28). There is some evidence that GABA has similar inhibitory effects on mammalial stretch receptors as on crustacean stretch receptors. Drakontides (29) observed that GABA, raised the threshold of the cat pulmonary stretch receptor, thus making i t less responsive to stretch deformation. Besides inhibit ing crustacean stretch receptors, GABA has shown actions resembling those caused by stimulating the cardio- inhibitory nerves when placed on crustacean heart ganglion (30). Pictrotoxin was found to block reversibly both the effects of stimulating the inhibitory nerve and the action of GABA on the CSR (30 and on the heart ganglion (30). This is a point of interest since picrotoxin is a potent analeptic agent in humans (32) and monkeys (33). Various extracellular microelectrode studies (34, 35, 36) have been undertaken to determine the effect of topically applied GABA to the mammalian cortex. The general observations were that direct application of GABA induced changes in the nature of the evoked potential recorded at the point of appl ication. The negative components of the evoked potential became positive after GABA was applied. The evoked responses recorded in deeper structures remained unchanged and the polarity of spindle burst was also reversed from a surface negative to a surface positive form (35). These findings were interpreted to mean that (6) 'GABA selectively blocks the depolarizing excitatory synapses, which represent the surface negative post-synaptic potentials in the cerebral cortex. The surface 17. positive potentials observed are hyperpolarizing post-synaptic potentials ordinari ly masked by depolarization. 1 A series of experiments (37, 38, 39) were performed by Curt is , in which recordings were made in or near motoneurones, interneurones and Renshaw cel ls from the cat cord while various substances were applied ionophoretically into or near these structures. Application of GABA depressed the act iv i ty of spinal neurones. Almost equally as active were beta-alanine and taurine. These substances appeared to cause a reduction in amplitude of both excitatory (EPSP) and inhibitory (IPSP) post-synaptic potentials, resulting in a membrane stabi l ized near i ts resting potential . The aIpha-carboxylation products of GABA, beta-alanine and taurine are glutamic ac id , aspartic acid and cysteic acid respectively. Administration of these alpha amino acids in the same manner caused an excitation of spinal neurones with the production of membrane depolarization. An inhibitory process (25) gener-a l ly induces a hyperpolarizing (IPSP) effect to the post-synaptic membrane. Curtis reasons that GABA is not a neurotransmitter because i t does not produce the IPSP. He expresses the view that GABA could more l ikely be a membrane ' s tab i l i ze r ' or neuronal modulator (40). 2. GABA as a Neurotransmitter. Florey (41) has enumerated a number of requirements which a chemical agent must f u l f i l in order to be considered as a neurotransmitter. These are: a) 'This substance must occur in detectable quantities in those neurons whose action i t is supposed to transmit. ' b) 'The neurons must contain an enzyme system capable of synthesiz-ing this compound.' c) 'The substance must Imitate the action of the neurons i f a r t i f i c i a l l y applied to the post-synaptic structures, preferably in low concentrations.' d) 'The post-synaptic structure should contain an enzyme system capable of inactivating the substance.' e) 'During or after stimulation of the pre-synaptic neuron, this substance should be detectable in the extracellular f lu id in the v ic in i ty of the synapse.' f) 'Drugs which potentiate the action of the neuron should also potentiate the action of the substance and vice versa. ' g) 'Drugs which block the action of the neuron should also block the action of the substance and vice versa. ' h) 'Drugs which interfere with the synthesis of the substance within the neuron should lead to a fa i lure of the neuron to act on the post-synaptic c e l l . ' In the l ight of the preceding c r i t e r i a , how much evidence is there which supports GABA as a neurotransmitter? Regarding point (a), i t has not yet been established that GABA occurs spec i f ica l ly in inhibitory neurones (41), although Roberts' (6) work indicates that i t is found in large amounts in grey matter of the CNS. Point (b): Albers (9) and Roberts (7) have given ample evidence that an enzyme system exists for the synthesis and degradation of GABA, but i t has not yet been established that this enzyme system occurs in inhibitory neurones. Point (c): Kuffler and Edwards (28) have shown that GABA imitates the action of stimulating the inhibitory f ibers when placed on the neurone 19. of the CSR. Curt is , on the other hand (39), found that GABA does not exactly mimic inhibit ion in the cat motoneurone. Point (d): A similar argument is advanced here as is given under point (b). Point (e): The release of GABA by the stimulation of inhibitory fibers has never been demonstrated (41). Point (f ) : No references have been made to experiments designed spec i f ica l ly to test the potentiation of an inhibitory process. Point (g): Florey (41) mentions that strychnine, although i t blocks inhibitory processes in the cord, only antagonizes rather than blocks the actions of GABA on CSR. Point (h): Killam and Bain (17) have correlated an increased seizure susceptibi l i ty (absence Of inhibition) with the inhibit ion of GAD with thiosemicarbazide, with a subsequent decrease in GABA levels, but Terzualo e_t aj_. (42) have shown thecevoked IPSP could s t i l l be recorded from cat motoneurones after GABA stores had been depleted by thiosemi-carbazide. This latter observation is strong evidence against GABA being a neurotransmitter agent in the cat . The evidence just given would rule out GABA as an inhibitory neuro-transmitter in mammalian systems. This leaves the poss ib i l i ty that in these systems at least GABA could act as a neuronal s tab i l i zer as suggested by Curtis (39), or i t could have no inhibitory role in vivo because i t may be isolated in an intracel lu lar compartment (24) which is not in contact with the receptor where the inhibitory process originates (41). GABA has been suggested as a transmitter agent of the inhibitory fibers impinging on the crustacean stretch receptor. This agent, designated 20. as the crustacean factor or substance I (43), has not been chemically identif ied but various analytic procedures have fa i led to locate GABA in extracts containing this substance (43). Recently, there has been some evidence that GABA may have some function in the regulation of hormones in the pituitary gland. Mori and Kosaka (44) observed that an intravenous injection of GABA administered to rabbits resulted in a large transitory reduction of adrenal ascorbic ac id . This effect did not appear when an hypophysectomy was performed prior to inject ion. These workers conclude that GABA exerts an effect on the adrenals via the hypophysis. Nelson (46) observed that the drop in adrenal ascorbic acid levels is a concomitant to the release of adrenal 17-0H-corticosteroid by c i r -culating ACTH. Investigations by Mori and Kosaka (45) have uncovered evidence that GABA accelerates the secretion of ACTH by the hypophysis, thus stimulating the adrenals to secrete 17-0H-corticosteroid. Another suggestion on the role of GABA in the CNS has been put forth by Pisano (47). He believes that a possib i l i ty exists that GABA may function as an intermediate in the synthesis of other physiologically active substances in mammals. Some of the compounds which may be possible products of GABA are Factor I, gamma guanidinobutyric acid , gamma aminobutyryl choline, homo-pantothenic ac id , gamma amino-beta-OH butyric, gamma aminobutyrobetaine, gamma aminobutyrylhistidine. These wil l be discussed in the next section. 3. GABA as a Precursor for Other CNS Metabolites. a) Factor I. Florey (48) found that extracts of beef brain had the ab i l i t y to abolish the discharge of the slow-adopting sensory neurone of 21. the crayfish stretch receptor organ. The active fraction of this extract was defined at Factor I. Bazemore et al_. ( 4 9 , 50) isolated crystal l ine material from Factor I preparations which was identif ied as GABA by paper chromatography and infrared techniques. There is a difference of opinion at the present time as to whether most of the act iv i ty of Factor I is due to GABA. E l l i o t and his coworkers (5i . 52) after quantitatively comparing the physiological act iv i ty on the CSR of Factor I per gram of brain with that of GABA, believe that v i r tual ly a l l the Factor I act iv i ty can be attributed to GABA. McLennan (53, 54), on the other hand, after a complex pur i f icat ion and extraction procedure, found no ninhydrin positive material in the active Factor I f ract ions. He believes, on the basis of a Sakaguchi posit ive test , that guanidino compounds make up at least part of this active extract. Rf values in various solvent systems led McLennan to the conclusion that these compounds are either gamma guanidinobutyric acid and/or beta guanidinopropionic ac id . GABA and Factor I have similar physiological actions on various assay systems (55). Some of these actions are: (i) Inhibition of crustacean stretch receptors, ( i i ) Inhibition of crustacean heart ganglion ( i i i ) Block-age of synaptic transmission in the autonomic ganglia of the cat and rabbit, and (iv) Prevention of strychnine convulsions in mice. McLennan (56, 57) found that GABA and Factor I d i f fe r in one resp-ect at least. The latter inhibits the monosynaptic knee-jerk reflex when i t is applied to an exposed cat cord. GABA, on the other hand, has no effect on this system even in concentrations as high as 10 mg per ml. 22. The controversy between McLennan and E l l i o t t is d i f f i c u l t to resolve since different methods have been used in both laboratories to obtain ,the active fract ions. A salient observation has been made recently (58) which has a great bearing on the assay of the inhibitory effects of brain extracts. Levin (58) notes that GABA levels in brain tissue can increase after death, presumably due to the effects of residual post-mortem GAD act i v i t y . (b) Gamma Guanidinobutyric Acid (GGB) . I rreverre et a_K (59) identif ied gamma guanidinobutyric acid (GGB) in the mammalian brain and other vertebrate t issues. Pisano and his group (60) observed that in mammalian tissue a reversible transamidination takes place between GABA and an ami dine donor such as arginine to produce GGB. The enzyme which catalyzes this reaction also is involved in the synthesis of gamma guanidinoacetic acid from glycine and arginine. This enzyme has been isolated from brain tissue (61). The existence of GGB in brain has been confirmed by Pisano (61) and Blass (62). McLennan (54) believes that GGB is part of the active fraction of Factor I. Some physiological act i v i t ies of GGB have been investigated. Kuffler and Edwards (28) observed that GGB is 3 to 13 times less effect ive than GABA in blocking the CSR preparation. Pisano (47) mentions that GGB ' inactivates the hyperpolarizing inhibitory axo-dendritic synapses' when i t is topically applied to the cortex. c) Gamma Aminobutyrylchol ine (GABA-Ch). Kuriaki et a [ . (63), in a chromatographic investigation of organic bases from dog brain, isolated a spot which ran to the same Rf as synthetic GABA-Ch. This spot, after elut ion, hydrolysis, and re-chromatography, revealed a ninhydrin positive spot which Kuriaki identif ied as GABA. Pisano (47) believes that the Rf 23. value of the ninhydrin positive spot was too high to draw this conclu-sion. Some interesting observations have been made on the physiological effects of GABA-Ch. Asano (64) reports that this material has cholinergic effects on the clam heart. It is a much more potent inhibitor of the crayfish neuromuscular junction than GABA and i t blocks neuromuscular transmission in the cat sciatic-gastrocnemius preparation. These effects are similar to a neuromuscular blocking agent. Takahashi (65) and his coworkers found that GABA-Ch, when applied topical ly to the rabbit cortex, resulted in an immediate decrease in the amplitude of both the surface negative and surface positive components of the evoked potential . These workers also observed that GABA-Ch was 500 to 1000 times as powerful as GABA in inhibit ing cortical spikes induced by metrazol. (d) Homopantothenic Acid (HPA). Homopantothenic acid has been reported in human urine (66). This compound di f fers from pantothenic acid in that i t contains GABA in place of beta-alanine. Pisano (47) sug-gests that a possib i l i ty could exist where homopantothenic acid might in part replace pantothenic acid in brain Coenzyme A. When Coenzyme A from dog and beef brain was hydrolysed, no GABA could be detected by enzymatic methods. During the puri f icat ion of Coenzyme A, GABA was detected in greater quantities in hydrolyzed protein free extracts than in the unhydro-lysed extracts. This observation indicates that a bound form of GABA may exist in the non-protein fraction of the brain. (e) Gamma Aminobutyrylhistidine (Homocarnosine). While attempting to isolate this bound form, Pisano (67) identif ied the dipeptide gamma Ik, aminobutyrylhistidine (homocarnosine). This compound was isolated from beef and rat brain in quantities of 0.5 - 1 mg/100 gm. It was not found in spleen, kidney, l iver or skeletal muscle. No data on the physiological effects of this dipeptide have been published. (f) Gamma Butyrobetaine. Hosein (68, 69) has reported that gamma butyrobetaine esters of CoA exist in the brain. Following dieldr in in -duced convulsions, free betaines were detected in rat brain. No betaines could be detected in controls. Hosein (69) noted that methyl, ethyl and choline esters of gamma butyrobetaine caused convulsions in rats with a concomitant r ise of cerebral ammonia leve l . Since no follow-up research has been done to confirm or disprove these observations, they must be taken with a certain amount of reservation. (g) Gamma Amino-Beta-Hydroxybutyric Acid (GABOB). GABOB has been mentioned as a constituent of rat brain by Hayashi (70). He quantita-t ively estimated i t to be 5.7 mg/10 gm of brain. The physiological effects he observed for this compound were a strong anticonvulsant act iv i ty in electr ical ly - induced seizures and an inhibitory property similar to Factor I. Hayashi (71) believes that stress should be laid on the importance of GABOB as the inhibitory principle of brain because (i) i t has a stronger inhibi tory acti vi ty in cerebral activity., and ( i i ) in con-trast to GABA which sometimes exerts excitory effects on the cortex, GABOB always has inhibitory actions. Ohara (72) found this compound in mice, rabbit, catt le and human brain. He estimated that GABOB levels were kB mg/100 gm in ox brain. Sacktor ejt aj_. (73) conclude from their work on brain mitochondrial preparations that the major pathway of GABA oxidation involves GABOB as an intermediate. 25. This pathway begins with an in i t ia l oxidation of GABA to gamma aminocrotonic acid which is then hydrated to GABOB, which is further oxidized to gamma amino-beta-ketobutyric ac id . Mitoma (74) has tried to demonstrate the formation of GABOB from GABA but has not been successful. He has also tried to confirm Ohara's quantitative estimation of GABOB in ox brain by the addition of C-14 labelled GABOB to pooled dog brains and re - iso lat ing the compound. If a s ignif icant amount of this compound occurred in the brain there would be a di lut ion of the 'hot' GABOB in re- isolated compounds. Such a di lut ion was not encountered. Mitoma believes that despite a l l claims in the l i terature there is no definite proof for the occurrence and formation of GABOB in the CNS. • _ Out of a l l the compounds mentioned, only a clearly established pathway to gamma guanidinobutyric acid has been proven, but a great effort is being made to apodictically characterize the other postulated metabolic pathways emanating from GABA. C. Functional Considerations. 1 . Ammoniaqenic Coma. One physiological condition whose chemical basis may be linked to amino acid metabolism is ammoniagenie coma. Mann (75) described a characterist ic set of neurological symptoms which occur when the l iver is seriously damaged or removed. The in i t i a l reaction to this insult is the establishment of a lethargic state leading progressively to a comatose state and f ina l l y to a terminal stage of convulsions and death. In humans (76) a direct correlation between this symptomatology, elevated blood ammonia levels and elevated brain glutamine 26. l eve ls (77) has been found. These increases could be potent iated and a concomitant comatose s ta te could be induced in pat ients with l i v e r d i s -ease, by the intravenous admin is t ra t ion of nitrogenous compounds. S ince ammonia is taken up by bra in from blood (79) i t has been suggested (78) that the r e s u l t i n g high i n t r a c e l l u l a r ammonia level in th is organ would tend to reverse the glutamic a c i d dehydrogenase step in the metabol ic conversion of glutamic ac id to alpha ke tog lu ta ra te . Under normal condi t ions there i s a tendency f o r glutamic ac id to be con -verted to alpha ketog lu ta ra te . The s t ress of an increased i n t r a c e l l u l a r ammonia level would reverse th is react ion r e s u l t i n g in a drop in i n t r a c e l l u l a r alpha keto -g lu ta ra te l e v e l s . T h i s , in tu rn , would i n i t i a t e a decrease in the leve ls of a l l Krebs c y c l e intermediates which depend upon alpha ke tog lu ta ra te . The outcome would be a decrease in the source of chemical f ree energy t ied up in these intermediates . The biochemical mechanisms which protect the dep le t ion of Krebs c y c l e intermediates are not operat i ve in bra in (80) . Blood born c i t r a t e and alpha ketog lutarate cannot penetrate the blood bra in b a r r i e r , and CO2 f i x a t i o n and g lu tamic -a lan ine transaminase do not occur in the b r a i n . A p o s s i b l e homeostatic p ro tec t ion mechanism in bra in could be the ox ida t ion of GABA to succ inate v i a the GAS, but , i f alpha ke tog lu ta r -ate were depleted there would be a concomitant decrease in the energy produced by th i s pathway s ince alpha ketog lutarate i s a s p e c i f i c rece iver o f the amino group which is transaminated in the f i r s t step of GABA o x i d a t i o n . Further evidence f o r the reversal of the glutamic ac id dehydro-27. genase reaction was implied from the evident increase in brain glutamine (78) following an intravenous dose of ammonia. Recent work using N'5 (76) labelled ammonium salt infusions has* established that the or ig in of the amide group in elevated brain glutamine was at least partly due to blood ammonia. An alternate theory (80) of the mechanism of ammonia toxicity has been proposed by Weil-Malherbe. He suggests that since the incorporation of ammonia into glutamic acid to form glutamine requires ATP, a high rate of operation of this pathway would put great stress oh the available stores of high energy phosphate. Bessman (80) believes that this theoretical model is inadequate, since i t cannot explain away the observed decrease in brain oxygen con-sumption during hepatic coma. He argues that these results are incompat-ible with a depletion of ATP stores since the increased formation of ADP and inorganic phosphate would stimulate the Krebs cycle and thus increase rather than decrease the oxygen consumption. Some further evidence (80) for the 'Krebs Cycle Depletion Theory' was obtained in Bessman's laboratory when the direct analysis of mice brains at different time intervals after these animals were given doses of ammonium acetate, showed a progressive f a l l in alpha ketoglutarate. One of the main points in this discussion on ammoniagenie coma is that a set of biochemical events is associated with an overt physio-logical ef fect . This point leads direct ly into the next section which wi l l discuss some of the biochemical changes which occur in brain during the state of convulsions. 28. 2. Amino Acid Levels, Vitamin B-6 and Seizures. Tower (81) was the f i r s t to compile enough evidence from the l i terature to assign tentatively a neurological role to the glutamic acid shunt (GAS). He suggested that the seizures related to Vitamin B-6 dysfunction were the result of an interference with one or more of the enzymatic steps of this metabolic pathway. His suggestion was based upon the following lines of evidence. (a) An absence of dietary Vitamin B-6 (B-6 deficiency) (82, 83) or an error in Vitamin B-6 u t i l i za t ion (B-6 dependency) (84), resulted in frank seizures resembling status epi lepticus. (b) The above seizures could be dramatically reversed with an intravenous dose of Vitamin B-6 (pyridoxal) (85). (c) The phosphate ester of Vitamin B-6 (pyridoxal phosphate) plays a major role in intermediary metabolism especially as a coenzyme for decarboxylases and transaminases (86). (d) Both the decarboxylase, glutamic acid decarboxylase (GAD) and the transaminase, GABA-alpha ketoglutaric transaminase (GABA-T), which catalyze the anabolism and catabolism of GABA, depend upon pyridoxal phos-phate (87). (e) GABA and GAD have been found in measurable quantities only in the central nervous system (87). In order to test his hypothesis further, Tower (81) attempted to duplicate the convulsion resulting from the natural depletion of B-6 stores by pharmacological means. By injecting a series of carbonyl trapping agents and Vitamin B-6 antimetabolites, he hoped to inactivate chemically the enzymes which depended upon pyridoxal phosphate. Concomit-29. ant seizures occurred after the injection of both classes of enzyme antogonists. The severity of the seizures depended upon the type of agent used, i ts dose, i ts route of administration, and the species of experi-mental animal. In general, the most potent carbonyl trapping agent was thiosemicarbazide (TSC), while methoxipyridoxine (MD-B6) was the most powerful of the Vitamin B-6 antagonists. However, no attempt was made, at that time, to test the effects of B-6 on seizures induced by these agents. Convulsions induced by isonicotonic acid hydrazide (isoniazid) and dexoxipyridoxine were reversed after the administration of Vitamin B-6 supplements. Tower could find no v is ib le damage in the brains taken from animals in convulsion. These latter two observations were further evidence to substantiate the existence of a biochemical lesion in seizures associated with Vitamin B-6 dysfunction. Killam and Bain (17) established further data which strengthen Towers' hypothesis. Their experiments showed that in rats GAD was inhibited during thiosemicarbazide seizures with a concomitant decrease in cerebral GABA by 30 to 50 per cent. Roberts and his coworkers (8) later confirmed this observation by measuring the decrease in the rate of conversion of radioactive glutamic acid to GABA during seizures induced by thiosemicarbazide. Following the biochemical studies, Killam et a[ . ( 8 8 , 89) designed an experiment which would answer the question as to whether the appear-ance of e lectr ical seizure act iv i ty was speci f ica l ly related to the changes in the act iv i ty of GAD. A series of cats were surgical ly prepared for cort ical and subcortical recordings. A plug of cort ical tissue contain-ing the electrodes was undercut and circumscribed with i ts blood supply 30. le f t intact. After thiosemicarbazide infusion there was an increased e lectr ica l act iv i ty within the cort ical plug. When a series of these plugs were analyzed manometrically (91), a correlation between a decrease in GAD act iv i ty and an increase In e lectr ical act iv i ty was observed. GABA administered topically and parentally reduced the frequency of seizures (90). Purpura ejt al_. (92), in a preliminary study, found that methoxipyri-doxine (M0-B6) seizures were refractory to subsequent injections of B-6, GABA and glutamic ac id . On the basis of these results additional studies were undertaken to define a relationship between brain amino acid levels and the development of methoxipyridoxine seizures. In a series of cats injected with M0-B6 (93) there was an 80 per cent decrease in cerebral GABA levels associated with the f i r s t general-ized seizure. After one to 3 hours of sustained seizures no further changes in GABA were detected, whereas glutamic acid decreased by 50 to 60 per cent and glutamine increased by 50 per cent. After injections of GABA and glutamic ac id , no effect was observed in the intensity or duration of the M0-B6 seizures, although the GABA content had increased by approxi-mately 100 per cent in brain tissue removed 30 minutes after GABA inject ion. No such increase in GABA was observed in tissue removed in the same time interval after glutamic acid injection. Topical application of M0-B6 also resulted in seizures but in contrast to systemically administered M0-B6 there was no observable change in cerebral amino acid levels. Gammon et a K (94) found that a parenteral ly administered dose of GABA protected mice from M0-B6 seizures. This protection corresponded -to an increase of endogenous GABA by 200 per cent. 31. "The case f o r a causal r e l a t i o n s h i p between the increases and decreases of s e i z u r e a c t i v i t y and the corresponding increases and de-creases of endogenous GABA l e v e l s would be strengthened i f the r a i s e d cere b r a l GABA l e v e l s would c o r r e l a t e w i t h e l e c t r i c a l patterns opposite to those found with c o n v u l s i v e hydrazides and B-6 a n t i m e t a b o l i t e s . " Baxter and Roberts (95) succeeded i n r a i s i n g GABA l e v e l s by pharmacological means. They observed that the carbonyl trapping agent, hydroxylamine, could i n h i b i t GABA-T a f t e r GAD had been p r e f e r e n t i a l l y r e a c t i v a t e d w i t h Vi tamin B -6. I n t e r p e r i t o n e a l i n j e c t i o n s o f hydroxylamine r e s u l t e d i n an e l e v a t i o n of cerebr a l GABA. This e f f e c t p e r s i s t e d f o r a t l e a s t 5 hours a f t e r the f i r s t i n j e c t i o n . The i n i t i a l p h y s i o l o g i c a l e f f e c t was con-v u l s i o n s , f o llowed by a period of le t h a r g y . The increase of GABA l e v e l s corresponded to the l a t t e r response. In order to determine whether the hydroxy I amine induced increase of GABA would have any e f f e c t on induced s e i z u r e a c t i v i t y , E i d e l b e r g et a l . (96) prepared a s e r i e s of cats w i t h s t i m u l a t i n g and recording e l e c t r o d e s i n s e r t e d i n t o the exposed c o r t e x . They observed that the du r a t i o n and spread of the recorded induced a f t e r - d i s c h a r g e s r e s u l t i n g from high v o l t a g e c o r t i c a l s t i m u l a t i o n was reduced s i g n i f i c a n t l y a f t e r hydroxylamine a d m i n i s t r a t i o n . This e l e c t r i c a l depression c o r r e l a t e d w i t h a r i s e i n ce r e b r a l GABA l e v e l s . The r i s e and f a l l of ce r e b r a l GABA, and the r e l a t e d behavioral and e l e c t r i c a l changes, add f u r t h e r weight to the suggestion that c e r t a i n types of convulsions and the r e s i s t a n c e to them depend upon the l e v e l s of GABA i n the mammalian b r a i n . If hydroxylamine increases the l e v e l of GABA, producing l e t h a r g y , and thiosemicarbazide decreases i t , producing 32. seizures, then a combination of both agents should theoretically keep GABA at a constant level with a subsequent balance in the act iv i ty of the neurones.. In order to test the val idi ty of this hypothesis, Baxter and Roberts (97) treated rats with both agents simultaneously. The results of this experiment were rather surprising. GABA levels were high but convulsions s t i l l occurred. These observations indicate that the main-tenance of high GABA levels was not necessarily the only factor involved in the resistance to seizures. Tower (98, 99, 100) introduced a new method which was highly successful in relating the metabolism of amino acids to the degree of seizure act iv i ty in brain t issue. He used the incubated tissue s l i ce technique (99) to demonstrate the in vitro biochemical differences between normal and excitable cort ical areas. Test tissue s l ices were obtained from cats in convulsions. These convulsions were produced with parental doses of thiosemicarbazide, megamid and methionine sulfoximine. Tissue sl ices were also prepared from human excised s l ices of cort ical f o c i . When glutamic ac id , glutamine and GABA were measured in test and control s l ices before and after incubation the following observations were made. (a) In a l l types of tissue s l ices fromkboth chemically induced and naturally occurring seizures, there is a decrease in the amount of glutamic acid with time, as compared to an increase in normal t issue. (b) GABA also decreased in time in a l l test s l ices except for megamid induced seizures where there was a sl ight increase. (c) These decreases cauld not be explained by assuming a net diffusion into the incubation media. Tower thought that these changes 33. were due to a 'metabolic loss' resulting from either a decreased rate, of formation or an increased rate of u t i l i za t ion of GABA and glutamic ac id . (d) When a l l types of test tissues were incubated with GABA they resembled the controls as far as glutamic acid metabolism was concerned. (e) 1-Asparagine or glutamine added to the incubation medium of methionine sulfoximine treated tissue and cortical focal s l ices also reversed the decrease in glutamic acid and GABA levels . (f) Vitamin B-6 reversed the glutamic acid and GABA changes in thiosemicarbazide treated s l i ces . On the basis of the following data, Tower did a series of prelim-inary c l in ica l t r ia ls to determine the efficacy of L-asparagine and GABA as anticonvulsant agents. Ini t ial results (101) showed that both compounds exerted some control of epi lept ic seizures, but not enough c l in i ca l data has been accumulated to draw any conclusions on their worth. Berl and his coworkers (102, 103) studied the in vivo metabolism of glutamic acid and related amino acids in a focal lesion produced in succinyl choline paralysed cats by local freezing with an ethyl chloride spray. These workers observed an overall decrease in glutamic ac id , glutamine and glutathione by 30 to 70 per cent in the lesion area, as compared to controls. GABA showed no signif icant changes in the lesion. Low frequency e lectr ical stimulation caused paroxysmal act iv i ty in the focal area. This act iv i ty did not correspond at a l l times to a decrease of the amino acids, although, in a general way, the extent of this decrease corresponded to the severity of the spiking discharges from the lesion. An intravenous injection of GABA resulted in a suppression of 34. paroxysmal discharges from the focus within 15 seconds. This decrease in e lectr ical act iv i ty could not be correlated with an increase in the amino acids at that particular time, although at approximately 15 minutes after injection GSH, glutamine and glutamic acid approached normal levels while GABA increased above i ts normal leve l . When the e lectr ical patterns from the focus were of a complex nature, injected GABA had no inhibitory ef fects, although i t increased four-fold above i ts normal concentration. Glutamic acid administered in the same manner as GABA showed similar biochemical and pharmacological ef fects, although to a lesser degree. Berl suggests three possible explanations for the measured bio-chemical changes. (a) GABA, although appearing unchanged in the focal lesion, is actually being metabolized at a rapid rate. An increasing demand for substrate to supply the decreasing GABA stores could account for the depletion of the other amino acids. (b) GABA is blocked, and the other ami no acids are shifted through the Krebs cycle to supply an increasing energy demand. (c) Ethyl chloride spray, besides producing focal lesions, also breaks down the blood brain barr ier . Glutamic ac id , glutamine and GSH 'leak 1 out, while GABA, which is more firmly bound within the c e l l , remains. In order to round out this discussion, a brief mention should be made on some of the work done to establish whether the effects on amino acid metabolism of other convulsive agents are similar to the effects seen during the seizure state induced by pyridoxine deficiency. Gammon et al_. (94) have measured the changes of some of the free 35. amino acids in mouse brain during convulsions induced by metrazol, picrotoxin, tripelJeraamine and electroshock. Generally, they found only small (20 per cent) changes in the levels of GABA, glutamine, glutamic acid or aspartic ac id , as compared to the large GABA changes found in M0-B6 seizures . ( Kamrin and Kamrin (104) in a much more sophisticated study on mice, have established essential ly the same results except that during tripellenamine induced seizures there was a decreased glutamine concentra-tion and during electroshock seizures glutamine levels were raised. The magnitude of these changes was comparable to Gammon's observations. These workers conclude that either the absolute level of GABA or the changing amino acid patterns cannot be used as an index of the general state of exc i tabi l i ty of brain, although they do not exclude the poss ib i l i ty that consistent localized changes in amino acids do take place during a l l types of convulsive act iv i ty . 36. PART 11. DEVELOPMENT OF METHOD A. Intravenous Injection of Control Animals. Two normal unanesthetized cats were each injected with 50 fxc of 1-glutamic acid-U-C-14 in 5 ml of d i s t i l l e d water through the right femoral vein. The animals were rendered unconscious by a blow to the head 3 and 12 minutes, respectively, after the inject ion. The chest cavity was rapidly opened, exposing the heart. The infer ior and superior vena cavae were cut. The animals were perfused with isotonic saline through a cannula inserted into the apex of the heart. Perfusion was continued until the perfusate issuing through the superior vena cava was c lear . The brains were removed from each animal, dissected into roughly thirty key anatomical areas and immediately frozen. The time interval between rendering the animal unconscious and freezing the tissue was approximately 60 minutes. B. Preparation of Tissue Extracts. Each brain area ( . 5 - 1.5 gm) was weighed while frozen, then rapidly homogenized in a 10% (w/v) solution of cold tr ichloroacetic acid (TCA) using 0.2 ml of TCA solution per gram of t issue. The homogenate was washed Into a centrifuge tube with 5 -6 ml of d i s t i l l e d water after which i t was centrifuged for 10 minutes. The clear supernatant f lu id was poured into a test tube, the precipitate was resuspended in 1-2 ml of d i s t i l l e d water and recentrifuged. Both super-natant fractions were combined, then lyophylized to dryness in a V i r t i s freeze drying unit . The residue was dissolved In . 5 ml of d i s t i l l e d 37. water. C. Chromatographic Separation of Amino Acids. Two dimensional descending paper chromatography was employed in the separation of aspartic ac id , glutamic ac id , glutamine and GABA from brain tissue extracts. In i t ia l l y , brain extract equivalent to approximately .1 gm of tissue was spotted on Whatman #1 chromatography paper and run bidimen-sional Iy in methanol/pyridine/ammonia/water (160/8/1/40) ( f i r s t dimen-sion), and butanol/acetic/water (4/1/1) (second dimension). Since a l l the aforementioned amino acids separated in this solvent system i t was adopted in the general procedure. The amino acids were identif ied i n i t i a l l y by a comparison of their Rf values with appropriate standards. More rigorous confirmation was attained by co-chromatography of each eluted amino acid spot with i ts standard in methanol/butanol/benzene/water (2/1/1/1), isopropyl/ammonia/water (8/1/1) and butanol/pyridine/water (1/1/1). D. Radioautography. A two dimensional chromatogram run in Me/Pyr/NHj and BuAc of labelled brain extract was stapled to a 14" x 171' sheet of l l fex safety base X -ray f i l m . This was left in an exposure holder, in the dark, for 7 months, after which i t was developed by standard procedures. E. A Note on Liquid Sc in t i l la t ion Counting. Sc in t i l la t ion counting depends, in the main, on the property of certain organic compounds called phosphors to absorb radiant energy in 38. either the sol id state or in solution, and to release this energy as a burst of photons. In a l iquid sc in t i l l a t ion counter, the phosphor is dissolved in solution together with the source of radiant energy. In the case of compounds labelled with C-14, sulfur 35 or tr it ium, the radiant energy is in the form of beta part ic les . In theory (113, 114) the energy dissipated by the beta part ic le is transferred through the solvent to the organic s c i n t i l l a t o r . This energy is absorbed by the sc in t i l l a to r resulting in the formation of excited atoms or molecules, which then return rapidly to their normal or ground state releasing as l ight (photons) the excess energy derived or ig inal ly from the nuclear radiation. The l ight emitted is l inearly related to the radiant energy dissipated. The emitted photons are detected and converted to e lectr ical energy by a pair of photomultipller tubes. This e lectr ical energy is also l inearly related to the energy of the l ight quanta. The e lectr ical energy is amplified, then fed into the scaler where the number of counts emitted from the radiation source are subsequently recorded. Any interruption of the energy transfer between the source of radiation and the photomultipiier tubes is defined as quenching. One way in which quenching can occur is the absorption of radiant energy by impurities and non-fluorescent material, such as acetone and water, in the sc in t i l l a t ion solution. Although these molecules are ex-c i ted , they return to the ground state by radiationless transition without concomitant emission of photons. This expenditure of radiation results in a decrease of fluorescence yield with a reduction of counting 39. eff ic iency . Another manner by which quenching can occur is by the process of photon absorption between the sc in t i l l a t ion phosphor and the photo-mult ipl ier tube. This process is called color quenching and occurs when colored material such as urine, hemoglobin, ninhydrin, e t c . , are d is -solved in the sc in t i l l a t ion solution. F. Assay of Labelled Amino Acids Separated by Paper Chromatography. Wang and Jones (115), Geiger and Wright (116), .Nunez and Jacquemin (117) have reported good experimental results in preliminary studies into the poss ib i l i t ies of employing the techniques of l iquid sc in t i l l a t ion to the assay of labelled compounds on f i l t e r paper. In order to apply these techniques to the analysis of radioactive compounds separated by paper chromatography, the following c r i te r ia should be met. 1. There should be no detectable loss in cpm (counts/minute) as a result of chromatographic separation. 2. There should be no detectable loss in cpm as a result of the process of spot location. 3. There should be a linear relationship between the amount of labelled compound spotted on the chromatogram and the total.measured- radio-act iv i ty after chromatography. k. The eff iciency of counting should not be affected by the size or amount of f i l t e r paper immersed in the sc in t i l l a t ion solution. 5. The cpm should be reproducible when equivalent amounts of the same extract are chromatographed in duplicate.! ' ' ko. 1• Experiments Designed to Test C r i te r ia , (a) To test whether there is a lOss of dpm during running in solvent systems. Method. Duplicate 50 X portions of extracts prepared from brain tissue of control cat injected with 50 JUC of C-14 glutamic acid were spotted on Whatman #1 f i l t e r paper and each run in duplicate unidimen-sional ly in each solvent system (BuAc and Methanol/Pyr/NH^). The chromatograms were cut into 1" x 1.5" segments and each segment was immersed in sc in t i l l a t ion mixture made with 4 gm of PPO and 100 mg of P0P0P dissolved in one l i t e r of toluene for 30 minutes. The background counts were subtracted and the counts from the papers run were totalled and compared to counts obtained from an equivalent amount of the same extract spotted on single 1" x 1.5" paper segments. Results. See Table 1. TABLE 1. Effect of Chromatography on the Recovery of Radioactivity. Cpm spotted Total cpm Total cpm not run from BuAc from MPN Run 1 270 300 304 Run 2 269 272 283 Therefore, no signif icant loss of counts could be measured during the process of paper chromatography. 2. Loss of Cpm During Spot Location. It was thought that a l ight ninhydrin spray could be used to locate the labelled amino acids after chromatographic separation without causing 41. a signif icant loss of counting eff iciency by color quenching. In order to test this possib i l i ty increasing amounts of extract were spotted on 1" x 1" segments of Whatman #1 papers in duplicate. One duplicate set of paper segments were counted for 30 minutes each, while the other set were l ightly sprayed with .1% ninhydrin solution in 95% ethanol, then counted in the same manner. The results shown in Graph A (page 42) indicate that under these experimental conditions the location of the amino acid spots for radioactive assay with ninhydrin wi l l v i t ia te the counts by color quenching. 3. Location of Amino Acids by Fluorescence. A method (118) for the detection of amino acids without the use of a chemical location reagent has been reported. This technique depends upon the property of amino acids on a paper chromatogram to fluoresce under UV l ight when subjected to heat above 100° C. The amino acids appear most intense as l ight blue fluorescent areas against a darker blue background when viewed at a wavelength of 3650 A. After heating (for 5 minutes at 105° C.) the fluorescent areas on a two dimensional chromato-gram could be easily delineated. The accuracy of location proved sat i s -factory when tested by ninhydrin spray. Effect of fluorescent location on cpm. One hundred )\ of aqueous labelled brain extract was spotted in duplicate on Whatman #1 paper and run in the regular bidimensional solvent system. After drying at room temperature the chromatograms were heated for 5 minutes at 105° C. Fluorescent areas corresponding to aspartic ac id , glutamic ac id , glutamine and GABA were cut out, placed in vials containing sc in t i l l a t ion mixture and counted for 30 minutes. The counts for each amino acid spot were 42. GRAPH A . Effect of Ninhydrin on the Eff iciency of Liquid Sc in t i l la t ion Counting. 401 43. recorded. The paper segments were removed from the v ia l s , dried and heated at 100 to 130° C. for one hour after which they were recounted. The results below show that there is no signif icant loss in cpm under the heating conditions of this experiment. TABLE 2. Effect of Heating on the Recovery of Radioactivity in Amino Acids on F i l t e r Paper Cpm after Cpm after 5 min. heat I hr. heat Aspartic acid 42 44 I I I I 39 40 Glutamic acid 110 114 I I I I 98 97 Glutamine 43 45 n 34 36 GABA 42 42 GABA 42 42 4. Proof of Linearity. Paterson (119) has shown that a linear relationship exists between the observed counting rates for a C-14 labelled thiolnosinate spot separ-ated by one dimensional paper chromatography and the amount applied to the chromatogram. This work was repeated using C-14 1-glutamic acid mixed with a cold trace of glutamic acid separated by one dimensional chromatography in Me/Pyr/NH3 and located by fluorescence. Results (Graph B, page 44) show l inear i ty . Since c r i te r ia one has been proven, i t is reasonable to assume GRAPH B. Liquid Sc in t i l la t ion Counting of C-)k labelled Glutamic on Paper After Chromatographic Separation. 45. that a l l labelled compounds wi l l show l inearity similar to glutamic ac id . 5. Effect of Paper Immersion in the Sc in t i l l a t ion Solution on  Counting Eff ic iency. There may be a possib i l i ty that the f i l t e r paper on which the spots-have been separated may act as a quenching agent in the sc in t i l l a t ion solution. If this is the case, the loss of counting eff iciency should be direct ly proportional to the area of each segment of paper counting C-14 labelled material . In order to determine i f paper, in fact , does quench, the following experiment was performed. Labelled C-14 glutamic acid was separated in duplicate from aqueous extract by two dimensional paper chromatography and located in the usual manner. Spots corresponding to glutamic acid were cut out of each of the duplicate chromatograms and coded as spot #1 and spot #2. Spot #1 was placed in a vial containing sc in t i l l a t ion solution. Three clean pieces of 1" x 2" Whatman #1 f i l t e r paper were placed around the inner surface of the v i a l . Spot #2 was cut in half (2a and 2b), each half was placed in a vial containing scint i1lat ion mixture, but without any extra f i l t e r ; paper. The three vials were counted for 30 minutes each. Counts were recorded (Table 3) after average background counts were subtracted. The results show that under the conditions of this experiment, paper wi l l not s ignif icant ly reduce the counting eff ic iency. 6. Reproducibility of Counts on Duplicate Chromatograms. Aqueous extracts prepared from different brain areas from a normal cat injected with 50/uc of labelled glutamic acid were spotted in duplicate on Whatman #1 paper. The amino acids were separated by two dimensional descending paper chromatography and located by fluorescence. 46. TABLE 3. Effect of Paper Immersion in Sc in t i l l a t ion Solution on Counting Ef f ic iency . Background Cpm minus Vial Cpm cpm background Spot #1 153 20 133 + paper Spot #2a 64 20 44 Spot #2b 100 20 80 124 Aspartic ac id , glutamic ac id , glutamine and GABA were cut from the papers, placed in sc in t i l l a t ion solution and counted for 30 minutes each. The results (Table 4) show that good reproducibil ity is achieved by this method. G. Procedure for Radioactive Analysis. Since a l l the points of the c r i te r ia have been ver i f ied , the f o l -lowing procedure was used to determine the radioactivity in aspartic ac id , glutamic ac id , glutamine and GABA after they had been separated from the aqueous brain extract of different anatomical areas by two dimensional paper chromatography and located by fluorescence. Fluorescent areas corresponding to the above amino acids were cut from duplicate chromatograms. Paper segments were placed in v ials con-taining sc in t i l l a t ion mixture, then each counted twice for 30 minutes. The background counts were subtracted, after which the mean of two deter-'-minations were recorded. The results were presented as dpm per amino acid per gram of wet t issue. 47. TABLE 4. Reproducibility of cpm in Amino Acids Separated by Paper Chromatography. Brain area Run Aspartic Acid Glutamic Acid Glutami ne GABA Rt. hippocampus 1 19 44 16 32 2 22 60 14 36 Rt. visual 1 21 77 37 29 cortex 2 19 70 27 27 Quadri gemi nal 1 22 27 11 61 plate 2 23 26 12 60 Lt . thalamus 1 9 45 12 18 2 9 44 9 19 Lt . basal 1 12 25 4 30 ganglia 2 12 26 8 30 H. Measurement of Amino Acid Pools. The method of Moore and Stein (120) was employed to measure the total amount of each labelled amino acid on the f i l t e r paper after the radioactivity was determined by l iquid sc in t i l l a t ion counting. This method was standardized by running a known quantity of each amino acid through the procedure as an internal standard. Prior to quantitative measurement, the amino acids were removed from the papers by centrifugal elut ion. Experiments showed that immersion of the paper segment in the sc in t i l l a t ion solution did not effect the analysis. Also, there was no signif icant loss of color intensity i f the papers were heated at 105° C. for less than 10 minutes during the location procedure (see graph ;.C. page 48) . 48. GRAPH C. Effect of Heat on Amino Acid Recovery. 100 gamma of each amino acid was used. 49. Following the suggestion of Fowden (121), the eluant was made basic with .1 ml of .IN NaOH, per 1 ml of eluant, then left in a vacuum desiccator over concentrated H2SO4 for 8 hours prior to its determination, in order to remove interfering ammonia. I. Animal Preparations. 1. Cat with Epileptogenic Focus. A cat with a focal lesion produced by the method of Wada and Cornelius (114) was injected with 45/ic of C-14 universally labelled 1-glutamic acid (specif ic act iv i ty 45 /uc/2 mg) in a 5 ml solution through the right femoral vein. The EE6 recording taken just prior to injection indicated an actively spiking focus in the lef t motor cortex. The animal was sacr i f iced after 7 minutes and the brain areas prepared in the same manner as the controls. The g l ia l scar from the focus adhered to the dura and was discarded. The labelled free amino acids were determined by the methods described previously. 2. Preparation of Cat for Study of the Time Changes of the Metabolism  of C-14 Glutamic Acid in the Cortex. The superior portion of the skull of a healthy cat was removed while the animal was under general anaesthetic. Twenty-four hours later , this cat was placed in a stereotaxic instrument and its brain exposed. Both the lef t and right femoral veins were cannulated. A 5 ml solution of 50/uc of C-14 1-glutamic acid (universally labelled) was injected into the lef t femoral vein. At set time intervals after this injection, cortical areas were removed, placed in clean p last ic v ials and immediately frozen in a dry ice/acetone mixture. A sample of blood was taken from the lef t femoral vein simultaneously with the removal of each cort ical area. 50. The labelled free amino acids were extracted from each cort ical area and measured by previously described methods. J . Post Mortem Changes in Labelling Patterns. In order to determine the post-mortem metabolic changes (58) occurring in tissue containing labelled glutamic ac id , the following experiment was performed. Two frozen cort ical areas from a control cat which had been given an intravenous dose of C-14 labelled glutamic acid were each cut in hal f . One half of each of the tissues was homogenized in cold TCA, while the other was lef t at room temperature for 2 hours. Both tissues were subjected to the procedure for removal and separation of free amino ac id . The radioactivity in each labelled amino acid was determined by l iquid sc in t i l l a t ion counting. 51. PART III. RESULTS AND DISCUSSION A. Qualitative Metabolism of Glutamic Acid . The pivotal position of glutamic acid between amino acid and carbohydrate metabolism suggests its importance in ce l lu lar function. In brain especial ly , as i l lustrated in Diagram I, i t occupies a key bio-chemical posit ion, mainly as a source of protein, as a source of free energy, and as a precursor of GABA and N-acetyl aspartic acid , two compounds found only in the CNS. Furthermore, i t can function also in the detoxification of cerebral ammonia through its conversion to gluta-mine. In order to determine which of the various routes glutamic acid takes after i t enters the brain from plasma, a radioautogram was prepared from a paper chromatogram on which the components of an aqueous extract of excised labelled brain tissue were separated. C-14 glutamic acid had been administered intravenously to a cat 7 minutes prior to sacr i f i ce . The results of this experiment, as shown f iguratively in Diagram II, indicate that labelled glutamic acid under these conditions is oonverted mainly to f ive components, three of which are aspartic acid , glutamine and GABA. The two unidentified compounds were ninhydrin negative and they did not fluoresce under UV light after heating. Thus, they were not amines. In general, similar observations were noted by Lajtha et a l . ( I l l ) and Roberts et aj_. (8) in vivo and by Tower (109) and McKhann et a K (13) in  vi tro. The high conversion of labelled glutamic to succinate (8) and to glutathione (111) could not be detected under the conditions of this study. ! DIAGRAM I . Major pathways of Glutamic Acid Metabolism in Brain. N- Rcetylaspartic Rsparhc acid Citrate Ox alo acetate. Log Protein Gluialhione 4 ex. ketoghtarate J fProline-t. hydmxi Q proline Glutamic setnialdehyde. Glulai 'mine Succinate ~*~ 75" MClQf TURN " 0 ONE STEP 4 ? NOT PROVEN IN BRH/W Qflfif] ? t- qammctcjuanidino butyric l^Histidine Homocarnosine Major pathways nf glutamic acid metabolism in brain DIAGRAM II. Chromatogram of Components in Aqueous Brain Extract. GLUTRMINE EJ - Non~ Labelled Ninhydrin positive spots 83 -labelled Ninhydrin positive spots -Labelled Ninhydrin negative spots ACID BiPBKTIC HOP 0) « Origin .Bo Be Hto <4/I/I ) 54. B. Quantitative Distribution of Labell ing. Only about 35% of the radioactivity in aqueous brain extracts was located in glutamic ac id , glutamine, GABA and aspartic ac id . Cursory measurements indicated that the two unidentified labelled compounds could account for only another 15%. Since i t has already been shown (Table 1) that there was no net loss of counts during paper chromato-graphic separation of the compounds detected, the rest of the radioactivity must be in the carbon skeletons of unstable compounds which are part of the other pathways of glutamic acid metabolism, or in stable compounds which escape detection because of low act i v i t y . The results of the systematic determination of label l ing in the identif ied amino acids throughout different areas in cat brain is shown in Table 5. In this study, cats were sacr i f iced at different time intervals after the intravenous administration of labelled glutamic ac id . This type of experiment wi l l be defined as a cross sectional time study. The data indicate that although the total radioactivity varies from brain area to brain area, its distr ibution between glutamic acid and its metabolites shows a consistent pattern with very few exceptions. These patterns i l lus t rate two things: (i) the turnover rates of glutamic acid and i ts products are similar throughout most of the brain, and ( i i ) some parts of brain take up glutamic acid more readily than others. C. Size of Amino Acid Pools in Brain. The absolute values of the amino acid pools in brain areas (Table 6) in contrast to their uniform distr ibution of label l ing, show marked var ia-tion throughout the brain and do not conform to any obvious pattern. This var iabi l i ty is reflected in their specif ic act iv i t ies (Table 7) which are TABLE 5. Comparisons of the Distribution of Labelling in Amino Acids in Different Brain Areas (Line one represents dpm/gm wet tissue; l ine two represents ratios of dpm/gm relative to glutamic acid) 3 Minute Cat 7 Minute Cat 12 Minute Cat Focal Lesion in Lt . Motor Cortex  Tissue Aspar-t ic Acid Gluta-mic Acid Gluta-mi ne GABA Aspar-t ic Acid Gluta-mi c Acid Gluta-mine GABA Aspar-t i c Acid Gluta-mic Acid Gluta-mine GABA Lt. Parietal 178 1273 165 165 404 1814 604 400 579 2756 827 689 Cortex .14 1 .13 .13 .22 1 .33 .22 .21 1 • 30 .25 Rt. Parietal 196 1308 131 170 302 1426 584 324 476 2267 227 385 Cortex .15 1 .10 .13 .21 1 .40 .23 .21 1 .10 .17 Rt. Motor J88 1172 281 316 302 1434 516 224 446 1783 1070 624 Cortex .16 1 .24 .27 .21 1 .36 .16 .25 1 .6 .35 Lt. Motor 274 1710 410 308 168 2240 1404 148" 270 1500 585 375 Cortex .16 1 .24 .18 .08 1 .63 .07 .18 1 .39 .25 Rt. Sensory 257 1609 434 257 _ _ 413 1059 339 7ie Cortex .16 I .27 .15 .39 1 .32 .67 Lt. Sensory 213 1422 284 256 246 1146 578 226 418 1045 460 690 Cortex .15 I .20 .18 .21 1 .50 .20 .40 1 .44 .66 Rt. Auditory 146 1120 236 56 308 1436 376 206 449 1951 761 648 Cortex .13 1 .21 .05 .22 1 .26 .14 .23 1 .39 .33 Lt. Auditory 132 1016 194 50 292 1456 468 228 389 1770 726 690 Cortex .13 1 .19 .05 .20 I .32 .16 .22 1 .41 .39 Rt. Visual 196 1312 354 170 360 1430 334 370 568 2103 904 799 Cortex .15 1 .27 .13 .25 1 .25 .26 .27 1 .43 .38 * focal lesion continued . . TABLE_5» cont'd. 3 Minute Cat Focal 7 Minute Cat Lesion in Lt . Motor Cortex 12 Minute Cat Ti ssue Aspar-t i c Acid Gluta-mi c Acid Gluta-mine GABA ,Aspar t ic Acid Gluta-mic Acid Gluta-mine GABA Aspar-t ic Acid „ Gluta-mic Acid Gluta-mi ne GABA j Lt . J/isual Cortex 256 .16 1600 1 384 .24 272 .17 356 .25 1412 I 778 .55 304 .22 640 .27 2370 1 758 .32 972 .41 Rt. Cingulum 236 .17 1386 1 290 .21 138 .10 238 .20 1190 1 416 .35 288 .24 643 .36 1786 1 589 .33 1054 .59 Lt . Cingulum 186 .15 1238 1 222 .18 148 .12 350 .23 1532 1 514 .34 300 .20 584 .35 1669 1 334 .20 1102 .66 Rt. Hippo-campus 154 .16 906 1 298 .33 100 .11 244 .26 950 1 410 .43 174 .18 330 .17 1940 I 252 .13 408 .21 Lt . Hippo-campus • -724 1 238 .33 60 .10 254 .28 910 1 444 .49 162 .18 Rt. Thalamus 210 .23 914 1 246 .27 374 .41 328 .21 1198 1 256 .21 352 .34 Lt . Thalamus 176 .20 882 1 194 .22 352 .40 260 .25 1030 1 410 .40 358 .34 Hypothalamus 198 .34 580 1 198 .34 238 .41 304 .22 1406 1 1026 .73 316 .23 Rt. Caudate 224 .23 970 1 224 .23 48 .05 254 .24 1252 1 368 .29 294 .24 510 .34 1498 1 954 .57 1018 .68 Lt . Caudate 44 .06 740 1 196 .25 44 .06 382 .21 1740 1 626 .36 388 .22 620 .30 2066 1 1426 .69 1342 .65 continued . . TABLE 5, cont'd. 3 Minute Cat Focal 7 Minute Cat Lesion in Lt . Motor Cortex 12 Minute Cat Ti ssue Aspar-t i c Acid Gluta-mi c Acid Gluta-mi ne GABA Aspar t i c Acid Gluta-mic Aci d Gluta-mine GABA Aspar-t i c Acid Gluta-mic Acid Gluta-mine GABA Rt. Putamen and globus pal 1idus 218 .25 870 1 156 .18 270 .31 222 .20 1110 356 .32 488 .44 Lt . Putamen and globus pal 1idus 332 .41 808 1 322 .41 282 .35 270 .26 1036 394 .38 172 .17 376 .71 488 1 488 1 732 1.5 Rt". Amygdala 164 .15 1090 1 208 .19 100 .1 260 .22 1016 508 .50 256 .22 Lt . Amygdala 170 .18 948 1 218 .23 104 .11 420 .25 854 350 .41 168 .20 Septal Region 180 .20 900 1 372 .37 208 .23 206 .21 962 420 .44 378 .39 Quadri gemi nal Plate 296 .19 1566 1 344 .22 1408 .9 298 .23 1292 350 .27 936 .73 785 .84 935 1 392 .42 2150 2.3 Medulla 213 .16 1330 1 306 .23 213 .16 535 .27 1004 432 .43 194 .19 165 .25 662 1 106 .16. 165 .25 Pons 172 .19 900 1 200 .22 136 .15 178 .31 540 236 .44 152 .28 Brain Stem 354 .25 1418 1 382 .27 554 .39 ( 212 .28 758 400 .53 308 .41 399 .30 1329 1 399 .30 412 .31 continued . TABLE 5, cont'd. 3 Minute Cat Focal 7 Minute Cat Lesion in Lt Motor Cortex 12 Minute Cat Ti ssue Aspar-t i c Acid Gluta-mi c Acid Gluta-mine GABA Aspar t i c Acid Gluta-mic Acid Gluta-ml ne GABA Aspar-t i c Acid gluta-mic Acid Gluta-mi ne GABA Cerebellum 222 .18 1230 1 222 .18 208 .17 280 .23 1206 1 530 .44 322 .27 Cervi cal Cord 300 .15 2003 1 422 .21 260 .13 178 .2 874 1 112 .13 128 .15 256 .38 674 1 256 .38 256 .38 TABLE 6. Concentrations (Micromoles/Gm) of Amino Acids in Different Brain Areas Ti ssue 3 Minute Cat Aspar- Gluta-t ic mic Gluta-Acid Acid mine GABA 7 Minute Cat Focal Lesion in Lt. Motor Cortex Aspar- Gluta-t ic mic Gluta-Aci d Acid mine GABA 12 Minute Cat Aspar- Gluta-t i c mic Gluta-Acid Acid mine GABA Lt . Parietal Cortex 2.5 10.8 .9 2.7 8.6 3.5 2.8 Rt. Parietal Cortex 3.0 14.1 3.2 1.8 8.4 1.5 Rt. Motor Cortex 2.8 7.5 4.7 3.0 2.2 9.1 3.3 2.1 1.3 5.5 3.4 1.2 Lt . Motor Cortex .8 7.2 2.; 2.0 1.5 4.4 5.8 1.3 2.5 6.7 4.3 2.6 Rt. Sensory Cortex 2.6 4.9 5.1 3.9 Lt . Sensory Cortex .2 5.4 1.7 1.9 7.3 3.8 1.2 2.0 5.6 2.1 3.7 Rt. Auditory Cortex .5 9.3 4.0 1.6 2.6 7.1 3.] Lt . Auditory 2.4 6.8 Cortex Rt. Visual 2.1 4.7 Cortex 2.1 3.0 6.9 2.7 2.6 2.5 1.5 7.6 4.2 2.0 3.4 8.2 2.0 3.6 continued . . TABLE 6, cont'd. 3 Minute Cat Focal 7 Minute Cat Lesion in Lt . Motor Cortex 12 Minute Cat Ti ssue Aspar-t i c Acid Gluta-mic Acid Gluta-mi ne GABA Aspar t ic Acid Gluta-mic Acid Gluta-mi ne GABA Aspar-t i c Aci d Gluta-mic Gluta-Acid mine GAB/ Lt . Visual Cortex 2.3 10.7 2.3 3.1 1.5 6.3 1.5 1.4 2.6 7.1 3.0( Rt. Cingulum 4.3 10.5 3.5 Lt. Cingulum 2.3 10.1 5.6 1.9 2.0 4.5 5.6 2.5 Rt. Hippocampus 1.1 9.9 3.3 1.3 8.9 3.1 2.4 .67 11.5 3.9 Lt. Hippocampus 1.5 8.4 3.2 2.2 1.3 10.2 3.5 1.6 Rt. Thalamus 1.8 7.1 4.6 3.3 Lt. Thalamus 1.7 10.0 3.0 2.4 12.2 4.1 Hypothalamus 1.0 8.5 3^ 0 4.7 2.1 5.6 4.1 5.3 Rt. Caudate 1.2 7.2 1.8 1.8 6.4 1.2 1.6 4.8 3.4 Lt. Caudate 1.0 7.7 3.8 2.3 Rt. Putamen and globus pal 1i dus 1.2 6.1 2.8 1.5 7.5 5.0 1.3 Lt . Putamen and g.p. Rt. Amygdala 1.0 3.1 6.4 11.2 5.6 4.0 1.8 1.5 8.0 10.3 3.1 1.8 1.8 7.4 13.6 7.9 Lt . Amygdala 1.1 8.2 3.0 1.8 9.1 2.2 conti nued TABLE 6, cont'd. 3 Minute Cat Focal 7 Minute Cat Lesion in Lt. Motor Cortex 12 Minute Cat Tissue Aspar-t ic Acid Gluta-mic Acid Gluta-mi ne GABA Aspar t ic Acid Gluta-mi c Acid Gluta-mi ne GABA Aspar-t ic Acid Gluta-mi c Acid Gluta-mine GAB/ Septal Region 2.0 9.8 3.9 1.4 6.5 - 1.0 Quadri geminal Plate 1.8 4.5 5.4 5.4 2.1 5.1 5.1 2.4 2.6 6.4 7.6 Medulla 1.8 4.8 4.0 1.6 2.2 5.0 3.0 1.5 Brain Stem 1.7 4.0 3.2 4.3 1.3 5.7 1.7 2.5 4.2 4.3 2.4 Cerebellum 1.4 5.5 2.7 1.5 5.1 1.4 Cervical 2.4 4.7 6.1 1.8 - 5.1 2.0 .87 Co rd TABLE 7-Specif ic Act iv i t ies (dpm/micromoies) of Amino Acids in Different Brain Areas. 3 Minute Cat Ti ssue Aspar- Gluta-t ic mic; Gluta-Acid Acid mi ne GABA 7 Minute Cat Focal Lesion in Lt . Motor Cortex Aspar- Gluta-t i c mic Gluta-Acid Acid mine GABA 12 Minute Cat Aspar- Gluta-t i c mic Gluta- GABA Acid Acid mine GABA Lt. Parietal Cortex Rt. Parietal Cortex Rt. Motor Cortex Lt . Motor Cortex Rt. Sensory Cortex Lt . Sensory Cortex Rt. Audi tory Cortex Lt . Audi tory Cortex Rt. Visual Cortex Lt . Visual Cortex 65.0 93 27.0 156 152.0 238 177 263.0 54 149 93 279.0 111.0 150 60 53 105 154 151 24.0 68.0 88 162.0 168.0 112 510 129.0 157 205.0 154.0 90 240.0 188.0 84 237.0 224.0 211 137.0 158.0 156.1 107 242 114 152 188 129 185 217 214 343 108 159 209 173 130 320 324 224 212 187 275 257 246.0 334.0 236 246 315 520 136 170 67 182 186 209 242 265 167.0 256.0 452 184 313.0 TABLE 7, cont'd. 3 Minute Cat Focal 7 Minute Cat Lesion in Lt . Motor Cortex 12 Mi nute Cat Ti ssue Aspar-t i c Acid Gluta-mic Acid Gluta-mine GABA Aspar t i c Acid Gluta-mic Acid Gluta-mi ne GABA Asapr-t i c Aci d Gluta-mic Acid Gluta-mine GABA Rt. Cingulum 55 132 40 Lt . Cingulum 152 152 92 150 292 334 67 441 Rt. Hippo-campus 140 92.0 30 188.0 107 132 72 - 169 105 Lt. Hippo-campus - 86.0 74.0 27.1 195.0 89 127 101 -Rt. Thalamus 117 130 53.0 113 Lt . Thalamus 104 88 117 108 84 - 87.0 Hypothalamus 198 68 66.0 51 144.0 251 66.0 51 .0 Rt. Caudate 187 135.0 27.0= 141 196.0 245 318.0 312 299 Lt . Caudate 382 226 166 169 Rt. Putamen and globus pal 1i dus 181.0 143.0 96.0 148.0 148.0 71 375 Lt . Putamen and globus pal 1idus 332.0 126 50.0 150.0 130.0 - 96 50 36 93 continued . . TABLE 7, c o n t ' d . 3 Minute Cat 7 Minute Cat 12 Minute Cat Focal Lesion in L t . Motor Cortex  Ti ssue Aspar -t i c Ac id G l u t a -mi c A c i d G l u t a -mi ne GABA Aspar -t i c Aci d Gluta^ mi c Ac id G l u t a -mi ne GABA Aspar -t i c A c i d G l u t a -mic Ac id G l u t a -mi ne GAB; R t . Amygdala 53.0 97.0 25 173 99 164 143 L t . Amygdala 155 116.0 35 233 94 - 76 Septal Region 90.0 92.0 53 147 148.0 - 378 Quadrigeminal P l a t e 164 348 64.1 261 142 253 184 327 360 61 282 Medulla 118 277 77 133.0 243 201 144 129 Brain Stem 208 355 119 129.0 163.0 133.0 181 160 317 93 172 Cerebellum 159 224 - 77 187 236 230 Cerv ica l 125.0 426.0 69 144 171 - 120 58. in sharp contrast to the clear cut specif ic act iv i ty patterns found by Berl et a l . (22, 24) in monkey brain areas after the intracisternal administration of labelled glutamic acid. When the size of the amino acid pools throughout the brain were averaged (Table 8 ) , they were comparable to the values obtained in other studies (112), although the range of part icular values was considerably broader. These variations may, therefore, be due to the lack of precision of the ninhydrin method used in their determination. TABLE 8. Average Brain Values of Amino Acid Pools (Micromoles/Gm). Cat Aspartic Acid Glutamic Acid Gl utamine GABA 3 Minute 1.9 7.5 3.4 2.9 7 Minute 1.7 7.8 3.4 1.8 12 Minute 2.3 6.7 3.8 2.9 Average of three brai ns 1.9 7.3 3.5 2.5 Values from (2.2) (8.7) (3.4) (2.3) 1 i terature (112) D. Changes in the Distribution of Labelling With Time. An experiment was designed to determine how glutamic acid metabolism proceeded in brain with time. This design was based on the following ration-ale: Since cort ical areas from each of the animals sacr i f iced at different 59. time intervals showed similar rates of conversion of labelled glutamic acid and its metabolites, then the prof i le of the label l ing changes of these compounds with time could be quantitativelydetermined. This determination can be made i f cort ical areas from a single cat are excised at different time intervals after the intravenous injection of labelled glutamic acid, then quantitatively analysed for radioactive metabolites. This type of experimental procedure is defined as a longitudinal time study. It d i f fers from the cross-sectional time study in that the brain could not be perfused and therefore the act iv i ty measured is the sum of the act iv i ty from tissue plus the act iv i ty from residual blood. Table 9 shows the apparent changes in the distr ibution of the radio-active metabolites extracted from brain tissue with time. (Figure D shows these changes graphically.) The longitudinal time study shows a considerably higher proportion of labelled glutamic acid and glutamine than would be expected from the comparable cross-sectional time study. This disparity is i l lustrated in Table 1 0 . This difference probably reflects the contribution of blood borne glutamic acid and glutamine to their total radioactivity. In order to examine quantitatively this possbil ity an estimate of the blood glutamic acid and glutamine was made. The data in Table 11 shows the rapid fa l l in labelled blood glutamic acid after 15 seconds and also the r ise of labelled blood glutamine. After 1 0 minutes the amount of radioactivity in these two compounds is approximately equal. The recipro-cal respective rise and fa l l of label l ing in blood glutamic acid and glutamine have been found repeated by Berl e_t a K ( 1 1 1 ) in similar experiments. TABLE 9. Longitudinal Time Study of Glutamic Acid Metabolism in Cat Cortex (Line one represents dpm/gm wet tissue; l ine two represents ratios of dpm/gm relative to glutamic acid) Excision Aspartic Glutamic Ti ssue Time Acid Acid Glutamine GABA Lt . Motor Cortex 15 sec. 288 .014 20,060 1 764 .038 344 .017 Rt. Motor Cortex 60 sec. 300 .029 10,062 1 936 .09 300 .031 Lt. Sensory Cortex 120 sec. 288 .030 9,556 1 1,838 .19 118 .012 Rt. Sensory Cortex 240 sec. 256 .055 4,616 1 1,098 .24 159 .034 Lt . Anterior Ecta and Suprasylvian 360 sec. ,224 .069 3,230 I 1,496 .46 134 .041 Rt. Anterior Ecta and Suprasylvian 600 sec. 404 .108 3,738 1 1,952 .52 178 .047 Post. Ecta and Suprasylvian 720 sec. 504 .14 3,578 1 2,400 .67 376 .105 Post Ecta and Suprasylvian 840 sec. 500 .083 5,980 1 2,244 .28 236 .04 Lt . Lateral is posterior 960 sec. 488 .20 2,462 1 1,032 .42 994 .075 Rt. Lateral is posterior 1110 sec. 550 .22 2,524 I 900 .36 220 .09 TABLE 9, cont'd. Tissue Excision Time Aspartic Acid Glutamic Acid Glutamine GABA Lt . Lateral is anterior 1260 sec. 263 .15 1,778 1 1129 .63 ]kk .08 Rt. Lateralis anterior 1500 sec. 516 .15 3,606 1 2,168 .6 282 .078 Lt . Posterior sylvian and 1800 sec. 1012 .18 5,500 1 3,172 .58 k3k .078 posterior inf . ecta sylvian GRAPH D. Changes in Labelling Patterns in Time in Cat Cortex. 10 15 Time (Minutes) TABLE 10. Comparison of Longitudinal and Cross Sectional Time Studies (dpm/gm). Longitudinal Time Study Cross-Sectional Time Study* Time Asp Glu Gli GABA 2" 288 9556 1838 118 6" 224 3230 1496 134 10" 404 3738 1952 178 Time Asp Glu Gli GABA 3" 196 1308 131 170 7" 306 1426 584 324 12" 477 2267 227 385 Rt. parietal cortex TABLE 11. Distribution of Radioactivity in Blood Glutamic Acid and Glutamine (dpm/gm of blood). Time Glutamic Acid Glutamine 15 Sec. 129,800 440 1 Min. 35,600 2240 2 Min. 37,000 1710 4 Min. 15,544 6352 6 Min. 14,000 9600 10 Min. 9,652 10,670 63. Knowing the amount of radioactivity in glutamic acid and gluta-mine in blood, and assuming that 10% of the weight of each excised cort ical area is retained blood; an estimate of the contribution of blood borne labelled glutamic acid and glutamine to the total radioactivity in brain was made. This estimation is shown in Table 12. TABLE 12. Estimated Contribution of Labelled Glutamic Acid and Glutamine in Blood, to the radioactivity in brain tissue (dpm/gm). Total Radioactivi ty Tissue Ami no Radioacti vi ty Contri buted mi nus Time Acid in Brain Tissue by Blood Blood 15 Sec. Glu 20,000 12,980 7000 Gli; 764 44 720 1 Min. Glu 10,062 3560 6502 Gli 936 224 712 2 Min. Glu 9,556 3700 5856 Gli 1,838 171 1667 4 Min. Glu 4,616 1554 3062 Gli 1,098 635 463 6 Min. Glu 3,230 1400 1830 Gli 1,496 960 536 10 Min. Glu 3,738 965 2773 Gli 1952 1067 885 Clearly, a very s ignif icant proportion of label l ing in tissue is contributed by blood borne glutamic acid and glutamine, assuming their 10% value. Even after these values were adjusted (Table 12), there was s t i l l a higher proportion of labelled glutamic acid and glutamine found in the longitudinal time study than in the cross sectional time study. 64. Exceptions to this occur at 6 minutes where the values were comparable after adjustment. Whether the high values found in the brain tissue are due entirely to retained blood or whether other factors are involved could not be deter-mined in this study. A salient point that must be considered in future experiments of this type is the contribution of blood borne labelled glutamine to its radio-act iv i ty in brain eel 1s, even after perfusion. Both the increase in the quantity of labelled glutamine in blood with time and its rapid uptake by brain (23) make i t l ikely that part of the labelled glutamine in brain is formed direct ly from labelled brain glutamic ac id , and part is taken into the brain from blood. E. Deviations from Consistent Labelling Patterns. Exceptions to the typical pattern of label l ing observed in most brain areas were found repeatedly in the quadrigeminal plate, thalamus and putamen-globus pallidus (Table 5). These areas show a higher proportion of label l ing in GABA. The putamen-globus pallidus also shows a high proportion in aspartic ac id . Evidence to support part ia l ly these results has been reported by Roberts (15) who found consistently high GABA pools in the quadrigeminal bodies and diencephalon of many species. Cursory studies by Okumura et a l . (16) have shown a higher GABA pool in the diencephalon of the human. These latter observations should be approached with a certain amount of reserva-tion since the brain areas analysed were post-mortem specimens taken at various times after death. Roberts (15) also found consistently low GABA pools in the pons and medulla. Table 5, however, shows no apparent decrease in the propor-tion of label l ing in GABA in these areas. Since GABA pools in grey matter are higher than in white matter (6), and since there is a smaller propor-tion of grey matter in the pons and medulla, the lower GABA pools in these areas, as found by Roberts, would be due to a decrease in the ratio of grey/white matter per gram of t issue. F. Labelling Patterns in the Focal Lesion. In the focal lesion (Table 5), the label l ing patterns are quite different than the patterns found in the rest of the brain areas of that particular cat . This area shows a relatively high proportion of act iv i ty in glutamine and glutamic acid and a relatively lower proportion in GABA and aspartic ac id , when compared to non-epileptogenic t issue. Whether the data indicate an apparent decrease in the rate of conversion of labelled glutamic acid to GABA and aspartic acid and an increase in this conversion to glutamine, or whether i t is due to experimental artefacts such as traces of blood borne glutamic acid and glutamine in t issue, cannot be judged by one determination. If these deviations in label l ing are contributed by metabolic changes in the lesion, the increase in the ratio of glutamic acid to GABA supports Roberts' (6) contention that a low relative GABA level would result in increased neuronal excitation due to a decrease of the inhibitory effects of this compound. The data also support Woodsbury and Espins* (105) suggestion that the degree of cerebral excitation is inversely proportional to the cerebral GABA levels, but contradicts their findings that i t is inversely propor-tional to the glutamine/giutamic acid rat io . Berl et a_L (102) and Bart e_t aj_. (122) have measured the changes 66. in amino acid pools in a paroxysmally discharging focal lesion produced by ethyl chloride spray. In this type of lesion, there appears to be no net increase in the GABA pools, but glutamic acid and glutamine levels are decreased. This data is qualitatively different in the direction of change from the results found in this study. Whether this difference is due to the different experimental procedures used to produce the lesion, or some other cause cannot be decided on the basis of the experimental evidence now avai lable. G. Labelling Patterns observed in a Cat in Status Epi lepticus. Table 13 shows the distr ibution of radioactivity in the brain of a cat in status epilepticus which was sacr i f iced 10 minutes after an intravenous injection of 50 juc of C-14 glutamic ac id . The condition of sustained seizures was progressively developed in a three year time period after an alumina cream focal lesion was produced in the right motor cortex. .Prior to freezing the brain areas were inadvertently lef t at room temperature for 3 to 4 hours. Due to technical d i f f i cu l t y the tissue was also thawed and left for periods of time up to 30 minutes before homo-genization. The standard procedures for the extraction, separation and measurement of the radioactive amino acids in brain were used in this study. The data (Table 13) show no apparent pattern of label l ing through-out the brain, except for a generally high label l ing in GABA. This is in sharp contrast to the ponsistent patterns observed in the controls. This var iabi l i ty could either be a metabolic property of brain TABLE 13. Distribution of Radioactivity in Amino Acids Throughout the Brain of a Cat in Status Epi lepticus. (Line one represents dpm/gm wet tissue) (Line two represents ratios of dpm/gm relative to glutamic acid) Area Aspartic Acid Glutamic Acid Glutamine GABA Rt. Auditory Cortex 1100 •31 3300 1 330 .1 1650 .5 Lt. Auditory Cortex 864 .46 1872 1 648 .33 . 1944 . 1 Rt. Visual Cortex 624 .28 2223 1 293 .13 1248 .57 Lt . Visual Cortex 1084 .32 3254 1 813 .24 2441 .66 Rt. Sensory Cortex 886 .5 1905 1 930 .5 886 .5 Lt . Sensory Cortex 695 .28 2320 1 732 .31 1336 .57 Rt. Cingulum 513 .17 2949 1 577 .19 449 .15 Lt. Cingulurn 1416 .26 5310 1 1416 .26 2242 .42 Rt. Basal Ganglion 913 .7 1304 1 1304 1 3520 2.7 Lt . Basal Ganglion 924 .48 1926 1 462 .24 2310 1.2 Rt. Thalamus-Hypothalamus 1540 .75 2060 1 600 .29 4800 2 .3 Lt. Thalamus Hypothalamus 960 .67 1440 1 300 .20 2800 2.0 continued . . . 68. TABLE 13, cont* d. Aspartic Glutamic Area Acid Acid Glutamine GABA Rt. Hippocampus 1392 3 6 1 9 1 0 4 4 2366 . 3 8 1 . 2 9 . 6 Lt . Hippocampus 1 5 0 0 3750 500 2 8 7 5 . 4 1 .13 . 7 7 Rt. Amygdala 1 1 7 5 2 6 6 7 1 3 5 6 2305 . 4 4 1 .51 . 8 6 Quadrigeminal Plate 1 8 9 4 1954 5 3 3 6 4 5 3 1 1 . 2 7 3 . 3 Cerebellum 9 4 8 2 8 0 8 6 3 2 1 2 6 5 . 3 1 . 2 3 . 4 5 Medulla 673 1154 3 8 5 1 9 2 4 . 5 8 1 . 3 3 1.7 Brain Stem 8 6 6 1 2 2 7 3 2 5 2 3 8 5 . 7 1 . 2 6 1 . 8 tissue under the conditions of status epilepticus or i t could be due to post mortem metabolic changes ( 5 8 ) that occurred when the tissue was lef t at room temperature. Enzyme act iv i ty is probably most intense after frozen tissue is thawed and left at room temperature. In i t ia l l y , under these conditions mitochondria and microsomes coinfa^ning the enzymes are part ia l ly or wholly broken down by the crysta l l i zat ion of the cytoplasm during freezing. After thawing the enzymes released from the disrupted intracel lu lar structure have a greater possib i l i ty of coming in contact with their sub-strates, thus increasing the rates of their conversion to products. • 6 9 . In order to test this possib i l i ty an experiment was performed (see Methods, (J)) in which previously labelled frozen brain tissue was thawed, then left at room temperature for 2 hours. The results of this experiment (Table 14) shows a qual i tat ive, yet variable increase in labelled GABA, mainly at the expense of labelled glutamic and aspartic acids. These data show that there is a possib i l i ty that the inconsist-encies found in the brain areas of the animal in status epilepticus could be due, in part, to post-mortem changes superimposed onto the metabolic changes occurring in vivo. The quantitative degree to which these changes contribute to the variable patterns under these experimental conditions could not be determined. TABLE 14. Changes In Labelling Pattern in Tissue Left at Room Temperature for 2 Hours (dpm/gm) Aspartic Acid Glutamic Acid Glutamine GABA Total Run #1 Time 0 1012 5500 3172 846 10,530 After 2 hrs. 800 4200 2860 1336 9,196 % Change After 2 hrs. - 21 - 27 - 11 + 58 Run #2 Time 0 1186 4126 2142 568 8,022 After 2 hrs. 726 3522 2282 1498 8,028 % Change - 38 - 7 + 7 + 164 After 2 hrs. 71. SUMMARY Certain metabolic products of intravenously administered C-14 glutamic acid were determined in various brain areas of several normal cats, a cat with an epileptogenic lesion in the le f t motor cortex pro-duced by alumina cream, and one cat in status epi lepticus. (1) Most non-epileptogenic brain areas showed similar conversion of glutamic acid to gamma aminobutyric acid (GABA), aspartic acid and glutamine. (2) Exceptions to this were found repeatedly in the quadrigeminal plate, diencephalon and putamen-globus pal l idus, where there was a higher conversion of glutamic acid to GABA. The putamen-globus pall idus also showed a higher conversion to aspartic ac id . - (3) In the epileptogenic lesion, there was a higher proportion of label l ing in glutamic acid and glutamine than in GABA and aspartic acid when compared with non-epileptogenic cort ical areas. (4) The cat in status epilepticus showed no apparent consistency in the degree of conversion of glutamic acid to GABA, aspartic acid and glutamine throughout the brain. 72. APPENDIX A. j ntroduction. Since toluene is one of the few non-quenching solvents for sc in t i l l a t ing phosphors, there is serious d i f f i cu l t y in accurately determining the radioactivity in labelled compounds that are insoluble or have limited solubi l i ty in toluene. One way to avoid this is to increase the solubi l i ty of the label -led material, in toluene by adding ethanol. Another manner, is to d i s t r i -bute uniformly the toluene-insoluble material on paper before counting. This latter method has been used in this study. Before a description can be given on how the counting eff iciency for this part icular method was determined, i t is f i r s t necessary to define some of the terms to be used. B. Defini t ions. 1. Counting Eff ic iency: the quotient of the sample counting rate (counts per minute, cpm) divided by the sample disintegration rate (dpm). 2. Non-Aqueous Counting Solution: a sc in t i l l a t ion solution for toluene-soluble sample materials made up using 4 gm of 2,5 diphenyl-oxazole (PPO) and 100 mg of 1,4 bis-(2-(5-phenyloxazoly1))-benzene (P0P0P) in one l i t e r of toluene. 3. Aqueous Counting Solution: a sc in t i l l a t ion solution for toluene-insoluble sample materials made using 10 volumes of non-aqueous counting solution and 4 volumes of absolute ethanol. C. Determination of the Counting Eff iciency of C-14 Labelled Toluene-Insoluble Material on Paper. The eff iciency of C-14 labelled glutamic acid on paper was 73. indirectly determined from a sealed standard of C-14 benzoic acid contain-ing 10^ dpm in non-aqueous countings solution. A l l of the following counting procedures were performed at the optimal gain setting for C-14 counting. Each sample was assayed for 3 minutes at a constant voltage setting of 355 (relative units) . 1. Eff ic iency in a Non-aqueous Counting Solution. cpm in sealed standard = 63,500 efficiency of sealed standard = 63,500 = 63.5% 105 2. Eff iciency in an Aqueous Counting Solution. (a) .1 ml of a stock solution of C-14 labelled cholesterol d i s -solved in toluene was counted in 20 ml of non-aqueous solution to deter-mine the dpm. cpm in C-14 cholesterol (non-aqueous) = 8530 cpm/.l ml dpm in C-14 cholesterol = cpm = 8,530 non-aqueous eff iciency .635 = 13,540 dpm .1 ml (b) .1 ml of stock C-14 cholesterol was counted in 14 ml of aqueous counting solution containing .1 ml of d i s t i l l e d water. cpm in C-14 cholesterol (aqueous) = 6,102/.1 ml eff iciency of C-14 in an aqueous counting solution = cpm dpm = 6,102 13,540 3. Eff iciency of C-14 Glutamic Acid (Toluene Insoluble) on Paper  in a Non-Aqueous Counting Solution. (a) .1 ml of an aqueous stock solution of C-14 glutamic acid was counted in an aqueous counting solution. 74. cpm in C-14 glutamic acid (aqueous) = 60,277 cpm .1 ml dpm in C-14 glutamic acid = cpm = 60,277 aqueous eff iciency .45 = 133,650 dpm .1 ml (b) .1 ml of aqueous C-14 glutamic acid solution was spotted on 1" x 1" segments of Whatman #1 paper in t r ip l i ca te . Each sample was counted. cpm in C-14 glutamic acid on paper (non-aqueous) = 77,900 Therefore, the eff iciency of C-14 glutamic acid on paper (non-aqueous) at a voltage setting of 355 is : cpm = 77,900 = 58.3% dpm 133,650 D. Eff iciency - Voltage Curves Graph E shows the relationship between the counting eff ic iencies and the voltage applied to the phototubes for C-14 labelled compounds in aqueous and non-aqueous systems. These graphs only apply when gain is set optimally for C -14. Using these curves one can determine the voltage setting needed to obtain the maximum efficiency using any counting solution. For example, i f one wishes to measure C-14 in an aqueous solution, he would set the voltage at 380 and count at a maximum efficiency of 55%. E. Background. Background is defined as the counting rate (cpm) over and above that contributed by the.radioactive sample being measured. The sources of background in l iquid sc in t i l la t ion counting are cosmic rays, K-40 in glass v i a l s , C-14 in paper and ethanol and electronic noise. Graph F shows how background varies as voltage is applied to the phototubes. GRAPH E. Eff iciency Curves for Different C-14 Counting Systems. 75. Efficiency Curves for  Different Ccounting systems. o 21 C a a u o — A — 0 — 70' 60 — C"non aqueous - C1* aqueous, —C'* on paper (non aqueous) 50 40 30 20 10 200 250 300 330 400 U50 Voltage Setting i I 76. GRAPH F. Background Curve of Paper Blank. I I Voltage Soiling 77. BIBLIOGRAPHY 1. Roberts, E., Frankel, S. and Harmen P.J. Proc. Soc. Exp. B i o l . Med., 2 4 : 383 (1950). 2. Roberts, E. and Frankel, S. J. 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