Influence of complete ischemia on glycolytic metabolites, citric acid cycle intermediates, and associated amino acids in the rat cerebral cortex

Brain Research, 80 (1974) 265-279 © Elsevier ScientificPublishingCompany,Amsterdam- Printed in The Netherlands

265

INFLUENCE OF COMPLETE ISCHEMIA ON GLYCOLYTIC METABOLITES, CITRIC ACID CYCLE INTERMEDIATES, AND ASSOCIATED AMINO ACIDS IN THE RAT CEREBRAL CORTEX

J. FOLBERGROVA*,B. LJUNGGREN, K. NORBERG ANDB. K. SIESJO Brain Research Laboratory, E-blocket, University Hospital, Lund (Sweden)

(Accepted June 24th, 1974)

SUMMARY

In order to evaluate the metabolism of citric acid cycle intermediates and amino acids in cerebral ischemia, brain tissue was sampled after occlusion of the cerebral circulation for 5 min and, in separate animals, after 15 min of recirculation, with subsequent analyses of cerebral cortex concentrations of citrate, a-ketoglutarate, succinate, fumarate, malate, glutamate, aspartate, GABA, alanine, glutamine, asparagine and ammonia. At the end of the ischemia the tissue was depleted of ct-ketoglutarate and (calculated) oxaloacetate, there were decreases in citrate, malate and fumarate, and a pronounced increase in succinate. The pool of citric acid cycle intermediates was increased to 120 % of normal, demonstrating that anaplerotic reactions more than compensated for loss of carbon skeletons due to ammonia detoxification. The ischemic brain showed no significant changes in the contents of glutamate, aspartate, glutamine, or asparagine but there were highly significant increases in GABA and in alanine. At the end of the recovery period the contents of citric acid cycle intermediates returned towards normal but the size of the pool remained elevated. The increase in GABA persisted, alanine was further increased, and there were decreases in glutamate and aspartate, and increases in glutamine and asparagine. The possible implication of the changes in glutamate, aspartate and GABA for the functional state of the tissue is emphasized. Since the increase in succinate during ischemia indicates a reversal of the terminal reactions of the citric acid cycle, the potential energetic contribution of such anaerobic reactions is discussed. It is concluded that these reactions do not contribute significantly to energy production in the ischemic brain.

* Present address: Institute of Physiology,CzechoslovakAcademy of Sciences,Prague, Czechoslovakia.

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INTRODUCTION

Although much information has been accumulated on the effect of total ischemia upon glycolytic metabolites and organic phosphates in the brain 1°,14,2~,27,3°,31,37,4°, relatively little is known about concomitant changes in the metabolism of citric acid cycle intermediates and of related amino acids. The only comprehensive account of changes in citric acid cycle metabolites is that of Goldberg et al. 1~, who measured all free intermediates of the Krebs cycle except succinyt coenzyme A in the mouse brain 5, 10 and 30 sec following decapitation. The changes observed by the authors were interpreted as showing the conversion of a steady state system to an equilibrium system. The authors observed a rapid decrease in the tissue level of a-ketoglutarate which was assumed to occur by means of reductive amination o f a-ketoglutarate to glutamate, a reaction which should channel carbon atoms from the Krebs cycle to the amino acid pools. However, there was an increase in the tissue level of succinate, assumed by the authors to arise from lactate via a pathway involving fumarate and the reversal of succinate dehydrogenase. These reactions should involve CO2 fixation and therefore replenish 'carbon skeletons'*; calculations based on the figures published by Goldberg et al. 15 also indicate that the pool of Krebs cycle intermediates remained close to normal. In the normal brain there is an intimate relationship between the citric acid cycle and the pools of amino acids, the main reactions being catalyzed by glutamate dehydrogenase and by the aspartate and alanine aminotransferases. However, although the amino acids involved show marked changes in severe hypoglycemia 21, they appear to change to a much lesser extent in ischemia. Thus, the tissue concentrations o f glutamate, glutamine and aspartate do not change 39,4°,4a, whereas GABA increases in ischemia6,2~, 39. Although alanine was reported to increase in one study 39, another group found no change in alanine after 10 min of ischemia 43. A further study of the metabolism of citric acid cycle intermediates and of associated amino acids in cerebral ischemia seems warranted, especially when the following two types of results are considered. (1) Certain invertebrate tissues appear to be able to increase the yield of high energy phosphate compounds under anaerobic conditions by reactions that involve carbon dioxide fixation and reversal o f the terminal steps of the citric acid cycle t7,a6. The end products of this sequence of events are succinate and alanine and since these intermediates accumulate in the brain in ischemia (see below) it seems warranted to explore the energetic yield of the reactions, if any. (2) When the brain has been ischemic, recirculation of the tissue is followed by normalization of the adenylate energy charge to within 1 ~ of normal even if the period o f ischemia is prolonged to 15-30 min 16,23. However, since functional recovery is not obtained within an observation period of 3 h, following ischemia of 5 min duration,

* In the present context, loss or gain of carbon skeletons is synonymous with a corresponding efflux or influx of citric acid cycle intermediates irrespective of the number of carbon atoms contained in these intermediates.

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or longer, it is conceivable that biochemical changes other than energy failure are responsible for the functional deficits. In the experiments quoted ~3 there was a delayed fall in glutamate and a rise in glutamine in the recovery period following ischemia of 7.5 or 15 min duration, indicating a deranged amino acid metabolism of possible functional significance. In the present experiments we have studied the influence of ischemia upon citric acid cycle metabolites and associated amino acids in the rat cerebral cortex. The period of ischemia chosen (5 min) was short enough to be theoretically compatible with eventual restitution of the energy state but long enough to induce long-lasting functional deficits 2a. A second group of animals was studied after a 15 min recovery period, following 5 min of ischemia. The objectives of the study were to explore the interrelationships between carbohydrate and amino acid metabolism, with particular emphasis on anaerobic energy production and the metabolism of amino acids of potential importance to the functional activity.

METHODS

The present experiments were identical to those reported in a previous communication from the laboratory24 ('normoglycemic group'), which gives details of the experimental procedures. In brief, the experiments were performed on 300-400 g male rats that were maintained artificially ventilated on 7 0 ~ N20 and 30 ~ 02. The body temperature was kept close to 37 °C, the arterial Pco2 at 35-40 mm Hg and the arterial Po2 above 100 mm Hg. In each animal, the blood pressure exceeded 120 mm Hg, and the hemoglobin concentration was at least 13 g/100 ml. Ischemia was induced for 5 min by increasing the intracranial CSF pressure to values exceeding the blood pressure. At the end of the ischemic period the brain was frozen in situ for subsequent biochemical analyses. In the 'recovery' group, the CSF pressure was reduced to normal at the end of the 5 min period of ischemia, and recirculation was allowed for 15 min before the tissue was frozen. The brains were stored at - - 80 °C before analysis. Cortical tissue from the parietal region was sampled, weighed and homogenized with HCl-methanol at --22 °C and subsequent neutralized perchloric acid extracts were than analyzed for metabolites using the enzymatic, fluorometric methods of Lowry and Passonneau 29. The citric acid cycle intermediates measured were: citrate, a-ketoglutarate (a-KG), succinate, fumarate and malate, whilst oxaloacetate was derived from the aspartate aminotransferase reaction, using an equilibrium constant of 6.7 (see ref. 18). The amino acids analyzed were glutamate, aspartate, glutamine, asparagine, alanine and 7-aminobutyric acid (GABA). The patterns of citric acid cycle intermediates and amino acids were compared to the values for glycogen, glucose, glucose-6-phosphate (G-6-P), pyruvate and lactate obtained from the same brains 24. In addition, and in order to allow characterization of the substrate pattern in the recovery group, the extracts were analyzed for fructose-l,6-diphosphate (FDP), dihydroxyacetone phosphate (DHAP) and 3-phosphoglycerate (3-PG). Analytical conditions for all metab-

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TABLE I ANALYTICAL CONDITIONS

Substrate

Buffer

Other additions

Enzymes

Fxtract (~d)

Reading time (re#l)

Heart lactate dehydrogenase, 15 #g/ml Muscle pyruvate kinase, 0.5/~g/ml Muscle pyruvate kinase, 10/~g/ml Succinyl thiokinase, 40/~g/ml Heart lactate dehydrogenase, 0.25 /~g/ml

50-100

15

Succinate Step I

Step 2

Imidazole, P-pyruvate, 15 mM 250 mM, pH 7.5 MgCI2, 15 mM KC1, 75 mM GTP, 2.0 mM NADH, 0.6 mM Imidazole, CoA, 0.1 rnM 150 mM, pH 7.5

60

Step 3

Imidazole, NADH, 0.003 mM 200 mM, pH 7.5

Fumarate

Hydrazine-HCl, NAD +, 0.2 mM 200 mM, pH 9.0 EDTA, 0.2 mM BSA, 0.01

Malic dehydroge50 nase*, 25 Izg/ml Fumarase*, 20/tg/ml

20

Alanine

Tris, 50 mM, pH 8.1

a-ketoglutarate, 0.05 mM NADH, 0.004 mM

Glutamate-alanine transaminase, 100/~g/ml Lactate dehydrogenase, 1 /~g/ml

25-50

35

Step 1

Tris, 100 raM, pH 8.9

tz-ketoglutarate, 3.2 mM NADP +, 0.5 mM dithiotreitol, 4 mM Na2SO4, 150 mM

Gabase-cell free (Pseadomonas fluorescens), 0,75 mg/ml

30

90

Step 2

Tris, 100 mM pH 7.9

20

GABA

100/~1 aliquot from the reaction mixture of step 1 (after 90 rain incubation at room temperature and subsequent heating at 100 °C for 3 rain)

Asparagine Step 1

lmidazole, 50 mM, pH 7.0

a-ketoglutarate, 0.05 mM NADH, 0.02 mM

Malic dehydrogenase*, 0.5/~g/ml Glutamate-aspartate transaminase,

NADH, 0.0015 mM

Malic dehydrogenase*, 0.5 #g/ml Glutamate-aspartate transaminase, 10 /~g/ml Asparaginase, 10 #g/rnl

50

45

10/~g/ml Step 2

20

* Stock suspensions of the enzymes were centrifuged to remove most of the (NH4)aSO4 and dissolved in neutral buffer.

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olites except succinate, fumarate, alanine, asparagine and GABA have been described in previous communications from the laboratory 11-1a. The analytical conditions for the remaining metabolites are given in Table I and the methods were as follows. Succinate was assayed by the method of Goldberg et al. 15, with small modifications; enzymes and unreacted NADH at step 1 and NADH-oxidizing activity in succinyl thiokinase at step 2, respectively, were destroyed by the addition of 5 M HC1 as described by Goldberg et al., but l0 rain later, the pH of the samples was adjusted back to the original value with 5 M NaOH before further treatment. Succinyl thiokinase, as lyophilized powder, was obtained from Sigma Chemical Co. (St. Louis, Mo.). Fumarate and alanine were measured according to Lowry and Passoneau29. GABA was determined by a modification of the enzymatic fluorimetric method described by Okada et al. a3, using a GABA-aminotransferase-succinic semialdehyde dehydrogenase system (Gabase-cell free, Pseudomonas fluorescens, from Worthington Biochemical Corp., New Jersey). In test tubes (6 mm × 75 mm), 30 #l of tissue extracts were incubated for 90 min at room temperature with 150 #l of the reagent specified in step 1 of Table I (including enzyme). After incubation the tubes were heated to 100 °C for 3 min and 100 #l aliquots were transferred to 1 ml of step 2 reagent of Table I. Fluorescence of step 2 reagent was read before and after addition of aliquots from step 1 and corrections were made for the blank values. Blanks and GABA standards were in each assay run at several levels in the range of the samples. Asparagine was assayed by an enzymatic fluorimetric method, using the enzyme asparaginase for hydrolysis of asparagine to aspartate which was measured according to Lowry and Passonneau 29. As the ratio of asparagine to aspartate contents in brain tissue is about 1:30, the determination of these two amino acids in the same sample a2 did not seem convenient for reliable measurements of asparagine in brain tissue. The present assay was conducted in two steps run in the same tube. In the first step, all aspartate present in the tissue extracts was removed. An aliquot (50 #l) of extract was incubated for 45 min at room temperature with 1 ml of the reagent specified in step 1 of Table I (including the two enzymes). The enzymes and unreacted NADH were then destroyed by adding 10 #1 of 5 M HC1. After standing for 10 rain at room temperature, the pH of the samples was adjusted back to the original value by adding 10 #l of 5 M NaOH. In the second step, asparagine present in the tissue extracts was determined. NADH, malic dehydrogenase and glutamate-aspartate transaminase, at the levels specified in step 2 of Table I were added. After an initial reading of the fluorescence, the reaction was started by addition of asparaginase (asparaginase, Escherichia coli from Boehringer, suspension in glycerine; step 2 of Table I). Blanks and standards in the range of the samples were run in each assay. Aspartate was measured separately, using 10 ktl of extracts and 1 ml of reagent as specified in the asparagine-step 1 of Table I, with the exception that the NADH concentration was 0.004 mM. The results were statistically evaluated using a two-sample t-test or AspinWelch's test. The following symbols are used: P < 0.05 = *, P < 0.01 = ** and P < 0.001 = ***

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J. FOLBERGROVA et GI. 300 200'

/

50

u

~ 100 n

I

I

I

I

I

Control values Gluc Pyr ~-KG Fum OAA in jumol/g 3.05 0.131 0.145 0.087 5.4.10 -3 wet weight _+0.15 _+0.005 ±0.610 ,0.003 ,0.2 (n=6 ) G.-6-P Citr Succ Mal 0.096 0.331 0.492 0.401 ±0.006 _+0.004 *_0.019 ,0.012

Fig. 1. Changes in glycolytic (glucose, G-6-P and pyruvate) and citric acid cycle intermediates (citrate, a-ketoglutarate, succinate, fumarate, malate and oxaloacetate) following an ischemic period of 5 rain duration. The values are given as percentage of controls ( ± S.E.M.). In this and in the other figures the statistical symbols have the following meaning: * = P < 0.05, ** = P < 0.01, *** -- P < 0.001.

RESULTS

Fig. 1 shows the percentage changes in the glycolytic and the citric acid cycle intermediates at the end of the 5 min period of ischemia. At this time the tissue was virtually depleted of glycogen, glucose and G-6-P (see ref. 24) and the very low pyruvate value illustrates that the increase in pyruvate immediately following decapitation, reported by Goldberg et al. 1~, is a transient phenomenon. The figure shows that 5 min of ischemia leads to marked changes in citric acid cycle metabolites. Thus, the tissue was depleted of a - K G and oxaloacetate, there were marked decreases in citrate and malate, a moderate fall in fumarate and an increase in succinate to 270 ~ of normal. A comparison with the results of Goldberg et al. 15 shows that when the ischemia is prolonged from 30 sec, as in their experiments, to 5 rain, in the present ones, the initial changes in citrate, a-KG, succinate and oxaloacetate (OAA) are exaggerated (with depletion of a-KG and OAA); in addition, decreases in fumarate and malate develop. However, in spite of the fact that the concentrations of most of the intermediates were lowered, the increase in succinate was sufficient to raise the size of the citric acid cycle pool to 120~ of normal, an increase of 0.3 mEq/kg (see Discussion). We have previously reported that a 15 min period of recirculation, following 5 min of ischemia, is accompanied by a normalization of the adenylate energy charge

271

INFLUENCE OF ISCHEMIA IN RAT CEREBRAL CORTEX

200~ ~ * *

15oi-\

50

0 I

Control vatues Gluc

in pmol/9 wet weight (n=6)

I

I

FDP

I

I

3-PG

I

I

Citr

I

I

Succ

I

I

I

Mal

3.05 0.109 0.034 0.331 0.492 0.401 _+0.15 _*0.002 _.0.001 _*0.00/, ±0.019 ±0.012 G-6-P DHAP Pyr o~-KG Fum OAA 0.096 0.021 0.131 0.145 0.087 5"4"10-3 ±0.006 _*0.001 _+0.005 ±0.010 -*0.003 -*0.2

Fig. 2. Changes in glycolytic and citric acid cycle intermediates at the end of a 15 rain period of recirculation, following an ischemic period of 5 rain duration. The values are given as percentage of controis (4- S.E.M.).

to within 1 ~ of the normal value, and by disappearance of most of the lactate accumulated during the ischemic period2Z, 24. Fig. 2 shows the pattern of glycolyticand citric acid cycle metabolites at the end of the recovery period. Previous results have shown that the oxygen consumption of the brain in the postischemic recovery period is slightly depressed 20. Furthermore, since at least part of the pyruvate equivalents oxidized by the brain in this period should come from lactate, the flux through the initial part of the glycolytic sequence should have been decreased. Thus, the markedly elevated G-6-P concentration reflects, in all probability, an inhibition of the phosphofructokinase reaction 19, possibly induced by the elevated citrate levels 2s. A similar pattern of changes was recently reported by Duffy et alp in the recovery period following hypoxia. It is shown in Fig. 2 that the decrease in the levels of citric acid cycle intermediates were reversed during the recovery phase and that the major part of the succinate accumulated during ischemia disappeared. However, the size of the citric acid cycle pool was still higher than normal (1.80 as compared to 1.46/~moles/g), indicating a preponderance of anaplerotic reactions (see Discussion). Fig. 3 illustrates the changes in amino acids and ammonia at the end of the ischemia (unbroken line) as well as following 15 min of recovery (interrupted line). There were no significant changes in glutamate, aspartate or glutamine at the end of the ischemic period. This is in confirmation of results obtained by others (see Introduction) and by ourselves2a. Furthermore, there were no changes in asparagine. The

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J. FOLBERGROVA ~'t a/. 500

e - - - - e Recovery Ischemia

400

300" tlI 1*** i i

250

J i i J J

200 /

i

i i

i

g u 150 "B u

0,.

100

50

0

t

Control values Glu in ~umol/g 13.20 wet weight <-0.12 (n=6)

I Asp 334 +0.13

I I I I Gin GABA Ala Aspn 5.65 1.79 0.484 0.097 _+0.21 :t0.06 :t0.011 +0.004

I NH4* 0.28/, +0.023

Fig. 3. Changes in the concentrations of some amino acids following an ischemic period of 5 min duration (unbroken line) and at the end of a 15 min period of recirculation following the ischemic interval (interrupted line). The values are means ~ S.E.M.

ischemia led to highly significant increases in GABA, alanine and ammonia. The increases in GABA confirm two previous reports25, 39 and our results on alanine corroborate those of Tews et al. 39 in showing an elevation of this amino acid in ischemia. The increase in ammonia concentration from a control value of 0.28 to an ischemic value of 1.12 #moles/g ( c f refs. 22 and 41) should be considered in relation to the minor changes in glutamine and asparagine (see Discussion). At the end of the recovery period there were significant decreases in glutamate, aspartate and ammonia, increases in glutamine, asparagine and in GABA, and a very marked elevation of the alanine content. Most of the changes in amino acid levels are explicable in terms of the relationships between the citric acid cycle intermediates and the amino acids (see Discussion). DISCUSSION

We will discuss in turn events occurring during ischemia and in the recovery

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INFLUENCE OF ISCHEMIA IN RAT CEREBRAL CORTEX TABLE II

RATIOS OF N A D - AND FAD-DEPENDENT SUBSTRATE COUPLES IN CONTROL GROUP, AFTER AN ISCHEMIC PERIOD OF 5 MIN DURATION (ISCHEMIC GROUP) AND FOLLOWING 15 MIN RESTITUTION AFTER AN IDENTICAL PERIOD OF ISCHEMIA (RECOVERY GROUP) V a l u e s g i v e n a r e m e a n s 4- S . E . M .

Ratio

Control

lschemia

Recovery

Citrate/a-ketoglutarate Succinate/fumarate Malate/oxaloacetate

2.3 ± 0.2 5.8 4, 0.3 75 4- 3

> 102 18.5 4. 1.3 > 10a

2.8 4, 0.2 8.2 4. 0.3 77 4, 5

phase before considering the possible implication of the amino acid changes for the function of the tissue and the energetic yield of anaerobic citric acid cycle reactions. Events occurring during ischemia Since the tissue stores of oxygen are virtually nil, all reactions occurring upon interruption of the blood flow are truly anaerobic. One can, therefore, anticipate that all substrate pairs that are coupled via redox reactions should change in accordance with the increases in the NADH/NAD + and FADH2/FAD ratios. This is evident from the results illustrated in Table II, which shows large increases in the ratios of citrate to a-KG, succinate to fumarate, and malate to oxaloacetate. However, since these ratios should also be influenced by the decrease in pH the actual increases observed have a complex relationship to the true redox changes. Apart from inducing relative changes in the levels of citric acid cycle intermediates ischemia may also influence the size of the pools of citric acid cycle metabolites and of ammonia equivalents. Ammonia is detoxified in the brain tissue mainly by means of reductive am±nation of a-KG and by amidation of glutamate to glutamine~-4. However, since the latter reaction requires ATP, the reductive am±nation of a-KG should dominate under ischemic conditions. This reaction is normally poised towards glutamate formation, and since ischemia is associated with increases in ammonia and NADH, and with a decrease in pH, depletion of a-KG is the expected result (see Goldberg et al.15). Any reductive am±nation of a-KG must deplete the citric acid cycle of 'carbon skeletons' but since the pool size of these actually increased (see above), anaplerotic reactions must be considered. As will be discussed below, CO2 fixation may be partly responsible for replenishing carbon atoms. However, a shift in the alan±he aminotransferase reaction would also provide carbon skeletons to the citric acid cycle. The observed increase in alanine indicates that the reaction

glutamate + pyruvate ~ alanine + a-KG is shifted to the right. During the early ischemic period the increased glycolytic rate provides pyruvate and the reaction should then be driven both by an increase in pyruvate and by a fall in a-KG. However, since pyruvate is subsequently lost, mainly via

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the lactate dehydrogenase reaction, the shift in the aminotransferase reaction may come to a halt due to depletion of intracellular pyruvate. In order to obtain an estimate of the drain of citric acid cycle intermediates, due to ammonia fixation, we have to make assumptions, the validity of which is rather uncertain. In general, the outflux o f carbon skeletons is equal to the influx minus the actual increase in pool size (about 0.3 #moles/g). If we assume that the influx from the alanine aminotransferase reaction is equal to the increase in the alanine concentration, and that the influx caused by CO2 fixation is equal to the increase in the sum of oxaloacetate, malate, fumarate, and succinate, we arrive at an outflux figure o f about 0.5 #moles/g. Admittedly, this is a very approximate figure, but it indicates that relatively small amounts of carbon skeletons are lost from the citric acid cycle due to ammonia detoxification. This would mean that the ischemic brain (which cannot amidate glutamate to glutamine due to depletion of ATP) is not capable of detoxifying ammonia to any appreciable extent. The main factor limiting this capability is the depletion ofpyruvate which prevents a-KG formation in the alanine aminotransferase reaction. In a previous communication z2 it was reported that 1 rain of ischemia was associated with an increase in ammonia content of about 0.4 #moles/g. Since the sum of adenine nucleotides was not changed from normal, it was suggested that the ammonia equivalents emanated from glutamine. In the present results, there was no significant decrease in glutamine at 5 min. However, in both the control and ischemic groups one single value for glutamine varied markedly from the rest. If these values are neglected, the glutamine content was significantly lower (P < 0.05) in the ischemic group. It thus appears probable that an increased glutamine breakdown during ischemia contributes to the increased tissue levels of ammonia. In the present experiments (5 min of ischemia) there was a decrease in the sum of adenine nucleotides 24 and, therefore, glutamine breakdown as well as deamination of AMP may have contributed to the increase in ammonia content. Events occurring in the recovery period At the end of the 15 min recovery period the ratios of NAD ~- and FAD-dependent substrate couples were again close to normal values (Table lI), as would be expected from a normalization of the redox state, but the size of the pool o f citric acid cycle intermediates remained elevated. At first sight, this may seem surprising in view o f the fact that about 0.9/~moles/g o f ammonia disappeared during the recovery period. Thus, if this amount of ammonia was detoxified by reductive amination to glutamate a corresponding amount of carbon skeletons should have been drawn out from the citric acid cycle. However, the major part of the ammonia was probably detoxified by amidation of glutamate, a conclusion which is supported by the elevated concentration of glutamine, and the decreased concentration of glutamate. Furthermore, two concomitant reactions should have provided carbon atoms to the citric acid cycle. The first of these is the alanine aminotransferase reaction, which apparently was shifted further in favor of alanine formation, probably because reoxidation of the cytoplasmatic N A D H / N A D ÷ system gave rise to pyruvate accumulation. Second-

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ly, the synthesis of adenine nucleotides from IMP occurs by the net reaction~ : IMP + aspartate + GTP ~ AMP + fumarate + GDP + Pi. Therefore, any net synthesis of AMP from IMP in the recovery period would trap ammonia in the adenine nucleotide pool and simultaneously replenish carbon skeletons in the citric acid cycle. The balance of ammonia equivalents in control, ischemic and recovery groups can be approximately obtained by adding together the tissue concentrations of the amino acids measured (taking the glutamine and asparagine concentrations twice), the adenine nucleotide pool, and the concentration of free ammonia. The values were 33.92 4- 0.51, 33.95 ± 0.30 and 34.86 ± 0.20 #moles/g, respectively (means i S.E.M.). If one grossly aberrant value (36.10 #moles/g) is excluded from the control group the mean value becomes 33.44 ! 0.25. This value is not significantly different from that of the ischemic group. However, in the recovery group the sum of 'ammonia equivalents' was significantly higher than in the control group (P < 0.01), indicating that amino acid equivalents are added to the pool during the course of the restitution. It is conceivable that the extra ammonia emanates from brain proteins, e.g. by splitting of amidic groups4% when circulation is restored to the ischemic brain. As stated above, there were postischemic decreases in glutamate and aspartate, and increases in glutamine, alanine and GABA compared to the normal situation. In view of the fact that these amino acids, and their related citric acid cycle intermediates are compartmentalized in the brain 1, it is hard to visualize the pattern of mechanisms involved. However, the data strongly suggest that the balance between the glutamine synthetase and glutaminase reactions is altered in favor of glutamine formation. Furthermore, the increase in alanine concentration is probably secondary to a shift in the alanine aminotransferase reaction, in all likelihood caused by an increased pyruvate concentration. Both an increased synthesis of glutamine, and a shift in the alanine aminotransferase reaction, would, therefore, contribute to the lowering of the glutamate concentration and, via the aspartate aminotransferase reaction, also to the decrease in aspartate. The elevation of the GABA concentration remains unexplained. It may be less meaningful to relate overall tissue concentrations of amino acids to the functional state of the tissue, but since glutamate and aspartate are excitatory substances, and since GABA is probably an inhibitory transmitterZ,3, a4, the changes in the tissue contents of these amino acids are at least in the right direction to explain a depressed function. It should also be recalled that changes in glutamate and glutamine are observed even if the recovery period is prolonged to 3 h and, since the magnitude of these changes are roughly proportional to the length of the preceding ischemia~a, it cannot be excluded that there is a correlation between the aberration in the amino acid metabolism and the functional deficits. Whether or not these changes represent reversible or irreversible events remains to be clarified.

Energetic yield of anaerobic pathways According to the established view, cerebral tissues deprived of oxygen can only obtain energy by degrading glucose (or glycogen) to lactic acid. In the classical glyco-

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lytic scheme, for 1 mole of glucose metabolized, a net production of 2 moles of ATP (occurring at the phosphoglycerate kinase and the pyruvate kinase steps) is obtained, and the system is in redox balance. It has recently been pointed out that helminths (e.g. Ascaris lumbricoides) and certain molluscs (such as the American oyster) have the ability to increase the yield of anaerobic energy production by utilizing some of the citric acid cycle pathways in the absence of oxygen 17,36. In these invertebrate tissues anaerobiosis promotes inhibition of pyruvate kinase and diverts phosphoenolpyruvate (PEP) to oxaloacetate via the PEP carboxykinase reaction. The oxaloacetate (OAA) produced by CO2 fixation is then reduced to malate via a reversal of the malate dehydrogenase reaction. These reactions would, like glycolysis, lead to the formation of 2 moles of ~ P/mole of glucose metabolized and leave the system in redox balance. However, since the carbohydrate formed is not lactate, but malate, there are possibilities for further production of energy. Thus, if malate is converted to succinate via a reversal of the fumarase and the succinate dehydrogenase steps, reduced flavoprotein (FADHz) would be oxidized (to FAD). It was considered by Saz 36 that the necessary reducing equivalents, in the form of NADPH, was provided by the malic enzyme reaction: malate + NADP ~ ~ pyruvate + CO2 + NADPH + H ~. This author concluded that about half of the malate produced was decarboxylated to pyruvate, the other half being converted to succinate, and assumed that ATP formation was coupled to electron transport, with fumarate taking the place of oxygen as the terminal electron acceptor. In the scheme proposed by Hochachka and Mustafa 17, pyruvate formed in the malic enzyme reaction reacts with glutamate in the alanine aminotransferase reaction to yield alanine and a-KG, the latter then being oxidized to succinate with the concomitant production of ~ P in the form of GTP. According to this scheme the path travelled by about half of the malate equivalents through the reverse reactions of the citric acid cycle to succinate would provide the NAD + necessary for oxidizing a-KG via the a-KG dehydrogenase reaction. The proposed sequence suggests that all malate equivalents end up as succinate, and requires a ready source of glutamate. Since brain tissue accumulates succinate and alanine during ischemia (see Results) the anaerobic reactions described for invertebrates seem to occur. The tissue also contains appreciable activities of PEP carboxykinase, pyruvate carboxylase and malic enzymea5,38, and CO2 fixation has been amply demonstrated. During ischemia there is a low HCO 3 and a high CO2 concentration24. Since the reactions catalyzed by PEP carboxykinase and malic enzyme utilize molecular CO2 (see refs. 5, 7 and 8) these reactions are probably responsible for COs fixation in ischemia. The pyruvate carboxylase reaction 5, on the other hand, can hardly contribute to CO2 fixation in ischemia. Thus, since HCO 3 and ATP are depleted the reaction must proceed towards pyruvate formation, and the reaction would channel carbon skeletons from the citric acid cycle. However, any pyruvate thus formed should be returned to the cycle via the alanine aminotransferase reaction. In vertebrates, a low activity of glutamate dehydrogenase favors oxidation of

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any a - K G formed in the alanine aminotransferase reaction 17. However, brain tissue contains appreciable amounts of glutamate dehydrogenase, and increases in ammonia, H + and N A D H during ischemia would make the reaction essentially irreversible in the direction of reductive amination of a-KG1L Thus, when conditions favor amination of a - K G a shift in the alanine aminotransferase reaction towards formation of alanine would provide a means of detoxifying ammonia, but the reaction can hardly carry any energetic advantage. It follows from the above discussion that, in the ischemic brain, CO2 fixation and reversal of the terminal reactions in the citric acid cycle can contribute to the anaerobic yield of energy only if F A D generated at the succinate dehydrogenase step is reduced by available N A D H with simultaneous oxidative phosphorylation. This is not necessarily the case but if one ATP is formed from A D P and Pi for each F A D produced, the extra ATP obtained should be equal to the rise in succinate concentration, or about 0.8 #moles/g. Since the total amount of ~ P released when the available stores of glucose and glycogen are metabolized to lactic acid is about 20-25/~moles/g (see ref. 24), it must be concluded that anaerobic citric acid cycle reactions do not contribute significantly to energy production in the ischemic brain. ACKNOWLEDGEMENTS

This study was supported by grants from the Swedish Medical Research Council (Projects No. 14X-263 and 14X-2179), from the Swedish Bank Tercentenary Fund, from U.S. PHS Grant No. 5 RO1 NS 07838-05 from N I H , from Helge and Manhild Johanssons Fund and Anna Lisa and Sven-Eric Lundgrens Stiftelse.

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