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406

Journal of Neuroscience Research 14:449-159 (1985)

Cerebral Citric Acid Cycle Enzymes inMethionine Sulfoximine ToxicityL. Ratnakumari, G.Y.C.V. Subbalakshmi, and Ch.R.K. Murthy

School of Life Sciences, University of Hyderabad, Hyderabad (L.R., Ch.R.K.M.),and Department of Neurochemistry, National Institute of Mental Health andNeurosciences, Bangalore, (G.Y.C.V.S.), India

The activity levels of pyruvate dehydrogenase. enzymes of the citric acid cycle,a.spartate and alanine aminotransferases, and NADP+-isocitrate dehydrogenasewere determined in the cerebral cortex, cerebellum, brain stem, corpus striatum.hippocampus, and midbrain regions of normal rats and rats injected with acuteand subacute doses of methionine sulfoximine (MSI). In both conditions there wasan elevation in the activities of pyruvate dehydrogenase and all the enzymes of thecitric acid cycle except malate dehydrogenase. whereas the activities of amino-transfera.ses and NADP^-isocitrate dehydrogenase were suppressed in all thecerebral regions. It is suggested that the operational rates of the citric acid cyclewould be enhanced in MSI-induced hyperammonemia and that there might be aderangement in the transport of reducing equivalents across mitochondnal mem-branes. It has been suggested that the convulsant action of the drug is due to itseffects on ionic gradients and may not be due to depletion of a-ketoglutarate fromthe citric acid cycle.

Key words: methionine sulfoximine. hyperammonemia. brain, citric acid cycle

INTRODUCTION

Methionine sulfoximine (MSI) is a potent convulsant used to study experimentalepilepsy, and the symptoms observed in experimental animals have been very closeto those encountered clinically in humans. The principle mechanism of action of MSIhas been shown to be irreversible inhibition of glutamine synthetase. an enzymeinvolved in detoxifying ammonia in extrahepatic tissues such as brain [Sellinger andWeiler. 1963; Larnar and Sellinger, 1965; Larnar, 1968; Ronzio et al. 1969], thusleading to a hyperammonemic state (Tews and Stone, 1964; Folbergrova et al, 1969;Subbalakshmi and Murthy. 1984).

Elevated ammonia levels in brain may interfere with the energy production inbrain by draining a-ketoglutarate. from the citric acid cycle, into glutamate formation

Address reprint requests lo Ch.R.K. Murthy. School ot" Lite Sciences. University of Hyderabad.Hyderabad 500 134, India.

Received November II, 1«84; accepted June 6. 1985.

S 1985 Alan R. Liss, Inc.

450 Kalnakumari, Subhalakxhmi, »ml Murthv

by reductive amination mediated by glutamate dehydrogenase |Bessman and Bess-man, 1955]. However, the evidence lor alteration in the levels of a-ketoglutarate anilATP in brain in hyperammonemic states is contradictory [Schenker et al. 1967:Himifclt and Scisjo, 1970; Hawkins et al. 1973; Vergara et al. 1974; McCandless andSchenker, 19811. Though much information is available on the content of the citricacid cycle intermediates in hyperammonemic states, less attention has been focusedon MSI toxicity.

We report an elevation in the activities of pynivate dehydrogenase and keyenzymes of the citric acid cycle except malale dehydrogenase in MSI toxicity.furthermore, we observed a suppression in the activities of aspartate and alanineaminotransferascs and N A D I ' 4 -dependent isocitrate dehydrogenase. These resultsindicated an increase, rather than a decrease, in the operational rates of cerebral citricacid cycle in MSI toxicity. It is also suggested that the transport of reducing equiva-lents across (he mitochondrial membranes might be impaired in MSI toxicity.

MATERIALS AND METHODS

Coenzyme A, acetyl coenzyme A, thiaminc pyrophosphate, sodium pyruvate,a-kctoglutarate, DL-dithiothrcitol, L-malic acid. DL-isocitratc, succinate, oxaloace-tatc. ADP. L-aspartatc, L alanine; lactate and malate dehydrogenascs; dithiobisnitrohenzoic acid, phenazine methosulfate, N A D 1 , N A D U , and N A D P ' were pur-chased from Sigma Chemical Company, St. Louis. Missouri. 2-(4-lodophenyl)-3-(4-nilrophenyl)-5-phenyl tetrazolium chloride ( INT) was from Loba Chemie, India.Dichlorophenol indophenol was purchased from V.P. Chest Institute, India and TritonX-I(X) was from Koch-Light Laboratories. UK. The rest of the chemicals were ofAnalaR or GR grade and were purchased locally. The commercial enzymes weredialyzed to remove ammonium sulfale and were reconstituted into 50% (v/v) glycerolto give a protein value of 0.5 ing/ml.

Adult Wistar rats of 250-300 g body weight and of either sex from an inbredcolony of the vivarium were used. Food (balanced pellet diet from Pragathi AnimalFeeds, India) and water was given ad libitum.

Drug Treatment

Methionine sulfoximinc was administered intrapcritoneally with saline as acarrier. Tor acute experiments a dose of 300 mg/kg body weight was used, for thesubacutc group a dose of ISOmg/kg body weight was used. Control animals receivednone. The animals in the acute group were decapitated at the end of 3.5 hr and thoseof the subacutc group at the end of 17.5 hr. Brains were quickly removed and washedwith ice-cold saline. Cerebral cortex, cerebellum, brain stem, hippocampus, andcorpus striatum were separated at 4°C; the rest of the brain designated as midbramincludes lhalamus. hypothalamus, and related structures. Finally, 10% homogenatcs(w/v) were prepared in 0.32 M sucrose containing 0.2% (v/v) Triton X-100.

In Vitro Experiments

To the homogenates of different cerebral regions that were prepared fromnormal animals, methionine sulfoximinc was added to a final concentration of I f i M /ml . and the en/ymc assays were performed with this preparation.

Energy Metabolism in Hyperammonemia 451

Enzyme Assays

In the assays of dehydrogenases (except succinate dehydrogenase) phenazinemethosuitate was used as intermediary electron acceptor and INT as the final electronacceptor. The reduction of INT was followed in a spectrophotometer at 500 nm. Inthe assay of succinate dehydrogenase the final electron acceptor was dicholorophenolindophenol. All assays were carried out at 37°C in a Gilford spectrophotometer witha thermoprogrammer.

Pyruvate dehydrogenase was assayed by the method of Hinman and Blass[1981]: citrate synthase by the method of Shepherd and Garland [1969]; NAD r -isocitrate dehydrogenase by the method of Plaut [1969]; a-ketoglutarate dehydrogen-ase by the method of Reed and Mukherjee [1969]; succinate dehydrogenase by themethod of Veeger et al [1969]; malate dehydrogenase by the method of Yoshida[1969]; aspartate, alanine aminotransferases, and NADP + -isocitrate dehydrogenaseby the method of Bergmeyer and Bernt [ 1974a,b.c]. In all the assays the final volumewas 250 id, and 10 /il of 10% (w/v) homogenate was used except for aminotransfer-ases. where only 1 /u.1 was used. Protein content was determined by the biuret methodas described by Varley [1969],

[NT was converted to formazan by both enzymatic and chemical methods. Inthe former, purified malate dehydrogenase was used and NAD1' concentrations werevaried from 0.01 to 0.1 pirn. In the chemical method INT concentration was variedfrom 20 to 100 nm, and reduction was carried by the addition of 10 /il of I % ascorbicacid and 10 /xl of I N NaOH in sodium phosphate buffer (12.5 im; pH 7.8). Astandard curve was prepared for NAD+ and INT by correlating the optical densityvalues obtained by these methods.

Statistical analysis of the method was carried out by the Student t test.

RESULTSBehavioral Changes

The behavioral changes in rats following the administration of MSI observed inthe present investigation were similar to those reported earlier [Subbalakshmi andMurthy. 1981, 1983. 1984; Subbalakshmi, 1981; 1984]. The preconvulsive phaseincluded lethargy, abnormalities in gait and posture, and loss of righting reflexes. Theonset of convulsions was noticed at the end of 3.5 hr after the administration of MSIin the acute group; in the subacute group it was at the end of 17.5 hr. Following this,the animals exhibited uncontrolled rolling along their body axis, and mortality afterthis period was high. Hence, the animals were sacrificed during convulsions.

Changes in Pyruvate Dehydrogenase and the Enzymes of the Citric AcidCycle

In the normal animals, the activity of malate dehydrogenase was the highest andthat of succinate dehydrogenase was the lowest. Activities of pyruvate. a-ketoglutar-ate, and malate dehydrogenases were higher in cerebral cortex, whereas brain stem,midbrain. and hippocampus had the highest levels of citrate synthase. NAD*-isocit-rate dehydrogenase, and succinate dehydrogenase, respectively (Tables I and II).

In animals injected with an acute dose of MSI, an increase in the activities ofpyruvate dehydrogenase was noticed along with the enzymes of the citric acid cycle

454 Ratnakumari, Subbalakshmi, and Mnrthy

except malate dchydrogenase and citrate synthasc. The magnitude of increase ob-served in the activity of pvruvate dchydrogenase was 17-fold in the corpus striatum,whereas in other regions, it was 12-fold to 13-fold except in cerebral cortex, wherethe change was only sevenfold (Table I). A statistically significant increase in theactivity of citrate synthase was noticed in cerebral cortex, cerebellum, and corpusstriatum, but in other regions the changes were not statistically significant (Table I).A fourfold elevation in the activity of NAD1 -isocitrate dchydrogenase was observedin all regions excepting in hippocampus (Table I). Elevation in the activity of a-ketoglutarate dchydrogenase was highest (sixfold) in the midbrain, whereas in otherregions it was threefold to fivefold over the controls (Table II). The magnitude ofincrease in the activity of succinate dchydrogenase was highest in the cerebellum andleast in the midbrain (Table II). In contrast to the elevation observed in the aboveen/.ymes of the citric acid cycle, the activity of malate dchydrogenase was suppressedby the administration of MSI. This effect was maximal in hippocampus and least incorpus striatum (Table II).

The overall pattern of changes observed in the activities of the enzymes of thecitric acid cycle in the subacute group of animals was more or less the same as in theacute group (Table I and II). However, the magnitudes of elevation in the activity ofpvruvate and isocitrate (NAD') dchydrogenases were less than in the acute group(Table I). The activity of citrate synthasc was suppressed in the brain of the subacutegroup of animals following the administration of MSI (Table I). Maximal increase inthe activity of NAD f-isocitrate dehydrogenase occurred in hippocampus and cerebel-lum and the least in midbrain (Table I). There was a twofold to fourfold stimulationin the activities of a-ketoglutarate dehydrogenase and succinate dchydrogenase fol-lowing the administration of a subacute dose of MSI (Table II). Malate dchydrogenasewas suppressed in all cerebral regions of the subacute group of animals (Table II)

Addition of MSI (in vitro) to the assay mixtures also resulted in similar changesin the activities of these enzymes except for citrate synthase, where an 80 90"inhibition was observed. The magnitude of activation was, however, less for pyruvaleand a-ketoglutarate dehydrogenases (Tables I and II).

Changes in Aminotransferases and NADP * -Isocitrate Dehydrogenase

The activities of both the atninotiansfcrases were higher in cerebral cortex,cerebellum, hippocampus, and midbrain than the other two regions in the normalanimals (Table III). In both normal and experimental animals the activity of aspartatcaminotransferasc was higher than that of alanine aminotransferase. The activities ofboth the aminotransferascs were inhibited in all the cerebral regions following theadministration of MSI. The enzyme activity was suppressed by a factor of about 3-4in the acute group of animals and about 1-2 in the subacule group. However, in vitroaddition of MSI to the assay mixture had only marginal effects on the activity of theseenzymes (Table III).

The activity level of NADP' -isocitrate dehydrogenase was more or less thesame in all regions of the brain except in hippocampus and midbrain, where it wasgreater (Table III). Administration of MSI. both in acute and subacute doses, sup-pressed the activity of this enzyme in all cerebral regions studied. However, the drughad no effect on this cnz.ymc when added in vitro except in cerebellum, where it wasstimulated (Table III).

456 Katnakiimari, Siibhiiliikshnii. and Murtliv

Changes in Total Protein Content

In normal rats cerebral cortex had the highest protein content and hippocampushad the lowest (Table IV) . Following the administration of an acute dose of MSI . asmall but significant decrease in the protein content was observed in cerebral cortexand corpus striatum. However, in brain stem and hippocampus an elevation in theprotein content was observed, whereas in cerebellum and midbrain there was nochange. The rise observed in hippocampus was greater than the changes observed inany other region (Table IV) .

In rats administered a subacute close of MSI , protein content was elevated in allcerebral regions. The observed increase was maximal in brain stem and least in thecerebral cortex (Table IV) .

DISCUSSION

Cerebral dependence on glucose for the sustenance of vital processes has beenrepeatedly documented in the past. A major portion of this energy (about one-third)is known to be diverted, through the enzyme Na4 , K*-ATPasc [Siesjo, I978|, forthe maintenance of ionic gradients, which are vital for cerebral functioning. Earlier,we reported an elevation in the Na+ . K + -ATPase activity in homogenates, ncuronalperikarya. glial cells, and synaptosomes following the administration of MSI [Subba-lakshmi and Murthy. 1981, 1983, 1984; Subbalakshmi, 1981. 1984|. Elevation in theactivity of this enzyme would not only affect the ionic gradients but also enhance theproduction of ADP. which is supposed to act as a positive modulator for glucoseutilization IMcIlwain and Bachelard. 1971; Siesjo, 19781. However, this hypothesiscontradicts the role of ammonia (accumulated owing to the inhibition of glutaminesynthctasc by MSI) in cerebral dysfunction, as proposed by Bessman and Bessman11955). As mentioned earlier, reductive amination of a-ketoglutarate to form gluia-mate during ammonia toxicity was supposed to interfere with the operation of thecitric acid cycle and energy production. As not much work has been done in the pastwith regard to the applicability of the Bessman hypothesis to MSI toxicity. an attempthas been made in the present investigation to study the changes in the activities of thecitric acid cycle enzymes in MSI toxicity.

The results indicated, in brief, an overall increase in the activities of theenzymes of the citric acid cycle in brain following the administration of MSI, whichsuggested an increase in the oxidation of pyruvate in MSI toxicity. The increaseobserved in pyruvate dehydrogenase activity suggested the channeling of more pyru-

Protein levels in milligrams per pram wet weigh! of tissue. For oiher details sec footnotes to Tahle I.Figures in parentheses indicate number of animals,

Energy Metabolism in Hj perammonemia 457

vate into the citric acid cycle and enhanced production of acetyl CoA. Owing to thelack of change in the activity of citrate synthase in many cerebral regions, it can beassumed that either this committed step of the citric acid cycle would be rate-limitingor the citrate formation might be proceeding at normal rates because of an enhancedavailability of acetyl CoA. Increased activity of isocitrate dehydrogenase (NAD*)would favor citrate formation by citrate synthase and simultaneously enhance theproduction of a-ketoglutarate.

The availability of a-ketoglutarate for the citric acid cycle depends on the rateof its utilization by aminotransferases and glutamate dehydrogenase. It was shownearlier that the equilibration of glucose carbon with that of glutamate and aspartatewould be through transamination rather than by reductive amination [Machiyama etal, 1970]. Besides the present report on the suppression of aminotransferases in thecerebral homogenates prepared from MSI-treated animals, we earlier demonstratedthe suppression of aminotransferases and glutamate dehydrogenase in the synapto-somes, which outnumber both neuronal and glial perikarya. in the MSI-intoxicatedanimals [Subbalakshmi and Murthy, 1984]. Thus the suppression of aminotransferaseswould spare both a-ketoglutarate and oxaloacetate for the citric acid cycle. Thissuggestion agrees well with the reported fall in the content of both glutamate andaspartate in MSI toxicity [Tews and Stone, 1964]. Furthermore, the elevation ob-served in the activity of a-ketoglutarate dehydrogenase would promote the utilizationof a-ketoglutarate for the operation of the citric acid cycle rather than for thetransamination pathway. A similar increase in the activity of this enzyme was reportedin both acute and chronic ammonia toxicity by Sadasivudu and Rangavalli [1981].Increased activity of succinate dehydrogenase would favor enhanced production offumarate and subsequently of malate.

The suppression of malate dehydrogenase observed in the present study wassurprising, as it would hinder the operation of the citric acid cycle by limitingoxaloacetate production. However, such a condition would be prevented by anapler-otic formation of oxaloacetate, especially from pyruvate by carbon dioxide fixation(mediated by pyruvate carboxylase), which was reported to be stimulated in hyper-ammonemic states [Berl, 1971]. Furthermore, it appears that there might be arelationship between this process and the stimulation of Na + , K + -ATPase, as boththe processes have been shown to be inhibited by ouabain [Cheng, 1971].

In addition to ADP, the rate of glucose utilization would also depend on theredox state of the cell, which is governed by the ratio of NAD(P)H to NAD(P)*.Mitochondrial NAD(P)H would be reoxidized by the electron transport system.However, owing to the absence of such a system in cytosol, and the relative imperme-ability of these compounds across mitochondrial membranes, the rate of reoxidalionof cytosolic NAD(P)H depends on other transport systems. Thus, in brain thereducing equivalents are transported by the operation of the malate-aspartate shuttle,NADP+-dependent isocitrate dehydrogenase, the pyruvate-alanine shuttle, and a-glycerophosphate dehydrogenase [Dennis and Clark, 1978; Siesjo. 1978]. Since theactivity of the last enzyme was shown to be negligible [Siesjo, 1978], the activities ofthe enzymes of the other two systems have been studied in the present work. Themalate-aspartate shuttle is made up of cytosolic and mitochondrial isozymes of malatedehydrogenase and aspartate aminotransferase. The results obtained indicated asuppression in the activities of malate dehydrogenase. aspartate and alanine amino-transferases, and NADP *-isocitrate dehydrogenase both in acute and subacute MSI

458 Ratnakumari, Subbalakshml, and Murthy

toxicity. These results suggested an impairment in the transport of reducing equiva-lents across mitochondria! membranes in MSI toxicity. Probably under these condi-tions the cytosol is in a more reduced state than normal.

Although the mechanism of action of MSI on the enzymes of citric acid cyclewas not elucidated in detail, the results obtained by in vitro fortification of thehomogcnates of normal rat brain wilh MSI indicated the direct action of the drug onthese enzymes at least in the acute stale. In the subacute state, however, besides theabove mechanism, increased synthesis of mitochondrial proteins might also be in-volved. This suggestion would be in agreement with the increase observed in theprotein content in the subacute state in this study and the ullrastructural evidencepresented by Gutierrez and Noreriberg |I975, I977|.

Our results thus indicate that in MSI induced hyperammonemia the operationalrates of the citric acid cycle might be enhanced by the elevated activity levels of theenzyme and that the ADP generated in the maintenance of ionic gradients (owing toelevated Na ' . K ' -ATPase activity) might be acting as a positive modulator. Theoxaloacetate required for this process might be generated by anaplerotic reactions.Furthermore, these results also indicate a possibility of derangement in the transportof reducing equivalents across mitochondrial membranes. The convulsant action ofthe drug may be due to its effects on glutamate metabolism and on ionic gradients[Subbalakshmi and Murthy, 1984] and may not be due to the depletion of a-keloglu-tarate from the citric acid cycle and interference with energy production by theaccumulated ammonia.

ACKNOWLEDGMENTS

Financial assistance for the study was provided by the Indian Council of MedicalResearch through a grant to Ch.R.K.M. G.Y.C.V.S. was a Senior Research Fellow.We thank Dr. S.L.N. Rao for his help in the preparation of mcthioninc sutfoximine.L.R. is a recipient of a U.G.C. fellowship.

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Bcrgmeycr MU. Bernl E (1974c): Glutamatc-pyruvale iransamina.se. In Bcrgmeycr HU (ed): "Methodsof enzymatic analysis." New York: Academic. Vol 2. pp 752-758.

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Cheng S-C (1971): CO3 fixation in the nervous tissue. Int Rev Neurobiol 14:125-157Dennis SC. Clark JB (1978): The regulation ol glutnmaic metabolism by tricarboxylic acid-cycle activity

in rat hrain mitochondria. Biochem J 172 155-162Folberginva J, Passonneau JV, Lowry OH. Schulz I)W (1969): Glycogcn, ammonia and related

metabolites in the brain during seizures evoked by mclhionine sulfoximinc. J Neurochcni 16:191-20.V

Gutierrez JA. Norenbcrg MD (1975): Alzheimer I] astrocylosis following mcthionine sulfoximine. ArchNeurol .12:123- 126.

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Gutierrez JA, Norenberg MD (1977): Ultraslructural study of melhionine sulfoximine-induced Alz-heimer Type II astrocytusis. Am J Pathol 86:285-299.

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Hindfelt B. Siesjo BK (1970): The effect of ammonia on the energy metabolism of the rat brain. Life Sci9:1021-1028.

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Lamar C Jr (1968): The duration of inhibition of glutamine synthetase by methionine sutfoximine.Biochem Pharmacol 17:636-640.

Lamar C Jr. Sellinger OZ (1965): The inhibition in vivo of cerebral glutamine syntheuse and glutaminetransferase by the eonvulsant melhionine sulfoximine. Biochem Pharmacol 14:489-506.

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Ol<n-(1IK6'S6 S3.U0+II00I1IM> Perpamtm Press Lid

ACUTE EFFECTS OF AMMONIA ON THE ENZYMES OFCITRIC ACID CYCLE IN RAT BRAIN

L. RATNAKUMARI*, G. Y. C V. SuBBALAKSHMit and C H . R. K. M U R T H Y ' J

•School of Life Sciences. University of Hyderabad. Hyderabad-SMI 134 andtDepartment of Neurochemistry, National Institute of Mental Health and Neuroscienccs.

Bangalore 560 029. India

{Received 25 January 1985; accepted 5 June 1985)

Abstract—Activities of the enzymes of citric acid cycle were determined along with aspartale and alanineaminotransferases and NADP'-isocitralc dehydrogenase in the brains of rats treated with an acute doseof ammonium acetate and compared with those of normal animals. Elevation in the activities of pyruvate.i-ketoglutarate and succinate dehydrogenases and citrate synthase was observed in hyperammonemicanimals. The activities of malate. NADP^-isocitrate dchydrogenases and aminotransferases decreasedunder these conditions. The results suggest that ammonia toxicity mighl not be due to the depletion ofa-keloglutarate from citric acid cycle.

Elevated concentration of ammonia either in bloodor in brain is known to be neurotoxic and producesconvulsions or coma. It was suggested that ammoniainterferes with cerebral energy metabolism by morethan one mechanism. The diversion ofa-keloglutarate for the detoxification of ammoniaresulting in the formation of glutamale (throughglutamate dehydrogenase reaction) was supposed todeplete the citric acid cycle intermediates, thusaffecting ATP production. This would be furtheraggravated by the loss of ATP during gluuimineformation [through glutamine synlhetase reaction](Bessman and Bcssman, 1955). This hypothesis re-ceived support from the observed reduction in cere-bral oxygen consumption in vivo; inhibition of theoxidation of keto acids such as pyruvate anda-ketoglutarate (McKhann and Tower, 1967): de-crease in cerebral a-kctoglutarale content (Bcssmanand Bessman, 1955; Clark and Eiseman, 1958) and afall in the levels of high energy phosphates in thebasal parts of brain and in reticular activating systemin hyperammonemic states (Schcnker et at.. 1967;Bessman and Pal, 1976; McCandless and Schenker.1981). However, these evidences were not un-equivocal as in later works no changes were noticedin the oxygen consumption of cerebral slices in thepresence of ammonium salts or in the rate of decar-boxylalion of either pyruvale or a-ketoglutarate.Similarly, ammonium ion induced depletion ofa-keloglularate (Clark and Eiseman. 1958) or ATPwas not observed (Shorey et a/., 1967; Schenker and

JTo whom correspondence should be addressed.

Mendelson. 1964: Hindfclt and Siesjo. 1970, 1971;Hawkins et a!., 1973: Hindfelt et al.. 1977). in fact, anelevation in the z-ketoglutarate content of brain wasreported (Hindfell and Siesjo, 1970). Despite thevoluminous information on the levels of variousKrebs. cycle intermediates, little information is avail-able on the enzymes of citric acid cycle in hyper-ammonemic slates. Presently, we report an elevationin the activities of pyruvate dehydrogenase. citratesynthasc. a-ketoglularate dehydrogenase and suc-cinate dehydrogenase in brain in acute hyper-ammonemic slates. Under these conditions, activitiesof malate dehydrogenase, aspartale and alaninc ami-nolransferases and NADP^-isocitrale dehydrogenasewere suppressed. These results were discussed inrelation to the operation of citric acid cycle and thetransport of reducing equivalents in brain underhyperammonemic states.

EXPERIMENTAL PROCEDURES

MaterialsCocnzyme A. aceiyl coenzyme A. thiamine pyro-

phosphate. sodium pyruvate. a-ketoglutarale. DL-dithio-thrcilol. l.-malic acid. DL-isocitric acid, disodium succinate.oxaloacetic acid. L-aspartic acid. 5.5 -dithiobis 2-nitroben-zoic acid. ADP. phenazine methosulfate. NAD+. NADH.NADP*. and lactate and malate dchydrogenases werepurchased from Sigma Chemical Co. 2-(4-lodophenyh-3-(4-nitrophenyl)-5-phenyl tetrazohum chloride (INT) wasfrom Loba Chemie. India. 2.f>-Dichlorophenol indophcnolwas purchased from V.P. Chest Institute, India. TritonX-100 was procured from Koch-Lighl Laboratories Ltd,U.K. The resl of the chemicals were of analytical gradeand were purchased locally. The commercial enzymeswere dialvzcd to remove ammonium sulfate and were

Svurovhem. Int. Vol K. N o . I, pp 115-120. I9K6Printed in Great Britain All rights reserved

lit. L. RAtNAKl'MARl el tli.

reconstituted into 50% (v/v) glycerol to a final proteinconccntralinn of 0.5 nig/ml.

Adult albino rats of Wistar strain (of either sex) weighing250 .'00 g body weight from Hie inbred colony of thevivarium were ined in this study. Pood (balanced pellet dietfrom Pragati Aninl.it feeds, India) and water were providedad libitum

Induction of hypertimrnonemia and preparation ft homage-mites (in vivo)

Experimental animals were injected inlraperiloncallv with25 mnK.I'kg body weight of ammonium acetate usimi salineas a carrier while the control animals received none. "Theanimals entered into convulsions at the end of Vs nun. 'I heywere decapitated at the end of 20 min (precoinulsive suite)or during convulsions. Brains were quickly removed andwashed with ice-cold saline. Cerebral cortex, cerebellum,brain stem, hippocampus, corpus striatum were dissectedout at 2 C and rest oflhe brain was designated as mid brain.Ten percent (w/v) homogenates were prepared in ice-cold(1.32 M sucrose. Triton X-10O was added to a final concen-tration of 0.2% (v/v).

In vitro experimentsTo the homngcnalcs of different cerebral regions which

were prepared from normal animals, ammonium acetatewas added to a final concentration of lOpmol and theenzyme assays were performed with this preparation

Enzyme waav*The dchydrogenascs of citric acid cycle were assayed by

dye reduction method using phena/ine niethosullsiie as :inintermediary electron acceptor and INT as final electronacceptor except in the case of succinate dchydrnyenasrwhere dichlorophenoi indophenol was used as final electronacceptor. The formation of formazan was followed atSO0 nm and all the enzymes were assayed at 37 C in (iilfordspectrophotometer using a thcrmoprogrammcr. The finalvolume of the assay mixture was always 250/; I and appropriatc blanks (without substrate) were run simultaneously

Pynivate ttehydrogenase was assayed by the method ofHinman and Mass (1481) and citrate synthase by themethod of Shepherd and Garland (1969). Assay ofNAD"-isocitrate dehydrogenase was carried out as de-scribed by Plant (1969); or-kctoglutarate dehydrogenase bythe method of Rccd and Mukhcrjee (1969) and malalrdehydrogenase by the method of Yoshida (1969) except thatphcna7inc mcthosulfate and INT were used as electronacceptors. Succinatc dehydrogenase was assayed by themethod of Vecgcr et at., (1969), asparlatc and alanincaminotransferases by the method of Bergmcyer and Bcrnl(1974). NADP'-Isocitrate dehydrogenase activity was de-termined by the method of Bcrgmeyer and Bcrnt (1974)using the aforesaid electron acceptors, in all the assays 10 >>'of 10% homogcnalc was used except for aspartate andalaninc aminotransferases where 10/d of 1% homoi'cnalcwas used Protein content was determined by the buiretmethod (Varlcy. 1969).

Preparation of forma:anForma7an was prepared from INT by chemical and

enzymatic methods. In the former, varying concentrationsof INT (20-I00nmol) was reduced with IO/II or 1%ascorbic acid and IO/iI of 0.1 N NaOH in a final volumeof 25O/il. In the en/ymatic reduction, purified malatcdchydrogenase (Sigma), malate and varying concentrations

of NAD' (10 100 nmol). in a final volume of 2S0jul, wereincubated along with INT and phena/ine mcthosulfate at37 C till the optical density remained constant. The valuesobtained in these experiments were used lo prepare aStandard curve for the estimation of NADH formed in theenzyme assays described above.

Statistical analysis of the results were carried out byStudent's (-test.

Following the administration of ammonium salts,rats entered into prcconvulsivc state in about15-20min, In this slate, the animals were lethargicand were less responsive These animals entered intoconvulsive slate by about 30 35 min and cxhibitcilholh clonic and tonic seizures. The in tiro dose ofammonium acetate used presently was higher thanthat used by Bcssman and Pal (1976) as it wasobserved that the dose recommended by them failedto elicit any behavioural response in our animals.

Pvrurate dchvdro^cnasc activity {liihlc I)

The activity levels of pynivate dehydrogenase wasmore or less the same in all the regions c*r brainstudied presently except in cerebral cortex where itwas twice to that of the other regions. Administrationof ammonium acetate resulted in an elevation in theactivity of this enzyme in all the regions bolh inprcconvulsivc and convulsive states. The magnitudeof elevation was. however, greater in the prccon-vulsive state than in convulsions. Of all the regions,maximal elevation was observed in corpus striatumand hippocampus in preconvulsivc and convulsivestates and minimal in cerebral cortex. In vitro, addi-tion of ammonium acetate also elevated the activityof this enzyme in all the regions of brain

Enzymes of citric acid cycle (Tables I and 2)

Of the enzymes of citric acid cycle studied, theactivity of inal.nr dehydrogenase was highest andthat of succinatc dehydrogenase was lowest in all theregions of brain. There was not much of regionalvariation in the distribution of the enzymes of citricacid cycle except succinatc dehydrogenase (lesser incerebellum than in other regions) and malatc dehy-drogenase (highest activity in hippocampus whencompared to other regions).

Following the administration of ammonium ace-tate, an elevation in the activity of citrate synlhascwas observed in cerebral cortex, corpus slriatum andmid brain regions while the change in cerebellum,hippocampus and brain stem was not statisticallysignificant in the prcconvulsivc state. In the con-vulsive state, the activity of this enzyme was elevated

KESI ITS

Citric acid cycle in acute hyperammoncmia 117

L. rUrNAKIIMARt tl 111-

vulrivc stales following (he administration of ammo-tiiuni acetate. As in the case of pyruvale dchydro-genase. the magnitude of change in the activity ofi-kctoglutaratc dehydrogenase declined followingthe onset of convulsions. Sticcinate dehydrogenastactivity was elevated under these conditions, (hechange being greater in the convulsive slate than inpreconvulsivp state in all the regions. However, ma-late dehydrogenase activity was suppressed by at least50% in all the cerebral regions in both the slates.Enrichment of homogenates by in vitro addition ofammonium acetate resulted in changes similar tothose described above except that the activity ofcitrate synthasc which was suppressed in all theregions.

Aminotransferases ami NADP'-isocitmtc dehydro-zoiase (Table .')

Both asparlatc and alaninc aminotransfcrascs weresuppressed in all the cerebral regions in ptcconvulsivcand convulsive stales, while the magnitude of de-crease was greater for alaninc aminotransfcrasc thanlor aspartatc aminotransferasc. A fall in the activityof NADP' -isocitrate dchydrogenasc was observed inall (he regions except in brain stem and corpusslriatum in Ihc prcconvulsive state while in convulsivestate significant fall was observed in cerebral cortex,hippocampus and mid brain. Under these conditions,the activity of NADP'-isocilrate dchydrogenasc waselevated in cerebellum. Unlike the enzymes of citricacid cycle, in vitro addition of ammonium acetatehad marginal effects on the aminotransferases andNADP'-isocitrate dchydrogenasc

DISCUSSION

In extrahepatic tissues, such as brain, ammoniawas shown to be detoxified chiefly by the formationof glutamale (a-kctoglutarate 4- ammonia^gluta-matc) and glulamine (glulamatc + ammonia + ATP—>glutamine) mediated by the enzymes glutamaledchydrogenasc and glutaminc synthclasc respectively

(Benjamin, 19X2; Kvammc. 1983). It was postulatedthat in hypcrammonemic states there would be anincreased utilization of oi-kctogliitaralc which wouldaffect the operation of citric acid cycle (therebyinterfere with energy production) and deplete cellularenergy stores (Bcssman and Bessman. 1955). Studieson metabolic compartmentation and histologicalchanges under these conditions indicated that astro-glia might he the sites of ammonia detoxification(Berl, 1971; Zamora et a/., 1973: Norenbcrg. 1977).

Results obtained presently indicated that elevatedammonia levels might be enhancing Ihc operationalrates of cerebral citric acid cycle. Increased activity ofpyruvale dehydrogenase would promote channellingof pyruvatc into the citric acid cycle in the form ofacetyl CoA. This observation was, however, in con-tradiction to Ihc suggested inhibition of Ihc oxidationofa-kclo acid in hypcrammonemic states (McKhannand Tower, 1967). Elevated activity of citrate syn-thasc, under these conditions, would promote theutilization of acetyl CoA and citralc formation incerebral cortex, corpus strialum and mid brain re-gions. In cerebellum and brain stem, where theactivity of this enzyme was unchanged, citrate for-mation might be rate limiting and proceed at normalrates. The increased utilization of o<-kctoglutaralc.due to elevated oc-kctoglularatc dchydrogenasc.would promote its formation from isocitrate al-though the activity of NAD1-isocitrate dchy-drogenasc was unchanged under these conditions.However, the availability ofa-ketoglutaratc to citricacid cycle depends on the activities of amino-transferases and glutamatc dchydrogenasc, which usethis metabolite.

Among the aminotransferases, Ihc activities ofaspartalc and alaninc aminotransfcrascs were re-ported to be highest and that a major portion ofglucose carbon enters the carbon skeleton of glu-tamatc through the reaction mediated by these en-zymes (Benuk et at,, 1971; Machiyama et a/., 1970).Decreased aclivity levels of these enzymes, observedpresently, would spare a-kctoglularatc, oxaloacctalc

Citric acid cycle in acute hypcrammoncmia 119

and pyruvate for citric acid cycle. The reportedinhibition of glulamate dehydrogenase in the aslro-glia and synaptosomes (which together outnumberneuronal perikarya) in acute hyperammonemiawould also spare »-kcloglularale for citric acid cycle(Subbalakshmi and Murthy. 1983, 1984). Thus, itappeared that in hypcrammoncmic states x-kcto-glutarate would be channelled more into citric acidcycle than for the formation of aspartate or gluta-mate. A similar increase in the activity of cerebrala-kcloglutarale dehydrogenase was reported earlierin acute ammonia toxicity (Sadasivudu and Ranga-valli, 19R1|. Increased activity of succinatc dehydro-genase would favour utilization of succinate andsubsequently fumarate.

Suppression of malate dehydrogenase in hyper-ammonemic states would result in the accumulationof malate and limit the formation of oxaloacetalewhich might interfere with the operation of citric acidcycle. Increased malate levels and a fall in the contentof oxaloacctate, reported earlier by Hawkins et ai,(1973) in acute hyperammonemic states, were inconcurrence with the present observation. However,this situation might be averted by anaplerotic replen-ishment of oxaloacctate b\ carbon dioxide fixation, aprocess stimulated in hyperammonemic states (Berl,1971).

Besides its participation in citric acid cycle, malatedehydrogenase along with aspartate amino transfer-ase is also involved in the transport of reducingequivalents across the mitochondria! membrane(Shank and Campbell. 1983). The other two systemsinvolved in this process arc alanine aminotransfcrase(alanine-pyruvale shuttle) and NADP'-isocilrale dc-hydrogena.se (Sicsjo. 1978). Following the adminis-tration of ammonium acetate, activities of these twoenzymes were found to be decreased suggesting animpairment in the transport of reducing equivalentsacross mitochondria. Under these conditions thecytosol may be in a more reduced slate than mito-chondria (where electron transport chain reoxidizesNAD(P)H generated). A similar observation wasmade by Hindfclt and Siesjo (1970) and Hawkinset at. (1973).

Thus, the results, obtained presently, were sug-gestive of enhanced oxidation of a-keto acids (pyru-vate and j-ketoglularatc) due to the stimulation ofthe enzymes of citric acid cycle. The ADP generateddue to an increase in gluiamine synthesis (Sub-balakshmi and Murthy. 1983: Benjamin. 1982:Kvamme. 1983) and Na*. K'-ATPase (Sadasivuduet a/., 1977; Subbalakshmi and Murthy. 1981) mightbe acting as a positive modulator. Oxaloacetate re-quired for the continuation of citric acid cycle would

be generated by carbon dioxide fixation. The resultsalso indicated an impairment in the transport ofreducing equivalents from cytosol to mitochondria.Hence ammonia might not be depletinga-ketoglutarate from citric acid cycle as suggestedearlier (Bessman and Bessman. 1955; Schenker et at..1967; Bessman and Pal. 1976).

Acknowledgements — We thank Indian Council of MedicalResearch for the financial assistance by a grant to R.K.M.G.V.C.V.S. was a CS1R Senior Research Fellow and L.R.is a recipient of UGC fellowship.

REFERENCES

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Bcnuck M.. Stern F. and Lajtha A. (1971) Transaminationof amino acids in homogenates of rat brain. J. Keuro-ehem. 18, 1555-1567.

Bergmeycr H. U. and Bernt E. (1974) Isocitrale dehydro-genase: glulamale oxaloacctate transaminase: gluta-mate pyruvate transaminase. In: Methods uf EnzymaticAnalysis (Bergmeyer H. U.. ed.). Vol. II. pp. 624-627.727-733. 752-758'. Academic Press. New York.

Berl S. (1971) Cerebral amino acid metabolism in hepaticcoma. In: NeuroehenUstry of Hepatic Coma. Exptl. BioiMed. (Polli E.. ed.), Vol". 4 pp. 71-89. Krager. Basel.

Bessman S P. and Bessman A. N. (1955) The cerebral andperipheral uptake of ammonia in liver disease, with anhypothesis for the mechanism of hepatic coma, J. din.forest 34. 622-628.

Bessman S. P. and Pal N. (1976) The Krebs cycle depictionthcor> of hepatic coma. In: I'rea Cycle (Grisolia S..Baguena R. and Maun F., eds), pp. 83-S8. John Wiley,London.

Clark G. M. and Eiseman B. 11958) Studies on ammoniametabolism. IV. Biochemical changes in hrain tissue ofdogs during ammonia induced coma. Sew Engl. J. Med.259, 178-180.

Hawkins R. A.. Miller A. L., Nielsen R. C. and VeechR. L. (1973) The acute action of ammonia on ral brainmetabolism in tiro. Biochem. J. 134, 1001-1008.

Hindfelt B. and Siesjo B. K. (1970) The effect of ammoniaon the energy metabolism of the rat brain. Life Sci. 9,1021-1028.

Hindfclt B. and Siesjo B. K. (1971) Cerebral effects of acuteammonia intoxication. II. The effect upon energy metab-olism. Stand. J. din. Lab. Invest. 28, 365-374.

Hindfelt B., Plum F. and Duffy T. E. (1977) Effect of a cuteammonia intoxication on cerebral metabolism in rats withportocaval shunts. J. elm. Invest. 59, 386-396.

Hinman 1. M. and Blass J. P. (1981) An NADH linkedspcctropholometric assay for pyruvate dehydrogenasecomplex in crude tissue homogenates. J. biol. Chcm. 256,6583-6586,

Kvamme E. (1983) Ammonia metabolism in the CNS. Prog.Nrunbiol. 20, 109-132.

Machiyama Y.. Balazs R.. Hammond B. J.. Julian T andRichter D. (1970) The metabolism of; -aminobutyric acidand glucose in potassium ion-stimulated brain tissue invitro. Bmchem. J. 116, 469-481.

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McCandleM n. W. and Schenker S. (1981) I l l c d uf acuteammonia intoxication on ihc cncrg\ Mores iti the cerebralrciicular activating system. E\pl Brain Rc\ 44. )25 J30.

McKhinnC M and Tower D.B (l%7) Ammonia toxicityand cerebral oxidative metabolism Am. ./. Phy.iM. ZCMI.42(1 4M

Norcnberg M, I). (I^77) A light and electron microscopicstudy of experimental portal systemic (ammonia) I'nccphalopalhy. J. rtoi. Lab. fatal, 36, 618 627.

Plaut G. W. E. (I969> lsocitralc dehydrogcnaK flll'N-ipeciAc) from bovine heart. In: Methods in &izymohgy(Uiwensicin .1. M., ed.), Vol. XIII, pp. .14 35. AcademicPress. New York.

Reed L. J and Mukherjce B. B (1969) i-Kctoglutaratedehydrogenase complex from Exchertchia coif. In: Meth-od* in Emymofagy (Lowcnstcin J . ed.), Vol XIII. pp55 58.

Sadasivudu B. and Rungavalli G (IDS I) Arphi-keloglutarate dehydrogenaie activity in the rat brain inconditions of ammonia toxicity. IRCS Mfd, S,i. V,245 24ft,

Sadasivndu B . Rao 1 I and MtirthyC R. K (1977) AcutenietaHolic elicits of ammonia in mouse brain. ,VVuni<Vi<>»i.AM. 2, ft.19 6S5.

Schenker S and Mendekon 1. H. (IW4) Cerebral adenoainetriphosph;He in rals v ith ammonia induced coma Am. JPhywl 2116. 1171 1176,

Schenker S.. McCnndlcss D W.. Brophy P and lewisM S (IV67) Studies on the intracerebial toxicily ofammonia. J. din. Imt'l. 46. 8.18 848.

Shepherd D and Garland P. B (I"W» Citrate synthase fromrat liver. In: Methods in Bmymnlogy (Lowcnstcin .1. M ,ed). Vol. XIII. pp. II 12. Academic Press. New York

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Cerebral i-kcloglutarate in ammonia intoxicationGmirnritltrology H, 7(16 7 1 1 .

Shank R. P and Campbell G. L. (1983) Olulamate In:Handbook <>l Nmmhcmhtrv (Lajtha A., ed). Vol. 5 (2ndedn) pp. 181 4(15. Plenum Press. New York.

Siesjo B. K. (1978) Hmin Energy Melabolilltt. John Wiley.New York.

Subbalakshmi G. Y C. V. and Murlhy Ch. R. K (1981)Pffects of mclhioninc sulfoximinc Ofl cerebral ATPasesBimhvm I'harrmir. .HI, 2127 2139.

Subhalakshmi O. Y. C. V. and Murthy Ch. R. K (1981)Acute metabolic eflecti of anuBonia on the enzymes oialulamale metabolism in isolated aitroglial cells. Neum-chrm. Int 5, 591 597.

Subbalakshmi G. Y. ('. V. and Mnrthy Ch. R. K. (1984)Suppression of the en/ymes of glulamale metabolism incortical synaplosomcs in melhioninc Milfoximinc loxicity.Life S,i. 35, 119 125.

Subbalakshmi G Y. C. V, (1984) Studies on the eiuymcsof cerebral cellular plutamatc metabolism in hypcr-ammoncmia. Ph.D. thesis. University of Hyderabad.Hyderabad.

Varlcy M. (1969) Practical Clinical Chemistry F.LBS andlleincmann Medical Books

Veegcr ('.. Dcrvarlanian D V and Zeylcmakcr W. P. (l%9)Succinate dehydrogenase In: Methods in &tzymology(Lowcnslein J M . ed I. Vol XIII. pp 81 B3. AcademicPress. New York.

Yoshida A. (1969) t.-Malate dehydrogenase from BacillussubtlILt, In: Methods in Eniynwlogy (Lowenitein .1 M .ed). Vol XIII. pp. 141 142. Academic Press. New York

Zamora A .1. Cavanagh J. B and Kyu M II. (1973)Ultiastructural response of astrocyte to portocaval anas-tomosis in the rat. ./. Nturol. Sei. 1H, 25 45.

"Scttrochcmieal Research, I '<>/. N, No. 3, 1989. pp. 221-228

Activities of Pyruvate Dehydrogenase, Enzymes of CitricAcid Cycle, and Aminotransferases in the SubcellularFractions of Cerebral Cortex in Normal andHyperammonemic Rats

L. Ratnakumari1 and Ch. R. K. Murthy1-2

(Accepted November 14, 1988)

Activity levels of pyruvate dehydrogenase. enzymes of citric acid cycle, aspartate and alanineaminotransferases were estimated in mitochondria, synaptosomes and cytosol isolated from brainsof normal rats and those injected with acute and subacute doses of ammonium acetate. In mito-chondria isolated from animals treated with acute dose of ammonium acetate, there was an elevationin the activities of pyruvate, isocitratc and succinaie dehydrogenases while the activities of malaiedehydrogenase (malate—»oxaloacetate), aspartate and alanine aminotransferases were suppressed.In subacute conditions a similar profile of change was noticed excepting that there was an elevationin the activity of a-ketoglutarate dehydrogenase in mitochondria. In the synaptosomes isolatedfrom animals administered with acute dose of ammonium acetate, there was an increase in theactivities of pyruvate, isocitrate, or-keioglutaratc and succinate dehydrogenases while the changesin the activities of malate dehydrogenase, aspartate and alanine amino transferascs were suppressed.In the subacute toxicity similar changes were observed in this fraction except that the activity ofmalate dehydrogenase (oxaloacetate—malate) was enhanced. In the cytosol, pyruvate dehydro-genase and other enzymes of citric acid cycle except malate dehydrogenase were enhanced in bothacute and subacute ammonia toxicity though their activities are lesser than that of mitochondria.In this fraction malate dehydrogenase (oxaloacetate—malate) was enhanced while activities ofmalate dehydrogenase (malate—oxaloacctate), aspartate and alanine aminotransferases were sup-pressed in both the conditions. Based on these results it is concluded that the decreased activitiesof malate dehydrogenase (malate-*oxaloacetate) in mitochondria and of aspartate aminotransfcrasein mitochondria and cytosol may be responsible for the disruption of malatc-aspartate shuttle inhyperammonemic state. Possible existence of a small vulnerable population of mitochondria inbrain which might degenerate and liberate their contents into cytosol in hyperammonemic statesis also suggested.

KEY WORDS: Citric acid cycle enzymes: hyperammonctnia; cytosol; mitochondria; synaptosomes..

I N T R O D U C T I O N

1 School of Lift Sciences, University of Hyderabad. Hyderabad - 5(X) Derangement in the cerebral energy metabolism was

134, India. proposed to be one of the manv mechanisms bv which• Address reprint requcsl 10: Dr. Ch. R. K. Murthv. School of Life K r J '

Sciences. Umversi.v of Hyderabad. HYDERABAD 500 134 AP. ammonia exerts toxic effects on the central nervous sys-lntija tern. It was hypothesized that removal of a-ketoglutarate

221(UM-31W/BWO3fflMC2ISfl6JJU*] £ I9W Plenum Publishing Curpuranon

222 Kiitniikumiiri and Murthv

from citric acid cycle for the purpose of ammonia de-toxification (in the glutamate dehydrogenase reaction),results in the formation of glutamale and (he conversionof the latter lo glutamine, would adversely affect thecerebral energy' stores (1-3). Moreover, by removingcytosolic pool of glutamate for the synthesis of gluta-mine, ammonia was postulated to interfere with the op-eration of malate-aspartale shuttle and thereby the transportof reducing equivalents from cytosol to mitochondria (4,5). Several conflicting reports were made in the past withrespect to the depiction of a-ketoglutaratc stores whileevidences have accumulated which strongly favour thelatter concept (6-9). It was observed that not many stud-ies were made in the past on the subcellular distributionand changes in the activities of the enzymes involved incarbohydrate metabolism in hyperammoncmia.

Earlier, we reported an elevation in the activities ofpyruvate dehydrogenase and enzymes of citric acid cycle,except malate dehydrogenase, in the homogenalcs pre-pared from different regions of brains of hypcrammo-nemic rats (10). We have also reported a fall in theactivities of aspartate and alanine aminotransfcrases,malate dehydrogenase and NADP-dependent isocitratedehydrogenase in these preparations and suggested thattransport of reducing equivalents across mitochondriamight be affected in hypcrammonemic states. As brainhas two types of milochondrial populations i.e., synapticand non-synaptic and both these have citric acid cycleenzymes, a study with homogenales will not revealwhether the changes in the activities of these enzymesare occurring in the synaptic or non-synpatic mitochon-dria. Moreover, enzymes involved in the transport ofreducing equivalents are present in both cylosol and mi-tochondria. Hence it becomes essential to localize thechanges (suppression) in the activities of the enzymes ofmalate-aspartatc shuttle in the brains of hyperammo-ncmic rats and such an attempt has been made in thepresent study.

Based on the results obtained in the present study,we suggest that (i) suppression of malaie-aspartate shut-tle may be due to decreased malale dehydrogenase activityin the direction of malate formation in the mitochondria(ii) there is a small population of mitochondria whichare vulnerable to patho-physiological ammonia concen-trations which degenerate/rupture and liberate their con-tents into the cytosol and (iii) the apparent increase incitric acid cycle enzymes observed in homogenales mayhe due to release of these enzymes into cytosol and lossof regulatory control over these enzymes in an alteredsubcellular environment.

EXPERIMENTAL PROCEDURE

Adult alhino rats of Wislar strain of 250-300 gms. body weight

werf maintained in groups (if 6--K per cape under natural light-darl;

cycles at a constant temperature. Food and water were provided id

libitum. These animals were divided into three groups with ten animal1.

each and two animals were used for each experiment. Animals in group

I were administered intrapcritoncally with 0.35 mmol of ammonium

acetate/100 g body weiphts (subacute experiments) and the animals in

group II received 2.5 mmol of ammonium acetate per 100 gms. bodv

weight (acute experiments) while group III animals received none and

served as controls. Animals in group I and II were sacrificed 25-3(1

min after the administration of ammonium acetate. Mitochondria, syn

aptosomes and cylosol were prepared by the method of Cotman (11)

as described by Subbalakshmi and Murthy (12). These fractions were

frozen overnight and Triton X-100 was added to a final concentration

of 0.19f v/v after thawing the preparations.

Protein, present in 20 u.1 aliquol of subcellular fractions, was

delermincd by the method of Lowry et al. (13). Ammonia content in

brains frozen in liquid nitrogen and in the scrum was determined as

described earlier (12).

Enzyme Assays. Activities of pyruvatc dehydrogenase. citrate

synthase, isocilrate dehydrogenase. a-ketogluiaraie dehydrogenase.

malale dehydrogenase (in the direction of oxaloacetate formation),

aspartate and alanine aminotransferases were assayed as described ear-

lier (10). Malale dehydrogenase activity, (in the direction of malate

formation) was assayed as suggested by Yoshida (14). Laciaic and

succinale dchydrogenases were assayed by the methods of Bcrgmeyer

and Bemt and Nandakumar et al. (15, 16) respectively. After detei-

mining the optimal concentrations of enzyme protein, substrate and

cofadors, suitable alterations were made (or each enzyme (Table II.

Statistical analysis of daia was performed by Student's / test.

RESULTS

Rats administered with subacute dose of ammoniumacetate (group I) showed no convulsions even uplo 10hours. However, they were sacrificed 25-30 min afterthe administration of ammonium acetate. This time pe-riod was chosen as the animals injected with acute doseentered into convulsions at this time period. Rats in-jected with acute dose of ammonium acetate (group 11)exhibited convulsions in about 25-30 min which wasusually the terminal phase. In this group all the animalsirrespective of their sex, succumbed to the toxic effectsof ammonia al about 45 min after the administration ofammonium acetate. Hence, they were sacrificed duringconvulsions and used for experimentation. After theadministration of ammonium acetate there was an in-crease in blood and brain ammonia levels (Table II).

In normal animals, activities of pyruvate, isocii-rate(NAD'), a-kcloglularate and succinate dehydrogen-ases were higher in mitochondria! fraction than in cylosolor synaptosomes. Activities of these enzymes were found

Citric Acid Cycle Enzymes in Hypcrammonemia 223

In all the above assays, except succinate dehydrogenase, final volume was 250 uJ. In the assay for succinate dehydrogenase, the final volume was1.0 ml and the assay was colorimeiric. The assay mixture for SDH was incubated for 15 mm and the reaction was arrested with 2 ml of glacialacetic acid. Colour produced due to the formation of formazan was extracted inlo 5 ml of toluene. In all the assays, the incubation temperaturewas 37°C. Corrections were made for non specific activity with suitable blanks. In the spectrophotomeiric assays changes in absorbance wererecorded at 15 sec intervals for 10 minutes and the values in linear kinetic zone were used for calculating enzyme activity. Relationship betweenformazan formed and NAD* reduced was established as earlier (10). Abbreviations: TPP: thiamine pyrophosphatc; DTT: dithiothreitol; PMS:phenazine methosulphate; INT: 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride; DTNB: 5-5'dithiobis(2-nitrobenzoic acid); CoA:coenzyme A; MDH: malate dehydrogenase; LDH: laciate dehydrogenase; ADP: adenosine diphosphate; NAD: nicotinamide adenine dinucleotide(oxidized); NADH: nicotinamide adenine dinucleotide (reduced). ( ) indicate the reference to the method.

to be higher in synaptosomes than in cytosol. However,in the case of malate dehydrogenase, the magnitude ofdifference between different fractions was not as high aswith other enzymes. Activity levels of aspartate ami-notransferase were observed to be higher in synaptoso-mal and cytosolic fractions than in mitochondria. Anopposite trend was observed in the distribution of alanineaminotransferase (Tables IV - VI).

Effects of Ammonia

Mitochondria (Table IV). Administration of sub-acute dose of ammonium salts (group I) resulted in amarginal elevation in the activity of pyruvate dehydro-genase without altering those of citrate synthase and iso-citrate dehydrogenase. Activities of a-ketoglutarate andsuccinate dehydrogenases were enhanced under theseconditions. In contrast to the above said enzymes, malatedehydrogenase activity, when measured in the directionof oxaloacetate formation, was suppressed in subacuteammonia toxicity. However, in the reverse direction,i.e., in the direction of malate formation, it was unal-tered. Activities of aspartate and alanine aminotransfer-ases decreased under these conditions.

Administration of an acute dose of ammonium ace-tate (group II) resulted in an elevation in the activitiesof pyruvate, isocitrate and succinate dehydrogenases inthe mitochondrial fraction. Under these conditions, ac-tivities of cirate synthase and a-ketoglutarate dehydro-genase were unaffected. Changes observed in the activities

224 Itiilnakiiniiiri and Murlhy

of malatc dchydrogenasc and aspartatc and alaninc ami-nolransfcrascs were similar lo those observed in subaculctoxicity. It is interesting to note that there is a small butstatistically significant increase in the mitochondrial pro-tein under these conditions (Table III).

Synaptosomes (Table V). Following the administra-tion of a subacutc dose of ammonium acetate, there wasan elevation in the activities of pyruvate, isocitrate, «-kctoglutaratc and succinate dehydrogenases while thai ofcitrate synthasc was unchanged in the synaptosomes.Malate dchydrogenase activity, in the direction of ox-aloacetate formation, was suppressed in synaptosomes

in subacute ammonia toxicily. However, in the reversedirection, activity of this enzyme was enhanced underthese conditions. Activities of both the aminotransfcr-ases decreased in the synaptosomes in subacutc ammoniatoxicity.

Pattern of changes in the activities of pyruvale dc-hydrogenase, citric acid cycle enzymes and the aspartalcand alanine aminotransfcrascs in synaplosomes in acuteammonia toxicity were similar lo those observed in thesubacute toxicity. Exceptions to this were seen in theactivity of malalc dchydrogenase in the direction of mal-ate formation which was suppressed in acute ammoniatoxicity. Though the activity of alaninc aminotransfcrascwas suppressed under these conditions, the change wasstatistically not significant. Protein content of corticalsynaptosomes decreased in acute ammonia toxicity whilein subacute toxicity this change was statistically not sig-nificant (Table I I I ) .

Cytosol (Table VI). Administration of a subacutedose of ammonium acetate resulted in an elevation inthe activities of pyruvate dchydrogenase, citrate syn-thase, isocitrate, a-ketoglutarate and succinate dehydro-genases in the cylosol. Eventhough the magnitude oftheir increase in cytosol was much higher than that inthe other two fractions, activities of these enzymes inthe cytosol were lower than that of mitochondria and

Table PV'. Effect of Ammonium Acetate on Pyruvate Dehydroeenase and Enzymes of Cilric Acid Cycle and of Borst Cycle in the Mitochondriaof Rat Cerebral Corlex

Activity is expressed as Mean ± SD.PDH: pyruvate dehyilropenase; CS: citrale synlhcuse: ICDH: isocilrate dehydropenase; a-KGDH: a-kcloplntarate dehydrnpenase; SDH: succinaicdchydrocenase; MDH: malale dehydrogenase; AAT: aspariale arninotransfernse: A1AT: abninc aminottansfcrase.Acli\if\ units for PDH, ICDH, «-KGDH, MDH(NAD) are p.mol of NAD reduced/mg protein/hr and for CS is |tmol of ciu.ne formed/mg proicin/hr, SDH is >imol of succinate oxidized/mg proicin/hr and for MDH(NADH). AAT and A1AT is jimol of NADU oxidized/mg protein/hr No. ofexperimfnls are $. For each experiment two animals were used.

Citric Acid Cycle Enzymes in Hyperammonemia 225

synaptosomes. As observed in the other two fractions,malate dehydrogenase activity in the direction of malaieto oxaloacetate was suppressed. However, activity ofthis enzyme in reverse direction was elevated. Activitiesof both the aminotransferases were suppressed in thissubcellular fraction following the administration of sub-acute dose of ammonium acetate.

Administration of acute dose of ammonium acetatebrought about changes in the activities of these enzymeswhich are similar to those described above. In the caseof pyruvate dehydrogenase, citrate synthase, isocitrate,a-ketoglutarate and succinate dehydrogenases and ala-nine aminotransferase, the magnitude of change underthese conditions was higher than that of subacute tox-icity.

DISCUSSION

In studies dealing with subcellular fractions, it iscustomary to establish the purity of the fractions by de-termining the marker enzymes. However, caution mustbe exerted in situations where the activities of markerenzymes are also altered in the experimental condition(17). In the present case, activities of the markers (lac-tate dehydrogenase and succinate dehydrogenase for cy-tosol and mitochondria respectively) were also alteredsubstantially in the hyperammonemic state (Table VII).Hence, the relative activities of the marker enzymes weretaken into consideration. Changes in the ratio of succi-

nate dehydrogenase activity between mitochondria andsynaptosomes were statistically not significant indicatingthat there were no alterations in the purity of these prep-arations. However, the cytosol/mitochondria ratio of lac-tate dehydrogenase was altered only in the subacutecondition. A statistically significant increase in the ratiofor succinate dehydrogenase in these two fractions wasobserved in hyperammonemic states. Though these re-sults indicated a contamination of mitochondria with cy-tosol, this is difficult to comprehend as the buoyantdensities of these two fractions are different and they areseparated at an early stage of preparation. Hence, thechanges in these ratios are due to drug induced changesin the activities of lactate and succinate dehydrogenasesrather than contamination of fractions.

Increased activity of pyruvate dehydrogenase ob-served presently in the conical mitochondria isolated fromthe brains of hyperammonemic rats is in agreement withour earlier reports in homogenates (10). Such an increasein the activity of this enzyme might permit channellingof more pyruvate into citric acid cycle. However, lackof change in the activities of citrate synthase and isocit-rate dehydrogenase in subacute conditions and citratesynthase and a-ketoglutarate dehydrogenase in acute statesmight limit the flow of carbons through this cycle.Suppression of malate dehydrogenase activity in the di-rection of oxaloacetate production lowers the formationof oxaloacetate and results in the accumulation of mal-ate. A fall in the production of oxaloacetate would affectthe rate of synthesis of citrate. Moreover, this would

226 Katnakumsiri and Murlhy

Upend as in Table IV.

also limit the amount of oxaloacetate available for tran-samination of glutamate, which together w i th thesuppression of aspartatc aminotransferase. would lowerthe production of aspartate in mitochondria. As mito-chondrial aspartate is exchanged for cytosolic glutamateduring the operation of malatc-aspartatc shuttle, reduc-tion in aspartate levels would affect the operation of thisshuttle. The reported fall in aspartate and increase inmalate level? in brain in hyperammonemic states are inagreement with this suggestion (9, 18). Though malatcdehydrogenase activity in the direction of malale for-mation is unaffected, it would be of little consequenceas malate is not the substrate for citrate synthase andaspartale aminotransferase.

It is quite possible that more than one mechanismmay be involved in bringing about the observed changesin the activities of different enzymes in hyperammo-nemic stales. These may be an increase in mitochondrialcontent (Table I I I); changes in the phosphorylation-de-phosphorylation of enzymes such as pyruvate. isocitratcand a-kctoglularatc dchydrogenases (19, 20) and changesin the membrane fluidity (21). Though an increase inthe mitochondrial protein content was observed in thepresent investigation, further studies are required in thisdirection.

Changes in the activities of malatc dehydrogenase(in the direction of OAA formation) and aspartatc ami-notransferasc in cytosol in hyperammoncmic states aresimilar to those of mitochondria. However, in cytosolmalatc dehydrogenase is supposed to be involved in the

synthesis of malate and it is interesting to note that theenzyme activity in the direction is enhanced in hypct-ammoncmic states. Despite this, production of malatc inthis compartment would be limited due to the reductionin the amount of asparlate available (c.f. above) and fallin the activity of aspartate aminotransferase in this com-partment. Thus, it appears that reduction of malate de-hydrogenase activity in mitochondria and of aspartaleaminotransferase in mitochondria and cytosol mighl bethe reasons for the failure of malate-aspartate shuttle inhyperammonemic states. Moreover, reduced aspartatcaminotransferase activity would affect the production ofglutamate which is required for the exchange with mi-tochondrial aspartate and also for glutamine synthesis.It is interesting to note that addition of cither glutamatcor aspartate normalized the malate-aspartate shuttle in theprimary cultures of astrocytes in the presence of pathophy-siological concentrations of ammonium chloride (22). Un-der in vivo conditions, such a situation is averted by theaugmented production of glulamale from the transamina-tion of branched chain amino acid and of oxaloacciatc bycarbon dioxide fixation (23, 24).

Changes observed in the cytosolic activities of py-ruvate dehydrogenase and citric acid cycle enzymes inhyperammonemic states are surprising as this fraction issupposed to be devoid of them. Activities of these en-zymes, though less in this fraction when compared lothe milochondria, were enhanced in hypcrnmmonennestate. Such an increase is difficult to explain unless it isassumed that at least some mitochondria have altered

Citric Acid Cycle Enzymes in Hyperammonemia 227

Tablt Ml . Acuvity Levels and Rdalive Percentages of Marker Enzymes in Subcellubr Fractionsof Normal and Hyperammonemic Rais

LDH : Laciaie dehydrogenase (1) pyruvaie - • laciate, activity is expressed as u.moles of NADHoxidized/mg protcin/hr: (2) lactate -• pyruvate. activity is expressed as pimoles of NAD reduced/mgproteiailr. Rest of the legend same as in Table IV.

their buoyant density due to swelling and sediment athigher centrifugal forces or a population of mitochondriadegenerate and release their contents in to cytosol inhyperammonemic states. It is interesting to note that suchchanges have been reported in the mitochondria in hy-perammonemic states (25). Despite these changes, avail-ability of substrates and NAD* required for these enzymesin cytosol might be inadequate and rate limiting. Hence,such changes may not influence cellular energy metab-olism under these conditions.

As synaptosomes used in the present study haveboth mitochondria and cytosol, changes observed in theactivities of pyruvate dehydrogenase and citric acid cycleenzymes might be similar to those described above. Asmall population of synaptic mitochondria might havealso degenerated under these conditions and liberatedtheir contents into synaptoplasm.

One pertinent point to be discussed at this junctureis whether the activities of enzymes measured in vitroespecially under optimal conditions serve as represen-tatives of in vivo changes. These enzymes from normaland hyperammonemic rats were measured under identi-cal assay conditions where optimal concentrations ofsubstrates and cofactors and pH are provided. It is ap-parent that the observed changes in the activities of theseenzymes are not experimental artifacts, but have oc-curred in situ and have survived the isolation procedureand hence they may represent changes that have takenplace in vivo.

Present study, thus, suggests a derangement in theoperation of malate-aspartate shuttle in the hyperam-monemic states might be due to the suppression of mal-ate dehydrogenase in mitochondria and of aspartateaminotransferase in mitochondria and cytosol and the

228 Uatnakiiiiniii sind Mm Hi\

possibility of exislcnce of a small population of mito-chondria which arc highly vulnerable to ammonia. Fur-ther studies are being conducted lo localise these changesin the specific cellular compartments of brain.

ACKNOWLEDGMENTS

Financial assistance of University Grants Commission in the formof Senior Research Fellowship to LRK ts gratefully acknowledged.

REFERENCES

1. Bessman. S. P.. and Bessman, A. N. 1955. The cerebral andperipheral uptake of ammonia in liver disease with an hypothesisfor the mechanism of hepatic coma. J. Clin. Invest. 34:622-628.

2. Bessman. S. P.. and Pal, N. 1976. The Krebs cycle depletionIheorv of hepatic coma. pp. 83-89. In. Grisolia. S., Baguena R.,and Mayer, F. (eds.). Urea Cycle, John Wiley and sons. NewYork.

3. McCandless. D. W.. and Schenker, S. 1981. Effect of acute am-monia intoxication of energy stores in the cerebral reticular acti-vating system. Exp. Brain Res. 44:325-330.

4. Mindlelt, B. 1983. Ammonia intoxication and brain energy me-tabolism, pp. 474-484, In, Kleinberger, G., and Deulsch, E.,(eds.). New Aspects of Clinical Nutrition, Katger. Basel.

5. Himlfelt. B., Plum. F., and Duffy. T. E. 1977. Effect of acuteammonia intoxication on cerebral metabolism in rats with porta-caval shunts. J. Clin. Invest. 59:386-3%.

6. Hindfell. B.. and Siesjo. B. K. 1971. Cerebral effects or acuteammonia intoxication. 1. The influence on extracellulat acid-baseparameters. Scand. I. Clin. Lab. Invest. 28:353-364.

7. Hindfell, B.. and Siesjo. B. K. 1971. Cerebral effects of acuteammonia intoxication. II. The effect upon energy metabolism.Scand. J. Clin. Lab. Invest. 28:365-374.

8. Hindfelt. B. 1975. On mechanisms in hvperammonrmic comawith paniculai reference lo hepatic encephalopathy. Ann. NewYork Acad. Sci. 252:116-123.

9. Hindfclt. B.. and Siesjo. B. K. 1970. The effect of ammonia onihe energy metabolism of the rat brain. Life Sci. 9:1021-1028.

10. Rainakumari, L.. Subbalakshmi, G. Y. C. V., and Mnrthy. Ch.R. K. 1986. Acule effects of ammonia on the enzymes of citricacid cycle in tat brain. Neurochem. Int. 8:115-120.

11. (otman, C. W. 1974. Isolation of synaplosomal and synaptic

plasma membrane fractions, pp. 445-452. in, Fleischer, S.. andPacker. L. (eds.), Methods in cnzymology. Vol. XXXI, Aca-demic Press, New York.

12. Subbalakshmi, G. Y. C. V., and Murthy, Ch. R. K. 1985. Iso-lation of Aslrocylcs. Neurons and Synaplosomcs of rat brain cor-tex: Distribution of enzymes of gtutamalc metabolism. Neurochcm.Res. 10:239-250.

13. Lowry. 0. H.. Rosenbrough. N. J., Farr, A. I... and Randall. R.J. 1951. Protein measurement with Ihe folin phenol reagcni. J.Biol. Chem. 193:265-275.

14. Yoshida. A. 1969. L-Malale dehydrogenase from Baalim subti-Iits. pp. 141-142, In, Lowenstcin. J. M. (ed.). Methods in cn-zymology. Vol. XIII, Academic press. New York.

15. Bergmeycr, II. U.. and Hernt. E. 1974. Glutamaie oxaloacetaletransaminase, Glulanialc pynivale Iransaniinasc. pp. 574-579. 727-733, In. Bergmeyer. H. U. (ed.). Methods of enzymatic analysis.Vol. II. Academic press. New York.

16. Nandakumar, N. V., Murlhy, Ch. R. K., Vijayakumari. D.. andSwami, K. S. 1973. Axonat protein charges and Succinatc dchydrogenase activity in Sheep medulla oblongata. Ind. J. Exptl.Biol. 11:525-528. '

17. Subbalakshmi. G. Y. C. V., and Murthy, Ch. R. K. 1985. Dif-ferential response of enzymes of glutamaie metabolism in neuronalperikarya and synaptosomes in acute hyperammonemia in rat Nu-erasci.Letl. 59:121-126.

18. Hawkins. R. A.. Miller, A. L., Nielsen. R. C. and Veech. R.L..1973. The acute action of ammonia on ral brain metabolism invivo. Biocticm. J. 134:1001-1008.

19. Randle. P. J. 1983. Milochondrial 2-oxo acid dehydrogenasecomplexes of animal tissues. Philos. Trans. R. Soc. London. Set.B. 302:47-57.

20. Laporle. D. C, and Koshland, Jr. D. E. 1983. Phosphorylaiionof isociiraic dehydrogenase as a demonstration of enhanced sensitivity in covalcnt regulation. Nature. 305:286-290.

21. O'Conner. J. E.. Guerri, C, and Grisolia. S. 1984. Effects ofammonia on synaplosomal membranes. Biochcm. Biophys. Res.Commun. 119:516-523. ' i ^ *

22. Murlhy. Ch. R. K.. and Hertz, L. Regulation of pyruvale decar-boxylation in astrocytes and in neurons in primary cultures in thepresence and absence of ammonia. Neurochcm. Res. 13:57-61.

23. Jessy, J., and Murthy, Ch. R. K. 1985. Elevation of transami-nalion of branched chain amino acids in brain in acule ammoniatoxiciry. Neurochem. Int. 7:1027-1031.

24. Berl, S. 1971. Cerebral amino acid metabolism in hepatic coma,pp. 71-89, In. Polli. E. E. (ed.), Neurochemistry of Hepatic Coma,Vol. 4, Krager, Basel.

25. Drewis, L. R.. and Leino, R. L. 1980. Neuron-specific mito-chondria! degeneration induced by hyperammonemic and ocianoicacidemia. Brain Res. 340:211-218.

Effect of methionine sulfoximine on pyruvatedehydrogenase, citric acid cycle enzymes andaminotransferases in the subcellular fractions

isolated from rat cerebral cortex

The etTect ofucute and subacute doses of L-methionine-PL-sultbximine iMSI j were studied on the activi-ties of pyruvute dehydrogenuxe. enzymes of citric acid cycle and asparlate and alanine aminotransferascsin i he mitochondria, synaptosomes and cytosol of rat brain. In general, the activities of pyruvate dehydro-tiena.se and of the citric acid cycle enzymes, except malate dehydrogenase (malate-*oxuloucetute), wereelevated in all 3 subcellular fractions. Malate dehydrogenase activity (malate->oxaloacctatel was sup-pressed in the mitochondria while the activity of this enzyme in the reverse direction was enhanced in thecytosol. Activities of aspartale and alanine aminotransferases were suppressed under these conditions. Asthe effects o\ MSI tin these enzymes were similar to those observed upon the administration of ammoniumsalts, it is suggested that the hyperammonemie stale induced by MSI might derange the operation of themalale-aspartate shuttle Increased activities of citric acid cycle enzymes in the cytosol suggested the exis-tence ofa small population ol mitochondria which was highly vulnerable either to ammonia or lo MSI.

Methionine sulphoximine is a potent convulsanl with prolonged latency period and

the onset of convulsions vanes with the dosage of the drug administered and the age

o( the animal [5. 6], Though it has been suggested earlier that the toxic effects of this

drug are primarily due to the inhibition of glutamine synthetase (GS). recent evidence

is not supportive of such a suggestion [5], However, the induction of hyperammone-

mic state by methionine sulphoximine (due to the inhibition of GS) has been con-

tirmed by several investigators [5. 6, 10].

Palhophysiologtcal concentrations of ammonia arc known to affect the cerebral

energy metabolism either by draining away the intermediates of citric acid cycle or

b\ depleting cerebral energy reserves [1]. Though there are numerous reports on the

levels of these intermediates in hyperammonemie states [2], very little information is

Cnrrespivtdeme: Ch R K. Murthy. School of Life Sciences. University of Hyderabad. Hyderabad 500 134,India.

0,1040940 90 S 0.1.50 ( I9»t) Hlseuer Scientific Publishers Ireland Lid.

available on the activities of enzymes and the specific mechanisms involved in thealteration of the levels of these intermediates in MSI induced hyperammonemicstates. In the present study, we report the changes in the activity levels of pyruvatedehydrogenase and citric acid cycle enzymes along with those of aminotransferasesin mitochondria, synaptosomes and cytosol isolated from the brains of rats treatedwith methionine sulphoximine and compared them with those of normal rats. Resultsof the present study indicated a generalized increase in the activities of pyruvatedehydrogenase and of citric acid cycle enzymes except that of malate dehydrogenasein the mitochondria and synaptosomes isolated from the brains of methionine sul-phoximine treated animals. Further, results of the present study also indicated sup-pression of the activities of malate dehydrogenase (in the direction of malate-»oxalo-acetate) and of the aminotransferases which is suggestive of a derangement in thetransport of reducing equivalents across the mitochondnal membranes through themalate-aspartate shuttle. It was also observed that the activities of these enzymeswere enhanced in the cytosol which is suggestive of the existence of a small popula-tion of mitochondria which degenerates and liberates their contents into the cytosolunder these conditions.

L-Methionine-DL-sulphoximine was dissolved in saline (pH 7.0) and was adminis-tered intraperitoneally to 6-month-old Wistar rats. The subacute group received 150mg ol the drug kg b.wt. while the acute group received 300 mg of the drug, kg b.wt.The subacute and acute groups of animals were decapitated at the end of 17.5 and3.5 h, respectively. Methods adopted for the preparation of the subcellular fractions(mitochondria, synaptosomes and cytosol) from the cerebral cortex and for theenzyme assays have been described earlier [8]. Ammonia content was determined asdescribed earlier [10] in the extracts of frozen (liquid nitrogen) brains. Protein contentwas determined by the method of Lowry et al. [4]. Statistical analysis of the data wasby Newman Keul's multiple-range analysis.

Behavioural changes observed prior to the onset of convulsions in the rats adminis-tered with MSI have been described earlier [5, 6, 9, 10]. Acute group of animalsentered into convulsions between 3.5 and 4 h while the onset of convulsions was

TABLE I

AMMONIA LEVELS IN BLOOD AND BRAINS OF NORMAL AND METH1ONINE SULFOXI-MINE TREATED RATS

Values are expressed in brain as /<mo! of ammonia g wet wt. and in blood as /*mol of ammonia/ml.Number in parentheses indieales number of experiments.

observed to be at 17.5 h in the subacule group. The mortality rale was very high inboth the groups alter the convulsions, hence the animals were sacrificed during theconvulsive phase. Mood and brain ammonia levels in the normal animals (Table I)were within the range ol reported values [2]. Following the administration of MSI.the ammonia levels in the brain and blood were enhanced. Ihe magnitude of eleva-tion in the ammonia levels in both the groups were observed to be higher in Ihe bloodthan in Ihe brain. As was expected, the increase in the blood and brain ammonialevels were higher in acute group of animals than in subacute group of animals.

TABLE II

EFFECT OF MlTHIONINI: SULFOXIMINE ON PYRUVATP DFHYDROGENASE ANDENZYMES OF CITRIC ACID CYCLE AND BORST CYCLE IN THh MITOCHONDRIA OF RATCEREBRAL CORTEX

Activity is expressed as mean ±S.D.PDH, pyruvatc dehydrogenase; CS. citrate synihciase; ICDH, isocilrate dehydrogemuc: a-KGDH, i-kc-Inglularale dehydrogenase: SDH. succinate dehydrogenaie; MDH. malatc dehydrogenase; AAI. aspar-tale aminotronsfcrase; AI\T. alanine aininotraiisfcrase. Activity units for PDII. ICDH. sc*KGD11.MDIKNAD' I are /mini of NAD' reduced, mg proleinh anil for CS/imol of citrate formed/mi protein/h.SDH is /rniol of succinate onkjiwd/ltlg prolcin h and Tor MDHINADIII. AAT and AIAT is /miol olNADU oadized/mg protein h. No. ol experiments is 5. For each experiment two animals were used

TABLE III

EFFECT OF METMONINE SULFOXIMINE ON PYRUVATE DEHYDROGESASE ANDENZYMES OF CITRIC ACID CYCLE AND BORST CYCLE IN THE SYNAPTOSOMES OF RATCEREBRAL CORTEX

Legend as in Table II.

Purity of the isolated subcellular fractions and the activity levels of the enzymesof citric acid cycle and of aminotransferases in the mitochondrial. synaptosomal andcytosolic fractions prepared from the normal animals have been described earlier [8].Activities of pyruvate, a-ketoglutarate and succinate dehydrogenases were enhancedin the mitochondrial fraction isolated from the brains of subacute group of animalswhen compared to the controls. Activity levels of citrate synthase. isocitrate dehydro-genase and malate dehydrogenase (oxaloacetate-»malate) were unaltered under theseconditions. However, activities of malate dehydrogenase (malate->oxaloacetate) andof aminotransferases were suppressed in the mitochondrial fraction isolated fromanimals injected with subacute dose of MSI.

In the synaptosomal fraction, activity levels of pyruvate. isocitrate. 2-ketoglutarateand succinate dehydrogenases were enhanced following the administration of a sub-acute dose of MSI. Citrate synthase activity was elevated marginally but not to asignificant extent. Malate dehydrogenase activity measured in the direction of malate

to oxaloacclale was suppressed while the activity of same in the reverse direction wasunaltered. Activities of both the aminotransfcrases were suppressed in the synaptoso-mal fractions prepared from the subacutc group ol animals.

In the cytosol more or less a similar profile of changes were observed and the mag-nitude of changes in the activities of pyruvatc dehydrogenasc and succinate dchydro-genase were much higher than those in the mitochondria. In contrast to the observa-tions made in the synaptosomal fractions, activity of malate dehydrogenase (oxaloa-cetate-> malate) was enhanced in cytosol. The magnitudes of changes in (he activitiesof aminotransfcrases in the cytosol were similar to those described above.

In all the 3 subcellular fractions isolated from the brains of rats administered withan acute dose of MSI. changes in the enzyme activities were more or less similar tothose observed in the subacutc state with few exceptions. In general, the magnitude

TABLE; iv

EFFECT OF METUIONINE SULFOXIMINi: ON PYRUVATE DEHYDROOF-NASE ANDE N Z Y M E S O F C ITRIC 1 A C I D i YC'LE A N D BORST C Y C L E I N T i l l C Y T O S O L O F R A T ( I K IB R A L C O R T E X

Legend as in Tahle I I

331

TABLE III

EFFECT OF METHIONINE SULFOXIMINE ON PYRUVATE DEHYDROGENASE ANDENZYMES OF CITRIC ACID CYCLE AND BORST CYCLE IN THE SYNAPTOSOMES OF RATCEREBRAL CORTEX

Legend as in Table II.

Purity of the isolated subcellular fractions and the activity levels of the enzymesof citric acid cycle and of aminotransferases in the mitochondrial. synaptosomal andcytosolic fractions prepared from the normal animals have been described earlier [8].Activities of pyruvate. a-ketoglutarate and succinate dehydrogenases were enhancedin the mitochondrial fraction isolated from the brains of subacute group of animalswhen compared to the controls. Activity levels of citrate synthase. isocitrate dehydro-genase and malate dehydrogenase (oxaloacetate—>malate) were unaltered under theseconditions. However, activities of malate dehydrogenase (malate-»oxaloacetate) andof aminotransferases were suppressed in the mitochondrial fraction isolated fromanimals injected with subacute dose of MSI.

In the synaptosomal fraction, activity levels of pyruvate, isocitrate, at-ketoglutarateand succinate dehydrogenases were enhanced following the administration of a sub-acute dose of MSI. Citrate synthase activity was elevated marginally but not to asignificant extent. Malate dehydrogenase activity measured in the direction of malate

to oxaloacetatc was suppressed while the activity of same in the reverse direction wasunaltered. Activities of both the aminotransfcrases were suppressed in the synaptoso-mal fractions prepared from the subacute group of animals.

In the cytosol more or less a similar profile of changes were observed and the mag-nitude of changes in the activities of pyruvate dehydrogenase and succinate dehydro-genase were much higher than those in the mitochondria. In contrast to the observa-tions made in the synaplosomal fractions, activity of nialate dehydrogenase (oxaloa-cetate-»malatc) was enhanced in cytosol. The magnitudes of changes in the activitiesof aminotranslcrases in the cytosol were similar to those described above.

In all the 3 suhccllular fractions isolated from the brains of rats administered withan acute dose of MSI. changes in the enzyme activities were more or less similar lothose observed in the subacute state with few exceptions. In general, the magnitude

TABLE IV

EFFECT OF METMON1NE SULFOX1M1NE ON PYRUVATE DEHYDROGENASE ANllENZYMES OF CITRIC ACID CYCLE AND BORST CYCLE IN TUT CYTOSOL OF RAT CERE-BRAL CORTEX

l e g e n d a s i n T a b l e I I .

•

333

of change was much higher in the acute group of animals than in the subacute. Theactivity of isocitrate dehydrogenase in the mitochondrial fraction isolated from acutegroup was enhanced significantly.

Results obtained in the present study on the enzymes of citric acid cycle and amino-transferases in the subcellular fractions were similar to those reported in the cerebralhomogenates prepared from rats treated either with MSI or ammonium acetate andto those in the subcellular fractions prepared from brains of rats treated with ammo-nium acetate [6-8]. Increased activity of pyruvate dehydrogenase in the mitochondriamight favour the channeling of pyruvate into the citric acid cycle. Similarly, increasedactivities of isocitrate, a-ketoglutarate and succinate dehydrogenases might enhancecarbon flux through the citric acid cycle in cerebral mitochondria in MSI toxicity.Though citrate synthetase activity in the mitochondria was not altered under theseconditions, increased activities of other enzymes might pull the reaction forward.However, reduction in malate dehydrogenase activity in the direction of oxaloacetateformation might act as a constraint and result in the accumulation of malate. Thoughmalate dehydrogenase activity in the direction of malate formation was unaltered,it will be of little importance as malate is not the substrate for citrate synthetase.Similar changes might also occur in the synaptosomal fraction as the synaptosomesare known to contain mitochondria. The decrease in the activity of malate dehydro-genase (malate-»oxaloacetate) would not only limit the oxaloacetate formation butalso results in accumulation of malate. Suppression in the aspartate aminotransferaseactivity together with that of malate dehydrogenase might lower the production ofaspartate in mitochondria. Hence, it appears that suppression of malate dehydroge-nase activity in mitochondria and aspartate aminotransferase both in mitochondriaand cytosol might be the reasons for the failure of the malate-aspartate shuttle inhyperammonemic states. The decreased activity of aspartate aminotransferase wouldlimit the formation of glutamate in cytosol which is required for the exchange withaspartate from mitochondria.

Enhancement in the activities of pyruvate dehydrogenase and of citric acid cycleenzymes in the cytosol were surprising as this subcellular fraction is supposed to befree of mitochondria. However, it must be mentioned that even under these condi-tions, activities of these enzymes were much lesser in the cytosol compared to themitochondria. Such an increase in the activities of these enzymes in the cytosol maybe due to altered buoyant density and/or fragmentation of mitochondria as a resultof which they do not sediment along with larger mitochondria. It might also be dueto the degeneration of the mitochondria and subsequent release of the enzymes intothe cytosol. It has been reported that in MSI toxicity mitochondrial number increasesand subsequently the mitochondria undergo degeneration [3]. It is quite possible thatsuch degenerating mitochondria might be releasing their contents into the cytosol.In an earlier study, we have reported similar changes in the activities of these enzymesin animals injected with an acute dose of ammonium acetate. Thus the observedchanges in the activities of enzymes of citric acid cycle and of aminotransferasesmight be due to the enhancement of cerebral ammonia levels in MSI toxicity.

Financial assistance was provided by University Granls Commission through asenior research fellowship to L.R.K.

1 Bessman, S.P. and Uessman. A N . The cerebral and peripheral uptake of ammonia in liver diseases

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