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Biochemical approaches in the study of epilepsy
BIOCHEMICAL
APPROACHES
IN THE STUDY OF EPILEPSY L
K. A. C. ELl.lo'rr Montreal Neurological Institute In the followin 9 discussion I shall consider only chemical factors possibly concerned in the initiation of epileptic activity in the brain of the epileptic subject. I think we have to consider three 9roups of possibilities: (1) T h e abnormal physiological and underlyin 9 chemical behavior of the nervous tissue in epilepsy may be a normal response of normal neurones reactin 9 to an abnormal environment. (2) T h e abnormal behavior may be due to changes in the chemical potentialities of the neurones. (3) Changes in the chemical potentialities of the neurones may occur but secondarily to the hyperactivity, as an adaptation to abnormal conditions, and may not be essential to the epileptic process. W e have made a study of the energy metabolism in vitro of a considerable number of samples of tissue excised by Dr. Penfield from human epileptic patients. No si.qn was found that the respiration rate, or effect of calcium on respiration rate. was different from what might be expected from normal human brain tissue. Values for normal human brain were estimated from a study of brains from a series of animals of different species. T h e r e was also no si.qn that the epilepto9enic tissue differed from the probable normal in respiratory quotient, or in rates of ~erobic or an~robic .q[ycolysis. Similar results were obtained with brain from a doq rendered epileptic by the "aqenized" diet and from an area of doq brain showing abnormal electrical activity foliowin 9 treatment with alumina cream. W e are inclined to believe that the first of the three possibilities mentioned above 1From the Department of Neuroloqy and Neurosur0ery. McGill University. and the Montreal Neurolo0ical Institute. Reprint no. 290.
may be true, namely that epileptogenic brain tissue contains normal neurones which are subjected to abnormal stimuli. O f course such a thesis rests on slim foundations as lon 9 as only energy metabolism studied in vitro is censidered. It is our immediate intention to study aspects of acetylcholine synthesis, liberation and destruction in normal and epileptogenic tissue since this type of metabolism is likely to be closely connected with function in nervous tissue. A couple of years ago Dr. A. Pope, workin9 in collaboration with a number of us in Montreal, obtained evidence that choline esterase activity was higher in brain tissue from epileptogenic loci than in samples of brain from other areas. But l think it possible that such an increase in activity may represent a secondary, adaptive, change and not be primary to the epileptic process. Dr. Donald T o w e r , workin 9 with Dr. M c E a c h e r n in Montreal, has found that usually a detectable amount of acetylcholine is to be found in the spinal fluid of epileptic patients and animals. This may indicate an increased acetylcholine release in the brain so that some of it finds its way into the subarachnoid space. T h e concentration, at least in the spinal fluid, is so far below the optimal for choline esterase activity that it would scarcely be affected by the esterase. Reliable detection of free acetylcholine in the brain tissue in situ is, at present, a very difficult problem. But it is possible that the level of free acetylcholine in the brain tissue is raised in epilepsy towards the threshold concentration for its action, so :hat any new release of acetylcholine as a result of normal processes will set off activity abnormally readily. H o w e v e r it seems to me that Dr. T o w e r ' s findin 9 may simply be the result of neuronal hyperactivity and may not indicate the basis for this activity.
[291
~0
IK. A. C. ELLIO'FT
T h e fact that such a wide variety of drugs and physiological conditions can produce convulsive seizures in normal animals does indicate, to my mind. that there ~s no need to postulate a change in the chemical potentialities of neurones to account for most epileptic conditions, On the contrary I feel that we need more than anything the type of understanding ot brain and nerve processes which is developing out of the work of Dr. Bronk and his colleagues. W h a t tvpes of factors will produce lowered thresholds to stimulation, or hyperactivity, in normal nervous tissue? Dr. Bronk's earlier studies on calcium deprivation have given us useful leads in this area. W h a t are the conditions with regard to supply and demand of oxygen in living brain and nerve tissue? T h e work of the Johnson Foundation is helping us rapidly in this regard. I feel that correlation of this type of information with in ~,itro studies will soon give us clearer ideas regarding the control of nervous activity. For instance, in ~,itro work has taught us that brain tissue is capable of rapid glycolysis, acid production, especially anaerobically. If the blood supply, and hence the oxygen tension, in a given local area falls below a certain low level, rapid glycolysis would set in. Acid would be produced intracellularly. It seems likely that a change, even temporary. in the relation of the intracellular pH to the extracellular p H would have considerable effect on the distribution of inorganic ions and might affect the liberation of acetylcholine, and so affect neuronal activity. (Quastel and his co-workers found that a fall in pH to 6.0 --- 6.5 accelerated the liberation of free acetylcholine from its precursor), T h e effects on the p H of extracellular fluid may be the basis for the action of the ketogenic diet in controlling some epilepsies and of hyperventilation in provoking seizures. M e a s u r e m e n t s of cortical pH have given us some necessacy information but so far I do not think they have given us specific information regarding epilepsy. Dr. Jasper and I find that the pH of the surface of the normal cortex varies from nearly equal to that of the venous blood to considerably
lower, T h e variability seems to be related to the proximity of large blood vessels, a situation similar to that found for oxygen tension by Dr. Bronk's group. T h e pH at a given point has been shown, by Dusser de Barenne. Nims and others, and by Jasper and Erickson, to undergo changes during seizures but these changes appear to be the result of. rather than primary to the seizure. T h e y can be explained on the basis of excessive acid production during hyperactivity, and reactive hyperemia which would tend to raise the pH toward the blood level. Possibly more closely relevant are the observations of Dusser de Barenne and McCulloch that the pH of the cortex seemed to be increased when the phenomenon of "facilitation" was observed and lowered during the period of e x t i n c tion". A group of us at Montreal found no obvious sign of abnormal pH in focal epileptogenic areas in monkeys and this type of study will need application of more refined methods if significant observations of differences in small local areas are to be understood. T h e studies of Dr. Bronk's group on the role of calcium in controlling nervous activity need to be borne in mind. Quastel and his co-workers noted that lack of Ca caused increased liberation of acetylcholine in brain, T h e availability of calcium /on is likely to be affected by pH changes and also by certain intermediary metabolites, notably citric acid which could conceivably accumulate when metabolism is interfered with by circulatory or other factors. W e are giving some attention to this possibility. C h a n g e s in oxygen supply and p H are not the only factors in structural and circulatory abnormalities which could affect nervous function. O n e must consider glucose concentration, CO~ tension and the possible accumulation of other metabolic end products. Deprivation of glucose, the normal substrate of brain respiration, naturally affects nervous function and this is seen in insulin stupor and convulsions. Feldberg found maximum acetylcholine synthesis, or rather release, in the presence of a low concentration of glucose, lower than that in
BIOCHEMICAl_. APPROACHES IN EPILEPSY normal blood. T h e normal blood concentration was inhibitory and he suooested that certain physiological processes mioht be controlled by glucose concentration. Quastel and his co-workers found a much 9reater effect of potassium on synthesis and release of acetylcholine by brain tissue in ~,itro in the presence of bicarbonate-CO._, buffer than without this buffer, but they did not elaborate on a physiolooical significance of this effect of CO~ or bicarbonate. Keto acids, like pyruvic acid have been found by Nachmanssohn and others to inhibit acetylcholine synthesis; the concentration of these might be affected by blood supply. T h e possibility that an abnormal amount of some blood constituent may be found and provide a clue to the epileptic mechanism has been considered by many workers but I do not believe that any real answer has come from such studies. Some years ago, M u r r a y and H o f f m a n n reported high basal values for substances estimated as 9uanidine in the blood of essential epileptics and found the value greatly increased at the time of seizures. This seemed specially interestin 9 since 9uanidme is itself a convulsant and it is structurally related to a number of compounds essential in normal metabolism, t i n -
31
tortunately, we have not been able to confirm this finding in any type of epileptic. I feel that the studies of Dr. Penfield and his colleagues indicatin 9 a probable impairment of local circulatory control in the neighborhood of epileptogenic loci is as likely a startin 9 point for chemical theories and experimentation as any we have at present. I have considered only factors concerned in clinical epilepsy. T h e r e are numerous chemical agents which can produce convulsive effects or epileptiform brain waves but in most cases we have scarcely any idea of the mechanisms of their actions. Acetylcholine and the choline esterase inhibitors perhaps cause an exaogeration of normal processes. Citrate immobilizes calcium ion. Fluoroacetate is known to interfere with acetic acid oxidation in tissues other than brain. But since, in the brains of species most studied. acetic acid is not normally oxidized anyway, the chemical action of fluoroacetate as a convulsant is not clear. I feel that more knowledge of the locus of chemical action of druos like metrazol, and of the biochemical basis of action of anticonvulsants, is one of the most essential requirements for the understandin 9 of the chemistry of epilepsy.
APPROACHES
IN THE STUDY OF EPILEPSY L
K. A. C. ELl.lo'rr Montreal Neurological Institute In the followin 9 discussion I shall consider only chemical factors possibly concerned in the initiation of epileptic activity in the brain of the epileptic subject. I think we have to consider three 9roups of possibilities: (1) T h e abnormal physiological and underlyin 9 chemical behavior of the nervous tissue in epilepsy may be a normal response of normal neurones reactin 9 to an abnormal environment. (2) T h e abnormal behavior may be due to changes in the chemical potentialities of the neurones. (3) Changes in the chemical potentialities of the neurones may occur but secondarily to the hyperactivity, as an adaptation to abnormal conditions, and may not be essential to the epileptic process. W e have made a study of the energy metabolism in vitro of a considerable number of samples of tissue excised by Dr. Penfield from human epileptic patients. No si.qn was found that the respiration rate, or effect of calcium on respiration rate. was different from what might be expected from normal human brain tissue. Values for normal human brain were estimated from a study of brains from a series of animals of different species. T h e r e was also no si.qn that the epilepto9enic tissue differed from the probable normal in respiratory quotient, or in rates of ~erobic or an~robic .q[ycolysis. Similar results were obtained with brain from a doq rendered epileptic by the "aqenized" diet and from an area of doq brain showing abnormal electrical activity foliowin 9 treatment with alumina cream. W e are inclined to believe that the first of the three possibilities mentioned above 1From the Department of Neuroloqy and Neurosur0ery. McGill University. and the Montreal Neurolo0ical Institute. Reprint no. 290.
may be true, namely that epileptogenic brain tissue contains normal neurones which are subjected to abnormal stimuli. O f course such a thesis rests on slim foundations as lon 9 as only energy metabolism studied in vitro is censidered. It is our immediate intention to study aspects of acetylcholine synthesis, liberation and destruction in normal and epileptogenic tissue since this type of metabolism is likely to be closely connected with function in nervous tissue. A couple of years ago Dr. A. Pope, workin9 in collaboration with a number of us in Montreal, obtained evidence that choline esterase activity was higher in brain tissue from epileptogenic loci than in samples of brain from other areas. But l think it possible that such an increase in activity may represent a secondary, adaptive, change and not be primary to the epileptic process. Dr. Donald T o w e r , workin 9 with Dr. M c E a c h e r n in Montreal, has found that usually a detectable amount of acetylcholine is to be found in the spinal fluid of epileptic patients and animals. This may indicate an increased acetylcholine release in the brain so that some of it finds its way into the subarachnoid space. T h e concentration, at least in the spinal fluid, is so far below the optimal for choline esterase activity that it would scarcely be affected by the esterase. Reliable detection of free acetylcholine in the brain tissue in situ is, at present, a very difficult problem. But it is possible that the level of free acetylcholine in the brain tissue is raised in epilepsy towards the threshold concentration for its action, so :hat any new release of acetylcholine as a result of normal processes will set off activity abnormally readily. H o w e v e r it seems to me that Dr. T o w e r ' s findin 9 may simply be the result of neuronal hyperactivity and may not indicate the basis for this activity.
[291
~0
IK. A. C. ELLIO'FT
T h e fact that such a wide variety of drugs and physiological conditions can produce convulsive seizures in normal animals does indicate, to my mind. that there ~s no need to postulate a change in the chemical potentialities of neurones to account for most epileptic conditions, On the contrary I feel that we need more than anything the type of understanding ot brain and nerve processes which is developing out of the work of Dr. Bronk and his colleagues. W h a t tvpes of factors will produce lowered thresholds to stimulation, or hyperactivity, in normal nervous tissue? Dr. Bronk's earlier studies on calcium deprivation have given us useful leads in this area. W h a t are the conditions with regard to supply and demand of oxygen in living brain and nerve tissue? T h e work of the Johnson Foundation is helping us rapidly in this regard. I feel that correlation of this type of information with in ~,itro studies will soon give us clearer ideas regarding the control of nervous activity. For instance, in ~,itro work has taught us that brain tissue is capable of rapid glycolysis, acid production, especially anaerobically. If the blood supply, and hence the oxygen tension, in a given local area falls below a certain low level, rapid glycolysis would set in. Acid would be produced intracellularly. It seems likely that a change, even temporary. in the relation of the intracellular pH to the extracellular p H would have considerable effect on the distribution of inorganic ions and might affect the liberation of acetylcholine, and so affect neuronal activity. (Quastel and his co-workers found that a fall in pH to 6.0 --- 6.5 accelerated the liberation of free acetylcholine from its precursor), T h e effects on the p H of extracellular fluid may be the basis for the action of the ketogenic diet in controlling some epilepsies and of hyperventilation in provoking seizures. M e a s u r e m e n t s of cortical pH have given us some necessacy information but so far I do not think they have given us specific information regarding epilepsy. Dr. Jasper and I find that the pH of the surface of the normal cortex varies from nearly equal to that of the venous blood to considerably
lower, T h e variability seems to be related to the proximity of large blood vessels, a situation similar to that found for oxygen tension by Dr. Bronk's group. T h e pH at a given point has been shown, by Dusser de Barenne. Nims and others, and by Jasper and Erickson, to undergo changes during seizures but these changes appear to be the result of. rather than primary to the seizure. T h e y can be explained on the basis of excessive acid production during hyperactivity, and reactive hyperemia which would tend to raise the pH toward the blood level. Possibly more closely relevant are the observations of Dusser de Barenne and McCulloch that the pH of the cortex seemed to be increased when the phenomenon of "facilitation" was observed and lowered during the period of e x t i n c tion". A group of us at Montreal found no obvious sign of abnormal pH in focal epileptogenic areas in monkeys and this type of study will need application of more refined methods if significant observations of differences in small local areas are to be understood. T h e studies of Dr. Bronk's group on the role of calcium in controlling nervous activity need to be borne in mind. Quastel and his co-workers noted that lack of Ca caused increased liberation of acetylcholine in brain, T h e availability of calcium /on is likely to be affected by pH changes and also by certain intermediary metabolites, notably citric acid which could conceivably accumulate when metabolism is interfered with by circulatory or other factors. W e are giving some attention to this possibility. C h a n g e s in oxygen supply and p H are not the only factors in structural and circulatory abnormalities which could affect nervous function. O n e must consider glucose concentration, CO~ tension and the possible accumulation of other metabolic end products. Deprivation of glucose, the normal substrate of brain respiration, naturally affects nervous function and this is seen in insulin stupor and convulsions. Feldberg found maximum acetylcholine synthesis, or rather release, in the presence of a low concentration of glucose, lower than that in
BIOCHEMICAl_. APPROACHES IN EPILEPSY normal blood. T h e normal blood concentration was inhibitory and he suooested that certain physiological processes mioht be controlled by glucose concentration. Quastel and his co-workers found a much 9reater effect of potassium on synthesis and release of acetylcholine by brain tissue in ~,itro in the presence of bicarbonate-CO._, buffer than without this buffer, but they did not elaborate on a physiolooical significance of this effect of CO~ or bicarbonate. Keto acids, like pyruvic acid have been found by Nachmanssohn and others to inhibit acetylcholine synthesis; the concentration of these might be affected by blood supply. T h e possibility that an abnormal amount of some blood constituent may be found and provide a clue to the epileptic mechanism has been considered by many workers but I do not believe that any real answer has come from such studies. Some years ago, M u r r a y and H o f f m a n n reported high basal values for substances estimated as 9uanidine in the blood of essential epileptics and found the value greatly increased at the time of seizures. This seemed specially interestin 9 since 9uanidme is itself a convulsant and it is structurally related to a number of compounds essential in normal metabolism, t i n -
31
tortunately, we have not been able to confirm this finding in any type of epileptic. I feel that the studies of Dr. Penfield and his colleagues indicatin 9 a probable impairment of local circulatory control in the neighborhood of epileptogenic loci is as likely a startin 9 point for chemical theories and experimentation as any we have at present. I have considered only factors concerned in clinical epilepsy. T h e r e are numerous chemical agents which can produce convulsive effects or epileptiform brain waves but in most cases we have scarcely any idea of the mechanisms of their actions. Acetylcholine and the choline esterase inhibitors perhaps cause an exaogeration of normal processes. Citrate immobilizes calcium ion. Fluoroacetate is known to interfere with acetic acid oxidation in tissues other than brain. But since, in the brains of species most studied. acetic acid is not normally oxidized anyway, the chemical action of fluoroacetate as a convulsant is not clear. I feel that more knowledge of the locus of chemical action of druos like metrazol, and of the biochemical basis of action of anticonvulsants, is one of the most essential requirements for the understandin 9 of the chemistry of epilepsy.