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Homocysteine induced convulsions: enhancement by vitamin B6 and inhibition by hydrazine
250
Brain Research, 209 (1981) 250-254 © Elsevier/North-Holland Biomedical Press
Homocysteine induced convulsions: enhancement by vitamin B6 and inhibition by hydrazine
RUSSELL W. H U R D , E D W A R D J. H A M M O N D and B. J. W I L D E R
Department of Neuroscience, College of Medicine, University of Florida, and Neurology and Medical Research Service, Veterans Administration Hospital, Gainesville, Fla. 32610 (U.S.A.) (Accepted October 30th, 1980)
Key words: convulsion - - homocysteine - - vitamin B6 - - hydrazine
The effects of pyridoxal phosphate, pyridoxine and hydrazine were studied on homocysteineinduced seizures in mice. Both of the Be vitamers significantly decreased the latency and increased the severity, lethality and duration of seizures induced by homocysteine. The Be inhibitor hydrazine sulfate, which is normally a convulsant, prevented the tonic component of the convulsions and increased the latency to the clonic component. This experiment indicates that a vitamin B~ dependent step is critically involved in the metabolic changes which precede homocysteine seizures.
The amino acid homocysteine is a metabolite of methionine involved in methyl group transfer reactions. Individuals with homocystinuria, in which increased plasma and brain levels of methionine and homocysteine are found, are characterized by various neurological defects, which include seizures in some patients14, is. Homocysteine sulphinic acid and homocysteic acid have been shown to be potent excitants of cortical neurons 7,12, and convulsive effects of i.p. injections of homocysteine have been describeda,lo,15,2~,23. In a series of experiments, Folbergrova and colleagues aAo have studied the cerebral metabolic changes which accompany homocysteine seizures. The actual mechanisms of seizure induction in both humans and experimental animals are, however, obscure. We 17 and others11,22,2z have studied the effects of substances which enhance or inhibit methyl transfer reactions; in general administration of substances which promote methylation of homocysteine, including betaine11,22,23 and vitamin B1217, tend to reduce its effectiveness as a convulsant. Homocysteine has been shown to inhibit glutamic acid decarboxylase (GAD) and ),-amino butyric acid aminotransferase (GABA-T)2a, 25. Pyridoxal phosphate (PLP) is a required coenzyme by GAD, GABA-T and other cerebral decarboxylase and amino transferases3. Hydrazides and other PLP-antagonists that induce seizures also inhibit GAD and GABAT 16.
In a majority of cases, homocystinuria is due to a defect in cystathionine synthetase14, another PLP-dependent enzyme. In this study, we report the effects of pretreatment with pharmacologic doses of pyridoxine, pyridoxal phosphate and the vitamin B6 inhibitor hydrazine sulfate on homocysteine-induced seizures in mice. The results suggest a significant interaction between homocysteine and vitamin B6.
251 Female CD-I mice (Charles River) 26-28 g were housed in animal quarters having 12:12 light-dark cycle, 07.00 h light on. Solutions of pyridoxal phosphate, pyridoxine HCI, hydrazine sulfate, and DL-homocysteine thiolactone, a stable form of homocysteine (Sigma Chemicals) were prepared in physiological saline immediately prior to use. The Be vitamers (50 mg/kg) and hydrazine (50 mg/kg) were given i.p. 1 h prior to homocysteine. Homocysteine thiolactone was administered i.p. at dosage levels of 4.5, 5.5 and 6.0 mmol/kg. The EDs0 for homocysteine in these animals is 5.5 mmol/kg and the EDa5 is 7.5 mmol/kg 22. Animals were placed 4-5 per clear plastic cage where two observers recorded manifestations of seizure development. Animals were observed for a period of 60 min following homocysteine injection. In previous studies if seizures were to occur at the dosages used, they would occur within this time period. Statistical significance of seizure incidence and latency differences was determined by two-tailed Mann-Whitney U or Fisher Exact Probability test. lntraperitoneal injections of homocysteine produced dose-related seizures as previously described10,15, 22. Initially the drug produced a somnolence lasting from 2 to 30 min depending upon dosage which was followed by brief tremors of the head and forepaws. In animals that convulsed the initial seizure usually consisted of clonic jerking of the limbs with loss of righting reflex lasting 5-15 sec (clonic seizure). After one or more clonic attacks generalized tonic-extensor seizures and/or running fits occurred, followed by a comatose state and sometimes death. However, animals receiving preinjections of pyridoxine HC1 or pyridoxal phosphate had significantly enhanced seizure activity manifested by increased incidence (Table I) and decreased latency (Table II) to both clonic- and tonic-extensor seizures. Seizures in the B6 treated groups were of extended duration and animals appeared to be in status epilepticus. Several series of clonic to tonic progression occurred in each Be treated animal until death. In contrast, hydrazine sulfate offered significant protection to homocysteine TABLE I Group
Homocysteine
Number of animals convulsing Clonic seizures Control
I I1 III IV Totals
4.5 mmol/kg 5.5 mmol/kg 5.5 mmol/kg 6.0 mmol/kg
Be§
Tonic seizures Hydrazine§§Control
5/8 8/10 9/10 9/10
7/8 10/10 10/10 10/10
0/8* 3/10" 3/10"* 7/10
1/8 4/10 6/10 9/10
31/38
37/38*
13/38"**
20/38
Bn
Hydrazine
6/8* 10/10"* 10/10" 10/10
0/8 1/10 0/10" 1/10"
36/38** 2/38***
* P < 0.05. ** P < 0.01. *** P < 0.001, Fisher Exact Probability Test. § Groups I and III received pyridoxine HCI, 50 mg/kg, Groups II and IV received pyridoxal phosphate, 50 mg/kg. §§ Group I received 25 mg/kg, Groups II, III, IV 50 rng/kg hydrazine sulfate.
252 TABLE II Treatments Pretre~tment
Homocysteine dosage
Saline B6
4.5 mmol/kg 4.5 mmol/kg
Hydrazine
Median latency to convulsions in min
Lethality
N
Clonic
Tonic
I h(%)
24 h(%)
8 8
60 15.2"
60 31.5"
0 0
0 0
4.5 mmol/kg
8
60
60
0
0
Saline Be Hydrazine
5.5 mmol/kg 5.5 mmol/kg 5.5 mmol/kg
20 20 20
18,9 49.0 11.3"** 21.4"** 60*** 60**
4 (20%) 8 (40%) 0
4 (20%) 9 (45 %) 0
Saline Be Hydrazine
6.0 mmol/kg 6.0 mrnol/kg 6.0 mmol/kg
10 10 10
12.3 5.5*** 37.0**
3 (30%) 7 (70%) 0
3 (30%) 8 (80%)* 0
* P<0.05;
** P < 0 . 0 1 ;
27.3 17.0" 60***
*** P < 0 . 0 0 1 , M a n n Whitney U Test.
seizures. Only 2 of 38 controls receiving hydrazine had tonic convulsions compared to 20 of 38 controls and 36 of 38 vitamin B6 treated animals. There were no deaths in the hydrazine pre-treated groups while homocysteine was lethal to 45 % of Bs-treated and 18 % of control animals. The enhancement of homocysteine seizures by pharmacological levels of B6 vitamers reported in this study was at first surprising since it is recognized that a variety of procedures producing a B6 deficiency result in seizures. Human infantile pyridoxine deficiency seizures have been described, which are not responsive to anticonvulsants and are only controlled by intake of vitamin B66. An increasing amount of evidence, however, implicates enhanced levels of vitamin B6 in lowering seizure thresholds in experimental animals. When injected intracerebrally, pyridoxal phosphate is a potent convulsant2, 9 and hydrazones formed from pyridoxine and hydrazines are 10-100 times more potent convulsants than hydrazides themselves s. Gailagher 1~ reported that i.p. pyridoxine HC1, pyridoxal-5-phosphate (PLP) and pyridoxamine significantly lowered the threshold to seizures induced by fluorothyl. The threshold to isonicotinic acid hydrazide seizures is also reduced by injections of B6 vitamers 5. Significant penetration of the B6 vitamers into rat brain occurs within 30 minutes of an i.p. injection 1. It is of interest that in this study, the convulsant effects of homocysteine were diminished by hydrazine sulfate, which is normally a convulsant. Both hydrazines and homocysteine are antagonists of PLP-dependent reactions. These reactions are subject to inhibition to a highly varying degree depending upon the differing affinity of the coenzyme for the apo-enzyme 18. Homocysteine acts as an antimetabolite to pyridoxal phosphate in vitro and the PLP-homocysteine complex is devoid of coenzyme activity 19. However, the hydrazone formed from complexing of hydrazine and PLP can have coenzyme activity ~6. Therefore, as the results of this study suggest, there was a decrease in PLP-mediated neural excitability as a result of the formation of the hydrazone complex. Since both hydrazine 1~ and homocysteine19,~4,2~ also result in
253 inhibition of GAD and GABA-T, measurement of the activities of these enzymes after homocysteine with and without hydrazine pretreatment may identify the changes necessary for seizure induction. In summary, we have shown that pharmacological levels of B6 vitamers increase the seizures induced by homocysteine. The mechanisms underlying these seizures are unknown but their enhancement by ]]6 vitamers and prophylaxis by a B6 antagonist suggest that a B6 dependent mechanism regulates neuronal excitability in these seizures. We propose that this finding may have clinical relevance to human epilepsy because a number of anticonvulsant drugs decrease pyridoxine levels in patients on long-term therapy 2°,2t. This research was supported by The Epilepsy Research Foundation of Florida.
1 Bain, J. A. and Williams, H. L., Concentrations of B6 vitamers in tissues and tissue fluids. In E. Roberts (Ed.), Inhibition in the Nervous System and Gamma-Amino Butyric Acid, Pergamon Press, New York, 1960, 275-293. 2 Balzer, H., Holtz, P. and Palm, D. Untersuchunger uberv die biochemischen Grundlagen der knovulsiven wirkung von hydraziden, Arch. exp. Path. Pharmak., 239 (1960) 520-552. 3 Bender, D. A. Amino Acid Metabolism, John Wiley, New York, N.Y., 1975, 35-58. 4 Blennow, G., Folbergrova, J., Nilsson, G. and Siesjo, B. K., Cerebral metabolic and circulatory changes in the rat during sustained seizures induced by DL-homocysteine convulsions, Brain Research, 179 (1979) 129-146. 5 Costa, E., Vitamin B6 accentuates in the mouse the convulsant action of isonicotinyl hydrazine, Boll. Soc. ital. Biol. sper., 28 (1952) 1015-1016. 6 Coursin, D. B., Seizures in vitamin B6 deficiency. In E. Roberts (Ed.), Inhibition in the Nervous System and Gamma-Aminobutyric Acid, Pergamon Press, New York, N.Y. 1960, 294-301. 7 Curtis, D. R. and Watkins, J. C., Acidic amino acids with strong excitatory actions on mammalian neurons, J. Physiol. (Lond.), 166 (1963) 1-14. 8 Dixon, R. H. and Williams, H. L., The toxicity of pyridoxal and pyridoxal phosphate hydrazones in mice, Fed. Proc., 21 (1962) 338. 9 Ebadi, M., Kouyoumdjian and Govitrapong, P., The specificity of pyridoxal and pyridoxal phosphate induced convulsions, Trans. Amer. Soc. Neurochem., 11 (1980) 202. 10 Folbergrova, J., Energy metabolism of mouse cerebral cortex during homocysteine convulsions, Brain Research, 81 (1974) 443454. 11 Freed, W. J., Gillin, J. C. and Wyatt, R. J., Anticonvulsant properties of betaine, Epilepsia, 20 (1979) 209-213. 12 Gaitonde, J. K., Sulphur amino acids. In A. Latha (Ed.), Handbook ofNeurochemistry, Fol. HI, Plenum Press, New York, N.Y., 1970, 225-287. 13 Gallagher, R. B., The influence of tyrosine phenylpyruvate and vitamin B6 upon seizure thresholds, J. Neurochem., 18 (1971) 799-808. 14 Gaull, G. E., Homocystinuria and other disorders of sulfur metabolism. In E. Goldensohn and S. Appel (Eds.), Scientific Approaches to Clinical Neurology, Lea and Febiger, Philadelphia, Pa, 1977, 46-54. 15 Hammond, E. J., Hurd, R. W., Wilder, B. J. and Thompson, F. J., Focal and generalized experimental seizures induced by homocysteine, Electroenceph. clin. Neurophys., 49 (1980) 184-186. 16 Holtz, P. and Palm, D., Pharmacological Aspects of Vitamin B6, Pharmacol. Rev., 13 (1964) 113-178. 17 Hurd, R. W., Hammond, E. J., Thompson, F. J. and Wilder, B. J., The use of homocysteineinduced seizures in the study of experimental epilepsy, Neurosci. Abstr., 5 (1979) 194. 18 Kang, S. S., Wong, P. W. K. and Becker, N., Protein-bound homocyst(e)ine in normal subjects and in patients with homocystinuria, Pediat. Res., 13 (1979) 1141-1143. 19 Pestana, A., Sandoval, I. V. and Sols, A., Inhibition by homocysteine of serine dehydratase and
254
20 21 22 23 24 25
other pyridoxal 5-phosphate enzymes of the rat through cofactor blockage, Arch. Biochem., 146 (1971) 373-379. Reinken, L., Hohenauer, L. and Zeigler, E. E., Activity of red cell glutamic oxalacetic transaminase in epileptic children under antiepileptic treatment, Clin. chim. Acta, (1972) 270--271. Reinken• L.• The in•uence •f anti•pi•eptic drugs •n vitamin B6 metab••ism• Acta vitamin enzyrn••.• 29 (1975) 252-254. Sprince, H., Parker, C. M. and Josephs, J. H., Homocysteine-induced convulsions in the rat: protection by homoserine, serine, betaine, glycine and glucose, Agents Act., 1 (1969) 9-13. Sprince, H., Parker, C. M., Josephs, J. A. and Magazino, J., Convulsant activity of homocysteine and other short-chain mercapto acids: protection therefrom. Ann. N.Y. Acid. Sci., 166 (1969) 323-325. Taberner, P. V., Pearce, M. J. and Watkins, J. C., The inhibition of mouse brain gutamate decarboxylase by some structural analogues of L-glutamic acid, Biochem. Pharmacol., 26 (1977) 345-349. Tunnicliff, G. and Ngo, T. T., The mode of action of homocysteine on mouse brain glutamic decarboxylase and 7-aminobutyrate aminotransferase, Canad. J. Biochem., 55 (1977) 1013-1018.
Brain Research, 209 (1981) 250-254 © Elsevier/North-Holland Biomedical Press
Homocysteine induced convulsions: enhancement by vitamin B6 and inhibition by hydrazine
RUSSELL W. H U R D , E D W A R D J. H A M M O N D and B. J. W I L D E R
Department of Neuroscience, College of Medicine, University of Florida, and Neurology and Medical Research Service, Veterans Administration Hospital, Gainesville, Fla. 32610 (U.S.A.) (Accepted October 30th, 1980)
Key words: convulsion - - homocysteine - - vitamin B6 - - hydrazine
The effects of pyridoxal phosphate, pyridoxine and hydrazine were studied on homocysteineinduced seizures in mice. Both of the Be vitamers significantly decreased the latency and increased the severity, lethality and duration of seizures induced by homocysteine. The Be inhibitor hydrazine sulfate, which is normally a convulsant, prevented the tonic component of the convulsions and increased the latency to the clonic component. This experiment indicates that a vitamin B~ dependent step is critically involved in the metabolic changes which precede homocysteine seizures.
The amino acid homocysteine is a metabolite of methionine involved in methyl group transfer reactions. Individuals with homocystinuria, in which increased plasma and brain levels of methionine and homocysteine are found, are characterized by various neurological defects, which include seizures in some patients14, is. Homocysteine sulphinic acid and homocysteic acid have been shown to be potent excitants of cortical neurons 7,12, and convulsive effects of i.p. injections of homocysteine have been describeda,lo,15,2~,23. In a series of experiments, Folbergrova and colleagues aAo have studied the cerebral metabolic changes which accompany homocysteine seizures. The actual mechanisms of seizure induction in both humans and experimental animals are, however, obscure. We 17 and others11,22,2z have studied the effects of substances which enhance or inhibit methyl transfer reactions; in general administration of substances which promote methylation of homocysteine, including betaine11,22,23 and vitamin B1217, tend to reduce its effectiveness as a convulsant. Homocysteine has been shown to inhibit glutamic acid decarboxylase (GAD) and ),-amino butyric acid aminotransferase (GABA-T)2a, 25. Pyridoxal phosphate (PLP) is a required coenzyme by GAD, GABA-T and other cerebral decarboxylase and amino transferases3. Hydrazides and other PLP-antagonists that induce seizures also inhibit GAD and GABAT 16.
In a majority of cases, homocystinuria is due to a defect in cystathionine synthetase14, another PLP-dependent enzyme. In this study, we report the effects of pretreatment with pharmacologic doses of pyridoxine, pyridoxal phosphate and the vitamin B6 inhibitor hydrazine sulfate on homocysteine-induced seizures in mice. The results suggest a significant interaction between homocysteine and vitamin B6.
251 Female CD-I mice (Charles River) 26-28 g were housed in animal quarters having 12:12 light-dark cycle, 07.00 h light on. Solutions of pyridoxal phosphate, pyridoxine HCI, hydrazine sulfate, and DL-homocysteine thiolactone, a stable form of homocysteine (Sigma Chemicals) were prepared in physiological saline immediately prior to use. The Be vitamers (50 mg/kg) and hydrazine (50 mg/kg) were given i.p. 1 h prior to homocysteine. Homocysteine thiolactone was administered i.p. at dosage levels of 4.5, 5.5 and 6.0 mmol/kg. The EDs0 for homocysteine in these animals is 5.5 mmol/kg and the EDa5 is 7.5 mmol/kg 22. Animals were placed 4-5 per clear plastic cage where two observers recorded manifestations of seizure development. Animals were observed for a period of 60 min following homocysteine injection. In previous studies if seizures were to occur at the dosages used, they would occur within this time period. Statistical significance of seizure incidence and latency differences was determined by two-tailed Mann-Whitney U or Fisher Exact Probability test. lntraperitoneal injections of homocysteine produced dose-related seizures as previously described10,15, 22. Initially the drug produced a somnolence lasting from 2 to 30 min depending upon dosage which was followed by brief tremors of the head and forepaws. In animals that convulsed the initial seizure usually consisted of clonic jerking of the limbs with loss of righting reflex lasting 5-15 sec (clonic seizure). After one or more clonic attacks generalized tonic-extensor seizures and/or running fits occurred, followed by a comatose state and sometimes death. However, animals receiving preinjections of pyridoxine HC1 or pyridoxal phosphate had significantly enhanced seizure activity manifested by increased incidence (Table I) and decreased latency (Table II) to both clonic- and tonic-extensor seizures. Seizures in the B6 treated groups were of extended duration and animals appeared to be in status epilepticus. Several series of clonic to tonic progression occurred in each Be treated animal until death. In contrast, hydrazine sulfate offered significant protection to homocysteine TABLE I Group
Homocysteine
Number of animals convulsing Clonic seizures Control
I I1 III IV Totals
4.5 mmol/kg 5.5 mmol/kg 5.5 mmol/kg 6.0 mmol/kg
Be§
Tonic seizures Hydrazine§§Control
5/8 8/10 9/10 9/10
7/8 10/10 10/10 10/10
0/8* 3/10" 3/10"* 7/10
1/8 4/10 6/10 9/10
31/38
37/38*
13/38"**
20/38
Bn
Hydrazine
6/8* 10/10"* 10/10" 10/10
0/8 1/10 0/10" 1/10"
36/38** 2/38***
* P < 0.05. ** P < 0.01. *** P < 0.001, Fisher Exact Probability Test. § Groups I and III received pyridoxine HCI, 50 mg/kg, Groups II and IV received pyridoxal phosphate, 50 mg/kg. §§ Group I received 25 mg/kg, Groups II, III, IV 50 rng/kg hydrazine sulfate.
252 TABLE II Treatments Pretre~tment
Homocysteine dosage
Saline B6
4.5 mmol/kg 4.5 mmol/kg
Hydrazine
Median latency to convulsions in min
Lethality
N
Clonic
Tonic
I h(%)
24 h(%)
8 8
60 15.2"
60 31.5"
0 0
0 0
4.5 mmol/kg
8
60
60
0
0
Saline Be Hydrazine
5.5 mmol/kg 5.5 mmol/kg 5.5 mmol/kg
20 20 20
18,9 49.0 11.3"** 21.4"** 60*** 60**
4 (20%) 8 (40%) 0
4 (20%) 9 (45 %) 0
Saline Be Hydrazine
6.0 mmol/kg 6.0 mrnol/kg 6.0 mmol/kg
10 10 10
12.3 5.5*** 37.0**
3 (30%) 7 (70%) 0
3 (30%) 8 (80%)* 0
* P<0.05;
** P < 0 . 0 1 ;
27.3 17.0" 60***
*** P < 0 . 0 0 1 , M a n n Whitney U Test.
seizures. Only 2 of 38 controls receiving hydrazine had tonic convulsions compared to 20 of 38 controls and 36 of 38 vitamin B6 treated animals. There were no deaths in the hydrazine pre-treated groups while homocysteine was lethal to 45 % of Bs-treated and 18 % of control animals. The enhancement of homocysteine seizures by pharmacological levels of B6 vitamers reported in this study was at first surprising since it is recognized that a variety of procedures producing a B6 deficiency result in seizures. Human infantile pyridoxine deficiency seizures have been described, which are not responsive to anticonvulsants and are only controlled by intake of vitamin B66. An increasing amount of evidence, however, implicates enhanced levels of vitamin B6 in lowering seizure thresholds in experimental animals. When injected intracerebrally, pyridoxal phosphate is a potent convulsant2, 9 and hydrazones formed from pyridoxine and hydrazines are 10-100 times more potent convulsants than hydrazides themselves s. Gailagher 1~ reported that i.p. pyridoxine HC1, pyridoxal-5-phosphate (PLP) and pyridoxamine significantly lowered the threshold to seizures induced by fluorothyl. The threshold to isonicotinic acid hydrazide seizures is also reduced by injections of B6 vitamers 5. Significant penetration of the B6 vitamers into rat brain occurs within 30 minutes of an i.p. injection 1. It is of interest that in this study, the convulsant effects of homocysteine were diminished by hydrazine sulfate, which is normally a convulsant. Both hydrazines and homocysteine are antagonists of PLP-dependent reactions. These reactions are subject to inhibition to a highly varying degree depending upon the differing affinity of the coenzyme for the apo-enzyme 18. Homocysteine acts as an antimetabolite to pyridoxal phosphate in vitro and the PLP-homocysteine complex is devoid of coenzyme activity 19. However, the hydrazone formed from complexing of hydrazine and PLP can have coenzyme activity ~6. Therefore, as the results of this study suggest, there was a decrease in PLP-mediated neural excitability as a result of the formation of the hydrazone complex. Since both hydrazine 1~ and homocysteine19,~4,2~ also result in
253 inhibition of GAD and GABA-T, measurement of the activities of these enzymes after homocysteine with and without hydrazine pretreatment may identify the changes necessary for seizure induction. In summary, we have shown that pharmacological levels of B6 vitamers increase the seizures induced by homocysteine. The mechanisms underlying these seizures are unknown but their enhancement by ]]6 vitamers and prophylaxis by a B6 antagonist suggest that a B6 dependent mechanism regulates neuronal excitability in these seizures. We propose that this finding may have clinical relevance to human epilepsy because a number of anticonvulsant drugs decrease pyridoxine levels in patients on long-term therapy 2°,2t. This research was supported by The Epilepsy Research Foundation of Florida.
1 Bain, J. A. and Williams, H. L., Concentrations of B6 vitamers in tissues and tissue fluids. In E. Roberts (Ed.), Inhibition in the Nervous System and Gamma-Amino Butyric Acid, Pergamon Press, New York, 1960, 275-293. 2 Balzer, H., Holtz, P. and Palm, D. Untersuchunger uberv die biochemischen Grundlagen der knovulsiven wirkung von hydraziden, Arch. exp. Path. Pharmak., 239 (1960) 520-552. 3 Bender, D. A. Amino Acid Metabolism, John Wiley, New York, N.Y., 1975, 35-58. 4 Blennow, G., Folbergrova, J., Nilsson, G. and Siesjo, B. K., Cerebral metabolic and circulatory changes in the rat during sustained seizures induced by DL-homocysteine convulsions, Brain Research, 179 (1979) 129-146. 5 Costa, E., Vitamin B6 accentuates in the mouse the convulsant action of isonicotinyl hydrazine, Boll. Soc. ital. Biol. sper., 28 (1952) 1015-1016. 6 Coursin, D. B., Seizures in vitamin B6 deficiency. In E. Roberts (Ed.), Inhibition in the Nervous System and Gamma-Aminobutyric Acid, Pergamon Press, New York, N.Y. 1960, 294-301. 7 Curtis, D. R. and Watkins, J. C., Acidic amino acids with strong excitatory actions on mammalian neurons, J. Physiol. (Lond.), 166 (1963) 1-14. 8 Dixon, R. H. and Williams, H. L., The toxicity of pyridoxal and pyridoxal phosphate hydrazones in mice, Fed. Proc., 21 (1962) 338. 9 Ebadi, M., Kouyoumdjian and Govitrapong, P., The specificity of pyridoxal and pyridoxal phosphate induced convulsions, Trans. Amer. Soc. Neurochem., 11 (1980) 202. 10 Folbergrova, J., Energy metabolism of mouse cerebral cortex during homocysteine convulsions, Brain Research, 81 (1974) 443454. 11 Freed, W. J., Gillin, J. C. and Wyatt, R. J., Anticonvulsant properties of betaine, Epilepsia, 20 (1979) 209-213. 12 Gaitonde, J. K., Sulphur amino acids. In A. Latha (Ed.), Handbook ofNeurochemistry, Fol. HI, Plenum Press, New York, N.Y., 1970, 225-287. 13 Gallagher, R. B., The influence of tyrosine phenylpyruvate and vitamin B6 upon seizure thresholds, J. Neurochem., 18 (1971) 799-808. 14 Gaull, G. E., Homocystinuria and other disorders of sulfur metabolism. In E. Goldensohn and S. Appel (Eds.), Scientific Approaches to Clinical Neurology, Lea and Febiger, Philadelphia, Pa, 1977, 46-54. 15 Hammond, E. J., Hurd, R. W., Wilder, B. J. and Thompson, F. J., Focal and generalized experimental seizures induced by homocysteine, Electroenceph. clin. Neurophys., 49 (1980) 184-186. 16 Holtz, P. and Palm, D., Pharmacological Aspects of Vitamin B6, Pharmacol. Rev., 13 (1964) 113-178. 17 Hurd, R. W., Hammond, E. J., Thompson, F. J. and Wilder, B. J., The use of homocysteineinduced seizures in the study of experimental epilepsy, Neurosci. Abstr., 5 (1979) 194. 18 Kang, S. S., Wong, P. W. K. and Becker, N., Protein-bound homocyst(e)ine in normal subjects and in patients with homocystinuria, Pediat. Res., 13 (1979) 1141-1143. 19 Pestana, A., Sandoval, I. V. and Sols, A., Inhibition by homocysteine of serine dehydratase and
254
20 21 22 23 24 25
other pyridoxal 5-phosphate enzymes of the rat through cofactor blockage, Arch. Biochem., 146 (1971) 373-379. Reinken, L., Hohenauer, L. and Zeigler, E. E., Activity of red cell glutamic oxalacetic transaminase in epileptic children under antiepileptic treatment, Clin. chim. Acta, (1972) 270--271. Reinken• L.• The in•uence •f anti•pi•eptic drugs •n vitamin B6 metab••ism• Acta vitamin enzyrn••.• 29 (1975) 252-254. Sprince, H., Parker, C. M. and Josephs, J. H., Homocysteine-induced convulsions in the rat: protection by homoserine, serine, betaine, glycine and glucose, Agents Act., 1 (1969) 9-13. Sprince, H., Parker, C. M., Josephs, J. A. and Magazino, J., Convulsant activity of homocysteine and other short-chain mercapto acids: protection therefrom. Ann. N.Y. Acid. Sci., 166 (1969) 323-325. Taberner, P. V., Pearce, M. J. and Watkins, J. C., The inhibition of mouse brain gutamate decarboxylase by some structural analogues of L-glutamic acid, Biochem. Pharmacol., 26 (1977) 345-349. Tunnicliff, G. and Ngo, T. T., The mode of action of homocysteine on mouse brain glutamic decarboxylase and 7-aminobutyrate aminotransferase, Canad. J. Biochem., 55 (1977) 1013-1018.