Suppression of neurotoxicity of ammonia by l -carnitine

328

Brain Research, 567 (1991) 328-331 (~) 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50

BRES 24969

Suppression of neurotoxicity of ammonia by L-carnitine Masato Matsuoka, Hideki Igisu, Kazuaki Kohriyama and Naohide Inoue Department of Environmental Toxicology, University of Occupational and Environmental Health, Yahatanishiku, Kitakyushu (Japan)

(Accepted 10 September 1991) Key words: L-Carnitine; Ammonia; Neurotoxin; Energy metabolism

Administration of ammonium acetate to mice caused seizures and alterations of brain energy metabolites. Pretreatment of animals with L-carnitine suppressed the frequency of the seizures and prolonged the latency to the first fit. When examined using the 'freeze clamp' method, brain energy metabolites were well preserved and the elevation of ammonia was less marked on administration of L-carnitine. Thus, L-camitine suppresses ammonia-induced seizures and biochemical alterations of the brain in mice. Hyperammonemia is associated with various conditions such as hepatic failure, inborn errors of urea cycle function, organic acidemia and Reye's syndrome, and is believed to be responsible for the dysfunctions of the central nervous system in these disorders 5. In our previous experiment, brain energy metabolism of mice could be impaired severely depending on the dose of ammonium acetate administered 16. L-Carnitine (fl-hydroxy-y-N-trimethylaminobutyrate) is widely distributed among tissues 4. In humans, it is synthesized in liver, kidney and brain 23. It is an essential cofactor for the transport of long-chain fatty acids or acetyl groups across the inner membrane Of mitochondria 4. Recently, it was reported that pretreatment with L-carnitine protected mice against acute ammonia toxicity 1s'19. However, some contradictory results have been presented s'9Ax'13, and the mechanism of the protection of the brain by L-carnitine, if any, against ammonia toxicity has not been clarified. In this communication, we present results indicating that L-carnitine can suppress seizures as well as biochemical alterations of the brain caused by hyperammonemia. L-Carnitine (inner salt) was purchased from Sigma Chemical Co. (St. Louis, MO). All other chemicals were reagent grade. Male ddY mice weighing 25-30 g, fed a standard chow ad libitum, were used. Animals were divided into 4 groups and injected intraperitoneaUy with two solutions 30 min apart: group I, injection with saline followed by the second saline injection; group II, saline and then 15 mmol ammonium acetate/kg b. wt.; group III, 10 mmol L-carnitine/kg b. wt. and 15 mmol ammonium acetate/kg b. wt.; group IV,

20 mmol L-carnitine/kg b. wt. and 15 mmol ammonium acetate/kg b. wt. The saline solution was 0.85% NaCl dissolved in double distilled water. L-Carnitine and ammonium acetate were dissolved in the saline solution. The concentration of L-carnitine was 10% (w/v, pH 7.5, for 10 mmol/kg b. wt. administration) or 20% (w/v, p H 7.7, for 20 mmol/kg b. wt.). The concentration of ammonium acetate was 0.8 M. Ten min after the injection of ammonium acetate or the second saline solution, the mouse was quickly immersed in liquid nitrogen for 5 min and kept at -80 °C. The behavior of each mouse injected with ammonium acetate was recorded with a Canon VM-E1 videotape recorder and analyzed later. The frozen brain was chiseled out, weighed, and powdered in a mortar chilled with liquid nitrogen. The powder was extracted with 4 vols. of 3.0 M perchloric acid at -15 °C using all glass PotterElvehjem homogenizer, and then diluted to 1.0 ml/75 mg tissue with 1 mM E D T A at 0 °C. The mixtures were centrifuged at 5000 g for 30 min at 0 °C, and the supernatants were neutralized with 2.0 M K H C O 3. After centrifugation at 3000 g for 15 min at 0 °C to remove the precipitated KCIO4, the supernatants were stored at -120 °C until analysis 14'16'22. The concentrations of ammonia 3, phosphocreatine (PCr) 2, ATP 2, A D P 2, AMP 2, creatine 2, glucose 17, pyruvate 3 and lactate 2 were all determined enzymatically. Brain energy charge potential (ECP) was calculated by the method of Atkinsonl; ECP = (ATP + ½ADP)/(ATP + A D P + AMP). In another set of experiments, we determined the concentration of blood ammonia. (Survival rate of animals in each group was calculated pooling the data obtained in

Correspondence: M. Matsuoka, Department of Environmental Toxicology, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishiku, Kitakyushu 807, Japan. Fax: (81) (93) 692-4790.

329 seizure and the n u m b e r of seizures were done by

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Wilcoxon test. Other differences of means were examined by one-way analysis of variance ( A N O V A ) and subsequent Tukey's procedures. P < 0.05 was considered as statistically significant.

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lenge by a m m o n i u m acetate did not differ among 3 groups: in group II, 20 (80.0%) out of 25 survived; in group III, 20 (90.9%) out of 22; in group IV, 19 (90.5%) out of 21. All mice (20 out of 20) injected with the second saline solution (group I) survived for 10 m i n after the injection.

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All mice in group II showed drowsiness first, and within 6.5 min after the injection of a m m o n i u m acetate, all developed short clonic seizures. The latency to the occurrence of the first seizure was significantly delayed

Fig. 1. Effects of L-carnitine on the latency to the first seizure (rain) (A), and number of the seizures during 10 rain observation (B). Each mouse was injected with saline solution with or without L-carnitine and then with 15 mmol ammonium acetate/kg b. wt. Bars indicate the mean value of each group (n = 7) and an asterisk significant difference (P < 0.01) from the untreated group (0 mmol L-carnitine/kg b. wt.) (Wilcoxon test).

(P < 0.01, Fig. 1A) in animals treated with L-carnitine (group III or IV) compared with group II. The n u m b e r of seizures in group III or IV was also reduced significantly ( P < 0.01, Fig. 1B), while there was no statistical difference between groups III and IV. A m m o n i a concentration in the brain was markedly

this and the preceding experiments.) Ten min after the injection of a m m o n i u m acetate or the second saline solution, mice were killed by decapitation. Blood was collected from cervical blood vessels, deproteinized immediately, and stored at -120 °C. The concentration of

elevated group I, whereas pyruvate

a m m o n i a was assayed by the method of O k u d a and Fujii21.

in group II (Table I). W h e n compared with PCr, A T P and E C P decreased significantly, creatine, ADP, AMP, lactate and lactate/ ratio increased in group II. O n the other hand,

in either group treated with L-carnitine (group III or IV), PCr, ATP, creatine, A D P and A M P levels and E C P were not different from those of group I. Besides, levels of

The data presented are from more than 7 mice in each group that survived for 10 min after the injection of a m m o n i u m acetate. All values are expressed as m e a n + S.E.M. Statistical analyses of the latency to the first

a m m o n i a and lactate in L-carnitine-treated animals (group III or IV) were significantly lower than in untreated animals (group II). W h e n groups III and IV were

TABLE I

Effects of L-carnitine on brain energy metabolites All values are expressed in terms of #mol/g tissue (wet wt.) except for ECP. Controls (group I) were injected with saline solutions only. Animals in other groups were injected with saline solution containing no carnitine (group II) or carnitine (groups III and IV), and then with 15 mmol ammonium acetate/kg b. wt. Data are mean --- S.E.M. values from 7 animals.

Ammonia Phosphocreatine ATP ADP AMP ECP Creatine Glucose Pyruvate Lactate Lactate/pyruvate

Control

L-Carnitine (mmol/kg b. wt.)

(group 1)

0 (group 1I)

10 (group Ill)

20 (group IV)

0.48 -+ 0.07 3.42 --- 0.11 2.72 -+ 0.10 0.574 -+ 0.023 0.049 --- 0.006 0.899 -+ 0.005 7.42 --- 0.17 2.05 -+ 0.11 0.104 +-- 0.011 1.82 --- 0.09 18.5 ± 1.8

5.95 2.33 2.21 0.706 0.239 0.815 8,43 1.61 0.107 4.48 41.7

3.38 -+ 0.22a'b 3.38 -+ 0.09b 2.77 -+ 0.07b 0.578 -+ 0.024b 0.081 - 0.015b 0.892 -+ 0.008b 7.52 --- 0.19b 1.64 ± 0.13 0.103 +-- 0.010 3.85 --- 0.I1 a'b 39.4 +-- 3.6~

3.08 -- 0.25a'b 3.74 -+ 0.05b 2.63 -+ 0.08b 0.536 -+ 0.013b 0.042 _+ 0.007b 0.903 +-- 0.002b 7.13 --- 0.17b 1.70 ± 0.14 0.098 +-- 0.009 3.21 - - 0 . 1 5 a ' b ' c 34.4 +- 3.9a

--- 0.28a -+ 0.12a +- 0.09~ -+ 0.036~ --- 0.027a - 0.010" -+ 0.28a ± 0.16 - 0.002 ± 0.20a --- 1.5a

aSignificantly different from group I (P < 0.05). bSignifieantlydifferent from group II (P < 0.05). cSignificantly different from group III (P < 0.05).

330

compared, lactate was lower in group IV than in group III (P < 0.05). The levels of glucose and pyruvate did not differ among the 4 groups. All mice given ammonium acetate showed significant elevation of blood ammonia compared with group I (Table II). However, ammonia concentrations in groups treated with L-carnitine (group III or IV) were lower than untreated group (group II) by approximately 40%. There was no significant difference between groups III and IV. In this study, we found that L-carnitine treatment delayed onset of the seizures, reduced the number of fits, preserved the brain energy metabolites, and lowered ammonia concentration in the brain. In the previous studies, clinical effects of carnitine in hyperammonemia were judged mainly by mortality rate, and the results were contradictory; while O'Connor et al. 18'19 found carnitine suppressed mortality caused by ammonia, Deshmukh et al. 8'9 found no such effect. We saw no significant effects of carnitine on mortality rate, probably because our observation period was short. However, detailed analyses of animal behavior recorded on a videotape revealed that carnitine clearly suppresses ammonia-induced seizures. Previous results of biochemical examinations, especially those on lactate and ammonia in the brain, were also contradictory 8'9'~9. It seems that brain must be processed properly to accurately measure ammonia or lactate (and energy metabolites). In the present study, we used the 'freeze clamp' method, immersing quickly the whole body of the mouse into liquid nitrogen. In some previous studies, the brain was immersed in liquid nitro-

gen after it was removed from the skull s'9'19. However, lactate levels can be affected profoundly within one minute after decapitation 15. Ammonia in the brain can also increase rapidly after decapitation 1°'24. These may underly the failure to observe the protective effects of L-carnitine against ammonia-induced biochemical alterations in the brain. The effects of L-carnitine are apparently systemic because treatment with L-carnitine lowered ammonia not only in the brain but also in the blood. Effect of simple dilution of ammonia in the peritoneal cavity, which was suggested previously 8'9, is excluded because we injected the control group with the same volume of saline. Mere neutralization of ammonia with carnitine solution is also unlikely because we used L-carnitine inner salt solution which is neutral, being different from highly acidic L-carnitine hydrochloride solution. L-Carnitine can stimulate the hepatic urea cycle, a major system to remove ammonia in the body 7'2°. In the brain, the removal of ammonia takes place mainly in astrocytes by amidation of glutamate to form glutamine, catalyzed by glutamine synthetase which requires ATP 5. And the present results show that carnitine can restore the lowered ATP and PCr levels in the brain. Besides, it has been reported that L-carnitine supplementation prevents mitochondrial damage in the rat choroid plexus induced by octanoic acid 12. Thus, it seems that L-carnitine can improve brain energy metabolism and stimulate the ammonia-removing system in the brains as well as in the liver. On the other hand, hyperammonemia was found to cause carnitine deficiency in tissues 6, and this might be corrected by carnitine administration. In the production of coma, the synergism between ammonia and fatty acids, particularly longer chain fatty acids, was reported 25. Hence, stimulation of fatty acid oxidation by L-carnitine, which may take place outside the brain, might further contribute toward improvement. Thus, while the precise mechanisms are still unknown, our results show that L-carnitine can counteract effects of ammonia and can improve clinical signs and biochemical alterations in the brain. Since the level of blood ammonia in this experiment was much higher than usually seen in human cases and because L-carnitine was administered before the induction of hyperammonemia, further studies are necessary to determine whether treatment with L-carnitine may be beneficial for patients with hyperammonemia.

1 Atkinson, D.E., The energy charge of the adenylate pool as a regulatory parameter: interaction with feedback modifiers, Biochemistry, 7 (1968) 4030-4034. 2 Bergmeyer, H.U. (Ed.), Methods of Enzymatic Analysis, Vols.

3, 4, 2nd edn., Academic Press, New York, 1974. 3 Bergmeyer, H.U., Bergmeyer, J. and Grassl, M. (Eds.), Methods of Enzymatic Analysis, Vols. 6-8, 3rd edn., Verlag Chemic, Weinheim, 1985.

TABLE II Effects of L-carnitine on blood ammonia concentration (ttmol/ml)

Controls (group I) were injected with saline solutions only. Animals in other groups were injected with saline solution containing no carnitine (group II) or carnitine (groups III and IV), and then with 15 mmol ammonium acetate/kg b. wt. Data are mean --- S.E.M. values from 8 animals. Control

L-Carnitine(mmol/kg b. wt.)

(group I)

0 (group 11)

0.06 -+ 0.00

4.18 -+ 0.18a 2.48 -+ 0.16~'b

10 (group III)

20 (group IV)

2.36 - 0.16a'b

aSignificantly different from group I (P < 0.01). bSignificantly different from group II (P < 0.01).

331 4 Bremer, J., Carnitine: metabolism and functions, Physiol. Rev., 63 (1983) 1420-1480. 5 Cooper, A.J.L. and Plum, F., Biochemistry and physiology of brain ammonia, Physiol. Rev., 67 (1987) 440-519. 6 Costeil, M., Miguez, M.P., O'Connor, J.E. and Grisolia, S., Effect of hyperammonemia on the levels of carnitine in mice, Neurology, 37 (1987) 804-808. 7 Costell, M., O'Connor, J.E., Higuez, H.P. and Grisolia, S., Effects of L-carnitine on urea synthesis following acute ammonia intoxication in mice, Biochem. Biophys. Res. Commun., 120 (1984) 726-733. 8 Deshmukh, D.R. and Rusk, C.D., Failure of L-carnitine to protect mice against ammonia toxicity, Biochem. Med. Metab. Biol., 39 (1988) 126-130. 9 Deshmukh, D.R., Singh, K.R., Meert, K. and Deshmukh, G.D., Failure of L-carnitine to protect mice against hyperammonemia induced by ammonium acetate or urease injection, Pediatr. Res., 28 (1990) 256-260. 10 Folbergrov~i, J., Passonneau, J.V., Lowry, O.H. and Schulz, D.W., Glycogen, ammonia and related metabolites in the brain during seizures evoked by methionine sulphoximine, J. Neurochem., 16 (1969) 191-203. 11 Hearn, T.J., Coleman, A.E., Lai, J.C.K., Griffith, O.W. and Cooper, A.J.L., Effect of orally administered L-carnitine on blood ammonia and L-carnitine concentrations in portacavalshunted rats, Hepatology, 10 (1989) 822-828. 12 Kim, C.S., Roe, C.R. and Ambrose, W.W., L-Carnitine prevents mitochondrial damage induced by octanoic acid in the rat choroid plexus, Brain Research, 536 (1990) 335-338. 13 Kloiber, O., Banjac, B. and Drewes, L.R., Protection against acute hyperammonemia: the role of quaternary amines, Toxicology, 49 (1988) 83-90. 14 Lowry, O.H. and Passonneau, J.V., A Flexible System of Enzymatic Analysis, Academic Press, New York, 1972, pp. 120128.

15 Lowry, O.H., Passonneau, J.V., Hasselberger, EX. and Schulz, D.W., Effect of ischemia on known substrates and cofactors of the glycolytic pathway in brain, J. Biol. Chem., 239 (1964) 18-30. 16 Matsuoka, M., Igisu, H., Kohriyama, K. and Inoue, N., Effects of ammonia on brain energy metabolites: dose-dependent alterations, J. Neurochem., 55 (1990) 354-355. 17 Miwa, I., Okuda, J., Maeda, K. and Okuda, G., Mutarotase effect on colorimetric determination of blood glucose with fl-Dglucose oxidase, Clin. Chim. Acta, 37 (1972) 538-540. 18 O'Connor, J.E., Costell, M. and Grisolia, S., Protective effect of L-carnitine on hyperammonemia, FEBS Lett., 166 (1984) 331-334. 19 O'Connor, J.E., Costell, M. and Grisolfa, S., Prevention of ammonia toxicity by L-carnitine: metabolic changes in brain, Neurochem. Res., 9 (1984) 563-570. 20 O'Connor, J.E., Costell, M., Mtguez, M,P., Portol6s, M. and Grisolia, S., Effect of L-carnitine on ketone bodies, redox state and free amino acids in the liver of hyperammonemic mice, Biochem. Pharmacol., 36 (1987) 3169-3173. 21 Okuda, H. and Fujii, S., Direct colorimetric determination of blood ammonia, Saishin lgaku, 21 (1966) 622-627. 22 Pulsinelli, W.A. and Duffy, T.E., Regional energy balance in rat brain after transient forebrain ischemia, J. Neurochem., 40 (1983) 1500-1503. 23 Rebouche, C.J. and Engel, A.G., Tissue distribution of carnitine biosynthetic enzymes in man, Biochim. Biophys. Acta, 630 (1980) 22-29. 24 Richter, D. and Dawson, R.M.C., The ammonia and glutamine content of the brain, Z Biol. Chem., 176 (1948) 1199-1210. 25 Zieve, F.J., Zieve, L., Doizaki, W.M. and Gilsdorf, R.B., Synergism between ammonia and fatty acids in the production of coma: implications for hepatic coma, J. Pharmacol. Exp. Ther., 191 (1974) 10-16.