Plasma urea and ammonia in epileptic patients and their relatives

239

Epilepsy Research, I3 (1992) 239-244 0920-121~~92/~05.~ Q 1992 Elsevier Science PubIishers B.V. Ail rights reserved

EPIRES 00520

Plasma urea and ammonia in epileptic patients and their relatives

Najma A, Janjuaa, Toshifumi Itanoa, Toshiaki Kugohb, Kiyoshi Hosokawab, Hideki Matsui”, Masaaki Tokudaa and Osamu Hatase” Departments of “Physiology and ‘Neuropsychiatry,

Kagawa Medical School, 1750-l Ikenobe, Miki-cho, Kagawa 761-07, Japan

(Received 6 April 1992; revision received 15 July 1992; accepted 25 July 1992) Key words: Plasma; Urea; Ammonia; Metabolic defect, genetic

The plasma levels of urea and ammonia were examined in patients with primary generalized epilepsy, patients with partial epilepsy and in the first-degree relatives of these subjects. The results show a significant decrease in plasma urea in both groups of patients and their first-degree relatives as compared to the non-epileptic controls. The plasma ammonia concentrations were signi~cantly higher in both groups of patients and in the relatives of generalized epilepsy patients as compared to the controls. The observed changes in plasma urea and ammonia were found not to be due to the effect of anticonvulsant drugs. The data suggest that a metabolic defect in urea synthesis may constitute one of the genetic components in the multifactorial etiologies of primary generalized and partial epilepsies.

Introduction A s~gni~cant increase in the plasma level of glutamic acid in epileptic patients and their relatives has been observed and a genetic basis for this increase has been suggested’1,‘2,2’. The m~hanisms underlying glutamic acid increase in the plasma, however, remain unknown. In a recent study we found no significant changes in leukocyte glutamate dehydrogenase activity in primary generatized or partial epilepsy patients and their first-degree relatives who had elevated plasma glutamic acid Ievelsr2. The present study reports for these subjects the plasma levels of urea and ammonia and of amino acid intermediates of the urea Correspondence to: Dr. Najma A. Janjua, department of Physiology, Kagawa Medical School, 1750-i Ikenobe, Mikicho, Kagawa 761-07, Japan.

cycle”. The main objective of the study was to investigate if urea synthesis was altered in epileptic patients and their relatives. A defective urea synthesis would produce hyperammonemia which in turn would favor the formation of glutamic acid’*. Since hyperammonemia and/or decreased urea synthesis have also been attributed to valproic acid therapy2593’4,plasma ammonia and urea levels were examined in patients both with and without valproic acid treatment. Methods Clinical

material

The clinical material comprised the following 5 groups of subjects: (A) 10 patients with primary generalized epilepsy including 6 males and 4 females, age range 1l-41 years, mean age (years i_ SD) 20.7 & 8.8; (B) 10 first-degree relatives of pri-

240

Fig. I. F&ma levels of urea and ammonia in (a) primary generalized epilepsy patients and their relatives and jb) partial epilepsy patients and their relatives. Values are mean + SD; number of cases in parentheses. ‘P
241

TABLE I Plasma levels of urginine, citrulline and ornithine in epileptic patients, their relatives and controls Group

Amino acids (mean f SD; ~rnoi/l~ _... __~ Arginine

(A) Primary 5.64 generalized epilepsy patients (PGEP) (N = 10) (B) Relatives of 6.86 PGEP (N = 10) (C) Partial epilepsy 5.80 patients (PEP) (N= 12) (D) Relatives of 7.34 PEP (N = 6) (E) Controls 7.23 (N=20)

Citrulline

ml)

Ornithine

f 1.40’ 2.19 k 0.82 5.37 f 1.14

analyzer (Hitachi 835). The plasma was deproteinized using picric acid as described in the operator’s manual for the amino acid analyzer. The ammonia dete~inations were carried out using the ‘Determiner’ kit (Kyowa Medics Co. Ltd., Tokyo) which employs an enzymatic assay using glutamate dehydrogenase. The statistical analysis was done using Student’s t-test. For comparisons involving unequal group variances, an approximation to t was computed.

& 2.76 2.44 f 0.99 6.83 + 2.35

Results f 1.78 2.57 f 0.96 7.36 + 1.59’

) 1.69 2.40 + 0.75 6.15 +_ 1.47 + 2.42 2.76 + 0.81 6.22 i_ i.33

‘P~0.05 as compared to controls {Student’s t-test).

mary generalized epilepsy patients including 2 fathers and 8 mothers, age range 34-55 years, mean age 46.2 t_ 6.8; (C) 12 patients with partial epilepsy including 8 males and 4 females, age range 24 43 years, mean age 32.6 f 6.8; (D) 6 first-degree relatives of partial epilepsy patients including 1 father, 4 mothers and 1 sister, age range 37-63 years, mean age 54.5 + 9.4; and (E) 20 non-epileptic controls including 13 males and 7 females, age range 20-36 years, mean age 24.6 + 5.0. The ascertainment and clinical details of these groups have been described previouslyi2, The liver and renal functions in the patients were normal as indicated by serum GOT (glutamate-oxaloacetate transaminase) and GPT (glutamate-pyruvate transaminase) activities, and serum creatinine levels, respectively3.

The mean plasma levels of urea and ammonia in primary generalized and partial epilepsy patients, their first-degree relatives and in controls are shown in Fig. 1. The urea levels were significantly decreased in both groups of patients and their relatives as compared to controls. Plasma ammonia concentrations, on the other hand, were signiticantly increased in both groups of patients and in the relatives of generalized epilepsy patients. Although the increase in ammonia in the relatives of partial epilepsy patients was not statistically significant as compared to controls, the levels did show a tendency to be higher. The plasma urea and ammonia levels showed no significant differences between males and females either within any of the groups studied or when the data for all patients and relatives were pooled. In the pooled data, the mean (& SD) plasma levels of urea were 333.69 t_ 198.88 for males (N= 17) and 385.78 f 275.83 for females (N= 21). The corresponding values for plasma ammonia in these subjects were 75.03 + 27.53 and 59.30 _t 22.96, respectively. In the control males (N = 13) TABLE II

Piasma urea, amino acid and ammonia determinations Venous blood samples from patients, relatives and controls were drawn in EDTA containing tubes after an overnight fast. The blood was centrifuged at 3000 rpm for 15 min and plasma stored at -80°C until urea, amino acid and ammonia determinations. The urea and amino acid levels were determined using an automatic amino acid

Plasma levels of urea and ~monia without valproic acid therapy

in epileptic putients with and

Group

Urea @mol/ 100 ml)

Ammonia @g/f00 ml)

Valproate (N=6) Other (N= 15)

390.89 + 194.15

67.39 + 26.12

262.55 f 155.42

79.13 + 24.92

Values are mean f SD.

and

females

346.26

and

(N = 21) the values 824.30

3:

379.36.

were 852.19 respectively,

k for

Hyperammonemia resulting from a defective urea synthesis also occurs in the inborn errors of’

urea, and 39.32 t_ 7.58 and 34.46 + 7.19. respec-

metabolism

tively, for ammonia.

clinical

Table I shows the plasma levels of arginine, citrulline and ornithine in the 2 groups of epileptic

tal coma from hyperammonemia

patients, their relatives and controls. The only significant changes were a decrease in arginine in the generalized

epilepsy

nithine in partial the controls.

patients

epilepsy

and an increase

patients,

in or-

as compared

to

The mean plasma levels of urea and ammonia in epileptic patients with and without valproic acid therapy are shown in Table II. There were no significant differences between the 2 groups for either urea or ammonia. In tact. the mean value of urea was numerically higher and that of ammonia lower in the group with valproate therapy as compared to the non-valproate

therapy

group.

Discussion The observation of a familial decrease in plasma urea and increase in plasma ammonia levels in primary generalized and partial epilepsy suggests the possibility of a hereditary defect in urea synthesis in these disorders. The observed alterations in urea and ammonia were found not to be related to valproic acid therapy. The changes can also not be due to the effect of other anticonvulsants since none of the relatives were receiving such drugs as they did not have seizures’*. For the latter reason, the findings cannot be ascribed to the neurophysiological sequelae of the seizures either. The dietary status of the subjects investigated in this study has previously been discussed12. In that study, no marked changes were observed in any of the essential amino acids in the patients or the relatives, thereby eliminating the possibility of a protein-rich or a protein-de~cient diet. Furthermore, under either of these dietary conditions, the plasma levels of both urea and ammonia would be expected to be altered in the same direction. In the present study, on the other hand, the decrease in urea is accompanied by increased ammonia levels, thus further supporting the involvement of a lnechanism within the urea cycIe rather than an external factor.

involving

findings

the urea cycleh. The main

in these disorders

include

neona-

and later protein

intolerance with mental deficit. Seizures and/or EEG abnormalities also occur frequentlyh.“‘. The hyperammonemia observed in the present study, however, is mild as compared to that in the classical urea cycle disorder@.‘“. As a result, the patients did not manifest the typical clinical symptoms related to ammonia toxicity. They did, however. manifest seizures suggesting that although the hyperammonemia is not high enough to precipitate the acute ammonia toxicity symptoms, it may be significant enough to lower the seizure threshold thereby increasing the susceptibility to have seizures. Such a possibility would also be in agreement with the fact that apart from their seizure disorder, most epileptic patients are in good physical health in general and would thus not be expectcd to have a metabolic abnormality which was not compatible with survival. Furthermore. epilepsy being a paroxysmal disorder, the patients are not sick most of the time. The underlying biochemical abnormality in epilepsy, therefore, is likely to be more subtle and unlike the classical inborn errors of metabolism which usually result in severalfold changes in metabolite levels”. The metabolic defects of the urea cycle reported thus far are all Mendelian traits6. In the present study, the ascertainment was incomplete to examine the intrafamily inheritance pattern of the individual values of plasma urea and ammonia. For most epilepsies, however, a multifactorial mode of inheritance has been postulated implying the existence of one or more major genes which would interact with other polygenes and/or environmental factors to produce the clinical phenotype’.‘7. The results of the present study are com~tible with this view. The observed changes in plasma urea and ammonia were found not to be sufficient for the clinical expression of epilepsy as the relatives of the patients showed relatively similar degrees of changes, at least with respect to the plasma urea levels, but did not manifest seizures. Thus it may be hypothesized that the metabohc abnormality in the urea cycle constitutes a genetic predis-

243

posing factor in the multifactorial etiologies of primary generalized and partial epilepsies whereas additional factors both genetic and non-genetic are necessary for the expression of the clinical phenotype. No marked changes were observed in 3 intermediates of the urea cycle, namely ornithine, citrulline and arginine, in epileptic patients and their relatives. The small alterations in arginine and ornithine do not appear meaningful as they are not suggestive of metabolic blocks at the sites preceding or following the 2 amino acids. Thus the site of the presumed metabolic defect would appear more likely to be carbamoyl phosphate synthetase-I, the enzyme which catalyzes the synthesis of carbamoyl phosphate from ammonia, bicarbonate and ATPi’. Measurements of carbamoyl phosphate synthetase-I in the liver tissue, however, would be necessary to verify this. How the metabolic consequences of the proposed metabolic defect in the urea cycle might be related to the mechanism of epilepsy is not clear at this stage. Although several hypotheses have been proposed to explain the biochemical basis of ammonia toxicity in genera16,‘*, little has been said more specifically about the relation between hyperammonemia and seizures. Results of the present study, in conjunction with previous observations of elevated plasma glutamic acid in epileptic subjects”*‘*,*’ suggest that the increase in glutamic acid may be due to an increase in ammonia’8. The elevated glutamic acid levels systemically, however, would not be expected to contribute directly to the neurophysiological mechanisms of seizures as glutamic acid

does not cross the blood-brain barrier4. Instead, due to high ammonia levels in the blood, the diffusion of ammonia out of the brain and into the blood will be slowed down, a process that normally keeps the brain ammonia below toxic levels?. This would result in increased ammonia levels in the brain which in turn would favor the formation of glutamic acid”. The latter being an excitatory neurotransmitter 13,its increased levels in the brain would produce hyperexcitability* thereby increasing the susceptibility to seizures. There is extensive neurochemical and neurophysiological evidence suggesting that the hyperexcitability in the epileptogenic brain may be due to elevated glutamic acid levels (reviews5’7910*20). According to the multifactorial view of the inheritance of epilepsy as already discussed above, however, the hyperexcitability resulting from the proposed defect in the urea cycle would not be sufficient for the clinical manifestation of seizures which would require additional endogenous and exogenous factors. Acknowledgements We are indebted to the patients, their family members and control subjects without whom this study would not have been possible. The technical assistance of Mr. A. Miyatake and Miss C. Shimizu was most appreciated. This work was supported by grants from the Ministry of Education, Science and Culture of Japan and the Japan Society for the Promotion of Science (JSPS). N.A.J. was also the recipient of a Research Scientist award from the JSPS.

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