Plasma glutamate decarboxylase activity in neuropsychiatry

fsychiurry Research, 6, 335-343 (1982) Elsevier Biomedical Press

Plasma Glutamate Neuropsychiatry

Decarboxylase

Hisanobu Kaiya, Masuyuki Namba, and Shigenobu Nakamura Received J&

335

Activity

Hiromichi

2, 1981; revised version received November

in

Yoshida,

6. 1981; accepted December

12.1981.

Abstract. Plasma glutamate decarboxylase (GAD) activity was measured in patients with endogenous psychoses and neurologic diseases. Unmedicated schizophrenic patients showed no difference in plasma GAD levels compared to controls. Administration of neuroleptics together with anticholinergic agents increased plasma GAD activity in schizophrenic patients. Compared to controls, patients with major depression and bipolar illness showed significantly lower GAD activity. No effect of antidepressants and minor tranquilizers on plasma GAD activity was found. Relatively lower GAD activity was shown in neurotic patients. The enzyme activity in plasma of patients with Huntington’s chorea (HC) was lower than control levels. The plasma GAD concentrations correlated with cerebrospinal fluid concentrations in five HC patients. Key Words. Plasma GAD, schizophrenia, roleptics, anticholinergic agents.

depression,

Huntington’s

chorea,

neu-

It is generally agreed that y-aminobutyric acid (GABA) is distributed throughout the brain and functions as an inhibitory neurotransmitter (Otsuka et al., 1971). GABA is also known to interact with many central neurotransmitters, such as dopamine, noradrenalin, serotonin, and acetylcholine (Pradhan and Bose, 1978). Because these biogenic amines and acetylcholine may be implicated in the pathophysiology of endogenous psychoses, the GABA-ergic mechanism in schizophrenic and affective disorders merits attention. Indeed, recent studies have examined the effects of agents that influence GABA metabolism in patients with schizophrenia (Tamminga et al., 1978) and mania (Emrich et al., 1980). Anxiolytic drugs with benzodiazepine structure have also been suggested to have effects in the GABA system (Costa et al., 1975). GABA is produced from glutamate with an enzyme, glutamic acid decarboxylase (GAD), whose distribution and content are thought to correlate with those of GABA (Roberts and Kuriyama, 1968). The present study examines plasma GAD levels in various neuropsychiatric diseases. Methods The sample comprised patients with schizophrenia, affective disorders, neurosis, organic brain disease, and Huntington’s chorea, as well as a group of normal control subjects. (See Table I for Hisanobu Kaiya, M.D., is Docent, Department of Neuropsychiatry, Gifu University School of Medicine; Masuyuki Namba, M.D., is Professor and Chief, Department of Neuropsychiatry, Gifu University School of Medicine; Hiromichi Yoshida, M.D., is Director, lnuyama Mental Hospital; Shigenobu Nakamura, M.D., is Assistant Professor, Department of Neurology, Kyoto University, Faculty of Medicine. (Reprint requests to Dr. H. Kaiya at Department of Neuropsychiatry, Gifu University School of Medicine, Tsukasamachi 40, 500 Gifu, Japan.)

0165-I 78

I I)82:0000-0000:

$02.75 0 Elaevier Biomedical

Press

336 details of the entire sample, and Table 2 for additional information concerning the patients with Huntington’s chorea.) Blood samples were obtained by venipuncture, using heparinized syringes, between 8 and I1 a.m. All samples were centrifuged at 4°C to separate the plasma and stored at -70°C until analysis. Blood from some patients with endogenous psychoses was sampled twice or more. In the patients with Huntington’s chorea, lumbar punctures were performed at the same time plasma samples were collected.

Table 1. Sample characteristics Mean age (years)

Age range (years)

25

19-32

7F 8M

25

17-40

3F 5M

37

29-52

8F 1M

46

20-60

13

11 F 2M

47

25-62

Tricyclic antidepressants such as imipramine, amitriptyline, and chlorimipramine, together with benzodiazepines

Bipolar illness

5

2F 3M

53

28-69

Lithium carbonate iin all but one bipolar patient)

Neurotic patients

11

7F 4M

26

1o-47

Organic brain diseases

13

3F 10 M

43

6-76

5

3F 2M

44

40-49

27 F 18 M

47

5-78

Diagnostic grow Schizophrenia1 Unmedicated2

n 33 10

Sex 2F

Treatment -

8M Short-term (4 5 years) medicated

15

Long-term (2 5 years) medicated

8

I

Affective disorders1 Unmedicated major depression Medicated major depression

Huntington’s chorea

Normal controls 1. 2. 3. 4.

27 9

45

Phenothiazines and/or butyrophenones in combination with anticholinergic agents

-

-

Drug therapy of different types Haloperidol, cloxazolazepam, clonazepam, thalamotomy”

Diagnosis based on DSM-I// 1,American Psychiatric Association, 19801 criteria. Either never medicated or off medication for at least 3 months before study. Including epilepsy, senile dementia, Parkinson’s disease, and brain injury. See Table 2 for breakdown, by subject, of treatment received.

-

337 Table 2. GAD activity in plasma and CSF of patients with Huntington’schorea

Subject

Duration of illness (years)2

GAD activity (pmole/ml/minute) Treatment

Age’

Sex

1

40

F

5

Haloperidol, cloxazolazepam

1.8

3.7

2

41

F

2

Haloperidol, cloxazolazepam

8.3

6.9

3

43

M

19

Thalamotomy

8.0

10.8

4

45

M

18

Thalamotomy, clonazepam

13.5

11.2

5

49

F

15

Haloperidol, cloxazolazepam

1.5

3.8

1. 2. 3. 4.

Mean Mean Mean Mean

k SD k SD k SD -CSD

= = = =

CSP

Plasma4

43.6 + 3.6. 11.6 k 7.0. 6.6 k 5.0. 7.3 i 3.6.

GAD enzyme activity was determined by measuring the rate of formation of WO, from ( I-r4C) glutamic acid (200,000 cpm, 10 PM) in the presence of specimen and lo-4M pyridoxal phosphate buffered by potassium phosphate (PH 6.6) as described by Susz et al. (1966). To minimize nonenzymatic decarboxylation, substrategl-1°C) glutamic acid-was purified by high performance liquid chromatography according to Nakamura et al. (1979). In addition, measurements were made under anaerobic conditions to prevent oxidative decarboxylation. GAD enzyme was purified from rat brain. The preparation, which lost its enzyme activity completely by heating at 65°C for 10 minutes in the presence of dithiothreitol (Fig. l), was used as a control sample. Student’s t test was used for statistical analyses.

Fig. 1. Heat inactivation

(5 minutes)

338

Results Plasma GAD activity in controls and psychiatric groups is presented in Fig. 2. Although there are intergroup differences in mean age, no significant relationship between age and GAD level is apparent, so it appeared justified to compare mean GAD levels in each group.

Fig. 2. Plasma GAD activity in various neuropsychiatric .

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diseases

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f . : .

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i

WI.

MO.1

N.S.

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sdn. I’M

sch.

Sch.

_P.

MD,

Il.ur.

MWS

MO5

Ms.

0.g.

Abbreviations: Cont. = controls, Sch.B.M. = schizophrenia before medication, Sch. MW5 = schizophrenia medicated within 5 years, Sch. MO5 = schizophrenia medwzated over 5 years, Dep. = major depression, MDI = bipolar depression, Neur. = neurosis, Org. = organic brain diseases. Mean plasma GAD actiwty

and standard

error are shown

by a large dot and a bar

The mean (k SD) level in controls (12.6 k 3.7 pmole/ml/minute) was not significantly different from that in unmedicated schizophrenic patients (10.3 f 3.4 pmole/ml/minute). However, schizophrenics in the short-term (< 5 years) medicated group showed a mean level of plasma GAD activity (14.6 k 4.3 pmole/ml/minute) that was nonsignificantly higher than that in controls and significantly (p < 0.02) higher than that in unmedicated schizophrenics. The higher level of plasma GAD activity seen in the short-term medicated schizophrenics was not found in the longterm (2 5 years) medicated group. Unmedicated patients with major depression had a significantly (p < 0.01) lower mean level of plasma GAD activity (8.6 IL 2.7 pmole/ml/minute) than controls. The

339 depressed group as a whole, regardless of medication status, also showed a significantly 0, < 0.01) reduced value (9.7 k 3.5 pmole/ml/minute). The reduction in GAD activity level was most marked in bipolar patients (7.0 * 3.3 pmole/ml/minute; p < 0.001). With the exception of one male patient, whose GAD activity level was normal, all bipolar patients were being treated with lithium carbonate. A relatively low mean GAD activity level was also found for neurotic patients @ < 0.05). The influence of psychotropic drugs on plasma GAD levels in schizophrenic and depressed patients is shown in Fig. 3. Dopamine blockers, in combination with anticholinergic agents, were associated with an increase in GAD activity that occurred within 4 months of the initiation of treatment; with the passage of time, however, GAD activity decreased to premeditation levels. There was a significant difference in GAD activity between unmedicated and medicated (both short- and long-term) schizophrenic patients @ < 0.02). Tricyclic antidepressants and benzodiazepines, on the other hand, had no apparent effect on the plasma GAD levels of depressed patients.

Fig. 3. Effects of drug therapy on plasma GAD activity

I .

0

Black dots are plasma GAD concentrations

1

3

2

of schizophrenic

patients

MDNT~ and white dots are those of depressed

340

Patients with Huntington’s chorea (see Table 2) had lower levels of plasma GAD activity (7.3 + 3.6 pmole/ml/minute) than controls @ < 0.01). GAD activity in cerebrospinal fluid (CSF) (6.6 + 5.0 pmole/ml/minute) was slightly lower than that in plasma, but not significantly so. Concentrations of GAD in plasma and CSF were significantly correlated (r = 0.91, p < 0.05). Discussion The validity of a GAD assay based on the production of 14C02 from (i4C-) glutamic acid has been questioned because non-GAD dependent decarboxylation of glutamate cannot be inhibited completely by Triton (McDonnell and Greengard, 1975). In the present study, however, non-GAD dependent decarboxylation was avoided as described in Methods. The use of a heat-inactivated sample as control confirmed the reliability of our GAD assay. Moreover, the significant correlation between levels of GAD activity measured in plasma and in CSF suggests that true GAD activity was being measured. In the present study, low plasma GAD levels found in patients with Huntington’s chorea were highly correlated with CSF levels. In post-mortem studies, brains of patients with Huntington’s chorea have frequently been reported to show decreased GABA-ergic activity (Perry et al., 1973; Bird and Iversen, 1974; Spokes et al., 1980). GABA has been found in kidney, liver, spleen (Drummond and Phillips, 1974), Langerhans’ cells of the pancreas (Okada et al., 1976), and Auerbach’s plexus of the intestine (Taniyama et al., 1981) but the content of systemic GABA seems to be negligible compared to that found in the central nervous system. It therefore appears that plasma GAD levels provide a reasonable index of central GABA-ergic activity. A GABA deficiency in the dopamine-rich mesolimbic area of the schizophrenic brain has been proposed (Roberts, 1972; Stevens et al., 1974; Fuxe et al., 1975; Van Kammen, 1977) in relationship to the dopamine hypothesis of schizophrenia. In a post-mortem study relevant to this question, Perry et al. (1979b) found an approximately 50% reduction of GABA content in the nucleus accumbens and the thalamus of 7 and 13 schizophrenics, respectively. Conversely, Cross et al. (1979) reported no difference in GABA content in the same regions between schizophrenics and controls. According to Spokes et al. (1980), who examined GABA concentration in 10 discrete areas of the brain in 42 psychotic patients, there were significant decreases in the amygdala and the nucleus accumbens. The reduction in the nucleus accumbens was seen only in the early-onset schizophrenics, while that in the amygdala was more generally observed, regardless of diagnostic category. Although Bird et al. (1977) found decreased GAD activity in the basal ganglia of more than 25 schizophrenic brains, the decrease disappeared (Cross and Owen, 1979) when post-mortem changes were taken into account (Perry et al., 1977). Finally, Bennett et al. (1979) found no reduction in GABA binding and GAD activity in the frontal cortex of schizophrenics. The present study revealed no differences in plasma GAD activity of unmedicated schizophrenic patients compared to normal controls. This finding corresponds to previously reported evidence that GABA levels in CSF of schizophrenics are not significantly different from those in controis (Lichtshtein et al., 1978; Gold et al., 1980, Zimmer et al., 1981). Although no significant difference was confirmed by Lichtshtein

341 et al. (1978), six of the seven lowest GABA levels in their study were from schizophrenic patients. It is possible that any abnormality in the GABA system of schizophrenic brain is too discrete to be reflected in measures of CSF or plasma. This problem needs further research. In the present study abnormalities in GAD activity were no more prominent in the affective disorders than in schizophrenia. There is some evidence suggesting that the GABA system is involved in the pathophysiology of affective disorders, Perry et al. (1977) found a significant reduction of GAD activity in the frontal cortex, occipital cortex, caudate nucleus, and substantia nigra of eight autopsied brains from unipolar depressives. These findings did not appear to be attributable to alterations arising from differences in times of death and types of terminal illness. Gold et al. (1980) reported that GABA levels in the lumbar CSF of depressed and schizoaffective patients were significantly lower than in a neurologic comparison group. There were no differences in GABA levels between unipolar and bipolar depressives. Recently, Gerner and Hare (1981) also found lower CSF GABA levels in depressive patients regardless of unipolar-bipolar or agitated-retarded distinctions. Although the present study failed to demonstrate any significant difference in GAD activity between bipolar and unipolar depressives, the reduction in bipolar patients was more marked. Recently, Emrich et al. (1980) examined the antimanic effect of valproate, which raises brain levels of GABA. They observed a noteworthy improvement in four of five manic patients and prophylactic effects over 1I/ to 3 years. This evidence should stimulate further research on the efficacy of manipulating GABA-ergic systems in the therapy of manic illnesses. In addition to the reduced levels of GAD activity found in depressive patients, the present study also showed decreased GAD activity in neurotic patients. Conceivably, the reduced level of plasma GAD activity in affective and neurotic patients might be related to a clinical symptom common to both disorders, such as anxiety. In this context, a recent suggestion that benzodiazepines exert their anxiolytic action through facilitation of GABA-ergic transmission is of interest (Costa et al., 1975). Combined treatment with antidepressants and benzodiazepines was not associated with changes in plasma GAD activity in the present study. However, combined treatment with neuroleptics and antiparkinsonian agents led, within 4 months of the initial dose, to significantly increased levels of plasma GAD, which normalized as treatment continued. To date, there are no experimental data suggesting that GAD activity in the brain can be influenced by neuroleptic drugs (Kim and Hassler, 1975; Perry et al., 1979~). In CSF studies, however, Lichtshtein et al. (1978) found a significant 120/, reduction of mean GABA levels in patients treated for a mean period of 2 months with chlorpromazine, fluphenazine, or perphenazine, in combination with anticholinergic agents. Zimmer et al. (198 I) also observed decreased CSF GABA levels during administration of various neuroleptics in combination with antiparkinsonian agents and minor tranquilizers. However, they demonstrated in a controlled study that sulpiride alone led to a significant increase of CSF GABA after 30 days of treatment. In view of these clinical observations and an experimental finding suggesting that simultaneous blockade of both dopamine and acetylcholine receptor is needed for enhancement of GABA turnover (Mao et al., 1978), antiparkinsonian agents would seem to have important actions on the GABA-ergic metabolism. It should be

342

noted that the change in plasma GAD with combined neuroleptic/anticholinergic treatment noted in the present study is in the opposite direction of the effect on CSF GABA reported in the clinical studies reviewed above. Finally, although the effects of lithium carbonate on plasma GAD levels were not systematically investigated in the present study, lithium-treated bipolar patients showed very low levels of GAD. The effects of lithium on plasma GAD should be examined in a future study.

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American

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