- Email: [email protected]
Glutamate dehydrogenase and its isozyme activity in olivopontocerebellar atrophy
Journal of the Neurological Sciences, 1986, 74:231-236
231
Elsevier JNS 2681
Glutamate Dehydrogenase and its Isozyme Activity in Olivopontocerebellar Atrophy Yoko Konagaya, Masaaki Konagaya and Tetsuya Takayanagi Department of Neurology, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634 (Japan)
(Received 7 October, 1985) (Revised, received 14 February, 1986) (Accepted 14 February, 1986)
SUMMARY Evaluation of glutamate dehydrogenase (GDH) in 12 patients with olivopontocerebellar atrophy showed deficiency of the enzyme in the group as well as in each patient. The activity of total GDH was 77.7~ of that in controls. We also demonstrated two components of this enzyme differentiated by their thermostabilities. The activity of the heat-labile component was remarkably reduced in patients although that of the heat-stable component showed the same magnitude as in controls. These data suggest that GDH deficiency is mainly caused by its heat-labile component deficiency, which might be related to the pathogenesis of this disease.
Key words: Glutamate dehydrogenase - Heat-labile component - l s o z y m e - Olivopontocerebellar atrophy
INTRODUCTION A partial deficiency of the enzyme glutamate dehydrogenase (GDH) in fibroblasts, leukocytes or platelets from some patients with the sporadic type of olivopontocerebellar atrophy (OPCA), has been reported repeatedly (Plaitakis et al. 1980a,b, 1983; Yamaguchi et al. 1982; Duvoisin et al. 1983, 1984). It is still unclear whether the enzyme deficiency relates to the pathogenesis of the disorder. GDH is an enzyme regulating the metabolism of glutamate, which is said to be an excitatory neurotransmitter in the Addressforcorrespondence:YokoKonagaya,DepartmentofNeurology,Nara MedicalUniversity, 840 Shijo-cho,Kashihara,Nara 634, Japan. 0022-510X/86/$03.50 © 1986Elsevier Science Publishers B.V.(BiomedicalDivision)
232 central n e r v o u s system, especially in the cerebellum. O t h e r n e u r o t r a n s m i t t e r s , such as G A B A or aspartate, m a y h a v e a m e t a b o l i c link with glutamate. Therefore, the r e d u c e d activity o f G D H might result in a b n o r m a l m e t a b o l i s m o f n o t o n l y g l u t a m a t e b u t also o f other a m i n o acid n e u r o t r a n s m i t t e r s , leading to f u n c t i o n a l d i s t u r b a n c e s in the central n e r v o u s system. I n this study, we investigated the activity o f total G D H as well as its i s o z y m e divided by t h e r m o s t a b i l i t y in leukocytes from p a t i e n t s with O P C A a n d their family members. SUBJECTS AND METHODS T w e l v e p a t i e n t s with clinically a n d radiologically d i a g n o s e d O P C A were studied. Their ages r a n g e d from 47 to 71 years (average 59.5 years). T h e s e patients p r e s e n t e d a v a r y i n g c o m b i n a t i o n o f extrapyramidal, cerebellar a n d p y r a m i d a l d y s f u n c t i o n a n d s o m e cases s h o w e d a b n o r m a l eye m o v e m e n t s or signs o f peripheral n e u r o p a t h y . T h e severity o f the s y m p t o m s or d u r a t i o n o f illness varied a m o n g the p a t i e n t s ( T a b l e 1). All p a t i e n t s s h o w e d m o r e or less severe a t r o p h y o f cerebellum, p o n s a n d b r a i n s t e m o n brain C T - s c a n . O n l y o n e p a t i e n t (case 11) h a d a n o b v i o u s family history, which i n d i c a t e d a u t o s o m a l recessive inheritance. W e e x a m i n e d 12 family m e m b e r s o f 5 patients i n c l u d i n g a d a u g h t e r o f case 11 ; n o n e o f t h e m were affected. C o n t r o l subjects were 30 subjects with m i n o r neurologic c o m p l a i n t s , b u t w i t h o u t neurological findings (average age 54.8 years). N o subjects t o o k drugs affecting the G D H activity prior to this study. L e u k o c y t e c o u n t s were w i t h i n the n o r m a l r a n g e in each patient. Leukocytepreparations: L e u k o c y t e pellets were isolated from h e p a r i n i z e d v e n o u s b l o o d ( F i c o l l - C o n r a y m e t h o d ) a n d stored at - 20 ° C u p to o n e m o n t h .
TABLE 1 CLINICAL FEATURES OF 12 PATIENTS WITH OPCA Case No.
Age (years)
Sex
1
67
M
2 3 4 5 6 7 8 9 10 11 12
65 58 57 47 71 69 60 60 58 53 49
M M M M F F F F F F F
Duration of illness (years)
Family history
Cerebellar features a
Parkinsonisma
6
-
4 10 5 5 6 3 1 4 5 6 12
+ -
++ ++ +++ + ++ ++ ++ + ++ ++ ++ ++
+++ + +++ + + ++ ++ _+ + +_ +_ +++
a Key: + = slightly, + = mild, + + = moderate, + + + = severe.
233
Enzyme assay: The pellets were disrupted by several times of freeze-thaw cycles and homogenized in 50 mM Tris HCI buffer (pH 7.4) with a motor-driven Teflon homogenizer (300 rpm) for 3 min at 0 °C. Assay of GDH was done by a modification of Plaitakis et al. (1984). The activity of the enzyme was measured fluorometricaUy by determining the rate of NAD production from NADH. The initial reaction mixture contained 1.8 ml of 50 mM triethanolamine buffer (pH 8.0), 0.03 ml of 5 mM NADH, 0.07 ml of 3 M ammonium acetate, 0.02 ml of 0.26 M EDTA and 2.5/~M rotenone, 1 mM ADP and 0.025 ~o Triton X-100. The fourty/~1 of 0.4 M sodium ~-ketoglutarate (pH 6.5) were added to 50/~1 of leukocyte homogenate to initiate GDH activity. Incubation was performed at 28 °C for 10 min. After adding 1 ml of 0.5 N HCI, 100 #1 was transferred to the fluorometric tube, and 0.2 ml of 9 N NaOH was added followed by another incubation at 60 °C for 10 min. The produced NAD was measured by a fluorometer. Estimation ofthermostability of the enzyme: The total GDH activity was measured as described above. Then, aliquots ofhomogenates were incubated at 48 °C for 60 min. After that, 50-#1 portions of the homogenates were removed and assayed again in the same manner. The activity found after heat incubation was considered to represent 'heat-stable' GDH. This was subtracted from the total activity to determine the activity of 'heat-labile' GDH. All assays were done in triplicate, of which the mean value was taken. Enzyme activity was expressed in nanomoles of NADH utilized/mg of protein/h. Protein concentrations were measured by Lowry's method (Lowry et al. 1951). Statistical analysis was done by Student's t-test. RESULTS
The total GDH activity in control subjects was 2054 + 240 nmoles of NADH/mg protein/h (mean + SD). In OPCA patients, the enzyme activity was 1596 + 222 and significantly reduced compared with that of the controls (P < 0.001). We failed to find a difference in activity between controls and family members of OPCA patients (Table 2). The lowest activity in 5 cases of OPCA was less than the mean minus 2 SD of that in controls (Fig. 1). TABLE 2 ACTIVITY OF GLUTAMATE D E H Y D R O G E N A S E IN LEUKOCYTES FROM SUBJECTS The results are expressed as the mean + SD of a triplicate study in each patient.
Subjects
Total G D H a (A)
Heat-stable GDH a
Heat-labile G D H ~ (B)
B/A
Controls OPCA Family
2054 + 240 1596 + 222* 2077 + 255
1459 + 274 1436 + 284 1392 + 285
596 + 200 160 + 149" 658 + 198
29.7 + 11.0 10.5 _+ 10.2" 33.2 _+ 9.2
a Nanomoles of N A D H utilized/mg protein/h. * P < 0.001 compared with the control values.
234
L
g c
7
2.500
I 16 =
2.000
I Z m 0 E c
t500
o I ~
tO00
5 CTR
OPCA
Fomily
Fig. 1. Activity of total GDH in leukocytes from subjects. CTR = control subjects; OPCA = olivopontocerebellar atrophy; Family = family members of OPCA. Bars represent the mean _+2 SD in each group.
Heat-stable G D H activity showed no differences among these three groups, being 1459 + 274, 1436 + 284 and 1392 + 285 in control subjects, OPCA and family members, respectively (Table 2). The activity of heat-labile G D H in O P C A patients was 160 + 149 and was remarkably reduced as compared with that in controls (596 + 200) (P < 0.001). This component activity in family members again failed to show the deficiency (Table 2). Ten out of 12 OPCA patients showed less activity of heat-labile G D H than the mean minus 2 S D of that in controls (Fig. 2). There were no correlations between severity of clinical features or duration of illness and activity of the enzyme. DISCUSSION In this study, the activity of total G D H was shown to be reduced in patients with OPCA as a group. The mean activity was about 77.7Yo of that in controls, which suggests the deficiency to be partial. In most of the previous reports (Plaitakis et al. 1980a,b, 1983; Yamaguchi et al. 1982; Duvoisin et al. 1983, 1984), some patients with OPCA showed partial G D H deficiency and others demonstrated normal activity. In contrast with those, our 12 patients with O P C A showed reduced activity of total G D H as a group and 5 of them showed less than the mean minus 2 SD of activity in controls.
235
"~ 1.200 o
'~ 1.00C
80C
gg
i •
600
... IIII
~
T~
4O0
:.
200
•
c
.6,
0 CTR
4OPCA
Fomily
Fig. 2. Activity of heat-labile G D H in leukocytes from subjects. CTR = control subjects; O P C A = olivopontocerebellar atrophy; Family = family members of OPCA. Bars represent the mean + 2 SD in each group.
In the study of G D H isozyme, Plaltakis and his colleagues divided the enzyme into particulate and soluble components (Plaitakis et al. 1984). The former corresponded with the heat-labile and the latter corresponded with the heat-stable component. They also reported that patients with total G D H deficiency showed remarkably low activity of the heat-labile component, which bound strongly to the mitochondrial membrane. The reduction of heat-labile G D H activity in our OPCA cases was more marked than that of total GDH, however, the activity of heat-stable G D H was of the same magnitude as that in controls. Therefore, the deficiency of total G D H activity is mainly caused by reduction of heat-labile G D H activity. Although family members studied showed no enzyme deficiency, the family study in this series was not of sufficient scope to allow further conclusion. The following data suggest that G D H deficiency is not a secondary phenomenon of OPCA but a primary genetic disorder. (1)There are no correlations between G D H activity and clinical features or duration of illness. (2) Clinical heterogeneity, suggesting a pleomorphic phenotype expression of the defect similar to this enzyme, was described in other well-defined enzymatic deficiencies (Johnson 1981). (3)An intermediate magnitude of enzymatic defect was reported in asymptomatic family members (Plaitakis et al. 1984). A recent histopathological report on OPCA with G D H deficiency revealed marked loss ofcerebellar granules and Purkinje cells (Chokroverty et al. 1984). Another report adduced neurophysiologlc evidence of sensorimotor neuropathy and peripheral auditory nerve dysfunction in GDH-deficient OPCA patients (Chokroverty et al.
236 1985). In addition, Plaitakis et al. (1982) have presented evidence o f a b n o r m a l regulation o f glutamate metabolism in G D H - d e f i c i e n t subjects. These d a t a suggest the enzymatic defect to be a p p a r e n t throughout the body. In the central nervous system, where the concentration o f glutamate is higher than at other sites, G D H deficiency in particular might result in dysfunction. The patients studied here showed r e d u c e d activity o f both total a n d heat-labile G D H , which m a k e s a b n o r m a l glutamate metabolism in their central nervous system very likely. ACKNOWLEDGEMENTS W e t h a n k Miss Y a s u k o M u r a y a m a for technical assistance and Dr. Yukio M a n o a n d Dr. Toshihiko Konishi for g o o d cooperation. REFERENCES Chokroverty, S., R. Khedekar, B. Derby, R. Sachdeo, C. Yook, F. Leopore, W. Nicklas and R.C. Duvoisin (1984) Pathology of olivopontocerebellar atrophy with glutamate dehydrogenase deficiency, Neurology (Cleveland), 34: 1451-1455. Chokroverty, S., R.C. Duvoisin, R. Sachdeo, J. Sage, F. Leopore and W. Nicklas (1985) Neurophysiologic study of olivopontocerebellar atrophy with or without glutamate dehydrogenase deficiency, Neurology, 35: 652-659. Duvoisin, R.C. and S. Chokroverty (1984) Clinical expression of glutamate dehydrogenase deficiency. In: R. C. Duvoisin and A. Plaitakis (Eds.), The Olivopontocerebellar Atrophies, Raven Press, New York, pp. 267-279. Duvoisin, R. C. and S. Chokroverty, F. Leopore and W. Nicklas (1983) Glutamate dehydrogenase deficiency in patients with olivopontocerebellar atrophy, Neurology (Cleveland), 33: 1322-1326. Johnson, W.G. (1981) The clinical spectrum of hexosaminidase deficiency diseases, Neurology (iVY), 31: 1453-1456. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall (1951) Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193: 265-275. Plaitakis, A., W.J. Nicklas and R.J. Desnick (1980a) Glutamate dehydrogenase deficiency in three patients with spinocerebellar syndrome, Ann. Neurol., 7: 297-303. Plaitakis, A., S. Bed, W.J. Nicklas and M.D. Yahr (1980b) Glutamate dehydrogenase deficiency in spinocerebellar degenerations - - Correlation with adult-onset recessive ataxia, Trans. Amer. Neural. Ass., 105: 476-478. Plaitakis, A., S. Berl and M. D. Yahr (1982) Abnormal glutamate metabolism in an adult-onset degenerative neurological disorder, Science, 216: 193-196. Plaitakis, A., W. Nicklas and M.D. Yahr (1983) The treatment of GDH-deficient olivopontocerebellar atrophy with branched chain amino acids, Neurology, 33 (Suppl. 2): 78 (Abstr.) Plaitakis, A., S. Berl and M. D. Yahr (1984) Neurological disorders associated with deficiency of glutamate dehydrogenase, Ann. Neurol., 15: 144-153. Yamaguchi, T., K. Hayashi, H. Murakami, K. Ota and S. Maruyama (1982) Glutamate dehydrogenase deficiency in spinocerebellar degenerations, Neurochem. Res., 7: 627-636.
231
Elsevier JNS 2681
Glutamate Dehydrogenase and its Isozyme Activity in Olivopontocerebellar Atrophy Yoko Konagaya, Masaaki Konagaya and Tetsuya Takayanagi Department of Neurology, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634 (Japan)
(Received 7 October, 1985) (Revised, received 14 February, 1986) (Accepted 14 February, 1986)
SUMMARY Evaluation of glutamate dehydrogenase (GDH) in 12 patients with olivopontocerebellar atrophy showed deficiency of the enzyme in the group as well as in each patient. The activity of total GDH was 77.7~ of that in controls. We also demonstrated two components of this enzyme differentiated by their thermostabilities. The activity of the heat-labile component was remarkably reduced in patients although that of the heat-stable component showed the same magnitude as in controls. These data suggest that GDH deficiency is mainly caused by its heat-labile component deficiency, which might be related to the pathogenesis of this disease.
Key words: Glutamate dehydrogenase - Heat-labile component - l s o z y m e - Olivopontocerebellar atrophy
INTRODUCTION A partial deficiency of the enzyme glutamate dehydrogenase (GDH) in fibroblasts, leukocytes or platelets from some patients with the sporadic type of olivopontocerebellar atrophy (OPCA), has been reported repeatedly (Plaitakis et al. 1980a,b, 1983; Yamaguchi et al. 1982; Duvoisin et al. 1983, 1984). It is still unclear whether the enzyme deficiency relates to the pathogenesis of the disorder. GDH is an enzyme regulating the metabolism of glutamate, which is said to be an excitatory neurotransmitter in the Addressforcorrespondence:YokoKonagaya,DepartmentofNeurology,Nara MedicalUniversity, 840 Shijo-cho,Kashihara,Nara 634, Japan. 0022-510X/86/$03.50 © 1986Elsevier Science Publishers B.V.(BiomedicalDivision)
232 central n e r v o u s system, especially in the cerebellum. O t h e r n e u r o t r a n s m i t t e r s , such as G A B A or aspartate, m a y h a v e a m e t a b o l i c link with glutamate. Therefore, the r e d u c e d activity o f G D H might result in a b n o r m a l m e t a b o l i s m o f n o t o n l y g l u t a m a t e b u t also o f other a m i n o acid n e u r o t r a n s m i t t e r s , leading to f u n c t i o n a l d i s t u r b a n c e s in the central n e r v o u s system. I n this study, we investigated the activity o f total G D H as well as its i s o z y m e divided by t h e r m o s t a b i l i t y in leukocytes from p a t i e n t s with O P C A a n d their family members. SUBJECTS AND METHODS T w e l v e p a t i e n t s with clinically a n d radiologically d i a g n o s e d O P C A were studied. Their ages r a n g e d from 47 to 71 years (average 59.5 years). T h e s e patients p r e s e n t e d a v a r y i n g c o m b i n a t i o n o f extrapyramidal, cerebellar a n d p y r a m i d a l d y s f u n c t i o n a n d s o m e cases s h o w e d a b n o r m a l eye m o v e m e n t s or signs o f peripheral n e u r o p a t h y . T h e severity o f the s y m p t o m s or d u r a t i o n o f illness varied a m o n g the p a t i e n t s ( T a b l e 1). All p a t i e n t s s h o w e d m o r e or less severe a t r o p h y o f cerebellum, p o n s a n d b r a i n s t e m o n brain C T - s c a n . O n l y o n e p a t i e n t (case 11) h a d a n o b v i o u s family history, which i n d i c a t e d a u t o s o m a l recessive inheritance. W e e x a m i n e d 12 family m e m b e r s o f 5 patients i n c l u d i n g a d a u g h t e r o f case 11 ; n o n e o f t h e m were affected. C o n t r o l subjects were 30 subjects with m i n o r neurologic c o m p l a i n t s , b u t w i t h o u t neurological findings (average age 54.8 years). N o subjects t o o k drugs affecting the G D H activity prior to this study. L e u k o c y t e c o u n t s were w i t h i n the n o r m a l r a n g e in each patient. Leukocytepreparations: L e u k o c y t e pellets were isolated from h e p a r i n i z e d v e n o u s b l o o d ( F i c o l l - C o n r a y m e t h o d ) a n d stored at - 20 ° C u p to o n e m o n t h .
TABLE 1 CLINICAL FEATURES OF 12 PATIENTS WITH OPCA Case No.
Age (years)
Sex
1
67
M
2 3 4 5 6 7 8 9 10 11 12
65 58 57 47 71 69 60 60 58 53 49
M M M M F F F F F F F
Duration of illness (years)
Family history
Cerebellar features a
Parkinsonisma
6
-
4 10 5 5 6 3 1 4 5 6 12
+ -
++ ++ +++ + ++ ++ ++ + ++ ++ ++ ++
+++ + +++ + + ++ ++ _+ + +_ +_ +++
a Key: + = slightly, + = mild, + + = moderate, + + + = severe.
233
Enzyme assay: The pellets were disrupted by several times of freeze-thaw cycles and homogenized in 50 mM Tris HCI buffer (pH 7.4) with a motor-driven Teflon homogenizer (300 rpm) for 3 min at 0 °C. Assay of GDH was done by a modification of Plaitakis et al. (1984). The activity of the enzyme was measured fluorometricaUy by determining the rate of NAD production from NADH. The initial reaction mixture contained 1.8 ml of 50 mM triethanolamine buffer (pH 8.0), 0.03 ml of 5 mM NADH, 0.07 ml of 3 M ammonium acetate, 0.02 ml of 0.26 M EDTA and 2.5/~M rotenone, 1 mM ADP and 0.025 ~o Triton X-100. The fourty/~1 of 0.4 M sodium ~-ketoglutarate (pH 6.5) were added to 50/~1 of leukocyte homogenate to initiate GDH activity. Incubation was performed at 28 °C for 10 min. After adding 1 ml of 0.5 N HCI, 100 #1 was transferred to the fluorometric tube, and 0.2 ml of 9 N NaOH was added followed by another incubation at 60 °C for 10 min. The produced NAD was measured by a fluorometer. Estimation ofthermostability of the enzyme: The total GDH activity was measured as described above. Then, aliquots ofhomogenates were incubated at 48 °C for 60 min. After that, 50-#1 portions of the homogenates were removed and assayed again in the same manner. The activity found after heat incubation was considered to represent 'heat-stable' GDH. This was subtracted from the total activity to determine the activity of 'heat-labile' GDH. All assays were done in triplicate, of which the mean value was taken. Enzyme activity was expressed in nanomoles of NADH utilized/mg of protein/h. Protein concentrations were measured by Lowry's method (Lowry et al. 1951). Statistical analysis was done by Student's t-test. RESULTS
The total GDH activity in control subjects was 2054 + 240 nmoles of NADH/mg protein/h (mean + SD). In OPCA patients, the enzyme activity was 1596 + 222 and significantly reduced compared with that of the controls (P < 0.001). We failed to find a difference in activity between controls and family members of OPCA patients (Table 2). The lowest activity in 5 cases of OPCA was less than the mean minus 2 SD of that in controls (Fig. 1). TABLE 2 ACTIVITY OF GLUTAMATE D E H Y D R O G E N A S E IN LEUKOCYTES FROM SUBJECTS The results are expressed as the mean + SD of a triplicate study in each patient.
Subjects
Total G D H a (A)
Heat-stable GDH a
Heat-labile G D H ~ (B)
B/A
Controls OPCA Family
2054 + 240 1596 + 222* 2077 + 255
1459 + 274 1436 + 284 1392 + 285
596 + 200 160 + 149" 658 + 198
29.7 + 11.0 10.5 _+ 10.2" 33.2 _+ 9.2
a Nanomoles of N A D H utilized/mg protein/h. * P < 0.001 compared with the control values.
234
L
g c
7
2.500
I 16 =
2.000
I Z m 0 E c
t500
o I ~
tO00
5 CTR
OPCA
Fomily
Fig. 1. Activity of total GDH in leukocytes from subjects. CTR = control subjects; OPCA = olivopontocerebellar atrophy; Family = family members of OPCA. Bars represent the mean _+2 SD in each group.
Heat-stable G D H activity showed no differences among these three groups, being 1459 + 274, 1436 + 284 and 1392 + 285 in control subjects, OPCA and family members, respectively (Table 2). The activity of heat-labile G D H in O P C A patients was 160 + 149 and was remarkably reduced as compared with that in controls (596 + 200) (P < 0.001). This component activity in family members again failed to show the deficiency (Table 2). Ten out of 12 OPCA patients showed less activity of heat-labile G D H than the mean minus 2 S D of that in controls (Fig. 2). There were no correlations between severity of clinical features or duration of illness and activity of the enzyme. DISCUSSION In this study, the activity of total G D H was shown to be reduced in patients with OPCA as a group. The mean activity was about 77.7Yo of that in controls, which suggests the deficiency to be partial. In most of the previous reports (Plaitakis et al. 1980a,b, 1983; Yamaguchi et al. 1982; Duvoisin et al. 1983, 1984), some patients with OPCA showed partial G D H deficiency and others demonstrated normal activity. In contrast with those, our 12 patients with O P C A showed reduced activity of total G D H as a group and 5 of them showed less than the mean minus 2 SD of activity in controls.
235
"~ 1.200 o
'~ 1.00C
80C
gg
i •
600
... IIII
~
T~
4O0
:.
200
•
c
.6,
0 CTR
4OPCA
Fomily
Fig. 2. Activity of heat-labile G D H in leukocytes from subjects. CTR = control subjects; O P C A = olivopontocerebellar atrophy; Family = family members of OPCA. Bars represent the mean + 2 SD in each group.
In the study of G D H isozyme, Plaltakis and his colleagues divided the enzyme into particulate and soluble components (Plaitakis et al. 1984). The former corresponded with the heat-labile and the latter corresponded with the heat-stable component. They also reported that patients with total G D H deficiency showed remarkably low activity of the heat-labile component, which bound strongly to the mitochondrial membrane. The reduction of heat-labile G D H activity in our OPCA cases was more marked than that of total GDH, however, the activity of heat-stable G D H was of the same magnitude as that in controls. Therefore, the deficiency of total G D H activity is mainly caused by reduction of heat-labile G D H activity. Although family members studied showed no enzyme deficiency, the family study in this series was not of sufficient scope to allow further conclusion. The following data suggest that G D H deficiency is not a secondary phenomenon of OPCA but a primary genetic disorder. (1)There are no correlations between G D H activity and clinical features or duration of illness. (2) Clinical heterogeneity, suggesting a pleomorphic phenotype expression of the defect similar to this enzyme, was described in other well-defined enzymatic deficiencies (Johnson 1981). (3)An intermediate magnitude of enzymatic defect was reported in asymptomatic family members (Plaitakis et al. 1984). A recent histopathological report on OPCA with G D H deficiency revealed marked loss ofcerebellar granules and Purkinje cells (Chokroverty et al. 1984). Another report adduced neurophysiologlc evidence of sensorimotor neuropathy and peripheral auditory nerve dysfunction in GDH-deficient OPCA patients (Chokroverty et al.
236 1985). In addition, Plaitakis et al. (1982) have presented evidence o f a b n o r m a l regulation o f glutamate metabolism in G D H - d e f i c i e n t subjects. These d a t a suggest the enzymatic defect to be a p p a r e n t throughout the body. In the central nervous system, where the concentration o f glutamate is higher than at other sites, G D H deficiency in particular might result in dysfunction. The patients studied here showed r e d u c e d activity o f both total a n d heat-labile G D H , which m a k e s a b n o r m a l glutamate metabolism in their central nervous system very likely. ACKNOWLEDGEMENTS W e t h a n k Miss Y a s u k o M u r a y a m a for technical assistance and Dr. Yukio M a n o a n d Dr. Toshihiko Konishi for g o o d cooperation. REFERENCES Chokroverty, S., R. Khedekar, B. Derby, R. Sachdeo, C. Yook, F. Leopore, W. Nicklas and R.C. Duvoisin (1984) Pathology of olivopontocerebellar atrophy with glutamate dehydrogenase deficiency, Neurology (Cleveland), 34: 1451-1455. Chokroverty, S., R.C. Duvoisin, R. Sachdeo, J. Sage, F. Leopore and W. Nicklas (1985) Neurophysiologic study of olivopontocerebellar atrophy with or without glutamate dehydrogenase deficiency, Neurology, 35: 652-659. Duvoisin, R.C. and S. Chokroverty (1984) Clinical expression of glutamate dehydrogenase deficiency. In: R. C. Duvoisin and A. Plaitakis (Eds.), The Olivopontocerebellar Atrophies, Raven Press, New York, pp. 267-279. Duvoisin, R. C. and S. Chokroverty, F. Leopore and W. Nicklas (1983) Glutamate dehydrogenase deficiency in patients with olivopontocerebellar atrophy, Neurology (Cleveland), 33: 1322-1326. Johnson, W.G. (1981) The clinical spectrum of hexosaminidase deficiency diseases, Neurology (iVY), 31: 1453-1456. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall (1951) Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193: 265-275. Plaitakis, A., W.J. Nicklas and R.J. Desnick (1980a) Glutamate dehydrogenase deficiency in three patients with spinocerebellar syndrome, Ann. Neurol., 7: 297-303. Plaitakis, A., S. Bed, W.J. Nicklas and M.D. Yahr (1980b) Glutamate dehydrogenase deficiency in spinocerebellar degenerations - - Correlation with adult-onset recessive ataxia, Trans. Amer. Neural. Ass., 105: 476-478. Plaitakis, A., S. Berl and M. D. Yahr (1982) Abnormal glutamate metabolism in an adult-onset degenerative neurological disorder, Science, 216: 193-196. Plaitakis, A., W. Nicklas and M.D. Yahr (1983) The treatment of GDH-deficient olivopontocerebellar atrophy with branched chain amino acids, Neurology, 33 (Suppl. 2): 78 (Abstr.) Plaitakis, A., S. Berl and M. D. Yahr (1984) Neurological disorders associated with deficiency of glutamate dehydrogenase, Ann. Neurol., 15: 144-153. Yamaguchi, T., K. Hayashi, H. Murakami, K. Ota and S. Maruyama (1982) Glutamate dehydrogenase deficiency in spinocerebellar degenerations, Neurochem. Res., 7: 627-636.