Folia Psychiatrica et Neurologica Japonica, Vol. 31, No. 4, 1977
Monoamine Oxidase Activity in Blood-Platelets From Autistic Children Saburo Takahashi, M.D., Hideko Kanai, M.D. and Yoshihiro Miyamoto, M.D. Department of Psychiatry and Neurology, Kyoto Prefectural University of Medicine, Kawaramachi and Hirokoji, Kyoto
INTRODUCTION In the first part of this studyz0 we have obtained the data generally concordant with the previous observations that autistic children have higher blood serotonin levels than normals.:’ lfi?* Elevated serotonin concentrations were also seen in some nonautistic individuals who were disturbed and hyperactive. We have, thus, summarized that elevated blood serotonin concentrations are not necessarily a specific biochemical finding for autistic children. Rather, they seem to be due to the children’s behavioral distinction. This prompted us to investigate the monoamine oxidase (MAO) activity, a major enzyme in the catabolism of serotonin, in the aliquots of blood platelets obtained from these subjects. Although M A 0 in blood platelets is a mitochodrial enzyme with some similarities to MA0 in brain,]? measurement of its activity still remains a somewhat controversial area in psychiatric researches. Some studies have found markedly reduced levels of blood platelet MA0 activity in bipolar primary affective disorders and in chronic schizophrenia, and the data obtained were interpreted as to have a relation to the etiology of these psychiatric disorders,1° 11 23 while other investigators did not always agree with their evidences in a similar group-
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Received for publication March 11, 1977.
ing of patients.6 l9 We have recently developed an assay for measuring M A 0 activity in human blood platelets in our laboratory,18and reported the data of MA0 activity levels in various psychiatric diso r d e r ~21. ~ It ~ is intriguing to see if there is a significant increase or decrease in M A 0 activity that correlates with their elevated serotonin levels and their behavioral distinction in autistic children. PATIENTS AND METHODS Patients for this study were 20 children with early infantile autism, 19 boys and a girl; and 39 nonautistic children with various psychiatric and neurological disorders, 22 boys and 17 girls, between the ages of one and 12 years, who were seen at the Child Psychiatry Clinic at the Kyoto Prefectural University Hospital. They were all outpatients, who were referred to us because of their mental disorders via the Kyoto Prefectural Child Guidance Center and the Department of Pediatrics, Kyoto Prefectural University Hospital. Their chronological ages and the outstanding features in their behavior and language are summarized in Table 1. The diagnoses were assigned with extreme caution. We understand that Kanner’s criteria for early infantile autism’ have been well accepted in Japan, while we recognize that the nomenclature of early infantile autism has become increasingly popular as a
598
S. Takahashi et al. Table I :
Name
Junji Ryosuke Kaoru Toshiyuki Yuji Hitoshi Hirokazu Essai Masaya Atsuro Kunitsugu Motoki Mieko Genki Tetsujin Shinichi Michitsugu Ryuji Takashi Shuji
* **
Summary of Clinical Data and Platelet M A 0 Activity Levels for 20 Children With a Diagnosis of Early Infantile Autism
Age Sex 2 m 2 r n 3 m 3 r n 3 m
3
m
3 m 3 r n 3 m 4 m 4 m 4 m 4 f 4 m 4 m 4 m 5 m 10 m 11
m
12
m
Hyperac.tive* Language** Blood Platelet MA0 Activity Behaviors Disturbances (nmol/mg proteinlhour) -2 -3 -3 -I -3 -3 -3 -1 -3 -2
+2 +2 +2 +2 $2 +2 +2 +I +2 0 +2 +2 -1 +2 +2 +2 +2 +2 +I +2
-1 -2 -2 -3 -3 -3 -2 -2 -2 -3
Activity in behavior was evaluated by 2, slightly hyperactive: hyperactive: hypoactive: 2. Predominant language disturbances were paired: - 1, moderately impaired: - 2,
+ -
3.89 4.65 4.67 7.33 3.96 4.26 4.55 8.57 4.43 5.84 3.87 5.65 7.94 5.60 6.54 7.30 3.73 4.68 1.96 5.50
Serotonin Level
(ng/mg protein)
960
-
693 1030 I360 1817 900 I188 641 957 1410 633 302 1568 670 548 I420 I100 765 580
multiple observations and scored for five grades. murkedly normal level: 0, slightly hypoactive: - 1, and markedly
+ 1,
ranked for four grades, normally developed: 0. slightly imand markedly impaired: - 3.
diagnosis with the concomitant dilution of its specificity. For this reason, Kanner’s original criteria for early infantile autism were carefully adhered to in terms of not only the outstanding features of autistic symptoms but also of early onset of the illness, i.e., we checked out all the patients if they had later onset and more varied symptoms. All the children selected for this study had such predominant symptoms as language disturbances, peculiar perceptual inconstancy, bizarre stereotyped motility and lack of ordinary relatedness to other people. They were seen by two staff psychiatrists of the Child Psychiatry Clinic (H.K. and Y.M.), who agreed unequivocally on the diagnosis of early infantile autism.*O Another group of patients consisted of 39 nonautistic children, including 10 with epilepsy, eight with mental retardation, four
with childhood schizophrenia suspected, and other patients with various disorders, such as school phobia, neurosis, facial tic, enuresis, neurotic polyuria, night terrors, anorexia nervosa, diabetes mellitus, brain injury, congenital glaucoma, dwarfism and cerebral palsy. Mentally retarded children with Down’s syndrome and those due to hypothyroidism were excluded. All subjects were free of intervening illnesses during the period of this study. They did not receive psychoactive drugs or any other drugs which might interfere with monoamine metabolism for a minimum of four weeks prior to the measurement of M A 0 activity. Subjects were instructed to be on an ordinary diet, but not specially on a diet low in tryptophan and serotonin. The group of normal controls consisted of 30 children, 23 boys and seven girls, obtained
Monoamine Oxidase Activity Table 2:
599
Monoamine Oxidase Activity in Blood Platelets From Children With Early Infantile Autism, Those With Various Psychiatric and Neurological Disorders and Normal Control Subjects ~~
Group
Autistic Children Boys Girls
Non-autistic Children Boys Girls
Normal Controls Boys Girls
No.
Age (years)
Platelet M A 0 Activity (nmol/mg protein/hour)
20 19 1 39 22 17 30. 23 7
4.5 jz 2.9 4.6 & 2.9 4, 7.5 f 3.4 7.5 f 3.4 7.5 f 3.4 5.4 f 3.2 5.6 f 3.5 4.91f12.0
5.24 f 1.67 5.10 f 1.63 7.94 6.35 f 2.42 5.66 f 2.03 7.18;f,2.72 5.10 f 2.09 4.85 fil.80 5.91 f 2.83
~
p*
n.s. n.s.
-
Comparison to the values for normal control suhjeds. Figures BIB shown hy Mean k S.D.
from the families of staff physicians and the siblings of patients. Eight ml of blood samples were collected between 10 a.m. and 1 1 a.m. by venipuncture into a plastic syringe and immediately transferred into a polypropylene centrifuge tube containing 1/3 of the blood volume of 0.5 % EDTA-Na, in physiological saline pH 7.4. Collected blood samples were kept at 4O C for 1-2 hours, then centrifuged at 130 G for 15 min. The supernatant was pipetted and recentrifuged at 130 G for 10 min to obtain platelet rich plasma, which was centrifuged at 1500 G for 15 min to obtain a platelet pellet. It was washed with 0.1 % EDTA-Na, in physiological saline, pH 7.4, centrifuged at 1500 G for 5 min, and stored at -2OO C until assayed. All operations were carried out at 4O C. The procedures for collecting blood platelet samples are described in detail in our previous communication.2o For the determination of MA0 activity, an aliquot of each platelet sample containing approx. 0.5 mg protein was used. The incubation media consisted of the platelets suspended homogenously in 0.6 ml of saline, 0.2 mi of 0.3 M phosphate buffer, pH 7.7, and 0.2 ml of 17 mM serotonin creatinine sulfate solution as substrate. They were incubated for 30 min at 37O C, and the reac-
tion was terminated by the addition of 2 ml of 0.3 M perchloric acid. 5-hydroxy-indoleacetic acid (5-HIAA) formed was separated by passage through the columns of Sephadex (3-10 and Amberlite CG-50 and finally measured spxtrofluorometrically in a medium of conc HCI. The fluorescence was read at 540 nm after activation at 295 nm on a Hitachi Fluorescence Spectrophotometer, Model MPF-4. Protein concentrations were measured according to the method of Lowry et d u Reaction was linear with respect to enzyme concentrations and time within 60 mIn. Measurement variation was f 9 "/o for duplicate samples. Details of the method for the blood platelet MA0 activity employed in this study are described elsewhere. RESULTS The mean value of M A 0 activity in blood platelets from autistic children was not significantly different from that in normal controls (Table 2). A wide range of M A 0 activity was recognized both in groups of autistic children (range: 1.96-8.57 nM/mg protein/hour) and of normal children (range: 2.10-12.10 nM/mg protein/hour). No evidence was obtained of markedly reduced MA0 activity in the subjects who suffered from autistic illnesses, while many of them
S. Takahashi et al.
600
I
0
X 0
0
0
O
.
0
.
0
m
em 0 0
0
0
0
O
00
X
0
0
X
X 0
0 0
0
I 2 4
6
8
10 12 AGE I N YEARS
Fig. 1 : Monoamine oxidase activity in blood platelets from children with a diagnosis of early infantile autism, those with childhood schizophrenia and normal control subjects. Solid circles and open circles represent the values of each individual of autistic and normal children respectively. Significant correlation between the age and platelet M A 0 activity was not found both in the autistic and the normal control groups (r- -0.299 and -0.149 respectively). Values for four individuals with a diagnosis of childhood schizophrenia were shown by a cross. The platelet samples were stored at -20°C and not thawed until assayed, Incubation was carried out for 30 minutes at 37”C, and 1.0ml of incubation mixture contained blood platelets of approximately 0.5 mg protein with serotonin creatinine sulfate (3.4X 104’M as free base) in a final concentration of 0.06 M phosphate buffer, pH7.7. Product formation was linear within time and enzyme concentration. exhibited elevated serotonin levels in the blood platelets (Table 1 ). Within the group of normal controls, boys exhibited slightly lower values of M A 0 activity than girls, although the difference was not significant and the sex difference of M A 0 activity in the child population was found to be even smaller than that seen in adults (Table 2). Age did not appear to account for the varia-
tion of M A 0 activity both in the autistic and the normal subjects who were under the age of preadolescence (r = - 0.299 and - 0.146 respectively) (Fig. 1 ) . Blood platelet M A 0 activity in the 39 nonautistic disturbed children also had a wide deviation of individual values (range: 3.09-12.83 nM/mg protein/hour). Mean value of this group was not significantly
Monoamine Oxidase Activity different from those in two other groups (Table 2). DISCUSSION
Evidences accumulated so far and claiming that autistic children have higher blood serotonin concentrations are still insufficient.'" 24 Our recent data on blood platelet serotonin concentrations in autistic children were partly concordant with this view.2o It is, however, still necessary to obtain large bodies of new data from a large population of subjects to draw a firm conclusion on this issue. As M A 0 is a major enzyme in the catabolism of serotonin, reduced M A 0 activity might cause the reduction of serotonin levels that was observed in our previous study. Findings obtained in this study, however, demonstrated that no reduction was recognized in M A 0 activity in these autistic subjects who exhibited an elevation of blood serotonin levels (Table 1). There was only one of 20 autistic children who showed markedly reduced level of blood platelet M A 0 activity, 1.96 nM/mg protein/hour (Table l ) , as low as the value of subjects who were on M A 0 inhibitors.ln Blood platelet M A 0 activity measured by our method did not appear to serve as a clue for the elevated serotonin levels in autistic children. Unfortunately, we are not aware of any data that have ever been reported on blood platelet M A 0 activity in these disorders, which might be useful for comparing with our findings. Individuals given a diagnosis of childhood schizophrenia ( N = 4) and neurotic disorders in childhood (N = 10) among 39 nonautistic ill patients also had M A 0 activity values similar to those in normal subjects (mean: 5.90 & 3.17 nM/mg protein/ hour, and 5.92 1.89 nM/mg protein/hour respectively). In case of Down's syndrome, blood platelet M A 0 activity levels not differing from those in control subjects were reported, even though endogenous serotonin content as well as the uptake of radiolabeled
*
601
serotonin in the blood platelets were markedly reduced.8 Therefore, there seems to be no possibility to give a specific meaning to blood platelet M A 0 activity in these psychiatric disorders in childhood. Several authors have shown some findings on the uptake, efflux of serotonin using blood platelets from autistic children, which are indicative of abnormal serotonin metabolism in this disorder and might have some relation to M A 0 activity levels.2 Factors which may affect the platelet M A 0 activity, such as medication, sex and age were carefully controlled in this study. Some previous data in adults indicated that sex difference as well as effects of sex steroids on the blood platelet M A 0 activity were evident.' Dominance of boys in the autistic group studied here can be ruled out when the data were compared with those from boys in other populations (Table 2). In case of affective disorders, we have previously reported that the blood platelet serotonin levels were significantly reduced in the primary unipolar depression. These individuals, however,. had no distinctive elevation of M A 0 activity.21 We have now a pessimistic perspective for this kind of biochemical study in various psychiatric disorders. M A 0 activity in blood platelets has been studied in schizophrenics by some investigators, who emphasized the significance of biochemical findings in the etiology of this illness showing markedly reduced levels of platelet enzyme MA0.loz3 Of course, such studies as showing decreased M A 0 activity against one substrate (tryptamine as substrate) in a small number of patients must be reexamined considering several @-I known biochemical factors whether this decrease is inherently due to a decrease in M A 0 itself, alterations in the matrix in which M A 0 is bound, changes in the number of mitochondria, or the presence of inhibitors of M A 0 enzyme, etc. This enzyme has also enough peculiar properties so that a difference using one substrate under one set of conditions does not necessarily mean
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that the same results will occur using a different set of conditions. Some recent works suggest that so-called A and B forms of the enzyme are interconvertible and dependent on the lipid matrix. Human platelet M A 0 is type B enzyme and not always the same as brain MAO, which comprises four isoenzymes of types A and B which differ from each other in their substrate specificities and sensitivities to M A 0 inhibitor^:^ By a multi-institutional study, different laboratories where the identical procedures employed gave different values of M A 0 activity using aliquots of the same blood platelet samples, and sufficient oxygen supply during incubation time gave enhanced M A 0 activity levels.22 Whatever data were obtained, it is immature to draw a conclusion that blood platelet M A 0 activity levels have no positive relation to possible underlying biochemical defect in early infantile autism. Data obtained in this study are only an evidence that M A 0 activity, under peculiar conditions and using serotonin as substrate, does not differentiate psychiatric populations. SUMMARY In order to evaluate the possible abnormality in monoamine oxidase (MAO) activity in early infantile autism, blood platelet samples were obtained from 20 autistic children, aged 2-12 years. M A 0 activity, measured fluorometrically using serotonin as substrate, was 5.24 k 1.65 (Mean k Standard Deviation) nM/mg protein/hour in these autistic children. This value was not significantly different from either that in 30 age-matched normal children or that in 39 nonautistic children with various psychiatric and neurological disorders, although autistic children had higher platelet serotonin concentrations than these nonautistic individuals. REFERENCES
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