Folia Psychiatrica et Neurologica Japonica, Vol. 31, No. 1, 1977

Monoamine Oxidase Activity in Blood Platelets From Manic and Depressed Patients Saburo Takahashi, M.D. Department o/ Psychiatry and Neurology, K y o t o Prefectural University of Medicine, Kyoto

INTRODUCTION

In the first part of this study, wc have obtaincd data showing that depressed patients had lower blood platelet serotonin levels than those in normal control subjects."' These data were, as a whole, concordant with previous observations,lx and this prompted us to investigate the monoamine oxidase (MAO) activity-a major enzyme in the catabolism of serotonin-in blood platelets from these subjects, as we have recently developed a sensitive and specific assay for measuring M A 0 activity in human blood platelets.'" M A 0 in blood platelets is a mitochondria1 cnzyme with some similarities to M A 0 in the brain."' Recent studies have found markedly reduced activity of platelet M A 0 in bipolar manic-depressive patients and in chronic schizophrenic patients, and their data were interpreted as to have a relation to the etiology of these psychiatric disorders,4 I ' although other investigators did not always agree with their evidences in a similar grouping of patients.? 2" Monozygotic twin studies with platelet M A 0 activity have shown that it is largcly under genetic contr01,l5 z2 while some recent works suggest that M A 0 activity is altered by biological milieu, such as menstrual cycle in women,l and that M A 0 in blood platelets consisted of so-called B form of the enzyme M A 0 - __ ___ Received for publication Aug. 9, 1976.

while M A 0 in the brain of A and B forms, thus, that they are not always identical with each other.lY Therefore, measurement of M A 0 activity in blood platelets may not represent inherent disposition of those subjects that are liable to suffer from these psychiatric disorders. Nevertheless, it is still useful as a strategy for exploration of affective disorders on the basis of serotonin deficiency hypothesis-that is, all the data previously reported employed assay procedures for M A 0 activity using tryptamine4 11 1222 or benzylaminelg as substrate which do not always represent the enzymatic activity for putative neurotransmitter serotonin, because relative activities of the oxidase are different for the substrate.5 l:' In this study, we measured M A 0 activity in blood platelets using serotonin as substrate in the aliquots of the same samples as we used for serotonin assay." Particular attention was paid to (a) whether correlation is noticed between the serotonin levels and M A 0 activity in the blood platelets, and (b) whether bipolar depressive patients, or any other subtypes of affective disorders, exhibit reduced or enhanced M A 0 activity. PATIENTS AND METHOD

Subjects Patients assigned to the study were selected from among the inpatients and outpatients at Kyoto Prefectural University

S. Takahashi

38

Table 1: Demographic Variables in Patients with Mania and Various Clinical Subtypes of Depression

Group Manic Patienlts Depressed Patients Bipolar depression

Previous History Male of vs Depression Female YesvsNo

No. of* Patients

Age in Years

14

38.4k18.3 (18-76)

4 : 10

63.3* 7.2 (5 1-76) 45.7k12.9 (24-67) 60.3k 7.4 (50-72) 34.8k 9.8 (27-47) 3 5 . 8 2 8.3 (2248) 47.7k14.6 (22-76)

5: 3

8: 0

8 : 10

18 : 0

5 : 12

4 : 13

5: 0

1: 4

11: 9

0:20

3 4 : 34

31 : 37

8

Unipolar depression

18

Involutional depression

17

Neurotic and chronic characterological depression First-episde depression

20

Total

68

5

10: 4

Months* * after the Onset of Present Episode 2.9-t 4.1

(0-9) 7.0k11.6 (0-24) 4 . 8 k 6.2 (148) 12.1k12.9 (1-36) 31.6k12.9 (14-48) 10.0213.5 (0-48) 10.5-tl3.1 (0-48)

Figures are shsown by the MeankS.D. wbth variable range in the parentheses.

* Aliquots of the same samples as used for the determination of serot,onin levels except for eight of them were available. the present episode in which the patient was examined for blood platelet MA0 aotivity levels.

* * Months after the onset of

Hospital. There were 80 patients with affective disorders, 38 men and 42 women between the ages of 18 and 76 years. They were all subjects from whom we obtained blood platelet samples for the determination of serotonin in our previous study,21 and aliquots of the same samples except for eight of them were available for measuring M A 0 activity. They consisted of 14 manic patients, four male and ten female patients, and 68 depressed patients of various clinical subtypes, 34 male and 34 female patients, of whom two bipolar patients were studied while they were both in manic and depressive states. Most of manic patients (eleven of 14 patients) were admitted to the general psy-

chiatric ward, but three were seen at the outpatients clinic, whereas 70% of depressive population (48 of 68 patients) consisted of outpatients. Ten of the manic patients were in a manic state of bipolar affective psychosis, and others with recurrent and first-episode mania. None of the manic patients was in a manic state associated with schizophrenic symptoms (schizoaffective). They were generally in a mildly or moderately manic state, although their manic behavior necessitated hospitalization and anti-manic medications. Manic symptoms and criteria for selecting patients were described in our previous studyS2' Th,e depressive symptoms were all characterized, as have been described in Hamil-

Monoamine Oxidase Activity in Blood Platelets ton's Deprcssion Scale," viz, the depressivc subjects selected were generally melancholic, retarded, hypochondriacal. Guilt, suicidal thoughts, sleep disturbances, loss of interests and worthless feelings were typical, and manifested anxiety, somatic complaints as well as diurnal variation of symptom intensity were present. The majority of the depressed patients were outpatients and eventually recovered from their depression with moderate doses of anti-deprcssivc agents, while some hospitalized patients were actually severely depressed patients, scoring more than 40 in Hamilton's Rating Scale. The diagnosis was established on the basis of interviews with the patients and their family members and reports from previous physicians, as well as our own medical records. Clinical diagnostic criteria, which have been widely used in most Japanese schools, were employed to classify these patients into several subcategories of depression.!' There were eight patients with bipolar depression (manic-depressive illness, circular), I8 with unipolar depression (recurrent endogenous depression), 17 with involutional depression, five with neurotic depression (depressive neurosis) and chronic characterological depression (constitutional depressive disposition) and 20 with firstepisode depression. First-episode depressives included all of those who were suffering from their first attack of depression before the age of 50 and diagnosed as endogenous depression, viz, aged patients suffering from their first episode with manifest psychotic depression patterns were classified as those with involutional depression, and young or middle-aged patientswith protracted depressive symptoms which had developed on the basis of their neurotic personality-as neurotic depression. Therefore, in this study, unipolar depression is exclusively defined as suffering from two or more episodes of depression, and all the

39

subjects who had any history of depressive episode before the age of 50 were checked out of the involutional depression group. All patients were free of intervening illnesses during the period of this study. Basic demographic variables for subtypes of depression are listed in Table 1. (The data is shown by the number of patients whose blood platelet samples were available for the determination of M A 0 activity, as a result, data in this table are not identical with those in the first part of the study.)" Out-patients were all unmedicated patients. Blood samples were collected on the days before psychoactive drugs wcre prescribed. In cases of in-patients who had been under pharmacotherapy, all the medication was withdrawn for seven consecutive days prior to admission to the study. Only samples from drug-free patients were included in this study, since psychoactive drugs may induce some effects on M A 0 activity. For example, some recent data have shown that tricyclic antidepressant drugs were found to inhibit human platelet M A 0 activity in vitro," and that blood platelet M A 0 activity increased during the lithium salt treatment period.' Patients were on an ordinary diet during the study. Normal control subjects consisted of 54 men and 67 women. They included staff physicians (N=25), psychologists (N= I), nurses (N= 13), medical students (N=3), nursing students (N= lo), attendants and aids (N=8) and healthy volunteers (N=61) ranging in age from 18 to 66. Control values reported represented the mean of samplings on two different days at 2-3 week interval. All blood samples were taken from outpatients and control subjects between 10 and 11 a.m., and from in-patients in a fasting state at 8 a.m. during the pretreatment period. Eight ml of blood samples were collected by venipuncture into a plastic disposable syringe and immediately transferred

40

S. Takahashi

into a polypropylene centrifuge tube containing 2.7 ml of 0.5% disodium ethylenediaminetetraacetic acid (Na,-EDTA) solution in physiological saline, pH 7.4, and inverted gently about ten times. The collected samples were kept at 4°C in a refrigerator for 1-2 hours.

70 adult subjects.) The incubation medium consisted of a platelet sample in 0.6-ml of physiological saline, 0.2-ml of 0.3 M Na, HP04/KH2P04buffer, pH 7.7, and 0.2-ml of serotonin creatinine sulfate solution, 17 pM/ml, of which pH was adjusted beforehand to 7.7 with NaOH. It was incubated for 30 min at 37°C and the reaction was Experimental terminated by placing in ice and mixing with 2-ml of 0.3 N perchloric acid. It was cenProcedures for collecting blood platelets trifuged at 12000xG at 0°C for 15 min, are described in our previous reports."' then, supernatant was passed through the Briefly, the procedures are as follows: blood column of Sephadex G-10, followed by 27samples were centrifuged twice at 1 3 0 x G ml of 0.02 N acetic acid for washing. After for 15 min and 10 min each to separate draining, each of Sephadex columns was red cells and leukocytes. The supernatant, placed above a column of Amberlite CG-50, platelet rich plasma, was divided into two and, 5-ml of 0.02 N NH40H containing parts, one for the determination of serotonin 0.01% cysteine was passed through the pairs content and the other for M A 0 activity, and of columns, so that absorbed 5-HIAA was centrifuged at 1500xG for 15 min. The eluted from the upper column and through sediment, a platelet pellet was then washed the lower one while excessive serotonin was once with 0.1 % Na,-EDTA in physiological completely trapped on the Amberlite resin. saline, pH 7.4, and collected again by centriThree ml of eluate was then mixed with fugation at 1500xG for five min. The I-ml of conc HCl containing 1% cysteine platelets were stored at -20°C until as- and determined spectrofluorometrically acsayed. M A 0 in the blood platelets was very cording to the method of Bogdanski et al." stable in this condition for as long as six Fluorescence intensity was read at 540-nm, months, and all the determination was perwhile exciting at 295-nm on a Hitachi formed within two months where no reduc- MPF-4 Type Fluorescence Spectrophototion of the enzyme activity was noticed. meter. A correction was made for the Enzyme assay procedures are described column blank by performing the entire proin detail in our previous report.'" A sensi- cedure without addition of incubated assay tive and specific procedure for M A 0 activity mixtures. Protein concentrations were meain human blood platelets has recently de- sured by the method of Lowry et d.l"using veloped in our laboratory, using serotonin as bovine serum albumin as standard. Enzyme substrate and formed 5-hydroxyindoleacetic activity was expressed in nM of substrate acid (5-HIAA) being separated by a double per mg platelet protein per hour at 37°C microcolumn technique on Sephadex G- 10 and a pH value of 7.7. A small reading as much as 0.3 nM/mg and Amberlite CG-50 and measured fluorometrically. The procedures are summarized proteinlhour was obtained by carrying boiled as follows. An aliquot containing 0.5-mg platelet through the whole procedures. Fiftyprotein, equivalent to approximately 4-ml percent inhibition (160) of M A 0 activity by of whole blood, was used for a single assay. addition of various drugs in this assay sys(Protein content in the precipitate with per- tem was in the orders of 10-GMM/l (Nialachloric acid was measured as 0.119+0.026 mide and Iproniazid) to 10-BM/I (Pargymg/ml of whole blood, the mean value for line), and a value of 0.3-0.4 nM/mg pro-

Monoamine Oxidase Activity in Blood Platelets

0

41

.

0 0

0

0

0 0 0

O0

8

0 0

OR -

oo

0

0

0

. 08

0 0

0

o o

80

0

I

I

I

20

30

40

I

I

I

50

60

70 Age in years

Fig. 1: Blood platelet monoamine oxidase activity in normal control subjects. Abscissa represents the age of subjects (years) and ordinate represents blood platelet monoamine oxidase activity (nmol/mg protein/ hour). Solid circles show the values for each male and open circles for each female individual of 121 normal adult subjects between the ages of 18-66. There was no significant correlation between the age and !blood platelet M A 0 activity values for each sex (r=0.057 and r= -0.032 for male and female respectively, Pearson's correlation coefficient). The platelet samples were stored at -20°C and not thawed until assayed. Incubation was carried out for 30 min at 37"C, and 1.0 ml of incubation mixture contained blood platelets of approximately 0.5 mg protein with serotonin creatinine sulfate ( 3 . 4 ~ 1 0 4M as free base) in a final concentration of 0.06 M phosphate buffer, pH 7.7. Product formation was linear with time and enzyme concentration.

tein/hour was obtained when the maximum inhibition of cnzyme activity by these M A 0 inhibiting drugs was measured. Therefore, data obtained may be corrected by subtracting 0.3 for enzyme blank. Reaction was linear with respect to enzyme concentration and time within 60min. Recovery of 5-

H I A A through the double columns of Sephadex (3-10 and Amberlite CG-50 was very satisfactory showing 98*2%. Duplicates of platelet samples agreed a t the level of *9%. (S.D. for 50 determinations) Samples obtained from the same healthy individual at 2-week-interval agreed within a

S. Takahashi

42

range of *20%. We have no data of seasonal or monthly variations in blood platelet M A 0 activity which may exist in healthy subjects in relation with the seasonal and menstrual changcs in their endocrine functions.' RESULTS

M A 0 activity in the blood platelet from normal control subjects varied in a wide range of 2.49-12.05 nM/mg protein/hour. There was no significant correlation between the age and M A 0 activity values, while the difference between male and female was evident (Fig. I , Table 2). Male subjects exhibited about 30% reduction in their M A 0 activity levels. The range for manic and depressed patients was similar to that for the former, 0.65 to 13.40nM/mg protein/ hour. Majority of depressed patients had blood platelet M A 0 activity within a range of normality, while a few patients showed exceptionally low values of enzyme activity

(Fig. 3). As a whole group, both manic and depressed patients showed the mean values that were not significantly different from those for normal controls (NS by Student's t test, Table 2). When the data were analysed for the distinction of each clinical subtype of depression, neither significant enhancement nor reduction was observed in the levels of blood platelet M A 0 activity for any of subcategories. The mean values for male and female bipolar patients had not outstandingly reduced as compared to those for other groups (Table 3). Ten of the 14 manic patients studied had previous episodes of depression, viz, bipolar patients, who also exhibited the M A 0 values of normality with the mean of 6.0122.76 nM/mg protein/ hour. Each of two bipolar patients did not exhibit any large variation of blood platelet M A 0 activity while the patient's mood swung between mania and depression, i.e., a patient, 64 y f, had the values of blood platelet M A 0 as 7.76 nM/mg protein/hour

Table 2: Monoamine Oxidase Activity in Blood Platelets from Manic and Depressed Patients Untreated, and Normal Control Subjects

Group

Manic Patients Men Women Depressed Patients Men Women Normal Controls Men Women '8

+Ii'

No. of Subjects 14 4 10 68 34 34 121 54 67

Age in Years

Blood Platelet Significant MA0 Activity Difference* (nmol/mg Compared with protein/hour) Normal Controls

46.8 k25.0 3 5 . 0 2 15.2

6.43 43.39 6.9343.30

NS NS

45.92 12.8 49.42 16.1

4.12r+1.81** 7.0342.30

NS NS

39.5k10.4 32.7+ 8.7

4.91 4 1.72** 6.88-t 1.99

Figures are shown by the MeantS.D. Difference from the mean value for the same sex of each group, by Student's t test. Si,gnificantly lower than the value for women at the significance level of p