44 BIOL PSYCHIATR>

1988.23:44-S?

Platelet MAO Deamination of Serotonin in Depressed Patients Changes after Imipramine Treatment and Clinical Correlations

Javier Quintana

Monoamine oxidase (MAO) in blood platelets has been used as a model to study MAO

in the central nervous system, where disorders in serotonergic systems are thought to

occur in depression. Inconsistent changes in platelet MAO of depressed patients have

been reported when several substrates other than serotonin (5-HT) have been used. To

correlate changes in platelet MAO activity with the enzyme activity in central serotonergic

systems, the platelet MAO activity of depressed patients (first unmedicated and then after

3 weeks and 2 months of imipramine treatment) and normal controls was measured using

5-HT as substrute. The results showed that there is a steady, measurable platelet MAO

uctivity with that substrate. This activity was signtficantly higher in unmedicated depressed

patients than in controls, and it decreased progressively with imiprumine treatment,

reaching a normul level when the patients were clinically recoveredfrom depression after

2 months of therapy.

Introduction

Disorders in the metabolism of biogenic amine neurotransmitters in the central nervous

system (CNS) have been implicated in the pathogenesis of affective disorders (Schildkraut 1965; Maas 1975). Monoamine oxidase (MAO) deamination is a major-although en- zymatically not limiting-mechanism for controlling the synaptic levels of those com- pounds. Some experiments in platelets (Paul et al. 198 1) suggest a structural and functional relationship between the uptake site for serotonin (5-HT) and the binding site for some tricyclic antidepressants (TCA), the most widely used therapy for endogenous depression, stressing the role of 5-HT. Platelets are generally considered to be a valid peripheral model of central serotonergic presynaptic terminals (Sneddon 1973; Pletscher 1978). Platelet MAO activity has been considered to be related to central 5-HT metabolism (Fowler et al. 1982). Changes in this activity are correlated with level changes of 5-HT metabolites in the CSF (Oreland et al. 1981), as it occurs with the cerebral MAO activity (Adolfsson et al. 1978). Platelet and neuronal MAO activity show similar K,,, and V,,,

values when the same substrate is used to measure them (Fowler et al. 198 1).

From the Department of Psychiatry and Brain Research Institute, UCLA School of Medicine, Los Angeles, CA.

Address reprint requests to Dr. 1. Qttintana, Department of Psychiatry and Brain Research Institute, UCLA School of Medicine.

University of California, 760 Westwood Plaza, Los Angeles, CA 90024.

Received August 25. 1986; revised March 23, 1987.

Platelet MAO Deamination of 5-HT in Depression BIOLPSYCHIATRY 45 1988:23:4&52

Published studies in which substrates other than 5-HT were used have reported a significant decrease in platelet MAO activity in bipolar, but not in unipolar, depressed patients (Murphy and Weiss 1972) when compared with normal volunteers. Those results were replicated in some subsequent studies (Murphy et al. 1974; Landowski et al. 1975); other groups, however, presented evidence of an increased platelet MAO activity in depressed patients as a whole (Nies et al. 1971), in unipolar depression (Buchsbaum et al. 1973; Nies et al. 1974), in bipolar patients (Belmaker et al. 1976), and in lithium- treated manic-depressive psychoses (Bockar et al. 1974). There is also evidence of a similar platelet MAO activity in depressed patients and normal volunteers (Friedman et al. 1974; Takahashi and Karasawa 1975; Edwards et al. 1978).

In this article, I present the results of studying platelet MAO activity levels, using serotonin as substrate, in a group of unmedicated endogenous depressed patients and a group, similar in age and sex distribution, of normal volunteers. The platelet MAO activity was measured before and after the patients had received imipramine treatment for 3 weeks; it was measured a third time when the treatment had been in continuation for 2 months. The results of the enzyme activity are discussed in relation to the patients’ clinical conditions.

Methods

Subjects

Fifty individuals were initially included in this study. Twenty-five were normal controls (age range 32-64 years, mean 52 + 7.8) and 25 were depressed patients diagnosed as endogenous subtype (age range 32-64 years, mean age 54 + 9.3). The age and sex (60% women) distribution was similar in both the patients and the controls. All the patients were admitted at the Adult Psychiatry Division, Bellvitge Hospital, University of Bar- celona, Spain, where they were found to meet RDC for major depressive disorders, endogenous subtype (Spitzer et al. 1978). After diagnosis and voluntary informed consent was obtained, they were included in the study and were maintained free of medication for 6 days. All of them had been drug-free for an additional week prior to their admission to the hospital, according to a screening that included a family interview. The patients were housed in the same unit and received the same standard hospital diet. Successive Hamilton tests for depression were administered to rank their clinical condition throughout the study. After a first blood sample was drawn, drug therapy was initiated (oral imi- pramine, 125-175 mg/day); a new sample was taken after 3 weeks and again after 2 months of therapy. No other drugs were administered, except for a few patients who received diazepam or barbiturates to help them sleep, and for one patient who was treated with cimetidine because of a gastric ulceration. Seven of the 25 patients were not available at the time of the third platelet MAO measurement. Thus, they were excluded from the study, and the results of their first two samplings are not presented here. At the time of that third measurement, the rest of the patients had been released from the hospital because they had clinically recovered from depression; to draw the third blood sample, they came in for an ambulatory control visit.

Control subjects consisted of healthy volunteers, carefully screened ambulatory pa- tients, and laboratory personnel. None of them suffered from any psychiatric illness, nor had they any previous history of personal or familial major psychiatric disorders. They

were also screened for medication intake, and those who had taken any drug during the 2 weeks prior to the blood extraction were excluded.

To initially characterize the platelet MAO assay used in this study, samples were also collected from more than 30 normal subjects, who were known to be free of medication for at least 1 week prior to the blood sampling.

Blood samples were drawn between 8:00 and 9:00 AM by venipuncture from patients and controls, who had fasted for at least 8 hr. The samples were collected in silicon-covered glass centrifuge tubes (about 5-8 ml for each test) containing 0.5 ml of a I-mM EDTA acid solution in 0.9% NaCl and were placed immediately over ice at 4°C. Platelets were harvested by cent~fugating the samptes at low speed (130 x g, 15 min, 4°C) and sep- arating the resulting platelet-rich plasma (PRP). The centrifugation procedure was repeated (130 X g. 10 min, 4”C), and the PRP separated and centrifugated again (750 x g, 10 min, 4°C). The resuIting pellet was then dissolved in 1 ml of 0.9% NaCl soiution containing 0.1% sodium-EDTA Iwt/vol), and finally centrifugated (750 x g, 5 min. 4°C). An aliquot of the PRP obtained after the second low-speed centrifugation was counted in a Throm~counter C (Coulter Electronics Ltd., Harpenden, Herts) to determine the platelet content of the samples.

The resulting pellet was then dissolved in bidistilled water up to a final concentration of IO’ platelets/ml and was frozen at -60°C. The protein content of the suspension was 0.67 mg/ml, as determined by the method of Lowry (Lowry et al. 1951). Within the next 2 weeks after its collection, the frozen suspension was thawed, then frozen and thawed 2 times more. Of this suspension, 0.2 ml was added to 0.2 ml of 0.2-M phosphate buffer (pH 7.8) containing 2 mM serotonin (creatinin-sulfate; Sigma Chemical Co.. St. Louis, MO), 0.5% (wtlvol) nonionic detergent Triton X-100 (Koch Light Labs.: scintillation grade), and L t..Ki/ymol JH-serotonin (2% ethanol solution, lo-20 Ciimmol; TRK 223, Amersham International Ltd.). The final reaction mixture pH was 7.4, with an 800 pM

serotonin concentration. Such a substrate concentration was chosen after a few preliminary experiments showed an apparent K,,, of the enzymatic activity of 1.6 X IO4 M. Finally. 0.1 ml of bidistilled water was added to the bath, which was then incubated at 37°C for 30 min. A parallel control tube was placed over ice for 30 min. On some occasions, the control tube was placed at 37°C. but the platelet aliquot was not added until the end of the 30-min incubation period. The tubes were then placed over ice, and 0.3 ml of 3 N HCl was added, followed by I .5 ml of a 50% mixture (vol/voi) of ethyl-acetate and Toluene. The tubes were immediately shaken for 10 set three times and centrifugated at 300 x g for 10 min. One milliliter of the upper organic phase was carefully collected. placed with 0.5 ml ethanol and 5 ml of Unisolve I (Koch Light Labs, Ltd.) scintillation liquid, and counted in a Beckman LS-7000 spectrometer. The MAO activity was calculated using the difference of activity between platelet aliquots incubated at 37°C and those of the control tubes, processed as described. No significant differences were seen between the two types of control tubes (i.e., those with platelets incubated at 4°C and those incubated at 37°C but with the platelets added only at the end of the 30 min, right before the organic extra&on). Measures of activity were expressed in nanomoles of substrate metabolized per 30 min per 10’ platelets, as the expression of the activity per milligram of platelet protein results in more variation (Jackman et al. 1979).

Platelet MAO Deamination of 5-HT in Depression BIOL PSYCHIATRY 47 1988;23:4&52

Statistical comparisons between the different groups of depressed patients and control subjects were made using a two-tailed Student’s r-test. Correlation coefficients and best- fit regression line equations were determined by regression analyses according to the method of least squares.

Results

The MAO assay described above rendered a signal-to-noise ratio of 3:l over the blanks (9: 1 after subtracting the “internal noise” common to the samples and the blanks). The coefficient of variation for the assay was less than 5% (n = 30). As illustrated in Figure 1, the platelet MAO activity using serotonin as substrate developed optimally at a final incubation mixture pH of 7.4; therefore, all routine assays were performed at such final reaction mixture pH. The platelet MAO activity depended on the incubation temperature; it increased progressively when the bath temperature was increased between 0°C and 37°C. The platelet MAO activity measured in this report was also dependent on the incubation time; it developed linearly (r = 0.98) throughout the first 40 min of incubation at 37”C, reaching a maximum plateau from that moment to 2 hr-the maximum incubation time at which that activity was measured in the experiments. No significant differences were observed between the average activities at 40, 50, 60, 90, and 120 min (means of 5 experiments). A 30-min incubation, still in the linear portion of the time course curve of product formation, was used in all routine assays. Finally, the MAO activity was linear

Figure 1. Effect of the pH of incubation media on human platelet MAO (‘H-serotonin as substrate) activity. Dots represent the means of five experiments.

BIOL PSYCHIATRY lYXX:23~41- 52

2- Y =2.57X - 0.02

r zO.996

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6

Platelet Protein (mg/ml)

Figure 2. Correlation between human platelet MAO (‘H-serotonin as substrate) and the amount of platelet protein in the sample. Activity is expressed in nanomoles of substrate metabolized per 30 min. Platelet protein is in milligrams per milliliter of suspension (see methods). Line represents regression of correlations, calculated by the least squares method. Spearman’s rank-order correlation coefficient was I.

with the amount of platelet sample, at least over the range of 0.18-1.5 mg platelet protein/ml explored, as demonstrated by the linear correlation (regression line equation, Y = 2.57X - 0.02; r = 0.996; n = 8) seen in Figure 2.

Table 1 shows the results of the platelet MAO activity measurements in normal subjects and in the group of depressed patients prior to and during two different steps of imipramine treatment. Platelet samples from normal volunteers showed an MAO activity of 1.78 & 0.05 nmol 5-HT/30 min/lO’ platelets (n = 25) at 37”C, using serotonin as substrate with the assay described in the methods section. Unmedicated (controlled drug-free for at least 6 days in the hospital) depressed patients showed a significant (p < 0.001) increase in platelet MAO activity using the same assay with respect to controls. Three weeks after the onset of the treatment, including a daily oral dose of imipramine of 125-175 mg, the platelet MAO activity of the same patients had decreased significantly (p < 0.037) with respect to the time of the first sampling (i.e., when they were unmedicated), but it was

still higher than in control subjects (p < 0.006). The continuation of the imipramine therapy reduced the platelet MAO activity even more (p < 0.033), which, after 2 months of treatment, was not significantly different (p < 0.347) from the group of normal sub- jects. The apparent clinical recovery from depression in the same group of patients paralleled the continuous decrease of their platelet MAO activity. After 3 or 4 weeks of therapy, the patients at large had left the hospital because of their recovery, which was

Platelet MAO Deamination of 5-HT in Depression BIOL PSYCHIATRY

1988;23:4452 49

Table 1. In Vivo Effects of Imipramine (125-175 mg/day orally) on Human Platelet Monoamine Oxidase Using Serotouin as Substrate

Monoamine oxidase (MAO) activity (nmoU30 min/109

platelets)

Number of subjects

Controls

1.78 + 0.05

25

Unmedicated depressed patients

5.30 ” 1.16”

18

DeplWCd Depressed

patients patients treated treated

3 weeks 2 months with imipramine with imipramine

(see text) (see text)

2.73 f 0.42”.b 1.70 2 0.226,’

18 18

“Statistically significant differences @ < 0.05) when compared to contmls.

bStatistically significant differences @ < 0.05) when compared to unmedicated patients.

‘Statistically significant differences @ < 0.05) when compared to patients treated 3 weeks with imipramine.

verified both by clinical evaluation and by the scores of the Hamilton test for depression. All patients included in this study whose platelet MAO activity was measured after 2 months of treatment had a complete clinical recovery from depression at that time.

Discussion

The present results show that using serotonin as a substrate, there is a steady, saturable, and temperature-dependent MAO activity in human platelets. The optimum pH for this activity is 7.4. The advantages of using such a substrate to measure platelet MAO in relation to central changes of the enzyme are obvious. For one thing, serotonin is a main substrate of MAO in the CNS. Changes in the central metabolism of serotonin have been repeatedly implicated in the origin of depression (Schildkraut 1965; Maas 1975). More- over, the use of the same substrate implies that the same specific enzymatic locus is used to metabolize the substrate. As many as four different loci of a unique form of the enzyme have been described as the active points of interactions with each of four different substrates (White and Wu 1975). A recent report (Young et al. 1986) indicated a lack of correlation between platelet MAO-B activity and the cerebral type A or B of the enzyme. However, it did not mention that any effort was made to study the correlation between the platelet MAO activity using serotonin as substrate and the cerebral type A enzyme. The assumption that there is no platelet MAO activity using 5-HT as substrate has been repeatedly shown to be erroneous (Boullin and O’Brien 1973; Kamoun et al. 1974; Takahashi and Karasawa 1975; Demish et al. 1976; Bond and Kundall 1977).

High levels of platelet MAO in unmedicated depressed patients have been also found in other studies (Buchsbaum et al. 1973; Bockar et al. 1974; Nies et al. 1974). However, others have found a decreased level of platelet MAO in bipolar patients (Murphy and Weiss 1972; Murphy et al. 1974; Landowski et al. 1975). A third group of studies did not find significant differences in platelet MAO between controls and depressive patients (Friedman et al. 1974; Takahashi and Karasawa 1975; Edwards et al. 1978), nor did they find differences between subtypes of depression (Takahashi and Karasawa 1975; Edwards et al. 1978). Differences in the results of these studies could be attributed to selection of patients, but are probably mainly due to the methodology and particularly to the substrate

so Bn_ PSYCMATR'I 1')88;23:46 52

.I. Quintana

used. The present study, which used serotonin as substrate and a new assay, complete and reaffirm the trend seen in previous studies on platelet MAO using type A substrates

(Takahashi and Karasawa 1975).

Tricyclic antidepressants (TCA) inhibit the platelet MAO activity in vitro (Edwards and Bums 1974; Roth and Gillis 1974; Sullivan et al. 1977) as well as the cerebral MAO (Roth and Gillis 1974). In vivo studies have demonstrated a correlation among the TCA administration, the decrease in platelet MAO activity, and the clinical improvement of anxiety and sleep-related disorders that occur in depression, but not of depression itself (Sullivan et al. 1978; Davidson et al. 1981). The results presented here, however, show evidence of a correlation between imipramine administration and both a decrease in platelet MAO activity to normal levels and clinical recovery from depression. Unfortunately, due mainly to the inability to evaluate the clinical condition in a way suitable for statistical analysis, it cannot be known which one, if any, of the two dependent variables (i.e., clinical recovery and decrease of platelet MAO activity) caused the other to change. However, there is evidence that a big decrease in platelet MAO levels, of an order of 80%, is needed to account for a clinical effect in the case of MAO inhibitor (MAOI) administration (Davidson et al. 1978; Raft et al. 198 l), as the enzyme is not a limiting step in monoamine metabolism, and therefore, it has to be strongly inhibited to produce

some increase in the central levels of neurotransmitters, which is the claimed therapeutic mechanism of antidepressant therapy (Green et al. 1977). Although TCAs inhibit platelet MAO activity, their effect cannot be explained by a decrease of 80% of the platelet enzyme activity. As a matter of fact, in vitro experiments not reported here showed that lo-’ M imipramine decreased the platelet MAO activity by 40% when the enzyme was measured according to the method described in this report. It could be argued that the platelet MAO activity decrease observed in patients treated with imipramine for 3 weeks may be due to the direct action of the drug present in the blood stream. A daily dose of 150-200 mg has been reported to produce plasma levels of 10m6 M imipramine (Modai et al. 1979). However, although this level is obtained after a few days of treatment, the patients in the present study, after 3 weeks of a similar daily imipramine dose, showed

a platelet MAO activity level still significantly higher than the controls, although it was significantly lower than the levels these patients showed before initiating imipramine therapy. The continuation of the treatment reduced the platelet MAO activity even more, whereas the drug’s plasma levels remained similar. Therefore, it is more reasonable to think that the platelet MAO activity decrease seen throughout the therapy would not be a primary cause for clinical recovery; rather, this decrease would be a TCA indirect effect

that parallels or follows the clinical improvement.

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