Platelet Monoamine Oxidase in Affective Disorders David J. Edwards, PhD; Duane G.
Spiker, MD;
David J.
Kupfer, MD;
(MAO) activity was determined tryptamine as substrate for 61 drug-free patients who had a primary major depressive disorder and for 32 normal controls. Although there were no significant differences between the mean platelet MAO activity of 19 bipolar patients (4.94 nmoles/hr/mg of protein), 42 unipolar patients (4.97 nmoles), and the controls (4.78 nmoles), an analysis of variance indicated that the variance of the bipolar group was significantly greater than that of the other groups. This suggests that there may be subgroups of bipolar patients that differ in their platelet MAO activity but that \s=b\ Platelet monoamine oxidase
with
appear to be distinct from the bipolar I vs bipolar II classification. (Arch Gen Psychiatry 35:1443-1446, 1978)
oxidase (MAO) has a key role in the degra¬ dation of a wide variety of biogenic amines. Since inhibitors of this enzyme are well known to possess antide¬ pressant properties, it was hypothesized that alterations in activity of MAO may occur in certain psychiatric disorders, and thereby exert a control on the metabolism of some crucial amine(s). The discovery that blood platelets contain this enzyme1 led to the possibility that the measurement of platelet MAO activity in humans might serve as an index of the activity of the enzyme in the CNS.2 Interest in this line of investigation was greatly spurred by the reported findings of reduced platelet MAO activity in schizophrenia ' and bipolar depression.1 Many laboratories have sought to replicate these findings, but most of these studies have dealt with schizophrenics. Results of those studies have been mixed,'11 but the most consistent finding has been a reduction of platelet MAO activity in chronic schizophre¬ nics but not in acute schizophrenics.
Monoamine
Accepted
for publication June 18, 1978. From the Department of Psychiatry, University of Pittsburgh School of Medicine, Western Psychiatric Institute and Clinic. Reprint requests to Department of Psychiatry, University of Pittsburgh School of Medicine, Western Psychiatric Institute and Clinic, 3811 O'Hara St, Pittsburgh, PA 15261 (Dr Edwards).
Downloaded from jamanetwork.com by Tulane University user on 02/03/2019
F. Gordon
Foster, MD; John F. Neil, MD; Leatrice Abrams
There are fewer reports and less agreement as to alterations of platelet MAO activity in affective disorders. Murphy and Weiss4 originally reported low platelet MAO activities in bipolar but not unipolar patients. However, Nies et al1" found the platelet MAO activity to actually be slightly, but significantly, higher in the platelets of depressed patients than of normal controls. The latter study did not divide the patients into unipolar and bipolar subtypes. Wyatt and Murphy11 have replicated their origi¬ nal study, using benzylamine as substrate. In their study, 14 of 49 bipolar I depressed patients but only three of 39 unipolar and 72 of 680 normal subjects had MAO activities below arbitrary cutoff levels. Leckman et al12 also found a significantly reduced (by approximately 25%) platelet MAO activity in bipolar I but not bipolar II patients as compared with controls. Two other studies1'·14 also found statistically lower platelet MAO activities in bipolar than in unipolar patients, although in those studies bipolar I and bipolar II patients were not distinguished. A study in Israel, however, found that the platelet MAO activity in bipolar patients was higher than in controls, whether benzylamine or tryptamine was used, although the differ¬ ences were significant only for the substrate benzylamine." These investigators suggested that this lack of reduced platelet MAO activity in bipolar patients may be due to cultural differences in patients in this study and the National Institute of Mental Health NIMH studies. In spite of these inconsistencies, the possible importance of platelet MAO activity as a biochemical correlate of subtypes of depressive disorder led us to reexamine this question in diagnostically well-defined and drug-free groups of patients who have a primary major depressive disorder. In the present study, we report on data obtained for 61 depressed patients diagnosed according to the Research Diagnostic Criteria (RDC) described by Spitzer et al." Only drug-free patients were included, since tricy¬ clic antidepressants inhibit platelet MAO activity and may therefore affect the results.1" Platelet MAO activity was measured with tryptamine as substrate.
specific activity
of 1.62 µ /µ ß. This stock substrate solution stored in frozen aliquote, which were thawed just prior to use. Each incubation mixture contained a final volume of 2 ml, including 1.6 ml of 0.2N phosphate buffer, with a pH of 7.5 (prepared from KH2P04 and Na,HP04), 0.2 ml of the stock substrate solution, and 0.2 ml of platelet sonicate. Blanks were obtained by adding buffer instead of the enzyme. All reactions were initiated by the addition of enzyme and were carried out for one hour at 37 °C. The reactions were stopped by the addition of 0.2 ml of 2N hydrochloric acid and the reaction products (primarily the neutral metabolite of tryptamine, indoleacetaldehyde2") were extracted into 3 ml of toluene by vortex mixing for 30 seconds. After the tubes were centrifuged for five minutes at 2,500 rpm, 2 ml of the toluene phase was removed and added to a glass scintillation vial containing 5 ml of cocktail (0.4% 2,5 diphenyloxazole and 0.005% l,4-Bis-2-(5-phenyloxazolyl) benzene in toluene). The radioactivity contained by the products was then determined by liquid scintillation, counting with a liquid scintillation spec¬ trometer. Protein concentrations of the platelet sonicates were determined by the Lowry procedure,21 which has been automated in our laboratory. Bovine serum albumin was used for the stan¬ dards. In the 61 patients included in this study, the average deviation in platelet MAO activity between replicate assays of the same platelet sonicate was 2.3%. This is similar to the 2% deviation observed by Murphy and his co-workers.19 In a separate, unpub¬ lished study, we determined the platelet MAO activity in ten patients, using 5 to 14 blood samples from each patient drawn twice per week. The average deviation of platelet MAO activity within the same patient was 13%, even though many of those patients were treated with medications. Similarly, Murphy et al19 reported an average deviation in platelet MAO activity of 17% in blood samples drawn one week apart from the same individual. The statistical significance of differences between the mean platelet MAO activities of different patient and control groups were assessed by use of a two-tailed Student's t test. Groups were also compared by analysis of variance using an F test.
SUBJECTS AND METHODS
was
All the patients studied were inpatients on the Clinical Research Unit at Western Psychiatric Institute and Clinic, Pittsburgh. They were interviewed by a staff psychiatrist, using a traditional format, and had a physical examination. They were then observed drug free for 14 days, during which they had routine laboratory tests, including an electroencephalogram and thyroid function tests, and any other tests indicated on the basis of their history and physical examination. Any patient who had an organic illness affecting the CNS was dropped from the study and is not included in this report. At the end of the two-week drug-free period, they were reinterviewed with the Schedule for Affective Disorder and Schizophrenia." This systematic research interview collects the data necessary to make diagnoses using the RDC.11 To be included in this study, the patient had to have a primary major depressive disorder as defined by the RDC. The sample excluded patients with schizo-affective disorder and included both bipolar and unipolar depression. Perris18 has shown that some patients who initially have a depressive episode will subsequently have a manic episode. However, if they have two or three separate depressive episodes, then the chances of ever becoming bipolar are very low. Consequently, to obtain a more homogeneous group of unipolar patients, our unipolar patients were further categorized as either nonrecurrent or recurrent. The latter category includes all of those patients who met the RDC criteria of "never having met the criteria for manic disorder, hypomanic disorder or schizo-affective disorder, manic type but who have had two or more episodes that met the criteria for probable or definite major depressive disorder each separated by at least two months of return to more or less usual functioning."15 All patients gave informed consent to this
project.
The control subjects in this study were employees, relatives, friends of employees, or students at Western Psychiatric Institute and Clinic. They all stated that they were alcohol and drug free, including aspirin, hormones, birth control pills, and marijuana, for at least two weeks prior to the time they had their blood drawn, and had never been psychiatrically ill. These subjects also gave informed consent and were paid a nominal fee for donating blood for the study. Blood was obtained by venipuncture between 7 and 8 AM from fasting patients, using three specimen tubes containing 1 ml of acid citrate, sodium citrate, and dextrose solution each as an anticoagulant. Platelets were obtained as described by Murphy et al." Monoamine oxidase activity was always assayed in triplicate, using unfrozen platelet sonicates within one to two hours after they were prepared. The stock substrate solution was prepared from tryptamine bisuccinate (side chain-2-14C), 53 µ /µ , and tryptamine hydrochloride to give a concentration of 617 µ and a
Platelet MAO
RESULTS The mean platelet MAO activities did not show any significant differences between the various patient groups and the normal controls (Table). Although the mean plate¬ let MAO activities in our unipolar patients (4.97 ± 1.83 nmoles/hr/mg of protein, 42) and normal controls (4.78 ± 1.76 nmoles, 32) were somewhat lower than those reported by Murphy and Weiss4 (6.92 and 6.21 nmoles/hr/mg of protein, respectively), the mean platelet =
=
Activity
in Patients and Controls
Platelet MAO
Platelet MAO
Platelet MAO
Activity, nmoles
Total
Women
Men
Age,
yr
Activity, nmoles
Age,
yr
Activity, nmoles
Age,
yr
Patients
Bipolar I Bipolar
4.20
±
4.20
±
All bipolar Recurrent
4.20 ± 5.47 ±1.43(10)t 3.71 ± 2.35(5) 4.88 ± 1.90(15) 3.56 ± 1.11(10)tî
unipolar Nonrecurrent unipolar All unipolar
Controls
*AII values represent the
tP
Spiker, MD;
David J.
Kupfer, MD;
(MAO) activity was determined tryptamine as substrate for 61 drug-free patients who had a primary major depressive disorder and for 32 normal controls. Although there were no significant differences between the mean platelet MAO activity of 19 bipolar patients (4.94 nmoles/hr/mg of protein), 42 unipolar patients (4.97 nmoles), and the controls (4.78 nmoles), an analysis of variance indicated that the variance of the bipolar group was significantly greater than that of the other groups. This suggests that there may be subgroups of bipolar patients that differ in their platelet MAO activity but that \s=b\ Platelet monoamine oxidase
with
appear to be distinct from the bipolar I vs bipolar II classification. (Arch Gen Psychiatry 35:1443-1446, 1978)
oxidase (MAO) has a key role in the degra¬ dation of a wide variety of biogenic amines. Since inhibitors of this enzyme are well known to possess antide¬ pressant properties, it was hypothesized that alterations in activity of MAO may occur in certain psychiatric disorders, and thereby exert a control on the metabolism of some crucial amine(s). The discovery that blood platelets contain this enzyme1 led to the possibility that the measurement of platelet MAO activity in humans might serve as an index of the activity of the enzyme in the CNS.2 Interest in this line of investigation was greatly spurred by the reported findings of reduced platelet MAO activity in schizophrenia ' and bipolar depression.1 Many laboratories have sought to replicate these findings, but most of these studies have dealt with schizophrenics. Results of those studies have been mixed,'11 but the most consistent finding has been a reduction of platelet MAO activity in chronic schizophre¬ nics but not in acute schizophrenics.
Monoamine
Accepted
for publication June 18, 1978. From the Department of Psychiatry, University of Pittsburgh School of Medicine, Western Psychiatric Institute and Clinic. Reprint requests to Department of Psychiatry, University of Pittsburgh School of Medicine, Western Psychiatric Institute and Clinic, 3811 O'Hara St, Pittsburgh, PA 15261 (Dr Edwards).
Downloaded from jamanetwork.com by Tulane University user on 02/03/2019
F. Gordon
Foster, MD; John F. Neil, MD; Leatrice Abrams
There are fewer reports and less agreement as to alterations of platelet MAO activity in affective disorders. Murphy and Weiss4 originally reported low platelet MAO activities in bipolar but not unipolar patients. However, Nies et al1" found the platelet MAO activity to actually be slightly, but significantly, higher in the platelets of depressed patients than of normal controls. The latter study did not divide the patients into unipolar and bipolar subtypes. Wyatt and Murphy11 have replicated their origi¬ nal study, using benzylamine as substrate. In their study, 14 of 49 bipolar I depressed patients but only three of 39 unipolar and 72 of 680 normal subjects had MAO activities below arbitrary cutoff levels. Leckman et al12 also found a significantly reduced (by approximately 25%) platelet MAO activity in bipolar I but not bipolar II patients as compared with controls. Two other studies1'·14 also found statistically lower platelet MAO activities in bipolar than in unipolar patients, although in those studies bipolar I and bipolar II patients were not distinguished. A study in Israel, however, found that the platelet MAO activity in bipolar patients was higher than in controls, whether benzylamine or tryptamine was used, although the differ¬ ences were significant only for the substrate benzylamine." These investigators suggested that this lack of reduced platelet MAO activity in bipolar patients may be due to cultural differences in patients in this study and the National Institute of Mental Health NIMH studies. In spite of these inconsistencies, the possible importance of platelet MAO activity as a biochemical correlate of subtypes of depressive disorder led us to reexamine this question in diagnostically well-defined and drug-free groups of patients who have a primary major depressive disorder. In the present study, we report on data obtained for 61 depressed patients diagnosed according to the Research Diagnostic Criteria (RDC) described by Spitzer et al." Only drug-free patients were included, since tricy¬ clic antidepressants inhibit platelet MAO activity and may therefore affect the results.1" Platelet MAO activity was measured with tryptamine as substrate.
specific activity
of 1.62 µ /µ ß. This stock substrate solution stored in frozen aliquote, which were thawed just prior to use. Each incubation mixture contained a final volume of 2 ml, including 1.6 ml of 0.2N phosphate buffer, with a pH of 7.5 (prepared from KH2P04 and Na,HP04), 0.2 ml of the stock substrate solution, and 0.2 ml of platelet sonicate. Blanks were obtained by adding buffer instead of the enzyme. All reactions were initiated by the addition of enzyme and were carried out for one hour at 37 °C. The reactions were stopped by the addition of 0.2 ml of 2N hydrochloric acid and the reaction products (primarily the neutral metabolite of tryptamine, indoleacetaldehyde2") were extracted into 3 ml of toluene by vortex mixing for 30 seconds. After the tubes were centrifuged for five minutes at 2,500 rpm, 2 ml of the toluene phase was removed and added to a glass scintillation vial containing 5 ml of cocktail (0.4% 2,5 diphenyloxazole and 0.005% l,4-Bis-2-(5-phenyloxazolyl) benzene in toluene). The radioactivity contained by the products was then determined by liquid scintillation, counting with a liquid scintillation spec¬ trometer. Protein concentrations of the platelet sonicates were determined by the Lowry procedure,21 which has been automated in our laboratory. Bovine serum albumin was used for the stan¬ dards. In the 61 patients included in this study, the average deviation in platelet MAO activity between replicate assays of the same platelet sonicate was 2.3%. This is similar to the 2% deviation observed by Murphy and his co-workers.19 In a separate, unpub¬ lished study, we determined the platelet MAO activity in ten patients, using 5 to 14 blood samples from each patient drawn twice per week. The average deviation of platelet MAO activity within the same patient was 13%, even though many of those patients were treated with medications. Similarly, Murphy et al19 reported an average deviation in platelet MAO activity of 17% in blood samples drawn one week apart from the same individual. The statistical significance of differences between the mean platelet MAO activities of different patient and control groups were assessed by use of a two-tailed Student's t test. Groups were also compared by analysis of variance using an F test.
SUBJECTS AND METHODS
was
All the patients studied were inpatients on the Clinical Research Unit at Western Psychiatric Institute and Clinic, Pittsburgh. They were interviewed by a staff psychiatrist, using a traditional format, and had a physical examination. They were then observed drug free for 14 days, during which they had routine laboratory tests, including an electroencephalogram and thyroid function tests, and any other tests indicated on the basis of their history and physical examination. Any patient who had an organic illness affecting the CNS was dropped from the study and is not included in this report. At the end of the two-week drug-free period, they were reinterviewed with the Schedule for Affective Disorder and Schizophrenia." This systematic research interview collects the data necessary to make diagnoses using the RDC.11 To be included in this study, the patient had to have a primary major depressive disorder as defined by the RDC. The sample excluded patients with schizo-affective disorder and included both bipolar and unipolar depression. Perris18 has shown that some patients who initially have a depressive episode will subsequently have a manic episode. However, if they have two or three separate depressive episodes, then the chances of ever becoming bipolar are very low. Consequently, to obtain a more homogeneous group of unipolar patients, our unipolar patients were further categorized as either nonrecurrent or recurrent. The latter category includes all of those patients who met the RDC criteria of "never having met the criteria for manic disorder, hypomanic disorder or schizo-affective disorder, manic type but who have had two or more episodes that met the criteria for probable or definite major depressive disorder each separated by at least two months of return to more or less usual functioning."15 All patients gave informed consent to this
project.
The control subjects in this study were employees, relatives, friends of employees, or students at Western Psychiatric Institute and Clinic. They all stated that they were alcohol and drug free, including aspirin, hormones, birth control pills, and marijuana, for at least two weeks prior to the time they had their blood drawn, and had never been psychiatrically ill. These subjects also gave informed consent and were paid a nominal fee for donating blood for the study. Blood was obtained by venipuncture between 7 and 8 AM from fasting patients, using three specimen tubes containing 1 ml of acid citrate, sodium citrate, and dextrose solution each as an anticoagulant. Platelets were obtained as described by Murphy et al." Monoamine oxidase activity was always assayed in triplicate, using unfrozen platelet sonicates within one to two hours after they were prepared. The stock substrate solution was prepared from tryptamine bisuccinate (side chain-2-14C), 53 µ /µ , and tryptamine hydrochloride to give a concentration of 617 µ and a
Platelet MAO
RESULTS The mean platelet MAO activities did not show any significant differences between the various patient groups and the normal controls (Table). Although the mean plate¬ let MAO activities in our unipolar patients (4.97 ± 1.83 nmoles/hr/mg of protein, 42) and normal controls (4.78 ± 1.76 nmoles, 32) were somewhat lower than those reported by Murphy and Weiss4 (6.92 and 6.21 nmoles/hr/mg of protein, respectively), the mean platelet =
=
Activity
in Patients and Controls
Platelet MAO
Platelet MAO
Platelet MAO
Activity, nmoles
Total
Women
Men
Age,
yr
Activity, nmoles
Age,
yr
Activity, nmoles
Age,
yr
Patients
Bipolar I Bipolar
4.20
±
4.20
±
All bipolar Recurrent
4.20 ± 5.47 ±1.43(10)t 3.71 ± 2.35(5) 4.88 ± 1.90(15) 3.56 ± 1.11(10)tî
unipolar Nonrecurrent unipolar All unipolar
Controls
*AII values represent the
tP
