Decreased Platelet Monoamine Oxidase Activity in Chronic Schizophrenia, Shown With Novel Substrates Wade H. Berrettini, MD; Walter Prozialeck, PhD;
Wolfgang H. Vogel,
\s=b\ Platelet monoamine oxidase (MAO) was kinetically evaluated in chronic schizophrenics and matched controls, using substrates of major physiologic importance and substrates of particular interest in the study of schizophrenia, such as serotonin (5-HT),N,N-dimethyltryptamine(DMT), 5-methoxytryptamine (5-MT), and dopamine (DA). Substrates were measured at six concentrations; values for maximal velocity (Vmax) and Michaelis constant (Km) were obtained by using Lineweaver-
Burk plots. TheVmax was decreased for all substrates in chronic schizophrenia and the Km was decreased for DA, 5-HT, and DMT, but ue remained unchanged for 5-MT.ThevalofKm/Vmax was similar for schizophrenics and normal persons when DA, 5-HT, and DMT were used as substrates, which may indicate that "uncompetitive" inhibition is responsible for the observed decrease in activity among chronic schizophrenics. The finding of a decreased Vmax but unchanged Km with 5-MT would be consistent with noncompetitive inhibition.
(Arch Gen Psychiatry 35:600-605, 1978) 1972 numerous investigators have described decreased platelet monoamine oxidase (MAO) activi¬ ty in chronic schizophrenia.19 Several investigators have not been able to duplicate these findings.1014 These discrepan¬ cies could be due to a variety of reasons. First, the existing studies have reported results obtained with different assays, subject groups, enzyme preparations, and sub¬ strates, making reliable comparisons difficult. Second, none of this work has been done with substrates of major physio¬ logic importance or with substrates of particular interest as regards schizophrenia. Third, the existing studies (with the exception of our own1) have relied on a determination of enzyme activity at a single substrate concentration. A large excess of substrate in such assays may lead to substrate inhibition; a substrate concentration less than twice the Michaelis constant (Km) may lead to falsely low values, obscuring significant differences. It is preferable to study the enzyme at several different substrate concentrations, in order to obtain Km and maximal velocity values (Vmax) that would permit a more reliable comparison. Only a convention of investigators in this field will solve the first difficulty. In an attempt to deal with the last two
Since
Accepted for publication Feb 22, 1978. From the Departments of Psychiatry (Dr Berrettini) and Pharmacology (Drs Prozialeck and Vogel), and the Schizophrenia Research and Treatment Center (Drs Berrettini and Vogel), Jefferson Medical College, Philadelphia. Reprint requests to Department of Pharmacology, Jefferson Medical College, 1020 Locust St, Philadelphia, PA 19107 (Dr Berrettini).
PhD
problems, we have studied platelet MAO kinetically in chronic schizophrenics and matched controls, using substrates of major physiologic importance (such as sero¬ tonin [5-HT]) and substrates of particular interest as regards schizophrenia, such as dopamine (DA) (the DA hypothesis15), N, iV-dimethyltryptamine (DMT), and 5methoxytryptamine (5-MT) (the transmethylation hypoth¬ esis16). The DA hypothesis of schizophrenia asserts that there exists a dopaminergic hyperactivity at certain critical sites in the brain. Data indicating that metabolism of this neurotransmitter, by platelet MAO from schizophrenics, is blocked would have bearing on this theory. The possibility that methylated indoleamines play a role in the pathogenesis of schizophrenia has been strengthened by the demon¬ stration that the necessary biosynthetic pathways for DMT exist in human brain.17 This finding is interesting in the light of the work of Narasimhachari et al, which
showed that very small amounts of this potent hallucino¬ genic molecule could be found in the blood and /or urine of some schizophrenics and controls.1819 Thus, there is evidence to suggest that minute amounts of DMT may be produced in the human brain. An inability to metabolize this compound would be an important finding in schizo¬ phrenia, with implications for the transmethylation hy¬ pothesis. Another methylated indoleamine that occurs naturally in the human brain,20 5-MT, has been shown to be behaviorally active and to be equipotent to LSD in disturbing the conditioned avoidance response in rats after intraventricular or intraperitoneal injection.21 The CSF levels of 5-MT were found to be elevated in schizophrenic patients, compared to controls.22 Thus, there is evidence to suggest that 5-MT may be involved in psychotic behavior. Again, a demonstration that metabolism of this compound by platelet MAO is decreased in schizophrenia would be
interesting.
METHODS Patient Selection The diagnosis of schizophrenia was made by applying diagnostic criteria selected from the World Health Organization's Report of the International Pilot Study of Schizophrenia." The criteria used were as follows: 1. Auditory hallucinations (defined as derogatory, threatening, or
command voices). 2. Delusions of thought alienation. 3. Delusions of thought broadcasting. 4. Delusions of thought control. 5. Delusions of persecution.
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6. Neologisms. 7. Looseness of associations. 8. Flat or blunted affect. All patients in this study gave
Enzyme Assay a
history
of
auditory
hallucina¬
tions and delusions of the type described above; all were consid¬ ered by two independent psychiatrists to have flat or blunted affect and looseness of associations. Seven patients were noted to use neologisms. Four patients had a family history of schizophre¬ nia. The patient was termed "chronic" if these symptoms and signs were present continuously, without remission, for a minimum period of two years prior to the study. A minimum of two previous hospitalizations within the last two years (with the diagnosis of
schizophrenia) was required. All patients were living in the community between hospitalizations, which were no longer than four months in duration. They were observed between hospitaliza¬ tions in community mental health centers. The diagnosis of chronicity was established by patient interview and by examina¬ tion of previous records. A patient was excluded from the study if the employment history indicated that any position was held for than one month within the last three years. time were these patients diagnosed as having affective disorder. All patients were free of concurrent disease. It was not considered necessary to limit the study to drug-free patients, since the major tranquilizers (such as the phenothiazines and butyrophenones) do not affect MAO activity.8·12 All patients had recent complete blood counts (CBCs) that showed no evidence of irondeficiency anemia,24 and that showed normal platelet count. Informed consent was obtained. Patients were selected from four different institutions in order to minimize the possible effects of environment. Two of these were acute-care inpatient units and two were day hospital programs. more
At
done with fresh platelet preparations. Blood or plasma was kept cool (4°C) and assays were done within four hours after venipuncture in all cases. For all substrates, the enzyme AU assays
no
Control Selection The control population was selected from healthy staff and laboratory personnel (not taking any medication for one month prior to the study) who had no personal or family history of psychiatric illness.
Enzyme Preparation venous blood, for each substrate, was Approximately collected in evacuated glass tubes, pretreated with 0.3 ml of 5% a, ethyldiaminetetra acetic acid (EDTA). The blood was then centri¬ fugea at 350 g for 30 minutes at 5°C. The platelet-rich plasma was withdrawn and centrifuged in a glass tube for 20 minutes at 1,100 g and at 5°C. The resultant platelet-poor plasma was discarded and the platelet plug washed twice with 2.0-ml volumes of Sorensen's phosphate buffer (0.05M; pH, 7.4). The plug was then suspended in a small volume of the buffer and homogenized in a Teflon and glass homogenizer using 50 strokes in one minute at 5°C. The protein content of each platelet homogenate was esti¬ mated using the method of Lowry et al.25 A control sample was assayed with each schizophrenic sample, as has been suggested by White et al.14
15 ml of
were
assay was a modification of the method of Wurtman and Axelrod.2" This method relies on the ability of an organic solvent to extract, reproducibly, a fraction of the metabolites of MAO oxidation from a highly acidic, aqueous phase. Table 1 lists the substrate, the metabolites, extraction solvent, and percentage of the metabolite extracted by a single aliquot (6 ml) of the solvent. This percentage was determined by multiple extractions of the same aqueous phase. All assays were shown to be linear for at least one hour. Blanks were prepared by pretreatment of the enzyme aliquot with 1 ml of 2N HC1. In all experiments, the organic phase was separated from the aqueous phase, after extraction, by freezing at —80°C for ten minutes. The organic phase was poured off into 10 ml of a standard fluor, and activity was measured in a standard scintillation counter. All substrates were assayed at six concentrations to provide data for Lineweaver-Burk plots. All incubations were carried out at 37°C in a Dubnoff metabolic shaker. All counts were corrected for the metabolites left in the aqueous phase by the single extraction. Assays were linear with protein (0.41 ± 0.24 mg mean ± SD added to each tube). Serotonin. Serotonin 2-'*C, as the oxalate salt (specific activity .050 mCi/30 mg) was diluted with 5-HT, as the oxalate salt, to a specific activity of .050 mCi/0.9 mg. Then 0.070 ml of platelet homogenate was added to test tubes containing various concentra¬ tions of 5-HT, ranging from 0.5 10'M to 3 10"3M in the phosphate buffer. The final volume of the assay was 0.1 ml. The reaction was stopped after 45 minutes by the addition of 1 ml of 2N HC1. These metabolites were extracted into 6 ml of toluene: ethyl acetate (1:1, v/v), as described previously.2' Dimethyltryptamine.—Dimethyltryptamine 2-1,C as the oxalate salt had a specific activity of 2.34 mCi/mmole). First 0.050 ml of platelet homogenate was added to test tubes containing final substrate concentrations, ranging from 2 10~4M to 6 x 10~4M in the phosphate buffer. The final volume was 0.1 ml. The reaction was stopped at one hour by the addition of 1 ml of 2N HC1. Metabolites were extracted into 6 ml of toluene. As this was a novel assay, substrate and metabolites were identified by radiochromatography, using silica gel with fluorescent indicator and methanolacetic acid (10:15) as solvent system. The substrate ratio of fronts (Rf) was identical to that of authentic DMT; indoleacetic acid and indoleacetaldehyde were identified as products. 5-Methyoxytryptamine.-As the radioactive-labeled amine was not available, the side-chain of 5-MT was labeled with 2-,4C and then synthesized according to the method of Kveder and McIsaac,28 using 14C (specific activity 60 mCi/mmole). The synthesized compound was characterized by its infrared spectrum, melting point (121°C to 122°C), by formation of a highly fluores¬ cent derivative with orthophthalaldehyde,2" by thin-layer radiochromatography using three different solvent systems (A, silica gel with CHCl.,:MeOH:lN NH, OH [12:7:1], B, silica gel with Nbutanohacetic acid:H20 [12:3:5], and C, cellulose with iV-butanohacetic acid:H,0 [12:3:5]) and by thin-layer radiochromatog=
=
Table 1 .—Substrates, Metabolites, and Extraction Solvents Used in MAO
Substrate DMT 5-MT 5-HT DA
"Large
amounts of the
Metabolite" Indolacetlc acid
Extraction Solvent Toluene Toluene Toluene: ethyl acetate 1:1 Toluene:THF 2:1
5-methoxyindolacetic acid 5-hydroxylndolacetlc acid 3,4-dihydroxyphenyl acetic acid
corresponding aldehyde
were
identified in each
Assays % of Products Recovered With 6 ml of Solvent in a Single Extraction 82 72 88 80
case.
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Table
2.-Vmax and Km Values for Platelet MAO Obtained With Dopamine as Substrate From Chronic Schizophrenics Patient/
Duration of
Illness,
Age,
Treatment*
Program
Sex 1/28/M 2/57/F 3/29/M 4/24/M
Eo
5/28/M 6/28/F 7/24/M 8/28/M 9/34/M 10/29/M 11/29/M 12/54/F 13/33/M 14/28/M 15/26/F 16/23/M
Eo
Jo Jo
y
nmole/Vm, mg/hr
K,„
5.9 7.4
29
3.0 6.2
5.9 3.7
3.8 1.5
4.7 4.2
4.3 5.9 3.3 5.0 2.8 4.0 3.7
6.8 6.9 5.8 4.6 6.0 4.8
14
27
10 «M 3.6
6.2 3.8 7.2 4.9
2.7 6.0 3.6 2.5
±
SD
31.6
10
5.6
±
1.2
3.9
±
3.-Vma„ and Km Values for Platelet MAO Obtained With Dopamine as Substrate From Controls
Control'/Age, yr/Sex_V^,_K„ _1/24/F_8J_7.1 _2/26/M_9J_5.9 _3/21/M_13_4.8 _4/40/M_12_7.4 _5/47/M_9J)_7.1 _6/39/F_6 _4.8 _7/20/M_9J5_6.6 _8/40/F_8_9_5.4 _9/29/M_8J_8.5 _10/47/M_9_3_7.7 11/29/M_7_9_5.0 _12/33/M_9J3_6.2 _13/47/M_8_8_5.3 _14/25/M_11_7.5 _15/27/F_11_7.5 16/25/M_8^7_3.4 _17/21/F_6_7_5.6 18/23/M_9J_3.7 19/23/M_8_9_5.8 Average
Aver¬
age
Table
1.3
*J, Thomas Jefferson University Hospital inpatient; Jo, Thomas Jeffer¬ University Day Hospital outpatient; E, Albert Einstein (Daroff Division) Hospital inpatient; Eo, Albert Einstein (Daroff Division) Day Hospital
±
SD 30.8
9.3
±
1.6
6.1
±
1.4
_P < .001_P < .001 •See footnote to Table 2.
son
outpatient.
Table
4—Comparison of Vm„
and
Km Values for Platelet MAO* Obtained With Serotonin
Duration of
Programf
Patient/Age, yr/Sex
Illness, yr
nmole/mg/hr
2/57/F
29
2.1 1.8
Jo
3/29/M 7/24/M 8/28/M 9/34/M 12/54/F
Treatment
Jo
Jo Jo
Average
±
SD
13/33/M 17/34/F 18/24/M 20/24/M 23/24/M 24/40/M 25/33/F 26/56/M 35
Km
2.1 2.2 14
27 11
1.6 2.2 2.2
4.8
0.3 0.9 2.0 1.0 1.0 1.1 1.4
0.6 1.3 1.3 23 30 14
2.3 1.8 2.2 1.8 ± .5
10 · 0.6 1.1 1.2 1.4
X
0.8 1.3
±
1.1
as
Substrate
Control/Age, yr/Sex 2/26/M 5/47/M 8/40/F 10/47/M 13/44/M 20/23/M 24/26/F 25/29/F 28/39/M 29/29/M 30/29/M 31/45/F 32/34/M 33/24/F Average ± SD 34.4
3.8 2.7 4.0 3.9
1.3 2.8 4.1 2.8
3.0 1.9 3.4 3.2 4.1
3.6 2.2
4.5 3.4 3.9 2.2 4.5 3.5
± 0.8 < .001
1.6 4.6 1.3 4.0
3.8 0.6 3.3 2.7 ± .13 < .005
"Obtained from chronic schizophrenics and controls. to Table 2.
fSee footnote
raphy
of its dansyl chloride derivative.30 These procedures indi¬ cated that the compound was greater than 95% radiochemically pure.
As the free base, 5-MT 2-14C, with specific activity 1,500 counts per minute nmole, was obtained as described above. Then 0.05 ml of platelet homogenate was added to test tubes containing final substrate concentrations, ranging from 0.4 10loM to 1.6 x 10 4M in the phosphate buffer. The final volume of the assay was 0.25 ml. The reaction was halted after 60 minutes by the addition of 1 ml of 2N HC1. These metabolites were extracted into 6 ml of toluene. As this was a novel assay, substrate and metabolites were identified by radiochromatography of the reac¬ tion mixture, using silica gel with fluorescent indicator and benzene:acetic acid (9:1). The substrate Rf was identical to that of
authentic 5-MT; the metabolites were identified as 5-methoxyindolacetic acid and the corresponding aldehyde. Dopamine.—Dopamine labeled with tritium (specific activity 11.363 Ci/mmole), as the free base, was diluted to a specific activity of 11.0 mCi/mmole, using dopamine HC1. Then 0.100 ml of the platelet homogenate was added to test tubes containing substrate concentrations ranging grom 3.0 x 10 4M to 10 x 10 4M in the phosphate buffer. The final volume of the assay was 0.250 ml. The reaction was halted after 30 minutes by the addition of 1 ml of 2N HC1. The metabolites were extracted into 6 ml of toluene:tetrahydrofuran (THF), 2:1, v/v. As this was a novel assay, substrate and metabolites were identified by radiochromatography of the reaction mixture, using cellulose with fluorescent indicator and A?-butanol:acetic acid:water (25:4:10). The substrate
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Rf
identical to that of authentic dopamine; the metabolites identified as 3,4-dihydroxyphenylacetic acid and the corre¬
was
were
sponding aldehyde. The statistical significance
of data was determined using the Student's t test. To determine whether an individual's Vmax, as measured with one substrate, correlated with Vmax, as measured with other substrates, a multiple rank correlation, using all individuals studied with 5-MT, 5-HT, and DMT, was performed. Because only two patients were studied with all four substrates, limiting the correlation to these three substrates allowed five patients (patients 3, 13,17, 18, and 20) and six controls (controls 2, 8, 10, 13, 20, and 25) to be included.
RESULTS
The results
are
shown in Tables 2 through 8. MAO
Table
Patient/Age,
Treatment
yr/Sex
Programf
Average
±
5—Comparison of Vm„ and Km
2/57/F 3/29/M 13/33/M 15/26/F 17/34/F 18/24/M 19/37/M 20/24/M 21/22/F 22/20/M 27/18/M 28/26/M 29/30/F 30/24/M 31/24/F 32/23/F 28.1
SD
Duration of Illness, yr 29
Values for Platelet MAO* Obtained With DMT
nmole/mg/r|r 1.3 0.91 1.7 0.91 0.50 0.48 0.67 0.83 1.5 1.1 0.57 0.71 1.2 1.7 0.93 0.83 1.0
"These values were obtained from chronic footnote to Table 2.
showed a significantly decreased Vmax and Km for all substrates in chronic schizophrenics, with the exception of the Km for 5-MT. The Vmax factor seemed to separate schizophrenics from controls somewhat better than did the Km index. Among the four substrates, DA seemed to separate the two groups best, followed by DMT, 5-HT, and 5-MT. The Km/Vmax value, which is the slope of the line in a Lineweaver-Burk plot, is similar for both groups in all instances, except for the 5-MT (see Table 6). Multiple rank correlations for the five schizophrenics indicated a high degree of correlation between the Vmax for the three substrates included in this study (P < .001). Similar results were obtained in rank correlations for the six controls (P < .04).
Kra X10
as
Substrate
Control/Age,
yr/Sex
'M
2.2 2.2 2.3
2/26/M 3/19/M
1.7 1.8
7/21/M
1.7 1.9
1.4 1.6 1.5
6/39/F 8/40/F 10/47/M 13/47/M 20/23/M 21/25/M 22/22/M 23/23/F 24/26/F 25/29/F 26/25/F 27/27/F
1.4
1.4 1.7 1.6 0.83 0.83 1.1 1.2 1.8 1.8
34/26/M ± SD 29.3
Average
4.6 2.5 1.3 1.7 1.9
1.3 1.3 1.2 1.5 1.1 1.7 2.4 1.3 1.4 1.5
4.5 1.6
1.9 1.5 ± .4 < .001
2.0 2.4 ± 1.3 < .0025
2.0
3.6 3.3 2.8 2.4 2.9 0.7 1.4
schizophrenics and controls.
fSee
Table Table 6.-Vmax and K,„ Values for Platelet MAO* Obtained With 5-MT as Substrate From Chronic Schizophrenics Duration of Treatment
Program*
Patient/
Age,
Illness,
V„,„
nmole/
yr/Sex_yr_mg/hr
K„,
X 10
'M
E_3/29/M_9_025_0.45 J_13/33/M_11_0_2J_0.24 J_15/26/F_5_O20_0.42 J_16/23/M_2_015_0.33 J_17/34/F_9_016_0.29 E_18/24/M_5_ _0.33 J_19/37/M_4_022_0.14 E_20/24/M_3_0_18_0.25 J_21/22/F_4_027_0.28 J_22/20/M_6_0_24_0.29 4_0_14_0.43 J_31/24/F J_32/23/F_4_0_T7_0.38
Aver-
age
±
SD_26J5_0.19
"See footnote to Table 2.
±
0.05 0.32
±
0.09
and Km Values for Platelet MAO Obtained With 5-MT as Substrate From Controls
7.-Vm„
Control'/Age, yr/Sex_V^_K„, _2/26/M_0_33_0.24 _3/20/M_037_0.36 _6/39/F_ 31_0.24 _7/21/M_033_0.20 _8/40/F_038_0.56
_10/47/M_017_0.37 _13/47/M_034_0.57 18/23/M_022_0.12
19/24/M_022_0.28 _20/23/M_0_19_0.23 _21/25/M_027_0.54 _22/21/M_022_0.22 _23/23/F_0_38_0.14 _24/26/F_0_34_0.22 _25/29/F_023_0.40 _26/24/F_026_0.25 _27/27/F_036_0.34 Average
±
SD 28.5
0.29
±
0.06
0.31
±
0.13
_P .001_NS
Wolfgang H. Vogel,
\s=b\ Platelet monoamine oxidase (MAO) was kinetically evaluated in chronic schizophrenics and matched controls, using substrates of major physiologic importance and substrates of particular interest in the study of schizophrenia, such as serotonin (5-HT),N,N-dimethyltryptamine(DMT), 5-methoxytryptamine (5-MT), and dopamine (DA). Substrates were measured at six concentrations; values for maximal velocity (Vmax) and Michaelis constant (Km) were obtained by using Lineweaver-
Burk plots. TheVmax was decreased for all substrates in chronic schizophrenia and the Km was decreased for DA, 5-HT, and DMT, but ue remained unchanged for 5-MT.ThevalofKm/Vmax was similar for schizophrenics and normal persons when DA, 5-HT, and DMT were used as substrates, which may indicate that "uncompetitive" inhibition is responsible for the observed decrease in activity among chronic schizophrenics. The finding of a decreased Vmax but unchanged Km with 5-MT would be consistent with noncompetitive inhibition.
(Arch Gen Psychiatry 35:600-605, 1978) 1972 numerous investigators have described decreased platelet monoamine oxidase (MAO) activi¬ ty in chronic schizophrenia.19 Several investigators have not been able to duplicate these findings.1014 These discrepan¬ cies could be due to a variety of reasons. First, the existing studies have reported results obtained with different assays, subject groups, enzyme preparations, and sub¬ strates, making reliable comparisons difficult. Second, none of this work has been done with substrates of major physio¬ logic importance or with substrates of particular interest as regards schizophrenia. Third, the existing studies (with the exception of our own1) have relied on a determination of enzyme activity at a single substrate concentration. A large excess of substrate in such assays may lead to substrate inhibition; a substrate concentration less than twice the Michaelis constant (Km) may lead to falsely low values, obscuring significant differences. It is preferable to study the enzyme at several different substrate concentrations, in order to obtain Km and maximal velocity values (Vmax) that would permit a more reliable comparison. Only a convention of investigators in this field will solve the first difficulty. In an attempt to deal with the last two
Since
Accepted for publication Feb 22, 1978. From the Departments of Psychiatry (Dr Berrettini) and Pharmacology (Drs Prozialeck and Vogel), and the Schizophrenia Research and Treatment Center (Drs Berrettini and Vogel), Jefferson Medical College, Philadelphia. Reprint requests to Department of Pharmacology, Jefferson Medical College, 1020 Locust St, Philadelphia, PA 19107 (Dr Berrettini).
PhD
problems, we have studied platelet MAO kinetically in chronic schizophrenics and matched controls, using substrates of major physiologic importance (such as sero¬ tonin [5-HT]) and substrates of particular interest as regards schizophrenia, such as dopamine (DA) (the DA hypothesis15), N, iV-dimethyltryptamine (DMT), and 5methoxytryptamine (5-MT) (the transmethylation hypoth¬ esis16). The DA hypothesis of schizophrenia asserts that there exists a dopaminergic hyperactivity at certain critical sites in the brain. Data indicating that metabolism of this neurotransmitter, by platelet MAO from schizophrenics, is blocked would have bearing on this theory. The possibility that methylated indoleamines play a role in the pathogenesis of schizophrenia has been strengthened by the demon¬ stration that the necessary biosynthetic pathways for DMT exist in human brain.17 This finding is interesting in the light of the work of Narasimhachari et al, which
showed that very small amounts of this potent hallucino¬ genic molecule could be found in the blood and /or urine of some schizophrenics and controls.1819 Thus, there is evidence to suggest that minute amounts of DMT may be produced in the human brain. An inability to metabolize this compound would be an important finding in schizo¬ phrenia, with implications for the transmethylation hy¬ pothesis. Another methylated indoleamine that occurs naturally in the human brain,20 5-MT, has been shown to be behaviorally active and to be equipotent to LSD in disturbing the conditioned avoidance response in rats after intraventricular or intraperitoneal injection.21 The CSF levels of 5-MT were found to be elevated in schizophrenic patients, compared to controls.22 Thus, there is evidence to suggest that 5-MT may be involved in psychotic behavior. Again, a demonstration that metabolism of this compound by platelet MAO is decreased in schizophrenia would be
interesting.
METHODS Patient Selection The diagnosis of schizophrenia was made by applying diagnostic criteria selected from the World Health Organization's Report of the International Pilot Study of Schizophrenia." The criteria used were as follows: 1. Auditory hallucinations (defined as derogatory, threatening, or
command voices). 2. Delusions of thought alienation. 3. Delusions of thought broadcasting. 4. Delusions of thought control. 5. Delusions of persecution.
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6. Neologisms. 7. Looseness of associations. 8. Flat or blunted affect. All patients in this study gave
Enzyme Assay a
history
of
auditory
hallucina¬
tions and delusions of the type described above; all were consid¬ ered by two independent psychiatrists to have flat or blunted affect and looseness of associations. Seven patients were noted to use neologisms. Four patients had a family history of schizophre¬ nia. The patient was termed "chronic" if these symptoms and signs were present continuously, without remission, for a minimum period of two years prior to the study. A minimum of two previous hospitalizations within the last two years (with the diagnosis of
schizophrenia) was required. All patients were living in the community between hospitalizations, which were no longer than four months in duration. They were observed between hospitaliza¬ tions in community mental health centers. The diagnosis of chronicity was established by patient interview and by examina¬ tion of previous records. A patient was excluded from the study if the employment history indicated that any position was held for than one month within the last three years. time were these patients diagnosed as having affective disorder. All patients were free of concurrent disease. It was not considered necessary to limit the study to drug-free patients, since the major tranquilizers (such as the phenothiazines and butyrophenones) do not affect MAO activity.8·12 All patients had recent complete blood counts (CBCs) that showed no evidence of irondeficiency anemia,24 and that showed normal platelet count. Informed consent was obtained. Patients were selected from four different institutions in order to minimize the possible effects of environment. Two of these were acute-care inpatient units and two were day hospital programs. more
At
done with fresh platelet preparations. Blood or plasma was kept cool (4°C) and assays were done within four hours after venipuncture in all cases. For all substrates, the enzyme AU assays
no
Control Selection The control population was selected from healthy staff and laboratory personnel (not taking any medication for one month prior to the study) who had no personal or family history of psychiatric illness.
Enzyme Preparation venous blood, for each substrate, was Approximately collected in evacuated glass tubes, pretreated with 0.3 ml of 5% a, ethyldiaminetetra acetic acid (EDTA). The blood was then centri¬ fugea at 350 g for 30 minutes at 5°C. The platelet-rich plasma was withdrawn and centrifuged in a glass tube for 20 minutes at 1,100 g and at 5°C. The resultant platelet-poor plasma was discarded and the platelet plug washed twice with 2.0-ml volumes of Sorensen's phosphate buffer (0.05M; pH, 7.4). The plug was then suspended in a small volume of the buffer and homogenized in a Teflon and glass homogenizer using 50 strokes in one minute at 5°C. The protein content of each platelet homogenate was esti¬ mated using the method of Lowry et al.25 A control sample was assayed with each schizophrenic sample, as has been suggested by White et al.14
15 ml of
were
assay was a modification of the method of Wurtman and Axelrod.2" This method relies on the ability of an organic solvent to extract, reproducibly, a fraction of the metabolites of MAO oxidation from a highly acidic, aqueous phase. Table 1 lists the substrate, the metabolites, extraction solvent, and percentage of the metabolite extracted by a single aliquot (6 ml) of the solvent. This percentage was determined by multiple extractions of the same aqueous phase. All assays were shown to be linear for at least one hour. Blanks were prepared by pretreatment of the enzyme aliquot with 1 ml of 2N HC1. In all experiments, the organic phase was separated from the aqueous phase, after extraction, by freezing at —80°C for ten minutes. The organic phase was poured off into 10 ml of a standard fluor, and activity was measured in a standard scintillation counter. All substrates were assayed at six concentrations to provide data for Lineweaver-Burk plots. All incubations were carried out at 37°C in a Dubnoff metabolic shaker. All counts were corrected for the metabolites left in the aqueous phase by the single extraction. Assays were linear with protein (0.41 ± 0.24 mg mean ± SD added to each tube). Serotonin. Serotonin 2-'*C, as the oxalate salt (specific activity .050 mCi/30 mg) was diluted with 5-HT, as the oxalate salt, to a specific activity of .050 mCi/0.9 mg. Then 0.070 ml of platelet homogenate was added to test tubes containing various concentra¬ tions of 5-HT, ranging from 0.5 10'M to 3 10"3M in the phosphate buffer. The final volume of the assay was 0.1 ml. The reaction was stopped after 45 minutes by the addition of 1 ml of 2N HC1. These metabolites were extracted into 6 ml of toluene: ethyl acetate (1:1, v/v), as described previously.2' Dimethyltryptamine.—Dimethyltryptamine 2-1,C as the oxalate salt had a specific activity of 2.34 mCi/mmole). First 0.050 ml of platelet homogenate was added to test tubes containing final substrate concentrations, ranging from 2 10~4M to 6 x 10~4M in the phosphate buffer. The final volume was 0.1 ml. The reaction was stopped at one hour by the addition of 1 ml of 2N HC1. Metabolites were extracted into 6 ml of toluene. As this was a novel assay, substrate and metabolites were identified by radiochromatography, using silica gel with fluorescent indicator and methanolacetic acid (10:15) as solvent system. The substrate ratio of fronts (Rf) was identical to that of authentic DMT; indoleacetic acid and indoleacetaldehyde were identified as products. 5-Methyoxytryptamine.-As the radioactive-labeled amine was not available, the side-chain of 5-MT was labeled with 2-,4C and then synthesized according to the method of Kveder and McIsaac,28 using 14C (specific activity 60 mCi/mmole). The synthesized compound was characterized by its infrared spectrum, melting point (121°C to 122°C), by formation of a highly fluores¬ cent derivative with orthophthalaldehyde,2" by thin-layer radiochromatography using three different solvent systems (A, silica gel with CHCl.,:MeOH:lN NH, OH [12:7:1], B, silica gel with Nbutanohacetic acid:H20 [12:3:5], and C, cellulose with iV-butanohacetic acid:H,0 [12:3:5]) and by thin-layer radiochromatog=
=
Table 1 .—Substrates, Metabolites, and Extraction Solvents Used in MAO
Substrate DMT 5-MT 5-HT DA
"Large
amounts of the
Metabolite" Indolacetlc acid
Extraction Solvent Toluene Toluene Toluene: ethyl acetate 1:1 Toluene:THF 2:1
5-methoxyindolacetic acid 5-hydroxylndolacetlc acid 3,4-dihydroxyphenyl acetic acid
corresponding aldehyde
were
identified in each
Assays % of Products Recovered With 6 ml of Solvent in a Single Extraction 82 72 88 80
case.
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Table
2.-Vmax and Km Values for Platelet MAO Obtained With Dopamine as Substrate From Chronic Schizophrenics Patient/
Duration of
Illness,
Age,
Treatment*
Program
Sex 1/28/M 2/57/F 3/29/M 4/24/M
Eo
5/28/M 6/28/F 7/24/M 8/28/M 9/34/M 10/29/M 11/29/M 12/54/F 13/33/M 14/28/M 15/26/F 16/23/M
Eo
Jo Jo
y
nmole/Vm, mg/hr
K,„
5.9 7.4
29
3.0 6.2
5.9 3.7
3.8 1.5
4.7 4.2
4.3 5.9 3.3 5.0 2.8 4.0 3.7
6.8 6.9 5.8 4.6 6.0 4.8
14
27
10 «M 3.6
6.2 3.8 7.2 4.9
2.7 6.0 3.6 2.5
±
SD
31.6
10
5.6
±
1.2
3.9
±
3.-Vma„ and Km Values for Platelet MAO Obtained With Dopamine as Substrate From Controls
Control'/Age, yr/Sex_V^,_K„ _1/24/F_8J_7.1 _2/26/M_9J_5.9 _3/21/M_13_4.8 _4/40/M_12_7.4 _5/47/M_9J)_7.1 _6/39/F_6 _4.8 _7/20/M_9J5_6.6 _8/40/F_8_9_5.4 _9/29/M_8J_8.5 _10/47/M_9_3_7.7 11/29/M_7_9_5.0 _12/33/M_9J3_6.2 _13/47/M_8_8_5.3 _14/25/M_11_7.5 _15/27/F_11_7.5 16/25/M_8^7_3.4 _17/21/F_6_7_5.6 18/23/M_9J_3.7 19/23/M_8_9_5.8 Average
Aver¬
age
Table
1.3
*J, Thomas Jefferson University Hospital inpatient; Jo, Thomas Jeffer¬ University Day Hospital outpatient; E, Albert Einstein (Daroff Division) Hospital inpatient; Eo, Albert Einstein (Daroff Division) Day Hospital
±
SD 30.8
9.3
±
1.6
6.1
±
1.4
_P < .001_P < .001 •See footnote to Table 2.
son
outpatient.
Table
4—Comparison of Vm„
and
Km Values for Platelet MAO* Obtained With Serotonin
Duration of
Programf
Patient/Age, yr/Sex
Illness, yr
nmole/mg/hr
2/57/F
29
2.1 1.8
Jo
3/29/M 7/24/M 8/28/M 9/34/M 12/54/F
Treatment
Jo
Jo Jo
Average
±
SD
13/33/M 17/34/F 18/24/M 20/24/M 23/24/M 24/40/M 25/33/F 26/56/M 35
Km
2.1 2.2 14
27 11
1.6 2.2 2.2
4.8
0.3 0.9 2.0 1.0 1.0 1.1 1.4
0.6 1.3 1.3 23 30 14
2.3 1.8 2.2 1.8 ± .5
10 · 0.6 1.1 1.2 1.4
X
0.8 1.3
±
1.1
as
Substrate
Control/Age, yr/Sex 2/26/M 5/47/M 8/40/F 10/47/M 13/44/M 20/23/M 24/26/F 25/29/F 28/39/M 29/29/M 30/29/M 31/45/F 32/34/M 33/24/F Average ± SD 34.4
3.8 2.7 4.0 3.9
1.3 2.8 4.1 2.8
3.0 1.9 3.4 3.2 4.1
3.6 2.2
4.5 3.4 3.9 2.2 4.5 3.5
± 0.8 < .001
1.6 4.6 1.3 4.0
3.8 0.6 3.3 2.7 ± .13 < .005
"Obtained from chronic schizophrenics and controls. to Table 2.
fSee footnote
raphy
of its dansyl chloride derivative.30 These procedures indi¬ cated that the compound was greater than 95% radiochemically pure.
As the free base, 5-MT 2-14C, with specific activity 1,500 counts per minute nmole, was obtained as described above. Then 0.05 ml of platelet homogenate was added to test tubes containing final substrate concentrations, ranging from 0.4 10loM to 1.6 x 10 4M in the phosphate buffer. The final volume of the assay was 0.25 ml. The reaction was halted after 60 minutes by the addition of 1 ml of 2N HC1. These metabolites were extracted into 6 ml of toluene. As this was a novel assay, substrate and metabolites were identified by radiochromatography of the reac¬ tion mixture, using silica gel with fluorescent indicator and benzene:acetic acid (9:1). The substrate Rf was identical to that of
authentic 5-MT; the metabolites were identified as 5-methoxyindolacetic acid and the corresponding aldehyde. Dopamine.—Dopamine labeled with tritium (specific activity 11.363 Ci/mmole), as the free base, was diluted to a specific activity of 11.0 mCi/mmole, using dopamine HC1. Then 0.100 ml of the platelet homogenate was added to test tubes containing substrate concentrations ranging grom 3.0 x 10 4M to 10 x 10 4M in the phosphate buffer. The final volume of the assay was 0.250 ml. The reaction was halted after 30 minutes by the addition of 1 ml of 2N HC1. The metabolites were extracted into 6 ml of toluene:tetrahydrofuran (THF), 2:1, v/v. As this was a novel assay, substrate and metabolites were identified by radiochromatography of the reaction mixture, using cellulose with fluorescent indicator and A?-butanol:acetic acid:water (25:4:10). The substrate
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Rf
identical to that of authentic dopamine; the metabolites identified as 3,4-dihydroxyphenylacetic acid and the corre¬
was
were
sponding aldehyde. The statistical significance
of data was determined using the Student's t test. To determine whether an individual's Vmax, as measured with one substrate, correlated with Vmax, as measured with other substrates, a multiple rank correlation, using all individuals studied with 5-MT, 5-HT, and DMT, was performed. Because only two patients were studied with all four substrates, limiting the correlation to these three substrates allowed five patients (patients 3, 13,17, 18, and 20) and six controls (controls 2, 8, 10, 13, 20, and 25) to be included.
RESULTS
The results
are
shown in Tables 2 through 8. MAO
Table
Patient/Age,
Treatment
yr/Sex
Programf
Average
±
5—Comparison of Vm„ and Km
2/57/F 3/29/M 13/33/M 15/26/F 17/34/F 18/24/M 19/37/M 20/24/M 21/22/F 22/20/M 27/18/M 28/26/M 29/30/F 30/24/M 31/24/F 32/23/F 28.1
SD
Duration of Illness, yr 29
Values for Platelet MAO* Obtained With DMT
nmole/mg/r|r 1.3 0.91 1.7 0.91 0.50 0.48 0.67 0.83 1.5 1.1 0.57 0.71 1.2 1.7 0.93 0.83 1.0
"These values were obtained from chronic footnote to Table 2.
showed a significantly decreased Vmax and Km for all substrates in chronic schizophrenics, with the exception of the Km for 5-MT. The Vmax factor seemed to separate schizophrenics from controls somewhat better than did the Km index. Among the four substrates, DA seemed to separate the two groups best, followed by DMT, 5-HT, and 5-MT. The Km/Vmax value, which is the slope of the line in a Lineweaver-Burk plot, is similar for both groups in all instances, except for the 5-MT (see Table 6). Multiple rank correlations for the five schizophrenics indicated a high degree of correlation between the Vmax for the three substrates included in this study (P < .001). Similar results were obtained in rank correlations for the six controls (P < .04).
Kra X10
as
Substrate
Control/Age,
yr/Sex
'M
2.2 2.2 2.3
2/26/M 3/19/M
1.7 1.8
7/21/M
1.7 1.9
1.4 1.6 1.5
6/39/F 8/40/F 10/47/M 13/47/M 20/23/M 21/25/M 22/22/M 23/23/F 24/26/F 25/29/F 26/25/F 27/27/F
1.4
1.4 1.7 1.6 0.83 0.83 1.1 1.2 1.8 1.8
34/26/M ± SD 29.3
Average
4.6 2.5 1.3 1.7 1.9
1.3 1.3 1.2 1.5 1.1 1.7 2.4 1.3 1.4 1.5
4.5 1.6
1.9 1.5 ± .4 < .001
2.0 2.4 ± 1.3 < .0025
2.0
3.6 3.3 2.8 2.4 2.9 0.7 1.4
schizophrenics and controls.
fSee
Table Table 6.-Vmax and K,„ Values for Platelet MAO* Obtained With 5-MT as Substrate From Chronic Schizophrenics Duration of Treatment
Program*
Patient/
Age,
Illness,
V„,„
nmole/
yr/Sex_yr_mg/hr
K„,
X 10
'M
E_3/29/M_9_025_0.45 J_13/33/M_11_0_2J_0.24 J_15/26/F_5_O20_0.42 J_16/23/M_2_015_0.33 J_17/34/F_9_016_0.29 E_18/24/M_5_ _0.33 J_19/37/M_4_022_0.14 E_20/24/M_3_0_18_0.25 J_21/22/F_4_027_0.28 J_22/20/M_6_0_24_0.29 4_0_14_0.43 J_31/24/F J_32/23/F_4_0_T7_0.38
Aver-
age
±
SD_26J5_0.19
"See footnote to Table 2.
±
0.05 0.32
±
0.09
and Km Values for Platelet MAO Obtained With 5-MT as Substrate From Controls
7.-Vm„
Control'/Age, yr/Sex_V^_K„, _2/26/M_0_33_0.24 _3/20/M_037_0.36 _6/39/F_ 31_0.24 _7/21/M_033_0.20 _8/40/F_038_0.56
_10/47/M_017_0.37 _13/47/M_034_0.57 18/23/M_022_0.12
19/24/M_022_0.28 _20/23/M_0_19_0.23 _21/25/M_027_0.54 _22/21/M_022_0.22 _23/23/F_0_38_0.14 _24/26/F_0_34_0.22 _25/29/F_023_0.40 _26/24/F_026_0.25 _27/27/F_036_0.34 Average
±
SD 28.5
0.29
±
0.06
0.31
±
0.13
_P .001_NS
