Psychopharmacology
Psychopharmacology 62, 17- 22 (1979)
9 by Springer-Verlag 1979
Cerebrospinal Fluid Cyclic AMP and Acid Monoamine Metabolites Following Probenecid: Studies in Psychiatric Patients Malcolm B. Bowers, Jr. and Robert E. Study Departments of Psychiatry and Pharmacology, Yale University School of Medicine, New Haven, Connecticut, U.S.A.
Abstract. At probenecid levels greater than 10 ~tg/ml, CSF cAMP was independent of CSF probenecid concentration. At these levels of probenecid, cAMP transport out of CSF is probably maximally blocked and cAMP levels reflect cAMP release into CSF. CSF cAMP was significantly higher in RDC-diagnosed schizophrenics than in other psychotics or depressives. A significant decrease in CSF cAMP was found in psychotic patients treated with chlorpromazine. No changes in CSF cAMP were observed in patients treated with tricyclic antidepressants or lithium. Key words: CSF cyclic AMP - Probenecid Homovanillic acid - 5-Hydroxyindoleacetic acid Chlorpromazine
In recent years, lumbar cerebrospinal fluid (CSF) monoamine metabolites have been measured in a variety of neuropsychiatric conditions. Homovanillic acid (HVA), 5-hydroxyindoleacetic acid (5 HIAA), and 3-methoxy, 4-hydroxyphenylglycol (MHPG) have been studied as possible direct indicators of the presynaptic activity of monoamine neurons in the central nervous system (Ashcroft and Sharman, 1960; Dencker et al., 1966; Gerbode and Bowers, 1968; Gordon and Oliver, 1971). The utility of this approach may be enhanced by the use of probenecid, which blocks the egress of acid monoamine metabolites from CSF and allows a more direct assessment of their formation (Olsson and Roos, 1968; Tamarkin et al., 1970; Korf et al., 1971; Bowers, 1972a). No reliable indicator of the postsynaptic activity of these neurotransmitter has been found; however, cyclic AMP (cAMP) levels in CSF may be related to such activity. Cyclic AMP is produced in postsynaptic cells as a result of the action ofdopamine (Kebabian et al., 1975; Krueger et al., 1976), norepinephrine (Siggins et al.,
1971), and perhaps serotonin (Schultz and Daly, 1975). A dopamine-sensitive adenylate cyclase has been shown to be a site of action of many neuroleptic drugs. Since abnormalities in monoaminergic systems are thought to be involved in certain neuropsychiatric conditions, measurement of CSF levels of cAMP may provide information about changes in the postsynaptic activity in these systems. Cyclic AMP is present in CSF, although its precise anatomic origin is not known. Sebens and Korf (1975) have shown in the rabbit that cisternal CSF cAMP most likely originates in the central nervous system and is increased by intracisternal injection of norepinephrine, isoprenaline, dopamine, and histamine, and by intraperitoneal injection of probenecid. Angel and coworkers (1976) have found increases in cisternal CSF cAMP and cGMP following probenecid in dogs. Using push-pull cannulae in the lateral ventricles of rats, Korf et al. (1976) found that norepinephrine, dopamine, and adenosine, but not serotonin or histamine, increased cAMP output. Cyclic AMP can be measured in lumbar CSF (Robison et al., 1970) and has been reported to increase two- to threefold following probenecid in neurologic patients and fourfold in mania (Cramer et al., 1972; Cramer et al., 1972b). Although several reports of baseline lumbar CSF cAMP values in neurologic conditions have appeared, there are few studies in psychiatric patients. Notable exceptions are found in the studies of Biederman et al. (1977a) in schizophrenia and Smith et al. (1976) in several patient groups. We have been interested in lumbar CSF cAMP in psychiatric patients as possible indicator of syndromerelated behavioral components and psychotropic drug effects. We have therefore examined CSF cAMP with regard to its relationship to CSF probenecid and lumbar CSF acid monoamine metabolites (5HIAA, HVA) in several groups of psychiatric patients before and during chemotherapy. Our findings indicate that
0033-3158/79/0062/0017/$01.20
18
probenecid blocks transport of cAMP from CSF and that this block can reach a maximum with clinically realizable probenecid levels. Using probenecid, we have also found a small but significant decrease in cyclic A M P levels in the lumbar CSF of patients during treatment with chlorpromazine.
Psychopharmacology 62 (1979) GSF cyclic AMP
(pmol/ml) 140
120
I00
Materials and Methods Patients and Procedures. A total of 72 patients (10 males and 62 females aged 16-67) participated in the study. All patients were admitted to the Clinical Research Unit, Connecticut Mental Health Center, for inpatient evaluation and treatment after informed consent was given by the patient and a responsible relative for the research procedures. Diagnoses were made according to research diagnostic criteria for schizophrenia 8 patients, for other psychoses, including schizoaffectiveillness 18 patients, and for primary affective disorder (depression or mania) 32 patients (Spitzer et at., 1975). Fourteen patients who did not meet criteria for one of these diagnoses were included only in the cAMP analysis of the total psychiatric group in relation to CSF probenecid. Medication that was being taken at the time of admission was discontinued during the 1st week. In the schizophrenic group three patients had taken medication previously and five had not. Subjects ate the regular ward diet, which restricted coffee, tea, and chocolate. At the end'of the following 3-week period, the probenecid test was performed on a total of 72 patients. Probenecid (80 - 100 mg/kg) was administered by mouth in six divided doses over a 20-h period. The lower dose was used if patients were subject to nausea during the first probenecid test. During this time patients were kept at bed rest. Four hours following the last probenecid dose a lumbar puncture was performed with the patient in the lateral position. The first 7 ml of CSF were obtained and frozen within 30 min. Patients were then treated with chlorpromazine, tricyclic antidepressants, or lithium. Four to eight weeks after specific chemotherapy was started, a second probenecid test was performed when the medication dose had been stabilized for at least a week. Pretreatment and during-treatment values were obtained in 13 patients with acute psychosis treated with chlorpromazine, 9 patients with primary affective disorder (unipolar depression) treated with tricyclic antidepressants, and 13 patients with primary affective disorder (bipolar depression or mania) treated with lithium. Ten CSF samples (without probenecid) were obtained from neurologic patients for measurement of baseline CSF cAMP. Behavioral change before and during treatment was quantified by means of the Brief Psychiatric Scale (BPRS). Biochemical Measures and Statistics. Cyclic AMP levels were mea-
sured using a protein-binding method (Brown et al., 1971). Immediately after thawing, CSF was prepared for cAMP measurement by heating for 3 min in a boiling water bath to destroy phosphodiesterase. CSF 5HIAA and HVA were measured according to Gerbode and Bowers (1968) and probenecid by the method of Korf and van Praag (1971). Correlations were calculated (Pearson r) between CSF probenecid and cAMP, 5HIAA, and HVA. CSF cAMP values for the entire psychiatric group (on probenecid) were compared to neurologic controls (no probenecid) using an unpaired t-test. Using the total psychiatric group, correlations were obtained between CSF probenecid, cAMP, 5HIAA, HVA, and age. This group was further subdivided into three groups schizophrenics, other psychotics, and primary affective depressed. Correlations among probenecid, cAMP, and the acid monoamine metabolites were also obtained for these subgroups. Cyclic AMP values were also compared in 10 male and 20 female patients with mixed diagnoses.
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PROBENEGID (#,g/ml )
Fig. 1. CSF cAMP and probenecid in psychiatric patients Pretreatment mean values for cAMP, 5HIAA, HVA, and probenecid were evaluated using an unpaired t-test (two-tailed) in the schizophrenic, other psychotic, and primary affective, depressed groups. Paired t-tests were used to assess differences in cAMP, 5HIAA, HVA, and probenecid before and during treatment in the three treatment groups. For all treatment groups, pretreatment, during-treatment, and change scores (where appropriate) for cAMP, 5HIAA, HVA, probenecid, and BPRS score were intercorrelated.
Results
Probenecid treatment increased cAMP levels in the CSF of the total psychiatric group to 74.1 + 1.0 pmol/ml as compared to 14.1 + 1.8 pmol/ml for a group of neurologic patients that did not receive probenecid. The baseline CSF cAMP values in the neurologic group were very similar to published values in psychiatric patients (Cramer et al., 1972a). The change in cAMP levels within the psychiatric group was not measured because spinal taps prior to probenecid treatment were not done. Previous studies (Robison et al., 1970; Cramer et al., 1972a; Cramer et al., 1972b; Smith et al., 1976) have indicated that probenecid increases cAMP levels in CSF, presumably by blocking transport of this acid out of the CSF. Probenecid was used in the studies reported here so that cAMP levels would better reflect the rate of entry into the CSF rather than a combination of entry and exit rates. When the level of probenecid in the CSF was compared to the levels of cAMP, 5HIAA, and HVA in the entire psychiatric group or the three subgroups before drug therapy, no correlations were found. It is likely that this
19
M. B. Bowers, Jr. and R. E. Study: Cyclic A M P in CSF Table 1. Cyclic A M P , 5HIAA, HVA, and probenecid in lumbar CSF in psychotic and depressed patients Diagnosis
N
Cyclic A M P a
5HIAA b
HVA b
Probenecid c
Depression Schizophrenia Other psychotic
19 8 18
67.5 _+ 5.6 89.3 _+ 9.5* 72.9 _+ 3.5
108.4 + 9.4 99.9 + 12.6 114.8 +_ 10.3
149.1 + 10.9 182.6 _+ 17.7"* i40.3 + 9.5
19.1 + 1.0 21.5 + 1.8 22.0 • 1.9
a b c * **
pmol/ml+SEM ng/ml+SEM gg/ml+SEM P < 0.05 compared to depression, P = 0.05 compared to other psychotic P < 0.05 compared to other psychotic
Table 2. CSF values before and during treatment Diagnosis
N
Cyclic A M P a
5HIAA b
HVA b
Probenecid c
Duration (days)
Psychosis Before treatment During treatment
13 13
82.9 • 6.3 66.6 _+ 5.0*
114.6 • 12.5 88.9 + 9.7*
164.0 + 14.2 198.8 _+ 12.5
21.5 + 1.7 19.2 _+ 1.8
38_+3
71.3 + 10.2 60.3 _+ 5.6
121.6 • 14.3 71.4 • 6.3***
160.6 + 15.0 143.0 + 13.7
20.3 _+ 1.5 17.4_+ 1.3"*
36_+3
99.6+_ 11.3 114.8 _+ 12.0
123.9 • 11.0 154.4 • 15.5
21.4 + 1.7 18.2 + 1.9"
Dose: 765 _+ 75 chlorpromazine (mg/day) Unipolar Depression Before treatment During treatment
9 9
Dose: 150 nortriptyline or amitriptyline (mg/day) Bipolar Depression or M a n i a Before treatment During treatment
13 13
70.1 _+ 5.3 71.5 _+ 7.7
Dose: 1550 + 83 lithium (mg/day) a b ~
pmol/ml _+ SEM ng/mI • SEM ~tg/ml _+ SEM
* P < 0.05 compared to pretreatment value ** P < 0.02 compared to pretreatment value *** P < 0.005 compared to pretreatment value
lack of correlation is due to a maximal blockade of metabolite transport by probenecid, resulting in metabolite levels that are not affected by a further increase in the level ofprobenecid. A plot of CSF cAMP values as a function of CSF probenecid is shown in the figure. The lack of correlation between CSF probenecid and cAMP therefore indicates that the transport blockade has reached a maximum at these probenecid levels. Cyclic AMP levels did not correlate with 5HIAA, HVA, or age in the total psychiatric group, nor did cAMP show any difference between the sexes in this group. Table 1 shows CSF values before treatment for the schizophrenic group, other psychotics, and the primary affective, depressed group. CSF cAMP values were significantly higher (0.05 level) in the schizophrenics than in other psychotics or depressives. CSF HVA
values were higher in the schizophrenics than in the other psychotics. Table 2 shows CSF values before and during drug treatment for three groups of patients: psychotic (including schizophrenic), unipolar depressed, and bipolar depressed or manic. Only in the psychotic group was the CSF cAMP significantly changed by drug treatment (P
Psychopharmacology 62, 17- 22 (1979)
9 by Springer-Verlag 1979
Cerebrospinal Fluid Cyclic AMP and Acid Monoamine Metabolites Following Probenecid: Studies in Psychiatric Patients Malcolm B. Bowers, Jr. and Robert E. Study Departments of Psychiatry and Pharmacology, Yale University School of Medicine, New Haven, Connecticut, U.S.A.
Abstract. At probenecid levels greater than 10 ~tg/ml, CSF cAMP was independent of CSF probenecid concentration. At these levels of probenecid, cAMP transport out of CSF is probably maximally blocked and cAMP levels reflect cAMP release into CSF. CSF cAMP was significantly higher in RDC-diagnosed schizophrenics than in other psychotics or depressives. A significant decrease in CSF cAMP was found in psychotic patients treated with chlorpromazine. No changes in CSF cAMP were observed in patients treated with tricyclic antidepressants or lithium. Key words: CSF cyclic AMP - Probenecid Homovanillic acid - 5-Hydroxyindoleacetic acid Chlorpromazine
In recent years, lumbar cerebrospinal fluid (CSF) monoamine metabolites have been measured in a variety of neuropsychiatric conditions. Homovanillic acid (HVA), 5-hydroxyindoleacetic acid (5 HIAA), and 3-methoxy, 4-hydroxyphenylglycol (MHPG) have been studied as possible direct indicators of the presynaptic activity of monoamine neurons in the central nervous system (Ashcroft and Sharman, 1960; Dencker et al., 1966; Gerbode and Bowers, 1968; Gordon and Oliver, 1971). The utility of this approach may be enhanced by the use of probenecid, which blocks the egress of acid monoamine metabolites from CSF and allows a more direct assessment of their formation (Olsson and Roos, 1968; Tamarkin et al., 1970; Korf et al., 1971; Bowers, 1972a). No reliable indicator of the postsynaptic activity of these neurotransmitter has been found; however, cyclic AMP (cAMP) levels in CSF may be related to such activity. Cyclic AMP is produced in postsynaptic cells as a result of the action ofdopamine (Kebabian et al., 1975; Krueger et al., 1976), norepinephrine (Siggins et al.,
1971), and perhaps serotonin (Schultz and Daly, 1975). A dopamine-sensitive adenylate cyclase has been shown to be a site of action of many neuroleptic drugs. Since abnormalities in monoaminergic systems are thought to be involved in certain neuropsychiatric conditions, measurement of CSF levels of cAMP may provide information about changes in the postsynaptic activity in these systems. Cyclic AMP is present in CSF, although its precise anatomic origin is not known. Sebens and Korf (1975) have shown in the rabbit that cisternal CSF cAMP most likely originates in the central nervous system and is increased by intracisternal injection of norepinephrine, isoprenaline, dopamine, and histamine, and by intraperitoneal injection of probenecid. Angel and coworkers (1976) have found increases in cisternal CSF cAMP and cGMP following probenecid in dogs. Using push-pull cannulae in the lateral ventricles of rats, Korf et al. (1976) found that norepinephrine, dopamine, and adenosine, but not serotonin or histamine, increased cAMP output. Cyclic AMP can be measured in lumbar CSF (Robison et al., 1970) and has been reported to increase two- to threefold following probenecid in neurologic patients and fourfold in mania (Cramer et al., 1972; Cramer et al., 1972b). Although several reports of baseline lumbar CSF cAMP values in neurologic conditions have appeared, there are few studies in psychiatric patients. Notable exceptions are found in the studies of Biederman et al. (1977a) in schizophrenia and Smith et al. (1976) in several patient groups. We have been interested in lumbar CSF cAMP in psychiatric patients as possible indicator of syndromerelated behavioral components and psychotropic drug effects. We have therefore examined CSF cAMP with regard to its relationship to CSF probenecid and lumbar CSF acid monoamine metabolites (5HIAA, HVA) in several groups of psychiatric patients before and during chemotherapy. Our findings indicate that
0033-3158/79/0062/0017/$01.20
18
probenecid blocks transport of cAMP from CSF and that this block can reach a maximum with clinically realizable probenecid levels. Using probenecid, we have also found a small but significant decrease in cyclic A M P levels in the lumbar CSF of patients during treatment with chlorpromazine.
Psychopharmacology 62 (1979) GSF cyclic AMP
(pmol/ml) 140
120
I00
Materials and Methods Patients and Procedures. A total of 72 patients (10 males and 62 females aged 16-67) participated in the study. All patients were admitted to the Clinical Research Unit, Connecticut Mental Health Center, for inpatient evaluation and treatment after informed consent was given by the patient and a responsible relative for the research procedures. Diagnoses were made according to research diagnostic criteria for schizophrenia 8 patients, for other psychoses, including schizoaffectiveillness 18 patients, and for primary affective disorder (depression or mania) 32 patients (Spitzer et at., 1975). Fourteen patients who did not meet criteria for one of these diagnoses were included only in the cAMP analysis of the total psychiatric group in relation to CSF probenecid. Medication that was being taken at the time of admission was discontinued during the 1st week. In the schizophrenic group three patients had taken medication previously and five had not. Subjects ate the regular ward diet, which restricted coffee, tea, and chocolate. At the end'of the following 3-week period, the probenecid test was performed on a total of 72 patients. Probenecid (80 - 100 mg/kg) was administered by mouth in six divided doses over a 20-h period. The lower dose was used if patients were subject to nausea during the first probenecid test. During this time patients were kept at bed rest. Four hours following the last probenecid dose a lumbar puncture was performed with the patient in the lateral position. The first 7 ml of CSF were obtained and frozen within 30 min. Patients were then treated with chlorpromazine, tricyclic antidepressants, or lithium. Four to eight weeks after specific chemotherapy was started, a second probenecid test was performed when the medication dose had been stabilized for at least a week. Pretreatment and during-treatment values were obtained in 13 patients with acute psychosis treated with chlorpromazine, 9 patients with primary affective disorder (unipolar depression) treated with tricyclic antidepressants, and 13 patients with primary affective disorder (bipolar depression or mania) treated with lithium. Ten CSF samples (without probenecid) were obtained from neurologic patients for measurement of baseline CSF cAMP. Behavioral change before and during treatment was quantified by means of the Brief Psychiatric Scale (BPRS). Biochemical Measures and Statistics. Cyclic AMP levels were mea-
sured using a protein-binding method (Brown et al., 1971). Immediately after thawing, CSF was prepared for cAMP measurement by heating for 3 min in a boiling water bath to destroy phosphodiesterase. CSF 5HIAA and HVA were measured according to Gerbode and Bowers (1968) and probenecid by the method of Korf and van Praag (1971). Correlations were calculated (Pearson r) between CSF probenecid and cAMP, 5HIAA, and HVA. CSF cAMP values for the entire psychiatric group (on probenecid) were compared to neurologic controls (no probenecid) using an unpaired t-test. Using the total psychiatric group, correlations were obtained between CSF probenecid, cAMP, 5HIAA, HVA, and age. This group was further subdivided into three groups schizophrenics, other psychotics, and primary affective depressed. Correlations among probenecid, cAMP, and the acid monoamine metabolites were also obtained for these subgroups. Cyclic AMP values were also compared in 10 male and 20 female patients with mixed diagnoses.
9 80
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9 8 gO
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j lid oooe
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80 9
40
20
o
r I o
I
I
I
I
I
l
1
I
5
I0
15
20
25
30
35
40
PROBENEGID (#,g/ml )
Fig. 1. CSF cAMP and probenecid in psychiatric patients Pretreatment mean values for cAMP, 5HIAA, HVA, and probenecid were evaluated using an unpaired t-test (two-tailed) in the schizophrenic, other psychotic, and primary affective, depressed groups. Paired t-tests were used to assess differences in cAMP, 5HIAA, HVA, and probenecid before and during treatment in the three treatment groups. For all treatment groups, pretreatment, during-treatment, and change scores (where appropriate) for cAMP, 5HIAA, HVA, probenecid, and BPRS score were intercorrelated.
Results
Probenecid treatment increased cAMP levels in the CSF of the total psychiatric group to 74.1 + 1.0 pmol/ml as compared to 14.1 + 1.8 pmol/ml for a group of neurologic patients that did not receive probenecid. The baseline CSF cAMP values in the neurologic group were very similar to published values in psychiatric patients (Cramer et al., 1972a). The change in cAMP levels within the psychiatric group was not measured because spinal taps prior to probenecid treatment were not done. Previous studies (Robison et al., 1970; Cramer et al., 1972a; Cramer et al., 1972b; Smith et al., 1976) have indicated that probenecid increases cAMP levels in CSF, presumably by blocking transport of this acid out of the CSF. Probenecid was used in the studies reported here so that cAMP levels would better reflect the rate of entry into the CSF rather than a combination of entry and exit rates. When the level of probenecid in the CSF was compared to the levels of cAMP, 5HIAA, and HVA in the entire psychiatric group or the three subgroups before drug therapy, no correlations were found. It is likely that this
19
M. B. Bowers, Jr. and R. E. Study: Cyclic A M P in CSF Table 1. Cyclic A M P , 5HIAA, HVA, and probenecid in lumbar CSF in psychotic and depressed patients Diagnosis
N
Cyclic A M P a
5HIAA b
HVA b
Probenecid c
Depression Schizophrenia Other psychotic
19 8 18
67.5 _+ 5.6 89.3 _+ 9.5* 72.9 _+ 3.5
108.4 + 9.4 99.9 + 12.6 114.8 +_ 10.3
149.1 + 10.9 182.6 _+ 17.7"* i40.3 + 9.5
19.1 + 1.0 21.5 + 1.8 22.0 • 1.9
a b c * **
pmol/ml+SEM ng/ml+SEM gg/ml+SEM P < 0.05 compared to depression, P = 0.05 compared to other psychotic P < 0.05 compared to other psychotic
Table 2. CSF values before and during treatment Diagnosis
N
Cyclic A M P a
5HIAA b
HVA b
Probenecid c
Duration (days)
Psychosis Before treatment During treatment
13 13
82.9 • 6.3 66.6 _+ 5.0*
114.6 • 12.5 88.9 + 9.7*
164.0 + 14.2 198.8 _+ 12.5
21.5 + 1.7 19.2 _+ 1.8
38_+3
71.3 + 10.2 60.3 _+ 5.6
121.6 • 14.3 71.4 • 6.3***
160.6 + 15.0 143.0 + 13.7
20.3 _+ 1.5 17.4_+ 1.3"*
36_+3
99.6+_ 11.3 114.8 _+ 12.0
123.9 • 11.0 154.4 • 15.5
21.4 + 1.7 18.2 + 1.9"
Dose: 765 _+ 75 chlorpromazine (mg/day) Unipolar Depression Before treatment During treatment
9 9
Dose: 150 nortriptyline or amitriptyline (mg/day) Bipolar Depression or M a n i a Before treatment During treatment
13 13
70.1 _+ 5.3 71.5 _+ 7.7
Dose: 1550 + 83 lithium (mg/day) a b ~
pmol/ml _+ SEM ng/mI • SEM ~tg/ml _+ SEM
* P < 0.05 compared to pretreatment value ** P < 0.02 compared to pretreatment value *** P < 0.005 compared to pretreatment value
lack of correlation is due to a maximal blockade of metabolite transport by probenecid, resulting in metabolite levels that are not affected by a further increase in the level ofprobenecid. A plot of CSF cAMP values as a function of CSF probenecid is shown in the figure. The lack of correlation between CSF probenecid and cAMP therefore indicates that the transport blockade has reached a maximum at these probenecid levels. Cyclic AMP levels did not correlate with 5HIAA, HVA, or age in the total psychiatric group, nor did cAMP show any difference between the sexes in this group. Table 1 shows CSF values before treatment for the schizophrenic group, other psychotics, and the primary affective, depressed group. CSF cAMP values were significantly higher (0.05 level) in the schizophrenics than in other psychotics or depressives. CSF HVA
values were higher in the schizophrenics than in the other psychotics. Table 2 shows CSF values before and during drug treatment for three groups of patients: psychotic (including schizophrenic), unipolar depressed, and bipolar depressed or manic. Only in the psychotic group was the CSF cAMP significantly changed by drug treatment (P
