- Email: [email protected]
Platelet Imipramine Binding in Children and Adolescents with Impulsive Behavior
·Platelet Imipramine Binding in Children and Adolescents with Impulsive Behavior BORIS BIRMAHER, M.D., MICHAEL STANLEY , PH.D., LAURENCE GREENHILL, M.D. , JANET TWOMEY, B.A. , ANTIGONY GAVRILESCU, M.D ., AND HARRIS RABINOVICH, M.D.
Abstract. ' . The serotonergic system has been implicated in the regulation of impulsive aggressive behavior either toward oneself or others. Imipramine binding sites were measured in the platelets of 23 impulsive aggressive children. Subject s ratings of total behavior, externalizing behavior, hostility, and aggression, as measured by the Child Behavior Checklist, were inversely correlated with the platelet imipramine binding. These findings are consistent with previous studies that suggest that decreased serotonergic activity is associated with impulsive aggressive behavior. J. Am. Acad . ChildAdolesc. Psychiatry, 1990,29,6:914-918. Key Words: platelet imipramine binding, impulsivity, aggression , children, serotonin . The serotonergic system has been reported to playa role in the regulation of impulsive aggressive behaviors (Asberg et al., 1987). Several investigators have shown an inverse correlation between impulsive aggressive behaviors and lower cerebrospinal fluid (CSF) 5-Hydroxy-indoleacetic acid (5HIAA) (Bioulac et al., 1980; Brown et al., 1982; Linnoila, et al., 1983; Lidberg et al. ; 1985; Virkkunen et al., 1987). This correlation between impulsive behaviors and CSF 5HIAA .has also been demonstrated in patients with borderline personality disorder (Brown et al., 1979) and in normal volunteers (Roy et al., 1988). Branchey et al. (1984) studied the ratio of tryptophan to other neutral amino acids in the serum of alcoholics . They found significant lower ratios, which suggest a deficiency of brain serotonin, in those subjects who had been arrested for assaultive behavior, when compared to other alcoholics or to nonalcoholic controls. Recently, Coccaro et al. (1989) reported an inverse correlation between the prolactin response to the 5-HT agonist, fenfluramine, and impulsive aggression in patients with personality disorder. Low CSF 5HIAA levels have also been reported in completed suicides and suicide attempters with different psychiatric diagnoses (Asberg et al. , 1987). Several postmortem studies have also reported low 5-HT and 5HIAA in the
hindbrain and certain raphe nuclei in suicide victims (Asberg et aI., 1987). Animal studies have shown that aggression can be regulated, at least in part, by the 5-HT system. These studies have shown that a decrease in central serotonin may predispose certain animal species for impulsive aggressive behavior (Eichelman, 1987). It has been demonstrated that high affinity imipramine binding sites (18) exist in the brain and in platelets of several species, including man (Briley et al., 1979; Raisman et al., 1979; Palkovits et al., 1981; Paul et aI. , 1981; Sette et al., 1981). In the brain , the 18 sites are distributed in the same pattern as 5-HT. In the platelet , IB is associated with the 5-HT uptake site (Briley et aI., 1979; Palkovits et aI., 1981; Paul et aI., 1981; Sette et aI., 1981). Stanley et al. (1982) and Stanley and Mann (1983) studied postmortem brain samples of patients who committed violent suicide and found reduced 18 sites in frontal cortices and an increase in postsynaptic 5HT-2 receptors, when compared with nonsuicide controls. Stoff et al. (1987) measured 18 sites in platelets of 10 drug free impulsive aggressive, conduct disorder children , ages 12 to 17 and 10 normal controls. Using the Child Behavior Checklist (CBCL), they observed a significant negative correlation between maximal platelet 18 and the factor for externalizing behavior (problems directed toward outside world) and aggression. However, Stoff et al. did not control for suicidality and the presence of depression variables which, by themselves, may have influenced the correlation they reported. In this study, 18 in platelets of children with impulsive and aggressive behaviors, who have had no history of suicidal attempts and affective disorders, were measured. It was hypothesized that platelet IB would be inversely correlated with the degree of aggression and/or impulsiveness.
Accepted June 28, 1990. Drs. Birmaher, Stanley, Greenhill, and Rabinovich are with the Department of Child Psychiatry, New York State Psychiatric Institute, and the Department of Psychiatry, Columbia University College of Physicians and Surgeons, New York. Drs. Birmaher, Gavrilescu, and Rabinovich are with the Manhattan Children's Psychiatric Center and Ms. Twomey is with the Department of Child and Adolescent Psychiatry, Western Psychiatric Institute and Clinic, University of Pittsburgh. Dr. Birmaher is now with the Department of Child and Adolescent Psychiatry, Western Psychiatric Institute and Clinic, University of Pittsburgh. Dr. Rabinovich is now with the Department of Child and Adolescent Psychiatry, University of Pennsylvania. This research was supported in part by Dr . Stanley's Grant: MH 42242 and MH 41847 from the U.S. Public Health Service and the Lowenstein Foundation. Reprint requests to Boris Birmaher, M.D ., Western Psychiatric Institute and Clinic, 3811 O'Hara Street, Pittsburgh, PA 15213. 0890-8567/9012906-0914$02 .00/0© 1990 by the American Academy of Child and Adolescent Psychiatry.
Method Children , ages 10 to 16, with a history of recurrent aggressive behavior and impulsivity were selected from the inpatient unit at Manhattan Children's Psychiatric Center and the Children's Day Hospital at New York Psychiatric Institute. Each patient was interviewed independently by 914
IMIPRAMINE BINDING AND IMPULSIVE BEHAVIOR
two child psychiatrists. After the interview, each psychiatrist reviewed the patient's records, and a joint decision was made regarding the patient's diagnosis. Diagnosis was made according to DSM -III criteria . Patients who had a history of recurrent aggression and/or impulsivity, who were not depressed and did not have a history of suicidal attempts , were accepted into the study . Inclusion also required that the patient and parents sign consent forms. Children were excluded from the study if they met any of the following criteria: (1) any significant medical illness; (2) IQ less than 70; (3) DSM-III diagnoses of affective disorder, schizophrenia, schizoaffective disorder, autism, or anorexia nervosa; (4) history of substance abuse within the past 6 months, and (5) history of suicide attempts. Patients were assessed with the following rating scales: CBCL (Achenbach and Edelbrock 1983), Hamilton Depression Rating Scale (HDRS) (Guy, 1976), Conner's Teacher Questionnaire (CTQ) (conduct and hyperactivity subscales) (Conners , 1976), and the Suicide Assessment Scale (SAS) (Stanley et aI., 1986). The HDRS and the SAS were administered by one of the child psychiatrists (B.B.); the CTQ was administered by the teacher and the CBCL was completed by the primary clinician who had managed the patient for at least 6 months. The CBCL has well documented psychometric properties and representative norms, reflecting direct observations of the most common complaints of children referred to child guidance clinics or mental health centers. Determination of Platelet IB The same week that the above rating scales were completed, patients had a single blood sample (30 ml) drawn for platelet lB. The blood was collected between 8:00 and 10:00 A.M., at room temperature, in 10 ml syringes containing ethylenediaminetetraacetate (EDTA) as an anticoagulant. Platelet rich plasma (PRP) was separated from erythrocytes and white blood cells by low speed centrifugation. Platelets were isolated on the day of collection (1 to 2 hours after being collected) and frozen until the IB assay. Platelet IB was determined in accordance with the methods described by Briley et al. (1979). PRP was spun for 10 minutes at 4C and 16,000 x1g. The pellet was rinsed 2 times with 3 ml 0.9% normal saline and centrifuged again. The pellet was frozen and not thawed until platelet membranes were prepared. The platelets were additionally lysed in 25 ml of 5mM Tris/HCL buffer, Ph 7.4, with 5mM EDTA and homogenized on a Polytron homogenizer for 15 seconds). The membranes were centrifuged at 4C, 39,000 x1g for 10 minutes. The membranes were rinsed twice in 25 ml of 70mM Tris/NCL, pH 7.4, with the same centrifugation. Final resuspension was in assay buffer 50 mM Tris/NCL, pH 7.4, with 120 mM NACL and 5mM KCL. Protein on the final homogenate was determined according to Lowry et al. (1951) and averaged approximately 2 mg/ml protein. Samples (175 f1L) of the homogenate were incubated in duplicate at 4C with 25 f1L of [3H] imipramine at a final concentration of 0.1 to 12 nM (specific activity, 55.4 Ci/ mmole from New England Nuclear) and with 25 f1L of assay J. Am .Acad. Child Adolesc. Psychiatry, 29:6, November 1990
buffer. Nonspecific binding was determined using a 10 f1M final concentration of desipramine. Total assay volume was 250 f1L. After 60 minutes, that incubate was diluted with 5 mL of ice-cold Trisma buffer and filtered through Whatman GF/C filters using a modified Brandell cell harvester. The filters were washed 2 more times with 5 ml of ice-cold Trisma buffer. The filters were counted in 10 ml Econofluor for 5 minutes on a Beckman LS 6800 liquid scintillation counter (counter efficiency better than 45%). The number of receptor sites (Bmax) and the affinity constant (Kd) were determined from the Scatchard plot for each individual's platelets. Statistical Analysis The data were tested for normality using the Shapiro and Wilk's W statistic. Kd did not follow a normal distribution, thus nonparametric statistics were used when Kd was involved. Differences between hospital status (inpatient versus day hospital) and medication status (psychotropic medications versus medication-free patients) on the IB parameters Bmax and Kd were tested by using the student r-test and Mann-Whitney, respectively. Bmax and Kd were correlated with each variable derived from the above named rating scales by using the Pearson Product Moment correlation and the Spearman Correlation, respectively. Whenever there were outlier subjects, correlations were made with and without these subjects. An outlier was defined as any subject whose standardized residual values were greater than two standard deviations from the mean of zero (Chatterjee and Price, 1977). Raw CBCL scores were used in all calculations. Sample Twenty-three boys were eligible for the study. Seventeen were inpatients, and six were from the day hospital. Eight of the 23 patients were taking medication during the period of the study. Three patients were taking lithium and a neuroleptic, and five were taking only neuroleptics (haloperidol, chlorpromazine or thioridazine); none of the day hospital patients were taking medications. Patients were on the average 12.6 SD ± 2.2 years old. All patients fulfilled DSM-III diagnostic criteria for conduct disorder, and 16 also fulfilled diagnostic criteria for attention deficit disorder with hyperactivity. The clinical picture of the patients ranged among moderate to severely hyperactive, impulsive, and physically or verbally aggressive behaviors. Relevant CBCL mean scores and standard deviations for this sample are as follows: total behavior: 62.2 ± 20.5; externalizing behavior (sum of scores for hostile, delinquent, aggressive arid hyperactive behaviors): 39 ± 12; aggression: 26.4 ± 10.2 , and hostility: 9.9 ± 4.8 . These mean scores were above the threshold considered pathological by the CBCL (38,21, 21, and 9, respectively). The mean HDRS score was 4.2 ± 4.6. The mean total score on the SAS was 24.4 ± 9.8. The SAS includes items for suicidality and impulsivity . The mean SAS suicidality subscale score was 0.76 ± 1.3, and the mean SAS impulsivity subscale score was 8.2 ± 2.8 . Neither medication nor hospitalization status statistically differentiated patients on the IB parameters (Table 1). Thus, all the groups were collapsed and the sample analyzed together. 915
BIRMAHER ET AL.
..
TABLE I. Platelet Imipramine Binding: Medication vs. Medication-Free Patients and Inpatients Ys. Day Hospital Patients
N
X
SD 1.19, p
Bmax- (no-meds) Bmax (meds)
13 675.83 10 587.98
193.38 155.00
1'":
Bmax (inpatient) Bmax (day-hospital) Kdb (no-meds)
17 615.07 6 711.68
190.05 135.20
t: 1.14, p
< 0.25
< 0.26
13 10
1.57 1.45
1.26 0.71
MWd: 66.0 , p
< 0.81
Kd (meds) Kd (inpatient) Kd (day-hospital)
17 6
1.63 1.19
1.19 0.38
MW: 45.0, p
< 0.55
-Bmax: Number of binding sites (f moles/mg protein). bKd: Affinity constant sites (nM). Cl: Two-tailed r-test, dMW: Mann-Whitney.
11 0 0
BfMX
Pearson correlation: N-23 R- - 0.48 P-O.019
Results The total mean Bmax was 683 ± 184 fm/mg of protein and the total mean Kd was 1.56 ± 1.07 nM. Bmax was significantly inversely correlated with the CBCL Total Behavior Score (R = -0.47 , P = 0.02), the Externalizing Behavior (R = -0.48 , P = 0.02) and the Hostility Scores (R = -0 .53, p = 0.01) (Figures 1, 2, 3). Figure 4 shows the correlation between CBCL Aggression scores and Bmax. There was a single outlier subject who was 3 S.D . greater than zero. After omitting this subject, the aggression score was found to be significantly inversely correlated with Bmax (R = -0.50, P = 0.02). There were no correlations between Bmax and HDRS , the CTQ, other CBCL subscales, and the SAS (impulsivity and/or suicide subscales). All the correlations between the different variables and Kd were statistically nonsignificant. There was no correlation between platelet IB parameters and age.
FI G. I. Correlation between Child Behavior Checklist Total Behavior Scores and imipramine binding (BMAX)
".. . .
oo--~-~--~-~--~-~--~-~ BfMX Pearsoncorrelation: N-23 R.. - 0.48 P-O.0I8
2. Correlation between Child Behavior Checklist Externalizing Scores and imipramine binding (BMAX)
FI G.
Discussion As the authors hypothesized and consistent with the finding of Stoff et al. (1987), the total behavior, externalizing, hostile, and aggressive scores as measured by the CBCL were inversely correlated with the IB binding sites (Bmax) . These results are consistent with previous findings that low central serotonergic activity is associated with aggression . and impulsive behavior. Low central serotonergic activity has also been reported to be associated with depression and suicidal behavior (Birmaher et al., 1990). In this sample of patients, there was no correlation between the Bmax and the degree of depression as rated by HDRS and depressive scores of the CBCL or suicidality as rated by the HDRS . However, the mean scores for depression and suicidality were low, consistent with the criteria for patient selection that excluded those with a history of a previous suicide attempt. These findings indicate that it is possible to observe an association between a measure of serotonergic function and aggressive/impulsive behaviors, independent of suicidal behavior.
916
The CTQ factors for hyperactivity and conduct problems did not significantly correlate with the Bmax. This finding is also consistent with a previous platelet IB study in children with attention deficit disorder (Weizman et al., 1988). Thirty-five percent of the patients were taking lithium and neuroleptic medications; however , there was no difference in platelet IB and Kd with the medication-free patients . To the authors' knowledge, there are no reports of the effects of neuroleptics on platelet lB. The Bmax of platelet IB has been found to increase after treatment with some antidepressant medications, especially those that block presynaptic serotonin reuptake (Asberg and Wagner, 1986; Mellerup and Plenge 1986; Poirier et al., 1987; Wagner et al. , 1987). Prophylactic treatment with lithium showed a transient decrease in the platelet Bmax in euthymic bipolar patients (Asberg and Wagner, 1986). A second study showed that short-term treatment with lithium has no effect on Kd or Bmax of platelet IB (Poirier et al., 1988). Thus, in this l.Am.Acad. Child Adolesc . Psychiatry, 29:6, November 1990
IMIPRAMINE BINDING AND IMPULSIVE BEHAVIOR
SMAX
Pearsoncorrelation: N...23 R- -0.53 p - 0.001
FIG. 3. Correlation between Child Behavior Checklist Hostility Scores and imipramine binding (BMAX)
SMAX
Pearson OOt1"8Iatlon: N-23 ft- -0.32 P-O.125 PeanlOn oonelaUon (wflhout upper "Outlier") : N-22 A .. -0.50 P-O.01l5
FIG. 4. Correlation between Child Behavior Checklist Aggression Scores and imipramine binding (BMAX)
study, it is unlikely that the findings can be attributed to the effects of medication on this measure. In summary, it was found that children with marked impulsive, hostile and aggressive behavior had lower platelet lB. This result is consistent with previous reports that indicate that decreased serotonergic function is associated with aggressive behavior. However, platelet IB is a peripheral measure of serotonergic functioning, and it cannot be assumed that these findings reflect changes in the central serotonergic system. Additional studies with larger samples and normal controls using the platelet IB, serotonin metabolites in eSF, and neuroendocrine probes, such as the fenfluramine challenges test, will be necessary to evaluate the role of the serotonergic system in aggressive and impulsive behaviors . References Achenbach, T. M. & Edelbrock, C. S. (1983), Manualfor the Child Behavior Checklist and Revised Child Behavior Profile. Burlington,
J.Am.Acad. Child Adolesc. Psychiatry, 29:6, November 1990
VT.: University Associates in Psychiatry. Asberg, M. & Wagner, A. (1986), Biochemical effects of antidepressant treatment-studies of monoamine metabolites in cerebrospinal fluid and platelet imipramine binding. Ciba Found. Symp .• 123:57-83. - - Schalling, D., Traksman-Bendz, L. & Wagner, A. (1987), Psychobiology of suicide , impulsivity, and related phenomena. In: Psychopharmacology: Third Generation of Progress, ed. H. Y. Meltzer. New York: Raven Press, pp. 655-688. Bioulac , B., Benezech, M . & Renaud, B. (1980), Serotonergic dysfunction in the 47,XYY syndrome . Biol . Psychiatry. 15:917-923. Birmaher , B. , Greenhill, L. L. & Stanley, M. (1990), Biochemical studies of suicide. In: Application of Basic Neuroscience to Child Psychiatry, ed. Steve Deutch et al. New York: Plenum Press, pp . 313-322. Branchey, L., Branchey, M., Shaw, S. & Lieber, C. S. (1984), Depression, suicide, and aggression in alcoholics and their relationship to plasma amino acids. Psychiatry Res., 12:219-226. Briley M., Raisman R. & Langer S. Z. (1979), Human platelets possess high affinity binding sites for titrated imipramine . Eur. J . Pharmacol. , 58:347-348. Brown, G. L., Goodwin, F. K., Ballenger, J. C., Goyer, P. F. & Major, L. F. (1979), Aggression in humans correlates with cerebrospinal fluid metabolites . Psychiatry Res., 1:131-139. - - Ebert , M. H. , Goyer, P. F., Jimerson, D. C., Klein , W. J., Bunney , W. E. & Goodwin, F. K. (1982), Aggression, suicide, and serotonin: relationships to CSF amine metabolites. Am . J . Psychiatry. 139:741-746. Chatterjee, S. & Price, B. (1977), Detection and correction of model violations: simple linear regression. In: Regression Analysis by Example. New York: John Wiley & Sons, pp. 19-50. Coccaro, E., Siever , L., Klar, H. et al. (1989), Serotonergic studies in patients with affective disorders and personality disorders. Arch. Gen. Psychiatry , 46:587-599. Conners , C. (1976), Rating scales for use in drug studies with children. Psychopharmacol . Bull . (Special Issue), 59. Eichelman, B. (1987), Neurochemical and psychopharmacologic aspects of aggressive behavior. In: Psychopharmacology: The Third Generation ofProgress, ed. H. Y. Meltzer . New York: Raven Press, pp. 697-704. Guy, W. (1976), ECDEU Manual for Psychopharmacology . (U.S . Department of Health , Education , and Welfare, Publication ADM, 76-338). Washington, DC.: U.S. Government Printing Office. Lidberg, L., Tuck, J. R., Asberg, M., Scalia-Tomba, G-P. & Bertilsson, L. (1985), Homicide, suicide and CSF 5-HIAA. Acta Psychiatr. Scand., 71:230-236. Linnoila, M., Virkkunen, M., Scheinin , M ., Nuutila , A., Rimon, R. & Goodwin , F. K. (1983), Low cerebrospinal fluid 5-hydroxyindolacetic acid differentiates impulsive from non-impulsive violent behavior. Life Sci .• 33:2609-2614. Lowry, 0 ., Rosebrough, J. , Farr, A. L. & Randell, R. J. (1951), Protein measurements with Folin phenol reagent. J . Bioi. Chem., 193:265-275 . Mellerup , E. T. & Plenge , P. (1986), Chlorimipramine but not imipramine, rapidly reduces imipramine binding in human platelet membranes. Eur. J. Pharmacol . 126:155-158. Palkovits, M., Raisman , R., Briley, M. S. & Langer S. Z. (1981), Regional distribution of titrated imipramine binding in rat brain. Brain Res., 210:493-498 . Paul, S. M., Rehavi, M., Skolnick, P., Ballenger, J. C. & Goodwin, F . K . (1981), Does high affinity titrated imipramine binding label serotonin reputake sites in the brain and platelet? Life Sci., 28, 27532760. . Poirier, M. F., Galzin , A. M. , Loo, H. et al. (1987), Changes in 5HT uptake and imipramine binding in platelets after chlorimipramine in healthy volunteers: comparison with maprotiline and amineptine. Bioi. Psychiatry, 22:287-302. - - Galzin, A. M ., Pimoule , C. et al. (1988) , Short-term lithium administration to healthy volunteers produces long-lasting pronounced changes in platelet serotonin uptake but not imipramine binding. Psychopharmacology, 94:521-526 . Raisman, R., Briley, M.S . & Langer, S. Z. (1979), High affinity
917
BIRMAHER ET AL.
titrated imipramine binding in rat cerebral cortex. European Journal of Psychopharmacology, 54:307-308. Roy, A., Adinoff, B. & Linoila, M. (1988), Acting out hostility in normal volunteers: negative correlation with levels of 5-HIAA in cerebrospinal fluid. Psychiatry Res., 24:187-194. Sette, M., Raisman, R., Briley, M. S. & Langer, S. Z. (1981), Localization of tricyclic antidepressant binding sites on serotonin nerve terminals. J. Neurochem., 37:4Q-42. Stanley, M. & Mann, 1. J. (1983), Increased serotonin-2-binding sites in frontal cortex of suicide victims. Lancet, 1:214-216. - - Viggilio, J. & Gershon, S. (1982), Tritiated imipramine binding sites are decreasedin the frontal cortex of suicides. Science, 216:13371339. Stanley, B., Traskman-Bendz, L. & Stanley, M. (1986), The Suicide Assessment Scale: a scale evaluating change in suicidal behavior.
918
Psychopharmacology Bull., 22:200-205. Stoff, D. M., Pollack, L., Vitiello, B., Behar, D. & Bridger, W. H. (1987), Reduction of 3-H-imipramine binding sites on platelets of conduct disordered children. Neuropsychopharmacology, 155:62. Virkkunen, M., Nuutila, A., Goodwin, F. K. & Linnoila, M. (1987), Cerebrospinal fluid metabolite levels in male arsonists. Arch. Gen. Psychiatry,44:241-247. Wagner, A., Asberg-Wistedt, A., Asberg, M., Bertilsson, L., Martensson, B. & Montero, D. (1987), Effects of antidepressants treatments on platelet tritiated imipramine binding in major depressive disorder. Arch. Gen. Psychiatry, 44:870-877. Weizman, A., Bemhout, E., Weitz, R., Tyano, S. & Rehavi, M. (1988), Imipramine binding to platelets of children with attention deficit disorder with hyperactivity. Bioi. Psychiatry, 23:491-496.
l.Am.Acad. ChildAdolesc. Psychiatry, 29:6, November 1990
Abstract. ' . The serotonergic system has been implicated in the regulation of impulsive aggressive behavior either toward oneself or others. Imipramine binding sites were measured in the platelets of 23 impulsive aggressive children. Subject s ratings of total behavior, externalizing behavior, hostility, and aggression, as measured by the Child Behavior Checklist, were inversely correlated with the platelet imipramine binding. These findings are consistent with previous studies that suggest that decreased serotonergic activity is associated with impulsive aggressive behavior. J. Am. Acad . ChildAdolesc. Psychiatry, 1990,29,6:914-918. Key Words: platelet imipramine binding, impulsivity, aggression , children, serotonin . The serotonergic system has been reported to playa role in the regulation of impulsive aggressive behaviors (Asberg et al., 1987). Several investigators have shown an inverse correlation between impulsive aggressive behaviors and lower cerebrospinal fluid (CSF) 5-Hydroxy-indoleacetic acid (5HIAA) (Bioulac et al., 1980; Brown et al., 1982; Linnoila, et al., 1983; Lidberg et al. ; 1985; Virkkunen et al., 1987). This correlation between impulsive behaviors and CSF 5HIAA .has also been demonstrated in patients with borderline personality disorder (Brown et al., 1979) and in normal volunteers (Roy et al., 1988). Branchey et al. (1984) studied the ratio of tryptophan to other neutral amino acids in the serum of alcoholics . They found significant lower ratios, which suggest a deficiency of brain serotonin, in those subjects who had been arrested for assaultive behavior, when compared to other alcoholics or to nonalcoholic controls. Recently, Coccaro et al. (1989) reported an inverse correlation between the prolactin response to the 5-HT agonist, fenfluramine, and impulsive aggression in patients with personality disorder. Low CSF 5HIAA levels have also been reported in completed suicides and suicide attempters with different psychiatric diagnoses (Asberg et al. , 1987). Several postmortem studies have also reported low 5-HT and 5HIAA in the
hindbrain and certain raphe nuclei in suicide victims (Asberg et aI., 1987). Animal studies have shown that aggression can be regulated, at least in part, by the 5-HT system. These studies have shown that a decrease in central serotonin may predispose certain animal species for impulsive aggressive behavior (Eichelman, 1987). It has been demonstrated that high affinity imipramine binding sites (18) exist in the brain and in platelets of several species, including man (Briley et al., 1979; Raisman et al., 1979; Palkovits et al., 1981; Paul et aI. , 1981; Sette et al., 1981). In the brain , the 18 sites are distributed in the same pattern as 5-HT. In the platelet , IB is associated with the 5-HT uptake site (Briley et aI., 1979; Palkovits et aI., 1981; Paul et aI., 1981; Sette et aI., 1981). Stanley et al. (1982) and Stanley and Mann (1983) studied postmortem brain samples of patients who committed violent suicide and found reduced 18 sites in frontal cortices and an increase in postsynaptic 5HT-2 receptors, when compared with nonsuicide controls. Stoff et al. (1987) measured 18 sites in platelets of 10 drug free impulsive aggressive, conduct disorder children , ages 12 to 17 and 10 normal controls. Using the Child Behavior Checklist (CBCL), they observed a significant negative correlation between maximal platelet 18 and the factor for externalizing behavior (problems directed toward outside world) and aggression. However, Stoff et al. did not control for suicidality and the presence of depression variables which, by themselves, may have influenced the correlation they reported. In this study, 18 in platelets of children with impulsive and aggressive behaviors, who have had no history of suicidal attempts and affective disorders, were measured. It was hypothesized that platelet IB would be inversely correlated with the degree of aggression and/or impulsiveness.
Accepted June 28, 1990. Drs. Birmaher, Stanley, Greenhill, and Rabinovich are with the Department of Child Psychiatry, New York State Psychiatric Institute, and the Department of Psychiatry, Columbia University College of Physicians and Surgeons, New York. Drs. Birmaher, Gavrilescu, and Rabinovich are with the Manhattan Children's Psychiatric Center and Ms. Twomey is with the Department of Child and Adolescent Psychiatry, Western Psychiatric Institute and Clinic, University of Pittsburgh. Dr. Birmaher is now with the Department of Child and Adolescent Psychiatry, Western Psychiatric Institute and Clinic, University of Pittsburgh. Dr. Rabinovich is now with the Department of Child and Adolescent Psychiatry, University of Pennsylvania. This research was supported in part by Dr . Stanley's Grant: MH 42242 and MH 41847 from the U.S. Public Health Service and the Lowenstein Foundation. Reprint requests to Boris Birmaher, M.D ., Western Psychiatric Institute and Clinic, 3811 O'Hara Street, Pittsburgh, PA 15213. 0890-8567/9012906-0914$02 .00/0© 1990 by the American Academy of Child and Adolescent Psychiatry.
Method Children , ages 10 to 16, with a history of recurrent aggressive behavior and impulsivity were selected from the inpatient unit at Manhattan Children's Psychiatric Center and the Children's Day Hospital at New York Psychiatric Institute. Each patient was interviewed independently by 914
IMIPRAMINE BINDING AND IMPULSIVE BEHAVIOR
two child psychiatrists. After the interview, each psychiatrist reviewed the patient's records, and a joint decision was made regarding the patient's diagnosis. Diagnosis was made according to DSM -III criteria . Patients who had a history of recurrent aggression and/or impulsivity, who were not depressed and did not have a history of suicidal attempts , were accepted into the study . Inclusion also required that the patient and parents sign consent forms. Children were excluded from the study if they met any of the following criteria: (1) any significant medical illness; (2) IQ less than 70; (3) DSM-III diagnoses of affective disorder, schizophrenia, schizoaffective disorder, autism, or anorexia nervosa; (4) history of substance abuse within the past 6 months, and (5) history of suicide attempts. Patients were assessed with the following rating scales: CBCL (Achenbach and Edelbrock 1983), Hamilton Depression Rating Scale (HDRS) (Guy, 1976), Conner's Teacher Questionnaire (CTQ) (conduct and hyperactivity subscales) (Conners , 1976), and the Suicide Assessment Scale (SAS) (Stanley et aI., 1986). The HDRS and the SAS were administered by one of the child psychiatrists (B.B.); the CTQ was administered by the teacher and the CBCL was completed by the primary clinician who had managed the patient for at least 6 months. The CBCL has well documented psychometric properties and representative norms, reflecting direct observations of the most common complaints of children referred to child guidance clinics or mental health centers. Determination of Platelet IB The same week that the above rating scales were completed, patients had a single blood sample (30 ml) drawn for platelet lB. The blood was collected between 8:00 and 10:00 A.M., at room temperature, in 10 ml syringes containing ethylenediaminetetraacetate (EDTA) as an anticoagulant. Platelet rich plasma (PRP) was separated from erythrocytes and white blood cells by low speed centrifugation. Platelets were isolated on the day of collection (1 to 2 hours after being collected) and frozen until the IB assay. Platelet IB was determined in accordance with the methods described by Briley et al. (1979). PRP was spun for 10 minutes at 4C and 16,000 x1g. The pellet was rinsed 2 times with 3 ml 0.9% normal saline and centrifuged again. The pellet was frozen and not thawed until platelet membranes were prepared. The platelets were additionally lysed in 25 ml of 5mM Tris/HCL buffer, Ph 7.4, with 5mM EDTA and homogenized on a Polytron homogenizer for 15 seconds). The membranes were centrifuged at 4C, 39,000 x1g for 10 minutes. The membranes were rinsed twice in 25 ml of 70mM Tris/NCL, pH 7.4, with the same centrifugation. Final resuspension was in assay buffer 50 mM Tris/NCL, pH 7.4, with 120 mM NACL and 5mM KCL. Protein on the final homogenate was determined according to Lowry et al. (1951) and averaged approximately 2 mg/ml protein. Samples (175 f1L) of the homogenate were incubated in duplicate at 4C with 25 f1L of [3H] imipramine at a final concentration of 0.1 to 12 nM (specific activity, 55.4 Ci/ mmole from New England Nuclear) and with 25 f1L of assay J. Am .Acad. Child Adolesc. Psychiatry, 29:6, November 1990
buffer. Nonspecific binding was determined using a 10 f1M final concentration of desipramine. Total assay volume was 250 f1L. After 60 minutes, that incubate was diluted with 5 mL of ice-cold Trisma buffer and filtered through Whatman GF/C filters using a modified Brandell cell harvester. The filters were washed 2 more times with 5 ml of ice-cold Trisma buffer. The filters were counted in 10 ml Econofluor for 5 minutes on a Beckman LS 6800 liquid scintillation counter (counter efficiency better than 45%). The number of receptor sites (Bmax) and the affinity constant (Kd) were determined from the Scatchard plot for each individual's platelets. Statistical Analysis The data were tested for normality using the Shapiro and Wilk's W statistic. Kd did not follow a normal distribution, thus nonparametric statistics were used when Kd was involved. Differences between hospital status (inpatient versus day hospital) and medication status (psychotropic medications versus medication-free patients) on the IB parameters Bmax and Kd were tested by using the student r-test and Mann-Whitney, respectively. Bmax and Kd were correlated with each variable derived from the above named rating scales by using the Pearson Product Moment correlation and the Spearman Correlation, respectively. Whenever there were outlier subjects, correlations were made with and without these subjects. An outlier was defined as any subject whose standardized residual values were greater than two standard deviations from the mean of zero (Chatterjee and Price, 1977). Raw CBCL scores were used in all calculations. Sample Twenty-three boys were eligible for the study. Seventeen were inpatients, and six were from the day hospital. Eight of the 23 patients were taking medication during the period of the study. Three patients were taking lithium and a neuroleptic, and five were taking only neuroleptics (haloperidol, chlorpromazine or thioridazine); none of the day hospital patients were taking medications. Patients were on the average 12.6 SD ± 2.2 years old. All patients fulfilled DSM-III diagnostic criteria for conduct disorder, and 16 also fulfilled diagnostic criteria for attention deficit disorder with hyperactivity. The clinical picture of the patients ranged among moderate to severely hyperactive, impulsive, and physically or verbally aggressive behaviors. Relevant CBCL mean scores and standard deviations for this sample are as follows: total behavior: 62.2 ± 20.5; externalizing behavior (sum of scores for hostile, delinquent, aggressive arid hyperactive behaviors): 39 ± 12; aggression: 26.4 ± 10.2 , and hostility: 9.9 ± 4.8 . These mean scores were above the threshold considered pathological by the CBCL (38,21, 21, and 9, respectively). The mean HDRS score was 4.2 ± 4.6. The mean total score on the SAS was 24.4 ± 9.8. The SAS includes items for suicidality and impulsivity . The mean SAS suicidality subscale score was 0.76 ± 1.3, and the mean SAS impulsivity subscale score was 8.2 ± 2.8 . Neither medication nor hospitalization status statistically differentiated patients on the IB parameters (Table 1). Thus, all the groups were collapsed and the sample analyzed together. 915
BIRMAHER ET AL.
..
TABLE I. Platelet Imipramine Binding: Medication vs. Medication-Free Patients and Inpatients Ys. Day Hospital Patients
N
X
SD 1.19, p
Bmax- (no-meds) Bmax (meds)
13 675.83 10 587.98
193.38 155.00
1'":
Bmax (inpatient) Bmax (day-hospital) Kdb (no-meds)
17 615.07 6 711.68
190.05 135.20
t: 1.14, p
< 0.25
< 0.26
13 10
1.57 1.45
1.26 0.71
MWd: 66.0 , p
< 0.81
Kd (meds) Kd (inpatient) Kd (day-hospital)
17 6
1.63 1.19
1.19 0.38
MW: 45.0, p
< 0.55
-Bmax: Number of binding sites (f moles/mg protein). bKd: Affinity constant sites (nM). Cl: Two-tailed r-test, dMW: Mann-Whitney.
11 0 0
BfMX
Pearson correlation: N-23 R- - 0.48 P-O.019
Results The total mean Bmax was 683 ± 184 fm/mg of protein and the total mean Kd was 1.56 ± 1.07 nM. Bmax was significantly inversely correlated with the CBCL Total Behavior Score (R = -0.47 , P = 0.02), the Externalizing Behavior (R = -0.48 , P = 0.02) and the Hostility Scores (R = -0 .53, p = 0.01) (Figures 1, 2, 3). Figure 4 shows the correlation between CBCL Aggression scores and Bmax. There was a single outlier subject who was 3 S.D . greater than zero. After omitting this subject, the aggression score was found to be significantly inversely correlated with Bmax (R = -0.50, P = 0.02). There were no correlations between Bmax and HDRS , the CTQ, other CBCL subscales, and the SAS (impulsivity and/or suicide subscales). All the correlations between the different variables and Kd were statistically nonsignificant. There was no correlation between platelet IB parameters and age.
FI G. I. Correlation between Child Behavior Checklist Total Behavior Scores and imipramine binding (BMAX)
".. . .
oo--~-~--~-~--~-~--~-~ BfMX Pearsoncorrelation: N-23 R.. - 0.48 P-O.0I8
2. Correlation between Child Behavior Checklist Externalizing Scores and imipramine binding (BMAX)
FI G.
Discussion As the authors hypothesized and consistent with the finding of Stoff et al. (1987), the total behavior, externalizing, hostile, and aggressive scores as measured by the CBCL were inversely correlated with the IB binding sites (Bmax) . These results are consistent with previous findings that low central serotonergic activity is associated with aggression . and impulsive behavior. Low central serotonergic activity has also been reported to be associated with depression and suicidal behavior (Birmaher et al., 1990). In this sample of patients, there was no correlation between the Bmax and the degree of depression as rated by HDRS and depressive scores of the CBCL or suicidality as rated by the HDRS . However, the mean scores for depression and suicidality were low, consistent with the criteria for patient selection that excluded those with a history of a previous suicide attempt. These findings indicate that it is possible to observe an association between a measure of serotonergic function and aggressive/impulsive behaviors, independent of suicidal behavior.
916
The CTQ factors for hyperactivity and conduct problems did not significantly correlate with the Bmax. This finding is also consistent with a previous platelet IB study in children with attention deficit disorder (Weizman et al., 1988). Thirty-five percent of the patients were taking lithium and neuroleptic medications; however , there was no difference in platelet IB and Kd with the medication-free patients . To the authors' knowledge, there are no reports of the effects of neuroleptics on platelet lB. The Bmax of platelet IB has been found to increase after treatment with some antidepressant medications, especially those that block presynaptic serotonin reuptake (Asberg and Wagner, 1986; Mellerup and Plenge 1986; Poirier et al., 1987; Wagner et al. , 1987). Prophylactic treatment with lithium showed a transient decrease in the platelet Bmax in euthymic bipolar patients (Asberg and Wagner, 1986). A second study showed that short-term treatment with lithium has no effect on Kd or Bmax of platelet IB (Poirier et al., 1988). Thus, in this l.Am.Acad. Child Adolesc . Psychiatry, 29:6, November 1990
IMIPRAMINE BINDING AND IMPULSIVE BEHAVIOR
SMAX
Pearsoncorrelation: N...23 R- -0.53 p - 0.001
FIG. 3. Correlation between Child Behavior Checklist Hostility Scores and imipramine binding (BMAX)
SMAX
Pearson OOt1"8Iatlon: N-23 ft- -0.32 P-O.125 PeanlOn oonelaUon (wflhout upper "Outlier") : N-22 A .. -0.50 P-O.01l5
FIG. 4. Correlation between Child Behavior Checklist Aggression Scores and imipramine binding (BMAX)
study, it is unlikely that the findings can be attributed to the effects of medication on this measure. In summary, it was found that children with marked impulsive, hostile and aggressive behavior had lower platelet lB. This result is consistent with previous reports that indicate that decreased serotonergic function is associated with aggressive behavior. However, platelet IB is a peripheral measure of serotonergic functioning, and it cannot be assumed that these findings reflect changes in the central serotonergic system. Additional studies with larger samples and normal controls using the platelet IB, serotonin metabolites in eSF, and neuroendocrine probes, such as the fenfluramine challenges test, will be necessary to evaluate the role of the serotonergic system in aggressive and impulsive behaviors . References Achenbach, T. M. & Edelbrock, C. S. (1983), Manualfor the Child Behavior Checklist and Revised Child Behavior Profile. Burlington,
J.Am.Acad. Child Adolesc. Psychiatry, 29:6, November 1990
VT.: University Associates in Psychiatry. Asberg, M. & Wagner, A. (1986), Biochemical effects of antidepressant treatment-studies of monoamine metabolites in cerebrospinal fluid and platelet imipramine binding. Ciba Found. Symp .• 123:57-83. - - Schalling, D., Traksman-Bendz, L. & Wagner, A. (1987), Psychobiology of suicide , impulsivity, and related phenomena. In: Psychopharmacology: Third Generation of Progress, ed. H. Y. Meltzer. New York: Raven Press, pp. 655-688. Bioulac , B., Benezech, M . & Renaud, B. (1980), Serotonergic dysfunction in the 47,XYY syndrome . Biol . Psychiatry. 15:917-923. Birmaher , B. , Greenhill, L. L. & Stanley, M. (1990), Biochemical studies of suicide. In: Application of Basic Neuroscience to Child Psychiatry, ed. Steve Deutch et al. New York: Plenum Press, pp . 313-322. Branchey, L., Branchey, M., Shaw, S. & Lieber, C. S. (1984), Depression, suicide, and aggression in alcoholics and their relationship to plasma amino acids. Psychiatry Res., 12:219-226. Briley M., Raisman R. & Langer S. Z. (1979), Human platelets possess high affinity binding sites for titrated imipramine . Eur. J . Pharmacol. , 58:347-348. Brown, G. L., Goodwin, F. K., Ballenger, J. C., Goyer, P. F. & Major, L. F. (1979), Aggression in humans correlates with cerebrospinal fluid metabolites . Psychiatry Res., 1:131-139. - - Ebert , M. H. , Goyer, P. F., Jimerson, D. C., Klein , W. J., Bunney , W. E. & Goodwin, F. K. (1982), Aggression, suicide, and serotonin: relationships to CSF amine metabolites. Am . J . Psychiatry. 139:741-746. Chatterjee, S. & Price, B. (1977), Detection and correction of model violations: simple linear regression. In: Regression Analysis by Example. New York: John Wiley & Sons, pp. 19-50. Coccaro, E., Siever , L., Klar, H. et al. (1989), Serotonergic studies in patients with affective disorders and personality disorders. Arch. Gen. Psychiatry , 46:587-599. Conners , C. (1976), Rating scales for use in drug studies with children. Psychopharmacol . Bull . (Special Issue), 59. Eichelman, B. (1987), Neurochemical and psychopharmacologic aspects of aggressive behavior. In: Psychopharmacology: The Third Generation ofProgress, ed. H. Y. Meltzer . New York: Raven Press, pp. 697-704. Guy, W. (1976), ECDEU Manual for Psychopharmacology . (U.S . Department of Health , Education , and Welfare, Publication ADM, 76-338). Washington, DC.: U.S. Government Printing Office. Lidberg, L., Tuck, J. R., Asberg, M., Scalia-Tomba, G-P. & Bertilsson, L. (1985), Homicide, suicide and CSF 5-HIAA. Acta Psychiatr. Scand., 71:230-236. Linnoila, M., Virkkunen, M., Scheinin , M ., Nuutila , A., Rimon, R. & Goodwin , F. K. (1983), Low cerebrospinal fluid 5-hydroxyindolacetic acid differentiates impulsive from non-impulsive violent behavior. Life Sci .• 33:2609-2614. Lowry, 0 ., Rosebrough, J. , Farr, A. L. & Randell, R. J. (1951), Protein measurements with Folin phenol reagent. J . Bioi. Chem., 193:265-275 . Mellerup , E. T. & Plenge , P. (1986), Chlorimipramine but not imipramine, rapidly reduces imipramine binding in human platelet membranes. Eur. J. Pharmacol . 126:155-158. Palkovits, M., Raisman , R., Briley, M. S. & Langer S. Z. (1981), Regional distribution of titrated imipramine binding in rat brain. Brain Res., 210:493-498 . Paul, S. M., Rehavi, M., Skolnick, P., Ballenger, J. C. & Goodwin, F . K . (1981), Does high affinity titrated imipramine binding label serotonin reputake sites in the brain and platelet? Life Sci., 28, 27532760. . Poirier, M. F., Galzin , A. M. , Loo, H. et al. (1987), Changes in 5HT uptake and imipramine binding in platelets after chlorimipramine in healthy volunteers: comparison with maprotiline and amineptine. Bioi. Psychiatry, 22:287-302. - - Galzin, A. M ., Pimoule , C. et al. (1988) , Short-term lithium administration to healthy volunteers produces long-lasting pronounced changes in platelet serotonin uptake but not imipramine binding. Psychopharmacology, 94:521-526 . Raisman, R., Briley, M.S . & Langer, S. Z. (1979), High affinity
917
BIRMAHER ET AL.
titrated imipramine binding in rat cerebral cortex. European Journal of Psychopharmacology, 54:307-308. Roy, A., Adinoff, B. & Linoila, M. (1988), Acting out hostility in normal volunteers: negative correlation with levels of 5-HIAA in cerebrospinal fluid. Psychiatry Res., 24:187-194. Sette, M., Raisman, R., Briley, M. S. & Langer, S. Z. (1981), Localization of tricyclic antidepressant binding sites on serotonin nerve terminals. J. Neurochem., 37:4Q-42. Stanley, M. & Mann, 1. J. (1983), Increased serotonin-2-binding sites in frontal cortex of suicide victims. Lancet, 1:214-216. - - Viggilio, J. & Gershon, S. (1982), Tritiated imipramine binding sites are decreasedin the frontal cortex of suicides. Science, 216:13371339. Stanley, B., Traskman-Bendz, L. & Stanley, M. (1986), The Suicide Assessment Scale: a scale evaluating change in suicidal behavior.
918
Psychopharmacology Bull., 22:200-205. Stoff, D. M., Pollack, L., Vitiello, B., Behar, D. & Bridger, W. H. (1987), Reduction of 3-H-imipramine binding sites on platelets of conduct disordered children. Neuropsychopharmacology, 155:62. Virkkunen, M., Nuutila, A., Goodwin, F. K. & Linnoila, M. (1987), Cerebrospinal fluid metabolite levels in male arsonists. Arch. Gen. Psychiatry,44:241-247. Wagner, A., Asberg-Wistedt, A., Asberg, M., Bertilsson, L., Martensson, B. & Montero, D. (1987), Effects of antidepressants treatments on platelet tritiated imipramine binding in major depressive disorder. Arch. Gen. Psychiatry, 44:870-877. Weizman, A., Bemhout, E., Weitz, R., Tyano, S. & Rehavi, M. (1988), Imipramine binding to platelets of children with attention deficit disorder with hyperactivity. Bioi. Psychiatry, 23:491-496.
l.Am.Acad. ChildAdolesc. Psychiatry, 29:6, November 1990