·Platelet Imipramine Binding in Children and Adolescentswith 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 behavioreither toward oneself or others. Imipramine binding sites were measured in the platelets of 23 impulsive aggressivechildren. Subjects ratings of total behavior, externalizing behavior, hostility, and aggression, as measured by theChild Behavior Checklist, were inversely correlated with the platelet imipramine binding. These findings areconsistent with previous studies that suggest that decreased serotonergic activity is associated with impulsiveaggressive behavior. J. Am. Acad . ChildAdolesc. Psychiatry, 1990,29,6:914-918. Key Words: platelet imipraminebinding, impulsivity, aggression , children, serotonin .

The serotonergic system has been reported to playa rolein the regulation of impulsive aggressive behaviors (Asberget al., 1987). Several investigators have shown an inversecorrelation between impulsive aggressive behaviors and lowercerebrospinal 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 CSF5HIAA .has also been demonstrated in patients with bor­derline personality disorder (Brown et al., 1979) and innormal volunteers (Roy et al., 1988). Branchey et al. (1984)studied the ratio of tryptophan to other neutral amino acidsin the serum of alcoholics . They found significant lowerratios, which suggest a deficiency of brain serotonin, inthose subjects who had been arrested for assaultive behavior,when compared to other alcoholics or to nonalcoholic con­trols. Recently, Coccaro et al. (1989) reported an inversecorrelation between the prolactin response to the 5-HT ag­onist, fenfluramine, and impulsive aggression in patientswith 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

Accepted June 28, 1990.Drs. Birmaher, Stanley, Greenhill , and Rabinovich are with the

Department ofChild Psychiatry, New York State Psychiatric Institute,and the Department of Psychiatry, Columbia University College ofPhysicians and Surgeons, New York. Drs. Birmaher, Gavrilescu, andRabinovich are with the Manhattan Children's Psychiatric Center andMs. 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 Ado­lescentPsychiatry, Western Psychiatric Institute and Clinic, Universityof Pittsburgh. Dr. Rabinovich is now with the Department of Childand Adolescent Psychiatry, University of Pennsylvania.

This research was supported in part by Dr . Stanley's Grant: MH42242 and MH 41847 from the U.S. Public Health Service and theLowenstein Foundation.

Reprint requests to Boris Birmaher, M.D ., Western PsychiatricInstitute and Clinic, 3811 O'Hara Street , Pittsburgh, PA 15213.

0890-8567/9012906-0914$02 .00/0© 1990by the American Academyof Child and Adolescent Psychiatry.

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hindbrain and certain raphe nuclei in suicide victims (Asberget aI., 1987).

Animal studies have shown that aggression can be reg­ulated, at least in part, by the 5-HT system. These studieshave 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 imipraminebinding sites (18) exist in the brain and in platelets of severalspecies, 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 samepattern as 5-HT. In the platelet , IB is associated with the5-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 brainsamples of patients who committed violent suicide and foundreduced 18 sites in frontal cortices and an increase in post­synaptic 5HT-2 receptors, when compared with nonsuicidecontrols.

Stoff et al. (1987) measured 18 sites in platelets of 10drug free impulsive aggressive, conduct disorder children ,ages 12 to 17 and 10 normal controls. Using the ChildBehavior Checklist (CBCL), they observed a significantnegative correlation between maximal platelet 18 and thefactor for externalizing behavior (problems directed towardoutside world) and aggression. However, Stoff et al. didnot control for suicidality and the presence of depressionvariables which, by themselves, may have influenced thecorrelation they reported.

In this study, 18 in platelets of children with impulsiveand aggressive behaviors, who have had no history of su­icidal attempts and affective disorders, were measured. Itwas hypothesized that platelet IB would be inversely cor­related with the degree of aggression and/or impulsiveness.

Method

Children , ages 10 to 16, with a history of recurrent ag­gressive behavior and impulsivity were selected from theinpatient unit at Manhattan Children's Psychiatric Centerand the Children's Day Hospital at New York PsychiatricInstitute. Each patient was interviewed independently by

two child psychiatrists. After the interview, each psychia­trist reviewed the patient's records, and a joint decision wasmade regarding the patient's diagnosis. Diagnosis was madeaccording to DSM-III criteria . Patients who had a historyof recurrent aggression and/or impulsivity, who were notdepressed and did not have a history of suicidal attempts ,were accepted into the study. Inclusion also required thatthe patient and parents sign consent forms. Children wereexcluded from the study if they met any of the followingcriteria: (1) any significant medical illness; (2) IQ less than70; (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 TeacherQuestionnaire (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 CTQwas administered by the teacher and the CBCL was com­pleted by the primary clinician who had managed the patientfor at least 6 months. The CBCL has well documentedpsychometric properties and representative norms, reflect­ing direct observations of the most common complaints ofchildren referred to child guidance clinics or mental healthcenters.

Determination of Platelet IB

The same week that the above rating scales were com­pleted, patients had a single blood sample (30 ml) drawnfor platelet lB. The blood was collected between 8:00 and10:00 A.M., at room temperature, in 10 ml syringes con­taining ethylenediaminetetraacetate (EDTA) as an antico­agulant. Platelet rich plasma (PRP) was separated fromerythrocytes and white blood cells by low speed centrifu­gation. Platelets were isolated on the day of collection (1to 2 hours after being collected) and frozen until the IBassay.

Platelet IB was determined in accordance with the meth­ods described by Briley et al. (1979). PRP was spun for 10minutes at 4C and 16,000 x1g. The pellet was rinsed 2 timeswith 3 ml 0.9% normal saline and centrifuged again. Thepellet was frozen and not thawed until platelet membraneswere prepared. The platelets were additionally lysed in 25ml of 5mM Tris/HCL buffer, Ph 7.4, with 5mM EDTA andhomogenized on a Polytron homogenizer for 15 seconds).The membranes were centrifuged at 4C, 39,000 x1g for 10minutes. The membranes were rinsed twice in 25 ml of70mM 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 onthe final homogenate was determined according to Lowryet al. (1951) and averaged approximately 2 mg/ml protein.

Samples (175 f1L) of the homogenate were incubated induplicate at 4C with 25 f1L of [3H] imipramine at a finalconcentration 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

IMIPRAMINE BINDING AND IMPULSIVE BEHAVIOR

buffer. Nonspecific binding was determined using a 10 f1Mfinal concentration of desipramine. Total assay volume was250 f1L. After 60 minutes, that incubate was diluted with5 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-coldTrisma buffer. The filters were counted in 10 ml Econofluorfor 5 minutes on a Beckman LS 6800 liquid scintillationcounter (counter efficiency better than 45%). The numberof receptor sites (Bmax) and the affinity constant (Kd) weredetermined from the Scatchard plot for each individual'splatelets.

Statistical AnalysisThe 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 versusday hospital) and medication status (psychotropic medica­tions versus medication-free patients) on the IB parametersBmax and Kd were tested by using the student r-test andMann-Whitney, respectively. Bmax and Kd were correlatedwith each variable derived from the above named ratingscales by using the Pearson Product Moment correlation andthe Spearman Correlation, respectively. Whenever there wereoutlier subjects, correlations were made with and withoutthese subjects. An outlier was defined as any subject whosestandardized residual values were greater than two standarddeviations from the mean of zero (Chatterjee and Price,1977). Raw CBCL scores were used in all calculations.

SampleTwenty-three boys were eligible for the study. Seventeen

were inpatients, and six were from the day hospital. Eightof the 23 patients were taking medication during the periodof 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 theaverage 12.6 SD ± 2.2 years old. All patients fulfilledDSM-III diagnostic criteria for conduct disorder, and 16 alsofulfilled diagnostic criteria for attention deficit disorder withhyperactivity. The clinical picture of the patients rangedamong moderate to severely hyperactive, impulsive, andphysically or verbally aggressive behaviors. Relevant CBCLmean scores and standard deviations for this sample are asfollows: 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 abovethe 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 . Themean SAS suicidality subscale score was 0.76 ± 1.3, andthe mean SAS impulsivity subscale score was 8.2 ± 2.8 .

Neither medication nor hospitalization status statisticallydifferentiated patients on the IB parameters (Table 1). Thus,all the groups were collapsed and the sample analyzed to­gether.

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BIRMAHER ET AL.

TABLE I. Platelet Imipramine Binding: Medication vs.Medication-Free Patients and Inpatients Ys. Day Hospital Patients

N X SD

Bmax- (no-meds) 13 675.83 193.38 1'": 1.19, p < 0.25Bmax (meds) 10 587.98 155.00

Bmax (inpatient) 17 615.07 190.05 t:1.14, p < 0.26Bmax (day-hospital) 6 711.68 135.20

Kdb (no-meds) 13 1.57 1.26 MWd: 66.0 , p < 0.81Kd (meds) 10 1.45 0.71

Kd (inpatient) 17 1.63 1.19 MW: 45.0, p < 0.55Kd (day-hospital) 6 1.19 0.38

-Bmax: Number of binding sites (f moles/mg protein).bKd: Affinity constant sites (nM).Cl: Two-tailed r-test,dMW: Mann-Whitney.

Results

The total mean Bmax was 683 ± 184 fm/mg of proteinand the total mean Kd was 1.56 ± 1.07 nM. Bmax wassignificantly inversely correlated with the CBCL Total Be­havior Score (R = -0.47, P = 0.02), the ExternalizingBehavior (R = -0.48, P = 0.02) and the Hostility Scores(R = -0.53, p = 0.01) (Figures 1, 2, 3). Figure 4 showsthe correlation between CBCL Aggression scores and Bmax.There was a single outlier subject who was 3 S.D . greaterthan zero. After omitting this subject, the aggression scorewas 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 (impulsivityand/or suicide subscales).

All the correlations between the different variables andKd were statistically nonsignificant. There was no corre­lation between platelet IB parameters and age.

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 CBCLwere inversely correlated with the IB binding sites (Bmax) .These results are consistent with previous findings that lowcentral serotonergic activity is associated with aggression .and impulsive behavior. Low central serotonergic activityhas also been reported to be associated with depression andsuicidal behavior (Birmaher et al., 1990). In this sample ofpatients, there was no correlation between the Bmax andthe degree of depression as rated by HDRS and depressivescores of the CBCL or suicidality as rated by the HDRS.However, the mean scores for depression and suicidalitywere low, consistent with the criteria for patient selectionthat excluded those with a history of a previous suicideattempt. These findings indicate that it is possible to observean association between a measure of serotonergic functionand aggressive/impulsive behaviors, independent of suicidalbehavior.

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..

11 0 0

BfMX

Pearsoncorrelation: N-23 R- - 0.48 P-O.019

FIG. I. Correlation between Child Behavior Checklist Total BehaviorScores and imipramine binding (BMAX)

"......oo--~-~--~-~--~-~--~-~BfMX

Pearsoncorrelation: N-23 R.. - 0.48 P-O.0I8

FIG. 2. Correlation between Child Behavior Checklist ExternalizingScores and imipramine binding (BMAX)

The CTQ factors for hyperactivity and conduct problemsdid not significantly correlate with the Bmax. This findingis also consistent with a previous platelet IB study in childrenwith attention deficit disorder (Weizman et al., 1988).

Thirty-five percent of the patients were taking lithium andneuroleptic medications; however , there was no differencein platelet IB and Kd with the medication-free patients . Tothe authors' knowledge, there are no reports of the effectsof neuroleptics on platelet lB. The Bmax of platelet IB hasbeen found to increase after treatment with some antide­pressant medications, especially those that block presynapticserotonin reuptake (Asberg and Wagner, 1986; Mellerupand 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 thatshort-term treatment with lithium has no effect on Kd orBmax of platelet IB (Poirier et al., 1988). Thus, in this

l.Am.Acad. Child Adolesc .Psychiatry, 29:6, November 1990

SMAX

Pearsoncorrelation: N...23 R- -0.53 p - 0.001

FIG. 3. Correlation between Child Behavior Checklist Hostility Scoresand imipramine binding (BMAX)

SMAX

Pearson OOt1"8Iatlon: N-23 ft- -0.32 P-O.125PeanlOn oonelaUon (wflhout upper "Outlier") : N-22 A .. -0.50 P-O.01l5

FIG. 4. Correlation between Child Behavior Checklist AggressionScores and imipramine binding (BMAX)

study, it is unlikely that the findings can be attributed tothe effects of medication on this measure.

In summary, it was found that children with marked im­pulsive, hostile and aggressive behavior had lower plateletlB. This result is consistent with previous reports that in­dicate that decreased serotonergic function is associated withaggressive behavior. However, platelet IB is a peripheralmeasure of serotonergic functioning, and it cannot be as­sumed that these findings reflect changes in the central se­rotonergic system. Additional studies with larger samplesand normal controls using the platelet IB, serotonin metab­olites in eSF, and neuroendocrine probes, such as the fen­fluramine challenges test , will be necessary to evaluate therole of the serotonergic system in aggressive and impulsivebehaviors .

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