Influence of clozapine on platelet serotonin, monoamine oxidase and plasma serotonin levels

Psychiatry Research 149 (2007) 49 – 57 www.elsevier.com/locate/psychres

Influence of clozapine on platelet serotonin, monoamine oxidase and plasma serotonin levels Aygun Ertugrul a,⁎, Gulberk Ucar b , Koray Basar a , Basaran Demir a , Samiye Yabanoglu b , Berna Ulug a a

Department of Psychiatry, Hacettepe University, Faculty of Medicine, Ankara 06100, Turkey b Department of Biochemistry, Hacettepe University, Faculty of Pharmacy, Turkey

Received 13 July 2005; received in revised form 4 October 2005; accepted 28 December 2005

Abstract The purpose of this study was to investigate the influence of clozapine on plasma serotonin, platelet serotonin and monoamine oxidase (MAO) levels in schizophrenic patients and to compare their results with those of unmedicated healthy controls. Groups of 20 outpatients with schizophrenia and 20 healthy controls matched for age, sex and smoking status were recruited for the study. Psychopathology, neurocognitive functioning, plasma serotonin, platelet serotonin and MAO levels were assessed after 1-week drug free interval, and 8 weeks after initiation of clozapine treatment in an open design. The mean clozapine dose at week 8 was 382.5 ± 96.4 (range: 250–600) mg/day. In the patient group, at baseline, plasma serotonin and platelet MAO levels were significantly lower, and platelet serotonin levels were significantly higher than in controls. After 8 weeks of clozapine treatment, plasma serotonin and platelet MAO levels increased significantly, while a significant decrease in platelet serotonin levels was detected compared with baseline values. Baseline platelet MAO levels explained 22% of the variance in Clinical Global Impression–Improvement (CGI–I) and improvement in attention, while baseline platelet serotonin predicted 23% of the variance in the improvement in positive symptoms during clozapine treatment. Our data indicate that clozapine may be reversing or compensating for a pre-existing alteration in serotonergic neurotransmission in schizophrenic patients. The prediction of response to clozapine through peripheral biochemical markers may have important clinical implications if repeated in larger samples. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Clozapine; Monoamine oxidase; Platelets; Serotonin; Schizophrenia

1. Introduction Clozapine is a prototypical atypical antipsychotic with higher affinity for 5-hydroxytryptamine 2 (5-HT2) than to dopamine 2 (D2) receptors (Meltzer et al., 1989). It has been shown to be effective in treating schizo-

⁎ Corresponding author. Tel.: +90 3123051873; fax: +90 3123101938. E-mail address: [email protected] (A. Ertugrul).

phrenic patients who are refractory to neuroleptics or have severe extrapyramidal side effects while taking other antipsychotics. Clozapine improves negative symptoms and various aspects of cognitive functions such as attention, verbal fluency and executive functions in patients with schizophrenia (Meltzer and McGurk, 1999). The therapeutic success of serotonergic-dopaminergic antagonists has focused attention on the role of serotonin in schizophrenia. Several studies have

0165-1781/$ - see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.psychres.2005.12.009

50

A. Ertugrul et al. / Psychiatry Research 149 (2007) 49–57

examined the hypothesis that a dysfunction in the central serotonergic system may be involved in its pathophysiology (Joyce et al., 1993; Grace, 2000). Blood platelets are widely used as an easily obtainable peripheral model for uptake, storage and release of serotonin, as well as 5-HT2A receptors in the central serotonergic neurons (Stahl, 1985). In clinical studies, platelet serotonin elements such as granular serotonin, membrane serotonin transporter, 5-HT2 receptor and monoamine oxidaseB (MAO-B) have been investigated as potential state or trait markers, or pharmacodynamic indicators in the course of treatment with serotonin-related drugs (Jernej et al., 2000). Platelet serotonin levels are found to be increased in patients with schizophrenia who were not receiving neuroleptic treatment (Borcsiczky et al., 1996; MuckSeler et al., 2004). The increased platelet serotonin levels are reported especially in chronic paranoid patients and in patients who have prominent positive symptoms (Muck-Seler et al., 1991; Jakovljevic et al., 1997; Pivac et al., 1997). However, there are contradictory findings regarding the effects of neuroleptics on whole blood and platelet serotonin levels. Some investigators claim that neuroleptics increase blood serotonin levels (Joseph et al., 1977) while others note the opposite (Garelis et al., 1975). Kaneda et al. (2001) found that neuroleptics decreased the platelet serotonin levels and this effect was not related to the severity of psychopathology. In a recent study in which patients were treated with atypical antipsychotics, plasma and platelet serotonin were reported to increase in poor responders and decrease in responders to treatment (van der Heijden et al., 2004). Clozapine was reported to increase blood serotonin levels (Banki, 1978). Clozapine-treated young patients have been found to have higher blood serotonin than neuroleptic-treated patients (Schulz et al., 1997). Fleischhaker et al. (1998) also reported an increase in serum serotonin levels from pretreatment levels after 6 weeks of clozapine treatment in children and adolescent patients with schizophrenia. In addition, they demonstrated that patients who became responders to clozapine showed a negative linear relationship between negative symptoms of schizophrenia and concentrations of plasma norepinephrine and serum serotonin. Conversely, Ichikawa et al. (1998) reported that clozapine and risperidone significantly increased extracellular serotonin concentrations in rat medial frontal cortex and nucleus accumbens, whereas olanzapine, another atypical neuroleptic, or the typical antipsychotic haloperidol induced no significant effects in either region.

Platelet monoamine oxidase-B (MAO-B) activity, another platelet serotonin measure, has been found to be significantly decreased in chronic schizophrenic patients (Jackman and Meltzer, 1980), especially in paranoid and hallucinating ones (Zureick and Meltzer, 1988; Buckman et al., 1990; Meszaros et al., 1998), although others did not confirm these results (MuckSeler et al., 1991). Regarding the effects of neuroleptics on platelet MAO-B activity, some investigators reported a decrease (Meltzer et al., 1982; Marcolin and Davis, 1992). Maj et al. (1987) reported that drug status (on neuroleptics vs. off neuroleptics) correctly classified 63.4% of patients where a higher percentage of patients on neuroleptics had low MAO. Other investigators also suggested that low platelet MAO values observed in chronic schizophrenia might be in part an effect of neuroleptic treatment (Del Vecchio et al., 1983). Haloperidol and its metabolites were found to inhibit MAO-B activity (Fang et al., 1995) while clozapine was shown not to inhibit intraneural MAO in schizophrenic patients (Elman et al., 1999). Moreover, in an in vitro study, a significant clozapine-related increase of MAO-B velocity was detected in hippocampal HT22 cells (Heiser et al., 2000). Collectively, the results from the studies cited above appear to be rather conflicting, and it is not yet completely clear what actions clozapine exerts on peripheral parameters of the serotonin system. Some other points that make it harder to reach a conclusion from the relevant literature are the differences in biochemical parameters studied, such as whole blood serotonin, serum serotonin, platelet serotonin, and the techniques used to measure them. The objectives of this study are to investigate the influence of clozapine on plasma serotonin, platelet serotonin and platelet MAO levels in schizophrenic patients and to study the role of these biochemical parameters in predicting the response to clozapine. Furthermore, the relationship of the change in these parameters to the change in positive or negative symptom dimensions and cognitive functions in clozapine is studied. 2. Methods 2.1. Subjects This open study was conducted in the Department of Psychiatry, Hacettepe University School of Medicine. The experimental comprised 20 consecutive outpatients who met DSM-IV criteria for schizophrenia whose treating psychiatrist decided to initiate clozapine due to

A. Ertugrul et al. / Psychiatry Research 149 (2007) 49–57

treatment refractoriness or intolerance of previous antipsychotics. In addition, 20 healthy controls who were matched for age, sex and smoking status were recruited for the study by local advertisement. Exclusion criteria were alcohol or drug abuse/dependency, chronic use of any other medication and any major medical or neurological disorder that would affect platelet and plasma serotonin levels. In addition, subjects in the control group did not have any psychiatric disorder. Informed consent was obtained from all subjects for the research in this study. There were 11 male and 9 female patients in each group. The mean age was 29.75 ± 5.55 in patients, 32.15 ± 9.90 in controls. The mean duration of illness was 8.05 ± 7.07 years in the patient group. Fourteen subjects were smoking, 6 were non-smoking in each group. All patients had paranoid schizophrenia and had a history of using multiple typical and atypical antipsychotics. Thirteen patients were on risperidone, five patients were on olanzapine, one patient was on amisulpiride and one was on pimozide at the time of the initiation of the study. 2.2. Procedure Patients were given clozapine treatment after 1 drugfree week. The clozapine dose was titrated according to the clinical status of the patient. The mean clozapine dose at week 8 was 382.5 ± 96.4 (range: 250–600) mg/ day. There were no dropouts during the 8 weeks treatment with clozapine. No serious adverse effect was reported. Psychopathology, neurocognitive functioning, plasma serotonin, platelet serotonin and platelet MAO levels were assessed at baseline and 8 weeks after initiation of clozapine treatment. Psychopathology was assessed with the Positive and Negative Syndrome Scale (PANSS). At week 8, improvement were made on the Clinical Global Impressions Scale. The Neurocognitive functions such as attention, executive functions, memory, verbal fluency and working memory were evaluated by a neuropsychological test battery. Plasma serotonin, platelet serotonin and platelet MAO levels of controls were also measured.

51

scale scored on the basis of a clinical interview lasting 30–45 min. It consists of three subscales: the positive syndrome scale, the negative syndrome scale and the general psychopathology scale. The reliability and validity of PANSS in the Turkish population have been tested (Kostakoğlu et al., 1999). 2.3.2. Clinical Global Impressions–improvement I (CGI–I) The clinical status of patients at week 8 is compared with the status at baseline, and the degree of change is rated on a 1 to 7 scale as follows: 1 = very much improved, 2 = much improved, 3 = minimally improved, 4 = no change, 5 = minimally worse, 6 = much worse, 7 = very much worse. 2.3.3. Digit span The Digit Span Test has two components: digit span forward and digit span backward. During the Digit Span Forward Test, the patients are asked to repeat immediately an increasing series of numbers read by the tester. During the Digit Span Backward Test, patients are asked to repeat the numbers in reverse order. The Digit Span Forward primarily measures attention, while the Digit Span Backward Tests mental control and working memory (Wechsler, 1987). 2.3.4. Auditory Consonant Trigram Test (ACT) The Turkish version of the ACT test was used to assess verbal working memory (Anil et al., 2003). A consonant trigram is delivered to the subject verbally at a rate of one letter per second followed immediately by a random number. The subject is asked to count out loud backwards from the number for interval delays of 3, 9, and 18 s used at random. Total number of letters correctly recalled is scored.

2.3. Instruments

2.3.5. Word and category fluency The purpose of the Word Fluency Test is to assess the spontaneous production of words beginning with a given letter or of a given class within a certain time period. In the word fluency test, the subject is asked to produce as many words as possible beginning with a letter in 1 min. Three letters are given, and the sum of all admissible words for the three letters is the score. In the Category Fluency Test, the patient is asked to produce as many words as possible within the categories of animal and human names (Benton and Hamsher, 1978).

2.3.1. Positive and Negative Syndrome Scale (PANSS) Symptoms were assessed by using the PANSS (Kay et al., 1987) at baseline and at week 8 of clozapine treatment. The PANSS is an interviewer-administered

2.3.6. Rey Auditory-Verbal Learning Test (RAVLT) Verbal learning and memory were assessed by the RAVLT (Lezak, 1995). The test consists of 15 nouns (List A) read aloud for five consecutive trials, each

52

A. Ertugrul et al. / Psychiatry Research 149 (2007) 49–57

trial followed by a free recall test. The order of presentation of words remains fixed across trials. Upon completion of trial 5, an interference list of 15 words (List B) is presented, followed by a free recall test of that list. Immediately following this, delayed recall of the first list is tested without further presentation of these words. After a 20-min delay period, each subject is again required to recall words from List A. The score for each trial is the number of words correctly recalled. Finally, recognition is tested by a matrix array in which patients must identify List A words from a list of 50 words containing all items from Lists A and B and 20 words phonemically and/or semantically similar to those in Lists A and B. This tests allows measurement of immediate memory span, new learning, susceptibility to interference and recognition memory. 2.3.7. Wechsler Visual Memory Scale–Visual Reproduction Scale In order to assess visual memory, the Visual Reproduction subtest of the Wechsler Memory Scalerevised (Wechsler, 1987) was given. This test requires the subject to draw from memory simple geometric figures that were exposed briefly. To measure delayed visual memory, the Visual Reproduction Scale is repeated after 30 min. 2.3.8. Wisconsin Card Sorting Test (WCST) Executive function was assessed by WCST (Heaton, 1981). The purpose of this test is to assess the ability to form abstract concepts, and to shift and maintain the set. During this test, patients are asked to match a series of cards according to sorting criteria (i.e., color, number or shape) that change each time 10 consecutive cards are correctly sorted. Performance is scored in a number of different ways. Categories achieved refer to the number of correct sorts, ranging from 0 to 6. Perseverative errors are also calculated, and it is the best measure of frontal lobe involvement. 2.4. Determination of plasma and platelet serotonin levels Blood samples were drawn by venipuncture after an overnight fast into tubes containing EDTA as anticoagulant. Ten milliliters of blood were divided into two portions. One portion was centrifuged for 5 min at 1000 ×g and the supernatant was kept as plasma. The next portion was centrifuged for 5 min at 10,000 ×g and 4 °C, to obtain platelet-rich plasma (PRP). Platelet counts were determined on aliquots of pooled PRP diluted in Isoton

II and counted twice on a thrombocounter (Coulter Electronics, STKS). After the platelet count, 2 ml PRP was centrifuged for 10 min at 2000 ×g. The supernatant was discarded and the pellet was suspended in 1 ml of a mixture containing 4% perchloric acid and 0.15% EDTA. The mixture was centrifuged for 10 min at 2000 ×g and 4 °C. The resulting supernatant was filtered through 0.45-μm Millipore filters by centrifugation for 5 min at 2000 ×g (Sigma, microcentrifuge filter ultrafree-CL, Durapore PVDF membrane) and 4 °C (Schmidt et al., 1997). The eluate was divided into two portions: one portion was used for the determination of platelet MAO activity while the rest was used for the determination of platelet serotonin level. The samples were kept frozen −80 °C until used. The portions kept for serotonin determination in plasma and PRP were thawed and centrifuged for 3 min at 2000 ×g. Plasma and platelet serotonin contents were determined according to a previous method (Schmidt et al., 1997). Aliquot of the supernatants was applied to the HPLC system equipped with a 5-μm C18 column. The elution buffer consisted of 50 mM potassium phosphate, pH 5.0 and 12% methanol, with flow rate of 1 ml min− 1 under isocratic conditions. The fluorescence detector (Dionex, RF 2000) was set at 230 nm excitation and 338 nm emission. Plasma and platelet serotonin contents were expressed as nanomole per liter and nanomole per 109 platelets, respectively. 2.5. Determination of platelet MAO activity Platelet MAO-B activity was measured in PRP samples by the method of Holt (Holt et al., 1997). A chromogenic solution, which consisted of 1 mM vanillic acid, 500 μM 4-aminoantipyrine, and 4 U ml − 1 peroxidase in 0.2 M potassium phosphate buffer, pH 7.6, was prepared daily. The assay mixture contained 167 μl chromogenic solution, 667 μl 500 μM benzylamine, and 133 μl KP buffer, pH 7.6. The mixture was preincubated at 37 °C for 10 min before the addition of enzyme. A reaction was initiated by the addition of the PRP (100 μl) and absorbance increase was monitored at 498 nm at 37 °C for 60 min. A molar absorption coefficient of 4654 M− 1. cm− 1 was used to calculate the initial velocity of the reaction. Results were expressed as nanomole per 109 platelets. 2.6. Reagents and equipment All chemicals were obtained from the Sigma Chemical Co. (Germany). Spectrophotometric measurements were

A. Ertugrul et al. / Psychiatry Research 149 (2007) 49–57

performed using a Shimadzu 1601 PC spectrophotometer, and HPLC measurements were performed using the HPLC system of Dionex, USA.

53

cantly different, although to a lesser extent (t = 2.5, df = 38, P b 0.05). 3.2. Comparison of biochemical and clinical parameters at baseline and at week 8 of clozapine treatment in the patient group

2.7. Clinical chemistry Biochemical parameters were measured in the plasma samples of the subjects in an autoanalyser (Roche Modular System) and the hematological parameters were determined using an electronic cell counter (Coulter STKS). 2.8. Statistical analysis Results are expressed as mean values with standard deviations (mean ± S.D.). Independent sample t tests and paired sample t tests are applied to test the statistical significance of differences between means. The relationship between the biochemical, clinical and cognitive parameters was assessed by Pearson correlation analysis. The predictive value of baseline biochemical parameters relative to improvement in clinical parameters was investigated with stepwise regression analysis. Level of significance was set as P b 0.05.

In order to detect the influence of clozapine treatment on plasma serotonin, platelet serotonin and platelet MAO measures in the patient group, baseline levels were compared with the values at week 8. Plasma serotonin and platelet MAO levels were found to be increased significantly (P b 0.001) while a significant decrease in platelet serotonin levels was detected (P b 0.001) (Table 1). There were significant improvements in the PANSS total score and in the positive, negative and general psychopathology subscores during the study. The mean CGI–I at week 8 was 2.95 ± 1.05. Regarding neurocognitive tests, digit span forward and word fluency scores were found to be significantly improved after 8 weeks of clozapine treatment. There were no significant differences in other cognitive measures (Table 2). 3.3. Relationship between the biochemical parameters, symptoms and cognitive functions

3. Results 3.1. Comparison of biochemical parameters between patient and control groups In the patient group, the baseline plasma serotonin and platelet MAO levels were significantly lower, and baseline platelet serotonin levels were significantly higher than in controls (P b 0.001) (Table 1). After 8 weeks of clozapine treatment, all biochemical results of patients approached those of controls. When the biochemical parameters of patients at week 8 of clozapine treatment were compared with those of controls, the difference with regard to plasma serotonin and platelet MAO levels was no longer significant, while platelet serotonin was still signifi-

The relationship of the changes in total PANSS, digit span forward and word fluency scores to the changes in plasma serotonin, platelet serotonin and platelet MAO levels after 8 weeks of clozapine treatment were assessed by Pearson correlation analyses which revealed no significant correlation between these parameters. The baseline platelet MAO level was negatively correlated with CGI–I scores (r = − 0.46, P = 0.04), and positively correlated with the change in digit span scores (r = 0.47, P = 0.04). High baseline platelet MAO was related to more improvement in both CGI and digit span score. On the other hand, baseline platelet serotonin was negatively correlated with improvement in positive symptoms (r = −0.48, P = 0.03).

Table 1 The biochemical results of patient and control groups (mean ± S.D.)

Plasma serotonin (nmol/l) Platelet serotonin (nmol/109 pl) Platelet MAO (nmol/109 pl)

Patient group before clozapine (N = 20)

Patient group after clozapine (8th week) (N = 20)

Control group (N = 20)

Before/after clozapine

Patient (before clozapine)/ control

6.36 ± 0.36 8.59 ± 1.37 29.65 ± 2.23

9.96 ± 0.60 6.30 ± 0.58 35 ± 1.68

10.16 ± 0.5 5.91 ± 0.4 35.3 ± 2.6

t = 28.1, df=19 ⁎ t = 8.45, df = 19 ⁎ t = 12.37, df = 19 ⁎

t = 27.65, df = 38 ⁎ t = 8.48, df = 38 ⁎ t = 7.34, df = 38 ⁎

* P b 0.001, MAO: monoamine oxidase.

54

A. Ertugrul et al. / Psychiatry Research 149 (2007) 49–57

Table 2 The clinical and neuropsychological results of patients before and after clozapine treatment (mean ± S.D.) Patient group Patient group Before/ before after clozapine after clozapine (8th week) (N = 20) clozapine (N = 20) PANSS total

79.55 ± 23.96

60.2 ± 21.53

PANSS positive symptoms PANSS negative symptoms PANSS general psychopathology Digit span forward

18.60 ± 6.55

14.15 ± 5.05

20.75 ± 8.77

16.6 ± 7.6

40.7 ± 12.76 29.55 ± 11.33 6.42 ± 2.48

7.21 ± 2.68

4.63 ± 1.89

4.95 ± 2.25

Digit span backward ACT

40.74 ± 10.29 44.26 ± 11.84

Word fluency

23.21 ± 12.28 28.32 ± 13.60

Category 16.53 ± 4.69 generation-animal Category 17.74 ± 5.84 generation-name RAVLT-immediate 5.32 ± 2.06 memory RAVLT-learning 1 8.95 ± 2.34

18.53 ± 6.65 19.84 ± 6.39 5.32 ± 1.99 9.53 ± 1.90

RAVLT-learning 2

36.63 ± 8.60

40.11 ± 9.77

RAVLT delayed memory WCST total category WCST percentage perseverative errors WMS-visual reproduction I WMS-visual reproduction II

6.58 ± 2.52

7.37 ± 2.79

3.53 ± 2.34

3.74 ± 2.45

21.38 ± 16.85 19.62 ± 12.05

30.10 ± 11.09 30.89 ± 10.4 25.05 ± 11.05 27.63 ± 11.04

t = 4.85, df = 19⁎⁎⁎ t = 3.72, df = 19⁎⁎⁎ t = 3.18, df = 19⁎⁎ t = 4.56, df = 19⁎⁎⁎ t = 2.28, df = 18⁎ t = 0.74, df = 18 t = 1.32, df = 18 t = 30.54, df = 18⁎⁎ t = 1.47, df = 18 t = 1.49, df = 18 t = 0, df = 18 t = 1.23, df = 18 t = 1.83, df = 18 t = 1.58, df = 18 t = 3.95, df = 18 t = 0.49, df = 18 t = 0.73, df = 18 t = 1.71, df = 18

*P b 0.05, **P b 0.01, ***Pb0.001, ACT: Auditory Consonant Trigram, PANSS: Positive and Negative Syndrome Scale, RAVLT: Rey's Auditory Verbal Learning Test, WMS: Wechsler Memory Scale, WCST: Wisconsin Card Sorting Test.

Stepwise regression analysis was done to assess the predictive value of baseline biochemical parameters to the improvement in clinical parameters. The independent variables were baseline platelet serotonin, platelet MAO and plasma serotonin levels. The analysis was repeated considering changes in PANSS total, positive symptom, negative symptom, general psychopathology, digit span forward, and word fluency and CGI–I scores as dependent variables one by one.

Regression analysis showed that baseline platelet MAO levels predicted the variability in CGI–I (R2 = 0.22, P = 0.04) and the change in digit span forward test scores (R 2 = 0.22, P = 0.04), whereas baseline platelet serotonin could predict the change in positive symptoms (R2 = 0.23, P = 0.03). Since there are some meaningful correlations between baseline biochemical parameters with improvement scores, we further explored potential differences in baseline biochemical status of responder vs. non-responder patients. For this, a 25% or more reduction in total PANSS scores during the study period was accepted as a criterion of response. Applying this rule, we did not find any significant difference in baseline biochemical values between responder vs. non-responder patients. 4. Discussion To our knowledge, this is the first study to assess the effect of clozapine on both platelet and plasma serotonin levels, in addition to platelet MAO levels, and their relation with symptoms and cognitive functions. In this study, at baseline, patients who were unmedicated for at least 1 week were found to have significantly higher platelet serotonin than controls. On the other hand, baseline plasma serotonin and platelet MAO activity level of our schizophrenic patients were significantly lower than those of controls. Higher platelet serotonin values in unmedicated schizophrenic patients are a replicated finding in the literature (Borcsiczky et al., 1996; Jakovljevic et al., 1997). The finding of low platelet MAO levels in schizophrenic patients is also in accordance with the observation of some previous researchers (Jackman and Meltzer, 1980). An important finding of this study is that clozapine treatment increased plasma serotonin and platelet MAO levels and decreased platelet serotonin levels. In other words, clozapine seems to have a normalizing effect on platelet serotonin, MAO and plasma serotonin levels, which after treatment approached those of controls. The platelet serotonin level is known to be dependent on a dynamic equilibrium between intraplatelet and free (plasma) serotonin, directly controlled by the platelet serotonin uptake mechanism (Jernej and Cicin-Sain, 1991). In contrast to neurons, platelets do not synthesize serotonin, and their amine content is a result of the transporter-mediated uptake from the surrounding blood plasma (Jernej et al., 2000). Therefore, the decreased serotonin levels in platelets and the increased serotonin level in plasma might be

A. Ertugrul et al. / Psychiatry Research 149 (2007) 49–57

due to decreased activity of the serotonin transporter in platelets, which is structurally identical to the brain serotonin transporter (Lesch et al., 1993). There have been many studies involving serotonin uptake in schizophrenic patients. The maximum number of serotonin transporter sites was found to be significantly higher in unmedicated schizophrenic patients than in controls (Gavitrapong et al., 2000). On the other hand, patients who had been treated with different types of antipsychotics were reported to have decreased platelet serotonin uptake (Arora and Meltzer, 1983; Stahl et al., 1983; Gavitrapong et al., 2000). In line with these results, clozapine was found to increase extracellular serotonin concentrations in the rat medial frontal cortex and nucleus accumbens (Ichikawa et al., 1998). Taking into account the results of the above cited articles, clozapine's effect in causing a decrease in platelet serotonin while increasing levels of plasma serotonin might be related to its potential effect on serotonin transporters platelets. An alternative explanation would be the effect of clozapine on MAO enzyme activity. Clozapine increased platelet MAO levels significantly compared with baseline. Increased platelet MAO activity after clozapine treatment may have led to an increase in serotonin catabolism and caused a decrease in platelet serotonin levels. This explanation seems unlikely as serotonin is a poor substrate for MAO-B and there is no direct evidence that the activation of MAO-B within neurons decreases the intraneuronal concentration of serotonin. Clozapine caused an improvement in all symptom dimensions, attention and verbal fluency as expected (Meltzer and McGurk, 1999). With respect to the relation of biochemical and clinical parameters, higher baseline platelet MAO was related with more improvement in both CGI scores and attention, explaining 22% of the variance in CGI–I and attention scores. Moreover, lower baseline platelet serotonin was related to more improvement in positive symptoms and predicted 23% of the variance in improvement in positive symptoms during clozapine treatment. Although there were no significant differences in baseline biochemical parameters of responders and nonresponders, certain clinical parameters of patients who had baseline platelet MAO and platelet serotonin levels nearer to those of controls improved more with clozapine treatment. Clozapine's normalizing effect on biochemical parameters and their relation to change in clinical parameters need further investigation. Low platelet MAO has been related to several psychiatric symptoms such as impulsivity, disinhibition

55

and cognitive impairment in which serotonergic dysfunction is believed to have a primary role (Bridge et al., 1984; Klinteberg et al., 1987; Lesch and Merschdorf, 2000; Oreland, 2004). In fact, platelet MAO is suggested to be a genetic marker for the functional capacity of the central monoamine systems and the serotonergic system in particular (Oreland, 2004). A positive association between platelet MAO activity and serotonin turnover is believed to occur via common gene promoter sequences and co-regulation of the platelet MAO gene and some unidentified gene-coding proteins important in serotonergic neurotransmission (Oreland and Hallman, 1995). According to this view, platelet MAO may be accepted as a marker of brain serotonin turnover rather than a primary effector of the above-mentioned symptoms. Therefore, we speculate that the prediction of improvement in CGI and attention scores by higher baseline platelet MAO in our study may actually be related to a possible increase in serotonin and monoamine turnover. In line with our results, the relation of platelet MAO activity to attention and concentration has been recently suggested (Kiive et al., 2005). The results of previous studies are not directly comparable to ours due to the differences in biochemical parameters and the techniques used to measure them (Muck-Seler et al., 1991; Jakovljevic et al., 1997; Pivac et al., 1997; Kaneda et al., 2001). Nevertheless, the observed changes in serotonin parameters, common in most studies, seem to be the result of a pre-existing alteration in serotonergic neurotransmission in schizophrenic patients. Clozapine, which mainly acts through a serotonergic mechanism, may be reversing or compensating for this disturbance in a way that is not yet clear. This study has some limitations. Patients in this study had a history of using multiple typical and atypical antipsychotics that might have caused long lasting changes in neurochemical parameters. Baseline assessments were done after a 1-week drug-free interval to minimize the effects of previous medication, but a comparison group of drug-naive schizophrenic patients would have been helpful in ruling out the chronic effects of antipsychotic treatment. In conclusion, this study showed an association between several peripheral serotonin indices and response to clozapine. As Meltzer (1996) reported, due to potential risk of agranulocytosis, the indication for clozapine is affected by risk-benefit considerations more than that for other antipsychotics. The ability to predict response to clozapine seems to be of special relevance to change the risk-benefit ratio. Therefore, our

56

A. Ertugrul et al. / Psychiatry Research 149 (2007) 49–57

results, which show that low baseline platelet serotonin can predict improvement in positive symptoms, while high baseline platelet MAO can predict improvement in CGI-I and attention scores, may have important clinical implications if repeated in larger samples in future studies. Acknowledgement This research project was supported by a grant from Hacettepe University Research Funds (03-G014). References Anil, A.E., Kivircik, B.B., Batur, S., Kabakci, E., Kitis, A., Guven, E., Basar, K., Turgut, T.I., Arkar, H., 2003. The Turkish version of the Auditory Consonant Trigram Test as a measure of working memory: a normative study. Clinical Neuropsychologist 17, 159–169. Arora, R.C., Meltzer, H.Y., 1983. Effects of chlorpromazine on serotonin uptake in blood platelets. Psychiatry Research 9, 23–28. Banki, C.M., 1978. Alterations of cerebrospinal fluid 5-hydroxyindoleacetic acid, and total blood serotonin content during clozapine treatment. Psychopharmacology (Berlin) 56, 195–198. Benton, A.L., Hamsher, K., 1978. Multilingual Aphasia Examination: Manual of Instructions. AJA Associates, Iowa City, IA. Borcsiczky, D., Tekes, K., Tarcalı, J., Meszaros, Z., Faludi, G.M., Magyar, K., 1996. Platelet rich plasma serotonin content in patients suffering from different psychiatric disorders. Acta Physiologica Hungarica 84, 403–404. Bridge, T.P., Phelps, B.H., Cutler, N.R., Jeste, D.V., Wyatt, R.J., 1984. Peripheral catecholamine enzyme function and cognitive impairment of elderly schizophrenics and controls. Journal of the American Geriatrics Society 32, 259–264. Buckman, T.D., Kling, A., Sutphin, M.S., Steinberg, A., Eiduson, S., 1990. Platelet glutathione peroxidase and monoamine oxidase activity in schizophrenics with CT scan abnormalities: relation to psychosocial variables. Psychiatry Research 31, 1–14. Del Vecchio, M., Maj, M., D'Ambrosio, A., Kemali, D., 1983. Low platelet MAO activity in chronic schizophrenics: a long-term effect of neuroleptic treatment? Psychopharmacology (Berlin) 79 (2–3), 177–179. Elman, I., Goldstein, D.S., Eisenhofer, G., Folio, J., Malhotra, A.K., Adler, C.M., Pickar, D., Breier, A., 1999. Mechanism of peripheral noradrenergic stimulation by clozapine. Neuropsychopharmacology 20, 29–34. Fang, J., Yu, P.H., Gorrod, J.W., Boulton, A.A., 1995. Inhibition of monoamine oxidases by haloperidol and its metabolites: pharmacological implications for the chemotherapy of schizophrenia. Psychopharmacology (Berlin) 118, 206–212. Fleischhaker, C., Schulz, E., Remschmidt, H., 1998. Biogenic amines as predictors of response to clozapine treatment in early-onset schizophrenia. Journal of Psychiatric Research 32, 325–333. Garelis, E., Gillin, J.C., Wyatt, R.J., Neff, N., 1975. Elevated blood serotonin concentrations in unmedicated chronic schizophrenic patients: a preliminary study. American Journal of Psychiatry 132, 184–186. Gavitrapong, P., Mukda, S., Turakitwanakan, W., Dumrongphol, H., Chindaduangratn, C., Sanvarinda, Y., 2000. Platelet serotonin

transporter in schizophrenic patients with and without neuroleptic treatment. Neurochemistry International 41, 209–216. Grace, A.A., 2000. Gating of information flow within the limbic system and the pathophysiology of schizophrenia. Brain Research Reviews 31, 330–341. Heaton, R.K., 1981. Wisconsin Card Sorting Test Manual. Psychological Assessment Resources, Odessa, FL. Heiser, P., Hausmann, C., Frey, J., Geller, F., Becker, R., Wesemann, W., Krieg, J.-C., Remschmidt, H., Vedder, H., 2000. Serotonergic effects of clozapine and its metabolites in hippocampal HT22 cells. Psychiatry Research 112, 221–229. Holt, A., Sharman, D.F., Baker, G.B., Palcic, M.M., 1997. A continuous spectrophotometric assay for MAO and related enzymes in tissue homogenates. Analytical Biochemistry 244, 384–392. Ichikawa, J., Kuroki, T., Dai, J., Meltzer, H.Y., 1998. Effect of antipsychotic drugs on extracellular serotonin levels in rat medial prefrontal cortex and nucleus accumbens. European Journal of Pharmacology 19, 163–171. Jackman, H.L., Meltzer, H.Y., 1980. Factors affecting determination of platelet monoamine oxidase activity. Schizophrenia Bulletin 6, 259–266. Jakovljevic, M., Muck-Seler, D., Pivac, N., Ljubicic, D., Bujas, M., Dodig, G., 1997. Seasonal influence on platelet 5-HT levels in patients with recurrent major depression and schizophrenia. Biological Psychiatry 41, 1028–1034. Jernej, B., Cicin-Sain, L., 1991. Genetic alterations of serotonin uptake kinetics in rat platelets. Biological Psychiatry 29, 601S. Jernej, B., Banovic, M., Cicin-Sain, L., Hranilovic, D., Balija, M., Oreskovic, D., Folnegovic-Smalc, V., 2000. Physiologic characteristics of platelet/circulatory serotonin: study on a large human population. Psychiatry Research 94, 153–162. Joseph, M.H., Owen, F., Baker, H.F., Bourne, R.C., 1977. Platelet serotonin concentration and monoamine oxidase activity in unmedicated chronic schizophrenic and in schizoaffective patients. Psychological Medicine 7, 159–162. Joyce, J.N., Shane, A., Lexow, N., Winokur, A., Casanova, M.F., Kleinman, J.E., 1993. Serotonin uptake sites and serotonin receptors are altered in the limbic system of schizophrenics. Neuropsychopharmacology 8, 315–336. Kaneda, Y., Fujii, A., Nagamine, I., 2001. Platelet serotonin concentrations in medicated schizophrenic patients. Progress in NeuroPsychopharmacology & Biological Psychiatry 25, 983–992. Kay, S.R., Fiszbern, A., Opler, L.A., 1987. The Positive and Negative Syndrome Scale (PANSS) for schizophrenia. Schizophrenia Bulletin 13, 261–276. Kiive, E., Merenakk, L., Harro, M., Harro, J., 2005. Changes in platelet monoamine oxidase activity, cholesterol levels and hyperactive behaviour in adolescents over a period of three years. Neuroscience Letters 384, 310–315. Klinteberg, B., Levander, S.E., Oreland, L., Asberg, M., Schalling, D., 1987. Neuropsychological correlates of platelet monoamine oxidase (MAO) activity in female and male subjects. Biological Psychology 24, 237–252. Kostakoğlu, E., Batur, S., Tiryaki, A., Göğüş, A., 1999. Reliability and validity of the Turkish version of the Positive and Negative Syndrome Scale. Türk Psikoloji Dergisi 14, 23–32. Lesch, K.P., Merschdorf, U., 2000. Impulsivity, aggression, and serotonin: a molecular psychobiological perspective. Behavioral Sciences and the Law 18, 581–604. Lesch, K.P., Wolzin, B.L., Murphy, D.L., Riederer, P., 1993. Primary structure of the human platelet serotonin uptake site: identity with

A. Ertugrul et al. / Psychiatry Research 149 (2007) 49–57 the brain serotonin transporter. Journal of Neurochemistry 60, 2319–2322. Lezak, M.D., 1995. Neuropsychological Assessment, 3rd edition. Oxford University Press, New York. Maj, M., Arena, F., Galderisi, S., Starace, F., Kemali, D., 1987. Factors associated with decreased platelet MAO activity in chronic schizophrenics. Progress in Neuro-Psychopharmacology & Biological Psychiatry 11, 79–86. Marcolin, M.A., Davis, J.M., 1992. Platelet monoamine oxidase in schizophrenia: a meta-analysis. Schizophrenia Research 7, 249–267. Meltzer, H.Y., 1996. Predictors of response to clozapine. Psychiatric Annals 26, 385–389. Meltzer, H.Y., McGurk, S.R., 1999. The effects of clozapine, risperidone, and olanzapine on cognitive function in schizophrenia. Schizophrenia Bulletin 25, 233–255. Meltzer, H.Y., Duncavage, M.B., Jackman, H., Arora, R.C., Tricou, B.J., Young, M., 1982. Effect of neuroleptic drugs on platelet monoamine oxidase in psychiatric patients. American Journal of Psychiatry 139, 1242–1248. Meltzer, H.Y., Matsubara, S., Lee, J.C., 1989. Classification of typical and atypical antipsychotic drugs on the basis of dopamine D-1, D-2 and serotonin-2 pKi values. Journal of Pharmacology and Experimental Therapeutics 251, 238–246. Meszaros, Z., Borcsiczky, D., Mate, M., Tarcali, J., Tekes, K., Magyar, K., 1998. MAO inhibitory side effects of neuroleptics and platelet serotonin content in schizophrenic patients. Journal of Neural Transmission Supplementum 52, 79–85. Muck-Seler, D., Jakovljevic, M., Deanovic, Z., 1991. Platelet serotonin subtypes of schizophrenia and unipolar patients. Psychiatry Research 38, 105–113. Muck-Seler, D., Pivac, N., Mustapic, M., Crncevic, Z., Jakovljevic, M., Sagud, M., 2004. Platelet serotonin and plasma prolactin and cortisol in healthy, depressed and schizophrenic women. Psychiatry Research 127, 217–226. Oreland, L., 2004. Platelet monoamine oxidase, personality and alcoholism: the rise, fall and resurrection. Neurotoxicology 25, 79–89.

57

Oreland, L., Hallman, J., 1995. The correlation between platelet MAO activity and personality: short review of findings and a discussion on possible mechanisms. In: Yu, M., Tipton, K., Boulton, A.A. (Eds.), Progress in Brain Research, vol. 106. Elsevier Science, Amsterdam, pp. 77–82. Pivac, N., Muck-Seler, D., Jakovljevic, M., 1997. Platelet 5-HT levels and hypothalamic-pituitary-adrenal axis activity in schizophrenic patients with positive and negative symptoms. Neuropsychobiology 36, 19–21. Schmidt, L.G., Dufeu, P., Heinz, A., Kuhn, S., Rommelspacher, H., 1997. Serotonergic dysfunction in addiction: effects of alcohol, cigarette smoking and heroin on platelet 5-HT content. Psychiatry Research 72, 177–185. Schulz, E., Fleischhaker, C., Clement, H.W., Remschmidt, H., 1997. Blood biogenic amines during clozapine treatment of early onset schizophrenia. Journal of Neural Transmission 104, 1077–1089. Stahl, S.M., 1985. Platelets as pharmacologic models for the receptors and biochemistry of monoaminergic neurons. In: Longenecker, G. L. (Ed.), Platelets: Physiology and Pharmacology. Academic Press, New York, pp. 307–340. Stahl, S.M., Woo, D.J., Mefford, I.N., Berger, P.A., Ciaranello, R.D., 1983. Hyperserotonemia and platelet serotonin uptake and release in schizophrenia and affective disorders. American Journal of Psychiatry 140, 26–30. van der Heijden, F.M.M.A., Tuinier, S., Fekkes, D., Sijben, A.E.S., Kahn, R.S., Verhoeven, W.M.A., 2004. Atypical antipsychotics and the relevance of glutamate and serotonin. European Neuropsychopharmacology 14, 259–268. Wechsler, D., 1987. Wechsler Memory Scale-Revised. The Psychological Corporation, New York. Zureick, J.L., Meltzer, H.Y., 1988. Platelet MAO activity in hallucinating and paranoid schizophrenics: a review and metaanalysis. Biological Psychiatry 24, 63–78.