Coagulation and inflammation markers during atypical or typical antipsychotic treatment in schizophrenia patients and drug-free first-degree relatives

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Schizophrenia Research 103 (2008) 83 – 93 www.elsevier.com/locate/schres

Coagulation and inflammation markers during atypical or typical antipsychotic treatment in schizophrenia patients and drug-free first-degree relatives Edgardo Carrizo a , Virginia Fernández a , Jesús Quintero a , Lissette Connell a , Zulay Rodríguez a , Mônica Mosquera b , Arnaldo Acosta c , Trino Baptista d,⁎ a

Institute of Clinical Research “Dr. Américo Negrette”, Zulia University Medical School, Maracaibo, Venezuela b University Psychiatric Hospital, Maracaibo, Venezuela c Department of Endocrinology, Francisco de Miranda University, Coro, Falcón State, Venezuela d Department of Physiology, Los Andes University Medical School, P.O. Box 93, Mérida, 5101-A, Venezuela Received 20 January 2008; received in revised form 2 March 2008; accepted 5 March 2008 Available online 23 April 2008

Abstract Background: Clinical studies suggest that the second generation antipsychotics (APs) clozapine and olanzapine and to a lesser extent the typical antipsychotics may be associated with a procoagulant and proinflammatory state that promotes venous thromboembolism. We evaluated here several blood factors associated with coagulation and inflammation in AP-treated schizophrenia patients and their first-degree relatives. Methods: Procoagulant factors (fibrinogen and plasminogen activator inhibitor [PAI-1]), the anticoagulant factor antithrombin III [AT-III], and inflammation-related factors (C-reactive protein [CRP] and leptin) were assessed in patients chronically treated with clozapine (n = 29), olanzapine (n = 29), typical APs (n = 30) and first degree relatives of clozapine (n = 23) and olanzapine subjects (n = 11). Results: The typical AP group had the highest CRP level (p = 0.013) in spite of having the lowest body mass index (BMI). Patients as a single group had higher CRP levels than relatives (p = 0.003). The typical AP group also had the highest AT-III levels (p = 0.021). Fibrinogen levels did not differ between the groups (p = 0.13). Olanzapine patients displayed the highest PAI-1 and leptin levels among the drug-treated subjects, but values were similar to those observed in their relatives, and were significantly correlated with the BMI. Conclusions: A homogeneous negative profile of high inflammation and procoagulant factors along with low levels of anticoagulants was not detected in any group. While preliminary, our results suggest that the observed abnormalities were not related to a direct drug effect, but to elevated BMI (high PAI-1 and leptin in olanzapine-treated patients). We speculate that the high CRP in the typical AP group might be related to poor lifestyle habits, but this must we confirmed in future studies. © 2008 Elsevier B.V. All rights reserved. Keywords: Antithrombin III; Clozapine; C-reactive protein; Fibrinogen; Leptin; Olanzapine; Plasminogen activator inhibitor; Typical antipsychotics

⁎ Corresponding author. Tel.: +58 416 6760320. E-mail address: [email protected] (T. Baptista). 0920-9964/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.schres.2008.03.004

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1. Introduction The elevated prevalence of the metabolic syndrome in psychiatric patients treated with some second generation antipsychotic drugs (SGAD) such as clozapine and olanzapine and to a lesser extent with quetiapine and risperidone, is an important problem in contemporary psychiatry (Newcomer, 2006, 2007). The most customary definitions of the metabolic syndrome comprise abdominal obesity, impaired glucose tolerance, dyslipidemia and blood hypertension (The International Diabetes Consensus, 2007; Third Report of the National Cholesterol Education Program, 2002). However, a proinflammatory and procoagulant state is also observed in subjects with the metabolic syndrome, which promotes venous and arterial thrombosis and thus, represents an additional risk for heart and brain vascular diseases (De Taeye et al., 2005; Fonseca and Jawa, 2005; Haffner, 2006; Nieuwdorp et al., 2005; Zimmet et al., 2005). The metabolic syndrome and more extreme cases of insulin resistance such as type 2 diabetes mellitus, are associated with: a) biochemical markers of inflammation and endothelial dysfunction (increased levels of creactive protein [CRP], proinflammatory cytokines interleukin (IL)-1, IL-6, α-tumor necrosis factor [α-TNF] and leptin, and low levels of nitric oxide); b) elevated levels of clotting-promoting factors (fibrinogen, tisular factor, factors VII and VIII, plasminogen activator factor inhibitor [PAI-1] and homocystein) and; c) decreased levels of anticoagulant factors involved in the fibrinolytic pathway (plasminogen activator factor, and antithrombin III [AT-III]) (Colwell, 2001; De Taeye et al., 2005; Nieuwdorp et al., 2005). In addition, platelets show high aggregability which is associated with insulin resistance, dyslipidemia and increased thromboxane A2 and soluble thrombomodulin levels (De Taeye et al., 2005; Nieuwdorp et al., 2005). Scarce information exists about the proinflammatory and procoagulant state in subjects treated with conventional or SGAD, but the available evidence suggests that it may be clinically relevant (Kudoh et al., 2001; Sarandol et al., 2007). For example, numerous cases of venous thromboembolism were reported by French and German authors between 1953 and 1984 in psychiatric patients treated with typical antipsychotics (reviewed in Hagg and Spigset, 2002). However, most of these studies were inconclusive by having a small sample size, lacking a proper control group and not controlling by specific mental disorder. In more recent epidemiological surveys, Zornberg and Jick (2002) assessed 29,952 patients treated with conventional APs and reported an adjusted odds ratio (OR) for developing venous thromboembolism of 7.1

[95% confidence interval (CI) 2.3 to 22.0] for current APs users compared with nonusers. Low-potency antipsychotic drugs such as chlorpromazine and thioridazine were more strongly associated with venous thromboembolism than high-potency drugs such as haloperidol. Parkin et al., (2003) (46) in a case–control study reported that recent use of antipsychotics (APs) was significantly associated with pulmonary thromboembolism, particularly with the conventional agent thioridazine. Since the SGAD clozapine and olanzapine have a stronger propensity than other typical and atypical APs to induce body weight gain (BWG) and the metabolic syndrome (Lieberman et al., 2005; Nemeroff, 1997; Newcomer, 2006, 2007), it has been hypothesized that these agents might be associated with a high rate of venous and pulmonary thromboembolism. In fact, numerous case reports have been published considering clozapine (reviewed in Hagg and Spigset, 2002) and to a minor extent regarding olanzapine and other SGAD (Hagg et al., 2003; Toki et al., 2004). An early report in 67,072 patients from the Clozaril National Registry in the US between 1991 and 1993, found that the risk of lethal pulmonary embolism was increased 5-fold among current clozapine users compared with past users (Walker et al., 1997). However, a survey published in 2000 and conducted in 35 psychiatric hospitals in Germany and Switzerland reported venous thromboembolism in 0.038% of 13,081 inpatients treated with clozapine. The figure was statistically similar to that observed among 59,637 inpatients treated with other APs (0.029%) and in 30,282 inpatients not treated with APs (0.026%) (Wolstein et al., 2000). In a recent retrospective cohort study where specific types of SGAD were described (19,940 new users and 112,078 nonusers) the rate of hospitalization due to venous thromboembolism was 0.91/100 person-years, and was considered as low. Seventy six percent of cases were venous tromboembolism and 22.4% were pulmonary embolism. Compared with nonusers, all the SGAD (risperidone, olanzapine, clozapine and quetiapine) increased the rate of hospitalization. Neither phenothiazines nor other conventional agents increased such a rate (Liperoti et al., 2005). In conclusion, available evidence, while inconclusive supports the concern about the increased risk of a procoagulant state during AP treatment, but further studies must clarify the differential propensity of specific agents to promote blood clotting and vascular inflammation. Few studies have been specifically designed to explore the mechanisms involved in the procoagulant and proinflammatory state during AP treatment. Besides the contributing effects of obesity, dyslipidemia, hypertension,

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sedation, physical restraint during excessive agitation and deleterious lifestyle factors often observed in schizophrenia patients [substance abuse, poor dietary and hygiene habits] (Hagg and Spigset, 2002), the following drug effects have been postulated: excessive circulating antiphospholipid antibodies (Ayuso and Ruiz, 1996; Davis et al., 1994; Metzer et al., 1994; Schwartz et al., 1998), increased platelet aggregation (Axelsson et al., 2007) and hyperprolactinemia (Toki et al., 2004). All this proposals wait for further confirmation in controlled, large-scale studies. In the present investigation we evaluated patients in chronic treatment with clozapine, olanzapine or conventional APs and in drug-free, first-degree relatives of the two former groups. These close relatives, free of psychotropic drug use, shared most lifestyle habits with their patients, and thus, represent additional controls to dissect the AP contribution to inflammation and coagulation anomalies. We determined the blood levels of some procoagulant (fibrinogen and PAI-1), and anticoagulant factors (AT-III), along with inflammation markers such as CRP and leptin, this last being and indicator of body adiposity which has been implicated in abnormal fibrinolysis (Söderberg et al., 1999). We hypothesized significantly higher (fibrinogen, PAI-1, CRP and leptin) and lower (AT-III) levels in clozapine or olanzapine-treated subjects than in conventional AP-treated patients and relatives. 2. Methods 2.1. Subjects This study was conducted in out- and inpatients at Catesfam (Center for the Attention of Schizophrenia Patients and their Families), and Zulia University Psychiatric Hospital, Maracaibo city, Zulia state, Venezuela. The ethics committees of each institution and from the Venezuelan government (FONACIT) approved the study. Voluntary participation was requested and a written informed consent was obtained from each participant. To enter the study the subjects had to be older than 18 years, free of hormone replacement and any chronic disease besides the mental disorder. Patients had to be on the same AP treatment for more than 6 consecutive months. Firstdegree relatives were free of any psychotropic medication. The psychiatric diagnosis was conducted by research psychiatrists or social workers using the Structured Clinical Interview for the DSM-IV (First et al., 1996). 2.2. Procedure Subjects were assessed in strict fasting conditions between 7:00 and 9:00 h. After the anthropometric assess-

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ment a cubital vein blood sample was taken and divided into two aliquots: one was placed in 3.8 sodium citratecoated tubes for coagulation factor quantification and the other in dry tubes for assessment of other serum variables. Plasma and serum were obtained and stored at −70 °C until analysis, except for glucose, lipids and fibrinogen that where immediately quantified. 2.3. Anthropometric and biochemical variables Body weight, ethnic group, body mass index (BMI = weight in kg/height squared) and waist circumference (WC, 1 cm above navel) were assessed along with the blood levels of following variables: glucose, insulin, leptin, high density lipoprotein cholesterol (HDL-C), triglycerides, fibrinogen, PAI-1, CRP and ATIII. The Homeostatic Model Assessment Index of insulin resistance (HOMA-IR) was calculated as follows: glucose (mmol/L) × insulin mIU/mL)/22.5. Subjects were classified as positive or negative for the metabolic syndrome according to the revised criteria of the Third Report of the National Cholesterol Education Program (2002). 2.4. Chemical analysis Serum glucose and lipids were quantified with an enzymatic method from Humans (Germany). Insulin and leptin were assessed by duplicate by radioimmunoassay with commercial kits from DPC, Los Angeles, CA, and Linco, MO, USA, respectively. The inter- and intra-assay variation was b 10%. Fibrinogen was assessed with the Von Claus method (Thrombin Teknika Organon) (detection limits 50 mg/dL). The CRP was assessed by an enzyme-linked immunosorbent assay from Bioline (Randolph, Massachusetts, USA), with a lower detection limit of 0.1 mg/L. PAI-1 levels were quantified by ELISA and ATIII by a colorimetric method with chemicals from American Diagnostica, Stanford, CO, USA. 2.5. Statistical analysis Sample size was calculated based on previous studies of our group on fibrinogen levels in olanzapine-treated patients, with the following criteria: power level: 80%; alpha level: 0.05; expected differences between the groups: 10%; expected standard deviation: 40. The number of subjects per group was estimated in 20. The SPSS 13.0 version (SPSS Inc. Chicago, Ilinois, USA) was used. Data normality was estimated with the one-sample Kolmogorov–Smirnov test and Levene's test for the equality of variances.

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For multiple comparisons, variables with normal distribution were analyzed with the one-way ANOVA followed by the Tukey test. Variables with a non-normal distribution were analyzed with the Kruskall–Wallis chi squared test followed by the Mann–Whitney test as a post hoc when necessary. Covariance analysis was conducted with the Univariate General Linear Model. Bivariate correlation analysis was conducted with the Pearson and Spearman coefficients. Frequencies were analyzed with the chi-squared test followed by the Ryan test as post hoc. Results were considered significant at the p b 0.05 level. 3. Results The minimal sample size was achieved for all the groups except for the relatives of olanzapine and typical AP-treated patients. This last group comprised patients from a very low social level and scarce family support. Hence, only 4 relatives were contacted, and such a small sample was deleted from the final analysis, which comprised 12 siblings and 22 parents (Table 1). In the typical AP group, 13 subjects received a combination of trifluoperazine and levomepromazine and 17 were treated with haloperidol plus levomepromazine. 3.1. Data distribution The following data followed a normal distribution: body weight, BMI, WC, AT-III, HDL-C, triglycerides, systolic and diastolic pressure (Kolmogorov–Smirnov test and Levene's test for the equality of variances N 0.05).

3.2. Demographic and clinical features Most clozapine patients were men (70%), whereas the olanzapine and typical AP groups had a similar gender distribution. Most relatives were women (84%) and as a whole, were significantly older than patients (p = 0.000) (Table 1). The clozapine group comprised 26 subjects with schizophrenia and 3 with schizoaffective disorder; the olanzapine group comprised 24 subjects with schizophrenia and 5 with schizoaffective disorder, and the typical AP group included 20 subjects with schizophrenia and 10 with schizoaffective disorder. None of the relatives was in psychiatric or psychological treatment or had a diagnosis of schizophrenia, major depression, bipolar disorders, brain disorders or dementia. One clozapine and one olanzapine relatives were diagnosed de novo as diabetic. Besides, one clozapine relative had severe hypertriglyceridemia and one olanzapine relative was found with moderate hypertension. Tobacco and alcohol consumption was common in patients and relatives, but its magnitude was not quantified. The ethnic distribution in the whole sample was as follows: whites: 57.4%; mixed: 29.5%; African-Americans: 7.4%; natives: 5.7%. Gender, age, BMI and frequency of the metabolic syndrome did not differ among the ethnic groups (data not shown). Besides, no differences in ethnicity were observed among the treatment groups (p N 0.05). Provided the small proportion of African-Americans and natives, no analysis was conducted in the biochemical variables among the ethnic groups.

Table 1 Sex, antipsychotic dose, age, anthropometric variables and frequency of the metabolic syndrome Treatment

Sex

Dose (mg/day)

Age (years) (a)

Body mass index (BMI, kg/m2) (b)

Waist circumference (WC, cm) (c)

Frequency of the metabolic syndrome (%) (d)

Clozapine (n = 29)

M: 20 F: 9 M: 13 F: 16 M: 15 F: 15 M: 2 F: 21 M: 3 F: 8

229 ± 120

39.7 ± 8.7 41.9 ± 10.9 43.6 ± 10.4 38.4 ± 14.6 41.9 ± 12.4 42.3 ± 11.3 58.0 ± 19.8 53.2 ± 14.0 51.3 ± 10.9 53.6 ± 12.4

27.1 ± 5.1

98.2 ± 12.5

32.1

29.9 ± 5.1

98.1 ± 11.8

27.6

24.2 ± 6.3

87.1 ± 13.5

13.3

27.2 ± 4.2

91.9 ± 10.9

52.2

29.6 ± 6

96.9 ± 16.6

54.5

Olanzapine (n = 29) Typical APS (n = 30) Clozapine relatives (n = 23) Olanzapine relatives (n = 11)

8.5 ± 3.9 774.2 ± 398.6. – –

M = masculine; F = feminine. Values represent mean ± standard deviation. The dose of typical APs represents mg of chlorpromazine. (a) (⁎) Both relative groups were older than all the patient groups: f (4, 120) = 6.6, p = 0.000. (b) Patients treated with conventional APs had the lowest BMI: f (4) = 4.7, p = 0.001; vs. olanzapine patients: p = 0.001; vs. olanzapine relatives: p = 0.039. (c) Patients treated with conventional APs had the lowest WC: f (4) = 4.1, p = 0.004; vs. clozapine patients: p = 0.009; vs. olanzapine patients: p = 0.01. (d) According to the Cholesterol Panel criteria (ATP-III): χ2 (4) = 11.8, p = 0.018. Ryan post hoc test: clozapine and olanzapine relatives groups vs. typical AP group: p b 0.05.

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Table 2 Glucose, lipids, insulin resistance index, blood pressure, coagulation and inflammation markers Clozapine (n = 29) Glucose (mg/dL) (a) HDL cholesterol (mg/dL) (b) Triglycerides (mg/dL) (c) Insulin Resistance Index (d) Systolic pressure (mm/Hg) (e) Diastolic pressure (mm/Hg) (f) C-Reactive Protein (mg/L) (g) Fibrinogen (mg/mL) (h) AT-III (%) (i) PAI-1 (ng/mL) (j) Leptin (pg/mL) (k)

Olanzapine (n = 29)

Typical APS (n = 30)

Clozapine (relatives, n = 23)

Olanzapine (relatives, n = 11)

87.3 ± 13.4

72.3 ± 9.5

73.1 ± 11.9

100.0 ± 70.4

84.2 ± 37.4

33.3 ± 8.3

36.1 ± 9.5

33.1 ± 7.4

41.1 ± 9.3

35.9 ± 6.2

151.7 ± 130.2

118.3 ± 48.6

115.4 ± 57.4

137.9 ± 133.6

146.3 ± 123.6

4.8 ± 3.9 115.7 ± 11.7

2.4 ± 0.8 109.6 ± 10.2

2.9 ± 1.7 110.7 ± 10.8

6.7 ± 9.3 128.6 ± 10.8

4.6 ± 4.8 124.5 ± 16.3

70.4 ± 7.4

68.9 ± 7.7

68.0 ± 9.6

76.1 ± 10.5

74.1 ± 10.7

5.2 ± 5.0

4.9 ± 2.9

6.9 ± 4.2

3.2 ± 2.6

3.6 ± 1.9

340.5 ± 74.9

312.9 ± 76.9

351.8 ± 66.9

342.2 ± 67.6

299.1 ± 81.6

109.4 ± 24.5

106.2 ± 23.8

121.2 ± 21.8

108.5 ± 24.4

95.5 ± 20.8

33.7 ± 13.9

54.3 ± 30.3

34.1 ± 10.8

36.8 ± 13.7

54.1 ± 32.6

9.5 ± 6.3

19.6 ± 11.1

9.6 ± 11.9

16.9 ± 7.4

18.6 ± 14.4

(a) Kruskall–Wallis χ2 (4) = 33.3, p = 0.000; post hoc analysis: clozapine vs. olanzapine, typical AP and olanzapine relative groups: p b 0.05; clozapine relatives vs. olanzapine and typical AP groups: p b 0.05. (b) f (4, 121) = 3.7, p = 0.007; post hoc analysis: clozapine relatives vs. clozapine and typical AP groups: p b 0.05. (c) f (4, 121) = 0.7, p = 0.5. (d) Kruskall–Wallis χ2 (4) = 23.2, p = 0.000; post hoc analysis: clozapine and clozapine relatives vs. olanzapine and typical AP groups: p b 0.05. (e) f (4, 119) = 12.1, p = 0.000; post hoc analysis: clozapine relatives vs. clozapine, olanzapine and typical AP groups: p b 0.05; olanzapine relatives vs. olanzapine and typical AP groups: p b 0.05. (f) f (4, 119) = 3.4, p = 0.01; post hoc analysis: clozapine relatives vs. olanzapine and typical AP groups: p b 0.05. (g)Kruskall–Wallis χ2 (4) = 12.7, p = 0.013; post hoc analysis: typical AP group vs. clozapine, clozapine and olanzapine relatives: p b 0.05; olanzapine group vs. clozapine relatives: p b 0.05. (h) Kruskall–Wallis χ2 (4) = 7.2, p = 0.13. (i) f(4, 121) = 3.0, p = 0.021; post hoc analysis: typical AP group vs. olanzapine relatives: p b 0.05. (j) Kruskall–Wallis χ2 (4) = 9.9, p = 0.04; post hoc analysis: olanzapine vs. clozapine and clozapine relatives: p b 0.05; olanzapine vs. typical APs: p = 0.052. (k) Kruskall–Wallis χ2 (4) = 28.0, p = 0.000; post hoc analysis: olanzapine vs. clozapine and typical AP groups: p b 0.05; clozapine and olanzapine relatives vs. clozapine and typical AP groups: p b 0.05.

3.3. Anthropometric and metabolic variables The BMI distribution was as follows: olanzapine N olanzapine relatives N clozapine relatives N clozapine N typical APs (p = 0.001). For WC it was: clozapine N olanzapine N olanzapine relatives N clozapine relatives N typical APs (p = 0.004) (Table 1). The BMI and WC did not significantly correlate with age in any group or in the total sample. Considering the biochemical variables entering the metabolic syndrome definition, clozapine-treated patients and clozapine relatives had the highest glucose levels (p = 0.000, Table 2). The clozapine and typical AP groups had the lowest HDL-C levels (p = 0.007, Table 2). No differences were observed in triglyceride levels between the groups (p = 0.5, Table 3). Clozapine

patients and their relatives had the highest HOMA-IR index (p = 0.000, Table 2). Clozapine and olanzapine relatives had the highest systolic (p = 0.000) and diastolic (p = 0.01) values (Table 2). Age positively correlated with systolic (r = 0.54, p = 0.000) and diastolic pressure (r = 0.28, p = 0.02) in the whole sample. In the group analysis only the systolic pressure showed a significant correlation: clozapine group: r = 0.36, p = 0.069; olanzapine group: r = 0.38, p =0.04; typical antipsychotic group: r = 0.03, p = 0.08; clozapine relatives: r = 0.52, p = 0.01; olanzapine relatives: r = 0 76, p = 0.007. The HOMA-R index and age did not significantly correlate either in the whole sample or in the group analysis (p N 0.5). The clozapine and olanzapine-relative groups had the highest frequency of the metabolic syndrome and the

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Table 3 Correlation matrix between the inflammation and coagulation markers, the HOMA-IR and the variables included in the definition of the metabolic syndrome CRP

FI

AT-III

PAI-1

Leptin

BMI

WC

Glucose

TG

HDL-C

SBP

DBP

HOMA-IR

CRP

–

FI

–

0.23 (0.012) –

AT-III

–

–

0.06 (0.4) − 0.05 (0.56) –

PAI-1

–

–

–

0.000 (0.99) 0.045 (0.68) − 0.13 (0.20) –

Leptin

–

–

–

–

0.02 (0.83) 0.028 (0.75) − 0.14 (0.13) 0.20 (0.061) –

0.26 (0.004) − 0.001 (0.98) − 0.12 (0.18) 0.26 (0.018) 0.63 (0.000)

0.23 (0.013) 0.035 (0.70) − 0.054 (0.6) 0.18 (0.10) 0.43 (0.000)

− 0.02 (0.83) 0.15 (0.10) − 0.15 (0.092) − 0.07 (0.53) − 0.012 (0.89)

0.15 (0.11) 0.05 (0.58) − 0.035 (0.70) 0.043 (0.69) 0.15 (0.095)

−0.33 (0.000) − 0.16 (0.075) 0.023 (0.80) − 0.08 (0.47) 0.12 (0.18)

− 0.07 (0.4) 0.11 (0.23) 0.06 (0.6) 0.061 (0.58) 0.12 (0.18)

− 0.002 (0.98) 0.039 (0.67) − 0.003 (0.97) 0.083 (0.45) 0.20 (0.028)

0.02 (0.83) 0.15 (0.091) −0.19 (0.037) 0.013 (0.9) 0.017 (0.067)

CRP = C-reactive protein; FI = fibrinogen; AT-III = antithrombin III; PAI-1 = plasminogen activator inhibitor-1; BMI = body mass index; WC = waist circumference; TG = triglycerides; HDL-C = high density cholesterol; SBP = systolic blood pressure; DBP = diastolic blood pressure; HOMA-IR = insulin resistance index. Values represent the Spearman's correlation coefficient and its associated probability in parenthesis. Significant figures are in bold letters.

typical AP the lowest (Ryan's post hoc test, p b 0.05) (Table 1). As expected, the frequency of the syndrome increased with age in all analysis (data not shown). 3.4. Coagulation and inflammation markers The bivariate correlation analysis between these markers, anthropometric and metabolic variables produced significant results of physiological relevance which are summarized as a correlation matrix in Table 3. No significant correlation was observed between levels of any variable and drug dose (data not shown). Correlation analysis between the variables blood levels and duration of drug treatment was not carried out because the latter could not be adequately quantified in a few subjects.

3.6. Fibrinogen Fibrinogen levels distributed as follows: typical APs N clozapine relatives N clozapine N olanzapine Nolanzapine relatives. However, no significant differences were observed between the groups (p = 0.13, Table 2) and between all patients vs. all relatives (p = 0.6). No differences were observed in fibrinogen levels among the genders in the whole sample or in the subgroup analysis (data not shown). In the bivariate correlation analysis in the total sample, fibrinogen correlated positively with CRP levels. Marginally significant (negative) correlation was observed with the HDL-C levels and positive with the HOMA-IR index (Table 3). 3.7. Antithrombin III [AT-III]

3.5. C-reactive protein [CRP] The CRP level distributed as follows: typical APs N clozapine N olanzapine N olanzapine relatives N clozapine relatives (p = 0.013, Table 2). Patients as a single group had CRP levels significantly higher than their relatives: 5.7 ± 4.2 mg/L vs. 3.4 ± 2.4 mg/L, t (118) = 3.03, p = 0.003. The patients vs. relatives difference was observed in women (p = 0.01) and men (p = 0.02). The CRP levels were similar in both genders in the total sample and in the sub-group analysis (data no shown). In the bivariate correlation analysis, CPR levels correlated positively with the fibrinogen, BMI and WC and negatively with the HDL-C levels (Table 3). The covariance analysis showed a significant effect only for the BMI: f(1, 77) = 6.1, p = 0.017. Specifically, the typical AP group had the lowest BMI among all groups (Table 1).

The AT-III activity was as follows: typical APs N clozapine N clozapine relatives N olanzapine N olanzapine relatives (p = 0.02, Table 2). No gender differences were observed, either in the total sample or in the subgroup analysis and between all patients vs. all relatives (p = 0.09). In the bivariate correlation analysis, only a negative correlation was observed between AT-III activity and the HOMA-IR index (Table 3). The covariance analysis showed a significant effect only for triglycerides: f(1, 77) = 4.1, p = 0.04 and HDLC levels: f = 4.2, p = 0.04. 3.8. Plasminogen activator inhibitor 1 (PAI-1) The PAI-1 average distribution was as follows: olanzapine N olanzapine relatives N clozapine relatives N

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typical APs N clozapine (p = 0.04, Table 2). The PAI-1 levels distributed similarly among the genders in all analysis. No differences were observed between the patients as a single group vs. all relatives (p = 0.86). The bivariate correlational analysis only showed a positive correlation between PAI-1 levels and the BMI (Table 3). However, the covariance analysis showed a marginally significant contribution of the BMI: f(1, 84) = 1.9, p = 0.16. 3.9. Leptin Leptin levels in the total sample distributed as follows: olanzapine N olanzapine relatives N clozapine relatives N typical APs N clozapine (p = 0.000, Table 2). Significant differences were observed when compar-

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ing olanzapine with clozapine and typical AP patients (Table 2). In the bivariate correlation analysis, leptin levels positively correlated with the BM, WC and diastolic pressure. A marginally significant correlation was observed with the HOMA-IR index (Table 3). The covariance analysis showed a significant contribution of the BMI: f(1, 77) = 16.0, p = 000; fibrinogen: f = 7.4, p = 0.009; systolic pressure: f = 4.4, p = 0.004 and AT-III, f = 5.3, p = 0.024. The critical effect of gender explains the low leptin levels in the clozapine group in spite of its high BMI: clozapine patients were mostly men, who usually have lower leptin levels than women, even when corrected for adiposity levels (Baptista and Beaulieu, 2002). In fact, the gender-based difference in leptin levels was maintained in an analysis of the whole sample: women (19.2 ± 11.1 pg/mL) vs. men (7.6 ± 6.2 pg/mL), t (120) = 6.8, p = 0.000. Fig. 1 also shows the close positive correlation between leptin levels and the BMI in both sexes. For this particular analysis, relatives were organized in a single group in order to increase the number of masculine subjects. Relatives had higher leptin levels than patients (p = 0.03), but this was completely accounted by the higher proportion of women in the former group (85% vs. 45%). 4. Discussion

Fig. 1. Serum leptin levels (pg/mL) and body mass index (BMI, kg/m2). (A) Women: for the BMI: f(3, 68) = 1.18, p = 0.32; for leptin: f = 2.7, p = 0.052. Correlation between BMI and leptin: Olanzapine: n = 16, r = 0.56, p = 0.025; All relatives: n = 29, r = 0.55, p = 0.002; Clozapine: n = 9, r = 0..4, p = 0.3; Typical APs: n = 15, r = 0.84, p = 0.000. (B) Men: for the BMI: f(3, 52) = 6.7, p = 0.001, typical AP group: p b 0.05 compared to all the other groups; for leptin: f = 8.1, p = 0.000, olanzapine group: p b 0.05 compared to clozapine and typical APs. Correlation between BMI and leptin: Olanzapine: n = 13, r = 0.83, p = 0.000; All relatives: n = 5, r = 0.64, p = 0.2; Clozapine: n = 20, r = 0.87, p = 0.000; Typical APs: n = 15, r = 0.71, p = 0.003.

Important limitations of this study were the uneven sex and ethnic group distribution, and the absence of relatives in the typical AP-group. In addition, even though a reasonable sample size was obtained, it was still inadequate to power specific analysis in gender and ethnicity effects. Since most biochemical factors are influenced by age, gender, anthropometric status and a multitude of endocrine and metabolic variables, an individual analysis (except correlation) and discussion of specific variables was preferred. The three groups of patients had a similar age, but relatives were older. The typical AP group had the lowest BMI, WC and frequency of the metabolic syndrome. However, as previously shown, this did not traduce in a uniformly better profile in the coagulation and inflammation variables. 4.1. C-reactive protein High serum levels of CRP are observed in most acute and chronic inflammatory disorders (Pepys and Hirschfield, 2003). In coronary atheroesclerosis, the elevated CRP reflects the endothelial dysfunction (Pearson et al.,

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2003). In obese subjects, the adipocytes are invaded by macrophages which trigger an inflammatory response that also increases CRP levels (Weisberg et al., 2003). Hence, CRP has been proposed as an estimator of cardiovascular risk, but recent evidence suggests that its predictive value has been overestimated (Danesh et al., 2004; Tall, 2004). Besides a pointer of inflammation, PCR might have a cytotoxic effect by promoting excessive low density cholesterol uptake by the endothelium (Chang et al., 2002). Hence, elevated CRP levels associated to AP treatment might be an additional factor in the metabolic dysfunction observed in schizophrenia and bipolar disorder. In this study, the expected positive correlation between CRP, BMI and fibrinogen was observed along with a negative correlation with HDL-C levels. Considering the BMI, the correlation with CRP, while significant, was relatively weak and thus, only explains 7% of the variance. In fact, in the subgroup analysis, only the clozapine group and its relatives achieved a significant figure (data not shown). The highest CRP levels were observed in the typical AP group, who had the lowest BMI and the highest fibrinogen level. Besides, the clozapine and olanzapine groups had numerically higher CRP levels than their relatives, in spite of having a similar BMI. Two provisional conclusions may be drawn from these results. First, antipsychotic treatment as a single group was associated with elevated CRP levels. It is presently unknown whether the elevated CRP levels observed in schizophrenia (Mazzarello et al., 2004; Shcherbakova et al., 2005) are independent from drug treatment, and if the APs may increase CRP through different mechanisms to those involved in excessive body weight gain (Baptista et al., 2007). Second, the high CRP levels in the typical AP in spite of its low BMI may be related to the low socioeconomic status of these subjects which may promote subclinical inflammation related to smoking, substance abuse, poor dietary and hygiene habits (Pepys and Hirschfield, 2003). Besides its role in body weight regulation and cardiovascular disease, elevated CRP levels are associated with more severe psychopathology (Fan et al., 2007) and cognitive impairment (Dickerson et al., 2007) in schizophrenia. Hence, this is a subject that deserves further investigation. 4.2. Fibrinogen Increased fibrinogen is a prominent factor in the procoagulant state observed in the metabolic syndrome (Reaven, 2005) Elevated fibrinogen levels are observed in subjects with obesity and insulin resistance and are

predictors for the development of type 2 diabetes (Festa et al., 1999, 2000, 2002). Insulin and the TNF-a are significant stimulating factors of fibrinogen synthesis (Grieninger et al., 1983). Even though no significant differences were observed in fibrinogen levels between the groups, some results are relevant. Fibrinogen positively correlated with CPR levels (p = 0.012) and the HOMA-IR index (p = 0.091) and negatively with HDL-C levels (p = 0.075). The typical AP group had the highest fibrinogen and CRP levels, and the lowest HDL-C levels among all groups. Hence, this particular set of risk factors (high fibrinogen and CRP and low HDL-C) were observed in our sample of subjects receiving typical APs in spite of having the lowest BMI and frequency of the metabolic syndrome. 4.3. Antithrombin III (AT-III) Antithrombin (or antithrombin III), is considered as the most important anticoagulant molecule in mammalian circulation systems (Quinsey et al., 2004). The ATIII activity is decreased in type 2 diabetes and other states of insulin resistance (Colwell, 2001; De Taeye et al., 2005; Nieuwdorp et al., 2005). In this study, subjects in the typical AP group had the highest AT-III activity, but specific comparisons were only significant in relation to the olanzapine relatives (Table 3). This result is concordant with the fact that subjects in the typical AP group had low BMI and HOMA-IR index. The pathophysiological relationship between AT-III activity and the general issue of insulin resistance is supported by our finding of a significant, negative correlation with the HOMA-IR index (Table 2). 4.4. Plasminogen activator inhibitor 1 (PAI-1) The PAI-1 is synthesized by adipose tissue, and its plasma levels are increased in obesity and reduced with weight loss. Besides their prothrombotic effects, PAI-1 is associated with obesity development and maintenance through several mechanisms, such as influencing insulin signaling, adipocyte differentiation and by regulating recruitment of inflammatory cells within adipose tissue. As a consequence, PAI-1 is considered by some authors as a true component of the metabolic syndrome (Alessi et al., 2007). In this study, olanzapine patients and their relatives had the highest PAI-1 levels. The BMI was the only variable that showed a significant, positive correlation with PAI-1 levels. Since the olanzapine patients and relatives had the highest BMI among all groups, the

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elevated PAI-1 levels may be parsimoniously attributed to enhanced adiposity. However, the BMI only explained 9% of the variance in men and 6% in women in the whole sample. Hence, others factors must be involved and must be explored in future studies. The small sample size in the specific subgroups × sex analysis precluded additional statistical exploration. 4.5. Leptin Leptin is a pleiotropic hormone considered as a proinflammatory cytokine belonging to the family of longchain helical cytokines with structural similarity with interleukin-6, prolactin, growth hormone, IL-12, IL-15, granulocyte colony-stimulating factor and oncostatin M. Its role in the modulation of immune response and inflammation has recently become evident (Otero et al., 2005). Leptin blood levels and tissue expression closely correlate with the percentage of body fat and the BMI, and are strongly influenced by gender. Hence, even after controlling by BMI, women have significantly higher leptin levels than men (Baptista and Beaulieu, 2002; Baptista et al., 2007). In this study, olanzapine-treated patients had the highest and the typical AP group the lowest leptin levels. Differences in BMI were important contributors and explained 71% of variance in men and 44% in women. These result could had been even stronger, but sample size was too small and the correlation did not achieve statistical significance in the clozapine group in women (n = 9) and in the relatives' group in men (n = 5). While most available evidence suggests that leptin elevation during SGAD administration is a consequence of weight gain, a direct drug effect on leptin levels and function has not been definitively ruled out. Specifically, it has been argued that leptin increase during olanzapine treatment might be relatively lower than expected, and thus, it might favor excessive body weight gain (Haupt et al., 2005). In any case, the results presented here agree with previous reports on elevated leptin levels during treatment with SGAD that promote obesity (Baptista and Beaulieu, 2002). This leptin increase is not innocuous since convergent evidence point to a pathogenic role in inflammatory conditions such as type 1 and 2 diabetes, rheumatoid arthritis, chronic bowel disease and cardiovascular disorders (Otero et al., 2005). Fibrinogen, AT-III and systolic pressure were significant covariates in leptin distribution as well. Leptin was not significantly correlated with any of these variables in any gender (data not shown). Hence, the impact of fibrinogen, AT-III and blood pressure on leptin levels may be mediated by the BMI and fat composition.

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5. General discussion and conclusions This study assumed that an abnormal profile regarding coagulation and inflammation should include high CRP, fibrinogen, leptin (corrected by gender) and PAI-1 levels and low AT-III activity. Such a homogeneous pattern was not observed here in any group. Patients in the typical AP group had the highest CRP and low levels of leptin. By contrast these subjects had the highest AT-III activity. The favorable profile regarding leptin and AT-III may be related to the normal BMI and HOMA-IR index. The high CRP levels may be a consequence of sub-clinical inflammation in subjects with low social status and lack of family support. Clozapine-treated patients had intermediate BMI and HOMA-IR, and high WC. They had significantly lower levels than olanzapine patients of PAI-1 and leptin levels, this last variable parsimoniously explained for the preponderance of masculine subjects. Compared to their relatives, clozapine patients were younger, had similar BMI, lower HOMA-IR and higher WC. Relatives had higher leptin levels, this last result related to their higher frequency of women. Olanzapine subjects had the highest BMI, high WC and the lowest HOMA-IR index. They displayed the highest PAI-1 and leptin levels. Compared to their relatives, olanzapine patients were younger, had similar BMI and WC, but lower HOMA-IR. Importantly olanzapine patients and relatives had similar PAI-1 and leptin levels. Even though this pilot study was not designed to test pathogenic hypothesis, we could not detect an abnormal result that could be safely attributed to any specific drug treatment. Elevated CRP levels in the typical AP group were not related to increased adiposity and BMI, and patients as a group had higher levels that relatives. Hence, AP treatment might have an impact per se on CRP regulation. The high PAI-1 and leptin levels detected in olanzapine patients were also observed in their relatives, and correlate well with elevated BMI and WC. Hence, these results suggest that abnormalities in the coagulation and inflammation factors explored here may be related to increased adiposity and subclinical inflammation, and not to a direct drug effect. However, this proposal, along with the correlation between these factors, clinical and metabolic changes, must be explored in prospective studies with more powered samples, homogeneous psychiatric diagnosis, quantitative psychopathological assessment, and matching patients and relatives by body composition, gender and age.

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Role of funding source No intervention in the study accomplishment, results analysis and manuscript preparation. Contributors Edgardo Carrizo1, Virginia Fernández1, Jesús Quintero1, Lissette Connell1, Zulay Rodríguez1, Mônica Mosquera2, Arnaldo Acosta3, Trino Baptista4,⁎ (1) Institute of Clinical Research “Dr. Américo Negrette”, Zulia University Medical School, Maracaibo, Venezuela. (2) University Psychiatric Hospital, Maracaibo, Venezuela. (3) Department of Endocrinology, Miranda University, Coro, Falcón State, Venezuela. (4) ⁎Corresponding author. Department of Physiology, Los Andes University Medical School, P.O. Box 93, Mérida, 5101-A, Venezuela. E-mail: [email protected]. Conflicts of interest None Acknowledgment This study was supported by FONACIT, Caracas, Venezuela, Grant 2005-000-384.

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