CLOZAPINE REMAINS AN ultimate option for. Serum levels of desacyl ghrelin in patients with schizophrenia on clozapine monotherapy.

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1 Psychiatry and Clinical Neurosciences 2014; 68: doi: /pcn Regular Article Serum levels of desacyl ghrelin in patients with schizophrenia on clozapine monotherapy Adam Wysokiński, MD, PhD, 1 * Marek L. Kowalski, MD, PhD 2 and Iwona Kłoszewska, MD, PhD 1 Departments of 1 Old Age Psychiatry and Psychotic Disorders and 2 Immunology, Rheumatology and Allergy, Medical University of Lodz, Lodz, Poland Aim: Desacyl ghrelin is a hormone that might be a functional inhibitor of ghrelin, a potent hungerstimulating peptide. Methods: We determined fasting serum desacyl ghrelin levels in 24 subjects with schizophrenia on clozapine monotherapy and 24 healthy, age- and sex-matched controls. Biochemical and anthropometric measurements were combined with body composition determined using bioelectric impedance analysis. Results: There were no differences in desacyl ghrelin levels between patients taking clozapine and the control group ( ± vs ± pg/ml, z = 0.17, P = 0.87). In the clozapine group, there were no differences between men and women for ghrelin levels ( ± vs ± pg/ml, z = 0.98, P = 0.32). In the clozapine group, fasting serum levels of ghrelin negatively correlated with waist-to-hip ratio (WHR) (r = 0.45, P = 0.03) and ionized calcium (r = 0.45, P = 0.03). Levels of ghrelin were lower in patients with WHR above World Health Organization-defined cut-off points ( ± [Q1 = , Q2 = , Q3 = ] vs ± [Q1 = , Q2 = , Q3 = 485.8] pg/ml, z = 2.52, P = 0.01). In the clozapine group, there were no correlation with age, height, weight, body mass index, abdominal circumference, waist circumference, hip circumference, WHR, blood pressure, total cholesterol, high-density lipoproteins, low-density lipoproteins, triglycerides, uric acid, homocysteine, glucose, insulin, clozapine dose, duration of treatment with antipsychotics, duration of treatment with clozapine, total fat, target fat, basal metabolic rate, target weight, lean weight, body water, homoeostasis model assessment of insulin resistance (HOMA) 1-IR, HOMA2-IR and quantitative insulin sensitivity check index. Conclusion: Based on our results, we cannot conclude that treatment with clozapine affects levels of desacyl ghrelin. Also, in our study population we did not confirm previously described associations between desacyl ghrelin and various metabolic parameters. Key words: clozapine, desacyl ghrelin, metabolic syndrome, schizophrenia, weight gain. CLOZAPINE REMAINS AN ultimate option for patients with treatment-resistant schizophrenia. 1 However, treatment with clozapine is associated with very detrimental effects on metabolic profile. 2 Clozapine-induced weight gain is very common, 3 and *Correspondence: Adam Wysokiński, MD, PhD, Department of Old Age Psychiatry and Psychotic Disorders, Medical University of Lodz, Czechosłowacka 8/10, Łódź, Poland. adam.wysokinski@gmail.com Received 6 February 2014; revised 19 March 2014; accepted 23 April so is impaired fasting plasma glucose levels. In patients with schizophrenia, weight gain is associated with impaired physical functioning and negative body appraisal. 4 Obesity is linked with various health problems (pressure overload on lungs, joints and bones) and is an important risk factor for life-threatening diseases, such as cardiovascular diseases, type 2 diabetes and certain cancers. Several mechanisms are considered as playing a role in clozapine-induced weight gain, including antagonism at histamine H1 receptors 5 and serotonin 5-HT1B and 5-HT2C receptors. Various receptors, including the aforementioned 833

2 834 A. Wysokiński et al. Psychiatry and Clinical Neurosciences 2014; 68: HT1B and 5-HT1C, regulate activity of the hypothalamic nuclei (and arcuate nucleus [ARC] in particular), which play the key role in appetite regulation. Activity of ARC is regulated by anorexigenic hormones: pancreatic polypeptide (PP), cholecystokinin (CCK), glucagon-like peptide-1 (GLP-1), oxyntomodulin (OXM), peptide YY (PYY) and the first discovered orexigenic substance ghrelin. 6 Ghrelin is a 28-amino acid hunger-stimulating peptide and hormone that is produced mainly by P/D1 cells lining the fundus of the human stomach and epsilon cells of the pancreas. It was discovered in 1999 by Kojima et al. 7 There are three forms of ghrelin: (i) the octanoylated form of ghrelin (acyl ghrelin) with orexigenic activity; (ii) the nonoctanoylated form (desacyl ghrelin), which is anorexigenic; and (iii) obestatin. 8 The octanoylation is performed by the ghrelin O-acyltransferase (GOAT) protein. Only acyl ghrelin can cross the blood brain barrier and bind to the central receptor (growth hormone secretagogue receptor [GHSR]), 9 and is thus known as the active form of ghrelin. The non-octanoylated form is known as desacyl ghrelin or the inactive form, and does not activate GHSR and thus does not release growth hormone like acyl ghrelin; however, it has its own effects: reduces fat mass, increases insulin resistance, inhibits ghrelin, and has trophic and protective effects on pancreatic β-cells. Recent studies indicate that desacyl ghrelin behaves like a separate hormone. An increasing number of studies suggest that desacyl ghrelin might be a functional inhibitor of ghrelin and that desacyl ghrelin can suppress ghrelin levels in humans. Therefore, desacyl ghrelin might be good candidates for future treatment of metabolic disorders or other conditions in which antagonism of active ghrelin actions could be beneficial, such as diabetes and obesity. 10 This study was undertaken to determine if patients with schizophrenia on clozapine monotherapy have lower levels of desacyl ghrelin compared to healthy controls. In order to provide more accurate measurements, biochemical and anthropometric measurements were combined with body composition determined using bioelectric impedance analysis (BIA), which provides accurate measurements of body fat, lean mass and body water. 11 To the best of our knowledge, this is the first study to investigate such combination of these parameters in subjects with schizophrenia. METHODS Data for 24 European Caucasian adult patients with paranoid schizophrenia ( according to DSM- IV, F20.0 according to ICD-10) were included in the study. These subjects were on clozapine monotherapy for at least 2 months prior to the assessments. The control group was 24 healthy subjects and was sex- and age-matched with patients in the clozapine group. Health status of the control subjects was determined on the basis of basic physical examination, including vital signs and an interview. All patients and volunteers included in the study were informed about the aims and methods of the study and expressed their written informed consent for participation in this study. The study protocol was approved by the local Bioethics Committee. There was no financial involvement from the industry. The blood samples for the chemistry panel were collected between hours and hours, after ensuring at least 8 h of overnight fasting. The samples were immediately transferred to the central laboratory where they were analyzed. Glucose, lipids, calcium and uric acid levels were measured using a Dirui CS-400 analyzer (Dirui, Changchun, China). Homocysteine chemiluminescence assessments were performed using an Immulite 2000 analyzer (Siemens, Munich, Germany), insulin immunochemistry assessments were performed using a Cobas E411 analyzer (Roche Diagnostics, Basel, Switzerland) and albumin levels were assessed using a Cobas Integra 800 analyzer (Roche Diagnostics). Levels of desacyl ghrelin were measured in blood serum using the enzyme-linked immunosorbent assay (ELISA) method. Prior to assays, serum samples were stored at 80 C for up to 6 months. ELISA assays were performed using commercial kits (Intra-Assay: CV < 10%, Inter-Assay: CV < 15%) manufactured by RayBiotech (Norcross, GA, USA), according to protocol provided by its manufacturer (all samples were twofold diluted). Metabolic syndrome and abdominal obesity were defined according to International Diabetes Foundation (IDF) criteria. 12 Impaired fasting glucose was defined as fasting plasma glucose 100 mg/dl. Body mass index (BMI) < 25 kg/m 2, kg/m 2 and 30 kg/m 2 were defined as normal weight, overweight and obesity, respectively. Raised triglycerides (TGA) level 150 mg/dl and/or total cholesterol (TC) 200 mg/dl and/or reduced high-density lipoprotein (HDL) cholesterol level <40 mg/dl for men

3 Psychiatry and Clinical Neurosciences 2014; 68: Desacyl ghrelin in patients on clozapine 835 and <50 mg/dl for women and/or raised low-density lipoprotein (LDL) cholesterol level 135 mg/dl were interpreted as dyslipidemia. Corrected calcium was calculated using the formula: corrected calcium (mg/dl) = measured total calcium (mg/dl) (4.0 serum albumin [g/dl]). Insulin resistance was estimated from fasting glucose and insulin results by homeostasis model assessment (HOMA) and quantitative insulin sensitivity check index (QUICKI) index, using the formula: HOMA1-IR = (fasting plasma glucose [mg/dl] insulin [mu/l])/405. HOMA2-IR index was calculated using a calculator downloaded from QUICKI index (lower numbers reflect greater insulin resistance) was calculated using the formula: 1/(log(fasting insulin [mu/l]) + log(fasting plasma glucose [mg/dl])). Insulin resistance was defined as HOMA1-IR > 2.0. Height was measured with a wall-mounted height measure to the nearest 0.5 cm. Weight was measured with a spring balance that was kept on a firm horizontal surface. Subjects wore light clothing, stood upright without shoes and weight was recorded to the nearest 0.5 kg. BMI was calculated as body weight in kg divided by height in meters squared (kg/m 2 ). Waist, abdominal and hip circumferences were measured using a non-stretchable fiber measuring tape. Waist-to-hip ratio (WHR) was calculated as waist circumference divided by hip circumference. WHR cutoff points were defined according to World Health Organization (WHO) recommendations (0.85 for women and 0.9 for men). Fat mass index (FMI) was calculated as total body fat in kg divided by height in meters squared (kg/m 2 ). Body composition was measured using a Maltron BF-906 Body Fat Analyser (Maltron, Rayleigh, UK) single-frequency bioelectrical impedance analyzer to determine resistance and reactance at 50 Hz. Standard operating conditions were observed by a trained operator, including preparation of the participant, electrode placement and operation. The measurement using BIA was taken immediately prior to anthropometry measurements with participants lying supine, in a rested state. Statistical procedures were performed with STATA 13.1 for OS X (StataCorp, College Station, TX, USA). Simple descriptive statistics (means and SD, median [Q2], 25% and 75% quartiles [Q1 and Q3]) were generated for all continuous variables. For discrete variables, number of patients and percentages are given. Inter-group differences were analyzed using the Mann Whitney U-test. The difference between proportions was analyzed by Fisher s exact test. Associations were tested by Spearman s correlation coefficient. The significant level was set at P < RESULTS The mean age was 38.8 ± 12.6 (Q1 = 28.0, Q2 = 38.5, Q3 = 47.5) for the clozapine-treated patients and 39.9 ± 12.3 (Q1 = 30.5, Q2 = 36.0, Q3 = 52.0) for the control group; there was no significant difference between the groups in age (P = 0.62). In both groups there were 12 men, that is, half of the group, and 12 women. In the clozapine group, 12 subjects (half of the group) smoked cigarettes and 8 in the control group (P = 0.38). The mean duration of monotherapy with clozapine was ± months (Q1 = 8.5, Q2 = 33.0, Q3 = 84.0) and mean clozapine dose was ± mg/day (Q1 = 237.5, Q2 = 300.0, Q3 = 425.0). Detailed results for anthropometric measurements and laboratory tests are shown in Table 1. IDF-defined metabolic syndrome was found in 12 (50.0%) patients from the clozapine group and in four (16.7%) subjects from the control group (P = 0.015, 1-sided Fisher s exact test). Insulin resistance was found in 12 (50.0%) patients from the clozapine group and in seven (29.2%) subjects from the control group (P = 0.12, 1-sided Fisher s exact test). We found no inter-group differences for body composition analysis. Detailed results for BIA analysis are shown in Table 2. Lean body mass was higher in men in the whole study sample (60.1 ± 6.4 [Q1 = 53.8, Q2 = 59.7, Q3 = 63.6] vs 43.8 ± 5.4 kg [Q1 = 41.3, Q2 = 43.9, Q3 = 46.4], z = 5.74, P < 0.001) and in the clozapine group (59.6 ± 5.7 [Q1 = 55.3, Q2 = 59.7, Q3 = 61.4] vs 45.3 ± 7.0 kg [Q1 = 42.5, Q2 = 46.4, Q3 = 49.1], z = 3.93, P < 0.001). Similarly, basal metabolic rate was higher in men in the whole study sample ( ± [Q1 = , Q2 = , Q3 = ] vs ± [Q1 = , Q2 = , Q3 = ] kg, z = 5.32, P < 0.001) and in the clozapine group ( ± [Q1 = , Q2 = , Q3 = ] vs ± [Q1 = , Q2 = , Q3 = ] kg, z = 3.87, P < 0.001). There were no differences in desacyl ghrelin levels between patients taking clozapine and the control group ( ± [Q1 = , Q2 = , Q3 = 366.7] vs ± [Q1 = , Q2 = 252.4, Q3 = ] pg/ml, z = 0.17, P = 0.87). In

4 836 A. Wysokiński et al. Psychiatry and Clinical Neurosciences 2014; 68: Table 1. Results of anthropometric measurements and laboratory tests Clozapine n = 24 Control n = 24 Weight (kg) 78.1 ± 14.2 (69.2, 78.5, 84.2) 72.6 ± 15.1 (62.5, 69.5, 82.7) 0.13 BMI (kg/m 2 ) 27.1 ± 3.6 (25.1, 26.2, 30.7) 24.8 ± 3.5 (22.8, 24.8, 26.8) 0.03 z = 2.16 FMI (kg/m 2 ) 8.9 ± 3.1 (7.6, 8.9, 10.5) 7.7 ± 3.0 (6.2, 7.2, 9.1) 0.08 z = 1.73 Abdominal circumference (cm) 96.5 ± 9.4 (89.0, 97.0, 103.5) 85.5 ± 11.6 (79.5, 86.0, 93.0) <0.001 z = 3.37 Waist circumference (cm) 91.1 ± 12.1 (82.5, 90.5, 100.5) 82.4 ± 10.6 (75.0, 83.0, 89.5) 0.01 z = 2.52 Hip circumference (cm) 99.0 ± 8.4 (92.5, 101.5, 104.5) 95.9 ± 7.1 (91.5, 97.0, 101.5) 0.12 WHR 0.92 ± 0.08 (0.87, 0.92, 0.98) 0.86 ± 0.08 (0.79, 0.86, 0.90) 0.01 z = 2.55 SBP (mm Hg) ± 13.6 (114.5, 123.5, 129.5) ± 17.9 (123.5, 138.5, 151.5) z = 2.98 DBP (mm Hg) 81.2 ± 8.5 (75.5, 80.0, 86.0) 82.8 ± 12.1 (73.5, 83.5, 89.5) 0.60 Uric acid (mg/dl) 4.5 ± 1.4 (3.5, 4.2, 5.0) 4.3 ± 1.3 (3.8, 4.4, 4.9) 0.86 Homocysteine (μmol/l) 14.5 ± 4.4 (11.3, 15.5, 17.7) 13.6 ± 5.0 (10.2, 13.5, 16.5) 0.48 TC (mg/dl) ± 53.2 (157.2, 184.4, 214.6) ± 65.3 (174.3, 205.8, 238.1) 0.17 HDL (mg/dl) 43.5 ± 12.6 (35.0, 39.9, 53.3) 55.1 ± 14.3 (46.3, 57.1, 64.6) z = 2.69 LDL (mg/dl) ± 41.9 (90.9, 115.4, 154.8) ± 39.7 (94.6, 123.4, 167.3) 0.59 TGA (mg/dl) ± (74.8, 105.1, 182.6) ± 81.4 (57.1, 85.4, 127.6) 0.19 FPG (mg/dl) ± 31.7 (87.4, 94.8, 112.9) 87.8 ± 11.7 (81.2, 85.9, 95.9) 0.04 z = 2.03 Insulin (mu/l) 11.8 ± 8.2 (6.7, 8.7, 12.3) 7.7 ± 3.2 (5.6, 7.0, 9.0) 0.08 HOMA1-IR 3.3 ± 3.4 (1.5, 2.0, 3.7) 1.7 ± 0.8 (1.2, 1.6, 2.1) 0.06 HOMA2-IR 1.6 ± 1.1 (0.9, 1.0, 1.7) 1.0 ± 0.4 (0.7, 0.9, 1.2) 0.06 QUICKI 0.15 ± 0.01 (0.14, 0.15, 0.16) 0.16 ± 0.01 (0.15, 0.15, 0.16) 0.06 Albumin (g/dl) 4.5 ± 0.5 (4.3, 4.5, 4.9) 4.7 ± 0.3 (4.5, 4.6, 4.8) 0.20 Total calcium (mg/dl) 9.0 ± 0.8 (8.5, 9.0, 9.6) 9.3 ± 0.7 (8.7, 9.3, 9.7) 0.39 Corrected calcium (mg/dl) 8.6 ± 0.9 (8.0, 8.7, 9.4) 8.7 ± 0.7 (8.3, 8.7, 9.3) 0.94 Data given as mean ± SD (25% quartile, median,75% quartile). BMI, body mass index; DBP, diastolic blood pressure; FMI, fat mass index; FPG, fasting plasma glucose; HDL, high-density lipoproteins; HOMA1-IR, homoeostasis model assessment of insulin resistance 1; HOMA2-IR, homoeostasis model assessment of insulin resistance 2; LDL, low-density lipoproteins; QUICKI, quantitative insulin sensitivity check index; SBP, systolic blood pressure; TC, total cholesterol; TGA, triglycerides; WHR, waist-to-hip ratio. P Table 2. Results of body composition analysis Clozapine (n = 24) Control (n = 24) P Total body fat (%) 32.6 ± 8.4 (29.0, 33.2, 38.5) 28.9 ± 7.1 (24.2, 28.9, 33.0) 0.06 Total body fat (kg) 25.6 ± 8.8 (20.1, 24.5, 31.5) 22.4 ± 8.7 (18.3, 21.2, 26.2) 0.12 Target body fat min (%) 21.6 ± 4.0 (20.0, 20.0, 24.0) 22.2 ± 3.6 (19.0, 22.5, 25.0) 0.57 Target body fat max (%) 27.6 ± 4.0 (26.0, 26.0, 30.0) 28.2 ± 3.6 (25.0, 28.5, 31.0) 0.57 Basal metabolic rate (kcal/day) ± (1388.5, , ) ± (1347.0, , ) 0.39 Target weight min (kg) 58.0 ± 9.1 (53.5, 58.0, 64.0) 56.6 ± 10.1 (49.0, 53.0, 66.0) 0.29 Target weight max (kg) 69.0 ± 10.3 (64.5, 69.0, 74.0) 67.9 ± 11.2 (59.5, 63.5, 78.0) 0.41 Lean body weight (kg) 52.5 ± 9.6 (61.5, 66.8, 70.9) 51.4 ± 10.8 (67.0, 71.0, 75.8) 0.48 Lean body weight (%) 67.6 ± 8.1 (46.4, 53.0, 59.7) 71.1 ± 7.1 (42.5, 47.7, 61.0) 0.07 Total body water (L) 38.4 ± 7.0 (33.9, 38.8, 43.7) 37.7 ± 7.9 (31.1, 34.9, 44.6) 0.48 Total body water (%) 49.9 ± 5.4 (46.5, 48.9, 52.0) 52.1 ± 5.2 (49.0, 52.0, 55.5) 0.09 Target body water min (%) 50.7 ± 3.2 (49.0, 52.0, 52.0) 50.1 ± 2.9 (48.0, 50.0, 53.0) 0.57 Target body water max (%) 57.7 ± 3.2 (56.0, 59.0, 59.0) 57.3 ± 2.8 (55.0, 57.0, 60.0) 0.66 Data given as mean ± SD (25% quartile, median,75% quartile).

5 Psychiatry and Clinical Neurosciences 2014; 68: Desacyl ghrelin in patients on clozapine 837 Serum desacyl ghrelin [pg/ml] Waist-to-hip ratio Figure 1. Correlation between fasting serum desacyl ghrelin level and waist-to-hip ratio in patients with schizophrenia., 95% CI;, Fitted values (r = 0.45, P = 0.03);, Serum desacyl ghrelin [pg/ml]. the clozapine group, there were no differences between men and women in desacyl ghrelin levels ( ± [Q1 = 124.1, Q2 = , Q3 = ] vs ± [Q1 = , Q2 = , Q3 = ] pg/ml, z = 0.98, P = 0.32). These differences between men and women were also not significant for the control group (z = 0.40, P = 0.68) and for the whole study population (z = 1.03, P = 0.30). In the clozapine group, levels of desacyl ghrelin negatively correlated with WHR (r = 0.45, P = 0.03) (see Fig. 1) and corrected calcium (r = 0.45, P = 0.03). Also, in the clozapine group, levels of desacyl ghrelin were lower in patients with WHR above WHO-defined cut-off points ( ± [Q1 = , Q2 = , Q3 = ] vs ± [Q1 = , Q2 = , Q3 = 485.8] pg/ml, z = 2.52, P = 0.01) (see Fig. 2). In the clozapine group, there was no correlation with age, height, weight, BMI, FMI, abdominal circumference, waist circumference, hip circumference, WHR, blood pressure, total cholesterol, HDL, LDL, triglycerides, uric acid, homocysteine, glucose, insulin, clozapine dose, duration of treatment with antipsychotics, duration of treatment with clozapine, total fat, target fat, basal metabolic rate, target weight, lean weight, body water, HOMA1-IR, HOMA2-IR and QUICKI. No correlations were significant for the control group or the whole study group. In the clozapine group, for levels of desacyl ghrelin, there were no differences in subjects with and without IDF-defined metabolic syndrome (P = 0.26), smokers and non-smokers (P = 0.15), subjects with BMI < 25 kg/m 2 and with BMI 25 kg/m 2 (P = 0.62), subjects with and without impaired fasting glucose (P = 0.46), subjects with and without abdominal obesity (P = 0.76), subjects with total body fat lower and higher than target maximum based on BIA (P = 0.41), subjects with and without dyslipidemia (P = 0.17), or subjects with and without insulin resistance (P = 0.75). In the control group, fasting serum levels of desacyl ghrelin were lower in subjects with dyslipidemia ( ± [Q1 = 116.6, Q2 = , Q3 = ] vs ± [Q1 = , Q2 = , Q3 = ] pg/ml, z = 2.60, P = 0.009). In the whole study group, levels of desacyl ghrelin were lower in subjects with dyslipidemia ( ± [Q1 = 126.8, Q2 = , Q3 = ] vs ± [Q1 = , Q2 = , Q3 = ] pg/ml, z = 2.85, 0.004). Other differences were not significant. Using one-way ANOVA, we have also analyzed if there are differences in levels of desacyl ghrelin depending on how many IDF criteria of metabolic syndrome are met. We have found no inter-group differences for the clozapine group (F = 2.25, P = 0.09), the control group (F = 0.36, P = 0.87) and the whole group (F = 1.59, P = 0.18). DISCUSSION To the best of our knowledge, this is the first study that assessed combination of fasting serum levels of Serum desacyl ghrelin [pg/ml] WHR normal WHR increased Figure 2. Serum levels of desacyl ghrelin in schizophrenia patients with normal and increased waist-to-hip ratio (WHR) (z = 2.52, P = 0.01).

6 838 A. Wysokiński et al. Psychiatry and Clinical Neurosciences 2014; 68: desacyl ghrelin and body composition in patients treated with clozapine. The main purpose of the present study was to observe whether there is a significant difference in levels of desacyl ghrelin between patients with schizophrenia on clozapine monotherapy and age- and sexmatched healthy controls. There were no differences in desacyl ghrelin levels between patients taking clozapine and the control group ( ± [Q1 = , Q2 = , Q3 = 366.7] vs ± [Q1 = , Q2 = 252.4, Q3 = ] pg/ml, z = 0.17, P = 0.87). In the clozapine group there were no differences between men and women in ghrelin levels ( ± [Q1 = 124.1, Q2 = , Q3 = ] vs ± [Q1 = , Q2 = , Q3 = ] pg/ml, z = 0.98, P = 0.32). We do not know pre-treatment values, so it is impossible to determine whether and how this parameter changed during therapy with clozapine. Nevertheless, this is interesting in relation to what we have expected as previous studies suggest that antipsychotics may affect serum levels of ghrelin. It was observed that levels of ghrelin are raised in patients treated with risperidone, 13 olanzapine, 14 clozapine and, interestingly, amisulpride. 15 In the last of these studies, Esen-Danaci et al. found that levels of ghrelin in patients treated with quetiapine did not differ from control subjects, but also the highest ghrelin levels were found in the subgroup taking amisulpride. This indicates there are complex and diverse mechanisms underlying treatment-induced weight gain and increased appetite. Palik et al. compared four groups of patients with schizophrenia taking clozapine, olanzapine, risperidone and quetiapine with healthy subjects. In this study it was found that levels of ghrelin were significantly higher in all four groups compared to control subjects. No changes were found between individual antipsychotics. 16 The same antipsychotics were studied by Birkas Kovats et al. 17 Their results were similar to results found by Palik et al. Togo et al. found that serum ghrelin concentrations did not increase, but rather decreased, in patients treated with olanzapine or risperidone in comparison with healthy volunteers and no significant difference was found in serum ghrelin concentration between patients treated with olanzapine and risperidone. 18 An excellent review published by Sentissi et al. 19 indicates that several results are inconclusive as some studies found decreased levels of ghrelin during treatment with antipsychotics. This applies mainly to olanzapine, which may result from the fact that this medication was studied most often (five out of eight analyzed studies). All these studies measured levels of active ghrelin (although we cannot verify that blood samples were collected in a way that prevents deacylation of ghrelin). Therefore, we may assume that this is the first study that assessed levels of desacyl ghrelin in patients on clozapine. Compared to healthy controls, WHR were higher in patients on clozapine (0.92 ± 0.08 [Q1 = 0.87, Q2 = 0.92, Q3 = 0.98] vs 0.86 ± 0.08 [Q1 = 0.79, Q2 = 0.86, Q3 = 0.90], z = 2.55, P = 0.01). In the clozapine group, fasting serum levels of desacyl ghrelin negatively correlated with WHR (r = 0.45, P = 0.03) and levels of ghrelin were lower in patients with WHR above WHO-defined cut-off points ( ± [Q1 = , Q2 = , Q3 = ] vs ± [Q1 = , Q2 = , Q3 = 485.8] pg/ml, z = 2.52, P = 0.01). As desacyl ghrelin might have anorexigenic properties, this raises the question whether decreased levels of ghrelin in patients with WHR exceeding cut-off points for central obesity play a role in the development of abdominal obesity (although we found no associations with other metabolic parameters). In order to confirm this hypothesis, a longitudinal study is required. According to Cansu et al., changes in ghrelin (and thus in desacyl ghrelin) levels may be secondary to weight gain induced by other neuropeptides (leptin, galanin, neuropeptide Y). 20 Thus, it would be interesting to assess the correlation between these factors. Asakawa et al. reported that desacyl ghrelin overexpressing mice exhibited a decrease in body weight and fat pad mass weight accompanied by moderately decreased linear growth. 21 Cederberg et al. reported that in humans, desacyl ghrelin is associated with changes in body weight and body composition during intensive long-term exercise intervention. 22 We have, however, found no correlation between desacyl ghrelin serum levels and body weight, BMI, FMI, abdominal circumference, waist circumference, hip circumference, WHR and body composition results (total fat, target fat, basal metabolic rate, target weight, lean weight, body water). There were also no differences in desacyl ghrelin levels between subjects with BMI < 25 kg/m 2 and with BMI 25 kg/m 2, subjects with and without abdominal obesity, and subjects with total body fat lower and higher than target maximum based on BIA. These results cannot be attributed to schizophrenia or clozapine, as there were

7 Psychiatry and Clinical Neurosciences 2014; 68: Desacyl ghrelin in patients on clozapine 839 also no differences in the control group and the total study group. Several authors reported that desacyl ghrelin may increase insulin sensitivity. Barazzoni et al. found in patients with metabolic syndrome that desacyl ghrelin was negatively correlated with plasma insulin and HOMA-IR. 23 We have found no correlation with plasma levels of glucose and insulin and with markers of insulin resistance (HOMA1-IR, HOMA2-IR and QUICKI). Moreover, no correlations were found in the subgroup of patients with metabolic syndrome. There were also no differences in serum levels of desacyl ghrelin between subjects with and without metabolic syndrome or subjects with and without insulin resistance. There are also reports that desacyl ghrelin may improve metabolism of lipids. 24 We found no correlation with total cholesterol, HDL, LDL or triglycerides. Interestingly, levels of desacyl ghrelin were lower in subjects with dyslipidemia in the control group and in the whole study group, but not in the schizophrenia group. There was a significant difference in BMI values between the clozapine group and the control group. However, we found no differences between both groups for FMI values. Compared to BMI, FMI is a better indicator of central obesity, as it is much more sensitive to body fat content, 25 while BMI may be increased by muscle mass. Moreover, there were no differences in any of the BIA results between the two groups. These indicate that there was a substantial similarity between both groups in terms of body fat content, which is important considering that ghrelin levels may affect or result more from the amount of adipose tissue than from body weight per se. Based on our results, we cannot conclude that treatment with clozapine affects levels of desacyl ghrelin. Also, in our study population we did not confirm previously described associations between desacyl ghrelin and various metabolic parameters. Lower levels of ghrelin in patients with increased WHR is an interesting finding and would require further explorations. The low number of study subjects limited the probability of finding inter-group differences due to lack of statistical power. Due to the cross-sectional study design, causal correlations cannot be established and effect of previous antipsychotic treatment cannot be excluded. Dual-energy X-ray absorptiometry could be used to measure body composition and percentage of fat more accurately. ACKNOWLEDGMENTS The authors are (partially) supported by the Healthy Ageing Research Centre (REGPOT , 7FP). None of the authors has anything to disclose. REFERENCES 1. Kane JM. Addressing nonresponse in schizophrenia. J. Clin. Psychiatry 2012; 73: e Newcomer JW. Second-generation (atypical) antipsychotics and metabolic effects: A comprehensive literature review. CNS Drugs 2005; 19 (Suppl 1): Wetterling T. Bodyweight gain with atypical antipsychotics. A comparative review. Drug Saf. 2001; 24: Bachmann CJ, Gebhardt S, Lehr D et al. Subjective and biological weight-related parameters in adolescents and young adults with schizophrenia spectrum disorder under clozapine or olanzapine treatment. Z. Kinder Jugendpsychiatr. Psychother. 2012; 40: ; quiz Kroeze WK, Hufeisen SJ, Popadak BA et al. H1-histamine receptor affinity predicts short-term weight gain for typical and atypical antipsychotic drugs. Neuropsychopharmacology 2003; 28: Druce MR, Small CJ, Bloom SR. Minireview: Gut peptides regulating satiety. Endocrinology 2004; 145: Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K. Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 1999; 402: Hosoda H, Kojima M, Matsuo H, Kangawa K. Purification and characterization of rat des-gln14-ghrelin, a second endogenous ligand for the growth hormone secretagogue receptor. J. Biol. Chem. 2000; 275: Wang H, Dey SK, Maccarrone M. Jekyll and Hyde: Two faces of cannabinoid signaling in male and female fertility. Endocr. Rev. 2006; 27: Delhanty PJ, Neggers SJ, van der Lely AJ. Mechanisms in endocrinology: Ghrelin: The differences between acyl- and des-acyl ghrelin. Eur. J. Endocrinol. 2012; 167: Bosy-Westphal A, Later W, Hitze B et al. Accuracy of bioelectrical impedance consumer devices for measurement of body composition in comparison to whole body magnetic resonance imaging and dual X-ray absorptiometry. Obes. Facts 2008; 1: Alberti KG, Zimmet P, Shaw J. Metabolic syndrome a new world-wide definition. A Consensus Statement from the International Diabetes Federation. Diabet. Med. 2006; 23: Murashita M, Inoue T, Kusumi I et al. Glucose and lipid metabolism of long-term risperidone monotherapy in patients with schizophrenia. Psychiatry Clin. Neurosci. 2007; 61:

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