Habitual binge/purge behavior influences circulating ghrelin levels in eating disorders

Transcription

1 Journal of Psychiatric Research 37 (2003) Habitual binge/purge behavior influences circulating ghrelin levels in eating disorders Muneki Tanaka a, *, Tetsuro Naruo a, Nobuatsu Nagai a, Nobutaka Kuroki a, Tomomi Shiiya b, Masamitsu Nakazato b, Shigeru Matsukura b, Shin-ichi Nozoe a a Department of Psychosomatic Medicine, Kagoshima University Hospital, Faculty of Medicine, Kagoshima University, Sakuragaoka , Kagoshima-City , Kagoshima, Japan b Department of Internal Medicine, Miyazaki Medical College, Miyazaki, Japan Received 6 May 2002; received in revised form 2 September 2002; accepted 19 September 2002 Abstract Previous studies have reported that fasting plasma ghrelin concentrations play an important role in the pathophysiology of eating disorders. The purpose of this study was to examine the relationship between plasma ghrelin levels and frequency of abnormal eating behaviors, nutritional parameters in eating disorders. Fasting blood samples were obtained in 40 female anorexia nervosa (AN) patients, 21 restricting type (AN-R) and 19 binge-eating/purging type (AN-BP), in 31 bulimia nervosa (BN) patients, 18 purging type (BN-P) and 13 nonpurging type (BN-NP), in 15 female healthy volunteers (control) before the initiation of active treatment. The fasting plasma ghrelin concentrations in all subjects were negatively correlated with nutritional parameters such as body mass index, percent body fat and serum cholinesterase concentration. The mean plasma ghrelin level in BN-P was higher than that in both BN-NP and controls despite similar nutritional parameters. The plasma ghrelin levels in both AN-R and AN-BP did not differ from BN-P despite difference of nutritional parameters. For both AN-BP and BN-P patients with habitual binge/purge behavior, there were significant correlations among plasma ghrelin values, frequencies of binge/purge cycles and serum amylase values. In BN-NP, there were no significant correlations among plasma ghrelin values, frequencies of binge-eating episodes and serum amylase values. These results suggest that habitual binge/purge behavior may have some influence on circulating plasma ghrelin levels in both BN-P and AN-BP. Habitual binge/purge cycles with vomiting as opposed to binge-eating episodes without vomiting may have a greater influence on fasting plasma ghrelin concentration in eating disorders. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Ghrelin; Eating disorders; Binge/purge cycles; Vomiting; Amylase 1. Introduction Ghrelin, a 28-amino acid peptide with an n-octanoylated serine 3, was originally discovered in the rat and human stomach, and stimulates growth hormone secretion (Kojima et al., 1999) and increases food intake and body weight in rodents (Tscho p et al., 2000; Wren et al., 2001b). This peptide is an orexigenic peptide that antagonizes leptin action has a role in the regulation of feeding behavior and energy metabolism in the central nervous system (Shintani et al., 2001; Nakazato et al., 2001). Regarding humans, ghrelin also stimulates growth hormone (Takaya et al., 2000) and enhances * Corresponding author. Tel.: ; fax: address: muneki@m3.kufm.kagoshima-u.ac.jp (M. Tanaka). appetite and increases food intake (Wren et al., 2001a). It has been reported that fasting plasma ghrelin concentrations from humans are negatively correlated with body mass index (BMI) (Shiiya et al., 2002), percent body fat, fasting leptin and insulin concentrations (Tscho p et al., 2001). The eating disorders are characterized by habitual abnormal eating behaviors and are divided into anorexia nervosa (AN) and bulimia nervosa (BN). Both AN (Otto et al., 2001) and BN (Tanaka et al., 2002) patients have been reported to increase fasting plasma ghrelin concentrations and decrease elevated plasma ghrelin of AN patients by weight gain (Otto et al., 2001). According to the Diagnostic and Statistical Manual of Mental Disorders, 4th edition (DSM-IV) (APA, 1994), AN are classified into two subtypes on the basis of the presence (AN-BP) or absence (AN-R) of habitual binge/purge /02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S (02)

2 18 M. Tanaka et al. / Journal of Psychiatric Research 37 (2003) behavior, and BN also can be classified into two subtypes on the basis of the presence (BN-P) or absence (BN-NP) of habitual purging behavior. Since our previous study (Tanaka et al., in press) suggested that differences in eating behavior might influence circulating plasma ghrelin, we therefore in this study measured fasting plasma ghrelin concentrations from both AN and BN subtypes in order to examine furthermore the relationship between plasma ghrelin levels and nutritional parameters, and frequencies of abnormal eating behaviors such as binge/purge cycles. 2. Methods 2.1. Subjects Twenty-one female AN-R patients, 19 female AN-BP patients, 18 female BN-P patients, 13 female BN-NP patients meeting DSM-IV and 15 sex-matched healthy volunteers (control) were the subjects in this study. Controls had no history of psychiatric illness or gastrointestinal disease. Patients were all outpatients who were examined to the Kagoshima University Hospital between August 2001 and April 2002, and excluded if they had a history of alcohol or substance abuse, or gastrointestinal disease, and assayed before the initiation of active treatment. To determine the neuroendocrinological aspect of eating behavior, both AN-BP and BN-P patients had habitual vomiting at least twice a week over the preceding 3 months and were excluded if they had only purging behavior without vomiting Procedures We collected blood samples from all subjects between 8.00 and 9.00 a.m. after an overnight fast. Subjects were interviewed to assess psychiatric and medical history and eating behavioral ratings in detail. We measured body weight and percent body fat and serum cholinesterase as nutritional parameters, when samples were obtained. Percent body fat was obtained by bioelectrical impedance analysis (Bolanowski and Nilson, 2001). To determine the relationship between frequencies of abnormal eating behaviors and plasma ghrelin levels, we further measured total serum amylase concentrations (Metzger et al., 1999; Kinzl et al., 1993; Walsh et al., 1990) and serum potassium (Wolfe et al., 2001). The Institutional Committee of Kagoshima University approved the protocol, and all subjects provided written informed consent before participation Laboratory methods Serum amylase, potassium and cholinesterase concentrations were determined in our laboratory. Blood was drawn into chilled tubes containing EDTA.2Na (1 mg/ml) and aprotinin (500 U/ml). Plasma ghrelin was measured using a RIA as described elsewhere (Shiiya et al., 2002). In brief, antiserum against the C-terminal region of human ghrelin was raised in New Zealand white rabbits immunized against synthetic human ghrelin [13-28] that had been coupled with maleimide-activated mariculture keyhole limpet hemocyanin. The antiserum recognized acylated ghrelin and non-acylated ghrelin equally on a molar basis. Human Tyr 0 -ghrelin [13-28] was radioiodinated by the lactoperoxidase method for use in the assay. Inter-and intra-assay variation was < 8 and < 6%, respectively. The limit of detection of this assay is 12 fmol/tube of human ghrelin. Two milliliters of plasma was diluted with 2 ml of 0.9% saline and applied to a Sep-Pak C-18 cartridge (Waters, Milford, MA) pre-equilibrated with 0.9% saline. The cartridge was washed first with saline and then with a 0.1% trifluoroacetic acid (TFA) solution, and peptides were eluted with a 60% acetonitrile (CH 3 CN) solution containing 0.1% TFA. The eluate was evaporated, reconstituted with RIA buffer, and subjected to RIA analysis. A diluted sample or a standard peptide solution (100 ml) was incubated for 24 h with 100 ml of the antiserum diluent (final dilution 1/20,000). The tracer solution (16,000 cpm/100 ml) was added, and the mixture incubated for 24 h. Bound and free ligands were separated by the second antibody method. All procedures were done at 4 C. Recovery of human ghrelin added to the plasma was % (n=6) Statistical methods Correction coefficients were calculated by linear regression analysis. The subject groups (mean SD) were compared using ANOVA and a post-hoc Fisher s test. P value of <0.05 was considered statistically significant. 3. Results The fasting plasma ghrelin concentrations in all subjects were negatively correlated with nutritional parameters such as BMI, percent body fat and serum cholinesterase concentration. There was positive correlation between plasma ghrelin value and serum amylase concentration in all subjects (Table 1). Physiological characteristics of subject groups were showed in Table 2. The frequencies of binge/purge cycles were /week in AN-BP group, / week in BN-P group. The frequency of binge-eating episodes was /week in BN-NP group. There were no significant differences in age and serum potassium concentration. The mean BMI, percent body fat and serum cholinesterase value in both AN-R and AN-BP groups were lower than those in controls

3 Table 1 Correlations of ghrelin values and physiological data M. Tanaka et al. / Journal of Psychiatric Research 37 (2003) r P Age (years) Duration of illness (years) Body mass index (kg/m 2 ) 0.49 a < Percent body fat (%) 0.46 a < Serum amylase (U/l) 0.57 a < Serum potassium (mmol/l) Serum cholinesterase (U/l) 0.31 a <0.005 a Significant correlations (P<0.05). respectively. Mean serum amylase level in AN-BP group was higher than that in AN-R, BN-NP and control groups (Table 2). The mean plasma ghrelin levels in AN-R ( pm, means.d.), AN-BP ( pm) and BN-P ( pm) were significantly higher than those in both BN-NP ( pm) and controls ( pm). Mean plasma ghrelin level in AN-BP was significantly higher than that in AN-R. The plasma ghrelin levels in both AN-R and AN-BP did not significantly differ from that in BN-P. There were no differences on plasma ghrelin levels between BN-NP and controls (Fig. 1). In both AN-BP and BN-P groups, the frequencies of binge/purge cycles were positively correlated with plasma ghrelin concentration (Fig. 2), and serum amylase value (AN-BP: r=0.56, P < 0.05; BN-P: r=0.65, P < 0.005). There was significant positive correlation between plasma ghrelin value and serum amylase value (Fig. 3). In BN-NP group, there were no significant correlations among frequencies of binge-eating episodes, plasma ghrelin concentrations and serum amylase values (P < 0.2). Serum cholinesterase value was significantly correlated with plasma ghrelin value (r= 0.57, P<0.05). Fig. 1. Comparison of plasma ghrelin concentration in female anorexia nervosa patients, restricting type (AN-R) and binge-eating/purging type (AN-BP), in female bulimia nervosa patients, purging type (BN-P) and nonpurging type (BN-NP), and in sex-matched healthy volunteers (controls). *P<0.05, ** P< Discussion The fasting plasma ghrelin concentrations in all subjects were negatively correlated with BMI and percent body fat. The mean plasma ghrelin levels in both AN-R and AN-BP patients were higher than healthy volunteers. These findings are in agreement with previous human studies (Shiiya et al., 2002; Tscho p et al., 2001; Otto et al., 2001). First, we have demonstrated that there was positive correlation between plasma ghrelin concentration and total serum amylase value in all subjects. Second, there was significantly difference on plasma ghrelin levels between BN subtypes. Third, there were significant correlations among plasma ghrelin concentrations, frequencies of binge/purge cycles and Table 2 Physiological characteristics of subject groups AN-R (N=21) AN-BP (N=19) BN-P (N=18) BN-NP (N=13) Control (N=15) Mean S.D. Mean S.D. Mean S.D. Mean S.D. Mean S.D. Age (years) Duration of illness (years) 2.6 b b 1.2 Body mass index (kg/m 2 ) ab ab ab 11 Percent body fat (%) ab ab ab 2.5 Serum amylase (U/l) 96.9 b b b 9.6 Serum potassium (mmol/l) Serum cholinesterase (U/l) a ab ab 56 a P<0.05 vs. AN-R. b P<0.05 vs. AN-BP, using ANOVA and post hoc Fisher s test.

4 20 M. Tanaka et al. / Journal of Psychiatric Research 37 (2003) Fig. 2. (A) Relationship of plasma ghrelin concentration and frequency of binge/purge cycles for anorexia nervosa patients with binge-eating/purging type (AN-BP). (B) Relationship of plasma ghrelin concentration and frequency of binge/purge cycles for bulimia nervosa patients with purging type (BN-P). Fig. 3. (A) Relationship of plasma ghrelin concentration and serum amylase concentration for anorexia nervosa patients with binge-eating/purging type (AN-BP). (B) Relationship of plasma ghrelin concentration and serum amylase concentration for bulimia nervosa patients with purging type (BN-P). serum amylase values in both AN-BP and BN-P patients. The mean plasma ghrelin in BN-P patients was higher than that found in BN-NP, though mean nutritional parameters between the groups were not significantly different. Eating behavior such as habitual purging with vomiting can only be used to discriminate the subtypes of BN patients. Mean plasma ghrelin in AN-BP patients was higher than that in AN-R despite similar nutritional parameters, as has been found in our previous research (Tanaka et al., in press). Also, habitual binge/purge cycles can only be used to discriminate the subtypes of AN patients. There were no significant differences between BN-P and subtypes of AN despite difference of BMI. Thus, we deem that habitual binge/purge behavior may have some influence on circulating plasma ghrelin levels. For both AN-BP and BN-P patients with habitual binge/purge behavior, there were significant correlations among plasma ghrelin values and frequencies of binge/ purge cycles, and serum amylase values. However, there were no significant correlations among frequencies of

5 M. Tanaka et al. / Journal of Psychiatric Research 37 (2003) binge-eating episodes, plasma ghrelin concentrations and serum amylase values in BN-NP. It has previously been reported that total serum amylase levels in BN patients were significantly correlated with frequencies of binge eating and vomiting (Metzger et al., 1999; Walsh et al., 1990) and furthermore vomiting repeatedly rather than binge-eating behavior that may increase amylase (Robertson and Millar, 1999). These findings suggest that habitual binge/purge cycles with vomiting as opposed to binge eating episodes without vomiting may have a greater influence on fasting plasma ghrelin concentration. Ghrelin is one of the gastrointestinal peptides and when administered intracerebroventricularly, stimulates gastric acid secretion by activating the vagus system (Date et al., 2001). As the regulation mechanism between gastrointestinal peptides and the vagus system has been reported to play a role (El-Salhy et al., 2000; Uvna s-moberg, 1983), circulating ghrelin levels may act as a feedback system via the vagal system. Several lines of evidence (Halmi and Sunday, 1991; Faris et al., 2000) have led us to postulate that afferent vagal hyperactivity could be an important factor in the pathophysiology of BN. Since BN has been reported to be due to hypervagal activity (Kennedy and Heslegrave, 1989), afferent vagal hyperactivity may increase circulating levels of plasma ghrelin in patients with habitual binge/purge behavior. In the present study, plasma ghrelin levels in the five subjects were demonstrated to vary even though serum potassium did not differ. Previous study has reported that total body potassium may be depleted even when serum potassium levels are normal (Powers et al., 1995). Another study concluded that routine electrolyte measurement was a poor screening method for purging behavior as the frequency of hypokalemia in the patients with eating disorders was found to occur in only 4.6% of the subjects (Greenfeld et al., 1995). Though hypokalemia in eating disorders may be an important as physiological factor, it is possible that plasma ghrelin concentration might be a better parameter of occult or denied binge/purge behavior with vomiting in eating disorders. Finally, habitual binge/purge behavior may influence circulating plasma ghrelin levels in both BN-P and AN-BP. Furthermore, habitual binge/purge cycles with vomiting as opposed to binge eating episodes without vomiting may have a greater influence on fasting plasma ghrelin concentration in eating disorders. Acknowledgements This study was supported by a research grant from the Japanese Ministry of Health, Labor and Welfare. References American Psychiatric Association. Diagnostic and statistical manual of mental disorders. 4th ed (DSM IV). Washington, DC: American Psychiatric Association; Bolanowski M, Nilson BE. Assessment of human body composition using dual-energy X-ray absorptiometry and bioelectrical impedance analysis. Medical Science Monitor 2001;7: Date Y, Nakazato M, Murakami N, Kojima M, Kangawa K, Matsukura S. Ghrelin acts in the central nervous system to stimulate gastric acid secretion. Biochemical and Biophysical Research Communications 2001;280: El-Salhy M, Danielsson H, Axelsson H, Qian BF. Neuroendocrine peptide levels in the gastrointestinal tract of mice after unilateral cervical vagotomy. Regulatory Peptides 2000;88: Faris PL, Kim SW, Meller WH, Goodale RL, Oakman SA, Hofbauer RD, et al. Effect of decreasing afferent vagal activity with ondansetron on symptoms of bulimia nervosa: a randomized, double-blind trial. Lancet 2000;355: Greenfeld D, Mickley D, Quinlan DM, Roloff P. Hypokalemia in outpatients with eating disorders. American Journal of Psychiatry 1995;152:60 3. Halmi KA, Sunday SR. Temporal patterns of hunger and fullness ratings and related cognitions in anorexia and bulimia. Appetite 1991;16: Kennedy SH, Heslegrave RJ. Cardiac regulation in bulimia nervosa. Journal of Psychiatric Research 1989;23: Kinzl J, Biebl W, Herold M. Significance of vomiting for hyperamylasemia and sialadenosis in patien ts with eating disorders. International Journal of Eating Disorders 1993;13: 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: Metzger ED, Levine JM, McArdle CR, Wolfe BE, Jimerson DC. Salivary gland enlargement and elevated serum amylase in bulimia nervosa. Biological Psychiatry 1999;45: Nakazato M, Murakami N, Date Y, Kojima M, Matsuo H, Kangawa K, Matsukura S. A role for ghrelin in the central regulation of feeding. Nature 2001;409: Otto B, Cuntz U, Fruehauf E, Wawarta R, Folwaczny C, Riepl RL, et al. Weight gain decrease elevated plasma ghrelin concentrations of patients with anorexia nervosa. European Journal of Endocrinology 2001;145: Powers PS, Tyson IB, Stevens BA, Heal AV. Total body potassium and serum potassium among eating disorder patients. International Journal of Eating Disorders 1995;18: Robertson C, Millar H. Hyperamylasemia in bulimia nervosa and hyperemesis gravidarum. International Journal of Eating Disorders 1999;26: Shiiya T, Nakazato M, Mizuta M, Date Y, Mondal MS, Tanaka M, et al. Plasma ghrelin levels in lean and obese humans and the effect of glucose on ghrelin secretion. Jounal of Clinical Endocrinology Metabolism 2002;87: Shintani M, Ogawa Y, Ebihara K, Aizawa-Abe M, Miyanaga F, Takaya K, et al. Ghrelin, an endogenous growth hormone secretagogue, is a novel orexigenic peptide that antagonizes leptin action through the activation of hypothalamic neuropeptide Y/Y1 receptor pathway. Diabetes 2001;50: Takaya K, Ariyasu H, Kanamoto N, Iwakura H, Yoshimoto A, Harada M, et al. Ghrelin strongly stimulates growth hormone release in humans. Journal of Clinical Endocrinology and Metabolism 2000; 85: Tanaka M, Naruo T, Daisuke Y, Yoshiki T, Nagai N, Shiiya T, Nakazato M, Matsukura S, Nozoe S. Fasting plasma ghrelin levels in subtypes of anorexia nervosa. Psychoneuroendocrinology [in press]. Tanaka M, Naruo T, Muranaga T, Yasuhara D, Shiiya T, Nakazato M, et al. Increased fasting plasma ghrelin levels in patients with

6 22 M. Tanaka et al. / Journal of Psychiatric Research 37 (2003) bulimia nervosa. European Journal of Endocrinology 2002;146: R1 R3. Tschöp M, Smiley DL, Heiman ML. Ghrelin induces adiposity in rodents. Nature 2000;407: Tschöp M, Weyer C, Tataranni AP, Devanarayan V, Ravussin E, Heiman ML. Circulating ghrelin levels are decreased in human obesity. Diabetes 2001;50: Uvnäs-Moberg K. Release of gastrointestinal peptides in response to vagal activation induced by electrical stimulation, feeding and suckling. Journal of the Autonomic Nervous System 1983;9: Walsh BT, Wong LM, Pesce MA, Hadigan CM, Bodourian SH. Hyperamylasemia in bulimia nervosa. Journal of Clinical Psychiatry 1990;51: Wolfe BE, Metzger ED, Levine JM, Jimerson DC. Laboratory screening for electrocyte abnormalities and anemia in bulimia nervosa: a controlled study. International Journal of Eating Disorders 2001;30: Wren AM, Seal LJ, Cohen MA, Brynes AE, Frost GS, Murphy KG, et al. Ghrelin enhances appetite and increased food intake in humans. Journal of Clinical Endocrinology and Metabolism 2001a;86:5952. Wren AM, Small CJ, Abbot CR, Dhillo WS, Seal LJ, Cohen MA, et al. Ghrelin causes hyperphagia and obesity in rats. Diabetes 2001b; 50: