Reduced cerebrospinal fluid levels of immunoreactive pro-opiomelanocortin related peptides (including beta-endorphin) in anorexia nervosa

Life Sciences, Vol. 41, pp. 2147-2155 Printed in the U.S.A.

Pergamon Journals

REDUCED C E R E B R O S P I N A L FLUID LEVELS OF IMMUNOREACTIVE PROOPIOMELANOCORTIN RELATED PEPTIDES (INCLUDING BETA-ENDORPHIN) IN ANOREXIA NERVOSA W.H. Kaye 1,2, W.H. Berrettini 2, H.E. Gwirtsman 2, M. Chretien 3, P.W.Gold 2, D.T. George 2, D.C. Jimerson 2, M.H. Ebert 4 iUniversity of Pittsburgh, Western Psychiatric Institute and Clinic, Pittsburgh, PA 15213; 2National Institute of Mental Health, Bethesda, Maryland 20892; 3Clinical Research Institute of Montreal, Montreal, Canada H2W,IR7; 4Department of Psychiatry, Vanderbilt University, Nashville, Tennessee 37232 (Received in final form September i0, 1987) Summarv The discovery that the endogenous opioid peptides contribute to the modulation of appetitive behavior and neuroendocrine function has raised questions as to whether disturbances of opioids contributes to the pathophysiology of eating disorders. To assess central nervous system (CNS) b e t a - e n d o r p h i n in patients with anorexia nervosa we measured cerebrospinal fluid (CSF) beta-endorphin concentrations before, and at intervals after weight correction. In addition, we measured three sister peptides (beta-lipotropin, adrenocorticotropic hormone (ACTH), and the N-terminal fragment) derived from the same precursor molecule, pro-opiomelanocortin (POMC) to determine whether possible disturbances might extend to sister peptides. Underweight anorectics (58 ± 5% of average body weight (ABW), n = i0) had significantly lower CSF concentrations of all 4 peptides compared to healthy controls (102 ± 10% ABW, n = Ii). CSF concentrations of all 4 POMCrelated peptides were found to be significantly increased when the same anorectics were restudied 4 to 6 weeks after weight gain (83 ± 4% ABW). After weight gain, levels of CSF beta-endorphin, betalipotropin, and ACTH were similar to controls, whereas levels of CSF N-POMC remained significantly less than controls. Another group of women, previously underweight with anorexia nervosa, but weight-restored (93 ± 11% ABW, n = 12) for greater than 1 year had CSF concentrations of all 4 POMCrelated peptides that were similar~to controls. We conclude that underweight anorectics have stateassociated disturbances of CNS beta-endorphin as well as other POMC-related peptides. These abnormalities are part of the neurobiological syndrome of anorexia nervosa and may contribute to the characteristic alterations in behavior and neuroendocrine function. 0024-3205/87 $3.00 + .00 Copyright (c) 1987 Pergamon Journals Ltd.

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Anorexia nervosa, which most often occurs in young women, is characterized by a fear of becoming obese, the relentless pursuit of thinness, and a resistance to weight gain (i). The gratification that anorectics display when refusing food and losing weight defies normal life-sustaining biological drives. Such an abnormal attitude toward eating and weight is the classic symptom of anorexia nervosa--and the most puzzling one. The pathophysiology of the appetitive disturbance in anorexia nervosa is not known, but a question has been raised as to whether altered endogenous opioid activity might contribute to disturbed feeding behavior (2-5). Such speculation has been fueled by considerable data, derived primarily from animal experimentation (6), which suggests that opioid agonists increase and opioid antagonists decrease food intake. In addition to the disturbance of appetite, anorexia nervosa is invariably associated with various forms of hypothalamic-pituitary dysfunction, including hypothalamic hypogonadism (7) and hypercortisolism (8). Opioids, probably acting through hypothalamic mechanisms (see reviews 9, i0), suppress pulsatile gonadotropin secretion in rats and humans and inhibit ACTH and cortisol release in man. It is possible that a disturbance of one or more of the endogenous opioid peptides could contribute to these neuroendocrine disturbances in anorexia nervosa. Little data is available regarding central nervous system opioid function in patients with anorexia nervosa (11-13), due in part to limitations placed on clinical research by the relative nonspecificity of pharmacologic probes and the inability to measure many of the endogenous opioid peptides in humans. CNS betaendorphin, however, is one opioid system that can be assessed in humans by measuring levels of immunoreactive beta-endorphin in CSF. In this study we report measurements of CSF beta-endorphin as well as CSF concentrations of its sister peptides that are derived from the same precursor molecule, POMC (14,15). These sister peptides include the following: the N-terminal fragment of POMC (N-POMC); ACTH, a peptide fragment located in the middle of the POMC precursor molecule; beta-lipotropin, which forms the C-terminal end. Beta-endorphin is the C-terminal fragment of beta-lipotropin. Our reason for measuring several related peptides derived from the same precursor molecule was to determine whether postulated disturbances of beta-endorphin in anorexia nervosa were also found in sister peptides derived from the same precursor molecule. Since it is likely that beta-endorphin is co-stored with its sister peptides, a systematic alteration in all POMCrelated peptide concentrations would provide supportive evidence that a disturbance existed in beta-endorphin and might implicate a pathologic process within the POMC system. Methods All subjects were hospitalized on the clinical research units at the National Institute of Mental Health and gave informed consent for the study. Patients fulfilled DSM-III criteria for anorexia nervosa (i). Each subject was free of any medication for a least six weeks before any study. Patients with anorexia nervosa (all female) were studied during three phases of illness; I0 patients (25 ± 3 years) were studied

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after having been chronically underweight (31.2 ± 3.7 kg) for six m o n t h s to 15 years; e a c h h a d b e e n at less t h a n 75 p e r c e n t of a v e r a g e b o d y weight, the c h r o n i c a l l y low w e i g h t phase, for at least 6 m o n t h s p r i o r to admission. All i0 of t h e s e p a t i e n t s w e r e r e s t u d i e d t h r e e to four w e e k s a f t e r w e i g h t g a i n (44.8 ± 3.0 kg), the s h o r t - t e r m r e c o v e r y phase. T w e l v e a d d i t i o n a l p a t i e n t s (23 ± 6 years) who had previously been diagnosed as h a v i n g anorexia n e r v o s a w e r e s t u d i e d one to t e n y e a r s (mean of 34 months) a f t e r c o r r e c t i o n of t h e i r w e i g h t loss (50.0 ± 6.7 kg), the l o n g - t e r m recovery phase. Underweight anorectic patients stayed in the hospital for four months or longer for a non-medication, behaviorally-oriented, w e i g h t - g a i n i n g program. Long-term weightr e s t o r e d a n o r e c t i c s and h e a l t h y c o n t r o l s w e r e h o s p i t a l i z e d for 3 to 4 d a y s on the same ward. C o n t r o l s (23 ± 3 years) c o n s i s t e d of ii a g e - m a t c h e d , drug-free, p a i d female v o l u n t e e r s (57.6 ± 7.1 kg) w h o r e s i d e d at the c l i n i c a l research units of t h e N a t i o n a l Institutes of H e a l t h Clinical Center. C o n t r o l s u b j e c t s w e r e free of m e d i c a l and n e u r o l o g i c a l problems. Controls were given a structured psychiatric interview by an e x p e r i e n c e d psychiatrist and found to be free of m e n t a l disorders. N o n e of the a n o r e c t i c s at low w e i g h t or at s h o r t - t e r m r e c o v e r y showed normal menses; all presumably manifested hypothalamic amenorrhea. S e v e n of the 12 l o n g - t e r m recovered patients had normal menses, 1 had oligomenorrhea, a n d 4 h a d amenorrhea. All normal s u b j e c t s had normal menses. In s u b j e c t s w h o h a d n o r m a l m e n s t r u a l cycles, the lumbar p u n c t u r e (LP) w a s c o n d u c t e d in the early f o l l i c u l a r p h a s e of the cycle. Patients f a s t e d overnite. L u m b a r p u n c t u r e s w e r e p e r f o r m e d at 9:00 a.m. CSF s a m p l e s t a k e n from the 12th to 15th ml of CSF w i t h d r a w n from the lumber spine w e r e i m m e d i a t e l y f r o z e n on dry ice and w e r e s t o r e d at -70 ° C. All R I A p r o c e d u r e s w e r e p e r f o r m e d by i n d i v i d u a l s w h o h a d no k n o w l e d g e of the i d e n t i t y of i n d i v i d u a l CSF samples. All d e t e r m i n a t i o n s for e a c h p e p t i d e w e r e d o n e in a s i n g l e assay. All d e t e r m i n a t i o n s w e r e d o n e in duplicate. The coefficient of v a r i a t i o n for d u p l i c a t e s w i t h i n a s s a y s w a s less than 15%. None of the antibodies used in these studies cross-reacted s i g n i f i c a n t l y w i t h any o t h e r m e a s u r e d p e p t i d e (16) e x c e p t for t h e b e t a - e n d o r p h i n antibody, w h i c h c r o s s - r e a c t e d w i t h b e t a - l i p o t r o p i n at the 10% level. Not all p e p t i d e f r a g m e n t s w e r e m e a s u r e d on e a c h l o n g - t e r m w e i g h t - r e s t o r e d anorectic, b e c a u s e the beta-endorphin and b e t a - l i p o t r o p i n assays w e r e c o m p l e t e d b e f o r e the e n t i r e p a t i e n t s a m p l e w a s r e c r u i t e d for study. BETA-ENDORPHIN RIA: A plasma beta-endorphin RIA kit from I m m u n o n u c l e a r C o r p o r a t i o n w a s a d a p t e d to use w i t h CSF (17). A 1.0 ml v o l u m e of CSF was used. The fluid w a s m i x e d for 4 h o u r s at 4 C in a s e p h a r o s e adsorption column. T h e c o l u m n w a s d r a i n e d and w a s h e d 3 t i m e s w i t h 1.0 ml a l i q u o t s of n o r m a l saline. The betae n d o r p h i n i m m u n o r e a c t i v i t y w a s e l u t e d w i t h 0.025 N HCI (0.5 mls). The e l u a t e w a s d i v i d e d into two equal fractions and e a c h was n e u t r a l i z e d w i t h 50 ul of 0.i N NaOH. The a n t i b o d y w a s a l l o w e d to react w i t h the e l u a t e for i0 h o u r s at 4 C b e f o r e t r a c e w a s added. The i n c u b a t i o n w a s c o n t i n u e d for an a d d i t i o n a l 14 h o u r s b e f o r e s e p a r a t i o n by the s e c o n d antibody. The d e t e c t i o n limit w a s 0.6 fmol/ml, and the r e c o v e r y of added s t a n d a r d w a s g r e a t e r t h a n 80%. Values for 4 low-weight anorectics were below the limit of detection. T h e s e p a t i e n t s w e r e a s s i g n e d a b e t a - e n d o r p h i n v a l u e of

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0.3 fmol/ml. A C T H - R I A : S e p - P a k c a r t r i d g e s w e r e w a s h e d w i t h 10 m l s of m e t h a n o l f o l l o w e d b y i0 m l s of d i s t i l l e d water. A l i q u o t s of 0.6 mls of CSF w e r e a p p l i e d to the c a r t r i d g e s w h i c h w e r e t h a n w a s h e d w i t h 10 mls of d i s t i l l e d water. Immunoreactive ACTH was eluted with a mixture of TEAF buffer (1% formic acid, pH 3.2 with addition of triethylamine) and acetonitrile (4:6, V/V). The eluent was l y o p h i l i z e d a n d the r e s i d u e t a k e n up in a s s a y b u f f e r (0.63 M Na 2 PO 4 and 0 . 1 3 M Na 2 EDTA, pH 7.4), c o n t a i n i n g 0.02% Na2NO2, 0.1% t r i t o n x - 1 0 0 and 250 K I U of a p r o t i n i n / m l . The r e c o v e r y of A C T H by this p r o c e d u r e is 85-95%. The a n t i b o d y was o b t a i n e d from IgG Corporation (Nashville, Tennessee, ACTH-1). B o t h s t a n d a r d and trace were ACTHI_39. T h e s p e c i f i c a c t i v i t y w a s less t h a n 450 Ci/g. The m e t h o d s of the R I A h a v e b e e n p r e v i o u s l y d e s c r i b e d (18). The d e t e c t i o n limit for this a s s a y was 0.8 fmol/ml. B~TA-LIPOTROPHIN RIA: RIA kits were obtained from New England Nuclear Corporation, Boston, M a s s a c h u s e t t s (19). A v o l u m e of 200 ul of CSF w a s u s e d in a final v o l u m e of 0.400 ml. R e c o v e r y was 85%; the d e t e c t i o n limit w a s 2 fmol/ml. N - p O M C RIA: N-POMC, a 76 a m i n o a c i d peptide, w a s d e t e r m i n e d in CSF and p l a s m a as p r e v i o u s l y described (20). The limit of d e t e c t i o n for CSF N - P O M C w a s 8.3 fmol/ml, a n d v a l u e s for 6 lowweight anorectics w e r e b e l o w this limit. These patients were a s s i g n e d a v a l u e of 4.2 fmol/ml. S T A T I S T I C A L PROCEDURES: T h e CSF levels of the 4 p e p t i d e s found in the u n d e r w e i g h t a n o r e c t i c s w e r e c o m p a r e d by a t w o - t a i l e d p a i r e d t-test with the levels found in these same patients after short-term weight-restoration. A o n e - w a y a n a l y s i s of v a r i a n c e was u s e d to c o m p a r e CSF levels of the p e p t i d e s in u n d e r w e i g h t anorectics, l o n g - t e r m w e i g h t - r e s t o r e d anorectics, a n d controls; it w a s a l s o u s e d to c o m p a r e the levels for s h o r t - t e r m w e i g h t - r e s t o r e d anorectics, long-term weight-restored anorectics, and controls. D i f f e r e n c e s w e r e e v a l u a t e d by the S c h e f f e p o s t h o c test. Values are e x p r e s s e d as m e a n ± SD. Results CSF c o n c e n t r a t i o n s of all 4 P O M C - r e l a t e d p e p t i d e s w e r e r e d u c e d in l o w - w e i g h t a n o r e c t i c s c o m p a r e d to the s a m e p a t i e n t s s t u d i e d 3 to 4 w e e k s a f t e r w e i g h t g a i n (beta-endorphin, t = 3.21, p = .01; beta-lipotropin, t = 4.78, p = .001; ACTH, t = 4.40, p = .001; N-POMC, t = 2.73, p = .02). (see F i g u r e One) One-way ANOVA between underweight anorectics, long-term weight-restored anorectics, and h e a l t h y c o n t r o l s s h o w e d s i g n i f i c a n t d i f f e r e n c e s for beta-endorphin (F = 5.04, df = 2, 25, p = .01), beta-lipotropin (F = 9.10, df = 2, 24, p = .001), A C T H (F = 10.2, df = 2, 29, p < .001), and N - P O M C (F = 17.25, df = 2, 30, p < .001). S c h e f f e p o s t h o c tests found t h a t low w e i g h t a n o r e c t i c s had significantly (p < .05) lower CSF c o n c e n t r a t i o n s of all 4 P O M C - r e l a t e d w h e n c o m p a r e d to controls. In addition, u n d e r w e i g h t a n o r e c t i c s h a d s i g n i f i c a n t l y l o w e r levels of CSF A C T H a n d N - P O M C c o m p a r e d to l o n g - t e r m w e i g h t - r e s t o r e d anorectics. One-way ANOVA between short-term weight-restored anorectics, long-term weightr e s t o r e d anorectics, and h e a l t h y c o n t r o l s found a s i g n i f i c a n t d i f f e r e n c e o n l y for N - P O M C (F = 5.15, df = 2, 30, p = .01).

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12 10

/~-endorphin (fmol/ml)

--

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,

1 CSF 6'~ '

*

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(fmol/ml)

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~:

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60

,

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Low Weight

Short Term Weight Restored

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J PATIENTS WITH ANOREXIA NERVOSA

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L e v e l s of the 4 CSF P O M C - r e l a t e d p e p t i d e s in all subjects. Boxes indicate mean levels, points indicate value for each subject, dashed lines indicate limit of detection of the assay. *indicates a significant d i f f e r e n c e b y p a i r e d ttest between low w e i g h t and short-term weightrestored anorectics. #indicates significant differences b y 1-way A N O V A and S c h e f f e Post Hoc Test.

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Scheffe post-hoc tests showed significant differences for N-POMC between short-term weight-restored anorectics and controls as well as between short- and long-term weight-restored anorectics. It should be mentioned that significant differences for other POMCrelated peptides between short-term weight-restored anorectics and controls may have been obscured by the large variance. Discussion We found that CSF beta-endorphin and other POMC-related peptides were significantly reduced in low-weight anorectics and normalized after weight-restoration. In contrast, in the only previous study of CSF beta-endorphin in anorexia nervosa, Gerner (12) reported normal amounts of beta-endorphin-like activity in anorexia nervosa. There were several differences between the patients in this study and those studied by Gerner that may explain this discrepancy. First, the mean weight of Gerner's patients was substantially greater than the mean weight of the underweight patients in this study. In fact, many of Gerner's patients were closer in weight to our recently weight-restored anorectics than to our underweight anorectics. Second, our underweight anorectic patients were studied before refeeding and weight gain whereas Gerner's patients were studied after they had begun to refeed and had gained several kilograms. One interpretation of these data considered together is that reduced CSF beta-endorphin levels are associated with extreme weight loss in anorexia nervosa and that these levels are increased by either improved nutrition and/or weight gain. The reduction in CSF beta-endorphin concentrations in underweight anorectics is contrary to data on total CSF opioid activity reported previously by our group (13). In this earlier study, we found that underweight anorectics had elevated total CSF opioid activity as measured by a radioreceptor assay. It is important to emphasize that beta-endorphin is only one of a surprisingly large number of peptides in the brain with opioid activity (21,22). In fact, in the present study, values of betaendorphin in CSF were found to be less than 1% of the values for total opioid activity measured by the radioreceptor assay. Other investigators have reported a discrepancy between measurements of beta-endorphin and total opioid activity (23,24). Thus, elevated concentrations of one or more of the other endogenous opioid peptide(s), such as dynorphin, may account for the radioreceptor assay results. Such a possibility remains to be explored. Beta-endorphin, beta-lipotropin and ACTH were present in CSF in approximately equimolar quantities. CSF N-POMC molar levels were approximately six to seven fold higher than any of the other CSF POMC fragments, most likely explained by data that indicates that the half-life of CSF N-POMC is higher than other fragments (25). It is less likely that these higher CSF N-POMC levels represented cross-reactivity with unrelated peptides since only one peak of CSF N-POMC immunoreactivity appeared on gel chromatography. A major finding in this study is the implication, on the basis of the consistency of the reduction in all 4 related peptides in underweight anorectics, that this reduction extends to the POMC precursor. The source of CSF POMC related peptides is not known, but POMC cell bodies are located in the arcuate nucleus,

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dorsolateral, and ventromedial hypothalamus (14,15) and presumably contribute to the CSF content of POMC-related peptides. It is unclear whether reduced POMC peptide fragment levels reflect a primary decrease in precursor synthesis or release into CSF, a secondary decrease in peptide turnover related to alterations in receptor activity, or depletion of peptide levels as a result of a persistent increase in turnover. Determining the sequence of cause and effect of neurobiologic alterations in anorexia nervosa is a major methodologic problem in studying this illness. Reduced CSF beta-endorphin in underweight anorectics could be secondary to malnutrition and/or weight loss. Alternatively, a defect in brain beta-endorphin pathways could be a pre-existing factor initiating the onset of anorexia nervosa. It is not practical to study a population at risk for anorexia nervosa to resolve this dilemma. We were, however, able to follow anorectics at intervals after weight-restoration. The correction of this disturbance by weight-restoration might imply that such disturbances are secondary to malnutrition and/or weight loss and not the cause. To our knowledge, cause and effect relationships between changes in weight or nutrition and CNS beta-endorphin activity have not been studied in humans. The effects of shortterm caloric deprivation on beta-endorphin have been studied in animals, but extrapolating from short-term animal studies to chronic human disease is problematic. Moreover, the effects of short term caloric deprivation on beta-endorphin in animals has been inconsistent (26-29), whether studied in the pituitary or brain regions. Reduced beta-endorphin activity, even if it occurs secondary to malnutrition, may still be relevant to the symptom of food refusal in anorexia nervosa. It has been theorized that a vicious cycle develops in anorexia nervosa in the sense that consequences of malnutrition perpetuate pathological behavior (30). Betaendorphin has been shown to stimulate feeding behavior in rats when injected intraventricularly or into the medial hypothalamus (31,32). Assuming that beta-endorphin activity contributes to feeding behavior in humans and that reduced CSF concentrations reflect decreased activity of this system, it is then possible that dieting behavior in vulnerable subjects creates a downward spiraling cascade whereby starvation diminishes appetite. Such a possibility may have heuristic value in developing new treatment approaches for this intractable illness. The relationship of beta-endorphin disturbances to neuroendocrine abnormalities is not clear. Baranowska et al. (ii) found that naloxone administration produced an increase in serum LH concentrations in ii of 24 underweight anorectics. Reduced levels of beta-endorphin argues against the possibility that betaendorphin inhibited gonadotrophin secretion (33) in anorectics, but raises the possibility that a disturbance of some other endogenous opioid peptide is responsible. Our group has recently reported (34) that underweight anorectics have increased concentrations of CSF corticotropin-releasing hormone (CRH) which are thought to contribute to the h y p e r c o r t i s o l i s m of anorexia nervosa (8). Reduced levels of CSF POMC related peptides suggests that underweight anorectics may have a dissociation of central and peripheral ACTH-opioid systems known to be related to CRH activity. It should be noted that there are two relatively

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independent ACTH-opioid systems, central and endocrine. In m e a s u r i n g plasma POMC-related peptides, one is assessing endocrine function of the HPA axis whereas CSF POMC-related peptides are thought to reflect the function of POMC neurons in the hypothalamus. It is possible that peripheral POMC-related peptide activity contribute to CSF concentrations of POMC-related peptides (14,15), although data suggesting that hypophysectomy does not alter brain ACTH or beta-endorphin argues against this possibility. On the other hand, since CNS opioids are thought to inhibit the peripheral secretion of ACTH and cortisol (9,10), reduced CNS beta-endorphin activity could contribute to hypercortisolism in underweight anorectics. Whether increased CRH contributes to the reduced levels of CSF POMC-related peptides is not known. While co-distribution patterns exist in the CNS between the CRH perikarya and POMC fibers (35), relatively little is known about the physiological relationships of these peptide systems in brain. In summary this finding adds to a growing body of knowledge characterizing disturbances in neurotransmitter function in anorexia nervosa. Such findings are likely to contribute to the appetitive and neuroendocrine abnormalities typically found in this illness. Acknowleduements We would like to thank Sandra Nicotra and Maureen Piraino for their editorial assistance and the staff of 3E at the National Institute of Health clinical Center. References i. 2.

A m e r i c a n Psychiatric Association, Diagnostic and statistical manual of mental disorders. Third edition, APA, Washington, DC. (1980). R. MOORE, I.H. MILLS, A. FORSTER, J. Royal Soc. Med. 74 129-

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T.P. DONOHOE, Life Sol. 3_~4203-218 (1984). G.I. SZMUKLER, D. TANTAM, Brit. J. Med. Psycho. 5_/7 303-310 (1984). M.A. MARRAZZI, E.D. LUBY, Inter. J. Eating Disor. 5 191-208 (1986). J.E. MORLEY, A.S. LEVINE, G.K. YIM, M.T. LOWRY, Neurosci. Biobehav. Rev. Z 281-305 (1983). R.A. VIGERSKY, D.L. LORIAUX, A.E. ANDERSEN, M.B. LIPSETT, clin. Endocrinol. Metab. 5 517-535 (1976). P.W. GOLD, H. GWIRTSMAN, P.C. AVGERINOS, L.K. NIEMAN, W.T. GALLUCCUI, W.H. KAYE, D. JIMERSON, M. EBERT, R. RITTMASTER, D.L. LORIAUX, G.P. CHROUSOS, New Engl. J. Med. 314 1335-1342 (1986). A. GROSSMAN, C l i n . E n d o c r i n o l . M e t a b . 12 7 2 5 - 7 4 6 ( 1 9 8 6 ) . A. PFEIFFER, A. HERZ, Horm. Metabol. Res. 16 386-397 (1984). B. BARANOWSKA, G. ROZBICKA, W. JESKE, A. ABDEL-FATTAH, J. Clin. Endocrinol. Metab. 59 412-416 (1984). R.H GERNER, B. SHARP, Brain Res. ~ 7 244-247 (1982). W.H. KAYE, D. PICKAR, D. NABER, M.H. EBERT, Am. J. Psych. i~9 643-645 (1982). D.T. KRIEGER, A.S. LIOTTA, M.J. BROWNSTEIN, E.A. ZIMMERMAN, Recent Prog. Horm. Res. 36 277-344 (1980).

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