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Influence of naloxone infusion on prolactin and growth hormone response to growth hormone-releasing hormone in anorexia nervosa
Psychoneuroendocrinology,Vol. 16, No. 6, pp. 499-504, 1991
0306-4530/91 $3.00+0.00 ©1992 Pergamon Press plc
Printed in Great Britain
INFLUENCE OF NALOXONE INFUSION O N PROLACTIN A N D GROWTH HORMONE RESPONSE TO GROWTH HORMONERELEASING HORMONE IN ANOREXIA NERVOSA L. DE MARINIS, A. MANCINI, C. D'AMICO, P. ZuPPI, A. TOFANI, S. DELLA CASA, A. SAPOROSI, P. SAMBO, C. FIUMARA, F. CALABRO, and A. BARBARINO Institute of Endocrinology,the Catholic University School of Medicine, Rome, Italy (Received 6 September 1988; in final form 24 December 1990)
SUMMARY Anorexia nervosa (AN) is frequently associated with anomalies of growth hormone (GH) and prolactin (PRL) secretion. We studied the GH and PRL responses to GHRH1_44 (50 ~g Iv) and the effect of a naloxone infusion (1.6 mg/hr), started 1 hr before GHRH administration, on this response in 12 female patients with AN, aged 15-30 yr, and in seven normal women, aged 19-27 yr, during the follicular phase as controls. In AN, GHRH induced an increase in GH levels similar to that observed in normal subjects. A significant inhibition of the GH response to GHRH was observed during naloxone infusion, similar to the inhibition in normal female subjects during the follicular phase. PRL levels showed a significant increment after GHRH alone and a slight, nonsignificant, PRL increment after GHRH during naloxone infusion in AN patients. In contrast a slight PRL decrease was observed after GHRH, both before and during naloxone infusion, in the normal subjects. Our study demonstrates that endogenous opioids play a role in influencing PRL secretion in patients with AN different from their role in normal subjects.
INTRODUCTION IN PATIENTSWITH ANOREXIANERVOSA(AN), malnutrition is secondary to a psychogenic disturbance of eating. Evidence for an opioid peptide (OP) involvement in the modulation of feeding behaviour has been shown in animals and in humans (Morley, 1987). Several investigators have attempted to elucidate the possible role of OP in the pathogenesis of AN and the potential role of OP antagonists in the treatment of the opposite condition, bulimia (Mitchell et al., 1986). In a preliminary investigation, [~-endorphin (BEP) levels were moderately reduced in patients with AN, and the BEP and ACTH responses to CRF were significantly blunted (Cavagnini et al., 1986). Since BEP is clearly involved in the regulation of pituitary hormone secretion, having a stimulatory effect on GH release in animals and humans (Morley, 1981) and on PRL release in animals (Krieger, 1985), we investigated the effect of a naloxone (NAL) infusion on the PRL Address correspondence and reprint requests to: Dr. L. De Marinis, Institute of Endocrinology, The Catholic University, School of Medicine, Largo A. Gemelli 8, 1-00168, Rome, ITALY. 499
500
L. DEMARINISet al.
and G H responses to G H R H in patients with AN and in normal female subjects, in order to further elucidate the endogenous OP tone in this condition. SUBJECTS AND METHODS Twelve patients with AN, ages 15-30 yr, and seven normal female volunteers, ages 19-27 yr, participated in this study. The diagnosis of AN was made with the DSM-III criteria of the American Psychiatric Association (1980). The history of AN lasted from 1-3 yr, five patients had previously participated in physical exercise, and three had suffered some form of family distress before the onset of the symptoms. All patients exhibited a marked food refusal, with an estimated caloric intake ranging from 700-1200 KCal. Five patients presented vomiting behaviour, and two had an excessive use of diuretics or laxatives. Their weight was 30-40% lower than their ideal body weight, and all had amenorrhea. All patients were studied when their weight was stable. They were not taking any medications. The following tests were performed: GHRH test: Following an overnight fast, at 0900h, all patients were placed at bed rest, with an indwelling venous cannula inserted in one arm and maintained open by a saline infusion. GHRH (Bachem, Switzerland) was administered as a bolus of 50 gg Iv at time 0. Blood samples were collected at -15, 0, 15, 30, 60, and 90 min. The mean values of the -15 and 0 min determinations were considered as basal levels. NAL + GHRH test: Following an overnight fast, the same test was repeated during the infusion of NAL (Narcan, Crinos, Italy), 1.6 mg/hr starting 1 hr before GHRH administration (time 0). Blood samples were collected at -60, -30, 0, 15, 30, 60, and 90 min. Time 0 values were considered as basal levels for the evaluation of GHRH effects. Saline test: Two milliliters of normal saline were administered in all patients, and blood was collected at the same intervals as after GHRH administration. The overnight fasting was well accepted by the patients, since it corresponded to their customary eating pattern. The tests were performed in random order, with an interval of at least three days between studies. As a control, GHRH, NAL + GHRH and saline tests were performed in seven normal women, tested during the follicular phase of their menstrual cycle. The three tests, in random order, were performed in the same phase of consecutive menstrual cycles. Blood samples were centrifuged within 2 hr after collection and plasma aliquots frozen at -20°C until assayed. GH and PRL were assayed, with a RIA procedure, with reagents furnished by Biodata, Milan, Italy. Normal ranges in our laboratory are: GH, 0 - 1 0 ng/ml and PRL, 5 - 2 0 ng/ml. Intra- and interassay coefficients of variation were 2.5% and 5.8% for GH and 2.1% and 4.6% for PRL, respectively. The cross reactivity of PRL with GH in our assay was negligible (0.13%). Other hormones also were determined in AN patients (T3, T4, TSH, FSH and LH after GnRH, cortisol, and 17-13-estradiol) by RIA. Normal ranges are indicated in Table III. Statistical analysis was performed with analysis of variance. RESULTS The results of G H R H a d m i n i s t r a t i o n , during N A L or saline i n f u s i o n , on G H levels are reported in Table I. G H R H elicited a G H response in the A N patients c o m p a r a b l e to that observed in the normal subjects, with a peak response at 30 m i n and a return to basal levels by 90 min. At the starting of NAL infusion (time -60), mean (+ SEM) G H levels were 8.4 + 2.7 n g / m l in the a n o r e c t i c p a t i e n t s a n d 1 5 . 1 + 6.7 n g / m l i n the n o r m a l s u b j e c t s . T h e y were n o t significantly modified by the 1-hr NAL infusion. NAL infusion completely blunted the G H response to GHRH, with a significant difference at 15, 30, 60 m i n in c o m p a r i s o n with the a d m i n i s t r a t i o n of G H R H alone, s i m i l a r l y in the anorectic and the normal women. The PRL response to GHRH, NAL + GHRH, and saline in the anorectic patients is shown in
501
NALOXONEANDPRL ANDGH RESPONSETOGHRFI IN ANOREXIANERVOSA
Table II. Basal PRL levels and peak responses are shown in the figure. GH1LH induced a significant PRL 14 increase when administered alone; the mean % peak in- ~" 12 10 crease was 247.3+45.5%. During the NAL infusion, the PRL response to GHRH was blunted: slight, non- #: significant, PRL increase was still present; the mean % peak increase was 140.6 + 9.2 (p < 0.05 vs. % peak after 0 BP BP BP BP GHRH alone). During saline infusion, a slight, nonGRF NAL+GRF GRF NAL+GRF significant, PRL decrease was observed (Table II); NORMALWOMEN ANORECTICWOMEN mean nadir values were 7 9 . 4 + 5 . 6 % of basal values Mean (± SEM} basal PRL levels and peak (i.e., a decrease of 20.5%) (p < 0.05 vs. % variation responses to GHRH alone or GHRH adafter NAL + GHRH, andp<0.01 vs. GHRH alone). ministered during a naloxone infusion in In normal subjects, a slight, nonsignificant, PRL de- normal women and patients with anorexia crease was present after GHRH both before and during nervosa. B = basal, P = peak response. *p< 0.05, significant difference between naloxone infusion (Fig.) and during saline infusion. basal levels and peak response. Other hormone levels in our AN patients are shown in Table HI. They exhibited low estradiol levels; the LH response to GnRH was depressed in eight patients and marked in four patients (causing a large SEM). TABLE I. PLASMA G H RESPONSE (MEAN+ SEM) IN NG/ML AFTER THE ADMINISTRATION OF G H R H 1 - 4 4 (50 ~G W), ALONE OR DURING NALOXONE (NAL) INFUSION (1.6 MG/HR), IN PATIENTS WITH ANOREXIA NERVOSA AND IN NORMAL SUBJECTS Basal v a l u e s t
15 m i n
30 m i n
60 m i n
90 m i n
17.1 + 5.7 9.6 + 4.6
39.9+ 8.2 6.7 + 2.5"
48.8+ 7.6 14.2 + 6.6
33.8+ 6.9 13.7 + 6.8*
18.5+ 5.2 13.3 + 6.4
10.9 + 3.4 8.5 + 3.7
42.9 + 8.6 7.2 + 3.0"*
40.9 + 7.2 7.0 + 2.4**
32.1 + 9.6 4.4 + 1.4"*
13.8 + 4.7 3.7+ 1.4
Anorectic Patients GHRH NAL + GHRH N o r m a l Subjects GHRH NAL + GHRH
? Mean of - 15 and 0 min values in GHRH test, 0 min values (after 1-hr NAL infusion) in NAt, + GHRH test. *p < 0.05, **p < 0.01, significant difference for GH levels after GHRH compared to NAL + GHRH.
TABLE II. PLASMA PRL RESPONSE (MEAN+ SEM) IN NG/ML AFTER THE ADMINISTRATION OF PLACEBO OR GHRI-I 1 - 4 4 (50 ~G W), ALONE OR DURING NALOXONE (NAL) INFUSION (1.6 MG/HR), IN PATIENTS WITH ANOREXIA NERVOSA Mean peak or n a d i r %
Basal v a l u e s
15 r a i n
30 rnin
60 m i n
90 m i n
of variation *
Placebo
(a)
6.3 + 0.8
5.9 + 0.7
5.7 + 0.7
5.5 + 0.9
5.7 + 0.9
79.4 + 5.6
GHRH
(b)
7.4 + 2.3
8.7 + 2.0
11.4 + 4.3
9.1 + 2.4
11.2 + 3.6
247.3 + 45.5
NAL + GHRH
(c)
7.1+2.0
8.1+1.8
9.3+2.3
8.2+1.7
7.5+1.5
* calculated with basal level as 100. p
140.6+9.2
502
L. DEMARINISet al.
TABLEIII. MEAN (+ SEM) CONCENTRATIONSOF OTHERHORMONES IN PATIENTSWITHANOREXIANERVOSA Basal T3 ( n g / m l )
T4 (l~g/dl) TSH (IxU/ml) FSH (mIU/ml) LH (mlU/ml) Cortisol (~tg/dl) 17-15-estradiol (pg/ml) *after GnRH I00 ~g Iv t follicular phase
90.8+ 20.1 6.1 + 0.93 2.04+ 0.65 9.78+ 0.5 6.36 + 0.84 20.0 + 1.67 19.2 -+4.9
Peak*
27.4+ 3.2 64.8_+ 33.7
Normal Range
52 - 160 4.2 - 11 0.5 - 5 5 - 25t 5 - 20t 7.5 -22 20 - 70t
DISCUSSION These experiments show that when GHRH is administered during NAL infusion in AN patients, the GH response is completely suppressed and the paradoxical PRL response is only partially suppressed. AN patients have elevated fasting GH levels, which have been related to the poor caloric intake of these subjects. However, the responses to provocative stimuli have been found to be normal (Brown et al., 1977; Vigersky & Loriaux, 1977) or blunted (Casper et al., 1977). The striking GH responses to GHRH are similar to those observed in normal female subjects tested during the follicular phase of their menstrual cycle (Gelato et al., 1983; Barbarino et al., 1987; De Marinis et al., 1988). Our present data suggest that GH levels are sustained by an appropriate central OP tone, despite the finding of low circulating levels of I]-endorphin in some patients with AN (Cavagnini et al., 1986). In addition, in normal women, the opioid tone and its effect on the GH response to GHRH is modulated by the steroid environment; naloxone infusion can block the GH response during the follicular and luteal phases, but not during the periovulatory period (Barbarino et al., 1987). A "paradoxical" PRL response to GHRH has been observed, in addition to AN (De Marinis et al., 1986; Casanueva et al., 1987), in acromegaly (De Marinis et al., 1986), GH-deficient adults (Borges et al., 1983), in normal subjects after high doses of GHRH (Gelato et al., 1984) and in normal women studied during the periovulatory phase of the menstrual cycle (De Marinis et al., 1989). A paracrine interaction between lactotroph and somatotroph cells has been suggested (Denef & Andries, 1983). Among paracrine signals, [3-endorphin has been indicated as a possible factor causing this phenomenon (Denef, 1986). We have suggested a possible involvement of opioid peptides in the paradoxical PRL response to GnRH present in normal women only during periovulatory phase; NAL infusion was able to blunt this paradoxical periovulatory response to GnRH (De Marinis et al., 1990). Our present data indicate that in AN patients, GHRH-induced PRL release is blunted, but not completely suppressed by NAL administration. It must be regarded as a sign of an abnormal hypothalamic-pituitary opioid regulation in these patients, together with the wellknown neuroendocrine alterations in gonadotropin, ACTH and GH dynamics (Casper et al., 1977; Ontjes et al., 1980). Other paradoxical responses, which are not present in normal
NALOXONEAND PRL AND G H RESPONSE TO G H R H IN ANOREXIANERVOSA
503
subjects, have been o b s e r v e d in A N patients. T h e y include a G H response to T R H ( M a e d a et al., 1976) and P R L response to G n R H (Beaumont et al., 1980), the latter evident during weight gain. It is difficult to establish whether these abnormalities are related to the weight loss or to the disease itself. The s a m e p a r a d o x i c a l responses have b e e n d e s c r i b e d in several conditions (Rubin et al., 1973; M a e d a et al., 1975; De Marinis et al., 1986), and they point to a dysfunction o f neurotransmitter systems in the hypothalamus. REFERENCES American Psychiatric Association (1980) Diagnostic and Statistical Manual of Mental Disorders, Third Edition. APA Press, Washington DC. Barbarino A, De Marinis L, Mancini A, D'Amico C, Passeri M, Zuppi P, Sambo P, Tofani A (1987) Sex-related naloxone influence on growth hormone-releasing hormone-induced growth hormone secretion in normal subjects. Metabolism 36: 105-109. Beaumont PV, Abraham SF, Turtle J (1980) Paradoxical prolactin response to gonadotropin-releasing hormone during weight gain in patients with anorexia nervosa. J Clin Endocrinol Metab 51: 1283-1285. Borges JLC, Gelato MC, Rogol AD, Vance ML, MacLeod RM, Loriaux DL, Rivier J, Blizzard RM, Furlanetto R, Evans VS, Kaiser DL, Merriam GR, Spiess J, Vale W, Thorner MO (1983) Effect of human pancreatic tumor growth hormone-releasing factor on growth hormone and somatomedin C levels in patients with idiopathic growth hormone deficiency. Lancet ii: 119-124. Brown GM, Garfinkel PE, Jeuniewic N, Moldofsky H, Stancer HC (1977) Endocrine profile in anorexia nervosa. In: Vigersky RA (Ed) Anorexia Nervosa. Raven Press, New York, pp 109-118. Casanueva FF, Borras CG, Burguera B, Lima L, Muruais C, Tresguerres JAF, Devesa J (1987) Growth hormone and prolactin secretion after growth hormone-releasing hormone administration, in anorexia nervosa patients, normal controls and tamoxifene-pretreated volunteers. Clin Endocrino127: 517-523. Casper RC, Davis JM, Pandey GN (1977) The effect of the nutritional status and weight changes on hypothalamic function tests in anorexia nervosa. In: Vigersky RA (Ed) Anorexia Nervosa. Raven Press, New York, pp 137-147. Cavagnini F, Invitti C, Dubini A, Silvestri G, Passamonti M, Magella A, Morabito F (1986) Endogenous opioids and hypothalamic-pituitary-adrenal function in obesity and anorexia nervosa. Psychiatry Res 16 (Suppl): 38-39. De Marinis L, Mancini A, D'Amico C, Passeri M, Sambo P, Zuppi P, Barbarino A (1986) Plasma prolactin response to GRF 1-44 in acromegaly and anorexia nervosa. In: Molinatti GM, Martini L (F_,ds)Endocrinology '85. Excerpta Medica, Amsterdam, pp 315-318. De Marinis L, Folli G, D'Amico C, Mancini A, Sambo P, Tofani A, Oradei A, Barbarino A (1988) Differential effects of feeding on the ultradian variation of the growth hormone (GH) response to GH-releasing hormone in normal subjects and patients with obesity and anorexia nervosa. J Clin Endocrinol Metab 66: 598-604. De Marinis L, Mancini A, D'Amico C, Sambo P, Tofani A, Calabr6 F, La Brocca A, Barbarino A (1989) Periovulatory plasma prolactin response to synthetic growth hormone-releasing hormone in normal women Metabolism 38: 275-277. De Marinis L, Mancini A, D'Amico C, Calabr6 F, Zuppi P, Fiumara C, Barini A (1990) Periovulatory plasma prolactin response to gonadotropin-releasing hormone: role of endogenous opiates. Neuroendocrinology 51: 717-720. Denef C (1986) Paracrine interactions in the anterior pituitary. In: Franchimont P (Ed) Clinics in Endocrinology and Metabolism. WB Saunders, London, pp 1-32. Denef C, Andries M (1983) Evidence for paracrine interaction between gonadotrophs and lactotrophs in pituitary cell aggregates. Endocrinology 112: 813-822. Gelato M, Pescovitz OH, Cassorla F, Loriaux DL, Merriam GR (1983) Effects of a growth hormone releasing factor in man. J Clin Endocrinol Metab 57: 674-676. Gelato M, Pescovitz OH, Cassorla F, Loriaux DL, Merriam GR (1984) Dose-response relationships for the effects of growth hormone-releasing factor-(1-44)NH2 in young adult men and women. J Clin Endocrinol Metab 59: 197-201.
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Krieger DT (1985) Neurocndocrinology. In: Ingbar SH (Ed) Contemporary Endocrinology (Vol 2). Plenum, New York, pp 1-26. Maeda K, Dato Y, Ohgo S, Chihara K, Yoshimoto Y, Yamaguchi N, Kuromaru S, Imura H (1975) Growth hormone and prolactin release after injection of thyrotrophin-releasing hormone in patients with depression. J Clin Endocrinol Metab 40: 501-505. Maeda K, Kato Y, Yamaguchi N, Chihara K, Ahga S, Iwasaki Y, Yoshimoto Y, Moridera K, Kuromaru S, Imura K (1976) Growth hormone release following thyrotrophin-mleasing hormone injection into patients with anorexia nervosa. Acta Endocrino181: 1-2. Mitchell JE, Laine DE, Morley JE, Levine AS (1986) Naloxone but not CCK-8 may attenuate binge-eating behavior in patients with the buliraia syndrome. Biol Psychiatry 21: 1399-1406. Morley JE (1981) The endocrinology of the opiates and opioid peptides. Metabolism 30: 195-209. Morley JE (1987) Neuropeptic~ regulation of appetite and weight. Endocr Rev 8: 256-287. Ontjes DA, Walton J, Nay RL (1980) Tests of anterior pituitary function. In: Bondy PK, Rosenberg LE (F_As) Metabolic Control and Disease. WB Sannders, Philadelphia, pp 1204-1210. Rubin AL, Levin SR, Bemestein RL, Tyrrel BJ, Noacco C, Forshara PH (1973) Stimulation of growth hormone by luteinizing hormone releasing hormone in active acromegaly. J Clin Endocrinol Metab 37: 160-162. Vigersky RA, Loriaux DM (1977) Anorexia nervosa as a model of hypothalamic dysfunction. In: Vigersky RA (Ed) Anorexia Nervosa. Raven Press, New York, pp 123-136.
0306-4530/91 $3.00+0.00 ©1992 Pergamon Press plc
Printed in Great Britain
INFLUENCE OF NALOXONE INFUSION O N PROLACTIN A N D GROWTH HORMONE RESPONSE TO GROWTH HORMONERELEASING HORMONE IN ANOREXIA NERVOSA L. DE MARINIS, A. MANCINI, C. D'AMICO, P. ZuPPI, A. TOFANI, S. DELLA CASA, A. SAPOROSI, P. SAMBO, C. FIUMARA, F. CALABRO, and A. BARBARINO Institute of Endocrinology,the Catholic University School of Medicine, Rome, Italy (Received 6 September 1988; in final form 24 December 1990)
SUMMARY Anorexia nervosa (AN) is frequently associated with anomalies of growth hormone (GH) and prolactin (PRL) secretion. We studied the GH and PRL responses to GHRH1_44 (50 ~g Iv) and the effect of a naloxone infusion (1.6 mg/hr), started 1 hr before GHRH administration, on this response in 12 female patients with AN, aged 15-30 yr, and in seven normal women, aged 19-27 yr, during the follicular phase as controls. In AN, GHRH induced an increase in GH levels similar to that observed in normal subjects. A significant inhibition of the GH response to GHRH was observed during naloxone infusion, similar to the inhibition in normal female subjects during the follicular phase. PRL levels showed a significant increment after GHRH alone and a slight, nonsignificant, PRL increment after GHRH during naloxone infusion in AN patients. In contrast a slight PRL decrease was observed after GHRH, both before and during naloxone infusion, in the normal subjects. Our study demonstrates that endogenous opioids play a role in influencing PRL secretion in patients with AN different from their role in normal subjects.
INTRODUCTION IN PATIENTSWITH ANOREXIANERVOSA(AN), malnutrition is secondary to a psychogenic disturbance of eating. Evidence for an opioid peptide (OP) involvement in the modulation of feeding behaviour has been shown in animals and in humans (Morley, 1987). Several investigators have attempted to elucidate the possible role of OP in the pathogenesis of AN and the potential role of OP antagonists in the treatment of the opposite condition, bulimia (Mitchell et al., 1986). In a preliminary investigation, [~-endorphin (BEP) levels were moderately reduced in patients with AN, and the BEP and ACTH responses to CRF were significantly blunted (Cavagnini et al., 1986). Since BEP is clearly involved in the regulation of pituitary hormone secretion, having a stimulatory effect on GH release in animals and humans (Morley, 1981) and on PRL release in animals (Krieger, 1985), we investigated the effect of a naloxone (NAL) infusion on the PRL Address correspondence and reprint requests to: Dr. L. De Marinis, Institute of Endocrinology, The Catholic University, School of Medicine, Largo A. Gemelli 8, 1-00168, Rome, ITALY. 499
500
L. DEMARINISet al.
and G H responses to G H R H in patients with AN and in normal female subjects, in order to further elucidate the endogenous OP tone in this condition. SUBJECTS AND METHODS Twelve patients with AN, ages 15-30 yr, and seven normal female volunteers, ages 19-27 yr, participated in this study. The diagnosis of AN was made with the DSM-III criteria of the American Psychiatric Association (1980). The history of AN lasted from 1-3 yr, five patients had previously participated in physical exercise, and three had suffered some form of family distress before the onset of the symptoms. All patients exhibited a marked food refusal, with an estimated caloric intake ranging from 700-1200 KCal. Five patients presented vomiting behaviour, and two had an excessive use of diuretics or laxatives. Their weight was 30-40% lower than their ideal body weight, and all had amenorrhea. All patients were studied when their weight was stable. They were not taking any medications. The following tests were performed: GHRH test: Following an overnight fast, at 0900h, all patients were placed at bed rest, with an indwelling venous cannula inserted in one arm and maintained open by a saline infusion. GHRH (Bachem, Switzerland) was administered as a bolus of 50 gg Iv at time 0. Blood samples were collected at -15, 0, 15, 30, 60, and 90 min. The mean values of the -15 and 0 min determinations were considered as basal levels. NAL + GHRH test: Following an overnight fast, the same test was repeated during the infusion of NAL (Narcan, Crinos, Italy), 1.6 mg/hr starting 1 hr before GHRH administration (time 0). Blood samples were collected at -60, -30, 0, 15, 30, 60, and 90 min. Time 0 values were considered as basal levels for the evaluation of GHRH effects. Saline test: Two milliliters of normal saline were administered in all patients, and blood was collected at the same intervals as after GHRH administration. The overnight fasting was well accepted by the patients, since it corresponded to their customary eating pattern. The tests were performed in random order, with an interval of at least three days between studies. As a control, GHRH, NAL + GHRH and saline tests were performed in seven normal women, tested during the follicular phase of their menstrual cycle. The three tests, in random order, were performed in the same phase of consecutive menstrual cycles. Blood samples were centrifuged within 2 hr after collection and plasma aliquots frozen at -20°C until assayed. GH and PRL were assayed, with a RIA procedure, with reagents furnished by Biodata, Milan, Italy. Normal ranges in our laboratory are: GH, 0 - 1 0 ng/ml and PRL, 5 - 2 0 ng/ml. Intra- and interassay coefficients of variation were 2.5% and 5.8% for GH and 2.1% and 4.6% for PRL, respectively. The cross reactivity of PRL with GH in our assay was negligible (0.13%). Other hormones also were determined in AN patients (T3, T4, TSH, FSH and LH after GnRH, cortisol, and 17-13-estradiol) by RIA. Normal ranges are indicated in Table III. Statistical analysis was performed with analysis of variance. RESULTS The results of G H R H a d m i n i s t r a t i o n , during N A L or saline i n f u s i o n , on G H levels are reported in Table I. G H R H elicited a G H response in the A N patients c o m p a r a b l e to that observed in the normal subjects, with a peak response at 30 m i n and a return to basal levels by 90 min. At the starting of NAL infusion (time -60), mean (+ SEM) G H levels were 8.4 + 2.7 n g / m l in the a n o r e c t i c p a t i e n t s a n d 1 5 . 1 + 6.7 n g / m l i n the n o r m a l s u b j e c t s . T h e y were n o t significantly modified by the 1-hr NAL infusion. NAL infusion completely blunted the G H response to GHRH, with a significant difference at 15, 30, 60 m i n in c o m p a r i s o n with the a d m i n i s t r a t i o n of G H R H alone, s i m i l a r l y in the anorectic and the normal women. The PRL response to GHRH, NAL + GHRH, and saline in the anorectic patients is shown in
501
NALOXONEANDPRL ANDGH RESPONSETOGHRFI IN ANOREXIANERVOSA
Table II. Basal PRL levels and peak responses are shown in the figure. GH1LH induced a significant PRL 14 increase when administered alone; the mean % peak in- ~" 12 10 crease was 247.3+45.5%. During the NAL infusion, the PRL response to GHRH was blunted: slight, non- #: significant, PRL increase was still present; the mean % peak increase was 140.6 + 9.2 (p < 0.05 vs. % peak after 0 BP BP BP BP GHRH alone). During saline infusion, a slight, nonGRF NAL+GRF GRF NAL+GRF significant, PRL decrease was observed (Table II); NORMALWOMEN ANORECTICWOMEN mean nadir values were 7 9 . 4 + 5 . 6 % of basal values Mean (± SEM} basal PRL levels and peak (i.e., a decrease of 20.5%) (p < 0.05 vs. % variation responses to GHRH alone or GHRH adafter NAL + GHRH, andp<0.01 vs. GHRH alone). ministered during a naloxone infusion in In normal subjects, a slight, nonsignificant, PRL de- normal women and patients with anorexia crease was present after GHRH both before and during nervosa. B = basal, P = peak response. *p< 0.05, significant difference between naloxone infusion (Fig.) and during saline infusion. basal levels and peak response. Other hormone levels in our AN patients are shown in Table HI. They exhibited low estradiol levels; the LH response to GnRH was depressed in eight patients and marked in four patients (causing a large SEM). TABLE I. PLASMA G H RESPONSE (MEAN+ SEM) IN NG/ML AFTER THE ADMINISTRATION OF G H R H 1 - 4 4 (50 ~G W), ALONE OR DURING NALOXONE (NAL) INFUSION (1.6 MG/HR), IN PATIENTS WITH ANOREXIA NERVOSA AND IN NORMAL SUBJECTS Basal v a l u e s t
15 m i n
30 m i n
60 m i n
90 m i n
17.1 + 5.7 9.6 + 4.6
39.9+ 8.2 6.7 + 2.5"
48.8+ 7.6 14.2 + 6.6
33.8+ 6.9 13.7 + 6.8*
18.5+ 5.2 13.3 + 6.4
10.9 + 3.4 8.5 + 3.7
42.9 + 8.6 7.2 + 3.0"*
40.9 + 7.2 7.0 + 2.4**
32.1 + 9.6 4.4 + 1.4"*
13.8 + 4.7 3.7+ 1.4
Anorectic Patients GHRH NAL + GHRH N o r m a l Subjects GHRH NAL + GHRH
? Mean of - 15 and 0 min values in GHRH test, 0 min values (after 1-hr NAL infusion) in NAt, + GHRH test. *p < 0.05, **p < 0.01, significant difference for GH levels after GHRH compared to NAL + GHRH.
TABLE II. PLASMA PRL RESPONSE (MEAN+ SEM) IN NG/ML AFTER THE ADMINISTRATION OF PLACEBO OR GHRI-I 1 - 4 4 (50 ~G W), ALONE OR DURING NALOXONE (NAL) INFUSION (1.6 MG/HR), IN PATIENTS WITH ANOREXIA NERVOSA Mean peak or n a d i r %
Basal v a l u e s
15 r a i n
30 rnin
60 m i n
90 m i n
of variation *
Placebo
(a)
6.3 + 0.8
5.9 + 0.7
5.7 + 0.7
5.5 + 0.9
5.7 + 0.9
79.4 + 5.6
GHRH
(b)
7.4 + 2.3
8.7 + 2.0
11.4 + 4.3
9.1 + 2.4
11.2 + 3.6
247.3 + 45.5
NAL + GHRH
(c)
7.1+2.0
8.1+1.8
9.3+2.3
8.2+1.7
7.5+1.5
* calculated with basal level as 100. p
140.6+9.2
502
L. DEMARINISet al.
TABLEIII. MEAN (+ SEM) CONCENTRATIONSOF OTHERHORMONES IN PATIENTSWITHANOREXIANERVOSA Basal T3 ( n g / m l )
T4 (l~g/dl) TSH (IxU/ml) FSH (mIU/ml) LH (mlU/ml) Cortisol (~tg/dl) 17-15-estradiol (pg/ml) *after GnRH I00 ~g Iv t follicular phase
90.8+ 20.1 6.1 + 0.93 2.04+ 0.65 9.78+ 0.5 6.36 + 0.84 20.0 + 1.67 19.2 -+4.9
Peak*
27.4+ 3.2 64.8_+ 33.7
Normal Range
52 - 160 4.2 - 11 0.5 - 5 5 - 25t 5 - 20t 7.5 -22 20 - 70t
DISCUSSION These experiments show that when GHRH is administered during NAL infusion in AN patients, the GH response is completely suppressed and the paradoxical PRL response is only partially suppressed. AN patients have elevated fasting GH levels, which have been related to the poor caloric intake of these subjects. However, the responses to provocative stimuli have been found to be normal (Brown et al., 1977; Vigersky & Loriaux, 1977) or blunted (Casper et al., 1977). The striking GH responses to GHRH are similar to those observed in normal female subjects tested during the follicular phase of their menstrual cycle (Gelato et al., 1983; Barbarino et al., 1987; De Marinis et al., 1988). Our present data suggest that GH levels are sustained by an appropriate central OP tone, despite the finding of low circulating levels of I]-endorphin in some patients with AN (Cavagnini et al., 1986). In addition, in normal women, the opioid tone and its effect on the GH response to GHRH is modulated by the steroid environment; naloxone infusion can block the GH response during the follicular and luteal phases, but not during the periovulatory period (Barbarino et al., 1987). A "paradoxical" PRL response to GHRH has been observed, in addition to AN (De Marinis et al., 1986; Casanueva et al., 1987), in acromegaly (De Marinis et al., 1986), GH-deficient adults (Borges et al., 1983), in normal subjects after high doses of GHRH (Gelato et al., 1984) and in normal women studied during the periovulatory phase of the menstrual cycle (De Marinis et al., 1989). A paracrine interaction between lactotroph and somatotroph cells has been suggested (Denef & Andries, 1983). Among paracrine signals, [3-endorphin has been indicated as a possible factor causing this phenomenon (Denef, 1986). We have suggested a possible involvement of opioid peptides in the paradoxical PRL response to GnRH present in normal women only during periovulatory phase; NAL infusion was able to blunt this paradoxical periovulatory response to GnRH (De Marinis et al., 1990). Our present data indicate that in AN patients, GHRH-induced PRL release is blunted, but not completely suppressed by NAL administration. It must be regarded as a sign of an abnormal hypothalamic-pituitary opioid regulation in these patients, together with the wellknown neuroendocrine alterations in gonadotropin, ACTH and GH dynamics (Casper et al., 1977; Ontjes et al., 1980). Other paradoxical responses, which are not present in normal
NALOXONEAND PRL AND G H RESPONSE TO G H R H IN ANOREXIANERVOSA
503
subjects, have been o b s e r v e d in A N patients. T h e y include a G H response to T R H ( M a e d a et al., 1976) and P R L response to G n R H (Beaumont et al., 1980), the latter evident during weight gain. It is difficult to establish whether these abnormalities are related to the weight loss or to the disease itself. The s a m e p a r a d o x i c a l responses have b e e n d e s c r i b e d in several conditions (Rubin et al., 1973; M a e d a et al., 1975; De Marinis et al., 1986), and they point to a dysfunction o f neurotransmitter systems in the hypothalamus. REFERENCES American Psychiatric Association (1980) Diagnostic and Statistical Manual of Mental Disorders, Third Edition. APA Press, Washington DC. Barbarino A, De Marinis L, Mancini A, D'Amico C, Passeri M, Zuppi P, Sambo P, Tofani A (1987) Sex-related naloxone influence on growth hormone-releasing hormone-induced growth hormone secretion in normal subjects. Metabolism 36: 105-109. Beaumont PV, Abraham SF, Turtle J (1980) Paradoxical prolactin response to gonadotropin-releasing hormone during weight gain in patients with anorexia nervosa. J Clin Endocrinol Metab 51: 1283-1285. Borges JLC, Gelato MC, Rogol AD, Vance ML, MacLeod RM, Loriaux DL, Rivier J, Blizzard RM, Furlanetto R, Evans VS, Kaiser DL, Merriam GR, Spiess J, Vale W, Thorner MO (1983) Effect of human pancreatic tumor growth hormone-releasing factor on growth hormone and somatomedin C levels in patients with idiopathic growth hormone deficiency. Lancet ii: 119-124. Brown GM, Garfinkel PE, Jeuniewic N, Moldofsky H, Stancer HC (1977) Endocrine profile in anorexia nervosa. In: Vigersky RA (Ed) Anorexia Nervosa. Raven Press, New York, pp 109-118. Casanueva FF, Borras CG, Burguera B, Lima L, Muruais C, Tresguerres JAF, Devesa J (1987) Growth hormone and prolactin secretion after growth hormone-releasing hormone administration, in anorexia nervosa patients, normal controls and tamoxifene-pretreated volunteers. Clin Endocrino127: 517-523. Casper RC, Davis JM, Pandey GN (1977) The effect of the nutritional status and weight changes on hypothalamic function tests in anorexia nervosa. In: Vigersky RA (Ed) Anorexia Nervosa. Raven Press, New York, pp 137-147. Cavagnini F, Invitti C, Dubini A, Silvestri G, Passamonti M, Magella A, Morabito F (1986) Endogenous opioids and hypothalamic-pituitary-adrenal function in obesity and anorexia nervosa. Psychiatry Res 16 (Suppl): 38-39. De Marinis L, Mancini A, D'Amico C, Passeri M, Sambo P, Zuppi P, Barbarino A (1986) Plasma prolactin response to GRF 1-44 in acromegaly and anorexia nervosa. In: Molinatti GM, Martini L (F_,ds)Endocrinology '85. Excerpta Medica, Amsterdam, pp 315-318. De Marinis L, Folli G, D'Amico C, Mancini A, Sambo P, Tofani A, Oradei A, Barbarino A (1988) Differential effects of feeding on the ultradian variation of the growth hormone (GH) response to GH-releasing hormone in normal subjects and patients with obesity and anorexia nervosa. J Clin Endocrinol Metab 66: 598-604. De Marinis L, Mancini A, D'Amico C, Sambo P, Tofani A, Calabr6 F, La Brocca A, Barbarino A (1989) Periovulatory plasma prolactin response to synthetic growth hormone-releasing hormone in normal women Metabolism 38: 275-277. De Marinis L, Mancini A, D'Amico C, Calabr6 F, Zuppi P, Fiumara C, Barini A (1990) Periovulatory plasma prolactin response to gonadotropin-releasing hormone: role of endogenous opiates. Neuroendocrinology 51: 717-720. Denef C (1986) Paracrine interactions in the anterior pituitary. In: Franchimont P (Ed) Clinics in Endocrinology and Metabolism. WB Saunders, London, pp 1-32. Denef C, Andries M (1983) Evidence for paracrine interaction between gonadotrophs and lactotrophs in pituitary cell aggregates. Endocrinology 112: 813-822. Gelato M, Pescovitz OH, Cassorla F, Loriaux DL, Merriam GR (1983) Effects of a growth hormone releasing factor in man. J Clin Endocrinol Metab 57: 674-676. Gelato M, Pescovitz OH, Cassorla F, Loriaux DL, Merriam GR (1984) Dose-response relationships for the effects of growth hormone-releasing factor-(1-44)NH2 in young adult men and women. J Clin Endocrinol Metab 59: 197-201.
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