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Effect of a test meal, duodenal acidification, and tetragastrin on the plasma concentration of β-endorphin-like immunoreactivity in man
Regulatory Peptides, 4 (1982) 173-181
173
Elsevier BiomedicalPress
Effect of a test meal, duodenal acidification, and tetragastrin on the plasma concentration of B-endorphin-like immunoreactivity in man M. Matsumura
a, N . F u k u d a a, S. S a i t o b a n d H . M o r i a
a Second Department oflnternal Medicine, School of Medicine, University of Tokushima, Tokushima 770, and b Department of Laboratory Medicine, School of Medicine, University of Tokushima, Tokushima 770, Japan
(Received 13 April 1982; accepted for publication 16 July 1982)
Summary The effects of various test materials on plasma/3-endorphin-like immunoreactivity (fl-EpLI) were investigated in man using a specific radioimmunoassay developed by the authors. Plasma/3-EpLI was determined after extraction by the acid/acetone method (recovery 73 ± 5%). The intraassay and interassay coefficients of variation were 5.0% and 7.6%, respectively. The plasma concentrations of human/3-EpLI in normal subjects were 11.6 --- 4.0 pmol/1 for men (n = 23) and 10.7 ± 4.8 p m o l / l for women (n = 27). Ingestion of a test meal (150 g of Campbell's condensed meat soup) resulted in a biphasic rise in plasma fl-EpLI from the basal level of 4.4 -+ 1.0 pmol/1 to 29.2 --- 1.9 pmol/1 after 5 min and 24.8 ± 6.7 pmol/1 after 90 rain. Intraduodenal infusion of 115 ml of 0.1 M HC1 over 10 min increased the plasma /3-EpLI level from 8.7 - 0.5 p m o l / l to 15.5 ± 0.4 pmol/1 at 10 min after the start of infusion, but the level rapidly returned to the initial value after the end of the infusion. Intramuscular injection of 4 /~g/kg body weight of tetragastrin markedly stimulated gastric acid output and B-EpLI release, but pretreatment with 10 mg of histamine H 2 receptor antagonist inhibited the gastric acid output and plasma fl-EpLI release induced by tetragastrin. These results indicate that/3-EpLI release is stimulated by ingestion of meat soup, duodenal acidification and tetragastrin administration. It is suggested that gastric acid participates, at least in part, in postprandial release of B-EpLI, probably from the gastrointestinal tract. /3-endorphin-like immunoreactivity; test meal; gastric acid; histamine H 2 receptor antagonist
0167-0115/82/0000-0000/$02.75 © 1982 ElsevierBiomedicalPress
174
Introduction
fl-Endorphin is an endogenous opioid known to be localized in the brain, pituitary gland, gastrointestinal tract, pancreas, and gonads [3,15,20,23,26]. After development of a radioimmunoassay for human fl-endorphin (flh-Ep), the plasma levels of human fl-endorphin-like immunoreactivity (flh-EpLI) have been determined in normal and diseased states [9,14,28-30], although the values reported differed, probably owing to differences in the antisera and assay procedures used. Ingestion of a meal has been reported to stimulate release of gut hormones including gastrin, secretin, motilin, gastric inhibitory polypeptide (GIP), enteroglucagon and vasoactive intestinal polypeptide (VIP) [1,4-6,18,24]. Duodenal acidification also stimulates the release of secretin, motilin and v I P [6,12,16]. As we demonstrated previously [15], flh-EpLI is present in human gut and is released in vitro by perfusion of human duodenal mucosa with solution of low pH, suggesting that gastric acid may be involved in release of/?h-EpLI. Therefore we investigated the effects of a test meal, duodenal acidification and tetragastrin on the release of /?-EpLI in man. The results suggest that gastric acid participates in postprandial release of/~-EpLI, probably from the gastrointestinal tract.
Materials and Methods
Subjects The fasting levels of plasma fl-EpLI were determined in 23 normal males aged 21-60 years and 27 females aged 20-60 years between 0800-1000 h. The response of fl-EpLI release to various tests was examined in normal male volunteers aged 19-21 years.
Experimental protocol The test meal used was Campbell's condensed meat soup (Campbell Soup Company, U.S.A.), containing 100 kcal, 7.4 g protein, 2.0 g fat, 13.3 g carbohydrates and 3.2 g minerals per 150 g. Five normal males were given 150 g of the soup orally and blood samples were collected 30 and 15 min before and 0, 15, 30, 60, 90 and 120 min after ingestion. Duodenal acidification was carried out by intraduodenal infusion of 115 ml of 0.1 M HCI over 10 min through a gastric tube. Blood samples were obtained at 0, 5, 10, 15, 20, 30, 45, and 60 min after the start of infusion. Tetragastrin (Sanayakuhin, Japan, 4 ~ g / k g body weight in 2 ml of physiological saline) was injected intramuscularly into three normal subjects. 7 days later, the same dose of tetragastrin was given 30 min after infusion of 10 mg histamine H 2 receptor antagonist (YM-11170, Yamanouchi Pharmaceutical Co., Japan) into the stomach through a gastric tube. In both experiments blood samples and gastric juice were collected at intervals as indicated in Results.
Radioimmunoassay of flh-Ep Anti-C/h-Ep serum was prepared by the method of Guillemin et al. [8] against
175
synthetic flh-Ep and proved to be highly specific for flh-Ep as reported previously [15]. It crossreacted only with human fl-lipotropin (flh-LPH, crossreactivity 4.5% on a molar basis) of the pro-opiocortin-related peptides tested, flh-Ep was radioiodinated by the chloramine T method [10], and I25I-labelled flh-Ep was separated on a column (1 × 18 cm) of Sephadex G-25 (medium). The procedure for flh-Ep radioimmunoassay was reported previously [15]. The binding of 125I-labelled flh-Ep to antiserum was displaced by the presence of unlabelled hormone in a dose-related manner in the range of 4-256 pg/tube.
Extraction of flh-EpLI from plasma Plasma flh-EpLI was extracted by a modification of the method of Robertson et al. [22]. 1 ml of plasma was mixed with 2 ml of acid/acetone (acetic acid/acetone, 4: 100, v / v ) and centrifuged for 30 rain at 3000 rpm. The resulting supernatant was washed twice with 4 ml of light petroleum. The upper layer was carefully removed and discarded. The remaining acetone was removed by vacuum evaporation and the aqueous portion lyophilized. The dry material was dissolved in 1 ml of the radioimmunoassay buffer. T h e r e c o v e r y in the extraction procedure was 73 ± 5% (mean ± S.E.M.) at a concentration of 28.6 pmol/1. In all cases the plasma values of fl-EpLI were corrected for recovery.
Gel chromatography The extract from 13 ml of plasma was chromatographed on a column (0.9 × 45 cm) of Sephadex G-50 (fine) with 0.1 M acetic acid containing 0.1% bovine serum albumin (BSA). Fractions of 1 ml were collected and lyophilized. The content of fl-EpLI in each fraction was measured by radioimmunoassay. Blue dextran (molecular mass > 2000000), synthetic flh-LPH, synthetic flh-Ep and Na125I were used for calibration. The mean recovery of 10 ng of synthetic flh-Ep added to plasma was 45 ± 4%.
Gastric juice analysis To determine acid output, gastric juice was collected with a gastric tube at 15-min intervals after removing the gastric contents for 30 rain using a continuous suction pump. The acid concentration of each sample was measured by titration with 0.1 M NaOH, with TOpher-Michaelis reagent and phenolphthalein as indicators.
Statistics Results for plasma flh-EpLI levels are quoted as mean ± S.E.M. The statistical significance of differences was analyzed by Student's t-test.
Results
Validity of assay The intra-assay coefficients of variation in 6 measurements at 3 concentrations (36, 72, and 143 pmol/1) were 7.0, 5.8, and 5.0%, respectively. The interassay
176
coefficients of variation in measurements on 10 days were 8.8 and 7.6% at concentrations of 36 and 72 pmol/1, respectively. When 14.3 and 143 p m o l / l of flh-Ep were added to plasma, the recoveries were 67.6 ± 4.3 and 85.8 ± 5.570, respectively. The normal values of plasma flh-EpLI for adults were 11.6 ± 4.0 pmol/1 (range 3.7-19.1) for men and 10.7 ± 4.8 p m o l / l (range 3.1-16) for women.
Effect of meat soup ingestion on plasma flh-EpL1 level Plasma fl-EpLI levels were increased from the basal level of 4.4 ± 1.0 to 29.2 ± 1.9 pmol/1 at 5 min and 24.8 ± 6.7 p m o l / l at 10 min ( P < 0 . 0 0 1 ) after ingestion of meat soup (150 g) and then returned to the initial level. The plasma fi-EpLI level showed a second rise (24.8 ± 6.7 pmol/1) after about 90 rain (Fig. 1). Effect of intraduodenal infusion of O.1 M HCI on the plasma flh-EpL1 level Intraduodenal infusion of 115 ml of 0.1M HCI over 10 min significantly increased the plasma flh-EpLI level from the basal level of 8.7 ± 0.5 p m o l / l to 15.3± 1.4 p m o l / l at 5 min and 15.5 ± 1.4 pmol/1 at 10 min after the start of infusion (Fig. 2). The plasma/3-EpLI level decreased rapidly to the initial level after the end of the infusion. Effect of tetragastrin on gastric acid output and the plasma flh-EpLl level As shown in Figure3, intramuscular injection of 4 /~g/kg body weight of tetragastrin markedly stimulated gastric acid output during the 60-min observation period. The plasma flh-EpLI level concomitantly increased from the basal level of 9.8 ± 0.2 pmol/1 to 20.7 -+ 1.4 pmol/1 after 30 min and then remained high.
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177
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Fig. 3. Effect of intramuscular injection of tetragastrin (4 /.tg/kg BW) on gastric acid output and the plasma fl-EpLI level with or without pretreatment with histamine H 2 receptor antagonist (10 mg, i.m.). • 0 , plasma fl-EpLI, without pretreatment with antagonist. O O, plasma fl-EpLI, with pretreatment with antagonist. [~, gastric acid output, without pretreatment with antagonist; EN, gastric acid output, with pretreatment with antagonist. Values are means+S.E.M. *P<0.01 vs. control. **P <0.02 vs. control. ***P <0.005 vs. control.
178
Effect of h&tamine H 2 receptor antagonist on tetragastrin-induced gastric acid output and fl-EpLl release When histamine H 2 receptor antagonist (10 rag) was infused into the stomach through a gastric tube 30 rain before tetragastrin injection, gastric acid output was completely inhibited and plasma flh-EpLI showed no significant increase (Fig, 3). Elution profiles on gel filtration of flh-EpLI extracted from plasma Plasma extracts were prepared from blood taken from two subjects before and 30 min after subcutaneous injection of 4 # g / k g body weight of tetragastrin, and applied to a column (0.9 × 45 cm) of Sephadex G-50. As shown in Figure4, two fractions of flh-EpLI were separated from each extract; the first peak was in the position of flh-LPH and the second peak in that of flh-Ep. The ratios of flh-LPH to flh-Ep in subject S.M. and in subject M.H. were 6.3: l and 7.7: 1, respectively, on a molar basis. The ratio of flh-LPH to flh-Ep in the extract from both subjects changed to 4.9:1 (S.M.) and 4.7:1 (M.H.) 30 rain after tetragastrin administration.
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Fig. 4. Elution profiles on Sephadex of plasma extracts from two normal men injected with tetragastrin. The lyophilized extracts from 13 ml of plasma were dissolved in 0.6 ml of 0.1 M acetic acid and applied to a column (0.9×45 cm) of Sephadex G-50. (A) basal state; (B) 30 rain after intramuscular injection of 4 # g / k g bodyweight tetragastrin. The column was calibrated with blue dextran, synthetic flh-LPH, flh-Ep and Na125I.
179
Discussion
Since Bradbury et al. [2] first isolated fl-endorphin from porcine pituitary, its regional distribution and action have been studied extensively. Recently, attempts have been made to determine the concentration of fl-Ep in biological fluids, but discrepant values have been reported, probably owing to differences in the antisera and radioimmunoassay procedures used. We developed a sensitive and specific radioimmunoassay for measuring plasma flh-Ep, in which specific anti-flh-Ep antiserum was used to obtain high specificity and acid/acetone extraction of plasma fl-EpLI was used to eliminate non-specific interference in radioimmunoassay. The recovery of plasma flh-Ep on extraction with acid/acetone is comparable with that by the silicic acid method [28], and the acid/acetone method is more convenient than the latter. The: .normal fasting values for plasma flh-EpLI obtained in our study are a little higher that~ those obtained by Wardlaw and Frantz [28] (6.2 -+ 2.2 pmol/l) by silicic acid extraction and by Nakao et al. [17] ( < 0.86-0.89 pmol/1) by chromatography, and lower than those reported by Wilkes et al. [30] (14.3-85.7 pmol/1) who did not use an extraction procedure. Wiedeman et al. [29] (1.4-12.9 pmol/1) reported similar values to ours. The difference between the value reported may be due to differences in antisera and methods for extraction of fl-Ep from plasma. However, no significant sex difference in plasma fl-Ep levels was noticed in any group. The plasma flh-Ep level in normal subjects is known to increase in response to various stimulants, such as metyrapone, vasopressin and insulin-induced hypoglycemia [17,28]. High levels of plasma fl-Ep are also found in some diseases such as Cushing's disease, Nelson's syndrome and Addison's disease [13,27]. These changes in plasma/3h-E p are associated with changes in plasma ACTH and fl-LPH, suggesting concomitant releases of these compounds from the pituitary. There are several reports on the distribution of pro-opiocortin-related peptides in tissues other than the central nervous system and pituitary [3,26]. Larsson [l l] demonstrated the presence of ACTH in the myenteric plexus, in epithelial cells of the small intestine and pancreatic islets by an immunohistochemical technique. Polak et al. [21] showed enkephalin-positive cells in both neural and epithelial sites immunohistochemically. Odell et al. [19] detected fl-LPH in human colon with a radioimmunoassay system that measures both fl-LPH and fl-MSH. Furthermore, fl-Ep was found in the rat gastrointestinal tract [20] and pancreas [3]. We reported recently that flh-EpLI is present in human stomach and duodenum and is released in vitro in response to various test meals [15]. These findings indicate that ACTH and related peptides are present in the gastroenteropancreatic system and may have a physiological role in the gut. Secretions of the gut hormones gastrin, secretin, motilin, GIP, enteroglucagon, VIP and somatostatin are known to be stimulated by ingestion of a meal [1,4-6,18,24]. In addition, duodenal acidification stimulates the release of secretin, motilin, GIP and VIP [6,12,16]. The present study demonstrated that the plasma flh-EpLI level increased biphasically in normal subjects after ingestion of condensed meat soup. The first peak may be due to nervous reflexes as seen in the case of postprandial release of plasma pancreatic polypeptide [7] or
180
related to the meal itself, but this remains to be elucidated. Since/3h-EpLI can be released from human duodenal mucosa in vitro by a solution of low pH [5] and by duodenal acidification as reported in the present paper, it seems very likely that gastric acid stimulates fl-EpLI release from the gut. In fact, we found that increase of the plasma/3-EpLI level was associated with increase in gastric acid output after tetragastrin administration. Furthermore, we found that tetragastrin-induced fl-EpLI release was prevented by pretreatment with histamine H 2 receptor antagonist, which completely inhibited tetragastrin-induced gastric acid output. This indicates that gastric acid may be related, at least in part, to fl-EpLI release. Gel-chromatographic analysis demonstrated that the fl-EpLI released after tetragastrin administration consisted of two fractions, namely the ratios of flh-LPH to flh-Ep in subject S.M. and in subject M.H. were 4.9:1 and 4.7:1, respectively, on a molar basis. In this study, biphasic release of/3-EpLI after ingestion of food was demonstrated in man. Duodenal acidification and tetragastrin administration were also shown to stimulate fl-EpLI release, but pretreatment with histamine H 2 receptor antagonist inhibited tetragastrin-induced gastric acid output and/3-EpLI release. These findings suggest that gastric acid participates, at least in part, in postprandial fl-EpLI release.
References 1 Boden, G., Essa, N. and Owen, O., Effects of intraduodenal amino acids, fatty acids, and sugars on secretin concentrations, Gastroenterology, 68 (1975) 722-727. 2 Bradbury, A.F., Smyth, D.G. and Snell, C,R., Biosynthesis of fl-MSH and ACTH. In: Chemistry Structure and Biology, R. Walter and J. Meiienhofer, (Eds.), Ann. Arbor Science Inc., 1975, pp. 609-615. 3 Bruni, J.F., Watkins, W.B. and Yen, S.S.C., fl-Endorphin in the human pancreas, J. Clin. Endocrinol. Metab., 49 (1979) 649-651. 4 Burhol, P.G., Waldum, H.L, Jorde, R. and Lygren, I., The effect of a test meal on plasma vasoactive intestinal polypeptide (VIP), gastric inhibitory polypeptide (GIP), and secretin in man, Scand. J. Gastroenterol., 14 (1979) 939-943. 5 ChayviaUe, J.-A., Miyata, M., Rayford, P.L. and Thompson, J.C., Effects of test meal, intragastric nutrients, and intraduodenal bile on plasma concentrations of immunoreactive somatostatin and vasoactive intestinal peptide in dog, Gastroenterology, 79 (1980) 844-852. 6 Ebeid, A.M., Escourtou, J., Murray, P. and Fischer, J.E., Pathophysiology of VIP, Gut Hormones, (1978) 479-483. 7 Ghatei, M.A. and Bloom, S.R., Enteroglucagon in man, Gut Hormones, (1981) 332-338. 8 Guillemin, R., Ling, N. and Vargo, T., Radioimmunoassay for a-endorphin and fl-endorphin, Biochem. Biophys. Res. Commun., 77 (1977) 361-366. 9 Hrllt, V., Miiller, O.A. and Fahlbusch, R.. fl-Endorphin in human plasma: basal and pathologically elevated levels, Life Sci., 25 (1979) 37-44. 10 Hunter, W,H. and Greenwood, F.A., Preparation of iodine-131 labelled human growth hormone of high specific activity, Nature 194 (1962) 495-496. 11 Larsson, L.I., Corticotropin-like peptides in central nerves and in endocrine cells of gut and pancreas, Lancet, 2 (1977) 1321-1323. 12 LeRoith, D., Spitz, I.M., Ebert, R., Liel, Y., Odes, S. and Creutzfeldt, W., Acid-induced gastric inhibitory polypeptide secretion in man, J. Clin. Endocrinol. Metab., 51 (1980) 1385 1389. 13 Liotta, A.S., Suda, T. and Krieger, D.T., fl-Lipotropin is the major opioid-like peptide of human pituitary and rat pars distalis: lack of significant fl-endorphin, Proc. Natl. Acad. Sci. U.S.A., 75 (1978) 2950-2954.
181 14 Malizia, E., Andreucci, G., Paolucci, D., Crescenzi, F., Fabbri, A. and Fraioli, F., Electroacupuncture and peripheral fl-endorphin and ACTH levels, Lancet, 2 (1979) 535-536. 15 Matsumura, M., Saito, S. and Fuijino, M., Effects of solution of low pH and taurocholate on release of fl-endorphin-like immunoreactivity from human duodenal mucosa in vitro, Regul. Peptides. 3 (1982) 173-181. 16 Mitznegg, P., Bloom, S.R., Domschke, W., Domschke, S., Wunsch, E. and Demling, L., Release of motilin after duodenal acidification, Lancet, 1 (1976) 888-889. 17 Nakao, K., Nakai, Y., Oki, S., Horii, K. and Imura, H., Presence of immunoreactive fl-endorphin in normal human plasma. A concomitant release of fl-endorphin with adrenocorticotropin after metyrapone administration, J. Clin. Invest., 62 (1978) 1395-1398. 18 Nilsson, G., Simon, J., Yalow, R.S. and Berson, S.A., Plasma gastrin and gastric acid responses to sham feeding and feeding in dogs, Gastroenterology, 63 (1972) 51-59. 19 Odell, W.D., Wolfsen, A.R., Bachelor, I. and Hirose, F.M., Ectopic production of lipotropin by cancer, Am. J. Med., 66 (1979) 631-638. 20 Orwall, E.S. and Kendall, J.E., fl-Endorphin and adrenocorticotropin in extrapituitary sites: gastrointestinal tract, Endocrinology, 107 (1980) 438-442. 21 Polak, J.M., Bloom, S.R., Sullivan, S.N., Facer, P. and Pearse, A.G.E., Enkephalin-like immunoreactivity in the human gastrointestinal tract, Lancet, 1 (1977) 972-974. 22 Robertson, G.L., Malhr, E.A., Athar, S. and Sinha, T., Development and clinical application of a new method for the radioimmunoassay of arginine vasopressin in human plasma, J. Clin. Invest., 52 (1973) 2340-2352. 23 Rossier, J., Vargo, T.M., Minick, S., Ling, N., Bloom, F.E. and Guillemin, R., Regional dissociation of fl-endorphin and enkephalin contents in rat brain and pituitary, Proc. Natl. Acad. Sci. U.S.A., 74 (1977) 5162-5165. 24 Saito, S., Ogawa, T., Saito, H., Ishimaru, K., Oshima, I, and Sonaka, Y., Regulation of motilin secretion in the postprandial state in man, Endocrinol. Japon., S.R. No. 1 (1980) 157-162. 25 Schwartz, T.W., Stenquist, B. and Olbe, L., Physiology of mammalian PP and the importance of vagal regulation, Gut Hormones, (1978) 261-264. 26 Sharp, B., Pekary, A.E., Meyer, N.V. and Hershman, J.M., /3-Endorphin in male rat reproductive organs, Biochem. Biophys. Res. Commun., 95 (1980) 618-623. 27 Suda, T., Liotta, A.S. and Krieger, D.T.,/3-Endorphin is not detectable in plasma from normal human subjects, Science, 202 (1978) 221-223. 28 Wardlaw, S.L. and Frantz, A.G., Measurement of/3-endorphin in human plasma, J. Clin. Endocrinol. Metab., 48 (1979) 176-180. 29 Wiedeman, E., Saito, T., Linfoot, J.A. and Li, C.H., Specific radioimmunoassay of human /3-endorphin in unextracted plasma, J. Clin. Endocrinol. Metab., 49 (1979) 478-480. 30 Wilkes, M.M., Stewart, R.D., Bruni, J.F., Quigley, M.E., Yen, S.S.C., Ling, N. and Chretien, M., A specific homologous radioimmunoassay for human /3-endorphin: direct measurement in biological fluids, J. Clin. Endocrinol. Metab., 50 (1980) 309-315.
173
Elsevier BiomedicalPress
Effect of a test meal, duodenal acidification, and tetragastrin on the plasma concentration of B-endorphin-like immunoreactivity in man M. Matsumura
a, N . F u k u d a a, S. S a i t o b a n d H . M o r i a
a Second Department oflnternal Medicine, School of Medicine, University of Tokushima, Tokushima 770, and b Department of Laboratory Medicine, School of Medicine, University of Tokushima, Tokushima 770, Japan
(Received 13 April 1982; accepted for publication 16 July 1982)
Summary The effects of various test materials on plasma/3-endorphin-like immunoreactivity (fl-EpLI) were investigated in man using a specific radioimmunoassay developed by the authors. Plasma/3-EpLI was determined after extraction by the acid/acetone method (recovery 73 ± 5%). The intraassay and interassay coefficients of variation were 5.0% and 7.6%, respectively. The plasma concentrations of human/3-EpLI in normal subjects were 11.6 --- 4.0 pmol/1 for men (n = 23) and 10.7 ± 4.8 p m o l / l for women (n = 27). Ingestion of a test meal (150 g of Campbell's condensed meat soup) resulted in a biphasic rise in plasma fl-EpLI from the basal level of 4.4 -+ 1.0 pmol/1 to 29.2 --- 1.9 pmol/1 after 5 min and 24.8 ± 6.7 pmol/1 after 90 rain. Intraduodenal infusion of 115 ml of 0.1 M HC1 over 10 min increased the plasma /3-EpLI level from 8.7 - 0.5 p m o l / l to 15.5 ± 0.4 pmol/1 at 10 min after the start of infusion, but the level rapidly returned to the initial value after the end of the infusion. Intramuscular injection of 4 /~g/kg body weight of tetragastrin markedly stimulated gastric acid output and B-EpLI release, but pretreatment with 10 mg of histamine H 2 receptor antagonist inhibited the gastric acid output and plasma fl-EpLI release induced by tetragastrin. These results indicate that/3-EpLI release is stimulated by ingestion of meat soup, duodenal acidification and tetragastrin administration. It is suggested that gastric acid participates, at least in part, in postprandial release of B-EpLI, probably from the gastrointestinal tract. /3-endorphin-like immunoreactivity; test meal; gastric acid; histamine H 2 receptor antagonist
0167-0115/82/0000-0000/$02.75 © 1982 ElsevierBiomedicalPress
174
Introduction
fl-Endorphin is an endogenous opioid known to be localized in the brain, pituitary gland, gastrointestinal tract, pancreas, and gonads [3,15,20,23,26]. After development of a radioimmunoassay for human fl-endorphin (flh-Ep), the plasma levels of human fl-endorphin-like immunoreactivity (flh-EpLI) have been determined in normal and diseased states [9,14,28-30], although the values reported differed, probably owing to differences in the antisera and assay procedures used. Ingestion of a meal has been reported to stimulate release of gut hormones including gastrin, secretin, motilin, gastric inhibitory polypeptide (GIP), enteroglucagon and vasoactive intestinal polypeptide (VIP) [1,4-6,18,24]. Duodenal acidification also stimulates the release of secretin, motilin and v I P [6,12,16]. As we demonstrated previously [15], flh-EpLI is present in human gut and is released in vitro by perfusion of human duodenal mucosa with solution of low pH, suggesting that gastric acid may be involved in release of/?h-EpLI. Therefore we investigated the effects of a test meal, duodenal acidification and tetragastrin on the release of /?-EpLI in man. The results suggest that gastric acid participates in postprandial release of/~-EpLI, probably from the gastrointestinal tract.
Materials and Methods
Subjects The fasting levels of plasma fl-EpLI were determined in 23 normal males aged 21-60 years and 27 females aged 20-60 years between 0800-1000 h. The response of fl-EpLI release to various tests was examined in normal male volunteers aged 19-21 years.
Experimental protocol The test meal used was Campbell's condensed meat soup (Campbell Soup Company, U.S.A.), containing 100 kcal, 7.4 g protein, 2.0 g fat, 13.3 g carbohydrates and 3.2 g minerals per 150 g. Five normal males were given 150 g of the soup orally and blood samples were collected 30 and 15 min before and 0, 15, 30, 60, 90 and 120 min after ingestion. Duodenal acidification was carried out by intraduodenal infusion of 115 ml of 0.1 M HCI over 10 min through a gastric tube. Blood samples were obtained at 0, 5, 10, 15, 20, 30, 45, and 60 min after the start of infusion. Tetragastrin (Sanayakuhin, Japan, 4 ~ g / k g body weight in 2 ml of physiological saline) was injected intramuscularly into three normal subjects. 7 days later, the same dose of tetragastrin was given 30 min after infusion of 10 mg histamine H 2 receptor antagonist (YM-11170, Yamanouchi Pharmaceutical Co., Japan) into the stomach through a gastric tube. In both experiments blood samples and gastric juice were collected at intervals as indicated in Results.
Radioimmunoassay of flh-Ep Anti-C/h-Ep serum was prepared by the method of Guillemin et al. [8] against
175
synthetic flh-Ep and proved to be highly specific for flh-Ep as reported previously [15]. It crossreacted only with human fl-lipotropin (flh-LPH, crossreactivity 4.5% on a molar basis) of the pro-opiocortin-related peptides tested, flh-Ep was radioiodinated by the chloramine T method [10], and I25I-labelled flh-Ep was separated on a column (1 × 18 cm) of Sephadex G-25 (medium). The procedure for flh-Ep radioimmunoassay was reported previously [15]. The binding of 125I-labelled flh-Ep to antiserum was displaced by the presence of unlabelled hormone in a dose-related manner in the range of 4-256 pg/tube.
Extraction of flh-EpLI from plasma Plasma flh-EpLI was extracted by a modification of the method of Robertson et al. [22]. 1 ml of plasma was mixed with 2 ml of acid/acetone (acetic acid/acetone, 4: 100, v / v ) and centrifuged for 30 rain at 3000 rpm. The resulting supernatant was washed twice with 4 ml of light petroleum. The upper layer was carefully removed and discarded. The remaining acetone was removed by vacuum evaporation and the aqueous portion lyophilized. The dry material was dissolved in 1 ml of the radioimmunoassay buffer. T h e r e c o v e r y in the extraction procedure was 73 ± 5% (mean ± S.E.M.) at a concentration of 28.6 pmol/1. In all cases the plasma values of fl-EpLI were corrected for recovery.
Gel chromatography The extract from 13 ml of plasma was chromatographed on a column (0.9 × 45 cm) of Sephadex G-50 (fine) with 0.1 M acetic acid containing 0.1% bovine serum albumin (BSA). Fractions of 1 ml were collected and lyophilized. The content of fl-EpLI in each fraction was measured by radioimmunoassay. Blue dextran (molecular mass > 2000000), synthetic flh-LPH, synthetic flh-Ep and Na125I were used for calibration. The mean recovery of 10 ng of synthetic flh-Ep added to plasma was 45 ± 4%.
Gastric juice analysis To determine acid output, gastric juice was collected with a gastric tube at 15-min intervals after removing the gastric contents for 30 rain using a continuous suction pump. The acid concentration of each sample was measured by titration with 0.1 M NaOH, with TOpher-Michaelis reagent and phenolphthalein as indicators.
Statistics Results for plasma flh-EpLI levels are quoted as mean ± S.E.M. The statistical significance of differences was analyzed by Student's t-test.
Results
Validity of assay The intra-assay coefficients of variation in 6 measurements at 3 concentrations (36, 72, and 143 pmol/1) were 7.0, 5.8, and 5.0%, respectively. The interassay
176
coefficients of variation in measurements on 10 days were 8.8 and 7.6% at concentrations of 36 and 72 pmol/1, respectively. When 14.3 and 143 p m o l / l of flh-Ep were added to plasma, the recoveries were 67.6 ± 4.3 and 85.8 ± 5.570, respectively. The normal values of plasma flh-EpLI for adults were 11.6 ± 4.0 pmol/1 (range 3.7-19.1) for men and 10.7 ± 4.8 p m o l / l (range 3.1-16) for women.
Effect of meat soup ingestion on plasma flh-EpL1 level Plasma fl-EpLI levels were increased from the basal level of 4.4 ± 1.0 to 29.2 ± 1.9 pmol/1 at 5 min and 24.8 ± 6.7 p m o l / l at 10 min ( P < 0 . 0 0 1 ) after ingestion of meat soup (150 g) and then returned to the initial level. The plasma fi-EpLI level showed a second rise (24.8 ± 6.7 pmol/1) after about 90 rain (Fig. 1). Effect of intraduodenal infusion of O.1 M HCI on the plasma flh-EpL1 level Intraduodenal infusion of 115 ml of 0.1M HCI over 10 min significantly increased the plasma flh-EpLI level from the basal level of 8.7 ± 0.5 p m o l / l to 15.3± 1.4 p m o l / l at 5 min and 15.5 ± 1.4 pmol/1 at 10 min after the start of infusion (Fig. 2). The plasma/3-EpLI level decreased rapidly to the initial level after the end of the infusion. Effect of tetragastrin on gastric acid output and the plasma flh-EpLl level As shown in Figure3, intramuscular injection of 4 /~g/kg body weight of tetragastrin markedly stimulated gastric acid output during the 60-min observation period. The plasma flh-EpLI level concomitantly increased from the basal level of 9.8 ± 0.2 pmol/1 to 20.7 -+ 1.4 pmol/1 after 30 min and then remained high.
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177
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178
Effect of h&tamine H 2 receptor antagonist on tetragastrin-induced gastric acid output and fl-EpLl release When histamine H 2 receptor antagonist (10 rag) was infused into the stomach through a gastric tube 30 rain before tetragastrin injection, gastric acid output was completely inhibited and plasma flh-EpLI showed no significant increase (Fig, 3). Elution profiles on gel filtration of flh-EpLI extracted from plasma Plasma extracts were prepared from blood taken from two subjects before and 30 min after subcutaneous injection of 4 # g / k g body weight of tetragastrin, and applied to a column (0.9 × 45 cm) of Sephadex G-50. As shown in Figure4, two fractions of flh-EpLI were separated from each extract; the first peak was in the position of flh-LPH and the second peak in that of flh-Ep. The ratios of flh-LPH to flh-Ep in subject S.M. and in subject M.H. were 6.3: l and 7.7: 1, respectively, on a molar basis. The ratio of flh-LPH to flh-Ep in the extract from both subjects changed to 4.9:1 (S.M.) and 4.7:1 (M.H.) 30 rain after tetragastrin administration.
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Fig. 4. Elution profiles on Sephadex of plasma extracts from two normal men injected with tetragastrin. The lyophilized extracts from 13 ml of plasma were dissolved in 0.6 ml of 0.1 M acetic acid and applied to a column (0.9×45 cm) of Sephadex G-50. (A) basal state; (B) 30 rain after intramuscular injection of 4 # g / k g bodyweight tetragastrin. The column was calibrated with blue dextran, synthetic flh-LPH, flh-Ep and Na125I.
179
Discussion
Since Bradbury et al. [2] first isolated fl-endorphin from porcine pituitary, its regional distribution and action have been studied extensively. Recently, attempts have been made to determine the concentration of fl-Ep in biological fluids, but discrepant values have been reported, probably owing to differences in the antisera and radioimmunoassay procedures used. We developed a sensitive and specific radioimmunoassay for measuring plasma flh-Ep, in which specific anti-flh-Ep antiserum was used to obtain high specificity and acid/acetone extraction of plasma fl-EpLI was used to eliminate non-specific interference in radioimmunoassay. The recovery of plasma flh-Ep on extraction with acid/acetone is comparable with that by the silicic acid method [28], and the acid/acetone method is more convenient than the latter. The: .normal fasting values for plasma flh-EpLI obtained in our study are a little higher that~ those obtained by Wardlaw and Frantz [28] (6.2 -+ 2.2 pmol/l) by silicic acid extraction and by Nakao et al. [17] ( < 0.86-0.89 pmol/1) by chromatography, and lower than those reported by Wilkes et al. [30] (14.3-85.7 pmol/1) who did not use an extraction procedure. Wiedeman et al. [29] (1.4-12.9 pmol/1) reported similar values to ours. The difference between the value reported may be due to differences in antisera and methods for extraction of fl-Ep from plasma. However, no significant sex difference in plasma fl-Ep levels was noticed in any group. The plasma flh-Ep level in normal subjects is known to increase in response to various stimulants, such as metyrapone, vasopressin and insulin-induced hypoglycemia [17,28]. High levels of plasma fl-Ep are also found in some diseases such as Cushing's disease, Nelson's syndrome and Addison's disease [13,27]. These changes in plasma/3h-E p are associated with changes in plasma ACTH and fl-LPH, suggesting concomitant releases of these compounds from the pituitary. There are several reports on the distribution of pro-opiocortin-related peptides in tissues other than the central nervous system and pituitary [3,26]. Larsson [l l] demonstrated the presence of ACTH in the myenteric plexus, in epithelial cells of the small intestine and pancreatic islets by an immunohistochemical technique. Polak et al. [21] showed enkephalin-positive cells in both neural and epithelial sites immunohistochemically. Odell et al. [19] detected fl-LPH in human colon with a radioimmunoassay system that measures both fl-LPH and fl-MSH. Furthermore, fl-Ep was found in the rat gastrointestinal tract [20] and pancreas [3]. We reported recently that flh-EpLI is present in human stomach and duodenum and is released in vitro in response to various test meals [15]. These findings indicate that ACTH and related peptides are present in the gastroenteropancreatic system and may have a physiological role in the gut. Secretions of the gut hormones gastrin, secretin, motilin, GIP, enteroglucagon, VIP and somatostatin are known to be stimulated by ingestion of a meal [1,4-6,18,24]. In addition, duodenal acidification stimulates the release of secretin, motilin, GIP and VIP [6,12,16]. The present study demonstrated that the plasma flh-EpLI level increased biphasically in normal subjects after ingestion of condensed meat soup. The first peak may be due to nervous reflexes as seen in the case of postprandial release of plasma pancreatic polypeptide [7] or
180
related to the meal itself, but this remains to be elucidated. Since/3h-EpLI can be released from human duodenal mucosa in vitro by a solution of low pH [5] and by duodenal acidification as reported in the present paper, it seems very likely that gastric acid stimulates fl-EpLI release from the gut. In fact, we found that increase of the plasma/3-EpLI level was associated with increase in gastric acid output after tetragastrin administration. Furthermore, we found that tetragastrin-induced fl-EpLI release was prevented by pretreatment with histamine H 2 receptor antagonist, which completely inhibited tetragastrin-induced gastric acid output. This indicates that gastric acid may be related, at least in part, to fl-EpLI release. Gel-chromatographic analysis demonstrated that the fl-EpLI released after tetragastrin administration consisted of two fractions, namely the ratios of flh-LPH to flh-Ep in subject S.M. and in subject M.H. were 4.9:1 and 4.7:1, respectively, on a molar basis. In this study, biphasic release of/3-EpLI after ingestion of food was demonstrated in man. Duodenal acidification and tetragastrin administration were also shown to stimulate fl-EpLI release, but pretreatment with histamine H 2 receptor antagonist inhibited tetragastrin-induced gastric acid output and/3-EpLI release. These findings suggest that gastric acid participates, at least in part, in postprandial fl-EpLI release.
References 1 Boden, G., Essa, N. and Owen, O., Effects of intraduodenal amino acids, fatty acids, and sugars on secretin concentrations, Gastroenterology, 68 (1975) 722-727. 2 Bradbury, A.F., Smyth, D.G. and Snell, C,R., Biosynthesis of fl-MSH and ACTH. In: Chemistry Structure and Biology, R. Walter and J. Meiienhofer, (Eds.), Ann. Arbor Science Inc., 1975, pp. 609-615. 3 Bruni, J.F., Watkins, W.B. and Yen, S.S.C., fl-Endorphin in the human pancreas, J. Clin. Endocrinol. Metab., 49 (1979) 649-651. 4 Burhol, P.G., Waldum, H.L, Jorde, R. and Lygren, I., The effect of a test meal on plasma vasoactive intestinal polypeptide (VIP), gastric inhibitory polypeptide (GIP), and secretin in man, Scand. J. Gastroenterol., 14 (1979) 939-943. 5 ChayviaUe, J.-A., Miyata, M., Rayford, P.L. and Thompson, J.C., Effects of test meal, intragastric nutrients, and intraduodenal bile on plasma concentrations of immunoreactive somatostatin and vasoactive intestinal peptide in dog, Gastroenterology, 79 (1980) 844-852. 6 Ebeid, A.M., Escourtou, J., Murray, P. and Fischer, J.E., Pathophysiology of VIP, Gut Hormones, (1978) 479-483. 7 Ghatei, M.A. and Bloom, S.R., Enteroglucagon in man, Gut Hormones, (1981) 332-338. 8 Guillemin, R., Ling, N. and Vargo, T., Radioimmunoassay for a-endorphin and fl-endorphin, Biochem. Biophys. Res. Commun., 77 (1977) 361-366. 9 Hrllt, V., Miiller, O.A. and Fahlbusch, R.. fl-Endorphin in human plasma: basal and pathologically elevated levels, Life Sci., 25 (1979) 37-44. 10 Hunter, W,H. and Greenwood, F.A., Preparation of iodine-131 labelled human growth hormone of high specific activity, Nature 194 (1962) 495-496. 11 Larsson, L.I., Corticotropin-like peptides in central nerves and in endocrine cells of gut and pancreas, Lancet, 2 (1977) 1321-1323. 12 LeRoith, D., Spitz, I.M., Ebert, R., Liel, Y., Odes, S. and Creutzfeldt, W., Acid-induced gastric inhibitory polypeptide secretion in man, J. Clin. Endocrinol. Metab., 51 (1980) 1385 1389. 13 Liotta, A.S., Suda, T. and Krieger, D.T., fl-Lipotropin is the major opioid-like peptide of human pituitary and rat pars distalis: lack of significant fl-endorphin, Proc. Natl. Acad. Sci. U.S.A., 75 (1978) 2950-2954.
181 14 Malizia, E., Andreucci, G., Paolucci, D., Crescenzi, F., Fabbri, A. and Fraioli, F., Electroacupuncture and peripheral fl-endorphin and ACTH levels, Lancet, 2 (1979) 535-536. 15 Matsumura, M., Saito, S. and Fuijino, M., Effects of solution of low pH and taurocholate on release of fl-endorphin-like immunoreactivity from human duodenal mucosa in vitro, Regul. Peptides. 3 (1982) 173-181. 16 Mitznegg, P., Bloom, S.R., Domschke, W., Domschke, S., Wunsch, E. and Demling, L., Release of motilin after duodenal acidification, Lancet, 1 (1976) 888-889. 17 Nakao, K., Nakai, Y., Oki, S., Horii, K. and Imura, H., Presence of immunoreactive fl-endorphin in normal human plasma. A concomitant release of fl-endorphin with adrenocorticotropin after metyrapone administration, J. Clin. Invest., 62 (1978) 1395-1398. 18 Nilsson, G., Simon, J., Yalow, R.S. and Berson, S.A., Plasma gastrin and gastric acid responses to sham feeding and feeding in dogs, Gastroenterology, 63 (1972) 51-59. 19 Odell, W.D., Wolfsen, A.R., Bachelor, I. and Hirose, F.M., Ectopic production of lipotropin by cancer, Am. J. Med., 66 (1979) 631-638. 20 Orwall, E.S. and Kendall, J.E., fl-Endorphin and adrenocorticotropin in extrapituitary sites: gastrointestinal tract, Endocrinology, 107 (1980) 438-442. 21 Polak, J.M., Bloom, S.R., Sullivan, S.N., Facer, P. and Pearse, A.G.E., Enkephalin-like immunoreactivity in the human gastrointestinal tract, Lancet, 1 (1977) 972-974. 22 Robertson, G.L., Malhr, E.A., Athar, S. and Sinha, T., Development and clinical application of a new method for the radioimmunoassay of arginine vasopressin in human plasma, J. Clin. Invest., 52 (1973) 2340-2352. 23 Rossier, J., Vargo, T.M., Minick, S., Ling, N., Bloom, F.E. and Guillemin, R., Regional dissociation of fl-endorphin and enkephalin contents in rat brain and pituitary, Proc. Natl. Acad. Sci. U.S.A., 74 (1977) 5162-5165. 24 Saito, S., Ogawa, T., Saito, H., Ishimaru, K., Oshima, I, and Sonaka, Y., Regulation of motilin secretion in the postprandial state in man, Endocrinol. Japon., S.R. No. 1 (1980) 157-162. 25 Schwartz, T.W., Stenquist, B. and Olbe, L., Physiology of mammalian PP and the importance of vagal regulation, Gut Hormones, (1978) 261-264. 26 Sharp, B., Pekary, A.E., Meyer, N.V. and Hershman, J.M., /3-Endorphin in male rat reproductive organs, Biochem. Biophys. Res. Commun., 95 (1980) 618-623. 27 Suda, T., Liotta, A.S. and Krieger, D.T.,/3-Endorphin is not detectable in plasma from normal human subjects, Science, 202 (1978) 221-223. 28 Wardlaw, S.L. and Frantz, A.G., Measurement of/3-endorphin in human plasma, J. Clin. Endocrinol. Metab., 48 (1979) 176-180. 29 Wiedeman, E., Saito, T., Linfoot, J.A. and Li, C.H., Specific radioimmunoassay of human /3-endorphin in unextracted plasma, J. Clin. Endocrinol. Metab., 49 (1979) 478-480. 30 Wilkes, M.M., Stewart, R.D., Bruni, J.F., Quigley, M.E., Yen, S.S.C., Ling, N. and Chretien, M., A specific homologous radioimmunoassay for human /3-endorphin: direct measurement in biological fluids, J. Clin. Endocrinol. Metab., 50 (1980) 309-315.