European Journal of Clinical Investigation (1979) 9,293-300
Effects of gastrointestinal hormones on fasting gallbladder storage patterns in man 0. G. BJORNSSON, T. E. ADRIAN, J. DAWSON, R. F. McCLOY, G. R. GREENBERG, S. R. BLOOM & V. S. CHADWICK, Gastroenterology Unit and Endocrinology Unit, Department of Medicine, Royal Postgraduate Medical School, London Received 23 October 1978 and in revised form 3 May 1979
Abstract. Gallbladder storage and emptying patterns were studied in fasting normal subjects by a duodenal perfusion technique using indocyanine green as a biliary marker. Fasting gallbladder storage patterns were very variable but a more uniform biliary output with net storage of about 40% of the biliary marker was observed during a simulated interprandial state ( 2 4 h after meals) produced by a low dose intravenous infusion of secretin and caerulein. With this background hormonal stimulation, infusion of bovine pancreatic polypeptide to achieve physiological interprandial levels promoted further gallbladder storage of bile. Bovine pancreatic polypeptide produced storage by a major effect on the gallbladder rather than on the liver, common bile duct or sphincter of Oddi since a reduction of biliary output was not observed during bovine pancreatic polypeptide infusion in cholecystectomized subjects. Bovine pancreatic polypeptide had a separate effect on the pancreas, reducing trypsin output in both normal and cholecystectomized subjects. Key words. Pancreatic polypeptide, secretin, caerulein, endogenous release of human pancreatic polypeptide, indocyanine green, gallbladder storage, cholecystectomy, biliary and pancreatic output, bile salts, trypsin, duodenal perfusion technique, sphincter of Oddi, common bile duct.
Introduction Cholecystokinin (CCK) is known to cause gallbladder emptying in man [ 11, but factors responsible for promoting gallbladder storage in the fasting state and between meals are not established. Recently pancreatic polypeptide (PP) has been suggested as a ‘candidate’ * Presented in part at the 2nd International Symposium on Gastrointestinal Hormones, 30 August to 2 September 1978, Oslo. Norway, and the Autumn Meeting of the British Society ofGastroenterology, Edinburgh, 20-23 September 1978. Correspondence: Dr Olafur Grimur Bjornsson, Department of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London W 12 OHS, England. 0014-2972/79/0800-0293$02.00 8 1979 Blackwell Scientific Publications
hormone for this function, since in dogs bovine pancreatic polypeptide (BPP) relaxed the gallbladder and increased the tone of the common bile duct [2] and in man the output of bilirubin into the duodenum fell markedly during intravenous BPP infusion [3-51. Pancreatic polypeptide is a linear peptide of thirty-six amino acid residues which has been isolated and sequenced from many mammalian species, including man, cattle and the pig [2]. In man PP is produced normally only by the pancreas [6-81. The biological activity of PP resides in the C-terminal hexapeptide of the molecule [2]. The C-terminal hexapeptide is identical in cattle and man [9]. To quantify gallbladder storage and emptying in man, the anionic dye indocyanine green (ICG) can be used as a biliary marker [lo]. ICG is infused intravenously at a constant rate and the appearance of ICG in duodenal bile quantified using a duodenal perfusion technique. After a steady state is established where hepatic excretion of ICG into bile is equal to intravenous input, the gallbladder storage fraction of ICG can be calculated over any period of time [lo]. The aim of this study was to validate in man the duodenal perfusion technique with indocyanine green, use this technique to establish gallbladder storage and emptying patterns in normal fasting volunteers, and to study the effects of secretin, caerulein and bovine pancreatic polypeptide (BPP) (simulating interprandial state) on these fasting patterns. Lastly, the effect of BPP on cholecystectomized subjects was studied to separate the effects of BPP on the gallbladder from the effects on the common bile duct and sphincter of Oddi. Bile salts and trypsin outputs were measured during the studies as endogenous markers of biliary and pancreatic secretion. Materials Preparations of secretin (pure natural porcine secretin, 3500 clinical units per mg, stabilized in cysteine hydrochloride, obtained from GIH Research Unit, Chemistry Department, Karolinska Institutet, Stockholm, Sweden), caerulein (Ceruletide, Farmitalia Ltd, Milan, Italy, distributed by Montedison Pharmaceuti293
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cals Ltd, Kingmarker House, Station Road, Barnet, Herts EN5 INU, England) and BPP (Lilly Research Laboratories, U.S.A.) were prepared immediately before intravenous infusion in a sterile NaCl (145 mmol/l) solution containing 200 pmol/l of human serum albumin (Lister Institute, Elstree, Herts, England). A commercial preparation of ICG (CardioGreen@,Hynson, Westcott and Dunning Inc., Baltimore, Maryland 21201, U.S.A.) was dissolved before use in manufacturer's solvent and diluted to appropriate concentrations in sterile NaCl (145 mmol/l) containing 750 pmol/l human albumin. Methods Radioimmunoassay of pancreatic polypeptide. Antiserum against BPP (Lilly Research Laboratories, 615-R110-1464) was raised in rabbits by the subcutaneous injection of a conjugate of BPP with albumin mixed with Freund's complete adjuvant [ 111. The antiserum was used at a final dilution of 1/1.6 x lo6. PP was iodinated by the conventional chloramine T method as previously described [6]. The labelled PP always gave a non-specific binding of < 8% and maximum binding of >90% in the presence of antibody excess. The specific activity of the label ranged from 1.8 to 2.0 nCi/fmol. Duplicate 0.8 ml assay tubes were set up containing 100 PI of plasma. Antisera was added to give final dilution of 1/1.6 x lo6 and bound 50% of the 5 pmol of HPP IZ5Ilabel. Standards were set up in hormone-free plasma prepared by charcoal extraction. Several such charcoaled plasmas have been tested and all produce superimposable standard curves as do plasma samples from patients who have undergone total surgical pancreatectomy. After an incubation period of 7 days at 4"C, antibody bound label was separated from free by the addition of 0.5 ml of a 40 g/l charcoal slurry (Hopkin & Williams, Norit GSX) coated with 4 g/l dextran (average molecular weight 70,000). After mixing and incubating at 4°C for 5 min the tubes were centrifuged at 2000 g (4°C) and the supernatant decanted; both free and bound fractions were counted. All dilutions were 0.5 mol/l sodium barbitone buffer at pH 8.0. HPP and BPP showed equal immunopotency in this assay system and addition of serial dilutions of plasma from patients with high basal PP concentrations resulted in parallel inhibition of binding of label to antibody. No displacement of label from antiserum was found with concentration of 10 nmol/l of insulin, glucagon, vasoactive intestinal peptide, somatostatin, little gastrin, secretin, bombesin and cholecystokinin. The amount of PP in the assay tube required to reduce the binding of labelled PP from 50% to 40% was 4.5 pmol/l plasma. The assay detected changes of PP concentration between adjacent samples of 8 pmol/l plasma with 95Xconfidence on the sensitive part of the standard curve. Intra-assay precision [(standard deviation x 1OO)/
mean] on ten estimates in one assay, at the PP concentrations of four separate plasma samples covering the range of the standard curve, was < 5%. These control samples were run in duplicate at every fiftieth sample throughout this assay to overcome any influence of drift. Interassay precision, calculated in a similar manner from two estimates in duplicate, one at each end of each of twenty-seven assays, of all four plasma samples, was
Effects of gastrointestinal hormones on fasting gallbladder storage patterns in man 0. G. BJORNSSON, T. E. ADRIAN, J. DAWSON, R. F. McCLOY, G. R. GREENBERG, S. R. BLOOM & V. S. CHADWICK, Gastroenterology Unit and Endocrinology Unit, Department of Medicine, Royal Postgraduate Medical School, London Received 23 October 1978 and in revised form 3 May 1979
Abstract. Gallbladder storage and emptying patterns were studied in fasting normal subjects by a duodenal perfusion technique using indocyanine green as a biliary marker. Fasting gallbladder storage patterns were very variable but a more uniform biliary output with net storage of about 40% of the biliary marker was observed during a simulated interprandial state ( 2 4 h after meals) produced by a low dose intravenous infusion of secretin and caerulein. With this background hormonal stimulation, infusion of bovine pancreatic polypeptide to achieve physiological interprandial levels promoted further gallbladder storage of bile. Bovine pancreatic polypeptide produced storage by a major effect on the gallbladder rather than on the liver, common bile duct or sphincter of Oddi since a reduction of biliary output was not observed during bovine pancreatic polypeptide infusion in cholecystectomized subjects. Bovine pancreatic polypeptide had a separate effect on the pancreas, reducing trypsin output in both normal and cholecystectomized subjects. Key words. Pancreatic polypeptide, secretin, caerulein, endogenous release of human pancreatic polypeptide, indocyanine green, gallbladder storage, cholecystectomy, biliary and pancreatic output, bile salts, trypsin, duodenal perfusion technique, sphincter of Oddi, common bile duct.
Introduction Cholecystokinin (CCK) is known to cause gallbladder emptying in man [ 11, but factors responsible for promoting gallbladder storage in the fasting state and between meals are not established. Recently pancreatic polypeptide (PP) has been suggested as a ‘candidate’ * Presented in part at the 2nd International Symposium on Gastrointestinal Hormones, 30 August to 2 September 1978, Oslo. Norway, and the Autumn Meeting of the British Society ofGastroenterology, Edinburgh, 20-23 September 1978. Correspondence: Dr Olafur Grimur Bjornsson, Department of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London W 12 OHS, England. 0014-2972/79/0800-0293$02.00 8 1979 Blackwell Scientific Publications
hormone for this function, since in dogs bovine pancreatic polypeptide (BPP) relaxed the gallbladder and increased the tone of the common bile duct [2] and in man the output of bilirubin into the duodenum fell markedly during intravenous BPP infusion [3-51. Pancreatic polypeptide is a linear peptide of thirty-six amino acid residues which has been isolated and sequenced from many mammalian species, including man, cattle and the pig [2]. In man PP is produced normally only by the pancreas [6-81. The biological activity of PP resides in the C-terminal hexapeptide of the molecule [2]. The C-terminal hexapeptide is identical in cattle and man [9]. To quantify gallbladder storage and emptying in man, the anionic dye indocyanine green (ICG) can be used as a biliary marker [lo]. ICG is infused intravenously at a constant rate and the appearance of ICG in duodenal bile quantified using a duodenal perfusion technique. After a steady state is established where hepatic excretion of ICG into bile is equal to intravenous input, the gallbladder storage fraction of ICG can be calculated over any period of time [lo]. The aim of this study was to validate in man the duodenal perfusion technique with indocyanine green, use this technique to establish gallbladder storage and emptying patterns in normal fasting volunteers, and to study the effects of secretin, caerulein and bovine pancreatic polypeptide (BPP) (simulating interprandial state) on these fasting patterns. Lastly, the effect of BPP on cholecystectomized subjects was studied to separate the effects of BPP on the gallbladder from the effects on the common bile duct and sphincter of Oddi. Bile salts and trypsin outputs were measured during the studies as endogenous markers of biliary and pancreatic secretion. Materials Preparations of secretin (pure natural porcine secretin, 3500 clinical units per mg, stabilized in cysteine hydrochloride, obtained from GIH Research Unit, Chemistry Department, Karolinska Institutet, Stockholm, Sweden), caerulein (Ceruletide, Farmitalia Ltd, Milan, Italy, distributed by Montedison Pharmaceuti293
294
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cals Ltd, Kingmarker House, Station Road, Barnet, Herts EN5 INU, England) and BPP (Lilly Research Laboratories, U.S.A.) were prepared immediately before intravenous infusion in a sterile NaCl (145 mmol/l) solution containing 200 pmol/l of human serum albumin (Lister Institute, Elstree, Herts, England). A commercial preparation of ICG (CardioGreen@,Hynson, Westcott and Dunning Inc., Baltimore, Maryland 21201, U.S.A.) was dissolved before use in manufacturer's solvent and diluted to appropriate concentrations in sterile NaCl (145 mmol/l) containing 750 pmol/l human albumin. Methods Radioimmunoassay of pancreatic polypeptide. Antiserum against BPP (Lilly Research Laboratories, 615-R110-1464) was raised in rabbits by the subcutaneous injection of a conjugate of BPP with albumin mixed with Freund's complete adjuvant [ 111. The antiserum was used at a final dilution of 1/1.6 x lo6. PP was iodinated by the conventional chloramine T method as previously described [6]. The labelled PP always gave a non-specific binding of < 8% and maximum binding of >90% in the presence of antibody excess. The specific activity of the label ranged from 1.8 to 2.0 nCi/fmol. Duplicate 0.8 ml assay tubes were set up containing 100 PI of plasma. Antisera was added to give final dilution of 1/1.6 x lo6 and bound 50% of the 5 pmol of HPP IZ5Ilabel. Standards were set up in hormone-free plasma prepared by charcoal extraction. Several such charcoaled plasmas have been tested and all produce superimposable standard curves as do plasma samples from patients who have undergone total surgical pancreatectomy. After an incubation period of 7 days at 4"C, antibody bound label was separated from free by the addition of 0.5 ml of a 40 g/l charcoal slurry (Hopkin & Williams, Norit GSX) coated with 4 g/l dextran (average molecular weight 70,000). After mixing and incubating at 4°C for 5 min the tubes were centrifuged at 2000 g (4°C) and the supernatant decanted; both free and bound fractions were counted. All dilutions were 0.5 mol/l sodium barbitone buffer at pH 8.0. HPP and BPP showed equal immunopotency in this assay system and addition of serial dilutions of plasma from patients with high basal PP concentrations resulted in parallel inhibition of binding of label to antibody. No displacement of label from antiserum was found with concentration of 10 nmol/l of insulin, glucagon, vasoactive intestinal peptide, somatostatin, little gastrin, secretin, bombesin and cholecystokinin. The amount of PP in the assay tube required to reduce the binding of labelled PP from 50% to 40% was 4.5 pmol/l plasma. The assay detected changes of PP concentration between adjacent samples of 8 pmol/l plasma with 95Xconfidence on the sensitive part of the standard curve. Intra-assay precision [(standard deviation x 1OO)/
mean] on ten estimates in one assay, at the PP concentrations of four separate plasma samples covering the range of the standard curve, was < 5%. These control samples were run in duplicate at every fiftieth sample throughout this assay to overcome any influence of drift. Interassay precision, calculated in a similar manner from two estimates in duplicate, one at each end of each of twenty-seven assays, of all four plasma samples, was
