Gut, 1970, 11, 206-211 Bio-assay evidence of abnormal secretin-like Gut: first published as 10.1136/gut.11.3.206 on 1 March 1970. Downloaded from and gastrin-like activity in tumour and blood in cases of 'choleraic diarrhoea' I. G. M. CLEATOR, CHRISTINE G. THOMSON, W. SIRCUSI, AND MIRANDA COOMBES From the Gastro-Intestinal Unit, and the Teaching and Research Centre, Western General Hospital, Edinburgh SUMMARY Methods for bio-assay of secretin-like humoral agents in both cat and dog are described. Bio-assay of tumour extracts and of plasma from patients with the pancreatic choleraic syndrome are described. The first patient was found to have choleretic and secretin- like activity in an extract of her pancreatic islet cell tumour and gastrin-like activity in her plasma. The second patient was found to have both secretin and gastrin-like activity in her plasma, as well as choleretic activity. It is concluded that at least part of the profuse, watery electrolyte diarrhoea of the 'pancreatic cholera' syndrome associated with peptide-secreting adenoma of the pancreas is likely to be a reflection of the excessive production of secretin, as well as of gastrin, and possibly also of a choleretic agent. http://gut.bmj.com/ The variety of clinical presentations associated chloride and ether for induction followed by with hyperplasia or tumour of the islet cells of 200 mg intravenous chloralose. No further the pancreas and with secretion of peptides has anaesthetic was required throughout the pro- been referred to (Sircus, Brunt, Walker, Small, cedure. A cuffed tracheotomy tube was inserted on October 1, 2021 by guest. Protected copyright. Falconer, and Thomson, 1970) and reviewed in to prevent aspiration of secretions. The main detail (Sircus, 1969). It has recently been sug- pancreatic duct was cannulated with a polythene gested (Zollinger, Tompkins, Amerson, Endahl, cannula, 0.63 mm in diameter, which was ligated Kraft, and Moore, 1968) that 'pancreatic cholera' in position. The common bile duct was separately may be caused by a secretin-like hormone cannulated using a 1.02 mm diameter polythene elaborated by an islet cell tumour. The purpose of cannula, and both cannulae were brought out this paper is to present the results, including bio- through stab wounds to establish dependent assay of plasma and of tumour extracts, ofstudies drainage. A cannula in the external jugular on two patients with choleraic diarrhoea, to- allowed a constant infusion of saline at the rate gether with an examination of the influence of of 0.076 ml/min using a Harvard continuous tumour extracts upon water and ion transport delivery pump. The cat's temperature was kept across the canine intestinal mucosa. constant at 38°C. The output from both cannulae was collected continuously into graduated tubes which were changed every 15 minutes. After an hour of basal Methods collections, the intravenous injections were made at intervals of 30 to 60 minutes using as a guide EXPERIMENTAL TECHNIQUE to procedure the volumes of secretions recovered. These injections were an extract of a neuro- Cat bio-assay blastoma, an extract from the adenoma removed A cat, 2.5 kg/bw, was anaesthetized using ethyl- from the first case of choleraic diarrhoea (E.S.), 'Member of the external scientific staff, Medical Research plasma from a subject with suspected lymphoma Council. of the small intestine but with a normal pancreas, 207 Bio-assay evidence ofabnormal secretin-like andgastrin-like activity in tumour and blood Gut: first published as 10.1136/gut.11.3.206 on 1 March 1970. Downloaded from and plasma from the second case of choleraic withdrawn into lithium-heparin tubes. After diarrhoea (A.G.). After the completion of these centrifuging at 5°C the resulting plasma was observations, secretin (Boots) was added to the stored at - 20°C, and just prior to assay, thawed saline and infused at the rate of 9.1 units/kg/bw/ at not greater than 30°C and recentrifuged at 5°C 60 minutes. When the volume output from the to remove fibrin. The resulting plasma was then pancreas had reached a steady state further assayed with no further manipulation. intravenous injections were made at intervals of After excision the tumour had been rapidly 30 to 60 minutes. These were an extract of the placed in a plastic bag and then dropped into a neuroblastoma, an extract of the adenoma from solid CO2 alcohol bath. This allowed rapid the first case of choleraic diarrhoea (E.S.), freezing of the tissue which was thereafter stored plasma from the second case of choleraic diar- at - 20C until required. The method used for rhoea (A.G.), plasma obtained from the first case tumour extraction was essentially that described of choleraic diarrhoea subsequent to removal of by Zollinger et al (1968). the adenoma (E.S.), and plasma from the patient Frozen tumour tissue (3.4 g) from patient E.S. with suspected intestinal lymphoma. was homogenized in increasing volumes of 0.1 M HCl until a final volume of 57 ml was Dog bio-assay achieved. The homogenizer vessel was chilled in A mongrel dog, 215 kg/bw, was initially anaes- ice water during this procedure. After filtration thetized with 10.5 ml of nembutal and subse- with suction on glass paper (Whatman GF/A), quently with hourly injections of 1-15 millitres. the filtrate was brought to 30% NaCl concentra- The airway was maintained with a cuffed endo- tion by the slow addition, with stirring, of solid tracheal tube. NaCl. A precipitate gradually formed and after The operation was a modification of that standing at 4°C for 20 minutes was centrifuged, described by Lin and Alphin (1962) and Zollinger to yield a light brown precipitate, the filtrate et al (1968). The main pancreatic duct was can- being clear. After decanting the filtrate, the nulated extraduodenally with a 1P02 mm poly- precipitate was stirred and then shaken with thene tube, and the common bile duct with a 6.0 ml ice-cold acetone in a stoppered tube. The 1.34 mm polythene tube ligated in position. Two acetone powder, which was pale cream in colour, accessory ducts were found and ligated. The was dried under suction and washed on the filter cystic duct was ligated to prevent the gallbladder with more acetone until completely dry. The final emptying during the course of the experiment, material (0.97 g) was then stored at - 20C in and the pylorus ligated to prevent stimulation of a desiccator until required for assay. Similar http://gut.bmj.com/ the duodenum by gastric contents. A cannula powders were prepared from other portions of the (1 cm diameter) was introduced into the fundus same tumour and also from the neuroblastoma of the stomach and this, as the other two, brought control. out through a stab wound and arranged to drain Just before assay, a 5 % aqueous suspension dependently into a graduated tube. A constant was prepared and filtered after shaking. Appro- infusion of saline at the rate of 0.076 ml/min was priate volumes (see Figs 1, 2, 3) were theninjected. begun using the right femoral vein. Tumour tissue was also extracted to test for on October 1, 2021 by guest. Protected copyright. The output of the three cannulae was collected gastrin-like activity using a modification of the continuously, the tubes being changed at 15- procedure of Gregory and Tracy (1964) known to minute intervals, and after two hours of basal yield active extracts from normal human antrum. collections a single injection was made of an extract from the pancreatic adenoma of the first case (E.S.). One and three quarter hours later, secretin was added to the saline and infused at COLLECTION OF SAMPLES the rate of 1.7 units/kg/bw/60 minutes. A steady In both experiments the tubes in which the juice output of pancreatic secretion was established from the cannulae was collected were capped with and the effect observed of, first, an intravenous a thin rubber diaphragm and partially immersed injection of an extract from the tumour of the in iced water to minimize the loss of bicarbonate. first case (E.S.) and, subsequently, ofplasma from Every 15 minutes the collecting tubes were case changed and the volume was made up to a the second (A.G.). manageable volume with distilled water. Each Rat bio-assays sample was then immediately drawn into glass Plasma from lithium-heparinized blood was syringes which were sealed temporarily, main- obtained from the first case (E.S.) before, during, tained at 0°C, and then stored at - 20C. and after the removal of the tumour. Multiple assays using a technique described elsewhere (Thomson, Cleator, and Sircus, 1970) were EFFECT ON WATER AND ION TRANSPORT subsequently carried out. These studies were done in the Department of Surgery of the University of Cardiff. Several PLASMA AND TUMOUR EXTRACTS extracts of the tumour of the first case were pre- Following an overnight fast venous blood was pared using different methods and injected intra- 208 I. G. M. Cleator, Christine G. Thomson, W. Sircus, and Miranda Coombes venously into dogs equipped with Thiry-Vella PANCREATIC SECRETION SECRETIN INFUSION Gut: first published as 10.1136/gut.11.3.206 on 1 March 1970. Downloaded from intestinal loops. 9.1 UNITS/KG BW/HIOUR IVI . CAT (1)4(2) [(3)1(4) [(24)(2) 4(4) (54)(3) Results ;°046'-4 n >0 0.2- CAT BIO-ASSAY The effect on basal secretion 1- No biliary secretion was observed throughout Z<w the whole experiment, nor did any change appear in the pancreatic flow after injection of the -z extract of a neuroblastoma, nor after either plasma.