Though considerable information is now available on the secretory

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1 212 J. Phy8iol. (1962), 162, pp With 1 pkae and 11 teat-figuree Printed in Great Britain BILE SECRETION IN THE SHEEP BY F. A. HARRISON* From the Agricultural Re8earch Council Institute of Animal Physiology, Babraham, Cambridge (Received 6 November 1961) Though considerable information is now available on the secretory activity of some of the digestive glands of the sheep, detailed studies of bile secretion in this animal are lacking. In the work to be described some of the factors which regulate the secretion of bile in the conscious sheep were studied. Some information on the composition of the secretion is also presented. A preliminary account of some of this work has been presented to the Physiological Society (Harrison & Hill, 1960). METHODS Surgical preparations Clun Forest sheep were brought indoors from pasture and allowed 2-3 weeks for adaptation to a dry diet. The sheep were maintained on a diet of 1000 g chopped hay and 200 g crushed oats, fed once daily. Anaesthesia was induced with Pentobarbitone sodium (Abbot Laboratories) and continued with cyclopropane-oxygen mixture in closed-circuit (Gregory, 1947). Rumen ft8tulae were established in all animals (Jarrett, 1948) and were fitted with polyvinyl cannulae (Welvic Paste, I.C.I. Ltd). Operatione for the exteriorization of the bile flow. By exteriorizing the flow of bile from the gall-bladder to the side arm of intestine, the technique used by Taylor (1960) was modified to permit the collection of bile unmixed with pancreatic juice (Text-fig. 1). Before the operation the rumen was emptied through the fistula and the contents were stored at 370 C. This procedure reduced the bulk of the abdominal contents and eliminated rumen distension by the accumulation of rumen gases during the period of anaesthesia. The abdominal cavity was opened through a right paracostal incision and the common bile duct was freed by blunt dissection for about 1 cm between the junction of the cystic and hepatic ducts and the point of entry of the pancreatic duct. After the common duct had been divided between ligatures, the cystic duct and artery were clamped and bile was removed from the gall-bladder with a syringe and needle. A purse-string suture was inserted into the fundus of the gall-bladder which was then incised, within the area defined by the suture, for the insertion of a stainlesssteel or Perspex cannula (Text-fig. 2). The anterior jejunum was transected between intestinal clamps and a cannula inserted in the caudal cut end and secured with a pursestring suture. The cranial cut end was anastomosed to the side arm of jejunum approximately 20 cm from the cannula. Both cannulae were brought out through stab wounds in the abdominal wall. The incision was closed in three layers and the contents returned to the rumen. Bile was allowed to drain continuously from the gall-bladder cannula for several days * Aleen Cust Fellow, Royal College of Veterinary Surgeons,

2 BILE SECRETION IN THE SHEEP 213 after operation. Subsequently, the gall-bladder cannula was connected to the jejunal cannula by vinyl tubing (P1. 1 a). Duodenal ftstulae were established, opposite the papilla of Vater, as described by Taylor (1960). Post-operative care. The vinyl tubing between the gall-bladder and jejunal cannulae and the inside of the gall-bladder cannula were cleaned frequently to remove bile-stained accretions. The preparations lasted for 89 to 319 days. Experiments Collection of bile secretion. The connexion between the gall-bladder and jejunal cannulae was detached and bile allowed to drain freely into a polythene container fastened to the animal's side (P1. lb). Normally the bile was water-clear in appearance, but on occasion encrusted debris from the gall-bladder cannula passed into the collecting bottle and was removed by straining through two thicknesses of surgical gauze. a Gall-bladder cannula JejunostomYd cannula. e Gall-bladder be f Anterior jejunum 101, ~~~~~~~~~ysticduct Hepatic duct Cmo bile duct Pancre g h 0'0'0 Duodenal(I cannula Text-fig. 1 Text-fig. 2 Text-fig. 1. Diagram of the gall-bladder fistula preparation. Text-fig. 2. Diagram of the stainless-steel or Perspex cannula used for the gallbladder fistula and the jejunostomy. a, I in. 20 mm; b, -A in. 8 mm diam. hole; c, i in. 10 mm ext., i in. 6 mm int. diam; d, I in. 16 mm ext. diam.; e, 1 in. 25 mm; f in. 13 mm x 16 B.S.F. thread; g, i in. ext., A7l in. 11 mm int. diam.; h, ini 45mm; i, 1iin. 38 mm.

3 214 F. A. HARRISON Intraduodenal infuioms. Continuous intraduodenal infusion of bile, bile-salt solutions and gastric juice was accomplished by means of a double syringe pump similar to that described by Armstrong & Blaxter (1957). The animal had complete freedom of movement within the pen during this procedure (P1. 1 c). Blood samples were drawn from the jugular vein into 20 rnl. syringes which had been 'wetted' with one drop of heparin ('Liquemin', 5000 i.u./ml.; Roche Products Ltd.). The samples were centrifuged at 4000 rev/min for 10 min and the plasma was used for analysis. Analytical Hydrogen-ion concentration was determined with a glass electrode (Pye Universal ph meter, Cambridge). Total 8olid8 (TS). 2 ml. portions of the secretion were evaporated in tared dishes and dried to constant weight at 1000 C. This method was accurate to + 2 % and the average of duplicate determinations which were within 2 % of each other has been used. Total chloride was estimated by the Whitehorn-Volhard titration method (Cole, 1941). Sodium and potasium were determined by flame photometry with an EEL photometer (Model A, Evans Electroselenium Ltd., Essex). Recovery experiments with bile to which NaCl and KCI had been added revealed no significant interference by bile constituents. Total nitrogen was estimated by a micro-kjeldahl method (Pregl, 1951) using the sodium selenate catalyst described by Chibnall, Rees &.,Williams (1943). RESULTS The composition of sheep gall-bladder bile Gall-bladder bile was obtained by gall-bladder puncture from slaughtered animals immediately after death and from anaesthetized sheep. Its composition, compared with that of sheep plasma, is shown in Table 1. TABLE 1. Composition of gall-bladder bile and sheep plasma Gall-bladder bile A Sheep plasma Mean S.E. Obs. Mean S.E. Obs. TS (g/100 ml.) (11) Cl- (m-equiv/l.) (10) (7) Na+ (m-equiv/l.) (5) (7) K+ (m-equiv/l.) (5) (7) N (mg/100 ml.) (2) Bile secretion in gall-bladder fistula sheep During non-return of bile to the duodenum. In 12 experiments on four animals the pattern of secretion was well defined and reproducible. The volume and TS decreased during the first 3 hr of collection and then continued at a relatively constant level which will be referred to subsequently as the 'steady state' of secretion. The average volume of secretion collected in the first hour was ml. (S.E. of mean; 12 obs.) with an average TS of g/100 ml. (12 obs.). The hourly volume of secretion during the 4-24 hr period in 8 of these experiments was ml. (163 obs.), which on a body-weight basis represented ml./kg. hr. The average TS over the 4-24 hr period was g/100 ml. (162 obs.). The total A

4 BILE SECRETION IN THE SHEEP Text-fig. 3. Bile secretion during non-return of bile to the duodenum. Sheep L 269, Expt O Text-fig. 4. Bile secretion during non-return of bile to the duodenum. Sheep K 81, Expt. 33.

5 216 F. A. HABBISON nitrogen content of the samples fell from a value of g/100 ml. (mean, 2 obs.) to a relatively constant level of mg/100 ml. (41 obs.) over the 4-24 hr period. The Na+ and K+ concentrations of m-equiv/l. (7 obs.) and m-equiv/l. (7 obs.), respectively, declined to m-equiv/l. Na+ (40 obs.) and m-equiv/l. K+ (40 obs.) during the 4-24 hr period in the two experiments for which complete analyses are available. The total chloride concentration decreased from m-equiv/l. (10 obs.) to an average of m-equiv/l. (139 obs.) for the 4-24 hr period in 7 experiments (Text-figs. 3 and 4). In Table 2 the analyses of bile obtained during the first hour and the 4-24 hr of secretion are presented for comparison with those of gall-bladder bile and plasma. TABLE 2. The composition of gall-bladder bile and plasma and of fistula bile secreted during non-return of secretion to the duodenum TS Cl- Na+ K+ N (g/100 ml.) (m-equiv/l.) (m-equiv/l.) (m-equiv/l.) (mg/100 Ml.) Plasma Gall-bladder bile st hr bile hr bile Fed Fed 8-0 I % _- a---v Time (hr) Text-fig. 5. Average ph of hourly samples of fistula bile collected in 5 experiments during non-return of bile to the duodenum. The hydrogen-ion concentration of the hourly samples of bile in individual experiments did not appear to form any regular pattern but the average results from 5 experiments indicated a trend towards increased alkalinity before and for a short time after feeding (Text-fig. 5). To investigate the true 'resting' or 'basal' secretion of the digestive glands of the sheep it is necessary to empty the reticulo-rumen of digesta. When this was done, bile secretion declined during the initial 3 hr of collection and then continued at a reduced, but relatively constant, rate of

6 BILE SECRETION IN THE SHEEP 217 0*243 ml./kg. hr (Text-fig. 6). This was lowerthanthe 'steady state' observed in experiments in which the rumen was not emptied. The solid content was reduced to an average of 1*81 + 0*06 (20 obs.) though a slight increase was observed from the 16th hour onwards. Feeding had no effect on the volume of bile secreted. The effect of prolonged drainage of bile on the hourly rate and composition of secretion was studied during the 8th day of continuous postoperative drainage in sheep L 183. The animal was kept without food on 801 -Fed 2Fed 6 1-, *, 120 E _ E _6 ii> 100 v- E 4 -o._ 0 80 Text-fig. 6. The effect of emptying the reticulo-rumen on the secretion of fistula bile. Sheep K81, Expt I---, cr E 1002 E 1_7 3gE bo 2 U, -v 1-0 tv o0 Time (hr) Text-fig. 7. The effect of prolonged drainage of bile, with non-return to the duodenum, on the hourly volume of secretion during the 8th day of post-operative drainage; note fall in chloride when sheep was fed after fasting for 24 hr. Sheep L 183, Expt. 83.

7 218 F. A. HARRISON the day of the experimeint and the secretory rate was ml./kg. hr with an average TS of g/100 ml. (21 obs.) (Text-fig. 7). The volume of secretion was lower than had previously been noted, whereas the average solid content was higher. In another sheep, M 139, when six consecutive hourly samples were collected during each of the 1st, 2nd and 3rd days of post-operative drainage, an increase in TS was observed over the 3 days. It seems possible that the greater solid content might have been caused by an increased secretion of bile-salts, since Light, Witmer & Vars (1959) have observed an increased output of cholic acid after continuous drainage of bile from rats. In experiment 83 (Text-fig. 7), a fall in the chloride concentration of the bile was observed when the animal was fed after a 24 hr fast. During the return of bile to the duodenum. The effect of returning bile to the animal was investigated in four experiments on three sheep. In the first of these experiments the volume of secretion collected from the fistula was noted at the end of each hour and an equivalent volume of pooled bile, collected during earlier experiments, was returned rapidly to the duodenum. There was considerable fluctuation in the volume of bile collected from the fistula, which was attributed to the intermittent return of a large volume of bile to the animal, and in subsequent experiments, therefore, a pump was used to infuse bile slowly, and at constant rates into the duodenum. The secretory activity during the first 3 hr was then similar to that observed in the non-return experiments, but thereafter continued at a higher level (Text-fig. 8). The volume output and composition of the bile varied in each experiment and was directly related to the rate of infusion and the composition of the bile infused (Text-fig. 9). Bile salts are known to be potent stimuli of bile secretion in man and a number of other animals and the fact that the return of bile to the duodenum of the sheep stimulated the flow of bile and caused an increase in TS, which was dependent upon the rate of infusion and the TS of the infused bile, indicated that a similar mechanism existed in the sheep. Additional evidence on this point was obtained from 2 experiments in which bile salts (solution of commercial bile-salt preparation 8 g/100 ml. distilled water; Oxo Ltd, Code L 55) were infused into the duodenum for a short period after the establishment of the 'steady state' of secretion. A marked increase in the volume of bile secreted and in the solid content occurred during the infusion and it was not until 2-3 hr after the infusion was stopped that secretion returned to the previous level. An infusion of distilled water had no effect on bile secretion, and it was concluded that the response described above was caused by the infused bile salts. A fall in chloride concentration of the 'steady state' secretion was again observed when the animal was fed after fasting for 24 hr (Text-fig. 10).

8 BILE SECRETION IN THE SHEEP 219 An infusion of distilled water at the rate of ml./hr for 2 hr, after the 'steady state' of secretion had been attained, had no effect on bile secretion, but sheep gastric juice (ph 1.5), infused at a similar rate, markedly stimulated bile secretion. This was accompanied by a pronounced fall in TS and total chloride concentration (Text-fig. 11). 140 _E E E _ o i > Time (hr) Text-fig. 8. Collection of fistula bile during the continuous infusion ofpooled bile to the duodenum; note decline in volume and total solids with a rise in chloride after the infusion was stopped. Sheep K81, Expt x = 240 w 0 'El 1-5 -o n 0._ / o~~~ 4, wo 0*5 CA. _ I I i0 Infused bile (ml./kg.hr x TS) Text-fig. 9. Relationship between the solid content and rate of secretion of fistula bile andthe solid content and rate of infusion ofpooled fistula bile. *, mean of 8 nonreturn experiments (4-24 hr averages); 0, 3 return experiments (4-24 hr averages).

9 F. A. HARRISON Infusion Distilled water Bile salts nb +~- 1-: *, 120 E -T *c Text-fig. 10. The effect of an infusion of bile salts into the duodenum on the secretion of fistula bile; note fall in Cl- when the animal wasvfed after fasting for 24 hr. Sheep L269, Expt. 84. Infusion Water Gastric juice Fed _ '10E4L0< E 100> :0CI t'io CO 120 3~~~~~~~~~~~~~0~ F Time (hr) Text-fig. 11. The effect ofan intraduodenal infusion of sheep gastric juice on the secretion of fistula bile; animal fasted to 15th hour. Sheep L269, Expt. 89.

10 BILE SECRETION IN THE SHEEP 221 DISCUSSION Consideration of the anatomy of the sheep's liver and biliary tract indicated that a prerequisite for a study of bile secretion in this animal was a procedure for the separation of bile and pancreatic juice. In the few recorded observations on conscious sheep and goats collection of bile has been achieved mainly by cannulation of the gall-bladder after ligating the common bile duct but, with the exception of the work of Schoregge (1933) and Kuimov (1955), no attempt has been made to return bile to the animal between experimental periods. The present observations were made by means of the gall-bladder fistula preparation in which the normal bile-salt circulation was maintained between experiments. In animals thus prepared the secretory pattern during drainage of bile, with non-return of secretion, was reproducible in that the volume and solid content of the bile secreted during the first 3 hr of collection always declined comparatively rapidly to a low level or 'steady state' which was maintained with little change during experiments continued for 30 hr. Schmidt & Ivy (1937) reported little or no difference in the bilirubin concentration of sheep gall-bladder and hepatic bile respectively and concluded that the gall-bladder had negligible absorptive activity. Nevertheless, the solid content of the samples of bile obtained in the first hour of the present experiments was less than that of gall-bladder bile, suggesting that either there had been dilution of the latter with hepatic bile or that the absorptive function of the gall-bladder had been interfered with. In one experiment four consecutive 10 ml. fractions collected during the 1st hour ofdrainage hadsolid contents of 6.04; 6 37; 5 75 and 5 41 g/100 ml., and the most likely explanation, therefore, of the difference in solid content of gall-bladder and fistula bile was that the absorptive function of the gall-bladder had been reduced by elimination of the controlling activity of the sphincter of Oddi on the flow of bile into the intestinal tract. The subsequent decline in volume and TS appeared to result from the removal of bile salts from the intestine and the consequent interruption of the entero-hepatic circulation, since the infusion of bile salts into the intestine caused an increased secretion of bile of higher solid content. Bidder & Schmidt's (1852) findings for the dry-residue content of three samples of bile obtained from each of four sheep also reveal a downward trend similar to that seen in the initial period of the present experiments, and it is unfortunate that, for the purpose of further comparison, these workers did not continue their observations for a longer period. Schoregge (1933) found that gall-bladder fistula bile from a goat, days after fistulation, had a solid content of g/100 ml., whilst that of bile obtained in drainage experiments was only g/100 ml. He evi-

11 222 F. A. HARRISON dently overlooked the significance of the reduction in solid content, though he indicated that the return of bile to the duodenum increased the volume of secretion. Likewise, Kuimov (1955) reported total solids of g/ 100 ml. in bile obtained during experiments lasting 6-24 hr in which some bile was returned to the duodenum. Although Kuimov associated differences in the total solid content of the bile with ingestion of different foods, in the work recorded here values extending over the same range were obtained during bile drainage experiments with the return of secretion when the animal's diet was unchanged. Reinhold & Wilson (1934) found that in dogs a reduction in bile-salt content of fistula bile was balanced by a decrease in sodium and an increase in chloride and bicarbonate output, and concluded that the secretion of bile salts was the dominant factor in the regulation of the acid-base composition of bile. An increase in chloride concentration during the drainage of bile, and hence depletion of bile salts, was found in the present experiments. Wheeler & Ramos (1960) have suggested that bile formation involves the secretion of two fractions: an electrolyte component, which resembles pancreatic secretion minus enzymes; and a bile-salt component. In the electrolyte component there is an inverse relationship between chloride and bicarbonate concentration with an increase in flow rate. In the present experiments, infusion of gastric juice into the sheep's duodenum increased the volume of bile secreted. This bile had a low chloride concentration and, by inference from the increase in ph, a high bicarbonate concentration, and these findings are compatible with an increase in the electrolyte component of bile. These results parallel those of Wheeler & Ramos in the dog and are presumably due to a release of secretin. Furthermore, as the secretion of gastric juice and the flow of digesta increase after feeding (Hill, 1960), the fall in chloride concentration of the bile observed when the sheep was fed after a 24 hr fast and its increased alkalinity in relation to feeding may signify alterations in the electrolyte component. Bile formation is probably due to a combination of spontaneous and continuous activity, as discussed by Babkin (1950) in relation to other digestive glands. The experiments reported here, in which bile was drained for long periods with and without return to the intestine, have demonstrated that the entero-hepatic circulation of bile salts is primarily responsible for the continuous secretion of bile in the ruminant, although it is evident that the presence of digesta in the reticulo-rumen also plays some part in maintaining secretion. The continued secretion of bile during the non-return of secretion, when the rumen and presumably most of the remainder of the tract were free of digesta, showed that spontaneous formation of bile also occurred.

12 BILE SECRETION IN THE SHEEP 223 It would seem that the most important factors regulating the secretion of bile by the sheep liver are the constant presence of digesta in the reticulorumen; the continuous passage of digesta through the abomasum and duodenum; and the entero-hepatic circulation of bile-salts. SUMMARY 1. A surgical preparation for the investigation of bile secretion in the conscious sheep is described. 2. Bile secretion has been studied in experiments of hr duration and the bile analysed for ph, total solid, total chloride, sodium, potassium and total nitrogen. 3. In fistulated sheep fed once daily continuous drainage of bile with non-return of secretion to the duodenum resulted in a fall in the volume and solid content during the first 3 hr of collection to a relatively constant level of secretion of 0*410 ml./kg. hr of bile with total solids of 2-84 g %. 4. The return of bile to the duodenum increased the volume and solid content of the bile secreted during the 4-24 hr period of collection and the magnitude of the response was related to the rate of infusion and the solid content of the infused bile. 5. Emptying the reticulo-rumen reduced the secretory rate of bile to 0*243 ml./kg.hr and the solid content to 181 g/100 ml. 6. The results are discussed in connexion with earlier work on ruminants and in relation to recent studies on bile formation in the dog. I wish to thank Dr K. J. Hill for suggesting the problem of bile secretion and for his help and advice during the course of this study; the A.R.C. and Dr J. H. Gaddum, F.R.S., Director of this Institute, for providing the facilities to carry out this work; Miss K. P. Bradley, S.R.N., and Miss M. Sheehy, S.R.N., for assistance with the operations; Miss V. H. Alabaster and Miss G. Lapham for their help with the analytical work and Mr A. L. Gallup for the photographs. This work formed part of a thesis for the degree of M.V.Sc. in the University of Liverpool. REFERENCES ARMSTRONG, D. G. & BLAXTER, K. L. (1957). The heat increment of steam-volatile fatty acids in fasting sheep. Brit. J. Nutr. 11, BABKIN, B. P. (1950). Secretory mechanisms of the digestive glands, 2nd ed. New York: P. B. Hoeber, Inc. BIDDER, F. & SCHMIDT, C. (1852). Die Verdauungssaefte und der Stoffwechsel. Eine physiologisch-chemische Untersuchung. Mitau und Leipsic: G. A. Reyhers Verlagsbuchhandlung. CHIBNALL, A. C., REES, M. W. & WILLIAMS, E. F. (1943). The total nitrogen content of egg albumin and other proteins. Biochem. J. 37, COLE, S. W. (1941). Practical Physiological Chemistry, 9th ed. Cambridge: Heffer and Sons. GREGORY, R. A. (1947). A technique of general anaesthesia in ruminants. Vet. Rec. 59, HARRISON, F. A. & HILL, J. K. (1960). Bile secretion in the conscious sheep. J. Physiol. 154, 61-62P. HILL, K. J. (1960). Abomasal secretion in the sheep. J. Physiol. 154, JARRETT, I. G. (1948). The production of rumen and abomasal fistulae in sheep. J. Coun. Sci. Indust. Res. Aust. 21,

13 224 F. A. HARRISON Kuimov, D. K. (1955). The secretion of bile in fine-woolled sheep. SechenovJ. Phy8iol. 41, LIGHT, H. G., WITMER, C. & VARS, H. M. (1959). Interruption of the entero-hepatic circulation and its effect on rat bile. Amer. J. Phy8iol. 197, PREGL, F. (1951). Quantitative Organic Microanalypi8 Based on the Method8 of Fritz Pregl, ed. GRANT, J., 5th ed. London: J. A. Churchill. REINHOLD, J. G. & WmsoN, D. W. (1934). The acid-base composition of hepatic bile. I. Amer. J. Physiol. 107, SCHMIDT, C. R. & Ivy, A. C. (1937). The general function of the gall-bladder. J. Cel. Coomp. Physiol. 10, SCHOREGGE, B. (1933). Beitrage zur Gallensekretion beim Wiederkauer. Arch. Tierendhr. 9, TAYLOR, R. B. (1960). A method for collection of pancreatic juice in the conscious sheep. Ree. vet. Sci. 1, WHEELER, H. 0. & RAMOS, 0. L. (1960). Determinants of the flow and composition of bile in the unanaesthetized dog during constant infusions of Na taurocholate. J. clin. Invest. 39, EXPLANATION OF PLATE a. The external connexion of the gall-bladder and jejunal cannulae; and (above) the duodenal cannula placed opposite the sphincter of Oddi. b. Collection of bile by drainage from the gall-bladder cannula. c. Collection of bile from the gall-bladder cannula during continuous infusion of fluid into the duodenum.

14 i The Journal of Physiology, Vol. 162, No. 2 Plate 1 I I/ 11.J! ti F. A. HARRISON (Facing p. 224)