Phospholipid degradation in, and protein content of, rat fistula bile

Journal of Hepatology, 1986;3:233-240

233

Elsevier HEP 000162

Phospholipid Degradation in, and Protein Content of, Rat Fistula Bile Contamination of Bile with Pancreatic Juice D a v i d B i l l i n g t o n 1, K h a l i d R a h m a n I , T e r e s s a W . J o n e s l, R o g e r C o l e m a n 2, I a n R . S y k e s 2 a n d K u l w a n t S. A u l a k 2 ~Department of Chemistry and Biochemistry, Liverpool Polytechnic, Byrom Street, Liverpool L3 3AF, and 2Department of Biochemistry, University of Birmingham, P. O. Box 363, Birmingham, B15 2 TT ( U.K. )

(Received 16 December, 1985) (Accepted 11 March, 1986)

Summary The extent to which pancreatic juice can contaminate bile collected from a rat with a biliary fistula has been investigated by cannulating the bile duct proximal to either the duodenum or the liver, and by stimulating pancreatic flow with secretin. Bile collected via a fistula proximal to the duodenum showed marked pancreatic contamination. Thus, bile collected via a fistula proximal to the duodenum has a higher flow rate, a greater total protein and amylase content and a different polypeptide profile than bile collected via a fistula proximal to the liver. The phospholipid content also differed in that phosphatidylcholine was converted enzymically to lysophosphatidylcholine. Secretin increased bile flow and the biliary output of total protein and amylase when the fistula was proximal to the duodenum, but had no effect upon these parameters when the fistula was proximal to the liver, or in the isolated perfused rat liver.

Introduction In addition to bile salts, lipids and bile pigments, it is now known that there are a considerable variety of proteins in mammalian bile. Many of these are derived from the plasma [1] whereas others are derived

from hepatocytes [2-4]. The mechanisms by which these proteins enter hepatic bile is currently the subject of much active research and several different mechanisms have been identified: (1) a receptor-mediated vesicle transport pathway for dimeric immunoglobulin A and several other proteins across the

This study was supported financiallyby the Medical Research Council. Correspondence: Dr. Billington,Tel. (051)207 3581, Ext. 2087. 0168-8278/86/$03.50© 1986 Elsevier SciencePublishers B.V. (BiomedicalDivision)

234 hepatocyte from blood to bile [4-6]; (2) pinocytosis and subsequent transcytosis from blood or perfusion fluid to bile [7]; (3) a paracellular pathway involving diffusion from blood to bile across hepatocyte tight junctions [7-10]; and (4) detergent extraction of ectoproteins of the bile canalicular membrane [2, 11, 12]. Much of this work has been carried out in the bile fistula rat. Remarkably, there is a 10-20-fold variation in reported values for the protein concentration of rat fistula bile: 1-2 mg/ml [13, 14], 2-5 mg/ml [3], 8-12 mg/ml [15, 16] and 16-20 mg/ml [17]. This discrepancy was recently highlighted in a review by Reuben [18] who stated that this variation was probably due to 'differences in protein assays, bile collection periods and the physiological state of the animals tested'. As part of another series of experiments [19] we noticed that rat fistula bile collected for 2 h at room temperature contained appreciable, but variable, amounts of lysophosphatidylcholine (20-90% of total biliary phospholipid). However, bile collected on ice and immediately solvent-extracted contained little (less than 10%) lysophosphatidylcholine. Evans et al. [20] and Kawamoto et al. [21] have reported that greater than 80% of rat biliary phospho• lipid is phosphatidylcholine and less than 1% is lysophosphatidylcholine, whilst we ourselves have reported that gallbladder bile of many species contains phosphatidylcholine as the predominant phospholipid [2]. It is known, but not always appreciated, that in the rat several pancreatic ducts deliver exocrine secretions into the common bile duct and hence to the duodenum (see [22]). Thus, if bile samples were contaminated by pancreatic juice, one possible explanation for the origin of iysophosphatidylcholine in rat fistula bile is hydrolysis of phosphatidylcholine by pancreatic phospholipase. Contamination of rat bile by pancreatic juice may also explain the wide variation in reported values for the protein concentration of rat bile. Therefore, in these studies we have attempted to assess possible pancreatic contamination of rat bile by collecting bile via fistulae in the bile duct either proximal to the duodenum (when pancreatic contamination would be expected to be maximal) or proxi-

D. BILLINGTON et al. mal to the liver (when pancreatic contamination would be expected to be minimal). In addition, we have examined the effect of secretin, a known stimulator of pancreatic secretion [23], in both the bile fistula rat and the isolated perfused rat liver.

Materials and Methods

Materials Antisera to rat immunoglobulin A (IgA) and rat serum albumin were obtained from Nordic Immunological Laboratories, Maidenhead, Berks., U.K. Secretin (porcine, practical grade), lysophosphatidylcholine, phosphatidylcholine, hydroxysteroid dehydrogenase (grade II, from Pseudomonas testostetoni) and other fine chemicals were obtained from Sigma (London) Chemical Co., Poole, Dorset, U.K. Sagatal was obtained from May and Baker, Dagenham, Essex, U.K. and heparin was made by Weddel Pharmaceuticals, London, U.K. All other chemicals and solvents were obtained from Fisons Scientific Apparatus Ltd., (Leics.), U.K. and were of the highest grade available. Cannulation tubing PP10 and PP25 was made by Portex Ltd., Hythe, Kent, U.K. Bile fistula rats Wistar rats weighing approximately 250 g were used throughout. Prior to the experiments, these had been maintained on a standard laboratory diet and under a constant light cycle. Bile duct cannulations were performed with PP10 tubing whilst the rats were under pentobarbital (Sagatal) anaesthesia. The fistula was inserted into the bile duct either proximal to the duodenum (i.e. within 2 mm of the entry of the bile duct into the duodenum) or proximal to the liver (i.e. within 5 mm of the bifurcation of the bile duct before it enters the liver). Bile was collected on ice for either 15 min or 1 h into pre-weighed tubes; bile volume was determined from the increase in weight of the collection tubes, assuming bile has a density of lg/ml. All bile samples were stored a t - 2 0 °C for up to 2 weeks before assay. In some animals, the femoral vein was cannulated with PP25 tubing immediately after cannulation of

PANCREATIC CONTAMINATION OF RAT BILE the bile duct. Secretin (0.2 Crick units/min in saline) or saline alone were infused 30 min after bile duct cannulation. In these experiments bile was collected on ice in 15-min aliquots. In another series of experiments the bile duct was cannulated and the abdominal incision closed and sealed. After recovery the animals were allowed free access to food and water. Bile was collected on ice in 6-h aliquots for up to 18 h.

Isolated perfused rat livers At 20 min after bile duct cannulation, livers were isolated essentially as described by Barnwell et al. [10]. Heparin (2 500 units) was injected into the inferior vena cava, the hepatic portal vein cannulated and the liver initially flushed free of blood by perfusion in situ with Ca2÷-free, Krebs-Ringer bicarbonate buffer, pH 7.4. The perfusion was then switched to a recycling medium of 100 ml of Krebs-Ringer bicarbonate buffer, pH 7.4, containing 2 mM Ca 2÷, 5 mM glucose, 1% (w/v) bovine serum albumin, a physiological amino acid mixture ([24]; but with 0.44 mM ornithine, 0.09 mM alanine and 0.1 mM arginine) and 20% (w/v) washed human erythrocytes. This medium was maintained at 37 + 1 °C and gassed continuously with O2/CO 2 (19:1). Secretin (0.2 Crick units/rain in saline) was infused into the liver via the hepatic portal cannula 60 min after liver isolation. Bile was collected on ice in 20-min aliquots and stored at -20 °C until assayed.

Assays Biliary protein was estimated by the method of Lowry et al. [25] using bovine serum albumin as standard. Previous work has shown that the addition of known amounts of protein to bile gives quantitative estimation and that low molecular weight biliary components do not interfere with the assay [15]. Immunoglobulin A and rat serum albumin in bile were determined by quantitative radial immunodiffusion [26] with specific antisera and using authentic rat serum albumin for standardisation. Immunoglobulin A, for which no standard was available, is expressed in arbitrary units relating to the diameter of the precipitation zone.

235 Total bile salt concentrations in bile were deiermined by using hydroxysteroid dehydrogenase as described previously [2]. Amylase activity in bile was assayed by two different methods. Activities presented in Table 1 were determined by dye release from amylose using the Phadebas test kit as supplied by Pharmacia Diagnostics, Uppsala, Sweden. Activities presented in Fig. 3 were determined as maltose liberation from starch. Maltose gives a quantitative colour reaction at 537 nm with methylamine hydrochioride (unpublished method, with permission of Dr. R.H. Michell, Department of Biochemistry, University of Birmingham).

Phospholipid chromatography and assay Lipids were extracted from bile by the method of Bligh and Dyer [27]. Phospholipids were determined in aliquots of this extract by the method of Bartlett [28] except that the samples, after drying down, were digested with 72% (w/v) HCIO4 [29]. Thin-layer chromatography of phospholipids was carried out on other aliquots of the lipid extract essentially as described by Skipski et al. [30] using silica gel H plates 20 cm x 20 cm and with a solvent composition of chloroform/methanol/acetic acid/water (75:45:12:15, v/v). Spots were detected with iodine vapour and identified by reference to authentic standards.

Polyacrylamide gel electrophoresis This was performed essentially as described by Weber and Osborn [31]. Protein was precipitated from bile by the addition of ethanol. The precipitate was dissolved at room temperature overnight in 8 M urea, 4% (w/v) sodium dodecylsulphate, 1% (w/v)flmercaptoethanol, 10 mM phosphate, pH 7.0, containing 0.1% (w/v) bromophenyl blue as tracking dye. Samples (equivalent to approximately 75/~g of biliary protein) were electrophoresed for 30 min at 1-2 mA/gel and then for 6 h at 4-6 mA/gel in cylindrical gels (0.4 x 14 cm) of 6.25% (w/v) polyacrylamide equilibrated in 0.1 M phosphate buffer, pH 7.0, containing 0.1% (w/v) sodium dodecylsulphate. Gels were stained with Coomassie Blue and calibrated according to molecular weight by reference to polypeptide standards.

236

D. BILLINGTON et al.

Results

Origin of biliary lysophosphatidylcholine A f t e r incubation at 37 °C for 4 h, rat bile obtained via a fistula placed in the bile duct proximal to the duodenum was shown by thin-layer c h r o m a t o g r a p h y to contain p r e d o m i n a n t l y ( > 8 0 % ) lysophosphatidylcholine, whilst bile from a fistula proximal to the liver contained p r e d o m i n a n t l y phosphatidylcholine. Incubation at 37 °C of rat bile collected 12-18 h after cannulation proximal to the d u o d e n u m (when biliary phospholipid is essentially absent [19]) with exogenous phosphatidyicholine (final concentration = 4 mM) led to its hydrolysis to lysophosphatidylcholine; this was complete after 4 h (Fig. 1). Pre-heating rat

0

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bile to 100 °C for 5 min p r e v e n t e d the hydrolysis suggesting it to be enzymically mediated. This activity was not shown by bile o b t a i n e d via a fistula proximal to the liver.

Biliary output in the bile fistula rat W h e n bile ducts were cannulated proximal to the d u o d e n u m , bile flow was 6 0 - 7 0 % greater than when bile ducts were cannulated proximal to the liver (Table 1). More strikingly, bile o b t a i n e d via a fistula proximal to the d u o d e n u m had a 5-fold greater protein concentration than bile o b t a i n e d via a fistula proximal to the liver. W h e n these results are expressed as protein output (mg/h/100 g body wt.) this difference is even m o r e e x a g g e r a t e d (Table 1). A m y lase, a classical exocrine pancreatic m a r k e r enzyme [32], was present in massive activity in bile o b t a i n e d via a fistula proximal to the d u o d e n u m but was present at less than 500-fold lower activity in bile obtained via a fistula proximal to the liver (Table 1). The output of bile salts and phospholipid was similar in bile o b t a i n e d via a fistula proximal to either the duo d e n u m or the liver (Table 1). A comparison of the p o l y p e p t i d e profile of bile collected from fistulae both proximal to the liver (gel a) ~nd to the d u o d e n u m (gel b) and from a fistula proximal to the d u o d e n u m with the u p p e r bile duct ligated

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BILIARY OUTPUT IN THE BILE FISTULA RAT Rat bile was collected for 1 h on ice via fistulae proximal to either the duodenum or the liver, Values are means + SEM of 4 observations.

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Fistula proximal to duodenum

origin

Fig. 1. Thin layer chromatogram demonstrating conversion of phosphatidylcholine (PC) to lysophosphatidylcholine (LPC) by rat bile. An aliquot of phosphatidylcholine in chloroform containing 4/~moles of phospholipid was evaporated to dryness in a test tube. To this was added 1 ml of bile collected 12-18 h after cannulation of the bile duct proximal to the duodenum and the mixture was shaken and incubated at 37 °C. Aliquots (0. l ml) were removed at the time intervals (h) indicated; lipids were extracted and subjected to thin-layer chromatography as described in the MATERIALSAND METHODS section.

Flow rate ~l/min/100 g) Protein concentration (mg/ml) Protein output (rag/h/100 g) Phospholipid output (,umol/h/100 g) Bile salt output (umol/h/100 g) Amylase (IU/I)

Fistula proximal to liver

11.73+0.32

7.04+0.74*

10.45 + 1 . 3 5

2.41+0.10"

7.34+0.89

1.01 +0.07*

1.81 +0.19

1.71 +0.12

13.3+ 1 . 4 3 2.5 + l0 s

10.56+0.67 607+ 1.66"

Significant differences (P < 0.05) are indicated by *.

P A N C R E A T I C C O N T A M I N A T I O N OF R A T BILE

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237 fect of secretin, a known simulator of exocrine pancreatic secretion, upon biliary output was investigated.

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'- ~ .o .E Fig. 2. Polypeptide profile of rat bile collected under various conditions. Sample preparation and electrophoresis were as described in the MATERIALSAND METHODS section, a: bile collected (0-15 min) via a fistula proximal to the liver, b: bile collected (0-15 min) via a fistula proximal to the duodenum, c: bile-collected (0-15 min) via a fistula proximal to the duodenum with the upper bile duct ligated (i.e. mainly pancreatic juice). Molecular weights (in kDaltons) were derived from comparison with polypeptide standards as indicated.

(gel c) (i.e. to exclude any hepatic contribution and is therefore mainly pancreatic juice) is shown in Fig. 2. It can be seen that the polypeptide profile of bile obtained from a fistula proximal to the duodenum contains polypeptides derived from both the liver and the pancreas. These results, therefore, strongly suggest a substantial pancreatic contamination of rat bile obtained via a fistula proximal to the duodenum. Thus the ef-

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Fig. 3. The effect of secretin upon (a) bile flow, (b) biliary protein output and (c) amylase output in the bile fistula rat. Bile ducts were cannulated at t = 0 and bile was collected on ice in ]5-min aliquots. Infusion of either saline or secretin (0.2 Crick units/min in saline) was started 30 min after cannulation and is indicated by the arrow, Symbols: ( 0 ) bile ducts cannulated proximal to the duodenum with secretin infusion; ( A ) bile ducts cannulated proximal to the duodenum with saline infusion; (O) bile ducts cannulated proximal to the liver with secretin infusion. Values are means of 4 observations _+ SEM except in the case of bile ducts cannulated proximal to the liver which are means of 2 observations.

238

D. B I L L I N G T O N et al.

a) Flow

16

Effect of secretin upon biliary output in the bile fistula rat

Infusion of secretin increased bile flow by about 60% when collected via a fistula proximal to the duodenum but had a much smaller (less than 10%) effect upon bile flow when collected via a fistula proximal to the liver (Fig. 3a). Secretin produced a greater effect upon total protein output; thus protein output into bile collected via a fistula proximal to the duodenum was stimulated 10-20 fold whilst protein output into bile collected via a fistula proximal to the liver was unaffected (Fig. 3b). Secretin had no effect upon bile salt output when bile ducts were cannulated proximal to either the duodenum or liver (results not shown). When bile ducts were cannulated proximal to the duodenum, secretin caused a 12-fold increase in the output of the pancreatic enzyme amylase (Fig. 3c).

Effect of secretin upon biliary output in the isolated perfused rat liver The simple advantage of using the isolated perfused liver is that, in contrast to the intact animal, any biliary output must originate from the liver. In this system, amylase was absent from bile, and secretin had no effect upon bile flow rate or biliary protein output (Fig. 4). Thin-layer chromatography showed that essentially all biliary phospholipid from the isolated perfused liver was phosphatidylcholine. In addition the bile contained no phospholipase activity towards exogenous phosphatidylcholine (results not shown).

Discussion It is clear from the results presented above that the composition of rat bile, as collected from a biliary fistula, varies according to the placement of cannula. It is also clear from the results with amylase and with secretin stimulation that the bile can be contaminated with pancreatic juice. At the extreme, substantial contamination with pancreatic juice occurg with a cannula proximal to the duodenum but not when the cannula is proximal to the liver (see also [33] and [34]).

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Time (min) Fig. 4. The effect of sccretin upon (a) bile flow rate and (b) biliary protein output in the isolated perfused rat liver. Bile ducts •were cannulated proximal to the duodenum at t = 0. Isolation and perfusion of the liver was commenced at 20 min. Secretin infusion (0.2 Crick units/min in saline) into the perfusion medium was commenced after 60 min and is indicated by the arrow. Values are means of 3 observations + SEM.

The degree of contamination is not related merely to the placement of the open tip of the cannula since the presence of amylase and a high biliary protein content may also result from a tying-in point being located below (i.e. closer to the duodenum) the point of entry of some of the pancreatic ducts, resulting in the reflux of pancreatic secretion up the space between the tie and the tip of the cannula. Thus for the collection of uncontaminated bile it is necessary to ensure that not only the tip of the cannula but also the upper tie-in point are set high up the bile duct, above the entry of the highest pancreatic duct. If the rat is not intended to recover at the end of the experiment it may be appropriate to strip pancreatic tissue from the bile duct to avoid the possibility of contamina-

PANCREATIC CONTAMINATION OF RAT BILE

239

tion, but if recovery of the animal is anticipated this measure is i n a p p r o p r i a t e . Bile samples t a k e n from an isolated perfused liver should not be subject to pancreatic contamination since the pancreas is non-functional u n d e r these conditions. A n i m a l s species possessing a gall-bladder, or in which the entry of pancreatic juice into the d u o d e num does not occur via the bile duct, are clearly able to provide bile samples without pancreatic contamination. T h e consequences and implications of pancreatic contamination of bile are numerous. Thus pancreatic contamination m a y contribute both extra v o l u m e (a bile-salt i n d e p e n d e n t flow) and extra c o m p o n e n t s , to the bile; this can be seen for e x a m p l e in terms of the a m o u n t of protein and in the n u m b e r s of p r o t e i n species p r e s e n t in fistula bile from intact animals (see Figs. 2 and 3). Pancreatic c o n t a m i n a t i o n m a y also contribute to an explanation for some of the variation seen in the r e p o r t e d values for the protein content of fistula bile from intact animals: e.g. 1 - 3 mg/ml (Table 1 and [13, 14]); 8 - 1 5 mg/ml (Table 1 and [15,

16]), and 16-20 mg/ml [17]. T h e presence in bile of pancreatic enzymes m a y result in post secretion modification of biliary components. This can be seen in its effect on phospholipids (Fig. 1) where phosphatidylcholine is d e g r a d e d to lysophosphatidylcholine. H e n c e , the phospholipid profile of bile m a y reflect its degree of pancreatic contamination and since lysophosphatidylcholine is a detergent in its own right [19] the biochemical and physiological p r o p e r t i e s of rat bile c o n t a m i n a t e d with pancreatic juice m a y be different from rat bile alone. H o w e v e r , in contrast to our results, B o u c r o t and Clement [35] have r e p o r t e d that rat bile phospholipid is resistant to the hydrolytic action of pancreatic and snake venom phospholipase A 2. In addition, although we have no specific evidence to support this, the presence of proteolytic enzymes may lead to processing of secreted biliary proteins and to possible deconjugation of c o n j u g a t e d organic anions. These effects m a y be differently a p p a r e n t in newly secreted bile, in relation to the extent of contamination, and would be magnified on storage.

References

9 Thomas P, Toth CA, Zamcheck N. The mechanism of biliary excretion of al-acid glycoprotein in the rat - - Evidence for a molecular weight dependent, nonreceptor mediated pathway. Hepatology 1982; 2: 800-803. 10 Barnwell SG, Godfrey PP, Low PJ, et al. Biliary protein output by isolated perfused rat livers - - Effects of bile salts. Biochem J 1983; 210: 549-557. 11 BiUington D, Evans CE, Godfrey PP, et al. Effects of bile salts on the plasma membranes of isolated rat hepatocytes. Biochem J 1980; 188: 321-327. 12 Lowe PJ, Barnwell SG, Coleman R. Rapid kinetic analysis of the bile salt dependent secretion of phospholipid, cholesterol and a plasma membrane enzyme into bile. Biochem J 1984; 222: 631-637. 13 Elias E, Boyer JL. Taurodehydrocholate choleresis increases low molecular weight proteins in bile and the penetration of ionic lanthanum into the bile canalicular tight junctions in the rat. Clin Res 1978; 26: 317A. 14 Reichen J, Paumgartner D, Berk PD, et al. Effects of bile acids on enzymes of the canalicular membrane and protein excretion into bile. In: G Paumgartner, A Stiel, and E Gerok (Eds.) Biological Effects of Bile Acids. M.T.P. Press, Lancaster, U.K., 1979: 25-26. 15 Godfrey PP, Warner MJ, Coleman, R. Enzymes and proteins in bile - - Variations in output in rat cannula bile during and after depletion of the bile salt pool. Biochem J 1981; 196: 11-16. 16 Rahman K, BiUington D. Effect of chenodeoxycholate

1 Mullock BM, Dobrota M, Hinton RH. Sources of the proteins of rat bile. Biochim Biophys Acta 1978; 543: 497-507. 2 Coleman R, Iqbal S, Godfrey PP, et al. Membranes and bile formation - - Composition of several mammalian biles and their membrane damaging properties. Biochem J 1979; 178: 201-208. 3 La Russo NF, Fowler S. Coordinate secretion of acid hydrolases in rat bile - - Hepatocyte exosytosis of lysosomal protein? J Clin Invest 1979; 64: 948-954. 4 Mullock BM, Hinton RH. Transport of proteins from blood to bile. Trends Biochem Sci 1981; 6: 188-191. 5 Goldman IS, Jones AL, Hradek GT et al. Hepatocyte handling of immunoglobulin A in the r a t - - T h e role of microtubules. Gastroenterology 1983; 85: 130-140. 6 Schiff JM, Fisher MM, Underdown, BJ. Receptor mediated biliary transport of immunogiobulin A and asialoglycoprotein - - Sorting and missorting of ligands revealed by two radiolabelling methods. J Cell Biol 1984; 98: 79-89. 7 Lowe PJ, Kan KS, Barnwell SG, et al. Transcytosis and paracellular movements of horseradish peroxidase across liver panenchymal tissue from blood to bile - - Effects of anaphthylisothiocyanate and colchicine. Biochem J 1985; 229: 529-537. 8 Thomas P. Studies on the mechanisms of biliary excretion of circulating glycoproteins - - The carcinoembryonic antigen. Biochem J 1980; 192: 837-843.

240 feeding upon the biliary output of plasma membrane enzymes in the rat. Biochem Pharmacol 1984; 33: 2231-2238. 17 Kakis G, Yousef IM. Protein composition of rat bile. Can J Biochem 1978; 56: 287-290. 18 Reuben A. Biliary proteins. Hepatology 1984; 4: 46S-50S. 19 Rahman K. Dihydroxy Bile Acids and the Hepatobiliary System of the Rat. Ph.D. Dissertation, C.N.A.A., 1984. 20 Evans WH, Kremmer T, Culvenor J. Role of membranes in bile formation - - Comparison of the composition of bile and a liver bile canalicular plasma membrane subfraction. Biochem J 1976; 154: 589-595. 21 Kawamoto T, Okano G, Akino T. Biosynthesis and turnover of individual molecular species of phosphatidylcholine in liver and bile. Biochim. Biophys Acta 1980; 619: 20-34. 22 Rowett HGQ. The Rat as a Small Mammal, 2nd edition, John Murray, London, 1968: 32-33. 23 Dockray GJ. Comparative biochemistry and physiology of gut hormones. Ann Rev Physiol 1979; 41: 83-95. 24 Seglen PO. Preparation of isolated rat liver cells. Methods Cell Biol 1976; 13: 29-83. 25 Lowry OH, Rosebrough N J, Farr AL, et al. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193: 265-275. 26 Mancini C, Carbonara AO, Heremans JF. Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochem 1965; 2: 235-254.

D. BILLINGTON et al. 27 Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol 1959; 37: 911917. 28 Bartlett GR. Phosphorous assay in column chromatography. J Biol Chem 1959; 234: 466-468. 29 Galliard T, Michell RH, Hawthorne JN. Incorporation of phosphate into diphosphoinositide by subcellular fractions from the liver. Biochim Biophys Acta 1965; 106: 551-563. 30 Skipski VP, Peterson RF, Barclay M. Quantitative analysis of phospholipids by thin layer chromatography. Biochem J 1964; 90: 374-378. 31 Weber K, Osborn M. The reliability of molecular weight determinations by dodecyl sulphate polyacrylamide gel electrophoresis. J Biol Chem 1969; 244: 4406-4411. 32 Lankisch, PG. Exocrine pancreatic function tests. Gut 1982; 23: 777-798. 33 Rutishauser SCB. An analysis of the reported choleretic effect of secretin in the rat. J Physiol 1976; 257: 59P. 34 Reuben A, Howell KE, Boyer JL. Effects of taurocholate on the size of mixed lipid micelles and their associations with pigment and proteins in rat bile. J Lipid Res 1982; 23: 1039-1052. 35 Boucrot P., Clement JR. Resistance to the effect of phospholipase A 2 of the biliary phospholipids during incubation of bile. Lipids 1971; 6: 652-656.