LIVER PHYSIOLOGY AND DISEASE

Transcription

1 GASTROENTEROLOGY 64: , 1973 Copyright by The Williams & Wilkins Co. Vol. 64, No. 6 Printed in U.S. A. LIVER PHYSIOLOGY AND DISEASE INTERRELATIONSHIP OF BILE SALTS, PHOSPHOLIPIDS, AND CHOLESTEROL IN BILE DURING MANIPULATION OF THE ENTEROHEPATIC CIRCULATION IN THE CONSCIOUS DOG R. D. SOLOWAY, M.D., K. M. POWELL, J. R. SENIOR, M.D., AND F. P. BROOKS, M.D., Sc.D. Departments of Medicine and Physiology, School of Medicine, University o f Pennsylvania, and the Gastrointestinal Research Laboratories, Philadelphia, General Hospital, Philadelphia, Pennsylvania The interrelationships of the biliary concentrations of bile salts, phospholipids, and cholesterol in the conscious dog were evaluated during complete interruption of the enterohepatic circulation (EHC), partial replacement of the EHC with intravenous taurocholate, or infusions of secretin, EHC interruption in the dog caused a fall in the concentrations and outputs of all three components; cholesterol concentration decreased relatively less than that of bile salts and phospholipids, but in contrast to man, enough of the latter components remained to keep cholesterol solubilized in micelles, Intravenous infusion of taurocholate increased bile salt, cholesterol, and phospholipid outputs. After cessation of the secretin infusion during EHC interruption, bile salt concentration was decreased while phospholipid and cholesterol concentrations were unchanged; outputs of all three components were lower than controls. However, no change in the outputs of these components occurred when secretin was infused together with taurocholate. Regression equations, relating bile salt output to cholesterol and phospholipid output, indicated that during EHC interruption a portion of biliary cholesterol, but not phospholipids, is secreted independently of bile salts. The lack of formation of lithogenic bile with EHC interruption, together with the normally low biliary concentration of cholesterol, may account for the rarity of gallstone formation in the dog. Unlike men, dogs rarely present with spontaneous gallstones. I In 1896, Naunyn 2 demonstrated that human gallstones dissolve when placed in the canine gallblad- Received August 6, Accepted January 29, Address requests for reprints to: Dr. R. D. Soloway, Gastrointestinal Section, Department of Medicine, Gates 7 East, Hospital of the University of Pennsylvania, Philadelphia, Pennsylvania Supported in part by Grants AM 05415, R CDA5K3, AM , and the Kinsey Thomas Foundat ion. The aut hors wish to thank Mrs. Elizabeth Foradori for help with the regression analyses der; this has been repeatedly confirmed. Bile salts and phospholipids are the principal solubilizing agents of cholesterol in bile 3 ; a decrease in the ratio of bile salts plus phospholipids to cholesterol ([BS + PL ]/C) in bile may leave excess cholesterol beyond the limits of mixed micellar solubility, a condition conducive to stone formation.4-6 We have studied the interrelationships of bile salts, phospholipids, and cholesterol in canine bile during acute interruption of the enterohepatic circulation, which, in primates and man H leads to striking decreases in the [BS + PL]lC ratio. The concentrations and outputs of

2 June 1973 INTERRELATIONSHIPS BETWEEN BILIARY LIPIDS 1157 these components were also measured during intravenous infusion of taurocholate, which increases canalicular bile flow, and of secretin which increases ductular flow.7, 8 Methods Cholecystectomy, lesser pancreatic duct ligation, and placement of Thomas cannulae 9 opposite the ampulla of Vater and in the dependent portion of the stomach were carried out in 2 male and 2 female mongrel dogs. Studies were begun 3 weeks after operation. After a 16-hr overnight fast, a polyethylene tube (Intramedic PE 190) was inserted about 5 cm into the common bile duct. Bile was collected by gravity drainage into graduated tubes in an ice bath and was stored at -15 C until determinations were performed. To minimize the release of endogenous secretin, gastric contents drained externally through the open gastric cannula during all experiments. Bile acid, phospholipid, and cholesterol concentrations were determined on bile samples on which other measurements were made and reported in another communication ' and the actual bile flows were reported there. Bile salts were measured by a modification of the hydroxysteroid dehydrogenase method as applied to the measurement of bile salts in bile. 3 The reaction mixture contained: 0.1 ml of a 1: 50 dilution of bile in methanol, 0.5 ml of hydroxysteroid dehydrogenase (1 mg per ml of purified material, Worthington Biochemical Corporation, Freehold, N. J.), 0.4 ml of nicotine adenine dinucleotide (41 mg per 10 mll, and 2 ml of a solution containing 7.5 g of glycine, 5.2 g of hydrazine sulfate, and 2.0 g of ethylenediaminetetraacetic acid per liter. Immediately after collection, lipids were extracted from bile by the Folch" technique. Phospholipids in the chloroform layer were determined by the method of Zilversmit and Davis. '2 Cholesterol was measured by the method of Abell et al. 13 Experimental Design Two control experiments in each of the 4 dogs were performed by collecting 30-min samples of bile for 7 hr without bile salt replacement. Three secretin experiments in each of the 4 dogs began with six 30-min periods of external bile drainage to reach a steady state, followed by infusion of secretin (Gastrointestinal Hormone Research Unit, Chemistry Department, Karolinska Institutet, Stockholm, Sweden) at the rate of 2 U per kg-hr for four 30-min periods, and ended with four 30-min collections after termination of the infusion. Each of the 4 dogs underwent one combined secretin-taurocholate experiment which consisted of complete interruption of the enterohepatic circulation for 2 hr followed by infusion of taurocholate (Calbiochern, Los Angeles, Calif.) at the rate of 14.8 JLmoles per min for the next 6 hr without return of the collected bile. After 2 hr of taurocholate administration, secretin infusion was added for 2 hr, followed by 2 final hr of taurocholate infusion alone. In comparing infusion periods, the first 30- min collection period was excluded to account for biliary dead space errors and to allow time for equilibration. All results are reported as the mean ± 1 standard error (SE). The regression analyses were performed on a PDP-8 computer. Results Controls. In control experiments, after interruption of the enterohepatic circulation at time zero, the outputs of bile salts (fig. 1), phospholipids (fig. 2), and cholesterol (fig. 3) all fell precipitously and. " " -!1 '" " '" o-..-,, o I II FIG. 1. Comparison of bile salt output during complete enterohepatic circulation jnterruption e--e, complete enterohepatic circulation with infusion of Gastrointestinal Hormone Research Unit secretin (2 J.I per kg-hr) , and partial enterohepatic circulation repletion by intravenous infusion of taurocholate (14.8 J.lmoles per min) with infusion of secretin ()... ().

3 1158 SOLOWA Y ET AL. Vol. 64, No.6 o I " FIG. 2. Comparison of phospholipid output during complete enterohepatic circulation interruption.--., complete enterohepatic circulation interruption with infusion of secretin t:.- - -t:., and partial enterohepatic circulation repletion with infusion of secretin A II 10 9 c:: , 5 4 <> 5 ::t I O+-., ro I " min. Co"ection Periods FIG. 3. Comparison of cholesterol output during complete enterohepatic circulation interruption.--., complete enterohepatic circulation interruption with infusion of secretin , and partial enterohepatic repletion with infusion of secretin 111, 11. reached a plateau after 3 hr of collection. Calculation of the ratio of bile salts plus phospholipids to cholesterol demonstrated that cholesterol fell to a lesser extent than phospholipids or bile salts. When the concentrations of all three components were summed, the percentage of total millimoles represented by cholesterol increased from an initial mean of 0.70 ± 0.04% to a value of 1.48 ± 0.15% after complete interruption of the enterohepatic circulation. These figures are well within the limits of the micellar solubility of cholesterol. 3 Secretin infusion. During infusion of secretin alone, an initial rise in the output of all three components was observed, most likely due to a washout of concentrated bile from the biliary tree. During the period 30 to 120 min after the termination of the secretin infusion (the post-secretin period), the concentrations of phospholipids (fig. 4) and cholesterol (fig. 5A) were not significantly different from controls. The mean bile salt concentration (12.8 ± 1.7 SE JLmoles per ml) reached levels significantly lower (P < 0.001) (fig. 6) than the mean control level (28.3 ± 2.8 Ilmoles per mi). However, due to a significant decrease in bile flow in the post-secretin period, 10 the outputs of all three components (figs. 1, 2, and 3) were significantly lower than controls (P < 0.05). Although the percentage of total millimoles represented by cholesterol (1.85 ± 0.6%) increased further in the E 22 <>. 20 '" 1: '" 18 " I FIG. 4. Comparison of phospholipid concentration during complete enterohepatic circulation interruption.--., complete enterohepatic circulation interruption with infusion of secretin t:.- - -t:., ancl partial enterohepatic circulation repletion with mfu sion of secretin 4... A.

4 June 1973 INTERRELATIONSHIPS BETWEEN BILIARY LIPIDS <l '" <> ISO e :I A O ' O 0 I.S I II IS FIG. 6. Comparison of bile salt concentration during complete enterohepatic circulation interruption e--e, complete enterohepatic circulation interruption with infusion of secretin , and partial enterohepatic circulation repletion with infusion of secretin ()... () <l 0.8 '" O.S ' t-r-.--r----r-'--'-' r-lr--o----r---r-, B 0 I S II IS 30 min Collection Periods FIG. 5. A, Comparison of cholesterol concentration during complete enterohepatic circulation interruption, complete enterohepatic circulation interruption with infusion of secretin , and B, partial enterohepatic circulation repletion with infusion of secretin [J.... [J. post-secretin period, it still remained well within the zone of cholesterol solubility. Taurocholate-secretin infusions. During infusion of taurocholate, there was an increase in the concentrations of all three components (figs. 4, 5, and 6) in bile. The increased outputs (figs. 1, 2, and 3) were due to augmented bile flow 1o as well as increased concentration. The addition of an infusion of secretin to that of taurocholate decreased the concentrations of bile salts (fig. 6), phospholipids (fig. 4), and cholesterol (fig. 5B) reciprocally to the increase in bile flow; therefore, the outputs were unchanged (figs. 1, 2, and 3). During the post-secretin period with continued taurocholate infusion, the outputs of the three components were unchanged in contrast with the decrease during the postsecretin period when bile salts were not replaced. Interrelationships. For each 30-min collection period, phospholipid and cholesterol outputs were plotted as a function of bile salt output (fig. 7). When controls were compared with samples obtained during taurocholate infusion, the slopes and Y intercepts of the regression equations were not significantly different. During control experiments, both relationships produced a hyperbolic curve reaching a maximum at a bile salt output of 1000 moles per 30 min. Seven periods with bile salt outputs above this level demonstrated no further increase in phospholipid or cholesterol output. To compare the relationship of the outputs of phospholipids and cholesterol to that of bile salts, regression equations were established using the linear part of the curves (all control periods with bile salt outputs of less than 1000 moles per

5 1160 SOLOWAY ET AL. Vol. 64. No. 6 PL I.B BS r : 0.93 (. o '-.L.L-"----;2OO:---c;'.:40c;;-O-60:;-;: O Bile 5011 (65) )l.moles per 30 min. FIG. 7. The relationship of bile salt (BS) output to the o utputs of cholesterol (C) 0 and phospholipids (PL) e. The outputs of all three lipid classes are pl,)tted for all control periods with a bile salt output of less than 1000 /Lmoles per 30 min. The cholesterol output is plotted on the left and the phospholipid output is plotted on the right abscissa. The Y intercept indicates the amount of C or PL secreted independently of BS. 30 min). The output of phospholipids rose more rapidly than that of cholesterol with increasing bile salt output (fig. 7). The ratio of the outputs of phospholipid to cholesterol at a bile salt output of 100 J.Lmoles per 30 min was 15.1 and at an output of 1000 J.Lmoles per 30 min was Extrapolating to zero bile salt output, the phospholipid output (1.88 /lmoles per 30 min) was not significantly different from zero and represented less than 1% of the level when the bile salt output was 1000 J.Lmoles per 30 min. In contrast, the cholesterol output (0.9 J.Lmoles per 30 min) was significantly different from zero (P < 0.001) and represented 6:4% of maximum. Discussion General. Divergent views on the bile secretory relationships of bile salts, phospholipids, and cholesterol have been based on a variety of animal models and techniques. This situation is comparable to that described for biliary water and electrolyte composition where extensive investigations have demonstrated significant differences between species. 14 Cholesterol. In mans' 6, 15, 16 enterohepatic circulation interruption led to concentrations of cholesterol that exceeded the limits of micellar solubility. In these experiments in the dog, the concentration of cholesterol did not parallel those of bile salts and phospholipids. However, even with the enterohepatic circulation interruption, the ratio of [BS + PL llc remained within the micellar zone. Canine bile has a much lower concentration of cholesterol than does human 17, 18 or simian 18 bile. Canine bile can solubilize much more added cholesterol than either simian 19 or human 20 bile. The ability of canine bile to dissolve human gallstones has been abolished by previous saturation with cholesterol. 17 These species differences may account for the presence or absence of cholesterol supersaturation or precipitation.4-6, 16 Phospholipids. In both these experiments (fig. 2) and in that of Swell et al.,2 1 after 2 hr of biliary diversion, the infusion of taurocholate progressively increased phospholipid output during the first 2 hr of administration. Our data also show that the phospholipid output paralleled the increasing output of bile salts during this period, and during the succeeding 4 hr when bile salt output was stable. Interrelationships, Using bile fistula rats and isolated, perfused rat livers, Kay and Entenman 22 showed that, after interruption of the enterohepatic circulation, the concentrations of all three components decreased with time. Cholic acid infusion then caused a dramatic increase in the outputs of bile salts and phospholipids but only a modest increase in cholesterol. The increase in the outputs of all three components in response to infused taurocholate in this study supports this observation (figs. 1, 2, and 3). However, in the dog, the output of cholesterol varied less relative to those of phospholipids and bile salts than in the rat. In isolated, perfused rat,23 and dog 21 livers, there was no significant secretion of cholesterol or phospholipids into bile until bile salts were administered. However, in these same preparations the use ofradioactive precursors demonstrated that these lipids progressively accumulated in the liver suggesting that they depend on bile salts for secretion into bile. Our data (fig. 7), and that from man 6, 15 and monkey, 4

6 June 1973 INTERRELATIONSHIPS BETWEEN BILIARY LIPIDS 1161 demonstrate that, with decreasing concentrations of bile salts in bile, cholesterol, more than phospholipids, is secreted into bile in increasing ratio to bile salts. In the rat,24 outputs of cholesterol and phospholipids increased linearly with low rates of taurocholate infusion, but reached a maximum with higher rates of bile salt administration. Our results and those of Wheeler and King 25 corroborate these findings in the dog. When the results of Wheeler and King 25 are multiplied to give output per 30 min, the outputs of cholesterol and phospholipids, at less than a bile salt output of 1000,umoles per 30 min, are very similar to these results. The different maximum outputs may be due to experimental design. The regression equations (fig. 7) indicate that under usual circumstances in the dog, a fraction of biliary cholesterol, but not phospholipids, is secreted independently of bile salts. Secretin. Present evidence suggests that bile salts, phospholipids, and cholesterol are secreted into the canaliculus. Secretin affects their concentration, but not their output, by adding a solution of salt and electrolytes to bile downstream at the level of the biliary ductules. 6, 8, 10 This mechanism of action for secretin clearly obtained when exogenous taurocholate was infused. In the bile salt-depleted dog, the postsecretin inhibition of bile flow with decreased outputs of bile salts, phospholipids, and cholesterol is more difficult to explain. Despite the decreased outputs of the three components in the postsecretin period, only the concentration of bile salts decreased significantly (from 28.3 ± 2.8 to 12.8 ± 2.4,umoles per ml) ; the concentrations of cholesterol and phospholipid were not significantly changed. This observation, as well as the demonstration of a bile salt-independent fraction for cholesterol by Wheeler and King 25 and ourselves, would support the hypothesis that, in vivo, neither phospholipids nor cholesterol are absolutely dependent on bile salt secretion for movement into bile. 24 In the absence of returning bile salts from extrahepatic sites, secretin appeared to impair either the synthesis and/or the secretin of bile salts. This demonstration agrees with recent findings that secretin has activity at sites other than those concerned with the secretion of water and electrolytes. Although proteinaceous canine gallstones have been produced by altering the diet, 26 under normal circumstances the dog produces bile unlikely to cause cholesterol precipitation,1 even with the stress of enterohepatic circulation interruption. The dog is a good model to contrast with man, whose bile is much less fortunately constituted. Further elucidation of the factors regulating canine bile flow and composition may suggest means for improving bile composition in man. REFERENCES 1. Martensson K: Studies on the etiology of gallstones. A subtilis-like bacilli-group as an etiologic factor. Acta Chir Scand (suppl) 62:1-227, Naunyn B: A treatise on cholelithiasis. London, New Sydenham Society, 1896, p Admirand WH, Small DM: The physicochemical basis of cholesterol gallstone formation in man. J Clin Invest 47: , Dowling RH, Mack E, Small DM: Biliary lipid secretion and bile composition following acute and chronic interruption of the enterohepatic circulation in the rhesus monkey. J Clin Invest 50: , Hauton JC, Lafont H, Tessier N, et al: Effet chez I'homme de l'interruption partielle du cycle entero-hepatique sur les concentrations biliares du cholesterol et des sels biliares. Clin Chim Acta 20: , Thureborn E: Human hepatic bile. Composition changes due to altered enterohepatic circulation. Acta Chir Scand (suppl) 303:1-63, Wheeler HO, Ramos OL: Determinants of the flow and composition of bile in the unanesthetized dog during constant infusions of sodium taurocholate. J Clin Invest 39: , O'Maillie ERL, Richards TG, Short AH: Factors determining the maximal rate of organic anion secretion by the liver and further evidence on the hepatic site of action of the hormone secretin. J Physiol 186: , Thomas JE: An improved cannula for gastric and intestinal fistulas. Proc Soc Exp Bioi Med 46: Soloway RD, Clark ML, Powell KM, et al: Effects of secretin infusions on bile composition and flow in the conscious dog. Am J Physiol 222: , Folch J, Lees M. Sloane Stanley GH: A simple method for the isolation and purification of total

7 1162 SOLOWA YET AL. Vol. 64, No. 6 lipides from animal tissues. J Bioi Chern 226: , Zilversmit DB, Davis AK: Microdetermination of plasma phospholipids by trichloracetic acid precipitation. J Lab Clin Med 35: , Abell LL, Levy BB, Brodie BB, et al: A simplified method for the estimation of total cholesterol in serum and demonstration of its specificity. J Bioi Chern 195: , Wheeler HO: Water and electrolytes in bile, Handbook of Physiology, sect 6: Alimentary Canal, vol 5: Bile, Digestion, Ruminal Physiology. Edited by CF Code. Washington DC, American Physiological Society, 1968, p Nilson S, Schersten T: Importance of bile acids for phospholipid excretion into human hepatic bile. Gastroenterology 57: , Swell L, Bell CC Jr: Influence of bile acids on biliary lipid excretion in man. Am J Dig Dis 13: , Pickens M, Spanner GO, Bauman L: The composition of gallstones and their solubility in dog bile. J Bioi Chern 95: , J ohnston CG, Nakayama F: Solubility of cholesterol and gallstones in metabolic material. Arch Surg 75: , Nakayama F, Johnston CG: Solubility of human gallstones in primate gallbladder. Proc Soc Exp Bioi Med 104:73-75, Nakayama F: Cholesterol holding capacity of bile in relation to gallstone formation. Clin Chim Acta 14: , Swell L, Bell CC Jr, Entenman C: Bile acids and lipid metabolism. III. Influence of bile acids on phospholipids in liver and bile of the isolated perfused dog liver. Biochim Biophys Acta 164: , Kay RE, Entenman C: Stimulation of taurocholic acid synthesis and biliary excretion of lipids. Am J Physiol 200: , Swell L, Entenman C, Leong GF, et al: Bile acids and lipid metabolism. IV. Influence of bile acids on biliary and liver organelle phospholipids and cholesterol. Am J Physiol 215: , Hardison WGM, Francis TI: The mechanism of cholesterol and phospholipid excretion in bile (abstr). Gastroenterology 56:1164, Wheeler HO, King KK: Biliary excretion of lecithin and cholesterol in the dog. J Clin Invest 51: , Harman CG, Englert E Jr, Wales EE Jr: Studies on the mechanism of diet-induced cholelithiasis in dogs (abstr). Clin Res 17:303, 1969