Influence of Cholic and Chenodeoxycholic Acid on Canalicular Bile Flow in Man

70:11 21-1124, 1976 Copyright © 1976 by The Williams & Wilkins Co.

Vol. 70, No. 6

GASTROENTEROLOGY

Printed in U.S.A.

INFLUENCE OF CHOLIC AND CHENODEOXYCHOLIC ACID ON CANALICULAR BILE FLOW IN MAN LEIF LINDBLAD,

M.D.,

AND TORE SCHERSTEN,

M.D.

Surgical Metabolic Research Laboratory, Department of Surgery, Sahlgrenska sjukhuset, University of Goteborg, Goteborg, Sweden

Hepatic bile flow was measured and hepatic [HC ]mannitol clearance was calculated during depletion of the bile acid pool and during duodenal administration of cholic or chenodeoxycholic acid in 10 patients 7 to 12 days after operation for uncomplicated gallstone disease. The relationship between [14 C ]mannitol clearance and bile flow was linear, with a regression coefficient which was not significantly different from unity. This finding supported the assumption that mannitol clearance can be used as a measure of canalicular bile flow in man. Linear relationships between bile acid secretion rate and bile flow and bile acid secretion rate and [HC ]mannitol clearance were found during depletion of the bile acid pool (secretion of cholic, chenodeoxycholic, and deoxycholic acids; relative concentrations, 1.0:1.0:0.5) as well as during cholic acid infusion (73 ± 3% cholic acid in the secreted hepatic bile) and during chenodeoxycholic acid infusion (88 ± 2% chenodeoxycholic acid in the secreted hepatic bile). The bile flow dependence on bile acids 0.011 ± 0.002 ml }.tmoles- 1) was not significantly different for "mixed" bile acid secretion, mainly cholic acid secretion, or mainly chenodeoxycholic acid secretion. Neither was the ductular bile flow (0.08 ± 0.03 ml min - 1) significantly influenced by the various experimental conditions. The canalicular flow-the bile acid-independent (0.17 ± 0.05 ml min - 1) and the bile acid-dependent flow-constituted 70 to 85% of the total bile flow. It is concluded that secretion of cholic acid and of chenodeoxycholic acid promote the same bile flow volume per micromole in man. The mechanisms controlling bi!e flow have been extensively studied in experimental animals. 1-6 The canalicular flow volume can be divided into at least two fractions-one bile acid-dependentS, 7-11 and one bile acid-independent, the latter of which has been proposed to be governed by an active inorganic ion transport. 4, 6, 12 Both of these fractions, however, seem to vary considerably in different animal species,6, 9 and evidence for different effects of different bile acids on the bile flow has also been presented. 6 The bile flow-determining factors in man are incompletely known, but convincing evidence for the existence of both the bile acid-dependent and the independent flow has been presented, 13-16 However, no information is available about the influence of the individual naturally occurring bile acids on the canalicular flow. Such information has become more important now in view of

the current interest in dissolution of cholesterol gallstones by treatment with chenodeoxycholic acid. 17 , 18 In the present study [l4C ]mannitol clearance is used as a measure of canalicular bile flow in man. The bile acid-dependent and -independent flow is determined during depletion of the bile acid pool (mixed bile acid secretion), during mainly cholic, and during mainly chenodeoxycholic acid secretion.

Material and Methods Clinical Material The study group comprised 10 patients, 5 women ages 58 to 68 years and 5 men ages 51 to 74 years, who were admitted to the hospital for operation of uncomplicated nonobstructive gallstone disease. All of the patients, who volunteered as subjects in the study, had normal liver function as judged from routine serum liver tests.19 None of them had taken any medicine known to affect the liver or had had acute cholecystitis or any other disease of importance. The patients were thoroughly informed about the details in the experiment and also about the aim of the study.

Received October 20, 1975. Accepted December 12, 1975. This paper was presented in part at the Second International Gstaad Symposium, The Liver. Quantitative Aspects of Structure and Function, September 2-4, 1975. Experimental Procedures Address correspondence and reprint requests to Tore Scherstim , At the time of operation the common bile duct was canM .D., Department of Surgery II, Sahlgrenska sjukhuset, S-413 45 nulated as described previously.'9 All of the patients had Goteborg, Sweden. This work was supported by the Swedish Medical Research Council, normal bile ducts and no choledocholithiasis as judged from Project 536, the Swedish Cancer Society, Project 557, and Assar preoperative cholangiograms. During the postoperative period, the patients were given intravenous infusions of glucose and Gabrielsson's Cancer Foundation. 1121

1122

Vol. 70,No.6

LINDBLAD AND SCHERSTEN

electrolyte solution the 1st day, whereupon they ate the ordinary hospital diet. No surgical complications were encountered. The experiment was performed 7 to 12 days after the operation. Until this time the enterohepatic circulation (EHC) was kept intact. Two days before the experiment a secondary cholangiography was performed to control the position of the catheters and also to determine that the bile ducts were normal. This time for the experiment was chosen because the liver function and the bile flow are considered to be restituted and stabilized in about 7 days after the operation. 16. 20 The patient was kept fasting during the 12-hr period immediately before and throughout the study. In the morning of the experimental day a duodenal catheter was inserted, and ["Clmannitol (New England Nuclear Corp., West Germany) was given intravenously as a single injection of 10 JLC, followed by a constant infusion of 0.2 JLC per hr in saline to a rate of 1.7 ml per min throughout the experiment. At the beginning of the experiment the EHC was interrupted by means of the balloon catheter (8 AM). At various points of time fluid was collected through the balloon catheter, which had the open tip below the inflated balloon. This fluid was uncolored, indicating that the EHC was completely interrupted. The hepatic bile was quantitatively collected through the other catheter in the common duct. ,. The bile production was measured continuously and hourly samples of bile were analyzed. The patient's loss of water during the experiment was compensated for by infusion of saline through the duodenal catheter. After depletion of the bile iicid pool (5 hr of interrupted EHC), chenodeoxycholic acid (CDCA) or cholic acid (CA) (Sigma grade II) was given through the duodenal catheter at a constant rate, 1.0 g per hr, for a further 4 hr. These bile acids, the purity of which (>95%) was analyzed by thin layer chromatography according to Eneroth,21 were given in saline. None of the patients had diarrhea during the experiment. Venous blood samples were taken every hour (10 ml) simultaneously with bile samples for determination of "C activity in plasma and bile. In three of the bile samples (9 AM, 1 PM, and 5 PM) the concentration of the individual bile acids and the ratio of glycine to taurine conjugates were determined. In the 9 AM bile sample the ratio CA:CDCA:deoxycholic acid (DCA) was 1.0:1.0 ± 0.07:0.5 ± 0.09 (SEM, n = 10). After a 5-hr interruption of the EHC, i.e., at 1 PM, only traces of DCA were detected in the hepatic bile, indicating that the bile acid pool was almost completely emptied. The mean ratio CA:CDCA in these 5-hr samples was 1.1 ± 0.2 (SEM). In all of the experiments the glycine to taurine ratio increased considerably during duodenal infusion of cholic or chenodeoxycholic acid. The mean glycine to taurine ratio (n = 10) before the start of the duodenal bile acid infusion was 3.5 ± 0.6 (SEM), and at the end of the experiments it was 17.5 ± 2.9 (SEM). After 4 hr of duodenal infusion of CA this acid constituted 73 ± 3% (SEM) of the total bile acids in bile. The corresponding value for CDCA in the chenodeoxycholic acid experiments was 88 ± 2% (SEM).

Analytical Procedures Biliary bile acids were extracted, analyzed, and determined as described previously.'· The radioactivity measurements were performed in a Packard Tri-Carb (3320) liquid scintillation spectrometer. Correction for quenching was performed by the external standard method. The biliary mannitol clearance was calculated as the product of bile flow and the bile (Bm) to plasma (Pm) ["Clmannitol ratio (Cl

=

F~:).

Statistical Methods Linear regressions were calculated according to the method of least squares. Differences between regression coefficients and intercepts were tested by analysis of variance.

Results The 14C activity in plasma was fairly constant without any rapid changes in all of the experiments (fig. 1). The biliary mannitol clearance correlated significantly with the bile flow (fig. 2), with a correlation coefficient of 0.92 and regression coefficient of 1.06, which was not significantly different from unity. The extrapolated flow at zero mannitol clearance was 0.07 ± 0.04 (SEM) ml per mm. During depletion of the bile acid pool, and during duodenal infusion of both cholic and chenodeoxycholic acid, the bile to plasma ratio of [14 C ]mannitol was significantly less than unity (0.74 ± 0.01 SEM), indicating that the mannitol clearance was lower than bile flow, which is illustrated in figure 3. The slopes of the regression lines for bile acid secretion rate versus bile flow and bile acid secretion rate versus mannitol clearance during depletion of the bile acid pool were 0.012 ± 0.001 (SEM) and 0.011 ± 0.001 (SEM) ml per Jlmole of bile acids, respectively. The corresponding values during cholic acid administration were 0.010 ± 0.03 and 0.007 ± 0.002, and during chenodeoxycholic acid administration 6 '(

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FIG. 1. "C-mannitol activity in serum during constant intravenous infusion of labeled mannitol. The activity is expressed as dpm ml- 1 x 0.5- 1 (± SEM) (n = 10).

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FIG. 2. The relationship between the biliary clearance of "C-mannitol and bile flow during depletion of the bile acid pool. Regression equation Y = 0.07 + 1.06X; SY% = 0.04, S. = 0.07. Correlation coefficient = 0.92 (n = 10).

June 1976

they were 0.012 ± 0.002 and 0.009 ± 0.003 ml per ~mole of bile acids. The regression coefficients were tested in all possible combinations for statistically significant differences, but none was found (F < 2.7 in all combinations, P > 0.05). The total and the canalicular bile acid-independent flow were obtained from the regression equations for the bile acid to flow and the bile acid to mannitol clearance relationships (fig. 3). The ductular flow was calculated Depletion of the bile acid pool

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CANALICULAR BILE FLOW IN MAN

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Bile aCid secretion rate,jJmol.min-' FIG. 3. The relationships between bile acid secretion rate and bile flow (e) and bile acid secretion rate and biliary clearance of "C-mannitol (A) during depletion of the bile acid pool, cholic acid (CA) infusion, and chenodeoxycholic acid (CDC A) infusion. Regression equations: Bile acid secretion rate (depletion) versus flow: Y = 0.24 + 0.012X, SYX = 0.07, S. = 0.001. Correlation coefficient r = 0.82 (N = 10). Bile acid secretion rate (depletion) versus mannitol clearance: Y = 0.17 + O.llX, SYX = 0.06, S. = 0.001, r = 0.83 (n = 10). Bile acid secretion rate (mainly CAl versus flow: Y = 0.25 + O.OlX, SYX = 0.07, S. = 0.003, r = 0.59 (n = 5). Bile acid secretion rate (mainly CAl versus mannitol clearance: Y = 0.19 + 0.07X, SYX = 0.04, S. = 0.002, r = 0.65 (n = 5). Bile acid secretion rate (mainly CDCA) versus flow: Y = 0.25 + 0.012X, SYX = 0.06, S. = 0.002, r = 0.76 (n = 5). Bile acid secretion rate (mainly CDCA) versus mannitol clearance: Y = 0.14 + 0.009X, SYX = 0.07, S. = 0.003, r = 0.56 (n = 5).

from the difference between these two fractions. No significant differences were found for the flow fractions among the three experimental conditions (table 1) F < 3.2 in all combinations, P> 0.05).

Discussion Most information on the physiology of bile formation is derived from animal studies. From these studies, however, it is apparent that there are marked species differences. Accordingly, it may not be possible to extrapolate directly to man the results obtained from the animal studies. One obvious limitation in human studies of the present type, which include sampling of hepatic bile, is that they can only be done in gallstone patients who recently have undergone an operation. It might be questioned whether the regulatory mechanisms for bile flow can be considered as normal in these patients. On the other hand, all of the present patients had normal bile ducts-no concretions, no inflammation, and no dilation of any part of the bile duct tree as judged from the peroperative and the postoperative cholangiograms. The biliary clearance of mannitol and erythritol has been used as measure of the canalicular bile flow in different species. 4, 7, 15, 16, 22 Recently, Barnhart and Combes 23 reported that in dogs secretin caused an increase of both erythritol and mannitol clearance, which may indicate that the canalicular flow could be overestimated by these methods. However, according to Boyer and Bloomer 15 this is probably not true fot man, since they did not find any significant increase of the biliary mannitol clearance after intravenous administration of secretin. In the present study the mannitol clearance was determined during constant intravenous infusion of labeled mannitol. The advantage of this technique is that the problem with the biliary transit time for mannitol is avoided, provided that the activity of [l4 C ]mannitol per plasma volume does not change rapidly during the study. In all of the present patients the activity of [l4 C ]mannitol per plasma volume was fairly constant throughout the experiment. One further indication of the validity of the present method for determination of canalicular bile flow in man was the linear relationship between mannitol clearance and bile flow, with a high correlation coefficient and a regression coefficient (1.06) not significantly different from unity. The present results are in good agreement with the previous report from this group,14 and with the reports from Boyer and Bloomer 15 and Prandi et al. 16 The average bile flow at intact enterohepatic circulation in the present patients, 0.43 ml per min, was almost the same as in these other studies-0.41, 14 0.43,15 and 0.46 16 ml per min. This study's estimated canalicular flow was 0.35 ml per min, and of that 0.17 ± 0.05 ml per min was the bile acid-independent fraction. Corresponding values in the report from Boyer and Bloomer were 0.27 and 0.13 ml per min and in the report from Prandi et al. they were 0.35 and 0.16 ml per min, respectively. The reason for slight difference between Boyer and Bloomer's and the present results is not clear, but may be related to

1124 TABLE

LINDBLAD AND SCHERSTEN 1. Bile fractions during bile acid pool depletion and cholic and chenodeoxycholic acid administration (mean procedures see Methods) Bile acid.dependent flow

CA administration CDCA administration

0.012 ± 0.001 (n = 44) 0.010 ± 0.003 (n = 22) 0.012 ± 0.002 (n = 22) 0.011 ± 0.002

differences in methodology-they used a bolus dose technique for the administration of [14 e lmannitol and were thus dependent on a correction for the mannitol biliary transit time. Both Boyer and Bloomer and Prandi et al. reported a slightly higher ductular flow than was found in this study. One possible explanation for this difference could be dilation of the bile ducts in their patients. 16 It has been postulated that the ductular flow is stimulated by biliary obstruction and bile duct dilation. 13 The present results , as well as these others obtained from human studies, 15 . 16 indicate that the canalicular flow in fasting man accounts for 70 to 85% of the total flow. These results show that the secretion of cholic acid and of chenodeoxycholic acid promote the same bile flow volume per micromole, which is not significantly different from that promoted by the secretion of "mixed" bile acids. The fact that duodenal infusion of cholic or chenodeoxycholic acid did not significantly modify the ductular flow may suggest that these bile acids do not cause release of secretin.

Total

1. Sperber I: Secretion of organic anions in the formation of urine and bile . Pharmacol Rev 11:109-134, 1959 2. Sperber I: Biliary excretion and choleresis. Proc 1st Int Pharmacol Mtg. 4:137- 143, 1963 3. Wheeler HO, Ramos OL: Determinants of the flow and composition of bile in the unanesthetized dog during constant infusion of sodium taurocholate. J Clin Invest 39:161-170, 1960 4. Wheeler HO , Ross ED, Bradley SE: Canalicular bile production in dogs . Am J Physiol 214:866-874, 1968 5. Presig R, Cooper HL, Wheeler HO: The relationship between taurocholate secretion rate and bile production in the unanesthetized dog during cholinergic blockade and during secretin administration . J Clin Invest 41 : 1152- 1162, 1962 6. Erlinger S, Dhumeaux D: Mechanisms and control of secretion of bile water and electrolytes. Gastroenterology 66:281-304, 1974 7. Forker EL : Two sites of bile formation as determined by mannitol

for experimental

Canalicular

Ductular

ml/min-'

0.24 ± 0.07 (n = 44) 0.25 ± 0.Q7 (n = 21) 0.25 ± 0.06 (n = 22) 0.25 ± 0.06

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12.

13.

14.

15.

16. REFERENCES.

± SEM;

Bile acid·independent flow

mi /Il mo/- '

Bile acid pool depletion

Mean

Vol. 70, No.6

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18. 19.

20. 21. 22. 23 .

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0.07

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and erythritol clearance in the guinea·pig. J Clin Invest 46: 1189-1195, 1967 Erlinger S, Dhumeaux D, Benhamou JP, et al; La secretion biliaire du lapin: preuves en faveur d'une importante fraction indepen· dante des sels biliaires. Rev Fr Etud Clin Bioi 14:144- 150, 1969 Wheeler HO: Secretion of bile acids by the liver and their role in the formation of hepatic bile. Arch Intern Med 130:533-541, 1972 Boyer JL: Canalicular bile formation in the isolated perfused rat liver. Am J Physiol 221:1156-1163, 1971 Dowling RH , Mack E , Picott I, et al : Experimental model for the study of the enterohepatic circulation of bile in Rhesus monkeys. J Lab Clin Med 72:169-176, 1968 Boyer JL, Klatskin G: Canalicular bile flow and bile secretory pressure. Evidence for a non·bile salt dependent fraction in the isolated perfused rat liver. Gastroenterology 59:853-859, 1970 Preisig R, Bircher H, Stirnemann 0, et al: Postoperative choleresis following bile duct obstruction in man. Rev Fr Etud Clin Bioi 14:151-158, 1969 Scherstim T, Nilsson S, Cahlin E, et al: Relationship between the biliary excretion of bile acids and the excretion of water, lecithin, and cholesterol in man. Eur J Clin Invest 1:242- 247, 1971 Boyer JL, Bloomer JR: Canalicular bile secretion in man. Studies utilizing the biliary clearance of "C· mannitol. J Clin Invest 54:773-781 , 1974 Prandi D, Erlinger S ., Glasinovic JC, et al: Canalicular bile production in. man .. E,ur JClin Invest 5: 1-6, 1975 Danzinger RG, Hofmann AF, Schoenfield LJ, et al: Dissolution of cholesterol gallstones by chenodeoxycholic acid. N Engl J Med 286:1- 8, 1972 Schoen field LJ : Medical therapy for gallstones. Gastroenterology 67:725-729, 1974 Nilsson S, Schersten T: Importance of bile acids for phospholipid secretion into human hepatic bile, Gastroenterology 57:525-532, 1969 Thureborn E: Human hepatic bile, Acta Chir Scand (Supp\) 303:1-63, 1962 Eneroth P: Thin·layer chromatography of bile acids, J Lipid Res 4:11-16, 1963 Forker EL, Hicklin T , Sorenson H: The clearance of mannitol and erythritol in rat bile. Proc Soc Exp Bioi Med 126:115- 119, 1967 Barnhart JL, Combes B: Comparison of the biliary clearance of erythritol and mannitol during taurocholate and secretin· induced choleresis in the dog (abstr). Gastroenterology 67 :A·3/780, 1974