J. Physiol. (1972), 224, pp. 259-269 259 With 6 text-ftgure8 Printed in Great Britain SPECIES DIFFERENCES IN THE CHOLERETIC RESPONSE TO BILE SALTS BY CURTIS D. KLAASSEN From the Clinical Pharmacology and Toxicology Center, Department of Pharmacology, University of Kansas Medical Center, Kansas City, Kansas 66103, U.S.A. (Received 11 October 1971) SUMHARY 1. C(holic, taurocholic and dehydrocholic acids were administered i.v. to rats, rabbits and dogs (3 12-100 mg/kg). With the higher doses of cholic acid, the bile-flo~w was increased sixfold in the dog and only 2 to 21- fold in the rat and rabbit. The choleretic response was also maintained for a longer time in the dog (90 min) than in the rat or rabbit (20-30 min). 2. Similar species differences in the choleretic response to taurocholic acid and dehydrocholic acid were observed. However, the bile flow returned to control rates more rapidly for cholic acid (10-15 min for rat and rabbit, 45 min for dog) and more slowly for dehydrocholic acid (25-40 min for rat and rabbit, 120 min for dog). 3. Cholic acid is conjugated more rapidly by the rabbit and rat than dog. 4. An increase in the biliary bile acid concentration was observed in all three species after the i.v. administration of the bile acid excreted by each species. 5. Control bile flow was much higher in the rat (50 #L./min. kg) and the rabbit (70 #1./min.kg) than in the dog (5 #L./min.kg). 6. Part of the difference in the choleretic response of the three species to the bile acids appears to be due to this difference in basal bile production. 7. Administration of taurocholic acid to the rat increases the biliary bile acid concentration in the bile but the bile flow may either increase or decrease, questioning the mechanism by which bile acids increase bile flow. INTRODUCTION Bile acids are an important constituent of bile and are involved in the absorption of fats from the gastro-intestinal tract. According to Sperber (1959, 1963, 1965), the secretion of bile acids into the canaliculi is the 11-2
260 CURTIS D. KLAASSEN driving force of bile production. The osmotic effect of these bile acids in the canaliculi then results in a flow of water and solutes into the canaliculi. This theory is supported by the fact that bile acids are considered to be one of the most potent choleretic drugs. The purpose of the present study is to compare the percentage increase and duration of bile flow produced by cholic, taurocholic and dehydrocholic acid in rats, rabbits and dogs in an attempt to further understand the importance of bile acid excretion in bile production. METHODS Animals. Simonsen Sprague-Dawley male rats (260-360 g), New Zealand white male rabbits (1-2 kg) and mongrel male dogs (7-13 kg) were used throughout. Anaethesia. The following anaesthetics were employed: rat-urethane, 900 mg/kg, I.P.; rabbit-urethane, 900 mg/kg, I.v.; dog-pentobarbitone sodium, 30 mg/kg, i.v. Rectal temperature of the animals was maintained at 370 C with a heat-lamp to prevent hypothermic alteration in hepatic function (Roberts, Klaassen & Plaa, 1967). Surgical procedure. In all three species, a femoral vein of each animal was cannulated for administering the bile salts. In the rat, PE-50 tubing was used and in the rabbit and dog PE-100 was employed. After ligation of the cystic duct in the rabbit and dog, the bile duct was cannulated with PE-50 and PE-100 respectively. In the rat the bile duct was cannulated with PE-10 tubing. Bile acids and administration. Cholic acid, taurocholic acid sodium salt, dehydrocholic acid, and glycodeoxycholic acid sodium salt (A grade) were obtained from Calbiochem (Los Angeles, Calif.). In the experiments to determine the dose-response effects of bile acids to increase bile flow, cholic, taurocholic, and dehydrocholic were administered at 3-12, 6-25, 12-5, 25, 50 and 100 mg/kg i.v. and bile flow recorded by a drop counter attached to a Gilson polygraph recorder. In the experiments in which the biliary bile acid concentrations were measured, bile was collected at 10-min intervals for 1 hr and then the bile acid was administered iv. (50 mg/kg) and bile again collected at 10-min intervals for an additional hour. Analytical methods. The 10-min bile samples were measured with a graduate pipette. The bile acid concentration in the bile was measured by the gas-liquid chromatographic method of Klaassen (1971 b). The amount of non-conjugated and total (non-conjugated plus conjugated) bile acids in the bile was measured without and with the addition of cholyl glycine hydrolase, respectively. The thin-laver chromatographic method of Hofmann (1962) was used to determine if the bile acid was conjugated with taurine or glycine. RESULTS Species difference in the choleretic response to cholic acid. Fig. 1 illustrates the increase in bile flow produced by a graded series of i.v. doses of cholic acid in the rat, rabbit and dog. In the dog, 3-12 mg/kg produced a 2--fold increase in bile flow and returned to control values in about 10 min. As the dosage was increased, the peak rate of bile flow increased as well as the duration of choleresis. With the highest dose of cholic acid (100 mg/kg),
BILE ACIDS AND CHOLERESIS a 6i-fold increase in bile flow was obtained and did not return to control values until about 100 min after injection. Cholic acid was much less choleretic in the rabbit than in the dog, in that only a twofold increase in bile flow was observed and a maximum duration of only about 30 mi was observed. With the two lower doses of cholic acid, 3412 and 6-25 mg/kg, a choleretic response was not observed in the rabbit. 3 2 261 1 2 0 1 -I C 47 0 0 =~5 4 3 I 10 20 40 60 80 100 Fig. 1. Bile flow in rats, rabbits and dogs after various doses of cholic acid. The choleretic response of the rat to cholic acid was also quite minimal. Again with cholic acid, 3-12 and 6-25 mg/kg, no detectable choleretic response was observed. As with the rabbit, only about a twofold increase in bile flow was observed after the higher doses of cholic acid and the bile flow returned to control values in a relatively short time. The 100 mg/ kg dose of bile acids were not given to the rats as was administered to rabbits and dogs, because this dose was often fatal for the rats.
262 CURTIS D. KLAASSEN Species difference in the choleretic response to taurocholic acid. Fig. 2 illustrates the choleretic response to taurocholic acid in the rat, rabbit and dog. Again the dog was very responsive to the choleretic properties of a bile acid. The highest dose of taurocholic acid produced a 62-fold increase in bile flow, very similar to that produced by cholic acid. The 2 1 2 _ 1 C 0 1._ U 0 L_ 7 #A Lr 4 31 1 10 20 30 40 50 Fig. 2. Bile flow in rats, rabbits and dogs after various i.v. doses of taurocholic acid. major difference in the choleresis produced by cholic and taurocholic acid was that the duration of choleresis was only about one-half as long with taurocholic acid as with cholic acid. Again the choleretic response of the rat and rabbit was much less than that observed for the dog. Only about a twofold increase in bile flow was observed and the duration of choleresis was relatively short. Species difference in the choleretic response to dehydrocholic acid. Dehydrocholic acid is a synthetic bile acid and considered to be the most choleretic bile acid. As can be seen (Fig. 3), dehydrocholic acid was more choleretic
o...dog BILE ACIDS AND CHOLERESIS 263 in the dog than the two other bile acids, cholic and taurocholic acid. Dehydrocholic acid produced a 7k-fold increase in bile flow in comparison to the 6j-fold increase observed with the other two bile acids. However, the most marked difference was in the duration of choleresis; after the 100 mg/kg dose, the bile flow rate did not return to control values until 150 min after administration of dehydrocholic acid, but returned in 50 and 100 min for taurocholic and cholic acid respectively. Dehydrocholic acid was also somewhat more choleretic in the rabbit and rat than the other two bile acids, dehydrocholic acid producing a Rat 3 2 p\. Rabbit 3 _,. 21 E 7 100~~~~~~ 1mg/kg o/ ~~~~~~~~~~~~~ 50 6 P~~~~~~ 12.5 50 Fig. 3. Bile flow in rats, rabbits and dogs after various i.v. doses of dehydrocholic acid.
264 CURTIS D. KLAASSEN 24- to 3-fold increase in bile flow while the other two bile acids produced a 2 to 22-fold increase. The choleretic response to dehydrocholic acid was much less in the rat and rabbit than in the dog as was observed for the other two bile acids. Bile flow and biliary bile acid concentration after cholic acid administration in the dog, rabbit and rat. Fig. 4 demonstrates the results when bile was collected at 10 min intervals for 1 hr after bile duct cannulation and for 25 E 15 5 u2 60 C 0 -c 45-0 U 20 40 60 80 100 120 Fig. 4. Bile flow and bile acid concentration in the dog for 60 min after bile duct cannulation and for an additional 60 min after cholic acid 50 mg/kg administered i.v. The shaded portion of each bar graph represents the amount of taurocholic acid and the unshaded portion the amount of cholic acid. another hour at 10 min intervals after the i.v. administration of cholic acid (50 mg/kg). The control bile flow rate in the dogs was 5,tl./min.kg and after the administration of the cholic acid the bile flow increased fivefold to 25 ul./min. kg. The major bile acid in dog bile is taurocholic acid and after the administration of cholic acid the total concentration of bile acids did not increase appreciably. Since the flow increased approximately five times and the concentration remained relatively constant, the excretion of bile acids also increased about five times. Within the hour
BILE ACIDS AND CHOLERESIS 265 after administration, cholic acid was excreted into the bile (approximately 25 mg/kg), approximately 50 % conjugated with taurine and the other half in an unconjugated form. 160 bt 120 0 - Q E 80 0-40 U C V 25. r_ U._ C 5 U -a.c 5 V.C-. 100 r OẸ 15 S l 20 40 60 80 100 120 Fig. 5. Bile flow and bile acid concentration in the rabbit for 60 min after bile duct cannulation and for an additional 60 min after cholic acid 50 mg/kg administered i.v. The shaded portion of each bar graph represents the amount of bile acid conjugated with glycine and the unshaded portion the amount of unconjugated bile acid. Fig. 5 illustrates the results of a similar experiment performed in rabbits. Bile flow in the rabbit is very high and in the present study the control bile flow was about 75 #1l./min.kg. The major bile acid present in rabbit bile is glycodeoxycholic acid and its concentration is relatively low, only 7 m-equiv/l. After the administration of cholic acid (50 mg/kg), the bile flow rate doubled and returned to control rates within 20 min.
266 CURTIS D. KLAASSEN The maximal concentration of cholic acid and its conjugate was detected in the first 10 min collection period. There was about a sevenfold increase in excretion of bile acids into the bile during this time interval over control rates while only a doubling in bile flow. The concentration of the total amount of cholic and glycocholic acid in the bile rapidly decreased. Within 1 hr after administration, approximately 30 mg/kg of the cholic acid 20 40 60 80 100 120 Fig. 6. Bile flow and bile acid concentration in the rat for 60 min after bile duct cannulation and for an additional 60 min after cholic acid 50 mg/kg administered i.v. The shaded portion of each bar graph represents the amount of taurocholic acid and the unshaded portion the amount of cholic acid. administered to the rabbit was excreted into the bile. Approximately 40 % of cholic acid was excreted as such into the bile and the other 60 % was conjugated with glycine. The effect of the administration of cholic acid on rat bile flow and biliary bile acid concentration is demonstrated in Fig. 6. The control bile flow in these rats was about 50,l./min. kg and the taurocholic acid concentration in the bile is about 13 m-equivfl. When cholic acid was administered i.v. into the rats, a very marked increase in biliary bile acid concentration was observed. Even though there was a fourfold increase in
BILE ACIDS AND CHOLERESIS 267 bile acid excretion only a 30 % increase in bile flow was observed. Within 1 hr after administration of the cholic acid, approximately 35 mg/kg was excreted into the bile. About 90 % of the cholic acid excreted into the bile of the rats during the hour was conjugated with taurine. Bile flow and biliary bile acid concentration in the dog, rabbit, and rat after administration of their major endogenous bile acid. Taurocholic acid, the major bile acid produced by dogs, was administered i.v. into the dogs and a fivefold increase in bile flow was observed which returned to control values in 50 min. The bile acid concentration in the bile doubled after administration whereas only a minor increase in bile acid concentration was observed after cholic acid administration. Glycodeoxycholic acid is the major bile acid produced by rabbits, and when it was administered i.v. into the rabbits (50 mg/kg) a threefold increase in bile flow was observed and it returned to control values within 30 min. A two- to threefold increase in biliary glycodeoxycholic acid concentration was also observed. When taurocholic acid was administered to rats and bile flow and biliary bile acid concentration measured, a decrease in bile flow was observed. The effect that bile acids had on bile flow in the rat was very erratic, sometimes a small decrease in bile flow was observed and other times a small increase in bile flow. Taurocholic acid concentration in the bile markedly increased after its administration, more than tripling within 20 min of its administration. DISCUSSION Marked species differences in bile production and biliary excretion of drugs have been reported. For example, rabbits and rats have a very high rate of bile production, from 40 to 90 #L./min.kg, and dogs have a low rate (5,d./min.kg). This difference in the rate of bile production appears to play an important role in determining the rate of biliary excretion of some compounds such as sulphobromophthalein (BSP). BSP is rapidly cleared from the plasma and excreted into the bile in rats and rabbits - species that have a high rate of bile production - and slowly in dogs (Klaassen & Plaa, 1967). Also, if bile flow of a dog is increased by the administration of bile acids, an enhanced BSP excretion rate is observed (O'Maille, Richards & Short, 1966). Thus, the rate of bile production appears to be an important factor in determining the rate at which some compounds are excreted into the bile, but the reason for such marked differences in the rates of biliary production in various species is not clearly understood. The present study demonstrates that these three species also respond
268 CURTIS D. KLAASSEN to the exogenous administration of bile acids differently. First of all, there is a difference in the conjugation of the bile acids by the three species. Both the rat and dog conjugate the bile acids with taurine and the rabbit conjugates it with glycine. The dog has a slow rate of conjugation for 1 hr after the 50 mg/kg administration of cholic acid; one half the cholic acid in the bile was in an unconjugated form. In contrast, the rabbit and rat appear to have a high capacity to conjugate cholic acid, for in 20 min after its administration less than one quarter of the cholic acid being excreted into the bile is in the unconjugated form. The most marked difference in the response of the three species to the administration of bile acids was that the percentage increase in bile flow produced and its duration was always greater in the dog than the rat and rabbit. The reason for this difference is not entirely clear. Part of the discrepancy is simply due to the difference in basal bile flow rates in the three species. Since the dog has a lower rate of bile production than the other two species, an equal increase in absolute bile flow in the dog as observed in the rabbit or rat would be interpreted as a much greater percentage increase in bile flow in the dog than the other two species. However, this does not appear to be the entire explanation. In the rat, for example, the effect that bile acids have on bile flow is very erratic - sometimes a small increase in bile flow is observed and other times a small decrease. If bile formation was exclusively and linearly dependent on bile acid secretion, then the bile salt concentration should be the same during normal bile flow and during choleresis produced by the injection of the bile acid normally excreted by that species. However, in the present study when taurocholic acid was administered to dogs, bile flow increased and the biliary bile acid concentration also increased. Also, when glycocholic acid was administered to rabbits, a marked increase in bile acid concentration was observed as the bile flow increased; in the rat, the bile flow even decreased while the bile acid concentration increased after taurocholic acid administered. Therefore, it would appear that the presence of bile acids in the bile of rats play a minor role in bile production as we previously concluded (Klaassen, 1971 a). It would also appear that biliary flow in the rabbit and dog is also not entirely dependent on bile acid secretion for bile formation as has been concluded by others (Erlinger, Dhumeaux, Berthelot & Dumont, 1970; Wheeler, Ross & Bradley, 1968) and that there is a bile-salt-independent fraction of bile formation. However, the real importance of bile acids in bile formation is open to conjecture. It seems somewhat conflicting to think that the bile acids in bile of some species can osmotically add fluid volume to the bile but that an increase in bile acid excretion does not always increase the bile flow of rats.
BILE ACIDS AND CHOLERESIS 269 The author acknowledges the very able technical assistance of Mrs Mary Reeves, Mr WV. H. Whittley and Mr Bill Kerr. This study was supported by funds from Public Health Service Grant GM 15956. The author is a recipient of a Public Health Service Career Development Award 1 K04 GM 30996. REFERENCES ERLINGER, S., DHUMEAUX, D., BERTHELOT, P. & DUmONT, M. (1970). Effect of inhibitors of sodium transport on bile formation in the rabbit. Am. J. Physiol. 219, 416-422. HOFMANN, A. F. (1962). Thin-layer absorption chromatography of free and conjugated bile acids on silicic acid. J. Lipid Res. 3, 127-128. KLAAS5EN, C. D. (1971 a). Does bile acid secretion determine canalicular bile production in rats? Am. J. Physiol. 220, 667-673. KLAAsSEN, C. D. (1971 b). Gas-liquid chromatographic determination of bile acids in bile. Clinica chim. Acta 35, 225-229. KLAASSEN, C. D. & PLAA, G. L. (1967). Species variation in metabolism storage, and excretion of sulfobromophthalein. Am. J. Physiol. 213, 1322-1326. O'MAiLLE, E. R. L., RICHARDS, T. G. & SHORT, A. H. (1966). Factors determining the maximal rate of organic anion secretion by the liver and further evidence of the hepatic site of action of the hormone secretin. J. Physiol. 186, 424-438. ROBERTS, R. J., KiAASSEN, C. D. & PLAA, G. L. (1967). Maximum biliary excretion of bilirubin and sulfobromophthalein during anesthesia-induced alteration of rectal temperature. Proc. Soc. exp. Biol. Med. 125, 313-316. SPERBER, I. (1959). Secretion of organic acids in the formation of urine and bile. Pharmac. Rev. 11, 109-134. SPERBER, I. (1963). Biliary excretion and choleresis. In First Proc. Int. Pharmac. Meeting, Stockholm, vol. 4, pp. 137.-143. SPERBER, I. (1965). Biliary excretion of organic anions and its influence on bile flow. In The Biliary System, pp. 457-467, ed. TAYLOR, WV. Oxford: Blackwell. WHEELER, H. O., Ross, E. D. & BRADLEY, S. E. (1968). Canalicular bile production in dogs. Am. J. Physiol. 214, 866-874.