J. Phygiol. (1975), 245, pp. 583-598 With 7 text-figurew Printed in Great Britain
583
COMPARATIVE EFFECTS OF SODIUM TAURODEOXYCHOLATE AND SODIUM TAUROCHOLATE ON BILE SECRETION IN THE RAT, DOG AND RABBIT
BY SIGRID C. B. RUTISHAUSER* AND THE LATE S. L. STONE From the Department of Physiology, University College, Cardiff CF1 1XL
(Received 28 May 1974) SUMMARY
1. The biliary effects of sodium taurodeoxycholate and sodium taurocholate were investigated in anaesthetized dogs and rats, and in the isolated perfused rat liver. 2. Both bile salts had similar qualitative and quantitative effects on the flow and composition of bile in the dog. 3. In the rat, the bile salts had similar effects on the ionic composition of bile, but differed in that bile flow was not always directly related to the rate of bile salt secretion in bile, in the experiments with sodium taurodeoxycholate. 4. Sodium taurodeoxycholate is hydroxylated to form sodium taurocholate by the liver of the rat but not by that of the dog. 5. The biliary effects of sodium taurodeoxycholate in the rat and in the dog are contrasted with its effects in the rabbit, and the differences between the three species are discussed. INTRODUCTION
The secretion of bile salt into the biliary canaliculus is believed to be an important driving force for the formation of bile. It has been suggested that the secretion of bile salt creates an osmotic force which results in the subsequent flow of water and dissolved electrolytes into the canalicular lumen (Sperber, 1959; Wheeler & Ramos, 1960). Bile salts differ in their efficiency in increasing the flow of bile. Thus, dehydrocholate, and cholate, have been shown to be more efficient as choleretics than sodium taurocholate (Sperber, 1959; O'Maille, Richards & Short, 1965). The choleretic efficiency of a bile salt is believed to depend on the number of osmotically * Address for correspondence: Dr S. C. B. Rutishauser, Department of Physiology, Stopford Building, University of Manchester, Manchester, M13 9PT.
584 S. C. B. RUTISHA USER AND THE LATE S. L. STONE effective particles in the canalicular lumen, and this, in turn, depends on the extent to which the bile salt molecules are aggregated into micelles. However, it has been shown recently, that sodium taurodeoxycholate is a more efficient choleretic than sodium taurocholate in the rabbit, and that this cannot be explained readily in terms of the known micellar properties of the two bile salts (Rutishauser & Stone, 1974, 1975). Sodium taurodeoxycholate, unlike sodium taurocholate, increased the bicarbonate concentration of rabbit bile, and it was suggested that sodium taurodeoxycholate may have stimulated a bicarbonate pump in the biliary system, and that this was the cause of its greater choleretic efficiency compared with sodium taurocholate. It was therefore of interest to examine the choleretic efficiency of these two bile salts in other species in order to determine whether sodium taurodeoxycholate had special choleretic properties in itself, or whether its effects were just peculiar to the rabbit. METHODS Dogs. Mongrel dogs of both sexes were anaesthetized with sodium pentobarbitone (30 mg/kg I.v.). The trachea was cannulated and the spleen was removed. The common bile duct was cannulated after ligation of the cystic duct and drainage of the gall-bladder. The ureters were cannulated. Bile and urine samples were collected serially as 10 or 20 min samples in 10 ml. tubes graduated in divisions of 0.1 ml. Infusions were given via a cannula lying in one of the femoral veins. Rats. Male Wistar rats (body weight 350-450 g) were anaesthetized with ether. The right jugular vein was cannulated, and anaesthesia was maintained by the i.v. injection of 0 02-010 ml. aliquots of sodium pentobarbitone (60 mg/ml. diluted 1:1 with 0-9 % NaCl). The bile duct was cannulated, the tip of the cannula lying just distal to the point at which the left and right hepatic ducts fuse. Bile was collected serially as 20 min samples in flat-based glass tubes of constant bore (3, 4, or 5 mm in diameter). The length of the column of bile was measured (± 0-5 mm) and the bile volume was calculated. All infusions were given via the right or left jugular vein. In some experiments, blood pressure was recorded from the left carotid artery. All animals were fasted overnight before the experiment, but were allowed free access to water. Body temperature was maintained during anaesthesia by surface warming. At the end of each experiment the liver was removed and weighed. Isolated perfused rat liver. The perfusion apparatus used was similar to that described by Curtis, Powell & Stone (1971). The livers from male rats (body weight 300 g) were removed under ether anaesthesia, and were perfumed via the portal vein. The perfusion medium consisted of 70 ml. of pooled rat blood diluted to -120 ml. with Krebs-Ringer bicarbonate solution, to which was added 2 g bovine albumin, 200 mg glucose, 20 mg streptomycin or penicillin, and a small amount of silicone anti-foam A. The Krebs-Ringer bicarbonate solution was gassed with 5 % C02-95 % 02 before use. In this perfusion system, after 1 hr of perfusion, perfusate flow was 5-6 ml./min. g liver, perfusion pressure was 18-20 cm H O. and bile flow was in the range 1.0-I6 ,sl./min. g liver. Bile salts. Sodium taurodeoxycholate (TDC) and sodium taurocholate (TC) were -
BILE SALTS AND BILE SECRETION
585
obtained from Maybridge Chemicals, Tintagel, Cornwall. For infusion, they were either dissolved in 0-9% NeCl, or in a mixture of 0 9% NaCl, 0X1 M-Na phosphate buffer pH 7-4, and bovine albumin (1-5%'). The addition of albumin reduced the haemolytic and cardiovascular effects of the bile salts. The concentration of bile salt in bile was determined using two methods. (a) By the use of the enzyme steroid dehydrogenase in a modification ofthe method detailed by Javitt & Emerman (1968). The mixture had to be incubated at 370 C for 1 hr before determination of the extinction of the solution at 340 nm. This was necessary because of the much slower reaction of TDC in this system as compared with TC. The enzyme steroid dehydrogenase determines all the bile salt present in bile bearing a 3-hydroxyl group, and this includes all the bile salts of known importance in the bile of rats and of dogs. Thus this enzymic method was taken to measure 'total bile salt' concentration in bile. (b) 'Cholate' in bile was determined by the method detailed by O'Maille et al. (1965). This method measures the amount of free and conjugated cholate in bile; deoxycholate is not reactive in this system. The difference between 'cholate' and 'total' bile salt was taken to be a measure of the concentration of dihydroxy bile salts in bile, and thus of deoxycholate in experiments where this bile salt predominated. Some bile was analysed by thin-layer chromatography. 1 ml. bile containing 25-50 mg of bile salt was hydrolysed in 12% KOH at 1200 C for 3 hr, and then extracted according to the method described by Iwata & Yamasaki (1964). The method of Siegfried & Elliott (1968) was used for thin-layer chromatography of the samples. 10 ,d. of the ethanolic extract containing 250-500 jug bile acid was applied to the plates, which were developed in an ascending system. In some experiments the individual bile acids were eluted from the plates with methanol, and the amounts of bile acid present were separately determined using the enzyme steroid
dehydrogenase. Ionic analysis. Sodium and potassium concentrations in bile were measured by flame photometry; chloride was determined by an electrometric titration method (Buchler-Cotlove chloridometer); bicarbonate concentration was measured using the micro-manometric method of Van Slyke (Natelson microgasometer); bile osmolality was determined cryoscopically (Advanced osmometer). Statistical analyses. Mean values are given together with their standard errors, unless otherwise specified in the text. The Student t test was used in the comparison of data. RESULTS
1. Dog Effects on bile flow Sodium taurocholate (TC), and sodium taurodeoxycholate (TDC) were dissolved in 0-9 % NaCl (2-0 limole/ml.) and were infused i.v. into seven dogs at rates ranging from 5 to 120 n-mole/min . g liver. Up to an infusion rate of 90 n-mole/min. g liver both bile salts increased bile flow, but at rates greater than this TDC caused cholestasis. In Fig. 1 the rate of bile salt secretion into bile is plotted against bile flow for each period of steadystate infusion. It can be seen that the two bile salts had similar quantitative effects on the rate of flow of bile. The choleretic efficiency of the bile salts was calculated from the slope of the regression lines determined from the
586 S. C. B. RUTISHA USER AND THE LATE S. L. STONE data shown in Fig. 1. The choleretic efficiency of TC was 8-5 ml./m-mole, and that for TDC was 9-6 ml./m-mole.
Bile salt metabolism In the infusion experiments, the increase in the rate of secretion of bile salt into bile was in each case approximately equal to the rate at which bile salt was infused (Fig. 2). The increase in the rate of secretion of bile salt into bile in the experiments with TDC was calculated as the increase in the secretion of 'non-cholate' bile salt into bile. Thus TDC is not appreciably converted into taurocholate in the liver of the dog. 1.20 1*00 _* ,L- O080_
to
_
W
z06 =
040_
020
4
20 80 40 100 60 120 Rate of bile salt secretion (n-mole/min.g liver)
Fig. 1. The relationship between the rate of bile flow and the rate of secretion of 'total' bile salt in bile, during the constant infusion of sodium taurodeoxycholate (A) or sodium taurocholate (e) in the dog. The regression equations are: y = 00096x+0 132 for TDC; and y = 00085x+0 151 for TC; where y = bile flow, and x = rate of bile salt secretion.
Similar results were obtained in experiments where TDC was given as a single injection (50 mg/kg body wt.). In this case, the mean recovery of the bile salt in bile was 88-2 + 2-6 0/ when calculated as 'non-cholate' bile salt, and 900 + 3-7 % when calculated as 'total' bile salt (mean + S.E. of mean in three experiments). Samples of bile from the infusion experiments were hydrolysed and
analysed by thin-layer chromatography. Only two bile acids could be shown to be present, and these had the mobilities of cholic and deoxycholic acids. During the infusion of TDC, 75-90 % of the bile salt present in bile could be shown by thin-layer chromatography to be deoxycholate.
BILE SALTS AND BILE SECRETION 587 It may be noted that appreciable amounts of 'non-cholate' bile salt (15.1+10-7%; mean+ S.D. of an observation), were found normally in samples of bile collected immediately after cannulation of the bile duct.
Effects on the ionic composition of bile Table 1 gives the mean net changes in the ionic composition of bile caused by the infusion of either TC or TDC at the rate of 40 n-mole/min . g liver. Both bile salts had essentially similar effects, in that the concentrations of sodium and of chloride in bile increased, and that of bicarbonate decreased. 140
120
.°0Z 100 -bo
0
80 60 _
0
_
*DA 00 U
20
An,&
^~~
60 40 80 100 20 120 Rate of bile salt infusion (n-molelmin.g liver)
140
Fig. 2. The relationship between the rate of infusion of bile salt, and the increase in the rate of secretion of bile salt into bile, during the constant infusion of sodium taurodeoxycholate (A) or sodium taurocholate (e) in the dog. The increase in the rate of bile salt secretion into bile was calculated as the increase in the 'cholate' fraction forthe experiments with taurocholate, and as the increase in the 'non-cholate' fraction for the experiments with taurodeoxycholate.
During the infusion of bile salt there was no appreciable difference between the sum of the anions and cations in bile, i.e. (sodium + potassium) (chloride + bicarbonate + bile salt). However, during the control period before and after infusion, there was a mean anion deficit of 18 + 7 mequiv/l. This accounts for the apparent discrepancy between the total anions and cations in Table 1.
588 S. C. B. RUTISHA USER AND THE LATE S. L. STONE TABiE 1. Net changes in the ionic composition of bile caused by the infusion of bile salt (TDC or TC -40 n-mole/min. g liver) in the dog. The figures give the mean difference in concentration + S.D. between the infusion period, and the control periods before and after infusion, in three experiments with each bile salt Taurodeoxycholate + 9-9 ± 2-9
Na
+0-55±0-26 +11-5 ± 8-7
K
Cl
Taurocholate +3-4+ 2-4 +0-78+ 102 + 8-2 + 8-4 -11-5±6-7 + 30-6 ± 7-3
,(m-equivfl.)
HCO3
-10-8 ± 8-4
Total bile salt)
+30-6±6-2
+1-6 0
+12 _bo
+08
0
F 0
c
x? +0-4
_
0
1-
A
A
0
*
*
. ._
-04 -
*
*
.,
.
*
*
.
*s
*
A.
120 60 Rate of bile salt infusion (n-mole/min.g liver)
180
C U
ok, -0-8
_-
z
-1*2
-16
A
a
_-
Fig. 3. The net changes in bile flow caused by the infusion of sodium taurodeoxycholate (A) and sodium taurocholate (e) in the anaesthetized rat.
2. Rat
Effects on bile flow Bile salts were dissolved in 0-9 % NaCl (25-50 ,umole/ml.) and were infused at rates ranging from 20 to 200 n-mole/min.g liver into anaesthetized rats. At low rates of infusion, both bile salts increased bile flow, but, as can be seen in Fig. 3, the relationship between bile flow and infusion rate was not a clearly linear one for TDC. At rates of infusion greater than 90 n-mole/min . g liver TDC caused cholestasis.
589 BILE SALTS AND BILE SECRETION It was found that a brief infusion of TDC (1 ml. of a 50 It-mole/ml. solution infused over 15 min) caused a reversible decrease in bile flow. This is illustrated in Fig. 4 where the effects of TDC are contrasted with the choleretic effect of an equimolar infusion of TC. It can be seen that the decrease in bile flow with TDC occurred in spite of a simultaneous increase in the rate of bile salt secretion into bile. TDC 30
TC
-
0
0
>=-
20o
E
150 _
150
-
0
0 c
E
2505o so
X
2 3 4 Time (hr) Fig. 4. The effects of a brief infusion of bile salt (50 molee over 15 min) on bile flow and bile salt secretion into bile in the anaesthetized rat. The effects of sodium taurodeoxycholate and sodium taurocholate are compared. Concentration of bile salt in bile: ' cholate ' (-; 'non-cholate ' (EA).
Effects on the ionic composition of bile 50 /czmole of either TC or TDC, dissolved in a phosphate buffer-albumin
solution, was infused intravenously over 15 min. Because of the very different effects of TC and TDC on the rate of flow of bile, bile was collected as timed samples of constant volume (0-38 ml.). If bile had been collected
590 S. C. B. RUTISHAUSER AND THE LATE S. L. STONE as 20 min samples, the peak ionic response to TC would have occurred in sample 1, whereas that for TDC would have occurred in samples 2 or 3. As the first sample of bile collected immediately after the infusion contains some 'dead-space' bile, it was thought that this could lead to underestimation of the effects of TC on the composition of bile. Accordingly, it was thought that collection of bile as timed samples of constant volume would enable a more accurate comparison of the ionic effects of the two bile salts to be made. The leaned data for 4 experiments with TDC and five experiments with TC are shown in Fig. 5. Both bile salts increased the concentrations of sodium and potassium in bile and decreased the concentrations of chloride and of bicarbonate. Although the peak concentrations of sodium and of bile salt in bile were almost the same for both bile salts, the concentration of sodium in bile thereafter was significantly greater in the experiments with TDC compared with those with TC (P < 0 0025). There was no change in the osmolality of bile after the infusion of TC, and the slight increase which occurred temporarily after the infusion of TDC amounted to no more than 3-4 m-osmole/kg. Biliary secretary pressures In these experiments bile was collected as 10 min samples for a period of 30 min. Biliary secretary pressure was then measured by affixing the filled bile cannula (internal bore = 0-28 mm) to a vertical 30 cm ruler and noting the maximum level to which the bile rose. 10 min after raising the cannula, with biliary stasis still imposed, an infusion of either TC or TDC, or the phosphate buffer-albumin solution, was begun at the same rate as in the previous experiments on the ionic composition of bile. The infusion was continued for 6 min (total dose of bile salt, 20 molee, the level of bile in the cannula being noted each minute during the infusion, and for 4 min thereafter. The total time from the interruption of normal flow to the end of the imposed biliary stasis was 20 min. The volume of bile collected in the 10 min immediately after free flow was resumed was measured in each case. After each infusion a further 50 min of free flow was allowed in order to give sufficient time for the infused bile salt to be eliminated, and for control conditions to be restored. The results of a typical experiment are shown in Fig. 6. In four experiments the mean control value for biliary secretary pressure before the infusion of bile salt was 218 + 09 cm H20 (S.E. of mean). A gradual decline from this value (1-3 cm in 10 min) was seen during the infusion of the phosphate buffer-albumin solution. Both TC and TDC initially increased the secretary pressure ( + 1 2 + 0 3 cm, and + 1I9 + 0 5 cm respectively), but a decrease in pressure then ensued. The rate of decrease
591 BILE SALTS AND BILE SECRETION was twice as great for TDC (IP04 + 0-06 cm/min) as for TC (0.57 + 0412 cm/ min). In every experiment, the final secretary pressure after the infusion of TDC was at least 3-5 cm less than the values recorded after the infusion Bile salt
I 2*0 0bo @4a-
1*0 lII I
I Il
I I
200
-
0s
150
tr
E 100 C
0
0 U U
.C 0
50s
I
1
I I1 I I I I I 2 3 4 5 6 7 8 Sample number
I
9
Fig. 5. The effects of the infusion of sodium taurodeoxycholate (- - - -) and sodium taurocholate ( ) (50 jimole over 15 min) on the flow and ionic composition of bile in the anaesthetized rat. The data represent the mean of four experiments with TDC and five experiments with TC. The S.E.'S of the means are indicated where these are meaningful. Bile was collected as timed samples of constant volume (038 ml.). Sodium (V), chloride (0), bicarbonate (E.), 'total' bile salt (x); open symbols, sodium taurodeoxycholate; filled symbols, sodium taurocholate.
592 S. C. B. RUTISHA USER AND THE LATE S. L. STONE of either TC or the buffer-albumin mixture at the corresponding time in the same rat. The maximum secretary pressure recorded 1 hr after the infusion of bile salt was not significantly different from the control value either after TC (22.2 + 0 3 cm) or after TDC (22.2 + 06 cm). This shows that no permanent change had been caused by the infusion of bile salt. Essentially similar quantitative results were obtained in the response to TC and TDC when a bile cannula of triple the internal diameter was used. Infusion
0
°I20 _
.
Ad
%I-J
5
10 Time (min)
15
20
Fig. 6. The effect of the infusion of either sodium taurodeoxycholate (A - - A), sodium taurocholate (e*-), or the infusion medium ( x ... x ), on biliary secretary pressures in the anaesthetized rat. At zero minutes the bile cannula was raised, and the height of the column of bile above the liver was recorded every minute for the next 20 min. The solutions were infused at the time indicated at the rate of 0.056 ml./min: total dose of bile salt = 20 molel.
The volume of bile collected in the 10 min period post-stasis also showed a consistent pattern. After the infusion of the buffer-albumin solution, 0 0074 + 0'0015 ml./g liver was obtained in excess of the expected flow rate. This represents the extra bile stored in the biliary system as a result of the distension caused by stopping the flow of bile. The extra volume obtained after TC was either the same as this or slightly less (0.0065 + 0.0011 ml./g liver). In contrast, after the infusion of TDC there was a volume deficit of 0-0028 + 0-0008 ml./g liver compared with the original control bile flow. Mixtures of TC and TDC in the isolated perfused rat liver The choleretic response to the infusion of bile salt was also examined in the isolated perfused rat liver. The same total dose of bile salt was given
593 BILE SALTS AND BILE SECRETION in each experiment (50 molele, but the proportions of TC and TDC in the infused solution were varied. 2*5 ml. of the prepared solutions (20 ,umole/ml. in phosphate buffer-albumin solution) was infused at the rate of 2-2 ml./ min, 1 hr after the start of the perfusion. Fig. 7 shows the net change in bile flow caused by the infusion of 50 #mole of bile salt in four experiments. It can be seen that the same total dose of bile salt produced very different volumes of bile, and that as the proportion of TDC in the mixture was increased, the volume of bile formed was reduced. 50 ,-mole bile salt
1+I5
4r
.E 01+10_ 50
0
.SQ -1*0_ -0-5 C
z
01. -15_
I
1
I
2 Time (hr)
I
3
4
Fig. 7. The net changes in bile flow caused by the infusion of four separate 50 jimole doses of bile salt, consisting of different proportions of sodium taurodeoxycholate (TDC) and sodium taurocholate (TC), in 4 individual experiments in the isolated perfused rat liver. The proportions of TC: TDC in themixturesinfusedwere: 100:0(0-*); 66:33 (0_-D); 60:40( x x 50:50 (v- -if).
It was possible in these experiments to accurately determine the recoveries of the infused bile salt in bile. In the isolated perfused rat liver, the rate of secretion of endogenous bile salt, as measured by the enzyme steroid dehydrogenase, is very low after 1 hr of perfusion (1-2 n-mole/ min.g liver). In calculating the recoveries of bile salt, the endogenous secretion rate was ignored, and this was estimated to introduce an error of only 1-2 % at most. In the four experiments illustrated in Fig. 7, the total recovery of bile 25
PHY 245
594 S. C. B. RUTISHAUSER AND THE LATE S. L. STONE salt in bile was virtually 100 % in each case, in spite of the different volumes of bile formed. The bile salt recoveries are listed in Table 2. It may be noted, that TDC was appreciably hydroxylated in its passage through the liver, in that the recovery of 'cholate' in bile was consistently greater than the amount infused, in those experiments in which TDC was included in the infusion mixture. TABiE 2. Amounts of bile salt recovered in bile after the infusion of 50 gmole of bile salt, consisting of different proportions of sodium taurodeoxycholate and sodium taurocholate in four experiments in the perfused rat liver
Amount infused
Amount recovered in bile
(Cmole)
(smole)
'Total' 50 50 50 50
'Cholate' 50 33 30 25
'Total'
'Cholate'
51-2 48-8 53-7 50 4
50.6 39*2 40*6 34 9
Increase in 'cholate' + 0.6 +6-2 +10.6 +9 9
Cardiovascular effects of the bile salts Both bile salts decreased blood pressure and heart rate when infused into anaesthetized rats, but TDC was more potent than TC. Clearly, a reduction in blood flow to the liver may affect the flow of bile, but it was observed that the cardiovascular effects of the bile salts did not always correlate with the biliary effects. In the isolated perfused rat liver both bile salts caused vasoconstriction. Again TDC was more effective than TC. In the perfusion system used, it was shown that bile flow was not affected by a reduction of blood flow until this fell below 2 5 ml./min.g liver, and this did not occur in the perfusion experiments described. DISCUSSION
The results have shown that sodium taurodeoxycholate is not a more efficient choleretic than sodium taurocholate in the rat and in the dog. This contrasts with the choleretic effects of this bile salt in the rabbit (Rutishauser & Stone, 1974, 1975), and suggests that sodium taurodeoxycholate is not inherently a good choleretic, but rather that it possesses special properties in the rabbit. The glycine conjugate of deoxycholate has also been shown to be an exceptionally good choleretic in this species, in that its choleretic efficiency is 70 ml./m-mole (Erlinger, Dhumeaux, Berthelot & Dumont, 1970). It would seem, therefore, that deoxycholate, which is the major bile salt present in rabbit bile, has an important and individual role to play in the formation of bile in this species.
595 BILE SALTS AND BILE SECRETION It is of interest that the biliary system of the rabbit has some other unusual features. The hormone, secretin, which has been shown to increase bile flow and bicarbonate excretion in the dog (Wheeler & Ramos, 1960), and in the guinea-pig (Forker, 1967), and to increase bile flow to a small extent in the rat (Forker, Hicklin & Sornson, 1967) is totally without effect on the biliary system of the rabbit (Scratcherd, 1965). As deoxycholate increases the output of bicarbonate in rabbit bile, it is possible that this secondary bile salt has in fact adopted the role of secretin in this particular species. As bile salts are detergents it is not unexpected that at high rates of infusion they should have toxic effects. Sodium taurodeoxycholate is clearly more potent in this than is sodium taurocholate, and this correlates with the observed susceptibility of red cells to lysis by these two bile salts. It is of interest that sodium taurodeoxycholate should cause cholestasis at about the same rate of bile salt infusion in both the rat and the dog, suggesting that the concentration of bile salt in the liver required to produce irreversible damage is about the same in both species. The rat, however, appeared to be peculiarly sensitive to sodium taurodeoxycholate. A large fraction of bile flow in the rat has been shown to be independent of bile salt secretion (Boyer & Klatskin, 1970; Klaassen, 1971) in that bile flow is maintained even when the rate of bile salt secretion into bile has decreased to very low levels. It has been suggested that this 'bile salt independent' fraction may be dependent on the activity of a sodium 'pump' sited at the cannaliculus as its formation may be depressed by inhibitors of the Na/K ATPase such as ouabain (Boyer, 1971). It has also been found in the rat, that the secretion of anions, such as taurocholate and bromsulphthalein, into bile does not always increase the rate of bile flow (Klaassen, 1972; Priestley & Plaa, 1970). The present results have shown that the secretion of sodium taurodeoxycholate into bile in the rat does not consistently increase bile flow, and indeed in some circumstances bile flow may decrease even though the rate of bile salt secretion into bile has increased. All these results suggest that the anions secreted may have reduced bile flow through affecting the formation of that fraction of bile which is independent of bile salt secretion. The Na/K ATPase which is extractable from rat liver, and which may be inhibited by ouabain, is dependent on the integrity of the cell membranes for full activity. Deoxycholate, which is highly effective in disrupting the plasma membranes of liver cells of the rat in vitro, has been shown to inhibit this enzyme. The concentration of bile salt required to disrupt plasma membranes in vitro is only about 20 m-equiv/l., a concentration which was readily achieved in the biliary system in the present experiments. The 'tight junctions' of the liver cell, which give the 25-2
596 S. C. B. RUTISHA USER AND THE LATE S. L. STONE canalicular network its inherent strength, in fact show some resistance to this concentration of lysin (Benedetti & Emmelot, 1968). The taurine conjugate of deoxycholate would not be expected to have precisely the same effects on the membranes of the biliary system as the unconjugated bile salt. Nevertheless, in view of the known properties of deoxycholate it is conceivable that sodium taurodeoxycholate may affect membrane structure and permeability in such a way as to interfere with the formation of that fraction of bile which is dependent on the activity of a sodium pump. It may be noted that sodium taurodeoxycholate and sodium taurocholate have broadly similar effects on the composition of rat bile and on biliary secretary pressures, suggesting that both bile salts act on the biliary system in essentially the same way but that at any given rate of bile salt secretion, sodium taurodeoxycholate is more potent than sodium taurocholate. In view of the different physiological effects of sodium taurodeoxycholate in rats, rabbits, and dogs, the occurrence and metabolism of deoxycholate in each of these species is of interest. The rapid hydroxylation of deoxycholic acid to give cholic acid in the rat liver was first described by Bergstr6m, Rottenberg & Sjovall (1953) and has been well documented (Bergstrom, Danielson & Samuelsson, 1960). In the dog, because of the presence of detectable amounts of deoxycholate in the bile, it has been inferred that the 7ac-hydroxylase system must be absent (Haslewood, 1964). The present results have confirmed that no appreciable conversion of deoxycholate to cholate occurs in the dog liver. Haslewood (1964) commented on the fact that the rat did not allow deoxycholate to accumulate, but rapidly converted it into cholate, whereas the rabbit seemed to specifically require the presence of deoxycholate. He suggested that the nature of the bile salt secreted into bile in a particular species might point to a special physiological advantage of that bile salt in that species. The results of this study have presented evidence in support of his statement. Clearly, deoxycholate possesses a distinct physiological advantage in the rabbit because of its special properties as a choleretic in that species, whereas the rat appears to take great pains to exclude the same bile salt from its biliary system. Here the hydroxylation of deoxycholate and its conversion to cholate has a protective function in view of the deleterious effects of taurodeoxycholate on the biliary system of the rat. In the dog, on the other hand, in which deoxycholate normally forms a small percentage of the total bile salts in bile, the biliary system is neither specially responsive to this bile salt, nor is there a mechanism designed to prevent its access into bile.
BILE SALTS AND BILE SECRETION
597
Anion-cation discrepancy in bile The existence of a discrepancy between the sum of the anions, and the sum of the cations in bile suggests the presence of another anion in bile undetected by the methods of analysis used. Klaassen (1971) has noted a similar discrepancy in rat bile, and this was confirmed in the present work. Soloway, Clark, Powell, Senior & Brooks (1972) have reported that measurement of the bile salt concentration of bile either by the enzyme steroid dehydrogenase, or by the difference (sodium + potassium) minus (chloride + bicarbonate), may not always be reliable. Clearly further studies are needed to evaluate the origin and significance of these
discrepancies. Thanks are due to Mr T. J. Surman, and Mr B. M. Shears, for their invaluable technical assistance. S.C.B.R. would like to thank the Medical Research Couincil for the award of a scholarship, and Dr J. S. Thomas for his help in the preparation of the manuscript. REFERENCES BENEDETTI, E. L. & EMMELOT, P. (1968). Structure and function of plasma membranes isolated from liver. In Ultrastructure in Biological Systems, vol. 4, The Membranes, ed. DALTON, A. J. & HAGENAU, F., pp. 33-120. New York and London: Academic Press. BERGSTROM, S., DANIELSSON, H. & SAMUELSSON, B. (1960). Formation and metabolism of bile acids. In Lipide Metabolism, ed. BLOCH, K., pp. 291--336. New York and London: Wiley. BERGSTROM, S., ROTTENBERG, M. & SJOVALL, J. (1953). rber den Stoffwechsel der Cholsaiire und Deoxycholsaiire in der Ratte. Hoppe-Seyler's Z. physiol. Chem. 295, 278-285. BOYER, J. L. (1971). Canalicular bile formation in the isolated perfused rat liver. Am. J. Physiol. 221, 1156-1163. BOYER, J. L. & KLATSKIN, G. (1970). Canalicular bile flow and bile secretary pressure. Gastroenterology 59, 853-859. CURTIS, C. G., POWELL, G. M. & STONE, S. L. (1971). Perfusion of the isolated rat liver. J. Physiol. 213, 14-15P. 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. FORKER, E. L. (1967). Two sites of bile formation as determined by mannitol and erythritol clearance in the guinea-pig. J. clin. Invest. 46, 1189-1195. FORKER, E. L., HicKIuN, T. & SORNSON, H. (1967). The clearance of mannitol and erythritol in rabbit bile. Proc. Soc. exp. Biol. Med. 126, 115-119. HASLEWOOD, G. A. D. (1964). The biological significance of chemical differences in bile salts. Biol. Rev. 39, 537-574. IWATA, T. & YAMASAKI, K. (1964). Enzymatic determination and thin layer chromatography of bile acids in blood. J. Biochem., Tokyo 56, 424-431. JAVITT, N. B. & EMERMAN, S. (1968). Effect of sodium taurolitlhocholate on bile flow and bile acid excretion. J. clin. Invest. 47, 1002-1014.
598 S. C. B. RUTISHAUSER AND THE LATE S. L. STONE KLAAssEN, C. D. (1971). Does bile acid secretion determine canalicular bile prpduction in rats? Am. J. Physiol. 220, 667-673. KLAASSEN, C. D. (1972). Species differences in the choleretic response to bile salts. J. Physiol. 224, 259-269. O'MMTL, E. R. L., RICEARDS, T. G. & SHORT, A. H. (1965). Acute taurine depletion and maximal rates of hepatic conjugation and secretion of cholic acid in the dog. J. Phyiol. 180, 67-79. PRIESTLY, B. G. & PTAA, G. L. (1970). Reduced bile flow after sulfobromophthalein administration in the rat. Proc. Soc. exp. Biol. Med. 138, 373-376. RuTISHAUSER, S. C. B. & STONE, S. L. (1974). Aspects of bile secretion in the rabbit. J. Physiol. 238, 46-47P. RuTSHAUSER, S. C. B. & STONE, S. L. (1975). Aspects of bile secretion in the rabbit. J. Physiol. 245, 567-582. SCRATCHERD, T. (1965). Electrolyte composition and control of biliary secretion in the cat and rabbit. In The Biliary System, ed. TAYLOR, W., pp. 515-528. Oxford: Blackwell. SIEGFRIED, C. M. & ELLIOTT, W. H. (1968). Separation of bile acids of rat bile by thin layer chromatography. J. Lipid Res. 9, 394-395. SOLOWAY, R. D., CLARK, M. L., POWELL, K. M., SENIOR, J. R. & BROOKS, F. P. (1972). Effects of secretin and bile salt infusion on canine bile composition and flow. Am. J. Physiol. 222, 681-686. SPERBER, I. (1959). Secretion of organic anions in the formation of urine and bile. Pharmac. Rev. 11, 109-134. WHEELER, H. 0. & Rsmos, 0. L. (1960). Determinants of the flow and composition of bile in the unanaesthetized dog during constant infusions of sodium taurocholate. J. cdin. Invest. 39, 161-170.
583
COMPARATIVE EFFECTS OF SODIUM TAURODEOXYCHOLATE AND SODIUM TAUROCHOLATE ON BILE SECRETION IN THE RAT, DOG AND RABBIT
BY SIGRID C. B. RUTISHAUSER* AND THE LATE S. L. STONE From the Department of Physiology, University College, Cardiff CF1 1XL
(Received 28 May 1974) SUMMARY
1. The biliary effects of sodium taurodeoxycholate and sodium taurocholate were investigated in anaesthetized dogs and rats, and in the isolated perfused rat liver. 2. Both bile salts had similar qualitative and quantitative effects on the flow and composition of bile in the dog. 3. In the rat, the bile salts had similar effects on the ionic composition of bile, but differed in that bile flow was not always directly related to the rate of bile salt secretion in bile, in the experiments with sodium taurodeoxycholate. 4. Sodium taurodeoxycholate is hydroxylated to form sodium taurocholate by the liver of the rat but not by that of the dog. 5. The biliary effects of sodium taurodeoxycholate in the rat and in the dog are contrasted with its effects in the rabbit, and the differences between the three species are discussed. INTRODUCTION
The secretion of bile salt into the biliary canaliculus is believed to be an important driving force for the formation of bile. It has been suggested that the secretion of bile salt creates an osmotic force which results in the subsequent flow of water and dissolved electrolytes into the canalicular lumen (Sperber, 1959; Wheeler & Ramos, 1960). Bile salts differ in their efficiency in increasing the flow of bile. Thus, dehydrocholate, and cholate, have been shown to be more efficient as choleretics than sodium taurocholate (Sperber, 1959; O'Maille, Richards & Short, 1965). The choleretic efficiency of a bile salt is believed to depend on the number of osmotically * Address for correspondence: Dr S. C. B. Rutishauser, Department of Physiology, Stopford Building, University of Manchester, Manchester, M13 9PT.
584 S. C. B. RUTISHA USER AND THE LATE S. L. STONE effective particles in the canalicular lumen, and this, in turn, depends on the extent to which the bile salt molecules are aggregated into micelles. However, it has been shown recently, that sodium taurodeoxycholate is a more efficient choleretic than sodium taurocholate in the rabbit, and that this cannot be explained readily in terms of the known micellar properties of the two bile salts (Rutishauser & Stone, 1974, 1975). Sodium taurodeoxycholate, unlike sodium taurocholate, increased the bicarbonate concentration of rabbit bile, and it was suggested that sodium taurodeoxycholate may have stimulated a bicarbonate pump in the biliary system, and that this was the cause of its greater choleretic efficiency compared with sodium taurocholate. It was therefore of interest to examine the choleretic efficiency of these two bile salts in other species in order to determine whether sodium taurodeoxycholate had special choleretic properties in itself, or whether its effects were just peculiar to the rabbit. METHODS Dogs. Mongrel dogs of both sexes were anaesthetized with sodium pentobarbitone (30 mg/kg I.v.). The trachea was cannulated and the spleen was removed. The common bile duct was cannulated after ligation of the cystic duct and drainage of the gall-bladder. The ureters were cannulated. Bile and urine samples were collected serially as 10 or 20 min samples in 10 ml. tubes graduated in divisions of 0.1 ml. Infusions were given via a cannula lying in one of the femoral veins. Rats. Male Wistar rats (body weight 350-450 g) were anaesthetized with ether. The right jugular vein was cannulated, and anaesthesia was maintained by the i.v. injection of 0 02-010 ml. aliquots of sodium pentobarbitone (60 mg/ml. diluted 1:1 with 0-9 % NaCl). The bile duct was cannulated, the tip of the cannula lying just distal to the point at which the left and right hepatic ducts fuse. Bile was collected serially as 20 min samples in flat-based glass tubes of constant bore (3, 4, or 5 mm in diameter). The length of the column of bile was measured (± 0-5 mm) and the bile volume was calculated. All infusions were given via the right or left jugular vein. In some experiments, blood pressure was recorded from the left carotid artery. All animals were fasted overnight before the experiment, but were allowed free access to water. Body temperature was maintained during anaesthesia by surface warming. At the end of each experiment the liver was removed and weighed. Isolated perfused rat liver. The perfusion apparatus used was similar to that described by Curtis, Powell & Stone (1971). The livers from male rats (body weight 300 g) were removed under ether anaesthesia, and were perfumed via the portal vein. The perfusion medium consisted of 70 ml. of pooled rat blood diluted to -120 ml. with Krebs-Ringer bicarbonate solution, to which was added 2 g bovine albumin, 200 mg glucose, 20 mg streptomycin or penicillin, and a small amount of silicone anti-foam A. The Krebs-Ringer bicarbonate solution was gassed with 5 % C02-95 % 02 before use. In this perfusion system, after 1 hr of perfusion, perfusate flow was 5-6 ml./min. g liver, perfusion pressure was 18-20 cm H O. and bile flow was in the range 1.0-I6 ,sl./min. g liver. Bile salts. Sodium taurodeoxycholate (TDC) and sodium taurocholate (TC) were -
BILE SALTS AND BILE SECRETION
585
obtained from Maybridge Chemicals, Tintagel, Cornwall. For infusion, they were either dissolved in 0-9% NeCl, or in a mixture of 0 9% NaCl, 0X1 M-Na phosphate buffer pH 7-4, and bovine albumin (1-5%'). The addition of albumin reduced the haemolytic and cardiovascular effects of the bile salts. The concentration of bile salt in bile was determined using two methods. (a) By the use of the enzyme steroid dehydrogenase in a modification ofthe method detailed by Javitt & Emerman (1968). The mixture had to be incubated at 370 C for 1 hr before determination of the extinction of the solution at 340 nm. This was necessary because of the much slower reaction of TDC in this system as compared with TC. The enzyme steroid dehydrogenase determines all the bile salt present in bile bearing a 3-hydroxyl group, and this includes all the bile salts of known importance in the bile of rats and of dogs. Thus this enzymic method was taken to measure 'total bile salt' concentration in bile. (b) 'Cholate' in bile was determined by the method detailed by O'Maille et al. (1965). This method measures the amount of free and conjugated cholate in bile; deoxycholate is not reactive in this system. The difference between 'cholate' and 'total' bile salt was taken to be a measure of the concentration of dihydroxy bile salts in bile, and thus of deoxycholate in experiments where this bile salt predominated. Some bile was analysed by thin-layer chromatography. 1 ml. bile containing 25-50 mg of bile salt was hydrolysed in 12% KOH at 1200 C for 3 hr, and then extracted according to the method described by Iwata & Yamasaki (1964). The method of Siegfried & Elliott (1968) was used for thin-layer chromatography of the samples. 10 ,d. of the ethanolic extract containing 250-500 jug bile acid was applied to the plates, which were developed in an ascending system. In some experiments the individual bile acids were eluted from the plates with methanol, and the amounts of bile acid present were separately determined using the enzyme steroid
dehydrogenase. Ionic analysis. Sodium and potassium concentrations in bile were measured by flame photometry; chloride was determined by an electrometric titration method (Buchler-Cotlove chloridometer); bicarbonate concentration was measured using the micro-manometric method of Van Slyke (Natelson microgasometer); bile osmolality was determined cryoscopically (Advanced osmometer). Statistical analyses. Mean values are given together with their standard errors, unless otherwise specified in the text. The Student t test was used in the comparison of data. RESULTS
1. Dog Effects on bile flow Sodium taurocholate (TC), and sodium taurodeoxycholate (TDC) were dissolved in 0-9 % NaCl (2-0 limole/ml.) and were infused i.v. into seven dogs at rates ranging from 5 to 120 n-mole/min . g liver. Up to an infusion rate of 90 n-mole/min. g liver both bile salts increased bile flow, but at rates greater than this TDC caused cholestasis. In Fig. 1 the rate of bile salt secretion into bile is plotted against bile flow for each period of steadystate infusion. It can be seen that the two bile salts had similar quantitative effects on the rate of flow of bile. The choleretic efficiency of the bile salts was calculated from the slope of the regression lines determined from the
586 S. C. B. RUTISHA USER AND THE LATE S. L. STONE data shown in Fig. 1. The choleretic efficiency of TC was 8-5 ml./m-mole, and that for TDC was 9-6 ml./m-mole.
Bile salt metabolism In the infusion experiments, the increase in the rate of secretion of bile salt into bile was in each case approximately equal to the rate at which bile salt was infused (Fig. 2). The increase in the rate of secretion of bile salt into bile in the experiments with TDC was calculated as the increase in the secretion of 'non-cholate' bile salt into bile. Thus TDC is not appreciably converted into taurocholate in the liver of the dog. 1.20 1*00 _* ,L- O080_
to
_
W
z06 =
040_
020
4
20 80 40 100 60 120 Rate of bile salt secretion (n-mole/min.g liver)
Fig. 1. The relationship between the rate of bile flow and the rate of secretion of 'total' bile salt in bile, during the constant infusion of sodium taurodeoxycholate (A) or sodium taurocholate (e) in the dog. The regression equations are: y = 00096x+0 132 for TDC; and y = 00085x+0 151 for TC; where y = bile flow, and x = rate of bile salt secretion.
Similar results were obtained in experiments where TDC was given as a single injection (50 mg/kg body wt.). In this case, the mean recovery of the bile salt in bile was 88-2 + 2-6 0/ when calculated as 'non-cholate' bile salt, and 900 + 3-7 % when calculated as 'total' bile salt (mean + S.E. of mean in three experiments). Samples of bile from the infusion experiments were hydrolysed and
analysed by thin-layer chromatography. Only two bile acids could be shown to be present, and these had the mobilities of cholic and deoxycholic acids. During the infusion of TDC, 75-90 % of the bile salt present in bile could be shown by thin-layer chromatography to be deoxycholate.
BILE SALTS AND BILE SECRETION 587 It may be noted that appreciable amounts of 'non-cholate' bile salt (15.1+10-7%; mean+ S.D. of an observation), were found normally in samples of bile collected immediately after cannulation of the bile duct.
Effects on the ionic composition of bile Table 1 gives the mean net changes in the ionic composition of bile caused by the infusion of either TC or TDC at the rate of 40 n-mole/min . g liver. Both bile salts had essentially similar effects, in that the concentrations of sodium and of chloride in bile increased, and that of bicarbonate decreased. 140
120
.°0Z 100 -bo
0
80 60 _
0
_
*DA 00 U
20
An,&
^~~
60 40 80 100 20 120 Rate of bile salt infusion (n-molelmin.g liver)
140
Fig. 2. The relationship between the rate of infusion of bile salt, and the increase in the rate of secretion of bile salt into bile, during the constant infusion of sodium taurodeoxycholate (A) or sodium taurocholate (e) in the dog. The increase in the rate of bile salt secretion into bile was calculated as the increase in the 'cholate' fraction forthe experiments with taurocholate, and as the increase in the 'non-cholate' fraction for the experiments with taurodeoxycholate.
During the infusion of bile salt there was no appreciable difference between the sum of the anions and cations in bile, i.e. (sodium + potassium) (chloride + bicarbonate + bile salt). However, during the control period before and after infusion, there was a mean anion deficit of 18 + 7 mequiv/l. This accounts for the apparent discrepancy between the total anions and cations in Table 1.
588 S. C. B. RUTISHA USER AND THE LATE S. L. STONE TABiE 1. Net changes in the ionic composition of bile caused by the infusion of bile salt (TDC or TC -40 n-mole/min. g liver) in the dog. The figures give the mean difference in concentration + S.D. between the infusion period, and the control periods before and after infusion, in three experiments with each bile salt Taurodeoxycholate + 9-9 ± 2-9
Na
+0-55±0-26 +11-5 ± 8-7
K
Cl
Taurocholate +3-4+ 2-4 +0-78+ 102 + 8-2 + 8-4 -11-5±6-7 + 30-6 ± 7-3
,(m-equivfl.)
HCO3
-10-8 ± 8-4
Total bile salt)
+30-6±6-2
+1-6 0
+12 _bo
+08
0
F 0
c
x? +0-4
_
0
1-
A
A
0
*
*
. ._
-04 -
*
*
.,
.
*
*
.
*s
*
A.
120 60 Rate of bile salt infusion (n-mole/min.g liver)
180
C U
ok, -0-8
_-
z
-1*2
-16
A
a
_-
Fig. 3. The net changes in bile flow caused by the infusion of sodium taurodeoxycholate (A) and sodium taurocholate (e) in the anaesthetized rat.
2. Rat
Effects on bile flow Bile salts were dissolved in 0-9 % NaCl (25-50 ,umole/ml.) and were infused at rates ranging from 20 to 200 n-mole/min.g liver into anaesthetized rats. At low rates of infusion, both bile salts increased bile flow, but, as can be seen in Fig. 3, the relationship between bile flow and infusion rate was not a clearly linear one for TDC. At rates of infusion greater than 90 n-mole/min . g liver TDC caused cholestasis.
589 BILE SALTS AND BILE SECRETION It was found that a brief infusion of TDC (1 ml. of a 50 It-mole/ml. solution infused over 15 min) caused a reversible decrease in bile flow. This is illustrated in Fig. 4 where the effects of TDC are contrasted with the choleretic effect of an equimolar infusion of TC. It can be seen that the decrease in bile flow with TDC occurred in spite of a simultaneous increase in the rate of bile salt secretion into bile. TDC 30
TC
-
0
0
>=-
20o
E
150 _
150
-
0
0 c
E
2505o so
X
2 3 4 Time (hr) Fig. 4. The effects of a brief infusion of bile salt (50 molee over 15 min) on bile flow and bile salt secretion into bile in the anaesthetized rat. The effects of sodium taurodeoxycholate and sodium taurocholate are compared. Concentration of bile salt in bile: ' cholate ' (-; 'non-cholate ' (EA).
Effects on the ionic composition of bile 50 /czmole of either TC or TDC, dissolved in a phosphate buffer-albumin
solution, was infused intravenously over 15 min. Because of the very different effects of TC and TDC on the rate of flow of bile, bile was collected as timed samples of constant volume (0-38 ml.). If bile had been collected
590 S. C. B. RUTISHAUSER AND THE LATE S. L. STONE as 20 min samples, the peak ionic response to TC would have occurred in sample 1, whereas that for TDC would have occurred in samples 2 or 3. As the first sample of bile collected immediately after the infusion contains some 'dead-space' bile, it was thought that this could lead to underestimation of the effects of TC on the composition of bile. Accordingly, it was thought that collection of bile as timed samples of constant volume would enable a more accurate comparison of the ionic effects of the two bile salts to be made. The leaned data for 4 experiments with TDC and five experiments with TC are shown in Fig. 5. Both bile salts increased the concentrations of sodium and potassium in bile and decreased the concentrations of chloride and of bicarbonate. Although the peak concentrations of sodium and of bile salt in bile were almost the same for both bile salts, the concentration of sodium in bile thereafter was significantly greater in the experiments with TDC compared with those with TC (P < 0 0025). There was no change in the osmolality of bile after the infusion of TC, and the slight increase which occurred temporarily after the infusion of TDC amounted to no more than 3-4 m-osmole/kg. Biliary secretary pressures In these experiments bile was collected as 10 min samples for a period of 30 min. Biliary secretary pressure was then measured by affixing the filled bile cannula (internal bore = 0-28 mm) to a vertical 30 cm ruler and noting the maximum level to which the bile rose. 10 min after raising the cannula, with biliary stasis still imposed, an infusion of either TC or TDC, or the phosphate buffer-albumin solution, was begun at the same rate as in the previous experiments on the ionic composition of bile. The infusion was continued for 6 min (total dose of bile salt, 20 molee, the level of bile in the cannula being noted each minute during the infusion, and for 4 min thereafter. The total time from the interruption of normal flow to the end of the imposed biliary stasis was 20 min. The volume of bile collected in the 10 min immediately after free flow was resumed was measured in each case. After each infusion a further 50 min of free flow was allowed in order to give sufficient time for the infused bile salt to be eliminated, and for control conditions to be restored. The results of a typical experiment are shown in Fig. 6. In four experiments the mean control value for biliary secretary pressure before the infusion of bile salt was 218 + 09 cm H20 (S.E. of mean). A gradual decline from this value (1-3 cm in 10 min) was seen during the infusion of the phosphate buffer-albumin solution. Both TC and TDC initially increased the secretary pressure ( + 1 2 + 0 3 cm, and + 1I9 + 0 5 cm respectively), but a decrease in pressure then ensued. The rate of decrease
591 BILE SALTS AND BILE SECRETION was twice as great for TDC (IP04 + 0-06 cm/min) as for TC (0.57 + 0412 cm/ min). In every experiment, the final secretary pressure after the infusion of TDC was at least 3-5 cm less than the values recorded after the infusion Bile salt
I 2*0 0bo @4a-
1*0 lII I
I Il
I I
200
-
0s
150
tr
E 100 C
0
0 U U
.C 0
50s
I
1
I I1 I I I I I 2 3 4 5 6 7 8 Sample number
I
9
Fig. 5. The effects of the infusion of sodium taurodeoxycholate (- - - -) and sodium taurocholate ( ) (50 jimole over 15 min) on the flow and ionic composition of bile in the anaesthetized rat. The data represent the mean of four experiments with TDC and five experiments with TC. The S.E.'S of the means are indicated where these are meaningful. Bile was collected as timed samples of constant volume (038 ml.). Sodium (V), chloride (0), bicarbonate (E.), 'total' bile salt (x); open symbols, sodium taurodeoxycholate; filled symbols, sodium taurocholate.
592 S. C. B. RUTISHA USER AND THE LATE S. L. STONE of either TC or the buffer-albumin mixture at the corresponding time in the same rat. The maximum secretary pressure recorded 1 hr after the infusion of bile salt was not significantly different from the control value either after TC (22.2 + 0 3 cm) or after TDC (22.2 + 06 cm). This shows that no permanent change had been caused by the infusion of bile salt. Essentially similar quantitative results were obtained in the response to TC and TDC when a bile cannula of triple the internal diameter was used. Infusion
0
°I20 _
.
Ad
%I-J
5
10 Time (min)
15
20
Fig. 6. The effect of the infusion of either sodium taurodeoxycholate (A - - A), sodium taurocholate (e*-), or the infusion medium ( x ... x ), on biliary secretary pressures in the anaesthetized rat. At zero minutes the bile cannula was raised, and the height of the column of bile above the liver was recorded every minute for the next 20 min. The solutions were infused at the time indicated at the rate of 0.056 ml./min: total dose of bile salt = 20 molel.
The volume of bile collected in the 10 min period post-stasis also showed a consistent pattern. After the infusion of the buffer-albumin solution, 0 0074 + 0'0015 ml./g liver was obtained in excess of the expected flow rate. This represents the extra bile stored in the biliary system as a result of the distension caused by stopping the flow of bile. The extra volume obtained after TC was either the same as this or slightly less (0.0065 + 0.0011 ml./g liver). In contrast, after the infusion of TDC there was a volume deficit of 0-0028 + 0-0008 ml./g liver compared with the original control bile flow. Mixtures of TC and TDC in the isolated perfused rat liver The choleretic response to the infusion of bile salt was also examined in the isolated perfused rat liver. The same total dose of bile salt was given
593 BILE SALTS AND BILE SECRETION in each experiment (50 molele, but the proportions of TC and TDC in the infused solution were varied. 2*5 ml. of the prepared solutions (20 ,umole/ml. in phosphate buffer-albumin solution) was infused at the rate of 2-2 ml./ min, 1 hr after the start of the perfusion. Fig. 7 shows the net change in bile flow caused by the infusion of 50 #mole of bile salt in four experiments. It can be seen that the same total dose of bile salt produced very different volumes of bile, and that as the proportion of TDC in the mixture was increased, the volume of bile formed was reduced. 50 ,-mole bile salt
1+I5
4r
.E 01+10_ 50
0
.SQ -1*0_ -0-5 C
z
01. -15_
I
1
I
2 Time (hr)
I
3
4
Fig. 7. The net changes in bile flow caused by the infusion of four separate 50 jimole doses of bile salt, consisting of different proportions of sodium taurodeoxycholate (TDC) and sodium taurocholate (TC), in 4 individual experiments in the isolated perfused rat liver. The proportions of TC: TDC in themixturesinfusedwere: 100:0(0-*); 66:33 (0_-D); 60:40( x x 50:50 (v- -if).
It was possible in these experiments to accurately determine the recoveries of the infused bile salt in bile. In the isolated perfused rat liver, the rate of secretion of endogenous bile salt, as measured by the enzyme steroid dehydrogenase, is very low after 1 hr of perfusion (1-2 n-mole/ min.g liver). In calculating the recoveries of bile salt, the endogenous secretion rate was ignored, and this was estimated to introduce an error of only 1-2 % at most. In the four experiments illustrated in Fig. 7, the total recovery of bile 25
PHY 245
594 S. C. B. RUTISHAUSER AND THE LATE S. L. STONE salt in bile was virtually 100 % in each case, in spite of the different volumes of bile formed. The bile salt recoveries are listed in Table 2. It may be noted, that TDC was appreciably hydroxylated in its passage through the liver, in that the recovery of 'cholate' in bile was consistently greater than the amount infused, in those experiments in which TDC was included in the infusion mixture. TABiE 2. Amounts of bile salt recovered in bile after the infusion of 50 gmole of bile salt, consisting of different proportions of sodium taurodeoxycholate and sodium taurocholate in four experiments in the perfused rat liver
Amount infused
Amount recovered in bile
(Cmole)
(smole)
'Total' 50 50 50 50
'Cholate' 50 33 30 25
'Total'
'Cholate'
51-2 48-8 53-7 50 4
50.6 39*2 40*6 34 9
Increase in 'cholate' + 0.6 +6-2 +10.6 +9 9
Cardiovascular effects of the bile salts Both bile salts decreased blood pressure and heart rate when infused into anaesthetized rats, but TDC was more potent than TC. Clearly, a reduction in blood flow to the liver may affect the flow of bile, but it was observed that the cardiovascular effects of the bile salts did not always correlate with the biliary effects. In the isolated perfused rat liver both bile salts caused vasoconstriction. Again TDC was more effective than TC. In the perfusion system used, it was shown that bile flow was not affected by a reduction of blood flow until this fell below 2 5 ml./min.g liver, and this did not occur in the perfusion experiments described. DISCUSSION
The results have shown that sodium taurodeoxycholate is not a more efficient choleretic than sodium taurocholate in the rat and in the dog. This contrasts with the choleretic effects of this bile salt in the rabbit (Rutishauser & Stone, 1974, 1975), and suggests that sodium taurodeoxycholate is not inherently a good choleretic, but rather that it possesses special properties in the rabbit. The glycine conjugate of deoxycholate has also been shown to be an exceptionally good choleretic in this species, in that its choleretic efficiency is 70 ml./m-mole (Erlinger, Dhumeaux, Berthelot & Dumont, 1970). It would seem, therefore, that deoxycholate, which is the major bile salt present in rabbit bile, has an important and individual role to play in the formation of bile in this species.
595 BILE SALTS AND BILE SECRETION It is of interest that the biliary system of the rabbit has some other unusual features. The hormone, secretin, which has been shown to increase bile flow and bicarbonate excretion in the dog (Wheeler & Ramos, 1960), and in the guinea-pig (Forker, 1967), and to increase bile flow to a small extent in the rat (Forker, Hicklin & Sornson, 1967) is totally without effect on the biliary system of the rabbit (Scratcherd, 1965). As deoxycholate increases the output of bicarbonate in rabbit bile, it is possible that this secondary bile salt has in fact adopted the role of secretin in this particular species. As bile salts are detergents it is not unexpected that at high rates of infusion they should have toxic effects. Sodium taurodeoxycholate is clearly more potent in this than is sodium taurocholate, and this correlates with the observed susceptibility of red cells to lysis by these two bile salts. It is of interest that sodium taurodeoxycholate should cause cholestasis at about the same rate of bile salt infusion in both the rat and the dog, suggesting that the concentration of bile salt in the liver required to produce irreversible damage is about the same in both species. The rat, however, appeared to be peculiarly sensitive to sodium taurodeoxycholate. A large fraction of bile flow in the rat has been shown to be independent of bile salt secretion (Boyer & Klatskin, 1970; Klaassen, 1971) in that bile flow is maintained even when the rate of bile salt secretion into bile has decreased to very low levels. It has been suggested that this 'bile salt independent' fraction may be dependent on the activity of a sodium 'pump' sited at the cannaliculus as its formation may be depressed by inhibitors of the Na/K ATPase such as ouabain (Boyer, 1971). It has also been found in the rat, that the secretion of anions, such as taurocholate and bromsulphthalein, into bile does not always increase the rate of bile flow (Klaassen, 1972; Priestley & Plaa, 1970). The present results have shown that the secretion of sodium taurodeoxycholate into bile in the rat does not consistently increase bile flow, and indeed in some circumstances bile flow may decrease even though the rate of bile salt secretion into bile has increased. All these results suggest that the anions secreted may have reduced bile flow through affecting the formation of that fraction of bile which is independent of bile salt secretion. The Na/K ATPase which is extractable from rat liver, and which may be inhibited by ouabain, is dependent on the integrity of the cell membranes for full activity. Deoxycholate, which is highly effective in disrupting the plasma membranes of liver cells of the rat in vitro, has been shown to inhibit this enzyme. The concentration of bile salt required to disrupt plasma membranes in vitro is only about 20 m-equiv/l., a concentration which was readily achieved in the biliary system in the present experiments. The 'tight junctions' of the liver cell, which give the 25-2
596 S. C. B. RUTISHA USER AND THE LATE S. L. STONE canalicular network its inherent strength, in fact show some resistance to this concentration of lysin (Benedetti & Emmelot, 1968). The taurine conjugate of deoxycholate would not be expected to have precisely the same effects on the membranes of the biliary system as the unconjugated bile salt. Nevertheless, in view of the known properties of deoxycholate it is conceivable that sodium taurodeoxycholate may affect membrane structure and permeability in such a way as to interfere with the formation of that fraction of bile which is dependent on the activity of a sodium pump. It may be noted that sodium taurodeoxycholate and sodium taurocholate have broadly similar effects on the composition of rat bile and on biliary secretary pressures, suggesting that both bile salts act on the biliary system in essentially the same way but that at any given rate of bile salt secretion, sodium taurodeoxycholate is more potent than sodium taurocholate. In view of the different physiological effects of sodium taurodeoxycholate in rats, rabbits, and dogs, the occurrence and metabolism of deoxycholate in each of these species is of interest. The rapid hydroxylation of deoxycholic acid to give cholic acid in the rat liver was first described by Bergstr6m, Rottenberg & Sjovall (1953) and has been well documented (Bergstrom, Danielson & Samuelsson, 1960). In the dog, because of the presence of detectable amounts of deoxycholate in the bile, it has been inferred that the 7ac-hydroxylase system must be absent (Haslewood, 1964). The present results have confirmed that no appreciable conversion of deoxycholate to cholate occurs in the dog liver. Haslewood (1964) commented on the fact that the rat did not allow deoxycholate to accumulate, but rapidly converted it into cholate, whereas the rabbit seemed to specifically require the presence of deoxycholate. He suggested that the nature of the bile salt secreted into bile in a particular species might point to a special physiological advantage of that bile salt in that species. The results of this study have presented evidence in support of his statement. Clearly, deoxycholate possesses a distinct physiological advantage in the rabbit because of its special properties as a choleretic in that species, whereas the rat appears to take great pains to exclude the same bile salt from its biliary system. Here the hydroxylation of deoxycholate and its conversion to cholate has a protective function in view of the deleterious effects of taurodeoxycholate on the biliary system of the rat. In the dog, on the other hand, in which deoxycholate normally forms a small percentage of the total bile salts in bile, the biliary system is neither specially responsive to this bile salt, nor is there a mechanism designed to prevent its access into bile.
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597
Anion-cation discrepancy in bile The existence of a discrepancy between the sum of the anions, and the sum of the cations in bile suggests the presence of another anion in bile undetected by the methods of analysis used. Klaassen (1971) has noted a similar discrepancy in rat bile, and this was confirmed in the present work. Soloway, Clark, Powell, Senior & Brooks (1972) have reported that measurement of the bile salt concentration of bile either by the enzyme steroid dehydrogenase, or by the difference (sodium + potassium) minus (chloride + bicarbonate), may not always be reliable. Clearly further studies are needed to evaluate the origin and significance of these
discrepancies. Thanks are due to Mr T. J. Surman, and Mr B. M. Shears, for their invaluable technical assistance. S.C.B.R. would like to thank the Medical Research Couincil for the award of a scholarship, and Dr J. S. Thomas for his help in the preparation of the manuscript. REFERENCES BENEDETTI, E. L. & EMMELOT, P. (1968). Structure and function of plasma membranes isolated from liver. In Ultrastructure in Biological Systems, vol. 4, The Membranes, ed. DALTON, A. J. & HAGENAU, F., pp. 33-120. New York and London: Academic Press. BERGSTROM, S., DANIELSSON, H. & SAMUELSSON, B. (1960). Formation and metabolism of bile acids. In Lipide Metabolism, ed. BLOCH, K., pp. 291--336. New York and London: Wiley. BERGSTROM, S., ROTTENBERG, M. & SJOVALL, J. (1953). rber den Stoffwechsel der Cholsaiire und Deoxycholsaiire in der Ratte. Hoppe-Seyler's Z. physiol. Chem. 295, 278-285. BOYER, J. L. (1971). Canalicular bile formation in the isolated perfused rat liver. Am. J. Physiol. 221, 1156-1163. BOYER, J. L. & KLATSKIN, G. (1970). Canalicular bile flow and bile secretary pressure. Gastroenterology 59, 853-859. CURTIS, C. G., POWELL, G. M. & STONE, S. L. (1971). Perfusion of the isolated rat liver. J. Physiol. 213, 14-15P. 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. FORKER, E. L. (1967). Two sites of bile formation as determined by mannitol and erythritol clearance in the guinea-pig. J. clin. Invest. 46, 1189-1195. FORKER, E. L., HicKIuN, T. & SORNSON, H. (1967). The clearance of mannitol and erythritol in rabbit bile. Proc. Soc. exp. Biol. Med. 126, 115-119. HASLEWOOD, G. A. D. (1964). The biological significance of chemical differences in bile salts. Biol. Rev. 39, 537-574. IWATA, T. & YAMASAKI, K. (1964). Enzymatic determination and thin layer chromatography of bile acids in blood. J. Biochem., Tokyo 56, 424-431. JAVITT, N. B. & EMERMAN, S. (1968). Effect of sodium taurolitlhocholate on bile flow and bile acid excretion. J. clin. Invest. 47, 1002-1014.
598 S. C. B. RUTISHAUSER AND THE LATE S. L. STONE KLAAssEN, C. D. (1971). Does bile acid secretion determine canalicular bile prpduction in rats? Am. J. Physiol. 220, 667-673. KLAASSEN, C. D. (1972). Species differences in the choleretic response to bile salts. J. Physiol. 224, 259-269. O'MMTL, E. R. L., RICEARDS, T. G. & SHORT, A. H. (1965). Acute taurine depletion and maximal rates of hepatic conjugation and secretion of cholic acid in the dog. J. Phyiol. 180, 67-79. PRIESTLY, B. G. & PTAA, G. L. (1970). Reduced bile flow after sulfobromophthalein administration in the rat. Proc. Soc. exp. Biol. Med. 138, 373-376. RuTISHAUSER, S. C. B. & STONE, S. L. (1974). Aspects of bile secretion in the rabbit. J. Physiol. 238, 46-47P. RuTSHAUSER, S. C. B. & STONE, S. L. (1975). Aspects of bile secretion in the rabbit. J. Physiol. 245, 567-582. SCRATCHERD, T. (1965). Electrolyte composition and control of biliary secretion in the cat and rabbit. In The Biliary System, ed. TAYLOR, W., pp. 515-528. Oxford: Blackwell. SIEGFRIED, C. M. & ELLIOTT, W. H. (1968). Separation of bile acids of rat bile by thin layer chromatography. J. Lipid Res. 9, 394-395. SOLOWAY, R. D., CLARK, M. L., POWELL, K. M., SENIOR, J. R. & BROOKS, F. P. (1972). Effects of secretin and bile salt infusion on canine bile composition and flow. Am. J. Physiol. 222, 681-686. SPERBER, I. (1959). Secretion of organic anions in the formation of urine and bile. Pharmac. Rev. 11, 109-134. WHEELER, H. 0. & Rsmos, 0. L. (1960). Determinants of the flow and composition of bile in the unanaesthetized dog during constant infusions of sodium taurocholate. J. cdin. Invest. 39, 161-170.
