AMERICAN JOURNAL OF PHYSIOLOGY
Vol. 229, No. 3, September
1975.
Printed in U.S.A.
Bile acid kinetics and bile secretion in the pony’ M. S. ANWER, R. R. GRONWALL, L. R. ENGELKING, Department of Physiological Sciences, College of Veterinary Medicine, Manhattan, Kansas 66506
ANWER, M. S.,R.R KLENTZ. Bile acid kinetics
R.D. J. Physiol. 229(3) : 592-597. 1975.-Bile acid pool size and synthesis rate were determined by both isotope-dilution and washout methods in ponies with chronic external biliary fistulas. Bile acid pool size (10.9 pmol/kg) and synthesis rate (11.2 pmol/day per kg) estimated by the isotope-dilution method did not differ significantly from pool size (9.4 pmol/kg) and synthesis rate (9.5 pmol/day per kg) estimated by washout method. Bile acid-dependent and -independent fractions of bile flow, determined by a method that circumvents any inevitable correlation of flow to bile acid secretion due to common factors in both parameters, did not differ from those values obtained by linear regression of bile flow versus bile acid secretion. The choleretic effect of infused chenodeoxycholic acid was higher than that of both endogenous bile acid and infused taurocholic acid. hepatic function;
GRONWALL, L.R. ENGELKING,AND and bile secretion in the pony. Am.
enterohepatic
circulation;
bile collection
l Contribution
no.
143. Department Experiment Station,
of Physiological Manhattan, Kan.
has not been related to bile acid secretion alone. A fraction of bile flow, independent of bile acid, has been suggested for rat cv, dog (1% man (16), and rabbit (5). Sodium transport may play an important role in the formation of that fraction of bile in rabbit (5) and in isolated, perfused rat liver (2). A quantitative estimation of such a fraction of bile flow has been obtained by plotting bile flow against bile acid secretion (23), but that method has recently been questioned (10). In the present investigation, the rate of biliary secretion of bile acid was varied by infusion of exogenous bile acids and by depletion of the endogenous bile acid pool in order to a) compare the choleretic effects of different bile acids with that of endogenous bile acids and b) evaluate the method of determining the bile acid-independent fraction of bile flow. METHODS
Three ponies with chronic external biliary fistulas were studied after complete recovery from surgery (6). They were fed native hay with grain supplement and were closely observed. Ponies had free access to feed and water during all experiments and stood in a stanchion without additional restraint during those experiments that required continuous sampling of bile. No drugs were administered to the animals after they recovered from surgery.
BILE ACIDS ARE SYNTHESIZED from cholesterol in the sliver. After being conjugated with either glycine or taurine, bile acids are actively secreted into the canaliculi and pass into the duodenum. More than 90 % of the secreted bile acid is reabsorbed by the intestine and returned to the liver via the portal vein. Most of the bile acid pool is thus in the enterohepatic circulation (EHC). A small amount escapes uptake by the liver from the portal blood and reaches the systemic circulation. In addition, only a small amount of the bile acid pool (about 5 % per enterohepatic circulation in man) is lost in the feces. The liver normally synthesizes enough bile acid to make up for that loss. An isotope-dilution technique has been used to determine the pool size and hepatic synthesis rate of bile acids (7). Recently those parameters were determined from a washout curve obtained during complete interruption of the EHC (4). In this study, both techniques were used to measure the parameters of bile acid metabolism in ponies with chronic external biliary fistulas, and the results from each technique were compared. Choleretic properties of bile acids are well known. The osmotic effect of actively secreted bile acids, which results in the flow of water and solutes into the canaliculi, is believed to be a principal mechanism behind the choleretic effect of bile acids (15 : 1’9-2 1). That view is supported by evidence that the choleretic properties of compounds depend on their osmotic activity (20, 21). All bile formation
Kansas Agricultural
AND R. D. KLENTZ Kansas State University,
Pool Size and Synthesis Rate Pool size and hepatic synthesis rate of bile acid were determined using two methods. Isotope-dilution method. Each pony was given a single intravenous dose ( 13- 16 &i) of [24-14C]chenodeoxycholic acid obtained from New England Nuclear Corporation, with a specific activity of 54 Ci/mol and purity of 99%. Bile was sampled every 8 h for 96 h by gravity drainage from the indwelling catheter. To ensure samples of freshly produced bile, the first 20-30 ml of bile were collected separately; then 1 ml was collected for study. To minimize alteration of the bile acid pool, the 20-30 ml of bile were infused back into the duodenum through the catheter and followed by 20 ml of saline. One milliliter of bile represented about 2 % of the total bile acid pool. Calculation of parameters of bile acid kinetics was based on the assumption of a first-order kinetic model as described by Lindstedt (12). The relationship of the logarithm of total bile acid specific activity to time was determined by least-squares regression (Fig. 1). All regressions were signincant (P < 0.01) with correlation coefficients of 0.9940.998. Total bile acid pool size and chenodeoxycholic acid
Sciences, 592
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BILE
ACID
KINETICS
AND
CHOLERETIC
EFFECT
0 a P 0
I 20
I
I 40
60
I 00
. 100
Hours FIG.
jection (-)
cient
1. Biliary bile acid specific activity following intravenous inof chenodeoxycholic acid-24J*C in 1 pony. Regression line where specific activity = 71.4 X e-“.04sxt (correlation coeffi0.998).
(CDC) synthesis rate were calculated from the dose, regression intercept, and slope (22). Since CDC represented 75 % of total bile acid in bile (see below), synthesis rate of CDC was calculated by multiplying regression slope times 75 % of total bile acid pool. Washout method. Bile was continuously collected in IO-min samplesfor 7 h with complete interruption of the EHC. Secretion of bile acid declined rapidly after lo-20 min of drainage and reached a low point in 2-3 h (Fig. 2). That rate was maintained for the remainder of the bile collection period and was taken to represent the hepatic synthesisrate. The amount of bile acid secretedfrom the time of interruption of the EHC until the low-level excretion was reached gave an estimate of circulating bile acid pool size (4). To obtain the normal secretion rate of bile acid, bile samples were taken twice a day (before feeding and during feeding) for 3 days prior to the washout study. That sampling required only short interruptions of the EHC. Choleretic Properties of Bile Acids
Major biliary bile acids in ponies are taurochenodeoxycholic acid and taurocholic acid and, thus, to study the choleretic properties of the normal bile acids of ponies, two bile acids, taurocholic acid (TC) and chenodeoxycholic acid, were used in these experiments. Bile samples were continuously collected by gravity drainage at lo-min intervals for 5 h with complete interruption of the EHC. After 2 h of bile drainage, bile acid excretion approached its low point. Bile acid (either TC (Nutritional Biochemical Corp.) or CDC (Sigma Chemical Co.)) dissolved in saline was then infused intravenously at three different infusion rates (4.7, 11.8, and 23.6 pmol min-’ for TC and 4.0, 10.2, and 20.3 pmol min-’ for CDC) for 1 h at each rate. Those infusion rates were within the normal bile acid secretion rates of the ponies. The relationship of bile flow, during an initial 2-h period, to bile acid secretion was determined by linear regressionto find choleretic effects of endogenous bile acids. Similar regression analyses were made for bile acid infusion periods. It has been argued that if bile flow is plotted against bile acid secretion, the two variables would be misleadingly
1
2
4
3
5
6
I
HOURS FIG. 2. A typical washout curveshowingchanges in bile acidsecretion rate in a pony during complete interruption of enterohepatic circulation. Secretion rate reached a low point in 2.5 h. Hepatic synthesis rate (-) was estimated from low point. Shaded area under curve, from interruption until low point, represents total bile acid pool.
correlated becauseflow is common to both axes (10). To avoid variables already correlated, an equation was developed using bile flow and biliary bile aEid concentration that would give the sameinformation asthe direct regression analysis of flow versus bile acid excretion: l/flow
= l/BIF
- (K/BIF)
X BC
where BIF = bile acid-independent fraction of bile flow, K = the constant relating bile flow and bile acid secretion, and BC = biliary bile acid concentration (see APPENDIX for details). If BIF is not zero and both BIF and K are constants, the inverse of flow would be linearly related to BC. BIF and K then can be calcttlated from the intercept and the slope of the regression line. Inverse of flow was plotted against BC during endogenous bile acid drainage and exogenousbile acid infusion. BIF and K were calculated from equations 4 and 5, respectively, in the APPENDIX. Analytical
Methods
Total bile acid concentration in bile and infusion solutions was determined by an enzymatic method (8) using Sol-hydroxysteroid dehydrogenase (Grade III, Sigma Chemical Co.). The reaction mixture contained 0.05 ml of bile (or diluted infusion solution), 0.45 ml of water, 1.2 ml of 0.2 M phosphate buffer, pH 9.9, 1.0 ml of 1 M hydrazine hydrate, and 0.2 ml of 5 mM NAD (Grade III, Sigma Chemical Co.). The reaction was started by adding 0.1 ml of hydroxysteroid dehydrogenase solution (2.5 Sigma Chemical Co. U/ml). The net increase in absorbance at 340 nm was determined using reagent blank after 30 min of incubation at room temperature. A bile acid standard was run parallel to each set of samples.The concentration of bile acid was determined from the net increase in absorbance and the molar extinction coefficient of NADH at 340 nm. The composition of primary bile acids in the bile and bile acid infusion solutions was determined using the method described by Bruusgaard (3). Bile acids were first separated
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594
ANWER,
by thin-layer chromatography, and then an enzymatic method (see above) was used to determine the concentrations of the separated bile acids. Radioactivity in bile was determined by dissolving 0.1 ml of bile in 10.0 ml of PCS solubilizer (Amersham/Searle Corp.) and counting in a liquid scintillation counter. A portion of the injected solution of [14C]chenodeoxycholic acid (50 ~1) was counted similarly to determine the injected dose. Counting efficiency for each sample was determined from the relationship of external standard ratio to efficiency for a standardized set of samples. Radioactivity in bile was expressed as microcuries per mole of bile acid.
GRONWALL,
ENGELKING,
AND KLENTZ
when bile acid was infused, both increased gradually (Fig. 3). Bile flow increased more during CDC infusion than during TC infusion. A significant regression of bile flow versus bile acid secretion with a positive slope and intercept was indicated by correlation coefficients ranging from 0.832 to 0.998 (Fig. 4). The intercepts of the regression represent the average bile acid-independent fraction of bile flow; the slopes (~1 of flow per pmol of bile acid) estimate the choleretic effects of the bile acids. A significant linear regression of the inverse of flow versus the corresponding BC (correlation coefficient from 0.784 to 0.99) with positive intercept and negative slope was found in all cases (Fig. 5). Values of BIF and K, calculated
RESULTS
Although labeled chenodeoxycholic acid was injected for the isotope-dilution studies, specific activities were determined for total bile acid. Thus, pool size calculated by that method represented total bile acid just as in the washout method. Total bile acid pool sizes did not differ significantly (P > 0.05) when compared between the methods (Table 1). Although CDC synthesis rate calculated from isotope-dilution method was less than total bile acid synthesis rate obtained from washout method, it did not differ significantly (P > 0.05). S ince CDC was the principal primary bile acid in the pony (see below), CDC synthesis rate would acco&t for most of the total bile acid synthesis rate in ponies. An estimate of the total bile acid synthesis rate, which was calculated by multiplying total bile acid pool with regression slope of the isotope-dilution data, did not differ significantly (P > 0.05) from that obtained from washout method (Table 1). Using average values from the washout method for each animal, a paired-t test, which is equivalent to two-way analysis of variance and should compensate for animal variation, also showed no significant difference between the two methods. During the initial 2 h of washout trials, when no bile acid was infused, both bile flow and bile acid secretion declined;
40
. . 30 l
9.53
Isotope
6.99
dilution
CDC
Total
6.25
8.34
6.63 9.18 8.66
7.86t 2
9.38
9.05
CDC
15.72
. . l
.
==...
O-t
I b-
1
Drainage
1
e
I C DC
1
1 Infusion
4
l
.
8
.
. .
l
l
. .
8 .
.
8
l
. 8
. l
0
l ==...=
.
.
l
l
I
I
I
I
1
1
2
3
4
5
FIG. 3. Bile flow and bile acid secretion rate during endogenous bile acid drainage (first 2 h) and chenodeoxycholic acid infusion (infusion rates were 4.0, 10.2, and 20.3 pmol/min for 3rd, 4th, and 5th h, respectively) in 1 pony. C henodeoxycholic
1
Taurocholic
Acid
Acid
Washout
6.89 8.94 10.11 8.37
B
0 E
I! z
6.74
8.98
6.99 11.85 10.80
12.13
16.18
11.69
9.38t 13.87
mm 8 . a
. .
l
.
.
10 -
60
8.58t
9.31 9.79
3
.
.
. .
Hepatic Synthesis Rate,* pmol/day per kg
Pool Size,* pmol/kg
I
l
20 -
1. Pool size and hepatic synthesis rates of bile acid in ponies
Washout
. l 8
TABLE
Isotope dilution
-
15
9.88t
= chenodeoxycholic acid. Total = total bile acid. * There was no significant difference (P > 0.05) between values derived from the two methods. t Average values obtained from washout method for each animal were used in the paired-t test.
b
0
.l
Bile
Acid
.2
Secretion,
0
I
1
.l
.2
yMoles/min/kg
FIG. 4. Regressions of bile flow versus bile acid endogenous bile acid depletion (- - - - -) and infusion cholic acid and taurocholic acid (--) in 3 ponies.
secretion during of chenodeoxy-
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BILE
ACID
KINETICS
AND
CHOLERETIC
EFFECT
595
.08
TABLE 2. Comparison of methods for calculation acid-independent fraction of bile and choleretic effect of bile acids in ponies
1
Bile Acid Depletion
of bile
Bile Acid Infusion
Pony Intercept*
1 BIFt
/ Slope*1
it
‘c”ap”t’;
1
BIFt
Chenodeoxycholic
1 Slope*
1
Rt
acid
I 2 3 Mean
0
f
0 Bile
1
1
I
I
.002
.004
.006
.008
Acid
Concentration
FIG. 5. Inverse of bile flow versus bile acid endogenous bile acid depletion (0) and infusion acid (m) for 1 typical trial. Regression lines (--).
p Moles/p1 concentration during of chenodeoxycholic
from the parameters of that regression, were not significantly different by the Student t test (P > 0.05) from the intercepts and slopes of the regressions of flow versus secretion (Table 2). Neither the intercept nor BIF differed significantly between endogenous bile acid depletion and exogenous bile acid infusion when tested by a paired-t test. The choleretic effect (slope or K) during CDC infusion differed significantly (P < 0.05) from that determined during both bile acid depletion and taurocholic acid infusion. (Mean choleretic effects of endogenous bile acids and infused TC were not significantly different.) Chenodeoxycholic acid was the major primary bile acid in bile. Taurochenodeoxycholic acid (71 %) and taurocholic acid (15 %) represented 86 % of the total biliary bile acid. A small amount of glycochenodeoxycholic acid (3-4 %) was always present. Bile obtained during CDC infusion contained primarily the taurine conjugate of CDC with no detectable increase in the proportion of unconjugated bile acid. Infusion of bile acids did not cause hemolysis in any of the ponies. DISCUSSION
Pool Size and Synthesis Rate Isotope-dilution methods have been widely used to determine pool size and synthesis rate of bile acid. First-order kinetics and a constant pool size during sampling period were assumed (12). On the other hand, the washout method involves direct measurement of bile acid pool and estimation of the synthesis rate (4). Thus, in this study, pool size and synthesis rate of bile acid were determined by two methods with different theoretical approaches. Neither of the two parameters differed when compared between the methods. Washout method would be preferred for animal
I 2 3 Mean
11.44 11.99 11.12 11.52
11.78 12.14 11.29 11.74
27.0 31.2 48.2 35.4
19.5 30.1 43.9 31.2
14.41 12.33 13.13 13.29
Taurocholic
acid
14.52 12.41 11.97 12.96
48.7 35.2 53.8 45.9
47.8 35.3 64.6 49.2
BIF = bile acid-independent fraction; K = the constant relating bile acid-dependent fraction with bile acid secretion; intercept = bile flow at zero bile acid secretion; slope = change in bile flow per change in bile acid secretion. There was no significant difference (P > 0.05) between the estimates of bile acid-independent fraction * Calcuor choleretic effect by the two calculation methods. t Callated from regressions of bile flow on bile acid secretion. culated from slope and intercept of regression of inverse of bile flow $ Significantly different (P < 0.05) on bile acid concentration. from the corresponding values of bile acid depletion and taurocholic acid infusion.
experiments because it is simple and direct, but does require quantitative bile collection from animals with previously intact EHC. acid was used In this study, only [‘“Cl c h enodeoxycholic to determine pool size and synthesis rate of total bile acid by isotope dilution. It has been reported that in man chenodeoxycholic acid and cholic acid have different synthesis rates (22), so that both primary bile acids must be studied by isotope-dilution method to accurately determine total bile acid synthesis rate (7). Since the washout method directly determined total bile acid synthesis, the similarity of this value to that obtained from isotope-dilution method indicated that total bile acid synthesis could be estimated in ponies using only [14C]chenodeoxycholic acid disappearance. The high percentage of chenodeoxycholic acid in ponies could account for the similarity. The pool size of total bile acid in ponies (11 pmol/kg) was relatively smaller than that of humans-l 23 pmol/kg (17), rats-181 pmol/kg (l), or monkey-ZOO pmol/kg (4). The synthesis rate of 9.5 pmol/clay per kg was similar to humans -14 pmol/day per kg ( 17) but less than that of rats-28 pmol/day per kg (1) or monkeys-l 10 pmol/day per kg (4). The relatively small pool size may be a species difference or a reflection of diet. The number of cycles of the total bile acid pool per day, calculated as the ratio of the normal daily secretion rate to pool size (17), was 38 per day in ponies, which was higher than that reported for either humans or monkeys. The high rate of enterohepatic cycling probably enables ponies to maintain intestinal bile acid needed for lipid digestion and absorption.
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596 Choleretic Properties of Bile Acids Both the customary direct estimate of bile acid-independent fraction and the choleretic effect of bile acids (bile flow versus bile acid secretion) and their estimate by method in the APPENDIX (inverse of flow versus bile acid concentration) gave essentially identical values. Thus, the apparent choleretic effect did not result from inevitable correlation because bile flow was common to both axes, and the bile acid-independent fraction of bile secretion must have been essentially constant. The choleretic effects of bile acids have been attributed to their osmotic properties (20, 21). Recently, Klaassen (11) objected to that view, reporting that in anesthetized dogs bile acids had different choleretic effects, which were not related to their different critical micellar concentrations. In this study, CDC had a significantly higher choleretic effect than endogenous bile acid or TC infusion (Table 2). Klaassen (10, 11) reported similar choleretic effects for both infused CDC and TC and a significantly greater choleretic effect for endogenous bile acids. Reason(s) for the differences in choleretic effects of bile acids are not clear. Based on current knowledge, dihydroxy bile acids, which tend to form larger micellar aggregates than do trihydroxy bile acids (18), should have a smaller choleretic effect than trihydroxy bile acids. Considering the micellar aggregate formation and cation associated with secreted bile acid, osmolarity of conjugated bile acids would approximately equal their molarity (13). However, we found CEC to have a greater choleretic effect than TC or endogenous bile acid, even though they were all conjugated. (Small unconjugated fraction of bile acids in bile did not markedly vary.) Therefore, the osmotic activity of secreted bile acid alone would not explain the differences in choleretic effects observed. This study demonstrated that bile acid-independent fraction of bile flow exists in ponies, that bile flow and bile acid secretion are quantitatively related, and that different bile acids have different choleretic effects. Possibilities for
ANWER,
GRONWALL,
ENGELKING,
AND
KLENTZ
the differences in choleretic effects include : a) the micellar properties of bile acids may be modified by other components of bile; and b) different bile acids may affect hepatocytes or their membranes differently, causing secretion of other osmotically active compounds. The latter possibility is indirectly supported by reports that the secretion rates of osmotically active ions like sodium, chloride, and bicarbonate were increased during bile acid infusion (10, 15). APPENDIX tion
Bile flow could be equated to the sum of bile acid-independent (BIF) and bile acid-dependent fraction (BDF) : flow
Assuming
BDF
related
= BIF
+ BDF
to bile acid secretion BDF
=KxBS = K x BC
frac(1)
(BS) by a constant
X flow
K, then
(2)
where BC is the biliary bile acid concentration. Substituting equation 2 into equation I, and rearranging, flow
= BIF/(l
-
K x
BC),
and l/flow
=
l/BIF
-
(K/BIF)
x BC
(3)
If BIF is constant and not zero, l/flow versus BC would be linear because, by definition, K is a constant. BIF and K could be calculated from the intercept and slope of the regression line as follows: BIF
=
1 /intercept
K = BIF
(a)
X slope
(5)
If flow is expressed as microliters per minute per kilogram and BC as micromoles per microliter, the unit of BIF would be microliters per minute per kilogram and that of K would be microliters per rnicromole. This investigation Grant AM1 1384. Received
for
publication
was supported 29 October
in part
by Public
Health
Service
1974.
REFERENCES 1. BEHER, W. T., B. RAO, M. E. BEHER, AND T. BERTASIUS. Bile acid synthesis in normal and hypophysectomized rats : a rate study using cholestyramine. Proc. Sot. Exptl. Biol. Med. 124: 1193-l 197, 1967. 2. BOYER, J. L., AND G. K~ATSKIN. Cannalicular bile flow and bile secretory pressure. Gastroenterology 59 : 853-859, 1970. 3. BRWSGAARD, A. Quantitative determination of the major 3-hydroxy bile acids in biological material after thin-layer chromatographic separation. C&n. Chim. Acta 28: 495-504, 1970. 4. DOWLING, R. H., E. MACK, AND D. M. SMALL. Effects of controlled interruption of enterohepatic circulation of bile salts by biliary diversion and by ileal resection on bile salt secretion, synthesis and pool size in the rhesus monkey. J. Clin. Invest. 49: 232-242, 1970. 5. ERLINGER, S., D. DHUMEAUX, AND P. BERTHELOT. Effect of inhibitors of sodium transport on bile formation in the rabbit. Am. J. Physiol. 219: 416-422, 1970. 6. GRONWALL, R. R., L. R. ENGELKING, M. S. ANWER, D. F. ERICHSEN, AND R. D. KLENTZ. Bile secretion in ponies with biliary fistulas. Am. J. Vet. Res. 36: 653-654, 1975. 7. HOFMANN, A. F., L. J. SCHOENFIELD, B. A. KOTTKE, AND J. R. POLEY. Methods for the description of bile acid kinetics in man. In : Methods in Medical Research, edited by R. E. Olson. Chicago: Year Book, vol. 12, 1970, p. 149-179. 8. JAVITT, N. B., AND S. EMERMAN. Effect of sodium taurolithocholate on bile flow and bile acid excretion. J. Clin. Invest. 47 : 1002-1014, 1968.
9. KLAASSEN, C. D. Studies on the increased biliary flow by phenobarbital in rats. J. Pharmacol. Exptl. Therap. 176 : 743-751, 1970. 10. KLAASSEN, C. D. Bile flow and composition during bile acid depletion and administration. Can J. Physiol. Pharmacol. 52: 334-348, 1974. 11. KLAASSEN, C. D. Comparison of the choleretic properties of bile acids. European J. Pharmacol. 23 : 270-275, 1973. 12. LINDSTEDT, S. The turnover of cholic acid in man. Acta Physiol. Stand. 40: l-9, 1957. 13. MOORE, E. W., AND J. M. DIETSCHY. Na and K activity coefficients in bile and bile salts determined by glass electrode. Am. J. Physiol. 206: 1111-1117, 1964. 14. NAHRWOOD, D. L., AND M. I. GROSSMAN. Secretion of bile in response to food with and without bile in the intestine. Gastroenterology 53: 11-17, 1967. 15. PREISIG, R., H. L. COPPER, AND H. 0. WHEELER. The relationship between taurocholate secretion rate and bile production in unanesthetized dog during cholinergic blockade and during secretin administration. J. Clin. Invest. 41: 1152-l 162, 1962. 16. SCHERSTEN, T., S. NILSSON, AND E. CAHLIN. Relationship between the biliary excretion of bile acids and the excretion of water, lecithin and cholesterol in man. European J. Clin. Invest. 1 : 242-247, 1971. 17. SMALL, D. M., R. H. DOWLING, AND R. N. REDINGER. The entero-
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ACID
KINETICS
AND
CHOLERETIC
597
EFFECT
hepatic circulation of bile salts. Arch. Intern. Med. 130: 552-573, 1972. 18. SMALL, D. R/I. A physical chemical approach to bile. In : Bile Salt Metabolism, edited by L. Schiff, J. B. Carey, and J. Dietschy. Springfield, Ill.: Thomas, 1969, p. 223-242. 19. SPERBER, I. Secretion of organic anions in the formation of urine and bile. Pharmacol. Rev. 11 : 109-l 34, 1959. 20. SPERBER, I. Biliary excretion and choleresis. Proc. Intern. Meeting, Pharmacol., Ist, Stockholm 4 : 137- 143, 196 1.
21.
SPERBER, I. Biliary secretion of organic anions and its influence on bile flow. In : The Biliary System, edited by W. Taylor. Oxford: Blackwell, 1965, p. 457-467. 22. VLAHCEVIC, 2. R.,J. R. MILLER, J. T. FARRAR, AND L. SWELL. Kinetics and pool size of primary bile acids in man. Gastroenterology 61: 85-90, 1971. 23. WHEELER, H. O., E. D. Ross, AND S. E. BRADLEY. Cannalicular bile production in dogs. Am. J. Physiol. 214: 866-874,1968.
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Vol. 229, No. 3, September
1975.
Printed in U.S.A.
Bile acid kinetics and bile secretion in the pony’ M. S. ANWER, R. R. GRONWALL, L. R. ENGELKING, Department of Physiological Sciences, College of Veterinary Medicine, Manhattan, Kansas 66506
ANWER, M. S.,R.R KLENTZ. Bile acid kinetics
R.D. J. Physiol. 229(3) : 592-597. 1975.-Bile acid pool size and synthesis rate were determined by both isotope-dilution and washout methods in ponies with chronic external biliary fistulas. Bile acid pool size (10.9 pmol/kg) and synthesis rate (11.2 pmol/day per kg) estimated by the isotope-dilution method did not differ significantly from pool size (9.4 pmol/kg) and synthesis rate (9.5 pmol/day per kg) estimated by washout method. Bile acid-dependent and -independent fractions of bile flow, determined by a method that circumvents any inevitable correlation of flow to bile acid secretion due to common factors in both parameters, did not differ from those values obtained by linear regression of bile flow versus bile acid secretion. The choleretic effect of infused chenodeoxycholic acid was higher than that of both endogenous bile acid and infused taurocholic acid. hepatic function;
GRONWALL, L.R. ENGELKING,AND and bile secretion in the pony. Am.
enterohepatic
circulation;
bile collection
l Contribution
no.
143. Department Experiment Station,
of Physiological Manhattan, Kan.
has not been related to bile acid secretion alone. A fraction of bile flow, independent of bile acid, has been suggested for rat cv, dog (1% man (16), and rabbit (5). Sodium transport may play an important role in the formation of that fraction of bile in rabbit (5) and in isolated, perfused rat liver (2). A quantitative estimation of such a fraction of bile flow has been obtained by plotting bile flow against bile acid secretion (23), but that method has recently been questioned (10). In the present investigation, the rate of biliary secretion of bile acid was varied by infusion of exogenous bile acids and by depletion of the endogenous bile acid pool in order to a) compare the choleretic effects of different bile acids with that of endogenous bile acids and b) evaluate the method of determining the bile acid-independent fraction of bile flow. METHODS
Three ponies with chronic external biliary fistulas were studied after complete recovery from surgery (6). They were fed native hay with grain supplement and were closely observed. Ponies had free access to feed and water during all experiments and stood in a stanchion without additional restraint during those experiments that required continuous sampling of bile. No drugs were administered to the animals after they recovered from surgery.
BILE ACIDS ARE SYNTHESIZED from cholesterol in the sliver. After being conjugated with either glycine or taurine, bile acids are actively secreted into the canaliculi and pass into the duodenum. More than 90 % of the secreted bile acid is reabsorbed by the intestine and returned to the liver via the portal vein. Most of the bile acid pool is thus in the enterohepatic circulation (EHC). A small amount escapes uptake by the liver from the portal blood and reaches the systemic circulation. In addition, only a small amount of the bile acid pool (about 5 % per enterohepatic circulation in man) is lost in the feces. The liver normally synthesizes enough bile acid to make up for that loss. An isotope-dilution technique has been used to determine the pool size and hepatic synthesis rate of bile acids (7). Recently those parameters were determined from a washout curve obtained during complete interruption of the EHC (4). In this study, both techniques were used to measure the parameters of bile acid metabolism in ponies with chronic external biliary fistulas, and the results from each technique were compared. Choleretic properties of bile acids are well known. The osmotic effect of actively secreted bile acids, which results in the flow of water and solutes into the canaliculi, is believed to be a principal mechanism behind the choleretic effect of bile acids (15 : 1’9-2 1). That view is supported by evidence that the choleretic properties of compounds depend on their osmotic activity (20, 21). All bile formation
Kansas Agricultural
AND R. D. KLENTZ Kansas State University,
Pool Size and Synthesis Rate Pool size and hepatic synthesis rate of bile acid were determined using two methods. Isotope-dilution method. Each pony was given a single intravenous dose ( 13- 16 &i) of [24-14C]chenodeoxycholic acid obtained from New England Nuclear Corporation, with a specific activity of 54 Ci/mol and purity of 99%. Bile was sampled every 8 h for 96 h by gravity drainage from the indwelling catheter. To ensure samples of freshly produced bile, the first 20-30 ml of bile were collected separately; then 1 ml was collected for study. To minimize alteration of the bile acid pool, the 20-30 ml of bile were infused back into the duodenum through the catheter and followed by 20 ml of saline. One milliliter of bile represented about 2 % of the total bile acid pool. Calculation of parameters of bile acid kinetics was based on the assumption of a first-order kinetic model as described by Lindstedt (12). The relationship of the logarithm of total bile acid specific activity to time was determined by least-squares regression (Fig. 1). All regressions were signincant (P < 0.01) with correlation coefficients of 0.9940.998. Total bile acid pool size and chenodeoxycholic acid
Sciences, 592
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BILE
ACID
KINETICS
AND
CHOLERETIC
EFFECT
0 a P 0
I 20
I
I 40
60
I 00
. 100
Hours FIG.
jection (-)
cient
1. Biliary bile acid specific activity following intravenous inof chenodeoxycholic acid-24J*C in 1 pony. Regression line where specific activity = 71.4 X e-“.04sxt (correlation coeffi0.998).
(CDC) synthesis rate were calculated from the dose, regression intercept, and slope (22). Since CDC represented 75 % of total bile acid in bile (see below), synthesis rate of CDC was calculated by multiplying regression slope times 75 % of total bile acid pool. Washout method. Bile was continuously collected in IO-min samplesfor 7 h with complete interruption of the EHC. Secretion of bile acid declined rapidly after lo-20 min of drainage and reached a low point in 2-3 h (Fig. 2). That rate was maintained for the remainder of the bile collection period and was taken to represent the hepatic synthesisrate. The amount of bile acid secretedfrom the time of interruption of the EHC until the low-level excretion was reached gave an estimate of circulating bile acid pool size (4). To obtain the normal secretion rate of bile acid, bile samples were taken twice a day (before feeding and during feeding) for 3 days prior to the washout study. That sampling required only short interruptions of the EHC. Choleretic Properties of Bile Acids
Major biliary bile acids in ponies are taurochenodeoxycholic acid and taurocholic acid and, thus, to study the choleretic properties of the normal bile acids of ponies, two bile acids, taurocholic acid (TC) and chenodeoxycholic acid, were used in these experiments. Bile samples were continuously collected by gravity drainage at lo-min intervals for 5 h with complete interruption of the EHC. After 2 h of bile drainage, bile acid excretion approached its low point. Bile acid (either TC (Nutritional Biochemical Corp.) or CDC (Sigma Chemical Co.)) dissolved in saline was then infused intravenously at three different infusion rates (4.7, 11.8, and 23.6 pmol min-’ for TC and 4.0, 10.2, and 20.3 pmol min-’ for CDC) for 1 h at each rate. Those infusion rates were within the normal bile acid secretion rates of the ponies. The relationship of bile flow, during an initial 2-h period, to bile acid secretion was determined by linear regressionto find choleretic effects of endogenous bile acids. Similar regression analyses were made for bile acid infusion periods. It has been argued that if bile flow is plotted against bile acid secretion, the two variables would be misleadingly
1
2
4
3
5
6
I
HOURS FIG. 2. A typical washout curveshowingchanges in bile acidsecretion rate in a pony during complete interruption of enterohepatic circulation. Secretion rate reached a low point in 2.5 h. Hepatic synthesis rate (-) was estimated from low point. Shaded area under curve, from interruption until low point, represents total bile acid pool.
correlated becauseflow is common to both axes (10). To avoid variables already correlated, an equation was developed using bile flow and biliary bile aEid concentration that would give the sameinformation asthe direct regression analysis of flow versus bile acid excretion: l/flow
= l/BIF
- (K/BIF)
X BC
where BIF = bile acid-independent fraction of bile flow, K = the constant relating bile flow and bile acid secretion, and BC = biliary bile acid concentration (see APPENDIX for details). If BIF is not zero and both BIF and K are constants, the inverse of flow would be linearly related to BC. BIF and K then can be calcttlated from the intercept and the slope of the regression line. Inverse of flow was plotted against BC during endogenous bile acid drainage and exogenousbile acid infusion. BIF and K were calculated from equations 4 and 5, respectively, in the APPENDIX. Analytical
Methods
Total bile acid concentration in bile and infusion solutions was determined by an enzymatic method (8) using Sol-hydroxysteroid dehydrogenase (Grade III, Sigma Chemical Co.). The reaction mixture contained 0.05 ml of bile (or diluted infusion solution), 0.45 ml of water, 1.2 ml of 0.2 M phosphate buffer, pH 9.9, 1.0 ml of 1 M hydrazine hydrate, and 0.2 ml of 5 mM NAD (Grade III, Sigma Chemical Co.). The reaction was started by adding 0.1 ml of hydroxysteroid dehydrogenase solution (2.5 Sigma Chemical Co. U/ml). The net increase in absorbance at 340 nm was determined using reagent blank after 30 min of incubation at room temperature. A bile acid standard was run parallel to each set of samples.The concentration of bile acid was determined from the net increase in absorbance and the molar extinction coefficient of NADH at 340 nm. The composition of primary bile acids in the bile and bile acid infusion solutions was determined using the method described by Bruusgaard (3). Bile acids were first separated
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594
ANWER,
by thin-layer chromatography, and then an enzymatic method (see above) was used to determine the concentrations of the separated bile acids. Radioactivity in bile was determined by dissolving 0.1 ml of bile in 10.0 ml of PCS solubilizer (Amersham/Searle Corp.) and counting in a liquid scintillation counter. A portion of the injected solution of [14C]chenodeoxycholic acid (50 ~1) was counted similarly to determine the injected dose. Counting efficiency for each sample was determined from the relationship of external standard ratio to efficiency for a standardized set of samples. Radioactivity in bile was expressed as microcuries per mole of bile acid.
GRONWALL,
ENGELKING,
AND KLENTZ
when bile acid was infused, both increased gradually (Fig. 3). Bile flow increased more during CDC infusion than during TC infusion. A significant regression of bile flow versus bile acid secretion with a positive slope and intercept was indicated by correlation coefficients ranging from 0.832 to 0.998 (Fig. 4). The intercepts of the regression represent the average bile acid-independent fraction of bile flow; the slopes (~1 of flow per pmol of bile acid) estimate the choleretic effects of the bile acids. A significant linear regression of the inverse of flow versus the corresponding BC (correlation coefficient from 0.784 to 0.99) with positive intercept and negative slope was found in all cases (Fig. 5). Values of BIF and K, calculated
RESULTS
Although labeled chenodeoxycholic acid was injected for the isotope-dilution studies, specific activities were determined for total bile acid. Thus, pool size calculated by that method represented total bile acid just as in the washout method. Total bile acid pool sizes did not differ significantly (P > 0.05) when compared between the methods (Table 1). Although CDC synthesis rate calculated from isotope-dilution method was less than total bile acid synthesis rate obtained from washout method, it did not differ significantly (P > 0.05). S ince CDC was the principal primary bile acid in the pony (see below), CDC synthesis rate would acco&t for most of the total bile acid synthesis rate in ponies. An estimate of the total bile acid synthesis rate, which was calculated by multiplying total bile acid pool with regression slope of the isotope-dilution data, did not differ significantly (P > 0.05) from that obtained from washout method (Table 1). Using average values from the washout method for each animal, a paired-t test, which is equivalent to two-way analysis of variance and should compensate for animal variation, also showed no significant difference between the two methods. During the initial 2 h of washout trials, when no bile acid was infused, both bile flow and bile acid secretion declined;
40
. . 30 l
9.53
Isotope
6.99
dilution
CDC
Total
6.25
8.34
6.63 9.18 8.66
7.86t 2
9.38
9.05
CDC
15.72
. . l
.
==...
O-t
I b-
1
Drainage
1
e
I C DC
1
1 Infusion
4
l
.
8
.
. .
l
l
. .
8 .
.
8
l
. 8
. l
0
l ==...=
.
.
l
l
I
I
I
I
1
1
2
3
4
5
FIG. 3. Bile flow and bile acid secretion rate during endogenous bile acid drainage (first 2 h) and chenodeoxycholic acid infusion (infusion rates were 4.0, 10.2, and 20.3 pmol/min for 3rd, 4th, and 5th h, respectively) in 1 pony. C henodeoxycholic
1
Taurocholic
Acid
Acid
Washout
6.89 8.94 10.11 8.37
B
0 E
I! z
6.74
8.98
6.99 11.85 10.80
12.13
16.18
11.69
9.38t 13.87
mm 8 . a
. .
l
.
.
10 -
60
8.58t
9.31 9.79
3
.
.
. .
Hepatic Synthesis Rate,* pmol/day per kg
Pool Size,* pmol/kg
I
l
20 -
1. Pool size and hepatic synthesis rates of bile acid in ponies
Washout
. l 8
TABLE
Isotope dilution
-
15
9.88t
= chenodeoxycholic acid. Total = total bile acid. * There was no significant difference (P > 0.05) between values derived from the two methods. t Average values obtained from washout method for each animal were used in the paired-t test.
b
0
.l
Bile
Acid
.2
Secretion,
0
I
1
.l
.2
yMoles/min/kg
FIG. 4. Regressions of bile flow versus bile acid endogenous bile acid depletion (- - - - -) and infusion cholic acid and taurocholic acid (--) in 3 ponies.
secretion during of chenodeoxy-
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BILE
ACID
KINETICS
AND
CHOLERETIC
EFFECT
595
.08
TABLE 2. Comparison of methods for calculation acid-independent fraction of bile and choleretic effect of bile acids in ponies
1
Bile Acid Depletion
of bile
Bile Acid Infusion
Pony Intercept*
1 BIFt
/ Slope*1
it
‘c”ap”t’;
1
BIFt
Chenodeoxycholic
1 Slope*
1
Rt
acid
I 2 3 Mean
0
f
0 Bile
1
1
I
I
.002
.004
.006
.008
Acid
Concentration
FIG. 5. Inverse of bile flow versus bile acid endogenous bile acid depletion (0) and infusion acid (m) for 1 typical trial. Regression lines (--).
p Moles/p1 concentration during of chenodeoxycholic
from the parameters of that regression, were not significantly different by the Student t test (P > 0.05) from the intercepts and slopes of the regressions of flow versus secretion (Table 2). Neither the intercept nor BIF differed significantly between endogenous bile acid depletion and exogenous bile acid infusion when tested by a paired-t test. The choleretic effect (slope or K) during CDC infusion differed significantly (P < 0.05) from that determined during both bile acid depletion and taurocholic acid infusion. (Mean choleretic effects of endogenous bile acids and infused TC were not significantly different.) Chenodeoxycholic acid was the major primary bile acid in bile. Taurochenodeoxycholic acid (71 %) and taurocholic acid (15 %) represented 86 % of the total biliary bile acid. A small amount of glycochenodeoxycholic acid (3-4 %) was always present. Bile obtained during CDC infusion contained primarily the taurine conjugate of CDC with no detectable increase in the proportion of unconjugated bile acid. Infusion of bile acids did not cause hemolysis in any of the ponies. DISCUSSION
Pool Size and Synthesis Rate Isotope-dilution methods have been widely used to determine pool size and synthesis rate of bile acid. First-order kinetics and a constant pool size during sampling period were assumed (12). On the other hand, the washout method involves direct measurement of bile acid pool and estimation of the synthesis rate (4). Thus, in this study, pool size and synthesis rate of bile acid were determined by two methods with different theoretical approaches. Neither of the two parameters differed when compared between the methods. Washout method would be preferred for animal
I 2 3 Mean
11.44 11.99 11.12 11.52
11.78 12.14 11.29 11.74
27.0 31.2 48.2 35.4
19.5 30.1 43.9 31.2
14.41 12.33 13.13 13.29
Taurocholic
acid
14.52 12.41 11.97 12.96
48.7 35.2 53.8 45.9
47.8 35.3 64.6 49.2
BIF = bile acid-independent fraction; K = the constant relating bile acid-dependent fraction with bile acid secretion; intercept = bile flow at zero bile acid secretion; slope = change in bile flow per change in bile acid secretion. There was no significant difference (P > 0.05) between the estimates of bile acid-independent fraction * Calcuor choleretic effect by the two calculation methods. t Callated from regressions of bile flow on bile acid secretion. culated from slope and intercept of regression of inverse of bile flow $ Significantly different (P < 0.05) on bile acid concentration. from the corresponding values of bile acid depletion and taurocholic acid infusion.
experiments because it is simple and direct, but does require quantitative bile collection from animals with previously intact EHC. acid was used In this study, only [‘“Cl c h enodeoxycholic to determine pool size and synthesis rate of total bile acid by isotope dilution. It has been reported that in man chenodeoxycholic acid and cholic acid have different synthesis rates (22), so that both primary bile acids must be studied by isotope-dilution method to accurately determine total bile acid synthesis rate (7). Since the washout method directly determined total bile acid synthesis, the similarity of this value to that obtained from isotope-dilution method indicated that total bile acid synthesis could be estimated in ponies using only [14C]chenodeoxycholic acid disappearance. The high percentage of chenodeoxycholic acid in ponies could account for the similarity. The pool size of total bile acid in ponies (11 pmol/kg) was relatively smaller than that of humans-l 23 pmol/kg (17), rats-181 pmol/kg (l), or monkey-ZOO pmol/kg (4). The synthesis rate of 9.5 pmol/clay per kg was similar to humans -14 pmol/day per kg ( 17) but less than that of rats-28 pmol/day per kg (1) or monkeys-l 10 pmol/day per kg (4). The relatively small pool size may be a species difference or a reflection of diet. The number of cycles of the total bile acid pool per day, calculated as the ratio of the normal daily secretion rate to pool size (17), was 38 per day in ponies, which was higher than that reported for either humans or monkeys. The high rate of enterohepatic cycling probably enables ponies to maintain intestinal bile acid needed for lipid digestion and absorption.
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596 Choleretic Properties of Bile Acids Both the customary direct estimate of bile acid-independent fraction and the choleretic effect of bile acids (bile flow versus bile acid secretion) and their estimate by method in the APPENDIX (inverse of flow versus bile acid concentration) gave essentially identical values. Thus, the apparent choleretic effect did not result from inevitable correlation because bile flow was common to both axes, and the bile acid-independent fraction of bile secretion must have been essentially constant. The choleretic effects of bile acids have been attributed to their osmotic properties (20, 21). Recently, Klaassen (11) objected to that view, reporting that in anesthetized dogs bile acids had different choleretic effects, which were not related to their different critical micellar concentrations. In this study, CDC had a significantly higher choleretic effect than endogenous bile acid or TC infusion (Table 2). Klaassen (10, 11) reported similar choleretic effects for both infused CDC and TC and a significantly greater choleretic effect for endogenous bile acids. Reason(s) for the differences in choleretic effects of bile acids are not clear. Based on current knowledge, dihydroxy bile acids, which tend to form larger micellar aggregates than do trihydroxy bile acids (18), should have a smaller choleretic effect than trihydroxy bile acids. Considering the micellar aggregate formation and cation associated with secreted bile acid, osmolarity of conjugated bile acids would approximately equal their molarity (13). However, we found CEC to have a greater choleretic effect than TC or endogenous bile acid, even though they were all conjugated. (Small unconjugated fraction of bile acids in bile did not markedly vary.) Therefore, the osmotic activity of secreted bile acid alone would not explain the differences in choleretic effects observed. This study demonstrated that bile acid-independent fraction of bile flow exists in ponies, that bile flow and bile acid secretion are quantitatively related, and that different bile acids have different choleretic effects. Possibilities for
ANWER,
GRONWALL,
ENGELKING,
AND
KLENTZ
the differences in choleretic effects include : a) the micellar properties of bile acids may be modified by other components of bile; and b) different bile acids may affect hepatocytes or their membranes differently, causing secretion of other osmotically active compounds. The latter possibility is indirectly supported by reports that the secretion rates of osmotically active ions like sodium, chloride, and bicarbonate were increased during bile acid infusion (10, 15). APPENDIX tion
Bile flow could be equated to the sum of bile acid-independent (BIF) and bile acid-dependent fraction (BDF) : flow
Assuming
BDF
related
= BIF
+ BDF
to bile acid secretion BDF
=KxBS = K x BC
frac(1)
(BS) by a constant
X flow
K, then
(2)
where BC is the biliary bile acid concentration. Substituting equation 2 into equation I, and rearranging, flow
= BIF/(l
-
K x
BC),
and l/flow
=
l/BIF
-
(K/BIF)
x BC
(3)
If BIF is constant and not zero, l/flow versus BC would be linear because, by definition, K is a constant. BIF and K could be calculated from the intercept and slope of the regression line as follows: BIF
=
1 /intercept
K = BIF
(a)
X slope
(5)
If flow is expressed as microliters per minute per kilogram and BC as micromoles per microliter, the unit of BIF would be microliters per minute per kilogram and that of K would be microliters per rnicromole. This investigation Grant AM1 1384. Received
for
publication
was supported 29 October
in part
by Public
Health
Service
1974.
REFERENCES 1. BEHER, W. T., B. RAO, M. E. BEHER, AND T. BERTASIUS. Bile acid synthesis in normal and hypophysectomized rats : a rate study using cholestyramine. Proc. Sot. Exptl. Biol. Med. 124: 1193-l 197, 1967. 2. BOYER, J. L., AND G. K~ATSKIN. Cannalicular bile flow and bile secretory pressure. Gastroenterology 59 : 853-859, 1970. 3. BRWSGAARD, A. Quantitative determination of the major 3-hydroxy bile acids in biological material after thin-layer chromatographic separation. C&n. Chim. Acta 28: 495-504, 1970. 4. DOWLING, R. H., E. MACK, AND D. M. SMALL. Effects of controlled interruption of enterohepatic circulation of bile salts by biliary diversion and by ileal resection on bile salt secretion, synthesis and pool size in the rhesus monkey. J. Clin. Invest. 49: 232-242, 1970. 5. ERLINGER, S., D. DHUMEAUX, AND P. BERTHELOT. Effect of inhibitors of sodium transport on bile formation in the rabbit. Am. J. Physiol. 219: 416-422, 1970. 6. GRONWALL, R. R., L. R. ENGELKING, M. S. ANWER, D. F. ERICHSEN, AND R. D. KLENTZ. Bile secretion in ponies with biliary fistulas. Am. J. Vet. Res. 36: 653-654, 1975. 7. HOFMANN, A. F., L. J. SCHOENFIELD, B. A. KOTTKE, AND J. R. POLEY. Methods for the description of bile acid kinetics in man. In : Methods in Medical Research, edited by R. E. Olson. Chicago: Year Book, vol. 12, 1970, p. 149-179. 8. JAVITT, N. B., AND S. EMERMAN. Effect of sodium taurolithocholate on bile flow and bile acid excretion. J. Clin. Invest. 47 : 1002-1014, 1968.
9. KLAASSEN, C. D. Studies on the increased biliary flow by phenobarbital in rats. J. Pharmacol. Exptl. Therap. 176 : 743-751, 1970. 10. KLAASSEN, C. D. Bile flow and composition during bile acid depletion and administration. Can J. Physiol. Pharmacol. 52: 334-348, 1974. 11. KLAASSEN, C. D. Comparison of the choleretic properties of bile acids. European J. Pharmacol. 23 : 270-275, 1973. 12. LINDSTEDT, S. The turnover of cholic acid in man. Acta Physiol. Stand. 40: l-9, 1957. 13. MOORE, E. W., AND J. M. DIETSCHY. Na and K activity coefficients in bile and bile salts determined by glass electrode. Am. J. Physiol. 206: 1111-1117, 1964. 14. NAHRWOOD, D. L., AND M. I. GROSSMAN. Secretion of bile in response to food with and without bile in the intestine. Gastroenterology 53: 11-17, 1967. 15. PREISIG, R., H. L. COPPER, AND H. 0. WHEELER. The relationship between taurocholate secretion rate and bile production in unanesthetized dog during cholinergic blockade and during secretin administration. J. Clin. Invest. 41: 1152-l 162, 1962. 16. SCHERSTEN, T., S. NILSSON, AND E. CAHLIN. Relationship between the biliary excretion of bile acids and the excretion of water, lecithin and cholesterol in man. European J. Clin. Invest. 1 : 242-247, 1971. 17. SMALL, D. M., R. H. DOWLING, AND R. N. REDINGER. The entero-
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BILE
ACID
KINETICS
AND
CHOLERETIC
597
EFFECT
hepatic circulation of bile salts. Arch. Intern. Med. 130: 552-573, 1972. 18. SMALL, D. R/I. A physical chemical approach to bile. In : Bile Salt Metabolism, edited by L. Schiff, J. B. Carey, and J. Dietschy. Springfield, Ill.: Thomas, 1969, p. 223-242. 19. SPERBER, I. Secretion of organic anions in the formation of urine and bile. Pharmacol. Rev. 11 : 109-l 34, 1959. 20. SPERBER, I. Biliary excretion and choleresis. Proc. Intern. Meeting, Pharmacol., Ist, Stockholm 4 : 137- 143, 196 1.
21.
SPERBER, I. Biliary secretion of organic anions and its influence on bile flow. In : The Biliary System, edited by W. Taylor. Oxford: Blackwell, 1965, p. 457-467. 22. VLAHCEVIC, 2. R.,J. R. MILLER, J. T. FARRAR, AND L. SWELL. Kinetics and pool size of primary bile acids in man. Gastroenterology 61: 85-90, 1971. 23. WHEELER, H. O., E. D. Ross, AND S. E. BRADLEY. Cannalicular bile production in dogs. Am. J. Physiol. 214: 866-874,1968.
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