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Physiologic determinants of biliary calcium secretion in the dog
GASTROENTEROLOGY
1984;87:664-73
Physiologic Determinants of Biliary Calcium Secretion in the Dog SUSAN A. CUMMINGS
and ALAN F. HOFMANN
Division of Gastroenterology, Department of Medicine. California at San Diego, San Diego, California
Studies were carried out to define the origins and physiologic determinants of biliary calcium secretion in the dog. CanaJicuJar bile flow was varied by infusions of several bile acids or a canalicular choleretic agent, SC2644. Three natural bile acids (cholyltaurine, chenodeoxycholyltaurine, and ursodeoxycholyltaurine) that have a low critical micelJar concentration and a synthetic hydroxy-oxo bile acid (12-dehydrocholyltaurine) with a much higher critical micellar concentration were used. Calcium output, bile acid output, and bile flow were determined; canalicular bile flow was assessed by measurement of erythritol clearance. Calcium output increased linearly with bile acid output for all three natural bile acids (r = 0.94; 0.026-0.035 PmoJAcaJcium/~moJAbiJe acid), but chenodeoxycholyltaurine and ursodeoxycholyltaurine infusions induced a slightly greater secretion of calcium for a given bile acid output and bile flow than cholyltaurine. CaJcium output also increased linearly with bile pow, Received February 29, 1984. Accepted April 10, 1984. Address requests for reprints to: Alan F. Hofmann, M.D., Department of Medicine, UCSD Medical Center, 225 Dickinson Street, San Diego, California 92103. This study was supported in part by National Institutes of Health Grants AM 21506, AM 32130, and RR 00827. S. A. Cummings is a fellow of the Medical Research Council of Canada. Her present address is Department of Medicine, Yale University, New Haven, Connecticut. A portion of this work was presented at the 1983 Annual Meeting of the American Gastroenterological Association and published in abstract form in Gastroenterology 1983;84:1131. The authors thank Dr. Joseph Steinbach, Dr. M. Sawkat Anwer, Dr. James Barnhart, Dr. Edward W. Moore, and Devorah Gurantz for helpful discussions; Dr. Ian Gilmore and Dr. Rudy Danzinger for bile samples collected previously; Linda Clayton for providing technical assistance; Dr. Gerhard Schrauzer of the Department of Chemistry for making available the atomic absorption spectrometer: Paul Schragg of the General Clinical Research Center for providing help in the use of the CLINFO computer; Dr. Adolf Krekel of Pharmazell, Diamalt, Redenfelden, Federal Republic of Germany for donating the bile acids; and Vicky Huebner for manuscript preparation. 0 1984 by the American Gastroenterological Association 00165085/84/$3.00
School
of Medicine,
University
of
whether bile flow was induced by the canalicular choleretic SC2644 [r = 0.93; 0.0018 PmoJAcaJciuml PlAwater) or the hydroxy-oxo bile acid (r = 0.99; 0.0021 ~moJAcaJcium/~JAwater). Because the calcium output induced by the three micelle-forming bile acids was greater than that which could be explained by the induced increase in biJe flow per se, it was deduced that canalicular calcium has two origins: a micellar component that is dependent on the presence of micelles or micelle-forming bile acids and an osmotic component that is dependent on osmotic forces, e.g., caused by secretion induced by SC2644 or bile acid molecules and possibly bile acid micelles. A small bile acid-independent output of calcium was also detected and should contribute to the osmotic fraction. To determine whether ductuJar modification of bile flow influenced biliary calcium secretion, ductular secretion was induced by secretin and ductular absorption was induced by somatostatin. Changes in calcium output followed water movements, increasing with ductular secretion and decreasing with ductular absorption. The relationship between changes in ductular bile flow and calcium output was identical during induced ductular secretion (r = 0.83; 0.0016 ~moJAcaJcium/ PJAwater) or induced ductular absorption (r = 0.92; 0.0019 ~moJAcaJcium/~Awater). Thus calcium movements in relation to water movements were identical for the osmotic component of canalicular bile and the ductular component of bile. The experimental manipulations in bile secretion caused anticipated changes in biliary calcium concentration which ranged from 1.6 to 6.4 mM. These results indicate that at the usual rates of bile acid secretion, most biliary calcium originates at the canaliculus and is bile acid-dependent. The biliary ductule is permeable to calcium, but the effect of ductular modification of bile flow on biliary calcium outputs is small.
Abbreviations centration
used
in this
paper:
CMC, critical
micellar
con-
September
BILIAKYCALCIUM SECRETION 665
1984
Precipitation of calcium in the biliary tree is a key event in the pathogenesis of human cholelithiasis, as calcium is the major cationic component of noncholesterol gallstones (l-3), and calcium salts often serve as a nidus for nucleation of cholesterol gallstones (43). For calcium precipitation to occur, the product of calcium activity and the activity of a calcium-sensitive anion (with appropriate exponents) must exceed the solubility product. Calcium activity in bile is determined by total calcium concentration minus that bound to micelles, anions, or proteins in bile. The total calcium concentration is determined by biliary calcium secretion in relation to biliary water secretion. Bile is formed in the canaliculus and modified in the ductules. It has been useful to divide canalicular bile flow into two components, the bile acid-dependent and the bile acid-independent fraction (reviewed in References 6 and 73, although the physiologic meaning of the bile acid-independent fraction continues to be questioned (8,9). Bile is further modified in the biliary ductules where the absorption or secretion of a bicarbonate-rich fraction may occur (10); these ductular modifications of bile may be influenced by pharmacologic doses of some of the gastrointestinal hormones such as secretin (ll,12), glucagon (12), or somatostatin (13). This paper reports studies aimed at defining the determinants of biliary calcium secretion in the bile fistula dog. Experiments were designed to distinguish canalicular from ductular events, and to divide canalicular events into the bile acid-dependent and bile acid-independent components. To alter bile acid-dependent flow, three natural bile acids (cholyltaurine, chenodeoxycholyltaurine, and ursodeoxycholyltaurine) were infused at varying rates. In addition, to gain further insight into the mechanism by which bile acids induced calcium secretion, a synthetic taurine-conjugated hydroxy-oxo bile acid (1Zdehydrocholyltaurine) was used. This bile acid has a critical micellar concentration (CMC) (40 mM) that
is considerably
higher
than
that
of the
common
bile acids (2-10 mM) (14). It was reasoned that the majority of the anions of this bile acid would be present in bile in nonmicellar form; thus, the use of this bile acid might permit the role of the micelle in biliary calcium secretion to be defined. Bile acidindependent flow was induced by means of a canalicular choleretic agent, SC2644 (SC is an abbreviation for Searle Compound, Searle Pharmaceuticals, Inc.. Chicago, Ill.). Ductular modification of bile flow was achieved by infusing secretin which causes ductular secretion of a bicarbonate-enriched component of bile (11,12), or somatostatin which causes ductular absorption of
a component of bile with a similar electrolyte position (13).
com-
Methods Experimental
Design
The experimental approach featured the collection of hepatic bile samples after the establishment of a steady state; individual components of biliary secretion were varied systematically by the use of c.holeretic agents. hormones, or various bile acids that were administered intravenously in random order. The experimental design was varied according to the agent [see below). Two animal preparations were used. Acute bile fistula dogs were prepared surgically and studied while anesthetized. Chronic bile fistula dogs had been prepared bl previous cholecystectomy and placement of a Thomas duodenal cannula (IS), permitting sampling of hepatic bile while the animal was awake and alert. The results from these two preparations were identical, permitting pooling of the data. Modification of canalicular bile pow. BILE ACIDDEPENDENT FLOW. Bile acid-dependent flow was varied b\ infusing bile acids over the physiologic: range of 0.5-4.5 pmolikg . min. The majority of studies were carried out with cholyltaurine, the predominant bile acid of the dog. Additional studies were carried out with chenodeoxycholyltaurine and ursodeoxycholyltaurine. Two experimental designs were used. The first design consisted of singledose experiments using the ABA’C design. A and A’ were control periods during which cholyltaurine was infused. B and C were periods in which chenodeoxycholyltaurine or ursodeoxycholyltaurine was infused in random order. The second design consisted of dose-response studies in which three doses of a given bile acid were infused in random order; in some studies, two doses of cholyltaurine were infused before and after the dose-response study of the test bile acid. Experimental details and data for bile flow, erythritol clearance, and biliary lipid secretion have been published previously for some of these studies (16,171. When data from these studies were analyzed, it was found that the induced calcium secretion was greater than that explicable by the increase in bile volume caused by the bile acid. These three bile acids are known to form micelles (14) when present above their CMC (2-10 mM), and bile acid micelles are known to bind calcium (8-20). Accordingly, it was elected to define the influence of a bile acid that would be present to a considerable extent in monomeric solution in bile rather than in micelles. 12Dehydrocholyltaurine (3a,i’Lu-dihydroxy-12-oxo-cholanoyltaurine) was chosen because this compound is known to have a relatively high CMC value of 40 mM (14) and is also known to be a powerful choleretic agent in the cat (21). This compound was studied at three doses, and its infusion was preceded and followed by infusions of two doses of cholyltaurine. BILE ACID-INDEPENDENT FLOW. Bile acid-independent flow was induced by administration of a substituted propionic acid derivative SC2644 which is known to be a powerful canalicular choleretic in the dog, inducing a striking
666
CUMMINGS AND HOFMANN
increase in bile volume associated with a parallel increase in erythritol clearance (22-25). The experimental design featured infusion of various doses of cholyltaurine followed by infusion of the canalicular choleretic agent; the choleretic had a prolonged effect on bile flow, so that period randomization was not possible. The infusion rate of SC2644 was 0.5 mgimin (0.06 Fmolimin . kg] for 60 min followed by 3 mgimin for 80 min. Modification of ductular j7ow. Ductular secretion was induced by secretin, 6 pgikg . h; and ductular absorption by somatostatin, 8 pgikg . h. The experimental design featured infusion of the hormones somatostatin or secretin in randomized order; each hormone period was preceded and followed by a 60-min control period. After a steady state was obtained, secretin or somatostatin was given for an 80-min period. Throughout the entire experiment cholyltaurine was infused at a dose of 1 pmolikg min. The final period featured the infusion of an additional 2-pmoli kg. min dose of cholyltaurine (total infusion rate of 3 pmolikg . min) to define the effect of cholyltaurine on calcium output under these experimental conditions.
Experimental
Procedure
Acute bile fistula dogs. Six adult male mongrel dogs, 21-32 kg, fasted for 16 h, were anesthetized with an intravenous bolus (30 mgikg) of sodium pentobarbital (Abbott Laboratories, North Chicago, 111.). Preparation included midline laparotomy, ligation of the renal pedicles (to prevent erythritol excretion), ligation of the cystic duct, and cannulations of both external jugular veins (Bard Biomedical I-Cath 16GA, Sunderland, U.K.) and lateral saphenous vein (Bard Biomedical I-Cath 18GA). The common bile duct was ligated close to its insertion in the duodenum and was cannulated proximal to this point using polyethylene tubing (Intramedic PE 190, Becton, Dickinson and Co., Parsippany, N.J.). An additional 200 mg of sodium pentobarbital was administered in 50-mg boluses over the ensuing 8 h to maintain adequate anesthesia. (The boluses were given only during control periods to minimize any possible effect of pentobarbital on bile flow.) Chronic bile fistula dogs. Three adult male mongrel dogs, 22-30 kg, had been prepared previously by cholecystectomy and insertion of a Thomas cannula in the duodenum. Dogs were studied after a 16-h fast. The duodenal cannula was opened and a polyethylene tube (Intramedic PE-190, OD 1.7 mm) was inserted 5-6 cm into the common bile duct and brought out through a small hole in the bung used to occlude the duodenal fistula. Both lateral saphenous veins were cannulated at the beginning of the study. The dogs were supported by a sling. Brief sedation was required during insertion of the cannula into the bile duct. Sodium thiamyl was used [Park-Davis, Detroit, Mich.). Throughout each experiment, the dogs received two continuous intravenous infusions delivered by two peristaltic pumps (Harvard Apparatus Co., Dover, Mass.). The first infusion contained the test agent, i.e., bile acid, SC2644, or the hormone. The second contained either cholyltaurine and [l*C]erythritol or [“Clerythritol alone, depending on the experiment. Bile was drained for 40 min before starting the experiment. Each agent was infused
GASTROENTEROLOGY
Vol. 87. No. 3
for 80 or 90 min, and bile was collected by gravity in preweighed vials at IO-min intervals, after a steady state of bile flow had been obtained. This was after 20 min or after 60 min, depending on the experiment. A 3-ml blood sample was drawn at the midpoint of most bile collections via a third intravenous line. In the chronic animals, a tracer priming dose of [‘“Clerythritol radioactivity, 3 &i, was injected as a bolus, followed by a maintenance dose of 0.04 &/min. In the acute animals, a 5-&i priming dose was given, followed by a maintenance dose of 0.15 &i/min.
Analytical
Procedures
Calcium was determined by atomic absorption spectrophotometry (Perkin-Elmer 373, Perkin-Elmer Corp., Norwalk, Conn.). Bile, 100 ~1, was added to 5 ml of diluent. The diluent contained 20 mM lanthanum chloride (50 ml of 0.4 M lanthanum chloride/L of diluent) and 0.45 M hydrochloric acid (25 ml of concentrated HCliL of diluent). The precipitate occurring when the acid diluent was added was removed by centrifugation at 2000 g for 20 min. Calcium determination was performed on the supernatant, as preliminary experiments had shown that calcium was not present in the precipitate. To validate this procedure, known concentrations of calcium were added to each bile solution. A standard curve was then obtained for each bile sample and the value of calcium in the unaltered bile sample could be calculated by extrapolation of the curve to the ordinate. Using 49 different bile samples, the correlation between this predicted value and that observed was r = 0.96. In other experiments, Triton X-100 (Sigma Chemical Co., St. Louis, MO.) 1% was added to the different samples to prevent the usual precipitation that occurred during dilution. Values for calcium were unchanged. Total bile acids were estimated by the 3-hydroxysteroid dehydrogenase assay (26). The biliary bile acid composition of representative bile samples was determined by gasliquid chromatography of the methyl ester acetates (27). [“C]Erythritol radioactivity was measured in bile and plasma by liquid scintillation counting. Samples of 0.5 ml were first bleached by the addition of 50-75 ~1 of 30% hydrogen peroxide. Samples were left to bleach overnight: the next day 10 ml of liquid scintillation fluid (Aquasol-2, New England Nuclear, Boston, Mass.) was added. Samples were then left 1 wk before counting because of chemiluminescence from the hydrogen peroxide. Quenching was so uniform that raw counts were used to calculate plasmai bile ratios of radioactivity. Chemicals,
Radiochemicals,
and
Hormones
Cholic acid was purchased from Aldrich Chemical Co. [Milwaukee, Wis.) and was purified by crystallization from 95% ethanol. Chenodeoxycholic acid, ursodeoxycholit acid, and l2-dehydrocholic acid (3a,7a-dihydroxp-12oxo-cholanoic acid) were generously donated by Pharmazell, Diamalt Aktiengesellschaft, Redenfelden, Federal Republic of Germany; we acknowledge the generous support of Dr. Adolf Krekel. All bile acids were of high purity and were used without further purification. The taurine
September
IA
Figure
BILIARY
1984
b-_--
1 2 Bile Acid Output,
3 pmol/kg.min
i-_--M
4
0 18
compound was converted to its sodium salt by titration with 1 N NaOH, and a solution containing 1.6 or 9.3 mM was infused at 1 mlimin. [U-‘“ClErythritol, specific activity 100 mCiimmo1 (Radiochemical Centre, Amersham, United Kingdom), was reported by the manufacturer to be 99% pure and was not purified further.
Data
Analysis
All results are expressed as mean * SD. The relationship between bile flow and calcium output and bile acid output was obtained by linear regression; equations were calculated and scatter plots were printed using the CLINFO computer of the UCSD General Clinical Research Center. Biliary calcium and bile acid outputs were expressed in micromoles per kilogram per minute. Erythrito1 clearance was calculated as the product of bile flow and the bile to plasma ratio of 14C activity. Differences between the slopes of lines were compared using an analysis of variance followed by multiple range testing (30).
Results Relationship Calcium
ids,
0 1C
Between
Bile
Acid
Output
and
Output
For all three calcium output
natural, increased
micelle-forming bile in direct proportion
acto
bile
acid
increment
output in
667
bile
1
2 3 Bile Acid Output, pmol/kg.min
-* 4
[J, = 0.019 * 0.02rix; r = 0.98). 1B. (y = 0.014 + 0.033s: r = 0.92). Bata from
1. Relationship between bile acid output and calcium output. 1A. cholyltaurine chrnodeoxycholgltaurine (y = 0.023 + 0.027~; r = 0.93). 1C. ursodeoxycholyltaurine all experiments are shown. (See Table 2 for p values.]
conjugates of these bile acids were synthesized by N. Abadi and E. Ljungwe of this laboratory from the unconjugated acids using a modification of the method of Tserng et al. (28). The product, after removal of impurities from the reaction mixture by precipitation or solvent extraction, was obtained by freeze-drying. The conjugated bile acids were >99% pure by thin-layer chromatography on silicic acid using a solvent system of isoamyl acetateipropionic acid/n-propanoliwater (4 : 3 : 2 : 1 volivol) (29). Bile acid solutions used for infusion were isotonic, and had a bile acid concentration ranging from 20 to 34 mM. Secretin was purchased from Squibb, New York, N.Y. Somatostatin was generously provided by Clin-Midy, Montpellier, France. Both peptides were diluted in normal saline. SC2644 (2,4-dimethoxy+cyclohexyl-benzoyl-propionic acid) was a generous donation from Dr. Henry 0. Wheeler, who had received it some years previously from The Searle Laboratories (courtesy of Dr. P. Klimstra).
SE:CRETION
_~ _~~. ~~_~~_~~._._~__
4
2 3 Bile Acid Output, Hmol/kg.min 1
CALUIJP\l
(r
=
acid
0.94). output,
For
each
there
micromole was
between
in calcium output; this relationship may be defined as the bile aciddependent calcium secretion. Calcium output also increased linearly with the output of the hydroxy0x0 bile acid (r = 0.99; 0.024 ~molAcalcium/~mol Abile acid), Calcium output, however, was not identical for the three natural bile acids (Figure 1 and Tables 1 and 2). Calcium outputs were slightly lower for cholyltaurine and chenodeoxycholyltaurine (slope 0.026 and 0.027, respectively); that induced by- ursodeoxycholyltaurine was significantly higher (slope 0.035). although the values overlapped. The differences between the three bile acids are more apparent if dog identity is maintained (Figure 2). The greater calcium output induced by ursodeoxycholyltaurino was not related to bile flow (Figure X). Bile acid outputs were essentially identical to the rate of bile acid infusion when the infusion rate was 1 or 2 pmolikg . min. At the lowest rate of bile acid infusion, 0.5 Fmolikg . min, bile acid output sometimes exceeded the infusion rate, presumably because of the contribution of endogenous cholyltaurine synthesis, which is 0.05-0.10 LLmolikg min in the dog (31). At the highest bile acid infusion rates (4 and 4.5 pmol/kg 3 min), outputs averaged only about 80% for cholyltaurine and 12dehydrocholyltaurine. Bile acid output and bile flow during the last 60 min of each period was constant. Analysis of biliary bile acids by gas-liquid chromatography indicated that >90% of recovered bile acids had the composition of the infused bile acid for cholyltaurine and chenodeoxycholyltaurine, except during the first infusion period where an 80%-90% recovery was obtained depending on the percentage of endogenous cholyltaurine remaining in the animal. 12-Dehydrocholyltaurine was excreted largely unchanged in hepatic bile, as >85% of the biliary bile acids was composed of the hydroxy-oxo compound. Ursodeoxycholyltaurine displayed an anomalous pattern of biliary secretion. Bile acid outputs were similar to the rate of bile acid infusion when 0.026
and
0.035
prnol
increase
668
CUMMINGS
Table
AND
HOFMANN
GASTROENTEROLOGY
1. Effect of Individual Bile Acids During Steady State Choleresis Infusion rate (pm011
Agent
on Bile Flow, in the Dog” Bile flow WI
kg. min)
Study
n
Erythritol
Clearance,
(PI/kg.
CT CT CT CT
1.0
C,A
3.0 4.0 4.5
c A c
23 2 5 2
13.95 2 1.72 23.80 33.05 + 3.45 30.07
CDCT CDCT CDCT
0.5 1.0 2.5
C C c
4 7 2
13.02 + 4.08 14.07 t 2.46 24.15
LJDCT UDCT UDCT UDCT
0.5 1.0 2.0 4.0
A A A A
1 3 3 3
7.80 13.43 + 3.14 18.08 _t 2.98 15.90 t 2.07
12-DHCT 1 ?-DHCT 12-DHCT
1.0
2.0 4.0
A A A
3 3 3
20.10 2 1.85 32.21 k 1.67 50.48 2 6.51
and
Calcium
[Bile acid]
min)
13.35 2 2.73 16.17 31.95 t 4.06 32.63
Calcium output
(pmoli kg. min)
(mM)
87. No. 3
Secretion
Bile acid output
Erythritol clearance
kg. min)
and Bile Acid
Vol.
[Calcium]
(pmofi kg. min)
(mMl
70.07 '-c13.10 99.89 101.85 + 11.02 97.82
0.96 k 0.16 2.38 3.34 t 0.21
3.37 5 0.34 3.30 3.14 t 0.34
0.047 + 0.006 0.079 0.103 2 0.007
2.94
3.44
0.103
73.26 + 37.37 74.79 '- 13.94 107.57
0.90 2 0.29 1.05 + 0.25 2.68
3.85 + 0.91 3.64 !I 0.41 3.88
0.049 + 0.013 0.050 k 0.005 0.094
8.36 13.58 + 2.31 17.67 2 2.02 15.77 '- 2.55
74.2 85.42 k 15.88 105.37 k 23.45 114.28 k 28.17
0.58 1.13 2 0.24 1.89 t 0.43 1.81 t 0.51
3.82 3.84 k 0.58 4.42 + 0.81 4.65 + 0.93
0.030 0.051 r 0.012 0.079 I?-0.014 0.074 t 0.017
19.53 t 1.07 29.85 2 2.71 47.92 k 5.89
53.28 t 3.14 60.58 t 3.15 72.70 5 5.44
1.07 t 0.13 1.95 t 0.17 3.67 2 0.58
2.06 + 0.12 2.03 " 0.05 2.06 k 0.08
0.041 + 0.004 0.065 + 0.003 0.104 k 0.014
n. number of steady state periods; each period had four IO-min collection periods, taken at the end of the 80- or 9O-min infusions. acute preparation; C, chronic preparation: CT, cholyltaurine; CDCT, chenodeoxycholyltaurine; UDCT, ursodeoxycholyltaurine; 11Each
DHCT, 12-dehydrocholyltaurine. experiments.
value
is either
the mean
the infusion rate was I or 2 pmolikg . min, but dropped to 50% at 4 pmolikg . min. The proportion of ursodeoxycholic acid in biliary bile acids was >88%, except during the first infusion period in 1
of two experimental
steady
states
or the mean
f
A, 12SD of multiple
dog when the proportion of ursodeoxycholic acid was only 72%. When the plot of calcium output versus bile acid output was extrapolated to the ordinate, a positive
Table 2. Relationships Between Calcium Output and Bile Acid Output, Calcium Output and Bile Flow, and Bile and
Bile Acid
Significance Slope
(~moli~mol) A. Calcium
y-Intercept (pmolikg . min)
r
CDCT
UDCT
NS
of observed
12. DHCT
differences
SC
SEC
SST
40.001
40.001
co.001
NS
NS NS
NS
output vs. bile acid output
Bile acid CT CDCT UDCT 12-DHCT B. Calcium output Agent CT CDCT UDCT 12-DHCT SC2644 Secretin Somatostatin
0.026 0.027 0.035 0.024
CT. choIyItaurine; 2644:
0.019
0.98
0.023 0.014 0.018
0.93
NS NS
0.92 0.99
vs. bile flow 0.0028 0.0037 0.0048 0.0021 0.0018 0.0016 0.0019
C. Bile flow vs. bile acid output Agent CT 8.46 CDCT 6.01 UDCT 5.46 12-DHCT 11.29
compound
Flow
OutDut
0.009 -0.001
-0.008 -0.001 0.009 0.024 0.021
4.48 8.03 7.26 9.14
CDCT, chenodeoxycholyltaurine; SEC, secretin; SST, somatostatin.
0.96
<0.005
0.90 0.86 0.99 0.93 0.83 0.91
co.005 10.001
NS NS -
0.96
0.86 0.79 0.99 UDCT,
ursodeoxycholyltaurine;
II-DHCT,
12-dehydrocholyltaurine;
SC,
Searle
September
1 Y84
.031
Figure
2
, 1 I “rsodeoxy Cnolyl CnenOdeOxy C”
Ratio of mean calcium output to mean bile acid output during infusion of 1 pmolikg min bile acid under steady state conditions in six separate dog experiments. Lines connect points from individual animals.
ural bile acids had an additional effect on calcium secretion unrelated simply to bile volume: A canalicular choleresis was also induced by infusion of the hydroxy-oxo bile acid. The induced calcium output with this bile acid (0.0021 PmolAcalcium/plAbile flow) had the same relationship to bile flow as that observed with SC2644 (0.0018 pmolhcalcium/&bile flow) (Figure 3B). The output of calcium per microliter of bile volume was significantly lower for the hydroxy-oxo bile acid than for that induced by infusions of the three micelle-forming bile acids, which ranged from 0.0028 to 0.0048 ~molAcalcium/~lAwater (Table 2B).
DuctuJar intercept was observed suggesting that calcium secretion would occur at zero bile acid output. Because bile acids induce bile acid-dependent bile flow by definition, this calcium output is the “bile acidindependent” calcium output, also by definition. and is present in the bile acid-independent bile flow.
Relationship Bile Flow
Between
Calcium
Output
and
The bile acid infusions also induced a linear increase in bile flow. The relationships between calcium output and bile flow induced by infusions of the individual bile acids are summarized in Figure 3C. The differing effects of the individual bile acids are apparent. During SC2644-induced choleresis, calcium output also increased linearly with bile flow despite there being little change in bile acid output. The data indicate that canalicular calcium secretion can be increased independent of any increase in bile acid secretion. For a given bile flow the calcium output induced with SC2644 was far less than that seen with cholyltaurine (Figure 3A) and the two other micelle-forming bile acids, indicating that these nat-
1
3A
Pigure
0
*
---+
Modification
During secretin-induced ductular choleresis, the average bile acid output remained unchanged as bile flow increased (Table 3). Caicium output rose as the bile flow increased (Figure 4A). The overall change in bile flow was small. and therefore the increase in calcium output was small. During somatostatin-induced anticholeresis, bile acid output remained unchanged as bile flow decreased. Calcium output fell as bile flow fell. Calcium output to bile flow ratios for both secretin and somatostatin (0.0016 and 0.0019 ~molAcalcium/~l~bile flow, respectively) were similar to that noted during SC2644-induced canalicular choleresis (O.O017~molAcalciumiplAbile tlow) (Figure 4B). In these studies, bile acid output was maintained by a continuous infusion of cholyltaurine. Although the average bile acid output was 1 pmolikg . min, the output rates varied randomly during the somatostatin and secretin experiments. To eliminate any possible influence of this cholyltaurine-induced calcium secretion on total calcium secretion, each bile acid output was multiplied by the slope shown in Figure 1A and the product was then subtracted from each calcium output. The resultant Icalcium output should be the influence of duct&r modification alone (Figure 4C). The slope lz’as essentially un-
_
20 40 Bile Flow, pl/kg.min
60 Bile Flow, pl/kg.min
Bile Flow, pl/kg.mln
3. Relationship between bile flow and calcium output. 3A. cholyltaurine (A) contrasted with the c,malic.ular c.holeretir. ~2644 (II]. 3B, 12.dehydrocholyltaurine (W) compared to SC2644 (II). XC. data for all agents used to modify canalic.ular flo~v, ursodeox~cholyltaurine (A), chenodeoxycholyltaurine (0). cholyltaurint: (A). 12.deh~drochol!ltallrine (W). ~~2644 (-I). (ore Table 2 for linear regression coefficients.)
670 CUMMINGS AND HOFMANN
GASTROENTEROLOGY
Vol.87,No. 3
Table 3. Effect of a Canalicular Choleretic (X2644), or Somatostatin- or Secretin-Induced Ductular Modification of Bile Flow on Erythritol Clearance, and Bile Acid and Calcium Secretion During Steady State Choleresis in the Dog”
Infusion rate
Agent
Study
n
Bile flow (&kg. min)
15.48 29.60
I. Induced SC2644 SC2644
canalicular choleresis 0.5 mgimin C 3 mgimin C
2 2
II. Induced Secretin Somatostatifl
ductular modification 6 Pgikg. h C 8 kg/kg. h C,A
2 5
a Values
given
are mean
6.31
of two experiments
20.30 + 1.19 or mean
[Calcium]
(mW
(mM1
kg. min]
23.92 35.26
36.85 24.97
0.58 0.73
2.96 2.15
0.040 0.063
11.85 10.64 t 1.09
40.36 133.49 i- 17.89
0.87 0.83 ? 0.06
2.71 5.34 t 0.42
Concentration
(Figure 5)
Calcium concentration ranged from 1.6 to 6.4 mM, reflecting the balance between induced calcium secretion and induced biliary water secretion. Calcium concentrations were higher when micelleforming bile acids induced bile flow or when ductular water absorption was induced by somatostatin. Calcium concentrations were lower during canalicular secretion induced by SC2644 or the hydroxy-
i
20 30 10 Bile Flow, pl/kg.min Figure
40
[Bile acid]
t SD of five experiments:
changed. A similar normalization procedure was carried out for data obtained during the SC2644 infusions, i.e., the bile acid-dependent component of calcium output was subtracted from the calcium output. The small difference between the effect of SC2644 and that of ductular modification was abolished after factoring out the bile acid component. This new line of bile acid-independent flow was contrasted to the line for bile acid-dependent flow that can also be deduced by subtracting the bile acidindependent calcium output (or y-intercept of calcium output versus bile acid output) from the total calcium output. The comparison of these two lines indicates more clearly that calcium output can be related to bile acid-depehdent flow and bile acidindependent flow. Calcium
48 0
Calcium output
Bile acid output (pmof/kg. min)
Erythritol clearance (/.&kg * min)
for definition
of column
0x0 bile acid and during induced by secretin.
(kmol/
0.033
headings.
see Table
ductular
dilution
0.055 + 0.004 1
of bile
Discussion The major conclusion to be drawn from these studies is that biliary calcium output in the dog has both canalicular and ductular components. CanaJicuJar
Factors
The results indicate that at least two components of canalicular bile influence the biliary output of calcium. The first component is most simply attributable to convective flow of water and electrolytes from plasma to bile induced by osmotic forces generated by active canalicular secretion of ions. The active secretion of ions may be that composing the bile acid-independent fraction of canalicular bile, that induced by a canalicular choleretic agent such as X2644, as well as bile acid anions in monomeric OF micellar form. We suggest the term “osmotic fraction” for this component of canalicular calcium output. Nonetheless, becatise of the extremely high correlation between bile acid output and bile flow, i.e., the constancy of the linkage between water and bile acid secretion rates, the bile acid-dependent
~*_~~~._ : : 10 30 20 Bile Flow, pl/kg.min
40
4C 0
10
20
30
40
Bile Flow, /.Nkg.min
4. Relitionship between bile flow and calcium output. 4A, effect of ductular modification (somatostatin. A; control, W; secretin, 0). 4B, effect of ductular modification; pooled values of somatostatin and secretin (W) compared to the effect of the canalicular choleretic SC2644 (0). 4C, calculated effect of the bile acid-dependent component of cholyltaurine-induced choleresis on calcium output (bold line), compared to the calculated effect of the bile acid-independent component during ductular modification of bile flow (m). or during canalicular choleresis induced by SC2644 (3) on calcium output.
September
Figure
BlL,IAKY
1~4
(:AI.(:ll’hl
SII(:KE’I‘ION
671
5. A, effect of agents influencing canalicular and ductular bile flolv on c.al[.ium I:clnc:entratlon. Bt\,. c.bolyltaurine: BA,,. XI. S(ZfX4: SSl‘. somatostatin: chenodeoxvc.holyltaurine: BA ,,,, ursodeoxycholyltaurine: BA,, , 12-tleh\:drochol~~ltaurinr: SFX:. secret-in; mean 2 SD. 58. relationship between calcium concentration and bile flo\v produced by +,ents causing tluc.tulal modification (somatostatin, A; control. ?? ; secretin, 0). 5C. relationship bet\veen bile flo\v and r:al[.ium LOIICentration, during infusion of cholyltaurine (01, which has a low critical micellar conc.rntration value. compared to th,lt of Ir-dt:llvdrochol~ltaurine (W) which has a relatively high critical micellar concentration value.
calcium secretion correlates equally well with bile volume or with bile acid output for the three micelleforming bile acids. The apparent calcium concentration of the osmotic fraction, 1.6-2.1 mM, can be determined from the relationship of calcium movement to water movement. This concentration is greater than the ionized calcium concentration in dog plasma (1.17-1.39 mM) (32). The observed concentration of calcium in the “osmotic fraction” reflects the algebraic sum of all calcium and all water movements in the biliary tree, and our data provide no direct information on the calcium concentration of the osmotic fraction of canalicular bile per se. The second component would appear to be related to the presence of micelles or micelle-forming bile acids in bile. This second canalicular fraction cannot be explained by osmotic forces, because for any given bile flow, calcium output was greater when the micelle-forming bile acids were infused. The “micellar” fraction appeared to be influenced by the bile acid type in our experiments in that the dihydroxy bile acids induced a greater calcium output than cholyltaurine. However, it is beyond the scope of the present experiments to define the nature of the forces involved in the micellar component. It is possible that both binding by micelles and binding by bile acid anions may be involved, because cholyltaurine, at least. binds calcium ions at a concentration below its critical micellar concentration. Bile acid micelles are known to bind calcium (l&20.3335), but only cholyltaurine has been studied in detail (19). The influence of biliary lipid which is also present is relatively unexplored, but it is clear that the addition of phospholipid to bile acid micelles increases their binding of calcium (33,35), and biliary phospholipid secretion might also influence biliary calcium secretion. The following equation is proposed to include the two components of canalicular calcium output: c I( ~z( ,,,_,/ A, + I:, ,,,I,+k,
(11
secretion of c.:alcium. (:.I = where c,~ = canalicular canalicuiar bile flow (plikg . min). k, = a phenor&ologic linkage coefficient relating calcium output to of canalicular bile flow (~moli~l), c:./,,, = secretion micelle-forming bile acids or micelles (pmol/ kg . min). and k,. = variable phenomenologic linkage coefficient relating calcium secretion to the secretion (or presence] of micelle-forming bile acids (pm01 calcium/pm01 micelle-forming molecules) or micelles. This partitioning of canalicular calcium outputs into an osmotic fraction and a micellar fraction contrasts with the usual partitioning of canalicular solute output into bile acid-independent and bile acid-dependent fractions, as is illustrated in Figure 6. We speculate that this new system of categorization will be useful for solutes that have appreciable binding to micelles. Such a scheme is not necessar! for solutes that do not associate \vith micelles such as erythritol. as the micellar fraction should be zero.
Ductular Fuctors The data show con\rincinglg that ductular modification of bile influences calcilum output in a predictable manner. Calcium output is diminished by ductular absorption and augmented by ductular secretion. The composition of the electrolyte fraction moving across the ductules in response to secrctin or
osmotic effect of transported ions Erythritol: WV-’ bile acid independent Calcium: Figure
-
osmotic ellect of bile acid monomers (and micelles)
additional effect of micelle-forming bile acids
bile acid dependent
Y osmotic
‘W
“micellar”
li. klech,inisnls used to dt!s~ rib12 c,illliii~.ulCir r~rythritol secreti cutl ~ompart~l \vith tl~os~, ~~CI~IWVI lor ~.dilalic:ular cal[.ium src.rc,tion
672
CUMMINGS
AND
HOFMANN
GASTROENTEROLOGY
somatostatin has been calculated by a number of authors (ll,l3), but calcium has not been included in these calculations. Our data indicate that the apparent calcium concentration of this fraction is about 1.7 mM, which is less than the total calcium concentration in bile collected more distally, and contrasts with the sodium concentration in this fraction which is equal to that present in bile. The simplest explanation of the low calcium concentration is that a fraction of biliary calcium is bound; in addition, the reflection coefficient of calcium ions may be greater than that of sodium ions. The ductular modification term can be added to the previous phenomenologic equation to give an equation for total biliary calcium secretion. Thus (21
+, &, tJ,‘*= CJY‘
where tJ. = total biliary calcium secretion and dJ. = ductui’ar calcium secretion (+) or ductular cair cium absorption (-). Combining equations (1) and (z), one obtains * k, + CJ,,,,, * k,. 2 d,,,?o *k,, r+,, = CJ&O
(31
where k3 is a phenomenologic linkage coefficient relating ductular water movements to calcium movements (micromoles per microliter). Note that in our studies in the mongrel dog k, = k,. Our data thus define for the first time the major physiologic determinants of calcium output in bile. Because the postulated osmotic forces influence both water and calcium similarly, calcium concentration remains relatively constant over a wide range of changes in osmotic flow, e.g., induced by SC2644 or the hydroxy-oxo bile acid. Because micelle-forming bile acids induce a calcium output in addition to that explicable by osmotic forces, calcium concentration increases when micelle-forming bile acids are infused. Lastly, because the electrolyte fraction moving into or out of the ductules is relatively low in calcium, calcium concentrations are decreased to some extent by ductular secretion and increased by ductular absorption. Limitations
of These Experiments
The data presented in these studies which comprise only calcium outputs and calcium concentrations do not bear directly on calcium precipitation in bile, as the majority of biliary calcium is bound to micelles, anions, or proteins (33,34,36). Nonetheless, elucidation of the physicochemical factors determining calcium activity in bile must begin with an understanding of the physiologic factors influencing overall calcium movements. References 1. Sutor DJ, Wooley SE. The nature and incidence containing calcium. Gut 1973;14:215-29.
of gallstones
Vol.
87, No. 3
2. WosiewitzU. Limy bile and radiopaque calcified gallstones: combination of analytical, radiographical,and micromorpho-
a
logical investigation. Path Res Pratt 1980;167:273-86, 3. Wosiewitz U, Wolpers C, Quint P. Roentgennegative Pigmentgallensteine. Leber Magen Darm 1978;8:353-60. 4. Been JM, Bills PM, Lewis D. Microstructure of gallstones. Gastroenterology 1979;76:548-55. 5. Bogren H. The composition and structure of human gall stones. Acta Radio1 (Stockh) 1964; Supplementum 226. 6. Boyer JL. New concepts of mechanisms of hepatocyte bile formation. Physiol Rev 1980;60:303-20. 7. Erlinger S. Hepatocyte bile secretion: current views and controversies. Hepatology 1981:1:352-59. 8. Balabaud C, Kron K, Gumucio JJ. The assessment of the bile salt-nondependent fraction of canalicular bile water in the rat. J Lab Clin Med 1977;89:393-9. 9. Baker AL, Wood AB, Moossa AR, Boyer JL. Sodium taurocholate modifies the bile acid-independent fraction of canalicular bile flow in the rhesus monkey. J Clin Invest 1979;64:312-20. 10. Forker EL. Mechanisms of hepatic bile formation. Ann Rev Physiol 1977;39:323-47. 11. Preisig R, Cooper HL, Wheeler HO. The relationship between taurocholate secretion rate and bile production in the unanesthetized dog during cholinergic blockade and during secretin administration. J Chn Invest 1962;41:1152-62. 12. Jones RS, Geist RE, Hall AD. The choleretic effects of glucagon and secretin in the dog. Gastroenterology 1971;60:64-8. 13. Rene E, Danzinger RG, Hofmann AF, Nakagaki M. Pharmacologic effect of somatostatin on bile formation in the dog. Gastroenterology 1983;84:120-9. 14. Roda A, Hofmann AF, Mysels KJ. The influence of bile salt structure on self-association in aqueous solutions. J Biol Chem 1983;258:6362-70. 15. Thomas JE. An improved cannula for gastric and intestinal fistulas. Proc Sot Exp Biol Med 1941;46:260-5. 16. Gilmore IT, Barnhart JL. Hofmann AF, Erlinger S. Effects of individual taurine-conjugated bile acids on biliary secretion and sucrose clearance in the unanesthetized dog. Am J Physiol 1982;5:G40-6. 17. Danzinger RG, Nakagaki M, Hofmann AF, Ljungwe EB. Differing effects of two hydroxy-7-oxo taurine conjugated bile acids on bile flow and biliary lipid secretion in the dog. Am J Physiol 1984;246:G166-72.. 18. Williamson BWA, Percy-Robb IW. The interaction of calcium ions with glycocholate micelles in aqueous solution. Biochem J 1979;181:61-6. 19. Moore EW, Celic L, Ostrow JD. Interactions between ionized calcium and sodium taurocholate: bile salts are important buffers for prevention of calcium-containing gallstones. Gastroenterology 1982;83:1079-89. 20. Rajagopalan N, Lindenbaum S. The binding of Ca’+ to taurine- and glycine-conjugated bile salt micelles. Biochim Biophys Acta 1982;711:66-74. 21. Sewell RB, Hoffman NE, Smallwood RA, Cockbain S. Bile acid structure and bile formation: a comparison of hydroxy and keto bile acids. Am J Physiol 1980;238:GlO-7. 22. Cook DL, Lawler CA, Green DM. Studies on the effect of hydrocholeretic agents on hepatic excretory mechanisms. J Pharmacol Exp Ther 1954;110:293-9. 23. Barnhart JL, Combes B. Characterization of SC2644-induced choleresis in the dog. Evidence for canalicular bicarbonate secretion. J Pharmacol Exp Ther 1978;206:190-7. 24. Gibson GE, Forker EL. Canalicular bile flow and bromosulfophthalein transport maximum. The effect of a bile independent choleretic, SC2644. Gastroenterology 1974:66:1046-53. 25. Wheeler HO, King KK. Biliary excretion of lecithin and cholesterol in the dog. J Clin Invest 1972;.51:1337-50. 26. Turley SD, Dietschy JM. Re-evaluation of the 3Lu-hydroxyster-
September
BILIAKY
1984
oid dehydrogenase assay for total bile acids in bile. J Lipid Res 1978;19:924-8. 27. Nakagaki M, Danzinger KG, Hofmann AF, Roda A. Hepatic biotransformation of two hydroxy-7-oxotaurine-conjugated bile acids in the dog. Am J Physiol 1983;245:G411-7. 28. Tserng K-Y, Hachey DL, Klein PD. An improved procedure for the synthesis of glycine and taurine conjugates of bile acids. J Lipid Res 1977;18:404-7. 29. Hofmann AF. Thin-layer adsorption chromatography of free and conjugated bile acids on silicic acid. J Lipid Res 1962; 3:127-a. 30. Zar JH. Biostatistical analysis. Englewood Cliffs, N.J.: Prentice-Hall. 1974:620. 31. Wollenweber J. Kottke BA, Owen CA Jr. Effect of nicotinic acid on pool size and turnover of taurocholic acid in normal
and
hypothyroid
dogs.
Proc
CALCltJM
Sot
Exp
SECKETION
Biol
Med
673
1966;
122:
1070-5. 32. Cummings S. Hofmann AF. Determinants of biliary ionized calcium in the dog (abstr). Gastroenterology 1983;84:1131. 33. Williamson BWA, Percy-Robb IW. Contribution of biliary lipids to calcium binding in bile. Gastroenterology 1980: 78:696-702. 34. Sutor DJ, Wilkie LI. Jackson MJ. Ionized calcium in pathological human bile. J Clin Path01 1980:33:86-8. 35. Jones C. Hofmann AF. Mysels KJ. Roda A. A physicochemical explanation for the rarity of precipitation of calcium bile salts in gallstones (abstr). Gastroenterology 1984;86:1325. 36. Sutor DJ. Wilkie LI. Calcium in bile and calcium gallstones. Clin Chim Acta 1977:79:119-2’7,
salts
in
1984;87:664-73
Physiologic Determinants of Biliary Calcium Secretion in the Dog SUSAN A. CUMMINGS
and ALAN F. HOFMANN
Division of Gastroenterology, Department of Medicine. California at San Diego, San Diego, California
Studies were carried out to define the origins and physiologic determinants of biliary calcium secretion in the dog. CanaJicuJar bile flow was varied by infusions of several bile acids or a canalicular choleretic agent, SC2644. Three natural bile acids (cholyltaurine, chenodeoxycholyltaurine, and ursodeoxycholyltaurine) that have a low critical micelJar concentration and a synthetic hydroxy-oxo bile acid (12-dehydrocholyltaurine) with a much higher critical micellar concentration were used. Calcium output, bile acid output, and bile flow were determined; canalicular bile flow was assessed by measurement of erythritol clearance. Calcium output increased linearly with bile acid output for all three natural bile acids (r = 0.94; 0.026-0.035 PmoJAcaJcium/~moJAbiJe acid), but chenodeoxycholyltaurine and ursodeoxycholyltaurine infusions induced a slightly greater secretion of calcium for a given bile acid output and bile flow than cholyltaurine. CaJcium output also increased linearly with bile pow, Received February 29, 1984. Accepted April 10, 1984. Address requests for reprints to: Alan F. Hofmann, M.D., Department of Medicine, UCSD Medical Center, 225 Dickinson Street, San Diego, California 92103. This study was supported in part by National Institutes of Health Grants AM 21506, AM 32130, and RR 00827. S. A. Cummings is a fellow of the Medical Research Council of Canada. Her present address is Department of Medicine, Yale University, New Haven, Connecticut. A portion of this work was presented at the 1983 Annual Meeting of the American Gastroenterological Association and published in abstract form in Gastroenterology 1983;84:1131. The authors thank Dr. Joseph Steinbach, Dr. M. Sawkat Anwer, Dr. James Barnhart, Dr. Edward W. Moore, and Devorah Gurantz for helpful discussions; Dr. Ian Gilmore and Dr. Rudy Danzinger for bile samples collected previously; Linda Clayton for providing technical assistance; Dr. Gerhard Schrauzer of the Department of Chemistry for making available the atomic absorption spectrometer: Paul Schragg of the General Clinical Research Center for providing help in the use of the CLINFO computer; Dr. Adolf Krekel of Pharmazell, Diamalt, Redenfelden, Federal Republic of Germany for donating the bile acids; and Vicky Huebner for manuscript preparation. 0 1984 by the American Gastroenterological Association 00165085/84/$3.00
School
of Medicine,
University
of
whether bile flow was induced by the canalicular choleretic SC2644 [r = 0.93; 0.0018 PmoJAcaJciuml PlAwater) or the hydroxy-oxo bile acid (r = 0.99; 0.0021 ~moJAcaJcium/~JAwater). Because the calcium output induced by the three micelle-forming bile acids was greater than that which could be explained by the induced increase in biJe flow per se, it was deduced that canalicular calcium has two origins: a micellar component that is dependent on the presence of micelles or micelle-forming bile acids and an osmotic component that is dependent on osmotic forces, e.g., caused by secretion induced by SC2644 or bile acid molecules and possibly bile acid micelles. A small bile acid-independent output of calcium was also detected and should contribute to the osmotic fraction. To determine whether ductuJar modification of bile flow influenced biliary calcium secretion, ductular secretion was induced by secretin and ductular absorption was induced by somatostatin. Changes in calcium output followed water movements, increasing with ductular secretion and decreasing with ductular absorption. The relationship between changes in ductular bile flow and calcium output was identical during induced ductular secretion (r = 0.83; 0.0016 ~moJAcaJcium/ PJAwater) or induced ductular absorption (r = 0.92; 0.0019 ~moJAcaJcium/~Awater). Thus calcium movements in relation to water movements were identical for the osmotic component of canalicular bile and the ductular component of bile. The experimental manipulations in bile secretion caused anticipated changes in biliary calcium concentration which ranged from 1.6 to 6.4 mM. These results indicate that at the usual rates of bile acid secretion, most biliary calcium originates at the canaliculus and is bile acid-dependent. The biliary ductule is permeable to calcium, but the effect of ductular modification of bile flow on biliary calcium outputs is small.
Abbreviations centration
used
in this
paper:
CMC, critical
micellar
con-
September
BILIAKYCALCIUM SECRETION 665
1984
Precipitation of calcium in the biliary tree is a key event in the pathogenesis of human cholelithiasis, as calcium is the major cationic component of noncholesterol gallstones (l-3), and calcium salts often serve as a nidus for nucleation of cholesterol gallstones (43). For calcium precipitation to occur, the product of calcium activity and the activity of a calcium-sensitive anion (with appropriate exponents) must exceed the solubility product. Calcium activity in bile is determined by total calcium concentration minus that bound to micelles, anions, or proteins in bile. The total calcium concentration is determined by biliary calcium secretion in relation to biliary water secretion. Bile is formed in the canaliculus and modified in the ductules. It has been useful to divide canalicular bile flow into two components, the bile acid-dependent and the bile acid-independent fraction (reviewed in References 6 and 73, although the physiologic meaning of the bile acid-independent fraction continues to be questioned (8,9). Bile is further modified in the biliary ductules where the absorption or secretion of a bicarbonate-rich fraction may occur (10); these ductular modifications of bile may be influenced by pharmacologic doses of some of the gastrointestinal hormones such as secretin (ll,12), glucagon (12), or somatostatin (13). This paper reports studies aimed at defining the determinants of biliary calcium secretion in the bile fistula dog. Experiments were designed to distinguish canalicular from ductular events, and to divide canalicular events into the bile acid-dependent and bile acid-independent components. To alter bile acid-dependent flow, three natural bile acids (cholyltaurine, chenodeoxycholyltaurine, and ursodeoxycholyltaurine) were infused at varying rates. In addition, to gain further insight into the mechanism by which bile acids induced calcium secretion, a synthetic taurine-conjugated hydroxy-oxo bile acid (1Zdehydrocholyltaurine) was used. This bile acid has a critical micellar concentration (CMC) (40 mM) that
is considerably
higher
than
that
of the
common
bile acids (2-10 mM) (14). It was reasoned that the majority of the anions of this bile acid would be present in bile in nonmicellar form; thus, the use of this bile acid might permit the role of the micelle in biliary calcium secretion to be defined. Bile acidindependent flow was induced by means of a canalicular choleretic agent, SC2644 (SC is an abbreviation for Searle Compound, Searle Pharmaceuticals, Inc.. Chicago, Ill.). Ductular modification of bile flow was achieved by infusing secretin which causes ductular secretion of a bicarbonate-enriched component of bile (11,12), or somatostatin which causes ductular absorption of
a component of bile with a similar electrolyte position (13).
com-
Methods Experimental
Design
The experimental approach featured the collection of hepatic bile samples after the establishment of a steady state; individual components of biliary secretion were varied systematically by the use of c.holeretic agents. hormones, or various bile acids that were administered intravenously in random order. The experimental design was varied according to the agent [see below). Two animal preparations were used. Acute bile fistula dogs were prepared surgically and studied while anesthetized. Chronic bile fistula dogs had been prepared bl previous cholecystectomy and placement of a Thomas duodenal cannula (IS), permitting sampling of hepatic bile while the animal was awake and alert. The results from these two preparations were identical, permitting pooling of the data. Modification of canalicular bile pow. BILE ACIDDEPENDENT FLOW. Bile acid-dependent flow was varied b\ infusing bile acids over the physiologic: range of 0.5-4.5 pmolikg . min. The majority of studies were carried out with cholyltaurine, the predominant bile acid of the dog. Additional studies were carried out with chenodeoxycholyltaurine and ursodeoxycholyltaurine. Two experimental designs were used. The first design consisted of singledose experiments using the ABA’C design. A and A’ were control periods during which cholyltaurine was infused. B and C were periods in which chenodeoxycholyltaurine or ursodeoxycholyltaurine was infused in random order. The second design consisted of dose-response studies in which three doses of a given bile acid were infused in random order; in some studies, two doses of cholyltaurine were infused before and after the dose-response study of the test bile acid. Experimental details and data for bile flow, erythritol clearance, and biliary lipid secretion have been published previously for some of these studies (16,171. When data from these studies were analyzed, it was found that the induced calcium secretion was greater than that explicable by the increase in bile volume caused by the bile acid. These three bile acids are known to form micelles (14) when present above their CMC (2-10 mM), and bile acid micelles are known to bind calcium (8-20). Accordingly, it was elected to define the influence of a bile acid that would be present to a considerable extent in monomeric solution in bile rather than in micelles. 12Dehydrocholyltaurine (3a,i’Lu-dihydroxy-12-oxo-cholanoyltaurine) was chosen because this compound is known to have a relatively high CMC value of 40 mM (14) and is also known to be a powerful choleretic agent in the cat (21). This compound was studied at three doses, and its infusion was preceded and followed by infusions of two doses of cholyltaurine. BILE ACID-INDEPENDENT FLOW. Bile acid-independent flow was induced by administration of a substituted propionic acid derivative SC2644 which is known to be a powerful canalicular choleretic in the dog, inducing a striking
666
CUMMINGS AND HOFMANN
increase in bile volume associated with a parallel increase in erythritol clearance (22-25). The experimental design featured infusion of various doses of cholyltaurine followed by infusion of the canalicular choleretic agent; the choleretic had a prolonged effect on bile flow, so that period randomization was not possible. The infusion rate of SC2644 was 0.5 mgimin (0.06 Fmolimin . kg] for 60 min followed by 3 mgimin for 80 min. Modification of ductular j7ow. Ductular secretion was induced by secretin, 6 pgikg . h; and ductular absorption by somatostatin, 8 pgikg . h. The experimental design featured infusion of the hormones somatostatin or secretin in randomized order; each hormone period was preceded and followed by a 60-min control period. After a steady state was obtained, secretin or somatostatin was given for an 80-min period. Throughout the entire experiment cholyltaurine was infused at a dose of 1 pmolikg min. The final period featured the infusion of an additional 2-pmoli kg. min dose of cholyltaurine (total infusion rate of 3 pmolikg . min) to define the effect of cholyltaurine on calcium output under these experimental conditions.
Experimental
Procedure
Acute bile fistula dogs. Six adult male mongrel dogs, 21-32 kg, fasted for 16 h, were anesthetized with an intravenous bolus (30 mgikg) of sodium pentobarbital (Abbott Laboratories, North Chicago, 111.). Preparation included midline laparotomy, ligation of the renal pedicles (to prevent erythritol excretion), ligation of the cystic duct, and cannulations of both external jugular veins (Bard Biomedical I-Cath 16GA, Sunderland, U.K.) and lateral saphenous vein (Bard Biomedical I-Cath 18GA). The common bile duct was ligated close to its insertion in the duodenum and was cannulated proximal to this point using polyethylene tubing (Intramedic PE 190, Becton, Dickinson and Co., Parsippany, N.J.). An additional 200 mg of sodium pentobarbital was administered in 50-mg boluses over the ensuing 8 h to maintain adequate anesthesia. (The boluses were given only during control periods to minimize any possible effect of pentobarbital on bile flow.) Chronic bile fistula dogs. Three adult male mongrel dogs, 22-30 kg, had been prepared previously by cholecystectomy and insertion of a Thomas cannula in the duodenum. Dogs were studied after a 16-h fast. The duodenal cannula was opened and a polyethylene tube (Intramedic PE-190, OD 1.7 mm) was inserted 5-6 cm into the common bile duct and brought out through a small hole in the bung used to occlude the duodenal fistula. Both lateral saphenous veins were cannulated at the beginning of the study. The dogs were supported by a sling. Brief sedation was required during insertion of the cannula into the bile duct. Sodium thiamyl was used [Park-Davis, Detroit, Mich.). Throughout each experiment, the dogs received two continuous intravenous infusions delivered by two peristaltic pumps (Harvard Apparatus Co., Dover, Mass.). The first infusion contained the test agent, i.e., bile acid, SC2644, or the hormone. The second contained either cholyltaurine and [l*C]erythritol or [“Clerythritol alone, depending on the experiment. Bile was drained for 40 min before starting the experiment. Each agent was infused
GASTROENTEROLOGY
Vol. 87. No. 3
for 80 or 90 min, and bile was collected by gravity in preweighed vials at IO-min intervals, after a steady state of bile flow had been obtained. This was after 20 min or after 60 min, depending on the experiment. A 3-ml blood sample was drawn at the midpoint of most bile collections via a third intravenous line. In the chronic animals, a tracer priming dose of [‘“Clerythritol radioactivity, 3 &i, was injected as a bolus, followed by a maintenance dose of 0.04 &/min. In the acute animals, a 5-&i priming dose was given, followed by a maintenance dose of 0.15 &i/min.
Analytical
Procedures
Calcium was determined by atomic absorption spectrophotometry (Perkin-Elmer 373, Perkin-Elmer Corp., Norwalk, Conn.). Bile, 100 ~1, was added to 5 ml of diluent. The diluent contained 20 mM lanthanum chloride (50 ml of 0.4 M lanthanum chloride/L of diluent) and 0.45 M hydrochloric acid (25 ml of concentrated HCliL of diluent). The precipitate occurring when the acid diluent was added was removed by centrifugation at 2000 g for 20 min. Calcium determination was performed on the supernatant, as preliminary experiments had shown that calcium was not present in the precipitate. To validate this procedure, known concentrations of calcium were added to each bile solution. A standard curve was then obtained for each bile sample and the value of calcium in the unaltered bile sample could be calculated by extrapolation of the curve to the ordinate. Using 49 different bile samples, the correlation between this predicted value and that observed was r = 0.96. In other experiments, Triton X-100 (Sigma Chemical Co., St. Louis, MO.) 1% was added to the different samples to prevent the usual precipitation that occurred during dilution. Values for calcium were unchanged. Total bile acids were estimated by the 3-hydroxysteroid dehydrogenase assay (26). The biliary bile acid composition of representative bile samples was determined by gasliquid chromatography of the methyl ester acetates (27). [“C]Erythritol radioactivity was measured in bile and plasma by liquid scintillation counting. Samples of 0.5 ml were first bleached by the addition of 50-75 ~1 of 30% hydrogen peroxide. Samples were left to bleach overnight: the next day 10 ml of liquid scintillation fluid (Aquasol-2, New England Nuclear, Boston, Mass.) was added. Samples were then left 1 wk before counting because of chemiluminescence from the hydrogen peroxide. Quenching was so uniform that raw counts were used to calculate plasmai bile ratios of radioactivity. Chemicals,
Radiochemicals,
and
Hormones
Cholic acid was purchased from Aldrich Chemical Co. [Milwaukee, Wis.) and was purified by crystallization from 95% ethanol. Chenodeoxycholic acid, ursodeoxycholit acid, and l2-dehydrocholic acid (3a,7a-dihydroxp-12oxo-cholanoic acid) were generously donated by Pharmazell, Diamalt Aktiengesellschaft, Redenfelden, Federal Republic of Germany; we acknowledge the generous support of Dr. Adolf Krekel. All bile acids were of high purity and were used without further purification. The taurine
September
IA
Figure
BILIARY
1984
b-_--
1 2 Bile Acid Output,
3 pmol/kg.min
i-_--M
4
0 18
compound was converted to its sodium salt by titration with 1 N NaOH, and a solution containing 1.6 or 9.3 mM was infused at 1 mlimin. [U-‘“ClErythritol, specific activity 100 mCiimmo1 (Radiochemical Centre, Amersham, United Kingdom), was reported by the manufacturer to be 99% pure and was not purified further.
Data
Analysis
All results are expressed as mean * SD. The relationship between bile flow and calcium output and bile acid output was obtained by linear regression; equations were calculated and scatter plots were printed using the CLINFO computer of the UCSD General Clinical Research Center. Biliary calcium and bile acid outputs were expressed in micromoles per kilogram per minute. Erythrito1 clearance was calculated as the product of bile flow and the bile to plasma ratio of 14C activity. Differences between the slopes of lines were compared using an analysis of variance followed by multiple range testing (30).
Results Relationship Calcium
ids,
0 1C
Between
Bile
Acid
Output
and
Output
For all three calcium output
natural, increased
micelle-forming bile in direct proportion
acto
bile
acid
increment
output in
667
bile
1
2 3 Bile Acid Output, pmol/kg.min
-* 4
[J, = 0.019 * 0.02rix; r = 0.98). 1B. (y = 0.014 + 0.033s: r = 0.92). Bata from
1. Relationship between bile acid output and calcium output. 1A. cholyltaurine chrnodeoxycholgltaurine (y = 0.023 + 0.027~; r = 0.93). 1C. ursodeoxycholyltaurine all experiments are shown. (See Table 2 for p values.]
conjugates of these bile acids were synthesized by N. Abadi and E. Ljungwe of this laboratory from the unconjugated acids using a modification of the method of Tserng et al. (28). The product, after removal of impurities from the reaction mixture by precipitation or solvent extraction, was obtained by freeze-drying. The conjugated bile acids were >99% pure by thin-layer chromatography on silicic acid using a solvent system of isoamyl acetateipropionic acid/n-propanoliwater (4 : 3 : 2 : 1 volivol) (29). Bile acid solutions used for infusion were isotonic, and had a bile acid concentration ranging from 20 to 34 mM. Secretin was purchased from Squibb, New York, N.Y. Somatostatin was generously provided by Clin-Midy, Montpellier, France. Both peptides were diluted in normal saline. SC2644 (2,4-dimethoxy+cyclohexyl-benzoyl-propionic acid) was a generous donation from Dr. Henry 0. Wheeler, who had received it some years previously from The Searle Laboratories (courtesy of Dr. P. Klimstra).
SE:CRETION
_~ _~~. ~~_~~_~~._._~__
4
2 3 Bile Acid Output, Hmol/kg.min 1
CALUIJP\l
(r
=
acid
0.94). output,
For
each
there
micromole was
between
in calcium output; this relationship may be defined as the bile aciddependent calcium secretion. Calcium output also increased linearly with the output of the hydroxy0x0 bile acid (r = 0.99; 0.024 ~molAcalcium/~mol Abile acid), Calcium output, however, was not identical for the three natural bile acids (Figure 1 and Tables 1 and 2). Calcium outputs were slightly lower for cholyltaurine and chenodeoxycholyltaurine (slope 0.026 and 0.027, respectively); that induced by- ursodeoxycholyltaurine was significantly higher (slope 0.035). although the values overlapped. The differences between the three bile acids are more apparent if dog identity is maintained (Figure 2). The greater calcium output induced by ursodeoxycholyltaurino was not related to bile flow (Figure X). Bile acid outputs were essentially identical to the rate of bile acid infusion when the infusion rate was 1 or 2 pmolikg . min. At the lowest rate of bile acid infusion, 0.5 Fmolikg . min, bile acid output sometimes exceeded the infusion rate, presumably because of the contribution of endogenous cholyltaurine synthesis, which is 0.05-0.10 LLmolikg min in the dog (31). At the highest bile acid infusion rates (4 and 4.5 pmol/kg 3 min), outputs averaged only about 80% for cholyltaurine and 12dehydrocholyltaurine. Bile acid output and bile flow during the last 60 min of each period was constant. Analysis of biliary bile acids by gas-liquid chromatography indicated that >90% of recovered bile acids had the composition of the infused bile acid for cholyltaurine and chenodeoxycholyltaurine, except during the first infusion period where an 80%-90% recovery was obtained depending on the percentage of endogenous cholyltaurine remaining in the animal. 12-Dehydrocholyltaurine was excreted largely unchanged in hepatic bile, as >85% of the biliary bile acids was composed of the hydroxy-oxo compound. Ursodeoxycholyltaurine displayed an anomalous pattern of biliary secretion. Bile acid outputs were similar to the rate of bile acid infusion when 0.026
and
0.035
prnol
increase
668
CUMMINGS
Table
AND
HOFMANN
GASTROENTEROLOGY
1. Effect of Individual Bile Acids During Steady State Choleresis Infusion rate (pm011
Agent
on Bile Flow, in the Dog” Bile flow WI
kg. min)
Study
n
Erythritol
Clearance,
(PI/kg.
CT CT CT CT
1.0
C,A
3.0 4.0 4.5
c A c
23 2 5 2
13.95 2 1.72 23.80 33.05 + 3.45 30.07
CDCT CDCT CDCT
0.5 1.0 2.5
C C c
4 7 2
13.02 + 4.08 14.07 t 2.46 24.15
LJDCT UDCT UDCT UDCT
0.5 1.0 2.0 4.0
A A A A
1 3 3 3
7.80 13.43 + 3.14 18.08 _t 2.98 15.90 t 2.07
12-DHCT 1 ?-DHCT 12-DHCT
1.0
2.0 4.0
A A A
3 3 3
20.10 2 1.85 32.21 k 1.67 50.48 2 6.51
and
Calcium
[Bile acid]
min)
13.35 2 2.73 16.17 31.95 t 4.06 32.63
Calcium output
(pmoli kg. min)
(mM)
87. No. 3
Secretion
Bile acid output
Erythritol clearance
kg. min)
and Bile Acid
Vol.
[Calcium]
(pmofi kg. min)
(mMl
70.07 '-c13.10 99.89 101.85 + 11.02 97.82
0.96 k 0.16 2.38 3.34 t 0.21
3.37 5 0.34 3.30 3.14 t 0.34
0.047 + 0.006 0.079 0.103 2 0.007
2.94
3.44
0.103
73.26 + 37.37 74.79 '- 13.94 107.57
0.90 2 0.29 1.05 + 0.25 2.68
3.85 + 0.91 3.64 !I 0.41 3.88
0.049 + 0.013 0.050 k 0.005 0.094
8.36 13.58 + 2.31 17.67 2 2.02 15.77 '- 2.55
74.2 85.42 k 15.88 105.37 k 23.45 114.28 k 28.17
0.58 1.13 2 0.24 1.89 t 0.43 1.81 t 0.51
3.82 3.84 k 0.58 4.42 + 0.81 4.65 + 0.93
0.030 0.051 r 0.012 0.079 I?-0.014 0.074 t 0.017
19.53 t 1.07 29.85 2 2.71 47.92 k 5.89
53.28 t 3.14 60.58 t 3.15 72.70 5 5.44
1.07 t 0.13 1.95 t 0.17 3.67 2 0.58
2.06 + 0.12 2.03 " 0.05 2.06 k 0.08
0.041 + 0.004 0.065 + 0.003 0.104 k 0.014
n. number of steady state periods; each period had four IO-min collection periods, taken at the end of the 80- or 9O-min infusions. acute preparation; C, chronic preparation: CT, cholyltaurine; CDCT, chenodeoxycholyltaurine; UDCT, ursodeoxycholyltaurine; 11Each
DHCT, 12-dehydrocholyltaurine. experiments.
value
is either
the mean
the infusion rate was I or 2 pmolikg . min, but dropped to 50% at 4 pmolikg . min. The proportion of ursodeoxycholic acid in biliary bile acids was >88%, except during the first infusion period in 1
of two experimental
steady
states
or the mean
f
A, 12SD of multiple
dog when the proportion of ursodeoxycholic acid was only 72%. When the plot of calcium output versus bile acid output was extrapolated to the ordinate, a positive
Table 2. Relationships Between Calcium Output and Bile Acid Output, Calcium Output and Bile Flow, and Bile and
Bile Acid
Significance Slope
(~moli~mol) A. Calcium
y-Intercept (pmolikg . min)
r
CDCT
UDCT
NS
of observed
12. DHCT
differences
SC
SEC
SST
40.001
40.001
co.001
NS
NS NS
NS
output vs. bile acid output
Bile acid CT CDCT UDCT 12-DHCT B. Calcium output Agent CT CDCT UDCT 12-DHCT SC2644 Secretin Somatostatin
0.026 0.027 0.035 0.024
CT. choIyItaurine; 2644:
0.019
0.98
0.023 0.014 0.018
0.93
NS NS
0.92 0.99
vs. bile flow 0.0028 0.0037 0.0048 0.0021 0.0018 0.0016 0.0019
C. Bile flow vs. bile acid output Agent CT 8.46 CDCT 6.01 UDCT 5.46 12-DHCT 11.29
compound
Flow
OutDut
0.009 -0.001
-0.008 -0.001 0.009 0.024 0.021
4.48 8.03 7.26 9.14
CDCT, chenodeoxycholyltaurine; SEC, secretin; SST, somatostatin.
0.96
<0.005
0.90 0.86 0.99 0.93 0.83 0.91
co.005 10.001
NS NS -
0.96
0.86 0.79 0.99 UDCT,
ursodeoxycholyltaurine;
II-DHCT,
12-dehydrocholyltaurine;
SC,
Searle
September
1 Y84
.031
Figure
2
, 1 I “rsodeoxy Cnolyl CnenOdeOxy C”
Ratio of mean calcium output to mean bile acid output during infusion of 1 pmolikg min bile acid under steady state conditions in six separate dog experiments. Lines connect points from individual animals.
ural bile acids had an additional effect on calcium secretion unrelated simply to bile volume: A canalicular choleresis was also induced by infusion of the hydroxy-oxo bile acid. The induced calcium output with this bile acid (0.0021 PmolAcalcium/plAbile flow) had the same relationship to bile flow as that observed with SC2644 (0.0018 pmolhcalcium/&bile flow) (Figure 3B). The output of calcium per microliter of bile volume was significantly lower for the hydroxy-oxo bile acid than for that induced by infusions of the three micelle-forming bile acids, which ranged from 0.0028 to 0.0048 ~molAcalcium/~lAwater (Table 2B).
DuctuJar intercept was observed suggesting that calcium secretion would occur at zero bile acid output. Because bile acids induce bile acid-dependent bile flow by definition, this calcium output is the “bile acidindependent” calcium output, also by definition. and is present in the bile acid-independent bile flow.
Relationship Bile Flow
Between
Calcium
Output
and
The bile acid infusions also induced a linear increase in bile flow. The relationships between calcium output and bile flow induced by infusions of the individual bile acids are summarized in Figure 3C. The differing effects of the individual bile acids are apparent. During SC2644-induced choleresis, calcium output also increased linearly with bile flow despite there being little change in bile acid output. The data indicate that canalicular calcium secretion can be increased independent of any increase in bile acid secretion. For a given bile flow the calcium output induced with SC2644 was far less than that seen with cholyltaurine (Figure 3A) and the two other micelle-forming bile acids, indicating that these nat-
1
3A
Pigure
0
*
---+
Modification
During secretin-induced ductular choleresis, the average bile acid output remained unchanged as bile flow increased (Table 3). Caicium output rose as the bile flow increased (Figure 4A). The overall change in bile flow was small. and therefore the increase in calcium output was small. During somatostatin-induced anticholeresis, bile acid output remained unchanged as bile flow decreased. Calcium output fell as bile flow fell. Calcium output to bile flow ratios for both secretin and somatostatin (0.0016 and 0.0019 ~molAcalcium/~l~bile flow, respectively) were similar to that noted during SC2644-induced canalicular choleresis (O.O017~molAcalciumiplAbile tlow) (Figure 4B). In these studies, bile acid output was maintained by a continuous infusion of cholyltaurine. Although the average bile acid output was 1 pmolikg . min, the output rates varied randomly during the somatostatin and secretin experiments. To eliminate any possible influence of this cholyltaurine-induced calcium secretion on total calcium secretion, each bile acid output was multiplied by the slope shown in Figure 1A and the product was then subtracted from each calcium output. The resultant Icalcium output should be the influence of duct&r modification alone (Figure 4C). The slope lz’as essentially un-
_
20 40 Bile Flow, pl/kg.min
60 Bile Flow, pl/kg.min
Bile Flow, pl/kg.mln
3. Relationship between bile flow and calcium output. 3A. cholyltaurine (A) contrasted with the c,malic.ular c.holeretir. ~2644 (II]. 3B, 12.dehydrocholyltaurine (W) compared to SC2644 (II). XC. data for all agents used to modify canalic.ular flo~v, ursodeox~cholyltaurine (A), chenodeoxycholyltaurine (0). cholyltaurint: (A). 12.deh~drochol!ltallrine (W). ~~2644 (-I). (ore Table 2 for linear regression coefficients.)
670 CUMMINGS AND HOFMANN
GASTROENTEROLOGY
Vol.87,No. 3
Table 3. Effect of a Canalicular Choleretic (X2644), or Somatostatin- or Secretin-Induced Ductular Modification of Bile Flow on Erythritol Clearance, and Bile Acid and Calcium Secretion During Steady State Choleresis in the Dog”
Infusion rate
Agent
Study
n
Bile flow (&kg. min)
15.48 29.60
I. Induced SC2644 SC2644
canalicular choleresis 0.5 mgimin C 3 mgimin C
2 2
II. Induced Secretin Somatostatifl
ductular modification 6 Pgikg. h C 8 kg/kg. h C,A
2 5
a Values
given
are mean
6.31
of two experiments
20.30 + 1.19 or mean
[Calcium]
(mW
(mM1
kg. min]
23.92 35.26
36.85 24.97
0.58 0.73
2.96 2.15
0.040 0.063
11.85 10.64 t 1.09
40.36 133.49 i- 17.89
0.87 0.83 ? 0.06
2.71 5.34 t 0.42
Concentration
(Figure 5)
Calcium concentration ranged from 1.6 to 6.4 mM, reflecting the balance between induced calcium secretion and induced biliary water secretion. Calcium concentrations were higher when micelleforming bile acids induced bile flow or when ductular water absorption was induced by somatostatin. Calcium concentrations were lower during canalicular secretion induced by SC2644 or the hydroxy-
i
20 30 10 Bile Flow, pl/kg.min Figure
40
[Bile acid]
t SD of five experiments:
changed. A similar normalization procedure was carried out for data obtained during the SC2644 infusions, i.e., the bile acid-dependent component of calcium output was subtracted from the calcium output. The small difference between the effect of SC2644 and that of ductular modification was abolished after factoring out the bile acid component. This new line of bile acid-independent flow was contrasted to the line for bile acid-dependent flow that can also be deduced by subtracting the bile acidindependent calcium output (or y-intercept of calcium output versus bile acid output) from the total calcium output. The comparison of these two lines indicates more clearly that calcium output can be related to bile acid-depehdent flow and bile acidindependent flow. Calcium
48 0
Calcium output
Bile acid output (pmof/kg. min)
Erythritol clearance (/.&kg * min)
for definition
of column
0x0 bile acid and during induced by secretin.
(kmol/
0.033
headings.
see Table
ductular
dilution
0.055 + 0.004 1
of bile
Discussion The major conclusion to be drawn from these studies is that biliary calcium output in the dog has both canalicular and ductular components. CanaJicuJar
Factors
The results indicate that at least two components of canalicular bile influence the biliary output of calcium. The first component is most simply attributable to convective flow of water and electrolytes from plasma to bile induced by osmotic forces generated by active canalicular secretion of ions. The active secretion of ions may be that composing the bile acid-independent fraction of canalicular bile, that induced by a canalicular choleretic agent such as X2644, as well as bile acid anions in monomeric OF micellar form. We suggest the term “osmotic fraction” for this component of canalicular calcium output. Nonetheless, becatise of the extremely high correlation between bile acid output and bile flow, i.e., the constancy of the linkage between water and bile acid secretion rates, the bile acid-dependent
~*_~~~._ : : 10 30 20 Bile Flow, pl/kg.min
40
4C 0
10
20
30
40
Bile Flow, /.Nkg.min
4. Relitionship between bile flow and calcium output. 4A, effect of ductular modification (somatostatin. A; control, W; secretin, 0). 4B, effect of ductular modification; pooled values of somatostatin and secretin (W) compared to the effect of the canalicular choleretic SC2644 (0). 4C, calculated effect of the bile acid-dependent component of cholyltaurine-induced choleresis on calcium output (bold line), compared to the calculated effect of the bile acid-independent component during ductular modification of bile flow (m). or during canalicular choleresis induced by SC2644 (3) on calcium output.
September
Figure
BlL,IAKY
1~4
(:AI.(:ll’hl
SII(:KE’I‘ION
671
5. A, effect of agents influencing canalicular and ductular bile flolv on c.al[.ium I:clnc:entratlon. Bt\,. c.bolyltaurine: BA,,. XI. S(ZfX4: SSl‘. somatostatin: chenodeoxvc.holyltaurine: BA ,,,, ursodeoxycholyltaurine: BA,, , 12-tleh\:drochol~~ltaurinr: SFX:. secret-in; mean 2 SD. 58. relationship between calcium concentration and bile flo\v produced by +,ents causing tluc.tulal modification (somatostatin, A; control. ?? ; secretin, 0). 5C. relationship bet\veen bile flo\v and r:al[.ium LOIICentration, during infusion of cholyltaurine (01, which has a low critical micellar conc.rntration value. compared to th,lt of Ir-dt:llvdrochol~ltaurine (W) which has a relatively high critical micellar concentration value.
calcium secretion correlates equally well with bile volume or with bile acid output for the three micelleforming bile acids. The apparent calcium concentration of the osmotic fraction, 1.6-2.1 mM, can be determined from the relationship of calcium movement to water movement. This concentration is greater than the ionized calcium concentration in dog plasma (1.17-1.39 mM) (32). The observed concentration of calcium in the “osmotic fraction” reflects the algebraic sum of all calcium and all water movements in the biliary tree, and our data provide no direct information on the calcium concentration of the osmotic fraction of canalicular bile per se. The second component would appear to be related to the presence of micelles or micelle-forming bile acids in bile. This second canalicular fraction cannot be explained by osmotic forces, because for any given bile flow, calcium output was greater when the micelle-forming bile acids were infused. The “micellar” fraction appeared to be influenced by the bile acid type in our experiments in that the dihydroxy bile acids induced a greater calcium output than cholyltaurine. However, it is beyond the scope of the present experiments to define the nature of the forces involved in the micellar component. It is possible that both binding by micelles and binding by bile acid anions may be involved, because cholyltaurine, at least. binds calcium ions at a concentration below its critical micellar concentration. Bile acid micelles are known to bind calcium (l&20.3335), but only cholyltaurine has been studied in detail (19). The influence of biliary lipid which is also present is relatively unexplored, but it is clear that the addition of phospholipid to bile acid micelles increases their binding of calcium (33,35), and biliary phospholipid secretion might also influence biliary calcium secretion. The following equation is proposed to include the two components of canalicular calcium output: c I( ~z( ,,,_,/ A, + I:, ,,,I,+k,
(11
secretion of c.:alcium. (:.I = where c,~ = canalicular canalicuiar bile flow (plikg . min). k, = a phenor&ologic linkage coefficient relating calcium output to of canalicular bile flow (~moli~l), c:./,,, = secretion micelle-forming bile acids or micelles (pmol/ kg . min). and k,. = variable phenomenologic linkage coefficient relating calcium secretion to the secretion (or presence] of micelle-forming bile acids (pm01 calcium/pm01 micelle-forming molecules) or micelles. This partitioning of canalicular calcium outputs into an osmotic fraction and a micellar fraction contrasts with the usual partitioning of canalicular solute output into bile acid-independent and bile acid-dependent fractions, as is illustrated in Figure 6. We speculate that this new system of categorization will be useful for solutes that have appreciable binding to micelles. Such a scheme is not necessar! for solutes that do not associate \vith micelles such as erythritol. as the micellar fraction should be zero.
Ductular Fuctors The data show con\rincinglg that ductular modification of bile influences calcilum output in a predictable manner. Calcium output is diminished by ductular absorption and augmented by ductular secretion. The composition of the electrolyte fraction moving across the ductules in response to secrctin or
osmotic effect of transported ions Erythritol: WV-’ bile acid independent Calcium: Figure
-
osmotic ellect of bile acid monomers (and micelles)
additional effect of micelle-forming bile acids
bile acid dependent
Y osmotic
‘W
“micellar”
li. klech,inisnls used to dt!s~ rib12 c,illliii~.ulCir r~rythritol secreti cutl ~ompart~l \vith tl~os~, ~~CI~IWVI lor ~.dilalic:ular cal[.ium src.rc,tion
672
CUMMINGS
AND
HOFMANN
GASTROENTEROLOGY
somatostatin has been calculated by a number of authors (ll,l3), but calcium has not been included in these calculations. Our data indicate that the apparent calcium concentration of this fraction is about 1.7 mM, which is less than the total calcium concentration in bile collected more distally, and contrasts with the sodium concentration in this fraction which is equal to that present in bile. The simplest explanation of the low calcium concentration is that a fraction of biliary calcium is bound; in addition, the reflection coefficient of calcium ions may be greater than that of sodium ions. The ductular modification term can be added to the previous phenomenologic equation to give an equation for total biliary calcium secretion. Thus (21
+, &, tJ,‘*= CJY‘
where tJ. = total biliary calcium secretion and dJ. = ductui’ar calcium secretion (+) or ductular cair cium absorption (-). Combining equations (1) and (z), one obtains * k, + CJ,,,,, * k,. 2 d,,,?o *k,, r+,, = CJ&O
(31
where k3 is a phenomenologic linkage coefficient relating ductular water movements to calcium movements (micromoles per microliter). Note that in our studies in the mongrel dog k, = k,. Our data thus define for the first time the major physiologic determinants of calcium output in bile. Because the postulated osmotic forces influence both water and calcium similarly, calcium concentration remains relatively constant over a wide range of changes in osmotic flow, e.g., induced by SC2644 or the hydroxy-oxo bile acid. Because micelle-forming bile acids induce a calcium output in addition to that explicable by osmotic forces, calcium concentration increases when micelle-forming bile acids are infused. Lastly, because the electrolyte fraction moving into or out of the ductules is relatively low in calcium, calcium concentrations are decreased to some extent by ductular secretion and increased by ductular absorption. Limitations
of These Experiments
The data presented in these studies which comprise only calcium outputs and calcium concentrations do not bear directly on calcium precipitation in bile, as the majority of biliary calcium is bound to micelles, anions, or proteins (33,34,36). Nonetheless, elucidation of the physicochemical factors determining calcium activity in bile must begin with an understanding of the physiologic factors influencing overall calcium movements. References 1. Sutor DJ, Wooley SE. The nature and incidence containing calcium. Gut 1973;14:215-29.
of gallstones
Vol.
87, No. 3
2. WosiewitzU. Limy bile and radiopaque calcified gallstones: combination of analytical, radiographical,and micromorpho-
a
logical investigation. Path Res Pratt 1980;167:273-86, 3. Wosiewitz U, Wolpers C, Quint P. Roentgennegative Pigmentgallensteine. Leber Magen Darm 1978;8:353-60. 4. Been JM, Bills PM, Lewis D. Microstructure of gallstones. Gastroenterology 1979;76:548-55. 5. Bogren H. The composition and structure of human gall stones. Acta Radio1 (Stockh) 1964; Supplementum 226. 6. Boyer JL. New concepts of mechanisms of hepatocyte bile formation. Physiol Rev 1980;60:303-20. 7. Erlinger S. Hepatocyte bile secretion: current views and controversies. Hepatology 1981:1:352-59. 8. Balabaud C, Kron K, Gumucio JJ. The assessment of the bile salt-nondependent fraction of canalicular bile water in the rat. J Lab Clin Med 1977;89:393-9. 9. Baker AL, Wood AB, Moossa AR, Boyer JL. Sodium taurocholate modifies the bile acid-independent fraction of canalicular bile flow in the rhesus monkey. J Clin Invest 1979;64:312-20. 10. Forker EL. Mechanisms of hepatic bile formation. Ann Rev Physiol 1977;39:323-47. 11. Preisig R, Cooper HL, Wheeler HO. The relationship between taurocholate secretion rate and bile production in the unanesthetized dog during cholinergic blockade and during secretin administration. J Chn Invest 1962;41:1152-62. 12. Jones RS, Geist RE, Hall AD. The choleretic effects of glucagon and secretin in the dog. Gastroenterology 1971;60:64-8. 13. Rene E, Danzinger RG, Hofmann AF, Nakagaki M. Pharmacologic effect of somatostatin on bile formation in the dog. Gastroenterology 1983;84:120-9. 14. Roda A, Hofmann AF, Mysels KJ. The influence of bile salt structure on self-association in aqueous solutions. J Biol Chem 1983;258:6362-70. 15. Thomas JE. An improved cannula for gastric and intestinal fistulas. Proc Sot Exp Biol Med 1941;46:260-5. 16. Gilmore IT, Barnhart JL. Hofmann AF, Erlinger S. Effects of individual taurine-conjugated bile acids on biliary secretion and sucrose clearance in the unanesthetized dog. Am J Physiol 1982;5:G40-6. 17. Danzinger RG, Nakagaki M, Hofmann AF, Ljungwe EB. Differing effects of two hydroxy-7-oxo taurine conjugated bile acids on bile flow and biliary lipid secretion in the dog. Am J Physiol 1984;246:G166-72.. 18. Williamson BWA, Percy-Robb IW. The interaction of calcium ions with glycocholate micelles in aqueous solution. Biochem J 1979;181:61-6. 19. Moore EW, Celic L, Ostrow JD. Interactions between ionized calcium and sodium taurocholate: bile salts are important buffers for prevention of calcium-containing gallstones. Gastroenterology 1982;83:1079-89. 20. Rajagopalan N, Lindenbaum S. The binding of Ca’+ to taurine- and glycine-conjugated bile salt micelles. Biochim Biophys Acta 1982;711:66-74. 21. Sewell RB, Hoffman NE, Smallwood RA, Cockbain S. Bile acid structure and bile formation: a comparison of hydroxy and keto bile acids. Am J Physiol 1980;238:GlO-7. 22. Cook DL, Lawler CA, Green DM. Studies on the effect of hydrocholeretic agents on hepatic excretory mechanisms. J Pharmacol Exp Ther 1954;110:293-9. 23. Barnhart JL, Combes B. Characterization of SC2644-induced choleresis in the dog. Evidence for canalicular bicarbonate secretion. J Pharmacol Exp Ther 1978;206:190-7. 24. Gibson GE, Forker EL. Canalicular bile flow and bromosulfophthalein transport maximum. The effect of a bile independent choleretic, SC2644. Gastroenterology 1974:66:1046-53. 25. Wheeler HO, King KK. Biliary excretion of lecithin and cholesterol in the dog. J Clin Invest 1972;.51:1337-50. 26. Turley SD, Dietschy JM. Re-evaluation of the 3Lu-hydroxyster-
September
BILIAKY
1984
oid dehydrogenase assay for total bile acids in bile. J Lipid Res 1978;19:924-8. 27. Nakagaki M, Danzinger KG, Hofmann AF, Roda A. Hepatic biotransformation of two hydroxy-7-oxotaurine-conjugated bile acids in the dog. Am J Physiol 1983;245:G411-7. 28. Tserng K-Y, Hachey DL, Klein PD. An improved procedure for the synthesis of glycine and taurine conjugates of bile acids. J Lipid Res 1977;18:404-7. 29. Hofmann AF. Thin-layer adsorption chromatography of free and conjugated bile acids on silicic acid. J Lipid Res 1962; 3:127-a. 30. Zar JH. Biostatistical analysis. Englewood Cliffs, N.J.: Prentice-Hall. 1974:620. 31. Wollenweber J. Kottke BA, Owen CA Jr. Effect of nicotinic acid on pool size and turnover of taurocholic acid in normal
and
hypothyroid
dogs.
Proc
CALCltJM
Sot
Exp
SECKETION
Biol
Med
673
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