215
Biochimica et Biophysics Acta, 398 (1975) 275-286 @ Elsevier Scientific Publishing Company, Amsterdam
-
Printed
in The Netherlands
BBA 56643
THE INTERMICELLAR BILE SALT CONCENTRATION WITH THE MIXED-MICELLES OF HUMAN BILE*
WILLIAM
IN EQUILIBRIUM
C. DUANE**
Phoenix Clinical Research Section, National Institute of Arthritis, Metabolism and Digestive Diseases, Phoenix Indian Medical Center, 4212 North 16th Street, Phoenix, (U.S.A.) (Received
February
llth,
Aria
1975)
Summary The intermicellar bile salt concentration in equilibrium with the bile saltlecithin-cholesterol mixed-micelle has been studied in human bile. Equilibriumdialysis, used to measure the biliary intermicellar bile salt concentration, has been validated as an applicable method by studying the cholate-lecithin mixedmicelle, for which intermicellar bile salt concentration values have previously been reported. The intermicellar bile salt concentration of bile was essentially independent of ionic strength in the range 0.05-0.15 M chloride. Simple dilution of bile lowered the intermicellar bile salt concentration (about 2/3 reduction for each two-fold dilution). This reduction occurred because of a simultaneous decrease in the molar ratio of bile salt/phospholipid in the micelle. Dilution of micelles with micellar bile salt/phospholipid held constant did not affect the intermicellar bile salt concentration. The relationship between intermicellar bile salt concentration and micellar bile salt /phospholipid, defined in the dilution studies, was linear in the range of study. For a composite of five biles, this relationship was described by the equation: intermicellar bile salt concentration = 1.27 (bile salt/phospholipid) + 0.538. Data obtained on an artificial bile agreed closely with the results obtained on bile suggesting that the other constituents of bile did not affect this analysis. These findings may be helpful in understanding the process of micellar cholesterol solubilization in bile.
* This work was presented in part at the National Meeting of the American Federation for Clinical Research. Atlantic City, N. J.. 1973. * * Present address: Department of Internal Medicine. Veterans Administration Hospital. 54th Street and 48th Avenue S,, Minneapolis, Minn. 55417 (U.S.A.) Address for reprint; Secretary: Phoenix Research Section, National Institute of Arthritis. Metabolism, and Digestive Diseases, Phoenix Indian Medical Center, 4212 N. 16th Street, Phoenix, Ariz. 85016, U.S.A.
276
Introduction Bile salt combines with lecithin and cholesterol to form mixed-micelles in bile [1,2]. This micellarization process serves to solubilize biliary cholesterol [2,3] and may play a role in the hepatic secretion of both cholesterol and lecithin [ 4,5] . Because of the physiological importance of biliary mixed-micelles, a large body of information has accumulated regarding critical micelle concentration (CMC), size, and structure of bile salt micelles [2]. Most of these data have been obtained using pure systems which are similar, but not identical, to bile. Although such data are useful, conclusions about biliary micelle phenomena would be strengthened by data obtained using bile as well. The equilibrium of mixed-micellar bile salt with intermicellar bile salt in the aqueous phase has not been studied in bile. Quantitation of this equilibrium would provide an estimate of the tendency for bile salt to form mixed-micelles in bile and refine our concepts of the dissolution of biliary cholesterol and lecithin. Shankland has studied this equilibrium for the cholate-lecithin mixed-micelle [ 61 . He employed a light-scattering technique to obtain values for intermicellar cholate concentrations (cholate monomer + simple cholate micelles) in pure sodium cholate-lecithin solutions; however, light-scattering and other established methods for micelle investigation are difficult to apply to bile because many biliary constituents interfere with application of these methods [2] . In the present investigation, this difficulty has been overcome by using equilibrium-dialysis to measure the intermicellar bile salt concentration (IMBC) in bile. This IMBC, like that in pure cholate-lecithin solutions, probably comprises both bile salt monomer and simple bile salt micelles, both of which are small enough to dialyze. The large mixed-micelles, however, are retained by the dialysis membrane. Thus, at dialysis equilibrium, the concentration of bile salt outside the dialysis bag is equal to the IMBC in equilibrium with the mixedmicelle. Validation of Lhis equilibriumdialysis method has been obtained using Shankland’s data on the cholate-lecithin mixed-micelle for comparison. Interference from other biliary constitutents has been indirectly ruled out by testing a pure artificial bile. Finally the method has been applied to bile for measurement of the bili‘ary IMBC as a function of ionic strength, dilution, and bile salt content of the mixed-micelle. Methods
Human gallbladder bile was obtained aseptically at uncomplicated cholecystectomy from Indian patients with gallstones and a visualizing gallbladder. Bile specimens were kept sterile at room temperature and centrifuged at 266() y g to remove cholesterol crystals. All specimens were studied within 12 h of surgery. Cholic: acid (Matheson, Coleman, and Belle; Norwood, Ohio), conjugated bile salts (Maybridge Research Chemicals; Lauceston, Cornwall, U.K., and Stcraloids, Inc.; I’awling, N.Y.), cholesterol (J.T. Baker, Phillipsburg, N.J.) and
a chromatographic preparation of egg lecithin (Sigma Chemical Co.; St. Louis, MO.) were obtained commercially. Cholic acid was converted to sodium cholate by neutralization with NaOH. These materials were tested for purity on thinlayer chromatography. All materials moved in a single band on thin-layer chromatography except for the glycine and taurine conjugates of chenodeoxycholate, both of which had a faint additional band at the solvent front. Since these chenodeoxycholate conjugates were to be used for confirmatory studies only, no attempt was made to purify them further. Chemical analyses Bile salts were measured by an automated procedure based on Talalay’s enzymatic assay [ 71. Phospholipids were determined as lipid-phosphorous using conventional methods [S] . Cholesterol was measured by an automated procedure based on the Liebermann-Burchard reaction [8] . Bile water content was measured gravimetrically after desiccation of a 0.20 ml bile sample at 100°C for 1 h. Chloride was determined by AgCl precipitation using a constant amperage chloride tritrator (American Instruments Co., Silver Spring, Md.). Dialyzate protein was measured by the Folin method [9] . Thin-layer chromatography of phospholipids was done on Silica Gel H in a solvent system of chloroform/methanol/water (65 : 25 : 4) and visualized with iodine vapor and molybdenum spray as described by Dittmer and Lester [lo]. Conjugated bile salt thin-layer chromatography was done on Silica Gel H in a solvent system of n-butanollacetic acid/water (10 : 1 : 1). Cholesterol thin-layer chromatography was done in a solvent system of ethyl ether/heptane (55 : 45). Dialysis procedure Because temperature in the range of 20-40°C has a negligible effect on the formation of most bile salt micelles [ll] , all dialysis were done at room temperature on a metabolic shaker. Dialysis tubing (Union Carbide) having a minimum molecular weight retention of about 12 000 was used throughout. Tubing was opened by gentle agitation in sterile distilled water and tied at one end. Bile samples were introduced into the open end of the tubing with a sterile syringe. The open end was then tied with surgical silk. Boiling this tubing for several hours prior to use did not affect the dialyzable bile salt concentration in three bile samples tested, so this procedure was not routineiy followed. Prior to dialysis, all bile samples were adjusted to pH 7.5 * 0.3 with 1 M NaOH or HCl and, unless otherwise specified, to a chloride concentration of 0.10 M with dry NaCl. Usually 1.0 ml aliquots of bile (or artificial bile) were loaded into the dialysis tubing and dialyzed against 6-10 volumes of 0.10 M NaCl. Dialysis equilibration time for this system was examined in five different bile specimens and in serial dilutions of two of these specimens. These kinetic studies covered the entire range of dialyzate volumes used for subsequent studies and were carried out for as long as 38 h. Bile samples for these experiments were put up in dialysis bags just as described above. Timed samples of the dialyzates were taken being careful to remove less than 2% of the total dialyzate volume with each sample. Similar dialysis kinetic studies were performed on a pure mixture of conjugated bile salts (without lecithin or choles-
218
terol) simulating the proportions found in bile (exact composition the same as described under Artificial System). On the basis of equilibration kinetics defined in these experiments (see Results) all subsequent samples were dialyzed for 12 h. In two of the kinetic studies, dialyzates were analyzed for phospholipid and cholesterol. This was done to insure that phospholipid and cholesterol were not escaping significantly into the dialyzate. Analysis of cholesterol in the dialyzates was done by gas-liquid chromatography as described by Miettinen, et al. [12]. Analysis of dialyzates for phospholipid was achieved by a semi-micro technique exactly the same as the conventional method but with all reagent volumes scaled down lo-fold. The sensitivity of this technique on lecithin standards is about 0.01 mg of phospholipid. In addition to these kinetic experiments, over 15 additional dialyzates have been analyzed for phospholipid and cholesterol after a 12-h dialysis. After dialysis, samples of dialyzate were taken for determination of bile salt and chloride concentrations. The contents of each bag were evacuated, and 6 O.lO-ml samples were taken for duplicate analysis of chloride, bile salt, phospholipid, and cholesterol. Total bile-water was determined on a 0.20-ml sample from the bag and used to correct the gross bile chloride concentration to bile-water chloride concentration. This correction usually amounted to a 515% upward adjustment of the gross bile chloride concentration. Dialyzate and bile-water chloride concentrations were used to monitor Donnan effects, which were negligible (bile-water chloride/dialyzate chloride, 0.95-1.0) in the samples studied. Consistent with negligible Donnan effects, the pH of bag contents was within 0.2 pH units of dialyzate pH and ranged from 7.2 to 7.7 in representative samples studied. From the above data, micellar bile salt concentration (mmol bile salt in mixed-micelles/l) was calculated by subtracting the dialyzate bile salt concentration (mmol/l) from the total bile salt concentration in the dialysis bag. This micellar bile salt concentration was then divided by the phospholipid concentration in the dialysis bag to obtain the molar micellar bile salt/phospholipid ratio. Method validation Because equilibrium-dialysis is not an established technique for the study of bile salt mixed-micellar systems, experiments were performed to test the validity of this method. For this purpose, the cholate-lecithin mixed-micelle was studied. Shankland has carefully defined the IMBC in equilibrium with this mixed-micelle and related the IMBC to the micellar bile salt/phospholipid ratio; however, his data were obtained by light-scattering [6], a technique quite different from equilibrium-dialysis. The basic working solution for these experiments was prepared by mixing pure sodium cholate in methanol with pure egg lecithin in hexane. The solvent was removed in vacua below O-C, and the dry residue taken up with sterile 0.12 M NaCl. The final clear solution was adjusted to about pH 10 with 1 M NaOH and contained 59.7 mmol cholate and 10.8 mmol lecithin per 1. Final composition of this solution was essentially identical to the composition of solutions used by Shankland 161.
2.44 2.75 2.71 2.74
IMBC
Bile S --“_.
28.2 13.5 7.61 3.80
Bag phosphalipid
--l--_
.._^
1.45 1.43 1.31 1.36
phospholipid 2.80 2.83 3.11 2.96
IMBC
1.37 1.50 1.36 1.48
32.5 15.8 8.36 4.34 66.0 36.6 20.6 12.3
Bag bile salt
HELD CONSTANT
2.51 323 3.20 &a6
XMBC
28.3 20.8 10.7 5.80
Bag phospholipid
1.65 1.60 1.63 I.54
Bile salt/ phospholipid
-_^____._-~“--
1: 8
Undiluted 1 : 2 1:4
_.._.
Dilution
47,3 18.7 6.00
1.49
8.20 2.16 I.45
0.96
IMBC Bag bile salt
Bile T (81
-__..--___
.__.__._~
3.01
1.10
3.38
14.4 11.2 7.79
,..^__.
28.7 18.3 7.85
3.00 2.26 1.64
_---“.-_
0.73
-_--..“-~
2.46 1.88 1.25
.*_-_---,
28.0 x4.3 7.26 4.01
1.27
1.02
2.51 2.12 1.46
-------.__-_-_1,
3.52
67.9 33.0 11.8
IMBC Bag bile salt
5.54
37.2 22.2 10.9
Bag phosphofipid
-_
..__
0.54
2.44 1.56 1.06
“,___
0.92
.Bag phospholipid
2.67
17.6 10.5 5.35
.__,_“__ 26.9 10.0 2.41
IMBC Bag bile sait
Artificial bile (10) -.~~~ -“-~.“_-“__-.
._,_ ______
.-_~---~-_____-_lll Bile B (101 ~ .__-
.-_l__~-_.__-..____
.-.----____
Bag phospholipid
---
-.._ _.__.
45.3 17.0 5.39
IMBC Bag bile sait
Bag phospholigid
IMBC Bag bile salt
~ Bile S (10)
-~
Bile N (6) --.---
.._ _ .,.I____---
5.48
26.2 17.2 8.13
Bag phospholipid
._
All concentrations in mmol/l. Numbers in parentheses denote dialyzate volume in ml. All bag volumes 1.0 ml. To put dilutions into prospective with respect to undiluted gallbladder bile, note that dialyzable bile salt is diluted not only by sample dilutions (first columnf but also by the dialyzate volume. Taking Bile T as on example, dialyzable bile salt in the undiluted sample is diluted 9-fold (1 ml of sample + 8 ml dialwateI; on the 1 : 2 sample, dialyzate bik salt is diluted Ill-fold, etcThe non-dialyzable mixed-micelies, however, are not diluted by the dialyzate.
SYSTEM
VOLUME
Bile salt/ ghospholipid
HELD CONSTANT
Bag phospholipid
BILE WITH TOTAL
47.3 26.6 14.5 9.3Q
IBag bile salt
I
BILE SALT/PHOSPHOLIPID
ON THE IMBC OF BILE AND ARTIFICIAL
43.3 22.0 12.7 1.89
bile salt
Bag
_-- ___. r_l____~.“l
EFFECT OF DILUTION
TABLE II
Undiluted 1: 2 I : 4 1:8
Dilution
______
Bile salt/
ON IMBC IN BILE WITH MICELLAR
All concentrations are expressed as mmol/l.
EFFECT OF DILUTION
TABLE I
CD
2
280
l-ml aliquots of this solution were dialyzed against varying volumes (1.0-12.0 ml) of 0.12 M NaCl for 12 h at room temperature. In kinetic experiments with three samples dialyzed for 24 h, it was found that complete dialysis equilibration occurred by the 12th hour. Varying the volume of dialyzate served to alter both IMBC and the ratio of bile salt/phospholipid in the mixedmicelle. strength The effect of ionic strength on the IMBC *as determined by adding dry NaCl to aliquots of gallbladder bile. One aliquot was made 0.55 M in chloride, another aliquot 0.10 M, and another aliquot 0.15 M. Each aliquot was then dialyzed against a solution of NaCl having the same molarity as the molarity of chloride in the bag. Dialyzate volumes and pH were held constant. In two bile specimens, the effect of divalent cations on the IMBC was evaluated. This was accomplished either by addition of EDTA to the dialyzate or by equivalent substitution of CaCl, for NaCl in the dialyzate (25 mequiv/l substituted).
Ionic
Dilution and micellar bile salt content Studies of the effect of dilution on the IMBC were done as follows: Chloride concentration and pH of each bile were adjusted as described under Dialysis procedure. The bile was then divided into aliquots one of which was left undiluted, another diluted 1 : 2 with 0.10 M NaCl, another diluted 1 : 4, and another diluted 1 : 8. Each aliquot was then dialyzed against 0.10 M NaCl. In some experiments the dialyzate volume for each dilution was held constant. This resulted in a progressive reduction of micellar bile salt/phospholipid with dilution. Consequently, these data could be used to define the relationship between the IMBC and micellar bile salt/phospholipid as well as the effect of simple dilution on the IMBC. Dialyzate volumes for these experiments are given in Table II. To put the dilutions in perspective with respect to undiluted gallbladder bile, note that dialyzable bile salt is diluted both in the original sample dilution and again by the dialyzate volume. III addition, similar dilution studies were performed attempting to hold micellar bile salt/phospholipid constant (Table I). It was hypothesized that IMBC would not change with dilution of micelles alone, since this is true in simplemicellar systems [13]. For any given bag volume, 2-fold dilution is equivalent to halving the total solute content (micelles and intermicell~ bile salt). However, by simultaneously reducing the dialyzate volume enough to halve the total system volume (bag volum e f dialyzate volume), the absolute quantity of intermicellar bile salt necessary to maintain a constant IMBC is also halved. This leaves micellar bile salt undisturbed and maintains micellar bile salt/phospholipid and its pre-dilution level. Therefore, progressive 2-fold dilutions of bile were made as above, but with each Z-fold dilution the dialyzate volume was reduced by the amount necessary to halve the total system volume. Artificial system To establish that the measurements done on bile were not affected by any of the other constituents of bile, (e.g. bilirubin and protein), an artificial bile
283
system was prepared from pure conjugated bile salts, egg lecithin, and cholesterol. Each of the three components was stored in ethanol or hexane at -20°C. At the ~~~ro~~ate time, volumes of each of the three components were cornbined. The sokent was evaporated in vacua below 0°C and the dry residue taken up with a volume of sterile 0.10 M NaCl calculated to give a final bile salt concentration of 60 mM. Molar percents in this artificial system were as follows: bile salt 64%, lecithin 24%, and cholesterol 12%. The bile salt fraction contained 29% glycocholate, 26% glycochenodeoxycholate~ 10% glycodeoxycholate, 15% ~u~ch~~a~* 14% ~ur~chen~eo~ycho~ate~ and 5% ta~r~eoxy~ cholate, The final sterile aqueous mixture was adjusted to pH ‘73 with sterile 1 M NaOH and allowed to equilibrate for 3 days at room temperature. One aliquot of this solution was left undiluted, another was diluted 1 : 2 with 0.10 M N&l, another diluted 1 : 4, and another diluted 1 : 8. Each aliquot was then dialyzed against 8.0 volumes of 0.10 M NaCl.
Dialysis procedure Studies of bile dialysis kinetics, shown in Fig. 1, demonstrated a plateau of dialyzate bile salt concentration by 12 h when the ~qu~l~br~~rn dialyzate bife salt concentration was 4 mM or lower. This was confirmed by dialysis of a mixture of pure conjugated bile salts (not containing lecithin or cholesterol) simulating the proportions typically found in bile, These studies showed a dialyzate bile salt concentration higher than 96% of the calculated equilibrium value at 12 h when the equilibrium dialyzate bile salt concentration was 7.8 mM, Equ~ib~~m was attained even more rapidly pihen the eq~~~ibr~~rn diafyzate bile salt concent~tio~ was below 7.8 mM.
Ttme fhrsf Fig. 1. K%.lyzate ISI& ~46, phasphaIipid. and cholesterol ~~n~entra~ans as a fun&lam of tinre. E~XZWX?~XS diatyzate bile salt c~ncentmtkms covering the Qntim? range of concentrations in this lieport are showr~ Open circled and closed ci.rck?s represent two different undiluted bilas. Open squares XQpKesent a 1 : 2 dilution af the former bile, while closed triangles and open tria.&es represent1 : 2 end ‘i : 4 dilutions respectively of the latter bile. Dialyzate bile salt plateaued by 12 h ia all cases. Phospholipid and cholesm terol concontmtions were meawrable but negligibly small in three dialyzates. In the remaining two dialyzates no phospholipid OX cholesterol could be de@cCed.
282
Kinetic studies of dialyzate phospholipid and cholesterol (Fig. 1) demonstrated little tendency for these two lipids to escape from the dialysis bag. In fact, dialyzates of two of the dilutions of bile shown in Fig. 1 contained no detectable phospholipid or cholesterol even after 38 h of dialysis. This nearly complete retention of phospholipid and cholesterol by the dialysis membrane was confirmed in more than 15 dialyzates tested only at 12,h. The highest concentrations of phospholipid and cholesterol measured in these dialyzates were 0.18 mM and 0.035 mM respectively. These concentrations correspond to less than 0.5% of the bag phospholipid and cholesterol and less than 3% of dialyzate bile salt concentration. Dialyzate protein, measured in four experiments, was less than 0.1 mg/ml suggesting nearly complete retention of biliary proteins by the dialysis membrane. Phospholipid thin-layer chromatography of six bag contents after dialysis demonstrated a single band visible by molybdenum spray. The position of this band corresponded to lecithin standards suggesting that no significant breakdown of phospholipid occurred during the study. Method
validation
Results of the method validation experiments on pure cholate-lecithin solutions are shown in Fig. 2. Measurements of the IMBC and micellar bile salt/phospholipid obtained by equilibrium-dialysis of these solutions correspond very closely to light-scattering measurements of these two parameters reported by Shankland [6]. Phospholipid concentrations were measured in all dialyzates in the cholate-lecithin experiments and were less than 0.04 mM after 12 h of dialysis. Dialysis equilibration time for this micellar system is even shorter than for bile. Thus, in three samples dialyzed for 24 h, 98% of equilibrium was attained by 9 h in two and by 11.5 h in the third. Consistent with these rapid dialysis kinetics, solutions of pure sodium cholate (not containing lecithin) reached
7
200
Chloride (mEq/L) Fig.
2.
pholipid land Fig.
3.
studies.
in cholate-lecithin
[I51 using
chloride but
Cholate-lecithin
IMBC
light-scattering as a function
concentrations.
the reduction
Equilibrium-dialysis
solutions (open of ionic All
is so small
bile
(closed circles, strength.
show
measurements
circles.
solid
dotted
line).
Four
different
a very
as to be negligible.
slight
line)
bile
reduction
of
IMBC
are compared
specimens in the
IMBC
and
micellar
to data
were with
dialyzed
bile
obtained
at three
increasing
ionic
salt/phosby Shank-
different strength.
283
dialysis equilibrium centration exceeded
by 12 h even when the equilibrium 15 mM.
dialyzate
cholate
con-
Ionic strength There was a consistent, but very small reduction in the IMBC with increasing ionic strength in the range of 0.05-0.15 M chloride (Fig. 3). This reduction was so small as to be negligible. Equivalent substitution of CaCl, for NaCl in the dialyzate and addition of EDTA to the dialyzate had no effect on the IMBC suggesting that divalent cations do not significantly influence this parameter. Dilution When micellar bile salt/phospholipid was held constant by appropriate adjustment of the dialyzate volume with each 2-fold dilution (see Methods), dilution did not affect the IMBC (Table II). This finding is consistent with the assumption used by previous investigators [6,14] that the escaping tendency of solubilizer from the mixed-micelle is independent of the concentration of micelles. * The above experiments, however, do not reflect the effect of simple dilution of bile which is essentially a process of halving the total solute for any given volume. Table II shows the effect of dilution when the total system volume is held constant, a situation analogous to simple dilution. In these experiments, any given 2-fold dilution reduced the IMBC, but by less than a factor of 2. Thus, at the instant of dilution both the micelle concentration and the IMBC fell by a factor of 2. The IMBC then tended to rise toward its previous level, but in doing so drew bile salt out of the mixed-micelle and lowered micellar bile salt/phospholipid. Therefore, the equilibrium IMBC was reduced by dilution because micellar bile salt/phospholipid was lowered. Micellar bile salt content The relationship between the IMBC and micellar bile salt/phospholipid shown in Fig. 4 was derived from the same samples used in the dilution experiments for which the dialyzate volume was held constant. There appears to be a linear relationship between IMBC and micellar bile salt/phospholipid with reasonable agreement among the bile specimens studied. It should be emphasized, however, that none of these points represent the IMBC or micellar bile salt/phospholipid of undiluted bile because dialyzate volumes ranged from 6 to 10 times the bag volume. It is possible that measurements on strictly undiluted bile would reveal a deviation from linearity at higher values of micellar bile salt/phospholipid *This assumption can be theoretically justified by considering an ideal mixed-micellar system. Suppose that 11 mol of the solubilizer, A, combine with VI mol of the solubikate. B. (otherwise insoluble), to form 1 mol of the mixed-micelle. M. Since B is insoluble, its chemical activity can be defined as 1.0, and the equilibrium constant for this reaction written: I< = [MI /LA]” where activity coefficients are neglcctrd. Then if solution 1 is diluted by a factor of two to obtain wllution 2:
Fig. 4. IMBC as a function of micrllar bile Salt/phospholipid. Serial dilutions of bile (or artificial bile) were dialyzed against equal volumr~s of dialyzate. Five different biles are shown (closed and open circles. closed and open squares, and closed triangles). Taken as a group, these five Specimens yield the depicted least-squaws regression: IMBC = 1.27 (bile salt/phospholipid) + 0.538. Open triangles represent the Samf’ studv performed on an artificial bile. These are indistinguishable from the data obtained on bile suggesting that the other constiturnts of bile do not affect IMBC or micellar bile Salt/phospholipid. Open Squares represent a bile dialyzed for 36 h. These points correspond closelv to the other data obtained by a 12 h dialysis and further Support the 12 h dialvsis time as being adequate for equilibration. since there is likely to be a point at which mixed-micelle becomes saturated with bile salt. Four of the points shown in Fig. 4 were obtained by dialyzing for 36 h. These appear to be in good agreement with the rest of the data obtained by dialyzing for 12 h. This observation provides additional evidence that dialysis equilibrium was achieved by 12 h. Artificial system Measurements of the IMBC as a function of micellar bile salt/phospholipid obtained in the artificial bile are also shown in Fig. 4. These measurements correspond closely to the data for bile obtained in similar experiments. This finding suggests that other constituents of bile, such as protein and bilirubin, do not have an appreciable effect on the IMBC. Discussion In the present investigation, the IMBC in equilibrium with the bile saltlecithin-cholesterol mixed-micelle has been studied in human bile. Lecithin and cholesterol, the other two lipid constituents of this mixed-micelle, presumably do not have appreciable chemical activity in the intermicellar aqueous phase since they are insoluble in water. Measurement of the IMBC has been achieved by the relatively direct technique of equilibrium-dialysis. Similar techniques have been used by other investigators for the study of detergent solutions [ 151 ; however, equilibriumdialysis apparently has not been previously applied to the study of bile salt mixedmicelles. To verify the applicability of this technique to IMBC measurements in bile salt mixed-micellar systems, the equilibrium-dialysis method has been applied to the cholate-lecithin mixed-micelle for which the IMBC has been previously defined using an independent method [6]. As shown in Fig. 2, the relationship between intermicellar cholate concentration and the micellar
285
cholate/lecithin ratio obtained in the present study using equilibriumdialysis corresponds very closely to the data obtained by Shankland using light-scattering. This close correspondence confirms Shankland’s observations and strongly suggests that equilibrium-dialysis is a valid technique for measurement of the IMBC in bile salt mixed-micellar systems. It should be emphasized, however, that dialysis equilibration time and egress of mixed-micelles through the dialysis membrane must be evaluated for each different mixed-micellar system being studied. Neither phospholipid nor cholest.erol escaped significantly from the dialysis bag in our experiments with pure cholate-lecithin solutions or with bile, but this may not be true for systems having low molecular weight mixed-micelles. Furthermore, as the bile salt concentration of pure bile salt solution (not containing lecithin or cholesterol) rises above the CMC, dialysis equilibrium time becomes progressively prolonged. The degree of prolongation apparently is proportional to the size of simple micelles formed by that species of bile salt. Thus, our solutions of pure cholate, even above the CMC (= 4.5 mM), had rather rapid dialysis kinetics and reached equilibrium at 12 h even when the equilibrium cholate concentration exceeded 15 mM. Presumably this rapid equilibrium occurred because the simple cholate micelles are small (Mr 2 2500) [ 111. Mixtures of pure conjugated bile salts simulating the proportions found in bile, (micelle M, 8000, CMC 0.7-1.4 mM) [ll] had slower dialysis kinetics reaching equilibrium at 12 h only when the equilibrium bile salt concentration was less than 7.5 mM. Consistent with these observations, bile dialysis specimens having an equilibrium dialyzate bile salt concentration of 4.1 mM or less reached dialysis equilibrium by 12 h, as shown in Fig. 1. Further evidence that equilibrium has been attained in such samples is provided by the fact that four bile dialyses carried out for 36 h yielded values for IMBC (as function of micellar bile salt/phospholipid) indistinguishable from values obtained in the other bile specimens dialyzed for 12 h. Theoretically, a high content of bile salt in the mixed-micelle should favor movement of bile salt out of the mixed-micelle (or conversely reduce the tendency for bile salt to enter the mixed-micelle). Thus, it is not surprising that the IMBC in bile and artificial bile vary directly with micellar bile salt/phospholipid as shown in Fig. 4. A similar direct proportion is apparent for the cholatelecithin mixed-micelle as reflected in the data reported by Shankland and confirmed in the present investigation (Fig. 2); however, for any given micellar bile salt/phospholipid, the IMBC in the cholate-lecithin system is higher than in the conjugated bile salt-lecithin-cholesterol system. This difference in IMBC levels may reflect some effect of bile salt conjugation or perhaps, the absence of cholesterol from the cholate-lecithin system. Alternatively, since cholate has a higher CMC (lower tendency to form simple-micelles) than the mixture of bile salts present in the bile [ll],cholate may have less tendency to form mixedmicelles as well. At least two reports of the CMC of bile have appeared in the literature [ 16,171. To paraphrase Mukerjee and Mysels, the CMC of a mixed-micellar system can be defined as that concentration of solubilizer (in this case bile salt) at which the solubilizate just begins to dissolve [13]. As they point out, this mixed-micelle CMC should theoretically be below the CMC of the pure SO~U-
286
bilizer. According to this definition, the CMC of bile would approximately correspond to the IMBC at zero micellar bile salt/phospholipid, obtained by extrapolating the data in Fig. 4. Such extrapolation yields a value of 0.54 mM for the average CMC of the five biles studied. This value is below the CMC of the mixture of bile salt found in bile (= 0.7 mM), as predicted by Mukerjee and Mysels, and is in fair agreement with the two previously reported bile CMC values of 1.5 and 1.45 mM. These two previously reported values for the CMC of bile may be imprecise because the methods employed in their measurement (ultracentrifugation and surface tension) are likely to be subject to considerable error when applied to a complex solution such as bile. In contrast, the nonmicellar constituents of bile would appear not to influence measurements obtained in the present study since our data in artificial bile correspond closely to data in similar bile specimens (Fig. 4). Because dialyzate volumes used in this study were 6-10 times larger than the volume of bile within the bag, the data reported here do not represent the IMBC of undiluted gallbladder bile. Such measurements would require a dialyzate volume that was negligibly small relative to the bag volume. Six bile specimens have been dialyzed against an equal volume of dialyzate; however, all of the 12 h dialyzate bile salt concentrations for those samples were above 7 mM, a concentration above which dialysis equilibration time is prolonged (as noted above). For this reason none of these data have been included in our results. In artificial bile systems, the maximum amount of cholesterol solubilized by any given amount of bile salt and lecithin is reduced by lowering the total solute concentration [ 181. Our observation that simple dilution of bile lowers micellar bile salt/phospholipid suggests that this reduction of cholesterol solubilizing capacity by dilution is a result of depleting the mixed-micelle of bile salt. Acknowledgments Cooperation from the surgical staff of the Phoenix Indian Medical Center in obtaining bile specimens is gratefully acknowledged. Thanks are also due to Mrs Marjorie Kennel and Mrs Harriet Rascon for careful secretarial assistance and Mr Robert Collins for technical assistance. References 1 Bourges, 2
Carey,
M..
3
Admirand.
4
Wheeler, Hard&n, Shankland, TaIaIay, Grundy,
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265-275
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57,
628-633
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and D.M.
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5 7
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Biochimica et Biophysics Acta, 398 (1975) 275-286 @ Elsevier Scientific Publishing Company, Amsterdam
-
Printed
in The Netherlands
BBA 56643
THE INTERMICELLAR BILE SALT CONCENTRATION WITH THE MIXED-MICELLES OF HUMAN BILE*
WILLIAM
IN EQUILIBRIUM
C. DUANE**
Phoenix Clinical Research Section, National Institute of Arthritis, Metabolism and Digestive Diseases, Phoenix Indian Medical Center, 4212 North 16th Street, Phoenix, (U.S.A.) (Received
February
llth,
Aria
1975)
Summary The intermicellar bile salt concentration in equilibrium with the bile saltlecithin-cholesterol mixed-micelle has been studied in human bile. Equilibriumdialysis, used to measure the biliary intermicellar bile salt concentration, has been validated as an applicable method by studying the cholate-lecithin mixedmicelle, for which intermicellar bile salt concentration values have previously been reported. The intermicellar bile salt concentration of bile was essentially independent of ionic strength in the range 0.05-0.15 M chloride. Simple dilution of bile lowered the intermicellar bile salt concentration (about 2/3 reduction for each two-fold dilution). This reduction occurred because of a simultaneous decrease in the molar ratio of bile salt/phospholipid in the micelle. Dilution of micelles with micellar bile salt/phospholipid held constant did not affect the intermicellar bile salt concentration. The relationship between intermicellar bile salt concentration and micellar bile salt /phospholipid, defined in the dilution studies, was linear in the range of study. For a composite of five biles, this relationship was described by the equation: intermicellar bile salt concentration = 1.27 (bile salt/phospholipid) + 0.538. Data obtained on an artificial bile agreed closely with the results obtained on bile suggesting that the other constituents of bile did not affect this analysis. These findings may be helpful in understanding the process of micellar cholesterol solubilization in bile.
* This work was presented in part at the National Meeting of the American Federation for Clinical Research. Atlantic City, N. J.. 1973. * * Present address: Department of Internal Medicine. Veterans Administration Hospital. 54th Street and 48th Avenue S,, Minneapolis, Minn. 55417 (U.S.A.) Address for reprint; Secretary: Phoenix Research Section, National Institute of Arthritis. Metabolism, and Digestive Diseases, Phoenix Indian Medical Center, 4212 N. 16th Street, Phoenix, Ariz. 85016, U.S.A.
276
Introduction Bile salt combines with lecithin and cholesterol to form mixed-micelles in bile [1,2]. This micellarization process serves to solubilize biliary cholesterol [2,3] and may play a role in the hepatic secretion of both cholesterol and lecithin [ 4,5] . Because of the physiological importance of biliary mixed-micelles, a large body of information has accumulated regarding critical micelle concentration (CMC), size, and structure of bile salt micelles [2]. Most of these data have been obtained using pure systems which are similar, but not identical, to bile. Although such data are useful, conclusions about biliary micelle phenomena would be strengthened by data obtained using bile as well. The equilibrium of mixed-micellar bile salt with intermicellar bile salt in the aqueous phase has not been studied in bile. Quantitation of this equilibrium would provide an estimate of the tendency for bile salt to form mixed-micelles in bile and refine our concepts of the dissolution of biliary cholesterol and lecithin. Shankland has studied this equilibrium for the cholate-lecithin mixed-micelle [ 61 . He employed a light-scattering technique to obtain values for intermicellar cholate concentrations (cholate monomer + simple cholate micelles) in pure sodium cholate-lecithin solutions; however, light-scattering and other established methods for micelle investigation are difficult to apply to bile because many biliary constituents interfere with application of these methods [2] . In the present investigation, this difficulty has been overcome by using equilibrium-dialysis to measure the intermicellar bile salt concentration (IMBC) in bile. This IMBC, like that in pure cholate-lecithin solutions, probably comprises both bile salt monomer and simple bile salt micelles, both of which are small enough to dialyze. The large mixed-micelles, however, are retained by the dialysis membrane. Thus, at dialysis equilibrium, the concentration of bile salt outside the dialysis bag is equal to the IMBC in equilibrium with the mixedmicelle. Validation of Lhis equilibriumdialysis method has been obtained using Shankland’s data on the cholate-lecithin mixed-micelle for comparison. Interference from other biliary constitutents has been indirectly ruled out by testing a pure artificial bile. Finally the method has been applied to bile for measurement of the bili‘ary IMBC as a function of ionic strength, dilution, and bile salt content of the mixed-micelle. Methods
Human gallbladder bile was obtained aseptically at uncomplicated cholecystectomy from Indian patients with gallstones and a visualizing gallbladder. Bile specimens were kept sterile at room temperature and centrifuged at 266() y g to remove cholesterol crystals. All specimens were studied within 12 h of surgery. Cholic: acid (Matheson, Coleman, and Belle; Norwood, Ohio), conjugated bile salts (Maybridge Research Chemicals; Lauceston, Cornwall, U.K., and Stcraloids, Inc.; I’awling, N.Y.), cholesterol (J.T. Baker, Phillipsburg, N.J.) and
a chromatographic preparation of egg lecithin (Sigma Chemical Co.; St. Louis, MO.) were obtained commercially. Cholic acid was converted to sodium cholate by neutralization with NaOH. These materials were tested for purity on thinlayer chromatography. All materials moved in a single band on thin-layer chromatography except for the glycine and taurine conjugates of chenodeoxycholate, both of which had a faint additional band at the solvent front. Since these chenodeoxycholate conjugates were to be used for confirmatory studies only, no attempt was made to purify them further. Chemical analyses Bile salts were measured by an automated procedure based on Talalay’s enzymatic assay [ 71. Phospholipids were determined as lipid-phosphorous using conventional methods [S] . Cholesterol was measured by an automated procedure based on the Liebermann-Burchard reaction [8] . Bile water content was measured gravimetrically after desiccation of a 0.20 ml bile sample at 100°C for 1 h. Chloride was determined by AgCl precipitation using a constant amperage chloride tritrator (American Instruments Co., Silver Spring, Md.). Dialyzate protein was measured by the Folin method [9] . Thin-layer chromatography of phospholipids was done on Silica Gel H in a solvent system of chloroform/methanol/water (65 : 25 : 4) and visualized with iodine vapor and molybdenum spray as described by Dittmer and Lester [lo]. Conjugated bile salt thin-layer chromatography was done on Silica Gel H in a solvent system of n-butanollacetic acid/water (10 : 1 : 1). Cholesterol thin-layer chromatography was done in a solvent system of ethyl ether/heptane (55 : 45). Dialysis procedure Because temperature in the range of 20-40°C has a negligible effect on the formation of most bile salt micelles [ll] , all dialysis were done at room temperature on a metabolic shaker. Dialysis tubing (Union Carbide) having a minimum molecular weight retention of about 12 000 was used throughout. Tubing was opened by gentle agitation in sterile distilled water and tied at one end. Bile samples were introduced into the open end of the tubing with a sterile syringe. The open end was then tied with surgical silk. Boiling this tubing for several hours prior to use did not affect the dialyzable bile salt concentration in three bile samples tested, so this procedure was not routineiy followed. Prior to dialysis, all bile samples were adjusted to pH 7.5 * 0.3 with 1 M NaOH or HCl and, unless otherwise specified, to a chloride concentration of 0.10 M with dry NaCl. Usually 1.0 ml aliquots of bile (or artificial bile) were loaded into the dialysis tubing and dialyzed against 6-10 volumes of 0.10 M NaCl. Dialysis equilibration time for this system was examined in five different bile specimens and in serial dilutions of two of these specimens. These kinetic studies covered the entire range of dialyzate volumes used for subsequent studies and were carried out for as long as 38 h. Bile samples for these experiments were put up in dialysis bags just as described above. Timed samples of the dialyzates were taken being careful to remove less than 2% of the total dialyzate volume with each sample. Similar dialysis kinetic studies were performed on a pure mixture of conjugated bile salts (without lecithin or choles-
218
terol) simulating the proportions found in bile (exact composition the same as described under Artificial System). On the basis of equilibration kinetics defined in these experiments (see Results) all subsequent samples were dialyzed for 12 h. In two of the kinetic studies, dialyzates were analyzed for phospholipid and cholesterol. This was done to insure that phospholipid and cholesterol were not escaping significantly into the dialyzate. Analysis of cholesterol in the dialyzates was done by gas-liquid chromatography as described by Miettinen, et al. [12]. Analysis of dialyzates for phospholipid was achieved by a semi-micro technique exactly the same as the conventional method but with all reagent volumes scaled down lo-fold. The sensitivity of this technique on lecithin standards is about 0.01 mg of phospholipid. In addition to these kinetic experiments, over 15 additional dialyzates have been analyzed for phospholipid and cholesterol after a 12-h dialysis. After dialysis, samples of dialyzate were taken for determination of bile salt and chloride concentrations. The contents of each bag were evacuated, and 6 O.lO-ml samples were taken for duplicate analysis of chloride, bile salt, phospholipid, and cholesterol. Total bile-water was determined on a 0.20-ml sample from the bag and used to correct the gross bile chloride concentration to bile-water chloride concentration. This correction usually amounted to a 515% upward adjustment of the gross bile chloride concentration. Dialyzate and bile-water chloride concentrations were used to monitor Donnan effects, which were negligible (bile-water chloride/dialyzate chloride, 0.95-1.0) in the samples studied. Consistent with negligible Donnan effects, the pH of bag contents was within 0.2 pH units of dialyzate pH and ranged from 7.2 to 7.7 in representative samples studied. From the above data, micellar bile salt concentration (mmol bile salt in mixed-micelles/l) was calculated by subtracting the dialyzate bile salt concentration (mmol/l) from the total bile salt concentration in the dialysis bag. This micellar bile salt concentration was then divided by the phospholipid concentration in the dialysis bag to obtain the molar micellar bile salt/phospholipid ratio. Method validation Because equilibrium-dialysis is not an established technique for the study of bile salt mixed-micellar systems, experiments were performed to test the validity of this method. For this purpose, the cholate-lecithin mixed-micelle was studied. Shankland has carefully defined the IMBC in equilibrium with this mixed-micelle and related the IMBC to the micellar bile salt/phospholipid ratio; however, his data were obtained by light-scattering [6], a technique quite different from equilibrium-dialysis. The basic working solution for these experiments was prepared by mixing pure sodium cholate in methanol with pure egg lecithin in hexane. The solvent was removed in vacua below O-C, and the dry residue taken up with sterile 0.12 M NaCl. The final clear solution was adjusted to about pH 10 with 1 M NaOH and contained 59.7 mmol cholate and 10.8 mmol lecithin per 1. Final composition of this solution was essentially identical to the composition of solutions used by Shankland 161.
2.44 2.75 2.71 2.74
IMBC
Bile S --“_.
28.2 13.5 7.61 3.80
Bag phosphalipid
--l--_
.._^
1.45 1.43 1.31 1.36
phospholipid 2.80 2.83 3.11 2.96
IMBC
1.37 1.50 1.36 1.48
32.5 15.8 8.36 4.34 66.0 36.6 20.6 12.3
Bag bile salt
HELD CONSTANT
2.51 323 3.20 &a6
XMBC
28.3 20.8 10.7 5.80
Bag phospholipid
1.65 1.60 1.63 I.54
Bile salt/ phospholipid
-_^____._-~“--
1: 8
Undiluted 1 : 2 1:4
_.._.
Dilution
47,3 18.7 6.00
1.49
8.20 2.16 I.45
0.96
IMBC Bag bile salt
Bile T (81
-__..--___
.__.__._~
3.01
1.10
3.38
14.4 11.2 7.79
,..^__.
28.7 18.3 7.85
3.00 2.26 1.64
_---“.-_
0.73
-_--..“-~
2.46 1.88 1.25
.*_-_---,
28.0 x4.3 7.26 4.01
1.27
1.02
2.51 2.12 1.46
-------.__-_-_1,
3.52
67.9 33.0 11.8
IMBC Bag bile salt
5.54
37.2 22.2 10.9
Bag phosphofipid
-_
..__
0.54
2.44 1.56 1.06
“,___
0.92
.Bag phospholipid
2.67
17.6 10.5 5.35
.__,_“__ 26.9 10.0 2.41
IMBC Bag bile sait
Artificial bile (10) -.~~~ -“-~.“_-“__-.
._,_ ______
.-_~---~-_____-_lll Bile B (101 ~ .__-
.-_l__~-_.__-..____
.-.----____
Bag phospholipid
---
-.._ _.__.
45.3 17.0 5.39
IMBC Bag bile sait
Bag phospholigid
IMBC Bag bile salt
~ Bile S (10)
-~
Bile N (6) --.---
.._ _ .,.I____---
5.48
26.2 17.2 8.13
Bag phospholipid
._
All concentrations in mmol/l. Numbers in parentheses denote dialyzate volume in ml. All bag volumes 1.0 ml. To put dilutions into prospective with respect to undiluted gallbladder bile, note that dialyzable bile salt is diluted not only by sample dilutions (first columnf but also by the dialyzate volume. Taking Bile T as on example, dialyzable bile salt in the undiluted sample is diluted 9-fold (1 ml of sample + 8 ml dialwateI; on the 1 : 2 sample, dialyzate bik salt is diluted Ill-fold, etcThe non-dialyzable mixed-micelies, however, are not diluted by the dialyzate.
SYSTEM
VOLUME
Bile salt/ ghospholipid
HELD CONSTANT
Bag phospholipid
BILE WITH TOTAL
47.3 26.6 14.5 9.3Q
IBag bile salt
I
BILE SALT/PHOSPHOLIPID
ON THE IMBC OF BILE AND ARTIFICIAL
43.3 22.0 12.7 1.89
bile salt
Bag
_-- ___. r_l____~.“l
EFFECT OF DILUTION
TABLE II
Undiluted 1: 2 I : 4 1:8
Dilution
______
Bile salt/
ON IMBC IN BILE WITH MICELLAR
All concentrations are expressed as mmol/l.
EFFECT OF DILUTION
TABLE I
CD
2
280
l-ml aliquots of this solution were dialyzed against varying volumes (1.0-12.0 ml) of 0.12 M NaCl for 12 h at room temperature. In kinetic experiments with three samples dialyzed for 24 h, it was found that complete dialysis equilibration occurred by the 12th hour. Varying the volume of dialyzate served to alter both IMBC and the ratio of bile salt/phospholipid in the mixedmicelle. strength The effect of ionic strength on the IMBC *as determined by adding dry NaCl to aliquots of gallbladder bile. One aliquot was made 0.55 M in chloride, another aliquot 0.10 M, and another aliquot 0.15 M. Each aliquot was then dialyzed against a solution of NaCl having the same molarity as the molarity of chloride in the bag. Dialyzate volumes and pH were held constant. In two bile specimens, the effect of divalent cations on the IMBC was evaluated. This was accomplished either by addition of EDTA to the dialyzate or by equivalent substitution of CaCl, for NaCl in the dialyzate (25 mequiv/l substituted).
Ionic
Dilution and micellar bile salt content Studies of the effect of dilution on the IMBC were done as follows: Chloride concentration and pH of each bile were adjusted as described under Dialysis procedure. The bile was then divided into aliquots one of which was left undiluted, another diluted 1 : 2 with 0.10 M NaCl, another diluted 1 : 4, and another diluted 1 : 8. Each aliquot was then dialyzed against 0.10 M NaCl. In some experiments the dialyzate volume for each dilution was held constant. This resulted in a progressive reduction of micellar bile salt/phospholipid with dilution. Consequently, these data could be used to define the relationship between the IMBC and micellar bile salt/phospholipid as well as the effect of simple dilution on the IMBC. Dialyzate volumes for these experiments are given in Table II. To put the dilutions in perspective with respect to undiluted gallbladder bile, note that dialyzable bile salt is diluted both in the original sample dilution and again by the dialyzate volume. III addition, similar dilution studies were performed attempting to hold micellar bile salt/phospholipid constant (Table I). It was hypothesized that IMBC would not change with dilution of micelles alone, since this is true in simplemicellar systems [13]. For any given bag volume, 2-fold dilution is equivalent to halving the total solute content (micelles and intermicell~ bile salt). However, by simultaneously reducing the dialyzate volume enough to halve the total system volume (bag volum e f dialyzate volume), the absolute quantity of intermicellar bile salt necessary to maintain a constant IMBC is also halved. This leaves micellar bile salt undisturbed and maintains micellar bile salt/phospholipid and its pre-dilution level. Therefore, progressive 2-fold dilutions of bile were made as above, but with each Z-fold dilution the dialyzate volume was reduced by the amount necessary to halve the total system volume. Artificial system To establish that the measurements done on bile were not affected by any of the other constituents of bile, (e.g. bilirubin and protein), an artificial bile
283
system was prepared from pure conjugated bile salts, egg lecithin, and cholesterol. Each of the three components was stored in ethanol or hexane at -20°C. At the ~~~ro~~ate time, volumes of each of the three components were cornbined. The sokent was evaporated in vacua below 0°C and the dry residue taken up with a volume of sterile 0.10 M NaCl calculated to give a final bile salt concentration of 60 mM. Molar percents in this artificial system were as follows: bile salt 64%, lecithin 24%, and cholesterol 12%. The bile salt fraction contained 29% glycocholate, 26% glycochenodeoxycholate~ 10% glycodeoxycholate, 15% ~u~ch~~a~* 14% ~ur~chen~eo~ycho~ate~ and 5% ta~r~eoxy~ cholate, The final sterile aqueous mixture was adjusted to pH ‘73 with sterile 1 M NaOH and allowed to equilibrate for 3 days at room temperature. One aliquot of this solution was left undiluted, another was diluted 1 : 2 with 0.10 M N&l, another diluted 1 : 4, and another diluted 1 : 8. Each aliquot was then dialyzed against 8.0 volumes of 0.10 M NaCl.
Dialysis procedure Studies of bile dialysis kinetics, shown in Fig. 1, demonstrated a plateau of dialyzate bile salt concentration by 12 h when the ~qu~l~br~~rn dialyzate bife salt concentration was 4 mM or lower. This was confirmed by dialysis of a mixture of pure conjugated bile salts (not containing lecithin or cholesterol) simulating the proportions typically found in bile, These studies showed a dialyzate bile salt concentration higher than 96% of the calculated equilibrium value at 12 h when the equilibrium dialyzate bile salt concentration was 7.8 mM, Equ~ib~~m was attained even more rapidly pihen the eq~~~ibr~~rn diafyzate bile salt concent~tio~ was below 7.8 mM.
Ttme fhrsf Fig. 1. K%.lyzate ISI& ~46, phasphaIipid. and cholesterol ~~n~entra~ans as a fun&lam of tinre. E~XZWX?~XS diatyzate bile salt c~ncentmtkms covering the Qntim? range of concentrations in this lieport are showr~ Open circled and closed ci.rck?s represent two different undiluted bilas. Open squares XQpKesent a 1 : 2 dilution af the former bile, while closed triangles and open tria.&es represent1 : 2 end ‘i : 4 dilutions respectively of the latter bile. Dialyzate bile salt plateaued by 12 h ia all cases. Phospholipid and cholesm terol concontmtions were meawrable but negligibly small in three dialyzates. In the remaining two dialyzates no phospholipid OX cholesterol could be de@cCed.
282
Kinetic studies of dialyzate phospholipid and cholesterol (Fig. 1) demonstrated little tendency for these two lipids to escape from the dialysis bag. In fact, dialyzates of two of the dilutions of bile shown in Fig. 1 contained no detectable phospholipid or cholesterol even after 38 h of dialysis. This nearly complete retention of phospholipid and cholesterol by the dialysis membrane was confirmed in more than 15 dialyzates tested only at 12,h. The highest concentrations of phospholipid and cholesterol measured in these dialyzates were 0.18 mM and 0.035 mM respectively. These concentrations correspond to less than 0.5% of the bag phospholipid and cholesterol and less than 3% of dialyzate bile salt concentration. Dialyzate protein, measured in four experiments, was less than 0.1 mg/ml suggesting nearly complete retention of biliary proteins by the dialysis membrane. Phospholipid thin-layer chromatography of six bag contents after dialysis demonstrated a single band visible by molybdenum spray. The position of this band corresponded to lecithin standards suggesting that no significant breakdown of phospholipid occurred during the study. Method
validation
Results of the method validation experiments on pure cholate-lecithin solutions are shown in Fig. 2. Measurements of the IMBC and micellar bile salt/phospholipid obtained by equilibrium-dialysis of these solutions correspond very closely to light-scattering measurements of these two parameters reported by Shankland [6]. Phospholipid concentrations were measured in all dialyzates in the cholate-lecithin experiments and were less than 0.04 mM after 12 h of dialysis. Dialysis equilibration time for this micellar system is even shorter than for bile. Thus, in three samples dialyzed for 24 h, 98% of equilibrium was attained by 9 h in two and by 11.5 h in the third. Consistent with these rapid dialysis kinetics, solutions of pure sodium cholate (not containing lecithin) reached
7
200
Chloride (mEq/L) Fig.
2.
pholipid land Fig.
3.
studies.
in cholate-lecithin
[I51 using
chloride but
Cholate-lecithin
IMBC
light-scattering as a function
concentrations.
the reduction
Equilibrium-dialysis
solutions (open of ionic All
is so small
bile
(closed circles, strength.
show
measurements
circles.
solid
dotted
line).
Four
different
a very
as to be negligible.
slight
line)
bile
reduction
of
IMBC
are compared
specimens in the
IMBC
and
micellar
to data
were with
dialyzed
bile
obtained
at three
increasing
ionic
salt/phosby Shank-
different strength.
283
dialysis equilibrium centration exceeded
by 12 h even when the equilibrium 15 mM.
dialyzate
cholate
con-
Ionic strength There was a consistent, but very small reduction in the IMBC with increasing ionic strength in the range of 0.05-0.15 M chloride (Fig. 3). This reduction was so small as to be negligible. Equivalent substitution of CaCl, for NaCl in the dialyzate and addition of EDTA to the dialyzate had no effect on the IMBC suggesting that divalent cations do not significantly influence this parameter. Dilution When micellar bile salt/phospholipid was held constant by appropriate adjustment of the dialyzate volume with each 2-fold dilution (see Methods), dilution did not affect the IMBC (Table II). This finding is consistent with the assumption used by previous investigators [6,14] that the escaping tendency of solubilizer from the mixed-micelle is independent of the concentration of micelles. * The above experiments, however, do not reflect the effect of simple dilution of bile which is essentially a process of halving the total solute for any given volume. Table II shows the effect of dilution when the total system volume is held constant, a situation analogous to simple dilution. In these experiments, any given 2-fold dilution reduced the IMBC, but by less than a factor of 2. Thus, at the instant of dilution both the micelle concentration and the IMBC fell by a factor of 2. The IMBC then tended to rise toward its previous level, but in doing so drew bile salt out of the mixed-micelle and lowered micellar bile salt/phospholipid. Therefore, the equilibrium IMBC was reduced by dilution because micellar bile salt/phospholipid was lowered. Micellar bile salt content The relationship between the IMBC and micellar bile salt/phospholipid shown in Fig. 4 was derived from the same samples used in the dilution experiments for which the dialyzate volume was held constant. There appears to be a linear relationship between IMBC and micellar bile salt/phospholipid with reasonable agreement among the bile specimens studied. It should be emphasized, however, that none of these points represent the IMBC or micellar bile salt/phospholipid of undiluted bile because dialyzate volumes ranged from 6 to 10 times the bag volume. It is possible that measurements on strictly undiluted bile would reveal a deviation from linearity at higher values of micellar bile salt/phospholipid *This assumption can be theoretically justified by considering an ideal mixed-micellar system. Suppose that 11 mol of the solubilizer, A, combine with VI mol of the solubikate. B. (otherwise insoluble), to form 1 mol of the mixed-micelle. M. Since B is insoluble, its chemical activity can be defined as 1.0, and the equilibrium constant for this reaction written: I< = [MI /LA]” where activity coefficients are neglcctrd. Then if solution 1 is diluted by a factor of two to obtain wllution 2:
Fig. 4. IMBC as a function of micrllar bile Salt/phospholipid. Serial dilutions of bile (or artificial bile) were dialyzed against equal volumr~s of dialyzate. Five different biles are shown (closed and open circles. closed and open squares, and closed triangles). Taken as a group, these five Specimens yield the depicted least-squaws regression: IMBC = 1.27 (bile salt/phospholipid) + 0.538. Open triangles represent the Samf’ studv performed on an artificial bile. These are indistinguishable from the data obtained on bile suggesting that the other constiturnts of bile do not affect IMBC or micellar bile Salt/phospholipid. Open Squares represent a bile dialyzed for 36 h. These points correspond closelv to the other data obtained by a 12 h dialysis and further Support the 12 h dialvsis time as being adequate for equilibration. since there is likely to be a point at which mixed-micelle becomes saturated with bile salt. Four of the points shown in Fig. 4 were obtained by dialyzing for 36 h. These appear to be in good agreement with the rest of the data obtained by dialyzing for 12 h. This observation provides additional evidence that dialysis equilibrium was achieved by 12 h. Artificial system Measurements of the IMBC as a function of micellar bile salt/phospholipid obtained in the artificial bile are also shown in Fig. 4. These measurements correspond closely to the data for bile obtained in similar experiments. This finding suggests that other constituents of bile, such as protein and bilirubin, do not have an appreciable effect on the IMBC. Discussion In the present investigation, the IMBC in equilibrium with the bile saltlecithin-cholesterol mixed-micelle has been studied in human bile. Lecithin and cholesterol, the other two lipid constituents of this mixed-micelle, presumably do not have appreciable chemical activity in the intermicellar aqueous phase since they are insoluble in water. Measurement of the IMBC has been achieved by the relatively direct technique of equilibrium-dialysis. Similar techniques have been used by other investigators for the study of detergent solutions [ 151 ; however, equilibriumdialysis apparently has not been previously applied to the study of bile salt mixedmicelles. To verify the applicability of this technique to IMBC measurements in bile salt mixed-micellar systems, the equilibrium-dialysis method has been applied to the cholate-lecithin mixed-micelle for which the IMBC has been previously defined using an independent method [6]. As shown in Fig. 2, the relationship between intermicellar cholate concentration and the micellar
285
cholate/lecithin ratio obtained in the present study using equilibriumdialysis corresponds very closely to the data obtained by Shankland using light-scattering. This close correspondence confirms Shankland’s observations and strongly suggests that equilibrium-dialysis is a valid technique for measurement of the IMBC in bile salt mixed-micellar systems. It should be emphasized, however, that dialysis equilibration time and egress of mixed-micelles through the dialysis membrane must be evaluated for each different mixed-micellar system being studied. Neither phospholipid nor cholest.erol escaped significantly from the dialysis bag in our experiments with pure cholate-lecithin solutions or with bile, but this may not be true for systems having low molecular weight mixed-micelles. Furthermore, as the bile salt concentration of pure bile salt solution (not containing lecithin or cholesterol) rises above the CMC, dialysis equilibrium time becomes progressively prolonged. The degree of prolongation apparently is proportional to the size of simple micelles formed by that species of bile salt. Thus, our solutions of pure cholate, even above the CMC (= 4.5 mM), had rather rapid dialysis kinetics and reached equilibrium at 12 h even when the equilibrium cholate concentration exceeded 15 mM. Presumably this rapid equilibrium occurred because the simple cholate micelles are small (Mr 2 2500) [ 111. Mixtures of pure conjugated bile salts simulating the proportions found in bile, (micelle M, 8000, CMC 0.7-1.4 mM) [ll] had slower dialysis kinetics reaching equilibrium at 12 h only when the equilibrium bile salt concentration was less than 7.5 mM. Consistent with these observations, bile dialysis specimens having an equilibrium dialyzate bile salt concentration of 4.1 mM or less reached dialysis equilibrium by 12 h, as shown in Fig. 1. Further evidence that equilibrium has been attained in such samples is provided by the fact that four bile dialyses carried out for 36 h yielded values for IMBC (as function of micellar bile salt/phospholipid) indistinguishable from values obtained in the other bile specimens dialyzed for 12 h. Theoretically, a high content of bile salt in the mixed-micelle should favor movement of bile salt out of the mixed-micelle (or conversely reduce the tendency for bile salt to enter the mixed-micelle). Thus, it is not surprising that the IMBC in bile and artificial bile vary directly with micellar bile salt/phospholipid as shown in Fig. 4. A similar direct proportion is apparent for the cholatelecithin mixed-micelle as reflected in the data reported by Shankland and confirmed in the present investigation (Fig. 2); however, for any given micellar bile salt/phospholipid, the IMBC in the cholate-lecithin system is higher than in the conjugated bile salt-lecithin-cholesterol system. This difference in IMBC levels may reflect some effect of bile salt conjugation or perhaps, the absence of cholesterol from the cholate-lecithin system. Alternatively, since cholate has a higher CMC (lower tendency to form simple-micelles) than the mixture of bile salts present in the bile [ll],cholate may have less tendency to form mixedmicelles as well. At least two reports of the CMC of bile have appeared in the literature [ 16,171. To paraphrase Mukerjee and Mysels, the CMC of a mixed-micellar system can be defined as that concentration of solubilizer (in this case bile salt) at which the solubilizate just begins to dissolve [13]. As they point out, this mixed-micelle CMC should theoretically be below the CMC of the pure SO~U-
286
bilizer. According to this definition, the CMC of bile would approximately correspond to the IMBC at zero micellar bile salt/phospholipid, obtained by extrapolating the data in Fig. 4. Such extrapolation yields a value of 0.54 mM for the average CMC of the five biles studied. This value is below the CMC of the mixture of bile salt found in bile (= 0.7 mM), as predicted by Mukerjee and Mysels, and is in fair agreement with the two previously reported bile CMC values of 1.5 and 1.45 mM. These two previously reported values for the CMC of bile may be imprecise because the methods employed in their measurement (ultracentrifugation and surface tension) are likely to be subject to considerable error when applied to a complex solution such as bile. In contrast, the nonmicellar constituents of bile would appear not to influence measurements obtained in the present study since our data in artificial bile correspond closely to data in similar bile specimens (Fig. 4). Because dialyzate volumes used in this study were 6-10 times larger than the volume of bile within the bag, the data reported here do not represent the IMBC of undiluted gallbladder bile. Such measurements would require a dialyzate volume that was negligibly small relative to the bag volume. Six bile specimens have been dialyzed against an equal volume of dialyzate; however, all of the 12 h dialyzate bile salt concentrations for those samples were above 7 mM, a concentration above which dialysis equilibration time is prolonged (as noted above). For this reason none of these data have been included in our results. In artificial bile systems, the maximum amount of cholesterol solubilized by any given amount of bile salt and lecithin is reduced by lowering the total solute concentration [ 181. Our observation that simple dilution of bile lowers micellar bile salt/phospholipid suggests that this reduction of cholesterol solubilizing capacity by dilution is a result of depleting the mixed-micelle of bile salt. Acknowledgments Cooperation from the surgical staff of the Phoenix Indian Medical Center in obtaining bile specimens is gratefully acknowledged. Thanks are also due to Mrs Marjorie Kennel and Mrs Harriet Rascon for careful secretarial assistance and Mr Robert Collins for technical assistance. References 1 Bourges, 2
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