Distinct effects of three bile salts on cholesterol solubilization by oleate-monoolein-bile salt micelles

289

Biochimica et Biophysics Acta, o Elsevier/North-Holland

575 (1979)

Biomedical

289-294

Press

BBA Report BBA 51259

DISTINCT EFFECTS OF THREE BILE SALTS ON CHOLESTEROL SOLUBILIZATION BY OLEATE-MONOOLEIN-BILE SALT MICELLES

J.C. MONTET,

M.O. REYNIER,

A.M. MONTET

and A. GEROLAMI

Unit8 de Recherches de Pathologie Digestive, U 31 INSERM, 46, Boulevard de la Gaye, 13009 Marseille (France) (Received

July 20th,

1979)

Key words: Cholesterol solubilization; Bile salt micelles; Lipid digestion; Phase equilibrium

Summary

Micellar cholesterol solubilities in bile salt-monoolein-oleic acid systems have been determined. Whatever the bile salt/oleyl compounds ratio, taurochenodeoxycholate solubilizes more cholesterol than taurocholate and much more than tauroursodeoxycholate. At pH 6.7, the cholesterol solubility limit is about the same with either oleate or monoolein. Cholesterol solubility falls in oleate-bile acid mixtures as the pH is raised. The capacity for supersaturation with cholesterol is greater for bile salt-monoolein than for bile salt-oleate micelles. For the latter it decreases as pH increases.

The role of bile salts in cholesterol solubilization in bile has been extensively studied [l-3] . These studies have shown particularly that taurochenodeoxycholate, taurocholate and taurodeoxycholate with lecithin have similar and better cholesterol solubilizing power [ 3-51 than tauroursodeoxycholate which was shown to be a poor detergent [6]. We know that the dissolving capacity of bile salts also plays an essential role in the intestinal absorption of lipids [ 71. Interactions between bile salts, cholesterol and lipolysis products have been studied especially by Hofmann [8,9] but the effects of various bile salts have not been compared. Initial studies however showed [lo] that the solvent properties of dihydroxy conjugates for monoolein were better than those of trihydroxy conjugates. In addition to their general interest these studies have now clinical importance since chenodeoxycholic and ursodeoxycholic acids are used in man for the treatment of cholesterol gallstones [ll, 123. This treatment modifies the bile composition [ 131 and consequently that of the intestinal content, and therefore may in-

290

fluence lipid absorption. The purpose of the present work is to delineate the interaction of lipids with various bile salt solutions in model systems the composition of which simulates the lipid composition of the intraluminal micellar phase of the small intestine. This work particularly studies cholesterol solubilization (metastabihty, equilibrium solubility at 37°C) by three bile salts: taurocholate, taurochenodeoxycholate and tauroursodeoxycholate. The micellar solubilization of lipids in bile salt solutions was investigated by the coprecipitation method [ 3, 141. The appropriate amounts of bile salts, monoolein, oleate and cholesterol were dissolved in organic solvents (methanol or chloroform). The solvent was evaporated in vacua over phosphorus pentoxide for 24 hours, then the dried mixtures were dissolved in phosphate buffer pH 6.7,0.13 M Na”, at 37°C so that each mixture had a content of 4 g/d1 total lipid. This total lipid concentration probably represents the upper limit of physiological concentrations found in the intestine. It was chosen however because accurate determination of solubility limits from Tyndall effect are obtained in these conditions. The tubes were flushed with nitrogen and then sealed. The separation of a second phase (cholesterol crystals, paracrystalline phases) from the isotopic mixtures was monitored by optical methods: polarizing microscope, Tyndall phenomenon. In a preliminary step, the solubilization of monoolein and oleic acid by the three bile salts, was examined at pH 6.7 which is near the intraluminal pH during fat absorption. Cholesterol solubilization was then measured in mixtures with various ratios of bile salt to oleate or monoolein or to both. Varying amounts of cholesterol were added to obtain series of mixtures with fixed ratios of bile salt to oleyl compounds and increasing amounts of cholesterol. Samples containing cholesterol were heated to 90°C for 3-40 min in order to prepare supersaturated solutions. The clear tubes were then cooled to 37°C. By this procedure were determined both the capacity for supersaturation at 37°C and the equilibrium solubility of cholesterol at 37°C. The changes in the physical state of the supersaturated solutions were followed daily for 15 days. The equilibrium solubility was reached when the upper limit of the micellar phase ceased to change with time. To check the results obtained by optical examination, the clear solutions containing the highest cholesterol concentrations were filtered through 0.1 pm Sartorius membranes and analyzed for monoolein, cholesterol and bile salt. Each experiment was carried out three times with very similar results in each case. ~ol~bili~~ of monoolein and oleic acid in bile salt solutions Bile salts disperse oleic acid-sodium oleate mixtures (pH = 6.7) into micellar solutions. At 37” C, taurocholate and taurochenodeoxycholate solubilize the acid-soap to the same extent i.e. 1.5 mol of oleate per mol of bile salt. These values agree well with earlier results [ 91. On the other hand, tauroursodeoxycholate has a lower dissolving power 1.04 mol of oleate per mol of bile salt. The solubilities of monoolein in bile salt solutions are slightly higher than for oleate: 1.63 mol of monoolein per mol of taurocholate or taurochenodeoxycholate. One mol of tauroursodeoxycholate can solubilize only 1.17 mol of monoolein. The solubility limits of a mixture oleate/monoolein

291

= 2 (molar ratio) are identical to that found with pure oleate. Egg lecithin has a similar solubility in bile salt solutions [ 31. Thus the number of dissolved fatty acid chains for a given bile salt concentration is twice with lecithin than what it is with oleate or monoolein.

Cholesterol

solubility

For each micellar system studied, the cholesterol solubility depends on the molecular structure of bile salt. Under all conditions, taurochenodeoxycholate solubilizes more cholesterol than taurocholate, and tauroursodeoxycholate is the poorest solubilizer. Bile salt-cholesterol system. At 37°C cholesterol solubility (moles per cent) is 1.96 with taurochenodeoxycholate and 1.47 with taurocholate. A great difference appears with tauroursodeoxycholate in which cholesterol solubility is 0.19. These results agree well with those of Hegardt and Dam [ 51 and Carey et al. [ 61. The slightly smaller values of solubility obtained are due to the fact that our solutions are more dilute. Effect of monoolein-oleate to bile salt ratio. The solubility limit of cholesterol (equilibrium values at 37” C) greatly depends on the ratio bile salt/oleyl compounds (Fig. 1). With a decreasing ratio bile salt/monoolein + oleate the hydrocarbon core of the mixed micelle is enlarged and the differences between the dissolving powers of taurochenodeoxycholate and taurocholate decrease (table I). Tauroursodeoxycholate shows a distinct behaviour since the 52/48 ratio approaches the micellar phase limit of the mixture monoolein + oleate in this bile salt. Whether the differences for cholesterol solubilization between taurocholate and taurochenodeoxycholate are explained by distinct values of their intermicellar bile salt concentration in presence of oleyl compounds is unknown. It was shown by Duane [15] that taurochenodeoxycholate in presence of lecithin has a lower intermicellar concentration than taurocholate. Ursodeoxycholic acid has an equatorial 7fl-hydroxyl group and probably the orientation at the air-water interface of the molecule is different from that of cholic or chenodeoxycholic acids. This particular orientation of ursodeoxycholic acid may induce a distinct micellar structure. It was found by Carey and Small [6] that the maximum cholesterol solPERCENT

MONOOLEIN

. OLEATE

*~~~~ so

60

70 PERCENT

60 BILE

50

40

30

SALT

Fig. 1. Equilibrium cholesterol solubility values at 37’C plotted on triangular coordinates for bile salt(oleate + monoolein) -cholesterol systems with oleate/monoolein = 2. Relative lipid compositions are plotted as mol per cent of total lipids, on the scales along the sides of truncated triangles. Limit of micel1a.rzone with taurocholate (- -_), taurochenodeoxycholate ( - - - - - - -) and tauroursodeoxycholate ( -), at pH 6.7.

292 TABLE I CHOLESTEROL

MICELLAR

SOLUBILIZATION

IN BILE SALT-OLEYL

COMPOUNDS MIXTURES

Micellar cholesterol solubilities (mol per cent of total lipids) are determined for the three bile salts at two temperatures 3?C and 90°C at pH = 6.7, in various systems: bile salt-monoolein-cholestexol, bile sit-monoole~-olea~~hole~erol with oleate~monoole~ = 2, and bile at-oleate-cholesterol. For each micellar system, two molar ratios bile salt/oleyl compounds are studied: 72128 (bile salt concentration = 62 mM) and 52/48 (bile salt concentration = 46.8 mM). ..--._.._____ __~~~. __.“_. Bide salt/Monoolein Bile salt/Monoolein + Bile saltloleate Bile salts oleate .___ __I--. _ .-~~___ - .._ _ 72128 52148 72128 52148 72/28 52148 -.. ____ - _-.. .__~.. - _~ ~~. 37Oc Taurocholate Taurochenodeoxycholate Tauro~sodeoxycbolate

3.95 4.85 3.73

7.24 7.44 4.22

4.54 5.65 3.69

6.61 7.05 2.17

4.36 5.55 3.41

6.56 7.05 2.17

90°C Taurocholate Taurochenodeoxycholate Tauroursodeoxycholate

8.95 11.17 6.59

9.97 13.04 5.25

7.30 9.08 4.70

8.14 5.52 9.93 6.96 2.48 4.51 -..-~~--__~--

7.42 8.83 2.38

ubility occurred at a molar ratio taurocholate/lecithin = 7/3. Here with the taurocholate-ol~yl compound mixtures, the solubility of cholesterol is more important for the molar ratio 52/48 than for the 72/28 ratio. Since oleyl compounds have one fatty acid per molecule instead of two as in lecithin, this may indicate that the maximum of cholesterol solubility depends on the ratio bile salt/fatty acid chains. It appears that for a given bile salt/fatty acid ratio, cholesterol solubility is about the same in presence of either egg lecithin or of other oleyl compounds. Solubility values at 90°C (Table I) are much higher than those obtained at 37*C, but taurochenodeoxycholate remains the best solubilizer and tauroursodeoxycholate the poorest. The increase of the monomer solubility of cholesterol and modifications of the structure of mixed micelles could explain this temperature effect. An increase of the micellar size at high temperature could take place as was shown for egg-lecithin by Mazer [ 161. These dissolving effects produced by the increase of temperature are much stronger than opposite effects such as the slight increase in the critical micellar concentration of bile salts [ 171. Effect of varyingrnonoolein-oleateratio. The amounts of cholesterol dissolved in bile salt-oleate, bile salt-monoolein and bile salt-oleate-monoolein (molar ratio oleate/monoolein = 2) micellar solutions are shown in Table I. At 37°C the cholesterol solubility does not depend very much on the nature of the lipid polar head. For the ratio bile salt/oleyl compounds = 72/28 the differences observed by Simmonds et al. [S] between fatty acid and monoglyceride were larger than ours, but the experimental conditions were not similar (pH, dilution), The role of the polar head of the lipid appears more clearly with supersaturation. With monoolein the maximum cholesterol solubility is very high and bile salts disperse the lipids very rapidly (3 to 5 min) into mice&r solutions. The solubilization obtained with oleate is slower (15 to 40 min) and lower whatever the type of bile salt, Effects of pH. The cholesterol solubilities -were compared with tauro-

293

cholate-oleate-cholesterol and taurochenodeoxycholate-oleate-cholesterol mixtures obtained at four different pH values with a ratio bile sa.lt/oleate = 72/28 (Table II). For a given bile salt, equilibrium solubility at 37’C is identical at pH = 6.0 and pH = 6.7 then it decreases when pH is increased. SuperTABLE

II

CHOLESTEROL pH

Solubilities Bile

SOLUBILITY

IN BILE

SALT-OLEATE-CHOLESTEROL

MICELLES

AT

VARIOUS

VALUES (mol

b/o) are given

salts

for

a bile

pH = 6.0

salt/ok&z

molar

ratio

= 12128

at 37’C

PH = 6.7 ~__

pH = 7.5 ___

pH = 9.2

and

90°C.

37Oc Taurocholate

4.36

4.36

3.38

3.08

Taurochenodeoxycholate

5.73

5.55

4.27

3.62

90°C Taurocholate Taurochenodeoxycholate

7.37 10.02

5.52

3.71

3.30

6.96

4.46

3.71

saturation is also dependent on pH. The solubility values are maxima for acidic pH. The decreased cholesterol solubility occurring in association with increased ionization of oleate may only reflect a decreased capacity of the mixed micelle for cholesterol. A more probable explanation is that modifications of micelle composition occur with pH. There must be an equilibrium between the fatty acid involved in the mixed micelle and the fatty acid remaining in the aqueous phase (monomer and micellar form). Increasing pH favors formation of oleate micellar solutions. Such micelles have a smaller capacity to solubilize cholesterol than the mixed bile-salt-oleate micelles. This assumption explains why, at 37°C cholesterol solubilization decreases only when pH exceeds the pK, (6.5) of oleic acid in bile salt solution [9]. The same mechanism may work during supersaturation since heating increases water solubility of fatty acids. In summary, it was shown that taurocholate, taurochenodeoxycholate and tauroursodeoxycholate have distinct capacities to solubilize cholesterol in presence of lipolysis products. Tauroursodeoxycholate has a lower ability than the others to solubilize oleic acid or monoolein. This is consistent with the results of Carey et al. [6] who used mixed micelles of tauroursodeoxycholate-lecithin-cholesterol. It must be recalled that our studies apply to a particular in vitro system. In vivo variability of total lipid composition possibly makes applicability of this system to intestinal content limited. It is interesting however, to compare these physical facts and physiological data on intestinal absorption of cholesterol. Apparently, the dissolving power of bile salt cannot completely explain the specific action of some bile salts on the rate of cholesterol absorption [lS, 191. It remains to be determined if bile salts react on other rate limiting factors or if structures other than mixed micelles play a role in lipid absorption [ 201. We would like to thank Professor Donal F. Magee for his help in the critical reading of the manuscript. J.C. Montet wishes to thank Professors D.G. Dervichian, D.M. Small and M.C. Carey for fruitful discussions during his stay in their laboratories.

294

This work was supported 58.78.90 and 60.78.92.

1 2

10 11 12 13 14 15 16 17 18 19 20

by grants from I.N.S.E.R.M.

78.5.232.7,

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