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Physicochemical Studies of Cholesterol Gallstone Formation
Vol. 52, No.3
GASTROENTEROLOGY
Printed in U.S.A.
Copyright © 1967 by The Williams & Wilkins Co.
COMMENTS Readers are invited to contribute Comments. If found suitable, they will be published promptly, subject to the usual editing. They should be typewritten double spaced and sent to the Editor.
PHYSICOCHEMICAL STUDIES OF CHOLESTEROL GALLSTONE FORMATION
Cholesterol gallstone formation may be divided into two processes: the oversaturation of bile with cholesterol and the formation of macroscopic stones from the oversaturated bile. The first of these processes is related to the change in the physical state of bile from a clear solution to a solution containing crystalline cholesterol. Physicochemically, this change may be regarded as the transformation of a one-phase system (liquid) to a two-phase system (liquid and solid) . For instance, this sort of change occurs when a solution of common table salt evaporates beyond its point of saturation and salt crystals appear in the liquid. By analogy one may say that in normal bile the concentration of cholesterol is less than the concentration at its saturation point, I while when cholesterol precipitates from bile its concentration must be above the saturation point. Although this analogy is probably correct, the situation as it pertains to bile is far more complicated. Cholesterol is virtually insoluble in water. I , 2 Therefore, the large amounts of cholesterol found in normal bile, e.g., 500 mg per 100 ml of bile, must be "solubilized" by some other biliary components. For many years it was thought that bile salts were responsible for holding cholesterol in solution, but during the last 15 years it has been conclusively shown that bile salts alone are not adequate and that lecithin is also necessary for this effect. a, 4 Address requests for reprints to: Dr. Donald M. Small, Department of Medicine, Research Building, Boston University Medical Center, 15 Stoughton Street, Boston, Massachusetts 02112. This work was in part supported by United States Public Health Service Research Grant AM 05589 and Training Grant 05025.
Recently phase equilibria techniques 5 , 6 and X-ray diffraction studies7 have been used to delineate the solubility of cholesterol in any and all combinations of bile salts, lecithin, and water. These studies showed that lecithin associates with cholesterol in a specific liquid crystalline phase and that bile salts break down this phase into micelle-sized aggregates, thus carrying the two insoluble components into solution. The bile salt and lecithin concentrations in normal bile are such that the limits of solubility of cholesterol are not surpassed. In contrast, bile from patients with cholesterol gallstones is oversaturated with respect to cholesterol. Since human bile behaves like the model system of bile salts, lecithin, cholesterol, and water with respect to cholesterol solubility, the other components in bile (bilirubin, inorganic ions, proteins, mucus, etc.) do not affect the solubility of cholesterol in bile. In recent years bile has been assumed to be a micellar solution. Thus, ultracentrifuge studies8,9 suggested that bile contained aggregates of micellar size, and light-scattering studies 10 demonstrated that these aggregates were roughly the same size as aggregates made up of combinations of bile salt, lecithin, and cholesterol in saline. Not all small lipoid aggregates, however, are micelles in the strict physicochemical sense of the word. Other optically clear liquids containing insoluble lipoid substances include microemulsions l l and polydisperse lamellar or helical aggregates formed by ultrasonic vibration of various phospholipids,12 as well as other colloidal suspensions, none of which behaves as a true micellar solution. Micelles
607
608
COMMENTS
Vol. 52, No.3
are mono disperse (i.e., the aggregates are to that shown in the figure for the bile all about the same size), spherical or rod- salt-lecithin-cholesterol micelle. shaped aggregates that form spontaneously Tamesue and Juniper, as reported elseand are made up of a group of molecules where in this journaI,16 have performed which are in constant rapid equilibrium the important task of demonstrating by with a given concentration of single, like surface tension methods that bile, abmolecules in the aqueous medium sur- normal as well as normal bile (excluding rounding the micelle. This concentration white bile), does have a CMC, thus prosurrounding the micelle is called the crit- viding excellent evidence that whole bile ical micellar concentration (CMC). (For is in fact a micellar solution. Tamesue and a lucid discussion see HofmannP) Juniper found that the first break in the surBile salts, free or conjugated, form true face tension-log concentration curve ocmicelles in water or weak salt solutions. cured at about 47 dynes per cm, which is ap(For a more extensive review of the micel- proximately the same as the surface tension lar properties of bile salts, see Hofmann exerted by a surface saturated with pure and Small. 14 ) Bile is not, however, a sim- bile salts. This strongly suggests that the ple micellar solution of bile salts, but a CMC of the mixed micelle of bile is due solution containing mixed micelles of to the concentration of bile salts in the bile salt and two insoluble components, medium around the micelle and not to lecithin and cholesterol (bile pigments and any appreciable quantities of lecithin or protein might also be a part of this mixed cholesterol. This CMC is lower than that micelle). A mixed micelle may be defined for pure bile salts and is similar to that as having more than one component; one found for mixed micelles of bile salt and of these must be soluble and able to form mono-olein, an insoluble amphipath. 15 Unmicelles by itself, while the other com- published work by us shows that the second break in the surface tension-log conponent may be either soluble or insoluble. Several types of mixed micelles are pos- centration curve noted by Tamesue and sible (fig. 1). (1) A completely insoluble Juniper can be obtained with micellar nonpolar compound is held in solution by a solutions of bile salt and lecithin in buffer detergent micelle and is present in the hy- (pH 7.4) and probably represents the redrocarbon center of the micelle, e.g., ben- placement of bile salt molecules by the zene in a soap micelle. (2) An insoluble more surface-active lecithin molecules; it amphipathic substance such as a long is probably not related to any change in chain alcohol is kept in solution by allow- the structure or size of the bile micelle. The second problem which must be ating it to lie side by side with the soap molecules. (3) Small lamellar aggregates tacked in gallstone formation concerns of insoluble amphipathic substances such the appearance of macroscopic stones, rather as lecithin and cholesterol are stabilized than just a suspension of cholesterol crysby a substance such as a bile salt. Each tals which could easily be washed out with of these types of mixed micelles has dif- a contraction of the gall bladder. Nucleaferent characteristics. For instance, the tion may play some role. Maki et alP have CMC of the first type is not altered sig- shown that certain bacteria containing (3nificantly by the incorporation of nonpolar glucuronidase can deconjugate bilirubin dihydrocarbon into its center. In contrast, glucuronide, permitting the formation of the CMC of the second type is markedly calcium bilirubinate. Calcium bilirubinate decreased by the presence of the inter- can form small black concretions in vivo. digitated insoluble amphipath. 15 Although Since the centers of "pure" cholesterol gallfew data exist on the third type of mixed stones often contain small black masses of micelle, one would predict on physical calcium bilirubinate, these may act as nugrounds that the CMC would be lower cleating centers for cholesterol in a bile than that of a pure bile salt micelle. In which has reached the saturation point. addition, there are grounds to suggest that Other substances, such as lithocholic the micelle in bile has a structure similar acid/so 19 bacteria,20 and possibly inorganic
609
COMMENTS
March 1967
Wa' " So/ub/, pa,' af ma/«u/. Lipid Sa/ubI. po,' o f m o/. cu/.
Detergent Molecule
Benzene
Long Chain Conjugated Alcohol Trihydroxy Bile Salt
2
Cholesterol
Lecithin
3
FIG. 1. The several types of lipid molecules mentioned in the text and the cross-sectional arrangement of these molecules in three types of mixed micelles are represented diagrammatically. Wavy lines, the lipid-soluble hydrocarbon chain part of the molecule; shaded area, the lipid-soluble cyclic hydrocarbon part of the molecule; 0, the polar head qf the detergent molecule; ., OH groups or ester groups, as the case may be; EEl and the positively and negatively charged ionic polar groups. The molecules have been tlepicted so that their water-soluble parts lie above the line and their fat-soluble parts lie below the line. Although the polar and nonpolar regions of most amphipathic substances are at opposite ends of the long axis of the molecule, it can be seen that bile salt is peculiar in that the lipophilic part of the ring is confined to one side, the other side being hydrophilic and spiked with several OH groups. This peculiar structure accounts for many of the properties of bile salts divergent from ordinary detergents. The lower part of the figure represents the cross section of three mixed micelles. 1, a mixed micelle of nonpolar benzene held in solution by a detergent. The benzene molecules lie in the lipid center of the micelle. Only the detergent molecules are in equilibrium with the molecules in the surrounding aqueous medium. 2, a mixed micelle of insoluble amphipath (long chain alcohol) and detergent. The alcohol with its OH group in the aqueous medium interdigitates with the detergent molecules. Again, only the detergent molecules are in equilibrium with like molecules in the surrounding aqueous medium. 3, a mixed micelle of bile salt, lecithin, and cholesterol. The proposed structure of this mixed micelle is a cylinder-shaped bimolecular disc of lecithin and cholesterol stabilized on its hydrophobic surface by the bile salts. Note that the entire surface of this micelle, like other micelles, is hydrophilic, and that the lipophilic parts of the molecules are in the interior of the micelle. Only the bile salts are in equilibrium with molecules in the aqueous medium surrounding the micelle. Since the height of the cylinder is very close to its diameter, this micelle would appear spherical by most physicochemical methods.
e
610
COMMENTS
salt crystals, proteins, polypeptides, or mucoproteins,21 might also act as nucleating agents. Therefore, some effort needs to be directed to the problem of nucleation of cholesterol from saturated or supersaturated mixed micellar solutions. It is also pertinent that some gallstones appear to be made up of many small crystals of cholesterol cemented together by some substance. More work is needed on the nature of this cement and the mode of its solidification. Further, once a stone has been formed, its mode of growth needs to be studied. Techniques familiar to the crystallographer and minerologist may give answers to the mechanisms of stone formation and stone growth. Donald M. Small, M.D. Department of Medicine Subdepartment of Gastroenterology Research Building Boston University Medical Center Boston, Massachusetts 02112 REFERENCES
8.
9.
10. 11.
2.
3. 4.
5.
818.
6. Small, D. M., M. Bourges, and D. G. Dervichian. 1966. Biophysics of lipidic associations. I. The ternary systems lecithin-bile salt-water. Biochim. Biophys. Acta 125: 563580.
7. Small, D. M., and M. Bourges. 1966. Lyotropic
paracrystalline phases obtained with ternary
and quaternary systems of amphiphilic substances in water; studies on aqueous systems of lecithin, bile salt and cholesterol. Molec. Crystals 1: 541-561. Juniper, K. 1965. Physicochemical characteristics of bile and their relation to gallstone formation. Amer. J. Med. 39: 98-107. Verschure, J. C. M., P. F. Mijnlieff, F. M. C. Hoefsmitt, and A. E. N. van der Hoeven. 1956. The dominating macromolecular complex of human gallbladder bile. Clin. Chim. Acta 1: 154-166. Furasawa, T. 1962. Surface-chemical studies on the stability of human bile. Fukuoka Igaku Zasshi 53: 124-165. Schulman, J. H., and J. Montagne. 1961. Formation of micro emulsions by aminoalkyl alcohols. Ann. N. Y. Acad. Sci. 92:
366-371. 12. Saunders, L. 1966. Molecular aggregation in
13.
14.
1. Ekwall, P., and L. Mandell. 1961. The solubil-
ity of cholesterol in sodium caprylate solutions at 20° C (solubility of cholesterol in water 20° C = 0.06--0.07 mgjL). Acta Chern. Scand. 15: 1404-1406. Bourges, M., D. M. Small, and D. G. Dervichian. 1967. Biophysics of lipidic associations. II. The ternary systems lecithin-cholesterol-water. Biochim. Biophys. Acta. In press. Isaksson, B. 1954. On the lipids and bile acids in normal and pathological bladder bile. Thesis, University of Lund. I-VI. Polonovski, M., and R. Bourillon. 1952. Les phospholipides de la bile. Bull. Soc. Chim. BioI. 34: 712-723. Small, D. M., M. Bourges, and D. G. Dervichian. 1966. Ternary and quaternary aqueous systems containing bile salt, lecithin, and cholesterol. Nature (London) 211: 816-
Vol. 52, No.3
15.
16.
17.
18.
19.
aqueous dispersions of phosphatidyl and lysophosphatidyl cholines. Biochim. Biophys. Acta 125: 70-74. Hofmann, A. F. 1965. Clinical implications of physicochemical studies on bile salts. Gastroenterology, 48: 484-494. Hofmann, A., and D. M. Small. 1967. Detergent properties of bile salts. Ann. Rev. Med. 18. Hofmann, A. F. 1963. The function of bile salts in fat absorption. The solvent properties of dilute micellar solutions of conjugated bile salts. Biochem. J. 89: 57-68. Tamesue, N., and K. Juniper. Concentrations of bile salts at the critical micellar concentration of human gall bladder bile. Gastroenterology 52: 473--479. Maki, T. 1966. Pathogenesis of calcium bilirubinate gallstones; role of E. coli, fJglucuronidase and coagulation by inorganic ions, poly-electrolytes and agitation. Ann. Surg. 164: 90-100. Jones, R. S., H. Solic, and F. Hirayama. 1965. Free bile acids in gallstones and bile of man (abstr.). Fed. Proc. 24: 167. Palmer, R. H., and Z. Hruban. 1966. Production of bile duct hyperplasia and gallstones by lithocholic acid. J. Clin. Invest.
45: 1255-1267. 20. Rains, A. J. H. 1962. Researches concerning
the formation of gallstones. Brit. Med. J. 2: 685--691. 21. Womack, N. A., R. Zeppa, and G. L. Irvin, III. 1963. The anatomy of gallstones. Ann. Surg. 157: 670--686.
GASTROENTEROLOGY
Printed in U.S.A.
Copyright © 1967 by The Williams & Wilkins Co.
COMMENTS Readers are invited to contribute Comments. If found suitable, they will be published promptly, subject to the usual editing. They should be typewritten double spaced and sent to the Editor.
PHYSICOCHEMICAL STUDIES OF CHOLESTEROL GALLSTONE FORMATION
Cholesterol gallstone formation may be divided into two processes: the oversaturation of bile with cholesterol and the formation of macroscopic stones from the oversaturated bile. The first of these processes is related to the change in the physical state of bile from a clear solution to a solution containing crystalline cholesterol. Physicochemically, this change may be regarded as the transformation of a one-phase system (liquid) to a two-phase system (liquid and solid) . For instance, this sort of change occurs when a solution of common table salt evaporates beyond its point of saturation and salt crystals appear in the liquid. By analogy one may say that in normal bile the concentration of cholesterol is less than the concentration at its saturation point, I while when cholesterol precipitates from bile its concentration must be above the saturation point. Although this analogy is probably correct, the situation as it pertains to bile is far more complicated. Cholesterol is virtually insoluble in water. I , 2 Therefore, the large amounts of cholesterol found in normal bile, e.g., 500 mg per 100 ml of bile, must be "solubilized" by some other biliary components. For many years it was thought that bile salts were responsible for holding cholesterol in solution, but during the last 15 years it has been conclusively shown that bile salts alone are not adequate and that lecithin is also necessary for this effect. a, 4 Address requests for reprints to: Dr. Donald M. Small, Department of Medicine, Research Building, Boston University Medical Center, 15 Stoughton Street, Boston, Massachusetts 02112. This work was in part supported by United States Public Health Service Research Grant AM 05589 and Training Grant 05025.
Recently phase equilibria techniques 5 , 6 and X-ray diffraction studies7 have been used to delineate the solubility of cholesterol in any and all combinations of bile salts, lecithin, and water. These studies showed that lecithin associates with cholesterol in a specific liquid crystalline phase and that bile salts break down this phase into micelle-sized aggregates, thus carrying the two insoluble components into solution. The bile salt and lecithin concentrations in normal bile are such that the limits of solubility of cholesterol are not surpassed. In contrast, bile from patients with cholesterol gallstones is oversaturated with respect to cholesterol. Since human bile behaves like the model system of bile salts, lecithin, cholesterol, and water with respect to cholesterol solubility, the other components in bile (bilirubin, inorganic ions, proteins, mucus, etc.) do not affect the solubility of cholesterol in bile. In recent years bile has been assumed to be a micellar solution. Thus, ultracentrifuge studies8,9 suggested that bile contained aggregates of micellar size, and light-scattering studies 10 demonstrated that these aggregates were roughly the same size as aggregates made up of combinations of bile salt, lecithin, and cholesterol in saline. Not all small lipoid aggregates, however, are micelles in the strict physicochemical sense of the word. Other optically clear liquids containing insoluble lipoid substances include microemulsions l l and polydisperse lamellar or helical aggregates formed by ultrasonic vibration of various phospholipids,12 as well as other colloidal suspensions, none of which behaves as a true micellar solution. Micelles
607
608
COMMENTS
Vol. 52, No.3
are mono disperse (i.e., the aggregates are to that shown in the figure for the bile all about the same size), spherical or rod- salt-lecithin-cholesterol micelle. shaped aggregates that form spontaneously Tamesue and Juniper, as reported elseand are made up of a group of molecules where in this journaI,16 have performed which are in constant rapid equilibrium the important task of demonstrating by with a given concentration of single, like surface tension methods that bile, abmolecules in the aqueous medium sur- normal as well as normal bile (excluding rounding the micelle. This concentration white bile), does have a CMC, thus prosurrounding the micelle is called the crit- viding excellent evidence that whole bile ical micellar concentration (CMC). (For is in fact a micellar solution. Tamesue and a lucid discussion see HofmannP) Juniper found that the first break in the surBile salts, free or conjugated, form true face tension-log concentration curve ocmicelles in water or weak salt solutions. cured at about 47 dynes per cm, which is ap(For a more extensive review of the micel- proximately the same as the surface tension lar properties of bile salts, see Hofmann exerted by a surface saturated with pure and Small. 14 ) Bile is not, however, a sim- bile salts. This strongly suggests that the ple micellar solution of bile salts, but a CMC of the mixed micelle of bile is due solution containing mixed micelles of to the concentration of bile salts in the bile salt and two insoluble components, medium around the micelle and not to lecithin and cholesterol (bile pigments and any appreciable quantities of lecithin or protein might also be a part of this mixed cholesterol. This CMC is lower than that micelle). A mixed micelle may be defined for pure bile salts and is similar to that as having more than one component; one found for mixed micelles of bile salt and of these must be soluble and able to form mono-olein, an insoluble amphipath. 15 Unmicelles by itself, while the other com- published work by us shows that the second break in the surface tension-log conponent may be either soluble or insoluble. Several types of mixed micelles are pos- centration curve noted by Tamesue and sible (fig. 1). (1) A completely insoluble Juniper can be obtained with micellar nonpolar compound is held in solution by a solutions of bile salt and lecithin in buffer detergent micelle and is present in the hy- (pH 7.4) and probably represents the redrocarbon center of the micelle, e.g., ben- placement of bile salt molecules by the zene in a soap micelle. (2) An insoluble more surface-active lecithin molecules; it amphipathic substance such as a long is probably not related to any change in chain alcohol is kept in solution by allow- the structure or size of the bile micelle. The second problem which must be ating it to lie side by side with the soap molecules. (3) Small lamellar aggregates tacked in gallstone formation concerns of insoluble amphipathic substances such the appearance of macroscopic stones, rather as lecithin and cholesterol are stabilized than just a suspension of cholesterol crysby a substance such as a bile salt. Each tals which could easily be washed out with of these types of mixed micelles has dif- a contraction of the gall bladder. Nucleaferent characteristics. For instance, the tion may play some role. Maki et alP have CMC of the first type is not altered sig- shown that certain bacteria containing (3nificantly by the incorporation of nonpolar glucuronidase can deconjugate bilirubin dihydrocarbon into its center. In contrast, glucuronide, permitting the formation of the CMC of the second type is markedly calcium bilirubinate. Calcium bilirubinate decreased by the presence of the inter- can form small black concretions in vivo. digitated insoluble amphipath. 15 Although Since the centers of "pure" cholesterol gallfew data exist on the third type of mixed stones often contain small black masses of micelle, one would predict on physical calcium bilirubinate, these may act as nugrounds that the CMC would be lower cleating centers for cholesterol in a bile than that of a pure bile salt micelle. In which has reached the saturation point. addition, there are grounds to suggest that Other substances, such as lithocholic the micelle in bile has a structure similar acid/so 19 bacteria,20 and possibly inorganic
609
COMMENTS
March 1967
Wa' " So/ub/, pa,' af ma/«u/. Lipid Sa/ubI. po,' o f m o/. cu/.
Detergent Molecule
Benzene
Long Chain Conjugated Alcohol Trihydroxy Bile Salt
2
Cholesterol
Lecithin
3
FIG. 1. The several types of lipid molecules mentioned in the text and the cross-sectional arrangement of these molecules in three types of mixed micelles are represented diagrammatically. Wavy lines, the lipid-soluble hydrocarbon chain part of the molecule; shaded area, the lipid-soluble cyclic hydrocarbon part of the molecule; 0, the polar head qf the detergent molecule; ., OH groups or ester groups, as the case may be; EEl and the positively and negatively charged ionic polar groups. The molecules have been tlepicted so that their water-soluble parts lie above the line and their fat-soluble parts lie below the line. Although the polar and nonpolar regions of most amphipathic substances are at opposite ends of the long axis of the molecule, it can be seen that bile salt is peculiar in that the lipophilic part of the ring is confined to one side, the other side being hydrophilic and spiked with several OH groups. This peculiar structure accounts for many of the properties of bile salts divergent from ordinary detergents. The lower part of the figure represents the cross section of three mixed micelles. 1, a mixed micelle of nonpolar benzene held in solution by a detergent. The benzene molecules lie in the lipid center of the micelle. Only the detergent molecules are in equilibrium with the molecules in the surrounding aqueous medium. 2, a mixed micelle of insoluble amphipath (long chain alcohol) and detergent. The alcohol with its OH group in the aqueous medium interdigitates with the detergent molecules. Again, only the detergent molecules are in equilibrium with like molecules in the surrounding aqueous medium. 3, a mixed micelle of bile salt, lecithin, and cholesterol. The proposed structure of this mixed micelle is a cylinder-shaped bimolecular disc of lecithin and cholesterol stabilized on its hydrophobic surface by the bile salts. Note that the entire surface of this micelle, like other micelles, is hydrophilic, and that the lipophilic parts of the molecules are in the interior of the micelle. Only the bile salts are in equilibrium with molecules in the aqueous medium surrounding the micelle. Since the height of the cylinder is very close to its diameter, this micelle would appear spherical by most physicochemical methods.
e
610
COMMENTS
salt crystals, proteins, polypeptides, or mucoproteins,21 might also act as nucleating agents. Therefore, some effort needs to be directed to the problem of nucleation of cholesterol from saturated or supersaturated mixed micellar solutions. It is also pertinent that some gallstones appear to be made up of many small crystals of cholesterol cemented together by some substance. More work is needed on the nature of this cement and the mode of its solidification. Further, once a stone has been formed, its mode of growth needs to be studied. Techniques familiar to the crystallographer and minerologist may give answers to the mechanisms of stone formation and stone growth. Donald M. Small, M.D. Department of Medicine Subdepartment of Gastroenterology Research Building Boston University Medical Center Boston, Massachusetts 02112 REFERENCES
8.
9.
10. 11.
2.
3. 4.
5.
818.
6. Small, D. M., M. Bourges, and D. G. Dervichian. 1966. Biophysics of lipidic associations. I. The ternary systems lecithin-bile salt-water. Biochim. Biophys. Acta 125: 563580.
7. Small, D. M., and M. Bourges. 1966. Lyotropic
paracrystalline phases obtained with ternary
and quaternary systems of amphiphilic substances in water; studies on aqueous systems of lecithin, bile salt and cholesterol. Molec. Crystals 1: 541-561. Juniper, K. 1965. Physicochemical characteristics of bile and their relation to gallstone formation. Amer. J. Med. 39: 98-107. Verschure, J. C. M., P. F. Mijnlieff, F. M. C. Hoefsmitt, and A. E. N. van der Hoeven. 1956. The dominating macromolecular complex of human gallbladder bile. Clin. Chim. Acta 1: 154-166. Furasawa, T. 1962. Surface-chemical studies on the stability of human bile. Fukuoka Igaku Zasshi 53: 124-165. Schulman, J. H., and J. Montagne. 1961. Formation of micro emulsions by aminoalkyl alcohols. Ann. N. Y. Acad. Sci. 92:
366-371. 12. Saunders, L. 1966. Molecular aggregation in
13.
14.
1. Ekwall, P., and L. Mandell. 1961. The solubil-
ity of cholesterol in sodium caprylate solutions at 20° C (solubility of cholesterol in water 20° C = 0.06--0.07 mgjL). Acta Chern. Scand. 15: 1404-1406. Bourges, M., D. M. Small, and D. G. Dervichian. 1967. Biophysics of lipidic associations. II. The ternary systems lecithin-cholesterol-water. Biochim. Biophys. Acta. In press. Isaksson, B. 1954. On the lipids and bile acids in normal and pathological bladder bile. Thesis, University of Lund. I-VI. Polonovski, M., and R. Bourillon. 1952. Les phospholipides de la bile. Bull. Soc. Chim. BioI. 34: 712-723. Small, D. M., M. Bourges, and D. G. Dervichian. 1966. Ternary and quaternary aqueous systems containing bile salt, lecithin, and cholesterol. Nature (London) 211: 816-
Vol. 52, No.3
15.
16.
17.
18.
19.
aqueous dispersions of phosphatidyl and lysophosphatidyl cholines. Biochim. Biophys. Acta 125: 70-74. Hofmann, A. F. 1965. Clinical implications of physicochemical studies on bile salts. Gastroenterology, 48: 484-494. Hofmann, A., and D. M. Small. 1967. Detergent properties of bile salts. Ann. Rev. Med. 18. Hofmann, A. F. 1963. The function of bile salts in fat absorption. The solvent properties of dilute micellar solutions of conjugated bile salts. Biochem. J. 89: 57-68. Tamesue, N., and K. Juniper. Concentrations of bile salts at the critical micellar concentration of human gall bladder bile. Gastroenterology 52: 473--479. Maki, T. 1966. Pathogenesis of calcium bilirubinate gallstones; role of E. coli, fJglucuronidase and coagulation by inorganic ions, poly-electrolytes and agitation. Ann. Surg. 164: 90-100. Jones, R. S., H. Solic, and F. Hirayama. 1965. Free bile acids in gallstones and bile of man (abstr.). Fed. Proc. 24: 167. Palmer, R. H., and Z. Hruban. 1966. Production of bile duct hyperplasia and gallstones by lithocholic acid. J. Clin. Invest.
45: 1255-1267. 20. Rains, A. J. H. 1962. Researches concerning
the formation of gallstones. Brit. Med. J. 2: 685--691. 21. Womack, N. A., R. Zeppa, and G. L. Irvin, III. 1963. The anatomy of gallstones. Ann. Surg. 157: 670--686.