ANNALS O F CLINICAL AND LABORATORY SCIENCE, Vol. 14, No. 4 Copyright © 1984, Institute for Clinical Science, Inc.

Pathogenesis of Gallstones HARRY F. WEISBERG, M.D. The Ida Soref-David and Ruth Coleman Department of Pathology and Laboratory Medicine, University o f Wisconsin Medical School, Milwaukee Clinical Campus, Mount Sinai Medical Center, Milwaukee, WI 53201

ABSTRACT The three lipids in bile, cholesterol, lecithin, and bile salts (about 90 percent of the dry weight of normal gallbladder bile) are amphipathic substances having both hydrophobic and hydrophilic functional groups. Knowledge of the physicochemical factors of gallstone formation (espe­ cially cholesterol stones) has increased in the past two decades. The ab­ solute amount of cholesterol supersaturation determines the extent of cho­ lesterol precipitation. The ionic strength of the bile and the types of bile salts present are minor factors, whereas the ratios of bile salts to lecithin at a particular concentration of total lipids are the major factors contrib­ uting to gallstone production. Bile acids (salts) form micelles which allow the lecithin and cholesterol to dissolve within the micelles. Thus the ad­ ministration of bile acids allows for non-invasive dissolution of some cho­ lesterol gallstones. Additional im portant risk factors are genetic and ethnic, sex (females predominate), obesity, diet (in contrast to animal pro­ tein and more refined carbohydrate diets, there is less lithogenicity with diets containing plant protein and unrefined carbohydrates), certain dis­ eases, and drug therapy. Pigment stones make up the majority of radi­ opaque stones and are predominant in the Orient; they are seen in certain diseases and in infections of the biliary tree. Introduction Gallstones have been recognized in man and animals since antiquity. The gallstones from a mummy of the priestess of Amenen (1500 B.C.) were lost when the mummy was destroyed during the bombing of London in World War II.2 Gallstones are common in herbivorous animals but rare in carnivera; stones have been found in both the elephant and 243

horse, animals without a gallbladder.3 The stones from cattle served as a major source of yellow pigment used by the an­ cient artists. It is estimated that from 1616 to 20 million6 people in the United States have gallstones. Annual cholecystecto­ mies vary from 80,000 in Canada14 to about one third of a million in the United States.2’16Annually, 5,000 to 8,000 deaths in the United States are attributed to gallstone disease.14,16

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In 1500, Paracelsus advanced the theory that certain chemical disturbances in the body initiated the precipitation of impurities in the biliary ducts.3 Various theories were promulgated since then. Gallstones are formed by the precipita­ tion of insoluble constituents of bile. By tradition, gallstones have been classified as (1) pure cholesterol or pigment (largely made of calcium bilirubinate), (2) mixed— two or three components of cho­ lesterol, calcium bilirubinate, and cal­ cium carbonate (usually more than 70 percent cholesterol), or (3) combination stones with a nucleus of one type and a shell of another substance.3 It is very rare to have a chemically pure gallstone.18 Analysis by infrared spectroscopy and Xray diffraction of gallstones found in the gallbladder and in the common bile duct revealed five major components,—cho­ lesterol, calcium bilirubinate, fatty acid calcium salts, inorganic calcium salts, and black pigment m aterial.21 Gall­ bladder stones contained black pigment and inorganic calcium salts more fre­ quently than did the common duct stones. However, the ductal stones had an incidence of 55.5 and 18.9 percent for calcium bilirubinate and fatty acid cal­ cium salts, respectively, in contrast to 8.7 and 1.2 percent for the gallbladder stones.21 Though relatively rare, the precipita­ tion of calcium carbonate in the gall­ bladder, as a separate mass or upon al­ ready existing stones, may give rise to difficulties in the proper interpretation of a cholangiogram. This whitish precipitate has been termed Kalkmilchgalle or milk of calcium bile or limey bile.3 White bile, however, denotes the absence of bile in the bile ducts; this has also been referred to as acholia, the term for lack of bile in the intestinal tract.5

(solids). Cholesterol gallstones usually contain more than 70 percent cholesterol and account for most of the gallstone dis­ ease seen in the Americas, Europe, and Africa.16 If such cholesterol stones con­ tain enough calcium, they may be radi­ opaque (33 percent) but 80 to 86 percent of the radiolucent stones consist of cho­ lesterol. 14 The solubility of cholesterol in bile fluid is limited and is found in the crys­ talline form in patients with cholesterol gallstones. Cholesterol is insoluble in water but normal human bile contains about 10 mmol of cholesterol dissolved in one liter of bile.14 Cholesterol is a C27, unsaturated, monohydroxylated, non­ polar compound; the hydroxyl group al­ lows for surface solubility on water but no solubility in water. The solubility of cholesterol depends on the relative con­ centrations of the three major lipid com­ ponents in bile: conjugated bile salts, phospholipids (at least 90 percent in the form of lecithin— phosphatidyl choline), and free cholesterol. These three lipids make up about 90 percent of the dry weight of normal gallbladder bile. The total solids in gallbladder bile can vary from 3 to 30 g per dl16 or 2.8 to 24.9 g per dl (average 12 g per dl).4 The con­ centration varies from patient to patient, from time to time in the same patient, and from one collection site to another. Hepatic bile is more dilute (1 to 4 g per dl) and the concentration in hepatic duct bile ranges from 0.2 to 7.9 g per dl (av­ erage 2.7 g per dl).4 The absolute solu­ bility of cholesterol varies with the con­ centration of total solids; in dilute hepatic bile it is about three mole percent whereas it is as high as 10 mole percent in a very concentrated gallbladder bile.16 The vast majority of normal and ab­ normal bile samples reported in the lit­ erature have relatively limited ranges of concentration.11 C h o lestero l S to nes Free or unconjugated bile acids are The word cholesterol is derived from the end product of cholesterol degrada­ the Greek, chol (bile or gall) and stereos tion (primary bile acids); they are C24,

PATHOGENESIS OF GALLSTONES

saturated, mono- or poly-hydroxylated, polar compounds with the carboxyl group located in the side chain. The ge­ neric term, bile salt, refers to bile acids conjugated in the liver with taurine or glycine or to bile alcohols esterified with a sulfate group.14 This conventional de­ scription has been changed since natu­ rally occurring bile acids are a more nu­ merous and diverse group than has been recognized. Thus, C27 bile acids are found in the bile, serum, and urine of infants with a familial form of cholestasis and intrahepatic bile duct anomalies and in the bile and serum of patients with Zellweger syndrom e.10 Patients with Zellweger syndrome also have a C29 dicarboxylic bile acid in their serum, and a C23 bile acid has been identified in the serum of an adult with cholestasis. The meconium of normal full-term infants has been shown to have several, short sidechain bile acids (C20, C21, and C22).10 The three lipid components of bile are amphipathic molecules, possessing groups with characteristically different properties, e.g., hydrophobic group at one end and hydrophilic group at the other end. The steroid nuclei of choles­ terol and the bile salts and the two longchained fatty acids of lecithin are hy­ drophobic and seek the oil phase. In contrast, the polar hydroxyl group(s) of cholesterol and the bile salts, the taurine and glycine conjugates of bile, and the phosphoryl choline and glycerol ester groups in lecithin are hydrophilic and are attracted to the water phase. Though lec­ ithin is insoluble in water, water pene­ trates between the hydrophilic choline groups, resulting in a swelling of the lec­ ithin—“liquid crystals.”14 The waterinsoluble cholesterol can interdigitate between the lecithin molecules with its steroid nucleus buried in the fatty acid interior of the lecithin molecule. A “mi­ celle” is a polymer of about 50 to 100 amphipathic molecules, e.g., bile salts, arranged spherically, usually with the hy­ drophobic end on the inside and the hy­

245

drophilic group on the outside. The ag­ gregation to form simple micelles occurs above specific concentrations of the bile salts in the water (the “critical micellar concentration”). Lecithin can dissolve in these bile-salt micelles, resulting in mixed micelles in which the cholesterol can dissolve in the hydrophobic core be­ tween the fatty acid chains of the leci­ thin. W ater associated with the polar groups of lecithin swells the micelles, al­ lowing the incorporation of more choles­ terol than could be contained by the simple bile-salt micelles alone.14 In figure 1 is shown a triangular phase diagram for plotting relative percent of the total molar concentrations (milli­ moles per liter) of the three major com­ ponents of bile. The original phase diagram1 was based on the average com­ position of normal bile — 90 percent water and 10 percent solids (bile salts, cholesterol, and lecithin). With biles ob­ tained at surgery or directly from the gallbladder or hepatic duct, one can de­ term ine the absolute concentration of solids and thus can estimate the true sol­ ubility of cholesterol. However, with bile samples obtained by duodenal drainage (cholecystokinin stimulation), the total bile lipids are diluted. Therefore one uses an arbitrary concentration (e.g., 10 percent total solids) to obtain a rough es­ timate of cholesterol solubility in duo­ denal bile.16 From a practical point of view, one should look for crystals in freshly collected bile; if present, they are separated by centrifugation and the re­ maining liquid phase analyzed. Centrif­ ugation of crystals which have appeared some time after collection gives an er­ roneous analysis. Biles are often frozen ( —20°C) for transport; this may alter the chemical and physical state of the bile.16 The concentration of the lipid compo­ nents is expressed in term s of “sub­ stance” concentration (millimoles per liter) rather than “mass” concentration (milligrams per deciliter). Bile has 3 to 23 mmol per 1 of cholesterol, 30 to 50

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B I L E S A L T S (% Total Moles)

Triangular phase diagram showing physical state of combinations of cholesterol, lecithin, and bile salts. ZONE I: One phase; micellar liquid. ZONE II: Two phases; micellar liquid and cholesterol monohydrate crystals. ZONE III: Three phases; micellar liquid, cholesterol monohydrate crystals, and liquid crystals of lecithin and cholesterol. ZONE IV: Two phases; liquid crystals of lecithin and cholesterol and isotropic liquid. Modified from Carey and Small.4 F i g u r e 1.

mmol per 1 of lecithin, and 40 to 145 mmol per 1of bile salts. Adding the lower values gives a total of 73 mmol with cor­ responding data of 4, 41, and 55 percent of total lipids for cholesterol, lecithin, and bile salts, respectively. Adding the upper values gives a total of 218 mmol with the corresponding values of 10, 23, and 67 percent of total lipids for choles­ terol, lecithin, and bile salts. Figure 1 is based on 20 g per dl solu­ tions of total lipids (cholesterol, lecithin, and sodium taurocholate) in 0.15 molar sodium chloride at room temperature.4 Studies with X-ray analysis and polar­ izing microscopy resulted in the descrip­ tion of the different number and types of equilibrium phases. The equilibrium phases for Zones II, III, and IV are found with concentrations of lecithin of less

than 20, between 20 to 40, and above 40 percent of total moles, respectively.4 In figure 2 is shown an enlargement of the clinically applicable portion of the phase diagram. It shows point P at a con­ centration of cholesterol at 5, bile salts at 80, and lecithin at 15 percent of total moles; the specimen is not saturated with cholesterol and lies in Zone I, a single phase of micellar liquid. Line ABC is the maximal effective solubility of cholesterol in varying mixtures of lecithin and bile salts. The line AB is too high, and the true limit of solubility of cholesterol at 37°C is shown by line DBC. Mixtures of bile with the value plot above line ABC contain readily precipitable excess cho­ lesterol. Mixtures falling in the metasta­ bile area (ABDA) have a slight excess of cholesterol; no precipitation occurs un­

PATHOGENESIS OF GALLSTONES

less the mixture is seeded or nucleated or it stands for long periods. In 1954 Isakkson7 reported that bile from patients with cholesterol gallstones had a ratio of cholesterol to the sum of bile salts and phospholipids greater than 1:11 whereas most patients without gall­ stones had a lower ratio. This was con­ firmed and extended by Admirand and Small,1 who reported the percent cho­ lesterol saturation.13 Similar expressions are the lithogenic index11 and the cho­ lesterol saturation index.17 The indices are expressed as a fraction of unity instead of percentage. The percent saturation is determ ined by extending a line from point P to the apex of the triangular dia­ gram (figure 2); the intersection (X) of this line with line ABC gives the relative cholesterol concentration at 100 percent effective saturation. Point X is at about 10 percent cholesterol; thus, the original relative concentration of 5 percent (point P) is divided by 10 and the result mul­ tiplied by 100 to give 50 percent satura­ tion; if expressed as the lithogenic index, the result is 0.50. A normal bile is less than 100 percent saturated with choles­ terol or has a lithogenic index less than 1.00; an index value above 1.00 denotes

24 7

a saturated (more than 100 percent cho­ lesterol saturation) or lithogenic bile.11 In contrast to drawing a line to the apex of the triangular coordinates (figures 1 and 2), Thomas and Hofmann19 modi­ fied the calculations of the lithogenic index by utilizing rectangular coordi­ nates; these are easily adapted to com­ puterized calculations. The relative per­ cent concentration of cholesterol is plotted on the ordinate and the abscissa depicts the ratio of the “percent” lecithin to the sum of the lecithin and bile salt percentages. They utilized the data of Admirand and Small1 and used regres­ sion analysis to obtain a third-degree polynomial; the equation for figure 3 is y = 4.86 + 39.3 x - 74.4 x2 + 0.88 x3. The original data for point P (figure 2) had concentrations of bile salts, lecithin, and cholesterol at 80, 15, and 5 percent, respectively, of the total moles. Thus the ratio of 15/15 + 80 = 0.158. In figure 3, the vertical line at 0.158 gives a choles­ terol concentration of about 9.5 at point X; thus 5/9.5 = 0.53 for the lithogenic index or 53 percent cholesterol satura­ tion. Carey and Small4 criticize this for­

F i c u r e 2. Triangular coordinate plot of maximal effective cholesterol solu­ bility line (ABC) and true equilibrium solubility line (DBC). C, cholesterol; L, lecithin; and BS, bile salts. Area within ABDA repre­ sents metastabile region. After Small.1-10,16

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F i g u r e 3. Curve for cholesterol saturation plotted on rectangular co­ ordinates. C, cholesterol; L, lecithin; and BS, bile salts. M odified from Thomas and Hofmann.19

mula, stating it is not as accurate as their fifth-degree polynomial expression; how­ ever, they have utilized that same rect­ angular plot of cholesterol on the ordi­ nate versus the ratio of lecithin to the sum of lecithin and bile salts on the ab­ scissa. They showed that, within physi­ ological bile salt:lecithin ratios at 37°C, the influence of type of bile salt and ionic strength is minor whereas the effects of bile salt ¡lecithin ratio and the total lipid concentration are major factors. Thus, utilizing cholesterol saturation values ap­ propriate to the total lipid concentration, all cholesterol stone patients have super­ saturated gallbladder biles,— 132 per­ cent for normal weight and 199 percent for morbidly obese individuals. For con­ trol and pigm ent stone patients, the mean values were 95 and 98 percent, re­ spectively, even though about half of the biles were supersaturated. Cholesterol crystals were seen in 83 percent in gall­ bladder and 58 percent in hepatic bile of cholesterol stone patients but were not seen in controls or pigm ent stone pa­ tients.4 Fasting biles of normal individ­ uals may be supersaturated with choles­ terol.

abolic defect leading to supersaturation; this can be subdivided into six types. Type 1 is the excessive loss of bile salts as seen in ileal disease or surgery or con­ genital loss of ileal active transport of bile salts. The decreased reabsorption leads to a decreased bile salt pool and bile salt secretion rate. Type 2 is an oversensitive bile acid feedback seen most usually in stone disease present in non-obese Cau­ casians. There is a relative depression of bile acid synthesis; the decreased hepatic return excessively inhibits bile acid syn­ thesis. Type 3 exhibits excessive choles­ terol secretion despite a normal bile salt secretion rate. This is noted in obese pa­ tients with increased synthesis of choles­ terol; the bile acid pool may be in the normal range. Type 4 is a mixture of Types 2 and 3 and is seen in the native American Indians and, perhaps, many Caucasians. The last two types are primarily extrahepatic in origin. Type 5 exhibits a rapid bile salt circulation seen in patients with a decreased bile acid pool, the small amount of normal bile in the gallbladder cannot compensate for abnormal bile en­ tering the gallbladder during fasting. Type 6 includes disorders of the gall­ bladder, ducts, or sphincters; aseptic or S tages bacterial cholecystitis may secondarily Cholesterol gallstone disease can be complicate other types of gallstone dis­ described in five stages.16 Stage I in­ ease, including pigment stone disease.16 volves the genetic, biochemical, or met­ Stage II is the chemical phase wherein

PATHOGENESIS OF GALLSTONES

the bile is supersaturated with choles­ terol but stones are absent. Studies of duodenal drainage reveal excess choles­ terol (figure 1) but no crystals are seen on microscopic examination of the fluid. The physical stage (III) shows cholesterol crystals on microscopy (nucleation, floc­ culation, and precipitation) but no stones are evident by cholecystography. Stones are present in Stages IV (growth) and V (clinical). In Stage IV macroscopic stones or a non-functioning gallbladder is seen on cholecystography. The duodenal drainage study usually reveals choles­ terol crystals or abnormal bile, but the patient is asymptomatic. In the clinical stage (V), signs and symptoms are present with the stones causing blockage of the cystic duct, cholecystitis, and jaun­ dice.16 R is k F a c t o r s 3-14>18

Genetic and Ethnic. The incidence of cholesterol gallstones varies from country to country and is related to the extent of cholesterol saturation of the bile (lithogenic index). The extremes are rep­ resented by the Masai tribe of East Africa who, despite a high fat diet and high cho­ lesterol absorption with bile saturation of about 50 percent, do not have cholesterol stones to the young Arizona Pima Indian women with bile saturation of about 110 percent and 80 percent prevalence of stones. Swedish individuals revealed about 60 percent and 150 percent bile saturation in 1954 and 1971 studies, re­ spectively, but both groups had a prev­ alence of gallstones at 50 percent.13 Younger female siblings of women with gallstones have more saturated bile than similar siblings of control patients without gallstones. Age. Gallstones have been reported from fetus to extreme old age but the average patient is in the fifth decade at the time of diagnosis or surgery. The in­ cidence increases with age.

24 9

Sex. The frequency of cholelithiasis is from two to four times greater in women and occurs at an earlier age in women. Pregnancy has been implicated in the higher incidence; with pregnancy there is an increase of the lithogenic index, ascribed to the effect of estrogen (estriol). Exogenous estrogens (for con­ traception and post-menopausal replace­ ment) increases the incidence of gall­ stones. In addition, in the last trimester of pregnancy, gallbladder emptying is impaired and hypercholesterolem ia is evident. Obesity. In obese individuals there is an increased biliary secretion of cho­ lesterol secondary to increased cholesterol synthesis. During active weight reduc­ tion, bile saturation increases as a result of decreased secretion of all biliary lipids; in some, secretion of bile acids decreases more than that of cholesterol. Once the weight is stabilized, the bile acid pool expands to normal but cholesterol secre­ tion remains low with resulting de­ creased cholesterol saturation. Diet. Urbanization and adoption of western dietary habits has changed the composition of gallstones in Japan,—cho­ lesterol stones are increasing and the pig­ m ent stones are decreasing. A higher caloric intake has been associated with individuals with gallstones and with the cholesterol concentration of T-tube bile in post-cholecystectomy patients. Other dietary factors that have been suggested are high cholesterol, polyunsaturated fats, high carbohydrate, and low vege­ table fiber. Animal protein (casein) is more litho­ genic and cholesterolem ic than plant protein (soy protein); a diet of equal amounts of animal and vegetable protein is only slightly more cholesterolem ic than a diet containing only plant pro­ tein.9 The addition of bran to the diet of patients with gallstones had reduced the lithogenic index of the bile; the reports are conflicting when bran is added to the

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diet of normal individuals. The type of carbohydrate consumed has been shown to affect the bile cholesterol saturation.20 Subjects with probable gallstones, given a refined carbohydrate diet (refined sugar, white flour, and white rice) had an increased saturation index (1.5 ± 0.10) whereas an unrefined carbohydrate diet (only whole grain products) resulted in an index of 1.2 ± 0.12.20 Long term par­ enteral nutrition results in the formation of biliary sludge and gallstones. Confir­ mation of these observations is reported in an animal model (prairie dog).12 Diseases. Malabsorption of bile acids from the ileum disturbs the enterohepatic circulation, diminishing the bile acid pool and the rate of secretion of bile; this results in the bile becoming lithogenic. This is seen in ileal disease or ileal resection, Crohn’s disease of the small bowel, and cystic fibrosis with pancreatic insufficiency. The higher incidence of gallstones in diabetics of both sexes is re­ lated to the obesity seen in some dia­ betics and the associated increase of cho­ lesterol secretion in the bile. Patients with hyperlipoproteinem ia, especially Type IV (hypertriglyceridemia), have a high incidence of cholesterol gallstones. Drugs. Clofibrate (ethyl chlorophenoxyisobutyrate), used to treat severe types of hyperlipidemia, lowers plasma cholesterol by inhibiting cholesterol syn­ thesis. It mobilizes cholesterol stores out of the tissues with increased secretion into the bile and also impairs bile salt synthesis; the net result is lithogenic bile with gallstone formation. The same may hold true for nicotinic acid. P ig m e n t S t o n e s

Pigment gallstones result from ab­ normal metabolism of bile pigment and are composed of bile pigment, calcium, and a matrix of organic material. The cholesterol content has been described as none,16 less than 5 percent,4 and as

“trace amounts (less than 20 percent).”14 Half of the pigment stones are opaque to X-rays, but two-thirds of the radiopaque stones are of the pigm ent variety.14 A small pigm ent stone is found in the center of almost every “mixed” choles­ terol gallstone. The center consists of cal­ cium bilirubinate as a protein-pigment complex, often containing copper, mixed with calcium phosphate and calcium car­ bonate.14 Even the “pure” cholesterol stone may have some pigm ent in its center. Pigment stones are predominant in the Orient; however, 5 to 15 percent,16 10 to 12 percent,14 or 27 to 33 percent18 of gallstones in Occidentals are of this va­ riety. Pigment gallstones are more common with increasing age but do not appear to be related to sex or obesity.18 Certain disease states are associated with an increased incidence of pigment stones: cirrhosis of the liver, particularly alco­ holic,14 and in severe chronic hemolytic anemias seen in sickle cell disease, thal­ assemia major, congenital spherocytosis, erythroblastosis fetalis or resulting from aortic valve replacement.15,18 Biliary in­ fection, especially with E. coli, or para­ sitic infections (e.g., ascaris) that cause stasis (and then infection) lead to decon­ jugation of the soluble bilirubin diglucuronide, resulting in the insoluble free bilirubin and glucuronic acid. The de­ conjugation is catalyzed by p-glucuronidase. The biliary tree is normally free of this lysozymal enzyme but coliform infectionS*fcan elaborate the enzyme or the enzyme may be produced by the gall­ bladder epithelium.14 Stasis per se can initiate nonenzymatic hydrolysis. The normal biliary tree contains glucaro-1, 4lactone (D-glutaric acid) which inhibits the (3-glucuronidase. In the presence of sufficient calcium, the free bilirubin is precipitated as calcium bilirubinate or similar compounds in the biliary tract. These compounds then polymerize to form a stone. Thus, the pigment stones

PATHOGENESIS OF GALLSTONES

may be bile pigment-calcium stones or pure pigment stones (non-bilirubin pig­ ments with calcium, copper, or iron).14

11. 12.

References 1.

W. H . and S m a l l , D. M.: The phys­ icochemical basis of cholesterol gallstone forma­ tion in man. J. Clin. Invest. 47:1043-1052, 1968. 2. B e r k , R. N.: Gallstones: Diagnostic imaging in historical perspective. The Pharos. Winter 1983, pp. 30-34. 3. B o c k u s , H . L . : Cholelithiasis. Gastroenterology. Philadelphia, W. B . Saunders Co., 2nd ed., Vol. Ill, 1965, pp. 746-782. 4. C a r e y , M. C . and S m a l l , D. M.: The physical chemistry of cholesterol solubility in bile: Rela­ tionship to gallstone formation and dissolution in man. J. Clin. Invest. 61:998-1026, 1978. 5. H a w t h o r n e , H . R. and S t e r l in g , J. A.: White bile in the common bile duct. Amer. J. Surg. 90:397-401, 1955. 6 . I n g e l f in g e r , F. J.: Digestive disease as a na­ tional problem: V. Gallstones. Gastroenterology. 559:102-104, 1968. 7. I sa k k s o n , B.: On the lipid constituents of bile from human gallbladder containing cholesterol gallstones, a comparison with normal human bladder bile. Acta Soc. Med. Upsalien. 259:277295, 1954. 8. J o r d a n , G. L .: Choledocholithiasis. Curr. Probl. Surg. i9:721-798, 1982. 9. K r it c h e v s k y , D. and K l u r f e l d , D. M.: Gall­ stone formation in hamsters: Effect of varying animal and vegetable protein levels. Amer. J. Clin. Nutrition. 37:802-804, 1983. 10. L e s t e r , R., et al: What is meant by the term A d m ir a n d ,

13. 14. 15. 16. 17. 18.

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“bile acid”? Amer. J. Physiol. 244:G107-G110, 1983. M e t z g e r , A. L., H e y m s f i e l d , S., and G r u n d y , S. M.: The lithogenic index — a numerical expression for the relative lithogenicity of bile. Gastroenterology 62:499-501, 1972. P itt , H. A. et al: Parenteral nutrition induces calcium bilirubinate sludge and gallstones in the prairie dog. Clin. Res. 31:243A, 1983. R e d i n g e r , R. N. and S m a l l , D. M.: Bile com­ position, bile salt metabolism and gallstones. Arch. Int. Med. 730:618-630, 1972. S h a f f e r , E. A.: Gallstones: Current concepts of pathogenesis and medical dissolution. Canad. J. Surg. 23:517-532, 557, 1980. S m a l l , D. M.: The etiology and pathogenesis of gallstones. Adv. Surg. 10:63-85, 1976. S m a l l , D. M.: Thé formation and treatment of gallstones. Diseases of the Liver. Schiff, L. and Schiif, E. R., eds. Philadelphia, J. B. Lippincott Co., 5th ed., 1982, pp. 151-166. S w e l l , L. et al: The cholesterol saturation index of human bile. Amer. J. Dig. Dis. i9:261—265, 1974. T a n , E. G. C. and W a r r e n , K. W . : Diseases of the gallbladder and bile ducts. Diseases of the Liver. Schiffj&k. and Schiff, E. R., eds. Phila­ delphia, J. B. Lippincott Co., 5th ed, 1982, pp. 1507-1559. T h o m a s , P. J. and H o f m a n n , A. F. : A simple cal­ culation of the lithogenic index of bile: Ex­ pressing biliary lipid composition on rectangular coordinates. Gastroenterology 65:698-700, 1973. T h o r n t o n , J. R. , E m m e t t , P. M., and H e a t o n , K. W. : Diet and gall stones: Effects of refined and unrefined carbohydrate diets on bile choles­ terol saturation and bile acid metabolism. Gut 24:2-6, 1983. W os i e W itz , U. et al: Investigations on common bile duct stones. Digestion 26:43-52, 1983.