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Carbohydrate diet-induced calcium bilirubinate sludge and pigment gallstones in the prairie dog
JOURNAL OF SURGICAL RESEARCH d&580-587
(1986)
Carbohydrate Diet-Induced Calcium Bilirubinate Sludge and Pigment Gallstones in the Prairie Dog’ ROBERT
L. CONTER, M.D., JOEL J. ROSLYN, M.D.,2,3 HENRY A. PITT, M.D., AND LAWRENCEDENBESTEN,M.D.
Surgical Research Service, VeteransAdministration Medical Center, Sepulveda, and Department of Surgery, University of California Medical Center, Los Angeles, California 90024
Presented at the Annual Meeting of the Association for Academic Surgery, Cincinnati, Ohio, November 10-13, 1985 Epidemiologic studiessuggestthat consumption of diets rich in carbohydratesmay, in part, be responsible for the increasing incidence of pigment gallstone disease.The mechanism by which these dietary components lead to pigment stone formation remains obscure. Furthermore, investigative efforts in this area have been hampered by the lack of a suitable animal model. The present study was undertaken to study the role of complex carbohydrates in pigment gallstone formation in the prairie dog. Two groups of eight animals each were maintained on either a control, nonlithogenic chow, or a high carbohydrate (35% sucrose,32% rich starch) diet for 2 months. Neither crystals nor gallstones were observed in any of the control animals. All of the carbohydrate fed animals (P < 0.005 vs control) had calcium bilirubinate crystals and sludge, while microscopic, black stoneswere present in six of these eight animals (P < 0.05 vs control). Although hepatic bile bilirubin was unchanged in the carbohydrate-fed group, these animals had a significant increase in hepatic bile calcium (P < 0.005) and phospholipids (P < 0.005) when compared to controls. Carbohydrate-fed animals also had a significant increase in gallbladder bile concentrations of phospholipids (P < O.OOl),calcium (P < O.OOl),unconjugated (P < 0.005), conjugated (P < 0.005), and total biibin (P < 0.001) as compared to controls. These data indicate that in the prairie dog, carbohydrate feeding results in (1) increasedbiliary concentrations of phospholipids, calcium and bilirubin, and (2) formation of calcium bilirubinate crystals, sludge and microscopic gallstones. Furthermore, these findings suggestthat the carbohydratsfed prairie dog provides a suitable model for studying pigment ga&tone disease. Q 1986 Academic Pw, Inc.
suggeststhat other factors are important in the pathogenesisof this disease. Pigment gallstone disease is being recogEpidemiologic studies suggestthat increased nized with increasing frequency in the United ingestion of certain dietary components States and now accounts for approximately (especially carbohydrates) may be responsible 30% of all patients with cholelithiasis [23,49]. for the changing pattern of pigment gallstone In the past, pigment stones in western coun- formation in this country and throughout the tries were assumed to be primarily related to world [35, 361. The mechanism by which dihemolytic disorders [3, 251. Recent studies etary and other factors lead to pigment stone have suggestedthat cirrhosis [6, 16, 371, ileal formation remains obscure. Efforts to clarify resection [5, 111, and prolonged parenteral the pathogenesisof pigment stones have been nutrition [41] are important risk factors for hampered by the lack of a suitable animal pigment gallstone formation. The absenceof model. Currently available models depend for these specific risk factors in the vast majority successon unphysiologic dietary manipulaof patients with pigment gallstones, however, tions: e.g., cholesterol in the dog [20, 211, guinea pig [28], and hamster models [ 14,461. Furthermore, recent studies suggestthat treat’ This work was supported by a VA researchgrant. ment with chenodeoxycholic or ursodeoxy2 Recipient of a Career Development Award, Research cholic acid decreasesthe incidence of pigment Service, Werans Administration. stones in hamsters [46]. 3To whom reprint requestsshould be addressed. INTRODUCTION
0022-4804186$1.50 Copyright 0 1986 by Academic Au rights of reproduction
F’ress, Inc. in my form men@.
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CONTER ET AL.: PIGMENT GALLSTONES
The present study, therefore, was undertaken to investigate carbohydrate-induced pigment gallstone formation in the prairie dog. This animal was selectedfor study becauseits biliary lipid composition is similar to that of man [ 171.Furthermore, recent reports demonstrate pigment sludge and stone formation in prairie dogs following ileal resection [ 5,421 and during the prolonged administration of total parenteral nutrition [41]. MATERIAL
AND METHODS
Study description. Adult male prairie dogs,4 weighing approximately 1 kg (range 73 1- 1339 g) were caged in thermoregulated (23°C) rooms. Groups of eight animals each were fed either a control, nonlithogenic chow’ or a carbohydrate-enriched chow6 for 8 weeks (see Table 1). The control diet is a standard rodent laboratory chow whose carbohydrate base is composedprimarily of cornstarch, with simple and complex sugars accounting for less than 1% of the total nutrient content. The carbohydrate diet is identical to the control chow except for the amount and type of carbohydrate base, such that the total carbohydrate content is increased and complex sugarscomprise 35% of the total nutrients. Both diets contain adequate calories and all animals maintained or gained weight. Prairie dogs fed the carbohydrate chow had no evidence of diarrhea, a frequent problem with other dietary models of pigment gallstones [ 13, 151. Prior to acute experiments, each animal was lightly sedated with ketamine, 50 mg/kg of body weight, and 0.4 &i of [‘4C]cholic acid was given intravenously to label the bile acid pool. Immediately thereafter, a gavagefeeding of a lipid-protein emulsion ( 1.6 g each of vegetable oil, peanut oil, and milk solids, suspended in 7 ml of water) wasgiven to stimulate gallbladder emptying. After a 16-hr fast, ani4 Cynomys ludivicianus, trapped in the wild state and obtained from Otto Marten Locke, New Branfels, Tex. ‘Purina Laboratory Chow, Ralston-Purina, Co., St. Louis, MO. 6 Teklad Test Diets, A Harlan Sprague-Dawley, Inc., Co., Madison, Wis.
TABLE 1 DIET COMPOSITION Control chow (%)
Carbohydrateenriched chow (96)
Carbohydrate Corn Wheat Sucrose Rice
45 33 11 1 0
61 0 0 35 32
Protein Fat Fiber Water Miscellaneous
23 5 5 10 12
18 4 1 I 3
mals were anesthetized with ketamine, 100 mg/kg of body weight and Valium 0.15 mg/kg of body weight. After a midline laparotomy, the cystic duct was ligated, the gallbladder completely aspirated and then removed, and the common bile duct cannulated with a Silastic catheter. One-hour hepatic bile specimens were collected in tared tubes and saved for further analysis. Gallbladders were opened and their mucosal surface caremlly inspected (using a dissecting microscope) for the presence of sludge and/or stones.Spun gallbladder bile was examined with light microscopy for crystals and/or stones. Analytic methods.Bile collected over ice was centrifuged for 5 min at 2000 rpm. Aliquots of hepatic and gallbladder bile were taken for subsequentanalysis and then stored at -20°C. Frozen bile was warmed to room temperature in a water bath at 37°C. Analyses for cholesterol [44], phospholipids [19], and bile salts [24] were performed using methodology previously described. Total and conjugated bilirubin were measured by a modification of the methods of Michaelsson [33] and Nosslin [39]. Total hepatic and gallbladder bile calciums were determined by the method of Anderegg et al. [2] as modified by Connerty and Briggs [9]. For determination of bile acid specific activity, aliquots of bile were suspended in a scintillation mixture and counted in a Beck-
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JOURNAL OF SURGICAL RESEARCH: VOL. 40, NO. 6, JUNE 1986
FIG. 1. Black and white photo (X33.6) of yellowish crystalline material from cabohydrate-fed prairie dog.
man LS-8000 liquid scintillation counter,7 as previously described [45]. Cholesterol saturation index (CSI) was calculated using Carey’s critical tables for cholesterol saturation based on the total lipid concentration [8]. Bile salt mass, and radioactivity in gallbladder bile, and the first-hour hepatic collections were measuredasdescribed above. The specific activity of each sample, defined as the ratio of radioactivity to mass, wasderived and the ratio of the specificactivity of gallbladder bile to first-hour hepatic bile was calculated. This value, Rsa, reflects the degree of equilibration of an intravenously administered radioisotope between gallbladder and hepatic subpools and has been shown to be an index of gallbladder stasis [45]. Statistical analysis. All data are expressed as the means + SEM. Statistical comparisons between dietary groups were made using Student’s t test for unpaired variables. Chi-square ’ Beckman Instruments, Fullerton, Calif.
analysis was used to compare the incidence of crystals and stones in the two groups of animals. RESULTS
Gallstone Formation Neither crystals nor gallstones were found in any of the animals fed the control chow. All eight of the carbohydrate-fed animals (P < 0.005 vs control) had microscopic, yellowish crystalline material and clusters of yellowishbrown material, consistent with calcium bilirubinate crystals and sludge identified in gallbladder bile (seeFig. 1). In addition, small microscopic stones(seeFig. 2) were found on the gallbladder musocal surface in six of eight prairie dogs maintained on the carbohydrateenriched diet (P < 0.05 vs control). Hepatic Bile Composition Data summarizing the effects of carbohydrate feeding on hepatic bile composition are
CONTER ET AL.: PIGMENT GALLSTONES
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FIG. 2. Black and white photo (X8.4) of yellowish-brown sludge and black pigment gallstones from prairie dog who received high carbohydrate diet for 8 weeks
listed in Table 2. Although hepatic bile concentrations of cholesterol and bile salts were unchanged in the two groups, there was a significant (P < 0.005) increase in phospholipids for carbohydrate-fed animals (9.4 + 1.5 pmole/ml) as compared to controls (4.2 + 0.4 pmole/ml). Despite this finding the mean CSI in carbohydrate-fed prairie dogs (0.49 f 0.04) was unchanged as compared to controls (0.46 zk0.05). In addition to alterations in biliary lipid composition, there was a significant (P < 0.00 1) increase in hepatic bile calcium concentration in carbohydrate-fed animals ( 12.9 + 0.6 mg/dl) as compared to controls (7.4 f 0.6 mg/dl). However, unconjugated, conjugated, and total bilirubin levels in hepatic bile were similar in both groups of animals. Gallbladder Bile Composition Analysis of gallbladder bile concentrations of biliary lipids, calcium and bilirubin are pre-
sented in Table 3. Again, there were no significant differences observed in cholesterol or bile acid concentrations between control and carbohydrate-fed animals. Gallbladder bilk-y phospholipid concentration was increased nearly threefold (P < 0.00 1) in carbohydratefed animals (38.1 + 3.8 pmole/ml) as compared to controls (13.3 f 1.5 pmole/ml). CSI was significantly (P < 0.05) decreasedin experimental animals. Although mean Rsa values were not significantly different between carbohydrate-fed animals and controls, four of the animals with carbohydrate-induced stones had evidence of gallbladder stasis as manifested by Rsa values of 0.26, 0.44, 0.5 1, and 0.8 1. In contrast, none of the controls had values less than 0.90. Gallbladder bile concentrations of calcium were significantly (P < 0.001) increased in carbohydrate-fed animals (33.4 + 3.7 mg/dl) as compared to controls (13.9 f 2.0 mg/dl). In addition, the carbohydrate group had in-
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JOURNAL OF SURGICAL RESEARCH: VOL. 40, NO. 6, JUNE 1986 TABLE 2
man diseaseremains uncertain. The lithogenic diet used in our current studies has several HEPATIC BILE COMPOSITION distinctive features. The caloric distribution of Control Carbohydrate the diet was equivalent to that which has been associated with black pigment gallstone forCholesterol mation in man [50]. Neither drugs nor cho@mole/ml) 0.6 kO.1 0.8 z!z0.1 lesterol were required to induce pigment gallBile acids stone formation in the prairie dog, as has been &mole/ml) 44.5 f 9.5 31.8 k4.9 Phospholipids the case with other dietary models [28, 461. (pmole/ml) 4.2 f 0.4 9.4 f 1.5’ Furthermore, 50% of the carbohydrates was CSI 0.49 * 0.04 0.46 f 0.05 derived from rice, a staple ingredient of diets Total calcium where pigment stones are endemic. 12.9 + 0.6’ 7.4 + 0.6 @g/W In the prairie dog, the feeding of a high carUnconjugated bilibohydrate diet results in the hepatic secretion 0.8 20.1 rubin (mg/dl) 1.0 f 0.2 of bile which has increased concentrations of Conjugated biliphospholipids, calcium, and normal levels of rubin (m&h) 4.4 + 0.5 3.1 kO.5 Total bilirubin cholesterol, bile salts, and bilirubin. In addi4.8 + 0.8 4.0 f 0.5 b.W) tion, gallbladder bile concentrations of phospholipids, calcium, and both unconjugated Note. Values are means f SEM. and total bilirubin, are all significantly in"P < 0.005,bP< 0.001 vscontrol. creasedin carbohydrate-fed animals. Increased hepatic bile phospholipids and calcium result creased levels of unconjugated (P < O.OOS), from carbohydrate-induced changesin hepatic conjugated (P c O.OOS),and total bilirubin (P metabolism. The observation that hepatic bile concentrations of bilirubin are normal sug< 0.001) versus controls. geststhat the increased gallbladder concentraDISCUSSION TABLE 3
Data from this study indicate that in the prairie dog, carbohydrate feeding results in the formation of pigment crystals, sludge and microscopic gallstones. Carbohydrate-fed animals have a significant increase in biliary concentrations of phospholipids, calcium, and bilirubin. These findings, coupled with the observation that the prairie dog has biliary lipid secretory kinetics similar to that of man, suggest that this animal may provide a model in which the discrete events in the evolution of pigment gallstones can be studied. The importance of dietary factors in the pathogenesis of pigment gallstones is gaining increasing support. Diets rich in refined, complex carbohydrates have been implicated as a potential etiologic factor in calcium bilirubinate gallstone formation [34, 351. While pigment gallstones have been induced in other animal models using specific dietary regimens [ 13-15, 20, 21, 28, 461 their relevance to hu-
GALLBLADDER BILE COMPOSITION
Control Cholesterol (pmole/ml) Bile acids @mole/ml) Phospholipids @mole/ml) CSI Rsa Total calcium OwJdU Unconjugated bilirubin (m&h) Conjugated bilirubin (m&U) Total bilirubin bwJdU
3.3 + 0.6 168.5 + 23.3
Carbohydrate
4.1 f
0.5
164.0 + 13.3
13.3 + 1.5 0.51 + 0.05 1.12 + 0.12
38.1 + 3.8b 0.37 + 0.04” 0.79 + 0.15
13.9 + 2.0
33.4 + 3.7c
I.0 + 0.2
3.2 k 0.5b
4.0 If 0.7
11.5 + 2.0b
5.1 + 0.9
14.6 f
Note. Values are means + SEM.
"P< O.O05,bP<0.005,'P < 0.001.
2.1’
CONTER ET AL.: PIGMENT GALLSTONES
tions of bilirubin are due to phenomena within the gallbladder, as opposed to hemolysis or alterations in the hepatic secretion of bilirubin. Our findings further suggestthat carbohydrateinduced increasesin biliary concentrations of phospholipids and calcium alter the solubility of unconjugated bilirubin, with resultant pigment precipitation and crystal formation. The gallbladder bile of carbohydrate-fed prairie dogs contained a sediment which microscopically appeared as yellowish-brown crystals in an amorphous matrix. In addition, there were larger, blackish stones which were noted in the bile as well as on the mucosal surface. The crystalline material appeared identical to the calcium bilirubinate sludge that has been describedby Juniper and Burson [26] and in prairie dogs maintained on TPN [41], or who had an ileal resection [5,42]. The stones were soft and consistent with the gross description of black pigment stones by Trotman and Soloway [XI]. Classically, black pigment stones contain minimal amounts of cholesterol [50]. Although we did not analyze stone composition in the present study, we assume that there was little cholesterol within them. This conjecture is basedon the absence of cholesterol in the diet and the low levels of biliary cholesterol in carbohydrate-fed animals. The appearance of the crystalline sludge and stones, coupled with the biochemical findings, strongly suggeststhat the carbohydrate-fed prairie dog is indeed a suitable model for studying the discrete events in the evolution of black (calcium bilirubinate) pigment gallstone formation. The hypothesis that phospholipids play an important etiologic role in carbohydrate-induced pigment gallstone. formation is supported by both clinical and experimental observations. Calcium bilirubinate sludge, asdetermined ultrasonographically, contains increased amounts of phospholipids [ 11.Recent reports have identified phospholipids within the central core of pigment stones [43]. A direct effect of phospholipids on the solubilization of unconjugated bilirubin is suggested by Ostrow’s [40] in vitro studies, in which he demonstrated a decreasein the solubility of unconjugated bilirubin with the ad-
585
dition of lecithin. The mechanism by which phospholipids alter the solubility of unconjugated bilirubin is not clear. Possible explanations include displacement of bilirubin from a bile salt-phospholipid micelIe, or direct binding with unconjugated bilirubin. The observed increase in hepatic bile phospholipids can be explained on the basis of carbohydrate-induced increases in hepatic lipogenesis.Consumption of diets rich in sucrose increases hepatic pyruvate kinase [22], glucose-6-phosphate dehydrogenase [32], malic synthetase [ 321,and fatty acid synthetase [7]. Stimulation of thesemetabolic pathways could lead to increased hepatic synthesis and secretion of phospholipids. The key to pigment gallstone formation is the solubilization of unconjugated bilirubin. Calcium bilirubinate precipitation may occur as a result of a relative increase in unconjugated bilirubin or calcium or by a change in the other factors (bile salts,phospholipids, etc.) which influence bilirubin solubility. The increasein total calcium observed in the current study should also lead to a similar change in ionized calcium aswell. Increased ionized calcium would alter the solubility product with unconjugated bilirubin, with the result being calcium bilirubinate precipitation. In a manner, analogous to cholesterol gallstone formation [3 11,the precipitate formed servesas a nidus for agglomeration and eventual stone growth. The mechanism by which carbohydrate feeding increasesbiliary calcium levels remains to be clarified. Although several authors have demonstrated enhanced intestinal absorption of calcium in animals [4,5 I] and humans [27, 381 maintained on high carbohydrate diets, whether or not this leads to increased hepatic bile concentrations of calcium is not clear. Hepatic secretion of calcium is influenced by bile acid composition and secretion [ 121.The possibility that this mechanism may account for the observed changes in calcium levels cannot be definitively determined from the current study since individual bile acids were not measured. Gallbladder stasishas been shown to be an important factor in the pathogenesis of cho-
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JOURNAL OF SURGICAL RESEARCH: VOL. 40, NO. 6, JUNE 1986
lesterol gallstones [ 18, 3 1, 471. In the present study there was a decreasein Rsa values (an index of gallbladder stasis), although these changes failed to achieve statistical significance. It is interesting to note, however, that four of the eight carbohydrate-fed animals had evidence of significant gallbladder stasisas indicated by low Rsa values. The failure to demonstrate stasis in the four remaining animals may be due to the effectsof accessorybile duct flow in this model [lo]. Further studies are needed to clarify the role of gallbladder stasis in carbohydrate-induced pigment gallstone formation. The pathogenesisof pigment gallstone diseaseis clearly multifactorial. Although the final common pathway is altered solubilization of unconjugated bilirubin with precipitation of calcium bilirubinate and insoluble salts, the mechanisms by which this is achieved are variable. Maki [30] first proposed that biliary infection promoted &glucuronidase activity resulting in hydrolysis of conjugated bilirubin. More recent studies suggestthat a similar processmay be initiated by nonbacterial factors [29]. Increased substrate (bilirubin) is the critical factor in pigment stone formation in patients with hemolytic disorders [48]. Altered bile salt metabolism has been implicated asan etiologic factor in pigment stones associated with cirrhosis [ 521.The absenceof thesefactors in the majority of patients with pigment stones, coupled with recent epidemiologic studies, suggeststhat dietary influences may be the leading cause of pigment gallstone formation. In summary, our animal study suggeststhat diets rich in complex carbohydrates alters hepatic metabolism of phospholipids and calcium. These changes in biliary composition alter bilirubin solubility, resulting in pigment sludge and microscopic gallstone formation. The carbohydrate fed prairie dog is a suitable model for studying the dietary induction of pigment gallstones and may provide further insight into the pathogenesis of this disease. REFERENCES 1. Allen, B., Bernhott, R., Blanckaert, N., Svanik, J., Filly, R., Gooding, G., and Way, L. Sludgeis calcium
bilirubinate associatedwith bile stasis.Am. J. Surg. 141: 51, 1981. 2. Anderegg, C., Flaschka, H., Sallmen, R., and Schwargenbach, G. M. Metallinkikatoren VII. Ein auf erdalkaliionen atsprechendesphtalein und seine analytische verwendung. Helv. Chim. Acta 37: 113, 1954. 3. Bates,G. C., and Brown, C. H. Incidence of gallbladder diseasein chronic hemolytic anemia (spherocytosis). Gastroenterology 21: 104, 1952. 4. Behar, J., and Kerstein, M. D. Intestinal calcium absorption: Dilherencesin transport between duodenum and ileum. Amer. J. Physiol. 23: 1255, 1976. 5. Bickerstaff,K. I., and Moosa, A. R. Effectsof resection or bypass of the distal ileum on the lithogenicity of bile. Amer. J. Surg. 145: 34, 1983. 6. Bouchier, J. A. D. Postmortem study of the frequency of gallstonesin patients with cirrhosis of the liver. Gut 10: 705, 1969. I. Bruckdorfer, K. R., Klan, I. H., and Yudkin, J. Fatty acid synthetaseactivity in the liver and adipose tissue of rats fed with various carbohydrates. Biochem. J. 124: 439, 1979. 8. Carey, M. C. Critical tables for calculating the cholesterol saturation of native bile. J. Lipid Rex 19: 945, 1978. 9. Connerty, H., and Briggs,A. Determination of serum calcium by means of orthocresolphthalein complex one. Amer. J. Clin. Pathol. 45: 290, 1966. 10. Conter, R. L., Roslyn, J. J., Porter-Fink, V., and DenBesten,L. Gallbladder absorption increasesduring early cholesterol gallstone formation. Amer. J. Surg., in press. 11. Coyle, J. J., Hoyt, D. B., and Sedaghat,A. Relationship of intestinal bypassoperationsand cholelithiasis.Surg. Forum 31: 139, 1980. 12. Cummings, S. A., and Hofmann, A. F. Physiologic determinants of biliary calcium secretion in the dog. Gastroenterology 87: 664, 1984. 13. Dam, H. Nutritional aspects of gallstone formation with particular referenceto alimentary production of gallstonesin laboratory animals. World Rev. Nutr. Diet 11: 199, 1969. 14. Dam, H., and Christensen,F. Alimentary production of gallstones in hamsters. Acta Pathol. Microbial. Stand. 30: 236, 1952. 15. Dam, H., and Christensen,F. Alimentary production of gallstones in hamsters. Inthrence of different carbohydrate sources on gallstone formation, diarrhea, and growth. 2. Emaehrungswiss 2: 36, 1961. 16. Davidson, J. F. Alcohol and cholehthiasis:A necropsy survey ofcirrhosis. Amer. J. Med. Sci. 244: 730, 1962. 17. DenBesten, L., Safaie-Shirazi, S., Connor, W. E., and Bell, S. Early changesin bile composition and gallstone formation induced by a high cholesteroldiet in prairie dogs. Gastroenterology 66: 1036, 1974. 18. Doty, J. E., Pitt, H. A., Kuchenbecker, S. L., and DenBesten, L. Impaired gallbladder emptying before gallstone formation in the prairie dog. Gastroenterology85: 168, 1983. 19 Dryer, R. L., Tammes, A. R., and Routh, R. The
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Lipid Res. 22: 229, 1981. 46. Setoguchi,T., Cohen, B. I., Mosbach, E. H., Soloway, 30. Maki, T. Pathogenesis of calcium bilirubinate gallR. D., Rzigalinski, B., May-Doneth, P., and Musberry, stones.Ann. Surg. 164: 90, 1966. C. K. A new hamster model of pigments cholelithiasis. 31. Meyer, P. D., DenBesten, L., and Gurll, N. J. Effects Gastroenterology 88: 1693, 1985. [Abstract] of cholesterol gallstone induction on gallbladder 47. Shalfer, E. A., McOrmond, P., and Duggan, H. Asfunction and bile salt pool size in the prairie dog sessmentof gallbladder filling and emptying and duomodel. Surgery 83: 599, 1978. denogastric reflux. Gastroenterology 79: 899, 1980. 32. Michaelis, 0. E., IV., and Szepesi,B. Effect of various 48. Soloway, R. D., Trotman, B. W., and Ostrow, J. D. sugarson hepatic glucoscd-phosphate dehydrogenase, Pigment gallstones. Gastroenterology 72: 167, 1977. malic enzyme and total liver lipid of the rat. J. Nutr. 49. Trotman, B. W., and Soloway, R. D. 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(1986)
Carbohydrate Diet-Induced Calcium Bilirubinate Sludge and Pigment Gallstones in the Prairie Dog’ ROBERT
L. CONTER, M.D., JOEL J. ROSLYN, M.D.,2,3 HENRY A. PITT, M.D., AND LAWRENCEDENBESTEN,M.D.
Surgical Research Service, VeteransAdministration Medical Center, Sepulveda, and Department of Surgery, University of California Medical Center, Los Angeles, California 90024
Presented at the Annual Meeting of the Association for Academic Surgery, Cincinnati, Ohio, November 10-13, 1985 Epidemiologic studiessuggestthat consumption of diets rich in carbohydratesmay, in part, be responsible for the increasing incidence of pigment gallstone disease.The mechanism by which these dietary components lead to pigment stone formation remains obscure. Furthermore, investigative efforts in this area have been hampered by the lack of a suitable animal model. The present study was undertaken to study the role of complex carbohydrates in pigment gallstone formation in the prairie dog. Two groups of eight animals each were maintained on either a control, nonlithogenic chow, or a high carbohydrate (35% sucrose,32% rich starch) diet for 2 months. Neither crystals nor gallstones were observed in any of the control animals. All of the carbohydrate fed animals (P < 0.005 vs control) had calcium bilirubinate crystals and sludge, while microscopic, black stoneswere present in six of these eight animals (P < 0.05 vs control). Although hepatic bile bilirubin was unchanged in the carbohydrate-fed group, these animals had a significant increase in hepatic bile calcium (P < 0.005) and phospholipids (P < 0.005) when compared to controls. Carbohydrate-fed animals also had a significant increase in gallbladder bile concentrations of phospholipids (P < O.OOl),calcium (P < O.OOl),unconjugated (P < 0.005), conjugated (P < 0.005), and total biibin (P < 0.001) as compared to controls. These data indicate that in the prairie dog, carbohydrate feeding results in (1) increasedbiliary concentrations of phospholipids, calcium and bilirubin, and (2) formation of calcium bilirubinate crystals, sludge and microscopic gallstones. Furthermore, these findings suggestthat the carbohydratsfed prairie dog provides a suitable model for studying pigment ga&tone disease. Q 1986 Academic Pw, Inc.
suggeststhat other factors are important in the pathogenesisof this disease. Pigment gallstone disease is being recogEpidemiologic studies suggestthat increased nized with increasing frequency in the United ingestion of certain dietary components States and now accounts for approximately (especially carbohydrates) may be responsible 30% of all patients with cholelithiasis [23,49]. for the changing pattern of pigment gallstone In the past, pigment stones in western coun- formation in this country and throughout the tries were assumed to be primarily related to world [35, 361. The mechanism by which dihemolytic disorders [3, 251. Recent studies etary and other factors lead to pigment stone have suggestedthat cirrhosis [6, 16, 371, ileal formation remains obscure. Efforts to clarify resection [5, 111, and prolonged parenteral the pathogenesisof pigment stones have been nutrition [41] are important risk factors for hampered by the lack of a suitable animal pigment gallstone formation. The absenceof model. Currently available models depend for these specific risk factors in the vast majority successon unphysiologic dietary manipulaof patients with pigment gallstones, however, tions: e.g., cholesterol in the dog [20, 211, guinea pig [28], and hamster models [ 14,461. Furthermore, recent studies suggestthat treat’ This work was supported by a VA researchgrant. ment with chenodeoxycholic or ursodeoxy2 Recipient of a Career Development Award, Research cholic acid decreasesthe incidence of pigment Service, Werans Administration. stones in hamsters [46]. 3To whom reprint requestsshould be addressed. INTRODUCTION
0022-4804186$1.50 Copyright 0 1986 by Academic Au rights of reproduction
F’ress, Inc. in my form men@.
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CONTER ET AL.: PIGMENT GALLSTONES
The present study, therefore, was undertaken to investigate carbohydrate-induced pigment gallstone formation in the prairie dog. This animal was selectedfor study becauseits biliary lipid composition is similar to that of man [ 171.Furthermore, recent reports demonstrate pigment sludge and stone formation in prairie dogs following ileal resection [ 5,421 and during the prolonged administration of total parenteral nutrition [41]. MATERIAL
AND METHODS
Study description. Adult male prairie dogs,4 weighing approximately 1 kg (range 73 1- 1339 g) were caged in thermoregulated (23°C) rooms. Groups of eight animals each were fed either a control, nonlithogenic chow’ or a carbohydrate-enriched chow6 for 8 weeks (see Table 1). The control diet is a standard rodent laboratory chow whose carbohydrate base is composedprimarily of cornstarch, with simple and complex sugars accounting for less than 1% of the total nutrient content. The carbohydrate diet is identical to the control chow except for the amount and type of carbohydrate base, such that the total carbohydrate content is increased and complex sugarscomprise 35% of the total nutrients. Both diets contain adequate calories and all animals maintained or gained weight. Prairie dogs fed the carbohydrate chow had no evidence of diarrhea, a frequent problem with other dietary models of pigment gallstones [ 13, 151. Prior to acute experiments, each animal was lightly sedated with ketamine, 50 mg/kg of body weight, and 0.4 &i of [‘4C]cholic acid was given intravenously to label the bile acid pool. Immediately thereafter, a gavagefeeding of a lipid-protein emulsion ( 1.6 g each of vegetable oil, peanut oil, and milk solids, suspended in 7 ml of water) wasgiven to stimulate gallbladder emptying. After a 16-hr fast, ani4 Cynomys ludivicianus, trapped in the wild state and obtained from Otto Marten Locke, New Branfels, Tex. ‘Purina Laboratory Chow, Ralston-Purina, Co., St. Louis, MO. 6 Teklad Test Diets, A Harlan Sprague-Dawley, Inc., Co., Madison, Wis.
TABLE 1 DIET COMPOSITION Control chow (%)
Carbohydrateenriched chow (96)
Carbohydrate Corn Wheat Sucrose Rice
45 33 11 1 0
61 0 0 35 32
Protein Fat Fiber Water Miscellaneous
23 5 5 10 12
18 4 1 I 3
mals were anesthetized with ketamine, 100 mg/kg of body weight and Valium 0.15 mg/kg of body weight. After a midline laparotomy, the cystic duct was ligated, the gallbladder completely aspirated and then removed, and the common bile duct cannulated with a Silastic catheter. One-hour hepatic bile specimens were collected in tared tubes and saved for further analysis. Gallbladders were opened and their mucosal surface caremlly inspected (using a dissecting microscope) for the presence of sludge and/or stones.Spun gallbladder bile was examined with light microscopy for crystals and/or stones. Analytic methods.Bile collected over ice was centrifuged for 5 min at 2000 rpm. Aliquots of hepatic and gallbladder bile were taken for subsequentanalysis and then stored at -20°C. Frozen bile was warmed to room temperature in a water bath at 37°C. Analyses for cholesterol [44], phospholipids [19], and bile salts [24] were performed using methodology previously described. Total and conjugated bilirubin were measured by a modification of the methods of Michaelsson [33] and Nosslin [39]. Total hepatic and gallbladder bile calciums were determined by the method of Anderegg et al. [2] as modified by Connerty and Briggs [9]. For determination of bile acid specific activity, aliquots of bile were suspended in a scintillation mixture and counted in a Beck-
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JOURNAL OF SURGICAL RESEARCH: VOL. 40, NO. 6, JUNE 1986
FIG. 1. Black and white photo (X33.6) of yellowish crystalline material from cabohydrate-fed prairie dog.
man LS-8000 liquid scintillation counter,7 as previously described [45]. Cholesterol saturation index (CSI) was calculated using Carey’s critical tables for cholesterol saturation based on the total lipid concentration [8]. Bile salt mass, and radioactivity in gallbladder bile, and the first-hour hepatic collections were measuredasdescribed above. The specific activity of each sample, defined as the ratio of radioactivity to mass, wasderived and the ratio of the specificactivity of gallbladder bile to first-hour hepatic bile was calculated. This value, Rsa, reflects the degree of equilibration of an intravenously administered radioisotope between gallbladder and hepatic subpools and has been shown to be an index of gallbladder stasis [45]. Statistical analysis. All data are expressed as the means + SEM. Statistical comparisons between dietary groups were made using Student’s t test for unpaired variables. Chi-square ’ Beckman Instruments, Fullerton, Calif.
analysis was used to compare the incidence of crystals and stones in the two groups of animals. RESULTS
Gallstone Formation Neither crystals nor gallstones were found in any of the animals fed the control chow. All eight of the carbohydrate-fed animals (P < 0.005 vs control) had microscopic, yellowish crystalline material and clusters of yellowishbrown material, consistent with calcium bilirubinate crystals and sludge identified in gallbladder bile (seeFig. 1). In addition, small microscopic stones(seeFig. 2) were found on the gallbladder musocal surface in six of eight prairie dogs maintained on the carbohydrateenriched diet (P < 0.05 vs control). Hepatic Bile Composition Data summarizing the effects of carbohydrate feeding on hepatic bile composition are
CONTER ET AL.: PIGMENT GALLSTONES
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FIG. 2. Black and white photo (X8.4) of yellowish-brown sludge and black pigment gallstones from prairie dog who received high carbohydrate diet for 8 weeks
listed in Table 2. Although hepatic bile concentrations of cholesterol and bile salts were unchanged in the two groups, there was a significant (P < 0.005) increase in phospholipids for carbohydrate-fed animals (9.4 + 1.5 pmole/ml) as compared to controls (4.2 + 0.4 pmole/ml). Despite this finding the mean CSI in carbohydrate-fed prairie dogs (0.49 f 0.04) was unchanged as compared to controls (0.46 zk0.05). In addition to alterations in biliary lipid composition, there was a significant (P < 0.00 1) increase in hepatic bile calcium concentration in carbohydrate-fed animals ( 12.9 + 0.6 mg/dl) as compared to controls (7.4 f 0.6 mg/dl). However, unconjugated, conjugated, and total bilirubin levels in hepatic bile were similar in both groups of animals. Gallbladder Bile Composition Analysis of gallbladder bile concentrations of biliary lipids, calcium and bilirubin are pre-
sented in Table 3. Again, there were no significant differences observed in cholesterol or bile acid concentrations between control and carbohydrate-fed animals. Gallbladder bilk-y phospholipid concentration was increased nearly threefold (P < 0.00 1) in carbohydratefed animals (38.1 + 3.8 pmole/ml) as compared to controls (13.3 f 1.5 pmole/ml). CSI was significantly (P < 0.05) decreasedin experimental animals. Although mean Rsa values were not significantly different between carbohydrate-fed animals and controls, four of the animals with carbohydrate-induced stones had evidence of gallbladder stasis as manifested by Rsa values of 0.26, 0.44, 0.5 1, and 0.8 1. In contrast, none of the controls had values less than 0.90. Gallbladder bile concentrations of calcium were significantly (P < 0.001) increased in carbohydrate-fed animals (33.4 + 3.7 mg/dl) as compared to controls (13.9 f 2.0 mg/dl). In addition, the carbohydrate group had in-
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JOURNAL OF SURGICAL RESEARCH: VOL. 40, NO. 6, JUNE 1986 TABLE 2
man diseaseremains uncertain. The lithogenic diet used in our current studies has several HEPATIC BILE COMPOSITION distinctive features. The caloric distribution of Control Carbohydrate the diet was equivalent to that which has been associated with black pigment gallstone forCholesterol mation in man [50]. Neither drugs nor cho@mole/ml) 0.6 kO.1 0.8 z!z0.1 lesterol were required to induce pigment gallBile acids stone formation in the prairie dog, as has been &mole/ml) 44.5 f 9.5 31.8 k4.9 Phospholipids the case with other dietary models [28, 461. (pmole/ml) 4.2 f 0.4 9.4 f 1.5’ Furthermore, 50% of the carbohydrates was CSI 0.49 * 0.04 0.46 f 0.05 derived from rice, a staple ingredient of diets Total calcium where pigment stones are endemic. 12.9 + 0.6’ 7.4 + 0.6 @g/W In the prairie dog, the feeding of a high carUnconjugated bilibohydrate diet results in the hepatic secretion 0.8 20.1 rubin (mg/dl) 1.0 f 0.2 of bile which has increased concentrations of Conjugated biliphospholipids, calcium, and normal levels of rubin (m&h) 4.4 + 0.5 3.1 kO.5 Total bilirubin cholesterol, bile salts, and bilirubin. In addi4.8 + 0.8 4.0 f 0.5 b.W) tion, gallbladder bile concentrations of phospholipids, calcium, and both unconjugated Note. Values are means f SEM. and total bilirubin, are all significantly in"P < 0.005,bP< 0.001 vscontrol. creasedin carbohydrate-fed animals. Increased hepatic bile phospholipids and calcium result creased levels of unconjugated (P < O.OOS), from carbohydrate-induced changesin hepatic conjugated (P c O.OOS),and total bilirubin (P metabolism. The observation that hepatic bile concentrations of bilirubin are normal sug< 0.001) versus controls. geststhat the increased gallbladder concentraDISCUSSION TABLE 3
Data from this study indicate that in the prairie dog, carbohydrate feeding results in the formation of pigment crystals, sludge and microscopic gallstones. Carbohydrate-fed animals have a significant increase in biliary concentrations of phospholipids, calcium, and bilirubin. These findings, coupled with the observation that the prairie dog has biliary lipid secretory kinetics similar to that of man, suggest that this animal may provide a model in which the discrete events in the evolution of pigment gallstones can be studied. The importance of dietary factors in the pathogenesis of pigment gallstones is gaining increasing support. Diets rich in refined, complex carbohydrates have been implicated as a potential etiologic factor in calcium bilirubinate gallstone formation [34, 351. While pigment gallstones have been induced in other animal models using specific dietary regimens [ 13-15, 20, 21, 28, 461 their relevance to hu-
GALLBLADDER BILE COMPOSITION
Control Cholesterol (pmole/ml) Bile acids @mole/ml) Phospholipids @mole/ml) CSI Rsa Total calcium OwJdU Unconjugated bilirubin (m&h) Conjugated bilirubin (m&U) Total bilirubin bwJdU
3.3 + 0.6 168.5 + 23.3
Carbohydrate
4.1 f
0.5
164.0 + 13.3
13.3 + 1.5 0.51 + 0.05 1.12 + 0.12
38.1 + 3.8b 0.37 + 0.04” 0.79 + 0.15
13.9 + 2.0
33.4 + 3.7c
I.0 + 0.2
3.2 k 0.5b
4.0 If 0.7
11.5 + 2.0b
5.1 + 0.9
14.6 f
Note. Values are means + SEM.
"P< O.O05,bP<0.005,'P < 0.001.
2.1’
CONTER ET AL.: PIGMENT GALLSTONES
tions of bilirubin are due to phenomena within the gallbladder, as opposed to hemolysis or alterations in the hepatic secretion of bilirubin. Our findings further suggestthat carbohydrateinduced increasesin biliary concentrations of phospholipids and calcium alter the solubility of unconjugated bilirubin, with resultant pigment precipitation and crystal formation. The gallbladder bile of carbohydrate-fed prairie dogs contained a sediment which microscopically appeared as yellowish-brown crystals in an amorphous matrix. In addition, there were larger, blackish stones which were noted in the bile as well as on the mucosal surface. The crystalline material appeared identical to the calcium bilirubinate sludge that has been describedby Juniper and Burson [26] and in prairie dogs maintained on TPN [41], or who had an ileal resection [5,42]. The stones were soft and consistent with the gross description of black pigment stones by Trotman and Soloway [XI]. Classically, black pigment stones contain minimal amounts of cholesterol [50]. Although we did not analyze stone composition in the present study, we assume that there was little cholesterol within them. This conjecture is basedon the absence of cholesterol in the diet and the low levels of biliary cholesterol in carbohydrate-fed animals. The appearance of the crystalline sludge and stones, coupled with the biochemical findings, strongly suggeststhat the carbohydrate-fed prairie dog is indeed a suitable model for studying the discrete events in the evolution of black (calcium bilirubinate) pigment gallstone formation. The hypothesis that phospholipids play an important etiologic role in carbohydrate-induced pigment gallstone. formation is supported by both clinical and experimental observations. Calcium bilirubinate sludge, asdetermined ultrasonographically, contains increased amounts of phospholipids [ 11.Recent reports have identified phospholipids within the central core of pigment stones [43]. A direct effect of phospholipids on the solubilization of unconjugated bilirubin is suggested by Ostrow’s [40] in vitro studies, in which he demonstrated a decreasein the solubility of unconjugated bilirubin with the ad-
585
dition of lecithin. The mechanism by which phospholipids alter the solubility of unconjugated bilirubin is not clear. Possible explanations include displacement of bilirubin from a bile salt-phospholipid micelIe, or direct binding with unconjugated bilirubin. The observed increase in hepatic bile phospholipids can be explained on the basis of carbohydrate-induced increases in hepatic lipogenesis.Consumption of diets rich in sucrose increases hepatic pyruvate kinase [22], glucose-6-phosphate dehydrogenase [32], malic synthetase [ 321,and fatty acid synthetase [7]. Stimulation of thesemetabolic pathways could lead to increased hepatic synthesis and secretion of phospholipids. The key to pigment gallstone formation is the solubilization of unconjugated bilirubin. Calcium bilirubinate precipitation may occur as a result of a relative increase in unconjugated bilirubin or calcium or by a change in the other factors (bile salts,phospholipids, etc.) which influence bilirubin solubility. The increasein total calcium observed in the current study should also lead to a similar change in ionized calcium aswell. Increased ionized calcium would alter the solubility product with unconjugated bilirubin, with the result being calcium bilirubinate precipitation. In a manner, analogous to cholesterol gallstone formation [3 11,the precipitate formed servesas a nidus for agglomeration and eventual stone growth. The mechanism by which carbohydrate feeding increasesbiliary calcium levels remains to be clarified. Although several authors have demonstrated enhanced intestinal absorption of calcium in animals [4,5 I] and humans [27, 381 maintained on high carbohydrate diets, whether or not this leads to increased hepatic bile concentrations of calcium is not clear. Hepatic secretion of calcium is influenced by bile acid composition and secretion [ 121.The possibility that this mechanism may account for the observed changes in calcium levels cannot be definitively determined from the current study since individual bile acids were not measured. Gallbladder stasishas been shown to be an important factor in the pathogenesis of cho-
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JOURNAL OF SURGICAL RESEARCH: VOL. 40, NO. 6, JUNE 1986
lesterol gallstones [ 18, 3 1, 471. In the present study there was a decreasein Rsa values (an index of gallbladder stasis), although these changes failed to achieve statistical significance. It is interesting to note, however, that four of the eight carbohydrate-fed animals had evidence of significant gallbladder stasisas indicated by low Rsa values. The failure to demonstrate stasis in the four remaining animals may be due to the effectsof accessorybile duct flow in this model [lo]. Further studies are needed to clarify the role of gallbladder stasis in carbohydrate-induced pigment gallstone formation. The pathogenesisof pigment gallstone diseaseis clearly multifactorial. Although the final common pathway is altered solubilization of unconjugated bilirubin with precipitation of calcium bilirubinate and insoluble salts, the mechanisms by which this is achieved are variable. Maki [30] first proposed that biliary infection promoted &glucuronidase activity resulting in hydrolysis of conjugated bilirubin. More recent studies suggestthat a similar processmay be initiated by nonbacterial factors [29]. Increased substrate (bilirubin) is the critical factor in pigment stone formation in patients with hemolytic disorders [48]. Altered bile salt metabolism has been implicated asan etiologic factor in pigment stones associated with cirrhosis [ 521.The absenceof thesefactors in the majority of patients with pigment stones, coupled with recent epidemiologic studies, suggeststhat dietary influences may be the leading cause of pigment gallstone formation. In summary, our animal study suggeststhat diets rich in complex carbohydrates alters hepatic metabolism of phospholipids and calcium. These changes in biliary composition alter bilirubin solubility, resulting in pigment sludge and microscopic gallstone formation. The carbohydrate fed prairie dog is a suitable model for studying the dietary induction of pigment gallstones and may provide further insight into the pathogenesis of this disease. REFERENCES 1. Allen, B., Bernhott, R., Blanckaert, N., Svanik, J., Filly, R., Gooding, G., and Way, L. Sludgeis calcium
bilirubinate associatedwith bile stasis.Am. J. Surg. 141: 51, 1981. 2. Anderegg, C., Flaschka, H., Sallmen, R., and Schwargenbach, G. M. Metallinkikatoren VII. Ein auf erdalkaliionen atsprechendesphtalein und seine analytische verwendung. Helv. Chim. Acta 37: 113, 1954. 3. Bates,G. C., and Brown, C. H. Incidence of gallbladder diseasein chronic hemolytic anemia (spherocytosis). Gastroenterology 21: 104, 1952. 4. Behar, J., and Kerstein, M. D. Intestinal calcium absorption: Dilherencesin transport between duodenum and ileum. Amer. J. Physiol. 23: 1255, 1976. 5. Bickerstaff,K. I., and Moosa, A. R. Effectsof resection or bypass of the distal ileum on the lithogenicity of bile. Amer. J. Surg. 145: 34, 1983. 6. Bouchier, J. A. D. Postmortem study of the frequency of gallstonesin patients with cirrhosis of the liver. Gut 10: 705, 1969. I. Bruckdorfer, K. R., Klan, I. H., and Yudkin, J. Fatty acid synthetaseactivity in the liver and adipose tissue of rats fed with various carbohydrates. Biochem. J. 124: 439, 1979. 8. Carey, M. C. Critical tables for calculating the cholesterol saturation of native bile. J. Lipid Rex 19: 945, 1978. 9. Connerty, H., and Briggs,A. Determination of serum calcium by means of orthocresolphthalein complex one. Amer. J. Clin. Pathol. 45: 290, 1966. 10. Conter, R. L., Roslyn, J. J., Porter-Fink, V., and DenBesten,L. Gallbladder absorption increasesduring early cholesterol gallstone formation. Amer. J. Surg., in press. 11. Coyle, J. J., Hoyt, D. B., and Sedaghat,A. Relationship of intestinal bypassoperationsand cholelithiasis.Surg. Forum 31: 139, 1980. 12. Cummings, S. A., and Hofmann, A. F. Physiologic determinants of biliary calcium secretion in the dog. Gastroenterology 87: 664, 1984. 13. Dam, H. Nutritional aspects of gallstone formation with particular referenceto alimentary production of gallstonesin laboratory animals. World Rev. Nutr. Diet 11: 199, 1969. 14. Dam, H., and Christensen,F. Alimentary production of gallstones in hamsters. Acta Pathol. Microbial. Stand. 30: 236, 1952. 15. Dam, H., and Christensen,F. Alimentary production of gallstones in hamsters. Inthrence of different carbohydrate sources on gallstone formation, diarrhea, and growth. 2. Emaehrungswiss 2: 36, 1961. 16. Davidson, J. F. Alcohol and cholehthiasis:A necropsy survey ofcirrhosis. Amer. J. Med. Sci. 244: 730, 1962. 17. DenBesten, L., Safaie-Shirazi, S., Connor, W. E., and Bell, S. Early changesin bile composition and gallstone formation induced by a high cholesteroldiet in prairie dogs. Gastroenterology 66: 1036, 1974. 18. Doty, J. E., Pitt, H. A., Kuchenbecker, S. L., and DenBesten, L. Impaired gallbladder emptying before gallstone formation in the prairie dog. Gastroenterology85: 168, 1983. 19 Dryer, R. L., Tammes, A. R., and Routh, R. The
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Nagase,M., Tanimura, H., Setoyama,M., and Hilcosa, Vlahcevic, Z. R. Bile acid metabolism in cirrhosis. Y. Presentfeaturesofgallstones in Japan. A collective IV. Characterization of the abnormality in deoxycholic review of 2 144 cases.Amer. J. Stag. 135: 788, 1978. acid metabolism. Gastroenterology 68: 335, 1975. determination of phosphorous and phosphatasewith N-phenyl-pphenylene diamine. J. Biol. Chem. 225: 171, 1956. 20. Englert, E., Jr. The dietdog model of pigment gallstone formation. Hepatoi. Rapid Lit. Rev. 11: 1702, 1981.