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Pigment Sludge and Stone Formation in the Acutely Ligated Dog Gallbladder
GASTROENTEROLOGY 1983 ;85: 1166-71
Pigment Sludge and Stone Formation in the Acutely Ligated Dog Gallbladder R. A. BERNHOFT, C. A. PELLEGRINI, W. C. BRODERICK, and L. W. WAY
Veterans Administration Medical Center and the Department of Surgery, Univers ity of California, San Francisco, California
We studied the effects of stasis of gallbladder bile in a dog model. Three days after cystic duct ligation, all gallbladders contained sludge, and the mucosa was covered by densely adherent mucus with solid particles 1-4 mm in diameter (gravel). Thirty percent of the animals developed stones (>4 mm), which appeared grossly like human pigment stones and microscopically like condensed biliary sludge. Centrifugation of bile yielded colorless pellets (3.B ± 3.2 mg/ml) at day a and pigmented pellets (33.1 ± 11.0 mg/ml) at day 3 (p < 0.05). Pellets contained 73 ± B% mucin by weight. Dissolved mucin in supernatant bile increased from 7.46 ± 1.19 mg/ml (day 0) to 27.36 ± 3.05 mg/ml (day 3) (p < 0.001), while bilirubin concentration decreased from 127 ± 12 mg/dl (day 0) to 71 ± 16 mg/dl (day 3) (p < 0.001). Cholesterol concentration increased but did not reach saturation, while the concentration of bile salt and phospholipid did not change. Mucin-bilirubin complexes formed and remained suspended as sludge initially. As bile mucin content increased , sludge particles coalesced , precipitated, and eventually formed gravel and stones. We suspect that stone formation in this setting occurs because of sequestration of biliary lipids by mucin. Biliary stasis has been shown to induce pigment stone formation in animals (1,2) and in patients with biliary stricture (3). Gallbladder stasis may be a factor in cholesterol gallstone formation after vagotomy (4). We have previously reported that gallbladder Received December 24, 1982. Accepted May 12, 1983. Address requests for reprints to : Carlos A. Pellegrini , M.D., Surgical Service (112), Veterans Administration Medical Center, 4150 Clement Street, San Francisco, California 94121. This work was supported by th e Veterans Administration and by a grant (MSC 72) from the University of California School of Medicine. The authors thank Anny O. Wong, K. Warren Hayashi, and James R. Gorring for technical ass istance. © 1983 by the American Gastroenterological Association 0016-5085/83/$3.00
stasis leads to biliary sludge formation in humans (5) and dogs (6). In related work, Englert et a1. (7) noted that dogs fed a high-cholesterol diet developed sludgelike material in their gallbladders, which eventually evolved into pigment stones. We have postulated that biliary sludge may provide the nidus necessary to initiate stone formation (5,6), because biliary sludge is similar to the material found at the core of pigment and cholesterol stones (8). Accordingly, accumulation of mucin, which is the main component of sludge, may be a primary event in the pathogenesis of gallstones. Since the gallbladder mucosa is the source of most of the mucin in bile, we developed a model of gallbladder stasis in the dog, which allowed us to study the mechanics of pigment stone formation.
Materials and Methods Twenty-four healthy mongrel dogs of both sexes were acclimated to the kennel. After an overnight fast, each dog underwent laparotomy ("day 0") under general anesthesia (Nembutal, Abbott Pharmaceuticals, North Chicago. Ill. . 10 mg/kg body wt) . The gallbladder and biliary tree were examined, and the cystic duct was ligated; the cystic artery was spared. The dome of the gallbladder was then punctured with a 2D-gauge Teflon catheter. Bile was allowed to flow back into the Teflon catheter until all air was eliminated. The catheter was then connected to a saline-filled tube connected to a strain gauge and a physiograph (Beckman R-611 Dynograph recorder, Beckman Instruments. Palo Alto, Calif.). Pressure within the gallbladder was measured in centimeters of water (cmH 2 0). Movement of bile out of the gallbladder into the cannula used for pressure measurements was minimal. Gallbladder bile was then completely aspirated. its volume was recorded. and a 3-ml aliquot was collected for study. The remainder of the bile was returned to the gallbladder. pressure was measured again. the Teflon catheter was removed, and the puncture site was closed with a Z-plasty of 6-0 silk. The laparotomy incision was closed. and the dog was returned to its kennel. Sterile technique was observed throughout the operation.
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The dogs were fed a liquid meal the evening of the operation and were given regular kennel dog food (Wayne Pro Mix Dog Food, Allied Mill s, Inc" Chicago, IlL) for the next 2 days, After an overnight fas t. each dog underwent a second laparotomy on the third day after ligation of the cystic duct ("day 3"), The gallbladder and biliary tree were inspected, the dome of the ga llbladde r was punctured , and meas urements of pressure and volume were performed as before, After the gallbladder was emptied , it was removed, opened, examined, photograp hed , and fixed in formalin , and the dog was killed , Bile specimens were treated identically, with immed iate separation of aliquots for aerobic an d anerobic culture and for light and polarized microscopy. The remainder of the bile was placed in nitrogen-filled sterile tubes in ice water (4°C), which were stored in the dark. Within 2 h of co ll ection , the specimens were centrifuged at 4500 rpm (7120 g) for 30 min at 4°C (Sorvall General Purpose RC-3 Automatic Refrigerated Centrifuge) . After centrifugation , the bile supernatant and pellet were separated. The supernatant volume was recorded , and aliquots were frozen at -60°C until assayed; aliquots of 500 JLl were frozen for cho lestero l assay (9). Twentymicroliter aliquots were collected for assay of bile salt (10), phospholipid (11), and bilirubin concentration (12). Mucin content was estimated by Peterson's modification of the Lowry protein assay (1 3), including decolorization of the pellet with hydrogen peroxide. The pellet , which microscopically looked like sludge, was washed three times w ith sterile water, weighed , and was then resuspended by sonication in a 1: 5 dilution with normal saline. The aliquots were coll ected for assay and were frozen in the same manner as described for the su pernatant. Fresh aliquots «2 h after removal from the gallbladder) of supernatant bile and pellet (200- 400 JLl) that had been kept at 4°C were used for measurement of bilirubin conjugates by thin-layer chromatograph y [14). Cholesterol saturation indi ces were calculated from the tables published by Carey (15). Residual micellar capacity (RMC , moles per deciliter) was defined as the arithmetic difference between chol esterol concentration at the point of theoretical micellar saturation and actual cholesterol concentration for each specimen. After the gallbladder was fixed in formalin, sections were taken through the dome, body, and neck for hematoxylin an d eosin (H&E) and periodic acid-Schiff (PAS) stains. The sections were examin ed for inflammatory infiltrate and for intestinal metaplasia of the ep ithelium. Data from 6 dogs were discarded-two because of unligated accessory cystic ducts , two for mucosal bleeding, and two for leakage from the asp iration site. Each animal was used as its own controL Data from day o were compared with data from day 3 using paired t-tests. Values are expressed as mean::!: SEM.
PIGMENT STONE FORMATION AND BILIARY STASIS
11 67
cally identical to biliary sludge as previously described (5). The gallbladder mucosa was covered by a layer of firmly adherent mucus. When the mucus was rinsed off the mucosa and examined (a procedure performed in 9 animals), it was seen to contain multiple solid particles of different sizes. Solid particles with a diameter > 4 mm will be referred to as stones, and those with a diameter between 1 and 4 mm will be referred to as grave /. Stones plus gravel were found in 3 of the 9 animals (Figure 1), and gravel alone was found in the remaining 6 animals. Wh en cru shed and examined microscopically, the solid particles contained granular pigmented material similar to sludge peripherally (Figure 2A), coalescing to homogenous sheets centrally (Figure 2B). Day 3 bile contained visible solid particles , which mi croscopically were identical to " bilirubinate granules" (16) and to what we have termed biliary sludge in previous reports (5 ,6) . No cholesterol crystals were present. No solid particles were visible in any day 0 bile specimen. Centrifugation of day 3 bile aspirates yielded a pigmented pellet of mean weight 33.1 ± 11.0 mg/ml spun bile. Centrifugation of day 0 bile yielded colorless pellets of 3.8 ± 3.2 mg/ml spun bile (p < 0.05) . Day 3 sludge pellet contents were as follows: mucin 73 ± 8%, bilirubin 0.9 ± 0.6%, cholesterol 2 ± 1%, bile salts 21 ± 7%, and phospholipids 3 ± 2% . The day 0 pellets were too small to assay. Changes in the concentrations of the constituents of supernatant bile and the mean total content per gallbladder for each constituent are listed in Table 1. Concentration and total gallbladder content of dissolved mucin increased, and bilirubin concentration and content decreas ed. Cholesterol concentration increased, but total chol esterol content did not change. Concentration and total co ntent of bile salt and phospholipid did not change.
Results At day 3, all 18 gallbladders contained copious amounts of viscous , black material, microscopi-
Figure 1. Opened gallbladder specimen 3 days after cystic duct ligation. A 5 x 6mm - pigment stone is present.
A
B
Figure 2. Photomicrograph of the periphery (A) and the center (B) of a crushed pigment stone formed in a dog gallbladder after ligation of the cystic duct (x400).
PIGMENT STONE FORMATION AND BILIARY STASIS
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1169
Table 1. Composition of Supernatant Bile in 18 Dogs Total gallbladder content (mg)
Bile concentration (mg/ml)
Day Mucin Bilirubin Cholesterol Phospholipid Bile salt Different from controls:
7.46:!: 1.27 ::!: 0.62 ::!: 44 .68 ::!: 85.47 ::!: a
a 1.19 0.12 0.04 2.23 3.65
Day a
Day 3 27.36 0.71 0.91 47.29 86.83
3.05" 0.16" ::!: 0.05" ::!: 2.85 ::!: 3.14 ::!: ::!:
106 18 11 678 1211
Day 3
18 2 ::!: 1 ::!: 78 ::!: 71
271 6 13 706 1207
::!: ::!:
::!: 57 b
1" 2 ::!: 91 ::!: 115
::!: ::!:
p < 0.001 ; b p < 0.005.
Some concentration of bile probably occurred, as suggested by a slight decrease in mean volume of gallbladder bile from 16.3 ± 1.42 ml (day 0) to 14.1 ± 2.01 ml (day 3) (p < 0.05). This did not change the total lipid concentration (13.1 ± 1 .3 g/dl day 0 vs. 13.5 ± 1.4 gldl day 3; NS) (Table 2). Cholesterol saturation indices in gallbladder bile increased significantly by day 3, while molar ratios of bilirubin to bile salt and of bilirubin to RMC decreased (Table 2). A small, but significant, degree of deconjugation of bilirubin was observed between day 0 and day 3 (Table 3). The pattern of conjugates present in the pellet was identical to that found in the supernatant bile. No change in intraluminal gallbladder pressure was observed (day 0: 3.2 ± 1.4 cmH 2 0; day 3 : 4.8 ± 1.6 cmH 2 0) . There was no gross evidence of cholecystitis. Histologic studies of the gallbladder wall revealed a minimal lymphocytic infiltrate and rare goblet cells; considerable PAS-positive material was present on the luminal surface. Five cultures of day 3 bile contained trace amounts of Staphylococcus aureus, probably resulting from contamination by proximity to the freshly reopened abdominal wound; no effect on bile chemistry was noted. A considerable amount of semisolid mucoid material was adherent to the gallbladder mucosa. It was quite viscous, resisted suspension in aqueous media by sonication, and was not included in the calculations of total gallbladder content.
Discussion The formation of pigment stones and gravel in the gallbladder within 3 days of ligation of the cystic duct has not been previously reported. The novelty of this observation is emphasized by the fact that stones or gravel were evident in all 9 animals whose mural layer of dense mucus was systematically examined. Bile in each case was apparently nonlithogenic (with regard to cholesterol) and sterile, and contained no other apparent impetus to stone formation. Pigment stone formation was associated with a
marked increase in total content and concentration of mucin in the gallbladder bile as estimated by Peterson's modification of Lowry's technique (13) . An exact assay for mucin does not exist. Techniques such as Alcian blue, which measure mucin hexoses , may substantially overestimate mucin content in bile because these techniques also measure other nonmucin hexoses. By contrast, our measurements of protein content correlated closely with pellet weight. Mucin was present in suspension (as sludge), in solution (as dissolved mucin), and as a semisolid precipitate densely adherent to the gallbladder mucosa. Mucin is probably also the main component of the solid particles. Although the solid particles resisted resuspension, their microscopic appearance was similar to that of sludge. We detected little stimulus to stone formation in these animals other than the accumulation of mucin. Cholesterol saturation was low; it increased slightly during the 3 days that the cystic duct was ligated (from 0.08 to 0.12)' but dog bile is typically low in cholesterol content, and cholesterol stones would not be expected to form with saturation levels in this range. The bilirubin concentration of supernatant bile was low, and the bilirubin consisted almost entirely of water-soluble conjugates (Table 3). Enough bilirubin was present in sludge, however, to confer pigmentation. The chemical factors controlling bilirubin precipitation (and solubilization) remain partly obscure. It has been assumed that bilirubin (conjugated or unconjugated) is carried within micelles in a manner
Table 2. Supernatant Saturation Indices Day Bilirubinlbile salt ratio Cholesterol saturation Total lipids (g/dl) Residual micellar capacity (RMC) Bilirubin/RMC
a
Day 3
p
0.011 ::!: 0.001 0.08 ::!: 0.01 13.1 ::!: 1.3
0.006 ::!: 0.001 0.12 ::!: 0.01 13.5 ::!: 1.4
< 0.05 < 0.001 NS
6.3 ::!: 0.2 0.13 ::!: 0.01
6.2 ::!: 0.2 0.08 ::!: 0.02
NS < 0.001
n = 18. Mean::!: SEM. NS = not s ignificant.
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GASTROENTEROLOGY Vol. 85, No. 5
BERNHOFT ET AL.
Table 3. Bilirubin Conjugation Day 0
Day 3
p
Supernatant bile Unconjugated bilirubin Monoconjugated bilirubin Diconjugated bilirubin
0.6 ± 0.1 13 ± 0.8 87 ± 0.7
4.3 ± 0.3 22 ± 2 74 ± 2
<0.005 <0.025 <0.01
Pellet Unconjugated bilirubin Monoconjugated bilirubin Diconjugated bilirubin
NP NP NP
8 ± 1.5 16 ± 5 76 ± 6
NS" NS" NS"
n = 18. Mean percent ± SEM. NP tant.
=
no pellet. " Versus superna-
analogous to that of cholesterol (17). Recently, Wosiewitz (18) has proposed that bilirubin is adsorbed to bile salt at the surface of the micelle. The bilirubinlbile salt ratio, cholesterol saturation, the concentration of divalent cations in bile, and bile pH are also believed to playa role in bilirubin solubility in bile (19,20). In our dogs, bilirubinlbile salt ratios were low at day 0, and were even lower at day 3. Given the apparent unsaturation of micelles in these bile specimens and the minimal degree of deconjugation of bilirubin, it would seem unlikely that bilirubin was present in the solid particles as the result of a primary abnormality in bilirubin solubility. Since mucin accumulation was the most striking change associated with stone formation in these animals, it is reasonable to postulate that the bilirubin was sequestered by the mucin. Mucins are high molecular weight molecules composed of many sequences of amino acids and sugars. They behave in many respects like polyelectrolyte resins, and can sequester other molecules (particularly lipids) within prosthetic groups (21). The relative amounts of biliary lipids present within sludge and stones may, therefore, be governed by the relative affinities of each for mucin and the relative concentrations of each within bile. If sequestration of bilirubin by mucin is the primary event in pigment gallstone formation, this would explain the failure of previous investigators to find bilirubin supersaturation in bile from patients with pigment stones. This might also explain the observation of Been and Lewis (8) that bilirubin in the core of cholesterol gallstones is always complexed with mucoproteins. In 1856, Meckel von Hemsbach (22) first proposed that gallstone formation occurs as a result of the accumulation of gallbladder mucin. More recently, Womack and others (23,24) argued that gallstones form by heterogenous nucleation of bile solids (cholesterol, bilirubin, or calcium salts) on a macroscopic lattice of mucin. Hulten (25) demonstrated cholesterol crystallization within globules of gallbladder mu-
cin, and Carey (26) showed that mucin secretion in prairie dog gallbladders is increased by exposure to lithogenic bile, and that suppression of mucin secretion with aspirin prevents gallstone formation. The sequence in stone formation in this model may begin with accumulation of mucin within the gallbladder. Mucin particles may form that sequester bilirubin, cholesterol, and calcium salts, yet initially remain suspended as sludge, As bile mucin content increases, some of these particles coalesce, precipitate, and adhere to the mucosa as a semisolid gel, which becomes increasingly viscous with time, eventually forming gravel and stones. All these stages coexisted in our dog gallbladders. They differed visually and microscopically only in viscosity; as yet, the technical difficulty of handling dense gallbladder mucin precludes quantitative proof of these assertions. The stimulus to mucin secretion in our animals is unclear. The bile was not saturated with cholesterol and the gallbladder was free of inflammation. Ligation of the cystic duct could be a factor, although it had no measurable effect on intraluminal pressure or vascular integrity. Perhaps the explanation is accumulation of mucin secreted at a normal rate. In summary, pigment gallstones and gravel formed within 3 days of ligation of the cystic duct in normal dogs in the absence of elevated concentrations of cholesterol or bilirubin. The stones appear grossly like human pigment stones and microscopically like condensed biliary sludge. The principal abnormality detected was the presence of large quantities of mucin. We suspect that stone formation in this setting, and perhaps in humans, occurs because of sequestration of biliary lipids by mucin.
References 1. Pikula JV, Dunphy JE. Some effects of stenosis of the terminal
common bile duct on the biliary tract and liver. N Engl I Med 1959;260:315-8.
2. Imamoglu K. Perry JF Ir, Wangensteen OH. Experimental production of gallstones by incomplete stricture of the terminal common bile duct. Surgery 1957;42:623-30. 3. Soloway RD, Trotman BW, Ostrow JD. Pigment gallstones. Gastroenterology 1977;72:167-82. 4. Tompkins RK, Kraft AR, Zimmerman E, et al. Clinical and biochemical evidence of increased gallstone formation after complete vagotomy. Surgery 1972;71:196-200. 5. Allen BA, Bernhoft RA, Svanvik BI, et al. Gallbladder sludge is calcium bilirubinate associated with biliary stasis. Am I Surg 1981;141:51-6. 6. Bernhoft RA, Allen BA, Pellergrini CA, et al. Characterization of sludge containing gallbladder bile (abstr). Gastroenterology 1980;78:1301.
7. Englert E Jr, Harman CG, Freston IW, et al. Studies on the pathogenesis of diet-induced dog gallstones. I Dig Dis 1977;22:305-14.
8. Been JM, Lewis D. Microstructure of gallstones. Gastroenterology 1979;76:548-55.
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9. Abell LL, Levy BB, Brodie BB, et al. A simplified method for estimation of total cholesterol in serum and demonstration of its specificity. J Bioi Chern 1952;195:357-69. 10. Talalay P. Enzymatic analysis of steroid hormones. Methods Biochem Anal 1960;8:119-26. 11. Fiske CH, Subbarow Y. The colorimetric determination of phosphorus. J Bioi Chern 1925;66:375-400. 12. Hogg CK, Meites S. A modification of the Malloy and Evelyn procedure for the micro-determination of total serum bilirubin. Am J Med TechnoI1959;25:281-6. 13. Peterson GL. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Ann Biochern 1977;83 :346-56. 14. Blanckaert N. Analysis of bilirubin and bilirubin mono- and di-conjugates. Biochem J 1980;185:115-28. 15. Carey Me. Critical tables for calculating the cholesterol saturation of native bile. J Lipid Res 1978;19:945-55. 16. Juniper K, Burson EN. Biliary tract studies. II. The significance of biliary crystals. Gastroenterology 1957;32:175-211. 17. Scharschmidt BF, Schmid R. The micellar sink. J Ciininvest 1978;62:1122-31. 18. Wosiewitz U. Common duct pigment stone and gallbladder pigment stones: a comparative morphological and analytical
PIGMENT STONE FORMA nON AND BILlARY STASIS
19.
20.
21. 22. 23. 24.
25. 26.
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investigation. In Falk Foundation, eds. Hepatology rapid literature reviews. Vol 11. Falk Foundation, 1981:1650-2. Ostrow JD, Devers TJ, Gallo D. Determinants of the solubility of unconjugated bilirubin in bile. In: Berk PD, Berlin Nl, eds. Chemistry and physiology of bile pigments. Chap 41. Washington, D.C.: National Institutes of Health, 1977:404-9. Nakama T, Furasawa T, Itoh H, et al. Correlation of cholesterol and bilirubin solubilization in bile salt solution. Gastroenterol Jpn 1979;14:565-72. Jacques LB. The nature of mucoploysaccharides. Med Hypotheses 1978;4:123-35. Meckel von Hemsbach JH. Mikrogeologie. Ueber die Concrement in thierischen Organismus. Berlin; Reimer, 1856. Womack NA, Zeppa R, Irvin GL III. The anatomy of gallstones. Ann Surg 1963;157:670-86. Maki T, Matsushiro T, Suzuki N, et al. Role of sulfated glycoprotein in gallstone formation. Surg Gynecol Obstet 1971; 132:846-54. Hulten O. Formation of gallstones I and II. Acta Chir Scand 1968;134:125-30, 557-61. Lee SP, Carey MC, LaMont JT. Aspirin prevention of cholesterol gallstone formation in prairie dogs. Science 1981; 211:1429-31.
Pigment Sludge and Stone Formation in the Acutely Ligated Dog Gallbladder R. A. BERNHOFT, C. A. PELLEGRINI, W. C. BRODERICK, and L. W. WAY
Veterans Administration Medical Center and the Department of Surgery, Univers ity of California, San Francisco, California
We studied the effects of stasis of gallbladder bile in a dog model. Three days after cystic duct ligation, all gallbladders contained sludge, and the mucosa was covered by densely adherent mucus with solid particles 1-4 mm in diameter (gravel). Thirty percent of the animals developed stones (>4 mm), which appeared grossly like human pigment stones and microscopically like condensed biliary sludge. Centrifugation of bile yielded colorless pellets (3.B ± 3.2 mg/ml) at day a and pigmented pellets (33.1 ± 11.0 mg/ml) at day 3 (p < 0.05). Pellets contained 73 ± B% mucin by weight. Dissolved mucin in supernatant bile increased from 7.46 ± 1.19 mg/ml (day 0) to 27.36 ± 3.05 mg/ml (day 3) (p < 0.001), while bilirubin concentration decreased from 127 ± 12 mg/dl (day 0) to 71 ± 16 mg/dl (day 3) (p < 0.001). Cholesterol concentration increased but did not reach saturation, while the concentration of bile salt and phospholipid did not change. Mucin-bilirubin complexes formed and remained suspended as sludge initially. As bile mucin content increased , sludge particles coalesced , precipitated, and eventually formed gravel and stones. We suspect that stone formation in this setting occurs because of sequestration of biliary lipids by mucin. Biliary stasis has been shown to induce pigment stone formation in animals (1,2) and in patients with biliary stricture (3). Gallbladder stasis may be a factor in cholesterol gallstone formation after vagotomy (4). We have previously reported that gallbladder Received December 24, 1982. Accepted May 12, 1983. Address requests for reprints to : Carlos A. Pellegrini , M.D., Surgical Service (112), Veterans Administration Medical Center, 4150 Clement Street, San Francisco, California 94121. This work was supported by th e Veterans Administration and by a grant (MSC 72) from the University of California School of Medicine. The authors thank Anny O. Wong, K. Warren Hayashi, and James R. Gorring for technical ass istance. © 1983 by the American Gastroenterological Association 0016-5085/83/$3.00
stasis leads to biliary sludge formation in humans (5) and dogs (6). In related work, Englert et a1. (7) noted that dogs fed a high-cholesterol diet developed sludgelike material in their gallbladders, which eventually evolved into pigment stones. We have postulated that biliary sludge may provide the nidus necessary to initiate stone formation (5,6), because biliary sludge is similar to the material found at the core of pigment and cholesterol stones (8). Accordingly, accumulation of mucin, which is the main component of sludge, may be a primary event in the pathogenesis of gallstones. Since the gallbladder mucosa is the source of most of the mucin in bile, we developed a model of gallbladder stasis in the dog, which allowed us to study the mechanics of pigment stone formation.
Materials and Methods Twenty-four healthy mongrel dogs of both sexes were acclimated to the kennel. After an overnight fast, each dog underwent laparotomy ("day 0") under general anesthesia (Nembutal, Abbott Pharmaceuticals, North Chicago. Ill. . 10 mg/kg body wt) . The gallbladder and biliary tree were examined, and the cystic duct was ligated; the cystic artery was spared. The dome of the gallbladder was then punctured with a 2D-gauge Teflon catheter. Bile was allowed to flow back into the Teflon catheter until all air was eliminated. The catheter was then connected to a saline-filled tube connected to a strain gauge and a physiograph (Beckman R-611 Dynograph recorder, Beckman Instruments. Palo Alto, Calif.). Pressure within the gallbladder was measured in centimeters of water (cmH 2 0). Movement of bile out of the gallbladder into the cannula used for pressure measurements was minimal. Gallbladder bile was then completely aspirated. its volume was recorded. and a 3-ml aliquot was collected for study. The remainder of the bile was returned to the gallbladder. pressure was measured again. the Teflon catheter was removed, and the puncture site was closed with a Z-plasty of 6-0 silk. The laparotomy incision was closed. and the dog was returned to its kennel. Sterile technique was observed throughout the operation.
November 1983
The dogs were fed a liquid meal the evening of the operation and were given regular kennel dog food (Wayne Pro Mix Dog Food, Allied Mill s, Inc" Chicago, IlL) for the next 2 days, After an overnight fas t. each dog underwent a second laparotomy on the third day after ligation of the cystic duct ("day 3"), The gallbladder and biliary tree were inspected, the dome of the ga llbladde r was punctured , and meas urements of pressure and volume were performed as before, After the gallbladder was emptied , it was removed, opened, examined, photograp hed , and fixed in formalin , and the dog was killed , Bile specimens were treated identically, with immed iate separation of aliquots for aerobic an d anerobic culture and for light and polarized microscopy. The remainder of the bile was placed in nitrogen-filled sterile tubes in ice water (4°C), which were stored in the dark. Within 2 h of co ll ection , the specimens were centrifuged at 4500 rpm (7120 g) for 30 min at 4°C (Sorvall General Purpose RC-3 Automatic Refrigerated Centrifuge) . After centrifugation , the bile supernatant and pellet were separated. The supernatant volume was recorded , and aliquots were frozen at -60°C until assayed; aliquots of 500 JLl were frozen for cho lestero l assay (9). Twentymicroliter aliquots were collected for assay of bile salt (10), phospholipid (11), and bilirubin concentration (12). Mucin content was estimated by Peterson's modification of the Lowry protein assay (1 3), including decolorization of the pellet with hydrogen peroxide. The pellet , which microscopically looked like sludge, was washed three times w ith sterile water, weighed , and was then resuspended by sonication in a 1: 5 dilution with normal saline. The aliquots were coll ected for assay and were frozen in the same manner as described for the su pernatant. Fresh aliquots «2 h after removal from the gallbladder) of supernatant bile and pellet (200- 400 JLl) that had been kept at 4°C were used for measurement of bilirubin conjugates by thin-layer chromatograph y [14). Cholesterol saturation indi ces were calculated from the tables published by Carey (15). Residual micellar capacity (RMC , moles per deciliter) was defined as the arithmetic difference between chol esterol concentration at the point of theoretical micellar saturation and actual cholesterol concentration for each specimen. After the gallbladder was fixed in formalin, sections were taken through the dome, body, and neck for hematoxylin an d eosin (H&E) and periodic acid-Schiff (PAS) stains. The sections were examin ed for inflammatory infiltrate and for intestinal metaplasia of the ep ithelium. Data from 6 dogs were discarded-two because of unligated accessory cystic ducts , two for mucosal bleeding, and two for leakage from the asp iration site. Each animal was used as its own controL Data from day o were compared with data from day 3 using paired t-tests. Values are expressed as mean::!: SEM.
PIGMENT STONE FORMATION AND BILIARY STASIS
11 67
cally identical to biliary sludge as previously described (5). The gallbladder mucosa was covered by a layer of firmly adherent mucus. When the mucus was rinsed off the mucosa and examined (a procedure performed in 9 animals), it was seen to contain multiple solid particles of different sizes. Solid particles with a diameter > 4 mm will be referred to as stones, and those with a diameter between 1 and 4 mm will be referred to as grave /. Stones plus gravel were found in 3 of the 9 animals (Figure 1), and gravel alone was found in the remaining 6 animals. Wh en cru shed and examined microscopically, the solid particles contained granular pigmented material similar to sludge peripherally (Figure 2A), coalescing to homogenous sheets centrally (Figure 2B). Day 3 bile contained visible solid particles , which mi croscopically were identical to " bilirubinate granules" (16) and to what we have termed biliary sludge in previous reports (5 ,6) . No cholesterol crystals were present. No solid particles were visible in any day 0 bile specimen. Centrifugation of day 3 bile aspirates yielded a pigmented pellet of mean weight 33.1 ± 11.0 mg/ml spun bile. Centrifugation of day 0 bile yielded colorless pellets of 3.8 ± 3.2 mg/ml spun bile (p < 0.05) . Day 3 sludge pellet contents were as follows: mucin 73 ± 8%, bilirubin 0.9 ± 0.6%, cholesterol 2 ± 1%, bile salts 21 ± 7%, and phospholipids 3 ± 2% . The day 0 pellets were too small to assay. Changes in the concentrations of the constituents of supernatant bile and the mean total content per gallbladder for each constituent are listed in Table 1. Concentration and total gallbladder content of dissolved mucin increased, and bilirubin concentration and content decreas ed. Cholesterol concentration increased, but total chol esterol content did not change. Concentration and total co ntent of bile salt and phospholipid did not change.
Results At day 3, all 18 gallbladders contained copious amounts of viscous , black material, microscopi-
Figure 1. Opened gallbladder specimen 3 days after cystic duct ligation. A 5 x 6mm - pigment stone is present.
A
B
Figure 2. Photomicrograph of the periphery (A) and the center (B) of a crushed pigment stone formed in a dog gallbladder after ligation of the cystic duct (x400).
PIGMENT STONE FORMATION AND BILIARY STASIS
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1169
Table 1. Composition of Supernatant Bile in 18 Dogs Total gallbladder content (mg)
Bile concentration (mg/ml)
Day Mucin Bilirubin Cholesterol Phospholipid Bile salt Different from controls:
7.46:!: 1.27 ::!: 0.62 ::!: 44 .68 ::!: 85.47 ::!: a
a 1.19 0.12 0.04 2.23 3.65
Day a
Day 3 27.36 0.71 0.91 47.29 86.83
3.05" 0.16" ::!: 0.05" ::!: 2.85 ::!: 3.14 ::!: ::!:
106 18 11 678 1211
Day 3
18 2 ::!: 1 ::!: 78 ::!: 71
271 6 13 706 1207
::!: ::!:
::!: 57 b
1" 2 ::!: 91 ::!: 115
::!: ::!:
p < 0.001 ; b p < 0.005.
Some concentration of bile probably occurred, as suggested by a slight decrease in mean volume of gallbladder bile from 16.3 ± 1.42 ml (day 0) to 14.1 ± 2.01 ml (day 3) (p < 0.05). This did not change the total lipid concentration (13.1 ± 1 .3 g/dl day 0 vs. 13.5 ± 1.4 gldl day 3; NS) (Table 2). Cholesterol saturation indices in gallbladder bile increased significantly by day 3, while molar ratios of bilirubin to bile salt and of bilirubin to RMC decreased (Table 2). A small, but significant, degree of deconjugation of bilirubin was observed between day 0 and day 3 (Table 3). The pattern of conjugates present in the pellet was identical to that found in the supernatant bile. No change in intraluminal gallbladder pressure was observed (day 0: 3.2 ± 1.4 cmH 2 0; day 3 : 4.8 ± 1.6 cmH 2 0) . There was no gross evidence of cholecystitis. Histologic studies of the gallbladder wall revealed a minimal lymphocytic infiltrate and rare goblet cells; considerable PAS-positive material was present on the luminal surface. Five cultures of day 3 bile contained trace amounts of Staphylococcus aureus, probably resulting from contamination by proximity to the freshly reopened abdominal wound; no effect on bile chemistry was noted. A considerable amount of semisolid mucoid material was adherent to the gallbladder mucosa. It was quite viscous, resisted suspension in aqueous media by sonication, and was not included in the calculations of total gallbladder content.
Discussion The formation of pigment stones and gravel in the gallbladder within 3 days of ligation of the cystic duct has not been previously reported. The novelty of this observation is emphasized by the fact that stones or gravel were evident in all 9 animals whose mural layer of dense mucus was systematically examined. Bile in each case was apparently nonlithogenic (with regard to cholesterol) and sterile, and contained no other apparent impetus to stone formation. Pigment stone formation was associated with a
marked increase in total content and concentration of mucin in the gallbladder bile as estimated by Peterson's modification of Lowry's technique (13) . An exact assay for mucin does not exist. Techniques such as Alcian blue, which measure mucin hexoses , may substantially overestimate mucin content in bile because these techniques also measure other nonmucin hexoses. By contrast, our measurements of protein content correlated closely with pellet weight. Mucin was present in suspension (as sludge), in solution (as dissolved mucin), and as a semisolid precipitate densely adherent to the gallbladder mucosa. Mucin is probably also the main component of the solid particles. Although the solid particles resisted resuspension, their microscopic appearance was similar to that of sludge. We detected little stimulus to stone formation in these animals other than the accumulation of mucin. Cholesterol saturation was low; it increased slightly during the 3 days that the cystic duct was ligated (from 0.08 to 0.12)' but dog bile is typically low in cholesterol content, and cholesterol stones would not be expected to form with saturation levels in this range. The bilirubin concentration of supernatant bile was low, and the bilirubin consisted almost entirely of water-soluble conjugates (Table 3). Enough bilirubin was present in sludge, however, to confer pigmentation. The chemical factors controlling bilirubin precipitation (and solubilization) remain partly obscure. It has been assumed that bilirubin (conjugated or unconjugated) is carried within micelles in a manner
Table 2. Supernatant Saturation Indices Day Bilirubinlbile salt ratio Cholesterol saturation Total lipids (g/dl) Residual micellar capacity (RMC) Bilirubin/RMC
a
Day 3
p
0.011 ::!: 0.001 0.08 ::!: 0.01 13.1 ::!: 1.3
0.006 ::!: 0.001 0.12 ::!: 0.01 13.5 ::!: 1.4
< 0.05 < 0.001 NS
6.3 ::!: 0.2 0.13 ::!: 0.01
6.2 ::!: 0.2 0.08 ::!: 0.02
NS < 0.001
n = 18. Mean::!: SEM. NS = not s ignificant.
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GASTROENTEROLOGY Vol. 85, No. 5
BERNHOFT ET AL.
Table 3. Bilirubin Conjugation Day 0
Day 3
p
Supernatant bile Unconjugated bilirubin Monoconjugated bilirubin Diconjugated bilirubin
0.6 ± 0.1 13 ± 0.8 87 ± 0.7
4.3 ± 0.3 22 ± 2 74 ± 2
<0.005 <0.025 <0.01
Pellet Unconjugated bilirubin Monoconjugated bilirubin Diconjugated bilirubin
NP NP NP
8 ± 1.5 16 ± 5 76 ± 6
NS" NS" NS"
n = 18. Mean percent ± SEM. NP tant.
=
no pellet. " Versus superna-
analogous to that of cholesterol (17). Recently, Wosiewitz (18) has proposed that bilirubin is adsorbed to bile salt at the surface of the micelle. The bilirubinlbile salt ratio, cholesterol saturation, the concentration of divalent cations in bile, and bile pH are also believed to playa role in bilirubin solubility in bile (19,20). In our dogs, bilirubinlbile salt ratios were low at day 0, and were even lower at day 3. Given the apparent unsaturation of micelles in these bile specimens and the minimal degree of deconjugation of bilirubin, it would seem unlikely that bilirubin was present in the solid particles as the result of a primary abnormality in bilirubin solubility. Since mucin accumulation was the most striking change associated with stone formation in these animals, it is reasonable to postulate that the bilirubin was sequestered by the mucin. Mucins are high molecular weight molecules composed of many sequences of amino acids and sugars. They behave in many respects like polyelectrolyte resins, and can sequester other molecules (particularly lipids) within prosthetic groups (21). The relative amounts of biliary lipids present within sludge and stones may, therefore, be governed by the relative affinities of each for mucin and the relative concentrations of each within bile. If sequestration of bilirubin by mucin is the primary event in pigment gallstone formation, this would explain the failure of previous investigators to find bilirubin supersaturation in bile from patients with pigment stones. This might also explain the observation of Been and Lewis (8) that bilirubin in the core of cholesterol gallstones is always complexed with mucoproteins. In 1856, Meckel von Hemsbach (22) first proposed that gallstone formation occurs as a result of the accumulation of gallbladder mucin. More recently, Womack and others (23,24) argued that gallstones form by heterogenous nucleation of bile solids (cholesterol, bilirubin, or calcium salts) on a macroscopic lattice of mucin. Hulten (25) demonstrated cholesterol crystallization within globules of gallbladder mu-
cin, and Carey (26) showed that mucin secretion in prairie dog gallbladders is increased by exposure to lithogenic bile, and that suppression of mucin secretion with aspirin prevents gallstone formation. The sequence in stone formation in this model may begin with accumulation of mucin within the gallbladder. Mucin particles may form that sequester bilirubin, cholesterol, and calcium salts, yet initially remain suspended as sludge, As bile mucin content increases, some of these particles coalesce, precipitate, and adhere to the mucosa as a semisolid gel, which becomes increasingly viscous with time, eventually forming gravel and stones. All these stages coexisted in our dog gallbladders. They differed visually and microscopically only in viscosity; as yet, the technical difficulty of handling dense gallbladder mucin precludes quantitative proof of these assertions. The stimulus to mucin secretion in our animals is unclear. The bile was not saturated with cholesterol and the gallbladder was free of inflammation. Ligation of the cystic duct could be a factor, although it had no measurable effect on intraluminal pressure or vascular integrity. Perhaps the explanation is accumulation of mucin secreted at a normal rate. In summary, pigment gallstones and gravel formed within 3 days of ligation of the cystic duct in normal dogs in the absence of elevated concentrations of cholesterol or bilirubin. The stones appear grossly like human pigment stones and microscopically like condensed biliary sludge. The principal abnormality detected was the presence of large quantities of mucin. We suspect that stone formation in this setting, and perhaps in humans, occurs because of sequestration of biliary lipids by mucin.
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