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Sustained gallbladder stasis promotes cholesterol gallstone formation in the ground squirrel
Sustained Gallbladder Stasis Promotes Cholesterol Gallstone Formation in the Ground Squirrel QI-WEI XU, MICHE`LE MANTLE, JU¨RGEN G. PAULETZKI,
Although gallbladder stasis exists in most patients with cholesterol gallstones, it is unknown whether stasis is a causative factor of gallstone disease or merely a consequence of it. We studied the impact of sustained gallbladder stasis induced by a cholecystokinin (CCK)-A receptor antagonist (MK329) on gallstone formation in ground squirrels fed either a trace or a high-cholesterol diet. MK-329 markedly inhibited gallbladder contraction in vitro in response to CCK (at EC100 , control: 3.6 { 0.5 vs. MK-329: 1.1 { 0.3 g; P õ .05) and increased gallbladder fasting volume in vivo (control: 462 { 66 vs. MK-329: 1,004 { 121 mL; P õ .05). Whereas the high-cholesterol diet alone (1%-cholesterol diet / placebo) increased the cholesterol saturation index (CSI) in control animals (trace-cholesterol diet / placebo), MK-329 significantly (P õ .05) decreased the CSI in both hepatic and gallbladder bile in animals on the trace-(trace-cholesterol diet / MK-329) as well as on the high-cholesterol diets (1%-cholesterol diet / MK-329). The mucin content of the mucus layer on the epithelial surface of the gallbladder wall more than doubled (P õ .05) with the high-cholesterol diet; adding MK329 to the latter group produced a further 82% increase (P õ .05). The cholesterol diet / MK-329 group had the highest (100%) incidence of cholesterol crystals that were evident in fresh gallbladder bile, coincident with a shortened nucleation time (2.5 { 0.6 days; P õ .05 vs. the cholesterol diet / placebo group, 5.8 { 1.0 days or the other 2 groups, ú21 days). Bile from animals on the trace-cholesterol diet, whether or not receiving MK-329, lacked crystals in bile and exhibited a normal nucleation time (ú21 days). Thus, stasis per se may lower the CSI, but its detrimental effect on the gallbladder predominates locally, and so accelerates cholesterol crystal formation in this model. (HEPATOLOGY 1997;26:831-836.) Gallbladder motility defects are present in most patients with cholesterol gallstones, yet the sequential events are unclear and the question remains whether gallbladder stasis is a causative factor of gallstone disease or merely a conse-
Abbreviations: CCK, cholecystokinin; CSI, cholesterol saturation index; PAS, periodic acid–Schiff. From the GI Research Group, Department of Medicine, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada. Received January 21, 1997; accepted June 11, 1997. Supported by a Medical Research Council of Canada grant (MT 69-1945). Dr. Xu was supported by a Canadian Liver Foundation Research Fellowship, and Dr. Pauletzki by an Alberta Heritage Foundation for Medical Research Clinical Fellowship. Address reprint requests to: Eldon A. Shaffer, M.D., Foothills Hospital, Department of Medicine, 1403 29th St. N.W., Calgary, Alberta T2N 2T9 Canada. Fax: (403) 6701095. Copyright q 1997 by the American Association for the Study of Liver Diseases. 0270-9139/97/2604-0004$3.00/0
AND
ELDON A. SHAFFER
quence of it.1-5 Identifying events in a chronological fashion is particularly difficult in humans before gallstone formation.5 Defective gallbladder emptying likely occurs before stone formation, followed by the appearance of cholesterol crystals.1,6,7 In one report on patients with cholesterol crystals alone (without gallstones), gallbladder emptying was impaired.6 Unfortunately, all the patients in this study had chronic cholecystitis, a confounding factor, which in itself could have adversely affected gallbladder motor function.1,5,8 Ground squirrels fed a high-cholesterol diet form cholesterol crystals and/or gallstones within a reasonably short period.9-13 Defective gallbladder contractility develops very early in these animals, concurrent with a rise in cholesterol saturation of bile and the appearance of cholesterol crystals, but before stone formation. A major limitation in this model is the inability to assess directly the primary role of gallbladder hypomotility in the process of gallstone formation because of the concurrent changes in both hepatic bile formation and gallbladder motility.13-14 Our previous study in the same animal model showed that a cholecystokinin (CCK)-A receptor antagonist (MK-329) suppressed gallbladder emptying, but reduced the cholesterol saturation in both hepatic and gallbladder bile.14 This apparent paradox challenges the traditional concept that gallbladder stasis is a primary and/or contributing factor in cholesterol gallstone formation.1-3,15-18 Gallbladder stasis is thought to facilitate nucleation; the retained precipitated microcrystals then would agglomerate into cholesterol gallstones in humans.1,5,17-19 We therefore hypothesize that, despite an improved cholesterol saturation of bile in certain circumstances,14,20-22 sustained gallbladder stasis may have a dominantly local effect in the gallbladder, i.e., in the presence of supersaturated bile, gallbladder stasis may cause an increased production and/or accumulation of mucin within the gallbladder, thereby providing both the residence time and the medium necessary for the nucleation of cholesterol crystals.17-19,23-28 To further explore the nature of gallbladder stasis in the whole process of cholesterol gallstone formation, we used a specific CCK-A receptor antagonist (MK-32914,29,30) to produce prolonged gallbladder hypomotility in ground squirrels on both a trace (standard) and a 1% high-cholesterol diet, while clarifying events within the gallbladder.9-14 MATERIALS AND METHODS Experimental Animals. Richardson ground squirrels (Spermophilus richardsoni) were trapped wild near Calgary, Alberta, Canada. They were males with an average body weight of 300 g. Animals were individually housed in thermoregulated rooms on a 12-hour day/ night light cycle with unlimited access to standard rat chow and water for a minimum of 1 month. One hundred twenty-eight experi-
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mental animals were then divided randomly into two dietary groups and maintained for an additional 4 weeks on either a control chow diet (n Å 64) with a trace (standard) cholesterol content (0.027%) (United States Biochemical, Cleveland, OH) or a high-cholesterol diet (n Å 64) comprised of an identical chow, enriched with 1% cholesterol by weight (United States Biochemical). Within each dietary group, two further subgroups (n Å 32 each) were created and treated with either a CCK-A receptor antagonist (MK-329, Merck Sharp & Dohme Research Laboratories, West Point, PA), 1 mg/kg three times per day, subcutaneously, prepared in a 4% dimethylsulfoxide/saline solution, or placebo (4% dimethylsulfoxide/saline). During the 4 weeks before the terminal experiments, all ground squirrels were allowed access to food and water ad libitum. This research protocol was approved by the Animal Care Committee of the University of Calgary. Experimental Design. The terminal experiments performed after a treatment period of 4 weeks had four objectives: 1) determine gallbladder contractility in vitro (n Å 8 each); 2) assess bile composition and cholesterol saturation (n Å 8 each); 3) examine the mucin content of gallbladder tissue and bile, as well as the adherent mucus layer at the mucosal surface of the gallbladder wall (n Å 8 each); and 4) measure the nucleation time of cholesterol crystals (n Å 16 each, including the bile samples from the contractility study). Gallbladder Contractility In Vitro. After a 16-hour fast, the cystic duct was exposed and ligated at laparotomy. The entire gallbladder was removed, and the gallbladder bile was aspirated as completely as possible, the volume measured, a drop examined under a polarizing microscope for cholesterol and liquid crystals, and the remainder stored at 0707C for later analysis. The preparation and setup for gallbladder contractility study in vitro have been described previously.12-13 Once each preparation exhibited a stable baseline tension, 1004 mol/L bethanechol was administered as a single bolus to verify its viability. Next, the gallbladder was washed thoroughly by changing the Krebs solution in the organ bath until the baseline tension was again attained. The tissue was then challenged with EC25 , EC50 , and EC100 of CCK-8. These concentrations were obtained from our preliminary dose-response curves, representing the concentrations required to produce the stated fraction (contractile response) of the maximum effect of CCK-8.12-14 Bile Collection and Analysis of Biliary Lipids. Hepatic bile collection by direct cannulation of the common bile duct was performed in terminal experiments following a specific dietary and/or treatment period as previously described.12-14 Bile samples were analyzed for total bile salts using the 3-a-hydroxysteroid dehydrogenase assay (Worthington Biochemical Company, Freehold, NJ), for phospholipid by an enzymatic assay for choline (Boehringer Mannheim, Mannheim, Germany) and for cholesterol by an enzymatic diagnostic kit (Ames Division, Miles Laboratories, Slough, UK). Serum cholesterol levels were also determined by this Ames cholesterol kit.12-14 The cholesterol saturation index (CSI) was determined using Carey’s critical tables for cholesterol solubility.31 Bile Collection and Extraction of Mucin. The intact gallbladder was carefully removed and the bile aspirated as completely as possible using a syringe. The bile volume was recorded. The empty gallbladder was carefully opened along one side and placed everted into 1 mL of TRIS-buffered saline (0.01 mol/L TRIS, 0.1 mol/L NaCI, 0.02% (wt/vol) NaN3 , pH 7.4) containing proteolytic inhibitor (1 mmol/L phenylmethanesulphonyl fluoride, 10 mmol/L N-ethylmaleimide, and 5 mmol/L Na2 ethylenediaminetetraacetic acid). After gentle vortexing at a low speed for 30 seconds to remove the adherent mucus layer on the epithelial surface,32 the gallbladder tissue was transferred into another 1-mL aliquot of the same buffer and homogenized for 60 seconds using a Polytron tissue solubilizer. Tissue homogenates and washings were stored at 0707C for later analysis. Samples of homogenate were later thawed and briefly homogenized. Aliquots were analyzed for DNA using the fluorometric method of Hinegardner.33 The mucin content of tissue homogenates
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and gallbladder washings was analyzed using a nitrocellulose slotblot assay stained with periodic acid–Schiff (PAS) reagent.32 Stained blots were scanned on a laser densitometer, and the amount of mucin present in each test sample was determined by comparing its peak area with standard curves obtained by applying purified rat intestinal mucin (0.3-25 mg) to the nitrocellulose blot. The mucin content of tissue homogenate and the mucus layer on the epithelial surface was expressed relative to the gallbladder DNA content and total recovery from the 1-mL tissue washings, respectively. For analysis of gallbladder bile, lipids were removed by chloroform:methanol extraction (2:1), and the residual material was solubilized in 1 mL of 2% (wt/vol) sodium dodecyl sulfate containing 0.2 mol/L 2-mercaptoethanol. Samples were boiled for 5 minutes, cooled to room temperature (237C), and applied to a Biogel P100 mini-column (0.4 1 10 cm) eluted with 2% sodium dodecyl sulfate to separate proteins and bile pigments (included) from mucin (excluded). The mucin-containing void volume fractions were collected, lyophilized, and sodium dodecyl sulfate was removed using anhydrous acetic:water (17:1:1:1, vol/vol/vol/vol). Samples were resolubilized in Tris-buffered saline to their original volume and analyzed for mucin by the PAS slotblot assay, expressed as micrograms of mucin per milliliter of bile. The recovery of mucin using this procedure was assessed in separate experiments in which a known amount of purified rat intestinal mucin was added to bile samples before processing. A recovery of ú90% was consistently achieved. To confirm that our assay was only detecting mucin in the above experiments, random samples of tissue homogenates and washings were tested by chromatography on a Sepharose CL-4B column and fractions were analyzed by the PAS assay.32 Only the void volume fractions, where mucin would be expected to elute, stained positively with PAS reagent. Thus, the changes in mucin content could not be attributed to the production of other PAS-positive glycoproteins during the course of cholesterol feeding and MK-329 treatment. Cholesterol Nucleation Time. Upon opening the abdomen, bile was aspirated from the gallbladder using a sterile needle and syringe. Particular care was taken to obtain a complete aspiration of the gallbladder bile, to avoid the interference of stratification.19,34 One drop was immediately examined by polarized light microscopy for cholesterol crystals, identified as birefringent, notched rhomboidal plates.19 The remaining bile was stored momentarily in sterile plastic syringes in the dark at 377C with all air expressed from the syringes. To determine the rate of de novo cholesterol microcrystal formation, isotropic (crystal-free) fractions of bile were processed as described by Holan et al.19 Throughout the course of each nucleation study, bile samples were constantly maintained at 377C. All instruments were sterile and prewarmed to 377C before manipulation of bile. Time zero of nucleation was defined as 1 hour after a thermal equilibration. At time zero and at daily intervals thereafter, 4 mL of each sample was examined with a polarizing microscopy to detect the earliest formation of cholesterol monohydrate. The interval between time zero and the first appearance of solid cholesterol crystals was designated the nucleation time (i.e., crystal observation time17,35). The nucleation time study was performed for 21 days, and those bile samples in which solid cholesterol crystals were not detected by the end of 21 days were classified as having no nucleation.19,34 Solutions and Drugs. The modified Krebs solution used in these experiments contained: 103 mmol/L NaCl, 4.7 mmol/L KCl, 5.6 mmol/L CaCl2 , 1.13 mmol/L MgCl2 , 25 mmol/L NaHCO3 , 1.15 mmol/L NaH2PO4 , 2.8 mmol/L D-glucose, 4.9 mmol/L Na pyruvate, 2.7 mmol/L Na fumarate, and 4.9 mmol/L Na glutamate. The pH was adjusted to 7.4. The CCK-A antagonist, MK-329, was kindly provided by Merck Sharp & Dohme Research Laboratories. CCK octapeptide was purchased from Peptide Institute, Inc. (Osaka, Japan), [14C]-cholic acid from New England Nuclear (Boston, MA), and halothane from Ayerst Laboratories (Montreal, Quebec, Canada). Statistics. Data are expressed as mean { SE. Means of multiple
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TABLE 1. Animal Characteristics and Effect of High-Cholesterol Diet and MK-329 on Gallbladder Volume and CSI Trace Diet Placebo (n Å 16)
Body weight (g) Serum cholesterol (mmol/L) Gallbladder volume (mL) Hepatic bile CSI Gallbladder bile CSI
403 5.8 462 98 79
{ { { { {
1%-Cholesterol Diet MK-329 (n Å 16)
28 0.6 66 6 4
423 5.2 1004 75 61
{ { { { {
27 0.7 121* 5* 5*
Placebo (n Å 16)
464 14.1 526 149 137
{ { { { {
35 1.8*,† 57† 11*,† 11*,†
MK-329 (n Å 16)
454 13.1 1242 109 110
{ { { { {
40 1.9*,† 169*,‡ 8†,‡ 6*,†,‡
NOTE. Data are means { SE. * P õ .05, compared with animals on the trace diet / placebo regime. † P õ .05, compared with animals on the trace diet / MK-329 regime. ‡ P õ .05, compared with animals on the high-cholesterol diet / placebo regime.
groups were evaluated by ANOVA. All calculations were performed using the computer program SYSTAT (SYSTAT Inc., Evanston, IL). Statistical values reaching probabilities of P õ .05 were considered significant. RESULTS
Animals appeared healthy with a shining coat and normal activity. No diarrhea was observed in any of the treatment regimes. In 1% cholesterol-fed animals, whether they received MK-329 or placebo, serum cholesterol levels were significantly elevated (P õ .05) compared with their respective controls on the trace cholesterol diet (Table 1). By conventional histological criteria, both liver and gallbladder histology were considered normal after either the high-cholesterol diet or MK-329 treatment. There was no evidence of inflammatory or fatty changes in these tissues. The 1% high-cholesterol diet did not significantly alter the gallbladder volumes measured after an overnight fast. Treatment with MK-329, however, markedly increased the gallbladder volumes in animals on both the trace and 1% high-cholesterol diets (Table 1). After 4 weeks of treatment, MK-329 significantly (P õ .05) suppressed gallbladder contractile responses to CCK in vitro in animals on both diets. The inhibitory effect on gallbladder contraction occurred at all CCK concentrations tested. This was more evident in animals on the 1% high-cholesterol diet (Table 2). In addition, the 1% high-cholesterol diet in itself (cholesterol plus placebo) moderately but significantly (P õ .05) inhibited CCK-induced gallbladder contraction at EC100 , thereby aggravating the effect of MK-329. The 1% high-cholesterol diet markedly increased the cholesterol saturation in both hepatic and gallbladder bile (P õ
TABLE 2. Effect of High-Cholesterol Diet and MK-329 on Gallbladder Contractility In Vitro Trace Diet
EC25 (g) EC50 (g) EC100 (g)
1%-Cholesterol Diet
Placebo
MK-329
Placebo
MK-329
0.9 { .07 1.7 { .18 3.6 { 0.5
0.2 { .04* 0.4 { .07* 1.1 { 0.3*
0.8 { .09† 1.3 { .15† 2.5 { 0.4*,†
0.1 { .03*,‡ 0.3 { .04*,‡ 0.4 { .06*,†,‡
NOTE. Data are means { SE. * P õ .05, compared with animals on the trace diet / placebo regime. † P õ .05, compared with animals on the trace diet / MK-329 regime. ‡ P õ .05, compared with animals on the high-cholesterol diet / placebo regime.
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.05, respectively) (Table 1). MK-329 treatment, on the other hand, significantly reduced the cholesterol saturation in hepatic bile, regardless of the diet (P õ .05) (Table 1). This was due to a higher molar percentage of bile salts (tracecholesterol diet / placebo: 78.2% { 1.6% vs. trace-cholesterol diet / MK-329: 79.9% { 1.2%; 1% high-cholesterol diet / placebo: 69.9% { 1.1% vs. 1% high-cholesterol diet / MK-329: 74.2% { 0.9%; P õ .05) and a lower molar percentage of cholesterol (trace-cholesterol diet / placebo: 4.6% { 0.2% vs. trace-cholesterol diet / MK-329: 3.3% { 0.4%; 1% high-cholesterol diet / placebo: 9.3% { 0.7% vs. 1% high-cholesterol diet / MK-329: 6.4% { 0.8%; P õ .05). MK-329 also significantly decreased the cholesterol saturation in gallbladder bile. Notably, the cholesterol saturation index (CSI) remained above 100% in animals on the 1% high-cholesterol diet receiving MK-329 (Table 1). The 1% high-cholesterol diet caused a significant (P õ .05) increase in the mucin content of gallbladder tissue homogenates compared with that from animals on the tracecholesterol (standard) diet. Adding MK-329 to this diet group had no additional impact (Fig. 1A). Likewise, MK329 in itself had little influence on the mucin content of gallbladder tissue in animals on the trace-cholesterol diet, when compared with their controls (trace / placebo group). The mucin content of gallbladder surface washings, reflecting the amount of mucus adherent to the gallbladder epithelium surface, was significantly increased (P õ .05) in animals either on the trace-cholesterol diet receiving MK-329 or on the 1% high-cholesterol diet receiving placebo. Administrating MK-329 to this latter group caused a further 82% increase in mucin content of the mucus layer (P õ .05) (Fig. 1B). In animals on the trace-cholesterol diet, MK-329 did not change the mucin concentration in the bulk phase of gallbladder bile (Fig. 1C). In cholesterol-fed animals, however, the mucin content was significantly (P õ .05) elevated in the bulk phase of gallbladder bile. MK-329 treatment produced a further 14% increase, but this difference failed to achieve statistical significance. Macroscopically, fresh gallbladder bile samples from animals on the trace-cholesterol diet (with or without MK-329) were clear without visible precipitates. In animals on the 1% high-cholesterol diet receiving MK-329, however, all bile samples were turbid and viscous, filled with numerous crystal-like precipitates. Under polarizing microscopic examination (n Å 16 in each group), no cholesterol crystals were found in the initial native gallbladder bile of any animals on the trace-cholesterol (standard) diet, regardless of whether
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or not they were on MK-329 treatment. In contrast, 9 of 16 cholesterol-fed animals had solid cholesterol crystals. Further, all animals simultaneously receiving the 1% high-cholesterol diet and MK-329 treatment developed solid cholesterol crystals upon initial microscopic examination. During the study period of 4 weeks, five animals on the 1% highcholesterol diet receiving MK-329 developed macroscopic stones, whereas no gallstones were found in any other groups. Nucleation did not occur within 21 days in any animals maintained on the trace-cholesterol (standard) diet, regardless of whether they received MK-329 (Table 3). The average
TABLE 3. Effect of High-Cholesterol Diet and MK-329 on Crystal Incidence and Nucleation Time Trace Diet
Crystal incidence Nucleation time (ds)
Placebo (n Å 16)
MK-329 (n Å 16)
0/16 ú21
0/16 ú21
1%-Cholesterol Diet Placebo (n Å 16)
MK-329 (n Å 16)
9/16* 5.8 { 1.0*
16/16*,† 2.5 { 0.6*,†
NOTE. Data are means { SE. * P õ .05, compared with animals on the trace diet / placebo regime. † P õ .05, compared with animals on the high-cholesterol diet / placebo regime.
nucleation time for animals on the 1%-cholesterol diet / placebo was 5.8 { 1.0 days, which was significant (P õ .05) compared with the two groups of animals on the tracecholesterol diet; the addition of MK-329 to this diet group further significantly shortened the nucleation time to 2.5 { 0.6 days (P õ .05 vs. animals on the 1%-cholesterol diet / placebo group) (Table 3). A general trend was observed in the nucleation time study. Liquid cholesterol crystals, as judged by their compressible shape with hallmark Maltese crosses,18,19,24 always occurred first. Then, over time, the number of the liquid crystals increased gradually to several hundred per high-power field. Many of them became larger and had an irregular warped shape. Soon after (usually a day or two), the first solid cholesterol crystals appeared, identified as birefringent, notched rhomboidal plates,18-19,24 with a marked decrease in the number of liquid crystals. DISCUSSION
This report again shows that MK-329 almost completely inhibits CCK-induced gallbladder contraction in vitro and markedly increases gallbladder fasting volume in vivo (Tables 1 and 2).14 MK-329, a specific CCK-A receptor antagonist, appears both effective and reliable in producing gallbladder stasis in this animal model of cholesterol gallstone disease.14,29,30 We also show that, despite an improved cholesterol saturation in bile, sustained suppression of gallbladder emptying with MK-329 increases the retention and accumulation of gallbladder mucin, thus enhancing the likelihood of cholesterol crystal formation in this model. Cholesterol gallstone formation is multifactorial, involving
b FIG. 1. (A) Mucin content of gallbladder tissue per DNA. The mucin content of gallbladder tissue increased significantly (P õ .05 vs. respective control group on the trace-cholesterol diet) in animals on the 1% highcholesterol diet, regardless of whether or not they received MK-329. MK329 alone had no further effect in either dietary group. (B) Mucin present on the epithelial surface of the gallbladder. The 1% high-cholesterol diet markedly increased (P õ .05 vs. trace-cholesterol / placebo group) the mucin contained in the mucus layer. Adding MK-329 caused a further 82% increase (P õ .05 vs. 1% high-cholesterol diet / placebo group). MK-329 treatment also significantly elevated the mucus layer in the animals on the trace-cholesterol (standard) diet (P õ .05 vs. trace-cholesterol diet / placebo group). (C) Mucin concentration in the bulk phase of gallbladder bile. The 1% high-cholesterol diet significantly increased the mucin concentration in gallbladder bile (P õ .05 vs. both groups of animals on trace diet, respectively). The addition of MK-329 produced a further 14% increase, but this difference did not achieve statistical significance.*P õ .05 versus animals on the trace-cholesterol (standard) diet.#P õ .05 versus animals on the 1% high-cholesterol diet / placebo.
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at least three tightly interrelated disturbances: biliary lipid secretion, cholesterol crystal nucleation, and gallbladder stasis.1-5,23-28,36-38 Although evidence is mounting that diminished gallbladder emptying, in response to a meal and/or CCK infusion, is present in a large proportion of patients with cholesterol gallstones, neither the sequential events nor the overall influence of gallbladder stasis on the whole process of stone formation is fully understood.1,5-7,11,17 Our recent report,14 which showed that gallbladder stasis actually lowers bile CSI, appeared to complicate this issue even further. We therefore designed the present study to determine how gallbladder hypomotility (stasis) may influence the initial stage of cholesterol gallstone formation with an emphasis on its local effect on the gallbladder.1,5,19,23-28 The present findings showed that sustained suppression of gallbladder emptying with MK-329 reduces the CSI in bile. This occurred in animals on both a trace-cholesterol (standard) diet and a 1% high-cholesterol diet, a necessary lithogenic challenge to initiate the formation of cholesterol gallstones in the ground squirrel.9-14 We have previously proposed that the MK-329–induced fall in CSI is largely caused by the increased hepatic secretion of bile salts.14 This, in turn, is mediated by the action of MK-329 on the enterohepatic circulation of bile salts. MK-329 substantially reduced the participation of the gallbladder in the enterohepatic circulation.14 Shortening of the enterohepatic circuit allowed the bile salt pool to cycle more frequently with a concomitant increase in bile salt secretion and decrease in the CSI.14,20-22 Potent and sustained suppression of gallbladder emptying may, therefore, have a ‘‘beneficial effect’’ on the enterohepatic circulation, indirectly rendering bile unsaturated. But, when the CSI is high and cannot be reduced to levels below 100%, such prolonged retention of supersaturated bile in the gallbladder may provide the residence time and medium necessary for cholesterol crystal formation, and eventually stone growth.1,5,17-19,23-28 The current findings support this concept. Compared with controls, all the measurements of mucin (in gallbladder tissue, the mucus layer, and gallbladder bile) were significantly elevated in animals on the 1%-cholesterol diet receiving placebo, an established animal model of cholesterol gallstone disease in which stasis develops early.9-14 The increased mucin content of gallbladders from animals on the 1%-cholesterol diet likely resulted from enhanced epithelial production.23-24 Adding MK-329 to this dietary group produced a further elevation in the mucin content of the surface mucus layer, the most significant site for promoting crystal nucleation in vivo.11,17-18,23-24 This was not caused by enhanced mucin production, because the addition of MK-329 did not further increase tissue levels in either dietary group (Fig. 1A). It is likely that the potent and sustained stasis induced by MK-329 leads to increased retention and then accumulation of mucin on the mucosal surface of the gallbladder wall due to reduced evacuation of gallbladder contents.1,5,17,36 This notion is supported by the finding that an increased surface mucus layer was observed in both dietary groups (Fig. 1B). As the gallbladders in both dietary groups had larger volumes on MK-329, they also had greater surface areas and presumably increased mucin exposure for crystal nucleation. The present studies, however, could not distinguish which component, a thicker mucus layer versus a greater surface, accounted for the observed increase in the mucin content of the mucus layer on the epithelial surface of the gallbladder.
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In either case, the abundant crystal formation (Table 3), despite reduced CSI (Table 1), signified that retention of mucin was a dominant force in stone formation in the cholesterol / MK-329 group. MK-329 was not only effective in inducing stasis in control animals, but clearly aggravated gallbladder stasis in animals on the 1% high-cholesterol diet, as evidenced by the greatest reduction in gallbladder contraction (Table 2) and largest gallbladder fasting volume (Table 1). This may explain why this group of animals had the highest mucin levels on the gallbladder surface. The failure of MK-329 to further increase the mucin content in the gallbladder tissue (Fig. 1A) might indicate that mucin production was already near maximal in the presence of bile saturated with cholesterol (Table 1). It may also suggest that stasis in itself or altered intraluminal pressure alone is less important for provoking gallbladder mucin production in this model. Evidence suggests that mucin is important for the initiation and subsequent growth of gallstones in humans and in experimental animals.23-28,34 The reduced evacuation of gallbladder contents,1,5,36 especially in animals on the 1%cholesterol diet treated with MK-329, may allow an increased accumulation of the mucus layer on the gallbladder epithelial surface, an ideal medium for heterogeneous nucleation to occur. Mucin is also the major structural protein of biliary sludge and forms the matrix for stones. It is the ‘‘glue’’ that binds the crystalline plates of cholesterol together.24-28,34 Cholesterol crystals therefore likely nucleate in the mucinous gel that adheres to the epithelial surface of the gallbladder, rather than in the bulk aqueous phase of the bile.17,18,24-28,34 This may explain why animals on the 1%-cholesterol diet treated with MK-329 possessed the most abundant cholesterol crystals (Table 3), even though animals on the 1%cholesterol diet alone had the highest CSI (Table 1). Despite MK-329 decreasing gallbladder CSI by nearly 25% in cholesterol-fed animals, the bile remained supersaturated with a CSI still above 100%. The potential for cholesterol gallstone formation persisted.1,17-18,38 Although animals simultaneously receiving the 1% cholesterol diet and MK-329 treatment had a shorter nucleation time (Table 3), their mucin content in the bulk aqueous phase of bile did not differ significantly from that of animals on the 1%-cholesterol diet alone (Fig. 1). It may indicate that other factors are actively involved in crystal nucleation. In addition to mucin, several nonmucin biliary glycoproteins promote cholesterol crystal nucleation, such as immunoglobulins (IgM, IgA, and IgG), haptoglobin, a1 -acid glycoprotein, phospholipase C, fibronectin, aminopeptidase N, and transferrin.1,17,35,39-41 In summary, prolonged stasis per se may actually decrease CSI in bile in certain circumstances. The impact of gallbladder stasis on bile composition, mucin secretion, and crystal nucleation differs, however. Potent and sustained gallbladder stasis in the presence of bile saturated with cholesterol leads to increased accumulation of mucin, which, when layered on the gallbladder epithelial surface, could entrap cholesterol crystals. This study reveals the importance of the gallbladder in the whole process of cholesterol gallstone formation. The influence of immobilizing the gallbladder within the enterohepatic cycling may produce, on one hand, an apparent benefit (lowered cholesterol saturation). This, however, does not offset its local detrimental effect—increased mucin accumulation on the gallbladder epithelium, which provides a site
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for nucleation to occur, and hence expedites the precipitation of cholesterol crystals in this model. Acknowledgment: The authors thank Dr. J. K. Kelly for his expert histological interpretation and D. Kirk for his excellent assistance. REFERENCES 1. Shaffer EA. Abnormalities in gallbladder function in cholesterol gallstone disease: bile and blood, mucosa and muscle—the list lengthens. Gastroenterology 1992;102:1808-1812. 2. Pomeranz IS, Shaffer EA. Abnormal gallbladder emptying in a subgroup of patients with gallstones. Gastroenterology 1985;88:787-791. 3. Behar J, Lee KY, Thompson WR, Biancani P. Gallbladder contraction in patients with pigment and cholesterol stones. Gastroenterology 1989; 97:1479-1484. 4. LaMont JT, Afdhal NH. Biliary tract. Curr Opin Gastroenterol 1993;9: 787-790. 5. LaMorte WW. Biliary motility and abnormalities associated with cholesterol cholelithiasis. Curr Opin Gastroenterol 1993;9:810-816. 6. Brugge WR, Brand DL, Arkins H, Lane B, Abel WG. Gallbladder dyskinesia in chronic acalculous cholecystitis. Dig Dis Sci 1986;31:461-467. 7. Spengler U, Sackmann M, Sauerbruch T, Holl J, Paumgartner G. Gallbladder motility before and after extracorporeal shock-wave lithotripsy. Gastroenterology 1989;96:860-863. 8. Kaminski DL. Arachidonic acid metabolites in hepatobiliary physiology and disease. Gastroenterology 1989;97:781-792. 9. Fridhandler TM, Davison JS, Shaffer EA. Defective gallbladder contractility in the ground squirrel and prairie dog during the early stages of cholesterol gallstone formation. Gastroenterology 1983;85:830-836. 10. MacPherson BR, Pemsingh RS, Scott GW. Experimental cholelithiasis in the ground squirrel. Lab Invest 1987;56:138-145. 11. Smith BF, LaMont JT, Small DM. The sequence of events in gallstone formation. Lab Invest 1987;56:125-127. 12. Xu QW, Shaffer EA. Cisapride improves gallbladder contractility and bile lipid composition in an animal model of gallstone disease. Gastroenterology 1993;105:1184-1191. 13. Xu QW, Scott RB, Tan D, Shaffer EA. Slow intestinal transit: a motility disorder contributing to cholesterol gallstone formation in the ground squirrel. HEPATOLOGY 1996;23:1664-1672. 14. Pauletzki JG, Xu QW, Shaffer EA. Biliary hypomotility decreases cholesterol saturation in bile in the Richardson ground squirrel. HEPATOLOGY 1995;22:325-331. 15. Roslyn JJ, DenBesten L, Pitt H, Kuchenbecker S, Polarek JW. Effect of cholecystokinin on gallbladder stasis and cholesterol gallstone formation. J Surg Res 1981;30:200-204. 16. Doty JE, Pitt HA, Porter-Fink V, DenBesten L. Cholecystokinin prophylaxis of parenteral nutrition-induced gallbladder disease. Ann Surg 1985;201:76-80. 17. Carey MC, Duane WC. Enterohepatic circulation. In: Arias IM et al., eds. The Liver: Biology and Pathobiology. 3rd ed. New York: Raven, 1994:719-768. 18. Carey MC, Cahalane MJ. Whither biliary sludge? Gastroenterology 1988;95:508-523. 19. Holan KR, Holzbach RT, Hermann RE, Cooperman A, Claffey NJ. Nucleation time: a key factor in the pathogenesis of cholesterol gallstone disease. Gastroenterology 1979;77:611-617. 20. Shaffer EA, Small DM. Biliary lipid secretion in cholesterol gallstone disease. The effect of cholecystectomy and obesity. J Clin Invest 1977; 59:828-840. 21. Berr F, Stellaard F, Pratschke E, Paumgartner G. Effects of cholecystectomy on the kinetics of primary and secondary bile acids. J Clin Invest 1989;83:1541-1550.
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22. Stolk MFJ, van Erpecum KJ, Renooij W, Portincasa P, van de Heijning BJM, van Berge-Henegouwen GP. Gallbladder emptying in vivo, bile composition and nucleation of cholesterol crystals in patients with cholesterol gallstones. Gastroenterology 1995;108:1882-1888. 23. Lee SP, LaMont JT, Carey MC. Role of gallstone mucus hypersecretion in the evolution of cholesterol gallstone: studies in a prairie dog. J Clin Invest 1981;67:1712-1723. 24. Lee SP, Maher K, Nicholls JF. Origin and fate of biliary sludge. Gastroenterology 1988;94:170-176. 25. Harvey PRC, Rupar CA, Gallinger S, Petrunka CN, Strasberg SM. Quantitative and qualitative comparison of gallbladder mucus glycoprotein from patients with and without gallstones. Gut 1985;27:374-381. 26. Halpern Z, Dudley MA, Kibe A, Lynn MP, Breuer AC, Holzbach RT. Rapid vesicle formation and aggregation in abnormal human bile. A time-lapse video-enhanced contrast microscopy study. Gastroenterology 1985;90:875-885. 27. Afdhal NH, Niu N, Gantz D, Small DM, Smith BF. Bovine gallbladder mucin accelerates cholesterol monohydrate crystal growth in model bile. Gastroenterology 1993;104:1515-1523. 28. Afdhal NH, Niu N, Nunes DP, Bansil R, Cao XX, Gantz D, Small DM, et al. Mucin-vesicle interactions in model bile: evidence for vesicle aggregation and fusion before cholesterol crystal formation. HEPATOLOGY 1995;22:856-865. 29. Chang RSL, Lotti V. Biochemical and pharmacological characterization of an extremely potent and selective non-peptide cholecystokinin antagonist. Proc Natl Acad Sci U S A 1986;83:4923-4926. 30. Liddle RA, Gertz BJ, Kanayama S, Beccaria L, Coker LD, Turnbull TA, Morita ET. Effects of a novel cholecystokinin (CCK) receptor antagonist, MK-329, on gallbladder contraction and gastric emptying in humans. J Clin Invest 1989;84:1220-1225. 31. Carey MC. Critical tables for calculating the cholesterol saturation of native bile. J Lipid Res 1978;19:945-955. 32. Mantle M, Thakore E, Atkins E, Mathison R, Davison JS. Effects of streptozotocin-diabetes on rat intestinal mucin and goblet cells. Gastroenterology 1989;97:68-75. 33. Hinegardner RT. An improved fluorometric assay for DNA. Anal Biochem 1971;39:197-201. 34. Jungst D, Lang T, Von Titter C, Pratschke E, Paumgartner G. Cholesterol nucleation time in gallstone bile of patients with solitary or multiple cholesterol gallstones. HEPATOLOGY 1992;15:804-808. 35. Harvey PRC, Strasberg SM. Will the real cholesterol nucleating and anti-nucleating proteins please stand up. Gastroenterology 1993;104: 646-650. 36. Jazrawi RP, Pazzi P, Petroni ML, Prandini N, Paul C, Adam JA, Gullini S, et al. Postprandial gallbladder motor function: refilling and turnover of bile in health and in cholelithiasis. Gastroenterology 1995;109:582591. 37. Kibe A, Dudley MA, Halpern Z, Lynn MP, Breuer AC, Holzbach RT. Factors affecting cholesterol monohydrate crystal nucleation time in model systems of supersaturated bile. J Lipid Res 1985;26:1102-1111. 38. Small MD, Rapo S. The source of abnormal bile in patients with cholesterol gallstones. N Engl J Med 1970;283:53-57. 39. Harvey PRC, Uphadya GA, Strasberg SM. Immunoglobulins as nucleating agents in the gallbladder bile of patients with cholesterol gallstones. J Biol Chem 1991;2266:13996-14003. 40. Abei M, Schwarzendrube J, Nuutinen H, Broughan TA, Kawczak P, Williams C, Holzbach RT. Cholesterol crystallization-promoters in human bile: comparative potencies of immunoglobulins, a1 -acid glycoprotein, phospholipase C and aminopeptidase N. J Lipid Res 1993;34:11411148. 41. Yamashita G, Corradini SG, Secknus R, Takabayashi A, Williams C, Hays L, Chernosky AL, et al. Biliary haptoglobin, a potent promoter of cholesterol crystallization at physiological concentrations. J Lipid Res 1995;36:1325-1333.
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Although gallbladder stasis exists in most patients with cholesterol gallstones, it is unknown whether stasis is a causative factor of gallstone disease or merely a consequence of it. We studied the impact of sustained gallbladder stasis induced by a cholecystokinin (CCK)-A receptor antagonist (MK329) on gallstone formation in ground squirrels fed either a trace or a high-cholesterol diet. MK-329 markedly inhibited gallbladder contraction in vitro in response to CCK (at EC100 , control: 3.6 { 0.5 vs. MK-329: 1.1 { 0.3 g; P õ .05) and increased gallbladder fasting volume in vivo (control: 462 { 66 vs. MK-329: 1,004 { 121 mL; P õ .05). Whereas the high-cholesterol diet alone (1%-cholesterol diet / placebo) increased the cholesterol saturation index (CSI) in control animals (trace-cholesterol diet / placebo), MK-329 significantly (P õ .05) decreased the CSI in both hepatic and gallbladder bile in animals on the trace-(trace-cholesterol diet / MK-329) as well as on the high-cholesterol diets (1%-cholesterol diet / MK-329). The mucin content of the mucus layer on the epithelial surface of the gallbladder wall more than doubled (P õ .05) with the high-cholesterol diet; adding MK329 to the latter group produced a further 82% increase (P õ .05). The cholesterol diet / MK-329 group had the highest (100%) incidence of cholesterol crystals that were evident in fresh gallbladder bile, coincident with a shortened nucleation time (2.5 { 0.6 days; P õ .05 vs. the cholesterol diet / placebo group, 5.8 { 1.0 days or the other 2 groups, ú21 days). Bile from animals on the trace-cholesterol diet, whether or not receiving MK-329, lacked crystals in bile and exhibited a normal nucleation time (ú21 days). Thus, stasis per se may lower the CSI, but its detrimental effect on the gallbladder predominates locally, and so accelerates cholesterol crystal formation in this model. (HEPATOLOGY 1997;26:831-836.) Gallbladder motility defects are present in most patients with cholesterol gallstones, yet the sequential events are unclear and the question remains whether gallbladder stasis is a causative factor of gallstone disease or merely a conse-
Abbreviations: CCK, cholecystokinin; CSI, cholesterol saturation index; PAS, periodic acid–Schiff. From the GI Research Group, Department of Medicine, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada. Received January 21, 1997; accepted June 11, 1997. Supported by a Medical Research Council of Canada grant (MT 69-1945). Dr. Xu was supported by a Canadian Liver Foundation Research Fellowship, and Dr. Pauletzki by an Alberta Heritage Foundation for Medical Research Clinical Fellowship. Address reprint requests to: Eldon A. Shaffer, M.D., Foothills Hospital, Department of Medicine, 1403 29th St. N.W., Calgary, Alberta T2N 2T9 Canada. Fax: (403) 6701095. Copyright q 1997 by the American Association for the Study of Liver Diseases. 0270-9139/97/2604-0004$3.00/0
AND
ELDON A. SHAFFER
quence of it.1-5 Identifying events in a chronological fashion is particularly difficult in humans before gallstone formation.5 Defective gallbladder emptying likely occurs before stone formation, followed by the appearance of cholesterol crystals.1,6,7 In one report on patients with cholesterol crystals alone (without gallstones), gallbladder emptying was impaired.6 Unfortunately, all the patients in this study had chronic cholecystitis, a confounding factor, which in itself could have adversely affected gallbladder motor function.1,5,8 Ground squirrels fed a high-cholesterol diet form cholesterol crystals and/or gallstones within a reasonably short period.9-13 Defective gallbladder contractility develops very early in these animals, concurrent with a rise in cholesterol saturation of bile and the appearance of cholesterol crystals, but before stone formation. A major limitation in this model is the inability to assess directly the primary role of gallbladder hypomotility in the process of gallstone formation because of the concurrent changes in both hepatic bile formation and gallbladder motility.13-14 Our previous study in the same animal model showed that a cholecystokinin (CCK)-A receptor antagonist (MK-329) suppressed gallbladder emptying, but reduced the cholesterol saturation in both hepatic and gallbladder bile.14 This apparent paradox challenges the traditional concept that gallbladder stasis is a primary and/or contributing factor in cholesterol gallstone formation.1-3,15-18 Gallbladder stasis is thought to facilitate nucleation; the retained precipitated microcrystals then would agglomerate into cholesterol gallstones in humans.1,5,17-19 We therefore hypothesize that, despite an improved cholesterol saturation of bile in certain circumstances,14,20-22 sustained gallbladder stasis may have a dominantly local effect in the gallbladder, i.e., in the presence of supersaturated bile, gallbladder stasis may cause an increased production and/or accumulation of mucin within the gallbladder, thereby providing both the residence time and the medium necessary for the nucleation of cholesterol crystals.17-19,23-28 To further explore the nature of gallbladder stasis in the whole process of cholesterol gallstone formation, we used a specific CCK-A receptor antagonist (MK-32914,29,30) to produce prolonged gallbladder hypomotility in ground squirrels on both a trace (standard) and a 1% high-cholesterol diet, while clarifying events within the gallbladder.9-14 MATERIALS AND METHODS Experimental Animals. Richardson ground squirrels (Spermophilus richardsoni) were trapped wild near Calgary, Alberta, Canada. They were males with an average body weight of 300 g. Animals were individually housed in thermoregulated rooms on a 12-hour day/ night light cycle with unlimited access to standard rat chow and water for a minimum of 1 month. One hundred twenty-eight experi-
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mental animals were then divided randomly into two dietary groups and maintained for an additional 4 weeks on either a control chow diet (n Å 64) with a trace (standard) cholesterol content (0.027%) (United States Biochemical, Cleveland, OH) or a high-cholesterol diet (n Å 64) comprised of an identical chow, enriched with 1% cholesterol by weight (United States Biochemical). Within each dietary group, two further subgroups (n Å 32 each) were created and treated with either a CCK-A receptor antagonist (MK-329, Merck Sharp & Dohme Research Laboratories, West Point, PA), 1 mg/kg three times per day, subcutaneously, prepared in a 4% dimethylsulfoxide/saline solution, or placebo (4% dimethylsulfoxide/saline). During the 4 weeks before the terminal experiments, all ground squirrels were allowed access to food and water ad libitum. This research protocol was approved by the Animal Care Committee of the University of Calgary. Experimental Design. The terminal experiments performed after a treatment period of 4 weeks had four objectives: 1) determine gallbladder contractility in vitro (n Å 8 each); 2) assess bile composition and cholesterol saturation (n Å 8 each); 3) examine the mucin content of gallbladder tissue and bile, as well as the adherent mucus layer at the mucosal surface of the gallbladder wall (n Å 8 each); and 4) measure the nucleation time of cholesterol crystals (n Å 16 each, including the bile samples from the contractility study). Gallbladder Contractility In Vitro. After a 16-hour fast, the cystic duct was exposed and ligated at laparotomy. The entire gallbladder was removed, and the gallbladder bile was aspirated as completely as possible, the volume measured, a drop examined under a polarizing microscope for cholesterol and liquid crystals, and the remainder stored at 0707C for later analysis. The preparation and setup for gallbladder contractility study in vitro have been described previously.12-13 Once each preparation exhibited a stable baseline tension, 1004 mol/L bethanechol was administered as a single bolus to verify its viability. Next, the gallbladder was washed thoroughly by changing the Krebs solution in the organ bath until the baseline tension was again attained. The tissue was then challenged with EC25 , EC50 , and EC100 of CCK-8. These concentrations were obtained from our preliminary dose-response curves, representing the concentrations required to produce the stated fraction (contractile response) of the maximum effect of CCK-8.12-14 Bile Collection and Analysis of Biliary Lipids. Hepatic bile collection by direct cannulation of the common bile duct was performed in terminal experiments following a specific dietary and/or treatment period as previously described.12-14 Bile samples were analyzed for total bile salts using the 3-a-hydroxysteroid dehydrogenase assay (Worthington Biochemical Company, Freehold, NJ), for phospholipid by an enzymatic assay for choline (Boehringer Mannheim, Mannheim, Germany) and for cholesterol by an enzymatic diagnostic kit (Ames Division, Miles Laboratories, Slough, UK). Serum cholesterol levels were also determined by this Ames cholesterol kit.12-14 The cholesterol saturation index (CSI) was determined using Carey’s critical tables for cholesterol solubility.31 Bile Collection and Extraction of Mucin. The intact gallbladder was carefully removed and the bile aspirated as completely as possible using a syringe. The bile volume was recorded. The empty gallbladder was carefully opened along one side and placed everted into 1 mL of TRIS-buffered saline (0.01 mol/L TRIS, 0.1 mol/L NaCI, 0.02% (wt/vol) NaN3 , pH 7.4) containing proteolytic inhibitor (1 mmol/L phenylmethanesulphonyl fluoride, 10 mmol/L N-ethylmaleimide, and 5 mmol/L Na2 ethylenediaminetetraacetic acid). After gentle vortexing at a low speed for 30 seconds to remove the adherent mucus layer on the epithelial surface,32 the gallbladder tissue was transferred into another 1-mL aliquot of the same buffer and homogenized for 60 seconds using a Polytron tissue solubilizer. Tissue homogenates and washings were stored at 0707C for later analysis. Samples of homogenate were later thawed and briefly homogenized. Aliquots were analyzed for DNA using the fluorometric method of Hinegardner.33 The mucin content of tissue homogenates
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and gallbladder washings was analyzed using a nitrocellulose slotblot assay stained with periodic acid–Schiff (PAS) reagent.32 Stained blots were scanned on a laser densitometer, and the amount of mucin present in each test sample was determined by comparing its peak area with standard curves obtained by applying purified rat intestinal mucin (0.3-25 mg) to the nitrocellulose blot. The mucin content of tissue homogenate and the mucus layer on the epithelial surface was expressed relative to the gallbladder DNA content and total recovery from the 1-mL tissue washings, respectively. For analysis of gallbladder bile, lipids were removed by chloroform:methanol extraction (2:1), and the residual material was solubilized in 1 mL of 2% (wt/vol) sodium dodecyl sulfate containing 0.2 mol/L 2-mercaptoethanol. Samples were boiled for 5 minutes, cooled to room temperature (237C), and applied to a Biogel P100 mini-column (0.4 1 10 cm) eluted with 2% sodium dodecyl sulfate to separate proteins and bile pigments (included) from mucin (excluded). The mucin-containing void volume fractions were collected, lyophilized, and sodium dodecyl sulfate was removed using anhydrous acetic:water (17:1:1:1, vol/vol/vol/vol). Samples were resolubilized in Tris-buffered saline to their original volume and analyzed for mucin by the PAS slotblot assay, expressed as micrograms of mucin per milliliter of bile. The recovery of mucin using this procedure was assessed in separate experiments in which a known amount of purified rat intestinal mucin was added to bile samples before processing. A recovery of ú90% was consistently achieved. To confirm that our assay was only detecting mucin in the above experiments, random samples of tissue homogenates and washings were tested by chromatography on a Sepharose CL-4B column and fractions were analyzed by the PAS assay.32 Only the void volume fractions, where mucin would be expected to elute, stained positively with PAS reagent. Thus, the changes in mucin content could not be attributed to the production of other PAS-positive glycoproteins during the course of cholesterol feeding and MK-329 treatment. Cholesterol Nucleation Time. Upon opening the abdomen, bile was aspirated from the gallbladder using a sterile needle and syringe. Particular care was taken to obtain a complete aspiration of the gallbladder bile, to avoid the interference of stratification.19,34 One drop was immediately examined by polarized light microscopy for cholesterol crystals, identified as birefringent, notched rhomboidal plates.19 The remaining bile was stored momentarily in sterile plastic syringes in the dark at 377C with all air expressed from the syringes. To determine the rate of de novo cholesterol microcrystal formation, isotropic (crystal-free) fractions of bile were processed as described by Holan et al.19 Throughout the course of each nucleation study, bile samples were constantly maintained at 377C. All instruments were sterile and prewarmed to 377C before manipulation of bile. Time zero of nucleation was defined as 1 hour after a thermal equilibration. At time zero and at daily intervals thereafter, 4 mL of each sample was examined with a polarizing microscopy to detect the earliest formation of cholesterol monohydrate. The interval between time zero and the first appearance of solid cholesterol crystals was designated the nucleation time (i.e., crystal observation time17,35). The nucleation time study was performed for 21 days, and those bile samples in which solid cholesterol crystals were not detected by the end of 21 days were classified as having no nucleation.19,34 Solutions and Drugs. The modified Krebs solution used in these experiments contained: 103 mmol/L NaCl, 4.7 mmol/L KCl, 5.6 mmol/L CaCl2 , 1.13 mmol/L MgCl2 , 25 mmol/L NaHCO3 , 1.15 mmol/L NaH2PO4 , 2.8 mmol/L D-glucose, 4.9 mmol/L Na pyruvate, 2.7 mmol/L Na fumarate, and 4.9 mmol/L Na glutamate. The pH was adjusted to 7.4. The CCK-A antagonist, MK-329, was kindly provided by Merck Sharp & Dohme Research Laboratories. CCK octapeptide was purchased from Peptide Institute, Inc. (Osaka, Japan), [14C]-cholic acid from New England Nuclear (Boston, MA), and halothane from Ayerst Laboratories (Montreal, Quebec, Canada). Statistics. Data are expressed as mean { SE. Means of multiple
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TABLE 1. Animal Characteristics and Effect of High-Cholesterol Diet and MK-329 on Gallbladder Volume and CSI Trace Diet Placebo (n Å 16)
Body weight (g) Serum cholesterol (mmol/L) Gallbladder volume (mL) Hepatic bile CSI Gallbladder bile CSI
403 5.8 462 98 79
{ { { { {
1%-Cholesterol Diet MK-329 (n Å 16)
28 0.6 66 6 4
423 5.2 1004 75 61
{ { { { {
27 0.7 121* 5* 5*
Placebo (n Å 16)
464 14.1 526 149 137
{ { { { {
35 1.8*,† 57† 11*,† 11*,†
MK-329 (n Å 16)
454 13.1 1242 109 110
{ { { { {
40 1.9*,† 169*,‡ 8†,‡ 6*,†,‡
NOTE. Data are means { SE. * P õ .05, compared with animals on the trace diet / placebo regime. † P õ .05, compared with animals on the trace diet / MK-329 regime. ‡ P õ .05, compared with animals on the high-cholesterol diet / placebo regime.
groups were evaluated by ANOVA. All calculations were performed using the computer program SYSTAT (SYSTAT Inc., Evanston, IL). Statistical values reaching probabilities of P õ .05 were considered significant. RESULTS
Animals appeared healthy with a shining coat and normal activity. No diarrhea was observed in any of the treatment regimes. In 1% cholesterol-fed animals, whether they received MK-329 or placebo, serum cholesterol levels were significantly elevated (P õ .05) compared with their respective controls on the trace cholesterol diet (Table 1). By conventional histological criteria, both liver and gallbladder histology were considered normal after either the high-cholesterol diet or MK-329 treatment. There was no evidence of inflammatory or fatty changes in these tissues. The 1% high-cholesterol diet did not significantly alter the gallbladder volumes measured after an overnight fast. Treatment with MK-329, however, markedly increased the gallbladder volumes in animals on both the trace and 1% high-cholesterol diets (Table 1). After 4 weeks of treatment, MK-329 significantly (P õ .05) suppressed gallbladder contractile responses to CCK in vitro in animals on both diets. The inhibitory effect on gallbladder contraction occurred at all CCK concentrations tested. This was more evident in animals on the 1% high-cholesterol diet (Table 2). In addition, the 1% high-cholesterol diet in itself (cholesterol plus placebo) moderately but significantly (P õ .05) inhibited CCK-induced gallbladder contraction at EC100 , thereby aggravating the effect of MK-329. The 1% high-cholesterol diet markedly increased the cholesterol saturation in both hepatic and gallbladder bile (P õ
TABLE 2. Effect of High-Cholesterol Diet and MK-329 on Gallbladder Contractility In Vitro Trace Diet
EC25 (g) EC50 (g) EC100 (g)
1%-Cholesterol Diet
Placebo
MK-329
Placebo
MK-329
0.9 { .07 1.7 { .18 3.6 { 0.5
0.2 { .04* 0.4 { .07* 1.1 { 0.3*
0.8 { .09† 1.3 { .15† 2.5 { 0.4*,†
0.1 { .03*,‡ 0.3 { .04*,‡ 0.4 { .06*,†,‡
NOTE. Data are means { SE. * P õ .05, compared with animals on the trace diet / placebo regime. † P õ .05, compared with animals on the trace diet / MK-329 regime. ‡ P õ .05, compared with animals on the high-cholesterol diet / placebo regime.
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.05, respectively) (Table 1). MK-329 treatment, on the other hand, significantly reduced the cholesterol saturation in hepatic bile, regardless of the diet (P õ .05) (Table 1). This was due to a higher molar percentage of bile salts (tracecholesterol diet / placebo: 78.2% { 1.6% vs. trace-cholesterol diet / MK-329: 79.9% { 1.2%; 1% high-cholesterol diet / placebo: 69.9% { 1.1% vs. 1% high-cholesterol diet / MK-329: 74.2% { 0.9%; P õ .05) and a lower molar percentage of cholesterol (trace-cholesterol diet / placebo: 4.6% { 0.2% vs. trace-cholesterol diet / MK-329: 3.3% { 0.4%; 1% high-cholesterol diet / placebo: 9.3% { 0.7% vs. 1% high-cholesterol diet / MK-329: 6.4% { 0.8%; P õ .05). MK-329 also significantly decreased the cholesterol saturation in gallbladder bile. Notably, the cholesterol saturation index (CSI) remained above 100% in animals on the 1% high-cholesterol diet receiving MK-329 (Table 1). The 1% high-cholesterol diet caused a significant (P õ .05) increase in the mucin content of gallbladder tissue homogenates compared with that from animals on the tracecholesterol (standard) diet. Adding MK-329 to this diet group had no additional impact (Fig. 1A). Likewise, MK329 in itself had little influence on the mucin content of gallbladder tissue in animals on the trace-cholesterol diet, when compared with their controls (trace / placebo group). The mucin content of gallbladder surface washings, reflecting the amount of mucus adherent to the gallbladder epithelium surface, was significantly increased (P õ .05) in animals either on the trace-cholesterol diet receiving MK-329 or on the 1% high-cholesterol diet receiving placebo. Administrating MK-329 to this latter group caused a further 82% increase in mucin content of the mucus layer (P õ .05) (Fig. 1B). In animals on the trace-cholesterol diet, MK-329 did not change the mucin concentration in the bulk phase of gallbladder bile (Fig. 1C). In cholesterol-fed animals, however, the mucin content was significantly (P õ .05) elevated in the bulk phase of gallbladder bile. MK-329 treatment produced a further 14% increase, but this difference failed to achieve statistical significance. Macroscopically, fresh gallbladder bile samples from animals on the trace-cholesterol diet (with or without MK-329) were clear without visible precipitates. In animals on the 1% high-cholesterol diet receiving MK-329, however, all bile samples were turbid and viscous, filled with numerous crystal-like precipitates. Under polarizing microscopic examination (n Å 16 in each group), no cholesterol crystals were found in the initial native gallbladder bile of any animals on the trace-cholesterol (standard) diet, regardless of whether
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or not they were on MK-329 treatment. In contrast, 9 of 16 cholesterol-fed animals had solid cholesterol crystals. Further, all animals simultaneously receiving the 1% high-cholesterol diet and MK-329 treatment developed solid cholesterol crystals upon initial microscopic examination. During the study period of 4 weeks, five animals on the 1% highcholesterol diet receiving MK-329 developed macroscopic stones, whereas no gallstones were found in any other groups. Nucleation did not occur within 21 days in any animals maintained on the trace-cholesterol (standard) diet, regardless of whether they received MK-329 (Table 3). The average
TABLE 3. Effect of High-Cholesterol Diet and MK-329 on Crystal Incidence and Nucleation Time Trace Diet
Crystal incidence Nucleation time (ds)
Placebo (n Å 16)
MK-329 (n Å 16)
0/16 ú21
0/16 ú21
1%-Cholesterol Diet Placebo (n Å 16)
MK-329 (n Å 16)
9/16* 5.8 { 1.0*
16/16*,† 2.5 { 0.6*,†
NOTE. Data are means { SE. * P õ .05, compared with animals on the trace diet / placebo regime. † P õ .05, compared with animals on the high-cholesterol diet / placebo regime.
nucleation time for animals on the 1%-cholesterol diet / placebo was 5.8 { 1.0 days, which was significant (P õ .05) compared with the two groups of animals on the tracecholesterol diet; the addition of MK-329 to this diet group further significantly shortened the nucleation time to 2.5 { 0.6 days (P õ .05 vs. animals on the 1%-cholesterol diet / placebo group) (Table 3). A general trend was observed in the nucleation time study. Liquid cholesterol crystals, as judged by their compressible shape with hallmark Maltese crosses,18,19,24 always occurred first. Then, over time, the number of the liquid crystals increased gradually to several hundred per high-power field. Many of them became larger and had an irregular warped shape. Soon after (usually a day or two), the first solid cholesterol crystals appeared, identified as birefringent, notched rhomboidal plates,18-19,24 with a marked decrease in the number of liquid crystals. DISCUSSION
This report again shows that MK-329 almost completely inhibits CCK-induced gallbladder contraction in vitro and markedly increases gallbladder fasting volume in vivo (Tables 1 and 2).14 MK-329, a specific CCK-A receptor antagonist, appears both effective and reliable in producing gallbladder stasis in this animal model of cholesterol gallstone disease.14,29,30 We also show that, despite an improved cholesterol saturation in bile, sustained suppression of gallbladder emptying with MK-329 increases the retention and accumulation of gallbladder mucin, thus enhancing the likelihood of cholesterol crystal formation in this model. Cholesterol gallstone formation is multifactorial, involving
b FIG. 1. (A) Mucin content of gallbladder tissue per DNA. The mucin content of gallbladder tissue increased significantly (P õ .05 vs. respective control group on the trace-cholesterol diet) in animals on the 1% highcholesterol diet, regardless of whether or not they received MK-329. MK329 alone had no further effect in either dietary group. (B) Mucin present on the epithelial surface of the gallbladder. The 1% high-cholesterol diet markedly increased (P õ .05 vs. trace-cholesterol / placebo group) the mucin contained in the mucus layer. Adding MK-329 caused a further 82% increase (P õ .05 vs. 1% high-cholesterol diet / placebo group). MK-329 treatment also significantly elevated the mucus layer in the animals on the trace-cholesterol (standard) diet (P õ .05 vs. trace-cholesterol diet / placebo group). (C) Mucin concentration in the bulk phase of gallbladder bile. The 1% high-cholesterol diet significantly increased the mucin concentration in gallbladder bile (P õ .05 vs. both groups of animals on trace diet, respectively). The addition of MK-329 produced a further 14% increase, but this difference did not achieve statistical significance.*P õ .05 versus animals on the trace-cholesterol (standard) diet.#P õ .05 versus animals on the 1% high-cholesterol diet / placebo.
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at least three tightly interrelated disturbances: biliary lipid secretion, cholesterol crystal nucleation, and gallbladder stasis.1-5,23-28,36-38 Although evidence is mounting that diminished gallbladder emptying, in response to a meal and/or CCK infusion, is present in a large proportion of patients with cholesterol gallstones, neither the sequential events nor the overall influence of gallbladder stasis on the whole process of stone formation is fully understood.1,5-7,11,17 Our recent report,14 which showed that gallbladder stasis actually lowers bile CSI, appeared to complicate this issue even further. We therefore designed the present study to determine how gallbladder hypomotility (stasis) may influence the initial stage of cholesterol gallstone formation with an emphasis on its local effect on the gallbladder.1,5,19,23-28 The present findings showed that sustained suppression of gallbladder emptying with MK-329 reduces the CSI in bile. This occurred in animals on both a trace-cholesterol (standard) diet and a 1% high-cholesterol diet, a necessary lithogenic challenge to initiate the formation of cholesterol gallstones in the ground squirrel.9-14 We have previously proposed that the MK-329–induced fall in CSI is largely caused by the increased hepatic secretion of bile salts.14 This, in turn, is mediated by the action of MK-329 on the enterohepatic circulation of bile salts. MK-329 substantially reduced the participation of the gallbladder in the enterohepatic circulation.14 Shortening of the enterohepatic circuit allowed the bile salt pool to cycle more frequently with a concomitant increase in bile salt secretion and decrease in the CSI.14,20-22 Potent and sustained suppression of gallbladder emptying may, therefore, have a ‘‘beneficial effect’’ on the enterohepatic circulation, indirectly rendering bile unsaturated. But, when the CSI is high and cannot be reduced to levels below 100%, such prolonged retention of supersaturated bile in the gallbladder may provide the residence time and medium necessary for cholesterol crystal formation, and eventually stone growth.1,5,17-19,23-28 The current findings support this concept. Compared with controls, all the measurements of mucin (in gallbladder tissue, the mucus layer, and gallbladder bile) were significantly elevated in animals on the 1%-cholesterol diet receiving placebo, an established animal model of cholesterol gallstone disease in which stasis develops early.9-14 The increased mucin content of gallbladders from animals on the 1%-cholesterol diet likely resulted from enhanced epithelial production.23-24 Adding MK-329 to this dietary group produced a further elevation in the mucin content of the surface mucus layer, the most significant site for promoting crystal nucleation in vivo.11,17-18,23-24 This was not caused by enhanced mucin production, because the addition of MK-329 did not further increase tissue levels in either dietary group (Fig. 1A). It is likely that the potent and sustained stasis induced by MK-329 leads to increased retention and then accumulation of mucin on the mucosal surface of the gallbladder wall due to reduced evacuation of gallbladder contents.1,5,17,36 This notion is supported by the finding that an increased surface mucus layer was observed in both dietary groups (Fig. 1B). As the gallbladders in both dietary groups had larger volumes on MK-329, they also had greater surface areas and presumably increased mucin exposure for crystal nucleation. The present studies, however, could not distinguish which component, a thicker mucus layer versus a greater surface, accounted for the observed increase in the mucin content of the mucus layer on the epithelial surface of the gallbladder.
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In either case, the abundant crystal formation (Table 3), despite reduced CSI (Table 1), signified that retention of mucin was a dominant force in stone formation in the cholesterol / MK-329 group. MK-329 was not only effective in inducing stasis in control animals, but clearly aggravated gallbladder stasis in animals on the 1% high-cholesterol diet, as evidenced by the greatest reduction in gallbladder contraction (Table 2) and largest gallbladder fasting volume (Table 1). This may explain why this group of animals had the highest mucin levels on the gallbladder surface. The failure of MK-329 to further increase the mucin content in the gallbladder tissue (Fig. 1A) might indicate that mucin production was already near maximal in the presence of bile saturated with cholesterol (Table 1). It may also suggest that stasis in itself or altered intraluminal pressure alone is less important for provoking gallbladder mucin production in this model. Evidence suggests that mucin is important for the initiation and subsequent growth of gallstones in humans and in experimental animals.23-28,34 The reduced evacuation of gallbladder contents,1,5,36 especially in animals on the 1%cholesterol diet treated with MK-329, may allow an increased accumulation of the mucus layer on the gallbladder epithelial surface, an ideal medium for heterogeneous nucleation to occur. Mucin is also the major structural protein of biliary sludge and forms the matrix for stones. It is the ‘‘glue’’ that binds the crystalline plates of cholesterol together.24-28,34 Cholesterol crystals therefore likely nucleate in the mucinous gel that adheres to the epithelial surface of the gallbladder, rather than in the bulk aqueous phase of the bile.17,18,24-28,34 This may explain why animals on the 1%-cholesterol diet treated with MK-329 possessed the most abundant cholesterol crystals (Table 3), even though animals on the 1%cholesterol diet alone had the highest CSI (Table 1). Despite MK-329 decreasing gallbladder CSI by nearly 25% in cholesterol-fed animals, the bile remained supersaturated with a CSI still above 100%. The potential for cholesterol gallstone formation persisted.1,17-18,38 Although animals simultaneously receiving the 1% cholesterol diet and MK-329 treatment had a shorter nucleation time (Table 3), their mucin content in the bulk aqueous phase of bile did not differ significantly from that of animals on the 1%-cholesterol diet alone (Fig. 1). It may indicate that other factors are actively involved in crystal nucleation. In addition to mucin, several nonmucin biliary glycoproteins promote cholesterol crystal nucleation, such as immunoglobulins (IgM, IgA, and IgG), haptoglobin, a1 -acid glycoprotein, phospholipase C, fibronectin, aminopeptidase N, and transferrin.1,17,35,39-41 In summary, prolonged stasis per se may actually decrease CSI in bile in certain circumstances. The impact of gallbladder stasis on bile composition, mucin secretion, and crystal nucleation differs, however. Potent and sustained gallbladder stasis in the presence of bile saturated with cholesterol leads to increased accumulation of mucin, which, when layered on the gallbladder epithelial surface, could entrap cholesterol crystals. This study reveals the importance of the gallbladder in the whole process of cholesterol gallstone formation. The influence of immobilizing the gallbladder within the enterohepatic cycling may produce, on one hand, an apparent benefit (lowered cholesterol saturation). This, however, does not offset its local detrimental effect—increased mucin accumulation on the gallbladder epithelium, which provides a site
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for nucleation to occur, and hence expedites the precipitation of cholesterol crystals in this model. Acknowledgment: The authors thank Dr. J. K. Kelly for his expert histological interpretation and D. Kirk for his excellent assistance. REFERENCES 1. Shaffer EA. Abnormalities in gallbladder function in cholesterol gallstone disease: bile and blood, mucosa and muscle—the list lengthens. Gastroenterology 1992;102:1808-1812. 2. Pomeranz IS, Shaffer EA. Abnormal gallbladder emptying in a subgroup of patients with gallstones. Gastroenterology 1985;88:787-791. 3. Behar J, Lee KY, Thompson WR, Biancani P. Gallbladder contraction in patients with pigment and cholesterol stones. Gastroenterology 1989; 97:1479-1484. 4. LaMont JT, Afdhal NH. Biliary tract. Curr Opin Gastroenterol 1993;9: 787-790. 5. LaMorte WW. Biliary motility and abnormalities associated with cholesterol cholelithiasis. Curr Opin Gastroenterol 1993;9:810-816. 6. Brugge WR, Brand DL, Arkins H, Lane B, Abel WG. Gallbladder dyskinesia in chronic acalculous cholecystitis. Dig Dis Sci 1986;31:461-467. 7. Spengler U, Sackmann M, Sauerbruch T, Holl J, Paumgartner G. Gallbladder motility before and after extracorporeal shock-wave lithotripsy. Gastroenterology 1989;96:860-863. 8. Kaminski DL. Arachidonic acid metabolites in hepatobiliary physiology and disease. Gastroenterology 1989;97:781-792. 9. Fridhandler TM, Davison JS, Shaffer EA. Defective gallbladder contractility in the ground squirrel and prairie dog during the early stages of cholesterol gallstone formation. Gastroenterology 1983;85:830-836. 10. MacPherson BR, Pemsingh RS, Scott GW. Experimental cholelithiasis in the ground squirrel. Lab Invest 1987;56:138-145. 11. Smith BF, LaMont JT, Small DM. The sequence of events in gallstone formation. Lab Invest 1987;56:125-127. 12. Xu QW, Shaffer EA. Cisapride improves gallbladder contractility and bile lipid composition in an animal model of gallstone disease. Gastroenterology 1993;105:1184-1191. 13. Xu QW, Scott RB, Tan D, Shaffer EA. Slow intestinal transit: a motility disorder contributing to cholesterol gallstone formation in the ground squirrel. HEPATOLOGY 1996;23:1664-1672. 14. Pauletzki JG, Xu QW, Shaffer EA. Biliary hypomotility decreases cholesterol saturation in bile in the Richardson ground squirrel. HEPATOLOGY 1995;22:325-331. 15. Roslyn JJ, DenBesten L, Pitt H, Kuchenbecker S, Polarek JW. Effect of cholecystokinin on gallbladder stasis and cholesterol gallstone formation. J Surg Res 1981;30:200-204. 16. Doty JE, Pitt HA, Porter-Fink V, DenBesten L. Cholecystokinin prophylaxis of parenteral nutrition-induced gallbladder disease. Ann Surg 1985;201:76-80. 17. Carey MC, Duane WC. Enterohepatic circulation. In: Arias IM et al., eds. The Liver: Biology and Pathobiology. 3rd ed. New York: Raven, 1994:719-768. 18. Carey MC, Cahalane MJ. Whither biliary sludge? Gastroenterology 1988;95:508-523. 19. Holan KR, Holzbach RT, Hermann RE, Cooperman A, Claffey NJ. Nucleation time: a key factor in the pathogenesis of cholesterol gallstone disease. Gastroenterology 1979;77:611-617. 20. Shaffer EA, Small DM. Biliary lipid secretion in cholesterol gallstone disease. The effect of cholecystectomy and obesity. J Clin Invest 1977; 59:828-840. 21. Berr F, Stellaard F, Pratschke E, Paumgartner G. Effects of cholecystectomy on the kinetics of primary and secondary bile acids. J Clin Invest 1989;83:1541-1550.
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