pigment gallstone formation in the cholesterol-fed guinea pig

0270-9139/85/0501-0021$02.00/0 HEPATOLOGY Copyright 0 1985 by the American Association for the Study of Liver Diseases

Vol. 5, NO. 1, pp. 21-27.1985 Printed in U. S. A.

Pigment Gallstone Formation in the Cholesterol-Fed Guinea Pig

WAYNE W. LAMORTE, ERICA A. BROTSCHI, THAYER E. SCOTT AND LESTER F. WILLIAMS, JR.

Division of Surgery, Boston University Medical Center, Boston, Massachusetts 021 18

Female Hartley guinea pigs fed a 0.5% cholesterol-supplemented diet were found to form pigmented gallstones after 6 weeks (17/23) and 12 weeks (1 1/1 l), while only 2 of 44 animals fed a trace cholesterol diet formed gallstones over a comparable period. The light brown stones consisted primarily of aggregates of fine granular crystals, morphologically similar to calcium bilirubinate crystals. The stones were soluble in 0.1 N sodium hydroxide and were found to contain a substance which co-migrated with unconjugated bilirubin during thin-layer chromatography.

Despite hypercholesterolemia (202 f 34 vs. 59 f 22 mg per dl in controls, p < 0.05) and fatty infiltration of the liver, cholesterol-fed animals had a lithogenic index of only 0.22 f 0.04 in gallbladder bile as compared to a lithogenic index of 0.02 f 0.01 in animals fed the trace cholesterol diet. Accordingly, no cholesterol monohydrate crystals were found in any animals. Hematocrits among cholesterol-fed animals (47.6 f 1.2%) were lower than those of controls (54.8 f 1.3%, p < 0.05) probably as a result of the cholesterol-induced hemolytic anemia which has been reported by others in this species.

Fasting gallbladder volume was greater in cholesterol-fed animals (2.4 f 0.18 ml) than in controls (1.7 f 0.11, p < 0.0025), and a comparable increase in gallbladder dry tissue mass was found. There was no evidence of biliary obstruction, however, and the gallbladder contractile response to octapeptide cholecystokinin was comparable in both groups. The increase in gallbladder volume appeared to result from: (a) an increase in hepatic bile salt secretion with an associated increase in bile flow and (b) a decrease in the capacity of the gallbladder to absorb fluid from hepatic bile (6.0 f 1.4 mg fluid per hr per mg tissue with cholesterol diet vs. 12.9 f 0.9 in controls, p < 0.0005). Total bile salt concentration was also decreased in gallbladder bile of cholesterol-fed animals (13.79 f 0.51 vs. 18.56 f 2.19 mmoles per liter in controls, p < 0.05), probably as a result of increased hepatic bile flow and decreased gallbladder absorption. The absence of cholesterol gallstones in this model is consistent with the failure to supersaturate bile with cholesterol. The reasons for pigment gallstone formation are less clear but their occurrence may be the result of a cholesterol-induced hemolytic anemia.

Because biliary calculi tend to occur in the gallbladder rather than in the extrahepatic ducts, it is believed that the modification of bile in the gallbladder plays a critical role in the pathogenesis of stones. Evidence to support this hypothesis has been provided for the most part from observations pertaining to cholesterol gallstone formation which suggest that gallbladder stasis (1-4), changes in gallbladder fluid absorption (5) and hypersecretion of mucin from the gallbladder mucosa (6) may contribute to cholesterol precipitation and/or stone growth.

Received May 11,1984; accepted September 19,1984. This study was supported by the National Institutes of Health Grant

AM 15304. Dr. La Morte was supported by a Research Fellowship from the

Medical Foundation, Inc., of Boston. Address reprint requests to: Wayne W. LaMorte, M.D., Division of

Surgery, L 908, Boston University Medical School, 80 East Concord Street, Boston, Massachusetts 02118.

Precipitation of pigment also tends to occur in the gallbladder rather than in the ducts, but relatively little is known of the factors responsible for this. Trotman et al. have speculated that stasis and the concentration of bile within the gallbladder are likely to be important in promoting pigment stone formation in the gallbladder (7). Bernhoft et al. recently reported the precipitation of pigmented material in the gallbladder of dogs following ligation of the cystic duct, and they concluded that gallbladder stasis and the resultant accumulation of mucin promoted stone formation (8).

Our preliminary studies showed that cholesterol-fed guinea pigs unexpectedly developed gallstones which appeared to be of the pigmented type, and gallbladder enlargement appeared to antedate the occurrence of mac- roscopic gallstones in these animals. This study was undertaken to investigate gallstone formation in the cholesterol-fed guinea pig and to determine the mecha- nism for gallbladder enlargement in this animal model.

21

LAMORTE ET AL. HEPATOLOGY 22

MATERIALS AND METHODS Female Hartley guinea pigs (Elm Hill Breeding Farm,

Chelmsford, Mass.) were obtained at an initial body weight of approximately 450 gm and caged in pairs. Animals were fed ad libitum and received either a trace cholesterol control diet (Purina guinea pig chow) or a diet composed of Purina Chow with 0.5% cholesterol (Teklad, Madison Wis.).

Animals were allowed only water for 24 hr prior to study. Anesthesia was obtained by intraperitoneal injec- tion of 2 gm per kg urethane (Sigma Chemical Co., St. Louis, Mo.) into the left lower quadrant of the abdomen. After midline laparotomy, the right lobe of the liver was retracted and small hemoclips were immediately applied to the cystic duct prior to excision of the gallbladder. After noting the presence or absence of stones, the gallbladder contents were aspirated through a needle intro- duced via the cystic duct, and the volume was measured to the nearest 0.1 ml.

ANALYSIS OF BILE Aliquots of gallbladder bile were centrifuged and ex-

amined for crystals by standard and polarized light microscopy. Gallstones were pooled in distilled water containing EDTA and ascorbic acid. These samples were then wrapped in tin foil and frozen at -20°C until analysis by thin-layer chromatography by the method of Blanckaert (9). Bilirubin IXLV (Porphyrin Products, Lo- gin, Utah) prepared by the method of McDonagh and Assisi (10) was used as a standard for unconjugated bilirubin.

Unspun samples of bile were stored -20°C and later analyzed for total bile salts by the hydroxysteroid dehy- drogenase method of Talalay (11) as modified by Admi- rand and Small (12). Aliquots of bile and serum were extracted in chloroform and methanol and analyzed for total cholesterol content by the method of Rude11 and Morris (13). Phospholipids were measured by the method of Bartlett (14) after extraction of bile in chloroform:methanol (2:l, v/v).

The viscosity of bile relative to distilled water was estimated by measuring the time required for 1.0 ml of bile to pass through a series of capillary tubes and comparing it to the time required for 1.0 ml of distilled water to pass. Glycoprotein content of gallbladder bile was measured by the method of Mantle and Allen (15). The pH of gallbladder and hepatic bile was measured within 10 min using a Fisher Accumet pH meter (model 620).

Upon completion of a study, blood was aspirated from the inferior vena cava. An aliquot of whole blood was used to measure hematocrit. Serum was frozen at -20°C for subsequent determination of cholesterol concentration. A few animals in each group were used to ob- tain specimens of liver and gallbladder for histologic examination.

TENSION CHANCES IN GALLBLADDER STRIPS IN VITRO

The gallbladder contractile response was assessed using a standard in uitro technique for measuring isometric

tension changes. After cholecystectomy, longitudinal full thickness strips of gallbladder measuring approximately 3 x 8 mm were immersed in a muscle bath chamber containing 140 ml of Krebs solution. The Krebs solution contained the following components in grams per liter: NaCl, 7.8; KCl, 0.23; NaHC03, 1.37; NaH2P04, 0.165; CaC12, 0.28; MgS04, 0.01; and glucose, 1.4. The muscle chamber was suspended in a water bath which was cir- culated and maintained at 37°C with a Thermomix 1420 (B. Braun and Co., San Francisco, Calif.). The Krebs solution was continuously bubbled with 95% 02/5% CO2, and the pH of the bubbled solution was 7.3 to 7.4. The gallbladder strip was suspended along its longitudinal axis from a Grass FT-03 force-displacement transducer (Grass Instrument Co, Quincy, Mass.). The lower end of the tissue was fixed to a moveable glass rod which was adjusted by micrometer to produce an initial tension of 0.5 to 0.75 gm. The strip was then equilibrated for 60 min prior to measuring tension changes in response to octapeptide cholecystokinin (Kinevac, Squibb Co, Princeton, NJ ) at bath concentrations of lo-’ - 2 X lo-’ M . Isometric tension changes in response to random drug concentrations were recorded as the peak change in tension compared to that observed immediately prior to drug administration; each response was observed for approximately 10 min, after which the muscle chamber was flushed with 6 volumes of fresh, prewarmed Krebs solution, and allowed to reequilibrate for 15 min prior to the next drug stimulation. Tension changes in response to a given concentration of cholecystokinin octapeptide were expressed as the mean ? S.E. for each group, and differences in response from one group to another were examined for significance using Student’s t test for in- dependent means.

I N VITRO GALLBLADDER FLUID ABSORPTION Fluid absorption was measured as described by Dia-

mond (16). A pool of guinea pig hepatic bile was obtained by pooling common hepatic duct bile collected from five animals receiving the control (trace cholesterol) diet. The hepatic bile pool had a total bile salt concentration of 3.26 mmoles per liter. Unconjugated [“C Jchenodeox- ycholic acid (CDCA) was added to the pool as a label. The pool contained 215,030 cpm per ml of bile (65,960 cpm per pmole of bile salts). After cholecystectomy and aspiration of bile, the interior of the gallbladder was rinsed with Krebs solution and a volume of pooled hepatic bile equal to 75% of the observed fasting gallbladder volume was infused. The gallbladder was then suspended in 30 ml Krebs solution bubbled with 95% 02/5% CO, and maintained at 37°C. At 5-min intervals, the gallbladder was removed from the bath, and fluid adherent to the serosal surface was gently removed by touching the gallbladder to the lip of a glass beaker 6 times as described by Diamond (16); the gallbladder was then suspended from a Metler balance, weighed to the nearest 0.1 mg and returned to the in vitro bath until the next reading. At 10-min intervals, a 100-pl sample was removed from the serosal solution, placed in 10 ml of scintillation fluid (Formula 963, New England Nuclear) and counted for [“C]. The rate of net fluid loss over a 1- hr period was calculated by linear regression of the

Vol. 5, No. 1,1985 PIGMENT GALLSTONES IN THE GUINEA PIG 23

cumulative weight change over time. Because of differences in gallbladder size, and therefore in absorptive area, absorption was expressed as fluid absorbed per milligram of gallbladder dry weight. Gallbladder dry weight was measured after drying the tissue overnight at 100°C. Results within groups are reported as the mean & S.E.

SECRETION OF COMMON HEPATIC DUCT BILE Samples of hepatic bile were obtained by cannulating

the common hepatic duct with a PE-60 catheter ligated in place. The total volume secreted was collected at 10- min intervals for a total of 1 hr. The volume of bile secreted in each 10-min period was measured volumet- rically to 0.1 ml. In some studies, hepatic secretory pressure was measured instead, by attaching the free end of the common hepatic duct cannula to a manometer and noting the maximum height of the bile column above the common bile duct after 60 min of observation.

RESULTS GENERAL EFFECTS OF CHOLESTEROL FEEDING

Guinea pigs maintained on the 0.5% cholesterol-supplemented diet appeared healthy during the first 6 weeks of treatment. There were no overt differences in activity level or coat quality among the cholesterol fed and control groups. Initial body weight was similar among the four groups as shown in Table 1, and after 6 weeks of dietary treatment, total body weight gain did not differ significantly, nor did mortality rates. After 12 weeks, mortality was greater in the cholesterol-fed animals, and weight gain in the cholesterol-fed group tended to be less. As reported by others (17), cholesterol feeding resulted in a significant anemia which was evident even at 6 weeks in this study. Cholesterol feeding also produced a significant elevation in serum cholesterol levels (Table 1).

FINDINGS A T LAPAROTOMY The most striking finding during laparotomy was the

presence of an enlarged, pale, friable liver in all animals receiving cholesterol supplementation. Histologic examination of liver sections showed moderate fatty infiltration (Figure 1). In addition, splenic enlargement was also invariably present in cholesterol-fed animals, although the spleens were normal in color.

The gallbladder of cholesterol-fed guinea pigs was usu- ally tense and enlarged. The dry weight of the gallbladder (infundibulum + fundus) was greater in animals fed cholesterol (Table 1). Aside from this increase in volume and tissue mass, the gallbladders appeared normal, both on gross and histologic examination. There were no obvious changes in other intraabdominal or intrathoracic organs.

EFFECTS OF CHOLESTEROL FEEDING ON BILE Cholesterol feeding resulted in the presence of a fine

light brown-yellow precipitate in the dependent portion of the gallbladder. Light brown to black concretions, measuring less than 2 mm in diameter, were found among the fine brown-yellow precipitate. On light microscopic inspection, the precipitate was similar in appearance to the calcium bilirubinate crystals described by Juniper and Burson (18). The black stones, when crushed, were shown to consist of an opaque golden yellow mateial which had the same appearance as the pigment stones described by Bernhoft et al. (8) (Figure 2). Thin-layer chromatography of the stone material demonstrated the presence of a single spot of pigmented material (Rf = 0.67) which co-migrated with unconjugated bilirubin I X a (Rf = 0.68). Cholesterol monohydrate crystals were not found in any gallbladder.

The composition of gallbladder and hepatic bile is

FIG. 1. Liver obtained from a cholesterol-fed guinea pig showing fatty infiltration (H & E stain).

TABLE 1. EFFECTS OF A 0.5% CHOLESTEROL-SUPPLEMENTED DIET IN THE GUINEA PIG (MEAN f S.E.) ~~~~ ~ ~~

Control fed (44) Cholesterol fed (39) Group (n)

6 weeks (22) 12 weeks (22) 6 weeks (23) 12 weeks (16)

Cumulative mortality Weight gain (gm) Hematocrit (%) Serum cholesterol (mg/dl) Gallbladder volume (ml) Gallbladder dry weight (mg) Gallstone incidence

2/44 2/44 0139 113 f 18 246 k 24 136 f 18

54.8 f 1.3 59.8 f 3.0 47.6 f 1.2' 59 f 22 - 202 f 34' 1.7 f 0.11 1.8 f 0.16 2.4 f 0.18*

0120 2/22 17/23d 21.6 f 2.3* 12.6 f 0.6 -

5/39" 183 * 33

38.0 f 4.5' 138 f 18' 3.5 f 0.58'

l l / l l d

Differs from control 12 weeks by x 2 analysis (p < 0.025).

Differs from control 12 weeks by Student's t test (p < 0.0025). Differs from control 6 weeks by xz analysis (p < 0.025).

' Differs from control 6 weeks by Student's t test (p < 0.025).

24 LAMORTE ET AL. HEPATOLOGY

presented in Table 2. Concentrations of both total bile salts and cholesterol were higher in hepatic bile collected from cholesterol-fed guinea pigs, while the concentration of phospholipids was unchanged. In contrast, the concentration of total bile salts in fasting gallbladder bile was significantly less after 6 weeks of cholesterol feeding, although cholesterol concentrations in gallbladder bile were significantly higher in the cholesterol-fed animals. Despite the increase in cholesterol concentration, gallbladder bile never became supersaturated with cholesterol, although the lithogenic indices calculated by the method of Carey and Small (19) did increase in the cholesterol-fed group (Table 3).

Consistent with the findings of others (20), the concentrations of total bile salts, phospholipids and cholesterol were markedly lower in the guinea pig than in humans, and bile salts accounted for more than 98% of biliary lipids. Lipid extractable phosphorus did not differ

FIG. 2. Gallbladder stones and crystals obtained from a cholesterol- fed guinea pig. Two types of precipitate are demonstrated in this specimen. The large clumps (large arrowhead) consist of aggregates of fine crystals similar to those described as calcium bilirubinate by Juniper and Burson (18). Amorphous golden-yellow material (small arrowhead), morphologically similar to the pigmented stones reported by Bernhoft et al. (8) was also present.

TABLE 2. CONCENTRATIONS OF TOTAL BILE SALTS,

HEPATIC BILE (MMOLES/LlTER f S.E.) PHOSPHOLIPIDS AND CHOLESTEROL IN GALLBLADDER AND

Cholesterol diet ~ - Control &et

'n = ') 6 weeks ( n = 9 ) 12 weeks ( n = 7)

Gallbladder bile Bile salts 18.56 -C 2.19 13.79 f 1.51" 15.62 f 1.85 Phospholipids 0.0894 f 0.0169 0.0857 f 0.0207 0.0816 f 0.0188 Cholesterol 0.0053 2 0.0011 0.0169 -C 0.002' 0.0171 f 0.0057"

Hepatic bile Bile salts 3.39 ? 0.33 5.91 ? 0.6gh 6.61 2 1.58" Phospholipids 0.059 ? 0.010 0.046 2 0.013 0.060 2 0.018 Cholesterol 0.0012 f 0.0004 0.0070 f 0.0005' 0.0043 k 0.0017"

Measurements for hepatic bile are from the hepatic effluent collected

"Differs from control (p < 0.05). 'Differs from control (p < 0.01).

over a 60-min period.

TABLE 3. RELATIVE COMPOSITION OF GALLBLADDER BILE (MEAN k S.E.)

6-week 6-week control diet cholesterol diet

Total bile salts ( % ) 99.58 f 0.10 99.16 f 0.22 Phospholipids (%) 0.39 k 0.10 0.71 f 0.22 Cholesterol (041) 0.03 f 0.01 0.13 f 0.02"

0.72 f 0.08' Total concentration' (gm/

PL/(PL+BS) 0.0065 f 0.0014 0.0105 0.0033 Calculated maximum solu- 0.67 f 0.03 0.54 -+ 0.07

Lithogenic indexd 0.04 2 0.01 0.22 f 0.04"

0.96 f 0.11 dl)

ble cholesterol (%)

The abbreviations used are: PL, phospholipid concentration (gm/ dl); BS, total bile salt concentration (gm/dl).

p < 0.005. Total concentration was calculated as the sum of total bile salts

p < 0.05. The maximum solubility of cholesterol, as a per cent of total biliary

lipids, was calculated using the fifth degree polynomial coefficients of Carey and Small (19).

and phospholipids and cholesterol in grams per deciliter.

as a result of cholesterol feeding. Glycoprotein concentrations in the gallbladder bile of cholesterol-fed guinea pigs at 6 weeks (2.00 f 0.38 pg per ml, n = 10) and at 12 weeks (1.65 f 0.48 pg per ml, n = 7) did not differ significantly from that in controls a t 6 weeks (1.64 +_

0.29, n = 8). Estimates of the viscosity of gallbladder bile relative to distilled water also were unchanged by cholesterol feeding. In addition, histologic sections of gallbladder stained with periodic acid-Schiff/Alcian blue demonstrated a similar density of glycoprotein granules in the mucosa of control and cholesterol-fed animals. The pH of both hepatic and gallbladder bile was consistently in the range of 8.14 to 8.20 in both the controls and the cholesterol-fed guinea pigs.

EFFECTS OF CHOLESTEROL FEEDING ON GALLBLADDER CONTRACTILITY Figure 3 illustrates dose-related changes in full thick-

ness gallbladder strips in response to octapeptide cholecystokinin. Cholesterol feeding did not significantly alter the contractile response to CCK-OP even after 12 weeks of cholesterol supplementation.

GALLBLADDER FLUID ABSORPTION IN VITRO Each animal studied showed a constant rate of absorp-

tion over the period of observation (Figure 4). Gallblad- der fluid absorption per milligram of tissue mass was markedly lower among cholesterol-fed animals (6.0 k 1.4 mg fluid per hr per mg dry weight) than in the control group (12.9 t- 0.9 mg fluid per hr per mg dry weight, p < 0.0005). Because of the greater gallbladder tissue mass among cholesterol fed animals, however, both groups absorbed approximately the same amount of fluid per gallbladder (134.8 k 8.1 mg fluid per hr among controls vs. 146.5 k 29.6 among cholesterol-fed animals, p > 0.30).

['*C]CDCA was translocated from the lumen to the serosal bath at a constant rate in both groups. The data relating counts per minute per milliliter of serosal fluid as a function of time were subjected to linear regression;

Vol. 5, No. 1, 1985 PIGMENT GALLSTONES IN THE GUINEA PIG

n cn E 1.5- c"

9) 0) C

Y

2 1.0.-

.- 5 0

cn C I-" 0.5-

0.0-

25

- 6 WK CONTROL O......O 6 WK CHOLESTEROL

0---0 12 WK CHOLESTEROL

z 15

w c " 2.5 I- -?

NORMAL / I

15 30 45 60

TIME (min) FIG. 4. Cumulative absorption of fluid from the guinea pig gallblad-

der in oitro. A pool of hepatic bile was used as the intraluminal fluid for animals on the control diet (n = 13) and for those on the 0.5% cholesterol diet (n = 6). Results are mean f S.E.

for each animal the slope was taken as a measured of the rate of [ 14C]CDCA absorption. Cholesterol-fed animals translocated [14C]CDCA at a rate of 0.057 k 0.008 cpm per min per mg dry tissue weight while the rate for animals fed trace cholesterol was 0.062 f 0.01 (p > 0.35). Unconjugated bile salts are absorbed from the gallbladder by passive ionic diffusion (21). Consequently, fluid absorption was significantly impaired, but passive ionic diffusion was unchanged.

HEPATIC BILE SECRETION Cholesterol feeding resulted in a marked increase in

bile flow from the liver (Figure 5), and in addition the concentration of total bile salts in hepatic bile was significantly higher during bile salt pool washout in animals receiving the cholesterol diet. Consequently, bile salt secretion rates were also elevated in the cholesterol-fed group (Figure 6). Bile secretory pressure in cholesterol-

- Control 6 Wk

10 20 30 40 50 60

TIME (min) FIG. 5. Bile flow from the common hepatic duct (CHD) during total

bile collection. The volume of bile secretion measured at 10-min intervals was consistently greater in guinea pigs that had received the cholesterol-supplemented diet (broken line, n = 11) than in controls (solid line, n = 10). Results represent mean f S.E. *, p < 0.005.

1' i * 0.52 Cholesterol Diet-6 Week

** T

\T' Control Diet -6 Week T

10 20 30 40 50 60

Time (min) FIG. 6. Total bile salt secretion during total bile collection. Each bile

specimen collected during the studies illustrated in Fig. 5 was assayed for total bile salt concentration, and bile salt secretion was then calculated at 10-minute intervals. Total bile salt secretion from cholesterol-fed guinea pigs (broken line) was greater than that of controls (solid line). Results represent mean f S.E. *, p < 0.05; **, p < 0.01.

fed animals (17.0 f 1.3 cm H20, n = 3) did not differ from controls (19.0 k 0.2 cm H20, n = 3).

DISCUSSION The major finding in this study was that supplemen-

tation of the guinea pigs diet with cholesterol unexpectedly resulted in the formation of pigment gallstones. Golden-brown to black pigmentation was apparent on gross inspection, and light microscopy showed that the stone material consisted primarily of masses of small granular crystals resembling the calcium bilirubinate crystals described by Juniper and Burson (18); lesser amounts of yellow amorphous solids were also present. Finally, the solubility of this material in sodium hydroxide and its ability to co-migrate with unconjugated bili-

26 LAMORTE ET AL. HEPATOLOGY

rubin during thin-layer chromatography further support the conclusion that these were bilirubinate stones.

In retrospect, the total absence of cholesterol crystals is not surprising. The secretion of biliary cholesterol was elevated, but bile salt secretion and total bile salt concentration in hepatic bile were markedly increased also. As a result, gallbladder bile remained unsaturated with respect to cholesterol. These observations suggest that the guinea pig has a remarkable capacity to convert excess cholesterol to bile acids. This view is supported by reports that 7tu-hydroxylase activity is dependent on vitamin C ( 2 2 ) , and only if marginally deficient in vitamin C, does the cholesterol-fed guinea pig’s choles- terolbile salt ratio become sufficiently elevated to result in cholesterol gallstone formation (23, 24).

Bilirubin concentrations were not measured in these studies, but increased bilirubin secretion may have oc- curred in the cholesterol-fed animals. Okey and Greaves reported that a 1% cholesterol diet produced hemolytic anemia in male and female guinea pigs (17), suggesting that both have a potential source of excess unconjugated bilirubin. The significant decline in hematocrit observed in our cholesterol-fed animals suggests that hemolysis was present. Trotman et al. have reported substantially increased concentrations of unconjugated bilirubin in hepatic bile from a strain of mice with an inherited hemolytic anemia (7 , 25, 26). Since hemolysis-induced gallstones form in the gallbladder rather than in the biliary ducts, these authors concluded that the solubility product of calcium bilirubinate in hepatic bile was not exceeded, and that precipitation did not occur until the abnormal bile entered the gallbladder where modification of bile produced conditions conducive to stone formation. They further suggested that gallbladder stasis and concentration of bile within the gallbladder promoted precipitation of calcium bilirubinate (7).

Gallstones were limited to the gallbladder in our study also, but we found no evidence of gallbladder stasis despite a significant increase in fasting gallbladder volume. First, there was no evidence that stone material had caused obstruction of the cystic duct or the chole- dochoduodenal junction. Second, in uitro contraction of gallbladder strips in response to octapeptide cholecystokinin appeared normal even after 12 weeks of cholesterol feeding. In addition, attempts to assess gallbladder stasis as described by Kuckenbecker et al. (27) suggested that cholesterol fed animals had less gallbladder stasis than controls (data not shown). However, bile flow rates were significantly increased during cholesterol feeding, raising the possibility that the apparent decrease in stasis was actually an artifact produced by the increased flow of hepatic bile.

Gallbladder distention can produce hyperplasia (28, 29), and it is likely that the marked increase in bile flow observed in these studies was responsible for the increase in gallbladder fasting volume and gallbladder tissue mass. A secondary factor which may have contributed to the volume increase was a significant decrease in the gallbladder’s ability to concentrate hepatic bile. In contrast, others have reported increases in gallbladder concentrat- ing ability prior to gallstone formation (5), and we are

unable to account for this difference. It should be noted that in our studies the greater gallbladder tissue mass probably compensated for the absorption defect to some extent. Regardless of their relative contributions, enhanced hepatic bile flow and depressed gallbladder fluid absorption were probably responsible for the observed increase in gallbladder volume. Additional support for this conclusion is provided by the observation that the concentration of bile salts within the gallbladder was decreased after 6 weeks of cholesterol feeding. This may also have resulted from absorption of bile salts from the gallbladder, as reported by others (30).

Others have demonstrated that stasis contributes to the precipitation of unconjugated bilirubin in some animal models (8), but the lack of evidence for stasis in the studies reported here suggests that the gallbladder contributed to precipitation in some other way. Secretion of mucin by the gallbladder epithelium might contribute to the precipitation of cholesterol and bilirubin (6 , 31). Our studies do not eliminate the possibility that small amounts of mucin in the guinea pig gallbladder enhanced precipitation of bilirubin. On the other hand, there are a number of objections to this explanation in our model. The concentration of glycoproteins in gallbladder bile was not increased after 6 weeks of cholesterol feeding, nor was there any change in bile viscosity. Neither of these measurements are specific for mucin, however, and it might also be argued that the small amount of mucous substances normally present in guinea pig bile might be sufficient to initiate precipitation of unconjugated bilirubin if, in fact, the concentration of unconjugated bilirubin were increased as a result of hemolysis, for example.

Micelles probably contribute to the solubility of unconjugated bilirubin (32) and calcium (33) in bile. For example, Bickerstaff and Moosa found that prairie dogs on a trace cholesterol diet formed pigmented gallstones after ileal resection or bypass (34). These bilirubin stones were associated with marked decreases in the concentration of bile salts and phospholipids in gallbladder bile, while cholesterol concentration was less dramatically decreased. It is also of interest that in Bickerstaff and Moosa’s study and in ours the lithogenic index had increased in the experimental groups, but not to the saturation point for cholesterol. Possibily, the solubility of unconjugated bilirubin and cholesterol are both dependent upon micelles and perhaps even compete for them. In the cholesterol-fed guinea pig, the increase in cholesterol concentration, combined with a less than normal concentration of bile salts in the gallbladder, might be sufficient to interfere with the solubilization of unconjugated bilirubin by micelles. Furthermore, a pro- gressive decline in the bile salt/cholesterol ratio might result in bilirubin precipitation before cholesterol precipitation. Although speculative, this explanation might also account for the common occurrence of calcium bilirubinate at the core of cholesterol gallstones.

The cholesterol-fed guinea pig may be a useful model for pigment gallstone formation and may provide an- swers for some of these issues. However, the studies reported here are limited to the extent that concentra-

Vol. 5, No. 1, 1985 PIGMENT GALLSTONES IN T H E GUINEA PIG 27

tions of conjugated and unconjugated bilirubin were not measured, and future studies will need to document changes in their concentration before gallstone pathogenesis can be fully understood in this model.

15. Mantle M, Allen A. A colorimetric assay for glycoproteins based on the periodic acid/Schiff stain. Biochem soc Trans 1978; 6607- 609.

16. Diamond JM. The reabsorptive function of the gallbladder. J Physiol 1962; 161:442-473.

17. Okey R, Greaves VD. Anemia caused by feeding cholesterol to guinea pigs. J Biol Chem 1939 129:lll-123.

18. Juniper K Jr, Burson EN Jr . Biliary tract studies. 11. The signifi-

19. Carey MC, Small DM. The physical solubility of cholesterol in bile:

Acknowledgments: We are indebted to Dr. J. T. La- Mont and Dr. B. F. Smith of the Gastroenterology Set-

advice cance of biliary crystals. Gastroenterlogy 1957; 32:175-211. University Boston' for and discussion during the preparation Of this manuscript. relationship of gallstone formation and dissolution in man. J Clin

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