Absorption of Water, Unconjugated Bilirubin, and Sodium Glycodeoxycholate by the Rabbit Gallbladder with Dietary-Induced Gallstones

Vol. 61 , No. 5 Printed in U.S.A.

GASTROENTEHOLOGY

Copyright © 1971 by The Williams & Wilkins Co.

ABSORPTION OF WATER, UNCONJUGATED BILIRUBIN, AND SODIUM GL YCODEOXYCHOLATE BY THE RABBIT GALLBLADDER WITH DIETARY-INDUCED GALLSTONES PATRICIA A. Kvo, AND IAN A. D. Bo uCHIER, M.D. , F.R.C .P.

The Medical Unit, The Royal Free Hospital, London, England

Rabbits fed a diet containing 40% casein, 15% oleic acid, and 45% laboratory pellets developed gallstones comprised mainly of the salts of glycoallodeoxycholic acid and glycodeoxycholic acid. This experimental model was used to determine whether cholelithiasis was accompanied by changes in gallbladder function. An in vitro everted sac technique was used, and the viability of the gallbladder was assessed by its ability to transport water. The absorption of 14 C-bilirubin and 14 Cglycodeoxycholate was demonstrated in control rabbit gallbladders. 2, 4-Dinitrophenol completely inhibited water absorption but had no effect on the absorption of bilirubin and bile salts, indicating a passive transport mechanism for both these compounds. Stone-forming gallbladders showed no difference in the rate of water absorption compared with control gallbladders, suggesting that the formation of gallstones did not impair the ability of the gallbladder to concentrate bile. Gallbladders with stones showed no change in bilirubin absorption compared with controls, but there was a significant decrease (1 c;. level) in the rate of glycodeoxycholate absorption. It is concluded that a change in gallbladder function, in particular an increase in bile salt absorption, is not a factor associated with the formation of gallstones in this model. Although gallstone disease in man is usually associated with the presence of a gallbladder it remains to be resolved whether altered gallbladder function contributes to, or is essential for , gallstone formation. The purpose of these studies was to find out whether the formation of gallstones in an animal model was associated with changes in the permeability of the gallbladder mucosa toward the organic constituents of bile. In particular, inReceived March 30, 1971. Accepted June 23, 1971. Address reprint requests to: Dr. I. A. D. Bouchier, Medical Unit, Royal Free Hospital, Gray's Inn Road, London W . C. 1., England. This work was supported by a grant from the Medical Research Council. The authors are grateful to Dr. B . H. Billing for much helpful advice, and to Mrs. J. Paxton and Mr. A. Shaw for technical assistance. Dr. H . Gregory kindly reported on the histology.

creased absorption of bile salts or phospholipids could reduce the cholesterol-holding capacity of gallbladder bile. A dietary model of cholelithiasis using the rabbit was chosen. This model was chosen not only because of the suitability of the rabbit gallbladder for absorption studies, but also because the gallstones formed were reported to contain cholesterol as a major constituent. 1 Until recently, investigations of gallbladder absorption have been confined principally to water and electrolyte uptake, although Ostrow 2 • 3 has demonstrated the ability of the gallbladder in vivo to absorb bilirubin 2 and bile salts. 3 In this study, the in vitro absorption of unconjugated bilirubin and sodium glycodeoxycholate, the major rabbit bile salt, by gallbladders of normal rabbits has been compared with absorption by gallbladders of rabbits with

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LIVER PHYSIOLOGY AND DISEASE

gallstones. At the same time, water absorption has been measured, not only as a check on viability, but also to determine whether the presence of gallstones was accompanied by impaired ability of the gallbladder to concentrate bile. An everted sac preparation was used because it permitted serial measurements to be made on the amounts of ''C-bilirubin or bile salt transferred to the serosal solution.

Materials and Methods Diets. Male New Zealand white rabbits, weighing 2.5 to 4.1 kg at the beginning of the experiment, were used . The control diet consisted of SG1 pellets [composition: 40 ~(, bran, 18% middlings, 4% ground oats, 10% white fish meal, 20% dry grass meal, and including 20.3)(; crude protein, 9.05 % root fiber, and 4.18% oil (Oxoid, London)]. The feeding of the lithogenic diet differed slightly in the three trials which were performed. The diet used in trial 1 was a modification of the diet used by Borgman' and consisted of 40% casein [casein, light white soluble, (British Drug House Chemicals, Ltd., Poole, England)], 15% oleic acid [oleic acid, 98 to 100.5% (British Drug House Chemicals, Ltd.)] and 45% SG 1 pellets. For trial 2 and trial 3 this was further modified by the addition of 6.8% sucrose and 6.5% glucose instead of SG1 pellets. This alteration increased the frequency of stone formation but had no discernible effects on the composition of the stones. Test rabbits were fed 100 g per day of the lithogenic diet for 16 weeks in trials 1 and 2, and for 8 weeks in trial 3. Control rabbits were fed SG1 pellets, ad libitum in trials 1 and 3, and in trial 2 they were pair-fed with a test rabbit. Preparation of "C-bilirubin. "C-labeled unconjugated bilirubin was prepared biosynthetically.' It was recrystallized • to constant specific activitJ:', 1.64 X 10' dpm per 1-Lmole. Preparatwn of "C-sodium glycodeoxycholate. Using the micromethod of Norman, • 98.4 mg of chromatographically pure deoxycholic acid were conjugated with 20.6 mg of glycine-2''C (100 !-LC) (The Radiochemical Centre, Amersham, England). "C-glycodeoxycholic acid was recrystallized from ethanol-water. The sodium salt was prepared • and recrystallized twice from 90% ethanol-ether (1: 1) . Trace impurities of deoxycholic acid were removed by purification on thin layer chromatography (TLC) using silica gel (silica gel, MFC, Hopkin and Williams, Ltd., Chadwell Heath, England) with the solvent system glacial acetic acid-ethyl-

Vol. 61 , No. 5

ene dichloride-water (10: 10 : 1, v/ v). The glycodeoxycholate band was detected with iodine vapor and eluted with hot 50% ethanol pH 10.0. The ''C-sodium glycodeoxycholate solution in 50% ethanol moved as a single spot on TLC. Determination of the total bile acid concentration 7 showed the specific activity to be 4.1 x 10 5 dpm per 1-Lmole. Preparation of everted gallbladder. Rabbits were fasted overnight in trials 1 and 3 but not in trial 2. They were anesthetized with Nembutal and ether and the gallbladder was carefully and rapidly removed. The bile was collected by drainage from the cystic duct and stored at -20 C. The gallbladder was everted and tied on the lower end of a glass cannula (fig. 1) using the method of Crane and Wilson. 8 Before adding the labeled solution for absorption studies, the gallbladder was incubated in oxygenated Krebs-Ringer bicarbonate buffer (KRB), pH 7.4, at 37 C. This delay was usually about 15 min. Incubation media. (1) For bilirubin: sufficient "C-bilirubin for each experiment was stored in the dark at - 20 C. It was dissolved in 0.1 ml of 0.1 N NaOH, and mixed with 7.9 ml of human gallbladder bile diluted 1:50 with KRB buffer, pH 7.4. The final pH was 7.65 and altered by less than 0.1 pH unit during incubation. "C-bilirubin did not precipitate

0

FIG. 1. Apparatus used for absorption studies. A, mucosal solution; B, serosal solution; C, mucosal surface of everted gallbladder; D, inlet tubing carrying 95 ~r 0, and 5 ~ i CO , ; E , glass cannula ; F, outlet tubing.

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LIVER PHYSIOLOGY AND DISEASE

from this solution as it did from buffer alone. The ' 4 C-bilirubin was more stable in human bile than in other suspending media tested (dilute bile salt solution and rabbit bile). The mucosal solution contained 0.52 JliDOle and 8.5 x 10• dpm of 14 C-bilirubin in a total volume of 8.0 mi. Eighty-six per cent of the radioactivity was recovered in the chloroform phase under acidic conditions after 2 hr of incubation. The serosal solution contained 6.25 mg per ml of reconstituted human serum albumin in KRB buffer, pH 7.4. The addition of albumin ensured removal of transported 14 C-bilirubin from the serosal surface. (2) For bile salts: the mucosal solution contained 0.1 mM 14 C-sodium glycodeoxycholate (3.28 x 10 5 dpm per 0.8 llmole per 8 ml). 14 Csodium glycodeoxycholate (0.8 Jlmole) in 50% ethanol was taken to dryness and redissolved in 8 ml of calcium-free KRB buffer, pH 7.4. The final pH was 7.8 and did not alter during the period of incubation of either the control or the test gallbladders. The serosal solution for bile salt absorption studies was calcium-free KRB buffer, pH 7.4. Absorption m ethod. The absorption of bili rubin and bile salts in vitro was measured by the rate of transfer of the 14 C-labeled compound from the mucosal to serosal surface of the gallbladder. The mucosal and serosal surfaces of the everted gallbladder were washed with KRB buffer. A small volume (0.1 to 0.5 ml) of the appropriate serosal solution was instilled into the gallbladder, and the whole cannula preparation was weighed after gently blotting the gallbladder. The preparation was then incubated (fig. 1) at 37 C for 15 to 20 min in the appropriate mucosal solution which was continuously gassed with 95% 0 2 -5% C0 2 • At the end of the incubation period the cannula preparation was removed from the mucosal solution and reweighed; the increase in weight was considered to be due to water absorption. The serosal solution was then aspirated into a counting vial, the serosal surface was washed with small volumes of fresh serosal solution, the washings were added to the counting vial, and the total radioactivity was counted. Fresh serosal solution was instilled into the gallbladder, the preparation was reweighed, and then reincubated for a further period in the mucosal solution. This procedure was repeated at 15- or 20-min intervals. Absorption of water and bilirubin or bile salt was measured for a total of 2 hr incubation time . The cumulative water absorbed and 14C radioactivity absorbed were plotted and the rates of absorption were calcu-

725

lated for each gallbladder from the linear portion of the curve. At the end of each experiment the everted gallbladder preparation was incubated in KRB containing Evan's blue dye -the appearance of blue color in the serosal solution would indicate a leak in the preparation. This was observed in only one experiment which was consequently discarded. The gallbladder was then removed from the cannula, blotted, and weighed. 14 C-bilirubin present in the gallbladder wall was extracted by homogenizing the gallbladder in 5 ml of chloroform-methanol (2: 1) and 1 ml of 4 N H 2 SO •. Mter centrifugation, the lower chloroform layer containing the extracted bilirubin was added to a counting vial and taken to dryness under N 2 • In the bile salt absorption experiments, the gallbladder was homogenized in 5 ml of 50 % ethanol adjusted to pH 10.0, and the bile salts were extracted at 70 C for 1 hr. Mter centrifugation the ethanolic extract was taken to dryness, redissolved in 2 ml of ethanol, and counted. Three experiments were performed to establish whether the radioactivity in the serosal fluid was present as 14 C-bilirubin or as breakdown products of bilirubin. At the end of the absorption experiments an aliquot each of mucosal fluid, serosal fluid, and gallbladder extract was counted immediately. Carrier bilirubin (1 mg) was added to another aliquot of each sample and the radioactivity of the total recrystallized bilirubin was determined. The percentage of the total radioactivity of each sample recovered in the recrystallized bilirubin fraction represented the percentage of 14 C remaining as 14 C-bilirubin. After correction for bilirubin lost during recrystallization (on average 5%) the percentages were: (a) mucosal fluid , 82.7% and 73.1%; (b) serosal fluid, 70.5%, 69.7%, and 57.9%; (c) gallbladder wall, 96.1% and 67.5 %. The missing values for mucosal fluid and gallbladder wall were due to an accident. Mucosal fluid incubated in the absence of a gallbladder preparation retained 85. 75(; of radioactivity as 14 C-bilirubin . These results suggest that, although some breakdown of 14 Cbilirubin in the mucosal and serosal fluids is inevitable, the great majority of radioactivity present in the serosal fluid and gallbladder wall was absorbed as 14C-bilirubin. Similar experiments were performed to establish whether the radioactivity absorbed in the bile salt experiments was still in the form of 14 C-glycodeoxycholate. The percentage of the total radioactivity recovered in the glycodeoxycholate band on TLC (see above) was deter-

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LIVER PHYSIOLOGY AND DISEASE

Vol. 61 , No. 5

with heptane-ether (1: 1, v/v) and assayed by mined at the end of absorption experiments: (a) mucosal fluid, 87% and 92%; (b) serosal the method of Zlatkis et al., 11 except that 2.0 fluid, 99% and 98%; (c) gallbladder wall, 108% ml of glacial acetic acid and 1.34 ml of color and 88%. These results suggest that very little reagent were used per test. Analysis of gallstones. The gallstones were deconjugation of glycodeoxycholic acid had occurred during incubation and that the radio- removed from the gallbiadder bile by centrifuactivity absorbed by the gallbladder was in the gation at 2000 rpm for 10 min. The gallstone precipitate was extracted with chloroformform of ''C-glycodeoxycholate. Radioactivity assay. Before measuring the methanol (1: 1, v/ v). Lipid separation of the activity of "C-bilirubin, color quenching had to chloroform layer using TLC on silica gel was be removed. Aliquots of the "C-bilirubin prep- performed with the solvent system heptanearation in chloroform were taken to dryness in a counting vial. To these vials and those con~ 400 taining the serosal fluid plus washings of the bilirubin absorption experiments were added 0.2 ml of NCS Solubilizer (Amersham/Searle Corp., Des Plaines, Ill.) and 0.2 ml of 30% w/v hydrogen peroxide. The vials were sealed and heated at 60 C for 1 hr. After cooling, 15 ml of scintillator [2 , 5-diphenyloxazole, 4 g per liter, and 1, 4-bis(2-(5-phenyloxazolyl) benzene, 50 mg per liter, dissolved in toluene]-Triton X-100 (British Drug House Chemicals, Ltd.) (2: 1) mixture were added and the vials counted in a Nuclear-Chicago liquid scintillation spectrometer using a channel ratio method. The efficiency of counting was 55 to 70%. In the bile salt absorption experiments the serosal fluid and washings were assayed for "C activity by the addition of 15 ml of scintillator120 60 90 30 Triton X-100 mixture, and were subsequently IN CUBATION TIME I mins J counted in a Packard Tri-Carb liquid scintilFIG. 2. A representafive .experiment of cumulative lator spectrometer. The radioactivity of the transport by the gallbladder of "C-bilirubin from "C-sodium glycodeoxycholate preparation was measured similarly. The efficiency of counting mucosal to serosal s.urfaces. was 77 to 87%. Calculations. From the radioactivity counts of the serosal fluid and washings, the total dis.,:;]Q: integrations per min of "C-bilirubin or "C.,"'0 glycodeoxycholate absorbed was plotted against time of incubation (figs. 2 and 3). The rate of " 30 00 ~ 14 absorption of "C-bilirubin or C-glycodeoxy" 0~ cholate was calculated from the linear portion co.. of the absorption curve and expressed as dpm "" "' per hr per 100 mg (wet weight) of gallbladder. e~ 20 00 "' ..."' The percentage of the total dose of "C-hili..,oo rubin or "C-glycodeoxycholate in the mucosal ~ -~ solution absorbed by each gallbladder was cal'-' :t I 000 culated from the total disintegrations per min ~ ;:: absorbed over the 2-hr incubation period plus that present in the gallbladder wall. It was ex" ~ pressed as a percentage per 100 mg (wet ~ weight) of gallbladder. " 90 120 60 30 Analysis of gallbladder biles. Mixed whole INCUBATION TIME I mins l biles were analyzed for total bile salts by the method of Iwata and Yamasaki. 7 Phospholipids Frc. 3. Cumulative transport of "C-glycodeoxywere measured by the method of King• after cholate from muco·s~l to serosal surfaces in a repreFolch extraction. 1 ° Cholesterol was extracted sentative experiment.

"

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LIVER PHYSIOLOGY AND DISEASE

ether-glacial acetic acid (60:40: 1, v/v). Bile salts in the upper layer were separated using the solvent system isooctane-isopropyl etherisopropyl alcohol-glacial acetic acid (2: 1 : 1 : 1, v/v). Histology . After the absorption studies, portions of the gallbladders were fixed in 10% formol-saline solution. Sections were stained with hematoxylin and eosin and periodic acidSchiff reagent. No differences were apparent between control and gallstone-containing gallbladders. The presence of gallstones was not associated with either mucosal damage or inflammation. Statistical analysis of results. The results of the analysis of gallbladder biles were analyzed using Student's t-test for the difference of the means. The results of absorption studies were analyzed using the Wilcoxon rank-sum test. A nonparametric test was used because of the small number of observations, and because the normality of their distribution was uncertain.

Results Incidence and composition of stones. None of the control animals developed gallstones. Gallstones were present in 21 of 24 test rabbits which had been fed the lith-

FIG .

ogenic diet (table 1) . The stones were not large concrements but appeared as numerous fine white pinhead-sized granules, clearly visible as discrete stones (fig. 4). In trial 3, when the lithogenic diet was fed for only 8 weeks, the gallstones were less numerous than in trials 1 and 2. The appearance of these gallstones closely resembled stones obtained by feeding rabbits cholestanol (0. 75 g per day) for 14 days. Lipid separation of chloroform-methanol extracts of the gallstone samples showed that cholesterol was present in trace quanTABLE

1. Incidence of gallstone formation No. of test

No. of test rabbits with ga llstones

Weeks

rabb its fed

Trial1" Trial 2•

16 16

11 7

9 6

Trial3•

8

6

6

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lithogeni c diet

Absorption stu di es undertaken

Bilirubin Sodium glycodeoxycholate Sodium glyco· deoxycholate

a Nature of diet: 40% casein, 15% oleic acid, 45% SG 1 pellets. • Nature of diet: 40% casein, 15% oleic acid, 6.8% sucrose, 6.5% glucose, 31.7% SG 1 pellets.

4. Representative example of the gallstones developed (X 450).

LIVER PHYSIOLOGY AND DISEASE

728

tities only. The major component of the stones was in the aqueous layer and appeared to be a bile acid with the same mobility as glycodeoxycholic acid. A much smaller spot with a mobility corresponding to that of glycolithocholic acid was obtained in some gallstone samples. The presence of cholesterol monohydrate could not be demonstrated by X-ray diffraction (kindly performed by Dr. J. Sutor, University College, London) , but the diffraction bands of gallstone samples were shown to be identical with the diffraction bands of gallstones from cholestanol-fed rabbits. Hofmann and Mosbach 12 have established that the major component of the latter stones is glycoallodeoxycholic acid. Composition of gallbladder biles. The biles of rabbits fed the lithogenic diets showed a significant increase in cholesterol content calculated as a percentage of the summation of bile salts, cholesterol, and phospholipids expressed as millimoles per liter (table 2). The relative percentages of phospholipids and bile salts were no different in the two groups in trials 1 and 2; in trial 3 phospholipids were significantly elevated in the test group and bile salts were significantly lowered compared with the control group. The significant differences observed in trial 3 reflect the smaller variations in bile composition between animals compared with trials 1 and 2. Water absorption. The cumulative net transport of water from mucosal to serosal TABLE

Trial

surface was linear with incubation time in all three trials. In some experiments, the rate of water absorptiOn fell off after 90 min of incubation. The rate of water transport was calculated from theJinear portion of the absorption curvi{arid was higher in trial 3 than in trials 1 imd 2 (fig. 5). An explanation for this is ri.ot.clear but may be due to an unknown improvement in technique. Another possibility is that the rate of water transport was .influenced by the differences in composition · of the mucosal and serosal solutions in trial 1 compared with trials 2 and 3. There was no difference in the rate of wat~i}ransport between the control group and test group in trial 1 (bilirubin absorption) (fig. 5A). The results of trials 2 and 3 (sodium glycodeoxycholate absorption) were combined to increase the numbers in each group, >and there was no significant difference .(Wilcoxon test) between the controLandtest groups (fig. 5B). Trial 1: absorption of bilirubin. 14 C-bilirubin appeared on the · serosal side of the gallbladder within 15 min of incubation. After an initial lag, the cumulative absorption of bilirubin was"linear with the time of incubation. A typ,ical example is shown in figure 2. The rate of absorption per hr was calculated from the. linear portion of the curve and expressed as disintegrations per min of 14 C-bilirubin per hr per 100 mg (wet weight). There was considerable variation in the 14 C-bilirubirt absorption between gallbladders (fig. ·6). There was no

2. Composition of gallbladder biles Percentage mol fracti ona

Diet group

No. in group Bile salt s

1

2

3

Vol. 61, No . 5

Control

10

Test

11

Control

5

Test

6

Control

6

Test

6

a Mean ± SD. • NS, not significant.

Phospholipid

Cholesterol

97.7 ± 0.9 (NS)' 96.0 ± 0.5

1.16 ± 0.42 (NS) 2.21 ± 2.20

1.17 ± 0.54 (P < 0.01) 1.80 ± 0.48

97.7 ± 0.5 (NS) 97.5 ± 0.5

1.39 ± 0.39 (NS) 1.42 ± 0 :3_3

0.80 ± 0.10 (P < 0.05) 1.08 ± 0.21

98.6 ± 0. 3 (P < 0.001) 97.2 ± 0.4

0.82 ± 0.24 (P < 0.01) 1.45 ± 0.39

0.61 ± 0.09 (P < 0.001) 1.31 ± 0.11

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LIVER PHYSIOLOGY AND DISEASE

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sorbed by each gallbladder over the 2-hr period, a mean of 33.0 5~ ± 5. 7 SE was recovered in the gallbladder wall and 67.0% ± 7.3 SE in the serosal fluid. Effect of dinitrophenol. 2, 4-dinitrophenol (DNP), a potent metabolic inhibitor, has been shown to inhibit water trans-

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significant difference between the control and test groups in the rate of bilirubin absorption per hr, or in the percentage of the total dose of 14 C-bilirubin absorbed over the 2-hr incubation period. Of the total 14 C-bilirubin absorbed during 2 hr of incubation, a mean of 30.4% ± 9.4 SE was recovered in the gallbladder wall and 69.6 )0 ± 9.4 SE in the serosal fluid. Trials 2 and 3: absorption of glycodeoxycholate. The total amount of 14 C-glycodeoxycholate absorbed was linear with the time of incubation, after an initial lag (fig. 3). As with 14 C-bilirubin absorption, there was considerable variation in the rates of 14 C-glycodeoxycholate absorption by individual gallbladders, both in the control and the test groups (fig. 7). The results of trials 2 and 3 were combined in order to increase the number of observations in the groups. The test group showed a significant decrease (1 % level) in the rate of absorption of 14 C-glycodeoxycholate (fig. 7A) compared with the control group. The percentage of the total dose of 14 C-glycodeoxycholate in the mucosal solution absorbed over the 2-hr incubation period was also significantly lower (1 % level) for the test group than for the control group (fig. 7B). Of the total 14 C-glycodeoxycholate ab-

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FIG. 5. Water absorption (H,O mg per hr per 100 mg of gallbladder wet weight) by control and test gallbladders. Control: trial 1 (0) , trial 2 (D), trial 3 (L::.). Test: trial 1 (e), trial 2 trial 3 (.6.). A, results of trial 1. B, results of trials 2 and 3. Note the difference in the scale between A and B . In this figure, as in figures 6 and 7, each point represents a single gallbladder.

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LIVER PHYSIOLOGY AND DISEASE

port by the gallbladder which is dependent on active anion-cation transport. 13 Its effect on 14 C-bilirubin and 14 C-glycodeoxycholate absorption was investigated in this study. The absorption experiment using 14 C-bilirubin or 14 C-glycodeoxycholate was begun as described, measuring the transport of radioactivity and the increase in weight of the gallbladder. After 90 min of incubation, 50 ,uliters of a solution of DNP in 50% ethanol were added to the mucosal solution to give a final concentration of 5 X 10 -s M. Water transport promptly ceased in all the gallbladders tested (fig. 8). The addition of DNP had no effect on the rate of absorption of either 14 C-bilirubin or 14 C-glycodeoxycholate . Twelve studies were performed with 14 C-glycodeoxycholate and two with 14 C-bilirubin, and typical results are shown in figure 8. The studies on bilirubin were performed on control gallbladders; those on 14 C-glycodeoxycholate were performed on six control and six test gallbladders which did not differ in their response to DNP.

that there are insufficient bile acid and phospholipid molecules available to solubilize all the cholesterol in mixed micelles and excess cholesterol precipitates. The source of the abnormal bile could be either the liver (by production of abnormal hepatic bile) or the gallbladder (by alteration of the composition of normal hepatic bile) . Studies by Small and Rapo, 1 5 and Vlahcevic et al. 16 of hepatic and gallbladder biles of patients with gallstones showed that the liver secreted . abnormal hepatic bile which was saturkted with cholesterol. On the other hand, Caldwell et al. , 17 • 18 using mice with ·di"et~in duced gallstones, demonstrated that . the ·gallbladder altered the composition of hepatic bile to produce gallbladder bile oversaturated with cholesterol. They argued .that in their model the bile salts, and to a lesser extent phospholipids, were selectively reabsorbed by the gallbladder. This study was an attempt to determine whether stone-forming gallbladders differ from normal gallbladders in their absorption of .bile constituents. The rabbit gallbladder was chosen because it is a convenient size ·and has been used in many previous studies ·of gallbladder ftmction. Gallbladder fun Ction was assessed in three ways: water absorption, bilirubin absorption, and bile·salt absorption. The mechanism of water absorption by

Discussion The current theory of cholesterol gallstone formation invokes the formation of bile with an increased cholesterol concentration relative to the concentration of bile acids and phospholipids. 14 It is believed

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November 1971

LIVER PHYSIOLOGY AND DISEASE

the gallbladder has been established as passive movement coupled to active anioncation transport. 1 3 • 19 The complete inhibition of net water transport by DNP in our studies is in agreement with the earlier report of Dietschy. 13 Water absorption is therefore energy-dependent, and was used in this study to assess the viability of the gallbladder preparations. The results from four gallbladders which failed to achieve a steady rate of transport were discarded. There was considerable variation between gallbladders, ranging from 20 to 150 mg of water per hr per 100 mg (wet weight) of gallbladder (fig. 5) but there was no difference between the control group and the group with gallstones, suggesting that cholelithiasis in this model is not associated with impaired ability of the gallbladder to concentrate bile . The results of this study suggest that both unconjugated bilirubin (if present) and glycodeoxycholate would be slowly absorbed by the normal rabbit gallbladder. Ostrow, 2 using an in vivo isolated guinea pig gallbladder, first demonstrated the ability of the gallbladder to absorb bile pigments. Unconjugated bilirubin was absorbed much faster than conjugated bilirubin, and a passive mechanism of non ionic diffusion was suggested. This is supported by the observation in these experiments that the addition of DNP had no effect on the rate of bilirubin absorption while completely inhibiting water transport. Previous studies with iodoacetate 2 demonstrated a similar effect, whereas DNP could not be shown to affect water absorption . The rate of bilirubin absorption is much slower in the everted sac preparation than in Ostrow's experiments, but this difference must be due in part to the lack of an intact circulation for the removal of transported bilirubin and possibly may be due to a species difference. The greater variation between in situ and in vitro studies could also be explained by these factors. The absorption of 14 C-glycodeoxycholate was also unaffected by DNP, suggesting a passive mechanism . Ostrow 3 found that the rate of absorption of a particular bile acid by the guinea pig gallbladder was roughly proportional to its

731

lipid solubility. Unconjugated bile acids were transported much faster than conjugated bile acids. We studied glycodeoxycholate absorption because it is the major bile acid of rabbit bile. 2 0 • 2 1 The total 14 Cglycodeoxycholate absorbed (1.0 to 4.0%) per 2 hr per gallbladder was of the same order as the in vivo results of Ostrow, 3 who used a similar dose of taurocholate. He suggested that deconjugation of bile acids, which would be rapidly absorbed, or mucosal damage to the gallbladder, leading to increased absorption of ionized conjugated bile acids, might be a factor in cholelithiasis by lowering the cholesterol-solubilizing capacity of bile. An increase in bile salt absorption was not found in our studies-rather, there was a significant decrease. Mucosal damage was not observed in our model of gallstone formation , and increased absorption of conjugated bile acids from this cause would not be expected. It is difficult to assess the importance of the decrease in glycodeoxycholate absorption observed in the gallbladders of rabbits with gallstones. Absorption of bile salts may have been inhibited by the increased production of mucus by the gallbladder mucosa observed in this type of gallstone model. 2 2 However, the results of these studies suggest that in this model altered gallbladder function is neither a factor in gallstone formation, nor a factor in the perpetuation of gallstones. The gallstones formed by this model of cholelithiasis were originally reported to contain a considerable proportion of cholesterol. However, in this study only trace quantities were detected and the gallstones were found to be identical both in composition and X-ray diffraction patterns to cholestanol-induced gallstones. Gallstones of similar composition were obtained by Bergman et al. 2 3 by feeding rabbits a diet containing 20 % casein, 20:'c oleic acid, and 33.6% sucrose. It seems likely that this type of diet in the rabbit induces the biosynthesis of cholestanol, the precursor of glycoallodeoxycholic acid, by an unknown mechanism . Cholestanol could be synthesized from cholesterol either by intestinal bacteria or by rabbit tissues, 24 with subsequent conversion to allocholic acid by the

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LNER PHYSIOLOGY AND DISEASE

liver. Bacterial dehydroxylation in the gut of the primary bile acid would then give allodeoxycholic acid. 21 Sodium glycoallodeoxycholate is known to be easily precipitated by calcium ions in the bile. 12 Experiments are in progress to investigate the pathway by which this cholestanol-free diet induces the formation of glycoallodeoxycholic acid stones in rabbits. REFERENCES 1. Borgman RF: Gallstone formation in rabbits as affected by dietary fat and protein. Amer J Vet Res 26: 1167-1171, 1965 2. Ostrow JD : Absorption of bile pigments by the gallbladder. J Clin Invest 46:2035-2052, 1967 3. Ostrow JD: Absorption by the gallbladder of bile salts, sulfobromophthalein, and iodipamide. J Lab Clin Med 74:482-494, 1969 4. Barrett PVD, Mullins FX, Berlin NI: Studies on the biosynthetic production of bilirubin-"C ; an improved method using o-aminolevulinic acid-4"C in dogs. J Lab Clin Med 68:905-912, 1966 5. Ostrow JD, Hammaker L, Schmid R: The preparation of crystalline bilirubin - "C. J Clin Invest 40:1442-1452, 1961 6. Norman A: Preparation of conjugated bile acids using mixed carboxylic acid anhydrides. Ark Kemi 8:331- 342, 1955 7. Iwata T, Yamasaki K: Enzymatic determination and thin-layer chromatography of bile acids in blood. J Biochem (Tokyo) 56:424-431, 1964 8. Crane RF, Wilson TH: In vitro method for the study of the rate of intestinal absorption of sugars. J Appl Physiol12 :145-6, 1958 9. King EJ: The colorimetric determination of phosphorus. Biochem J 26:292-297, 1932. 10. Folch J, Lees M, Sloane Stanley GH: A simple method for the isolation and purification of total lipides from animal tissues. J Bioi Chern 226:497509, 1957 11. Zlatkis A, Zak B, Boyle AJ: A new method for

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the direct determination :of serum cholesterol. J Lab Clin Med 41:486-492, 1953 Hofmann AF, Mosbach EH: .Identification of allodeoxycholic acid · as the major component of gallstones induced in the rabbit by 5a-cholestan3,8-ol. J Bioi Chem239:2813-'2821, 1964 Dietschy JM: Water .and solute movement across the wall of the e~erted rabbit gallbladder. Gastroenterology 47:395,-408, 1964 Admirand WH, Small DM : The physicochemical basis of cholesterol gallstone formation in man. J Clin Invest 47:1043-1052, 1968 Small DM, Rapo S: Source of abnormal bile in patients with cholesterol gallstones. New Eng J Med 283:53-57, 1970 Vlahcevic ZR, Bell cc·, Swell L: Significance of the liver in the production of lithogenic bile in man. Gastroenterology 59:62-69, 1970 Caldwell FT, Levitsky K , Rosenberg B: Dietary production and dissolution . of cholesterol gallstones in the mouse. Amer J Physiol 209:473-478, 1965 Caldwell FT, Levitsky .K: The gallbladder and gallstone formation. Ann Surg 166:753-758, 1967 Diamond JM: Transport of salt and water in rabbit and guinea pig gallbladder. J Gen Physiol 48:1- 14, 1964 Hellstrom K , Sjovall J: Turnover of deoxycholic acid in the rabbit. J Lipid Res 3:397-404, 1962 Hofmann AF, Bokkenheuser V, Hirsch RL, et al: Experimental cholelithiasis in the rabbit induced by cholestanol fe'eciirtg: effect of neomycin treatment on bile composition and gallstone formation. J Lipid Res 9:244-253, 1968 · Freston JW, Bouchier lAD; Newman J : Biliary mucous substances in dihydrocholesterol-induced cholelithiasis. Gastroenterology 57:670-678, 1969 Bergman F, Bogren H, Lindelof G, et al: Influence of the carb.ohydrate source of the diet on gallstone formation in .rabbits and mice. Acta Chir Scand 132:715-723, 1966 Shefer S, Milch S, Mosbach EH: Biosynthesis of 5a-cholestan-.3,8,ol in the rabbit and guinea pig. J Bioi Chern 239:1731-1736, 1964