Impaired human gallbladder lipid absorption in cholesterol gallstone disease and its effect on cholesterol solubility in bile

GASTROENTEROLOGY 2000;118:912–920

Impaired Human Gallbladder Lipid Absorption in Cholesterol Gallstone Disease and Its Effect on Cholesterol Solubility in Bile STEFANO GINANNI CORRADINI,* WALTER ELISEI,* LUCA GIOVANNELLI,* CRISTINA RIPANI,* PAOLA DELLA GUARDIA,* ALESSANDRO CORSI,‡ ALFREDO CANTAFORA,§ LIVIO CAPOCACCIA,* VINCENZO ZIPARO,\ VINCENZO STIPA,\ PIERO CHIRLETTI,\ ROBERTO CARONNA,\ DAVIDE LOMANTO,¶ and ADOLFO FRANCESCO ATTILI* *Divisione di Gastroenterologia del Dipartimento di Medicina Clinica, ‡Sezione di Anatomia Patologica del Dipartimento di Medicina Sperimentale e Patologia, \Prima Clinica Chirurgica, and ¶Seconda Clinica Chirurgica, Universita´ ‘‘La Sapienza,’’ Rome, Italy; and §Istituto Superiore di Sanita´, Rome, Italy

Background & Aims: The role of the gallbladder in gallstone pathogenesis is still unclear. We examined the effects of gallbladder mucosal lipid absorption on lipid composition and cholesterol crystallization in bile. Methods: The in vitro–isolated, intra-arterially perfused gallbladder model was used (1) to compare the absorption rates of lipids from standard bile by gallbladders obtained from 7 patients with cholesterol gallstones and 6 controls; and (2) to measure the microscopic cholesterol crystal detection time in cholesterolenriched pig bile before and after lipid absorption by the pig gallbladder. Results: Control gallbladders, but not cholesterol gallstone gallbladders, significantly reduced cholesterol (P F 0.02) and phospholipid (P F 0.01) and increased bile salt (P F 0.01) molar percentages in bile over a 5-hour period by efficient and selective cholesterol and phospholipid absorption. A histomorphometric study of the epithelial cells showed significantly higher values for nuclear density (P F 0.01) and nuclear (P F 0.05) and cytoplasmic (P F 0.05) areas in the cholesterol gallstone than the control group. Sequential microscopy of cholesterolenriched pig bile showed significantly shorter cholesterol filament (P F 0.01) and typical cholesterol plate (P F 0.02) detection times before than after exposure of bile to the gallbladder lipid absorption. Conclusions: In cholesterol gallstone disease, the human gallbladder epithelium loses its capacity to selectively and efficiently absorb cholesterol and phospholipids from bile, even if it is hyperplastic and hypertrophic. This epithelial dysfunction eliminates the positive effect that the normal gallbladder exerts on cholesterol solubility in bile and might be a pathogenetic cofactor for cholesterol gallstone formation.

free subjects.1 It is still debated whether the gallbladder epithelium could reduce biliary metastability by secreting proteins with excessive cholesterol crystallizationpromoting activity in cholesterol gallstone patients.2–5 In a previous study, we hypothesized that a dysfunction of the gallbladder mucosa to absorb lipids from bile was a further mechanism in gallstone pathogenesis.6 In fact, we claimed that the differences in the molar distribution of lipids in fasting gallbladder bile between gallstone-free nonobese subjects on one side, and nonobese gallstone patients and obese subjects with and without gallstones on the other, were mainly the result of more efficient gallbladder mucosal cholesterol and phospholipid absorption in the nonobese gallstone-free subjects than in all other groups.6 However, this hypothesis was indirectly deduced from a correlation-plot analysis of biliary lipid concentrations in gallbladder bile samples obtained at cholecystectomy and, thus, needed confirmation by a more direct methodological approach. In the present study, to directly measure biliary lipid absorption by the intact gallbladder, we used a recently validated in vitro–isolated, intra-arterially perfused gallbladder model developed in our laboratory7 to investigate (1) whether the absorptive capacity of the biliary lipids by the gallbladder epithelium is different in cholesterol gallstone patients and controls; (2) whether the differences in the net disappearance rates of lipids from bile induced by gallbladder absorption can differently affect the molar distribution of lipids in the bile in the same groups; and (3) whether normal lipid absorption function of the

iliary metastability is reduced during storage in the gallbladder compared with that in hepatic bile, only in patients with cholesterol gallstones excluding gallstone-

Abbreviation used in this paper: CSI, cholesterol saturation index. r 2000 by the American Gastroenterological Association 0016-5085/00/$10.00 doi:10.1053/gg.2000.7034

B

May 2000

gallbladder leads to improved cholesterol solubility in bile.

Materials and Methods The study protocol complied with our institute’s ethical guidelines for animal and human studies, and consent was obtained from the patients.

Patients Gallbladders were obtained at laparotomy from 7 patients with cholesterol gallstones (3 solitary and 4 multiple) submitted to elective cholecystectomy and from 6 patients without cholesterol gallstones. The latter were considered as controls and included 4 gallstone-free subjects submitted to prophylactic cholecystectomy during gastrectomy for gastric neoplasms, and 2 patients submitted to elective cholecystectomy for small, multiple, black-pigment gallstones. Evidence showing non-neoplastic liver disease, cholestasis, cystic duct obstruction, biliary sepsis, or gross appearance of acute or chronic gallbladder inflammation were considered as exclusion criteria. Native bile composition and measurement of biliary lipid absorption by the human gallbladder in the presence or absence of cholesterol gallstone disease. Total bile salt,

phosphatidylcholine, and cholesterol concentrations in native gallbladder bile were measured as described previously.7 To compare the absorptive efficiency of lipids from standard bile by the human gallbladder in the presence or absence of cholesterol gallstone disease, in vitro–isolated, intra-arterially perfused human gallbladder was prepared, after complete aspiration of native bile, as recently described.7 We have already demonstrated, and confirmed in this study also on cholesterol gallstone organs, that organ viability is excellent throughout the experimental period in the in vitro–isolated, intra-arterially perfused gallbladder experiments.7 In addition, no difference in lactate dehydrogenase release and oxygen consumption was found between the control and cholesterol gallstone gallbladders (data not shown). Measurement of the rate of lipid absorption from the bile contained in human gallbladder lumen required large amounts of human bile, which were prepared, frozen in aliquots, and used on the day of each experiment, after cholesterol and phosphatidylcholine radiolabeling, as previously described.7 Bile composition and specific activities at the beginning of each experiment were the same in all the studies performed on human organs, as described previously.7 Before each study, the presence of cholesterol crystals was excluded at polarizing microscope. The net mass of bile salts, cholesterol, and phosphatidylcholine that disappeared from bile was calculated by measuring at certain intervals the volume of the totally aspirated bile and the lipid concentrations, as described previously.7 At the end of each study, the recovery by aspiration of a known volume of modified Ringer’s bicarbonate solution7 instilled in the gallbladder lumen was found to be excellent in both control and cholesterol gallstone gallbladders (99.55% 6 0.40% vs. 99.26% 6 0.45%, respectively [mean 6 SD]).

GALLBLADDER LIPID ABSORPTION AND GALLSTONES

913

The recovery of radioactive cholesterol and phosphatidylcholine in the perfusate and gallbladder tissue was assessed as previously described.7 At the end of the experiments, a part of the gallbladder was used for histology, morphometric analysis, and, in selected cases, for electron microscopy. Gallstones were washed in saline, dried, and weighed. A fraction was crushed thoroughly, weighed, and solubilized in isopropanol. The cholesterol content of gallstones was determined enzymatically to classify them. Cholesterol gallstones were considered those with a cholesterol content .70% of dry weight and pigment stones ,15%.8 The isopropilic extract of gallstones was counted to measure the recovery of radioactive cholesterol and phosphatidylcholine from gallstones. Human gallbladder morphology and morphometry.

At the end of the in vitro–isolated, intra-arterially perfused human gallbladder experiments, a part of the gallbladders used for morphology and morphometry was immediately fixed in 4% paraformaldehyde, buffered at pH 7.2 with 0.1 mol/L phosphate buffer. Three wall samples obtained by perpendicular sectioning were routinely embedded in paraffin. The specimens were properly oriented to entirely section the gallbladder wall. From each sample, 4-µm sections were cut and stained with H&E. Small samples of the mucosa from 4 gallbladders (2 with cholesterol gallstones and 2 without gallstones) were carefully dissected after fixation, postfixed in 1% osmium tetroxide, dehydrated in graded ethanol solutions, and embedded in epoxy resin (Araldite; Agar Scientific Ltd., Stanstead, Essex, England) for ultrastructural examination. Semithin sections were stained with Azur II-Methylene Blue (Merck, Darmstadt, Germany) to select the appropriate fields (i.e., the epithelial-lining cells). Ultrathin sections were cut with diamond knives, placed on uncoated grids, contrasted with uranyl acetate and lead citrate, and observed using transmission electron microscopy with a CM10 Philips electron microscope (Philips, Eindhoven, Netherlands). Histological sections were used to compare the status of each gallbladder by measuring 7 parameters (cholesterolosis, fibrosis, muscular thickening, Rokitansky–Aschoff sinuses, inflammatory cell infiltrate, plasma cells, and eosinophils), as previously described.9 A score ranging from 0 to 4 was assigned to each parameter; the final histological score was calculated from the total of each individual value. The same histological sections were used to measure, by a semiautomatic image analyzer (IAS 2000; Delta System, Rome, Italy), the epithelial nuclear density, which is the number of epithelial cell nuclei per millimeter of basal membrane, and the epithelial nuclear and cytoplasmic areas considering 100 cells at random as previously established.10 Measurements were performed at a microscopic objective magnification of 253, which is equivalent to an on-screen area of 0.031 mm2. Observations were performed in a blind fashion. Effect of the exposure of bile to gallbladder mucosal lipid absorption on cholesterol crystal formation. To investi-

gate whether the changes of the molar lipid percent composition of bile caused by selective gallbladder epithelial lipid absorption were associated with any change in cholesterol

914

GINANNI CORRADINI ET AL.

crystallization in bile, 5 experiments with in vitro–isolated, intra-arterially perfused pig gallbladder obtained from cadaveric animals were performed as described previously,7 with the following minor modifications. The cystic artery was perfused with gas-saturated (95% oxygen and 5% carbon dioxide) pig plasma in a recirculating system, and air was introduced via the cystic duct catheter to maintain an intraluminal pressure of 3 cm H20. These conditions were chosen because, in preliminary experiments, they were found to minimize transepithelial net fluid movements. One pool of pig gallbladder bile was prepared, centrifuged at 3000g for 15 minutes, and frozen in aliquots. On the day of each experiment, bile was thawed, centrifuged at 3000g for 15 minutes, transferred to a tube in which cholesterol (Eastman Kodak, Rochester, NY) solubilized in ethanol had been previously dried under nitrogen, and incubated at 50°C under shaking for 1 hour. During incubation, bile was frequently vortexed. The amount of cholesterol added to bile was calculated to achieve a final concentration in bile similar to that reported by Juste et al.11 in the gallbladder bile of cholesterol-fed pigs. Bile was then cooled at 37°C and filtered (0.45-µm pore size) using sterile filters preheated at 37°C after 30 minutes. Native bile in the gallbladder lumen was substituted with 15 mL of the cholesterol-enriched bile via the cystic duct catheter and carefully mixed. The time of this event was considered as the beginning of the pig gallbladder perfusion experiment. The volume of the totally aspirated bile was measured by weight at the beginning of the experiments and after 4 hours (end of the experiments), while samples were taken for lipid measurements and crystallographic studies at the same times. In 3 cases, the gallbladder had secreted fluid during the experiment (0.170, 0.100, and 0.500 mL, respectively); in 2 cases, fluid absorption had occurred (2.540 and 1.100 mL, respectively). Because the fluid content of bile is known to influence cholesterol crystallization, at the end of each experiment, saline was added to the bile sample with the lower fluid content (that obtained at the beginning or that sampled at the end) to obtain the same dilution of the other. To measure the microscopic detection times of cholesterol crystals before and after exposure of bile to the gallbladder epithelial lipid absorption, the bile samples obtained at the beginning and end of these pig gallbladder experiments were incubated in screw-capped sterile flasks under nitrogen at 37°C with no shaking. One drop (5 µL) of bile was examined by polarizing microscopy (Zeiss Axioplan photomicroscope; Zeiss, Jena, Germany; at 6403 magnification) at the time of incubation and every day thereafter to detect the appearance of the different cholesterol crystal habits. At the time of incubation, none of the samples contained cholesterol crystal even at 16003 magnification. The filament and typical plate cholesterol monohydrate crystal detection times were considered as the earliest detection of filamentous structures and of typical (plate-like) cholesterol monohydrate crystals, respectively, as described previously.11,12 To measure the changes in biliary lipid composition induced by exposure of bile to the gallbladder mucosa, the total bile salt, phosphatidylcholine, and cholesterol concentrations in

GASTROENTEROLOGY Vol. 118, No. 5

bile at the beginning and end of experiments were measured by enzymatic methods as previously described.7 Bile composition at the beginning of experiments was 7.2 6 1.2 mmol/L cholesterol, 11.1 6 0.3 mmol/L phosphatidylcholine, and 149.4 6 1.0 mmol/L bile salts (mean 6 SD). Effect of bile composition on the rates of cholesterol and phosphatidylcholine absorbed by the gallbladder. To

investigate whether the cholesterol molar percentage of the bile that enters the gallbladder lumen influences the ratio at which cholesterol and phosphatidylcholine are absorbed by the gallbladder, we performed 2 groups (A and B) of experiments using in vitro–isolated, intra-arterially perfused pig gallbladder prepared from cadaveric animals as previously described.7 Human bile was instilled in the gallbladder lumen in each group of experiments. Bile composition at the beginning of the pig experiments in group A (n 5 5) was 5.8 mmol/L cholesterol, 19.6 mmol/L phosphatidylcholine, and 100.5 mmol/L bile salts. In group B (n 5 6), the same bile was added with cholesterol to achieve a final cholesterol concentration of 11 mmol/L. Consequently, bile B had a higher cholesterol molar percentage (8.4%) than bile A (4.6%). The rates of radiolabeled cholesterol and phosphatidylcholine disappearance from bile were measured as previously described.7 Specific activities in bile at the beginning of the experiments were 50,000 dpm/µmol and 4000 dpm/µmol for cholesterol and phosphatidylcholine, respectively.

Statistical Analysis The mean values were compared by the Student t test. The repeated measurements of the cumulative disappearance of lipids from bile obtained in the human experiments were analyzed by 1-way analysis of variance using the raw data at each time point. If the analysis of variance indicated statistical significance (P , 0.05), the unpaired t test was used to compare groups at each time point. Regression lines were fitted to the data using simple linear regression. Statistical analyses were performed on the GraphPad InStat version 3.00 software (GraphPad Software, San Diego, CA), and the significance level was set at 0.05.

Results Native Gallbladder Bile Lipid Composition in Controls and Cholesterol Gallstone Patients The mean age (55.9 6 11.2 vs. 58.2 6 5.3 years [mean 6 SD]) and sex distribution (men/women, 3/3 vs. 3/4) were comparable in the control and cholesterol gallstone groups. For native gallbladder bile composition, the cholesterol molar percentage was significantly higher in the cholesterol gallstone group than in the control group (10.3% 6 3.3% vs. 6.6% 6 2.3%, respectively [mean 6 SD]; P , 0.05). The cholesterol saturation index (CSI) was also higher in the cholesterol gallstone group than in controls (1.62 6 0.49 vs. 1.04 6

May 2000

0.31, respectively [mean 6 SD]; P , 0.05). The bile salt molar percentage was lower in the cholesterol gallstone group than in controls (70.0% 6 7.4% vs. 74.5% 6 6.1%, respectively [mean 6 SD]), although the difference was not statistically significant. The phosphatidylcholine molar percentage did not differ between groups (19.8% 6 5.1% vs. 18.9% 6 4.3% in the cholesterol gallstone and the control group, respectively [mean 6 SD]).

GALLBLADDER LIPID ABSORPTION AND GALLSTONES

915

the end of each experiment of the 2 groups (Figure 2). At the end of experiments performed on control gallbladders, bile had significantly lower cholesterol (P , 0.02) and phosphatidylcholine (P , 0.01) and significantly higher bile salt (P , 0.01) molar percentages than bile at the beginning. On the contrary, an opposite trend was found in the experiments on cholesterol

Gallbladder Biliary Lipid Absorption Rates and Their Effects on Bile Composition in the Presence or Absence of Cholesterol Gallstone Disease No intergroup difference was found in the mean gallbladder mucosal surface (42.05 6 12.27 vs. 43.17 6 6.97 cm2 in control and cholesterol gallstone groups, respectively [mean 6 SD]). Figure 1 shows the mean cumulative disappearance curves of each lipid class from bile during the in vitro–isolated, intraarterially perfused gallbladder experiments performed on control and cholesterol gallstone organs. For bile salts, a trend toward lower values was found in the experiments on cholesterol gallstone gallbladders, but no statistical difference was present between the 2 groups (Figure 1A). In both groups, no plateau was reached at the end of the experiments. For phosphatidylcholine, the disappearance from bile was significantly lower in the experiments on cholesterol gallstone organs than on control organs (Figure 1B). Cholesterol disappearance from bile was also significantly lower in the experiments using cholesterol gallstone organs than control organs (Figure 1C). Both phosphatidylcholine and cholesterol disappearance curves reached a plateau at a time point of 240 minutes in the cholesterol gallstone group, but not in control group. A positive correlation was found between the cumulative disappearance of cholesterol from bile at the end of the experiments and that of phosphatidylcholine (R 5 0.859, P , 0.0005); no correlation was found between the disappearance of bile salts and that of cholesterol or phosphatidylcholine. In preliminary studies, we ascertained that the specific activities of cholesterol and phosphatidylcholine did not differ in the biliary lipid carriers isolated by fast protein liquid chromatography (Superose 6 column; Pharmacia, Uppsala, Sweden), as described previously,13 when bile had previously been preincubated with radiolabeled cholesterol and phosphatidylcholine for 1 hour (data not shown). Thus, our correlation plots of the lipids absorbed from bile would indirectly suggest that the cholesterol/ phospholipid vesicles, rather than the mixed micelles, are the main final source of the cholesterol and phosphatidylcholine moieties absorbed. To assess the effect that the disappearance of lipids from bile had on bile composition, biliary lipid molar percentages in the standard bile instilled in gallbladder lumen at the beginning of the experiments were compared with those in the same bile at

Figure 1. Cumulative disappearance curves of each lipid class from bile (means 6 SEM) as an effect of gallbladder absorption during in vitro– isolated perfused gallbladder experiments using organs from control subjects (m; n 5 6) and patients with cholesterol gallstones (h; n 5 7). Disappearance of (A) bile salts, (B) phosphatidylcholine, and (C) cholesterol. In the cholesterol gallstone group, phosphatidylcholine and cholesterol disappearance rates reached a plateau at 240 minutes. *P , 0.03, **P , 0.01 compared with cholesterol gallstone gallbladders at the same time point.

916

GINANNI CORRADINI ET AL.

GASTROENTEROLOGY Vol. 118, No. 5

gallstones were present, less than 0.2% of the total radioactivity was recovered from the stones in each experiment.

Morphology and Morphometry of the Human Gallbladders

Figure 2. Biliary lipid molar percentages (means 6 SEM) in the standard bile used at the beginning of each experiment and in bile at the end of in vitro–isolated perfused gallbladder experiments using organs from controls (n 5 6) and cholesterol gallstone patients (n 5 7). At the end of control gallbladder experiments, the bile contained significantly lower cholesterol and phosphatidylcholine and significantly higher bile salt molar percentages than at the beginning of studies (paired Student t test). At the end of the experiments on cholesterol gallstone gallbladders, no statistical difference was found vs. bile at the beginning of studies (paired Student t test). At the end of control gallbladder experiments, the bile had significantly lower cholesterol and phosphatidylcholine and significantly higher bile salt molar percentages than bile at the end of experiments on gallbladders from cholesterol gallstone patients (unpaired Student t test).

gallstone gallbladders; however, the differences were not statistically significant. When biliary lipid molar percentages at the end of the 2 groups of experiments were compared (Figure 2), bile had significantly higher cholesterol (P , 0.05) and phosphatidylcholine (P , 0.05) and significantly lower bile salt (P , 0.05) molar percentages in the studies on cholesterol gallstone gallbladders than in those performed on control gallbladders. The mean percent recovery of radioactive cholesterol and phosphatidylcholine in gallbladder tissue and in perfusate in the human experiments is shown in Table 1. Intraepithelial recovery of both lipids was significantly lower in the cholesterol gallstone gallbladders than in controls. Both cholesterol (0.57% and 0.74%) and phosphatidylcholine (6.02% and 7.28%) recoveries in the epithelial cells of the 2 gallbladders of the control group containing pigment stones were higher than the maximal corresponding epithelial recovery found in the cholesterol gallstone group (0.32% and 2.84% for cholesterol and phosphatidylcholine, respectively). Recovery in the gallbladder wall underneath the mucosa for both lipids was significantly lower in cholesterol gallstone gallbladders than in controls. The percentage recovered in the perfusate throughout the experiments did not differ in the 2 groups. When

Representative histological samples obtained from a cholesterol gallstone and a control gallbladder are shown in Figure 3. No acute inflammation or cholesterolosis was found in any gallbladder. The degree of histological score of the gallbladder wall was slightly higher in the cholesterol gallstone than control gallbladders (10.17 6 2.14 vs. 7.67 6 2.73, respectively [mean 6 SD]), but the difference was not statistically significant (P 5 0.11). Light and dark epithelial cells were observed in all the gallbladders examined by electron microscopy.14,15 The former cells were evident in all sections, but the latter ones were less constant. Some features were more frequently associated with the presence of cholesterol gallstones. These included multilayering of the epithelial cells (Figure 4A), which was also detected by histology and morphometry; a more variable appearance of the microvillous border being constituted by microvilli substantially normal in size, number, and distribution in some cells, but rather short and rarefied in others (Figure 4B); and occasional protrusions of the cytoplasm devoid of organelles extending from the luminal surface into the lumen, similar to those described by Fox14 and Gilloteaux et al.16 (Figure 4C). Mucous droplets, frequently with the dissolution of the limiting membrane (Figure 4D), and dense bodies, including multivesicular ones (Figure 4E), were evident in all the examined gallbladders. The morphometric analysis showed that the epithelial nuclear density (243.77 6 22.64 vs. 181.07 6 27.45 nuclei/ mm; P , 0.01), nuclear area (30.79 6 2.05 vs. 27.01 6 3.45 µm2; P , 0.05), and cytoplasmic area (133.15 6 10.92 vs. 102.14 6 22.66 µm2; P , 0.05) of the epithelial cells were significantly higher in the cholesterol gallstone organs than the Table 1. Percent Recovery of Radioactive Cholesterol and Phosphatidylcholine From Gallbladder Tissue and Perfusate at the End of In Vitro–Isolated Perfused Gallbladder Experiments in Cholesterol Gallstone and Control Groups Cholesterol (% dpm)

Control

Cholesterol gallstone

Phosphatidylcholine (% dpm)

Control

Epithelial cells 0.83 6 0.2 a 0.16 6 0.5 6.9 6 1.2 b GB wall below epithelium 13.9 6 2.5 a 4.9 6 1.6 17.0 6 1.8 a Perfusate 0.63 6 0.3 0.85 6 0.3 4.8 6 1.4

Cholesterol gallstone 1.4 6 0.4 8.6 6 2.5 3.1 6 0.3

NOTE. Data are calculated as the percentage of total radioactivity for each experiment. Values are means 6 SEM. GB, gallbladder. aP , 0.03, bP , 0.01 vs. cholesterol gallstone group; Student 2-tailed t test.

May 2000

GALLBLADDER LIPID ABSORPTION AND GALLSTONES

917

much more typical plate monohydrate crystals per microscopic field than bile sampled at the end of the experiments in all cases (data not shown).

Effect of Bile Composition on the Rates of Cholesterol and Phosphatidylcholine Absorbed by the Pig Gallbladder Mucosa The mean cholesterol/phosphatidylcholine molar ratio of the lipids that had disappeared from bile at the end of the in vitro–isolated, intra-arterially perfused pig gallbladder experiments was significantly (P , 0.03) higher in group B (in which cholesterol-enriched bile was used at the beginning of the experiments) than in group A (0.347 6 0.062 vs. 0.149 6 0.028, respectively [mean 6 SD]).

Discussion

Figure 3. Histology of (A and B) a cholesterol gallstone and (C and D) a control gallbladder. In both samples, the mucosa shows slight chronic inflammatory cellularity (A and C). In the epithelial lining of the cholesterol gallstone gallbladder (B), nuclei are at different levels (pseudostratification), and nuclear density is higher than in the control organ (D).

control ones. A positive correlation was found between the cholesterol molar percentage in native bile and the nuclear density of epithelium (R 5 0.738, P , 0.01).

Effect of the Exposure of Bile to Gallbladder Mucosal Lipid Absorption on Cholesterol Crystal Formation As shown in Table 2, bile had significantly lower cholesterol (P , 0.05) and phosphatidylcholine (P , 0.02) and significantly higher bile salt (P , 0.01) molar percentages after 4-hour exposure to the absorption of the pig gallbladder epithelium compared with bile at the beginning. With regard to cholesterol crystallization, 2 habits of cholesterol crystals appeared during bile incubation in all cases: filaments followed by typical monohydrate plates. Both the filament (P , 0.01) and the typical plate (P , 0.02) detection times were significantly shorter in the bile samples harvested from the gallbladder at the beginning of these in vitro–isolated, intraarterially perfused pig gallbladder experiments compared with the bile samples at the end of the experiments. In addition, after 30 days of incubation, bile samples harvested from the gallbladder at the beginning of the experiments clearly showed

The main finding of this study, performed using the isolated, intra-arterially perfused gallbladder model, was that gallbladder cholesterol absorption and phosphatidylcholine absorption from standard bile were markedly reduced in organs obtained from cholesterol gallstone patients compared with those from controls, whereas bile salt absorption was less affected. Consequently, over the experimental period of 5 hours, the control gallbladders induced a significant reduction of cholesterol and phospholipid, and a significant increase of bile salt molar percentage in bile, whereas the gallbladders from cholesterol gallstone patients slightly modified the biliary lipid molar percentages in an opposite way. When bile composition at the end of the experiments in the 2 groups was compared, significantly higher cholesterol and phosphatidylcholine and significantly lower bile salt molar percentages were found in the cholesterol gallstone studies than control experiments. Comparison of these lipid absorption data with the molar percent distribution of lipids in native gallbladder bile in the same patients directly supports our previous hypothesis that the efficiency of biliary lipid absorption by the gallbladder is a major determinant of gallbladder bile lipid composition.6 However, fasting native gallbladder bile composition is influenced in vivo by the efficiency of gallbladder lipid absorption, as well as by the composition of hepatic bile, which is different for each subject. This might explain why, although in this study the control gallbladders showed a higher capacity to absorb phosphatidylcholine in vitro, the phosphatidylcholine molar percentage in native gallbladder bile did not differ between groups. The key issue is whether the impaired gallbladder epithelial cholesterol and phosphatidylcholine absorption from bile is one of the pathogenetic factors of cholesterol gallstone disease. The mucosal defective lipid absorption should fulfill 2 criteria to be considered pathogenetically important: it should (1) favor gallstone formation and (2) occur before gallstone formation. The first criterion implies that the changes of bile lipid percent composition induced by the efficient lipid absorption of the normal gallbladder improve cholesterol solubility in bile. On the other hand, when lipid absorption is impaired and lipid

918

GINANNI CORRADINI ET AL.

GASTROENTEROLOGY Vol. 118, No. 5

Figure 4. Representative ultrastructural features of epithelial cells. (A) Nuclear multilayering, (B) loss of microvillous border (lumen*), and (C) cytoplasmic protrusion were more frequently associated with the presence of cholesterol gallstones. By contrast, (D) mucous droplets and (E ) multivesicular bodies were observed in gallbladders both with and without cholesterol gallstones. (Original magnifications: [A] 52003, [B] 11,5003, [C] 52,5003, [D] 22,2503, [E ] 22,2503.)

composition is not affected by the gallbladder, cholesterol solubility does not improve and gallstone formation is favored. To prove this theory, we measured the detection times of cholesterol crystals in cholesterol-enriched pig bile before and after exposure to normal pig gallbladder epithelial absorption. We used certain experimental conditions so that the bile samples incubated for the crystallization studies had the same degree of dilution (a variable known to influence cholesterol crystallization) before and after being exposed to the gallbladder epithelial function. The data showed a drastic prolongation of the detection times of both cholesterol filaments and typical cholesterol plates after exposure of bile to the gallbladder epithelium compared with bile not exposed to the gallbladder’s

action.11,12 This inhibition of cholesterol crystallization was associated with changes of bile lipid molar percentages after exposure to the pig gallbladder compared with bile at the beginning of the experiments, similar to those induced by the efficient lipid absorption of the normal human gallbladder. The reduced cholesterol molar percentage favors cholesterol solubility by decreasing the CSI. Efficient phosphatidylcholine absorption from bile can also provide protection from gallstone formation, because cholesterol vesicle formation is minimal when the bile salt-phospholipid molar ratio is increased.17 However, if cholesterol and phosphatidylcholine were always absorbed at the same ratio, such as in our in vitro experiments performed on human gallbladders, this would have a negative

Table 2. Biliary Lipid Molar Percent Composition and Crystal Detection Times in Bile Harvested From the Gallbladder at the Beginning and End of In Vitro–Isolated Perfused Pig Gallbladder Experiments Beginning of experiments

End of experiments

Experiment no.

Ch (mol% )

PC (mol% )

BS (mol% )

Filament DT (h)

TP-ChM DT (h)

Ch (mol% )

PC (mol% )

BS (mol% )

Filament DT (h)

TP-ChM DT (h)

1 2 3 4 5 Mean SD

3.8 3.6 3.9 5.2 4.8 4.3 0.7

6.7 6.5 6.4 6.8 6.5 6.6 0.2

89.5 89.9 89.7 88 88.6 89.1 0.8

12 12 12 12 12 12 0

72 72 72 24 24 52.8 26.3

2.9 1.9 3.8 2.4 3.2 2.9 a 0.8

5.1 5.1 4.8 2.4 4.7 4.4 b 1.1

92 93 91.4 95.2 92.1 92.7 b 1.5

136 120 41 144 144 117 c 43.6

360 336 120 180 160 231.2 b 109.1

Ch, cholesterol; PC, phosphatidylcholine; BS, bile salts; Filament DT, filament detection time; TP-ChM DT, typical plate cholesterol monohydrate crystal detection time. aP , 0.05, bP , 0.02, cP , 0.01 vs. the respective value at the beginning of the experiments by Student 2-tailed paired t test.

May 2000

effect on the CSI of bile, especially when the hepatic bile that enters the gallbladder lumen in vivo has a very high cholesterol molar percentage. A second set of in vitro–isolated, intraarterially perfused pig gallbladder studies showed that this is not the case. In fact, the cholesterol-phosphatidylcholine molar ratio of lipids subtracted from bile by gallbladder absorption increased as the cholesterol molar percentage of bile instilled in the lumen increased. This study was not aimed at investigating the mechanism of lipid absorption; however, we found an excellent correlation between the disappearance rate of cholesterol from bile and that of phosphatidylcholine, whereas no correlation was found between the disappearance of bile salts and that of cholesterol or phosphatidylcholine. This would indirectly suggest that cholesterol/phospholipid vesicles are the main final source of the absorbed cholesterol and phosphatidylcholine moieties. It can thus be speculated that a high cholesterol molar percentage in bile increases the cholesterol-phosphatidylcholine molar ratio of lipids absorbed by the gallbladder by increasing the cholesterol-phosphatidylcholine molar ratio of vesicles. Thus, the normal gallbladder continuously improves cholesterol solubility in bile and provides protection from cholesterol gallstone formation by absorbing cholesterol from vesicles, the ultimate source of cholesterol for crystal formation, and by changing the cholesterol-phosphatidylcholine molar ratio of lipids absorbed according to bile composition. The gallbladder mucosal defect of cholesterol gallstone patients is probably not only at the lipid absorption level, but also involves other aspects of epithelial function, such as increased secretion of mucin or other proteins that promote cholesterol crystallization, decreased secretion of anticrystallization proteins, and changes in the concentration of ionized calcium. Unfortunately, we did not study the kinetics of cholesterol crystallization in the native bile and cannot exclude different gallbladder mucosal defects, other than impaired lipid absorption, in our cholesterol gallstone patients. In addition, it cannot be excluded that other gallbladder anticrystallization factors could be involved in our pig experiments showing an inhibiting effect of the gallbladder epithelial function on biliary cholesterol crystallization associated with efficient lipid absorption. Notwithstanding the above limitations, we think that the defective gallbladder lipid absorption is an important factor that favors gallstone formation. In agreement with the above, only cholesterol saturation and mucin gel, of the numerous biliary variables able to influence cholesterol crystallization, have been recently shown to play a major role in lithogenesis.4,5 The second criterion to judge the pathogenetic relevance of the gallbladder mucosal defect for cholesterol gallstones is that it should occur before gallstone formation and not be caused by the presence of stones. Our study adds to this hypothesis by demonstrating that the epithelial absorption of cholesterol and phosphatidylcholine of the 2 control group gallbladders containing pigment stones was good. We have also recently found indirect evidence for impaired gallbladder mucosal function in obese, gallstone-free subjects, who have an increased risk for

GALLBLADDER LIPID ABSORPTION AND GALLSTONES

919

cholesterol gallstone development.6 Finally, the proliferative changes of the gallbladder epithelium that we found to be associated with the reduced mucosal lipid absorption have previously been shown to occur before gallstone formation.18,19 Our data indicate that the defect of biliary lipid absorption in cholesterol gallstone disease begins at the gallbladder epithelial level. In fact, the recovery of biliary cholesterol and phosphatidylcholine in the gallbladder epithelial cells was 5-fold less in cholesterol gallstone organs than control group organs. The lower epithelial lipid absorptive efficiency in the cholesterol gallstone group was associated with a higher value of epithelial nuclear density in the cholesterol gallstone group. This finding is well related to the established higher epithelial cell turnover and DNA synthesis index leading to hyperplasia in gallbladders with cholesterol stones rather than without stones or with pigmentary stones.20–22 The proliferative changes of the gallbladder epithelium in cholesterol gallstone disease have been related to a factor existing in bile rather than to mechanical irritation caused by the stones or inflammation.21 In agreement with this, in our study, the histological score was not significantly different between the cholesterol gallstone group and the control group. Furthermore, our finding of a positive correlation between the cholesterol molar percentage in native gallbladder bile and the nuclear density of the epithelium could allow for speculation that the relative enrichment of bile with cholesterol is the stimulus for epithelial proliferation, which in turn represents the organ’s attempt, even if still insufficient, to increase the absorbing surface and compensate for the impaired lipid absorption capacity at each single cell level. The poor cellular function could be caused by a previous excessive cholesterol accumulation in the epithelial cells, in analogy to the pathogenesis of gallbladder muscle cell malfunction demonstrated in both the prairie dog cholesterol gallstone model and in cholesterol gallstone patients.23,24 In this study, we also found higher values of the epithelial nuclear and cytoplasmic areas in the cholesterol gallstone group than control group. These signs of gallbladder epithelial hypertrophy, which coexist with hyperplasia at the same level, have already been shown in several gallstone animal models and need further investigation.18,19 In light of the limited number of samples examined by electron microscopy in this study, the features and cytoplasmic content of the epithelial cells in gallbladders with and without cholesterol gallstones cannot be established at this time. Also, our histological and ultrastructural data can neither confirm nor deny the previously reported higher abundance of dark epithelial cells in pathological rather than normal gallbladder epithelium.14 The proportion of cells with light and dark cytoplasm can in fact be influenced by the suboptimal conditions of fixation,15 as used in the present study (gallbladders were fixed 6 hours after surgical removal). In conclusion, this study directly demonstrates that, in the presence of cholesterol gallstone disease, the human gallbladder epithelium absorbs cholesterol and phospholipids from bile less efficiently. This defect might be an additional cofactor in the pathogenesis of cholesterol gallstones.

920

GINANNI CORRADINI ET AL.

References 1. Nakano K, Chijiiwa K. Reduced cholesterol metastability of hepatic bile and its further decline in gallbladder bile in patients with cholesterol gallstones. Gut 1993;34:702–707. 2. Portincasa P, Van de Meeberg P, Van Erpecum KJ, Palasciano G, Van Berge-Henegouwen. An update on the pathogenesis and treatment of cholesterol gallstones. Scand J Gastroenterol 1997; 32(suppl 223):60–69. 3. Keulemans YCA, Mok KS, Gouma DJ, Groen AK. The role of the concanavalin A–binding fraction in cholesterol crystallization in native human bile. J Hepatol 1997;27:1041–1050. 4. Miquel JF, Nu`nez L, Amigo L, Gonza`lez S, Raddatz A, Rigotti A, Nervi F. Cholesterol saturation, not proteins or cholecystitis, is critical for crystal formation in human gallbladder bile. Gastroenterology 1998;114:1016–1023. 5. Wang DQH, Cohen DE, Lammert F, Carey MC. No pathophysiologic relationship of soluble biliary proteins to cholesterol crystallization in human bile. J Lipid Res 1999;40:415–425. 6. Ginanni Corradini S, Yamashita G, Nuutinen H, Chernosky A, Williams C, Hays L, Shiffman ML, Walsh RM, Svanvik J, Della Guardia P, Capocaccia L, Holzbach RT. Human gallbladder mucosal function. Effects on intraluminal fluid and lipid composition in health and disease. Dig Dis Sci 1998;43:335–343. 7. Ginanni Corradini S, Ripani C, Della Guardia P, Giovannelli L, Elisei W, Cantafora A, Codacci Pisanelli M, Tebala GD, Nuzzo G, Corsi A, Attili AF, Capocaccia L, Ziparo V. The human gallbladder increases cholesterol solubility in bile by differential lipid absorption: a study using a new in vitro model of isolated intra-arterially perfused gallbladder. Hepatology 1998;28:314–322. 8. Trotman BW, Soloway RD. Pigment gallstone disease: summary of the National Institutes of Health–International Workshop. Hepatology 1982;2:879–884. 9. Nahrwold DL, Rose RC, Ward SP. Abnormalities in gallbladder morphology and function in patients with cholelithiasis. Ann Surg 1976;184:415–421. 10. Nakajo S, Yamamoto M, Tahara E. Morphometric analysis of gallbladder adenocarcinoma: discrimination between carcinoma and dysplasia. Virchows Arch A Pathol Anat 1989;416:133–140. 11. Juste C, Catala I, Riottot M, Andre´ M, Parquet M, Lyian B, Be´guet F, Fe´re´zou-Viala J, Se´rougne C, Domingo N, Lutton C, Lafont H, Corring T. Inducing cholesterol precipitation from pig bile with b-cyclodextrin and cholesterol dietary supplementation. J Hepatol 1997;26:711–721. 12. Konikoff FM, Chung DS, Donovan JM, Small DM, Carey MC. Filamentous, helical, and tubular microstructures during cholesterol crystallization from bile. Evidence that cholesterol does not nucleate classic monohydrate plates. J Clin Invest 1992;90:1155– 1160.

GASTROENTEROLOGY Vol. 118, No. 5

13. Ginanni Corradini S, Arancia G, Calcabrini A, Della Guardia P, Baiocchi L, Nistri A, Giacomelli L, Angelico M. Lamellar bodies coexist with vesicles and micelles in human gallbladder bile. Ursodeoxycholic acid prevents cholesterol crystal nucleation by increasing biliary lamellae. J Hepatol 1995;22:642–657. 14. Fox H. Ultastructures of the human gallbladder epithelium in cholelithiasis and chronic cholecystitis. J Pathol 1972;108:157– 164. 15. Ghadially FN. The dark cell/light cell phenomenon. In: Ultrastructural pathology of the cell and matrix. 4th ed. London: Butterworth Heinemann, 1997:1014–1019. 16. Gilloteaux J, Karkare S, Kelly TR, Hawkins WS. Ultrastructural aspects of human gallbladder ephitelial cells in cholelithiasis. Production of anionic mucus. Microsc Res Tech 1997;38:643– 659. 17. Halpern Z, Dudley MA, Lynn MP, Nader JM, Breuer AC, Holzbach RT. Vesicle aggregation in model systems of supersaturated bile: relation to crystal nucleation and lipid composition of the vesicular phase. J Lipid Res 1986;27:295–306. 18. Pemshing RS, Mac Pherson BR, Scott GW. Morphological observations on the gallbladder of ground squirrels fed a lithogenic diet. J Pathol 1987;152:127–135. 19. Scott AJ. Epithelial cell proliferation in diverse models of experimental cholelithiasis. Gut 1978;19:558–562. 20. Lamote J, Putz P, Francois M, Willems G. DNA synthesis index: higher for human gallbladders with cholesterol gallstones than with pigment gallstones. J Natl Cancer Inst 1983;71:449–453. 21. Lamote J, Willems G. DNA synthesis, cell proliferation index in normal and abnormal gallbladder epithelium. Microsc Res Tech 1997;38:609–615. 22. Marsch-Ziegler U, Palme G. The influence of cholic acid and cholesterol on cell proliferation in the gallbladder mucosa of the mouse. Virchows Arch B Cell Pathol 1982;39:217–228. 23. Yu P, Chen Q, Biancani P, Behar J. Membrane cholesterol alters gallbladder muscle contractility in prairie dogs. Am J Physiol 1996;34:G56–G61. 24. Chen Q, Amaral J, Biancani P, Behar J. Excess membrane cholesterol alters human gallbladder muscle contractility and membrane fluidity. Gastroenterology 1999;116:678–685. Received March 4, 1999. Accepted December 23, 1999. Address requests for reprints to: Stefano Ginanni Corradini, M.D., Ph.D., Via Asmara 9-B, 00199 Rome, Italy. e-mail: corradini@ uniroma1.it; fax: (39) 06-4453319. The authors thank Giuseppe Di Lullo for technical assistance in electron microscopy, Marina Pines for revising the English text, and the veterinary surgeons of the Tor Cervara abattoir of Rome, Italy, for providing the pig gallbladders.