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Increased acylcoa-cholesterol ester acyltransferase activity in gallbladder mucosa in patients with gallbladder cholesterolosis
THE AMERICAN JOURNAL OF GASTROENTEROLOGY Copyright © 1998 by Am. Coll. of Gastroenterology Published by Elsevier Science Inc.
Vol. 93, No. 9, 1998 ISSN 0002-9270/98/$19.00 PII S0002-9270(98)00354-2
Increased AcylCoA-Cholesterol Ester Acyltransferase Activity in Gallbladder Mucosa in Patients With Gallbladder Cholesterolosis Fumitoshi Watanabe, M.D., Hiroyuki Hanai, M.D., Ph.D., and Eizo Kaneko, M.D., Ph.D. First Department of Medicine, Hamamatsu University School of Medicine, Hamamatsu-City, Japan
Objective: Although cholesterolosis of the human gallbladder is a relatively common disease, its etiology has not been fully understood. The aim of this study was to determine this etiology. Methods: The lipid composition of the gallbladder mucosa and gallbladder bile and the enzyme activities (acylCoA-cholesterol ester acyltransferase [ACAT] and cholesterol ester hydrolase [CEH]) of the gallbladder mucosa were measured in control subjects, patients with cholesterolosis, and patients with cholesterol gallstone disease. Results: Levels of cholesterol ester in gallbladder mucosa in patients with cholesterolosis (n 5 12) were higher than those in control subjects (n 5 8). With regard to the lipid content in gallbladder bile, no differences were found in concentrations of cholesterol, phospholipids, and bile acids among control subjects (n 5 11), patients with cholesterolosis (n 5 13), and those with cholesterol gallstone disease (n 5 15). In gallbladder mucosa, ACAT activity was significantly higher in patients with cholesterolosis (n 5 10) than in control subjects (n 5 8), whereas CEH activity did not differ between the two groups. As a result, the ACAT/CEH activity ratio was higher in patients with cholesterolosis than in control subjects. Conclusions: It would be suggested that cholesterol ester synthesis of gallbladder mucosa might play an etiological role in the development of cholesterolosis. (Am J Gastroenterol 1998;93:1518 –1523. © 1998 by Am. Coll. of Gastroenterology)
Cholesterolosis is characterized histologically by the accumulation of lipids in the lamina propria of the gallbladder (3, 4). These lipid deposits consist mostly of cholesterol ester (4, 5) and triglycerides (6). Despite the many histological studies of cholesterolosis, its etiology has not been fully understood. The aim of the present study is to examine the etiology of cholesterolosis by measuring lipid contents in the gallbladder mucosa and the gallbladder bile and the activities of enzymes (acylCoA-cholesterol ester acyltransferase [ACAT] and cholesterol ester hydrolase [CEH]) that which regulate the formation and hydrolysis of cholesterol ester. MATERIALS AND METHODS The gallbladders of patients who had undergone cholecystectomy were assessed as normal, having cholesterolosis, or having cholesterol gallstone based on macroscopic and microscopic examination. The gallbladder which revealed a characteristic yellow-spotted or reticular pattern mucosa macroscopically and lipid droplets in the lamina propria microscopically was diagnosed as cholesterolosis. Samples of bile were aspirated from the gallbladder of each patient during surgery. Lipid content of gallbladder mucosa The body portion of the gallbladder resected during cholecystectomy was rinsed with saline to remove bile, and the mucosa of the gallbladder was separated from the muscle layer with the surgical knife. The control group included eight subjects (three men, five women; mean age 51.0 6 13.2 yr) who underwent surgery for either gastric cancer or chronic pancreatitis. The cholesterolosis group included 12 subjects (nine men, three women; mean age 49.0 6 12.6 yr). After homogenization of pieces of the mucosa with a Potter glass homogenizer, lipids were extracted using the method described by Folch et al. (7) and isolated using thin layer chromatography (TLC). Free cholesterol and phospholipids were isolated by applying samples to silica gel G glass plates, whereas cholesterol ester and triglycerides were isolated by applying samples to silica gel aluminium plates. The plates were developed in n-hexane-diethyl ether-
INTRODUCTION Cholesterolosis of the human gallbladder is a common disease (in surgical studies its prevalence has varied from 9% to 24%), and is characterized by accumulation of lipids. The polypoid form of cholesterolosis is characterized by a cholesterol polyp of the gallbladder that is most frequently detected by ultrasound examination. Cholesterolosis of the gallbladder was first described by Moynihan in 1909 (1) and called “strawberry gallbladder” by MacCarty in 1910 (2). Received Dec. 29, 1997; accepted May 11, 1998. 1518
AJG – September 1998 acetic acid (8:2:0.1) to isolate free cholesterol, cholesterol ester, and triglycerides, and in chloroform-methanol-water (6:4:1) to isolate phospholipids. After lipids had been isolated, free cholesterol was detected by spraying CrSO4 (85% H2SO4 involved with 0.6% Cr) and heating for 30 min at 100 –120°C (8), phospholipids by staining with molybdenum blue (9), and triglycerides by staining with Coomassie blue. The lipid content was measured by standardized densitometry (high-speed TLC scanner, CS-920 Shimazu, Tokyo, Japan). Lipid content of gallbladder bile Based on appearance during surgery, gallbladders were assessed as either being normal controls, having cholesterolosis, or having cholesterol stone disease. The control group included 11 subjects (five men, six women; mean age 61.2 6 10.7 yr), the cholesterolosis group included 13 subjects (five men, eight women; mean age 50.1 6 14.4 yr), and the cholesterol stone group included 15 subjects (seven men, eight women; mean age 53.0 6 14.1 yr). Cholesterol and phospholipids in the gallbladder bile (aspirated by needle during surgery) were extracted using the method described by Folch et al. (7) and separated by TLC. Lipid content was measured using the same method as for gallbladder mucosa. After the gallbladder bile had been diluted with methanol and filtrated, bile acid content was quantitated by high performance liquid chromatography (HPLC) and 3 a-hydroxysteroid dehydrogenase (3 a-HSD) was immobilized in column form (using 5 b-pregnane-3 a-17 a-2 a-triol as an internal standard) (10). Assay of ACAT and CEH activity in gallbladder mucosa The control group included eight subjects (five men, three women; mean age 58.9 6 13.9 yr), and the cholesterolosis group included 10 subjects (five men, five women; mean age 43.6 6 10.0 yr). The portion of the gallbladder resected during cholecystectomy was carefully rinsed with saline, and the mucosa of the gallbladder was separated from the muscle layer with the surgical knife. After homogenizing pieces of the mucosa using a Potter homogenizer, an aliquot was used to measure protein content and the remainder was used for the enzyme assay. Protein was measured using the method described by Bradford (11). To ensure accurate measurements of enzyme activity in human gallbladder mucosa, we determined the optimal conditions for each assay through time course and dose-response experiments. Assay of ACAT activity The assay used was a modified version of that described earlier (12). Each assay sample contained 0.2 mg fatty acid-free bovine serum albumin in 0.04 mol/L KH2PO4
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buffer (pH 7.4), with 0.05 mol/L KCl, 0.03 mmol/L EDTA, and 0.3 mol/L sucrose. The total volume was 0.2 ml. The reaction was started by adding 0.1 mg of gallbladder mucosa protein and 10 nmol [1-14C]oleoyl-CoA(0.005 mCi/ nmol), and the mixture was incubated for 30 min at 37°C. The assay was stopped by adding chloroform/methanol (2: 1). Lipid was extracted using the method described by Folch et al., and the [1-14C]cholesterol oleate formed was separated in n-hexane-diethyl ether-acetic acid (8:2:0.1) using TLC. The band corresponding to cholesterol esters was scraped into scintillation vials and counted in a Beckman liquid scintillation counter. Assay of CEH activity The assay used was a modified version of that described earlier (13). Each assay contained 0.05 mol/L Tris-HCl buffer (pH 7.1) with 5 mol/L MgCl2, 50 mmol/L KCl, 0.2 mmol/L EDTA, 0.4% fatty acid-free bovine serum albumin and 0.5% Triton 100-X. The total volume was 0.2 ml. The reaction was started by adding 0.1 mg of gallbladder mucosa protein and 30 mmol/L [14C]cholesteryl oleate (5.0 mCi/mmol), and the mixture was incubated for 15 min at 37°C. The assay was stopped by adding methanol. After addition of 1-N HCl and n-hexane, lipids was extracted from the methanol solution using the method described by Folch et al., and the [14C] cholesterol formed was separated in n-hexane-diethyl ether-acetic acid using TLC. The band corresponding to cholesterol was scraped into scintillation vials and counted in a Beckman liquid scintillation counter. All data about biliary lipids, mucosal lipids and mucosal enzymes were obtained from each individual. The significance of the differences was determined using Student’s t-test. RESULTS Lipid content of gallbladder mucosa The lipid contents of the gallbladder mucosa are shown in Table 1. The concentration of cholesterol ester in mucosa was higher in patients with cholesterolosis than in control subjects (890.1 6 293.1 mg/mg z protein vs 57.2 6 21.6 mg/mg z protein, p , 0.05). However, levels of free cholesterol, phospholipids, and triglycerides in mucosa did not differ significantly between patients with cholesterolosis and control subjects (free cholesterol: 51.9 6 5.8 mg/mg z protein vs 41.9 6 10.9 mg/mg z protein; phospholipids: 45.9 6 14.4 mg/mg z protein vs 109.7 6 57.4 mg/mg z protein; triglycerides: 299.5 6 102.7 mg/mg z protein vs 446.8 6 186.5 mg/mg z protein). Lipid content of gallbladder bile The concentration of cholesterol did not differ among control subjects, patients with cholesterolosis, and patients with cholesterol gallstone disease (9.2 6 3.8 vs 8.8 6 5.6 vs 9.6 6 4.6 mmol/L) (Fig. 1). Similarly, the concentration of phospholipids did not dif-
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TABLE 1 Lipid Content of Gallbladder Mucosa in Control Subjects and Patients With Cholesterolosis
Control (n 5 8) Cholesterolosis (n 5 12)
CHE (mg/mg z protein)
FCH (mg/mg z protein)
PL (mg/mg z protein)
TG (mg/mg z protein)
57.2 6 21.6*
41.9 6 10.9
109.7 6 37.4
299.5 6 102.7
890.1 6 293.1*
14.9 6 4.0
45.9 6 10.4
446.8 6 186.5
CHE, cholesterol ester; FCH, free cholesterol; PL, phospholipid; TG, triglyceride. * p , 0.05
FIG. 1. Lipid composition of gallbladder bile in control subjects, patients with cholesterolosis, and patients with gallstone (mmol/L and percentage of total mmoles).
fer among these three groups (43.8 6 17.7 vs 35.6 6 18.9 vs 28.5 6 19.0 mmol/L), nor did that bile acids (178.8 6 68.8 vs 180.0 6 40.8 vs 141.2 6 67.2 mmol/L) (Fig. 1). The molar percentages of cholesterol [(moles of cholesterol/total moles of biliary lipid) 3 100] were higher in patients with cholesterol gallstone disease than in both con-
trol subjects (6.2 6 1.8 vs 4.0 6 1.3%, p , 0.05) and patients with cholesterolosis (6.2 6 1.8 vs 4.2 6 1.3% p , 0.05) (Fig. 1). The molar percentages of phospholipids and bile acids were similar in the three groups; no significant differences were found among them. (phospholipids: 18.4 6 6.4 vs
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TABLE 2 Molar Percentage of Free Cholesterol and Cholesterol Ester, Composition of Bile Acids, and Lithogenic Index of Gallbladder Bile in Control Subjects, Patients With Cholesterolosis, and Patients With Cholesterol Gallstone
Control (n 5 11) Cholesterolosis (n 5 13) Cholesterol gallstone (n 5 15)
Cholesterol (Mole %) FCH
CHE
Bile Acids (Wt%) UDCA
CA
CDCA
DCA
LCA
99.1 6 0.8 98.9 6 0.9
0.9 6 0.8 1.1 6 0.9
6.3 6 4.1 5.2 6 3.4
35.8 6 8.1 37.8 6 9.5
44.7 6 8.5 43.0 6 121
10.6 6 6.2 12.6 6 8.4
0.7 6 0.6 0.4 6 0.2
0.42 6 0.13* 0.47 6 0.12*
99.3 6 0.7
0.8 6 0.7
9.0 6 5.8
34.0 6 11.3
37.8 6 7.7
18.1 6 8.8
1.4 6 0.9
0.95 6 0.17*
LI
FCH, free cholesterol; CHE, cholesterol ester; UDCA, ursodeoxycholic acid; CA, cholic acid; CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; LCA, lithocholic acid; LI, lithogenic index. * p , 0.05.
15.8 6 5.9 vs 17.0 6 5.8%; bile acids: 78.1 6 7.3 vs 82.3 6 7.4 vs 80.7 6 5.2%) (Fig. 1). Total cholesterol consisted of .99% free cholesterol in all three groups, and no differences were found among the groups in cholesterol ester content (Table 2). The molar percentages of individual bile acids in total bile acids did not differ among the three groups (Table 2). The lithogenic index (14) was higher in patients with cholesterol gallstone disease than in control subjects and in patients with cholesterolosis, wheres no differences were found between control subjects and those with cholesterolosis (Table 2). ACAT and CEH activity in gallbladder mucosa The optimal conditions for assay of ACAT and CEH are illustrated in Figures 2 and 3, respectively. The ACAT activity was linear at 0.1 mg protein over a period of 45 min (Fig. 2A). ACAT activity was linear up to 0.3 mg protein for a period of 30 min (Fig. 2B). The CEH activity was linear at 0.1 mg protein over a period of 15 min (Fig. 3A). CEH activity was linear up to 0.5 mg protein for a period of 15 min (Fig. 3B). In gallbladder mucosa, ACAT activity was significantly higher in patents with cholesterolosis than in control subjects, but CEH activity did not differ between these two groups. As a result, the ACAT/CEH activity ratio was higher for patients with cholesterolosis than for control subjects (Table 3). DISCUSSION Cholesterolosis of the human gallbladder is characterized by the accumulation of lipids in the epithelial mucosa and lamina propria, and cells containing these lipids are called “foamy cells” (4). The pathological features of cholesterolosis have been studied by microscopy (3, 4, 15) and electron microscopy (16), but the pathogenesis of this condition is not fully understood. The accumulated lipids have been reported to consist mainly of cholesterol ester (4, 5). In the present study, the gallbladder mucosa had markedly elevated concentrations of cholesterol ester in patients with cholesterolosis, but no differences were found in the con-
FIG. 2. Assay of ACAT activity in gallbladder mucosa. The assay was a modified version of one described previously (10). Each assay sample contained 0.2 mg fatty acid-free bovine serum albumin in 0.04 mol/L KH2PO4 buffer (pH 7.4) with 0.05 mol/L KCl, 0.03 mmol/L EDTA, and 0.3 mol/L sucrose. The total volume was 0.2 ml. (A) Protein dependence: The reaction was started by adding various amounts of gallbladder mucosa protein and 10 nmol [1-14C]oleoyl-CoA(0.005 mCi/nmol), and the mixture was incubated for 30 min at 37°C. The assay was stopped by adding chloroform/methanol (2:1). Lipid was extracted and separated as described in Materials and Methods. (B) Time dependence: Assays were performed as above. The amount of protein was 0.1 mg.
centrations of free cholesterol, phospholipids, and triglycerides between patients with cholesterolosis and control subjects. In an analysis of gallbladder bile (17, 18), patients with cholesterolosis were found to have high biliary cholesterol
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FIG. 3. Assay of CEH activity in gallbladder mucosa. The assay was a modified version of one described previously (11). Each assay sample contained 0.05 mol/L Tris-HCl buffer (pH 7.1) with 5 mmol/L MGCl2, 50 mmol/L KCL, 0.2 mmol/L EDTA, 0.4% fatty acid-free bovine serum albumin, and 0.5% Triton 100-X. The total volume was 0.2 ml. (A) Protein dependence: The reaction was started by adding various amounts of gallbladder mucosa protein and 30 mmol/L [14C]cholesteryl oleate (5.0 mCi/ mmol), and the mixture was incubated for 15 min at 37°C. The assay was stopped by adding methanol. Lipids was extracted and separated as described in Materials and Methods. (B) Time dependence: Assays were performed as above. The amount of protein was 0.1 mg. TABLE 3 ACAT and CHE Activities in Gallbladder Mucosa in Control Subjects and Patients With Cholesterolosis
Control (n 5 8) Cholesterolosis (n 5 10)
ACAT (pmol/mg z protein z min)
CEH (pmol/mg z protein z min)
ACAT/CEH Ratio
13.7 6 4.7*
12.9 6 6.4
1.4 6 0.6†
47.5 6 3.8*
14.9 6 4.0
3.9 6 1.9†
ACAT, acyl CoA cholesterol acyltransferase; CEH, cholesterol ester hydrolase. * p , 0.01; † p , 0.05
content and supersaturated bile. On the other hand, supersaturated bile was found to be present in both cholesterol gallstone and those with cholesterolosis (19), but was also commonly found in healthy subjects. The present data showed that the concentrations of cholesterol, phospholipids, and bile acid in gallbladder bile did not differ between
control subjects and patients with cholesterolosis. Most of the cholesterol in gallbladder bile consisted of free cholesterol, and the molar percentage of cholesterol did not differ among control subjects, patients with cholesterolosis, and patients with cholesterol gallstones. The high degree of variation in the incidences of cholesterolosis and cholesterol gallstone suggests that the pathogenesis of cholesterolosis might be unrelated to that of cholesterol gallstones. Supersaturation of bile cholesterol may not be the major etiological factor in cholesterolosis. Instead, altered hepatic cholesterol metabolism, altered uptake (20) and mucosal esterification of free sterols from the bile, may result in cholesterolosis (6). In guinea pigs (20) and humans (21), gallbladders mucosa is embryonically related to intestinal mucosa and has been shown to absorb small amounts of free cholesterol from bile through the epithelium by a passive as well as a partially active process. In a study of cholesterol uptake by human gallbladder using a modified Ussing technique, neither the uptake of cholesterol by the mucosa nor the appearance of cholesterol in the serosal fluid differed between a normal gallbladder and one with cholesterolosis (22). However, the mechanism by which absorbed cholesterol reaches the lamina propria of the gallbladder and how it is stored have not yet been established. In any case, cholesterol metabolism in the gallbladder mucosa is the most important factor in understanding the etiology. Our data suggested that accumulated lipids in patients with cholesterolosis consisted mainly of cholesterol ester. This cholesterol ester may have originated in bile or may have been synthesized de novo in the gallbladder mucosa. Our findings suggest that it is not the mechanism by which the degree of saturation of bile causes cholesterolosis. Enzymes participating in intracellular cholesterol metabolism include acyl CoA cholesterol acyltransferase (ACAT), cholesterol ester hydrolase (CEH), 3-hydroxy 3-methylglutary coenzymeA reductase (HMG-CoA reductase) and acid lipase, which have been studied in human liver (23), intestine (24), adrenal cortex (25), and ovary (26). The presence of mucosal-sterol-esterifying enzyme (probably ACAT) has been found in the guinea pig (27) and human (28) gallbladder epithelium, but no reports have been made about CEH of human gallbladder. We postulate the pathways of cholesterol in the gallbladder mucosa in patients with cholesterolosis (Fig. 4). For storage of cholesterol ester in the cell, cholesterol ester flux into the cell or cholesterol synthesis de novo is required. Furthermore, cholesterol ester formed from cholesterol by ACAT is converted to free cholesterol by CEH. The imbalance between activity of ACAT and that of CEH could thus cause the disorder of cholesterol metabolism in gallbladder mucosa. In this study, both ACAT and CEH activities were measured in the human gallbladder mucosa. ACAT activity was significantly higher in patients with cholesterolosis than in control subjects, but CEH activity did not differ between these two groups. It would be suggested that the deposition of cholesterol ester could be
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FIG. 4. Postulated pathways of cholesterol in gallbladder mucosa in patients with cholesterolosis. CHE was accumulated in gallbladder mucosa because ACAT activity was increased, but CEH activity was not increased compared with that of control subjects. ACAT, acylCoA cholesterol acyltransfenase; CEH, cholesterol ester hydrolase; FCH, free cholesterol; CHE, cholesterol ester; HMG-CoA, 3-hydroxy-3methylglutaryl CoA; LDL, low density lipoprotein.
accelerated in the gallbladder mucosa in patients with cholesterolosis. The origin of the cholesterol that was transferred to the free-cholesterol pool in the cell has not been identified, because we have not measured the activity of HMG-CoA reductase (located in the microsome for catalysis of the rate-limiting step in cholesterol synthesis) or that of acid lipase (located in the lysosome for hydrolysis of cholesterol ester). It has previously been suggested that patients with cholesterolosis of the gallbladder might have increased hepatic cholesterol synthesis (6). More recently, however, Sahlin et al. reported that cholesterolosis of the gallbladder mucosa is not associated with increased levels of HMG-CoA reductase activity in the liver and gallbladder mucosa (28). Foamy cells originates from macrophages, and the macrophages possess ACAT (29). These findings also support our suggestion that the increase in ACAT activity in gallbladder mucosa could be the cause of cholesterolosis. Reprint requests and correspondence: Fumitoshi Watanabe, M.D., First Department of Medicine, Hamamatsu University School of Medicine, 3600 Handa-cho, Hamamatsu-City, 431-3192 Japan.
REFERENCES 1. Moynihan BGA. A disease of the gallbladder requiring cholecystectomy. Ann Surg 1909;50:1265–72. 2. MacCarty WC. The pathology of the gallbladder and some associated lesions. Ann Surg 1910;51:651– 69. 3. Mackey WA. Cholesterosis of the gallbladder. A further contribution to the histology of this condition. Br J Surg 1941;28:462.
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4. Boyd W. Studies in gallbladder pathology. Br J Surg 1922;10:337–56. 5. Akitoshi K, Satoru T, Masaya N. Electron microscopic observations on the cholesterosis of the gallbladder, with special reference to its pathogenesis. Jpn J Gastroenterol 1974;71:1085–1101. 6. Tilvis RS, Aro J, Strandberg TE, et al. Lipid composition of bile and gallbladder mucosa in patients with acalculous cholesterolosis. Gastroenterology 1982;82:607–15. 7. Folch J, Less M, Sloane Stanley GH. A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 1957;226:497–509. 8. Wiklund B, Eliason. A micromethod for the quantitative determination of lipids by use of thin-layer chromatography, photodensitometry and an internal standard. J Chromatogr 1972;68:153–9. 9. Heyeman RA, Bernard DM, Vercauteren RE. Direct fluorometric microdetermination of phospholipids on thin-layer chromatograms. J Chromatogr 1972;68:285– 8. 10. Sumihiko O. Analysis of free, glycine- and taurine-conjugated individual bile acids using high performance liquid chromatography and immobilized 3 a-hydroxysteroid dehydrogenase in column form. Jpn J Clin Pathol 1981;5:446 –58. 11. Bradford M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248 –54. 12. Jeffrey FF, Cooper AD, Erickson SK. Regulation of rabbit intestinal acyl coenzyme A-cholesterol acyltransferase in vivo and in vitro. Gastroenterology 1982;83:873– 80. 13. Brody AL, Black VH. Acyl-coenzyme A cholesterol acyltransferase and cholesterol ester hydrolase in the outer and inner cortices of the guinea pig adrenal: Effects of adrenocorticotropin and dexamethasone. Endocrinology 1988;122:1722–31. 14. Holzbach RT, Marsh M, Olszewski M, et al. Cholesterol solubility in bile. J Clin Invest 1973;52:1467–79. 15. Illingworth CFW. Cholesterosis of the gallbladder: A clinical and experimental study. Br J Surg 1929;17:208. 16. Akitoshi K. Fine structure of the human gallbladder with cholesterosis with special reference to the mechanism of lipid accumulation. Br J Exp Pathol 1985;66:605–11. 17. Luciano L. Morphological aspects of cholesterol storage in the human gallbladder. Cells Tissues 1989;269 –75. 18. Agalovschi M, Dumitrascu D, Grigorescu M, et al. Pathogenetic interrelations between cholesterolosis and cholesterol gallstone disease. Rev Roum Med-Med Int 1983;21:175–9. 19. Teruyoshi Y. A study of cholesterol gallstone formation in cases of cholesterolosis of the gallbladder. Jpn J Gastroenterol 1979;76:91–102. 20. Jacyna MR, Ross PE, Hopwood D, et al. Absorption of cholesterol by the human gallbladder. Gut 1985;26:A1132. 21. Neiderhiser DH, Harmon CK, Roth HP. Absorption of cholesterol by the gallbladder. J Lipid Res 1976;17:117–24. 22. Jacyna MR, Ross PE, Bakar MA, et al. Characteristics of cholesterol absorption by human gallbladder: Relevance to cholesterolosis. J Clin Pathol 1987;40:524 –9. 23. Einarsson K, Benthin L, Ewerth S, et al. Studies on acyl-coenzyme A: Cholesterol acyltransferase activity in human liver microsomes. J Lipid Res 1989;30:739 – 46. 24. Norum K, Lilljeqvist A, Helgerud P, et al. Esterification of cholesterol in human small intestine: The importance of acyl-CoA:cholesterol acyltransferase. Eur J Clin Invest 1979;9:55– 62. 25. Tetsuo N, Keiji M, Yasushi S, et al. Functional differences in cholesterol ester hydrolase and acyl-coenzyme-A/cholesterol acyltransferase between the outer and inner zones of the guinea pig adrenal cortex. Endocrinology 1988;122:877– 83. 26. Veldhuis JD, Stuauss JF, Silavin SL. The role of cholesterol esterification in ovarian steroidogenesis. Endocrinology. 1985;116:25–30. 27. Subbian MT, Dicke BA. Presence of cholesterol ester synthetase activity in guinea pig gallbladder epithelium. Biochem Biophys Res Commun 1977;74:1273–9. 28. Sahlin S, Stahlberg D, Einarsson K. Cholesterol metabolism in liver and gallbladder mucosa of patients with cholesterolosis. Hepatology 1995;21:1269 –75. 29. Brown MS, Goldstein JL. Lipoprotein metabolism in the macrophage: Implications for cholesterol deposition in atherosclerosis. Annu Rev Biochem 1983;52:223– 61.
Vol. 93, No. 9, 1998 ISSN 0002-9270/98/$19.00 PII S0002-9270(98)00354-2
Increased AcylCoA-Cholesterol Ester Acyltransferase Activity in Gallbladder Mucosa in Patients With Gallbladder Cholesterolosis Fumitoshi Watanabe, M.D., Hiroyuki Hanai, M.D., Ph.D., and Eizo Kaneko, M.D., Ph.D. First Department of Medicine, Hamamatsu University School of Medicine, Hamamatsu-City, Japan
Objective: Although cholesterolosis of the human gallbladder is a relatively common disease, its etiology has not been fully understood. The aim of this study was to determine this etiology. Methods: The lipid composition of the gallbladder mucosa and gallbladder bile and the enzyme activities (acylCoA-cholesterol ester acyltransferase [ACAT] and cholesterol ester hydrolase [CEH]) of the gallbladder mucosa were measured in control subjects, patients with cholesterolosis, and patients with cholesterol gallstone disease. Results: Levels of cholesterol ester in gallbladder mucosa in patients with cholesterolosis (n 5 12) were higher than those in control subjects (n 5 8). With regard to the lipid content in gallbladder bile, no differences were found in concentrations of cholesterol, phospholipids, and bile acids among control subjects (n 5 11), patients with cholesterolosis (n 5 13), and those with cholesterol gallstone disease (n 5 15). In gallbladder mucosa, ACAT activity was significantly higher in patients with cholesterolosis (n 5 10) than in control subjects (n 5 8), whereas CEH activity did not differ between the two groups. As a result, the ACAT/CEH activity ratio was higher in patients with cholesterolosis than in control subjects. Conclusions: It would be suggested that cholesterol ester synthesis of gallbladder mucosa might play an etiological role in the development of cholesterolosis. (Am J Gastroenterol 1998;93:1518 –1523. © 1998 by Am. Coll. of Gastroenterology)
Cholesterolosis is characterized histologically by the accumulation of lipids in the lamina propria of the gallbladder (3, 4). These lipid deposits consist mostly of cholesterol ester (4, 5) and triglycerides (6). Despite the many histological studies of cholesterolosis, its etiology has not been fully understood. The aim of the present study is to examine the etiology of cholesterolosis by measuring lipid contents in the gallbladder mucosa and the gallbladder bile and the activities of enzymes (acylCoA-cholesterol ester acyltransferase [ACAT] and cholesterol ester hydrolase [CEH]) that which regulate the formation and hydrolysis of cholesterol ester. MATERIALS AND METHODS The gallbladders of patients who had undergone cholecystectomy were assessed as normal, having cholesterolosis, or having cholesterol gallstone based on macroscopic and microscopic examination. The gallbladder which revealed a characteristic yellow-spotted or reticular pattern mucosa macroscopically and lipid droplets in the lamina propria microscopically was diagnosed as cholesterolosis. Samples of bile were aspirated from the gallbladder of each patient during surgery. Lipid content of gallbladder mucosa The body portion of the gallbladder resected during cholecystectomy was rinsed with saline to remove bile, and the mucosa of the gallbladder was separated from the muscle layer with the surgical knife. The control group included eight subjects (three men, five women; mean age 51.0 6 13.2 yr) who underwent surgery for either gastric cancer or chronic pancreatitis. The cholesterolosis group included 12 subjects (nine men, three women; mean age 49.0 6 12.6 yr). After homogenization of pieces of the mucosa with a Potter glass homogenizer, lipids were extracted using the method described by Folch et al. (7) and isolated using thin layer chromatography (TLC). Free cholesterol and phospholipids were isolated by applying samples to silica gel G glass plates, whereas cholesterol ester and triglycerides were isolated by applying samples to silica gel aluminium plates. The plates were developed in n-hexane-diethyl ether-
INTRODUCTION Cholesterolosis of the human gallbladder is a common disease (in surgical studies its prevalence has varied from 9% to 24%), and is characterized by accumulation of lipids. The polypoid form of cholesterolosis is characterized by a cholesterol polyp of the gallbladder that is most frequently detected by ultrasound examination. Cholesterolosis of the gallbladder was first described by Moynihan in 1909 (1) and called “strawberry gallbladder” by MacCarty in 1910 (2). Received Dec. 29, 1997; accepted May 11, 1998. 1518
AJG – September 1998 acetic acid (8:2:0.1) to isolate free cholesterol, cholesterol ester, and triglycerides, and in chloroform-methanol-water (6:4:1) to isolate phospholipids. After lipids had been isolated, free cholesterol was detected by spraying CrSO4 (85% H2SO4 involved with 0.6% Cr) and heating for 30 min at 100 –120°C (8), phospholipids by staining with molybdenum blue (9), and triglycerides by staining with Coomassie blue. The lipid content was measured by standardized densitometry (high-speed TLC scanner, CS-920 Shimazu, Tokyo, Japan). Lipid content of gallbladder bile Based on appearance during surgery, gallbladders were assessed as either being normal controls, having cholesterolosis, or having cholesterol stone disease. The control group included 11 subjects (five men, six women; mean age 61.2 6 10.7 yr), the cholesterolosis group included 13 subjects (five men, eight women; mean age 50.1 6 14.4 yr), and the cholesterol stone group included 15 subjects (seven men, eight women; mean age 53.0 6 14.1 yr). Cholesterol and phospholipids in the gallbladder bile (aspirated by needle during surgery) were extracted using the method described by Folch et al. (7) and separated by TLC. Lipid content was measured using the same method as for gallbladder mucosa. After the gallbladder bile had been diluted with methanol and filtrated, bile acid content was quantitated by high performance liquid chromatography (HPLC) and 3 a-hydroxysteroid dehydrogenase (3 a-HSD) was immobilized in column form (using 5 b-pregnane-3 a-17 a-2 a-triol as an internal standard) (10). Assay of ACAT and CEH activity in gallbladder mucosa The control group included eight subjects (five men, three women; mean age 58.9 6 13.9 yr), and the cholesterolosis group included 10 subjects (five men, five women; mean age 43.6 6 10.0 yr). The portion of the gallbladder resected during cholecystectomy was carefully rinsed with saline, and the mucosa of the gallbladder was separated from the muscle layer with the surgical knife. After homogenizing pieces of the mucosa using a Potter homogenizer, an aliquot was used to measure protein content and the remainder was used for the enzyme assay. Protein was measured using the method described by Bradford (11). To ensure accurate measurements of enzyme activity in human gallbladder mucosa, we determined the optimal conditions for each assay through time course and dose-response experiments. Assay of ACAT activity The assay used was a modified version of that described earlier (12). Each assay sample contained 0.2 mg fatty acid-free bovine serum albumin in 0.04 mol/L KH2PO4
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buffer (pH 7.4), with 0.05 mol/L KCl, 0.03 mmol/L EDTA, and 0.3 mol/L sucrose. The total volume was 0.2 ml. The reaction was started by adding 0.1 mg of gallbladder mucosa protein and 10 nmol [1-14C]oleoyl-CoA(0.005 mCi/ nmol), and the mixture was incubated for 30 min at 37°C. The assay was stopped by adding chloroform/methanol (2: 1). Lipid was extracted using the method described by Folch et al., and the [1-14C]cholesterol oleate formed was separated in n-hexane-diethyl ether-acetic acid (8:2:0.1) using TLC. The band corresponding to cholesterol esters was scraped into scintillation vials and counted in a Beckman liquid scintillation counter. Assay of CEH activity The assay used was a modified version of that described earlier (13). Each assay contained 0.05 mol/L Tris-HCl buffer (pH 7.1) with 5 mol/L MgCl2, 50 mmol/L KCl, 0.2 mmol/L EDTA, 0.4% fatty acid-free bovine serum albumin and 0.5% Triton 100-X. The total volume was 0.2 ml. The reaction was started by adding 0.1 mg of gallbladder mucosa protein and 30 mmol/L [14C]cholesteryl oleate (5.0 mCi/mmol), and the mixture was incubated for 15 min at 37°C. The assay was stopped by adding methanol. After addition of 1-N HCl and n-hexane, lipids was extracted from the methanol solution using the method described by Folch et al., and the [14C] cholesterol formed was separated in n-hexane-diethyl ether-acetic acid using TLC. The band corresponding to cholesterol was scraped into scintillation vials and counted in a Beckman liquid scintillation counter. All data about biliary lipids, mucosal lipids and mucosal enzymes were obtained from each individual. The significance of the differences was determined using Student’s t-test. RESULTS Lipid content of gallbladder mucosa The lipid contents of the gallbladder mucosa are shown in Table 1. The concentration of cholesterol ester in mucosa was higher in patients with cholesterolosis than in control subjects (890.1 6 293.1 mg/mg z protein vs 57.2 6 21.6 mg/mg z protein, p , 0.05). However, levels of free cholesterol, phospholipids, and triglycerides in mucosa did not differ significantly between patients with cholesterolosis and control subjects (free cholesterol: 51.9 6 5.8 mg/mg z protein vs 41.9 6 10.9 mg/mg z protein; phospholipids: 45.9 6 14.4 mg/mg z protein vs 109.7 6 57.4 mg/mg z protein; triglycerides: 299.5 6 102.7 mg/mg z protein vs 446.8 6 186.5 mg/mg z protein). Lipid content of gallbladder bile The concentration of cholesterol did not differ among control subjects, patients with cholesterolosis, and patients with cholesterol gallstone disease (9.2 6 3.8 vs 8.8 6 5.6 vs 9.6 6 4.6 mmol/L) (Fig. 1). Similarly, the concentration of phospholipids did not dif-
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TABLE 1 Lipid Content of Gallbladder Mucosa in Control Subjects and Patients With Cholesterolosis
Control (n 5 8) Cholesterolosis (n 5 12)
CHE (mg/mg z protein)
FCH (mg/mg z protein)
PL (mg/mg z protein)
TG (mg/mg z protein)
57.2 6 21.6*
41.9 6 10.9
109.7 6 37.4
299.5 6 102.7
890.1 6 293.1*
14.9 6 4.0
45.9 6 10.4
446.8 6 186.5
CHE, cholesterol ester; FCH, free cholesterol; PL, phospholipid; TG, triglyceride. * p , 0.05
FIG. 1. Lipid composition of gallbladder bile in control subjects, patients with cholesterolosis, and patients with gallstone (mmol/L and percentage of total mmoles).
fer among these three groups (43.8 6 17.7 vs 35.6 6 18.9 vs 28.5 6 19.0 mmol/L), nor did that bile acids (178.8 6 68.8 vs 180.0 6 40.8 vs 141.2 6 67.2 mmol/L) (Fig. 1). The molar percentages of cholesterol [(moles of cholesterol/total moles of biliary lipid) 3 100] were higher in patients with cholesterol gallstone disease than in both con-
trol subjects (6.2 6 1.8 vs 4.0 6 1.3%, p , 0.05) and patients with cholesterolosis (6.2 6 1.8 vs 4.2 6 1.3% p , 0.05) (Fig. 1). The molar percentages of phospholipids and bile acids were similar in the three groups; no significant differences were found among them. (phospholipids: 18.4 6 6.4 vs
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TABLE 2 Molar Percentage of Free Cholesterol and Cholesterol Ester, Composition of Bile Acids, and Lithogenic Index of Gallbladder Bile in Control Subjects, Patients With Cholesterolosis, and Patients With Cholesterol Gallstone
Control (n 5 11) Cholesterolosis (n 5 13) Cholesterol gallstone (n 5 15)
Cholesterol (Mole %) FCH
CHE
Bile Acids (Wt%) UDCA
CA
CDCA
DCA
LCA
99.1 6 0.8 98.9 6 0.9
0.9 6 0.8 1.1 6 0.9
6.3 6 4.1 5.2 6 3.4
35.8 6 8.1 37.8 6 9.5
44.7 6 8.5 43.0 6 121
10.6 6 6.2 12.6 6 8.4
0.7 6 0.6 0.4 6 0.2
0.42 6 0.13* 0.47 6 0.12*
99.3 6 0.7
0.8 6 0.7
9.0 6 5.8
34.0 6 11.3
37.8 6 7.7
18.1 6 8.8
1.4 6 0.9
0.95 6 0.17*
LI
FCH, free cholesterol; CHE, cholesterol ester; UDCA, ursodeoxycholic acid; CA, cholic acid; CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; LCA, lithocholic acid; LI, lithogenic index. * p , 0.05.
15.8 6 5.9 vs 17.0 6 5.8%; bile acids: 78.1 6 7.3 vs 82.3 6 7.4 vs 80.7 6 5.2%) (Fig. 1). Total cholesterol consisted of .99% free cholesterol in all three groups, and no differences were found among the groups in cholesterol ester content (Table 2). The molar percentages of individual bile acids in total bile acids did not differ among the three groups (Table 2). The lithogenic index (14) was higher in patients with cholesterol gallstone disease than in control subjects and in patients with cholesterolosis, wheres no differences were found between control subjects and those with cholesterolosis (Table 2). ACAT and CEH activity in gallbladder mucosa The optimal conditions for assay of ACAT and CEH are illustrated in Figures 2 and 3, respectively. The ACAT activity was linear at 0.1 mg protein over a period of 45 min (Fig. 2A). ACAT activity was linear up to 0.3 mg protein for a period of 30 min (Fig. 2B). The CEH activity was linear at 0.1 mg protein over a period of 15 min (Fig. 3A). CEH activity was linear up to 0.5 mg protein for a period of 15 min (Fig. 3B). In gallbladder mucosa, ACAT activity was significantly higher in patents with cholesterolosis than in control subjects, but CEH activity did not differ between these two groups. As a result, the ACAT/CEH activity ratio was higher for patients with cholesterolosis than for control subjects (Table 3). DISCUSSION Cholesterolosis of the human gallbladder is characterized by the accumulation of lipids in the epithelial mucosa and lamina propria, and cells containing these lipids are called “foamy cells” (4). The pathological features of cholesterolosis have been studied by microscopy (3, 4, 15) and electron microscopy (16), but the pathogenesis of this condition is not fully understood. The accumulated lipids have been reported to consist mainly of cholesterol ester (4, 5). In the present study, the gallbladder mucosa had markedly elevated concentrations of cholesterol ester in patients with cholesterolosis, but no differences were found in the con-
FIG. 2. Assay of ACAT activity in gallbladder mucosa. The assay was a modified version of one described previously (10). Each assay sample contained 0.2 mg fatty acid-free bovine serum albumin in 0.04 mol/L KH2PO4 buffer (pH 7.4) with 0.05 mol/L KCl, 0.03 mmol/L EDTA, and 0.3 mol/L sucrose. The total volume was 0.2 ml. (A) Protein dependence: The reaction was started by adding various amounts of gallbladder mucosa protein and 10 nmol [1-14C]oleoyl-CoA(0.005 mCi/nmol), and the mixture was incubated for 30 min at 37°C. The assay was stopped by adding chloroform/methanol (2:1). Lipid was extracted and separated as described in Materials and Methods. (B) Time dependence: Assays were performed as above. The amount of protein was 0.1 mg.
centrations of free cholesterol, phospholipids, and triglycerides between patients with cholesterolosis and control subjects. In an analysis of gallbladder bile (17, 18), patients with cholesterolosis were found to have high biliary cholesterol
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FIG. 3. Assay of CEH activity in gallbladder mucosa. The assay was a modified version of one described previously (11). Each assay sample contained 0.05 mol/L Tris-HCl buffer (pH 7.1) with 5 mmol/L MGCl2, 50 mmol/L KCL, 0.2 mmol/L EDTA, 0.4% fatty acid-free bovine serum albumin, and 0.5% Triton 100-X. The total volume was 0.2 ml. (A) Protein dependence: The reaction was started by adding various amounts of gallbladder mucosa protein and 30 mmol/L [14C]cholesteryl oleate (5.0 mCi/ mmol), and the mixture was incubated for 15 min at 37°C. The assay was stopped by adding methanol. Lipids was extracted and separated as described in Materials and Methods. (B) Time dependence: Assays were performed as above. The amount of protein was 0.1 mg. TABLE 3 ACAT and CHE Activities in Gallbladder Mucosa in Control Subjects and Patients With Cholesterolosis
Control (n 5 8) Cholesterolosis (n 5 10)
ACAT (pmol/mg z protein z min)
CEH (pmol/mg z protein z min)
ACAT/CEH Ratio
13.7 6 4.7*
12.9 6 6.4
1.4 6 0.6†
47.5 6 3.8*
14.9 6 4.0
3.9 6 1.9†
ACAT, acyl CoA cholesterol acyltransferase; CEH, cholesterol ester hydrolase. * p , 0.01; † p , 0.05
content and supersaturated bile. On the other hand, supersaturated bile was found to be present in both cholesterol gallstone and those with cholesterolosis (19), but was also commonly found in healthy subjects. The present data showed that the concentrations of cholesterol, phospholipids, and bile acid in gallbladder bile did not differ between
control subjects and patients with cholesterolosis. Most of the cholesterol in gallbladder bile consisted of free cholesterol, and the molar percentage of cholesterol did not differ among control subjects, patients with cholesterolosis, and patients with cholesterol gallstones. The high degree of variation in the incidences of cholesterolosis and cholesterol gallstone suggests that the pathogenesis of cholesterolosis might be unrelated to that of cholesterol gallstones. Supersaturation of bile cholesterol may not be the major etiological factor in cholesterolosis. Instead, altered hepatic cholesterol metabolism, altered uptake (20) and mucosal esterification of free sterols from the bile, may result in cholesterolosis (6). In guinea pigs (20) and humans (21), gallbladders mucosa is embryonically related to intestinal mucosa and has been shown to absorb small amounts of free cholesterol from bile through the epithelium by a passive as well as a partially active process. In a study of cholesterol uptake by human gallbladder using a modified Ussing technique, neither the uptake of cholesterol by the mucosa nor the appearance of cholesterol in the serosal fluid differed between a normal gallbladder and one with cholesterolosis (22). However, the mechanism by which absorbed cholesterol reaches the lamina propria of the gallbladder and how it is stored have not yet been established. In any case, cholesterol metabolism in the gallbladder mucosa is the most important factor in understanding the etiology. Our data suggested that accumulated lipids in patients with cholesterolosis consisted mainly of cholesterol ester. This cholesterol ester may have originated in bile or may have been synthesized de novo in the gallbladder mucosa. Our findings suggest that it is not the mechanism by which the degree of saturation of bile causes cholesterolosis. Enzymes participating in intracellular cholesterol metabolism include acyl CoA cholesterol acyltransferase (ACAT), cholesterol ester hydrolase (CEH), 3-hydroxy 3-methylglutary coenzymeA reductase (HMG-CoA reductase) and acid lipase, which have been studied in human liver (23), intestine (24), adrenal cortex (25), and ovary (26). The presence of mucosal-sterol-esterifying enzyme (probably ACAT) has been found in the guinea pig (27) and human (28) gallbladder epithelium, but no reports have been made about CEH of human gallbladder. We postulate the pathways of cholesterol in the gallbladder mucosa in patients with cholesterolosis (Fig. 4). For storage of cholesterol ester in the cell, cholesterol ester flux into the cell or cholesterol synthesis de novo is required. Furthermore, cholesterol ester formed from cholesterol by ACAT is converted to free cholesterol by CEH. The imbalance between activity of ACAT and that of CEH could thus cause the disorder of cholesterol metabolism in gallbladder mucosa. In this study, both ACAT and CEH activities were measured in the human gallbladder mucosa. ACAT activity was significantly higher in patients with cholesterolosis than in control subjects, but CEH activity did not differ between these two groups. It would be suggested that the deposition of cholesterol ester could be
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FIG. 4. Postulated pathways of cholesterol in gallbladder mucosa in patients with cholesterolosis. CHE was accumulated in gallbladder mucosa because ACAT activity was increased, but CEH activity was not increased compared with that of control subjects. ACAT, acylCoA cholesterol acyltransfenase; CEH, cholesterol ester hydrolase; FCH, free cholesterol; CHE, cholesterol ester; HMG-CoA, 3-hydroxy-3methylglutaryl CoA; LDL, low density lipoprotein.
accelerated in the gallbladder mucosa in patients with cholesterolosis. The origin of the cholesterol that was transferred to the free-cholesterol pool in the cell has not been identified, because we have not measured the activity of HMG-CoA reductase (located in the microsome for catalysis of the rate-limiting step in cholesterol synthesis) or that of acid lipase (located in the lysosome for hydrolysis of cholesterol ester). It has previously been suggested that patients with cholesterolosis of the gallbladder might have increased hepatic cholesterol synthesis (6). More recently, however, Sahlin et al. reported that cholesterolosis of the gallbladder mucosa is not associated with increased levels of HMG-CoA reductase activity in the liver and gallbladder mucosa (28). Foamy cells originates from macrophages, and the macrophages possess ACAT (29). These findings also support our suggestion that the increase in ACAT activity in gallbladder mucosa could be the cause of cholesterolosis. Reprint requests and correspondence: Fumitoshi Watanabe, M.D., First Department of Medicine, Hamamatsu University School of Medicine, 3600 Handa-cho, Hamamatsu-City, 431-3192 Japan.
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