CASE REPORTS
Increased Secondary Bile Acids in a Choledochal Cyst Possible Role in Biliary Metaplasia and Carcinoma R. MATTHEW and GREGORY
REVEILLE, GREGORY T. EVERSON
VAN STIEGMANN,
Division of Gastroenterology, Department of Medicine, and Department of Surgery, University of Colorado Health Sciences Center, Denver, Colorado
Choledochal cysts are uncommon congenital or acquired lesions of the hiliary tree. The incidence of biliary tract carcinoma in patients with choledochal cysts is 5-35 times greater than that of the general population. Factors responsible for the increased risk of carcinoma are unknown. The case of a young woman who underwent excision of a choledochal cyst 16 years after initial diagnosis and treatment by choledochocystduodenostomy is reported. Metaplasia of the epithelial lining of the cyst was found in the resected specimen. The relative composition of bile acids in cyst contents was as follows: lithocholate, 2%; deoxycholate, 66%; chenodeoxycholate, 5%; and cholate, 5 70. Virtually all bile acids were recovered in unconjugated form. In contrast, the bile acid composition of hepatic bile was as follows: lithocholate, 0%; deoxycholate, 34%; chenodeoxycholate, 43%; and cholate, 23%. Bile acids were fully conjugated. These data suggest that stasis of bile within choledochal cysts contributes to bacterial overgrowth and generation of unconjugated secondary bile acids.
C
holedochal cysts are uncommon anomalies of the biliary tree and are associated with a high risk for development of cholangiocarcinoma and carcinoma of the gallbladder. The prevalence of biliary carcinoma increases with age (l-8) and develops earlier in patients who have previously undergone cyst-enteric drainage operations (2,6,7). Histopathologic studies of excised cysts have demonstrated the frequent occurrence of metaplasia of the lining epithelium. The prevalence of such metaplasia increases with age (4). Despite these observations, there is no direct evidence linking metaplasia to the development of biliary carci-
noma (45). It has been suggested that stasis and generation of abnormal bile acids may contribute to the development of metaplasia and carcinoma in these patients (9). Thus, we report the results of bile acid analysis performed in a patient with a choledochal cyst. Case Report A 32-year-old woman was evaluated at the University of Colorado Health Sciences Center for right upper quadrant pain. At age 16, the patient had undergone exploratory laparotomy for chronic right upper quadrant pain; a type I choledochal cyst was found, and the patient underwent cholecystectomy and choledochocystduodenostomy. Her health was good until 1 year before evaluation. At that time she described episodes of constant, dull, aching pain in the right upper quadrant lasting 5-10 days and bearing no relationship to meals, time of day, bowel habits, or physical activity. Her abdomen was soft and nontender and there were no palpable masses. Abdominal ultrasound demonstrated filling defects within the choledochal cyst. Similar defects were also observed at endoscopic retrograde cholangiopancreatography (ERCP] but not further characterized. A 2-cm stricture of the distal common bile duct was seen. Results of routine laboratory studies, including liver enzyme profile, were normal. Because of persistent pain and the need to exclude neoplastic transformation within the cyst, the patient underwent excision of the cyst and a Roux-en-Y hepatodochojejunostomy. The cyst contained a single calculus and debris with no gross tumor. A sample of bile was obtained intraoperatively from the proximal common hepatic duct after its Abbreviations used in this paper: CA, cholic acid; CDCA, chenodeoxychok add; DC& deoxycholicadd; ERCP, endoscopic retrograde cholangiopamreatography; LCA, lithachollc acid. 0 1990by the American Gastraenterologkal Mtion 0016-5085/99/$3.00
526 REVEILLE ETAL.
GASTROENTEROLOGYVol.99,No.2
division from the cyst, and a sample of bile and debris was also obtained from within the cyst. Both samples were quickly frozen and stored at -20°C until they were analyzed. The patient recovered uneventfully and remains completely asymptomatic. Histological examination of the excised cyst showed typical features of metaplasia, including cellular atypia, a predominance of mucous cells with scattered goblet cells, and villoglandular transformation of the epithelium. There was no evidence of carcinoma. The relative bile acid composition was determined by capillary gas chromatography. Samples and standards were either acidified and extracted with diethyl ether (unconjugated fraction) or subjected to alkaline hydrolysis (total bile acid fraction) prior to extraction with diethyl ether. After extraction, all samples were dried under N, and methylated, and trimethyl silyl ether derivatives were formed. An aliquot of each sample (1-2 ML)was injected onto a capillary gas chromatography system (HP 5970, Hewlett Packard, Englewood, CO) (10).The column was a fused silica column, 30 m x0.25 mm (DB-1, J & W Scientific, Ranch0 Cordova, CA) coated with 0.25pm film thickness of SE-30. All bile acids eluted from the column within 16 minutes [lithocholic acid (LCA), 13.6 minutes: deoxycholic acid (DCA), 14.6 minutes; chenodeoxycholic acid (CDCA], 15.0 minutes; and cholic acid (CA], 15.3 minutes]. Composition was determined from the GC peak areas of samples relative to those of a standard bile acid mixture (Figure 1). The composition of hepatic bile was normal; DCA, CDCA, and CA were the only bile acids found. All bile acids were completely conjugated. In contrast, CDCA and CA represented only 10% of total bile acid in the sample from the choledochal cyst. Deoxycholic acid (88%) accounted for most of the cyst bile acid. Furthermore, virtually all of the bile acid recovered from the cyst was unconjugated. Of particular interest was the presence of a small but significant amount of LCA (2%) within the cyst contents, despite its complete absence from hepatic bile.
% 100
1
a0 60 -
40 20 0
I LCA
DCA
I
I
CDCA
CA
Figure I. The relative composition of bile acid (%) was measured fbom hepatic bile (?)? and the debris within the cyst [m).
Discussion We determined the bile acid composition of hepatic bile and cyst contents in a patient with a choledochal cyst. The cyst contained a predominance of unconjugated deoxycholate and lithocholate. The implication of these findings relative to the pathogenesis of biliary metaplasia and carcinoma in patients with choledochal cyst is discussed. The reported prevalence of carcinoma in choledochal cysts varies between 2.5% and 17.5% (1-8). In contrast, the incidence of extrahepatic biliary tract carcinoma is 0.007% -0.04% of all hospital admissions (11); 0.012%-0.048% of the general population (8); and 0.05% of a large autopsy series (12). Thus, the incidence of biliary carcinoma in patients with choledochal cysts may be 5-35 times greater than that of the population at large. In a review of 1433 cases of choledochal cysts, the incidence of carcinoma at initial operation was 0.7% in children aged t10 years, 6.8% in patients aged 11-20 years, and 14.3% in adults aged >20 years (3). The age-related increase in risk of carcinoma has been confirmed in a second study suggesting that by age 50, the frequency of carcinoma may be -50% (6). Patients who have had previous internal drainage operations may develop carcinoma at an earlier age (31. Epithelial metaplasia occurs commonly in choledochal cysts. Rossi et al. (5) observed simple columnar epithelium in 17 cases, proliferative changes of the epithelium in seven cases, and both mural ulcerations and goblet cell hyperplasia occurring in four cases. In 24 adult cases, Komi et al. (4) observed mucous glands (82%), goblet cells (42%), and argyrophil cells (27%). Thus, the frequency of both metaplasia and carcinoma arising in choledochal cysts increases with increasing age, and internal drainage operations may accelerate the development rate of carcinoma. Bile stasis within choledochal cysts has been proposed as a contributing factor to the development of metaplasia and carcinoma. Evidence supporting stasis within cysts includes the presence of stones and debris in 13.8%-25% of cases of choledochal cysts complicated by the development of carcinoma (6.7). However, many cases of carcinoma arising in cysts are not associated with stone formation. An additional consequence of bile stasis is bacterial overgrowth. Internal drainage operations which may accelerate the development of carcinoma should reduce bile stasis but may increase bacterial overgrowth as a consequence of the direct communication of the cyst cavity and the bowel. These data suggest that bacterial overgrowth rather than stasis is the important variable in the development of transformed epithelinm within cysts.
August 1990
One consequence of bacterial overgrowth is the generation of unconjugated secondary bile acids. Although the contents of our patient’s cyst were not cultured, we found a predominance of unconjugated secondary bile acids, DCA and LCA, within the cyst. Because hepatic bile was devoid of LCA and had a much lower proportion of DCA than cyst contents, it is most likely that the LCA and increased DCA found in the cyst were formed by bacteria and were not derived from enterohepatic return of these bile acids from the intestine. The finding that all bile acids in the cyst were completely unconjugated also supports the hypothesis that bacterial overgrowth generates secondary bile acids in choledochal cysts. Although primary bile acids are not mutagenic, secondary bile acids may be. Lithocholic acid or its conjugates are mutagenic when incubated with 2-aminoanthracene (13). Lithocholic and deoxycholic acids are co-mutagenic when incubated with 1,2-dimethylhydrazine in a standard Ames assay using the TAlOO strain of Salmonella typhimurium (14,~). Secondary bile acids also possess comutagenic activity in the presence of 2-acetylaminofluorene and benzo(a)pyrene (13). Interestingly, Bull et al. (IS]found that a stone from a patient with a choledochal cyst was mutagenic, but gallbladder stones from seven other patients were not. There is a reasonable correlation between in vitro mutagenic activity and the carcinogenic capacity of agents when tested in experimental animal models. It is possible that secondary bile acids or an as yet unidentified bile acid metabolite or bacterial product may participate in the development of metaplasia and carcinoma in choledochal cysts. Perhaps further studies of bile composition, coupled with histopathologic examination, microbiological studies, and tests of mutagenicity, will yield insights into the pathogenesis of metaplasia and carcinoma arising in choledochal cysts. References 1. Bloustein PA. Association
2.
of carcinoma with congenital cystic conditions of the liver and bile ducts. Am J Gastroenterol 1977:67:40-46. Todani T. Tabuchi K. Watanabe Y. Kobayashi Y. Carcinoma arising in the wall of congenital bile duct cysts. Cancer 1979$ 1134-1141.
BILE ACID ANALYSIS OF A CHOLEDOCHAL
CYST
527
LH. Carcinoma in 3. Voyles CR, Smadja C, Shands WC, Blmart choledochal cysts: age related incidence. Arch Surg 1963;118:966988. 4. Komi N, Tamura T, Miyoshi Y, Hino M, Yada S, Kawahara H, Udaka H, Takehara H. Histochemical and immunohistochemical studies on development of biliary carcinoma in forty-seven patients with choledochal cyst: special reference to intestinal metaplasia in the biliary duct. Jpn J Surg 1985;15:273-278. 5. Rossi RL, Silverman ML, Braasch JW, Munson JL, ReMine SG. Carcinoma arising in cystic conditions of the bile ducts. A clinical and pathologic study. Ann Surg 1987;205:377-384. 6. Todani T, Watanabe Y, Toki A, Urushihara N. Carcinoma related to choledochal cysts with internal drainage operations. Surg Gynecol Obstet 1987;164:61-64. 7. Flanigan OP. Biliary carcinoma associated with biliary cysts. Cancer 1988;4o:aao-883. a. Kagawa Y, Kashihara S, Kuramoto S, Maetani S. Carcinoma arising in a congenitally dilated biliary tract. Report of a case and review of the literature. Gastroenterology 197&74:X2861294. 9. Lowenfels A. Does bile promote extra-colonic cancer? Lancet i978;2:239-241. 10. Everson, G. Steady-state kinetics of serum bile acids in healthy human subjects: single and dual isotope techniques using stable isotopes and mass spectrometry. J Lipid Res 1987;28:238-252. 11.Sako K, Seitzinger GL, Garside E. Carcinoma of the extrahepatic bile ducts. Surgery 1957;41:416-437. 12.Edmondson HA. Tumors of the gallbladder and extrahepatic bile ducts. Washington, DC: Armed Forces Institute of Pathology, 1967:93. 13.Silverman SJ, Andrews AW. Bile acids: co-mutagenic activity in the salmonella-mammalian-microsome mutagenicity test. JNCI 1977;50:1557-1559. 14.Ames BN, McCann J, Yamasaki E. Methods for detecting carcinogens and mutagens with the salmonella/mammalianmicrosome mutagenicity test. Mutat Res 1975;31:347-364. 15.Wilpart M, Mainguet P, Maskens A, Roberfroid M. Structureactivity relationship amongst biliary acids showing co-mutagenic activity towards 1,2-dimethylhydrazine. Carcinogenesis i983;4:1239-1241. 16.Bull P, Guzman S, Nervi F. Mutagenic activity in stones from a patient with a congenital choledochal cyst. J Cancer Res Clin Oncol1984;107:61-63.
Received October 2.1989. Accepted February 15.1990. Address requests for reprints to: Gregory T. Everson. M.D., Division of Gastroenterology, University of Colorado Health Sciences Center, 4200 East 9 Avenue, Box B-158, Denver, Colorado 80262. The authors thank Teresa Boulay for secretarial work, Clare Trombley for bile acid analysis, and the Hepatobiliary Center GC/MS Core Laboratory for providing analytical capability.
Increased Secondary Bile Acids in a Choledochal Cyst Possible Role in Biliary Metaplasia and Carcinoma R. MATTHEW and GREGORY
REVEILLE, GREGORY T. EVERSON
VAN STIEGMANN,
Division of Gastroenterology, Department of Medicine, and Department of Surgery, University of Colorado Health Sciences Center, Denver, Colorado
Choledochal cysts are uncommon congenital or acquired lesions of the hiliary tree. The incidence of biliary tract carcinoma in patients with choledochal cysts is 5-35 times greater than that of the general population. Factors responsible for the increased risk of carcinoma are unknown. The case of a young woman who underwent excision of a choledochal cyst 16 years after initial diagnosis and treatment by choledochocystduodenostomy is reported. Metaplasia of the epithelial lining of the cyst was found in the resected specimen. The relative composition of bile acids in cyst contents was as follows: lithocholate, 2%; deoxycholate, 66%; chenodeoxycholate, 5%; and cholate, 5 70. Virtually all bile acids were recovered in unconjugated form. In contrast, the bile acid composition of hepatic bile was as follows: lithocholate, 0%; deoxycholate, 34%; chenodeoxycholate, 43%; and cholate, 23%. Bile acids were fully conjugated. These data suggest that stasis of bile within choledochal cysts contributes to bacterial overgrowth and generation of unconjugated secondary bile acids.
C
holedochal cysts are uncommon anomalies of the biliary tree and are associated with a high risk for development of cholangiocarcinoma and carcinoma of the gallbladder. The prevalence of biliary carcinoma increases with age (l-8) and develops earlier in patients who have previously undergone cyst-enteric drainage operations (2,6,7). Histopathologic studies of excised cysts have demonstrated the frequent occurrence of metaplasia of the lining epithelium. The prevalence of such metaplasia increases with age (4). Despite these observations, there is no direct evidence linking metaplasia to the development of biliary carci-
noma (45). It has been suggested that stasis and generation of abnormal bile acids may contribute to the development of metaplasia and carcinoma in these patients (9). Thus, we report the results of bile acid analysis performed in a patient with a choledochal cyst. Case Report A 32-year-old woman was evaluated at the University of Colorado Health Sciences Center for right upper quadrant pain. At age 16, the patient had undergone exploratory laparotomy for chronic right upper quadrant pain; a type I choledochal cyst was found, and the patient underwent cholecystectomy and choledochocystduodenostomy. Her health was good until 1 year before evaluation. At that time she described episodes of constant, dull, aching pain in the right upper quadrant lasting 5-10 days and bearing no relationship to meals, time of day, bowel habits, or physical activity. Her abdomen was soft and nontender and there were no palpable masses. Abdominal ultrasound demonstrated filling defects within the choledochal cyst. Similar defects were also observed at endoscopic retrograde cholangiopancreatography (ERCP] but not further characterized. A 2-cm stricture of the distal common bile duct was seen. Results of routine laboratory studies, including liver enzyme profile, were normal. Because of persistent pain and the need to exclude neoplastic transformation within the cyst, the patient underwent excision of the cyst and a Roux-en-Y hepatodochojejunostomy. The cyst contained a single calculus and debris with no gross tumor. A sample of bile was obtained intraoperatively from the proximal common hepatic duct after its Abbreviations used in this paper: CA, cholic acid; CDCA, chenodeoxychok add; DC& deoxycholicadd; ERCP, endoscopic retrograde cholangiopamreatography; LCA, lithachollc acid. 0 1990by the American Gastraenterologkal Mtion 0016-5085/99/$3.00
526 REVEILLE ETAL.
GASTROENTEROLOGYVol.99,No.2
division from the cyst, and a sample of bile and debris was also obtained from within the cyst. Both samples were quickly frozen and stored at -20°C until they were analyzed. The patient recovered uneventfully and remains completely asymptomatic. Histological examination of the excised cyst showed typical features of metaplasia, including cellular atypia, a predominance of mucous cells with scattered goblet cells, and villoglandular transformation of the epithelium. There was no evidence of carcinoma. The relative bile acid composition was determined by capillary gas chromatography. Samples and standards were either acidified and extracted with diethyl ether (unconjugated fraction) or subjected to alkaline hydrolysis (total bile acid fraction) prior to extraction with diethyl ether. After extraction, all samples were dried under N, and methylated, and trimethyl silyl ether derivatives were formed. An aliquot of each sample (1-2 ML)was injected onto a capillary gas chromatography system (HP 5970, Hewlett Packard, Englewood, CO) (10).The column was a fused silica column, 30 m x0.25 mm (DB-1, J & W Scientific, Ranch0 Cordova, CA) coated with 0.25pm film thickness of SE-30. All bile acids eluted from the column within 16 minutes [lithocholic acid (LCA), 13.6 minutes: deoxycholic acid (DCA), 14.6 minutes; chenodeoxycholic acid (CDCA], 15.0 minutes; and cholic acid (CA], 15.3 minutes]. Composition was determined from the GC peak areas of samples relative to those of a standard bile acid mixture (Figure 1). The composition of hepatic bile was normal; DCA, CDCA, and CA were the only bile acids found. All bile acids were completely conjugated. In contrast, CDCA and CA represented only 10% of total bile acid in the sample from the choledochal cyst. Deoxycholic acid (88%) accounted for most of the cyst bile acid. Furthermore, virtually all of the bile acid recovered from the cyst was unconjugated. Of particular interest was the presence of a small but significant amount of LCA (2%) within the cyst contents, despite its complete absence from hepatic bile.
% 100
1
a0 60 -
40 20 0
I LCA
DCA
I
I
CDCA
CA
Figure I. The relative composition of bile acid (%) was measured fbom hepatic bile (?)? and the debris within the cyst [m).
Discussion We determined the bile acid composition of hepatic bile and cyst contents in a patient with a choledochal cyst. The cyst contained a predominance of unconjugated deoxycholate and lithocholate. The implication of these findings relative to the pathogenesis of biliary metaplasia and carcinoma in patients with choledochal cyst is discussed. The reported prevalence of carcinoma in choledochal cysts varies between 2.5% and 17.5% (1-8). In contrast, the incidence of extrahepatic biliary tract carcinoma is 0.007% -0.04% of all hospital admissions (11); 0.012%-0.048% of the general population (8); and 0.05% of a large autopsy series (12). Thus, the incidence of biliary carcinoma in patients with choledochal cysts may be 5-35 times greater than that of the population at large. In a review of 1433 cases of choledochal cysts, the incidence of carcinoma at initial operation was 0.7% in children aged t10 years, 6.8% in patients aged 11-20 years, and 14.3% in adults aged >20 years (3). The age-related increase in risk of carcinoma has been confirmed in a second study suggesting that by age 50, the frequency of carcinoma may be -50% (6). Patients who have had previous internal drainage operations may develop carcinoma at an earlier age (31. Epithelial metaplasia occurs commonly in choledochal cysts. Rossi et al. (5) observed simple columnar epithelium in 17 cases, proliferative changes of the epithelium in seven cases, and both mural ulcerations and goblet cell hyperplasia occurring in four cases. In 24 adult cases, Komi et al. (4) observed mucous glands (82%), goblet cells (42%), and argyrophil cells (27%). Thus, the frequency of both metaplasia and carcinoma arising in choledochal cysts increases with increasing age, and internal drainage operations may accelerate the development rate of carcinoma. Bile stasis within choledochal cysts has been proposed as a contributing factor to the development of metaplasia and carcinoma. Evidence supporting stasis within cysts includes the presence of stones and debris in 13.8%-25% of cases of choledochal cysts complicated by the development of carcinoma (6.7). However, many cases of carcinoma arising in cysts are not associated with stone formation. An additional consequence of bile stasis is bacterial overgrowth. Internal drainage operations which may accelerate the development of carcinoma should reduce bile stasis but may increase bacterial overgrowth as a consequence of the direct communication of the cyst cavity and the bowel. These data suggest that bacterial overgrowth rather than stasis is the important variable in the development of transformed epithelinm within cysts.
August 1990
One consequence of bacterial overgrowth is the generation of unconjugated secondary bile acids. Although the contents of our patient’s cyst were not cultured, we found a predominance of unconjugated secondary bile acids, DCA and LCA, within the cyst. Because hepatic bile was devoid of LCA and had a much lower proportion of DCA than cyst contents, it is most likely that the LCA and increased DCA found in the cyst were formed by bacteria and were not derived from enterohepatic return of these bile acids from the intestine. The finding that all bile acids in the cyst were completely unconjugated also supports the hypothesis that bacterial overgrowth generates secondary bile acids in choledochal cysts. Although primary bile acids are not mutagenic, secondary bile acids may be. Lithocholic acid or its conjugates are mutagenic when incubated with 2-aminoanthracene (13). Lithocholic and deoxycholic acids are co-mutagenic when incubated with 1,2-dimethylhydrazine in a standard Ames assay using the TAlOO strain of Salmonella typhimurium (14,~). Secondary bile acids also possess comutagenic activity in the presence of 2-acetylaminofluorene and benzo(a)pyrene (13). Interestingly, Bull et al. (IS]found that a stone from a patient with a choledochal cyst was mutagenic, but gallbladder stones from seven other patients were not. There is a reasonable correlation between in vitro mutagenic activity and the carcinogenic capacity of agents when tested in experimental animal models. It is possible that secondary bile acids or an as yet unidentified bile acid metabolite or bacterial product may participate in the development of metaplasia and carcinoma in choledochal cysts. Perhaps further studies of bile composition, coupled with histopathologic examination, microbiological studies, and tests of mutagenicity, will yield insights into the pathogenesis of metaplasia and carcinoma arising in choledochal cysts. References 1. Bloustein PA. Association
2.
of carcinoma with congenital cystic conditions of the liver and bile ducts. Am J Gastroenterol 1977:67:40-46. Todani T. Tabuchi K. Watanabe Y. Kobayashi Y. Carcinoma arising in the wall of congenital bile duct cysts. Cancer 1979$ 1134-1141.
BILE ACID ANALYSIS OF A CHOLEDOCHAL
CYST
527
LH. Carcinoma in 3. Voyles CR, Smadja C, Shands WC, Blmart choledochal cysts: age related incidence. Arch Surg 1963;118:966988. 4. Komi N, Tamura T, Miyoshi Y, Hino M, Yada S, Kawahara H, Udaka H, Takehara H. Histochemical and immunohistochemical studies on development of biliary carcinoma in forty-seven patients with choledochal cyst: special reference to intestinal metaplasia in the biliary duct. Jpn J Surg 1985;15:273-278. 5. Rossi RL, Silverman ML, Braasch JW, Munson JL, ReMine SG. Carcinoma arising in cystic conditions of the bile ducts. A clinical and pathologic study. Ann Surg 1987;205:377-384. 6. Todani T, Watanabe Y, Toki A, Urushihara N. Carcinoma related to choledochal cysts with internal drainage operations. Surg Gynecol Obstet 1987;164:61-64. 7. Flanigan OP. Biliary carcinoma associated with biliary cysts. Cancer 1988;4o:aao-883. a. Kagawa Y, Kashihara S, Kuramoto S, Maetani S. Carcinoma arising in a congenitally dilated biliary tract. Report of a case and review of the literature. Gastroenterology 197&74:X2861294. 9. Lowenfels A. Does bile promote extra-colonic cancer? Lancet i978;2:239-241. 10. Everson, G. Steady-state kinetics of serum bile acids in healthy human subjects: single and dual isotope techniques using stable isotopes and mass spectrometry. J Lipid Res 1987;28:238-252. 11.Sako K, Seitzinger GL, Garside E. Carcinoma of the extrahepatic bile ducts. Surgery 1957;41:416-437. 12.Edmondson HA. Tumors of the gallbladder and extrahepatic bile ducts. Washington, DC: Armed Forces Institute of Pathology, 1967:93. 13.Silverman SJ, Andrews AW. Bile acids: co-mutagenic activity in the salmonella-mammalian-microsome mutagenicity test. JNCI 1977;50:1557-1559. 14.Ames BN, McCann J, Yamasaki E. Methods for detecting carcinogens and mutagens with the salmonella/mammalianmicrosome mutagenicity test. Mutat Res 1975;31:347-364. 15.Wilpart M, Mainguet P, Maskens A, Roberfroid M. Structureactivity relationship amongst biliary acids showing co-mutagenic activity towards 1,2-dimethylhydrazine. Carcinogenesis i983;4:1239-1241. 16.Bull P, Guzman S, Nervi F. Mutagenic activity in stones from a patient with a congenital choledochal cyst. J Cancer Res Clin Oncol1984;107:61-63.
Received October 2.1989. Accepted February 15.1990. Address requests for reprints to: Gregory T. Everson. M.D., Division of Gastroenterology, University of Colorado Health Sciences Center, 4200 East 9 Avenue, Box B-158, Denver, Colorado 80262. The authors thank Teresa Boulay for secretarial work, Clare Trombley for bile acid analysis, and the Hepatobiliary Center GC/MS Core Laboratory for providing analytical capability.
