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Transpapillary biopsy of the gallbladder disease
April 1998 observed with the bile samples of the UDCA-treated group (2.18_ 0.16 vs. 0.85 _+0.72 times control; p < 0.01). Conclusion: UDCA treatment of patients with CGD results in reduction of lipid peroxidation and mucin secretagogue activity of bile. Thus, the beneficial effects of UDCA in cholesterol gallstone patients may be mediated by reducing lipid peroxidation and mucin concentration of bile. Supported by grants from the Else Kr6ner-Fresenius Foundation and the Deutsche Forschungsgemeinschaft (DFG Ri-584/3). G2245 RAPID DIAGNOSIS OF CHOLEDOCHOLITHIASIS USING BIOCHEMICAL TESTS IN THE ERA OF LAPAROSCOPIC CHOLECYSTECTOMY. CH Wang, LR Mo, RC Lin, JY Kuo, KK Chang. Dept. of Internal Medicine, Tainan Municipal Hospital, Tainan, Taiwan A number of invasive and/or non-invasive methods were used to examine common bile duct (CBD) stones prior to laparoscopic cholecystectomy (LC). The aim of this study was to define a simple, accurate biochemical test to predict CBD stones, which could therefore avoid unnecessary, costly and risky procedures. Methods: From January 1995 to May 1997, endoscopic retrograde cholangiopancreatography was performed on 350 patients with suspected CBD stones by biochemical tests and nltrasonography. The patients were operated on with LC for symptomatic gallstones. The patients with viral hepatitis, alcoholic liver disease, and other liver were excluded. The remaining 306 patients (male 127, female 179, mean age 56 -+ 14 yrs) enrolled into the study. The biochemical test included aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase (AP), total bilirubin (T-Bil), direct bilirubin (D-Bil), ,/-glutamyltransferase (GGT), and albumin. Resolts: Univariate analysis identified Alb, AP, T-Bil, D-Bil, and GGT as independent predictors of CBD stones. The above five significant parameters were included in the multivariate logistic regression analysis which identified AP (p=0.0366) as only independent factor to differentiate between patients with and without CBD stones. Furthermore, a receiver operator characteristic (ROC) curve was constructed according to different levels of AP to reveal that the best level of AP to diagnose CBD stones was that greater than 300 IU/L. A model of predicted probability (PP) of following formula of 1/[1 + e-z] was developed, in which z = 0.5797 - 0.4425 (Alb) + 0.0012 (AP) + 0.5049 (D-Bil) + 0.0007 (GGT) - 0.2297 (T-Bil). If PP is greater than 0.5, the patient would be predicted to have CBD stones. The model correctly classified patients as having CBD stones or not in 70% of the patients. Conclusions: A > 300 IU/L of AP is the best cutoff point in ROC curve for rapidly diagnosing CBD stones. The formula of the study was another predicting model to exclude CBD stones. This research was made possible by partial grant from Tainan Municipal Hospital. • G2246 TRANSPAPILLARY BIOPSY OF THE GALLBLADDER DISEASE. Y.Watanabe, H.Goto, Y.Hirooka, A.Itoh, S.Hayakawa, Y.Ishiguro, S.Kojima, S.Hashimoto, T.Hayakawa, Y.Naitoh*, 2nd Department of Internal Medicine and * Clinical Laboratory Medicine, Nagoya University School of Medicine, Nagoya, Japan. Introduction: Transpapillary gallbladder(GB) biopsy is thought to be difficult because of anatomical problems. For the gastrointestinal diseases, endoscopic biopsy is the routine procedure, similarly histopathological diagnosis is desirable in the GB disease. We have reported the clinical utility of Intraductal Ultrasonography (IDUS) in GB disease last year in AGA meeting. This time we tried the transpapillary GB biopsy applying IDUS method. Subjects and methods: We tried transpapillary GB biopsy in 6 patients with GB disease (4 elevated and 2 wall thickened lesions). As for the location, 5 were at the fundus and one was at the body of the hepatic bed side. After inserting the guidewire into the GB applying IDUS method, we inserted the sheath along the guidewire into the GB, then, withdraw the guidewire and inserted the biopsy forceps (lmm in diameter, GIP, Grassau, Germany) into the lumen of the sheath (6F in diameter) without sphincterotomy. According to above-stated process, we tried to get the specimens. The evaluated points were success rate of getting specimens and accuracy of histopathological diagnosis. Result: We could get enough specimens in 5 of 6 cases(83.3%). They were diagnosed histopathologically (2 carcinoma: moderately differentiated adenocarcinoma and small cell carcinoma, 1 adenoma and 2 adenomyomatosis). As for the locations in successful cases, four were at fundus and one was at the GB body of the hepatic bed side. We could not get the specimen in one case, because the diameter of the cystic duct lumen was narrower than that of the sheath. Complication did not occur in all cases. Discussion: In this study, all lesions were located at GB fundus and the hepatic bed side of the GB body, So we could get the specimens at the rate of 83.3%. Because of its anatomic features, biopsy at the peritoneal side would be more difficult than the hepatic bed side of the GB. In this study, we could diagnose one case as a small cell carcinoma before operation. There was a report that chemotherapy was a first choice and operation was a supported therapy in small cell carcinoma. So in this case, the histnpathological diagnosis was an important factor to determine the therapeutic strategy. Therefore transpapillary GB biopsy without sphincterotomy has a significant meaning in the diagnosis of the GB disease. Conclusion: The possibility of the transpapillary GB biopsy is demonstrated and it will become a useful diagnostic method for the GB disease.
Biliary Disorders A549 G2247 THE PATHOGENESIS OF GALLBLADDER ADENOMATOUS POLYPS IS DI~'ERENT FROM THAT OF GALLBLADDER CARCINOMA. I Wistuba, JF Miquel, AF Gazdar and J Albores-Saavedra. UT-Southwestem Medical Center, Dallas, TX, and Departamento de Gastroenterologia, P.Universidad Cat61ica de Chile.
Introduction: In patients with polyps in their gallbladder larger than 1.5 cm or in association with gallstones, cholecystectomy is warranted. However, it remains uncertain if gallbladder adenomas (GBAs) possess malignant potential. In contrastl the sequence of preneoplastic changes involved in the development of gallbladder carcinoma arising in flat mucosa are well defined, and some of their sequential molecular abnormalities known (Cancer Res. 55,2511,1995), with P53 inactivation and 9p LOH as frequent and early events. To further clarify the malignant potential of GBAs, we studied their molecular abnormalities and correlated them with their histological type and size. Methods: We studied 14 GBAs between 0.5 and 5 cm size. Eighty six percent were pyloric type and one of them was seen adjacent to an invasive carcinoma. DNA was extracted from precisely microdissected tissue to study loss of heterozygosity (LOH) at 6 chromosomal regions (5q APC-MCC, 3p14.2 FHIT, 13q RB, 9p p15/p16, 18q DCC and 17p P53) frequently deleted in gallbladder carcinoma, using a PCR-based assay. For the P53 mutations study, SSCP analysis in exon 4 to 8 was performed. K- and N-ras mutations detection was performed by designed RFLP. Mutations were confirmed by sequencing. Results: K-ras mutations were the most frequent molecular change detected in GBA (4/14, 29%; 2 in codon 12 and 2 in codon 61). Only a single LOH (at 5q) was present in a gallbladder adenoma. No molecular alterations were detected in the GBA adjacent to an invasive carcinoma with LOH at different loci. No correlation was found between the size of GBAs and the presence of molecular changes. Conclusion: Molecular abnormalities were found in 36% of the GBAs. The pattern of molecular changes detected in GBAs differed from those present in gallbladder carcinomas arising in flat mucosa, but resemble those present in colonic adenomas (5q LOH and K-ras mutations). Our findings suggest that GBAs have a different pathogenesis to invasive gallbladder carcinoma. Supported in part by Fondecyt 1971100. G224g MECHANISMS OF PREGNANCY-INDUCED GALLBLADDER HYPOMOTILITY IN GUINEA PIGS. Z.L. Xiao, Q. Chen, P. Biancani and J. Behar. Department of Medicine, Rhode Island Hospital and Brown University School of Medicine, Providence, RI. We have previously shown that gallbladders from progesterone treated guinea pigs exhibit impaired muscle contraction in response to receptor-G protein dependent agonists such as CCK and ACh when compared to those from control animals. The muscle defect associated with progesterone appears to reside at the G protein level (Gastroenterology 108: A583, 1995). The present study was therefore designed to investigate the effect of pregnancy on gallbladder motility in pregnant guinea pigs. Gallbladder muscle cells were isolated from adult third-trimester pregnant guinea pigs and nonpregnant female guinea pigs (control) by enzymatic digestion with collagenase and permeabilized by brief exposure to saponin. Cell contraction was expressed as percent shortening of initial control cell length. Contraction in response to CCK-8 (0.1 pM to 0.1 laM) was significantly reduced in gallbladder muscle cells from pregnant guinea pigs with a maximal shortening of only 12.02 + 1.1% at 10 nM when compared to 23.4 _+ 1.8% shortening in control animals (p<0.01, by ANOVA). Contraction in response to direct G protein activation with GTP3,S was also significantly reduced in gallbladder muscle ceils from pregnant guinea pigs. GTP'/S at 10 laM caused a maximal shortening of only 12.7-+ 0.9% as opposed to 20.5 + 0.8% shortening in controls. However, the contractile response to potassium chloride, which is a receptor-G protein independent agonist, was not significantly different from control. These data suggested that pregnancy might affect the function of G proteins in gallbladder muscle. To further confirm this hypothesis, the function of certain G proteins in gallbladder muscle from pregnant and control guinea pigs was examined by using [35S]GTPTS binding. Crude membranes derived from gallbladder muscle were incubated with 30 nM [35S]GTPTS in the presence or absence of CCK-8. A panel of specific antibodies to ct subunits of Gq/ll, Gs, Gil-2 and Gi3 (1:1000 dilution) was applied to ELISA wells precoated with anti-rabbit IgG goat antibody to immunoprecipitate the complex of [35S]GTPTS-G protein subunits. CCK-8 at 1 laM caused a significant increase in [35S]GTP3,S binding to Gict3 binding over basal level (without CCK), but not to Gq/llet, Gictl-2 or Gsct. The stimulation of Gict3 binding induced by CCK-8, however, was significantly reduced in gallbladder muscle from pregnant guinea pigs when compared to that from control animals (p<0.05, by t test). In conclusion, pregnancy downregulates certain contraction-linked G proteins in gallbladder muscle such as Gict3 resulting in impaired contraction. These effects may be primarily due to the actions of progesterone. This research was supported by NIH grant R01-DK27389.
Biliary Disorders A549 G2247 THE PATHOGENESIS OF GALLBLADDER ADENOMATOUS POLYPS IS DI~'ERENT FROM THAT OF GALLBLADDER CARCINOMA. I Wistuba, JF Miquel, AF Gazdar and J Albores-Saavedra. UT-Southwestem Medical Center, Dallas, TX, and Departamento de Gastroenterologia, P.Universidad Cat61ica de Chile.
Introduction: In patients with polyps in their gallbladder larger than 1.5 cm or in association with gallstones, cholecystectomy is warranted. However, it remains uncertain if gallbladder adenomas (GBAs) possess malignant potential. In contrastl the sequence of preneoplastic changes involved in the development of gallbladder carcinoma arising in flat mucosa are well defined, and some of their sequential molecular abnormalities known (Cancer Res. 55,2511,1995), with P53 inactivation and 9p LOH as frequent and early events. To further clarify the malignant potential of GBAs, we studied their molecular abnormalities and correlated them with their histological type and size. Methods: We studied 14 GBAs between 0.5 and 5 cm size. Eighty six percent were pyloric type and one of them was seen adjacent to an invasive carcinoma. DNA was extracted from precisely microdissected tissue to study loss of heterozygosity (LOH) at 6 chromosomal regions (5q APC-MCC, 3p14.2 FHIT, 13q RB, 9p p15/p16, 18q DCC and 17p P53) frequently deleted in gallbladder carcinoma, using a PCR-based assay. For the P53 mutations study, SSCP analysis in exon 4 to 8 was performed. K- and N-ras mutations detection was performed by designed RFLP. Mutations were confirmed by sequencing. Results: K-ras mutations were the most frequent molecular change detected in GBA (4/14, 29%; 2 in codon 12 and 2 in codon 61). Only a single LOH (at 5q) was present in a gallbladder adenoma. No molecular alterations were detected in the GBA adjacent to an invasive carcinoma with LOH at different loci. No correlation was found between the size of GBAs and the presence of molecular changes. Conclusion: Molecular abnormalities were found in 36% of the GBAs. The pattern of molecular changes detected in GBAs differed from those present in gallbladder carcinomas arising in flat mucosa, but resemble those present in colonic adenomas (5q LOH and K-ras mutations). Our findings suggest that GBAs have a different pathogenesis to invasive gallbladder carcinoma. Supported in part by Fondecyt 1971100. G224g MECHANISMS OF PREGNANCY-INDUCED GALLBLADDER HYPOMOTILITY IN GUINEA PIGS. Z.L. Xiao, Q. Chen, P. Biancani and J. Behar. Department of Medicine, Rhode Island Hospital and Brown University School of Medicine, Providence, RI. We have previously shown that gallbladders from progesterone treated guinea pigs exhibit impaired muscle contraction in response to receptor-G protein dependent agonists such as CCK and ACh when compared to those from control animals. The muscle defect associated with progesterone appears to reside at the G protein level (Gastroenterology 108: A583, 1995). The present study was therefore designed to investigate the effect of pregnancy on gallbladder motility in pregnant guinea pigs. Gallbladder muscle cells were isolated from adult third-trimester pregnant guinea pigs and nonpregnant female guinea pigs (control) by enzymatic digestion with collagenase and permeabilized by brief exposure to saponin. Cell contraction was expressed as percent shortening of initial control cell length. Contraction in response to CCK-8 (0.1 pM to 0.1 laM) was significantly reduced in gallbladder muscle cells from pregnant guinea pigs with a maximal shortening of only 12.02 + 1.1% at 10 nM when compared to 23.4 _+ 1.8% shortening in control animals (p<0.01, by ANOVA). Contraction in response to direct G protein activation with GTP3,S was also significantly reduced in gallbladder muscle ceils from pregnant guinea pigs. GTP'/S at 10 laM caused a maximal shortening of only 12.7-+ 0.9% as opposed to 20.5 + 0.8% shortening in controls. However, the contractile response to potassium chloride, which is a receptor-G protein independent agonist, was not significantly different from control. These data suggested that pregnancy might affect the function of G proteins in gallbladder muscle. To further confirm this hypothesis, the function of certain G proteins in gallbladder muscle from pregnant and control guinea pigs was examined by using [35S]GTPTS binding. Crude membranes derived from gallbladder muscle were incubated with 30 nM [35S]GTPTS in the presence or absence of CCK-8. A panel of specific antibodies to ct subunits of Gq/ll, Gs, Gil-2 and Gi3 (1:1000 dilution) was applied to ELISA wells precoated with anti-rabbit IgG goat antibody to immunoprecipitate the complex of [35S]GTPTS-G protein subunits. CCK-8 at 1 laM caused a significant increase in [35S]GTP3,S binding to Gict3 binding over basal level (without CCK), but not to Gq/llet, Gictl-2 or Gsct. The stimulation of Gict3 binding induced by CCK-8, however, was significantly reduced in gallbladder muscle from pregnant guinea pigs when compared to that from control animals (p<0.05, by t test). In conclusion, pregnancy downregulates certain contraction-linked G proteins in gallbladder muscle such as Gict3 resulting in impaired contraction. These effects may be primarily due to the actions of progesterone. This research was supported by NIH grant R01-DK27389.