- Email: [email protected]
Detection of Helicobacter pylori by PCR after amplification of 16S RRNA from gallstone
April 2000
AGAA9
296
298
ASSOCIATION BETWEEN LITHOGENESIS AND ANATOMICAL CHARACTERISTICS OF CYSTIC DUCT AND JUXTAPAPILLARY DUODENAL DIVERTICULUM. Hyun Seung Kim, Chang Young Park. Yong Kyun Cho, Woo Kyu Chun, Jung II Son, Byung Ik Kim, Eul Soon Jung, Jun Ho Shin, Hwa Young Lee. Sang Jong Lee, Kangbuk Samsung Hosp Sungkyunkwan Univ Sch of Medicine, Seoul, South Korea; CHA Gen Hosp Pochon CHA Univ Coli of Medicine, Seoul, South Korea. BackGround: There are many factors, that influence on lithogenesis such as age, sex. nutrition, diet, homones, drugs, hereditary, infection, etc. The anatomical characteristics such as morphology, implantaion site, diameter, length of cystic duct may also influence on lithogenesis. It is also Known that Juxapapillary duodenal diverticulum is associated with lithogenesis. This study investigated correlation between lithogenesis and the anatomical charateristics of cystic duct and juxtapapillary duodenal diverticulum. Methods : In the Department of Gastroenterology of the Sungkyunkwan University Kangbuksamsung Hospital, between January 1, 1996, and April 31. 1999,461 endoscopic retrograde cholangiopancreatography were performed. We examined these cases and excluded 371cases from the study. patients excluded from the study were those with previous cholecystectomy, carcinoma of the pancreas or biliary tract, benign stenosis of the sphincter of Oddi, lack of visualization of the entire cystic duct, and biliary endoprotheses. Finally, 90 patients were included and datas were obtained from the ERCP in the prone position. The length and diameter of cystic duct was measured by means of string. The patients were divided into three groups.(GroupI, absence of lithiasis in the biliary tract and gallbladder; GroupII, presence of lithiasis in the gallbladder but not in the CBD; Group III, lithiasis in the CBD with or without gallbladder lithiais.) Results : There was significant diferences between groups in sex. age, triglyceride level, site of implantation, length of cystic duct, morphology, existence of juxtapapillary duodenal diverticulum but not in diameter. The patients with gallstones or CBD stones has sinificantly longer than others. The insertion of the cystic duct on the left margin of the CBD, which is associated with a length greater than 3cm and spiral morphology. Conclusion: Age, sex, level of triglyceride, Implantation site, morphology, length of the cystic duct and juxtapapillary duodenal diverticulum is associated with lithiasis. It seems that left marginal insertion of cystic duct can piay an important role in lithogenesis by increasing cystic duct resistance ahd causing a reduced washout effect of the gallbladder contents Key Words: Lithogenesis, morphology, Diameter and Length of cystic duct, Implantation site, Juxtapapillary duodenal diverticulum
DETECTION OF HELICOBACTER PYWRI BY PCR AFTER AMPLIFICATION OF 16S RRNA FROM GALLSTONE. Don H. Lee, In H. Kim, Jin W. Lee, Sun H. Kim, Won Choi, Pum S. Kim, Hyung G. Kim, Young S. Kim, Keon Y. Lee, Seok H. Shin, Mi S. Choi, Inha Univ Hosp, Inchon, South Korea. Background/Aim: The Helicobacterpylori (H. pylori) causes chronic gastritis and peptic ulcer and is a risk factor of gastric MALToma and stomach cancer. Among the genus Helicobacter species that colonize the intestinal tract, Hepatic Helicobacterspecies have been found in gallbaldder tissue or bile of Chilean patients with chronic cholecystitis. The bacteria closely resembling H. pylori was detected in resected gallbladder mucosa and H. pylori was also found in bile juice of gallstone patients. But these studies were performed with bile, not with gallstone itself. To further explore the possible relation between H. pylori and gallstone formation we have investigated their presence in the gallstones. PatientslMethods: Stones were sampled from 70 patients (male 32, female 38) with gallstones on ultrasound. Mean age was 52.8 years (range,27-78 years). The gallstones and bile were obtained under aseptic condition during cholecystectomy. We ground the stone to powder from the interior of the stone by scraping with a surgical blade asepically. The DNA was extracted by a modification of the method of Swidsinsky et al (Gastroenterology, 1995) from 100 mg grounded stone. Polymerase chain reaction (PCR) was used to detect bacterial16S rRNA. The presence of H. pylori was revealed by PCR using primer for 26kDa surface antigen of H. pylori. Results: Twenty stone samples were positive for bacterial 16S rRNA. Among these 20 samples, PCR products for 26 kDa surface antigen of H. pylori were detected in 2 stone samples (10%). The detected stones were all brown pigment stones. Conclusion: H. pylori could be detected in the gallstone by PCR after amplification of 16S rRNA. The role of H. pylori and their products in gallstone formation will be elucidated.
297 HELICOBACTER SPECIES IN BILE FROM KOREAN PATIENTS WITH GALLBLADDER STONES. Dong Ki Lee, S.H. Ha, 1.B. Kim, K.H. Kim, T.W. Kim, K.H. Kim, J.H. Jung, J.W. Kim, H.S. Kim, S.K. Baik, S.O. Kwon, Wonju Coli of Medicine, Wonju, South Korea; Coli Health Sci, Wonju, South Korea. Recent reports on the association of Helicobacter sp. with gallbladder disease show discrepant results. This discrepancy may be explained by regional differences in the distribution of Helicobacter sp.. According to some reports, the Helicobacter sp. may be the culprit in the pathogenesis of some biliary tract disease. Hence, we have attempted to detect the Helicobacter sp. in the bile of patients in Korea, a country in which Helicobacter pylori is highly prevalent (>70% in adults). And to document the origin of Helicobacter sp., we tested stomach samples simultaneously. Methods: Gallbladder stones, bile and gallbladder tissue were obtained using strict aseptic technique from 33 patients with symptomatic gallbladder stones. Concomitantly gastric mucosa and gastric juice samples were obtained before cholecystectomy. DNA was extracted from crushed gallstones, gallbladder epithelium, bile, as well as the gastric mucosa and gastric juice. Helicobacter genus-specific and Helicobacter pylori-specific oligonucleotide primers were designed based on the nucleotide sequences of the 16S ribosomal DNA of Helicobacter pylori These primers were used to amplify a DNA fragment using the polymerase chain reaction (PCR). The specificity of the PCR products was examined by Southern hybridization. Results: PCR with Helicobacter pylori-specific primers produced amplicons in 30.3% (10/33) of the gallstones, 36% (9/25) of the bile and 6.2% (2/32) of the gallbadder epithelium samples. In 41% (13/32) of the patients, no Helicobacter genus PCR amplicon was produced from the samples of gallstone, bile or gallbladder epithelium. PCR produced evidence of Helicobacter pylori in 97% (32/33) of the gastric mucosa and/or gastric juice samples. The specificity of each of amplicons was verified by Southern hybridization. PCR amplicons for Helicobacter sp. were present in all of the corresponding gastric samples of the patients whose biliary samples were positive for Helicobacter genus. A PCR amplicon for a Helicobacter genus other than Helicobacter pylori was identified in 3% (1/32) of the gallstone and 16% (4/25) of the gallbladder epithelium samples. Conclusion: Helicobacter sp. in the biliary tract seems to have migrated from the stomach. Despite such a high detection rate of Helicobacter sp. in the gallstones or bile of patients in a Helicobacter pylori endemic country,there is little evidence of Helicobacter sp. actually colonizing the gallbladder. Nevertheless, the role of Helicobacter sp. in biliary tract disease warrants further study.
299 ACCURATE QUANTIFICATION OF MICELLAR AND VESICULAR PHASES IN CHOLESTEROL·SUPERSATURATED MODEL SYSTEMS: COMPARISON OF GEL FILTRATION AND ULTRACENTRIFUGATION-FILTRATION-DIAL YSIS METHODS. Antonio Moschetta, Erik R. Eckhardt, Martin B. de Smet, Gerard P. vanBerge-Henegouwen, Willem Renooij, Karel 1. van Erpecum, Gastrointestinal Research Unit, Univ Med Ctr, Utrecht, Netherlands; Semeiotica Medical, DIMIMP, Univ of Bari, Bari, Italy. Gel filtration with bile salts at intermixed micellar-vesicular concentration (IMC) in the eluant has been proposed to separate vesicles and micelles. However, this method is only suitable in highly diluted and slightly supersaturated model biles, since the presence of vesicular aggregates in more concentrated and supersaturated systems precludes accurate separation (Donovan JM, Hepatol 1998;27:641). We developed a new method for state-of-art separation. Methods: First aggregated vesicles and -if present- cholesterol crystals were pelleted by ultracentrifugation of supersaturated model biles (tot. lipid cone. 2.4 and 7.3 g/dL: CSI 1.7 resp. 1.4: EYPC/(EYPC + taurocholate) =0.3). Amounts of aggregated vesicles and cholesterol crystals were quantified by determining cholesterol content in the pellet both without and with excess added taurodeoxycholate (complete dissolution of aggregates). Unilamellar vesicles and micelles in the supernatant were separated by gel filtration with bile salts at IMC in the eluant ("gel filtration method"), by filtration of micelles through highly selective 300 kDa filter + (for unilamellar vesicles) dialysis against bile salts at IMC ("ultrafiltration-dialysis method") or estimated by subtraction of lipid contents in filtered micelles from lipid contents in (unilamellar vesicIes+micelles containing) supernatant ("subtraction method"). Results: =30% of cholesterol and 5-10% of phospholipids were solubilized in vesicular aggregates. Ultrafiltration-dialysis and subtraction methods yielded identical lipid solubilization in micelles vs unilamellar vesicles and identical chollPL ratios. In contrast, gel filtration method yielded much more lipids in micelles and less in unilamellar vesicles, with much higher vesicular chollPL ratios (1.98±0.56 vs 0.99±0.I). When vesicles obtained by dialysis were analized by gel filtration, vesicular chollPL ratios increased from 0.99 to 1.98, despite correct IMC values for bile salts in the eluant. Subsequent extraction of column material showed considerable amounts of lipids. Conclusions: gel filtration may underestimate vesicular lipids and overestimate vesicular chollPL ratios, supposedly because of lipids remaining attached to the column. Combined uItracentrifuagtionultrafiltration-dialysis should be considered state-of-art methodology for quantification of cholesterol carriers in model systems.
AGAA9
296
298
ASSOCIATION BETWEEN LITHOGENESIS AND ANATOMICAL CHARACTERISTICS OF CYSTIC DUCT AND JUXTAPAPILLARY DUODENAL DIVERTICULUM. Hyun Seung Kim, Chang Young Park. Yong Kyun Cho, Woo Kyu Chun, Jung II Son, Byung Ik Kim, Eul Soon Jung, Jun Ho Shin, Hwa Young Lee. Sang Jong Lee, Kangbuk Samsung Hosp Sungkyunkwan Univ Sch of Medicine, Seoul, South Korea; CHA Gen Hosp Pochon CHA Univ Coli of Medicine, Seoul, South Korea. BackGround: There are many factors, that influence on lithogenesis such as age, sex. nutrition, diet, homones, drugs, hereditary, infection, etc. The anatomical characteristics such as morphology, implantaion site, diameter, length of cystic duct may also influence on lithogenesis. It is also Known that Juxapapillary duodenal diverticulum is associated with lithogenesis. This study investigated correlation between lithogenesis and the anatomical charateristics of cystic duct and juxtapapillary duodenal diverticulum. Methods : In the Department of Gastroenterology of the Sungkyunkwan University Kangbuksamsung Hospital, between January 1, 1996, and April 31. 1999,461 endoscopic retrograde cholangiopancreatography were performed. We examined these cases and excluded 371cases from the study. patients excluded from the study were those with previous cholecystectomy, carcinoma of the pancreas or biliary tract, benign stenosis of the sphincter of Oddi, lack of visualization of the entire cystic duct, and biliary endoprotheses. Finally, 90 patients were included and datas were obtained from the ERCP in the prone position. The length and diameter of cystic duct was measured by means of string. The patients were divided into three groups.(GroupI, absence of lithiasis in the biliary tract and gallbladder; GroupII, presence of lithiasis in the gallbladder but not in the CBD; Group III, lithiasis in the CBD with or without gallbladder lithiais.) Results : There was significant diferences between groups in sex. age, triglyceride level, site of implantation, length of cystic duct, morphology, existence of juxtapapillary duodenal diverticulum but not in diameter. The patients with gallstones or CBD stones has sinificantly longer than others. The insertion of the cystic duct on the left margin of the CBD, which is associated with a length greater than 3cm and spiral morphology. Conclusion: Age, sex, level of triglyceride, Implantation site, morphology, length of the cystic duct and juxtapapillary duodenal diverticulum is associated with lithiasis. It seems that left marginal insertion of cystic duct can piay an important role in lithogenesis by increasing cystic duct resistance ahd causing a reduced washout effect of the gallbladder contents Key Words: Lithogenesis, morphology, Diameter and Length of cystic duct, Implantation site, Juxtapapillary duodenal diverticulum
DETECTION OF HELICOBACTER PYWRI BY PCR AFTER AMPLIFICATION OF 16S RRNA FROM GALLSTONE. Don H. Lee, In H. Kim, Jin W. Lee, Sun H. Kim, Won Choi, Pum S. Kim, Hyung G. Kim, Young S. Kim, Keon Y. Lee, Seok H. Shin, Mi S. Choi, Inha Univ Hosp, Inchon, South Korea. Background/Aim: The Helicobacterpylori (H. pylori) causes chronic gastritis and peptic ulcer and is a risk factor of gastric MALToma and stomach cancer. Among the genus Helicobacter species that colonize the intestinal tract, Hepatic Helicobacterspecies have been found in gallbaldder tissue or bile of Chilean patients with chronic cholecystitis. The bacteria closely resembling H. pylori was detected in resected gallbladder mucosa and H. pylori was also found in bile juice of gallstone patients. But these studies were performed with bile, not with gallstone itself. To further explore the possible relation between H. pylori and gallstone formation we have investigated their presence in the gallstones. PatientslMethods: Stones were sampled from 70 patients (male 32, female 38) with gallstones on ultrasound. Mean age was 52.8 years (range,27-78 years). The gallstones and bile were obtained under aseptic condition during cholecystectomy. We ground the stone to powder from the interior of the stone by scraping with a surgical blade asepically. The DNA was extracted by a modification of the method of Swidsinsky et al (Gastroenterology, 1995) from 100 mg grounded stone. Polymerase chain reaction (PCR) was used to detect bacterial16S rRNA. The presence of H. pylori was revealed by PCR using primer for 26kDa surface antigen of H. pylori. Results: Twenty stone samples were positive for bacterial 16S rRNA. Among these 20 samples, PCR products for 26 kDa surface antigen of H. pylori were detected in 2 stone samples (10%). The detected stones were all brown pigment stones. Conclusion: H. pylori could be detected in the gallstone by PCR after amplification of 16S rRNA. The role of H. pylori and their products in gallstone formation will be elucidated.
297 HELICOBACTER SPECIES IN BILE FROM KOREAN PATIENTS WITH GALLBLADDER STONES. Dong Ki Lee, S.H. Ha, 1.B. Kim, K.H. Kim, T.W. Kim, K.H. Kim, J.H. Jung, J.W. Kim, H.S. Kim, S.K. Baik, S.O. Kwon, Wonju Coli of Medicine, Wonju, South Korea; Coli Health Sci, Wonju, South Korea. Recent reports on the association of Helicobacter sp. with gallbladder disease show discrepant results. This discrepancy may be explained by regional differences in the distribution of Helicobacter sp.. According to some reports, the Helicobacter sp. may be the culprit in the pathogenesis of some biliary tract disease. Hence, we have attempted to detect the Helicobacter sp. in the bile of patients in Korea, a country in which Helicobacter pylori is highly prevalent (>70% in adults). And to document the origin of Helicobacter sp., we tested stomach samples simultaneously. Methods: Gallbladder stones, bile and gallbladder tissue were obtained using strict aseptic technique from 33 patients with symptomatic gallbladder stones. Concomitantly gastric mucosa and gastric juice samples were obtained before cholecystectomy. DNA was extracted from crushed gallstones, gallbladder epithelium, bile, as well as the gastric mucosa and gastric juice. Helicobacter genus-specific and Helicobacter pylori-specific oligonucleotide primers were designed based on the nucleotide sequences of the 16S ribosomal DNA of Helicobacter pylori These primers were used to amplify a DNA fragment using the polymerase chain reaction (PCR). The specificity of the PCR products was examined by Southern hybridization. Results: PCR with Helicobacter pylori-specific primers produced amplicons in 30.3% (10/33) of the gallstones, 36% (9/25) of the bile and 6.2% (2/32) of the gallbadder epithelium samples. In 41% (13/32) of the patients, no Helicobacter genus PCR amplicon was produced from the samples of gallstone, bile or gallbladder epithelium. PCR produced evidence of Helicobacter pylori in 97% (32/33) of the gastric mucosa and/or gastric juice samples. The specificity of each of amplicons was verified by Southern hybridization. PCR amplicons for Helicobacter sp. were present in all of the corresponding gastric samples of the patients whose biliary samples were positive for Helicobacter genus. A PCR amplicon for a Helicobacter genus other than Helicobacter pylori was identified in 3% (1/32) of the gallstone and 16% (4/25) of the gallbladder epithelium samples. Conclusion: Helicobacter sp. in the biliary tract seems to have migrated from the stomach. Despite such a high detection rate of Helicobacter sp. in the gallstones or bile of patients in a Helicobacter pylori endemic country,there is little evidence of Helicobacter sp. actually colonizing the gallbladder. Nevertheless, the role of Helicobacter sp. in biliary tract disease warrants further study.
299 ACCURATE QUANTIFICATION OF MICELLAR AND VESICULAR PHASES IN CHOLESTEROL·SUPERSATURATED MODEL SYSTEMS: COMPARISON OF GEL FILTRATION AND ULTRACENTRIFUGATION-FILTRATION-DIAL YSIS METHODS. Antonio Moschetta, Erik R. Eckhardt, Martin B. de Smet, Gerard P. vanBerge-Henegouwen, Willem Renooij, Karel 1. van Erpecum, Gastrointestinal Research Unit, Univ Med Ctr, Utrecht, Netherlands; Semeiotica Medical, DIMIMP, Univ of Bari, Bari, Italy. Gel filtration with bile salts at intermixed micellar-vesicular concentration (IMC) in the eluant has been proposed to separate vesicles and micelles. However, this method is only suitable in highly diluted and slightly supersaturated model biles, since the presence of vesicular aggregates in more concentrated and supersaturated systems precludes accurate separation (Donovan JM, Hepatol 1998;27:641). We developed a new method for state-of-art separation. Methods: First aggregated vesicles and -if present- cholesterol crystals were pelleted by ultracentrifugation of supersaturated model biles (tot. lipid cone. 2.4 and 7.3 g/dL: CSI 1.7 resp. 1.4: EYPC/(EYPC + taurocholate) =0.3). Amounts of aggregated vesicles and cholesterol crystals were quantified by determining cholesterol content in the pellet both without and with excess added taurodeoxycholate (complete dissolution of aggregates). Unilamellar vesicles and micelles in the supernatant were separated by gel filtration with bile salts at IMC in the eluant ("gel filtration method"), by filtration of micelles through highly selective 300 kDa filter + (for unilamellar vesicles) dialysis against bile salts at IMC ("ultrafiltration-dialysis method") or estimated by subtraction of lipid contents in filtered micelles from lipid contents in (unilamellar vesicIes+micelles containing) supernatant ("subtraction method"). Results: =30% of cholesterol and 5-10% of phospholipids were solubilized in vesicular aggregates. Ultrafiltration-dialysis and subtraction methods yielded identical lipid solubilization in micelles vs unilamellar vesicles and identical chollPL ratios. In contrast, gel filtration method yielded much more lipids in micelles and less in unilamellar vesicles, with much higher vesicular chollPL ratios (1.98±0.56 vs 0.99±0.I). When vesicles obtained by dialysis were analized by gel filtration, vesicular chollPL ratios increased from 0.99 to 1.98, despite correct IMC values for bile salts in the eluant. Subsequent extraction of column material showed considerable amounts of lipids. Conclusions: gel filtration may underestimate vesicular lipids and overestimate vesicular chollPL ratios, supposedly because of lipids remaining attached to the column. Combined uItracentrifuagtionultrafiltration-dialysis should be considered state-of-art methodology for quantification of cholesterol carriers in model systems.