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The efficacy of treatment of benign biliary stricture by intralesional steroid injection through choledochoscope
Biliary Disorders A541
April 1998 G2212 REGULATION OF RAT HEPATIC 313-HYDROXYSTEROL AT-REDUCT ASE ACTIVITY. S. Shefer, G. Salen, A.K. Batta, G.S. Tint, A. Honda, Newark, NJ and East Orange, NJ
To investigate the mechanism for the catalytic reduction of the double bond at C-7(8) in 7-dehydrocholesterol by 313-hydroxysterol AT-reductase, we tested structurally related sterols as substrates and potential inhibitors and identified hepatic smooth endoplasmic reticulum as the site of enzyme activity. All putitive substrates, contained 27 carbons, but differed from 7-dehydrocholesterol by the addition of either an ethyl substituent at C-24 (7-dehydrositosterol), a double bond at C-22 with a methyl substituent at C24 (ergosterol), epimerization of the hydroxyl from the 313- to 3orconfiguration (7-dehydroepicholesterol) or a saturated double bond at C-5(6) (lathosterol). Two non-steroidal compounds that inhibit 31]- hydroxysterol AT-reductase in vivo (AY 9944 and BM 15.766) were also tested. Ergosterol, 7-dehydrositosterol and 7-dehydroepicholesterol were reduced at C-7(8) to form brassicasterol, sitosterol and epicholesterol, respectively, but 75% less efficient than 7-dehydrocholesterol. Increasing concentrations of these sterols competitively inhibited 313-hydroxy sterol AT-reductase activity. The double bond at C-7(8) in lathosterol was not reduced. AY 9944 and BM 15.766 inhibited 313-hydroxysterol AT-reductase activity non-competitively. 313Hydroxysterol AT-reductase activity declined after microsomes were exposed to alkaline phosphatase, and enzyme activity was restored by phosphorylation with protein kinase, Mg ÷, and ATP. These results demonstrate that the reduction of the double bond at C-7(8) requires binding of the enzyme protein with the B-ring of the sterol substrate that contains a double bond at C-5(6). The reaction is hindered by substituents located on the apolar side-chain and epimerization of the hydroxyl group in ring A to a 3or-configuration. AY 9944 and BM 15.766 non-competatively inhibit 313-hydroxysterol AT-reductase activity by combining with the enzyme protein at a site different from the active site, and the blockage cannot be overcome by increasing the substrate concentration. 313-Hydroxysterol AT-reductase exists in two forms: an active phosphorylated form and an inactive dephosphorylated form. This research was funded by VA Research Service, and US Public Health Service grants HL 17818 and DK 26756. • G2213 HUMAN BILIARY GLYCOPROTEIN AND CALCIUM BIL1RUBINATE BOTH CHANGE MOLECULAR CONFIGURATION FOLLOWING BINDING IN SOLUTION IN VITRO. G-R Shen, R.D. Sol0way, Y-H Shen, S-F Weng, W-H Li, J-G Wu. Department of Chemistry, Peking University, Beijing, China, and Division of Gastroenterology, The University of Texas Medical Branch, Galveston, TX Infrared spectroscopy can detect changes in the molecular configuration of molecules following binding to ligands by identifying spectral peak shifts and changes in band intensities. Measurement of these changes allows identification of the atomic groups within the molecule that are involved in binding and in configurational changes. The aim of this study was to examine the infrared spectra of a biliary glycoprotein fraction and calcium bilirubinate (Ca2BR), prior to and after binding, to identify the portions of the molecules that changed conformation following binding. Ca2BR was synthesized by reacting CaCI 2 and bilirubin in nonaqueous solution. The precipitate was dessicated and then added to a 3.3% aqueous solution of a biliary glycoprotein fraction (MW 66,000) and then stirred for 10 hrs. The solutions were then centrifuged at 3000 rpm for 10 min and the supernate (sup) and precipitate (ppt) were examined by infrared spectroscopy. The supernate became pigmented indicating some of the Ca2BR dissolved and some of the protein in the supernate bound to the remaining precipitate. The secondary structure of the protein was measured using deconvolution and curve fitting in the amide 1 region (1615-1700 cm'l). Group Glycoprotein (GP) Ca2BR sup [GP+Ca2BR]-Ca2BR ppt [GP+Ca2BR]-Ca2BR
N-H 3295 3394 3289 3396
CHst 2954 2964 2958 2958
C=O, lactam, COOa.~ 1736,1657, 1547 1662,1663,1571 1732,1656, 1543 1693,1663,1572
COOs C-O-C 1167 1416 1055 1411 1087 1421 1042
Glycoprotein conformational changes after binding with Ca2 BR. Protein Protein+Ca2BR Protein+Ca2BR Extended chain sheet Random coil a-helix turn 13sheet
1615 1638
1.6% 19.9%
1657 1672 1694
51.6% 4.0% 0.85%
1612 1633 1640 1657 1674 1699
0.22% 6.4% 12.6% 63.2% 10.0% 0.0%
1614 1635 1656 1672 1699
0.03% 14.4% 73.0% 9.7% 0.0%
We conclude that both Ca2BR and the glycoprotein were changed by binding in the supemate as well as the precipitate. This study confirms that soluble Ca2BR complexes are predominantly composed of protein while precipitated complexes are predominantly composed of Ca2BR. Ca2+ predominantly bound to the oligosaccharide portion of the protein molecule. Protein binding in the study suggests a mechanism for solubilization of some of the insoluble
Ca2BR that could be operative in bile. Supernatant protein configuration was changed by binding. G2214 DETECTION OF MOST SPECIES OF PATHOGENIC BACTERIAL DNA AND HELICOBACTER PYLORI DNA IN BILIARY STONES. C,S. Shim, J.H. Moon, Y.D. Cho, S.J. Hong, LO. Kim, J.Y. Cho, Y.S. Kim, J.S. Lee, M.S. Lee, S.G. Hwang. Institute for Digestive Research, Department of Internal Medicine, Soon Chun Hyang University, Seoul, Korea Background: Bacteria is very important in the pathogenesis of brown pigment gallstones and bile duct stones. Cholesterol gallstones formation is believed to be unrelated to the presence of bacteria, but composite stones have cholesterol and pigment portion, bacteria will be find within the pigment area or center. In Korea, bile duct stones are common and Helicobacter pylori infection rates are higher than the Western countries. Aims: The aim of this study was to evaluate the presence of bacterial DNA and Helicobacter pylori DNA in many types and sites of gallstones. Materials and Method: Stones were obtained using strict aseptic method from 24 symptomatic patients with biliary stones at operation or percutaneous choledochoscopic lithotripsy. The stones were divided into two groups, 7 gallbladder stones and 17 bile duct stones(3 extrahepatic bile duct(EHD) stones, 8 intrahepatic bile duct(IHD) stones, 6 EHD and IHD stones). These stones consisted of 3 cholesterol, 10 brown, 5 black, 8 mixed stones as visually determined. DNA was extracted from pulverized stones and oligonucleotide primers for the conserved 16S rRNA gene of pathogenic bacteria(product size: 367 bp) and Helicobacter pylori (product size: 522 bp) were used to amplify a DNA fragment using the PCR. Results: 1) Pathogenic bacterial DNA could be amplified from biliary stones of 24 patients, and Bacterial DNA was found 14 patients(58.3%). 2) Among the 7 patients with gallbladder stones, bacterial DNA was found in 4(57.1%). 3) Among the 17 bile duct stones, 10(58.8%) of them were positive. 4) There was no significant difference of bacterial DNA detection rate for extrahepatic duct stones(66.7%) and intrahepatic duct stones(50%). 5) Helicobacterpylori DNA was not detected in biliary stones of 24 patients. Contusion: Above half of gallbladder and bile duct stones showed pathogenic bacterial DNA, but, no Helicobacter pylori DNA was found in the biliary stones.
G2215 THE EFFICACY OF TREATMENT OF BENIGN BILIARY STRICTURE BY INTRALESIONAL STEROID INJECTION THROUGH CHOLEDOCHOSCOPE. C.S. Shim. E.J. Kim, S.J. Hong, J.H. Moon, Y.D. Cho, J.O. Kim, J.Y. Cho, Y.S. Kim, M.S. Lee, S.G. Hwang. Institute for Digestive Research. Department of Internal Medicine, Soon Chun Hyang Universty, Seoul, Korea Background: Benign biliary stricture is the most common serious benign disease of the biliary tract and, if left untreated, can lead to recurrent cholangitis and secondary biliary cirrhosis, cholangiocarcinoma. Benign biliary stricture has been treated with endoscopic or percutaneous balloon dilatation with or without stent placement, or surgical repair, although unsatisfactory. The mechanism of ACTH and synthetic corticosteroid has not been fully understood, but they apparently interfere with the formation of mucopolysaccharides necessary for collagen synthesis and more potent antiinflammatory corticosteroid, triamcinolone acetonide(9 alpha-fluorohydrocortisone acetonide) has been used intralesionally upon keloids, hypertropic scars, and bum contractures with almost no systemic corticoid effects because of its slow absorption, which remains in high local concentration. Recently, successful treatment of benign esophageal strictures using intralesional steroid injection has been reported in clinical and experimental study. Aims: This study was carried out to evaluate the efficacy of treatment of benign biliary stricture by intralesional steroid injection through choledochoscope. Materials and Methods: Eight patients with benign biliary stricture and stones diagnosed by choledochoscope(M:F = 3:5, mean age: 50 years old) were studied. The sites of strictures were as follows: Right hepatic ducts in two patients, left hepatic ducts in three patients, intrahepatic duct and common hepatic ducts in two patients and common bile duct in one patient. Patients were treated with local steroid injection via a 23 gauge needle with triamcinolone acetonide in 0.5 ml-l.0 ml(5 - 10 mg) increments within the stricture segments, for a total of 20-40rag per stricture through choledochoscope. After steroid injection, repeat cholangiography was conducted afterward at 2 weeks, 4 weeks, 3 months intervals and stricture diameter was measured. Results: Number of mean session of local steroid injection was 1.6 and mean follow up period was 4 weeks. Mean diameter of stricture site before triamcinolone acetonide injection was 3.2 mm and it was 7.2 mm after local steroid injection. So mean diameter of dilated bile duct was 4.0 mm(p<0.001). Complications such as bleeding, pain, perforation and fever were not noted during the steroid injections at the stricture sites. Conclusions: Local triamcinolone acetonide injections on the stricture segments might be a safe method and improve the clinical outcome in the patients with benign biliary stricture. More cases and longer follow up periods seem to be required.
April 1998 G2212 REGULATION OF RAT HEPATIC 313-HYDROXYSTEROL AT-REDUCT ASE ACTIVITY. S. Shefer, G. Salen, A.K. Batta, G.S. Tint, A. Honda, Newark, NJ and East Orange, NJ
To investigate the mechanism for the catalytic reduction of the double bond at C-7(8) in 7-dehydrocholesterol by 313-hydroxysterol AT-reductase, we tested structurally related sterols as substrates and potential inhibitors and identified hepatic smooth endoplasmic reticulum as the site of enzyme activity. All putitive substrates, contained 27 carbons, but differed from 7-dehydrocholesterol by the addition of either an ethyl substituent at C-24 (7-dehydrositosterol), a double bond at C-22 with a methyl substituent at C24 (ergosterol), epimerization of the hydroxyl from the 313- to 3orconfiguration (7-dehydroepicholesterol) or a saturated double bond at C-5(6) (lathosterol). Two non-steroidal compounds that inhibit 31]- hydroxysterol AT-reductase in vivo (AY 9944 and BM 15.766) were also tested. Ergosterol, 7-dehydrositosterol and 7-dehydroepicholesterol were reduced at C-7(8) to form brassicasterol, sitosterol and epicholesterol, respectively, but 75% less efficient than 7-dehydrocholesterol. Increasing concentrations of these sterols competitively inhibited 313-hydroxy sterol AT-reductase activity. The double bond at C-7(8) in lathosterol was not reduced. AY 9944 and BM 15.766 inhibited 313-hydroxysterol AT-reductase activity non-competitively. 313Hydroxysterol AT-reductase activity declined after microsomes were exposed to alkaline phosphatase, and enzyme activity was restored by phosphorylation with protein kinase, Mg ÷, and ATP. These results demonstrate that the reduction of the double bond at C-7(8) requires binding of the enzyme protein with the B-ring of the sterol substrate that contains a double bond at C-5(6). The reaction is hindered by substituents located on the apolar side-chain and epimerization of the hydroxyl group in ring A to a 3or-configuration. AY 9944 and BM 15.766 non-competatively inhibit 313-hydroxysterol AT-reductase activity by combining with the enzyme protein at a site different from the active site, and the blockage cannot be overcome by increasing the substrate concentration. 313-Hydroxysterol AT-reductase exists in two forms: an active phosphorylated form and an inactive dephosphorylated form. This research was funded by VA Research Service, and US Public Health Service grants HL 17818 and DK 26756. • G2213 HUMAN BILIARY GLYCOPROTEIN AND CALCIUM BIL1RUBINATE BOTH CHANGE MOLECULAR CONFIGURATION FOLLOWING BINDING IN SOLUTION IN VITRO. G-R Shen, R.D. Sol0way, Y-H Shen, S-F Weng, W-H Li, J-G Wu. Department of Chemistry, Peking University, Beijing, China, and Division of Gastroenterology, The University of Texas Medical Branch, Galveston, TX Infrared spectroscopy can detect changes in the molecular configuration of molecules following binding to ligands by identifying spectral peak shifts and changes in band intensities. Measurement of these changes allows identification of the atomic groups within the molecule that are involved in binding and in configurational changes. The aim of this study was to examine the infrared spectra of a biliary glycoprotein fraction and calcium bilirubinate (Ca2BR), prior to and after binding, to identify the portions of the molecules that changed conformation following binding. Ca2BR was synthesized by reacting CaCI 2 and bilirubin in nonaqueous solution. The precipitate was dessicated and then added to a 3.3% aqueous solution of a biliary glycoprotein fraction (MW 66,000) and then stirred for 10 hrs. The solutions were then centrifuged at 3000 rpm for 10 min and the supernate (sup) and precipitate (ppt) were examined by infrared spectroscopy. The supernate became pigmented indicating some of the Ca2BR dissolved and some of the protein in the supernate bound to the remaining precipitate. The secondary structure of the protein was measured using deconvolution and curve fitting in the amide 1 region (1615-1700 cm'l). Group Glycoprotein (GP) Ca2BR sup [GP+Ca2BR]-Ca2BR ppt [GP+Ca2BR]-Ca2BR
N-H 3295 3394 3289 3396
CHst 2954 2964 2958 2958
C=O, lactam, COOa.~ 1736,1657, 1547 1662,1663,1571 1732,1656, 1543 1693,1663,1572
COOs C-O-C 1167 1416 1055 1411 1087 1421 1042
Glycoprotein conformational changes after binding with Ca2 BR. Protein Protein+Ca2BR Protein+Ca2BR Extended chain sheet Random coil a-helix turn 13sheet
1615 1638
1.6% 19.9%
1657 1672 1694
51.6% 4.0% 0.85%
1612 1633 1640 1657 1674 1699
0.22% 6.4% 12.6% 63.2% 10.0% 0.0%
1614 1635 1656 1672 1699
0.03% 14.4% 73.0% 9.7% 0.0%
We conclude that both Ca2BR and the glycoprotein were changed by binding in the supemate as well as the precipitate. This study confirms that soluble Ca2BR complexes are predominantly composed of protein while precipitated complexes are predominantly composed of Ca2BR. Ca2+ predominantly bound to the oligosaccharide portion of the protein molecule. Protein binding in the study suggests a mechanism for solubilization of some of the insoluble
Ca2BR that could be operative in bile. Supernatant protein configuration was changed by binding. G2214 DETECTION OF MOST SPECIES OF PATHOGENIC BACTERIAL DNA AND HELICOBACTER PYLORI DNA IN BILIARY STONES. C,S. Shim, J.H. Moon, Y.D. Cho, S.J. Hong, LO. Kim, J.Y. Cho, Y.S. Kim, J.S. Lee, M.S. Lee, S.G. Hwang. Institute for Digestive Research, Department of Internal Medicine, Soon Chun Hyang University, Seoul, Korea Background: Bacteria is very important in the pathogenesis of brown pigment gallstones and bile duct stones. Cholesterol gallstones formation is believed to be unrelated to the presence of bacteria, but composite stones have cholesterol and pigment portion, bacteria will be find within the pigment area or center. In Korea, bile duct stones are common and Helicobacter pylori infection rates are higher than the Western countries. Aims: The aim of this study was to evaluate the presence of bacterial DNA and Helicobacter pylori DNA in many types and sites of gallstones. Materials and Method: Stones were obtained using strict aseptic method from 24 symptomatic patients with biliary stones at operation or percutaneous choledochoscopic lithotripsy. The stones were divided into two groups, 7 gallbladder stones and 17 bile duct stones(3 extrahepatic bile duct(EHD) stones, 8 intrahepatic bile duct(IHD) stones, 6 EHD and IHD stones). These stones consisted of 3 cholesterol, 10 brown, 5 black, 8 mixed stones as visually determined. DNA was extracted from pulverized stones and oligonucleotide primers for the conserved 16S rRNA gene of pathogenic bacteria(product size: 367 bp) and Helicobacter pylori (product size: 522 bp) were used to amplify a DNA fragment using the PCR. Results: 1) Pathogenic bacterial DNA could be amplified from biliary stones of 24 patients, and Bacterial DNA was found 14 patients(58.3%). 2) Among the 7 patients with gallbladder stones, bacterial DNA was found in 4(57.1%). 3) Among the 17 bile duct stones, 10(58.8%) of them were positive. 4) There was no significant difference of bacterial DNA detection rate for extrahepatic duct stones(66.7%) and intrahepatic duct stones(50%). 5) Helicobacterpylori DNA was not detected in biliary stones of 24 patients. Contusion: Above half of gallbladder and bile duct stones showed pathogenic bacterial DNA, but, no Helicobacter pylori DNA was found in the biliary stones.
G2215 THE EFFICACY OF TREATMENT OF BENIGN BILIARY STRICTURE BY INTRALESIONAL STEROID INJECTION THROUGH CHOLEDOCHOSCOPE. C.S. Shim. E.J. Kim, S.J. Hong, J.H. Moon, Y.D. Cho, J.O. Kim, J.Y. Cho, Y.S. Kim, M.S. Lee, S.G. Hwang. Institute for Digestive Research. Department of Internal Medicine, Soon Chun Hyang Universty, Seoul, Korea Background: Benign biliary stricture is the most common serious benign disease of the biliary tract and, if left untreated, can lead to recurrent cholangitis and secondary biliary cirrhosis, cholangiocarcinoma. Benign biliary stricture has been treated with endoscopic or percutaneous balloon dilatation with or without stent placement, or surgical repair, although unsatisfactory. The mechanism of ACTH and synthetic corticosteroid has not been fully understood, but they apparently interfere with the formation of mucopolysaccharides necessary for collagen synthesis and more potent antiinflammatory corticosteroid, triamcinolone acetonide(9 alpha-fluorohydrocortisone acetonide) has been used intralesionally upon keloids, hypertropic scars, and bum contractures with almost no systemic corticoid effects because of its slow absorption, which remains in high local concentration. Recently, successful treatment of benign esophageal strictures using intralesional steroid injection has been reported in clinical and experimental study. Aims: This study was carried out to evaluate the efficacy of treatment of benign biliary stricture by intralesional steroid injection through choledochoscope. Materials and Methods: Eight patients with benign biliary stricture and stones diagnosed by choledochoscope(M:F = 3:5, mean age: 50 years old) were studied. The sites of strictures were as follows: Right hepatic ducts in two patients, left hepatic ducts in three patients, intrahepatic duct and common hepatic ducts in two patients and common bile duct in one patient. Patients were treated with local steroid injection via a 23 gauge needle with triamcinolone acetonide in 0.5 ml-l.0 ml(5 - 10 mg) increments within the stricture segments, for a total of 20-40rag per stricture through choledochoscope. After steroid injection, repeat cholangiography was conducted afterward at 2 weeks, 4 weeks, 3 months intervals and stricture diameter was measured. Results: Number of mean session of local steroid injection was 1.6 and mean follow up period was 4 weeks. Mean diameter of stricture site before triamcinolone acetonide injection was 3.2 mm and it was 7.2 mm after local steroid injection. So mean diameter of dilated bile duct was 4.0 mm(p<0.001). Complications such as bleeding, pain, perforation and fever were not noted during the steroid injections at the stricture sites. Conclusions: Local triamcinolone acetonide injections on the stricture segments might be a safe method and improve the clinical outcome in the patients with benign biliary stricture. More cases and longer follow up periods seem to be required.