A Correlation Study On The Pancreaticobiliary And Duodenal-biliary Refluxes In Biliary Tract Diseases

PrefaceIn 1966, Elmslie put forward the theory of pancreaticobiliary reflux.Elmslie foundthat in addition to pancreaticobiliary reflux may cause the development of CBD cystwhich progresses into cholangiocarcinoma or gallbladder carcinoma, it may alsoundermine bile colloidal stability, thus relating to cholelithiasis. The pancreaticobiliaryreflux is commonly seen with PBM because of the elongated pancreaticobiliaryjunction (＞15mm) and it is not within the SO contraction scope, thus causingpancreatic juice regurgitation. Inagaki M, Horaguchi J and etal have suggested thepresence of occult pancreaticobiliary reflux in non-PBM patients. Even withoutanomalous pancreaticobiliary ductal union morphology, the entry of pancreatic juiceinto bile ducts can also cause PBM-like ductal pathological changes.When enterokinase is involved in the pancreaticobiliary reflux, this may wellinduce an enzymatic cascade reaction which may in turn speed up above-describedpathological changes. Such reaction is also seen in the duodenal-biliary reflux asduodenal fluid enters the bile ducts, which needs to be differentiated.Recent diagnostic methods for the two refluxes showed improvements onsensitivity and specificity, such as the detection of bile radioactivity after oral RN induodenal-biliary reflux, and secretin-induced ductal width changes under MRIobservation in pancreaticobiliary reflux. These methods are not widely accepted due toits limitations and drawbacks-unable to reflect physiologic reflux and such. Hencelow-specific bile amylase level testing is still in use.Our study not only performed the typical RN and AMY tests, in addition, forcomparison we introduced a new innovative approach to observe the presence of thetwo refluxes in biliary diseases-the Western blot test for enterokinase and trypsin-1detection. To determine the correlation of the two refluxes in biliary diseases. Patients and MethodsPatient151 patients with biliary disease hospitalized at Shenjing Hospital of ChinaMedical University from the times of January 2007 to August 2008 are as follows:Including Gall stone, gall polyps, ductal stone, ductal pigment stone, ductal residualstone, CBD cyst and hilar cholangiocarcinoma underwent percutaneous transhepaticcholangial drainage(PTCD ) and obstructive jaundice underwent endoscopicnasobilliary drainage(ENBD) patients.The diagnosis of biliary diseases is based on clinical symptoms, radiology,endoscopic or intraoperative findings, and pathological histology results. Gallstonesfound in the ductal residual stone and ductal pigment stone groups were comprised ofsandy and brown pigment stones. Patients with ENBD drainage also had endoscopicsphincterotomy (EST) performed on them, whilst others had intacted sphincter of Oddi.The Collection of Samples(一)Sample Collection72 gallbladder bile and 31 bile duct bile samples were obtained intraoperatively, withremaining ductal bile samples collected from patients carrying either T tube, PTCD, orENBD drainages whom had fasted over 12 hours.(二)Sample PreservationPrior to sample analysis, all the specimens were preserved in -80℃conditionfollowing withdrawal.三,Targets detection(一)The measuring of bile AMY using biochemical methodThe measuring of AMY activities used Gal-G2-CNP matrix method. The normalblood AMY level ranges are 20～115 U/L.(二)The measuring of bile AMY and LPS activities using biochemicalmethodThe measuring of LPS activities used DGGMR matrix methods. 93/106 ductal bileand 60/72 gall bile samples were chosen for LPS activity test. The normal blood LPS level ranges are 23～300 U/L.(三) Western bloting detection of bile Trypsin-1Bile specimens were collected as described above: removal of residuals,desalination, and Immunoprecipitation.Western bloting: Detection using Enhanced Chemiluminescene method, thepresence of specific band at 25KD is said to be Trypsin-1 positive.(四) Detection of bile radioactivity after oral ^99mTc-DTPA42 intrahepatic and extrahepatic bile duct stone patients underwent bile ductexploration and T tube drainage received this test. The patients were asked to fastovernight before the test.185 MBq (5 mCi) of technetium 99mdiethy-lenetri-aminepentaacetatic acid(99mTc-DTPA, Mr 549 000) orally followed by a 240 mL of water and immediate bedrest in supine position. From the time of ingestion, a 2 hour 20 mL bile was collectedthough the T-tubes to determine duodenal-biliary reflux using RM905 radioactivitymeter. The duodenal-biliary reflux diagnosis was made upon findings of bileradioactivity.(五) Western blotting detection of bile EKBile preparation method same as previously described.Western bloting: Detection using Enhanced Chemiluminescence, ECL, method, thepresence of a specific band when 300KD is said to be EK positive.四,Statistical AnalysisStatistical analysis was carried out using statistical software package SPSS 13.0.Statistical analysis was the appropriate use of the Pearson correlation analysis,ANOVA, Kruskal-Wallis, kappa, Fisher exact test and chi square test. A P value＜0.05was considered to be statistically significant.ResultsConsistency test for the Trypsin-1 and bile AMY and LPSactivities of detecting pancreaticobiliary reflux(一)measuring of bile AMY and LPS activities Detection of AMY level, 63(59.43%) ductal bile samples were determined aspositive for pancreaticobiliary reflux with＞115U/L; in gall bile, 36(50%) cases werepancreaticobiliary reflux positive.Detection of LPS level, 53(56.99%) ductal bile samples were determined aspositive for pancreaticobiliary reflux with＞300U/L; in gall bile, 16(26.67%) caseswere pancreaticobiliary reflux positive.Correlative analysis was performed after logarithmic transformation of AMY andLPS levels. In bile duct, r=0.812(P＜0.001); in gallbladder, r=0.775(P＜0.001).(二)Bile Trypsin-1 DetectionIn ductal bile, western blotting tested 55 (51.89%) cases with Trypsin-1 specificbands and thus determined as positive for pancreaticobiliary reflux; as for gall bile, 26(36.11%) cases were tested positive.(三)Consistency test for diagnostic methods of pancreaticobiliaryrefluxIn gallbladder, Kappa value for Trypsin-1 and AMY tests came about 0.611(P＜0.001); Kappa value for Trypsin-1 and LPS tests was 0.624(P＜0.001); In bile duct,Kappa value for Trypsin-1 and AMY tests was 0.696(P＜0.001); Kappa value forTrypsin-1 and LPS tests was 0.806(P＜0.001).Consistency was found between pancreaticobiliary reflux AMY/LPS and Trypsin-1diagnostic methods (P＜0.001) . Ductal bile LPS detection result was approachingTrypsin-1.Consistency test for the EK and Oral 99mTc-DTPA Test ofdetecting duodenal-biliary reflux(一)Oral ^99mTc-DTPA TestIn 42 bile exploratory T-tube drainage cases, 9(21.43%)of which were diagnosedwith duodenal-biliary reflux based on radioactivity detected. In that 9 cases, 2 hour20mL bile Technetium activity came about 175.9±129.2KBq, 2 hour bile drainagevolume was 41.7±12.2mL; the remaining 33 cases with 2 hour bile drainage volume of46.4±19.2mL. No significant differences were found between the 2 groups (P＞0.05). (二)Bile Enterokinase DetectionIn 42 bile exploratory T-tube drainage cases, 17(40.48%) of which underwentwestern bloting and bound with EK specific band thus diagnosed with duodenal-biliaryreflux.(三)Consistency test for the EK and Oral 99mTc-DTPA Test of detectingduodenal-biliary refluxKappa value for EK and Oral ^99mTc-DTPA tests was 0.466(P＜0.001). Oral^99mTc-DTPA test (9/42)is comparatively less sensitive than EK test (17/42) , with nostatistical significance (P＞0.05) .EK and Trypsin-1 Detections to Evaluate Pancreatico- biliary andDuodenal-biliary Reflux Rates in Biliary DiseasesIn ductal bile, EK positive-rates were significantly higher in the residual stone,CBD pigment stone and ENBD groups than that of PTCD and CBD cyst groups (P＜0.05); Trypsin-1 positive-rate for the PTCD group was significantly lower than other 4groups (P 0.05).In gall bile, EK positive-rate has no obvious significance among the groups (P＜0.05); Trypsin-1 positive rate for the congenital CBD cyst group was significantlyhigher than other groups (P＜0.05).Conclusions1,Western bloting detection of bile EK and Trypsin-1 is comparatively moresensitive and more specific in the determination of duodenal-biliary andpancreaticobiliary refluxes over oral Oral ^99mTc-DTPA test and AMY level tests.2,Bile AMY/LPS primarily come from the pancreas.3,Pancreatic enzyme in the ductal pigment stone and T-tube drainage groups comefrom duodenal-biliary reflux. Duodenal-biliary reflux positive rate and bile origins arenot associated in any ways, perhaps a correlation between duodenal-biliary reflux andpigment stone is present.4,In CBD cyst, bile originates from pancreaticobiliary reflux.5,Occult pancreaticobiliary reflux is found present in non-congenital CBD cyst and biliary diseases.