Association Of Duodenal-Biliary Reflux And Pigment Calculus In Bile Duct And Significance Of Cholecyst GUSB MRNA Expression In Cholecystolithiasis

PART 1 Methods of determination of duodenal-biliary refluxObjectiveUp to now, the methods used to examine the duodenal-biliary reflux mostly depend on complicated and expensive instruments ( such as, X-ray emission, ultrasound detector, SPECT) , and some of the results can only be inspected by the radiologists experts. The large fluctuation of individual determination and low sensitivity lead to its difficulty of evaluation. New methods are at urgent to be discovered to farther the research on duodenal-biliary reflux. This study tested some new methods and compared their individual characteristics.MethodsPatients;45 resident patients were included from May 2005 to March 2006. They were all suffered from bile duct residual stones, with 19 males and 26 females. All patients accepted cholecystectomy and choledochotomy and " T" tube drainage. Another 11 patients suffered from bile duct malignant tumor accepted PTCD, with 8 males and 3 females, whose end of choledochus had been fully obstructed. All patients were free from fever and bile duct infection with normal blood white cell counts.2. Methods2.1 Determination of duodenal-biliary reflux by nuclideThe patients with T tube were kept fasting over 12 hours and 1ml of ^99mTc-DTPA (diethylene triamine pentaacetic acid) with 185 MBq was taken orallyand they rinsed out with 240ml water. Then they lied down in horizontal position. Bile in the following 2h was collected from T tube or PTCD tube and 20ml was tested with RM905 radioactive measure equipment. Duodenal-biliary reflux was identified if radioactivity existed in bile.2.2 Determination of duodenal-biliary reflux by ICGStandardized 1 jig/fil of ICG was prepared. Then it was added into the bile juice with the concentration tilter 1 (xg/ml. The standardized curve was prepared with the absorbed degree at wavelength of 806nm. The patients were kept fasting over 12 hours and ICG was taken orally after collection of 20ml of T tube bile. After rinsing out with 240ml water , they lied down in horizontal position . Bile in the following 2h was collected from T tube and 20ml was tested with photometer immediately at wavelength of 806nm. The actual count of ICG was calculated according to the standardized curve. Duodenal-biliary reflux was identified if ICG existed in the bile juice.2. 3 Determination of duodenal-biliary reflux by pancreatic enzyme20 ml T tube bile was collected, the amount of pancreatic amylase and li-pase were measured respectively by 7170A full auto biochemical analyses instrument. The upper normal range of blood amylase and lipase were used as the test benchmark (for pancreatic amylase 100U/L, for pancreatic lipase 60U/L). Duodenal-biliary reflux was identified if the amount of these two enzymes in bile exceeds the range above.Results1. Results of determination of T tube duodenal-biliary reflux by nuclide Duodenal-biliary reflux existed in 16 of the 45 patients (35. 56% ) , the radioactivity count in these 16 patients was 99. 7 ± 16. 24KBq, and the 2h bile drainage amount was 38. 5 ± 17.2ml. The radioactivity count in the rest 29 patients was zero, the 2h bile drainage amount was 35.9 ±20.0ml.2. Results of determination of PTCD tube duodenal-biliary reflux by nuclide The radioactivity count in all the 11 patients accepting PTCD was zero andthe 2h bile drainage amount was 43.9 ± 19.7ml.3. Comparisons between the results of T tube duodenal-biliary reflux by nuclide and ICGNuclide method detected 4 cases of reflux (33. 33% ) , while ICG methods detected 2 cases (16.67% ) in the same group of 12 patients. That is to say the positive detection rate of reflux of ICG method is hah0 of that of nuclide method and the ICG positive result can be gained only when the reflux amount is relative large.4. Comparisons between the results of T tube duodenal-biliary reflux by nuclide and pancreatic enzyme12 cases (46.15% ) of positive reflux were detected by the marker pancreatic amylase, which is slightly higher than the nuclide 10 cases (38.46% ). 22 cases (84. 61% ) of positive reflux were detected by the marker pancreatic li-pase, which is far higher than the nuclide 10 cases (38.46% ).Conclusion1. Nuclide method is a good way to determine duodenal-biliary reflux with satisfied sensitivity and specificity which possess priority up to now.2. ICG method is a brand-new way to determine duodenal-biliary reflux which is worthy of further study and possess bright prospect.3. Pancreatic enzymes method lack specificity in the determination of duodenal-biliary reflux, it can not be used alone to determine the existence of duodenal-biliary reflux.PART 2 The relationship between duodenal-biliary reflux and bilepigment calculus in bile ductObjectiveCholelithiasis is common in China. There are many patients diagnosed as pigment calculus in bile duct which seldom attack in west countries. No significant advances have caught about the pathogenesis of pigment calculus for a long period. Researchers have put more attention on the role of the duodenal-biliary reflux in the bile pigment calculus pathogenesis. We think that the formation of bile pigment calculus in bile duct may relate with duodenal-biliary reflux. We studied patients with cholelithiasis to observe the relationship between cholelithiasis and duodenal-biliary reflux and its mechanisms. Based on the following line that there is an increase of duodenal contents because of the duodenal-biliary reflux and it induces the enzyme reaction of bile, bacteria and endotoxin, resulting in the pathogenesis of pigment calculus, we studied the bacteria and endotoxin in bile from patients with pigment calculus and the reflux was evaluated by radioactivity . The patients diagnosed as pigment calculus that undergone choledochot-omy with T tube are included to analyze the amylase, lipase, (3-glucuronidase in bile to evaluate the relationship between pigment calculus and reflux to discover the pathogenesis of pigment calculus.Methods1. Patients: 89 resident patients were included from May. 2005 to Oct. 2005. They were not caught fever, no evidence of bile duct infection and blood white blood cell was in normal range. These cases were divided into groups as following: 10 cases of gallbladder polyp, 27 cases of cholecystolithiasis, 11 cases of pigment calculus in bile duct, and 26 cases with T tube. There were no significant differences in these groups. It was average 62.3(59 -66) d of postoper-ation period in patients with T tube who need choledochoscopy.2. Methods: The bile was collected from gallbladder and bile duct by puncturing in operation. The bile was cultured and endotoxin was tested. 26 ran-domed cases with T tube were divided into reflux group and non-reflux group based on the results of radioactivity and bile amylase, lipase and B-glucuroni-dase were tested. Bacteria was cultured with sheep blood agar and China blue mediums and identified by whole auto bacterial identified system. The bile was collected with depyrogenic tube and divided into 2 parallel tubes. The endotoxin was tested by dynamic turbidity method with BET-32B bacterial endotoxin equipment. The patients with T tube were kept fasting over 12 hours and lml of "mTc-DTPA (diethylene triamine pentaacetic acid) with 185 MBq was taken orally and they rinsed out with 240ml water. Then they lied down in horizontal position. Bile in the following 2h was collected from T tube and 20ml was tested with RM905 radioactive measure equipment. Duodenal-bile reflux was identified if radioactivity existed in bile. The endogenous and exogenous S-glucuronidase was measured separately within 550nm wave length with PH4.6 or PH 6. 8. Bile amylase and lipase were measured with Hitachi 7170A full auto biochemical analyses instrument.Statistical analyze;Dates were analyzed with SPSS11.5, expressed as mean± SD and processed ^2 test, t-test . It was considered statistical significant if P<0.05.ResultsThe bacteria in bile of pigment calculus in bile duct group are all intestinal bacteria including 5 Escherichia coli, 2 Klebsiella oxytoca, 1 Enterobacter aero-genes and 1 Enterobacter cloacae and the positive rate is 81. 8%. The positive rate of bacterial culture were 0% in polypi of gallbladder group and cholecystoli-thiasis group. The different is significant between them. The endotoxin in bile is consistent with bacteria and it was 10.12 ±4.49 EU/ml in pigment calculus in bile duct group and much higher than 0. 003 ±0. 004 EU/ml in polypi of gallbladder group or 0.01 ±0.02 EU/ml in cholecystolithiasis group. It means that the calculus of bile duct is related with bacteria and endotoxin. 10 cases in 26patients with T tube show duodenal-biliary reflux (38.46% ). There were no radioactivities in the other 16 cases. And then the patients with T tube were divided into.reflux group and non-reflux group. Activity of bile amylase, lipase and P-glucuronidase were measured . There was no significant different in sex, age and the amount of drainage between them. The levels of bile amylase, lipase and exogenous p-glucuronidase in reflux group is much higher than that in non-reflux group ( amylase: 4792. 50 ± 5468. 05U/L vs 34. 94 ± 55. 67U/L, P < 0.01, lipase: 5165.57 ±3494.60U/L vs 367. 35 ±449.93U/L,P <0.01, exogenous p-glucuronidase: 1667.01 ±830.76 Fishman Unit vs 921. 98 ±451. 88 Fishman Unit, P<0.01).Conclusion1. There is close relationship between bile pigment calculus and duodenal-biliary reflux.2. Bacteria and endotoxin play an important role in the formation of pigment calculus during duodenal-biliary reflux.3. Duodenal-biliary reflux may promote pigment calculus formation through bacteria, endotoxin, amylase, lipase and exogenous p-glucuronidase.PART 3 Significance of Cholecyst GUSB mRNA expressionin cholecystolithiasisObjectivep-glucuronidase may hydrolyze the conjugated bilirubin in bile to insoluble calcium bilirubinate, which is the common source of cholecystolithiasis. GUSBgene is encoding gene of endogenous p-glucuronidase. There are no reports a-bout whether GUSB gene relate to the formation of cholecystolithiasis. This study approaches the formation mechanism of cholecyst bile pigment calculus and cholesterol calculus, from the activity of p-glucuronidase in bile and the expression of GUSB mRNA in mucosa of cholecyst.Method1. Patients*. 39 resident patients were included from April 2005 to July 2005. They were all suffered from cholecystolithiasis or cholecystpolypus, with 11 males and 28 females. All the patients were free from fever and bile duct infection with normal blood white cell counts. Another 9 gallbladders of subscribers were from hepatic transplantation. All specimens divided into cholecystpolypus group, bile pigment calculus group, cholesterol calculus group and hepatic transplanting donation group.2. Methods2. 1 Activity of endogenous p-glucuronidase of bileImproved Fishman method, with pH 4. 6 and shade-selected wavelength 550nm detect the activity of endogenous (3-glucuronidase of bile.2. 2 Expression of GUSB mRNA in cholecyst mucosaHuman GUSB mRNA sequence, gene bank serial numobr NM-000181, primers are designed by software, and half-quantitated RT-PCR detect the expression of GUSB mRNA in cholecyst mucosa.Statistical treatment: All data are expressed by mean ± square deviation, a-nalysis by SPSS 11,5, significant disparity if P <0.05.Results1. Activity of endogenous (3-glucuronidase of bileThe activity of endogenous (3-glucuronidase was in cholecystpolypus group (192.03 ±49.65 Fishman unit) , cholesterol calculus group (259. 66 ±61. 60 Fishman unit) and bile pigment calculus group (373. 19 ±74.43 Fishman u-nit). It is much higher in cholesterol calculus group and bile pigment calculus group than in cholecystpolypus group ( P < 0. 01) and much higher in pigment calculus group than in cholesterol calculus group (P<0.01).2. Expression of GUSB mRNA in cholecyst mucosaThe expression of GUSB mRNA in cholecyst mucosa are in hepatic transplanting donation group (0. 675 ± 0. 275 ) , cholecystpolypus group (101. 4 ± 0.070) , cholesterol calculus group(1.280 ±0. 099) and bile pigment calculus group (1. 668 ±0.053). It is much higher in cholecystpolypus groupNcholesterol calculus group and bile pigment calculus group than in hepatic transplanting donation group ( P <0.01). It is much higher in cholesterol calculus group and bile pigment calculus group than in cholecystpolypus group (P<0.01). It is much higher in pigment calculus group than in cholesterol calculus group ( P < 0.01).3. There is correlation between the expression of GUSB mRNA in cholecyst mucosa and endogenous p-glucuronidase of bile, r= 0.902 (P < 0.01).Conclusion1. Endogenous (3-glucuronidase plays an important part in bile pigment calculus in cholecyst.2. Endogenous |3-glucuronidase plays a part in cholesterol calculus in cholecyst.3. Endogenous p-glucuronidase in bile is designed by GUSB mRNA in cholecyst mucosa.4. High expression GUSB mRNA relate to formation of bile pigment calculus in cholecyst.5. High expression GUSB mRNA relate to formation of cholesterol calculus in cholecyst.6. High expression GUSB mRNA may relate to formation of cholesterol cholecystpolypus.