胆道疾病中胰胆反流和肠胆反流现象的观察及相关性的研究
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摘要
前言
1966年Elmslie首次提出胰胆反流的学说,发现它可引起胆管上皮化生、胆总管囊肿,可发展为胆管或胆囊癌。胰胆反流还可以破坏胆汁胶态稳定性,推测与胆石形成有关。胰胆反流常见于胰胆管合流异常(Pancreaticobiliary maljunction;PBM),即胰胆管合流部较长(＞15mm),超过Oddi括约肌(Sphincter of Oddi;SO)收缩范围,胰液经胰胆管共同通道反流。Inagaki M和Horaguchi J等提出,在非PBM患者也存在隐匿性胰胆反流,即没有胰胆管合流形态学异常但胰液可反流入胆管而引起类似PBM的病理改变。
胰胆反流如有肠激酶参与则迅速激活胰蛋白酶原,引起酶级联反应而加速胆管的刺激损害等一系列病理过程。此过程与十二指肠液反流入胆管即肠胆反流相似,因此需要区别胰胆和肠胆反流。
诊断两种反流的方法均有改进,如判断肠胆反流通过口服核素后检测胆汁中放射性活度的方法,判断胰胆反流通过磁共振动态观察胰泌素诱导后胆管直径的变化等方法,其敏感度和特异度明显提高。但目前尚存在应用范围局限和难以反映生理性反流等缺点而不能得以广泛应用,因此仍沿用测胆汁中淀粉酶(amylase;AMY)水平的方法,但其特异性较差。
本研究通过口服核素方法和测胆汁中AMY、脂肪酶(lypase;LPS)水平的方法分别观察各胆道疾病中肠胆和胰胆反流的存在,同时采用蛋白印迹(westernblot)方法检测肠激酶(enterokinase;EK)和胰蛋白酶原-1(Trypsin-1)的存在来观察反流现象,并相互对比。用较好的诊断方法研究胆道疾病中两种反流的存在及与疾病的相关性。
方法
一、实验对象
收集2007年1月至2008年8月,中国医科大学附属盛京医院收治的共151例胆道疾病患者,包括胆囊结石,胆囊息肉,胆管色素结石,胆管残石,胆总管囊肿和肝门部胆管癌行经皮经肝胆管穿刺(percutaneous transhepatic cholangialdrainage;PTCD)和梗阻性黄疸行内镜下鼻胆管引流(endoscopic nasobilliarydrainage;ENBD)患者。
上述诊断是依据临床症状、放射学,内窥镜检查或手术所见和病理组织学检查的主要诊断。胆管残石和胆管色素结石组中的结石均呈泥沙样、棕色色素结石。ENBD管引流的病例行十二指肠镜乳头切开术(endoscopic sphincterotomy;EST),其余患者SO完整。
二、标本采集及保存
(一)标本采集
72例胆囊胆汁和31例胆管胆汁在术中获取,其余的胆管胆汁分别通过T管、经皮经肝胆管引流(percutaneous transhepatic cholangial drainge;PTCD)管、ENBD管引流获取。所有胆汁样本在空腹超过12h时获取。
(二)保存
获得的胆汁立即保存在-80℃,直至分析。
三、检测指标
(一)生物化学方法测量胆汁中AMY活力
所有胆汁样本用日立7170A生化分析仪,通过Gal-G2-CNP基质法测定AMY水平。血液内AMY正常值范围为20～115U/L,超过正常值上限判定为胰胆反流。
(二)生物化学方法测量胆汁中LPS的活力
在106例胆管胆汁随机抽取93例、在72例胆囊胆汁随机抽取60例,用日立7170A生化分析仪,通过DGGMR基质法测定LPS水平。血液内LPS的正常值范围为23～300U/L,超过正常值上限判定为胰胆反流。
(三)western blot检测胆汁中Trypsin-1
上样前处理:去除胆汁内碎石及各种残渣、脱盐洗涤,此后免疫沉淀。
Western blot:检测用ECL化学发光法,25KD水平出现特异性条带者判定为Trypsin-1阳性。
(四)口服放射性核素后检测胆汁中放射性活度
对42例胆囊切除、胆道探查取石、T管引流术后的患者,入院后放开T管超过24h,保证T管引流通畅。检查前禁食一夜,口服1 mL含有185 MBq(5mCi)的99mTc-DTPA水,接着240mL水漱服,患者立即平卧位。经T管收集2h胆汁,取其中20mL,立即检测放射性活度。如果胆汁中可以检测到放射性活度,则判定该患者存在十二指肠胆道反流。
(五)western blot检测胆汁中EK
上样前处理同(三)。
Western blot:检测用ECL化学发光法,300KD水平处出现特异性条带者判定为EK阳性。
四、统计分析
所得数据使用SPSS13.0统计软件进行统计,对各组数据应用Pearson相关分析、单因素方差分析、Kruskal-Wallis检验、Fisher精确检验以及卡方检验,设定P＜0.05为有统计学意义。
结果
一、判定胰胆反流,检测Trypsin-1与AMY和LPS方法之间的一致性检验
(一)胆汁中AMY和LPS
检测AMY水平,在106例胆管胆汁63例判定为胰胆反流阳性(59.43%);在72例胆囊胆汁36例判定为胰胆反流阳性(50.00%)。
检测LPS水平,在93例胆管胆汁53例判定为胰胆反流阳性(56.99%);在60例胆囊胆汁16例判定为胰胆反流阳性(26.67%)。
AMY和LPS水平对数转换后进行相关性分析,在胆管r=0.812(P＜0.001),在胆囊r=0.775(P＜0.001)。
(二)检测胆汁中Trypsin-1
55例胆管胆汁经western blot检测到Trypsin-1的特异性条带而判定为胰胆反流阳性(51.89%);26例胆囊胆汁经western blot检测到Trypsin-1的特异性条带而判定为胰胆反流阳性(36.11%)。
(三)判定胰胆反流,检测Trypsin-1与AMY/LPS方法之间的一致性检验
在胆囊胆汁,Trypsin-1与AMY方法,Kappa值为0.611(P＜0.001);检测Trypsin-1与LPS方法,Kappa值为0.624(P＜0.001);在胆管胆汁,Trypsin-1与AMY方法,Kappa值为0.696(P＜0.001);检测Trypsin-1与LPS方法,Kappa值为0.806(P＜0.001)。
诊断胰胆反流的AMY、LPS和Trypsin-1方法检测之间存在一致性(P＜0.001),在胆管胆汁LPS检测结果接近Trypsin-1。
二、判定肠胆反流,检测EK与口服核素方法之间的一致性检验
(一)口服核素检测
42例行胆道探查T管引流术后的患者,有9例检测到放射性活度而判定为十二指肠胆道反流阳性(21.43%)。此9例患者2h引流的20mL胆汁中锝放射性活度为175.9±129.2KBq,2h胆汁引流量41.7±12.2mL;其余33例2h胆汁引流量46.4±19.2mL,两组间无显著性差异(P＞0.05)。
(二)检测胆汁中EK
42例行胆道探查T管引流术后的患者,有17例用western blot检测到EK的特异性条带而判定为肠胆反流阳性(40.48%)。
(三)判定肠胆反流,检测EK与口服核素方法之间的一致性检验
EK检测与核素检测方法,Kappa值为0.466(P＜0.001),核素检测(9/42)较EK检测方法(17/42)敏感度差,但无统计学意义(P＞0.05)。
三、检测EK和Trypsin-1方法评价胆道疾病中胰胆反流和肠胆反流率
在胆管胆汁,EK阳性率在残石组、胆总管色素结石组和ENBD组较PTCD组和胆总管囊肿组显著高(P＜0.05);Trypsin-1阳性率在PTCD组较其余四组显著低(P＜0.05)。
在胆囊胆汁中,EK阳性率在各组之间无明显差异(P＞0.05);Trypsin-1阳性率在先天胆总管囊肿组较其他组显著高(P＜0.05)。
结论
1、Western blot测定胆汁EK和Trypsin-1方法同口服核素方法和AMY测定方法,也可以有效地反映肠胆反流和胰胆反流的存在。
2、胆汁内AMY/LPS主要来源于胰腺。
3、在胆管色素结石和T管引流组,胆汁中胰酶主要来源于肠胆反流,肠胆反流率与胆汁获取途径无关,考虑肠胆反流与色素结石形成有关。
4、在胆总管囊肿组,胆汁中胰酶主要来自胰胆反流。
5、在非先天胆总管囊肿、胆道疾病患者中隐匿性胰胆反流较常见。
Preface
In 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 Methods
Patient
151 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 Collection
72 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 Preservation
Prior to sample analysis, all the specimens were preserved in -80℃conditionfollowing withdrawal.
三、Targets detection
(一)The measuring of bile AMY using biochemical method
The 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 biochemicalmethod
The 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-1
Bile 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 ~(99m)Tc-DTPA
42 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 EK
Bile 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 Analysis
Statistical 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.
Results
Consistency 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 Detection
In 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 pancreaticobiliaryreflux
In 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 ~(99m)Tc-DTPA Test
In 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 Detection
In 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 reflux
Kappa value for EK and Oral ~(99m)Tc-DTPA tests was 0.466(P＜0.001). Oral~(99m)Tc-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 Diseases
In 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).
Conclusions
1、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 ~(99m)Tc-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.
1966年Elmslie首次提出胰胆反流的学说,发现它可引起胆管上皮化生、胆总管囊肿,可发展为胆管或胆囊癌。胰胆反流还可以破坏胆汁胶态稳定性,推测与胆石形成有关。胰胆反流常见于胰胆管合流异常(Pancreaticobiliary maljunction;PBM),即胰胆管合流部较长(＞15mm),超过Oddi括约肌(Sphincter of Oddi;SO)收缩范围,胰液经胰胆管共同通道反流。Inagaki M和Horaguchi J等提出,在非PBM患者也存在隐匿性胰胆反流,即没有胰胆管合流形态学异常但胰液可反流入胆管而引起类似PBM的病理改变。
胰胆反流如有肠激酶参与则迅速激活胰蛋白酶原,引起酶级联反应而加速胆管的刺激损害等一系列病理过程。此过程与十二指肠液反流入胆管即肠胆反流相似,因此需要区别胰胆和肠胆反流。
诊断两种反流的方法均有改进,如判断肠胆反流通过口服核素后检测胆汁中放射性活度的方法,判断胰胆反流通过磁共振动态观察胰泌素诱导后胆管直径的变化等方法,其敏感度和特异度明显提高。但目前尚存在应用范围局限和难以反映生理性反流等缺点而不能得以广泛应用,因此仍沿用测胆汁中淀粉酶(amylase;AMY)水平的方法,但其特异性较差。
本研究通过口服核素方法和测胆汁中AMY、脂肪酶(lypase;LPS)水平的方法分别观察各胆道疾病中肠胆和胰胆反流的存在,同时采用蛋白印迹(westernblot)方法检测肠激酶(enterokinase;EK)和胰蛋白酶原-1(Trypsin-1)的存在来观察反流现象,并相互对比。用较好的诊断方法研究胆道疾病中两种反流的存在及与疾病的相关性。
方法
一、实验对象
收集2007年1月至2008年8月,中国医科大学附属盛京医院收治的共151例胆道疾病患者,包括胆囊结石,胆囊息肉,胆管色素结石,胆管残石,胆总管囊肿和肝门部胆管癌行经皮经肝胆管穿刺(percutaneous transhepatic cholangialdrainage;PTCD)和梗阻性黄疸行内镜下鼻胆管引流(endoscopic nasobilliarydrainage;ENBD)患者。
上述诊断是依据临床症状、放射学,内窥镜检查或手术所见和病理组织学检查的主要诊断。胆管残石和胆管色素结石组中的结石均呈泥沙样、棕色色素结石。ENBD管引流的病例行十二指肠镜乳头切开术(endoscopic sphincterotomy;EST),其余患者SO完整。
二、标本采集及保存
(一)标本采集
72例胆囊胆汁和31例胆管胆汁在术中获取,其余的胆管胆汁分别通过T管、经皮经肝胆管引流(percutaneous transhepatic cholangial drainge;PTCD)管、ENBD管引流获取。所有胆汁样本在空腹超过12h时获取。
(二)保存
获得的胆汁立即保存在-80℃,直至分析。
三、检测指标
(一)生物化学方法测量胆汁中AMY活力
所有胆汁样本用日立7170A生化分析仪,通过Gal-G2-CNP基质法测定AMY水平。血液内AMY正常值范围为20～115U/L,超过正常值上限判定为胰胆反流。
(二)生物化学方法测量胆汁中LPS的活力
在106例胆管胆汁随机抽取93例、在72例胆囊胆汁随机抽取60例,用日立7170A生化分析仪,通过DGGMR基质法测定LPS水平。血液内LPS的正常值范围为23～300U/L,超过正常值上限判定为胰胆反流。
(三)western blot检测胆汁中Trypsin-1
上样前处理:去除胆汁内碎石及各种残渣、脱盐洗涤,此后免疫沉淀。
Western blot:检测用ECL化学发光法,25KD水平出现特异性条带者判定为Trypsin-1阳性。
(四)口服放射性核素后检测胆汁中放射性活度
对42例胆囊切除、胆道探查取石、T管引流术后的患者,入院后放开T管超过24h,保证T管引流通畅。检查前禁食一夜,口服1 mL含有185 MBq(5mCi)的99mTc-DTPA水,接着240mL水漱服,患者立即平卧位。经T管收集2h胆汁,取其中20mL,立即检测放射性活度。如果胆汁中可以检测到放射性活度,则判定该患者存在十二指肠胆道反流。
(五)western blot检测胆汁中EK
上样前处理同(三)。
Western blot:检测用ECL化学发光法,300KD水平处出现特异性条带者判定为EK阳性。
四、统计分析
所得数据使用SPSS13.0统计软件进行统计,对各组数据应用Pearson相关分析、单因素方差分析、Kruskal-Wallis检验、Fisher精确检验以及卡方检验,设定P＜0.05为有统计学意义。
结果
一、判定胰胆反流,检测Trypsin-1与AMY和LPS方法之间的一致性检验
(一)胆汁中AMY和LPS
检测AMY水平,在106例胆管胆汁63例判定为胰胆反流阳性(59.43%);在72例胆囊胆汁36例判定为胰胆反流阳性(50.00%)。
检测LPS水平,在93例胆管胆汁53例判定为胰胆反流阳性(56.99%);在60例胆囊胆汁16例判定为胰胆反流阳性(26.67%)。
AMY和LPS水平对数转换后进行相关性分析,在胆管r=0.812(P＜0.001),在胆囊r=0.775(P＜0.001)。
(二)检测胆汁中Trypsin-1
55例胆管胆汁经western blot检测到Trypsin-1的特异性条带而判定为胰胆反流阳性(51.89%);26例胆囊胆汁经western blot检测到Trypsin-1的特异性条带而判定为胰胆反流阳性(36.11%)。
(三)判定胰胆反流,检测Trypsin-1与AMY/LPS方法之间的一致性检验
在胆囊胆汁,Trypsin-1与AMY方法,Kappa值为0.611(P＜0.001);检测Trypsin-1与LPS方法,Kappa值为0.624(P＜0.001);在胆管胆汁,Trypsin-1与AMY方法,Kappa值为0.696(P＜0.001);检测Trypsin-1与LPS方法,Kappa值为0.806(P＜0.001)。
诊断胰胆反流的AMY、LPS和Trypsin-1方法检测之间存在一致性(P＜0.001),在胆管胆汁LPS检测结果接近Trypsin-1。
二、判定肠胆反流,检测EK与口服核素方法之间的一致性检验
(一)口服核素检测
42例行胆道探查T管引流术后的患者,有9例检测到放射性活度而判定为十二指肠胆道反流阳性(21.43%)。此9例患者2h引流的20mL胆汁中锝放射性活度为175.9±129.2KBq,2h胆汁引流量41.7±12.2mL;其余33例2h胆汁引流量46.4±19.2mL,两组间无显著性差异(P＞0.05)。
(二)检测胆汁中EK
42例行胆道探查T管引流术后的患者,有17例用western blot检测到EK的特异性条带而判定为肠胆反流阳性(40.48%)。
(三)判定肠胆反流,检测EK与口服核素方法之间的一致性检验
EK检测与核素检测方法,Kappa值为0.466(P＜0.001),核素检测(9/42)较EK检测方法(17/42)敏感度差,但无统计学意义(P＞0.05)。
三、检测EK和Trypsin-1方法评价胆道疾病中胰胆反流和肠胆反流率
在胆管胆汁,EK阳性率在残石组、胆总管色素结石组和ENBD组较PTCD组和胆总管囊肿组显著高(P＜0.05);Trypsin-1阳性率在PTCD组较其余四组显著低(P＜0.05)。
在胆囊胆汁中,EK阳性率在各组之间无明显差异(P＞0.05);Trypsin-1阳性率在先天胆总管囊肿组较其他组显著高(P＜0.05)。
结论
1、Western blot测定胆汁EK和Trypsin-1方法同口服核素方法和AMY测定方法,也可以有效地反映肠胆反流和胰胆反流的存在。
2、胆汁内AMY/LPS主要来源于胰腺。
3、在胆管色素结石和T管引流组,胆汁中胰酶主要来源于肠胆反流,肠胆反流率与胆汁获取途径无关,考虑肠胆反流与色素结石形成有关。
4、在胆总管囊肿组,胆汁中胰酶主要来自胰胆反流。
5、在非先天胆总管囊肿、胆道疾病患者中隐匿性胰胆反流较常见。
Preface
In 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 Methods
Patient
151 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 Collection
72 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 Preservation
Prior to sample analysis, all the specimens were preserved in -80℃conditionfollowing withdrawal.
三、Targets detection
(一)The measuring of bile AMY using biochemical method
The 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 biochemicalmethod
The 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-1
Bile 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 ~(99m)Tc-DTPA
42 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 EK
Bile 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 Analysis
Statistical 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.
Results
Consistency 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 Detection
In 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 pancreaticobiliaryreflux
In 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 ~(99m)Tc-DTPA Test
In 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 Detection
In 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 reflux
Kappa value for EK and Oral ~(99m)Tc-DTPA tests was 0.466(P＜0.001). Oral~(99m)Tc-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 Diseases
In 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).
Conclusions
1、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 ~(99m)Tc-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.
引文
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