| BackgroundIntrahepatic bile duct stone is stones occured in the bile duct branches above the confluence of the left and right hepatic duct.This disease is rare in western, mainly concentrates in Asian countries, such as China, South Korea, Japan, etal. A very large proportion of these patients is chinese.To intrahepatic bile duct stone,its incidence is high,usually its condition is seriously.lt is difficulty to treat and easy to recurrence. Environmental, diet, infection, bile deposition, parasitic, genetic and other factors are closely related to the occurrence of intrahepatic bile duct stones. These factors lead to bile cholesterol saturated and even over saturated state, or non conjugated bilirubin in bile is relatively increased.The clinical manifestations of it are recurrent abdominal pain, fever and jaundice. However, cholesterol and bile pigment calcium salt deposition can not form stones directly, bile glycoprotein and metal ions play a very important role.Intrahepatic bile duct stones can cause serious complications, such as recurrent cholangitis, portal hypertension, liver failure, even liver cancer, influencea the health and life of patients seriously. However, intrahepatic bile duct stones is not a malignant tumor, is a benign disease, should be able to prevent and cure.Part I. The Determination of Bile Glycoprotein Content and Its Significance To Intrahepatic Bile Duct Stones PatientsObjectiveIn this study we determined bile glycoprotein content.By comparison of intrahepatic bile duct stone patients with extrahepatic bile duct stone patients with bile glycoprotein content we obtained more convincing research results. We hoped to find the factors associated with the multiple and recurrent of intrahepatic bile duct stones.With these important basis we can guide the patients to pay attention to the related matters in the life and work to prevent it from occurring, and can instruct the doctor how to choose the appropriate treatment method to obtain the better curative results.Methods1. General information:We choosed 38 cases of patients with gallstone admitted to hospital in December 2012-February 2014.17 cases with intrahepatic bile duct stones group,21 cases with extrahepatic bile duct stone group,18 cases of non gallstone patients was used as a control group,56 cases of patients in all.2. Specimen collection:For 17 patients with intrahepatic bile duct stones and 21 cases with extrahepatic bile duct stones,20 ml bile samples were collected at 3 days after operation (or EST) via T-tube drainage tube or endoscopic nasobiliary duct, in sterile tubes, preservated in-20℃ refrigerator.3. Method:After bile specimens were thawed, was centrifuged,dissoluted protein precipitation with distilled water.Then extracted protein to dechloridate quickly.The dechloridated bile protein was to prepare for SDS-PAGE electrophoresis. The molecular weight of the protein bands under 200KD were transferred to nitrocellulose membrane.We used glycoprotein determination kit for qualitative test of glycoprotein by periodic acid Schiff reagent staining (PAS) method. Quantitative determination of protein was made by Lowry method,and serum albumin as the standard, the molecular weight of the glycoprotein content under 200KD was calculated.4. Statistical processing:The bile glycoprotein content of each group were calculated respectively, and the data were expressed by X±S. The data were processed by SPSS 17.0 software.By means of independent sample test,the P values of the corresponding variances are taken by the variance if equal. P< 0.05 for the difference was statistically significant.Results1. The content of bile glycoprotein in patients with intrahepatic bile duct stones was higher than that in patients with extrahepatic bile duct stones. The content of bile glycoprotein in patients with extrahepatic bile duct stones was higher than that of the control group.2. In the mean comparison of the content of bile glycoprotein between patients with intrahepatic bile duct stones and patients with extrahepatic bile duct stones, the difference between the two groups of data was significant. In the mean comparison of the content of bile glycoprotein between patients with intrahepatic bile duct stones and the control group,the difference between the two groups of data was significant. In the mean comparison of the content of bile glycoprotein between patients with extrahepatic bile duct stones and the control group,the difference between the two groups of data was significant.Dicussion1. The importance of study the pathogenesis of intrahepatic bile duct stones: Intrahepatic bile duct stone can cause serious complications which influencea the health and life of patients seriously. However, intrahepatic bile duct stones is not a malignant tumor, is a benign disease, should be able to prevent and cure.So it is important to provide an important basis for the patients or doctors to treat this disease.2. The mechanism of glycoprotein in the formation of intrahepatic bile duct stones:Other studies found that the cohesion function of glycoprotein is the main factor of gallstone formation. Many experimental results showed that a range of molecular weight glycoprotein play a promoting role in the process of gallstone formation. Glycoprotein or glycoprotein bile pigment compound can be used as the core, make cholesterol or bile pigment calcium salt to deposite and formate stones. The connection of each other between glycoprotein molecules formate a kind of jelly on bile duct mucosa surface, which provides a substrate for the stone formation. In the colloid network structure formated by glycoprotein, such as the movement of micro bubble is restricted. Mmicro bubble contains cholesterol molecules, dehydrate, and form cholesterol crystals. The viscosity of glycoprotein colloid is very high, bile pigment molecules and calcium ions are adhesion in the gel, combined to form bile pigment calcium compounds. In glycoprotein gels, cholesterol crystal or bile pigment calcium salt continuously depose and adhesion around the glycoprotein compounds or glycoprotein bile pigment complex, and form stones.3. Studies on glycoprotein:Many scholars who had studied about glycoprotein agreed that glycoprotein plays an important role in the process of gallstone formation.4. Characteristics of this study:The subjects in our study were patients with intrahepatic bile duct stones.The specimens were bile. These were different from the former related research.We hoped to find the factors associated with the multiple and recurrent of intrahepatic bile duct stones in a deeper level. After being separated by SDS-PAGE electrophoresis and stained by PAS, the bile glycoprotein content is determined through a series of processing calculation. This method is a more advanced method of determination of glycoprotein, economic, reliable and easy. Glycoprotein that can promote the formation of bile duct stones are under the molecular weight of 200KD, so this study calculated the content of the bile glycoprotein under 200 kdConclusionThe results of this study showed that intrahepatic bile duct stone is a spe special type of bile duct stone. In which the formation mechanism of the stone, has not only the basic role of bile duct stone formation mechanism, but also has its own characteristics. The determination of bile glycoprotein content for patients of intrahepatic bile duct stones is important.Part II. The Determination of Bile Calcium ion Content and Its Significance To Intrahepatic Bile Duct Stones PatientsObjectiveIn this study we determined bile calcium ion content. By comparison bile calcium ion content of intrahepatic bile duct stone patients with extrahepatic bile duct stone patients, we obtained more convincing research results. We hoped to find the factors associated with the multiple and recurrent of intrahepatic bile duct stones. With these important basis we can guide the patients to pay attention to the related matters in the life and work to prevent it from occurring, and can instruct the doctor how to choose the appropriate treatment method to obtain the better curative results.Methods1. general information and specimen collection are the same as Part Ⅰ2. Bile specimen after thawed was added to the buffer solution of acid chrome blue K as chromogenic agent. Bile calcium and acid chrome blue K formed a complex, the maximum absorption wavelength of the complex is 496 nm. Using spectrophotometry at the wavelength of 496 nm, reduction of acid chrome blue K solution absorbance was measured,the content of calcium ion was at 0 60mol/L.This method is simple and rapid,bile magnesium, zinc and biliverdin do not interfere with the determination.3. Statistical processing:The bile calcium ion content of each group were calculated respectively,and the data were expressed by X±S. The data were processed by SPSS17.0 software.By means of independent sample test,the P values of the corresponding variances are taken by the variance if equal.P< 0.05 for the difference was statistically significant.Results1. The content of bile calcium ion in patients with intrahepatic bile duct stones was higher than that in patients with extrahepatic bile duct stones. The content of bile calcium ion in patients with extrahepatic bile duct stones was higher than that of the control group.2. In the mean comparison of the content of bile calcium ion between patients with intrahepatic bile duct stones and patients with extrahepatic bile duct stones, the difference between the two groups of data was significant. In the mean comparison of the content of bile calcium ion between patients with intrahepatic bile duct stones and the control group,the difference between the two groups of data was significant. In the mean comparison of the content of bile calcium ion between patients with extrahepatic bile duct stones and the control group,the difference between the two groups of data was significant.Discussion1. Characteristics of intrahepatic bile duct stones:The morbidity of intrahepatic bile duct stones is high in our country. Environmental, diet, infection,etal are closely related to the occurrence of intrahepatic bile duct stones. How does gallstone formate, following aspects are involved:(1)Free bilirubin concentration increased in bile; (2) Formation of the core of the stones;(3) the cohesion function of glycoprotein and the participation of metal ions. The composition of intrahepatic bile duct stones is bilirubin calcium complex.,usually accompanied by cholestasis and infection of biliary tract. It attack Intermittently and recurrently is clinical feature of it. It is difficulty to treat and easy to recurrence.2 The particularity of the mechanism:The composition of intrahepatic bile duct stones is bilirubin calcium complex. Free bilirubin concentration increased in bile can combine with the calcium in bile, formate insoluble calcium bilirubin and precipitate,then formate stones in hepatic.3 The mechanism of calcium ion in the formation of intrahepatic bile duct stones:That cholesterol crystallization, bile pigment and others combine metal ions is an important step in the formation of gallstones, the most important one is calcium ion. Bile calcium ion is easily combined with bile pigment, fatty acid, mucin and form refractory compound calcium salt. The microstructure of gallstone is a network structure formed by high molecular polymer, and calcium ion play a role in the formation of network structure as a ion bridge.4 Characteristics of this study:Direct specimens are bile, which is different from the former related research.We hoped to find the factors associated with the multiple and recurrent of intrahepatic bile duct stones in a deeper level.Calcium ion was determined by the method of acid chrome blue K spectrophotometry. The method can determine trace calcium in bile, the error is small, and the method is simple and accurate.ConclusionThe results of this study showed that intrahepatic bile duct stone is a spe special type of bile duct stone. In which the formation mechanism of the stone has not only the basic role of bile duct stone formation mechanism, but also has its own characteristics. The determination of bile glycoprotein content for patients of intrahepatic bile duct stones is important.In Summary1. Introduction of intrahepatic bile duct stones:Intrahepatic bile duct stone is rare in western, but the morbidity of intrahepatic bile duct stones is high in our country. Intrahepatic bile duct stone can cause obstruction and infection of bile duct, serious complications, such as recurrent cholangitis, portal hypertension, infective shock,liver failure and even liver cancer. The treatment of intrahepatic bile duct stones include surgical resection of calculi lesions and stenosis of intrahepatic bile ducts, endoscopic treatment (choledochoscope stone or duodenumscope), or iver transplantation,etal. To intrahepatic bile duct stone,its incidence is high, usually its condition is seriously, can cause serious complications,and influencea the health and life of patients seriously. However, intrahepatic bile duct stones is not a malignant tumor, is a benign disease, should be able to prevent and cure.2. Findiings of this study:Intrahepatic bile duct stone is a spe special type of bile duct stone. In which the formation mechanism of the stone has not only the basic role of bile duct stone formation mechanism, but also has its own characteristics.3. The significance of prevention:In clinic, we can determine the content of bile glycoprotein or bile calcium ion for patients with high risk factors of intrahepatic bile duct stones. If the result of the determination is high, these patients should pay special attention to the habits, diet, environment and other matters in life and work, or take some preventive measures such as the application of some drugs to protect liver and bile duct, etc, as far as possible to reduce or avoid the occurrence of the disease.4. The significance of the treatment:For those intrahepatic bile duct stones patients with high bile glycoprotein or calcium content, more positive and effect treatment methods should be adopted; shoud reserve the way for the treatment of the following treatment; the comprehensive treatment measures shoud be used; follow-up is necessary.5. Comparison with related studies:In these related studies, the mechanism and the practical significance of the formation of the stone are in agreement with our research.The results are consistent with the general rule of the formation mechanism of stone.Inconsistent individual results, which may be related to the regional differences, ethnic differences, measurement method and statistics method. It is the results are not consistent,but not the essence or rule, and it does not affect our founding that the formation of the specific location of the stone has a special mechanism.6. Deficiency:All the processes of this study are in strict accordance with the requirements of the subject and the design of experiments.But after all, the sample size is limited, the experimental results are still need multi-center> large sample and randomly contrast experiments to confirm furtherly. |
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