Study On Cholesterol Gallstones In Vitro

Human bile mainly contains bile salts, lecithin and cholesterol. Cholesterol wasdissolved, nucleated and crystallized in the bile which were affected by the change in thecontent of these substances. Previous studies of cholesterol gallstones were concentrated invitro simulation study, the model bile prepared was similar to human bile, and initially got themechanism of cholesterol gallstones.The manuscript mainly studied from these aspects, i.e. cholesterol dissolved in modelbile, mechanisms of cholesterol nucleated in model bile and the factors may affect thecrystallization.Firstly, prepare quaternary system (bile salt-cholesterol-lecithin-water) model bile wasnecessary, and then studied cholesterol solubility (the concentration of cholesterol that modelbile can dissolved) in biles, so the conclusion we got is lecithin and bile salt concentrations aswell as total lipid concentration (total sum of bile salt, cholesterol and lecithin) would havesome impact on cholesterol solubility. Among that, the contents of every component wasmeasured by high performance liquid chromatography (HPLC). From the experiment welearned that when the bile salt concentration is higher, the greater cholesterol solubility is; asto bile salt/lecithin molar ratio, the cholesterol solubility would be increased with increasingmolar ratio, when the ratio is between0.5-1.77, the solubility reached maximum, thencontinued to increase the ratio, whereas cholesterol solubility would be decreased. In addition,with increasing the total lipid concentration, the ability that model bile dissolved cholesterolenhanced gradually. At the same time, cholesterol saturation solubility curves in the ternarydiagram also drafted in the experiment.And then, we studied changes of cholesterol in model bile by microscope and based onthe light scattering theory, and investigated the particle size distribution of the dispersed phase(including micelles and vesicles). We found the contents changed of every component inmodel bile meant different phase would present in bile. And the measured vesicle averageparticle diameter was mainly about455.5nm, while the measured micelle was about5.7nm.Moreover, we observed that cholesterol in bile would experienced various stages until itnucleated and crystallized. Vesicles existed in bile and started to aggregate, then cholesterolembedded into vesicles to nucleate, eventually formed liquid crystal or crystal.Lastly, as Human bile composition was more complex, not only contains the above threemain substances, but includeed calcium, protein, bilirubin, bacteria, other major elements andtrace elements. So we chose calcium salt and protein as factors to study.For calcium, calcium chloride was added into model bile to composed five-componentsystem (bile salt-lecithin-cholesterol-water-inorganic salt) in the experiment and chosequaternary system model bile to be reference bile. Then we concluded that when Ca2+was added, nucleation time got shorter, and cholesterol solubility decreased. And the higher theconcentration of Ca2+, the lower cholesterol solubility, the shorter nucleated time, withdifferent total lipids concentration, furthermore, cholesterol nucleated faster as the total lipidsconcentration increased. In addition, the solubility curve was also drew which thesingle-phase region became smaller. Therefore we concluded that Ca2+plays the role ofpromoting cholesterol gallstones from cholesterol nucleation time and solubility study. Andthe vesicle average particle diameter got to be larger of model bile contained Ca2+by particlesize distribution analysis.As to the effect of protein, the experiment also chose bovine serum albumin (BSA) asbile protein, BSA replaced calcium salt was added in model bile composed anotherfive-component system, and then we learned BSA combined with lecithin throughelectrostatic and hydrophobic interaction, thus the stability of lecithin vesicle was affected,and cholesterol solubility also decreased with increasing BSA concentration. After addingBSA, cholesterol nucleation time has no significant change in model bile, so we initiallyconcluded that BSA only has effect on cholesterol solubility, impact on cholesterol nucleationjust a little.