AIM: To explore the role of bile liquid crystal in the process of gallbladder stone formation and to provide bases for preventing and treating cholelithiasis.

METHODS: 46 guinea pigs, half males and half females, were randomly divided into control group and stone-causing group. Normal feed and stoneleading feed were used respectively to raise guinea pigs in the control group and stone-causing group. The guinea pigs were killed in three batches during the raising period. Under polarizing microscope, the pattern changes of bile liquid crystal in the gallbladder biles of the guinea pigs in the control group and stone-causing group were dynamicly observed respectively in single-blind trial.

RESULTS: It was found that there were few crystals in the guinea pigs’ biles of the control group, and their Malta cross was small and scattered, and existed in single form. With the increase of the feeding days, bile liquid crystals grew and Malta cross became bigger with their distribution densified, denser somewhere, but always existed in single form. While those of the stone-causing group had more bile liquid crystals, Malta cross was big and merged in strings. With the increase of the feeding days, bile liquid crystals grew in amount and strings of Malta cross increased and became bigger. The crosses in strings were arranged more and more regularly and they gradually changed into stone crystals.

CONCLUSION: Formation of gallbladder stone is a process of nucleation from different substances, and the causing-stone gallbladder bile is a constantly supersaturated solution, and bile liquid crystal is a nucleation factor in the formation of gallbladder stones. The process of nucleation includes gathering, merging and phase-changing of bile liquid crystals. The process of gathering, merging of bile liquid crystal is the key to nucleation.

Bile liquid crystal is a type of liquid crystal in the living beings’ bile. Since American scholar Olszewski et al[1] found the substance of liquid crystal in the bile liquid of human body in 1973, some scholars at home and abroad have explored the matter of liquid crystal in the living beings’ bile[2-4], but the relation between bile liquid crystal and formation of gallbladder stone has not been clarified so far. In order to explore the role of bile crystal in the process of gallbladder-stone formation and to provide certain bases for the prevention and treatment of gallbladder stone, we made dynamic observations and studied on the nucleation of bile liquid crystal in gallbladder stone through animal tests in guinea pigs.

The cause of gallbladder stone is very complicated, various factors are involved[5-9]. The basic factor is now considered to be the change of compositions and physical-chemistry characteristics of biles, which results in cholesterol in the state of super-saturation in the bile. And it is very likely to form crystals, which is then deposited to form gallbladder stones. It also causes imbalance between nucleation-leading factors and antinucleation factors and abnormal function of the gallbladder and so on[10-19]. From the viewpoint of crystallogeny, in a system of solution, the precondition for solute to be separated out and to form crystal is that the solute in the system must be in the state of super-saturation, which is an unstable state in thermodynamics. Only in this state, can the solute be crystallized and separated out. The formation of crystals has two steps of nucleation and crystallization, it firstly forms a crystalline nucleus containing hundreds of molecules, then crystals grow. In a system of solution, spontaneous nucleation of solute often requires a very high degree of saturation, the supersaturated solution in this state is called unstably supersaturated solution. When a certain type of special nucleating factors is added, supersaturated solution of a relatively low degree also can produce nucleation, which is called heterogeneous nucleation, and the supersaturated solution in this state is called substably supersaturated solution[20,21]. Homogeneous nucleation of bile cholesterol requires a very high degree of saturation (estimated at about 300%), which is not possible to occur in human body, and even less possible in guinea pigs’ body. So the formation of gallbladder-stone should be a process of heterogeneous nucleation, stone-causing gallbladder bile should be a substably supersaturated solution, and bile liquid crystal should be a type of nucleating factors for gallbladder stones.

As we observed under the polarizing microscope, there were many substances of liquid crystal in the gallbladder bile of the guinea pigs in the stone-causing group, which were big and string of Malta crosses. With the increase of the feeding days, bile liquid crystals grew in number, more and more strings of Malta cross appeared and steadily became bigger and bigger, and their arrangement became more and more regular, and gradually phase-changed into stone crystals. These have proved that the formation of gallbladder stone is indeed a process of heterogeneous nucleation, and stone-causing gallbladder bile is a type of substably supersaturated solution, and that bile liquid crystal is a type of nucleating factors in gallbladder-stone. Its process of nucleation is such that bile liquid crystals get together, merge and phase change. In another word, single Malta crosses assemble into strings, then merge as slices, and finally phase-change into crystals. The assemblage and merger of bile liquid crystals are the key for nucleation. Although there were also substances of liquid crystal existing in gallbladder biles in the control group and bile crystals also increased with feeding time, they neither assembled nor merged, and Malta crosses always existed in single forms which were unable to nucleate to form stone. If some way can be found to prevent or retard the assemblage and merger of bile liquid crystals, i. e., formation of the nucleus, it will be of a great significance in theory and practice for prevention and treatment of cholelithiasis.

Cholesterol can not dissolve in water, but it can dissolve in bile. The concentration of cholesterol in bile is about 10-20 mmol/L, this concentration is about 10⁶ times[22] denser than that of the solubility of cholesterol in water. Studies concerned pointed out that cholesterol in bile maintained its state of dissolution in forms of micelle and vesicle. Micelle is the polymer of cholesterol, phosphatide and cholate, while vesicle is the compound of cholesterol and phosphatide[23-31]. These two form a system of thermodynamic balance in bile so that they are interlinked and interconverted, and play the role of modulation for dissolution and separation of cholesterol. Under the effect of nucleation-leading factors and antinucleation factors in bile, vesicles in bile assemble, merge and separate out crystalline nuclei of cholesterol which then gradually grow up to form stones[32]. As we observed under the polarizing microscope, single Malta cross in bile liquid crystal of guinea pigs in stone-causing group, gradually assembled into strings, then merged into slices, and finally phase-changed into stone crystals. The present study supports the above viewpoints from the studies of morphology. Whether bile crystal is such a kind of vesicles or vesicles aggregate and what factors cause or stop bile liquid crystal assembling and merging, still need to be further studied.