Direct visualization of lipid aggregates in native human bile by light- and cryo-transmission electron-microscopy

AbstractThe evolution of microstructures present in human gallbladder and hepatic bile was observed simultaneously by video-enhanced light microscopy (VELM) and transmission electron microscopy of vitrified specimens (cryo-TEM), as a function of time after withdrawal from patients. Fresh centrifuged gallbladder bile samples contained small (6 nm) spherical micelles in coexistence with vesicles (40 nm). Out of the seven bile samples investigated four contained, in addition, two types of elongated aggregates that have not been previously described. Uncentrifuged gallbladder bile also contained a mixture of ribbon- and plate-like crystals seen by VELM, but not by cryo-TEM. In aged (3–6-week-old) gallbladder bile samples VELM also revealed spiral and helical crystal structures. No such crystals were present in hepatic bile samples, although microcrystals, not observable by VELM were seen by cryo-TEM in addition to micelles and vesicles. The similarity of these observations to those observed in bile models lends strong support for the validity of the model systems. Furthermore, the presence of microcrystals in hepatic bile samples, apparently devoid of crystals by light microscopy, indicates that under certain conditions the common criterion of ‘nueleation time’ (NT), based on light microscopy, does not represent the real time of nucleation. In the human bile samples investigated in this study the dissociation between NT and the time of observation of microcrystals was seen in hepatic but not in gallbladder bile samples. Hence, crystal growth may be rate limiting only in dilute biles

Microstructural analysis of bile: relevance to cholesterol gallstone pathogenesis

The study of physical-chemical factors and pathways leading to cholesterol crystallization in bile has important clinical relevance. The major processes in cholesterol gallstone formation can be subdivided into nucleation, formation and precipitation of solid crystals (crystallization), crystal growth, crystal agglomeration and stone growth. A clear understanding of the microstructural events occurring during the earliest stages of these processes in bile is crucial for the identification of factors possibly delaying or preventing precipitation of cholesterol crystals and, therefore, gallstone formation in bile.Detection and characterization of microstructures in native and model biles can be achieved by both direct and indirect techniques. Direct imaging techniques provide more readily interpretable information, but sample preparation problems, particularly for electron microscopy, are a source of artifacts. Moreover, microscopic techniques provide only qualitative data without the possibility to quantitate or to analyse the composition of microstructures. Several indirect techniques have been used to obtain additional microstructural information about nucleating bile. These techniques have the disadvantage of often being model dependent in addition to constraints specific for each method.The systematic, judicious use of a combination of complementary direct and indirect techniques have led to a comprehensive understanding of the various microstructural processes and interactions occurring during bile secretion, flow in the biliary tract and storage in the gallbladder. This forms the basis for our current understanding of cholesterol nucleation, crystallization and gallstone formation

Use of novel cationic bile salts in cholesterol crystallization and solubilization in vitro

Unnatural bile salts have been synthesized with a cationic group at the side chain of natural bile acids. These cationic bile salts aggregate in water and aqueous salt solutions in a manner similar to their natural counterparts. The critical micellar concentrations of the cationic bile salts were measured using a fluorescence method. Cationic bile salts aggregated at a concentration lower than natural deoxycholic acid. Since dihydroxy bile salt micelles are well known for cholesterol dissolution/removal, the dissolution in the cationic micelles has been evaluated. The cationic analogs dissolve approximately 70 mg/dL of cholesterol, which is comparable to taurochenodeoxycholate micelle under identical bile salt concentrations. Cholesterol dissolution in cationic bile salt micelle enhanced upon adding various amounts of PC. Cholesterol crystallization was studied in model bile at various cationic bile salt concentrations. The addition of 5, 15 and 30 mM of the cationic bile salts attenuated the crystallization process, without influencing the crystal observation time or decreasing the final amount of crystals formed. All these effects were comparable to those observed with cholic acid. These findings suggest that cationic bile salts have physico-chemical properties analogous to those of natural anionic bile salts, and thus may have therapeutic potential