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

Intermediate structures in the cholate-phosphatidylcholine vesicle-micelle transition

The vesicle-micelle transition of egg phosphatidylcholine (PC) and sodium cholate was described by comparing cryo-transmission electron microscopic (cryo-TEM) images of the structures formed to the associated turbidity changes. These experiments were designed to identify the morphology of the intermediates between vesicles and small spheroidal mixed micelles. With increasing cholate concentration, the vesicular structures changed size and more multilamellar vesicles were seen. Between the apparent upper and lower phase boundaries, three structures were observed: open vesicles, large bilayer sheets (twenty to several hundred nanometers in diameter), and long (150-300 nm) flexible cylindrical micelles. The cylindrical micelles evolved from the edges of the bilayer sheets. At higher relative cholate concentration, the phase boundary was sharply defined by optical clarification of the egg PC-cholate mixtures. Cryo-TEM revealed only small spheroidal mixed micelles at this transition. These results provide the first direct evidence of the structural pathway or of molecular intermediates between a lamellar and a micellar state. Understanding these specific intermediates and the transitions between them is essential to developing reconstitution protocols and properly analyzing either activity or structural data obtained from cholate-dispersed membrane proteins