Abstract
Using a system of phosphatidylcholine-cholesterol vesicles to model the vesicle phase of mammalian bile (1:1 molar ratio) we evaluated whether very small amounts of C. perfringens phospholipase C activity (0.5-6.5 nmol/min per ml) could lead to vesicle fusion, a precursor step for cholesterol precipitation in gallbladder bile. Quasielastic light scattering spectroscopy (QLS) was used to monitor vesicle growth and aggregation in model bile (0.89 mM total lipid) in the presence of phospholipase C. Vesicle growth over 2 h could be detected with phospholipase activity as little as 0.5 nmol/min per ml. Vesicle growth was sustainable over days in the absence of Ca2+ once as little as 3-7 mol% diacylglycerol had been generated as a result of the initial phospholipase C treatment. The presence of fusion intermediates was confirmed using transmission electron microscopy. In addition, kinetically slow vesicle fusion with intravesicle content mixing and minimal leakage was also confirmed by fluorescence spectroscopy using two populations of vesicles containing 5 mM TbCl3 or 50 mM dipicolinic acid. Efficient fusion (40% maximum fluorescence) was obtained at 30 min at 25 degrees C with phospholipase C activity. This level of enzyme activity approximates that found in human gallbladder bile (1.2 nmol/min per ml). We conclude that the hydrolysis products of phospholipase C activity can, in very small amounts (3-7 mol% diacylglycerol), lead to destabilization and fusion of cholesterol-saturated biliary vesicles. A reappraisal of the importance of phospholipase C hydrolysis products in the pathogenesis of cholesterol gallstones is warranted based on these observations.
Highlights
Using a system of phosphatidylcholine-cholesterol vesicles to model the vesicle phase of mammalian bile (1:l molar ratio) we evaluated whether very small amounts of C. perfnngem phospholipase C activity (0.5-6.5 nmol/min per ml) could lead to vesicle fusion, a precursor step for cholesterol precipitation in gallbladder bile
Vesicle size was measured as a function of time after addition of increasing concentrations of phospholipase C to a 890 fiM solution of sonicated lipid vesicles in the presence of 5 mM Ca2+.In the absence of phospholipase C, vesicles were stable for several weeks
The kinetics of crystal formation were not determined in the present experiments, cholesterol monohydrate crystals with the typical notched rhomboidal plate configuration were observed with polarizing microscopy in samples containing phospholipase C
Summary
Using a system of phosphatidylcholine-cholesterol vesicles to model the vesicle phase of mammalian bile (1:l molar ratio) we evaluated whether very small amounts of C. perfnngem phospholipase C activity (0.5-6.5 nmol/min per ml) could lead to vesicle fusion, a precursor step for cholesterol precipitation in gallbladder bile. Quasielastic light scattering spectroscopy (QLS) was used to monitor vesicle growth and aggregation in model bile (0.89 mM total lipid) in the presence of phospholipase C. We explored the possibility that phospholipase C could lead to the important phase transitions in model bile vesicles that are known to be preliminary steps in cholesterol nucleation. In the present study quasielastic light scattering spectroscopy (QLS), transmission electron microscopy (EM), and fluorescence spectroscopy were used to characterize phospholipase C-induced fusion in cholesterol-saturated model bile vesicles
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