E. R.M. Eckhardt

Accurate separation of vesicles, micelles and cholesterol crystals in supersaturated model biles by ultracentrifugation, ultrafiltration and dialysis.

Gel filtration with bile salts at intermixed micellar/vesicular concentrations (IMC) in the eluant has been proposed to isolate vesicles and micelles from supersaturated model biles, but the presence of vesicular aggregates makes this method unreliable. We have now validated a new method for isolation of various phases. First, aggregated vesicles and - if present - cholesterol crystals are pelleted by short ultracentrifugation. Cholesterol contained in crystals and vesicular aggregates can be quantitated from the difference of cholesterol contents in the pellets before and after bile salt-induced solubilization of the vesicular aggregates. Micelles are then isolated by ultrafiltration of the supernatant through a highly selective 300 kDa filter and unilamellar vesicles by dialysis against buffer containing bile salts at IMC values. Lipids contained in unilamellar vesicles are also estimated by subtraction of lipid contents in filtered micelles from lipid contents in (unilamellar vesicle+micelle containing) supernatant (subtraction method). Ultrafiltration-dialysis and subtraction methods yielded identical lipid solubilization in unilamellar vesicles and identical vesicular cholesterol/phospholipid ratios. In contrast, gel filtration yielded much more lipids in micelles and less in unilamellar vesicles, with much higher vesicular cholesterol/phospholipid ratios. When vesicles obtained by dialysis were analyzed by gel filtration, vesicular cholesterol/phospholipid ratios increased strongly, despite correct IMC values for bile salts in the eluant. Subsequent extraction of column material showed significant amounts of lipids. In conclusion, gel filtration may underestimate vesicular lipids and overestimate vesicular cholesterol/phospholipid ratios, supposedly because of lipids remaining attached to the column. Combined ultracentrifugation-ultrafiltration-dialysis should be considered state-of-the-art methodology for quantification of cholesterol carriers in model biles.

Lipid solubilization in human gallbladder versus hepatic biles

BACKGROUND/AIMSnCholesterol crystallizes more rapidly in gallbladder than in hepatic biles, supposedly due to formation of cholesterol-supersaturated vesicles in concentrated gallbladder biles because of preferential micellization of phospholipids compared to cholesterol. We therefore aimed to compare lipid solubilization in hepatic and gallbladder biles.nnnMETHODSnMixed micellar and vesicular phases were separated from hepatic and associated gallbladder biles of seven cholesterol gallstone patients by using state-of-the-art gel filtration with bile salts at intermixed micellar/intervesicular compositions and concentrations in the eluant.nnnRESULTSnVesicles were found in 6 out of 7 hepatic biles, but only in 2 of the corresponding gallbladder biles. Both percentage (7.8+/-5.1 vs. 36.3+/-7.6%; p = 0.01) and amount (0.9+/-0.2 vs. 1.7+/-0.3 mM; p = 0.06) of vesicular cholesterol were lower in gallbladder biles. Similar results were found for vesicular phospholipids (1.3+/-0.8 vs. 11.6+/-6.0%; p = 0.05; and 0.3+/-0.1 vs. 1.1+/-0.5 mM; p = 0.07). The vesicular cholesterol/ phospholipid ratio was 1.7+/-0.5 in hepatic bile but 4.3 and 1.8 in the 2 gallbladder biles which contained vesicles. Mixed micelles in gallbladder biles had a higher cholesterol saturation index than mixed micelles in hepatic biles (1.43+/-0.11 vs. 1.15+/-0.07; p = 0.02).nnnCONCLUSIONSnConcentration of bile in the gallbladder leads to decreased vesicular lipid contents. The finding of supersaturated mixed micelles in the absence of vesicles in a significant number of patients points to the possibility of non-vesicular modes of crystallization.

Asymmetric distribution of phosphatidyicholine (PC) and spingomyelin (SM) between micellar and vesicular phases: potential implications for canalicular bile formation

Both phosphatidylcholine (PC) and sphingomyelin (SM) are the major phospholipids in the outer leaflet of the hepatocyte canalicular membrane. Yet, the phospholipids secreted into bile consist principally (>95%) of PC. In order to understand the physical;-chemical basis for preferential biliary PC secretion, we compared interactions with bile salts (taurocholate) and cholesterol of egg yolk (EY)SM (mainly 16:0 acyl chains, similar to trace SM in bile), buttermilk (BM)SM (mainly saturated long (>20 C-atoms) acyl chains, similar to canalicular membrane SM) and egg yolk (EY)PC (mainly unsaturated acyl chains at sn-2 position, similar to bile PC). Main gel to liquid-crystalline transition temperatures were 33. 6 degrees C for BMSM and 36.6 degrees C for EYSM. There were no significant effects of varying phospholipid species on micellar sizes or intermixed-micellar/vesicular bile salt concentrations in taurocholate-phospholipid mixtures (3 g/dL, 37 degrees C, PL/BS + PL = 0.2 or 0.4). Various phases were separated from model systems containing both EYPC and (EY or BM)SM, taurocholate, and variable amounts of cholesterol, by ultracentrifugation with ultrafiltration and dialysis of the supernatant. At increasing cholesterol content, there was preferential distribution of lipids and enrichment with SM containing long saturated acyl chains in the detergent-insoluble pelletable fraction consisting of aggregated vesicles. In contrast, both micelles and small unilamellar vesicles in the supernatant were progressively enriched in PC. Although SM containing vesicles without cholesterol were very sensitive to micellar solubilization upon taurocholate addition, incorporation of the sterol rendered SM-containing vesicles highly resistant against the detergent effects of the bile salt. These findings may have important implications for canalicular bile formation.