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 concentrati ons (IMC) in the eluant has been proposed to isolate vesicles and micelles from supersaturated model biles, but the presence of vesicular aggregates m akes this method unreliable. We have now validated a new method for isolati on of various phases. First, aggregated vesicles and - if present cholester ol crystals are pelleted by short ultracentrifugation. Cholesterol containe d in crystals and vesicular aggregates can be quantitated from the differen ce of cholesterol contents in the pellets before and after bile salt-induce d solubilization of the vesicular aggregates. Micelles are then isolated by ultrafiltration of the supernatant through a highly selective 300 kDa filt er and unilamellar vesicles by dialysis against buffer containing bile salt s at IMC values. Lipids contained in unilamellar vesicles are also estimate d by subtraction of lipid contents in filtered micelles from lipid contents in (unilamellar vesicle+micelle containing) supernatant ('subtraction meth od'). 'Ultrafiltration-dialysis' and 'subtraction' methods yielded identica l lipid solubilization in unilamellar vesicles and identical vesicular chol esterol/phospholipid ratios. In contrast, gel filtration yielded much more lipids in micelles and less in unilamellar vesicles, with much higher vesic ular cholesterol/phospholipid ratios. When vesicles obtained by dialysis we re analyzed by gel filtration, vesicular cholesterol/phospholipid ratios in creased strongly, despite correct IMC values for bile salts in the eluant. Subsequent extraction of column material showed significant amounts of lipi ds. In conclusion, gel filtration may underestimate vesicular lipids and ov erestimate vesicular cholesterol/ phospholipid ratios, supposedly because o f lipids remaining attached to the column. Combined ultracentrifugation-ult rafiltration-dialysis should be considered state-of-the-art methodology for quantification of cholesterol carriers in model biles. (C) 2001 Elsevier S cience B.V. All rights reserved.