Ultracentrifugation systematically overestimates vesicular cholesterol levels in bile

To accurately determine the cholesterol (Ch) distribution between mixed micelles and vesicles in lithogenic bile, both ultracentrifugation and gel chromatography with the correct intermixed micellar/vesicular bile salt concentration (IMC) have been proposed. We have systematically compared both separation techniques with physiological model biles to ascertain their quantitative separation ability. After determination of optimal ultra-centrifugation conditions in systems containing only micelles or vesicles, Ch-supersaturated model biles [3-10 g/dL, 10 mol percent Ch, taurocholate (TC)]/([TC + egg yolk phosphatidylcholine (EYPC)] = 0.6 and 0.7) were adjusted to a density of 1.03 g/mL, and ultracentrifuged at 42,000 rpm and 37 degrees C for 13 hours. Identical model biles were subjected to gel chromatography with the correct IMC, either directly or after remixing and incubation at 37 degrees C after ultracentrifugation. By ultracentrifugation, 31 percent +/- 2 percent (TC/(TC + EYPC) = 0.6) and 40 percent +/- 5 percent (TC/(TC + EYPC) = 0.7) of total Ch were found in vesicles (Ch/EYPC molar ratios = 1.0 and 1.3, respectively). However, by gel chromatography, only 19 percent +/- 2 percent (Ch/EYPC = 1.0) and 22 percent +/- 2 percent (Ch/EYPC = 1.5) of total Ch were found in the corresponding biles. Gel chromatography of biles (TC/(TC + EYPC) = 0.7) ultracentrifuged for various durations showed a progressive increase in vesicular Ch to 41 percent after 13 hours. On incubation for 11.5 hours after ultracentrifugation, vesicular Ch decreased to 31 percent, thus approaching the initial (gel chromatography) value. Quasielastic light scattering also demonstrated formation of vesicles in ultracentrifuged Ch-unsaturated model bile (cholesterol saturation index (CSI) approximately 0.97). As compared with gel chromatography, ultracentrifugation systematically elevates vesicular Ch, possibly because of induced shifts in lipids between lipid aggregates caused by variation in local bile salt concentration. Because ultracentrifugation can alter the phases present in bile, gel chromatography with the correct IMC more accurately represents the distribution of Ch in biliary lipid aggregates.