ULTRACENTRIFUGATION SYSTEMATICALLY OVERESTIMATES VESICULAR CHOLESTEROL LEVELS IN BILE

To accurately determine the cholesterol (Ch) distribution between mixe d micelles and vesicles in lithogenic bile, both ultracentrifugation a nd gel chromatography with the correct intermixed micellar/vesicular b ile salt concentration (IMC) have been proposed. We have systematicall y compared both separation techniques with physiological model biles t o ascertain their quantitative separation ability. After determination of optimal ultracentrifugation conditions in systems containing only micelles or vesicles, Ch-supersaturated model biles [3-10 g/dL, 10 mol % Ch, taurocholate (TC)]/([TC + egg yolk phosphatidylcholine (EYPC)] = 0.6 and 0.7) were adjusted to a density of 1.03 g/mL, and ultracentri fuged at 42,000 rpm and 37 degrees C for 13 hours. Identical model bil es were subjected to gel chromatography with the correct IMC, either d irectly or after remixing and incubation at 37 degrees C after ultrace ntrifugation. By ultracentrifugation, 31% +/- 2% (TC/(TC + EYPC) = 0.6 ) and 40% +/- 5% (TC/(TC + EYPC) = 0.7) of total Ch were found in vesi cles (Oh EYPC molar ratios = 1.0 and 1.3, respectively). However, by g el chromatography, only 19% +/- 2% (Ch/EYPC = 1.0) and 22% +/- 2% (Ch/ EYPC = 1.5) of total Ch were found in the corresponding biles. Gel chr omatography of biles (TC/(TC + EYPC) = 0.7) ultracentrifuged for vario us durations showed a progressive increase in vesicular Oh to 41% afte r 13 hours. On incubation for 11.5 hours after ultracentrifugation, ve sicular Ch decreased to 31%, thus approaching the initial (gel chromat ography) value. Quasielastic light scattering also demonstrated format ion of vesicles in ultracentrifuged Ch-unsaturated model bile (cholest erol saturation index (CSI) approximate to 0.97). As compared with gel chromatography, ultracentrifugation systematically elevates vesicular Ch, possibly because of induced shifts in lipids between Lipid aggreg ates caused by variation in local bile salt concentration. Because ult racentrifugation can alter the phases present in bile, gel chromatogra phy with the correct IMC more accurately represents the distribution o f Ch in biliary Lipid aggregates.