Yuet P K, Blankschtein D, Donovan J M
Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, USA.
Hepatology. 1996 Apr;23(4):896-903. doi: 10.1002/hep.510230434.
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.
为准确测定致石性胆汁中混合微胶粒与囊泡之间胆固醇（Ch）的分布，有人提出采用超速离心法和凝胶色谱法，并确保微胶粒/囊泡混合胆汁盐浓度（IMC）正确。我们系统地将这两种分离技术与生理模型胆汁进行比较，以确定它们的定量分离能力。在确定了仅含有微胶粒或囊泡的系统中的最佳超速离心条件后，将Ch超饱和模型胆汁[3 - 10 g/dL，10 mol% Ch，牛磺胆酸盐（TC）]/（[TC + 蛋黄磷脂酰胆碱（EYPC）] = 0.6和0.7）调整至密度为1.03 g/mL，并在42,000 rpm和37℃下超速离心13小时。相同的模型胆汁直接或在超速离心后于37℃重新混合并孵育后，采用正确的IMC进行凝胶色谱分析。通过超速离心，在囊泡中发现总Ch的31%±2%（TC/（TC + EYPC） = 0.6）和40%±5%（TC/（TC + EYPC） = 0.7）（Ch/EYPC摩尔比分别为1.0和1.3）。然而，通过凝胶色谱法，在相应胆汁中仅发现总Ch的19%±2%（Ch/EYPC = 1.0）和22%±2%（Ch/EYPC = 1.5）。对不同时长超速离心的胆汁（TC/（TC + EYPC） = 0.7）进行凝胶色谱分析，结果显示囊泡Ch在13小时后逐渐增加至41%。超速离心后孵育11.5小时，囊泡Ch降至31%，从而接近初始（凝胶色谱法）值。准弹性光散射也证明在超速离心的Ch不饱和模型胆汁（胆固醇饱和指数（CSI）约为0.97）中形成了囊泡。与凝胶色谱法相比，超速离心系统性地提高了囊泡Ch的含量，这可能是由于局部胆汁盐浓度变化导致脂质聚集体之间脂质发生诱导性转移所致。由于超速离心会改变胆汁中的相态，采用正确IMC的凝胶色谱法能更准确地反映Ch在胆汁脂质聚集体中的分布。
