Smith B F, LaMont J T
J Clin Invest. 1985 Aug;76(2):439-45. doi: 10.1172/JCI111991.
The goals of this study were to isolate and characterize the nonlipid matrix of human cholesterol gallstones. The lipid portion of gallstones was dissolved in ethanol/ether, leaving an insoluble, granular, brown-black matrix that constituted 12.5% of solitary large stones and 3.5% of multiple small stones. The matrix was partially solubilized by sonication and studied by exclusion gel chromatography and density gradient ultracentrifugation. On Sepharose 2B column chromatography, bile pigment eluted with glycoprotein in the void volume, suggesting the presence of a high molecular weight complex (Mr greater than 2 X 10(6)). The identity of mucin in this complex was confirmed by its typical buoyant density during ultracentrifugation. The major bile pigments in the matrix were identified as bilirubin (84%) and bilirubin monoglucuronide (15%) by thin-layer chromatography. Because of their ability to solubilize mucin-type glycoproteins, we tested the ability of the reducing agents 2-mercaptoethanol (2ME) and N-acetylcysteine (NAcCys) to solubilize gallstone matrix. Both reducing agents caused a two- to threefold enhancement of matrix dissolution after 4 d compared to aqueous buffer alone (P less than 0.01). Sepharose 2B chromatography revealed that 2ME released a high molecular weight mucin-bilirubin complex as well as unbound pigment from the insoluble matrix. We also tested the effect of reducing agents on dissolution of matched cholesterol gallstones by monooctanoin, a cholesterol solvent. Both 2ME and NAcCys significantly accelerated gallstone dissolution in monooctanoin. Matched human cholesterol stones (n = 10) incubated for 4 d in monooctanoin plus either 2ME or NAcCys (1 M final concentration) weighed approximately half as much (P less than 0.01 for each) as stones incubated in monooctanoin alone. This study describes, for the first time, the isolation of a bilirubin-mucin complex in the insoluble matrix of human cholesterol gallstones. The ability of reducing agents to dissolve the matrix and thereby accelerate gallstone dissolution by monooctanoin in vitro may be relevant to gallstone dissolution in humans.
本研究的目的是分离并鉴定人类胆固醇胆结石的非脂质基质。胆结石的脂质部分溶解于乙醇/乙醚中，留下一种不溶性的、颗粒状的棕黑色基质，其在单个大结石中占12.5%，在多个小结石中占3.5%。通过超声处理使该基质部分溶解，并采用排阻凝胶色谱法和密度梯度超速离心法进行研究。在琼脂糖2B柱色谱上，胆汁色素与糖蛋白一起在空体积中洗脱，表明存在一种高分子量复合物（分子量大于2×10⁶）。通过超速离心过程中其典型的浮力密度证实了该复合物中粘蛋白的身份。通过薄层色谱法鉴定出该基质中的主要胆汁色素为胆红素（84%）和胆红素单葡萄糖醛酸酯（15%）。由于它们能够溶解粘蛋白型糖蛋白，我们测试了还原剂2-巯基乙醇（2ME）和N-乙酰半胱氨酸（NAcCys）溶解胆结石基质的能力。与单独的水性缓冲液相比，4天后两种还原剂均使基质溶解增加了2至3倍（P<0.01）。琼脂糖2B色谱显示，2ME从不溶性基质中释放出一种高分子量的粘蛋白-胆红素复合物以及未结合的色素。我们还测试了还原剂对单辛脂（一种胆固醇溶剂）溶解匹配的胆固醇胆结石的影响。2ME和NAcCys均显著加速了单辛脂中胆结石的溶解。在单辛脂加2ME或NAcCys（终浓度1M）中孵育4天的匹配人类胆固醇结石（n=10）的重量约为单独在单辛脂中孵育结石的一半（每种情况P<0.01）。本研究首次描述了在人类胆固醇胆结石的不溶性基质中分离出胆红素-粘蛋白复合物。还原剂溶解基质从而在体外加速单辛脂溶解胆结石的能力可能与人类胆结石的溶解有关。
