Smith J L, Lutton C
Department of Surgery, The University of Queensland, Royal Brisbane Hospital, Herston, Australia.
J Gastroenterol Hepatol. 1997 Dec;12(12):877-86. doi: 10.1111/j.1440-1746.1997.tb00387.x.
Acyl-coenzyme A-cholesterol acyltransferase (ACAT) catalyses the esterification of cholesterol with long-chain fatty acyl-coenzyme A derivatives and has been implicated in the development of cholesterol gallstones. In this study we have examined several key components of the hepatic ACAT assay in order to develop a reliable and sensitive ACAT assay for LPN hamsters, a breed of golden Syrian hamster which has been characterized recently by this laboratory as a particularly good model for studying the pathogenesis of cholesterol gallstones. The newly developed ACAT assays were subsequently used to examine whether hepatic ACAT activity is altered in this animal model. Important new methodological findings were: (i) ACAT activity displayed two pH optima, one at 7.0 when assayed using endogenous cholesterol as substrate, and the other at about pH 8.5-9.0 when assayed in the presence of exogenous cholesterol; (ii) ACAT activity increased markedly when exogenous cholesterol was delivered to ACAT in Tween 80 (125-fold) or hydroxypropyl-beta-cyclodextrin (200-fold) in contrast to the use of cholesterol/phosphatidylcholine liposomes (9-fold); (iii) the addition of dithiothreitol, but not reduced glutathione, to the assay mixture resulted in a marked decrease in ACAT activity. Using the optimal assay conditions (exogenous cholesterol added), hepatic ACAT activity was shown to be significantly reduced in hamsters fed a high sucrose lithogenic diet compared with controls (587+/-42 vs 737+/-44 pmol/min per mg; P=0.025). In contrast, ACAT activity measured using endogenous cholesterol as a substrate was greater in sucrose-fed hamsters compared with controls (22.3+/-2.5 vs 13.2+/-2.9 pmol/min per mg; P= 0.030). These results highlight the importance of using an ACAT activity assay which has been well characterized and supports the hypothesis that the pathogenesis of cholesterol gallstones in LPN hamsters is related to an altered hepatic cholesterol metabolism.
酰基辅酶A胆固醇酰基转移酶（ACAT）催化胆固醇与长链脂肪酰基辅酶A衍生物的酯化反应，并与胆固醇胆结石的形成有关。在本研究中，我们检测了肝脏ACAT检测的几个关键成分，以便为LPN仓鼠（一种金色叙利亚仓鼠，本实验室最近将其鉴定为研究胆固醇胆结石发病机制的特别好的模型）开发一种可靠且灵敏的ACAT检测方法。随后，使用新开发的ACAT检测方法来检测该动物模型中肝脏ACAT活性是否发生改变。重要的新方法学发现如下：（i）ACAT活性表现出两个pH最佳值，以内源性胆固醇为底物进行检测时，一个在pH 7.0，在存在外源性胆固醇的情况下进行检测时，另一个在约pH 8.5 - 9.0；（ii）与使用胆固醇/磷脂酰胆碱脂质体（9倍）相比，当外源性胆固醇以吐温80（125倍）或羟丙基-β-环糊精（200倍）递送至ACAT时，ACAT活性显著增加；（iii）向检测混合物中添加二硫苏糖醇而非还原型谷胱甘肽会导致ACAT活性显著降低。使用最佳检测条件（添加外源性胆固醇），与对照组相比，喂食高蔗糖致石性饮食的仓鼠肝脏ACAT活性显著降低（587±42对737±44 pmol/分钟每毫克；P = 0.025）。相比之下，与对照组相比，以蔗糖喂养的仓鼠使用内源性胆固醇作为底物测得的ACAT活性更高（22.3±2.5对13.2±2.9 pmol/分钟每毫克；P = 0.030）。这些结果突出了使用经过充分表征的ACAT活性检测方法的重要性，并支持了LPN仓鼠中胆固醇胆结石发病机制与肝脏胆固醇代谢改变有关的假说。
