Validation and application of a novel cholesterol efflux assay using immobilized liposomes as a substitute for cultured cells
Validation and application of a novel cholesterol efflux assay using immobilized liposomes as a substitute for cultured cells
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DOI：
10.1042/bsr20180144
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发表时间：
2018-04-27
影响因子：
4
通讯作者：
Tozuka, Minoru
中科院分区：
文献类型：
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作者：
Horiuchi, Yuna;Lai, Shao-Jui;Tozuka, Minoru
高密度脂蛋白（HDL）的功能以及数量的估计是需要预测心血管疾病的发展风险。胆固醇外排能力（CEC）是衡量HDL抗动脉粥样硬化功能的关键指标。然而，目前用于计算CEC的测定方法对于临床使用并不理想，因为它们需要培养细胞。在本研究中，我们开发了一种新的CEC检测使用固定化脂质体结合的凝胶珠（ILG），含有荧光标记的胆固醇，作为培养细胞的替代品。当用聚乙二醇沉淀法去除载脂蛋白B-100的血清作为胆固醇受体时，该方法的基本特性，如HDL-胆固醇的有效范围、流出温度和时间以及归一化参数，表明该方法足以估计CEC。此外，用该ILG方法获得的CEC值也与用THP-1巨噬细胞衍生的泡沫细胞和3 H-胆固醇作为示踪剂的常规方法获得的CEC值相关（r = 0.932）。总的来说，这种新的胆固醇流出测定方法是该领域现有方法的现实和有效的替代方法，同时也更容易在临床实验室中使用，因为既不需要细胞培养，放射性同位素也不需要超离心。
Estimation of the function as well as the amount of high-density lipoprotein (HDL) is required to predict the risk of cardiovascular disease development. Cholesterol efflux capacity (CEC) is the key metric for determining the antiatherosclerotic function of HDL. However, the assay methods currently used to calculate CEC are not ideal for clinical use as they require the culture of cells. In the present study, we developed a novel CEC assay using immobilized liposome-bound gel beads (ILGs), containing fluorescently labeled cholesterol, as a substitute for cultured cells. When apolipoprotein B-100 depleted serum, obtained by polyethylene glycol precipitation, was used as the cholesterol acceptors, the basic properties of this method, such as the available range of HDL-cholesterol, efflux temperature and time, and normalization parameters, indicate that this method is sufficient to estimate CEC. Furthermore, the CEC values obtained with this ILG method were also correlated with those obtained with a conventional method using THP-1 macrophages derived foam cells and 3H-cholesterol as a tracer (r = 0.932). Overall, this novel cholesterol efflux assay method is a realistic and effective alternative to current methods in the field while also being easier to use in clinical laboratories as neither cell culture, radioisotope nor ultracentrifugation is required.