Facultat de Medicina i Ciències de la Salut, Departament de Medicina i Cirurgia, Grup Nutrició Funcional, Oxidació i Malalties Cardiovasculars (NFOC-Salut), Universitat Rovira i Virgili, Reus, Spain.
Hospital Universitari Sant Joan de Reus, Reus, Spain.
Methods Mol Biol. 2022;2419:257-274. doi: 10.1007/978-1-0716-1924-7_15.
Cholesterol efflux (ChE) capacity is associated with the incidence of cardiovascular events and has been proposed as an emerging cardiovascular risk factor. ChE has been traditionally assessed by in vitro radioactive methods but these are not appropriate when assessing a large number of samples. Therefore, alternative, reproducible nonradioactive methods have been developed. This chapter describes a robust nonradioactive method using a fluorescent tracer to assess ChE in vitro.The measurement of ChE in vitro requires three main components: a cholesterol-loaded donor cell, a cholesterol tracer, and a cholesterol acceptor. This method involves labeling of murine macrophage J774A.1 cells using the fluorescent sterol dipyrromethene boron difluoride (BODIPY)-cholesterol. The cholesterol acceptors from humans or animals include lipid-free apolipoprotein (ApoA)-1, high-density lipoprotein (HDL), HDL and HDL subfractions, serum, plasma or ApoB-depleted serum or plasma. While lipid-free ApoA-1 mediates ChE via only ATP-binding cassette (ABC)A1 transporter, the remaining acceptors mediate ChE via ABCA1 , ABCG1 and scavenger receptor class B type 1 (SRB1) transporters. The reproducibility of this BODIPY-ChE assay is excellent as the intra-assay coefficients of variation (CVs) were <10% (30 replicates on the same day) and the interassay CVs were <14% (10 experiments performed on different days, with 3 replicates each). The fluorescent method therefore represents a reproducible, safe and useful tool to evaluate ChE as an emerging cardiovascular risk factor.
胆固醇外排（ChE）能力与心血管事件的发生有关，并已被提议作为一种新兴的心血管风险因素。ChE 传统上通过体外放射性方法进行评估，但在评估大量样本时，这些方法并不合适。因此，已经开发出了替代的、可重复的非放射性方法。本章描述了一种使用荧光示踪剂体外评估 ChE 的稳健非放射性方法。体外 ChE 的测量需要三个主要组成部分：负载胆固醇的供体细胞、胆固醇示踪剂和胆固醇接受体。该方法涉及使用荧光甾醇二吡咯甲川（BODIPY）-胆固醇标记鼠巨噬细胞 J774A.1 细胞。人类或动物的胆固醇接受体包括无脂载脂蛋白（ApoA）-1、高密度脂蛋白（HDL）、HDL 和 HDL 亚组分、血清、血浆或 ApoB 耗尽的血清或血浆。虽然无脂 ApoA-1 通过仅 ABC 转运蛋白 A1（ABCA1）介导 ChE，但其余接受体通过 ABCA1、ABCG1 和清道夫受体 B 型 1（SRB1）转运蛋白介导 ChE。BODIPY-ChE 测定的重现性非常好，因为日内变异系数（CVs）<10%（同一天 30 个重复），日间变异系数（CVs）<14%（不同天进行 10 次实验，每个实验重复 3 次）。因此，荧光法是一种可重复、安全且有用的工具，可用于评估 ChE 作为一种新兴的心血管风险因素。
