Cholesterol Efflux and Reverse Cholesterol Transport: Experimental Approaches

Cholesterol efflux as a key event in reverse cholesterol transport (RCT) is considered now as both diagnostic tool and a promising target for the treatment of atherosclerosis. Radioactive in vitro cholesterol efflux assay (CEA) is the gold standard for determination of efflux at cellular level. Fluorescent tracers and stable isotope-labeled cholesterol gradually come into use as convenient tools for non-radioactive CEAs.

We review the use of various tracer-based and tracer-free methods for CEAs and for measuring RCT with focus on macrophage-specific cholesterol efflux. CEA utilizing stable isotope-labeled cholesterol is equally reliable with radioactive assay and especially well suited for the determination of both cholesterol efflux and net cholesterol flux. Fluorescent tracers cannot fully mimic cholesterol; however, they are successfully applied in CEA in specific well-defined conditions. Fluorescent CEAs can be high throughput and can provide unique information on efflux from fast cholesterol pools or with single cell resolution. Enzymatic and chromatographic CEAs are net cholesterol flux assays, and they can be applied as efflux assays when used with specific acceptors only. In vivo tests are suited for studies of cholesterol efflux and RCT at the level of the organism. They include injection of tracer-loaded macrophages, a method suitable at present for animal models only, and recently invented modification of whole body tracer kinetics with multicompartment modeling that is capable to determine cholesterol efflux from macrophages.

Despite the decisive role of in vitro assays in our understanding of cholesterol efflux mechanism, the in vivo assays are highly desired to study cholesterol efflux in atherosclerotic lesions and RCT in whole body.