Background: 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.
Results: 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
Conclusion: 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.