Abstract 17232: Comparison of Methods for the Measurement of Cholesterol Efflux Capacity of Plasma HDL With J774 Mouse Macrophages Reveals Superiority of the Radioactive Cholesterol Method over the BODIPY-Cholesterol-2 Method

Recent clinical studies suggest that raising HDL-cholesterol (HDL-C) concentration is insufficient to lower cardiovascular risk and that protection derives from other characteristics of HDL particles. One such characteristic that can be measured in vitro is the cholesterol efflux capacity of plasma, an assay where HDL accepts labelled cholesterol from macrophages. Recently, a method for the measurement of cholesterol efflux with a fluorescent tracer, BODIPY-cholesterol-2 (BC2), has been proposed as an alternative to the radioactive method. We undertook a systematic comparison of BC2 and 3H-cholesterol methods with J774 macrophages in basal and cAMP-stimulated conditions with purified acceptors, plasma from healthy volunteers and patients with myocardial infarction of the Montreal Heart Institute Biobank. Dose-response curves show higher affinity of BC2 vs. 3H cholesterol with apoA-I, HDL and apoB-depleted plasma (all p<0.01) and a higher maximal efflux for HDL and depleted plasma (both p<0.001) in stimulated conditions. This was reflected in a faster kinetics of BC2 efflux compared to 3H-cholesterol efflux (time for half-maximal efflux 4.1h vs. 10.2 h) and resulted in a 2.5-fold increase of BC2 maximal efflux (p<0.01). With 50 normolipidemic plasmas (2.8%, 4h efflux), the two methods were not correlated in basal conditions (r2=0.079) and strongly correlated in stimulated conditions (r2=0.830). BC2 values did not correlate with HDL-C in all conditions, contrary to 3H cholesterol values (p<0.01). Using more complex samples from patients with MI (n=115), the correlations were modest in basal (r2=0.219) and stimulated (r2=0.400) conditions. Differences between controls and MI cases showed reduced significance with BC-2 compared to 3H cholesterol. Lastly, in macrophages examined under the confocal microscope, BC2 labels vesicular structures colocalized with filipin, a free cholesterol marker, suggesting endosomal loading of BC2. Thus, the BC2 method is not equivalent to the classic 3H cholesterol method for cholesterol efflux measurement.