Importance of Evaluating Cell Cholesterol Influx With Efflux in Determining the Impact of Human Serum on Cholesterol Metabolism and Atherosclerosis

OBJECTIVE: Cholesterol efflux relates to cardiovascular disease but cannot predict cellular cholesterol mass changes. We asked whether influx and net flux assays provide additional insights. APPROACH AND RESULTS: Adapt a bidirectional flux assay to cells where efflux has clinical correlates and examine the association of influx, efflux, and net flux to serum triglycerides (TGs). Apolipoprotein B-depleted (high-density lipoprotein-fraction) serum from individuals with unfavorable lipids (median [interquartile range]; high-density lipoprotein-cholesterol=39 [32-42], low-density lipoprotein-cholesterol=109 [97-137], TGs=258 [184-335] mg/dL; n=13) promoted greater ATP-binding cassette transporter A1-mediated [1,2-(3H)] cholesterol efflux (3.8±0.3%/4 hour versus 1.2±0.4%/4 hour; P<0.0001) from cyclic 3',5'-amp(CTP-amp)-treated J774 macrophages than from individuals with favorable lipids (high-density lipoprotein-cholesterol=72 [58-88], low-density lipoprotein-cholesterol=111 [97-131], TGs=65 [56-69] mg/dL; n=10). Thus, high TGs associated with more ATP-binding cassette transporter A1 acceptors. Efflux of cholesterol mass (μg free cholesterol/mg cell protein per 8 hour) to serum was also higher (7.06±0.33 versus 5.83±0.48; P=0.04). However, whole sera from individuals with unfavorable lipids promoted more influx (5.14±0.65 versus 2.48±0.85; P=0.02) and lower net release of cholesterol mass (1.93±0.46 versus 3.36±0.47; P=0.04). The pattern differed when mass flux was measured using apolipoprotein B-depleted serum rather than serum. Although individuals with favorable lipids tended to have greater influx than those with unfavorable lipids, efflux to apolipoprotein B-depleted serum was markedly higher (6.81±0.04 versus 2.62±0.14; P<0.0001), resulting in an efflux:influx ratio of ≈3-fold. Thus both serum and apolipoprotein B-depleted serum from individuals with favorable lipids promoted greater net cholesterol mass release despite increased ATP-binding cassette transporter A1-mediated efflux in samples of individuals with high TGs/unfavorable lipids. CONCLUSIONS: When considering the efficiency of serum specimens to modulate cell cholesterol content, both influx and efflux need to be measured.