Abstract Background Cholesterol efflux capacity (CEC), a key anti-atherosclerotic function of high-density lipoprotein (HDL), plays a crucial role in preventing cardiovascular disease (CVD). HDL reduces lipid burden in atherosclerotic lesions via cholesterol efflux. While HDL-cholesterol (HDL-C) levels, a measure of HDL quantity, have been widely used to assess CVD risk due to HDL’s role in mitigating atherosclerosis, numerous studies have demonstrated that HDL-C levels do not always correlate with CVD risk. In contrast, CEC, which reflects HDL’s functional quality, is considered a superior predictor of cardiovascular events and holds promise as a novel CVD risk marker. Although numerous clinical studies have demonstrated the utility of CEC assessment, the CEC assay lacks standardization, and the impact of measurement conditions remains insufficiently understood. This study investigated the effect of efflux duration on CEC values. Methods Serum samples were collected from 15 healthy human volunteers, and CEC and HDL-C levels were measured. CEC was assessed using two commonly employed cell lines, THP-1 and J774, in a cellular assay. Cells were loaded with BODIPY-cholesterol, a fluorescent-labeled cholesterol, and incubated with apolipoprotein B-depleted serum (BDS). To induce ATP-binding cassette transporter A1 (ABCA1) expression, THP-1 cells were cultured with a Liver X receptor (LXR) agonist, and J774 cells were treated with cyclic adenosine monophosphate (cAMP). The fluorescence intensity of the supernatant was measured after 4 and 24 hours of incubation to determine CEC at different efflux durations. Changes in cellular and cholesterol acceptor characteristics during efflux were assessed by evaluating ABCA1 expression levels and HDL particle size distribution using electrophoresis and western blotting. Results In both THP-1 and J774 cells, ABCA1 expression levels increased with efflux duration, while the HDL particle size distribution remained unchanged. CEC values measured after 4 hours of incubation showed no significant correlation between THP-1 and J774 cells (r = 0.500, p 0.05). However, after 24 hours, CEC values from the two cell lines exhibited a significant correlation (r = 0.737, p 0.05). Furthermore, at 4 hours, no significant correlation was observed between CEC and HDL-C in either THP-1 cells (r = 0.252, p 0.05) or J774 cells (r = 0.491, p 0.05). In contrast, after 24 hours, significant correlations with HDL-C were observed for both THP-1 cells (r = 0.703, p 0.05) and J774 cells (r = 0.682, p 0.05). Conclusions CEC measured at short efflux durations is influenced by cell-related factors beyond HDL-C levels, and these factors vary between cell lines. However, with prolonged efflux durations, CEC becomes more reflective of HDL-C levels, irrespective of the cell line used, suggesting a time-dependent shift in the determinants of CEC. This shift in CEC is primarily attributed to alterations in cellular properties, given the greater temporal changes observed in cellular properties compared to acceptor characteristics. These findings highlight the significant impact of cellular assay measurement conditions on CEC values and their interpretation. Therefore, further investigation into optimal measurement conditions and the establishment of standardized methods are essential for accurate CEC assessment.
Akiyama et al. (Wed,) studied this question.