A-238 Influence of different apolipoprotein B-depleted serum preparation methods on high-density lipoprotein cholesterol efflux capacity assays

Abstract

Background Cholesterol efflux capacity (CEC), a critical anti-atherosclerotic function of high-density lipoprotein (HDL), is a promising biomarker for cardiovascular disease (CVD) risk. However, conventional CEC assays utilizing cultured cells are complex, hindering clinical application. To address this limitation, we previously developed a cell-free CEC assay, the immobilized liposome-bound gel beads (ILG) method. Both the ILG and conventional methods employ apolipoprotein B (apoB)-depleted serum (BDS) as the measurement sample. However, the optimal BDS preparation method remains unclear, as various methods exist, including polyethylene glycol (PEG), heparin sodium (Hep), and dextran sulfate (Dex). This study aimed to investigate the impact of different BDS preparation methods on both ILG and conventional cell-dependent assays. Methods Three types of BDS were prepared by mixing serum (set as 100 parts by volume) with the following reagents: (1) 20% PEG 6000 (200 mM glycine solution, pH 7.4) at a ratio of 100:40, (2) 1.3 mg/mL Hep in saline and 1M MnCl2 at 100:40:50, and (3) 5% Dex solution and 2M MgCl2 at 100:2:2.5. After centrifugation, the supernatants were collected as PEG-BDS, Hep-BDS, and Dex-BDS, respectively. The apoB-containing precipitates were dissolved in saline. Total cholesterol (TC) and HDL-C concentrations of each supernatant and precipitate solution were determined enzymatically. Apolipoprotein profiles were analyzed using SDS-PAGE and Native-PAGE followed by western blotting. CEC was measured using both the ILG method and conventional assay employing THP-1 cells and BODIPY-labeled cholesterol. To assess ATP-binding cassette transporter A1 (ABCA1)-mediated CEC, cells were cultured with and without a Liver X receptor agonist to modulate ABCA1 expression. Results While the three BDS exhibited different profiles, these differences did not significantly affect CEC measurements using the ILG method. TC concentrations in PEG-BDS and Dex-BDS were comparable to HDL-C concentrations in the original serum, whereas Hep-BDS exhibited significantly higher TC levels. SDS-PAGE revealed no differences in apoA-I and apoE profiles across the BDS samples, and barely detected apoB in any BDS, with the detected amount corresponding to less than 10% of serum. ApoA-I was present in all precipitates, with no significant differences in its relative abundance. However, Native-PAGE demonstrated a distinct distribution of HDL particles in Hep-BDS compared to the original serum. Importantly, the ILG method yielded comparable CEC values between the original serum and all BDS. In contrast, the cell-dependent assay revealed a significant reduction in ABCA1-mediated CEC in PEG-BDS compared to the original serum (approximately 56%). Furthermore, the Hep-BDS interfered with the cell-dependent assay by causing turbidity in the medium. Conclusion This study demonstrates that the choice of BDS preparation method does not significantly influence CEC measurements using the ILG method. However, cell-dependent assay exhibits substantial sensitivity to the BDS preparation method, with significant reductions in ABCA1-mediated CEC observed in PEG-BDS and medium turbidity by Hep-BDS. These findings highlight the critical importance of carefully selecting the BDS preparation method for CEC assays, particularly those reliant on cell-based assays. Further research is needed to investigate the effect of BDS preparation reagents on CEC assays and to determine whether these methodological differences influence the CVD risk assessment.