Comparison of a novel cholesterol efflux assay using immobilized liposome-bound gel beads with the conventional method

Abstract

Cholesterol efflux capacity (CEC) is an atheroprotective function of high-density lipoprotein (HDL). CEC is currently measured using artificially prepared foam cells composed of cultured macrophage and 3H-cholesterol. However, this conventional method is not suitable for clinical laboratory use due to poor repeatability, complexity, and low safety. Recently, we reported a novel CEC assay, called the immobilized liposome-bound gel beads (ILG) method. The ILG method is an alternative to foam cells, comprised of gel beads and BODIPY-cholesterol instead of macrophage and 3H-cholesterol, respectively. The ILG method has shown adequate basic properties and strong correlation with the conventional method. Here, we aimed to compare this new ILG method with the conventional method in-depth. When apoB-depleted serum was used as the cholesterol acceptor, the ILG method had far better reproducibility than the conventional method. The CEC of major HDL subclasses HDL2 and HDL3 had similar results in both the ILG and conventional method. However, the ILG method did not reflect the CEC of apolipoprotein A-I and a minor HDL subclass which uses ATP-binding cassette transporter A1 on foam cells. Superior reproducibility of the ILG method, which is a limitation of the conventional method, and similar CEC results for major HDL subclasses in the ILG and conventional methods, provide further evidence that the ILG method is promising for measuring CEC clinically. However, some HDL subclasses or apolipoproteins might have poor CEC correlation between these methods. Further research is therefore needed to confirm the clinical significance of estimating CEC by the ILG method.

Development and validation of novel automatable assay for cholesterol efflux capacity

“…In the ILG method, it is known that globulin has the ability to efflux cholesterol [ 15 ]. In addition, previous reports have described the effect of serum proteins, especially albumin, on CEC obtained using BDS as the cholesterol acceptor [ 16 , 20 , 21 ]. Hence, these serum proteins might have extracted cholesterol from the ILM, and this deviation may have occurred.…”

“…As for the relationship between beads-based methods (the ILG and ILM methods) and the conventional cell-based method, the ILM method does not involve transporter-mediated efflux, which is reflected in cellular methods. However, it has already been reported that the CEC of the conventional cellular method is well correlated with the ILG method, and CEC values of BDS reflect the HDL-CEC [ 15 , 16 ]. Moreover, when CEC values obtained by the ILG and conventional methods were compared between HDL 2 and HDL 3 , similar trends (HDL 2 > HDL 3 ) in both methods were observed, which suggested that the specificities of the ILG method and cell-based method to HDL were similar, whereas the ILG method showed different behavior in CEC values of apolipoprotein E-containing HDL and free apoA-I from the cell-based method: CEC values by the ILG method were quite lower than those by the cell-based method [ 16 ].…”

“…However, it has already been reported that the CEC of the conventional cellular method is well correlated with the ILG method, and CEC values of BDS reflect the HDL-CEC [ 15 , 16 ]. Moreover, when CEC values obtained by the ILG and conventional methods were compared between HDL 2 and HDL 3 , similar trends (HDL 2 > HDL 3 ) in both methods were observed, which suggested that the specificities of the ILG method and cell-based method to HDL were similar, whereas the ILG method showed different behavior in CEC values of apolipoprotein E-containing HDL and free apoA-I from the cell-based method: CEC values by the ILG method were quite lower than those by the cell-based method [ 16 ]. Since the ILM and ILG methods have a same specificity to HDL as mentioned above, CEC by the ILM method is also thought to generally reflect that of the conventional method using the major HDL subclasses such as HDL 2 and HDL 3 , but the ILM method has some limitations such that it does not reflect CEC of lipid-free apoA-I.…”

“…To solve this problem, we developed a novel radioisotope- and cell-free CEC assay, the immobilized liposome-bound gel beads (ILGs) method [ 15 ], to replace the conventional method, and demonstrated that the ILG method showed a good correlation with the conventional cell-based method [ 15 ]. Moreover, this method was extremely superior to the conventional method in aspects of repeatability and shorten turnaround time [ 16 ]. Thus, the ILG method enables safer, simpler, and more accurate measurements than the conventional method to evaluate CEC of serum from large numbers of subjects for running health screening or making diagnosis to predict the CVD risk.…”

Can “Bad” HDL Be Turned Good Again?

“…The CEC assay, however, is a tedious cell-based assay that typically uses radiolabeled cholesterol and is, therefore, not suitable for routine diagnostic testing. The fact that the AER assay and other recently developed cell-free surrogate assays for HDL function 13,20,21 also closely correlate with the CEC assay and ASCVD events, it may be possible to develop one of these newer fluorescent and potentially automatable assays into a routine diagnostic test. Like the well-known aphorism says, if one waits long enough, even bad things can sometimes turn good.…”