Development and validation of novel automatable assay for cholesterol efflux capacity

Abstract

During the past decade, evaluation of high-density lipoprotein (HDL) functionality has been well studied for predicting cardiovascular disease risk. Cholesterol efflux capacity (CEC) is the strongest candidate as the biomarker out of various HDL anti-atherosclerotic functions. However, CEC has not yet been introduced clinically because of several technical issues, including the use of radioactive materials and differentiated cells in the assay. Previously, our laboratory developed a radioisotope- and cell-free CEC assay called the immobilized liposome-bound gel beads (ILGs) method to replace the conventional method. However, the separation process of the supernatant was not suitable for installation in an automatic analyzer. This study aims to develop a new method that is easier to operate. We assumed that the use of magnetic beads instead of gel beads would enable the skip of the centrifugal process. First, similar to the ILG method, porous magnetic beads were treated with liposomes containing fluorescently labeled cholesterol. Fluorescence was observed inside the magnetic beads, and almost the same amount of liposomes as in the ILG method was immobilized successfully. These immobilized liposome-bound magnetic beads (ILMs) were available for CEC assay when HDL and apolipoprotein B-100 depleted serum were used as cholesterol acceptors. The ILM method showed sufficient basic performance and a good correlation with the ILG method. Furthermore, when the CEC of 15 serum samples from healthy subjects was measured, a good correlation between HDL-cholesterol level and the ILG method was confirmed. Thus, it was confirmed that the ILM method was successfully developed and could be automated.

A commentary on the paper, ‘Development and validation of a novel automatable assay for cholesterol efflux capacity’

“…The authors of the research we are referencing, note that the traditional CEC measurement technique necessitates cultured cells and radioisotope-labeled cholesterol, both of which are impractical in clinical settings given large number of clinical samples to be analyzed. In their prior study, they created a CEC assay to circumvent this issue by using gel beads and apolipoprotein B-100-depleted serum (BDS) in place of cells and radioisotopes [ 22 ]. Unfortunately, their earlier procedure required the centrifugation step to spin down the gel beads and to separate cholesterol from supernatant that was fluorescently labeled [ 23 ].…”

“…Before ILM is used in the laboratory diagnostic settings, its stability should also be examined. The authors, therefore, assessed the ILM’s stability and their findings showed that the ILM solution was stable for 45 days at 4 degrees [ 22 ].…”

“…The present study by Mutsuda et al showed the liposomes containing cholesterol that was fluorescently tagged and successfully immobilized on magnetic beads [ 22 ]. They developed a novel CEC assay, which does not require the centrifugal step.…”