Improvement in bilirubin influence on cholesterol efflux capacity evaluation using the immobilized liposome-bound gel beads method.

INTRODUCTION: High-density lipoprotein (HDL) has a cholesterol efflux capacity (CEC) that protects against atherosclerosis. Recently, we developed an assay for CEC evaluation, named the immobilized liposome-bound gel beads (ILG) method, which is a highly accurate, simple, and safe method for CEC evaluation because it uses liposomes and BODIPY-labeled cholesterol instead of cultured cells and radioactive substances, respectively. Although the ILG method can be implemented in clinical settings, our previous study revealed that bilirubin causes a positive error in the CEC value. Therefore, in the present study, we attempted to improve the influence of bilirubin levels on the ILG method. METHODS: To investigate why bilirubin caused a positive error in CEC values when using the ILG method, 3D fluorescence spectra of BODIPY-labeled cholesterol and bilirubin were measured. To avoid the fluorescence emitted by bilirubin, CEC was measured using the ILG method with shifting of excitation wavelength for BODIPY-labeled cholesterol quantification. In addition, we used bilirubin oxidase to oxidize bilirubin during the incubation time of the ILG method to weaken bilirubin fluorescence. RESULTS: We found that bilirubin emitted fluorescence at the measurement setting of the ILG method. By shifting the excitation wavelength, the positive error caused by bilirubin was improved by approximately 70%. Furthermore, by utilizing bilirubin oxidase, the false-high values of CEC were improved by approximately 80%. CONCLUSIONS: Bilirubin interferes with CEC assay using BODIPY-cholesterol, but we successfully improved the influence of bilirubin on CEC evaluation using the ILG method. These improvements will promote the clinical application of the ILG method.

Development and validation of novel automatable assay for cholesterol efflux capacity.

During the past decade, evaluation of high-density lipoprotein (HDL) functionality has been well studied for predicting cardiovascular disease (CVD) risk. Cholesterol efflux capacity (CEC) is the strongest candidate as the biomarker out of various HDL antiatherosclerotic 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. The present 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 (BDS) 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.