A-209 Application of Bilirubin Oxidase for Improvement of False Positive Error Caused by Bilirubin on Cholesterol Efflux Capacity Assay by Immobilize Liposome-Bound Gel Beads method
Authors: Miyakoshi T , Mutsuda Y , Horiuchi Y , Kameda T , Tozuka M , Ohkawa R 
Published: September 2023

Abstract

Background
High density lipoprotein (HDL) exerts various atheroprotective functions including cholesterol efflux capacity (CEC). HDL can attenuate lipid burden at atherosclerotic lesion by CEC. Based on the HDL`s role for moderate atherosclerosis, HDL-cholesterol level which indicates the amount of HDL has been evaluated to assess the risk of cardiovascular disease (CVD). Nevertheless, many clinical studies demonstrated that HDL-cholesterol level does not always reflect the risk of CVD, and CEC which represents the quality of HDL is considered as surrogate biomarker for CVD. CEC is prospective biomarker for CVD risk assessment, however, CEC measurement encounters barriers for clinical application because of its requirements of cultured cell and radioactive substance for conventional CEC assay. To overcome these problems, we invented a novel CEC assay called immobilized liposome-bound gel beads (ILG) method. This method is cell-free assay and utilizes a fluorescence substance as cholesterol tracer. Although the ILG method is useful assay for clinical application, previous study revealed that bilirubin caused false positive error on CEC value. Hence, current ILG method might produce incorrect CEC evaluation for patients with hyperbilirubinemia and even healthy subjects within the range of biological variation of bilirubin. In this study, we improved the influence of bilirubin for ILG method by using bilirubin oxidase (BOD).

Methods
BOD (Asahi Kasei Corp.) was added to 10 mM Tris-HCl (pH 7.4) containing 150 mM of NaCl and 1 mM of Na2EDTA (Buffer A). Bilirubin from ‘Interference check A Plus’ (Sysmex Inc.) was added to serum (free-: 10 mg/dL, conjugated-:9.7 mg/dL), and apoB-depleted serum (BDS) was derived from the bilirubin additive serum using polyethylene glycol. After the prepared bilirubin additive BDS was incubated with BOD-containing Buffer A for 16 h, the absorbance spectrum was analyzed. In addition, the time course of bilirubin decrement was monitored during 16 h-incubation. We further measured the fluorescence intensity of bilirubin additive BDS incubated with BOD-containing Buffer A. Using the newly developed BOD-containing Buffer A for ILG method, CEC of bilirubin additive serum was evaluated. Finally, we measured CEC of 4 healthy individuals by conventional and the improved ILG method. The CEC value was standardized by fluorescence intensity of reference sample which was measured in every assay.

Results
BOD contained in Buffer A could convert bilirubin to biliverdin from results of absorbance spectrum. According to the time course of bilirubin decrement, both free- and conjugated-bilirubin were oxidized to biliverdin in approximately 6 h. By converting bilirubin to biliverdin, fluorescence light was weakened greatly. We confirmed that there was no significant difference of CEC values between non bilirubin-additive serum and bilirubin additive serum. Compared healthy individual CEC values measured by conventional and improved ILG method, all subjects showed significant decrease of CEC values from the improved method, and false positive error caused by bilirubin was ameliorated.

Conclusion
In this study, we achieved to improve the positive error caused by bilirubin on CEC assay using ILG method. This improvement would contribute to more accurate CEC assessment by ILG method and advance the clinical application of this method.