Department of Analytical Laboratory Chemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), Tokyo, Japan.
Department of Clinical Laboratory Technology, Faculty of Medical Sciences, Juntendo University, 6-8-1, Hinode, Urayasu, Chiba 279-0013, Japan.
Biosci Rep. 2023 Jun 28;43(6). doi: 10.1042/BSR20230393.
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.
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.
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%.
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.
高密度脂蛋白（HDL）具有胆固醇外排能力（CEC），可预防动脉粥样硬化。最近，我们开发了一种用于 CEC 评估的测定法，命名为固定化脂质体结合凝胶珠（ILG）法，这是一种高度准确、简单且安全的 CEC 评估方法，因为它分别使用脂质体和 BODIPY 标记的胆固醇代替培养细胞和放射性物质。尽管 ILG 法可在临床环境中实施，但我们之前的研究表明胆红素会导致 CEC 值出现正误差。因此，在本研究中，我们试图改善胆红素水平对 ILG 法的影响。
为了研究为什么在使用 ILG 法时胆红素会导致 CEC 值出现正误差，我们测量了 BODIPY 标记的胆固醇和胆红素的 3D 荧光光谱。为了避免胆红素发出的荧光，我们通过改变 ILG 法中 BODIPY 标记的胆固醇定量的激发波长来测量 CEC。此外，我们在 ILG 法孵育时间内使用胆红素氧化酶氧化胆红素，以减弱胆红素的荧光。
我们发现胆红素在 ILG 法的测量设置下发出荧光。通过改变激发波长，胆红素引起的正误差提高了约 70%。此外，通过利用胆红素氧化酶，CEC 的假性高值提高了约 80%。
胆红素会干扰使用 BODIPY-胆固醇的 CEC 测定，但我们成功地改善了 ILG 法中胆红素对 CEC 评估的影响。这些改进将促进 ILG 法的临床应用。
