Novel cholesterol efflux assay using immobilized liposome-bound gel beads: Confirmation and improvement for application in clinical laboratory
Novel cholesterol efflux assay using immobilized liposome-bound gel beads: Confirmation and improvement for application in clinical laboratory
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使用固定化脂质体结合凝胶珠的新型胆固醇流出测定：临床实验室应用的确认和改进
DOI：
10.1177/00045632211054406
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发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Ohkawa Ryunosuke
中科院分区：
文献类型：
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作者：
Horiuchi Yuna;Lai Shao-Jui;Kameda Takahiro;Tozuka Minoru;Ohkawa Ryunosuke
胆固醇外排能力（CEC）是高密度脂蛋白的一种抗动脉粥样硬化功能，有望成为心血管疾病的潜在生物标志物。然而，由于使用细胞和放射性物质的常规CEC测定的复杂性，CEC尚未被广泛引入临床实验室中应用。以前，我们开发了一种新的CEC测定使用固定化脂质体结合的凝胶珠（ILG），解决了这些问题。我们的目的是确认的验证，并进一步提高ILG方法的应用在clinical setting.MethodsCholesterol efflux capacity值由ILG方法测定较短的孵育时间（4小时）进行了比较，那些测定为16小时（我们以前的ILG方法）。为了研究可校正ILG生产批次之间差异的参比物质，对牛血清白蛋白、人γ-球蛋白和球蛋白复合物进行了评价。CEC值也估计在血浆中获得与不同的抗凝剂，血清处理与冻融cycle，和血清混合有几个干扰substances. ResultsCEC的4-和16-h孵育时间有很好的相关性。球蛋白复合物可用作参比物质。血浆可用作标本。血清和标本保存温度对CEC无明显影响。血红蛋白和乳糜对CEC没有影响，而高胆红素血清显示CEC升高。胆红素的影响几乎可以通过减去基础荧光强度来抵消。结论ILG方法进一步满足了临床实验室的应用要求。该方法简便、可靠，有望用于临床CEC的评价。
ObjectivesCholesterol efflux capacity (CEC), an atheroprotective function of high-density lipoprotein, is expected to be a potential biomarker for cardiovascular disease. However, CEC has not been widely introduced for application in clinical laboratories because of the complexity of the conventional CEC assay using cells and radioactive materials. Previously, we developed a novel CEC assay using immobilized liposome-bound gel beads (ILG), which solves these issues. We aimed to confirm the validation and further improve the ILG method for application in the clinical setting.MethodsCholesterol efflux capacity values by the ILG method assayed for shorter incubation time (4 h) were compared to those assayed for 16 h (our previous ILG method). To investigate a reference material that can correct the variation between ILG manufacturing lots, bovine serum albumin, human gamma-globulins, and globulin complexes were evaluated. CEC values were also estimated in plasmas obtained with different anticoagulants, serum treated with freeze-thaw cycles, and serum mixed with several interference substances.ResultsThe CEC of 4- and 16-h incubation times were well correlated. Globulin complexes may be used as a reference material. Plasma can be used as the specimen. The serum and stored temperature of the specimen did not largely affect CEC. Hemoglobin and chyle did not have an effect on CEC, whereas high-bilirubin serum showed elevated CEC. The effect of bilirubin was nearly canceled by subtracting basal fluorescence intensity.ConclusionsPresent ILG method further fulfills some requirements for application in clinical laboratory. Using this reliable simple method, evaluation for clinical significance of CEC is expected.