Novel cholesterol efflux assay using immobilized liposome-bound gel beads: Confirmation and improvement for application in clinical laboratory

Objectives: Cholesterol 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. Methods: Cholesterol 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. Results: The 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. Conclusions: Present ILG method further fulfills some requirements for application in clinical laboratory. Using this reliable simple method, evaluation for clinical significance of CEC is expected.