Development of a Novel Homogeneous Assay for Remnant Lipoprotein Particle Cholesterol

Abstract

Background: Quantification of remnant lipoprotein particle cholesterol (RLP-C) by automated assay is useful in routine clinical laboratories to assess coronary artery disease risk and diagnose type III hyperlipoproteinemia. Methods: Enzymes and surfactants were screened to establish a homogeneous RLP-C assay using the chylomicron-VLDL, LDL, and HDL fractions isolated by ultracentrifugation, along with the RLP fraction isolated by immunoaffinity gel. All data were generated using a Hitachi analyzer. Results: A specific cholesterol esterase with a polyoxyethelene styrenated phenyl ether derivative (surfactant) was used for the establishment of a homogeneous RLP-C assay. This cholesterol esterase with subunits of >40 kDa (H-CE) was found to react with lipoproteins other than RLP, whereas this enzyme with subunits of <40 kDa (L-CE) reacted with RLP. H-CE was applied for the first reaction step with the specific surfactant to decompose non-RLP lipoproteins, degrading non-RLP cholesterol into water and oxygen in the presence of cholesterol oxidase and catalase. For the second step, L-CE was applied to release cholesterol from RLP, and then the released RLP-C was determined in a standard cholesterol oxidase and peroxidase system. This new homogeneous assay exhibited good correlation with the RLP-C immunoseparation method. Conclusions: We established a simple, rapid, automated homogeneous assay for RLP-C. The assay can determine RLP-C levels in 10 min in a fully automated manner, processing a large number of samples in routine clinical laboratories.