Received on May 21, 2007
Accepted on September 5, 2007

Lipids, Lipoproteins, and Cardiovascular Risk Factors

Development of a Homogeneous Assay to Measure Remnant Lipoprotein Cholesterol

¹ Scientific and Technical Affairs Department, Kyowa Medex Co., Ltd., Tokyo, Japan
² Research and Development Department, Kyowa Medex Co., Ltd., Tokyo, Japan
³ Division of Health Sciences, Osaka University Graduate School of Medicine, Osaka, Japan
⁴ Research Laboratory Department, Kyowa Medex Co., Ltd., Shizuoka, Japan
⁵ Kumamoto Health Science University, Kumamoto, Japan
⁶ Faculty of Medical and Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan
⁷ Laboratory of Clinical Nutrition and Medicine, Kagawa Nutrition University, Saitama, Japan
⁸ Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Osaka, Japan

^* To whom correspondence should be addressed. E-mail: norihiko.kayahara{at}kyowa.co.jp.

Background: Quantification of triglyceride-rich lipoprotein (TRL) remnants is useful for risk assessment of coronary artery disease and the diagnosis of type III hyperlipoproteinemia. Although an immunoseparation procedure for remnant-like particle cholesterol has been evaluated extensively in recent years, available methods for measuring TRL remnants have not achieved wide use in routine laboratory practice, suggesting a need for a homogeneous assay that can measure TRL remnant cholesterol in serum or plasma without pretreatment.

Methods: We screened for suitable surfactants that exhibited favorable selectivity toward the VLDL remnant (VLDLR) fraction, including intermediate-density lipoproteins (IDLs). We investigated the principal characteristics of this assay by gel filtration of lipoproteins and their particle size distribution. We developed a simple assay and evaluated its performance with the Hitachi-7170 analyzer.

Results: Polyoxyethylene-polyoxybutylene block copolymer (POE-POB) exhibited favorable selectivity toward VLDLR and IDL fractions. POE-POB removed apolipoprotein (apo) E and apo C-III from IDL particles in the presence of cholesterol esterase (CHER), and the particle size distribution of IDLs became smaller after the reaction. These results revealed that IDL particles are specifically modified in the presence of CHER and POE-POB, making their component cholesterol available for enzymatic assay. Addition of phospholipase D improved the reactivity toward chylomicron remnants (CMRs). We found a high correlation [y = 1.018x– 0.01 mmol/L[d1], r = 0.962 (n = 160)] between the proposed assay and the immunoseparation assay in serum from healthy individuals.

Conclusion: The homogeneous assay described in this report can measure TRL remnant cholesterol, including CMRs, VLDLRs, and IDLs, with high sensitivity and specificity.