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Journal of Lipid Research, Vol. 49, 1130-1136, May 2008
Copyright © 2008 by American Society for Biochemistry and Molecular Biology

Methods

A simple and precise method for measuring HDL-cholesterol subfractions by a single precipitation followed by homogenous HDL-cholesterol assay

Tsutomu Hirano^1,*, Kyoko Nohtomi^*, Shinji Koba, Ayako Muroi and Yasuki Ito

^* First Department of Internal Medicine, Showa University, School of Medicine, Tokyo, Japan
Third Department of Internal Medicine, Showa University, School of Medicine, Tokyo, Japan
Research and Development Department, Denka Seiken Co., Ltd., Tokyo, Japan

Published, JLR Papers in Press, January 27, 2008.

¹ To whom correspondence should be addressed. e-mail: hirano{at}med.showa-u.ac.jp

HDL consists of two major subfractions, HDL₂ and HDL₃. This paper describes a simple method for assaying HDL subspecies by combining a single precipitation with a direct high density lipoprotein-cholesterol (HDL-C) assay. A precipitation reagent (0.06 ml) containing 1,071 U/ml heparin, 500 mmol/l MnCl_2, and 12 mg/ml dextran sulfate was added to a serum (0.3 ml). The sample was incubated and centrifuged at 10,000 rpm for 10 min. HDL₃-C was measured by a homogenous HDL-C assay in the supernatant, and HDL₂-C was estimated by subtracting the HDL₃-C from the direct HDL-C. The HDL₃-C and HDL₂-C values determined by the precipitation method were identical to those determined by ultracentrifugation, and there were excellent correlations between the methods in the measurements of HDL₃-C and HDL₂-C (r = 0.933 and 0.978, respectively; n = 102). The two methods also proved to be highly correlated in the measurement of apolipoprotein A-I and A-II in HDL subfractions. The HDL-C subfractions determined by ultracentrifugation were more closely associated with the homogenous HDL-C assay than with the total cholesterol assay, especially in the hypertriglyceridemic samples. Our method is far simpler and more precise than the classical dual precipitation method for HDL-C subfractions, and it can be easily performed in a routine chemical laboratory

Supplementary key words high density lipoprotein-cholesterol • direct homogenous assay • triglyceride

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