J. Lipid Res.
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Originally published In Press as doi:10.1194/jlr.D700027-JLR200 on January 27, 2008

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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 Hirano1,*, Kyoko Nohtomi*, Shinji Koba{dagger}, Ayako Muroi§ and Yasuki Ito§

* First Department of Internal Medicine, Showa University, School of Medicine, Tokyo, Japan
{dagger} 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 MnCl2, 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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