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

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

First Department of Internal Medicine, Showa University School of Medicine, Tokyo 142-8666

Corresponding Author: 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 HDL-cholesterol (C) assay. A precipitation reagent (0.06 ml) containing 1,071 units/ml heparin, 500 mmol/l MnCl2, and 12 mg/ml dextran sulfate (DS) 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=0.938 and 0.979, respectively). The two methods also proved to be highly correlated in the measurement of apolipoprotein AI and AII 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.

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