J. Lipid Res.
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Originally published In Press as doi:10.1194/jlr.D300003-JLR200 on May 1, 2003

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Journal of Lipid Research, Vol. 44, 1404-1412, July 2003
Copyright © 2003 by American Society for Biochemistry and Molecular Biology

Methods

Measurement of cholesterol of major serum lipoprotein classes by anion-exchange HPLC with perchlorate ion-containing eluent

Yuji Hirowatari1,*, Hiroshi Yoshida{dagger}, Hideo Kurosawa§, Ken-iti Doumitu§ and Norio Tada{dagger}

* Scientific Instruments Division, TOSOH Corp., 2743-1 Hayakawa Ayase-shi, Kanagawa 252-1123, Japan
{dagger} Department of General Medicine, Kashiwa Hospital, Jikei University School of Medicine, 163-1 Kashiwashita Kashiwa-shi, Chiba 277-8567, Japan
§ Department of Clinical Laboratory, Kashiwa Hospital, Jikei University School of Medicine, 163-1 Kashiwashita Kashiwa-shi, Chiba 277-8567, Japan

1 To whom correspondence should be addressed. e-mail: hirowata{at}tosoh.co.jp

We have developed a high-performance liquid chromatography (HPLC) method for measurement of cholesterol in the major classes of serum lipoproteins, i.e., HDL, LDL, IDL, VLDL, and chylomicrons. Lipoproteins in serum were separated on a column containing diethylaminoethyl-ligand nonporous polymer-based gel by elution with a step gradient of sodium perchlorate concentration, and detected by post-column reaction with a reagent containing cholesterol esterase and cholesterol oxidase. The within-day assay and between-day assay coefficients of variation for cholesterol concentration in lipoproteins were in the ranges of 0.9–6.4% and 1.1–11.9%, respectively. The correlation coefficients between the values of HDL, LDL, IDL, VLDL, and chylomicron cholesterol measured by the HPLC method and those estimated by an ultracentrifugation method were 0.892, 0.921, 0.840, 0.930, and 0.873, respectively. Values of remnant-like particle cholesterol measured by an immunoseparation technique (Japan Immunoresearch Laboratories, Japan) were significantly correlated with VLDL and chylomicron cholesterol values measured by the HPLC method (r = 0.883 and r = 0.729, respectively).

This rapid and accurate HPLC method was successfully applied to the analysis of plasma lipoproteins of patients with hyperlipidemia.

Abbreviations: CV, coefficient of variation; CAD, coronary artery disease; RLP, remnant-like particle; TC, total cholesterol; TG, triglyceride

Supplementary key words hyperlipidemia • high-performance liquid chromatography • lipoprotein remnant-like particle


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