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Journal of Lipid Research, Vol. 43, 805-814, May 2002
Copyright © 2002 by Lipid Research, Inc.
Methods |
A new on-line dual enzymatic method for simultaneous quantification of cholesterol and triglycerides in lipoproteins by HPLC
* Department of Biochemistry and Biophysics, Graduate School of Allied Health Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan
Department of Human Life Sciences, Jissen Women's University, 4-1-1 Ohsakaue, Hino-shi, Tokyo 191-8510, Japan
Laboratory of Chemistry, College of Liberal Arts and Sciences, Tokyo Medical and Dental University, 2-8-30, Kohnodai, Ichikawa-shi, Chiba 272-0827, Japan
1 To whom correspondence should be addressed. e-mail: okazaki.las{at}tmd.ac.jp
We describe an on-line dual detection method using HPLC for lipoprotein analysis that allows simultaneous determination of cholesterol and triglyceride profiles from a single injection of sample. Two different gel permeation columns, TSKgel LipopropakXL and Superose 6HR, were applied to the dual detection system, evaluating analytical performance of the proposed method and the columns by analyzing serum samples from human and nonhuman subjects. Both TSK and Superose columns produced good within-day imprecision values less than 4.7% for cholesterol and 4.2% for triglyceride determination. Linear regression analysis showed the results from the Superose column (y) correlated well with those from the TSK column (x): y = 0.969x + 5.44 (r = 0.990) for total cholesterol (mg/dl), y = 1.08x - 11.14 (r = 0.985) for total triglycerides (mg/dl), and y = 1.093x - 0.06 (r = 0.978) for the ratios of triglycerides to cholesterol (mg/mg). Furthermore, the cholesterol and triglyceride profiles elucidated the differences in the resolution ability of the columns, which have not been apparent from a single lipid profile. We conclude that the dual detection concept with proper choice of column and enzymic reagents specific to the objectives of the particular study can facilitate studies of lipoprotein metabolism.—Usui, S., Y. Hara, S. Hosaki, and M. Okazaki. A new on-line dual enzymatic method for simultaneous quantification of cholesterol and triglycerides in lipoproteins by HPLC. J. Lipid Res. 2002. 43: 805–814.
Abbreviations: 4-AA, 4-aminoantipyrine; apo, apolipoprotein; CHO, cholesterol oxidase; CM, chylomicron; CV, coefficient of variation; EMSE, N-ethyl-N-(3-methylphenyl)-N'-succinylethylendiamine; FG, free glycerol; GPO, glycerol-3-phosphate oxidase; POD, peroxidase; R1, reagent 1; R2, reagent 2; TBA, Tris-buffered acetate
Supplementary key words gel permeation column • lipoprotein profile • hyperlipidemia • chylomicron
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