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Anion‐exchange HPLC separation of five major rabbit lipoproteins using a nonporous diethylaminoethyl‐ligated gel with a perchlorate‐containing eluent
Author(s) -
Hirowatari Yuji,
Kon Mika,
Shimura Yuko,
Hirayama Satoshi,
Miida Takashi
Publication year - 2012
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.1683
Subject(s) - chemistry , chromatography , lipoprotein , chylomicron , elution , cholesterol , column chromatography , hyperlipidemia , reagent , very low density lipoprotein , high density lipoprotein , size exclusion chromatography , ion chromatography , high performance liquid chromatography , ion exchange , biochemistry , ion , enzyme , endocrinology , organic chemistry , medicine , diabetes mellitus
Rabbits are often used as experimental animals in studies of atherosclerosis and hyperlipidemia. Although rabbit lipoproteins can be quantitated by sequential ultracentrifugation, a simpler and more reproducible method is desirable for detailed analyses. The current study describes a method to analyze rabbit lipoproteins in plasma by anion‐exchange high‐performance liquid chromatography using a column filled with nonporous, diethylaminoethyl‐ligated polymers. A solution of NaClO 4 was used to adjust the ionic strength of the eluent. The method required only 15 μL of plasma and analysis was completed in 23 min. Five lipoprotein fractions (high‐density lipoprotein, low‐density lipoprotein, intermediate‐density lipoprotein, very‐low‐density lipoprotein and chylomicrons) were eluted with step‐wise increases in a concentration of NaClO 4 . The post‐column eluate was reacted with an enzymatic reagent to determine total cholesterol, and the lipoprotein‐cholesterol fraction was calculated according to relative peak areas in the chromatogram. The within‐day and between‐day assay coefficients of variation for lipoprotein cholesterol levels ranged between 0.436 and 7.143% and between 2.905 and 10.526%, respectively. Administering a high‐fat diet increased lipoprotein‐cholesterol concentrations by 6‐ to 77‐fold. The method described here was nevertheless able to quantitate levels of lipoprotein‐cholesterol in plasma samples from these rabbits. These results indicate that this method may be applied to lipoprotein studies using hyperlipidemic rabbit models. Copyright © 2011 John Wiley & Sons, Ltd.