title: Anion-exchange HPLC separation of five major rabbit lipoproteins using a nonporous diethylaminoethyl-ligated gel with a perchlorate-containing eluent.
abstract: 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 NaClO₄ 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 NaClO₄. 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.
authors.affiliation: Bioscience Division, Tosoh Corporation, 2743-1 Hayakawa Ayase-shi, Kanagawa 252-1123, Japan. yuujihirowatari-bx@tosoh.co.jp
authors.forename: Yuji
authors.initials: Y
authors.lastname: Hirowatari
authors.forename: Mika
authors.initials: M
authors.lastname: Kon
authors.forename: Yuko
authors.initials: Y
authors.lastname: Shimura
authors.forename: Satoshi
authors.initials: S
authors.lastname: Hirayama
authors.forename: Takashi
authors.initials: T
authors.lastname: Miida
mesh_terms: D000818:Animals; D002784:Cholesterol / Q000097:blood / Q000302:isolation & purification; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004048:Diethylamines / Q000737:chemistry; D008074:Lipoproteins / Q000097:blood* / Q000302:isolation & purification*; D008297:Male; D010472:Perchlorates / Q000737:chemistry; D016062:Porosity; D011817:Rabbits; D012680:Sensitivity and Specificity
chemical_list: D004048:Diethylamines; D008074:Lipoproteins; D010472:Perchlorates; D002784:Cholesterol; C494474:perchlorate