Original Articles

An improved anion-exchange high-performance liquid chromatography method for measuring oxidized form of LDLs in human plasma

Soichi Kitano1, Yuichiro Higashimoto2, Shoji Harada1, Masaru Sano3, Tsunehiko Kurata3, Yu Yamaguchi4, Masaru Kunitomo4, Jun Haginaka5 and Sho-ichi Yamagishi6

1 SRL Inc, Hino, Tokyo; 2 Department of Chemistry, Kurume University School of Medicine, Kurume, Fukuoka; 3 Performance Products Segments, Specialty Chemicals Research Center, Mitsubishi Chemical Corporation, Yokohama, Kanagawa; 4 Department of Pharmacology; 5 Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, Mukogawa Women’s University, Nishinomiya, Hyogo; 6 Department of Pathophysiology and Therapeutics of Diabetic Vascular Complications, Kurume University School of Medicine, 67 Asahi-machi, Kurume, Fukuoka 830-0011, Japan

Corresponding author: Sho-ichi Yamagishi. Email: [email protected]

Background: Circulating oxidized low-density lipoproteins (LDLs) (ox-LDLs)could be a sensitive marker to predict future cardiovascularevents. However, a method to evaluate oxidized forms of LDLssystemically in human plasma is not yet established. In thisstudy, we developed a novel and convenient high-performanceliquid chromatography (HPLC) method for measuring ox-LDL levelsin humans.

Methods: Human plasma lipoproteins were separated by a modified HPLCmethod using a diethylaminoethyl-type anion-exchange gel columnwith stepwise elution. Ox-LDLs were detected by postcolumn reactionwith a regent containing cholesterol esterase and cholesteroloxidase. Particle size of each LDL fraction separated by HPLCwas determined in 61 healthy subjects.

Results: Our HPLC method separated LDLs into three fractions, which weredesignated as LDL-1, LDL-2 and LDL-3, on the basis of theirnegative charges, with LDL-3 the most strongly retained fractionmigrating fastest in the anodic direction, a property that reflectsthe net negative charge of the molecule. Western blot analysisrevealed that apolipoprotein B100 in LDL-3 fraction was themost fragmented and oxidatively modified. When LDLs were oxidizedin vitro by Cu2+ or 2,2-azo-bis (2-aminopropane)-2HCl or modifiedby various aldehydes, all of the LDL fractions migrated at theposition of LDL-3. Further, among three fractions, particlesize was smallest in LDL-3 fraction.

Conclusion: Here, we developed a convenient HPLC method and identified LDL-3as oxidized LDL fractions, although ox-LDLs were present inLDL-2 fraction, albeit lesser concentrations than in LDL-3 subfraction.Measuring ox-LDL levels in human plasma by this method may beuseful to evaluate atherosclerotic disorders.