An improved anion-exchange high-performance liquid chromatography method for measuring oxidized form of LDLs in human plasma.
BACKGROUND
Circulating oxidized low-density lipoproteins (LDLs) (ox-LDLs) could be a sensitive marker to predict future cardiovascular events. However, a method to evaluate oxidized forms of LDLs systemically in human plasma is not yet established. In this study, we developed a novel and convenient high-performance liquid chromatography (HPLC) method for measuring ox-LDL levels in humans.


METHODS
Human plasma lipoproteins were separated by a modified HPLC method using a diethylaminoethyl-type anion-exchange gel column with stepwise elution. Ox-LDLs were detected by postcolumn reaction with a reagent containing cholesterol esterase and cholesterol oxidase. Particle size of each LDL fraction separated by HPLC was determined in 61 healthy subjects.


RESULTS
Our HPLC method separated LDLs into three fractions, which were designated as LDL-1, LDL-2 and LDL-3, on the basis of their negative charges, with LDL-3 the most strongly retained fraction migrating fastest in the anodic direction, a property that reflects the net negative charge of the molecule. Western blot analysis revealed that apolipoprotein B100 in LDL-3 fraction was the most fragmented and oxidatively modified. When LDLs were oxidized in vitro by Cu2+ or 2,2-azo-bis (2-aminopropane)-2HCl or modified by various aldehydes, all of the LDL fractions migrated at the position of LDL-3. Further, among three fractions, particle size was smallest in LDL-3 fraction.


CONCLUSIONS
Here, we developed a convenient HPLC method and identified LDL-3 as oxidized LDL fractions, although ox-LDLs were present in LDL-2 fraction, albeit lesser concentrations than in LDL-3 subfraction. Measuring ox-LDL levels in human plasma by this method may be useful to evaluate atherosclerotic disorders.
Kitano
Soichi
S
SRL Inc, Hino, Tokyo, Japan. shoichi@med.kurume-u.ac.jp
Higashimoto
Yuichiro
Y
Harada
Shoji
S
Sano
Masaru
M
Kurata
Tsunehiko
T
Yamaguchi
Yu
Y
Kunitomo
Masaru
M
Haginaka
Jun
J
Yamagishi
Sho-ichi
S
D000328:Adult; D001774:Blood Chemical Analysis / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D005260:Female; D006801:Humans; D008077:Lipoproteins, LDL / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification; D008297:Male; D010316:Particle Size; D015203:Reproducibility of Results; D013997:Time Factors
D008077:Lipoproteins, LDL; C099252:oxidized low density lipoprotein