Analyzing of high-density lipoprotein subfractions and low-density lipoprotein subfractions in human serum with anion-exchange chromatography.
OBJECTIVE
We previously established a separation method for five lipoprotein classes (HDL, LDL, IDL, VLDL and chylomicrons) in human serum by using anion-exchange high-performance liquid chromatography (AEX-HPLC). It was thought that analyzing of the subfractions of HDL or LDL is important for study of the atherogenic lipoproteins in atherosclerotic diseases, because a large number of studies were reported regarding them. Therefore, we estimated a separation mode of AEX-HPLC for analyzing cholesterol levels of two HDL-subfractions and two LDL-subfractions.


METHODS
In this method, the lipoproteins were separated by a step gradient of sodium perchlorate concentration, and the separated lipoproteins were detected by using a post-column reactor with a reagent containing cholesterol esterase and cholesterol oxidase.


RESULTS
The major component of earlier-eluting fraction in HDL (HDL-f) and later-eluting fraction in HDL (HDL-s) were HDL3 and HDL2, respectively. The retention time of peak of earlier-eluting fraction in LDL (LDL-f) was changed into that of later-eluting fraction in LDL (LDL-s) by the oxidization, incubating 2 micromol/l copper ion in PBS for 3h at 37 degrees C. The within- and between-day assay coefficients of variation (CV) for cholesterol levels of the lipoprotein subfractions were in the ranges of 0.85-19.23% and 2.55-23.50%, respectively. The cholesterol levels of HDL-f and HDL-s were lower in patients with coronary heart disease (CHD) than in healthy controls, while LDL-s cholesterol levels were higher in CAD patients than in healthy controls.


CONCLUSIONS
This accurate and convenient assay of the lipoprotein subfractions may be useful for studying the atherogenic lipoproteins.
Hirowatari
Yuji
Y
Bioscience Division, Tosoh Corporation, 2743-1 Hayakawa Ayase-shi, Kanagawa 252-1123, Japan. hirowata@tosoh.co.jp
Tsunoda
Yuko
Y
Ogura
Yutaka
Y
Homma
Yasuhiko
Y
D000328:Adult; D000368:Aged; D000837:Anion Exchange Resins; D015415:Biomarkers / Q000097:blood; D016022:Case-Control Studies; D002784:Cholesterol / Q000097:blood; D008076:Cholesterol, HDL / Q000097:blood*; D008078:Cholesterol, LDL / Q000097:blood*; D015243:Cholesterol, VLDL / Q000097:blood; D002851:Chromatography, High Pressure Liquid / Q000592:standards; D002852:Chromatography, Ion Exchange / Q000379:methods* / Q000592:standards; D002914:Chylomicrons / Q000097:blood; D003327:Coronary Disease / Q000097:blood*; D005260:Female; D006801:Humans; D008074:Lipoproteins / Q000097:blood; D008077:Lipoproteins, LDL / Q000097:blood; D008297:Male; D008875:Middle Aged; D015203:Reproducibility of Results; D014461:Ultracentrifugation
D000837:Anion Exchange Resins; D015415:Biomarkers; D008076:Cholesterol, HDL; D008078:Cholesterol, LDL; D015243:Cholesterol, VLDL; D002914:Chylomicrons; D008074:Lipoproteins; D008077:Lipoproteins, LDL; C030104:lipoprotein cholesterol; C099252:oxidized low density lipoprotein; D002784:Cholesterol