title: Analyzing of high-density lipoprotein subfractions and low-density lipoprotein subfractions in human serum with anion-exchange chromatography.
abstract: 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.
authors.affiliation: Bioscience Division, Tosoh Corporation, 2743-1 Hayakawa Ayase-shi, Kanagawa 252-1123, Japan. hirowata@tosoh.co.jp
authors.forename: Yuji
authors.initials: Y
authors.lastname: Hirowatari
authors.forename: Yuko
authors.initials: Y
authors.lastname: Tsunoda
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Ogura
authors.forename: Yasuhiko
authors.initials: Y
authors.lastname: Homma
mesh_terms: 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
chemical_list: 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