An optimized fast-performance liquid chromatography method for analyzing lipoprotein profiles using microliter volumes of serum.
Plasma or serum lipoprotein analysis is commonly carried out with a conventional size-exclusion fast-performance liquid chromatography method that requires large sample volumes (1-2 ml). To determine lipoprotein profiles of mice with this method, plasma or serum samples have to be pooled from a group of animals, which often requires sacrificing animals. Here we report an optimized anion-exchange chromatography method with simplified cholesterol collection and detection system. After 5-10 microl serum was injected for anion-exchange chromatography, a stepwise gradient was applied and fractions were collected on a 96-well plate. Cholesterol content in each well was measured using a fluorescence-based detection method. With this method, distinct lipoprotein peaks corresponding to high-density lipoprotein, low-density lipoprotein, and very-low-density lipoprotein, can be easily separated and identified with excellent resolution. The entire high-performance liquid chromatography run takes about 30min and the results are reproducible with a low variability. The small sample size allows analyzing the lipoprotein profile in a given mouse at a given time point with nonterminal bleeding. The method is simple to set up with commercially available parts and convenient to run.
Kim
Ki Won
KW
Department of Metabolic Disorders, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA.
McCormick
Jocelyn
J
Helmering
Joan
J
Véniant
Murielle M
MM
Wang
Minghan
M
D000818:Animals; D015153:Blotting, Western; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D008074:Lipoproteins / Q000097:blood*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D011817:Rabbits; D051381:Rats; D015203:Reproducibility of Results
D008074:Lipoproteins