Analysis of plasma lipoproteins is very important for the diagnosis of hyperlipidemia and the investigation of atherosclerosis. In is this protocol, we'll introduce the basic method using ultracentrifugation to isolate plasma lipoproteins. This method allows the researchers to use one milliliter of plasma to isolate seven lipoprotein fractions.
These fractions can be used for the of lipids, apolipoproteins, as well as well-functional studies This method can be applied for both human and animal samples for the diagnosis of dyslipidemia and the related disease. Begin by transferring one milliliter of plasma into each polycarbonate ultracentrifuge tube. Then load the tubes in a fixed angle rotor and centrifuge the plasma at 356, 000 times G for 2.5 hours at four degrees Celsius.
Adjust the position of the blade in the tube slicer to a level between the top fraction and the bottom fraction. Cut the tubes using a slicer and collect the top fraction, approximately 200 microliters, into a new micro tube. Collect the remaining bottom fraction into a new polycarbonate ultracentrifuge tube for the next separation.
Adjust the total volume to 800 microliters by adding the same density solution. Adjust the bottom fraction to a density of 1.02 grams per milliliter by adding 58.9 microliters of 1.21 grams per milliliter solution, and 141.1 microliters of 1.02 grams per milliliter solution for a total volume of one milliliter. Load these tubes in the fixed angle rotor and centrifuge at 356, 000 times G for 2.5 hours at four degrees Celsius.
Continue collecting top fractions, adjusting the density of bottom fractions, and centrifuging for fraction densities of 1.02, 1.04, 1.06, 1.08, 1.1, and 1.21 grams per milliliter. For the final ultracentrifugation, centrifuge at 513, 000 times G for four hours at four degrees Celsius. Lipoprotein profiles of rabbits fed either a normal standard diet or a high cholesterol diet are shown here.
Rabbits are herbivores, so their plasma TC, TG, and PL levels are generally lower than those of humans and mice. In normal standard diet-fed rabbits, TC is mainly distributed in HDL3 and LDL. In wild type rabbits on a normal standard diet, 39%of plasma TG are distributed in VLDLs, whereas 57%of plasma PL is contained in HDL3.
When rabbits were challenged with a high-cholesterol diet, they rapidly developed hypercholesterolemia. Their lipoprotein profiles were characterized by marked elevation of VLDLs. Seven lipoprotein fractions from wild type rabbits fed either a normal standard or a high-cholesterol diet were run on an SDS polyacrylamide gel and visualized with CBB staining.
On a high-cholesterol diet, both ApoB-100 and ApoE contents in VLDLs, IDLs, and LDLs were markedly elevated. Western blotting was used to compare apolipoproteins and lipoprotein profiles of three different hyperlipidemic rabbits:high-cholesterol diet-fed wild type rabbits, ApoE knockout rabbits, and Watanabe Heritable Hyperlipidemic rabbits with LDL receptor deficiency. The ApoB containing particles of high-cholesterol diet-fed wild type rabbits are characterized by increased ApoB-100 and ApoE contents, whereas ApoE knockout rabbits showed an increase of ApoB-48 along with the appearance of ApoA-1.
Watanabe Heritable Hyperlipidemic rabbits are genetically deficient in LDL receptor functions, so a marked increase of ApoB-100 in LDLs accompanied by reduced ApoA-1 in HDLs was observed, similar to human familial hypercholesterolemia patients. Sample recovery is the most critical in this procedure. To minimize sample loss, one should it be careful to collect fraction and completely dissolve viscous precipitation in the bottom fraction.
After the collection of seven lipoprotein fractions, dialysis is require for the sorting. Once dialysis is complete, the lipoproteins samples for analysis using SDS page, western blotting, and cell-based study. In the clinical setting, ultracentrifugation usually separates lipoproteins into four fractions.
The current method can isolate seven lipoproteins fractions, which provides more detail of lipoproteins for investigation of hyperlipidemia.
