Several methods have been used for analyzing plasma lipoproteins; however, ultracentrifugation is still one of the most popular and reliable methods. Here, we describe a method regarding how to isolate lipoproteins from plasma using sequential density ultracentrifugation and how to analyze the apolipoproteins for both diagnostic and research purposes.
Analysis of plasma lipoproteins and apolipoproteins is an essential part for the diagnosis of dyslipidemia and studies of lipid metabolism and atherosclerosis. Although there are several methods for analyzing plasma lipoproteins, ultracentrifugation is still one of the most popular and reliable methods. Because of its intact separation procedure, the lipoprotein fractions isolated by this method can be used for analysis of lipoproteins, apolipoproteins, proteomes, and functional study of lipoproteins with cultured cells in vitro. Here, we provide a detailed protocol to isolate seven lipoprotein fractions including VLDL (d<1.006 g/mL), IDL (d=1.02 g/mL), LDLs (d=1.04 and 1.06 g/mL), HDLs (d=1.08, 1.10, and 1.21 g/mL) from rabbit plasma using sequential floating ultracentrifugation. In addition, we introduce the readers how to analyze apolipoproteins such as apoA-I, apoB, and apoE by SDS-PAGE and Western blotting and show representative results of lipoprotein and apolipoprotein profiles using hyperlipidemic rabbit models. This method can become a standard protocol for both clinicians and basic scientists to analyze lipoprotein functions.
Dyslipidemia is the major risk factor of atherosclerotic disease in the world. High levels of low-density lipoproteins (LDLs) and low levels of high-density lipoproteins (HDLs) are closely associated with a high risk of coronary heart disease (CHD)1,2. In the clinical setting, both LDL-cholesterol (LDL-C) and HDL-cholesterol (HDL-C) are routinely measured using an automated analyzer in a clinical laboratory3,4. Despite this, it is essential to analyze lipoprotein profiles in details for the diagnosis of dyslipidemia and the study of lipid metabolism and atherosclerosis in human and experimental animals. Several methods have been reported to analyze plasma lipoproteins such as ultracentrifugation, size exclusion chromatography [fast protein liquid chromatography (FPLC) and high performance liquid chromatography (HPLC)], electrophoresis by agarose and polyacrylamide gels, nuclear magnetic resonance, and selective chemical precipitation using polyanions and divalent cations or other chemicals. In 1950s, Havel’s group first proposed the concept of lipoproteins defined by densities using ultracentrifugation and classified them into chylomicrons (CM), very-low-density lipoproteins (VLDL), intermediate-density lipoproteins (IDL), LDL, and HDL5 and later on, the method was further modified by other groups6,7. Until now, ultracentrifugation is the most popular and reliable method while the practical protocol is still not available. In this paper, we attempted to describe an easy-to-use protocol for isolating a small scale of plasma using sequential density floating ultracentrifugation originally described previously8. Isolation of seven plasma lipoprotein fractions [VLDL (d<1.006 g/mL), IDL (d=1.02 g/mL), LDLs (d=1.04 and 1.06 g/mL), HDLs (d=1.08, 1.10, and 1.21 g/mL)] enables researchers to make an extensive analysis of both lipoproteins and their compositional apolipoproteins9,10,11. The intact seven consecutive density lipoproteins can be used for analyzing lipoprotein functions and also serving to cell-based in vitro strategies. This protocol should be useful for both clinical diagnosis and basic research. Here we used rabbit plasma as an example to demonstrate this technique while plasma from other species can be applied in the same way.
All procedures for rabbit studies were performed with approval of University of Yamanashi Institutional Animal Care and Use Committee (Approved number: A28-39).
1. Plasma separation from rabbit blood
2. Isolation of plasma lipoproteins
NOTE: The schematic procedure is shown in Figure 1. The preparation method of potassium bromide (KBr) density solutions is shown in Table 1.
3. Dialysis
NOTE: Because the density fractions (except d<1.006 g/mL fraction) contain high concentrations of KBr, it is necessary to remove them by dialysis.
4. Analysis of lipoproteins
NOTE: After dialysis, these lipoproteins are ready for different analyses. Lipoproteins can be evaluated by measuring either lipids or apolipoproteins or both. For measuring lipid contents such as total cholesterol (TC), triglycerides (TG), phospholipids (PL), and free cholesterol (FC) in each fraction, we use commercial enzymatic colorimetric assay kits. For analysis of apolipoproteins, we use SDS-PAGE visualized with CBB staining or Western blotting.
Using this protocol, we isolated rabbit lipoproteins using 1 mL of plasma and obtained seven density fractions. Isolated density fractions are enough for measuring lipids and apolipoproteins as described above for most research purposes. The same procedure can also be used for isolating plasma lipoproteins from human and other species. For small-sized animals such as mice, pooled plasma is required. Figure 3 shows lipoprotein profiles of wild-type (WT) rabbits fed either a normal standard (NS) diet or high cholesterol (HC) diet. Rabbits are herbivore animals so their plasma TC, TG, and PL levels are generally lower than humans and mice. In NS diet-fed rabbits, TC is mainly distributed in HDL3 (d=1.21 g/mL) and followed by LDL (d=1.04 g/mL) (Figure 3A). On a NS diet, 39% of plasma TG are distributed in VLDLs whereas 57% of plasma PL is contained in HDL3 (Figure 3A). When rabbits were challenged with a diet supplemented with high cholesterol, they rapidly developed into hypercholesterolemia. As shown in Figure 3B, the lipoprotein profiles are characterized by marked elevation of VLDLs (165-fold↑ in VLDL-TC, 1.5-fold↑ in VLDL-TG, and 30-fold↑ in VLDL-PL compared with NS-fed rabbits). Because VLDLs isolated from cholesterol-fed rabbits are rich in cholesteryl esters and move to the β position on agarose gel electrophoresis, they are often called β-VLDLs to distinguish them from normal VLDLs which move to pre-β position.
In addition to lipids, apolipoproteins can be simply analyzed by SDS-PAGE either by CBB staining or Western blotting (Figure 4). Seven lipoprotein fractions from WT rabbits fed either a NS or a HC diet were run on a 4-20% SDS-polyacrylamide gel and visualized with CBB staining (Figure 4A). On a HC diet, both apoB-100 and apoE contents in VLDLs (d<1.006 g/mL), IDLs (d=1.02 g/mL), and LDLs (d=1.04 g/mL) were markedly elevated compared with rabbits on a NS diet. Furthermore, we also compared apolipoprotein and lipoprotein profiles of three different hyperlipidemic rabbits: HC diet-fed WT rabbits, apoE knockout (KO) rabbits, and Watanabe heritable hyperlipidemic (WHHL) rabbits with LDL receptor deficiency by Western blotting (Figure 4B). Plasma lipoproteins were fractionated by 4-20% SDS-PAGE and followed by Western blotting with antibodies against apoE, apoB, and apoA-I. The apoB-containing particles (VLDLs, IDLs and LDLs) of HC diet-fed WT rabbits are characterized by increased apoB-100 and apoE contents whereas apoE KO rabbits showed marked increase of apoB-48 along with the appearance of apoA-I. WHHL rabbits are genetically deficient in LDL receptor functions so there is a marked increase of apoB-100 in LDLs accompanied by reduced apoA-I in HDLs, which is similar to human familial hypercholesterolemia.
Figure 1: Schematic illustration of sequential floating ultracentrifugation. Please click here to view a larger version of this figure.
Figure 2: Representative images of plasma lipoprotein isolation. (A) Plasma of normolipidemic and hyperlipidemic rabbits. (B) Floated top VLDL (d<1.006 g/mL) and bottom fraction (d>1.006 g/mL) after ultracentrifugation. (C) Tube slicing and collection of the top fraction by a pipette. (D) Collection of the bottom fraction. Please click here to view a larger version of this figure.
Figure 3: Quantitation of lipid contents in each lipoprotein fraction isolated from normal rabbits (A) and cholesterol-fed rabbits (B). Plasma lipoproteins were separated by sequential floating ultracentrifugation from plasma of wild-type rabbits on either a normal standard diet (A) or a high cholesterol diet (B). Total cholesterol (TC), triglycerides (TG) and phospholipids (PL) were measured. Data are expressed as mean ± SEM (n=6). This figure has been modified from Yan H et al.12. Please click here to view a larger version of this figure.
Figure 4: Comparison of apolipoprotein distribution using CBB staining (A) and Western blotting (B). Plasma was isolated by ultracentrifugation and lipoproteins were fractionated by 4-20% SDS-PAGE. (A) Apolipoprotein profiles of wild-type (WT) rabbit fed on a normal standard (NS) diet and a high cholesterol (HC) diet were visualized by CBB staining. Compared with NS diet-fed rabbits (left), HC diet-fed rabbits (right) showed increased apoB-100 and apoE contents in VLDLs, IDLs, and LDLs. (B) Comparison of apolipoprotein features of WT rabbits on a NS or a HC diet, apoE KO (knockout) rabbits on a HC diet, and WHHL rabbits on a NS diet. Western blotting was performed with “cocktail antibodies” against apoE, apoB, and apoA-I. Compared with normolipidemic rabbits (WT rabbits on a NS diet, first left), other three hyperlipidemic rabbits exhibit unique and different apolipoprotein profiles. The apoB-containing particles (VLDLs, IDLs and LDLs) of HC-fed WT rabbits are characterized by increased apoB-100 and apoE contents whereas apoE KO rabbits showed marked increase of apoB-48 along with the appearance of apoA-I. WHHL exhibited marked increase of apoB-100 in LDLs accompanied by reduced apoA-I in HDLs. This figure has been modified from Niimi M et al.10. Please click here to view a larger version of this figure.
Density solution (g/mL) | KBr (g) | Distilled water (mL) |
d=1.006 | 8.404 | 1000 |
d=1.02 | 28.271 | 1000 |
d=1.04 | 57.261 | 1000 |
d=1.06 | 86.958 | 1000 |
d=1.08 | 117.365 | 1000 |
d=1.10 | 148.490 | 1000 |
d=1.21 | 333.394 | 1000 |
Table 1: Preparation for potassium bromide (KBr) density solutions. The weight (g) of KBr add to 1000 mL distilled water is shown.
Hyperlipidemia is one of the most important risk factors of atherosclerotic disease. Thus, analysis of plasma lipoproteins is not only essential for diagnosis of dyslipidemia patients but also important for investigation of molecular mechanisms of lipoprotein metabolism and atherosclerosis. In this study, we described the protocol of isolation and analysis of plasma lipoproteins which can be applied in the laboratories where ultracentrifugation is available. Information obtained by this method is comprehensive and straightforward therefore it is recommended for both clinical and basic research scientists. Isolated lipoproteins can also be used for investigating many other facets of lipoprotein functions such as negative-stain electron microscopy13,14, oxidizability9,15, proteomics16, cell culture based in vitro study such as cholesterol efflux assay9 and lipoprotein uptake assay17.
It should be pointed out; however, there are several weak points that need to take considerations. First, this method is relatively time-consuming (three days at least) compared with other methods. If using the latest tabletop ultracentrifuge, high-speed spin (150,000 rpm) can shorten separating time 50–140 min for each lipoprotein fraction18. Second, sample loss may happen during the isolation. To minimize sample loss, the viscous precipitant in the bottom fraction should be dissolved and collected carefully by pipetting. The centrifuge tubes need to fix tightly with a slicer to avoid leaking of the top fraction. In addition, the samples should be recovered carefully from dialysis bags. The recovery can be calculated by comparing the total lipoprotein cholesterol with the original plasma cholesterol. According to our experience, general recovery rate will be ≈ 80%19. Third, this protocol is limited for small number of the samples because one rotor can only run twelve tubes a time. To perform a large number of samples, it may be suitable to use other methods such as precipitation method for HDL preparation4 and automated HPLC lipoprotein analysis20.
In summary, this protocol provides a guide for researchers to isolate plasma lipoproteins using sequential density ultracentrifugation and the analysis of lipids and apolipoproteins.
The authors have nothing to disclose.
This work was supported in part by a research grants from JSPS KAKENHI Grant Number JP 20K08858, the National Natural Science Foundation of China (No. 81941001 and 81770457), JSPS-CAS under the Japan-China Research Cooperative Program.
22-gauge needle | Terumo | NN-2232S | For blood collection |
96-well microplate | greiner bio-one | 655101 | For lipids measurment |
Anti-apolipoprotein A-I antibody | LifeSpan BioSciences | LS-C314186 | For Western blottng, use 1:1,000 |
Anti-apolipoprotein B antibody | ROCKLAND | 600-101-111 | For Western blottng, use 1:1,000 |
Anti-apolipoprotein E antibody | Merck Millipore | AB947 | For Western blottng, use 1:1,000 |
CBB staining kit | FUJIFILM Wako Pure Chemical | 299-50101 | For apolipoprotein analysis |
Centrifuge | HITACHI | himac CF15RN | |
Closure | Spectrum | 132736 | For lipoprotein dialysis |
Dialysis tubing | FUJIFILM Wako Pure Chemical | 043-30921 | For lipoprotein dialysis, MWCO 14,000 |
Dry heat block | Major Science | MD-01N | For SDS-PAGE sample preparation |
ECL Western blotting detection reagents | GE Healthcare | RPN2209 | For Western blotting |
Electrophoresis Chamber | BIO-RAD | Mini-PROTEAN Tetra Cell | |
Ethylenediamine-N,N,N',N'-tetraacetic acid (EDTA) disodium salt dihydrate | FUJIFILM Wako Pure Chemical | 345-01865 | For anticoagulant (0.5 M), for dialysis (1 mM) |
Filter paper | ADVANTEC | 590 | For Western blotting |
Fixed angle ultracentrifuge rotor | BECKMAN COULTER | 357656 | TLA-120.2 |
Fixing solution | For SDS-PAGE (50% Methanol/ 10% Acetic acid) | ||
Immun-Blot PVDF menbrane | BIO-RAD | 1620177 | For Western blotting |
Lumino image analyzer | GE Healthcare | For Western blotting, ImageQuant LAS 4000 | |
Magnetic stirrer | ADVANTEC | SR-304 | For lipoprotein dialysis |
Microplate reader iMARK | BIO-RAD | For lipids measurment | |
Microtube | INA-OPTIKA | SC-0150 | |
Orbital agitator USBDbo | Stovall Life Science | ||
Peroxidase congugated anti goat IgG antibody | Jackson ImmunoResearch | 705-035-003 | For Western blotting, use 1:2,000 |
Peroxidase congugated anti mouse IgG antibody | Jackson ImmunoResearch | 715-035-150 | For Western blotting, use 1:2,000 |
Phospholipids assay kit | FUJIFILM Wako Pure Chemical | 433-36201 | For lipids measurment |
Polycarbonate ultracentrifuge Tubes | BECKMAN COULTER | 343778 | |
Potassium Bromide | FUJIFILM Wako Pure Chemical | 168-03475 | For density solution |
Power Supply | BIO-RAD | For SDD-PAGE and Western blotting, PowerPac 300, PowerPac HC | |
Protein standards Precidion Plus Protein Dual Xtra | BIO-RAD | 161-0377 | For SDS-PAGE and Western blotting |
Rabbit restrainer | Natsume Seisakusho | KN-318 | For blood collection |
Rotor | HITACHI | T15A43 | |
SDS-PAGE running buffer | 25 mM Tris/ 192 mM Glycine/ 0.1% SDS | ||
SDS-PAGE sample buffer (2x) | 0.1M Tris-HCl (pH 6.8)/ 4% SDS/ 20% glycerol/ 0.01% BPB/12% 2-merpaptoethanol | ||
SDS-polyacrylamide gel | 4-20% gradient polyacrylamide gel | ||
Skim milk powder | FUJIFILM Wako Pure Chemical | 190-12865 | For Western blotting blocking buffer (5% skim milk/ 0.1% Tween 20/ PBS) |
Total cholesterol assay kit | FUJIFILM Wako Pure Chemical | 439-17501 | For lipids measurment |
Triglyicerides assay kit | FUJIFILM Wako Pure Chemical | 432-40201 | For lipids measurment |
Tube slicer for thick-walled tube | BECKMAN COULTER | 347960 | For lipoprotein isolation |
Tween 20 | SIGMA-ALDRICH | P1379 | For Western blotting washing buffer (0.1% Tween 20/ PBS) |
Ultracentrifuge | BECKMAN COULTER | A95761 | Optima MAX-TL |
Western blotting wet transfer system | BIO-RAD | Mini Trans-Blot Cell |