Isolation and Analysis of Plasma Lipoproteins by Ultracentrifugation

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 laboratory^3,4. 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 groups^6,7. 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 apolipoproteins^9,10,11. 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.

Protokół

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

1. Prepare 1.5 mL microtubes containing 15 µL of 0.5 M EDTA (pH 8.0) for blood collection.
2. Put a rabbit in a restrainer and puncture an auricular intermediate artery using a 22 gauge needle and collect blood into a tube. Mix the blood with EDTA gently and put them on ice.
3. Centrifuge the blood tubes at 1,500 x g for 20 min at 4 °C and collect plasma to a new microtube.
   NOTE: 3 mL of blood is enough for collecting 1 mL plasma. If you collect blood from mice or other small animals, you need to pool them.

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.

1. Transfer 1 mL of plasma to a polycarbonate ultracentrifuge tube (Figure 2A).
2. Load these tubes in a fixed angle rotor and centrifuge the plasma at 356,000 x g for 2.5 h at 4 °C (Figure 2B).
   NOTE: For Beckman TLA 120.2 rotor, 356,000 x g (average relative centrifugal field, Av RCF) corresponds to 100,000 rpm.
3. Cut the tubes using a slicer and then collect the top fraction [VLDL (d<1.006 g/mL)], approximately 200 µL into a new microtube (Figure 2C). Collect the remaining bottom fraction into a new polycarbonate ultracentrifuge tube for the next separation (Figure 2D). Measure the volume and make the total volume to 800 µL by adding the same density solution (d=1.006 g/mL).
   NOTE: Set up a blade to the tube slicer. Adjust the position of the blade at a level between the top fraction (200 µL) and the bottom fraction (800 µL). The viscous precipitant in the bottom fraction should be collected carefully and completely by pipetting in all following steps. If pause, do so after separating the top and bottom fraction in all steps. Store the sample at 4°C until the next centrifugation.
4. Adjust the bottom fraction (total 800 µL) to d=1.02 g/mL by adding 58.9 µL of d=1.21 g/mL solution and 141.1 µL of d=1.02 g/mL solution (the total volume is 1 mL).
5. Load these tubes in fixed angle rotor and centrifuge at 356,000 x g for 2.5 h at 4 °C.
6. Cut the tubes using a slicer and then collect the top fraction [IDL (d=1.02 g/mL)] approximately 200 µL into a new microtube. Collect the remaining bottom fraction into a new polycarbonate ultracentrifuge tube for the next separation. Measure the volume and make the total volume to 800 µL by adding the same density solution (d=1.02 g/mL).
7. Adjust the bottom fraction (total 800 µL) to d=1.04 g/mL by adding 94.1 µL of d=1.21 g/mL solution and 105.9 µL of d=1.04 g/mL solution (the total volume is 1 mL).
8. Load these tubes in a fixed angle rotor and centrifuge at 356,000 x g for 2.5 h at 4 °C.
9. Cut the tubes using a slicer and then collect the top fraction [LDL (d=1.04 g/mL)] approximately 200 µL into a new microtube. Collect the remaining bottom fraction into a new polycarbonate ultracentrifuge tube for the next separation. Measure the volume and make the total to 800 µL by adding the same density solution (d=1.04 g/mL).
10. Adjust the bottom fraction (total 800 µL) to d=1.06 g/mL by adding 106.7 µL of d=1.21 g/mL solution and 93.3 µL of d=1.06 g/mL solution (the total volume is 1 mL).
11. Load these tubes in a fixed angle rotor and centrifuge at 356,000 x g for 2.5 h at 4 °C.
12. Cut the tubes using a slicer and then collect the top fraction [LDL (d=1.06 g/mL)] approximately 200 µL into a new microtube. Collect the remaining bottom fraction into a new polycarbonate ultracentrifuge tube for the next separation. Measure the volume and make the total to 800 µL by adding the same density solution (d=1.06 g/mL).
13. Adjust the bottom fraction (total 800 µL) to d=1.08 g/mL by adding 123.1 µL of d=1.21 g/mL solution and 76.9 µL of d=1.08 g/mL solution (the total volume is 1 mL).
14. Load these tubes in a fixed angle rotor and centrifuge at 356,000 x g for 2.5 h at 4 °C.
15. Cut the tubes using a slicer and then collect the top fraction [HDL₂ (d=1.08 g/mL)] approximately 200 µL into a new microtube. Collect the remaining bottom fraction into a new polycarbonate ultracentrifuge tube for the next separation. Measure the volume and make the total to 800 µL by adding the same density solution (d=1.08 g/mL).
16. Adjust bottom fraction (total 800 µL) to d=1.10 g/mL by adding 145.5 µL of d=1.21 g/mL solution and 54.5 µL of d=1.10 g/mL solution (the total volume is 1 mL).
17. Load these tubes in a fixed angle rotor and centrifuge at 356,000 x g for 2.5 h at 4 °C.
18. Cut the tubes using a slicer and then collect the top fraction [HDL₂ (d=1.10 g/mL)] approximately 200 µL into a new microtube. Collect the remaining bottom fraction into a new polycarbonate ultracentrifuge tube for the next separation. Measure the volume and make the total to 800 µL by adding the same density solution (d=1.10 g/mL).
19. Adjust bottom fraction (total 800 µL) to d=1.21 g/mL by adding 0.140 g of potassium bromide (KBr) powder and dissolve it completely. Measure the volume and make them to 1 mL by adding d=1.21 g/mL solution.
20. Load these tubes in fixed angle rotor and centrifuge at 513,000 x g for 4 h at 4 °C.
    NOTE: For Beckman TLA 120.2 rotor, 513,000 x g (Av RCF) corresponds to 120,000 rpm.
21. Cut the tubes using a slicer and then collect the top fraction [HDL₃ (d=1.21 g/mL)] approximately 200 µL into a new microtube. This is the last step for ultracentrifugation.

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.

1. Transfer the density fractions to a dialysis tubing and clip both ends with closures.
2. Dialyze against 2 L of dialysis buffer such as PBS/ 1 mM EDTA for 6 h at 4 °C with agitation using a magnetic stirrer.
3. Replace with new buffer and dialyze again for another 6 h at 4 °C.
4. Collect the density fractions and measure the volume. Adjust all of the density fractions to the same volume (e.g., 250 µL).
   NOTE: You can calculate the concentrated factor from 1 mL of original plasma (e.g., 250 µL of density fraction corresponds to four times concentration).

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.

1. Lipid analysis
   NOTE: Lipids such as TC, TG, PL and FC in the lipoprotein fraction can be measured using commercially available measurement kits. The procedures depend on the reagents used, so follow the instructions of each kit used. Here we show a typical microplate assay using a commercial enzymatic assay kit.
   1. Apply 8 μL of lipoprotein sample and standard calibration substance on 96-well microplate.
   2. Add 240 μL of assay reagent and mix by pipetting.
   3. Incubate at 37 °C for 10 min.
   4. Measure the OD with a microplate reader and calculate lipid concentrations.
2. Apolipoprotein analysis
   1. SDS-PAGE and CBB staining
      1. Sample preparation: Add 10 μL of 2x Sample buffer to 10 μL of lipoprotein fraction. Heat the mixture at 80 °C for 5 min using a dry heat block.
      2. Prepare 4-20% gradient SDS-polyacrylamide gel and set up the gel on the electrophoresis chamber filled with running buffer.
      3. Load lipoprotein sample (10 μL/lane) and protein standards (5 μL/ lane) on the stacking gel.
         NOTE: If the samples are two times concentrated, an equivalent amount of 20 μL plasma will be analyzed per lane.]
      4. Run electrophoresis at 20–40 mA constant current.
      5. CBB staining: Soak the gel twice in fixing solution with gentle shaking for 10 min. Stain the gel with CBB staining solution for 30 min. Rinse the gel in distilled water to remove the excess stain. The gel is ready for photographing.
   2. Western blotting
      1. Perform electrophoresis in the same procedure as described above.
         NOTE: The volume of lipoproteins loaded for the Western blotting is less than for CBB staining described above (e.g. 1–5 μL).
      2. Place the gel and PVDF membrane between transfer buffer-soaked filter paper and form pad. Set up the sandwich in a holder cassette and place in a tank filled with transfer buffer.
      3. Perform electroblotting at 100 mA constant current for 3 h at 4 °C.
      4. Blocking: Incubate the membrane in blocking buffer for 1 h at room temperature or overnight at 4 °C.
      5. Primary Ab reaction: Incubate the membranes in the primary Abs diluted with blocking buffer for 1 h at room temperature or overnight at 4 °C with mild shaking.
         NOTE: Suggested primary Ab dilution is shown in the Table of Materials. Three kinds of primary Abs against apolipoproteins can be used singly or in cocktails.
      6. Wash the membrane three times in washing buffer with agitation, 5 min per wash.
      7. Secondary Ab reaction: Incubate the membrane in the secondary Abs diluted with blocking buffer for 1 h at room temperature with agitation.
         NOTE: Suggested secondary Ab dilution is shown in the Table of Materials.
      8. Wash the membrane three times in washing buffer with agitation, 5 min per wash.
      9. ECL detection: Place the membrane on a plastic wrap. Add ECL detection reagents and incubate for 1 min. Drain excess liquid and seal the membrane in a bag.
      10. Visualize the signals using an image analyzer.

Wyniki

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 (N...

Dyskusje

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 straig...

Materiały

Name	Company	Catalog Number	Comments
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