Name: isolation and analysis of plasma lipoproteins by ultracentrifugation
Uploaded: 2021-01-28T15:00:00.000+00:00
Duration: 6 min 47 s
Description: Watch this Scientific Journal Video about Isolation and Analysis of Plasma Lipoproteins by Ultracentrifugation at JoVE.com

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.

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
<ol>
	<li>Prepare 1.5 mL microtubes containing 15 &#181;L of 0.5 M EDTA (pH 8.0) for blood collection.</li>
	<li>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.</li>
	<li>Centrifuge the blood tubes at 1,500 x g for 20 min at 4 &#176;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.</li>
</ol>
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.
<ol>
	<li>Transfer 1 mL of plasma to a polycarbonate ultracentrifuge tube (Figure 2A).</li>
	<li>Load these tubes in a fixed angle rotor and centrifuge the plasma at 356,000 x g for 2.5 h at 4 &#176;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.</li>
	<li>Cut the tubes using a slicer and then collect the top fraction [VLDL (d&#60;1.006 g/mL)], approximately 200 &#181;L 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 &#181;L 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 &#181;L) and the bottom fraction (800 &#181;L).&#160;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&#176;C until the next centrifugation.</li>
	<li>Adjust the bottom fraction (total 800 &#181;L) to d=1.02 g/mL by adding 58.9 &#181;L of d=1.21 g/mL solution and 141.1 &#181;L of d=1.02 g/mL solution (the total volume is 1 mL).</li>
	<li>Load these tubes in fixed angle rotor and centrifuge at 356,000 x g for 2.5 h at 4 &#176;C.</li>
	<li>Cut the tubes using a slicer and then collect the top fraction [IDL (d=1.02 g/mL)] approximately 200 &#181;L 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 &#181;L by adding the same density solution (d=1.02 g/mL).</li>
	<li>Adjust the bottom fraction (total 800 &#181;L) to d=1.04 g/mL by adding 94.1 &#181;L of d=1.21 g/mL solution and 105.9 &#181;L of d=1.04 g/mL solution (the total volume is 1 mL).</li>
	<li>Load these tubes in a fixed angle rotor and centrifuge at 356,000 x g for 2.5 h at 4 &#176;C.</li>
	<li>Cut the tubes using a slicer and then collect the top fraction [LDL (d=1.04 g/mL)] approximately 200 &#181;L 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 &#181;L by adding the same density solution (d=1.04 g/mL).</li>
	<li>Adjust the bottom fraction (total 800 &#181;L) to d=1.06 g/mL by adding 106.7 &#181;L of d=1.21 g/mL solution and 93.3 &#181;L of d=1.06 g/mL solution (the total volume is 1 mL).</li>
	<li>Load these tubes in a fixed angle rotor and centrifuge at 356,000 x g for 2.5 h at 4 &#176;C.</li>
	<li>Cut the tubes using a slicer and then collect the top fraction [LDL (d=1.06 g/mL)] approximately 200 &#181;L 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 &#181;L by adding the same density solution (d=1.06 g/mL).</li>
	<li>Adjust the bottom fraction (total 800 &#181;L) to d=1.08 g/mL by adding 123.1 &#181;L of d=1.21 g/mL solution and 76.9 &#181;L of d=1.08 g/mL solution (the total volume is 1 mL).</li>
	<li>Load these tubes in a fixed angle rotor and centrifuge at 356,000 x g for 2.5 h at 4 &#176;C.</li>
	<li>Cut the tubes using a slicer and then collect the top fraction [HDL2 (d=1.08 g/mL)] approximately 200 &#181;L 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 &#181;L by adding the same density solution (d=1.08 g/mL).</li>
	<li>Adjust bottom fraction (total 800 &#181;L) to d=1.10 g/mL by adding 145.5 &#181;L of d=1.21 g/mL solution and 54.5 &#181;L of d=1.10 g/mL solution (the total volume is 1 mL).</li>
	<li>Load these tubes in a fixed angle rotor and centrifuge at 356,000 x g for 2.5 h at 4 &#176;C.</li>
	<li>Cut the tubes using a slicer and then collect the top fraction [HDL2 (d=1.10 g/mL)] approximately 200 &#181;L 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 &#181;L by adding the same density solution (d=1.10 g/mL).</li>
	<li>Adjust bottom fraction (total 800 &#181;L) 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.</li>
	<li>Load these tubes in fixed angle rotor and centrifuge at 513,000 x g for 4 h at 4 &#176;C. 
	NOTE: For Beckman TLA 120.2 rotor, 513,000 x g (Av RCF) corresponds to 120,000 rpm.</li>
	<li>Cut the tubes using a slicer and then collect the top fraction [HDL3 (d=1.21 g/mL)] approximately 200 &#181;L into a new microtube. This is the last step for ultracentrifugation.</li>
</ol>
3. Dialysis
NOTE: Because the density fractions (except d&#60;1.006 g/mL fraction) contain high concentrations of KBr, it is necessary to remove them by dialysis.
<ol>
	<li>Transfer the density fractions to a dialysis tubing and clip both ends with closures.</li>
	<li>Dialyze against 2 L of dialysis buffer such as PBS/ 1 mM EDTA for 6 h at 4 &#176;C with agitation using a magnetic stirrer.</li>
	<li>Replace with new buffer and dialyze again for another 6 h at 4 &#176;C.</li>
	<li>Collect the density fractions and measure the volume. Adjust all of the density fractions to the same volume (e.g., 250 &#181;L). 
	NOTE: You can calculate the concentrated factor from 1 mL of original plasma (e.g., 250 &#181;L of density fraction corresponds to four times concentration).</li>
</ol>
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.
<ol>
	<li>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.
	<ol>
		<li>Apply 8 &#956;L of lipoprotein sample and standard calibration substance on 96-well microplate.</li>
		<li>Add 240 &#956;L of assay reagent and mix by pipetting.</li>
		<li>Incubate at 37 &#176;C for 10 min.</li>
		<li>Measure the OD with a microplate reader and calculate lipid concentrations.</li>
	</ol>
	</li>
	<li>Apolipoprotein analysis
	<ol>
		<li>SDS-PAGE and CBB staining
		<ol>
			<li>Sample preparation: Add 10 &#956;L of 2x Sample buffer to 10 &#956;L of lipoprotein fraction. Heat the mixture at 80 &#176;C for 5 min using a dry heat block.</li>
			<li>Prepare 4-20% gradient SDS-polyacrylamide gel and set up the gel on the electrophoresis chamber filled with running buffer.</li>
			<li>Load lipoprotein sample (10 &#956;L/lane) and protein standards (5 &#956;L/ lane) on the stacking gel. 
			NOTE: If the samples are two times concentrated, an equivalent amount of 20 &#956;L plasma will be analyzed per lane.]</li>
			<li>Run electrophoresis at 20&#8211;40 mA constant current.</li>
			<li>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.</li>
		</ol>
		</li>
		<li>Western blotting
		<ol>
			<li>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.&#160;1&#8211;5 &#956;L).</li>
			<li>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.</li>
			<li>Perform electroblotting at 100 mA constant current for 3 h at 4 &#176;C.</li>
			<li>Blocking: Incubate the membrane in blocking buffer for 1 h at room temperature or overnight at 4 &#176;C.</li>
			<li>Primary Ab reaction: Incubate the membranes in the primary Abs diluted with blocking buffer for 1 h at room temperature or overnight at 4 &#176;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.</li>
			<li>Wash the membrane three times in washing buffer with agitation, 5 min per wash.</li>
			<li>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.</li>
			<li>Wash the membrane three times in washing buffer with agitation, 5 min per wash.</li>
			<li>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.</li>
			<li>Visualize the signals using an image analyzer.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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.&#160;Isolated&#160;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&#8211;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 &#8776; 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.

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&#8217;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&#60;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&#160;cell-based in vitro strategies. This protocol should be useful for both clinical diagnosis and basic research. Here we used&#160;rabbit plasma as an example to demonstrate this technique while plasma from other species can be applied in the same way.

<table><tbody><tr><td>22-gauge needle</td><td>Terumo</td><td>NN-2232S</td><td>For blood collection</td></tr><tr><td>96-well microplate</td><td>greiner bio-one</td><td>655101</td><td>For lipids measurment</td></tr><tr><td>Anti-apolipoprotein A-I antibody</td><td>LifeSpan BioSciences</td><td>LS-C314186</td><td>For Western blottng, use 1:1,000</td></tr><tr><td>Anti-apolipoprotein B antibody</td><td>ROCKLAND</td><td>600-101-111</td><td>For Western blottng, use 1:1,000</td></tr><tr><td>Anti-apolipoprotein E antibody</td><td>Merck Millipore</td><td>AB947</td><td>For Western blottng, use 1:1,000</td></tr><tr><td>CBB staining kit</td><td>FUJIFILM Wako Pure Chemical</td><td>299-50101</td><td>For apolipoprotein analysis</td></tr><tr><td>Centrifuge</td><td>HITACHI</td><td></td><td>himac CF15RN</td></tr><tr><td>Closure</td><td>Spectrum</td><td>132736</td><td>For lipoprotein dialysis</td></tr><tr><td>Dialysis tubing</td><td>FUJIFILM Wako Pure Chemical</td><td>043-30921</td><td>For lipoprotein dialysis, MWCO 14,000</td></tr><tr><td>Dry heat block</td><td>Major Science</td><td>MD-01N</td><td>For SDS-PAGE sample preparation</td></tr><tr><td>ECL Western blotting detection reagents</td><td>GE Healthcare</td><td>RPN2209</td><td>For Western blotting</td></tr><tr><td>Electrophoresis Chamber</td><td>BIO-RAD</td><td></td><td>Mini-PROTEAN Tetra Cell</td></tr><tr><td>Ethylenediamine-N,N,N&#039;,N&#039;-tetraacetic acid (EDTA) disodium salt dihydrate</td><td>FUJIFILM Wako Pure Chemical</td><td>345-01865</td><td>For anticoagulant (0.5 M), for dialysis (1 mM)</td></tr><tr><td>Filter paper</td><td>ADVANTEC</td><td>590</td><td>For Western blotting</td></tr><tr><td>Fixed angle ultracentrifuge rotor</td><td>BECKMAN COULTER</td><td>357656</td><td>TLA-120.2</td></tr><tr><td>Fixing solution</td><td></td><td></td><td>For SDS-PAGE (50% Methanol/ 10% Acetic acid)</td></tr><tr><td>Immun-Blot PVDF menbrane</td><td>BIO-RAD</td><td>1620177</td><td>For Western blotting</td></tr><tr><td>Lumino image analyzer</td><td>GE Healthcare</td><td></td><td>For Western blotting, ImageQuant LAS 4000</td></tr><tr><td>Magnetic stirrer</td><td>ADVANTEC</td><td>SR-304</td><td>For lipoprotein dialysis</td></tr><tr><td>Microplate reader iMARK</td><td>BIO-RAD</td><td></td><td>For lipids measurment</td></tr><tr><td>Microtube</td><td>INA-OPTIKA</td><td>SC-0150</td><td></td></tr><tr><td>Orbital agitator USBDbo</td><td>Stovall Life Science</td><td></td><td></td></tr><tr><td>Peroxidase congugated anti goat IgG antibody</td><td>Jackson ImmunoResearch</td><td>705-035-003</td><td>For Western blotting, use 1:2,000</td></tr><tr><td>Peroxidase congugated anti mouse IgG antibody</td><td>Jackson ImmunoResearch</td><td>715-035-150</td><td>For Western blotting, use 1:2,000</td></tr><tr><td>Phospholipids assay kit</td><td>FUJIFILM Wako Pure Chemical</td><td>433-36201</td><td>For lipids measurment</td></tr><tr><td>Polycarbonate ultracentrifuge Tubes</td><td>BECKMAN COULTER</td><td>343778</td><td></td></tr><tr><td>Potassium Bromide</td><td>FUJIFILM Wako Pure Chemical</td><td>168-03475</td><td>For density solution</td></tr><tr><td>Power Supply</td><td>BIO-RAD</td><td></td><td>For SDD-PAGE and Western blotting, PowerPac 300, PowerPac HC</td></tr><tr><td>Protein standards Precidion Plus Protein Dual Xtra</td><td>BIO-RAD</td><td>161-0377</td><td>For SDS-PAGE and Western blotting</td></tr><tr><td>Rabbit restrainer</td><td>Natsume Seisakusho</td><td>KN-318</td><td>For blood collection</td></tr><tr><td>Rotor</td><td>HITACHI</td><td></td><td>T15A43</td></tr><tr><td>SDS-PAGE running buffer</td><td></td><td></td><td>25 mM Tris/ 192 mM Glycine/ 0.1% SDS</td></tr><tr><td>SDS-PAGE sample buffer (2x)</td><td></td><td></td><td>0.1M Tris-HCl (pH 6.8)/ 4% SDS/ 20% glycerol/ 0.01% BPB/12% 2-merpaptoethanol</td></tr><tr><td>SDS-polyacrylamide gel</td><td></td><td></td><td>4-20% gradient polyacrylamide gel</td></tr><tr><td>Skim milk powder</td><td>FUJIFILM Wako Pure Chemical</td><td>190-12865</td><td>For Western blotting blocking buffer (5% skim milk/ 0.1% Tween 20/ PBS)</td></tr><tr><td>Total cholesterol assay kit</td><td>FUJIFILM Wako Pure Chemical</td><td>439-17501</td><td>For lipids measurment</td></tr><tr><td>Triglyicerides assay kit</td><td>FUJIFILM Wako Pure Chemical</td><td>432-40201</td><td>For lipids measurment</td></tr><tr><td>Tube slicer for thick-walled tube</td><td>BECKMAN COULTER</td><td>347960</td><td>For lipoprotein isolation</td></tr><tr><td>Tween 20</td><td>SIGMA-ALDRICH</td><td>P1379</td><td>For Western blotting washing buffer (0.1% Tween 20/ PBS)</td></tr><tr><td>Ultracentrifuge</td><td>BECKMAN COULTER</td><td>A95761</td><td>Optima MAX-TL</td></tr><tr><td>Western blotting wet transfer system</td><td>BIO-RAD</td><td></td><td>Mini Trans-Blot Cell</td></tr></tbody></table>

isolation and analysis of plasma lipoproteins by ultracentrifugation

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&#60;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.

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&#160;(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&#8593; in VLDL-TC, 1.5-fold&#8593; in VLDL-TG, and 30-fold&#8593; in VLDL-PL compared with NS-fed rabbits). Because VLDLs isolated from cholesterol-fed rabbits are rich in cholesteryl esters and move to the &#946; position on agarose gel electrophoresis, they are often called &#946;-VLDLs to distinguish them from normal VLDLs which move to pre-&#946; 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&#60;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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61790/61790fig01.jpg" /> 
Figure 1: Schematic illustration of sequential floating ultracentrifugation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61790/61790fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61790/61790fig02.jpg" /> 
Figure 2: Representative images of plasma lipoprotein isolation.&#160;(A) Plasma of normolipidemic and hyperlipidemic rabbits. (B) Floated top VLDL (d&#60;1.006 g/mL) and bottom fraction (d&#62;1.006 g/mL) after ultracentrifugation. (C) Tube slicing and collection of the top fraction by a pipette. (D) Collection of the bottom fraction. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61790/61790fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61790/61790fig03.jpg" /> 
Figure 3: Quantitation of lipid contents in each lipoprotein fraction isolated from normal rabbits (A) and cholesterol-fed rabbits (B).&#160;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 &#177; SEM (n=6). This figure has been modified from Yan H et al.12. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61790/61790fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61790/61790fig04.jpg" /> 
Figure 4: Comparison of apolipoprotein distribution using CBB staining (A) and Western blotting (B).&#160;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 &#8220;cocktail antibodies&#8221; 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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61790/61790fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Density solution (g/mL)</td>
			<td>KBr (g)</td>
			<td>Distilled water (mL)</td>
		</tr>
		<tr>
			<td>d=1.006</td>
			<td>8.404</td>
			<td>1000</td>
		</tr>
		<tr>
			<td>d=1.02</td>
			<td>28.271</td>
			<td>1000</td>
		</tr>
		<tr>
			<td>d=1.04</td>
			<td>57.261</td>
			<td>1000</td>
		</tr>
		<tr>
			<td>d=1.06</td>
			<td>86.958</td>
			<td>1000</td>
		</tr>
		<tr>
			<td>d=1.08</td>
			<td>117.365</td>
			<td>1000</td>
		</tr>
		<tr>
			<td>d=1.10</td>
			<td>148.490</td>
			<td>1000</td>
		</tr>
		<tr>
			<td>d=1.21</td>
			<td>333.394</td>
			<td>1000</td>
		</tr>
	</tbody>
</table>
Table 1: Preparation for potassium bromide (KBr) density solutions. The weight (g) of KBr add to 1000 mL distilled water is shown.

Watch this Scientific Journal Video about Isolation and Analysis of Plasma Lipoproteins by Ultracentrifugation at JoVE.com

Isolation and Analysis of Plasma Lipoproteins by Ultracentrifugation

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 ...

isolation-and-analysis-of-plasma-lipoproteins-by-ultracentrifugation

Nanyang Technological University

Research

JoVE Journal

Biochemistry

10.9K Views.  University of Yamanashi. Analyse van plasma lipoproteïnen is zeer belangrijk voor de diagnose van hyperlipidemie en het onderzoek van atherosclerose. In dit protocol introduceren we de basismethode met behulp van ultracentrifugatie om plasma lipoproteïnen te isoleren. Met deze methode kunnen de onderzoekers één milliliter plasma gebruiken om zeven lipoproteïnefracties te isoleren.Deze fracties kunnen worden gebruikt voor lipiden, apolipoproteïnen en goed functionele studies Deze methode kan worden toegep...

Video: Isolation and Analysis of Plasma Lipoproteins by Ultracentrifugation

Isolation of High-density Lipoproteins for Non-coding Small RNA Quantification

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most interestingly, sRNAs are also found outside of cells and are stably present in all biological fluids. As such, extracellular sRNAs represent a novel class of disease biomarkers and are likely involved in cell signaling and intercellular communication networks. To assess their potential as biomarkers, sRNAs can be quantified in plasma, urine, and other fluids. Nevertheless, to fully understand the impact of extracellular sRNAs as endocrine signals, it is important to determine which carriers are transporting and protecting them in biological fluids (e.g., plasma), which cells and tissues contribute to extracellular sRNA pools, and cells and tissues capable of accepting and utilizing extracellular sRNA. To accomplish these goals, it is critical to isolate highly pure populations of extracellular carriers for sRNA profiling and quantification. We have previously demonstrated that lipoproteins, particularly high-density lipoproteins (HDL), transport functional microRNAs (miRNA) between cells and HDL-miRNAs are significantly altered in disease. Here, we detail a new protocol that utilizes tandem HDL isolation with density-gradient ultracentrifugation (DGUC) and fast-protein-liquid chromatography (FPLC) to obtain highly pure HDL for downstream profiling and quantification of all sRNAs, including miRNAs, using both high-throughput sequencing and real-time PCR approaches. This protocol will be a valuable resource for the investigation of sRNAs on HDL.

This protocol describes the isolation and quantification of high-density lipoprotein small RNAs.

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most ...

Isolation of Cellular Lipid Droplets: Two Purification Techniques Starting from Yeast Cells and Human Placentas

Lipid droplets are dynamic organelles that can be found in most eukaryotic and certain prokaryotic cells. Structurally, the droplets consist of a core of neutral lipids surrounded by a phospholipid monolayer. One of the most useful techniques in determining the cellular roles of droplets has been proteomic identification of bound proteins, which can be isolated along with the droplets. Here, two methods are described to isolate lipid droplets and their bound proteins from two wide-ranging eukaryotes: fission yeast and human placental villous cells. Although both techniques have differences, the main method - density gradient centrifugation -&#160;is shared by both preparations. This shows the wide applicability of the presented droplet isolation techniques.

In the first protocol, yeast cells are converted into spheroplasts by enzymatic digestion of their cell walls. The resulting spheroplasts are then gently lysed in a loose-fitting homogenizer. Ficoll is added to the lysate to provide a density gradient, and the mixture is centrifuged three times. After the first spin, the lipid droplets are localized to the white-colored floating layer of the centrifuge tubes along with the endoplasmic reticulum (ER), the plasma membrane, and vacuoles. Two subsequent spins are used to remove these other three organelles. The result is a layer that has only droplets and bound proteins.

In the second protocol, placental villous cells are isolated from human term placentas by enzymatic digestion with trypsin and DNase I. The cells are homogenized in a loose-fitting homogenizer. Low-speed and medium-speed centrifugation steps are used to remove unbroken cells, cellular debris, nuclei, and mitochondria. Sucrose is added to the homogenate to provide a density gradient and the mixture is centrifuged to separate the lipid droplets from the other cellular fractions.

The purity of the lipid droplets in both protocols is confirmed by Western Blot analysis. The droplet fractions from both preps are suitable for subsequent proteomic and lipidomic analysis.

Two techniques for isolating cellular lipid droplets from 1) yeast cells and 2) human placentas are presented. The centerpiece of both procedures is density gradient centrifugation, where the resulting floating layer containing the droplets can be readily visualized by eye, extracted, and quantified by Western Blot analysis for purity.

Lipid droplets are dynamic organelles that can be found in most eukaryotic and certain prokaryotic cells. Structurally, the droplets consist of a core of ...

Bioengineering

Cell Fractionation of U937 Cells in the Absence of High-speed Centrifugation

In this protocol we detail a method to obtain subcellular fractions of U937 cells without the use of ultracentrifugation or indiscriminate detergents. This method utilizes hypotonic buffers, digitonin, mechanical lysis and differential centrifugation to isolate the cytoplasm, mitochondria and plasma membrane. The process can be scaled to accommodate the needs of researchers, is inexpensive and straightforward. This method will allow researchers to determine protein localization in cells without specialized centrifuges and without the use of commercial kits, both of which can be prohibitively expensive. We have successfully used this method to separate cytosolic, plasma membrane and mitochondrial proteins in the human monocyte cell line U937.

Here, we present a protocol to isolate the plasma membrane, cytoplasm and mitochondria of U937 cells without the use of high-speed centrifugation. This technique can be used to purify subcellular fractions for subsequent examination of protein localization via immunoblotting.

In this protocol we detail a method to obtain subcellular fractions of U937 cells without the use of ultracentrifugation or indiscriminate detergents. ...

Enrichment of Native Lipoprotein Particles with microRNA and Subsequent Determination of Their Absolute/Relative microRNA Content and Their Cellular Transfer Rate

Lipoprotein particles are predominately transporters of lipids and cholesterol in the bloodstream. Furthermore, they contain small amounts of strands of noncoding microRNA (miRNA). In general, miRNA alters the protein expression profile due to interactions with messenger-RNA (mRNA). Thus, knowledge of the relative and absolute miRNA content of lipoprotein particles is essential to estimate the biological effect of cellular particle uptake. Here, a quantitative real-time polymerase chain reaction (qPCR)-based protocol is presented to determine the absolute miRNA content of lipoprotein particles&#8212;exemplified shown for native and miRNA-enriched lipoprotein particles. The relative miRNA content is quantified using multiwell microfluidic array cards. Furthermore, this protocol allows scientists to estimate the cellular miRNA and, thus, the lipoprotein particle uptake rate. A significant increase of the cellular miRNA level is observable when using high-density lipoprotein (HDL) particles artificially loaded with miRNA, whereas incubation with native HDL particles yields no significant effect due to their rather low miRNA content. In contrast, the cellular uptake of low-density lipoprotein (LDL) particles&#8212;neither with native miRNA nor artificially loaded with it&#8212;did not alter the cellular miRNA level.

Here, a quantitative real-time polymerase chain reaction-based protocol is presented for the determination of the native micro-RNA content (absolute/relative) of lipoprotein particles. In addition, a method for increasing the micro-RNA level, as well as a method for determining the cellular uptake rate of lipoprotein particles, is demonstrated.

Lipoprotein particles are predominately transporters of lipids and cholesterol in the bloodstream. Furthermore, they contain small amounts of strands of ...

Enrichment of Bacterial Lipoproteins and Preparation of N-terminal Lipopeptides for Structural Determination by Mass Spectrometry

Lipoproteins are important constituents of the bacterial cell envelope and potent activators of the mammalian innate immune response. Despite their significance to both cell physiology and immunology, much remains to be discovered about novel lipoprotein forms, how they are synthesized, and the effect of the various forms on host immunity. To enable thorough studies on lipoproteins, this protocol describes a method for bacterial lipoprotein enrichment and preparation of N-terminal tryptic lipopeptides for structural determination by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Expanding on an established Triton X-114 phase partitioning method for lipoprotein extraction and enrichment from the bacterial cell membrane, the protocol includes additional steps to remove non-lipoprotein contaminants, increasing lipoprotein yield and purity. Since lipoproteins are commonly used in Toll-like receptor (TLR) assays, it is critical to first characterize the N-terminal structure by MALDI-TOF MS. Herein, a method is presented to isolate concentrated hydrophobic peptides enriched in N-terminal lipopeptides suitable for direct analysis by MALDI-TOF MS/MS. Lipoproteins that have been separated by Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis (SDS-PAGE) are transferred to a nitrocellulose membrane, digested in situ with trypsin, sequentially washed to remove polar tryptic peptides, and finally eluted with chloroform-methanol. When coupled with MS of the more polar trypsinized peptides from wash solutions, this method provides the ability to both identify the lipoprotein and characterize its N-terminus in a single experiment. Intentional sodium adduct formation can also be employed as a tool to promote more structurally informative fragmentation spectra. Ultimately, enrichment of lipoproteins and determination of their N-terminal structures will permit more extensive studies on this ubiquitous class of bacterial proteins.

The enrichment of bacterial lipoproteins using a non-ionic surfactant phase partitioning method is described for direct use in TLR assays or other applications. Further steps are detailed to prepare N-terminal tryptic lipopeptides for structural characterization by mass spectrometry.

Lipoproteins are important constituents of the bacterial cell envelope and potent activators of the mammalian innate immune response. Despite their ...

Immunology and Infection

Fast and Simplified Method for High Through-put Isolation of miRNA from Highly Purified High Density Lipoprotein

Small non-coding RNAs (miRNAs) have been implicated in a variety of human diseases including metabolic syndromes. They may be utilized as biomarkers for diagnosis and prognosis or may serve as targets for drug development, respectively. Recently it has been shown that miRNAs are carried in lipoproteins, particularly high density lipoproteins (HDL) and are delivered to recipient cells for uptake. This raises the possibility that miRNAs play a critical and pivotal role in cellular and organ function via regulation of gene expression as well as messenger for cell-cell communications and crosstalk between organs. Current methods for miRNA isolation from purified HDL are impractical when utilizing small samples on a large scale. This is largely due to the time consuming and laborious methods used for lipoprotein isolation. We have developed a simplified approach to rapidly isolate purified HDL suitable for miRNA analysis from plasma samples. This method should facilitate investigations into the role of miRNAs in health and disease and in particular provide new insights into the variety of biological functions, outside of the reverse cholesterol transport, that have been ascribed to HDL. Also, the miRNA species which are present in HDL can provide valuable information of clinical biomarkers for diagnosis of various diseases.

MicroRNAs play an important regulatory role and are emerging as novel therapeutic targets for various human diseases. It has been shown that miRNAs are carried in high density lipoproteins. We have developed a simplified method to rapidly isolate purified HDL suitable for miRNA analysis from human plasma.

Small non-coding RNAs (miRNAs) have been implicated in a variety of human diseases including metabolic syndromes. They may be utilized as biomarkers for ...

Biology

Rapid Lipid Droplet Isolation Protocol Using a Well-established Organelle Isolation Kit

Lipid droplets (LDs) are bioactive organelles found within the cytosol of the most eukaryotic and some prokaryotic cells. LDs are composed of neutral lipids encased by a monolayer of phospholipids and proteins. Hepatic LD lipids, such as ceramides, and proteins are implicated in several diseases that cause hepatic steatosis. Although previous methods have been established for LD isolation, they require a time-consuming preparation of reagents and are not designed for the isolation of multiple subcellular compartments. We sought to establish a new protocol to enable the isolation of LDs, endoplasmic reticulum (ER), and lysosomes from a single mouse liver.
Further, all reagents used in the protocol presented here are commercially available and require minimal reagent preparation without sacrificing LD purity. Here we present data comparing this new protocol to a standard sucrose gradient protocol, demonstrating comparable purity, morphology, and yield. Additionally, we can isolate ER and lysosomes using the same sample, providing detailed insight into the formation and intracellular flux of lipids and their associated proteins.

This protocol establishes a novel method of lipid droplet isolation and purification from mouse livers, using a well-established endoplasmic reticulum isolation kit.

Lipid droplets (LDs) are bioactive organelles found within the cytosol of the most eukaryotic and some prokaryotic cells. LDs are composed of neutral ...

Isolation and Characterization of Human Adipocyte-Derived Extracellular Vesicles using Filtration and Ultracentrifugation

Extracellular vesicles (EVs) are lipid enclosed envelopes that carry biologically active material such as proteins, RNA, metabolites and lipids. EVs can modulate the cellular status of other cells locally in tissue microenvironments or through liberation into peripheral blood. Adipocyte-derived EVs are elevated in the peripheral blood and show alterations in their cargo (RNA and protein) during metabolic disturbances, including obesity and diabetes. Adipocyte-derived EVs can regulate the cellular status of neighboring vascular cells, such as endothelial cells and adipose tissue resident macrophages to promote adipose tissue inflammation. Investigating alterations in adipocyte-derived EVs in vivo is complex because EVs derived from peripheral blood are highly heterogenous and contain EVs from other sources, namely platelets, endothelial cells, erythrocytes and muscle. Therefore, the culture of human adipocytes provides a model system for the study of adipocyte derived EVs. Here, we provide a detailed protocol for the extraction of total small EVs from cell culture media of human gluteal and abdominal adipocytes using filtration and ultracentrifugation. We further demonstrate the use of Nanoparticle Tracking Analysis (NTA) for quantification of EV size and concentration and show the presence of EV-protein tumor susceptibility gene 101 (TSG101) in the gluteal and abdominal adipocyte derived-EVs. Isolated EVs from this protocol can be used for downstream analysis, including transmission electron microscopy, proteomics, metabolomics, small RNA-sequencing,&#160;microarrays and can be&#160;utilized in functional in vitro/in vivo studies.

We describe the isolation of human adipocyte-derived extracellular vesicles (EVs) from gluteal and abdominal adipose tissue using filtration and ultracentrifugation. We characterize the isolated adipocyte-derived EVs by determining their size and concentration by Nanoparticle Tracking Analysis and by western blotting for the presence of EV-protein tumor susceptibility gene 101 (TSG101).

Extracellular vesicles (EVs) are lipid enclosed envelopes that carry biologically active material such as proteins, RNA, metabolites and lipids. EVs can ...

Isolation and Analysis of Traceable and Functionalized Extracellular Vesicles from the Plasma and Solid Tissues

Circulating and tissue-resident extracellular vesicles (EVs) represent promising targets as novel theranostic biomarkers, and they emerge as important players in the maintenance of organismal homeostasis and the progression of a wide spectrum of diseases. While the current research focuses on the characterization of endogenous exosomes with the endosomal origin, microvesicles blebbing from the plasma membrane have gained increasing attention in health and sickness, which are featured by an abundance of surface molecules recapitulating the membrane signature of parent cells. Here, a reproducible procedure is presented based on differential centrifugation for extracting and characterizing EVs from the plasma and solid tissues, such as the bone. The protocol further describes subsequent profiling of surface antigens and protein cargos of EVs, which are thus traceable for their derivations and identified with components related to potential function. This method will be useful for correlative, functional, and mechanistic analysis of EVs in biological, physiological, and pathological studies.

The present protocol describes a method to extract extracellular vesicles from the peripheral blood and solid tissues with subsequent profiling of surface antigens and protein cargos.

Circulating and tissue-resident extracellular vesicles (EVs) represent promising targets as novel theranostic biomarkers, and they emerge as important ...

Cardiovascular Biomarker Studies Using Plasma Extracellular Vesicle Proteome

Isolation, Characterization, and Proteomic Analysis of Plasma-Derived Extracellular Vesicles for Cardiovascular Biomarker Discovery

Extracellular vesicles (EV) are cell-derived, lipid bilayer-enclosed, non-replicable nanoparticles. EV currently gain attention in cardiovascular research due to their role in regulating intercellular communication, potentially serving as valuable biomarkers for cardiovascular disease. However, the EV proteome and its potential as a biomarker in cardiovascular diagnostics remain poorly understood. This protocol presents a standardized method for the isolation and quantification of plasma-derived EV and the analysis of their protein cargo using plasma samples from patients presenting to the Chest Pain Unit of a large university hospital. Following routine phlebotomy, EV are isolated from plasma by differential ultracentrifugation. The enrichment of specific EV marker proteins in EV isolates is visualized by immunoblotting, and average size distribution and plasma EV concentrations are quantified by nanoparticle tracking analysis. Finally, ultra-performance liquid chromatography-tandem mass spectrometry is employed for label-free analysis of the EV proteome. This protocol thus provides a comprehensive approach to study and use plasma-derived EV as potential carriers of critical biological information as well as to explore their potential as novel biomarkers.

Author Spotlight: Advancing the Analysis of Plasma Extracellular Vesicle Proteome for Cardiovascular Biomarker Studies

This article describes a methodology for the isolation, characterization, and quantification of human plasma-derived extracellular vesicles (EV) and presents a workflow for label-free analysis of the EV proteome using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Extracellular vesicles (EV) are cell-derived, lipid bilayer-enclosed, non-replicable nanoparticles. EV currently gain attention in cardiovascular research ...

Isolation and Analysis of Plasma Lipoproteins by Ultracentrifugation

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