Application of Polyacrylamide Gel Electrophoresis in Analyzing Lipoprotein Subfractions Relevant to Atherosclerotic Cardiovascular Disease

Summary

This protocol aims to elaborate a lipoprotein subfraction analysis method based on polyacrylamide gel electrophoresis, which provides an important diagnostic basis for cardiovascular disease risk assessment. It can divide LDL into seven subfractions. LDL1 and LDL2 are characterized as large particles, while LDL3 to LDL7 are characterized as small particles.

Abstract

Low-density lipoprotein cholesterol (LDL-C) is a key factor in the development of atherosclerotic cardiovascular disease (ASCVD). Elevated levels of LDL-C are closely linked to the formation of atherosclerotic plaques and the risk of cardiovascular events. However, the heterogeneity of LDL significantly influences its ability to promote atherosclerosis. Research indicates that Low-density lipoprotein (LDL) particles can be further categorized into small dense LDL (sdLDL) and large buoyant LDL (lbLDL). Among these, sdLDL is more likely to penetrate vascular endothelium and has stronger oxidative modification activity, which is particularly associated with ASCVD. In patients with metabolic syndrome, diabetes, and hypertriglyceridemia, LDL-C levels may be normal, but the proportion of sdLDL is elevated, leading to an underestimation of cardiovascular risk. Therefore, accurately detecting LDL subfractions is crucial for stratifying ASCVD risk and optimizing intervention strategies.

Traditional LDL-C detection methods, such as the Friedewald formula and direct measurement, only reflect total cholesterol levels and cannot differentiate between subfractions. Methods for detecting LDL-C subfractions include ultracentrifugation (UC), high-performance liquid chromatography (HPLC), and nuclear magnetic resonance (NMR) techniques. However, these methods have high technical requirements, complex operations, or expensive equipment, making them impractical for widespread clinical use.

Polyacrylamide gel electrophoresis (PAGE) technology separates lipoprotein particles through the molecular sieve effect and charge differences. This technique uses non-denaturing gradient gel systems, combined with specific stains like Sudan black, offering high-resolution, strong reproducibility, and ease of operation. Lipoprotein typing using PAGE technology can provide a more comprehensive and precise assessment of patients' lipoprotein levels, aiding in clinical treatment decisions. This article has presented five representative results. The mean LDL-particle size of sample A, B, C, D, E are 275.3 Å, 266.6 Å, 258.2 Å , 257.4 Å, and 252.9 Å, indicating a low, marginal, mild, moderate, and severe risk of cardiovascular disease due to dyslipidemia, respectively.

Introduction

According to 2021 World Health Organization report, cardiovascular disease (CVD) remains the leading cause of death among human diseases. The report indicates that 17.9 million people worldwide died from CVD in 2019, accounting for nearly one-third of global deaths¹. ASCVD, a type of CVD, is the leading cause of death among urban and rural residents in China, making up over 40% of all deaths². Atherosclerosis is a chronic inflammatory vascular disease driven by both traditional and non-traditional risk factors³. It is characterized by lipid deposition in the arterial wall⁴, fibrous tissue proliferation and calcification, ultimately leading to thickening of the vessel wall, reduced elasticity and narrowing of the lumen. Dyslipidemia is the most significant risk factor for ASCVD, with LDL-C being a pathogenic risk factor for ASCVD, playing a crucial role in ASCVD development⁵. Numerous epidemiological studies, mendelian randomization studies, and randomized controlled trials have consistently shown that plasma LDL-C levels are positively correlated with the risk of ASCVD. Reducing LDL-C levels can lower the risk of ASCVD⁶.

LDL is the primary lipoprotein responsible for transporting endogenous cholesterol in the body⁷. Plasma LDL is heterogeneous, consisting of a variety of particles with different sizes, densities, and chemical compositions⁸. LbLDL particles have a lower density and larger size, while sdLDL particles have higher density and smaller size. LDL heterogeneity underlies its varied biological effects⁹. LbLDL particles are larger in diameter (≥ 25.5 nm), lower in density (close to 1.02 g/mL) and loose in morphology, consisting of LDL1 and LDL2. LbLDL particles contain a higher amount of cholesterol esters. They are less likely to be oxidized or penetrate the vascular endothelium, making them relatively safer. In contrast, sdLDL particles have a smaller diameter (< 25.5 nm)¹⁰, higher density (close to 1.06 g/mL) and dense structure, consisting of LDL3 to LDL7. SdLDL particles contain lower amount of cholesterol ester but higher triglyceride and apolipoprotein B (ApoB) levels compared to lbLDL. Their physical properties make them more likely to penetrate the arterial endothelial barrier. Under the subendothelium, sdLDL particles are oxidized by reactive oxygen species (ROS) to form oxidized LDL (ox-LDL)¹¹. Ox-LDL inhibits endothelial nitric oxide synthase (eNOS) activity, reduces NO production and induces endothelial cells to express VCAM-1/MCP-1, promoting monocyte adhesion and migration¹². The migrating endothelial mononuclear cells differentiate into macrophages, which take up ox-LDL without limit through the scavenger receptors. The accumulation of intracellular cholesterol ester transforms into foam cells, which are the core components of atherosclerotic lipid streaks. Foam cells secrete inflammatory factors such as IL-1β and TNF-α, which further expand oxidative stress and endothelial damage, forming a positive feedback loop¹³. It can activate endothelial cell inflammation, promotes foam cell formation and accelerates plaque progression. Two large cohort studies have shown that sdLDL-C has greater clinical reference value than LDL-C in predicting and assessing the potential risks of ASCVD^14,15. But traditional LDL-C detection method cannot test lipoprotein subfractions due to the limitation of the method.

LDL particles (LDL-P) reflect the number of LDL particles per unit volume of blood and serve as carriers for LDL molecules, each containing one ApoB molecule and multiple cholesterol molecules. LDL-C represents the total mass of cholesterol carried by these particles. LDL-C levels are influenced by the number of LDL particles and cholesterol density, which may not fully reflect the actual risk¹⁶. For example, patients with metabolic syndrome, diabetes, or hypertriglyceridemia may have normal LDL-C levels but abnormally elevated LDL-P (due to smaller particles and lower cholesterol content). Even LDL-C levels meet the target, high LDL-P can still increase residual cardiovascular risk. Studies have shown that LDL-P is more predictive than any other parameter related to LDL¹⁷. Analysis of lipoprotein subfractions is critical for understanding the pathogenesis of ASCVD, assessing residual risk, and guiding precision treatment. In recent years, domestic guidelines have also recommended LDL subfractions as indicators for ASCVD risk assessment or treatment monitoring¹⁸.

ASCVD is the leading cause of death globally. Its prevention and control strategies heavily depend on lipid management. Currently, the screening of individuals with dyslipidemia in clinical practice primarily relies on traditional four lipid tests, including triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and LDL-C. Additionally, other lipid markers such as lipoprotein (a), apolipoprotein A1 (ApoA1), and ApoB are mainly used for high-risk populations of ASCVD¹⁹, such as diabetic patients, hypertensive patients, and smokers or those whose LDL-C levels have been controlled but still pose a high risk.

As research has progressed, the limitations of traditional lipid testing have become increasingly apparent, particularly in early warning effectiveness and explaining residual risk. A considerable number of ASCVD patients are initially diagnosed with normal lipid levels, making LDL-C ineffective for primary warning. Epidemiological data show that about 20% of patients with acute coronary syndrome have LDL-C levels within the normal range (<3.4 mmol/L) upon admission, suggesting that traditional lipid testing may miss high-risk individuals. Moreover, even after achieving LDL-C targets, ASCVD patients may still face a high residual cardiovascular risk. In these patients, most of the residual coronary artery events cannot be explained by a reduction in LDL-C levels. Therefore, simple LDL-C testing may not fully meet the clinical needs for ASCVD diagnosis and treatment, because routine lipid tests cannot differentiate between lbLDL-C and sdLDL-C.

Multiple studies have confirmed a clear correlation between lipoprotein subfractions and ASCVD. Abnormal results from lipoprotein subfraction testing can serve as a warning for primary prevention of ASCVD in individuals with normal lipid levels, helping to identify hidden high-risk groups. For secondary prevention of ASCVD, where LDL-C levels are already within target ranges, lipoprotein subfraction testing can help assess residual risk and guide further treatment²⁰. Therefore, the detection of novel LDL subfractions is expected to provide new targets for the prevention and treatment of dyslipidemia and ASCVD compared with traditional lipid tests.

The detection and analysis system in our laboratory primarily uses PAGE to separate lipoproteins subfractions based on their size and charge. Lipoprotein particles are separated primarily by size due to molecular sieving in the polyacrylamide matrix by PAGE. Small molecules migrate fast while large molecules are hindered by steric obstruction and migrate slowly. And the more charge a molecule carries, the faster it migrates. This method can quickly divide lipoproteins into twelve subfractions. LDL is divided into seven subfractions, labeled as LDL1 to LDL7. They all have distinct sizes, densities, physicochemical properties, metabolic behaviors and atherosclerotic potential. LDL1 and LDL2 are classified as lbLDL, while LDL3 to LDL7 are categorized as sdLDL. Lipoprotein subfraction detecting techniques include UC, HPLC and NMR. However, due to high equipment requirements, complex operation and time consumption, their clinical application is relatively limited²¹. The PAGE method efficiently separates various subfractions of LDL, making it more user-friendly compared to other techniques. The equipment used is cost-effective and portable, making it more suitable for widespread use in clinical routine laboratories. This study aims to validate PAGE as a rapid, cost-effective method for LDL subfraction analysis in serum samples to support clinical decision-making.

Protocol

All samples in this study are evaluated and approved by the Ethics Review Committee of Guangdong Provincial People's Hospital (Approval Number: KY2025-435-01). All participants signed written informed consent before the experiment.

1. Kit preparation

1. Preparation of the gel buffer
   1. Preparation of 30% acrylamide (ACR): Weigh 30 g of acrylamide, add 80 mL of deionized water, stir to dissolve, and adjust to 100 mL. Store at 4 °C in the dark until use.
   2. Preparation of 1% bisacrylamide (BIS): Weigh 1 g of bisacrylamide, add 80 mL of deionized water, stir to dissolve, and adjust to 100 mL. Store at 4 °C in the dark until use.
   3. Preparation of 25% sucrose: Weigh 25 g of sucrose, add 70 mL of deionized water, stir to dissolve, and adjust to 100 mL for storage at 4 °C.
   4. Preparation of 10% ammonium persulfate (APS): Dissolve 0.1 g ammonium persulfate in 1 mL of deionized water and use immediately.
   5. Preparation of 1M Tris (pH = 6.8): Weigh 121.14 g of trishydroxymethylaminomethane, add 800 mL of deionized water, stir to dissolve, and adjust the pH value to 6.8, then make up to 1000 mL.
   6. Preparation of 1M Tris (pH = 8.6): Weigh 121.14 g of trishydroxymethylaminomethane, add 800 mL of deionized water, stir to dissolve, and adjust the pH value to 8.6, then make up to 1000 mL.
      NOTE: When handling acrylamide, wear butyl rubber gloves, safety goggles, protective clothing, and operate in a fume hood. In case of skin contact, immediately wash with soap and water, then seek medical attention. In case of inhalation, move to fresh air.
2. Preparation of separation gel
   1. Take a clean glass tube and seal the lower layer with a sealing film.
   2. Add 8.29 mL of 30% ACR, 17.76 mL of 1% BIS, 10 mL of 25% sucrose, 1 mL of 10% APS, 10 mL of 1M Tris (pH = 8.6), 0.1 mL of tetramethylethylenediamine (TEMED), and 32.85 mL of deionized water into a clean beaker in sequence. Mix well to get 80 mL of mother liquor (with polyacrylamide concentration of 3.3%) and add to the glass tube at 1.2 mL per root. Polymerize at room temperature for 60 min and seal the liquid with purified water to ensure a flat liquid surface.
3. Preparation of concentrated glue
   1. Add 4.07 mL of 30% ACR, 3.05 mL of 1% BIS, 5 mL of 25% sucrose, 0.5 mL of 10% APS, 5 mL of 1M Tris (pH = 6.8), 0.05 mL of TEMED, and 32.33 mL of deionized water into a clean beaker in sequence, mix well to get 50 mL of mother liquor (with polyacrylamide concentration of 3.05%), add to the glass tube at 0.2 mL per root. Polymerize at room temperature for 60 min and seal the liquid with purified water to ensure a flat liquid surface.
4. Preparation of the preservation solution
   1. Weigh 6.4 g of Tris, 75 g of sucrose, 2.5 mL of glycerin, and 0.3 mL of PC-300 (antibacterial preservative) and add 800 mL of deionized water, stir to dissolve, adjust the PH value to 6.8, and finally dilute to 1000 mL. The concentration of Tris-HCl is about 52 mM.
5. Preparation of Sudan black staining solution
   1. Weigh 0.03 g of Sudan black and dissolve it in a mixture of 2 mL of ethylene glycol, 6 mL of anhydrous ethanol, and 2 mL of dimethyl sulfoxide. After filtration, divide it into 1 mL per vial.
      NOTE: When handling Sudan black and dimethyl sulfoxide, wear nitrile gloves and protective goggles to prevent direct contact. Operations must be conducted in a ventilated environment. Immediately rinse skin and eyes with water after exposure, then seek medical attention if necessary.
6. Inner packing
   1. Complete packaging according to the kit composition requirements.

2. Kit composition

1. Ensure the product contains prefabricated columns, preservative solution, and Sudan black staining solution. The prefabricated columns should contain acrylamide, bisacrylamide, sucrose, ammonium persulfate, Tris, and tetramethylethylenediamine. The prefabricated column's length is 75 ± 0.5 mm, inner diameter is 5.5 ± 0.02 mm, and outer diameter is 7.0 ± 0.02 mm. The preservative solution should contain Tris, sucrose, glycerin, and preservatives. The Sudan black staining solution should contain Sudan black, ethylene glycol, anhydrous ethanol, and dimethyl sulfoxide. Ensure that the packaging specifications are different according to the quantity of prefabricated columns. One serving of reagent must have one prefabricated column. Do not combine reagent kits of different batches, as this may affect experimental results.

3. Sample preparation

1. Patient preparation: Ensure the patient is on an empty stomach while drawing blood samples.
2. Use serum separation gel sampling tubes and carry out the sample collection in accordance with the Standard Operating Procedure. The blood must be separated as soon as possible after blood collection to avoid hemolysis.
3. Test the serum samples after separation as soon as possible at room temperature. If immediate testing is not possible, store the samples at 2-8 °C for up to 7 days or at -20 °C for up to 30 days.
4. Restore the samples to room temperature and fully mix before use, especially for the frozen and thawed samples. Repeated freeze-thawing is not permitted as it may compromise sample integrity. Both lipemia and hemolysis samples may affect test results. If it's a mild lipemia or hemolysis sample, it is recommended to perform high-speed centrifugation at 14,462 x g at room temperature for 15 min. For lipemia samples, use a pipette to aspirate the entire clear layer from the bottom. For hemolysis samples, collect the supernatant. Then transfer it to a new centrifuge tube and stain. However, if the lipemia or hemolysis is very severe, it is recommended to draw blood again.

4. Operation steps

1. Preparation
   1. Take out prefabricated columns and samples to be tested. Use a pipette to aspirate the residual preservation liquid from the sample port of prefabricated columns. Balance prefabricated columns and samples to room temperature and dissolve 14.3 g electrophoretic buffer powder with 800 mL deionized water.
2. Dye
   1. Take clean centrifuge tubes and number them. The number of tubes is consistent with the order of the blood collection tubes. Add the centrifuge tubes with a ratio of 50 µL sample to 10 µL Sudan black staining solution, mix well, and let it stand for 30 min at room temperature to stain.
      NOTE: The staining principle of Sudan black is based on its lipophilic property. It can induce specific chromogenic reactions with lipid substances such as neutral fats, phospholipids, and steroids within cells, resulting in brownish-black or dark black particles. In this system, we have optimized the staining time and dosage using a single-factor experimental design with the following protocol. Three LDL-C serum samples (high, medium, and low concentrations) are selected, with corresponding staining volumes of 5 µl, 10 µl, 15 µl, and 20 µl, respectively. The staining durations are set at 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 45 min, and 60 min. Electrophoretic experiments are conducted, followed by grayscale analysis of the electrophoretic bands to verify staining effectiveness. The optimal conditions are determined to be 10 µl staining volume with 30 min ± 5 min staining duration. Lower staining volume and duration result in faintly stained lipoprotein cholesterol. While the optimal conditions have achieved balance, higher staining volume and duration are not necessary.
3. Ionophortic separation
   1. Insert prebalanced columns into the rubber socket of the electrophoresis tank. The sample port of the gel column is about 2 cm away from the rubber stopper. Ensure that it is tightly closed and no leakage occurs. When the number of sample gel columns is not full, fill the empty space with a holeless rubber plug.
   2. Then pour electrophoresis buffer into the upper and lower tanks. Remove the bubbles at the mouth of prefabricated columns. Add 25 µL of stained samples to the sample port of prefabricated columns.
   3. Cover the lid, connect the power switch, and set the parameters of voltage (U = 100 V), current (I = 3 mA/tube), power (P = 120 W), time (T = 70 min) for electrophoresis separation.
      NOTE: Voltage, power, and time are fixed parameters. Current needs to be set according to the number of test samples (e.g., 10 sample tubes, I = 3 mA x 10 tubes = 30 mA).
   4. After electrophoresis is completed, turn off the power, remove the lid and pour out electrophoresis buffer on the upper part of the electrophoresis device. Take out the gel columns in turn and place them on the fixing frame, pour out the excess electrophoresis buffer on the upper surface and analyze in the scanning analysis system.
4. Scanning analysis
   NOTE: The type of analyzer is DY-03, and the scan software is KBL 3.3.9 GD.
   1. Turn on the power switch of the scanning analyzer, double-click the desktop system software, and click Collection to enter the software interface. Click Export Sample Tray in the software interface, place gel columns on the sample rack with the sampling end facing towards the interior of the device. Ensure gel columns are placed in the corresponding numbered positions. Positions 25 and 26 are designated for quality control testing only.
   2. Click Import Sample tray on the software interface to import the sample tray into the device. Click Scan to obtain sample scan images. The device has a built-in auto-focus system. It automatically adjusts the focus between the scanning module and the gel tube to ensure clear imaging. The software automatically identifies the initial position (VLDL) and termination position (HDL) of the electrophorogram. When obvious peaks are detected at these positions, the software locks onto the designated analysis area for spectral analysis.
   3. After entering patients' information and scanning the barcode, click Analysis and processing in the software interface, the system will automatically analyze and generate test reports. Click Upload LIS to transfer test reports to LIS system.
   4. Take out prefabricated columns after scanning and analysis and treat them according to medical waste specifications.

5. Quality control

1. Perform a quality control test of two levels before daily sample testing. The operation steps of the quality control test are the same as those of a sample test. Type A reference is the average particle diameter greater than 268 Å. Type B reference is the average particle diameter lesser than 265 Å.
   NOTE: Result analysis: TYPE A, the mean LDL-particle size is > 268 Å, LDL1 and LDL2 account for more, indicating a low risk of CVD. TYPE INTERMEDIATE (TYPE INT), the mean LDL-particle size is 265 to 268 Å, indicating that the proportion of LDL3 to LDL7 is above the critical value. TYPE B, the mean LDL-particle size is < 265 Å, LDL3 to LDL7 account for more, indicating a high risk of CVD.

6. Technical improvements

1. To ensure accurate diagnostic outcomes, the following technical improvements should be implemented.
   1. Pre-examination: Ensure that the patients are fully fasting before blood collection. Use serum or EDTA anticoagulant plasma instead of heparin anticoagulant plasma. Ensure that the pipette is accurate in its uptake. Avoid severe chylous or hemolytic samples.
   2. During examination: Set electrophoresis parameters of voltage (U = 100 V), current (I = 3 mA/tube), power (P = 120 W), time (T = 70 min) according to standard protocols. Wear sterile gloves to prevent contamination of glass tubes that might affect the ability to take photos and scan.
   3. After examination: When issuing reports, review abnormal chromatograms for potential interference and perform reanalysis to avoid providing incorrect diagnostic conclusions.
      1. The normal chromatogram: At the initial position of VLDL and at the termination position of HDL, both have a distinct peak (see Representative results section).
      2. The anomaly chromatogram: At the initial position of VLDL or the termination position of HDL has no or more than one peak.

Results

This study aims to invent a method using PAGE to detect lipoprotein subfractions in participants' serum. The goal is to determine whether the LDL-C subfractions are primarily large particles (LDL1 and LDL2) or small particles (LDL3 to LDL7). After staining with Sudan black, the samples are separated by the electrophoresis device in a polyacrylamide gel. Lipoproteins are then separated into distinct bands within the gel tubes, and subfraction analysis is performed using a scanning analysis system.

Discussion

Although the role of sdLDL as a cardiovascular risk factor in the development of atherosclerosis has been confirmed by research, there is currently no established method to identify LDL subfractions. Over the past few decades, significant progress has been made in developing detection methods for LDL subfractions. UC is considered the gold standard. It can separate LDL subfractions based on their density differences, distinguishing between sdLDL and lbLDL with high-resolution. However, this method requires expensive ultr...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Electrophoretic buffer powder	Guangxi Kangbolai Technology Company	25030401	For preparing buffer solutions and providing electrophoresis conditions
High value quality control products	Guangxi Kangbolai Technology Company	25030301	For monitoring accuracy and precision
Lipoprotein sample density separation solution (gradient gel electrophoresis)	Guangxi Kangbolai Technology Company	LSS60-SB1-A1  25030301	For separating lipoprotein subtype components from serum samples
Low value quality control products	Guangxi Kangbolai Technology Company	25030301	For monitoring accuracy and precision
Sudan black staining solution	Guangxi Kangbolai Technology Company	LSS60-SB1-A1  25030301	For sample staining