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

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 10....

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