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Published on: March 14, 2021
High-Density Lipoprotein-Specific Phospholipid Efflux Assay
Edward B Neufeld1, Masaki Sato2, Alan T Remaley2
1Lipoprotein Metabolism Laboratory, National Heart, Lung, and Blood Institute, National Institutes of Health; neufelde@nhlbi.nih.gov.
Insights
A new cell-free assay measures high-density lipoprotein (HDL) function in removing arterial plaque lipids. This HDL-specific phospholipid efflux (HDL-SPE) assay predicts cardiovascular disease risk effectively.
Area of Science:
- Cardiovascular Science
- Biochemistry
- Lipid Metabolism
Background:
- Plasma high-density lipoprotein cholesterol (HDL-C) is a key cardiovascular disease (CVD) risk metric.
- HDL's anti-atherogenic function involves lipid removal from arterial plaques.
- HDL-mediated cholesterol efflux is a better CVD risk predictor than HDL-C levels.
Purpose of the Study:
- To describe a cell-free assay for measuring HDL functionality in lipid mobilization.
- To enable researchers to assess HDL-mediated phospholipid efflux.
- To provide a high-throughput method for CVD risk assessment and therapeutic screening.
Main Methods:
- Developed and validated the HDL-specific phospholipid efflux (HDL-SPE) assay.
- The assay measures HDL apolipoprotein-mediated removal of fluorescent phospholipids.
- Utilized cell-free, high-throughput methodology for standardization.
Main Results:
- The HDL-SPE assay predicts incident CVD risk.
- Performance is comparable or superior to the gold-standard cell-based cholesterol efflux assay.
- Demonstrated HDL-SPE's utility in clinical case-control studies.
Conclusions:
- The HDL-SPE assay is a simple, standardized, cell-free method for assessing HDL function.
- This assay can be used for basic research into HDL lipid efflux mechanisms.
- HDL-SPE serves as a valuable tool for clinical CVD risk assessment and drug discovery.
Abstract:
Plasma levels of high-density lipoprotein (HDL)-cholesterol (HDL-C) is currently a key metric for the clinical assessment of cardiovascular disease (CVD) risk. HDL-mediated removal of plaque lipids from cells and extracellular deposits in the arterial wall is one of several anti-atherogenic functions of HDL that may account for its inverse association with CVD risk. The HDL-mediated removal of cellular cholesterol in vitro has recently proven to be an even better predictor of CVD risk than HDL-C. HDL is composed of a heterogeneous population of particles, which perform different functions. HDL particles involved in the removal of atherosclerotic plaque lipids contain exchangeable apolipoproteins, mostly apolipoprotein A-I, that dissociate from the particle and then remove plaque lipids. Nascent HDL is formed by the solubilization and removal of both phospholipids and cholesterol from ABCA1-generated cellular plasma membrane domains by HDL-derived exchangeable apolipoproteins. This process plays a critical role in both the prevention and regression of atherosclerotic plaque. Herein, we describe the protocol for the cell-free, HDL-specific phospholipid efflux (HDL-SPE) assay, which we have previously shown can predict incident CVD risk. This assay specifically measures HDL apolipoprotein-mediated removal of a non-exchangeable fluorescent phospholipid from a lipid donor particle. Our case-control clinical studies have established that the HDL-SPE assay performs as well as and potentially even better than the "gold-standard" cell-based cholesterol efflux capacity assay, a laborious and technically complex assay that often utilizes radioactive cholesterol. The goal of this protocol is to enable basic and clinical researchers alike to assess the functionality of HDL in lipid mobilization, using a simple, standardized, cell-free, high-throughput assay. This assay can be used by basic researchers to gain insights into mechanisms underlying HDL-mediated lipid efflux and for screening new therapeutic agents that enhance HDL functionality. HDL-SPE can also serve as a clinical laboratory diagnostic assay for CVD risk assessment.
