Cell-free Biochemical Fluorometric Enzymatic Assay for High-throughput Measurement of Lipid Peroxidation in High Density Lipoprotein

Summary

We describe here a fluorometric cell-free biochemical assay for determination of HDL-lipid peroxidation. This rapid and reproducible assay can be used to determine HDL function in large scale studies and can contribute to our understanding of HDL function in human disease.

Abstract

Low high-density lipoprotein cholesterol (HDL-C) levels are one of the most powerful independent negative predictors of atherosclerotic cardiovascular disease (CVD). The structure and function of HDL rather than HDL-C may more accurately predict atherosclerosis. Several HDL protein and lipid compositional changes that impair HDL function occur in inflammatory states such as atherosclerosis. HDL function is usually determined by cell based assays such as cholesterol efflux assay but these assays have numerous drawbacks lack of standardization. Cell-free assays may give more robust measures of HDL function compared to cell-based assays. HDL oxidation impairs HDL function. HDL has a major role in lipid peroxide transport and high amount of lipid peroxides is related to abnormal HDL function. Lipid-probe interactions should be considered when interpreting the results of non-enzymatic fluorescence assays for measuring the lipid oxidative state. This motivated us to develop a cell-free biochemical enzymatic method to assess HDL lipid peroxide content (HDLox) that contributes to HDL dysfunction. This method is based on the enzyme horseradish peroxidase (HRP) and the fluorochrome Amplex Red that can quantify (without cholesterol oxidase) the lipid peroxide content per mg of HDL-C. Here a protocol is describedfor determination of HDL-lipid peroxidation using the fluorochrome reagent. Assay variability can be reduced by strict standardization of experimental conditions. Higher HDLox values are associated with reduced HDL antioxidant function. The readout of this assay is associated with readouts of validated cell-based assays, surrogate measures of cardiovascular disease, systemic inflammation, immune dysfunction, and associated cardiovascular and metabolic risk phenotypes. This technical approach is a robust method to assess HDL function in human disease where systemic inflammation, oxidative stress and oxidized lipids have a key role (such as atherosclerosis).

Introduction

Atherosclerotic cardiovascular disease (CVD) is the leading cause of death worldwide^1,2. Epidemiological studies have shown that low levels of high-density lipoprotein (HDL) cholesterol are generally inversely associated with the risk for the development of atherosclerosis^1,2. Although several studies support an atheroprotective role for HDL^1,2, the mechanism by which HDL attenuates the initiation and progression of atherosclerosis is complex ^3,

Protocol

All experiments using human biological samples were performed with ethics approval from the University of California Los Angeles, Los Angeles and the Alfred Hospital Human Ethics committee, Melbourne.

NOTE: There are many variations of the fluorochrome HDL function Assay (see discussion)³². Below we will describe the protocol that gives the most consistent and reproducible results. An overview of the assay is shown in Figure 2.

1. Specimen Processing

1. Use fresh, non-hemolyzed serum (can be collected in serum separator tubes) or citrate plasma obtain....

Results

50 µL of each HDL sample are added into each well as in step 7.3. 50 µL of HRP solution 5 U/mL (0.25 U) are then added into each well as in step 7.5. Samples are incubated for 30 min at 37 °C as in step 7.6. 50 µL of fluorochrome reagent are then added into each well as in step 7.7 (final concentration of 300 µM). The fluorescent readout (in dark) is then assessed every minute over 120 minutes at 37 °C with a fluorescent plate reader (530/590 nm filters). Representative fluorescence data .......

Discussion

The protocol described here offers a robust tool to answer important research questions regarding the role of HDL function in atherosclerosis and human disease. The assay quantifies the HDL lipid peroxide content per mg of HDL-C using enzymatic amplification (HRP). This approach avoids known limitations of prior HDL function assays (e.g. the cholesterol efflux assay) including significant heterogeneity with regards to types of cells used, type of readout reported, lack of standardization and confounding effects of trigly.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Experimental Reagents
HDL PEG (Polyethylene Glycol) Precipitating Reagent	Pointe Scientific	H7511
Amplex Red reagent.	Life Technologies, Grand Island, NY	A12216	Amplex Red Cholesterol Assay Kit. • ≤–20°C • Desiccate • Protect from light
DMSO.	Life Technologies, Grand Island, NY	A12216	Amplex Red Cholesterol Assay Kit. • ≤–20°C • Desiccate • Protect from light
Horse Radish Peroxidase (HRP)	Life Technologies, Grand Island, NY	A12216	Amplex Red Cholesterol Assay Kit. • ≤–20°C • Desiccate • Protect from light
Cholesterol Esterase.	Life Technologies, Grand Island, NY	A12216	Amplex Red Cholesterol Assay Kit. • ≤–20°C • Desiccate • Protect from light
Cholesterol Reference standard	Life Technologies, Grand Island, NY	A12216	Amplex Red Cholesterol Assay Kit. • ≤–20°C • Desiccate • Protect from light
Resorufin fluorescense Reference standard	Life Technologies, Grand Island, NY	A12216	Amplex Red Cholesterol Assay Kit. • ≤–20°C • Desiccate • Protect from light
5x Reaction Buffer.	Life Technologies, Grand Island, NY	A12216	Amplex Red Cholesterol Assay Kit. • ≤–20°C • Desiccate • Protect from light
HDL Cholesterol Automated Reagent	ThermoFisher Scientific Co., San Jose, CA, USA.	TR39601
Name	Company	Catalog Number	Comments
Plasticware
96-well plates (polypropylene, flat bottom, clear).	Sigma Aldrich	M0687
96-well plates (polypropylene, flat bottom, black).	Sigma Aldrich	M9936
1.5 mL Eppendorf tubes	Eppendorf	0030 125.150
ClipTip 200, sterile	ThermoFisher Scientific Co., San Jose, CA, USA.	14-488-058
Thermo Scientific Multichannel Pipettes, 8-channel, 125	ThermoFisher Scientific Co., San Jose, CA, USA.	14-387--955
Name	Company	Catalog Number	Comments
Software
Gen5 2.01 software	Biotek, Vermont, USA	NA
Name	Company	Catalog Number	Comments
Equipment
Gen5 Plate reader	Biotek, Vermont, USA	NA