Cell-free biochemical fluorometric enzymatic assay for high-throughput measurement of lipid peroxidation in high density lipoprotein

Roy, S, Nguyen, H, Angelovich, T, Hearps, A, Huynh, D, Jaworowski, A and Kelesidis, T 2017, 'Cell-free biochemical fluorometric enzymatic assay for high-throughput measurement of lipid peroxidation in high density lipoprotein', Journal of Visualized Experiments, vol. 2017, no. 128, pp. 1-10.


Document type: Journal Article
Collection: Journal Articles

Title Cell-free biochemical fluorometric enzymatic assay for high-throughput measurement of lipid peroxidation in high density lipoprotein
Author(s) Roy, S
Nguyen, H
Angelovich, T
Hearps, A
Huynh, D
Jaworowski, A
Kelesidis, T
Year 2017
Journal name Journal of Visualized Experiments
Volume number 2017
Issue number 128
Start page 1
End page 10
Total pages 10
Publisher Journal of Visualized Experiments
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. Cellfree 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
Subject Cardiology (incl. Cardiovascular Diseases)
Keyword(s) Amplex red
Cell-free assay
Fluorometric method
HDL function
Immunology
Issue 128
Lipid peroxidation
Oxidized HDL
DOI - identifier 10.3791/56325
Copyright notice © 2017 Journal of Visualized Experiments.
ISSN 1940-087X
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