A High Throughput Biochemical Fluorometric Method for Measuring Lipid Peroxidation in HDL

There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

Abstract

Current cell-based assays for determining the functional properties of high-density lipoproteins (HDL) have limitations. We report here the development of a new, robust fluorometric cell-free biochemical assay that measures HDL lipid peroxidation (HDLox) based on the oxidation of the fluorochrome Amplex Red. HDLox correlated with previously validated cell-based (r = 0.47, p<0.001) and cell-free assays (r = 0.46, p<0.001). HDLox distinguished dysfunctional HDL in established animal models of atherosclerosis and Human Immunodeficiency Virus (HIV) patients. Using an immunoaffinity method for capturing HDL, we demonstrate the utility of this novel assay for measuring HDLox in a high throughput format. Furthermore, HDLox correlated significantly with measures of cardiovascular diseases including carotid intima media thickness (r = 0.35, p<0.01) and subendocardial viability ratio (r = −0.21, p = 0.05) and physiological parameters such as metabolic and anthropometric parameters (p<0.05). In conclusion, we report the development of a new fluorometric method that offers a reproducible and rapid means for determining HDL function/quality that is suitable for high throughput implementation.

Related collections

Most cited references 54

Record: found
Abstract: not found
Article: not found

The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum.

R J Havel, H A Eder, J BRAGDON (1955)

0 comments Cited 418 times – based on 0 reviews      Review now

Bookmark

Record: found
Abstract: found
Article: not found

HDL and cardiovascular disease: atherogenic and atheroprotective mechanisms.

Mohamad Navab, Srinivasa T. Reddy, Brian Van Lenten … (2011)

The lipoprotein HDL has two important roles: first, it promotes reverse cholesterol transport, and second, it modulates inflammation. Epidemiological studies show that HDL-cholesterol levels are inversely correlated with the risk of cardiovascular events. However, many patients who experience a clinical event have normal, or even high, levels of HDL cholesterol. Measuring HDL-cholesterol levels provides information about the size of the HDL pool, but does not predict HDL composition or function. The main component of HDL, apolipoprotein A-I (apo A-I), is largely responsible for reverse cholesterol transport through the macrophage ATP-binding cassette transporter ABCA1. Apo A-I can be damaged by oxidative mechanisms, which render the protein less able to promote cholesterol efflux. HDL also contains a number of other proteins that are affected by the oxidative environment of the acute-phase response. Modification of the protein components of HDL can convert it from an anti-inflammatory to a proinflammatory particle. Small peptides that mimic some of the properties of apo A-I have been shown in preclinical models to improve HDL function and reduce atherosclerosis without altering HDL-cholesterol levels. Robust assays to evaluate the function of HDL are needed to supplement the measurement of HDL-cholesterol levels in the clinic. © 2011 Macmillan Publishers Limited. All rights reserved

0 comments Cited 175 times – based on 0 reviews      Review now

Bookmark

Record: found
Abstract: found
Article: not found

Fluorescent and luminescent probes for measurement of oxidative and nitrosative species in cells and tissues: progress, pitfalls, and prospects.

Peter Wardman (2007)

Chemical probes for free radicals in biology are important tools; fluorescence and chemiluminescence offer high detection sensitivity. This article reviews progress in the development of probes for "reactive oxygen and nitrogen" species, emphasizing the caution needed in their use. Reactive species include hydrogen peroxide; hydroxyl, superoxide, and thiyl radicals; carbonate radical-anion; and nitric oxide, nitrogen dioxide, and peroxynitrite. Probes based on reduced dyes lack selectivity and may require a catalyst for reaction: despite these drawbacks, dichlorodihydrofluorescein and dihydrorhodamine have been used in well over 2,000 studies. Use in cellular systems requires loading into cells, and minimizing leakage. Reactive species can compete with intracellular antioxidants, changes in fluorescence or luminescence possibly reflecting changes in competing antioxidants rather than free radical generation rate. Products being measured can react further with radicals, and intermediate probe radicals are often reactive toward antioxidants and especially oxygen, to generate superoxide. Common probes for superoxide and nitric oxide require activation to a reactive intermediate; activation is not achieved by the radical of interest and the response is thus additionally sensitive to this first step. Rational use of probes requires understanding and quantitation of the mechanistic pathways involved, and of environmental factors such as oxygen and pH. We can build on this framework of knowledge in evaluating new probes.

0 comments Cited 175 times – based on 0 reviews      Review now

Bookmark

All references

Author and article information

Contributors

Partha Mukhopadhyay: Role: Editor

Journal

Journal ID (nlm-ta): PLoS One

Journal ID (iso-abbrev): PLoS ONE

Journal ID (publisher-id): plos

Journal ID (pmc): plosone

Title: PLoS ONE

Publisher: Public Library of Science (San Francisco, USA )

ISSN (Electronic): 1932-6203

Publication date Collection: 2014

Publication date (Electronic): 4 November 2014

Volume: 9

Issue: 11

Electronic Location Identifier: e111716

Affiliations

[1 ]Department of Medicine, University of California Los Angeles, Los Angeles, California, United States of America

[2 ]Exercise and Metabolic Disease Research Laboratory, Translational Sciences Section, School of Nursing, University of California Los Angeles, Los Angeles, California, United States of America

[3 ]Department of Obstetrics and Gynecology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States of America

[4 ]Department of Molecular and Medical Pharmacology, University of California Los Angeles, Los Angeles, California, United States of America

[5 ]Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States of America

National Institutes of Health, United States of America

Author notes

* E-mail: tkelesidis@ 123456mednet.ucla.edu

Competing Interests: STR is a principal in Bruin Pharma. Other authors have no conflicts of interest to declare. This manuscript is related to the Provision Patent Application UCLA Case UCLAP123P/2014-425-1 entitled “High Throughput Biochemical Fluorometric Method for Measuring HDL Redox Activity”. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

Conceived and designed the experiments: TK STR OY. Performed the experiments: TK DH CKR. Analyzed the data: TK CKR. Contributed reagents/materials/analysis tools: TK CKR JSC OMM STR OY. Wrote the paper: TK STR OY.

Article

Publisher ID: PONE-D-14-31425

DOI: 10.1371/journal.pone.0111716

PMC ID: 4219769

PubMed ID: 25368900

SO-VID: 6928ecd8-59e5-4758-ba96-28eb475fcdfa

License:

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

History

Date received : 15 July 2014

Date accepted : 2 October 2014

Page count

Pages: 17

Funding

This work was supported by RO1 grants HL095132 (JSC) and HL082823 and HL71776 (STR), K08 grant K08AI08272 (TK) and UCLA AIDS Institute and the UCLA Center for AIDS Research (AI28697). Partial funding for laboratory work was provided by the University of Washington's CVD and Metabolic Complications of HIV/AIDS Data Coordinating Center (5R01HL095126). The exercise work was supported by the American Heart Association (BGIA 0765139Y to CKR), the National Heart, Lung and Blood Institute (P50 HL105188 to CKR), and the National Institute of Diabetes and Digestive and Kidney Diseases (DK090406 to CKR). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health or other granting agencies. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Custom metadata

Data Availability The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.

A High Throughput Biochemical Fluorometric Method for Measuring Lipid Peroxidation in HDL

Read this article at

Abstract

Related collections

PLOS Climate

Most cited references 54

The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum.

HDL and cardiovascular disease: atherogenic and atheroprotective mechanisms.

Fluorescent and luminescent probes for measurement of oxidative and nitrosative species in cells and tissues: progress, pitfalls, and prospects.

Author and article information

Contributors

Journal

Affiliations

Author notes

Article

History

Page count

Funding

Categories

Custom metadata

Comments

Comment on this article

Similar content 242

Cited by 7

Most referenced authors 1,254