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.
Highlights
There is a continuing search for new biomarkers of atherosclerotic disease
Using this approach the fluorescence readout that corresponds to HDL lipid peroxidation (HDLox) can be expressed as the rate of formation of specific amount of resorfurin per specific unit of time and this readout can be directly compared among different experiments especially since all the reagents used in the Amplex red assay, including all buffers, are commercially available and accessible to all researchers (Fig. S7)
Amplex Red can reliably measure lipid peroxidation of a specific amount of high-density lipoproteins (HDL) isolated using immunoaffinity capture of HDL. Using this methodology with different matrices we demonstrate that HDL from patients with dysfunctional HDL has a higher rate of lipid peroxidation of a specific amount of HDL compared to HDL from healthy patients (Fig. 7)
Summary
Manipulation of HDL has great potential in reducing cardiovascular risk; studies in humans suggest a complex relationship between HDL and atherosclerosis [1,2]. HDL function rather than absolute level may be a more accurate indicator of cardiovascular risk [1,2]. Due to the complexity of the HDL particles, measurement of HDL function has not been studied extensively in humans [3,4]. Robust assays to evaluate the function of HDL are needed to supplement the measurement of HDL cholesterol levels in the clinic. HDL functional properties are most often determined by cell-based assays including the measurement of cholesterol efflux capacity [5,6,7,8]. Several limitations render these cell-based assays inaccessible to many researchers and difficult to scale up for large-scale clinical trials and/or routine clinical use [9]
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