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A high throughput biochemical fluorometric method for measuring lipid peroxidation in HDL.

Kelesidis T, Roberts CK, Huynh D, Martínez-Maza O, Currier JS, Reddy ST, Yang OO - PLoS ONE (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of California Los Angeles, Los Angeles, California, United States of America.

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.

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The Amplex Red Assay of HDL function can detect acute phase HDL in vivo in subjects previously shown to have dysfunctional HDL.ApoB depleted serum was isolated by PEG precipitation from 50 healthy subjects and 100 patients with HIV infection and that have previously been shown to have acute phase HDL (Lipids Health Dis 2012; 11: 87). The Amplex Red oxidation rate (AROR) as a marker of HDL redox activity (HDLox) was determined as described in Figure 2 and Figure S10. The HIV-infected subjects had significantly higher HDLox (1.59±0.53) compared to the uninfected subjects 1.01±0.31) (p<0.001).
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pone-0111716-g004: The Amplex Red Assay of HDL function can detect acute phase HDL in vivo in subjects previously shown to have dysfunctional HDL.ApoB depleted serum was isolated by PEG precipitation from 50 healthy subjects and 100 patients with HIV infection and that have previously been shown to have acute phase HDL (Lipids Health Dis 2012; 11: 87). The Amplex Red oxidation rate (AROR) as a marker of HDL redox activity (HDLox) was determined as described in Figure 2 and Figure S10. The HIV-infected subjects had significantly higher HDLox (1.59±0.53) compared to the uninfected subjects 1.01±0.31) (p<0.001).

Mentions: To further validate the new method we used the Amplex Red assay to detect dysfunctional HDL in conditions in which dysfunctional HDL is known to be present such as established animal models of atherosclerosis [12], [14], and Human Immunodeficiency Virus infection (HIV) [12]. The Amplex Red assay could detect established effect of statins on functional properties of HDL in animal models of atherosclerosis such as LDLR−/− (Fig. 3a) and ApoE−/− mice (Fig. 3b). In addition, using samples from a previous cohort of HIV patients known to have dysfunctional HDL by using previously established assays of HDL function [12], [22], the Amplex Red assay confirmed that these patients had higher HDLox compared to healthy controls (Fig. 4). Thus, using this methodology we demonstrate that HDL from patients with dysfunctional HDL has a higher rate of lipid peroxidation of a specific amount of HDL (HDLox) compared to HDL from healthy patients.


A high throughput biochemical fluorometric method for measuring lipid peroxidation in HDL.

Kelesidis T, Roberts CK, Huynh D, Martínez-Maza O, Currier JS, Reddy ST, Yang OO - PLoS ONE (2014)

The Amplex Red Assay of HDL function can detect acute phase HDL in vivo in subjects previously shown to have dysfunctional HDL.ApoB depleted serum was isolated by PEG precipitation from 50 healthy subjects and 100 patients with HIV infection and that have previously been shown to have acute phase HDL (Lipids Health Dis 2012; 11: 87). The Amplex Red oxidation rate (AROR) as a marker of HDL redox activity (HDLox) was determined as described in Figure 2 and Figure S10. The HIV-infected subjects had significantly higher HDLox (1.59±0.53) compared to the uninfected subjects 1.01±0.31) (p<0.001).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4219769&req=5

pone-0111716-g004: The Amplex Red Assay of HDL function can detect acute phase HDL in vivo in subjects previously shown to have dysfunctional HDL.ApoB depleted serum was isolated by PEG precipitation from 50 healthy subjects and 100 patients with HIV infection and that have previously been shown to have acute phase HDL (Lipids Health Dis 2012; 11: 87). The Amplex Red oxidation rate (AROR) as a marker of HDL redox activity (HDLox) was determined as described in Figure 2 and Figure S10. The HIV-infected subjects had significantly higher HDLox (1.59±0.53) compared to the uninfected subjects 1.01±0.31) (p<0.001).
Mentions: To further validate the new method we used the Amplex Red assay to detect dysfunctional HDL in conditions in which dysfunctional HDL is known to be present such as established animal models of atherosclerosis [12], [14], and Human Immunodeficiency Virus infection (HIV) [12]. The Amplex Red assay could detect established effect of statins on functional properties of HDL in animal models of atherosclerosis such as LDLR−/− (Fig. 3a) and ApoE−/− mice (Fig. 3b). In addition, using samples from a previous cohort of HIV patients known to have dysfunctional HDL by using previously established assays of HDL function [12], [22], the Amplex Red assay confirmed that these patients had higher HDLox compared to healthy controls (Fig. 4). Thus, using this methodology we demonstrate that HDL from patients with dysfunctional HDL has a higher rate of lipid peroxidation of a specific amount of HDL (HDLox) compared to HDL from healthy patients.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of California Los Angeles, Los Angeles, California, United States of America.

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.

Show MeSH
Related in: MedlinePlus