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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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Increased HDL redox activity (HDLox), as measured by the Amplex Red Method and the immunoaffinity capture, is independently associated with progression of atherosclerosis in HIV-1- infected subjects in vivo.Scatter plot of the Rate of Change in Carotid intima–media thickness (CIMT) (ΔCIMT) and HDLox for 55 HIV-infected subjects (solid circles) and 36 uninfected controls (white circles). HDL ELISA kit was used to capture HDL in 96-well plates (kit B) as described in Methods. HDLox was determined as described in Figure 2 and Figure S10. The values from HDLox for each subject are plotted against ΔCIMT. In multivariate analysis of the HIV-infected subjects, higher baseline HDLox was associated with the ΔCIMT increasing by 2.3 mm/year (95% CI  =  (0.24, 5.6); p = 0.03) but no association between ΔCIMT and HDLox was seen in the controls (not shown).
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pone-0111716-g009: Increased HDL redox activity (HDLox), as measured by the Amplex Red Method and the immunoaffinity capture, is independently associated with progression of atherosclerosis in HIV-1- infected subjects in vivo.Scatter plot of the Rate of Change in Carotid intima–media thickness (CIMT) (ΔCIMT) and HDLox for 55 HIV-infected subjects (solid circles) and 36 uninfected controls (white circles). HDL ELISA kit was used to capture HDL in 96-well plates (kit B) as described in Methods. HDLox was determined as described in Figure 2 and Figure S10. The values from HDLox for each subject are plotted against ΔCIMT. In multivariate analysis of the HIV-infected subjects, higher baseline HDLox was associated with the ΔCIMT increasing by 2.3 mm/year (95% CI  =  (0.24, 5.6); p = 0.03) but no association between ΔCIMT and HDLox was seen in the controls (not shown).

Mentions: To validate the utility of the new assay to measure HDLox as a marker of disease in humans, HDLox was measured blindly using plasma samples from a previously described cohort of 55 HIV infected subjects and 36 uninfected matched controls [28] and the Amplex Red assay. We found that HDLox was independently associated with progression of subclinical atherosclerosis in HIV-infected subjects (Fig. 9).


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)

Increased HDL redox activity (HDLox), as measured by the Amplex Red Method and the immunoaffinity capture, is independently associated with progression of atherosclerosis in HIV-1- infected subjects in vivo.Scatter plot of the Rate of Change in Carotid intima–media thickness (CIMT) (ΔCIMT) and HDLox for 55 HIV-infected subjects (solid circles) and 36 uninfected controls (white circles). HDL ELISA kit was used to capture HDL in 96-well plates (kit B) as described in Methods. HDLox was determined as described in Figure 2 and Figure S10. The values from HDLox for each subject are plotted against ΔCIMT. In multivariate analysis of the HIV-infected subjects, higher baseline HDLox was associated with the ΔCIMT increasing by 2.3 mm/year (95% CI  =  (0.24, 5.6); p = 0.03) but no association between ΔCIMT and HDLox was seen in the controls (not shown).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111716-g009: Increased HDL redox activity (HDLox), as measured by the Amplex Red Method and the immunoaffinity capture, is independently associated with progression of atherosclerosis in HIV-1- infected subjects in vivo.Scatter plot of the Rate of Change in Carotid intima–media thickness (CIMT) (ΔCIMT) and HDLox for 55 HIV-infected subjects (solid circles) and 36 uninfected controls (white circles). HDL ELISA kit was used to capture HDL in 96-well plates (kit B) as described in Methods. HDLox was determined as described in Figure 2 and Figure S10. The values from HDLox for each subject are plotted against ΔCIMT. In multivariate analysis of the HIV-infected subjects, higher baseline HDLox was associated with the ΔCIMT increasing by 2.3 mm/year (95% CI  =  (0.24, 5.6); p = 0.03) but no association between ΔCIMT and HDLox was seen in the controls (not shown).
Mentions: To validate the utility of the new assay to measure HDLox as a marker of disease in humans, HDLox was measured blindly using plasma samples from a previously described cohort of 55 HIV infected subjects and 36 uninfected matched controls [28] and the Amplex Red assay. We found that HDLox was independently associated with progression of subclinical atherosclerosis in HIV-infected subjects (Fig. 9).

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