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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 in combination with immunoaffinity capture of HDL can detect acute phase HDL in vivo in subjects previously shown to have dysfunctional HDL.HDL was isolated using immunoaffinity capture as described in Methods from 30 healthy subjects and 30 patients with HIV infection that have previously been shown to have acute phase HDL (Lipids Health Dis 2012; 11: 87). The following different matrices were added in 96 well plates for immunoaffinity capture of HDL: a) purified HDL isolated by ultracentrifugation (5 µg of HDL cholesterol as determined by cholesterol assay), b) apo-B depleted serum (5 µg of HDL cholesterol as determined by cholesterol assay) c) apo-B depleted serum (100 µl) d) plasma (100 µl). In the latter two methods, the fluorescent readout (that corresponds to HDLox) was normalized to the HDL cholesterol concentration (measured by the clinical lab). ApoB depleted serum and plasma was isolated by PEG precipitation and HDL was also isolated by ultracentrifugation as described in methods. 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 (A: 1.66±0.37; B: 1.54±0.32; C: 1.40±0.33; D: 1.32±0.32) compared to the uninfected subjects (A: 1.05±0.28; B: 0.95±0.23; C: 0.81±0.24; D: 0.73±0.24) (p<0.01 for all comparisons).
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pone-0111716-g007: The Amplex Red Assay of HDL function in combination with immunoaffinity capture of HDL can detect acute phase HDL in vivo in subjects previously shown to have dysfunctional HDL.HDL was isolated using immunoaffinity capture as described in Methods from 30 healthy subjects and 30 patients with HIV infection that have previously been shown to have acute phase HDL (Lipids Health Dis 2012; 11: 87). The following different matrices were added in 96 well plates for immunoaffinity capture of HDL: a) purified HDL isolated by ultracentrifugation (5 µg of HDL cholesterol as determined by cholesterol assay), b) apo-B depleted serum (5 µg of HDL cholesterol as determined by cholesterol assay) c) apo-B depleted serum (100 µl) d) plasma (100 µl). In the latter two methods, the fluorescent readout (that corresponds to HDLox) was normalized to the HDL cholesterol concentration (measured by the clinical lab). ApoB depleted serum and plasma was isolated by PEG precipitation and HDL was also isolated by ultracentrifugation as described in methods. 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 (A: 1.66±0.37; B: 1.54±0.32; C: 1.40±0.33; D: 1.32±0.32) compared to the uninfected subjects (A: 1.05±0.28; B: 0.95±0.23; C: 0.81±0.24; D: 0.73±0.24) (p<0.01 for all comparisons).

Mentions: 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). However, since the proteome of dysfunctional HDL has not been fully elucidated [19], it is possible that dysfunctional HDL may bind less efficiently to different commercially available HDL antibodies and the total amount of HDL protein may not be directly compared between different subjects. Thus, to determine whether the approach of immunoaffinity capture of HDL depends on the quality of commercially available HDL antibodies, we used 2 different commercially available HDL kits with the Amplex Red assay (kit A and B) to measure HDLox in 60 samples. We found comparable results (Fig. 8) which indicate that the detected differences in HDLox are real and not artificial secondary to variations in binding of HDL to the antibody. Thus, the suggested method may allow high throughput isolation of HDL and in situ detection of HDLox that is associated with HDL function.


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 in combination with immunoaffinity capture of HDL can detect acute phase HDL in vivo in subjects previously shown to have dysfunctional HDL.HDL was isolated using immunoaffinity capture as described in Methods from 30 healthy subjects and 30 patients with HIV infection that have previously been shown to have acute phase HDL (Lipids Health Dis 2012; 11: 87). The following different matrices were added in 96 well plates for immunoaffinity capture of HDL: a) purified HDL isolated by ultracentrifugation (5 µg of HDL cholesterol as determined by cholesterol assay), b) apo-B depleted serum (5 µg of HDL cholesterol as determined by cholesterol assay) c) apo-B depleted serum (100 µl) d) plasma (100 µl). In the latter two methods, the fluorescent readout (that corresponds to HDLox) was normalized to the HDL cholesterol concentration (measured by the clinical lab). ApoB depleted serum and plasma was isolated by PEG precipitation and HDL was also isolated by ultracentrifugation as described in methods. 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 (A: 1.66±0.37; B: 1.54±0.32; C: 1.40±0.33; D: 1.32±0.32) compared to the uninfected subjects (A: 1.05±0.28; B: 0.95±0.23; C: 0.81±0.24; D: 0.73±0.24) (p<0.01 for all comparisons).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4219769&req=5

pone-0111716-g007: The Amplex Red Assay of HDL function in combination with immunoaffinity capture of HDL can detect acute phase HDL in vivo in subjects previously shown to have dysfunctional HDL.HDL was isolated using immunoaffinity capture as described in Methods from 30 healthy subjects and 30 patients with HIV infection that have previously been shown to have acute phase HDL (Lipids Health Dis 2012; 11: 87). The following different matrices were added in 96 well plates for immunoaffinity capture of HDL: a) purified HDL isolated by ultracentrifugation (5 µg of HDL cholesterol as determined by cholesterol assay), b) apo-B depleted serum (5 µg of HDL cholesterol as determined by cholesterol assay) c) apo-B depleted serum (100 µl) d) plasma (100 µl). In the latter two methods, the fluorescent readout (that corresponds to HDLox) was normalized to the HDL cholesterol concentration (measured by the clinical lab). ApoB depleted serum and plasma was isolated by PEG precipitation and HDL was also isolated by ultracentrifugation as described in methods. 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 (A: 1.66±0.37; B: 1.54±0.32; C: 1.40±0.33; D: 1.32±0.32) compared to the uninfected subjects (A: 1.05±0.28; B: 0.95±0.23; C: 0.81±0.24; D: 0.73±0.24) (p<0.01 for all comparisons).
Mentions: 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). However, since the proteome of dysfunctional HDL has not been fully elucidated [19], it is possible that dysfunctional HDL may bind less efficiently to different commercially available HDL antibodies and the total amount of HDL protein may not be directly compared between different subjects. Thus, to determine whether the approach of immunoaffinity capture of HDL depends on the quality of commercially available HDL antibodies, we used 2 different commercially available HDL kits with the Amplex Red assay (kit A and B) to measure HDLox in 60 samples. We found comparable results (Fig. 8) which indicate that the detected differences in HDLox are real and not artificial secondary to variations in binding of HDL to the antibody. Thus, the suggested method may allow high throughput isolation of HDL and in situ detection of HDLox that is associated with HDL function.

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