Second scoring network inclusive of up-regulated genes (score = 40) in LOCK vs RUN.

<p>Nodes represent genes/molecules. Shading is proportional to fold change size (red: upregulated). Direct and indirect relationships are denoted with solid and dashed lines, respectively. White nodes denote network members that were not altered in the network. Lines ending in an arrow or blunt end indicate known direction of molecular activation or inhibition, respectively. Different shapes of genes represent different gene functions.</p

PRAT adipocyte size distribution.

<p>Adipocyte diameters were classified in 10-μm categories [category #, adipocyte diameter range in μm (10: 0.0–9.0μm, 20: 10.0–19.9μm, 20: 20.0–29.9μm, etc.] on the x-axis; and the percentage of total adipocytes in a 10-μm category was plotted on y-axis. At least 300 adipocyte diameters were measured for each tissue sample. Different letters denote differences among groups at each 10-μm category, p<0.05, as determined by Student’s t-test.</p

Protein expression of A) Col1a1 and B) Cd36 in PRAT from continued wheel access (RUN) and wheel locking (LOCK) rats (n = 5 per group).

<p>Protein expression was normalized for Ponceau S.</p

Daily food intake over the final 14 days.

<p>The vertical dashed line represents the day of wheel locking (LOCK). Different letters denote significance among groups at p < 0.05, as determined by Student’s t-test.</p

Top scoring network inclusive of genes expressed only in LOCK (score = 63).

<p>Nodes represent genes/molecules. Shading is proportional to RPKM value. Direct and indirect relationships are denoted with solid and dashed lines, respectively. White nodes denote network members that were not altered in the network. Lines ending in an arrow or blunt end indicate known direction of molecular activation or inhibition, respectively. Different shapes of genes represent different gene functions.</p

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.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111716#s2" target="_blank">Methods</a>. HDLox was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111716#pone-0111716-g002" target="_blank">Figure 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111716#pone.0111716.s010" target="_blank">Figure S10</a>. 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).</p

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

<div><p>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.</p></div

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.

<p>HDL was isolated using immunoaffinity capture as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111716#s2" target="_blank">Methods</a> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111716#s2" target="_blank">methods</a>. The Amplex Red oxidation rate (AROR) as a marker of HDL redox activity (HDLox) was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111716#pone-0111716-g002" target="_blank">Figure 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111716#pone.0111716.s010" target="_blank">Figure S10</a>. 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).</p

The Amplex red assay of HDL function can detect established effect of statins on functional properties of HDL in animal models of atherosclerosis.

<p>A: By using FPLC, HDL was isolated from three pooled plasma samples from LDLR^−/− mice on Western diet (LDLR^−/− WD) for two weeks and from three pooled plasma samples from LDLR^−/− mice on Western diet for two weeks that were also treated with pravastatin 12.5 µg/ml for two weeks. Each plasma sample was pooled from 4 mice (12 mice in total). Oxidation of Amplex Red was assessed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111716#pone-0111716-g002" target="_blank">Figure 2</a>, using 2.5 µg (cholesterol) of added HDL. The oxidation slope of Amplex Red in the presence of HDL from LDLR^−/− WD + statin was normalized to the oxidation slope of Amplex Red in the presence of HDL from LDLR^−/− WD, and the percent relative differences are shown. The data represent the average of measurements from three independent experiments. There was a statistically significant reduction in the oxidation slope of Amplex Red in the presence of HDL isolated from LDLR^−/− WD + statin mice compared with the oxidation slope of DHR in the presence of HDL isolated from LDLR^−/− WD mice (** <i>P</i> = 0.01) B: By using FPLC, HDL was isolated from three pooled plasma samples from ApoE^−/− female mice on Western diet (ApoE^−/− WD) for two weeks and from three pooled plasma samples from ApoE^−/− female mice on Western diet for two weeks that were also treated with pravastatin 12.5 µg/ml for two weeks. Each plasma sample was pooled from 4 mice (12 mice in total). Oxidation of Amplex Red was assessed as in A. There was a statistically significant reduction in the oxidation slope of Amplex Red in the presence of HDL isolated from ApoE^−/− WD + statin mice compared with the oxidation slope of Amplex Red in the presence of HDL isolated from ApoE^−/− WD mice (** <i>P</i> = 0.01).</p

The HDLox as measured with the novel assay is significantly associated with numerous anthropometric, metabolic and physiological parameters in humans.

<p>HDLox was measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111716#pone-0111716-g002" target="_blank">Figure 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111716#pone.0111716.s010" target="_blank">Figure S10</a> in a previous cohort of 100 humans looking into the effect of exercise on metabolic and other physiological parameters. The values from HDLox for each subject are plotted against representative physiological parameters such as Body Mass Index (BMI), subendocardial viability ratio (SEVR), a noninvasive measure of subendocardial perfusion, C reactive protein (CRP) and oxidized Low Density Lipoprotein (ox-LDL).</p