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

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.

Figure 1. Principle of the Amplex Red assay of HDL function.

1. The acute-phase (AP) reaction favors the formation of dysfunctional HDL. In the basal state, HDL contains apoA-I and apoJ as well as 4 enzymes, paraoxonase (PON) and platelet-activating factor acetylhydrolase (PAF-AH), lecithin: cholesterol acyltransferase (LCAT), and plasma reduced glutathione selenoperoxidase (GSH peroxidase) that can prevent the formation of or inactivate the LDL-derived oxidized phospholipids found in oxidized LDL. As a result, in the basal state, HDL may be considered anti-oxidant. As previously published (Navab M et al. Arterioscler Thromb Vasc Biol 2001; 21: 481–488), during the acute-phase reaction, A-I may be displaced by the pro-oxidant acute-phase reactant Serum amyloid A (SAA). Another pro-oxidant acute-phase reactant, ceruloplasmin, associates with HDL as does the anti-oxidant acute phase reactant apoJ. PON, PAF-AH, and LCAT decrease in HDL during the acute-phase reaction, and the lipid hydroperoxides (LOOH) 5-hydroperoxyeicosatetraenoic acid (HPETE), hydroperoxyoctadecadienoic acid (HPODE), and cholesteryl linoleate hydroperoxide (CE-OOH) increase in HDL. The net effect of the changes in HDL during the acute-phase reaction is the production of pro-oxidant, HDL particles (AP-HDL or dysfunctional HDL). 3. HDL can be isolated using different methods such as ultracentrifugation, PEG precipitation and immunoaffinity capture. Using immunoaffinity capture of HDL and commercially available antibodies against total human HDL, HDL is isolated from a specific volume (e.g. 100 ul) of either a) non EDTA plasma b) serum or c) apoB depleted serum 4. Amplex Red (N-acetyl-3, 7-dihydroxyphenoxazine) reagent is a colorless substrate that reacts with hydrogen peroxide (H₂O₂) in the presence of horseradish peroxidase (HRP) with a 1∶1 stoichiometry to produce highly fluorescent resorufin (excitation/emission maxima  = 570/585 nm). This highly stable, sensitive and specific fluorogenic substrate for HRP has been widely used to develop a variety of fluorogenic assays for enzymes that produce hydrogen peroxide. For example Amplex Red reagent coupled with the enzymes cholesterol oxidase and HRP permit the ultrasensitive quantitation of HDL cholesterol based on lipid peroxidation. The biochemistry of lipid peroxidation has been well established (Free Radical Research 2010∶44, 1098–1124) and during this reaction there is formation of reactive oxygen species (ROS) such as peroxyl and alkoxyl radicals (ROO-, RO-; LOOH-) that have previously been shown to react with the Amplex Red reagent to form fluorescent resorufin ((J. Biol. Chem. 284, 46–55; J Biol Chem. 2010 May 28; 285(22): 16599–605). Resorufin is produced by the reaction of the Amplex Red reagent with H₂O₂ produced from the cholesterol oxidase-catalyzed oxidation of cholesterol. In the absence of cholesterol oxidase, the “endogenous” hydroperoxide content of a specific amount of HDL cholesterol can be quantified in the presence of HRP and Amplex Red. High hydroperoxide content of a specific amount of HDL cholesterol has previously been shown to be significantly associated with abnormal HDL function. The background production of hydroxyradicals as a result of air oxidation of the buffer (based on the readout of the blank well that contains Amplex Red reagent and buffer) is subtracted from the fluorescent readout of each well. In addition, to better evaluate the LOOH-dependent oxidation, catalase (not shown in figure) may be added in the medium to remove rapidly the formed H2O2 so that the detected increase in fluorescence may be attributed mainly to lipid LOOH release.

Figure 2. Assay performance.

A. Linearity of the Amplex Red Assay of HDL lipid peroxidation when ≤10 ug of HDL cholesterol is added. HDL was isolated by ultracentrifugation from 3 HIV infected patients known to have acute phase HDL (AP-HDL) and 3 patients with normal HDL (as determined using a previous assay of HDL function; J Lipid Res. 2011; 52: 2341–51). HDL was then added in varying concentrations (cholesterol) to 300 µM Amplex Red in a 96 well flat bottom plate and the rate of change in fluorescence was measured as in Figure S2 in the presence of 4 U/ml of HRP. The rates of change in fluorescence were normalized against the added HDL cholesterol amount (in µg of cholesterol as determined by a cholesterol assay) and are plotted (means and standard deviations) against the amounts of added HDL. Similar results were observed when HDL cholesterol isolated by PEG precipitation was added to the reaction. B. The linearity of the assay was also demonstrated using in vitro oxidation of HDL by adding increasing amounts of pre-formed lipid peroxides. In vivo studies have shown that 13(S)-H(P)ODE is an in vivo generated lipid oxidant that has a key role in atherogenesis and contributes to formation of dysfunctional HDL (Drug Metab Lett. 2010; 4: 139–48). HDL from 3 different healthy subjects (1 µg) was oxidized in vitro with increasing amounts of HPODE as previously described (Drug Metab Lett. 2010; 4: 139–48). Increasing amounts of 13(S)-H(P)ODE linearly increased the fluorescence readout using the Amplex Red assay as described in A.

Figure 3. The Amplex red assay of HDL function can detect established effect of statins on functional properties of HDL in animal models of atherosclerosis.

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 Figure 2, 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 (** P = 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 (** P = 0.01).

Figure 4. 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).

Figure 5. The readout from the Amplex Red Assay of HDL function correlates significantly to the readout of a previously validated cell based assay of HDL function.

Thirty samples of FPLC-purified HDL were assessed for their HDL redox activity (HDLox) using the Amplex Red assay as shown in Figure 2, and their HDL inflammatory index was determined in a cell-based assay as described in Materials and Methods. The values from each assay are plotted against each other.

Figure 6. The readout from the Amplex Red Assay of HDL function correlates significantly to the readout of a previously validated biochemical cell free assay of HDL function.

ApoB depleted serum was isolated by PEG precipitation from 50 healthy subjects and 100 patients with HIV infection that have previously been shown to have acute phase HDL (Lipids Health Dis 2012; 11: 87). HDL redox activity (HDLox) was determined with the Amplex Red assay as described in Figure 2 and with the dihydrorhodamine (DHR) assay as described in Methods. Non cryopreserved apoB depleted serum was used for the DHR assay and the readout was normalized by the readout of a pooled control as described in Figure S8. The values from each assay are plotted against each other (r = 0.46, p<0.001).

Figure 7. 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).

Figure 8. Use of different commercially available antibodies does not affect significantly the immunoaffinity capture of HDL and determination of HDLox using the Amplex Red assay.

HDL was isolated using immunoaffinity capture as described in Methods and Figure 7 from 30 healthy subjects (white circles) and 30 patients with HIV infection (solid circles). Two different antibodies were used (kit A and Kit B) as described in Methods. The Amplex Red oxidation rate (AROR) as a marker of HDLox was determined as described in Figure 2 and Figure S10. The values from each assay are plotted against each other.

Figure 9. 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).

Figure 10. The Amplex Red assay of HDL function can detect previously established favorable effects of exercise on HDL function.

HDLox was measured as described in Figure 2 and Figure S10 in a cohort of 90 humans looking into the effect of exercise on metabolic and other physiological parameters. In this study we found that high-intensity resistance training (RT) improved central and brachial blood pressures in the overweight untrained (OU) group, while having no effect on major indices of arterial stiffness in overweight/obese young men, without weight loss. Using the samples from this study we found that HDLox was significantly lower in both trained groups compared to the untrained group (LT vs. OU: 0.65±0.12 vs. 0.91±0.17, p = <0.001; OT vs. OU: 0.68±0.11vs. 0.91±0.17, p = 0.003), and LT and OT were not significantly different (p = 0.12).

Figure 11. The HDLox as measured with the novel assay is significantly associated with numerous anthropometric, metabolic and physiological parameters in humans.

HDLox was measured as described in Figure 2 and Figure S10 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).