A biochemical fluorometric method for assessing the oxidative properties of HDL
2011; Elsevier BV; Volume: 52; Issue: 12 Linguagem: Inglês
10.1194/jlr.d018937
ISSN1539-7262
AutoresTheodoros Kelesidis, Judith S. Currier, Diana Huynh, David Meriwether, Christina Charles‐Schoeman, Srinivasa T. Reddy, Alan M. Fogelman, Mohamad Navab, Otto O. Yang,
Tópico(s)Retinoids in leukemia and cellular processes
ResumoMost current assays of HDL functional properties are cell-based. We have developed a fluorometric biochemical assay based on the oxidation of dihydrorhodamine 123 (DHR) by HDL. This cell-free assay assesses the intrinsic ability of HDL to be oxidized by measuring increasing fluorescence due to DHR oxidation over time. The assay distinguishes the oxidative potential of HDL taken from different persons, and the results are reproducible. Direct comparison of this measurement correlated well with results obtained using a validated cell-based assay (r2= 0.62, P < 0.001). The assay can be scaled from a 96-well format to a 384-well format and, therefore, is suitable for high-throughput implementation. This new fluorometric method offers an inexpensive, accurate, and rapid means for determining the oxidative properties of HDL that is applicable to large-scale clinical studies. Most current assays of HDL functional properties are cell-based. We have developed a fluorometric biochemical assay based on the oxidation of dihydrorhodamine 123 (DHR) by HDL. This cell-free assay assesses the intrinsic ability of HDL to be oxidized by measuring increasing fluorescence due to DHR oxidation over time. The assay distinguishes the oxidative potential of HDL taken from different persons, and the results are reproducible. Direct comparison of this measurement correlated well with results obtained using a validated cell-based assay (r2= 0.62, P < 0.001). The assay can be scaled from a 96-well format to a 384-well format and, therefore, is suitable for high-throughput implementation. This new fluorometric method offers an inexpensive, accurate, and rapid means for determining the oxidative properties of HDL that is applicable to large-scale clinical studies. There is a continuing search for new biomarkers of increased risk for atherosclerotic disease. High-density lipoproteins (HDL) can become dysfunctional during acute-phase responses (1.Navab M. Reddy S.T. Van Lenten B.J. Anantharamaiah G.M. Fogelman A.M. The role of dysfunctional HDL in atherosclerosis.J. Lipid Res. 2009; 50: S145-S149Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 2.Navab M. Berliner J.A. Watson A.D. Hama S.Y. Territo M.C. Lusis A.J. Shih D.M. Van Lenten B.J. Frank J.S. Demer L.L. et al.The Yin and Yang of oxidation in the development of the fatty streak. A review based on the 1994 George Lyman Duff Memorial Lecture.Arterioscler. Thromb. Vasc. Biol. 1996; 16: 831-842Crossref PubMed Scopus (614) Google Scholar). We have previously shown a correlation between the lipid hydroperoxides produced by oxidized lipids such as low-density lipoproteins (LDL) in cell culture supernatant of macrophages and the recruitment of inflammatory mediators that are conducive to atherosclerosis (1.Navab M. Reddy S.T. Van Lenten B.J. Anantharamaiah G.M. Fogelman A.M. The role of dysfunctional HDL in atherosclerosis.J. Lipid Res. 2009; 50: S145-S149Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 2.Navab M. Berliner J.A. Watson A.D. Hama S.Y. Territo M.C. Lusis A.J. Shih D.M. Van Lenten B.J. Frank J.S. Demer L.L. et al.The Yin and Yang of oxidation in the development of the fatty streak. A review based on the 1994 George Lyman Duff Memorial Lecture.Arterioscler. Thromb. Vasc. Biol. 1996; 16: 831-842Crossref PubMed Scopus (614) Google Scholar). Thus, oxidized lipids produce reactive oxygen species (ROS) and can induce monocyte migration, which is an important event in atherosclerosis (1.Navab M. Reddy S.T. Van Lenten B.J. Anantharamaiah G.M. Fogelman A.M. The role of dysfunctional HDL in atherosclerosis.J. Lipid Res. 2009; 50: S145-S149Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 2.Navab M. Berliner J.A. Watson A.D. Hama S.Y. Territo M.C. Lusis A.J. Shih D.M. Van Lenten B.J. Frank J.S. Demer L.L. et al.The Yin and Yang of oxidation in the development of the fatty streak. A review based on the 1994 George Lyman Duff Memorial Lecture.Arterioscler. Thromb. Vasc. Biol. 1996; 16: 831-842Crossref PubMed Scopus (614) Google Scholar). Normally functioning HDL removes ROS from low-density lipoproteins (LDL), reducing both the oxidation of LDL and the recruitment of inflammatory mediators that are important in the pathogenesis of atherosclerosis (3.Navab M. Hama S.Y. Cooke C.J. Anantharamaiah G.M. Chaddha M. Jin L. Subbanagounder G. Faull K.F. Reddy S.T. Miller N.E. et al.Normal high density lipoprotein inhibits three steps in the formation of mildly oxidized low density lipoprotein: step 1.J. Lipid Res. 2000; 41: 1481-1494Abstract Full Text Full Text PDF PubMed Google Scholar, 4.Navab M. Hama S.Y. Anantharamaiah G.M. Hassan K. Hough G.P. Watson A.D. Reddy S.T. Sevanian A. Fonarow G.C. Fogelman A.M. Normal high density lipoprotein inhibits three steps in the formation of mildly oxidized low density lipoprotein: steps 2 and 3.J. Lipid Res. 2000; 41: 1495-1508Abstract Full Text Full Text PDF PubMed Google Scholar). However, dysfunctional HDL is defective in this function (1.Navab M. Reddy S.T. Van Lenten B.J. Anantharamaiah G.M. Fogelman A.M. The role of dysfunctional HDL in atherosclerosis.J. Lipid Res. 2009; 50: S145-S149Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar); this HDL can be pronounced in some persons, including those with chronic inflammatory conditions that predispose to atherosclerosis (1.Navab M. Reddy S.T. Van Lenten B.J. Anantharamaiah G.M. Fogelman A.M. The role of dysfunctional HDL in atherosclerosis.J. Lipid Res. 2009; 50: S145-S149Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 5.Ansell B.J. Navab M. Hama S. Kamranpour N. Fonarow G. Hough G. Rahmani S. Mottahedeh R. Dave R. Reddy S.T. et al.Inflammatory/antiinflammatory properties of high-density lipoprotein distinguish patients from control subjects better than high-density lipoprotein cholesterol levels and are favorably affected by simvastatin treatment.Circulation. 2003; 108: 2751-2756Crossref PubMed Scopus (529) Google Scholar, 6.Ansell B.J. Fonarow G.C. Fogelman A.M. The paradox of dysfunctional high-density lipoprotein.Curr. Opin. Lipidol. 2007; 18: 427-434Crossref PubMed Scopus (168) Google Scholar, 7.Navab M. Anantharamaiah G.M. Reddy S.T. Van Lenten B.J. Fogelman A.M. HDL as a biomarker, potential therapeutic target, and therapy.Diabetes. 2009; 58: 2711-2717Crossref PubMed Scopus (93) Google Scholar). Thus, although plasma HDL cholesterol levels are inversely related to risk across large populations, HDL function rather than absolute level may be a more accurate indicator for risk of developing atherosclerosis (1.Navab M. Reddy S.T. Van Lenten B.J. Anantharamaiah G.M. Fogelman A.M. The role of dysfunctional HDL in atherosclerosis.J. Lipid Res. 2009; 50: S145-S149Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). Currently, HDL functional properties are most often determined by cell-based assays (8.Navab M. Hama S.Y. Hough G.P. Subbanagounder G. Reddy S.T. Fogelman A.M. A cell-free assay for detecting HDL that is dysfunctional in preventing the formation of or inactivating oxidized phospholipids.J. Lipid Res. 2001; 42: 1308-1317Abstract Full Text Full Text PDF PubMed Google Scholar, 9.Patel S. Drew B.G. Nakhla S. Duffy S.J. Murphy A.J. Barter P.J. Rye K.A. Chin-Dusting J. Hoang A. Sviridov D. et al.Reconstituted high-density lipoprotein increases plasma high-density lipoprotein anti-inflammatory properties and cholesterol efflux capacity in patients with type 2 diabetes.J. Am. Coll. Cardiol. 2009; 53: 962-971Crossref PubMed Scopus (168) Google Scholar, 10.Undurti A. Huang Y. Lupica J.A. Smith J.D. DiDonato J.A. Hazen S.L. Modification of high density lipoprotein by myeloperoxidase generates a pro-inflammatory particle.J. Biol. Chem. 2009; 284: 30825-30835Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 11.Van Lenten B.J. Wagner A.C. Navab M. Anantharamaiah G.M. Hama S. Reddy S.T. Fogelman A.M. Lipoprotein inflammatory properties and serum amyloid A levels but not cholesterol levels predict lesion area in cholesterol-fed rabbits.J. Lipid Res. 2007; 48: 2344-2353Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 12.Watson C.E. Weissbach N. Kjems L. Ayalasomayajula S. Zhang Y. Chang I. Navab M. Hama S. Hough G. Reddy S.T. et al.Treatment of patients with cardiovascular disease with L-4F, an apo-A1 mimetic, did not improve select biomarkers of HDL function.J. Lipid Res. 2011; 52: 361-373Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). However, the limitations and labor of these cell-based assays render them inaccessible to many researchers, and they are difficult to scale up for large-scale clinical trials or routine clinical use. Previously a cell-free assay that measures functional properties of HDL by testing the effect of HDL on the production of reactive oxygen species after oxidation and conversion of dichlorodihydrofluorescein diacetate (DCF-DA) to fluorescent DCF (2',7'-dichlorofluorescein) was developed to provide an alternative to cell-based assays (8.Navab M. Hama S.Y. Hough G.P. Subbanagounder G. Reddy S.T. Fogelman A.M. A cell-free assay for detecting HDL that is dysfunctional in preventing the formation of or inactivating oxidized phospholipids.J. Lipid Res. 2001; 42: 1308-1317Abstract Full Text Full Text PDF PubMed Google Scholar). This was based on our demonstration that levels of reactive oxygen species (such as lipid hydroperoxides produced from oxidation of lipoproteins) are significantly associated with the monocyte chemotactic activity and functional properties of HDL that are measured by a cell-based assay (8.Navab M. Hama S.Y. Hough G.P. Subbanagounder G. Reddy S.T. Fogelman A.M. A cell-free assay for detecting HDL that is dysfunctional in preventing the formation of or inactivating oxidized phospholipids.J. Lipid Res. 2001; 42: 1308-1317Abstract Full Text Full Text PDF PubMed Google Scholar). Thus this measurement of reactive oxygen species after HDL exposure as reflected by the increased DCF fluorescence reflects the oxidative properties of different types of HDL that vary in their capacity to engage in redox cycling (1.Navab M. Reddy S.T. Van Lenten B.J. Anantharamaiah G.M. Fogelman A.M. The role of dysfunctional HDL in atherosclerosis.J. Lipid Res. 2009; 50: S145-S149Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). This approach promised to allow a direct biochemical assessment of functional properties of HDL without relying on a biologic readout. Although this assay yielded results that correlated to the cell-based assay, it did not achieve widespread usage due to i) the oxidative instability of DCF-DA and the resulting tendency for minor variations in experimental conditions to cause inconsistency of the assay and ii) the sensitivity of the assay to any degree of hemolysis. Here we describe an approach to quantify the oxidative activity of HDL in a cell-free, biochemical assay. The products of redox cycling are detected as time-dependent oxidation of the fluorogenic probe dihydrorhodamine 123 (DHR) to fluorescent rhodamine 123 (13.Esposito B.P. Breuer W. Sirankapracha P. Pootrakul P. Hershko C. Cabantchik Z.I. Labile plasma iron in iron overload: redox activity and susceptibility to chelation.Blood. 2003; 102: 2670-2677Crossref PubMed Scopus (378) Google Scholar). The rate of DHR oxidation in the presence of HDL reflects the oxidative/antioxidative activity of HDL. This assay provides a readout that is highly correlated to a validated cell-based assay (5.Ansell B.J. Navab M. Hama S. Kamranpour N. Fonarow G. Hough G. Rahmani S. Mottahedeh R. Dave R. Reddy S.T. et al.Inflammatory/antiinflammatory properties of high-density lipoprotein distinguish patients from control subjects better than high-density lipoprotein cholesterol levels and are favorably affected by simvastatin treatment.Circulation. 2003; 108: 2751-2756Crossref PubMed Scopus (529) Google Scholar, 11.Van Lenten B.J. Wagner A.C. Navab M. Anantharamaiah G.M. Hama S. Reddy S.T. Fogelman A.M. Lipoprotein inflammatory properties and serum amyloid A levels but not cholesterol levels predict lesion area in cholesterol-fed rabbits.J. Lipid Res. 2007; 48: 2344-2353Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 12.Watson C.E. Weissbach N. Kjems L. Ayalasomayajula S. Zhang Y. Chang I. Navab M. Hama S. Hough G. Reddy S.T. et al.Treatment of patients with cardiovascular disease with L-4F, an apo-A1 mimetic, did not improve select biomarkers of HDL function.J. Lipid Res. 2011; 52: 361-373Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), and it is highly reproducible and amenable to high-throughput implementation. Blood samples were collected from patients with coronary artery disease (CAD) or equivalent as defined by National Cholesterol Education Program Adult Treatment Panel III criteria (14.14., Expert panel on detection, evaluation, and treatment of high blood cholesterol in adults. 2001. Executive summary of the third report of the National Cholesterol Education Program (NCEP) expert panel on detection, evaluation, and treatment of high blood cholesterol in adults (adult treatment panel III). JAMA. 285: 2486–2497.Google Scholar) and were collected from patients referred to the cardiac catheterization laboratory at the Center for Health Sciences at the University of California, Los Angeles (UCLA). After signing a consent form approved by the Human Research Subject Protection Committee of UCLA, patients donated a fasting blood sample collected in a heparinized tube. Plasma samples were also randomly selected from pretreatment samples remaining from a previously described study in which all patients had coronary artery disease or equivalent (12.Watson C.E. Weissbach N. Kjems L. Ayalasomayajula S. Zhang Y. Chang I. Navab M. Hama S. Hough G. Reddy S.T. et al.Treatment of patients with cardiovascular disease with L-4F, an apo-A1 mimetic, did not improve select biomarkers of HDL function.J. Lipid Res. 2011; 52: 361-373Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). All of these patients were on a statin (12.Watson C.E. Weissbach N. Kjems L. Ayalasomayajula S. Zhang Y. Chang I. Navab M. Hama S. Hough G. Reddy S.T. et al.Treatment of patients with cardiovascular disease with L-4F, an apo-A1 mimetic, did not improve select biomarkers of HDL function.J. Lipid Res. 2011; 52: 361-373Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Rheumatoid arthritis (RA) patients were recruited from the rheumatology offices at UCLA via flyers posted in the offices and in the UCLA Medical Center. All RA patients met the American College of Rheumatology criteria for RA, which was verified by chart review. Human immunodeficiency virus (HIV)-infected subjects had HIV-1 RNA ≥ 10,000 copies/ml; were not receiving antiretroviral therapy; had no documented coronary atherosclerosis; had normal total cholesterol (200 mg/dl), LDL cholesterol (130 mg/dl), HDL cholesterol (males, >45 mg/dl; females, >50 mg/dl), and triglycerides ( 1.0 is considered proinflammatory, and a value <1.0 is considered anti-inflammatory. The DCF-based cell-free assay was performed as previously described (8.Navab M. Hama S.Y. Hough G.P. Subbanagounder G. Reddy S.T. Fogelman A.M. A cell-free assay for detecting HDL that is dysfunctional in preventing the formation of or inactivating oxidized phospholipids.J. Lipid Res. 2001; 42: 1308-1317Abstract Full Text Full Text PDF PubMed Google Scholar, 16.Charles-Schoeman C. Khanna D. Furst D.E. McMahon M. Reddy S.T. Fogelman A.M. Paulus H.E. Park G.S. Gong T. Ansell B.J. Effects of high-dose atorvastatin on antiinflammatory properties of high density lipoprotein in patients with rheumatoid arthritis: a pilot study.J. Rheumatol. 2007; 34: 1459-1464PubMed Google Scholar). Quadruplicates of HDL (5 µg of cholesterol, unless otherwise specified) were added to 96-well plates (polypropylene, flat bottom, black, Fisher Scientific). HBS was added to each well to a final volume of 150 µl, followed by addition of 25 µl of the working solution of 50 µM DHR, for a total volume of 175 µl (final DHR concentration of 7 µM). Immediately following DHR addition, the plate was protected from light and placed in a fluorescence plate reader. The fluorescence of each well was assessed at two minute intervals for an hour with either a DTX 800/880 Multimode Detector (Beckman Coulter, CA) or Synergy 2 Multi-Mode Microplate Reader (Biotek, VT), using a 485/538 nm excitation/emission filter pair with the photomultiplier sensitivity set at medium. Both readers gave equivalent readings (r2= 0.99). Using Microsoft Excel software, the oxidation rate was calculated for each well as the slope for the linear regression of fluorescence intensity between 10 and 50 min, expressed as FU minute−1 (fluorescence units per minute). HDL oxidative function was calculated as the mean of quadruplicates for the wells containing the HDL sample. The assay was also run in a 384-well format (polypropylene, flat bottom, black, Fisher Scientific), using 1.5-3 µg of HDL cholesterol with 15 µl of the DHR working stock in a total volume of 100 µl of HBS per well. LC/MS/MS was performed using a 4000 QTRAP quadruple mass spectrometer (Applied Biosystems) equipped with an electrospray ionization source. The HPLC system utilized an Agilent 1200 series LC pump equipped with a thermostatted autosampler (Agilent Technologies). Chromatography was performed using a Luna C-18(2) column (3 µm particle, 150 × 3.0 mm, Phenomenex) with a security guard cartridge (C-18; Phenomenex) at 40°C. Mobile phase A consisted of 0.1% formic acid in water, and mobile phase B consisted of 0.1% formic acid in acetonitrile. The autosampler was set at 4°C. The injection volume was 20 µl, and the flow rate was controlled at 0.4 ml/min. The gradient program was as follows: 0-8 min, linear gradient 0-95% B; 8-9 min, 95% B; 9-9.15, 95-0% B; 9-12 min, 0% B. Data acquisition and instrument control were accomplished using Analyst 1.4.2 software (Applied Biosystems). Detection was accomplished by using the multiple reaction monitoring (MRM) mode with positive ion detection. The parameter settings used were the following: ion spray voltage = 5500 V; curtain gas = 22 (nitrogen); ion source gas 1 = 34; ion source gas 2 = 25; ion source gas 2 temperature = 250°C. Collision energy, declustering potential, and collision cell exit potential were optimized to obtain optimum sensitivity. The transition monitored was m/z 347.05 to m/z 315.1 for DHR. HDL from control and CAD-patient donors was combined with DHR, and the amount of DHR remaining in the samples after 2 h incubation was determined by LC/MS/MS. First, UC- and FPLC-isolated HDLs were combined in triplicate with DHR. Each sample contained 2.5 μg HDL and 50 μM DHR in a final volume of 175 μl HEPES-buffered saline. The samples were incubated in the dark for 2 h. Urate was then added (final concentration 25 μM) to slow further oxidation of DHR (22.Kooy N.W. Royall J.A. Ischiropoulos H. Beckman J.S. Peroxynitrite-mediated oxidation of dihydrorhodamine 123.Free Radic. Biol. Med. 1994; 16: 149-156Crossref PubMed Scopus (676) Google Scholar). The samples were transferred in minimal light to autosampler vials (Fisher Scientific) for LC/MS/MS analysis. The samples were ordered so that HDL-control samples were measured last, to ensure that any additional air oxidation of DHR did not result in a false positive as regards the difference between HDL-CAD and HDL-Ct with respect to DHR. In a separate experiment, UC-isolated HDL from a healthy donor was serially diluted, in triplicate, from 10 to 0.625 μg cholesterol. The dilutions were combined with DHR and incubated in the dark for 2 h as above. After incubation, urate was added, the samples were transferred to autosampler vials, and the amount of DHR remaining was determined by LC/MS/MS. Statistical analysis was performed using Excel (Microsoft Corp., Seattle, WA). Group means were compared using Student t-test for unpaired variates with P < 0.05 considered statistically significant. Correlation coefficients between variables were calculated using least-squares linear regression. To assess whether the oxidation rate of DHR is affected by the properties of HDL, HDL samples were tested for their effect on DHR oxidation rate. Alone, DHR exposed to air became oxidized (and therefore fluorescent) at a linear rate between 10 and 50 min (Fig. 1). The rate of oxidation of DHR was significantly less with added HDL, and the two HDL samples (an example of anti-inflammatory HDL (aHDL) compared with proinflammatory HDL (pHDL)] showed clearly different effects in this regard (Fig. 1A). Furthermore, when the amount of added HDL was varied, there was a clear dose dependence in the oxidative effects on DHR that was linear in the range 2.5-15 µg (cholesterol) of added HDL per well in the assay (Fig. 1C). We observed a significant reduction in fluorescence signal and oxidation rate of DHR after addition of HDL (Fig. 1). To determine whether the reduction in fluorescence signal of DHR after addition of HDL is caused by antioxidant effect of HDL or nonspecific lipid-probe interactions, we tested the effect of addition of different lipids with known oxidative properties on the oxidation rate of DHR. We found that addition of LDL, HDL (Fig. 1, supplementary Fig. I), and other lipids that are known not to be antioxidant, such as oxidized L-α-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (oxPAPC) (data not shown), leads to significant decrease in oxidation signal of DHR. In addition, coincubation of a stable amount of DHR with different concentrations of two lipids simultaneously (HDL with LDL, supplementary Fig. I; HDL with oxPAPC, data not shown) caused significant decrease in the oxidation signal of DHR. Similar lipid-probe interactions were confirmed with DCFH (data not shown). The lipid-probe interaction between DHR and HDL was confirmed with an independent experiment (supplementary Fig. II). To determine whether the lipid-probe interactions were dose dependent, increasing doses of DHR (1-50 μM) were added to specific amount of HDL or LDL (2.5 μg) (supplementary Fig. III). We found that decreasing concentrations of DHR resulted in decreasing the oxidation rate of both LDL and HDL and that the oxidation rate of LDL was higher than HDL (supplementary Fig. III). To increase the sensitivity of the assay and to detect smaller differences in oxidative properties of lipoproteins, we used a concentration of 50 μM of DHR for all our experiments; this concentration has also been previously used to quantify redox activity (13.Esposito B.P. Breuer W. Sirankapracha P. Pootrakul P. Hershko C. Cabantchik Z.I. Labile plasma iron in iron overload: redox activity and susceptibility to chelation.Blood. 2003; 102: 2670-2677Crossref PubMed Scopus (378) Google Scholar). To further investigate the dose-dependent interaction between DHR and lipoproteins, increasing concentrations of lipoproteins (HDL and LDL) were incubated with 50 μM of DHR. Fig. 1C and supplementary Fig. I show that the fluorescent signal generated by HDL was dose dependent but that increasing the concentrations of lipoproteins resulted in a statistically significant decrease in fluorescence for each concentration of lipoprotein used. The correlation observed between the quantity of lipoprotein added and DHR fluorescence and oxidation rate was inverse and highly significant (Fig. 1C, supplementary Fig. I-B). These results are consistent with lipid-probe interactions and possible fluorescence quenching as explanation of reduction in fluorescence signal of DHR after addition of lipids. To ensure that the differences we observed with respect to fluorescence corresponded to real differences in the degree of oxidation of DHR, we subjected samples of HDL-CAD and HDL-non-CAD that had been combined with DHR in the manner of the fluorometric assay to LC/MS/MS analysis to determine the amount of DHR remaining in each (Fig. 2). Regardless of whether the HDLs were isolated by sequential UC (Fig. 2A) or by FPLC (Fig. 2B), HDL from healthy non-CAD donors contained significantly more DHR than HDL from CAD-patient donors after an initial 2 h incubation with 50 μM DHR (P = 0.001, n = 3 for each pair). As the am
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