Dysfunctional high-density lipoproteins have distinct composition, diminished anti-inflammatory potential and discriminate acute coronary syndrome from stable coronary artery disease patients

There is a stringent need to find means for risk stratification of coronary artery diseases (CAD) patients. We aimed at identifying alterations of plasma high-density lipoproteins (HDL) components and their validation as dysfunctional HDL that could discriminate between acute coronary syndrome (ACS) and stable angina (SA) patients. HDL2 and HDL3 were isolated from CAD patients’ plasma and healthy subjects. ApolipoproteinAI (apoAI), apoAII, apoCIII, malondialdehyde (MDA), myeloperoxidase (MPO), ceruloplasmin and paraoxonase1 (PON1) were assessed. The anti-inflammatory potential of HDL subfractions was tested by evaluating the secreted inflammatory molecules of tumor necrosis factor α-activated endothelial cells (EC) upon co-incubation with HDL2 or HDL3. We found in ACS versus SA patients: 40% increased MPO, MDA, apoCIII in HDL2 and HDL3, 35% augmented apoAII in HDL2, and in HDL3 increased ceruloplasmin, decreased apoAII (40%) and PON1 protein and activity (15% and 25%). Co-incubation of activated EC with HDL2 or HDL3 from CAD patients induced significantly increased levels of secreted inflammatory molecules, 15–20% more for ACS versus SA. In conclusion, the assessed panel of markers correlates with the reduced anti-inflammatory potential of HDL subfractions isolated from ACS and SA patients (mostly for HDL3 from ACS) and can discriminate between these two groups of CAD patients.


Results
Subjects. The clinical characteristics (anthropometric and clinical data, medication) and plasma biochemical parameters of the CAD patients enrolled in the study are summarized in Tables 1 and 2. The subjects included in the study have ages with a mean of 61.7 ± 8.0 for stable angina (SA), 62.9 ± 9.1 for acute coronary syndrome (ACS), and 41.8 ± 9.5 for healthy (N) subjects. A significant increase in the body mass index (BMI) and glucose concentrations in the sera of all CAD patients (15%, p = 0.0003 and 16%, p = 0.048 respectively, for SA, and 12%, p = 0.003 and 21%, p = 0.021 respectively, for ACS) compared to N subjects was found ( Table 2).
In addition, we analyzed PON1 activity in connection with HDL-C and apoAI levels in the sera of CAD patients and N subjects, and found a positive correlation between HDL-C or apoAI levels and PON1 activity (r = 0.273, p = 0.004 and r = 0.221, p = 0.013, respectively) (Figure S1a, S1b). No correlation was found between PON1 protein and HDL-C or apoAI levels.
We performed a binary logistic regression (BLR) analysis to assess the relationship between the oxidative markers and HDL parameters in the cohort under study in order to find an approach to discriminate between SA and ACS patients. The BLR model showed that a panel of parameters comprising PON1 activity and mass protein, TBARS, MPO protein and apoAI levels, adjusted for age and gender, could significantly distinguish between ACS and SA patients, with an accuracy of over 82% (p = 0.034, Table S1). The highest contribution to this BLR model was brought by the ratio MPO protein/PON1 activity (odd ratio expB = 1723.33, Table S2), in good correlation  We also assessed some relevant pro-inflammatory molecules present in the plasma of CAD patients and N subjects. The soluble vascular cell adhesion molecule 1 (sVCAM-1) concentration was increased in all CAD patients' compared to N subjects (SA, 17%, p = 0.041; ACS, 38%, p = 0.0072) (Fig. 2a). Monocyte chemoattractant protein 1 (MCP-1) levels were higher in ACS compared to SA and N subjects (120%, p = 0.026, and 170%, p = 0.0063 respectively) (Fig. 2b). The C reactive protein (CRP) levels were considerably augmented in CAD  Characterization of HDL subfractions isolated from the plasma of ACS and SA patients. HDL 2 and HDL 3 subfractions separated by ultracentrifugation were collected based on the density of the fractions, the protein and cholesterol profiles ( Figure S3). We performed seven ultracentrifugation isolations using between 6-8 patients/pool of plasma, and a total of 40 SA and 53 ACS patients. Characterization of HDL 2 and HDL 3 was performed by measuring the size of the particles, apolipoproteins (apoAI, apoAII, apoCIII, apoE and CP) and lipid content (C, TG, and phospholipids-PL). The results showed no statistically significant differences in the size, lipid content and apoE levels of HDL 2 or HDL 3 between the studied groups (Table 3).
In summary, HDL 3 from ACS versus SA patients have statistically significant higher levels of apoCIII, MPO protein, conjugated dienes, 4-HNE-apoAI, MDA-apoAI and lower levels of apoAII, PON1 protein and activity.  Table 3. Characteristic parameters of HDL 2 and HDL 3 subfractions isolated from sera of normal subjects (N), stable angina patients (SA) and acute coronary syndrome (ACS) patients. N -healthy normal subjects, SA -stable angina patients, and ACS -acute coronary syndrome patients; C -cholesterol, TG -triglycerides, PL -phospholipids, ApoE -apolipoprotein E. Data are expressed as means ± SD and analyzed with Independent Student's T-test (n = 7).
SCIENtIFIC RePoRTS | 7: 7295 | DOI:10.1038/s41598-017-07821-5 The anti-inflammatory effect of HDL subfractions on TNFα-activated EC. We aimed at the evaluation of the anti-inflammatory potential of HDL subfractions by co-incubating TNFα-activated EC with HDL 2 or HDL 3 , followed by the assessment of the synthesis and secretion of several well-known pro-inflammatory molecules, VCAM-1, MCP-1 and CRP. HDL 2 from N subjects induced a 15% (p = 0.014) decrease of VCAM-1 protein expression compared to TNFα-exposed EC (Fig. 5a). HDL 2 from either of the two CAD groups exhibited no protective effect on activated EC in terms of cellular protein expression of VCAM-1; however, VCAM-1 levels were significantly higher in the culture media of HDL 2 from ACS compared to SA (10%, p = 0.044) (Fig. 5a). The levels of soluble VCAM-1 (sVCAM-1) secreted by TNFα-exposed EC were significantly reduced by HDL 2 from SA (33%, p = 0.004) and to a lesser extent by HDL 2 from ACS (23%, p = 0.016), the difference between the effect of HDL 2 from ACS and SA being statistically significant (15%, p = 0.043) (Fig. 5b). HDL 3 from both CAD groups induced a diminished VCAM-1 protein expression in activated EC (17%, p = 0.0035 for SA; 10%, p = 0.034 for ACS), and a 9% reduction in SA versus ACS (p = 0.041) (Fig. 5a). HDL 3 from ACS induced no significant decrease of sVCAM-1 in the culture medium of activated EC compared to HDL 3 from SA (26%, p = 0.026) (Fig. 5b).
It was reported that the active form of disintegrin and metalloprotease domain 17 (ADAM17) is responsible for cleaving VCAM-1 from the EC membrane to give sVCAM-1 in the culture medium 29 . We found that ADAM17 protein levels were significantly reduced in HDL 2 from ACS (10%, p = 0.046) and SA (27%, p = 0.02) compared to TNFα-activated EC (Fig. 5d). The determined increased levels of sVCAM-1 in the case of HDL 2 from CAD compared to N subjects correlated with the increased levels of ADAM17 protein. ADAM17 protein levels were significantly increased in HDL 2 from ACS compared to SA (20%, p = 0.048), in good correlation with the higher levels sVCAM-1 (Fig. 5d). HDL 3 from SA induced a significant decrease compared to TNFα-exposed EC (20%, p = 0.038), while the HDL 3 from ACS patients had no effect on ADAM17 protein expression, that is in accordance with the sVCAM-1 protein levels in the culture medium (Fig. 5d). The protein expression of ADAM17 pro-form followed the same trend as the active form (Fig. 5c).
Co-incubation of EC with HDL 2 from SA patients induced a 27% (p = 0.003) decrease of the secreted MCP-1 level compared to HDL 2 from N subjects (45%, p = 0.0015) both figures expressed relative to TNFα-activated EC. In contrast, HDL 2 from ACS induced only a modest, statistically insignificant decrease of the secreted MCP-1 level (Fig. 5e). HDL 3 from SA had no protective effect on activated EC in terms of MCP-1 secretion, while HDL 3 from ACS exhibited a significant pro-inflammatory effect by inducing a 23% (p = 0.0023) increase of the secreted MCP-1 by activated EC. As expected, HDL 3 from N subjects induced a significant reduced secretion of MCP-1 (22%, p = 0.022) (Fig. 5e).
In summary, the anti-inflammatory potential of HDL 2 from ACS versus SA in activated EC determined significantly higher levels of sVCAM-1, ADAM-17, MCP-1 and CRP, while HDL 3 induced a pro-inflammatory effect in the case of all measured inflammatory molecules.

Discussions
In physiological conditions, HDL have anti-atherosclerotic, anti-inflammatory and anti-thrombotic properties 8 . Our hypothesis was that in CAD patients modifications of the apolipoproteins and enzymes in HDL are associated with progressive functional alterations that could explain the differences between ACS and SA patients. Together with other known biomarkers, these changes in HDL composition could be further used for a better prediction of the major adverse cardiac events (MACE) in CAD patients.
The novel findings of our study are: (i) HDL 2 and HDL 3 from CAD patients are dysfunctional as revealed by the increased apoCIII, MPO and oxidatively-modified apoAI levels; these alterations are more pronounced in HDL 3 from ACS than from SA patients; (ii) apoAII levels are increased in HDL 2 (ACS > SA), in contrast with their decrease in HDL 3 (ACS < SA); (iii) PON1 protein and activity are decreased, and CP is increased in HDL 3 from ACS versus SA patients; (iv) the anti-inflammatory effect of HDL 2 and HDL 3 assessed on the activated EC is lower for ACS versus SA, as reflected by the increase in ADAM17-mediated sVCAM-1, MCP-1 and CRP levels.  These modifications were detected despite of the extensive medication that substantially ameliorated some of the other plasma biochemical parameters, such as total cholesterol and LDL-C.
The concept of dysfunctional HDL emerged from the observations that CAD patients with normal HDL-C, but low PON1 activity and increased lipid peroxides in plasma and in HDL, compared to healthy subjects, have decreased plasma anti-oxidant potential that favors the oxLDL-induced inflammation 10 . The anti-inflammatory and antioxidant potentials of HDL are closely related and appear to depend on complex, not yet deciphered, interactions between their apolipoproteins and enzymes.
It is accepted that oxidized apoAI is a marker of dysfunctional HDL 30 , and our data show for the first time that MDA-apoAI and 4-HNE-apoAI levels are significantly increased in HDL 2 and HDL 3 (HDL 3 > HDL 2 ) from ACS compared to SA. The oxidative modifications of HDL subfractions are additionally supported by the increased level of conjugated dienes in ACS compared to SA, particularly in HDL 3 . MDA-apoAI appearance could be due to the increase of MPO/PON1 ratio in HDL 2 and HDL 3 (HDL 3 > HDL 2 ) from ACS compared to SA, in accordance with the TBARS and MPO levels measured in plasma (ACS > SA) 30 . Here we report for the first time that ceruloplasmin is firmly associated with HDL 3 and very little with HDL 2 from CAD patients and healthy subjects (ACS > SA > N). This could explain the increased oxLDL in plasma of ACS > SA, since we have previously demonstrated that ceruloplasmin has a significant oxidant potential against LDL at neutral pH 31 .
It is largely accepted that PON1 is one of the major proteins which determines the antioxidant potential of HDL and its reduced activity contributes to the impairment of HDL quality 32 . Several studies have shown that the concentration and activity of PON1 are decreased in CAD patients compared to healthy subjects 23,33 . These results are confirmed and extended by the present study in which we report that the significant decrease of PON1 activity in CAD patients' sera is characteristically more prominent in ACS than SA. Our data are in contrast with the study of Yunoki et al. conducted on patients with stable and unstable angina showing that PON1 activity and protein do not differ between these two groups 34 . Our statistical analysis evidenced a significant positive correlation between PON1 activity, HDL-C and apoAI levels in sera of the studied groups that is in good agreement with other recent reports 33,35 . Our data show for the first time that the levels of PON1 protein and activity are significantly lower in HDL 3 from ACS < SA < N subjects. Moreover, the decrease of PON1 activity in HDL 3 from ACS patients exceeded the decrease of its protein level suggesting the existence of dysfunctional PON1. The firm attachment of PON1 to HDL particles is mediated by its interaction with phospholipids and apoAI, and is influenced by the presence of MPO and apoAII 36 . It was reported that MPO may form a ternary complex with PON1 and HDL, and determine the oxidation of PON1, the ratio between these two enzymes in serum being proposed as marker for HDL functionality 37,38 . Our data show that the ratio MPO protein/PON1 activity is increased by 85% in ACS versus SA sera, in good agreement with the former reports 37,38 .
The role of apoAII in stabilizing the HDL structure and function is still controversial, being reported that it can impede PON1 binding to HDL 39 . Our data show for the first time that the levels of apoAII are increased in HDL 2 from ACS versus SA patients, in contrast with HDL 3 , where apoAII levels are decreased in ACS versus SA. Thus, an explanation for the decreased levels of PON1 protein in HDL 2 compared to HDL 3 could be the increase of apoAII in CAD versus N subjects that favors PON1 displacement by MPO, which in turn oxidizes apoAI and weakens the PON1-apoAI link. Compared to SA, HDL 3 from ACS has higher MPO levels in association with higher oxidized-apoAI levels compared to HDL 2 , which can explain the more reduced PON1 activity/PON1 protein ratio.
ApoCIII is a protein involved in HDL remodeling by lipases, known to increase plasma TG through inhibition of lipoprotein lipase 40 . There are no data about the relationship between apoCIII and MPO or PON1 in HDL. Our results show for the first time that apoCIII is increased in HDL 2 and HDL 3 (HDL 2 > HDL 3 ) from ACS compared to SA and N. The higher levels of apoCIII in HDL 2 compared to HDL 3 could be associated with the higher content of TG in HDL 2 versus HDL 3 , as was previously reported 41 . It is known that functional HDL inhibit or attenuate the oxidation of LDL and of the membrane phospholipids 18,42 . In accordance with the latter reports, our data show significant increases of oxLDL, TBARS, and free 4-HNE in ACS versus SA plasma. The presence of oxidative stress markers in higher amounts in ACS compared to SA supports the existence of dysfunctional HDL 2 and HDL 3 , more prominent in ACS than in SA, despite of the intensive treatments of patients with statins, known to have anti-oxidant properties.
Atherosclerosis is an inflammatory disease and activation of the endothelium by various risk factors that trigger the EC to secrete pro-inflammatory molecules is among the most important processes in the inception and evolution of the atheroma 43 . It is accepted that the TNFα signaling is a central pro-inflammatory pathway involved in atherosclerosis progression 44 . To validate the functional significance of the demonstrated biochemical alterations of HDL, we assessed the anti-inflammatory potential of HDL 2 and HDL 3 from ACS and SA patients by measuring their effect on the inflammatory molecules secreted by TNFα-activated EC. We found that in ACS compared to SA, the levels of VCAM-1 protein expression and secretion in the presence of HDL 2 and HDL 3 were significantly higher, indicating a lower anti-inflammatory potential for both HDL subfractions. We show for the first time that the increased secretion of sVCAM-1 is due to a significantly increased expression of the metalloprotease ADAM17 in TNFα-exposed EC, and that HDL 2 and HDL 3 from ACS determine a smaller inhibition of ADAM17 protein expression than HDL from N subjects or SA patients. In addition, MCP-1 and CRP levels in the culture medium of TNFα-exposed EC incubated with HDL 2 from ACS were increased compared to those from SA and N, strengthening the evidence for a more prominent dysfunction of HDL 2 in ACS versus SA patients. HDL 3 from ACS compared to SA induced higher levels of MCP-1 and CRP in the culture media of activated EC. These data suggest that HDL 3 from the ACS group have pro-inflammatory action on EC, increasing the pro-inflammatory effect of TNFα. Our previous data and those from other laboratories show that VCAM-1 and MCP-1 expression is induced by activation of NF-kB in EC 45,46 . It was reported that apoCIII per se is able to activate NF-kB in EC and increase the adhesion of monocytic cells 47 . Our results reveal an association between the increased apoCIII in HDL subfractions and the diminution of their anti-inflammatory properties. In good association with the in vitro data, we measured a significant increase of sVCAM-1, MCP-1 and CRP levels in the plasma of CAD patients (ACS > SA > N).
Taken together, our data show that HDL 3 from CAD patients are more pro-oxidatively altered than HDL 2 (increased oxidized-apoAI, MPO/PON1 and ceruloplasmin). Consequently, HDL 3 from CAD patients are converted to pro-inflammatory lipoproteins, despite of the intensive statin treatment of the patients. These data are in agreement with our previous results showing that hyperlipidemia induces oxidation of PON1 and apoAI and their down-regulation in the small intestine and liver of hamsters, but the treatment with probiotics diminishes the oxidative stress 16,48 . It is known that HDL play a key role in RCT and this function is affected by oxidative modifications, as was previously reported 49, 50 . Gibson C.M. et al. and Kallend D.G. et al. showed that the infusion of apoAI-based compound CS112 and MDCO-216 improved plasma cholesterol efflux, but did not reduce MACE and did not improve plaque regression 51,52 . Taken together with our results, we can conclude that it is not only the level of apolipoprotein or enzyme, but also its quality that contributes to MACE.
In conclusion, the assessed panel of markers (apoCIII, MPO, oxidized-apoAI, ceruloplasmin, and PON1) correlates with the reduced anti-inflammatory potential of HDL 2 and HDL 3 (mostly HDL 3 from ACS) isolated from ACS and SA patients and can discriminate between these groups of CAD patients. Approaches to develop new therapies for treatment of CAD patients at risk based on the stimulation of the liver and small intestine to produce functional HDL are the future challenge.

Methods
Reagents. All reagents used were from Sigma-Aldrich Co., MO, USA or Cayman Chemicals, MI, USA.

Subjects. Our study includes 121 subjects
Lipoprotein isolation. For lipoproteins isolation, equal amounts of sera from patients of each group were randomly pooled; the procedure was performed on 7 independent pools of sera from each group (6-8 samples per pool). We performed isolation and characterization of HDL sub-fractions (HDL 2 and HDL 3 ) from the patients' group-pooled sera as described 53 . Briefly, an aliquot of 1.25 mL pooled sera from each N, SA, and ACS groups was adjusted to a density of 1.23 g/mL with KBr (Sigma-Aldrich, St. Louis, MO, USA) and then overlaid with 2 mL of 1.21 g/mL KBr, 5 ml of 1.063 g/mL KBr, 1 mL of 1.019 g/mL KBr, and 1 mL of phosphate buffered saline (PBS). The mixture was ultracentrifuged for 18 h in a SW-41 Ti rotor at 30,000 rpm (154,000x g) in an Optima L-80XP ultracentrifuge (Beckman Coulter International SA, Nyon, Switzerland). Ten fractions (1 mL each) were collected from each tube and dialyzed against PBS, pH 7.4, at 4 °C in the dark. The fractions number 7-8 and 9 were of interest, corresponding to HDL 2 (d = 1.06-1.12 g/ml) and HDL 3 (d = 1.12-1.25 g/ml) subpopulations. The protein in the collected fractions was assessed by a modified Lowry method using bovine serum albumin as standard 54 .

Serum parameters determination and HDL characterization.
Total cholesterol (C) and triglycerides (TG) levels were determined employing automated biochemical analyzers. HDL cholesterol (HDL-C) and low density lipoproteins cholesterol (LDL-C) levels were measured by a commercially available kit (Dialab Gmbh., Neudorf, Austria). Phospholipids (PL) levels were determined by using a commercially available kit (Wako Chemicals GmbH, Neuss, Germany). ApoAI, apoAII, apoCIII, apoE, PON1, MPO, VCAM-1 and MCP-1 levels were determined by enzyme-linked immunosorbent assay kits commercially available (apoAI and apoE -Mabtech, Sweden, apoAII and apoCIII -Abnova Corporation, Taoyuan, Taiwan, PON1, MPO, VCAM-1 and MCP-1 -R&D Systems, Minneapolis, Minnesota, USA). CETP activity was determined by using a commercial available kit (Biovision Inc., CA, USA). The CRP levels were measured by a turbidimetric method using commercial available kit (Wako Chemicals GmbH, Neuss, Germany). PON1 activity was assessed by measuring the capacity of the enzyme to hydrolyze the paraoxon substrate using a method described by Rozenberg et al. 55 . Briefly, 10 µL serum samples prediluted (1:2) or undiluted HDL sub-fractions were added to 95 µL buffer solution (100 mM Tris-HCl, 2 mM CaCl 2 , pH 8.0) and then 95 µl of 5.5 mM paraoxon-ethyl (Sigma-Aldrich, Canada). PON1 activity was calculated based on the absorbance recorded at 412 nm during enzymatic reaction kinetic. Blanks without enzyme were used to correct for the spontaneous hydrolysis of the substrate. The dimension of the HDL particles were measured using the nanoparticle size analyzer (Agilent Nicomp-380), based on the dynamic light scattering principle. The density of lipoproteins fractions separated by density gradient ultracentrifugation was determined based of refraction indices measured with an Abbe refractometer.
Determination of lipid oxidation products in plasma and HDL. The levels of thiobarbituric acid reactive substances (TBARS) were determined by alkaline hydrolysis of protein bound aldehydes from plasma using an UHPLC system (Agilent Technologies 1290 Infinity) and malondialdehyde (MDA) as standard 56 . The levels of free 4-HNE were determined using a method based on serum samples derivatization in accordance to the procedure suggested 57 , using GC MSMS analysis (Agilent Technologies 7000 A GC/MS Triple Quad direct interfaced with a gas chromatograph GC 7890 A). Plasma oxidized LDL (oxLDL) concentration was determined with Mercodia competitive ELISA kit (Uppsala, Sweden). The measurement of MDA and 4-hydroxy-2-nonenal (4-HNE) associated to apoAI, protein in HDL subpopulations was performed by WB using specific antibodies (see Reagents section). The levels of conjugated dienes in the HDL subfractions were determined by measuring the absorbance at 234 nm.
Cell culture and in vitro tests for HDL function. All experiments to evaluate the HDL function used human umbilical vein endothelial cells (EA.hy926 cell line) (HEC) from ATCC (Manassas, VA, USA), grown after the manufacturer instructions. At confluence, after 6 h starvation (exposure to the serum free medium), HEC were pre-incubated with 80 µg protein/ml HDL 2 or HDL 3 without fetal calf serum, for 18 h. Cells were further exposed to additional 10 µM tumor necrosis factor (TNFα). In parallel, cells without HDL pre-incubation were exposed to TNFα in the same condition. After 6 h, the culture media were collected and cells were harvested by lysing them with radio-immunoprecipitation assay (RIPA) buffer. The protein level in cell lysates was quantified by bicinchoninic acid assay following the manufacturer instructions (Sigma-Aldrich Co., MO, USA).

Western blot analysis.
Equal volumes of HDL sub-fractions or culture medium and 40 µg total protein of EC lysate were loaded on a 8/12% SDS-PAGE, transferred to nitrocellulose membrane, blocked with 5% skimmed milk for 1 h, then incubated for 16 h with the primary antibody of the following: ceruloplasmin, 4-HNE-apoAI and MDA-apoAI adducts for HDL subfractions and VCAM-1, ADAM17, MCP-1, and CRP for EC lysate/culture medium samples. Specific antibodies were used as mentioned in Reagents section. The immunostaining detection was made by using the ECL kit (AppliChem GmbH, Darmstadt, Germany) in an LAS4000 analyzer and densitometry of the blots was performed with ImageQuant TL 7.0 software (both from GE Healthcare Bio-Sciences AB, Uppsala, Sweden). The relative level of 4-HNE-apoAI adducts in SA and ACS was expressed as percentage relative to values from N subjects. The protein expression measured in EC was estimated relative to β-actin, and the proteins secreted to the culture medium were expressed relative to cellular protein (BCA assay). The WB images were processed by cropping the gel lanes and using dividing lines where the order of the samples on WB was different that in the final graph. Statistical analysis. Statistical software SPSS for Windows v21.0 (IBM SPSS, IBM Ireland, Dublin, Ireland) and GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, CA, USA) were used. The continuous distributed quantitative variables were expressed either as means ± standard deviation (SD, for small datasets <10 values, such as HDL-biochemical composition and cell culture data) or as means ± standard error of the mean (SEM, for large datasets related to patients' plasma); these data were analysed by two-tailed Oneway ANOVA with Least Significant Difference (LSD) Post-hoc test (for the plasma data in the three groups of subjects) or by Independent Student's T-test with Levene's test for equality of variances (for differences between HDL biochemical composition, and for cell culture data for two groups of datasets). Patients' biochemical data were tested for normal distribution with Shapiro-Wilk test. Crosstabs analysis with chi-squared (χ 2 ) function was performed to evaluate the differences between binary logistic data (gender, obesity, diabetes, hypertension, medication). Linear regression curves were plotted for scatterplots between continuous distributed parameters with parametric bivariate correlation analysis performed using the Pearson's function and its corresponding p-value. A binary logistic regression model (BLR) was performed with the enter iteration method, considering SA group as reference category, and ACS groups as risk category, with serum PON1 activity and mass protein, TBARS, MPO and apoAI levels as covariates, with adjustments for age and gender. The threshold for statistical significance was set to 5% (p-values lower than 0.05).
Data availability. We state that the materials, data and associated protocols used in the present work will be made promptly available to readers without undue qualifications in material transfer agreements. We disclose no restriction on the availability of materials or information in this manuscript.