Oxidized low-density lipoprotein (oxLDL) and β2-glycoprotein I (β2GPI) have been identified in human atherosclerotic lesions and when complexed have been implicated as a pro-atherothrombotic antigen. We examined the association of free oxLDL and oxLDL-β2GPI complex in patients with coronary artery disease who underwent elective cardiac catheterization. Serum was collected from patients with suspected coronary artery disease immediately before elective cardiac catheterization who were either treated (n = 385) or not treated (n = 150) with statins and from healthy volunteers (n = 134). OxLDL and oxLDL-β2GPI complex levels were determined by enzyme-linked immunosorbent assay. Disease severity was defined angiographically as none-minimal (<20%), moderate (20% to 75%), and severe (>75%) luminal diameter obstruction of any major coronary vessel. Both oxLDL and oxLDL-β2GPI complex were lower in patients on statins (p <0.001). In statin-naive patients, oxLDL-β2GPI complex, but not free oxLDL, was associated with severe coronary artery disease (p = 0.036). However, no association was observed in patients on statins. LDL 4 and triglycerides increased with oxLDL-β2GPI complex quartiles (p = 0.001). OxLDL-β2GPI complex (>0.32 U/ml) was predictive of severe atherosclerosis by receiver-operating characteristic curve analysis in statin-naive patients (area under the curve 0.66, p = 0.002). In conclusion, oxLDL-β2GPI appears more predictive of coronary artery disease severity than oxLDL alone in statin-naive patients.
Given the vast burden of coronary artery disease (CAD) and the survival advantage observed from timely intervention of critical coronary lesions, the search of noninvasive markers to assess the risk and predict the severity of CAD for an early characterization of high-risk population is imperative. An added value to the prospective biomarker would be its modification by therapeutic intervention that might influence the assessment of disease progression and management. Although numerous serologic markers of inflammation have been studied, their usefulness in routine patient management remains controversial. In this study, we aimed to compare and evaluate the association of CAD severity with oxidized low-density lipoprotein (oxLDL) and oxLDL-β2-glycoprotein I (β2GPI) complex in patients on statin therapy and statin naive who underwent elective cardiac catheterization.
Methods
Consecutive patients >18 years old with suspected CAD who underwent elective cardiac catheterization being treated with statin therapy (n = 385) or statin naive (n = 150) were enrolled in the Multi-analyte, Thrombogenic, and Genetic Markers of Atherosclerosis (NCT01276678) study after obtaining written informed consent and approval by the Institutional Review Board of the Sinai Hospital of Baltimore. Patients were referred for elective cardiac catheterization for the following reasons: (1) a positive stress test with no angina, (2) a positive stress test with angina, and/or (3) a positive computed tomography angiography. Patients were excluded from the study for any of the following reasons: pregnancy; acute infection; experimental drug therapy; blood donation within 8 weeks; any coagulopathy or bleeding disorder; ongoing treatment for neoplastic, autoimmune, or connective tissue disease, HIV, or hepatitis C infection; or patients with any abnormal laboratory value or physical finding that according to the investigator may interfere with interpretation of the study results. Additionally, blood samples were collected from 134 healthy volunteers not on statin therapy. The study performed conforms to the Declaration of Helsinki.
Blood samples were collected before cardiac catheterization and from healthy volunteers during the day. Patients fasted for >12 hours before cardiac catheterization and took concomitant medications including aspirin and lipid-lowering therapy before arrival (in the statin treatment group). Blood was collected from the antecubital vein or indwelling catheter into one 3.2% trisodium citrate tube and 2 serum separator Vacutainer tubes (Becton-Dickinson, Franklin Lakes, New Jersey), after discarding the initial 3 ml of blood. Serum was separated by centrifugation at 2,000 g for 15 minutes and stored at −70°C. Serum samples were batched and shipped on dry ice for analysis by Atherotech Diagnostics Lab (Birmingham, Alabama) for vertical auto profile (VAP) cholesterol testing to Corgenix Medical Corp. (Broomfield, Colorado) for measurements of oxLDL-β2GPI complex (Atherox) and to Cleveland Heart Lab (Cleveland, Ohio) for oxLDL.
Coronary artery disease severity was based on angiographically defined luminal diameter obstruction of any major coronary vessel as follows: none/minimal (<20%), moderate (20% to 75%), and severe (>75%). Patients with a history of percutaneous intervention or coronary artery bypass grafting surgery were classified as severe CAD. Disease severity was adjudicated by an independent cardiologist to confirm initial angiographic assessment. Statin-naive patients were defined as those who had never been exposed to statin therapy. Statin therapy was divided into low-intensity, moderate-intensity, and high-intensity therapy as per the 2013 guidelines for cholesterol management.
The VAP is a comprehensive lipoprotein cholesterol profile that is based on a well-established method of ultracentrifugation incorporating a vertical rotor and single-density gradient centrifugation. Cholesterol concentrations of high-density lipoprotein (HDL-C), low-density lipoprotein (LDL-C), very low–density lipoprotein (VLDL-C), lipoprotein(a) (Lp(a)-C), intermediate-density lipoprotein (IDL-C), HDL subclasses (HDL 2 -C and HDL 3 -C), LDL subclasses (LDL 1 -C, LDL 2 -C, LDL 3 -C, and LDL 4 -C), and VLDL subclasses (VLDL 1 -C, VLDL 2 -C, and VLDL 3 -C) were determined. The VAP cholesterol test also characterizes each patient into 1 of 3 patterns, depending on which type of LDL predominates. The larger, more buoyant LDL pattern A comprises LDL 1,2 , and the smaller, denser LDL pattern B comprises LDL 3,4 . Patients can also present with a mixed pattern A/B, where neither pattern predominates.
Serum OxLDL was measured at the Cleveland Heart Lab using the Mercodia OxLDL ELISA Test Kit and oxLDL-β2GP1 complex (AtherOx) by Corgenix Medical Corp. using the AtherOx ELISA Test Kit (Broomfield, Colorado). Corgenix and Cleveland Heart Lab were blinded to patient data.
The Mercodia OxLDL ELISA Test Kit used 2 monoclonal antibodies directed against separate antigenic determinants on the oxidized human apolipoprotein B molecule. OxLDL in the sample reacts with the anti-oxLDL monoclonal antibody (4E6) on the microwell. After washing, a peroxidase-conjugated anti-apolipoprotein B monoclonal antibody binds the oxLDL bound to the microwell. Color is developed by tetramethylbenzidine, the reaction stopped with 0.5 M sulfuric acid, and read spectrophotometrically. Results are expressed in units per liter.
The AtherOx (oxLDL-β2GPI complex) test kit uses a monoclonal antibody (3H3) against human β2GPI coated onto microplates. 3H3 is an IgG2b murine monoclonal antibody against β2GPI and used in this assay to capture oxLDL-β2GPI complexes through its reactivity with β2GPI. The assay procedure is as follows: 100 μL of patient serum samples diluted 1:100 are added to the appropriate microwells for incubation at room temperature for 1 hour. The microwells are washed 4 times with phosphate-buffered saline. Biotinylated 2E10 antibody (IgG murine monoclonal anti-human Apo B-100) is added to the microwells and incubated for 30 minutes at room temperature, followed by horseradish peroxidase-streptavidin for 30 minutes. Color is developed with tetramethylbenzidine/H 2 O 2 for 30 minutes and the reaction stopped with 0.36 N sulfuric acid. Optical density is read at a wavelength of 450 nm (650 nm reference). Serum oxLDL-β2GPI complex concentration (expressed in U/ml) is calculated against a reference curve.
Categorical variables were expressed as percentage and continuous variables as mean ± SD, with p ≤0.05 considered statistically significant. Fisher’s exact test was used for comparison of categorical variables. Student’s t test was used for normally distributed continuous data sets, whereas Welch’s t test was used for continuous data sets that did not follow a normal distribution. A receiver operator curve analysis was performed in statin-naive patients to determine criterion associated with moderate and severe CAD. ANOVA was performed comparing LDL 4 and triglyceride level quartiles to oxLDL-β2GPI. Univariate logistic regression analysis was performed to determine the relation of quartiles oxLDL-β2GPI and free oxLDL to severe CAD. The cutoffs used for oxLDL-β2GPI were <0.19, 0.19 to 0.29, 0.30 to 0.66, and >0.66 U/ml and that for free oxLDL was <36, 37 to 43, 44 to 53, and >54 U/L. Multivariate analysis was performed with all significant co-morbidities, oxLDL-β2GPI and oxLDL. Analyses were performed with MedCalc Software (version 13.1.2; Medcalc Statistical Software, Ostend, Belgium).
Results
After approval by the institutional review board, a total of 585 subjects were included in our study. The subjects comprised patients with CAD on a statin (n = 385), not on statin therapy (n = 150) and healthy volunteers (n = 134). Among patients receiving statin therapy, 6% were on low-intensity, 60% on moderate-intensity, and 34% were on high-intensity statin therapy. The patient demographics are listed in Table 1 . Progressively worsening angina was observed in 37% patients not on statin therapy and 39% patients on statin therapy (p = 0.47). A thrombus was observed in 2% of statin-naive patients compared with 1% patients on statin therapy (p = 0.94). In patients with severe CAD, the number of lesions with >50% stenosis and number of diseased vessels with >50% obstruction were comparable between statin-naive patients and those on statin therapy (1.6 ± 0.6 vs 1.5 ± 0.8, respectively, for number of lesions with >50% stenosis, p = 0.65; 1.9 ± 0.9 vs 1.9 ± 0.8, respectively, for number of diseased vessels with >50% stenosis, p = 0.90). Healthy volunteers were free from cardiovascular risk factors, had a mean age of 32 ± 11 years, and 52% were men.
| Variables | Total group | Statin Therapy | p-value | |
|---|---|---|---|---|
| No (n=150) | Yes (n=385) | |||
| Age (years, mean ±SD) | 64 ± 10 | 62 ± 11 | 65 ± 10 | 0.003 |
| Men | 64% | 57% | 67% | 0.04 |
| White | 77% | 79% | 76% | 0.53 |
| Body mass index ( kg/m2, mean ± SD) | 31 ± 7 | 31 ± 7 | 31 ± 7 | 1.0 |
| Hypercholesterolemia | 78% | 48% | 90% | <0.0001 |
| Hypertension | 78% | 68% | 82% | 0.0007 |
| Diabetes mellitus | 33% | 22% | 38% | 0.0006 |
| Myocardial Infarction | 21% | 14% | 24% | 0.02 |
| Percutaneous Coronary Intervention | 29% | 13% | 35% | 0.0001 |
| Coronary Artery Bypass Grafting | 14% | 6% | 17% | 0.002 |
| Stroke | 7% | 3% | 9% | 0.03 |
| Smoker (Current or Past) | 40% | 38% | 41% | 0.59 |
| Aspirin | 100% | 100% | 100% | 1.0 |
| P2Y 12 receptor inhibitors | 30% | 15% | 36% | <0.0001 |
| Beta blockers | 60% | 58% | 61% | 0.59 |
| Calcium channel blockers | 100% | 23% | 23% | 1.0 |
| Angiotensin converting enzyme inhibitors | 33% | 18% | 39% | <0.0001 |
| White blood cells, (×1,000/μL) | 7.3 ± 2.6 | 7.5 ± 3.8 | 7.3 ± 2.3 | 0.46 |
| Hematocrit, (%) | 40 ± 6 | 41 ± 4 | 40 ± 5 | 0.06 |
| Platelets, (×1,000/μL) | 230 ± 63 | 234 ± 60 | 227 ± 66 | 0.25 |
| Creatinine, (g/dL) | 0.99 ± 0.29 | 0.95 ± 0.23 | 1.02 ± 0.31 | 0.01 |
| Blood urea nitrogen, (g/dL) | 18 ± 6 | 17 ± 6 | 18 ± 7 | 0.12 |
Mean oxLDL-β2GPI complex was significantly higher in statin-naive and statin-treated patients compared with the healthy controls (0.17 ± 0.01 U/ml). Compared with statin treated, statin-naive patients had significantly higher oxLDL-β2GPI complexes (0.48 ± 0.47 vs 0.30 ± 0.29 U/ml, p <0.0001, Figure 1 ) and free oxLDL (45.2 ± 15.8 vs 37.8 ± 13.3 U/L, p = 0.002, Figure 2 ).
OxLDL-β2GPI was significantly elevated in statin-naive compared with statin-treated patients across CAD severity groups (0.36 ± 0.29 U/ml, p <0.001, for none-minimal CAD; 0.36 ± 0.0.38 U/ml, p <0.001, for moderate CAD, and 0.58 ± 0.56 U/ml, p <0.0001, for severe CAD, Figure 3 ). Statin-naive patients with severe CAD had the highest oxLDL-β2GPI levels compared with those statin naive with none-minimal or moderate CAD severity (p = 0.036). However, no differences were observed in statin-treated patients between all CAD severity groups.
