Clinical Chemistry 54: 1975-1981, 2008.
First published October 2, 2008; 10.1373/clinchem.2008.107359
(Clinical Chemistry. 2008;54:1975-1981.)
© 2008 American Association for Clinical Chemistry, Inc.
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Lipids, Lipoproteins, and Cardiovascular Risk Factors |
Association of Lipoprotein-Associated Phospholipase A2 Mass and Activity with Coronary and Aortic Atherosclerosis: Findings from the Dallas Heart Study
Emmanouil S. Brilakis1,2,a,
Amit Khera1,
Bilal Saeed1,2,
Subhash Banerjee1,2,
Darren K. McGuire1,
Sabina A. Murphy3 and
James A. de Lemos1
1 The Donald W. Reynolds Cardiovascular Clinical Research Center at the University of Texas Southwestern Medical Center, Dallas, TX; 2
Dallas VA Medical Center, Dallas, TX; 3
Brigham and Women’s Hospital, Boston, MA.
aAddress correspondence to this author at: Dallas VA Medical Center (111A), 4500 South Lancaster Road, Dallas, TX 75216. Fax (214) 302-1341; e-mail emmanouil.brilakis{at}utsouthwestern.edu.
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Abstract
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Background: Our aim was to characterize the association of lipoprotein-associated phospholipase A2 (Lp-PLA2) with coronary and aortic atherosclerosis in a large population-based study.
Methods: Lp-PLA2 mass and activity were measured in 2171 subjects 30–65 years old participating in the Dallas Heart Study. We examined the association of Lp-PLA2 levels with 3 atherosclerosis phenotypes: coronary artery calcium (CAC) measured by electron-beam computed tomography and abdominal aortic plaque (AAP) and aortic wall thickness (AWT) measured by magnetic resonance imaging.
Results: CAC and AAP were detected in 21% and 40% of subjects, respectively, and mean AWT (SD) was 1.70 (0.32) mm. In univariable analyses, Lp-PLA2 mass (but not activity) was higher in both men (P = 0.04) and women (P = 0.02) with detectable CAC. Lp-PLA2 mass and activity were higher (P = 0.004 and P = 0.01, respectively) and AWT was greater (P < 0.001 and P = 0.02, respectively) in women with aortic atheroma, but not in men. After adjustment for traditional atherosclerosis risk factors and C-reactive protein concentrations, Lp-PLA2 mass and activity were not associated with AAP or AWT in either sex, but Lp-PLA2 mass remained modestly associated with detectable CAC only in men (odds ratio 1.20 per 1 standard deviation increase, 95% CI 1.01–1.42, P = 0.04).
Conclusions: Although Lp-PLA2 mass was independently associated with CAC in men, it was not associated with AAP or AWT in men or with any of the atherosclerosis phenotypes in women. These findings suggest that if Lp-PLA2 independently influences clinical events, it does so by promoting atherosclerotic plaque instability rather than by stimulating atherogenesis.
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Introduction
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Lipoprotein-associated phospholipase A2 (Lp-PLA2)1
is an enzyme that circulates largely bound to low-density lipoproteins (1). Although higher Lp-PLA2 levels have been associated with an increased incidence of cardiovascular events in several (but not all (2)) studies, as summarized in a recent metaanalysis (1), it remains unclear whether Lp-PLA2 is a marker of atherosclerosis development and progression (i.e., "burden"), the biological activity and instability of existing plaque (3), or both.
We measured circulating Lp-PLA2 mass and activity in a large, multiethnic, population-based cohort from Dallas, Texas, to determine: a) the association of Lp-PLA2 with coronary artery calcification (CAC) as measured by electron beam computed tomography (EBCT); and b) the association of Lp-PLA2 measures with abdominal aortic atherosclerosis and wall thickness as measured by magnetic resonance imaging (MRI).
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Materials and Methods
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study population
The Dallas Heart Study is a population-based, probability sample of 6101 subjects in Dallas County that was designed to study early stages of cardiovascular disease, as described in detail (4). Briefly, a stratified random sample of Dallas County residents age 18–65 years was obtained from a pool of 841 943 eligible subjects using the US Postal Service Delivery Sequence File, with deliberate oversampling of African Americans. An initial visit for 6101 participants included a detailed in-home interview for demographic and health-related data, as well as measurements of weight, heart rate, and blood pressure (5 sequential measures). All subjects between the ages of 30 and 65 who completed the initial visit were invited to participate in a second in-home visit to collect fasting venous blood and urine samples and, if they completed the second visit, a third detailed clinic visit that included abdominal aortic MRI and cardiac EBCT to assess CAC. A total of 3399 subjects completed the second visit, and 2171 subjects completed the third visit. In the present study, we included all 2171 subjects who had complete data from Lp-PLA2 measurements, cardiac EBCT, and aortic MRI. The study was approved by our Institutional Review Board, and every patient gave written informed consent.
lp-pla2 assays
Blood samples were obtained in EDTA tubes and were stored for 
4 h at 4 °C before processing, with plasma aliquots stored at –80 °C. Lp-PLA2 activity was measured with a colorimetric activity method provided by GlaxoSmithKline (5). The mean duplicate CV was 2.94% for the low controls and 3.85% for the high controls. The mean duplicate CV for samples was 3.87% (5).
Lp-PLA2 mass was measured by diaDexus, Inc., using a dual monoclonal antibody immunoassay standardized to recombinant Lp-PLA2, as described (5). The mean duplicate CV was 2.89% for the low controls and 3.23% for the high controls. Because of limited plasma quantities, Lp-PLA2 mass measurements were run singly (5). There was a strong correlation between Lp-PLA2 mass and activity (Spearman 
= 0.69, P < 0.001) (5). All analyses were performed using a Molecular Devices microplate reader, and the day-to-day CV for LP-PLA2 activity and mass ranged from 2.9% to 3.9% and from 2.1% to 4.2%, respectively.
computed tomography
We performed EBCT using an Imatron 150 XP, 30 cm FOV, 512 matrix with sharp kernel reconstruction, with measurements obtained at 80% of the RR interval. Calcium scoring followed the protocol of the Multi Ethnic Study of Atherosclerosis (MESA) (6). Detection of calcium was based on a focus of calcium with 
3 contiguous pixels and a CT threshold of 130 Hounsfield units (HU). EBCT scores were expressed in Agatston units, and the mean of 2 consecutive scans was used as the final EBCT score, unless only 1 scan was obtained. To minimize false-positive classifications of CAC prevalence due to tissue-associated artifact, we used a mean Agatston score >10 to define prevalent CAC, a data-derived threshold above which interscan concordance exceeded 95% (7).
magnetic resonance imaging
We performed abdominal MRI using a 1.5-T whole-body system (Intera; Philips Medical Systems) including 6 total slices of the infrarenal abdominal aorta using a free-breathing, electrocardiogram-gated, T2-weighted turbo spin-echo (black-blood) sequence. For each slide, adventitial and luminal borders were drawn using a freehand manual contour drawing tool. Areas of increased signal intensity, luminal protrusion, and focal wall thickening were identified as AAP and categorized dichotomously as present or absent (8). For each slide, adventitial and luminal borders were drawn using a freehand manual contour drawing tool, and aortic wall area was calculated as the difference between the adventitial and luminal areas. We defined AWT as the ratio of the area of the aortic wall/mean aortic circumference. The mean value of the 6 slices was reported.
statistical analysis
Nominal data are reported as percentiles and continuous data as mean values with SDs or as median values with interquartile ranges (25th to 75th percentile) for non–normally distributed variables. Subjects were divided into sex-specific quartiles based on Lp-PLA2 levels, as there are sex differences in Lp-PLA2 distribution (5). Baseline demographic variables and cardiovascular risk factors were compared across quartiles of Lp-PLA2 using the 
2 trend test for nominal variables and the test for trend or the Kruskall–Wallis test across ordered groups for continuous variables. We determined associations between Lp-PLA2 levels and the presence of detectable CAC or AAP using a series of univariable and multivariable logistic regression models, with adjustment for traditional risk factors (age, sex, race, LDL cholesterol, HDL-cholesterol, diabetes, smoking, hypertension), C-reactive protein (CRP) concentration, and statin use. Lp-PLA2 mass and activity were analyzed in these models as continuous parameters, and odds ratios (ORs) were expressed per SD increment. We determined the association between Lp-PLA2 levels and AWT (a continuous variable) using linear regression models adjusting for the same variables described above. All comparisons were 2-sided, and P < 0.05 was considered statistically significant.
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Results
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Age was 45 (9) years, and 46% were men. The mean (SD) and median (interquartile range) Lp-PLA2 mass values were 191 (58) µg/L and 187 (156–220) µg/L, respectively. Lp-PLA2 activity values were 148 (39) and 145 (121–173) µmol/min/L. Prevalent CAC and AAP were present in 21% and 40% of subjects, respectively. The mean (SD) and median aortic wall thickness was 1.70 (0.32) mm and 1.67 mm. Lp-PLA2 mass was 205 (61) µg/L in men and 180 (54) µg/L in women, and Lp-PLA2 activity was 163 (39) µmol/min/L in men and 135 (34) µmol/min/L in women. The association of baseline characteristics of the study population with sex-specific quartiles of Lp-PLA2 mass is shown in Table 1
, and with Lp-PLA2 activity, in Table 2
.
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Table 1. Baseline characteristics of the study population stratified according to sex-specific Lp-PLA2 mass quartiles.
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Table 2. Baseline characteristics of the study population stratified according to sex-specific Lp-PLA2 activity quartiles.
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In univariable analyses, Lp-PLA2 mass was associated with detectable CAC in both men (P = 0.04) and women (P = 0.02) (Table 3
; Fig. 1
). After multivariable adjustment, Lp-PLA2 mass remained modestly associated with detectable CAC in men (P = 0.04) but not women (Table 3
). In contrast, Lp-PLA2 activity was not associated with detectable CAC in univariable or multivariable analyses in either sex (Table 3
).
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Table 3. Univariable and multivariable models examining the association between Lp-PLA2 mass and activity with 3 atherosclerosis phenotypes.
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Figure 1. Lp-PLA2 mass and activity in men and women classified according to the presence of detectable CAC.
The upper, middle, and lower lines of the shaded box correspond to the 75th percentile, median, and 25th percentile values. The upper line at the end of the whisker coming out of the box on the upper side is the minimum value of either (a) the maximum value in the data set or (b) the largest value in the data set that is less than the upper quartile value plus 1.5 times the interquartile range (IQR). The lower line at the end of the whisker coming out of the box on the lower side is the maximum value of either (a) the minimum value in the data set or (b) the smallest value in the data set that is more than the lower quartile value minus 1.5 times the IQR.
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In univariable analyses, higher Lp-PLA2 mass and activity were associated with prevalent AAP in women (P = 0.004 and P = 0.01, respectively) but not men (Fig. 2
; Table 3
). After multivariable adjustment, neither Lp-PLA2 mass or activity was associated with AAP in men or women (Table 3
).
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Figure 2. Lp-PLA2 mass and activity in men and women classified according to the presence of detectable aortic plaque.
The upper, middle, and lower lines of the shaded box correspond to the 75th percentile, median, and 25th percentile values, respectively. The upper line at the end of the whisker coming out of the box on the upper side is the minimum value of either (a) the maximum value in the data set or (b) the largest value in the data set that is less than the upper quartile value plus 1.5 times the interquartile range (IQR). The lower line at the end of the whisker coming out of the box on the lower side is the maximum value of either (a) the minimum value in the data set or (b) the smallest value in the data set that is more than the lower quartile value minus 1.5 times the IQR.
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Finally, in univariable analysis, Lp-PLA2 mass and activity were both associated with AWT in women (P 0.02 for each) but not in men (P > 0.2). In linear regression analyses in which AWT was the dependent variable, adjusting for standard risk factors, CRP, and statin use, Lp-PLA2 mass and activity were no longer associated with AWT in men or women (Table 3
).
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Discussion
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Our study of a multiethnic population-based subject cohort shows that although Lp-PLA2 activity and mass were associated modestly with selected atherosclerosis phenotypes in univariable analyses, most of these associations were attenuated or abolished after adjustment for standard risk factors. The only independent association observed was a modest one between Lp-PLA2 mass and CAC that was evident only among men.
Our study is the largest reported to date to evaluate the associations between LP-PLA2 and CAC, and the first to examine the association of Lp-PLA2 levels with AAP and AWT. Although Lp-PLA2 mass and activity were higher in women with abdominal aortic atherosclerosis and a thicker abdominal aortic wall, the association was no longer significant after adjusting for other atherosclerosis risk factors. Few data are available regarding AWT as a marker of atherosclerosis, but it may provide information in the aorta similar to that of carotid intima-media thickness in the carotid arteries. Perrson et al. (9) recently reported a modest, yet statistically significant, independent association of Lp-PLA2 mass and activity with carotid intima-media thickness measured by ultrasonography, but included only age and sex in the multivariable analysis.
Although Lp-PLA2 mass and activity have been independently associated with cardiovascular clinical events in multiple (but not all (2)) studies (1), the association with measures of atherosclerosis burden has been less consistent. Most studies have examined the association between Lp-PLA2 levels and extent of coronary artery disease assessed by coronary angiography. Lp-PLA2 mass and activity have been associated with the severity of angiographic coronary artery disease in univariable analyses in all published studies (10)(11)(12)(13). The association of Lp-PLA2 mass with angiographic coronary artery disease persisted after adjustment in 1 study (14), but was no longer significant in another study (12).
Three studies have examined the association of Lp-PLA2 with CAC. In the Rotterdam population-based study, Lp-PLA2 activity was measured in 520 subjects >55 years of age; although Lp-PLA2 activity was associated with CAC in univariable analysis, the association did not persist after adjustment for HDL and non-HDL cholesterol (15). In the Coronary Artery Risk Development in Young Adults (CARDIA) nested case-control study, 266 subjects with CAC were compared with 266 controls (men comprised 71% of both groups) (3). Both Lp-PLA2 mass and activity were associated with CAC in univariable analysis; after adjusting for age, educational attainment, smoking, alcohol consumption, body mass index, waist circumference, diabetes, hypertension, LDL and HDL cholesterol, triglycerides, and CRP, Lp-PLA2 mass maintained its association with detectable CAC (OR 1.28 per SD, 95% CI 1.03–1.60), whereas Lp-PLA2 activity did not (OR 1.09 per SD, 95% CI 0.84–1.42) (3). Finally, in a small study of 100 American (research volunteers) and 100 Japanese (population-based) men, Lp-PLA2 activity was not associated with CAC in American men and was inversely associated with CAC in Japanese men, although no multivariable adjustment was performed (16).
The findings of our study are very similar to the above studies, with the further finding that Lp-PLA2 mass was independently associated with CAC in men but not women. This may be due to the higher CAC prevalence and significantly higher Lp-PLA2 levels seen in men compared with women (5). The lack of independent association of Lp-PLA2 activity with CAC in both CARDIA (3) and our study could be in part due to the stronger association of Lp-PLA2 activity (Spearman’s correlation coefficient = 0.42) compared to Lp-PLA2 mass (Spearman’s correlation coefficient = 0.33) with LDL cholesterol. These findings do not support an incremental role of LP-PLA2 activity over mass for the identification of subclinical atherosclerosis.
Although Lp-PLA2 levels do not appear to be strongly associated with atherosclerotic burden, higher levels have been associated with an increased incidence of cardiovascular events in multiple studies (17). This has also been described for other biomarkers, such as CRP (18), and suggests that Lp-PLA2 may be a better marker of atherosclerosis activity and vulnerability rather than atherosclerosis burden. In the Dallas Heart Study, higher CRP was associated with more extensive coronary and aortic atherosclerosis in men; on multivariable analysis, however, the association was no longer significant (18).
Lp-PLA2 activity measurement was performed on frozen rather than fresh specimens, but Lp-PLA2 has been shown to be stable in repeated freeze-thaw cycles (12). Because limited amount of available plasma, repeat measurements could not be performed for Lp-PLA2 mass, and therefore we could not measure the duplicate CV for samples, although the duplicate CV for controls was low. The finding of an association only between Lp-PLA2 mass and coronary atherosclerosis and only in the subgroup of men could be the result of chance. All observations were cross-sectional, since follow-up for clinical events is not yet available on the Dallas Heart Study cohort.
In conclusion, Lp-PLA2 mass and activity were not found to be independently associated with abdominal aortic atherosclerosis or with abdominal aortic wall thickness. Lp-PLA2 activity was not found to be independently associated with detectable CAC, whereas Lp-PLA2 mass was independently associated with detectable CAC in men but not women. Previously reported associations between Lp-PLA2 and clinical cardiovascular events (17) may be related to atherosclerotic plaque instability rather than atherosclerotic burden.
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Acknowledgments
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Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.
Authors’ Disclosures of Potential Conflicts of Interest: Upon submission, all authors completed the Disclosures of Potential Conflict of Interest form. Potential conflicts of interest:
Employment or Leadership: None declared.
Consultant or Advisory Role: D.K. McGuire, Tethys Bioscience, CV Therapeutics, Johnson and Johnson, and AstraZeneca; J.A. de Lemos, Biosite/Inverness, Roche, and Tethys.
Stock Ownership: None declared.
Honoraria: D.K. McGuire, Pfizer, and Takeda. J.A. de Lemos, GlaxoSmithKline and Roche.
Research Funding: The current study was supported in part by a research grant from GlaxoSmithKline. The Dallas Heart Study was funded by the Donald W. Reynolds Foundation (Las Vegas, NV) and was partially supported by US Public Health Service General Clinical Research Centers grant M01-RR00633 from NIH, National Center for Research Resources, Clinical Research. J.A. de Lemos received research funding from Biosite/Inverness.
Expert Testimony: None declared.
Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.
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Footnotes
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1 Nonstandard abbreviations: Lp-PLA2, lipoprotein-associated phospholipase A2; CAC, coronary artery calcification; EBCT, electron beam computed tomography; MRI, magnetic resonance imaging; CRP, C-reactive protein; OR, odds ratio; AAP, abdominal aortic plaque; AWT, aortic wall thickness. 
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Abstract
Introduction
