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American Journal of Clinical Nutrition, Vol. 88, No. 3, 630-637, September 2008
© 2008 American Society for Nutrition

ORIGINAL RESEARCH COMMUNICATION

Lipoprotein-associated phospholipase A2 activity is associated with coronary artery disease and markers of oxidative stress: a case-control study1,2,3

Ji Young Kim1, Yae Jung Hyun1, Yangsoo Jang1, Byoung Kwon Lee1, Jey Sook Chae1, So Eui Kim1, Hyun Yang Yeo1, Tae-Sook Jeong1, Dong Woon Jeon1 and Jong Ho Lee1

1 From the Yonsei University Research Institute of Science for Aging (JYK, YJH, YJ, JSC, and JHL), the National Research Laboratory of Clinical Nutrigenetic/Nutrigenomics (JYK, YJH, JSC, and JHL), the Department of the Graduate Program in Science for Aging (SEK and HYY), and the Cardiovascular Genome Center, Yonsei Medical Institute (YJ), Yonsei University, Seoul, Korea; the Cardiology Division, Yonsei Cardiovascular Center, Yonsei University College of Medicine, Seoul, Korea (YJ); the Division of Cardiology, Sanggyepaik Hospital, Inje University College of Medicine, Seoul, Korea (BKL); the National Research Laboratory of Lipid Metabolism and Atherosclerosis, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea (T-SJ); and the Division of Cardiology, Department of Internal Medicine, Cardiovascular Center, National Health Insurance Corporation Ilsan Hospital, Goyang-si, Korea (DWJ). JYK and YJH contributed equally to the study and the manuscript

2 Supported by Korea Science and Engineering Foundation (KOSEF) grant no. M10642120002-06N4212-00210, Ministry of Science and Technology; National Research Laboratory project no. R0A-2005-000-10144-0, Ministry of Science and Technology; and Korea Health 21 R&D Project no. A000385, Ministry of Health and Welfare.

3 Reprints not available. Address correspondence to JH Lee, Department of Food and Nutrition, Yonsei University, 134 Shinchon-Dong, Sudaemun-Gu, Seoul, 120-749, Korea. E-mail: jhleeb{at}yonsei.ac.kr.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background:Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a lipoprotein-bound enzyme that can release atherogenic isoprostanes from esterified phospholipids and that may be involved in inflammation and atherosclerosis.

Objective:This study investigates the association between Lp-PLA2 activity and coronary artery disease (CAD) in relation to oxidative stress markers, in particular urinary 8-epi-prostaglandin F2{alpha} (8-epi-PGF2{alpha}).

Design:We conducted a case-control study in which the cross-sectional relation between Lp-PLA2 activity, lipoproteins, and oxidative stress markers was determined in 799 patients with angiographically confirmed CAD and 925 healthy controls.

Results:Lp-PLA2 activity was significantly (P < 0.001) higher in CAD cases than in controls (32.9 ± 0.46 and 29.7 ± 0.42 nmol · mL–1 · min–1, respectively). Both elevated Lp-PLA2 activity and urinary excretion concentrations of 8-epi-PGF2{alpha} were associated with greater CAD risk (P for trend < 0.001). Odds ratios for the upper quartiles of Lp-PLA2 activity and 8-epi-PGF2{alpha}.excretion were 2.47 (95% CI: 1.79, 3.40) and 2.19 (1.52, 3.15), respectively, after adjustment for sex, age, BMI, blood pressure, smoking and alcohol consumption status, and LDL and HDL cholesterol. When we examined the additive effect of both markers for CAD risk, the relation between 8-epi-PGF2{alpha} and CAD was weakened above the second quartile of Lp-PLA2 activity. Moreover, Lp-PLA2 activity was positively correlated with urinary excretion concentrations of 8-epi-PGF2{alpha} in controls (r = 0.277, P < 0.001) and cases (r = 0.202, P < 0.001) and with the tail moment of lymphocyte DNA (r = 0.213, P < 0.001) in controls.

Conclusion:This study shows an association of elevated Lp-PLA2 activity with CAD risk in relation to oxidant stress and thus supports a proatherogenic role of Lp-PLA2.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The biological role of lipoprotein-associated phospholipase A2 (Lp-PLA2) is controversial: whereas initial reports indicate an antiatherogenic effect (1), there is growing evidence of a role for Lp-PLA2 as a proinflammatory molecule and an independent risk factor for coronary artery disease (CAD) (2–6). Lp-PLA2 is a lipoprotein-bound enzyme that catalyzes the hydrolysis of oxidized phospholipids, generating bioactive oxidized free fatty acids and lysophosphatidylcholine, which are potent proinflammatory and proatherogenic products (7). In addition, plasma Lp-PLA2 is responsible for the release of F2-isoprostanes, the end-products of lipid peroxidation, into the bloodstream (8).

Some studies have indicated that F2-isoprostanes are probably the most valid in vivo markers of oxidative stress (9, 10). There is evidence that cardiovascular risk is related, at least in part, to greater systemic oxidative stress, and oxidative damage is known to be involved in the pathogenesis of atherosclerosis and CAD (11, 12), in which it is closely associated with the inflammatory response and bioactive lipid formation. To determine the oxidation status of a person, a set of oxidative markers is recommended, including ≥1 marker each for lipid peroxidation, protein oxidation, and total antioxidative status and, ideally, 1 marker for DNA damage (13).

This case-control study was designed to determine the relation between Lp-PLA2 activity and angiographically proven CAD. In addition, we wanted to explore the relation between Lp-PLA2 and a variety of oxidative stress markers, in particular urinary 8-epi-prostaglandin F2{alpha} (8-epi-PGF2{alpha}), to shed further light on the pathogenic role of this emerging biomarker for CAD risk.


   SUBJECTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects
This study involved 1724 unrelated Korean subjects (1520 men and 204 women) aged 31–83 y. Subjects were divided into 2 subgroups: a control group of 925 persons (805 men and 120 women) with no history or clinical evidence of CAD, who were recruited either from the Health Service Center during the course of routine check-up visits or by advertisements briefly describing the study design, and a case group of 799 patients (715 men and 84 women) with CAD, who were recruited from the Cardiovascular Center at Yonsei University Severance Hospital (Seoul, Korea) or National Health Insurance Corporation Ilsan Hospital (Goyang-si, Korea).

The inclusion criteria for controls were no history or diagnosis of atherosclerosis, vascular disease, chronic heart failure and arrhythmias, diabetes mellitus, or cancer and no pathologic electrocardiogram patterns. The inclusion criteria for CAD cases were angiographic evidence of ≥50% occlusion of ≥1 major coronary artery or previously confirmed myocardial infarction according to the World Health Organization criteria for symptoms, enzyme elevation, or electrocardiographic changes; absence of nonatherogenic occlusion, such as osteal stenosis and spasm; and no history or diagnosis of diabetes mellitus or any diagnosis of thyroid or pituitary disease. Exclusion criteria included abnormal liver or renal function (ie, serum aminotransferase activity > 40 IU/L and serum creatinine concentrations > 1.2 mg/dL); cancer (diagnosed clinically or by anamnesis); extreme weight loss or gain over the previous 6 mo; thyroid or pituitary disease; infection determined by medical questionnaire and complete blood count; and acute or chronic inflammatory disease.

The purpose of the study was carefully explained to all participants, and their written informed consent was obtained. The study protocol was approved by the Institutional Review Board of Yonsei University, and the study was carried out in accordance with the Helsinki Declaration.

Survey method, anthropometric measurements, and blood collection
All subjects completed a standardized questionnaire administered by a specially trained interviewer to provide information on lifestyle factors, current medication use, and medical history. For CAD patients, information was also taken from the hospital medical records. Briefly, subjects were categorized into current smokers, ex-smokers, and never smokers and current alcohol consumers, ex-consumers, or nonconsumers. Data on the frequency of use of all medications, in particular the antihypertensive, hypoglycemic, antidyslipidemic, and antiplatelet drugs, were collected.

Body weight and height were measured in the morning, while the participants were unclothed and not wearing shoes. Body mass index (BMI; in kg/m2) was calculated. Waist and hip circumferences were measured with the use of a paper tape placed horizontally at the umbilicus while the participants were in a standing position after normal expiration. Blood pressure was measured from the left arm of seated participants with the use of an automatic blood pressure monitor (TM-2654; A&D, Tokyo, Japan) after 20 min of rest. The average of 3 measurements was recorded for each subject.

Venous blood specimens were collected in EDTA-treated or plain tubes after a 12-h fast. The tubes were immediately covered with aluminum foil and placed on ice until they arrived at the laboratory room (within 1–3 h), where they were stored at –70 °C until they were analyzed.

Serum lipid profile and C-reactive protein concentraion
Fasting serum concentrations of total cholesterol and triglycerides were measured by using commercially available kits on a Hitachi 7150 Autoanalyzer (Hitachi Ltd, Tokyo, Japan). After precipitation of serum chylomicron conducted with dextran sulfate magnesium, the concentrations of LDL and HDL cholesterol in the supernatant fluid were measured by using an enzymatic method. LDL cholesterol was indirectly estimated in participants with serum triglyceride concentrations < 4.52 mol/L (400 mg/mL) by using the Friedewald formula. In participants with serum triglyceride concentrations ≥ 4.52 mol/L, LDL cholesterol was directly measured by using an enzymatic method on a Hitachi 7150 Autoanalyzer. Serum apolipoprotein A-I and B concentrations were measured by using turbidometry at 340 nm with a specific antiserum (Roche, Basel, Switzerland). Serum high-sensitivity C-reactive protein (hs-CRP) concentrations were measured with an Express plus autoanalyzer (Chiron Diagnostics Co, Walpole, MA) by using a commercially available hs-CRP Latex (II) X2 kit (Seiken Laboratories Ltd, Tokyo, Japan) that allowed detection of CRP concentrations in the range of 0.001 to 32 mg/dL.

Lipoprotein-associated phospholipase A2 activity and plasma oxidized LDL concentrations
Lp-PLA2 activity, also known as platelet-activating factor (PAF)–acetylhydrolase, was measured by using a modification of a previously described high-throughput radiometric activity assay (4). Briefly, a micelle substrate was prepared with unlabeled PAF and [3H]PAF (100 µCi/mL, 21.5 Ci/mmol; NET 910; PerkinElmer, Waltham, MA) in 10 mmol phosphate-buffered saline (PBS)/L (pH 7.4), containing 2.7 mmol EDTA/L (PBS-EDTA). The reaction mixture, containing 20 µL human plasma and 140 µL PBS-EDTA, was incubated at 37 °C for 15 min. The reaction was initiated by the addition of 40 µL micelle substrate (0.05 µCi, final concentration 80 µmol PAF/L) to measure the initial Lp-PLA2 activity. The reaction was stopped by mixing by vortex with 600 µL CHCl:MeOH (2:1), and the CHCl3 and aqueous layers were separated by centrifugation at 1500 rpm for 3 min at 4 °C. The aqueous layer (300 µL) was removed and mixed by vortex with 300 µL CHCl. The aqueous layer (100 µL) was again removed and added to 3 mL scintillation cocktail (Lumagel; Lumac Co, Groningen, Netherlands) in a scintillation vial. The [3H] acetate counts were determined by using a liquid scintillation counter (1450 Microbeta Trilux; Qallac Oy, Turku, Finland). The raw counts were corrected for background by using an enzyme-noncontaining blank and were expressed as nmol degraded PAF · min–1 · mL–1 plasma. The intraassay and interassay CVs were 11.27% and 8.71%, respectively.

Plasma oxidized LDL (ox-LDL) was measured by using an enzyme immunoassay (Mercodia, Uppsala, Sweden). The resultant color reaction was read at 450 nm with a Wallac Victor2 multilabel counter (Perkin Elmer Life Sciences, Turku, Finland).

Lipid peroxidation: urinary 8-epi-prostaglandin F2{alpha}and plasma malondialdehyde
Urine was collected in polyethylene bottles containing 1% butylated hydroxytoluene after a 12-h fast. The tubes were immediately covered with aluminum foil and stored at –70 °C until they were analyzed. We measured 8-epi-prostaglandin F2{alpha} (8-epi-PGF2{alpha}) by using an enzyme immunoassay (BIOXYTECH urinary 8-epi-PGF2{alpha} assay kit; OXIS International Inc, Portland, OR) and the resulting color reaction was read at 650 nm by using a Wallac Victor2 multilabel counter. Urinary creatinine was measured by using the alkaline picrated (Jaffe) reaction. Urinary 8-epi-PGF2{alpha} concentrations were expressed as pmol/mmol creatinine. Plasma malondialdehyde (MDA) was assayed by using the fluorometric method described by Buckingham (14).

Alkaline comet assay for DNA damage
For the comet assay, 120 µL whole blood was mixed with 900 µL PBS, and the mixture was poured gently over 150 µL lymphocyte separation solution (Histopaque-1077; Sigma-Aldrich Korea Ltd, Yong-In, Korea). After a 4-min centrifugation at 1450 rpm and 4 °C, lymphocytes were removed with a pipette and transferred to another tube. DNA damage was analyzed as described by Green et al (15).

Statistical analysis
Statistical analyses were performed with SPSS for WINDOWS software (version 12.0; SPSS Inc, Chicago, IL). Differences in biomarkers between CAD cases and controls were analyzed by using Student's t test. A chi-square test was used to test whether there was a difference in sex distribution, cigarette smoking, alcohol consumption, and medication therapy between CAD cases and controls. Pearson and partial correlation coefficients were used to examine the relation of Lp-PLA2 activity and markers of oxidative stress. In addition, Bonferroni correction for multiple testing was used with {alpha} = 0.005 after the 11 different laboratory variables tested were taken into account.

Plasma Lp-PLA2 activity or urinary 8-epi-PGF2{alpha} concentrations were divided into quartiles according to their distribution in the control group. Next, multiple logistic regression analysis including CAD as a dependent variable and Lp-PLA2 activity or urinary excretion concentration of 8-epi-PGF2{alpha} as an independent variable was applied to estimate the independent association of elevated Lp-PLA2 activity or urinary 8-epi-PGF2{alpha} excretion concentration with the presence of CAD; simultaneous control for confounding factors was exerted. Results were presented as odds ratio (ORs) and 95% CIs estimated from the logistic regression model with initial control for sex, age, and BMI and subsequent further control for blood pressure, cigarette smoking, alcohol consumption, lipid profiles, and statin therapy. Finally, the Lp-PLA2 activity x urinary excretion concentration of 8-epi-PGF2{alpha} interaction for CAD risk was evaluated by using multiple logistic regression analysis. Each OR was estimated with comparison of the reference group in the lowest quartiles for both variables after adjustment for sex, age, BMI, blood pressure, and smoking and alcohol consumption status.

We examined whether each variable presented a normal distribution before statistical testing, and then we performed logarithmic transformation on the skewed variable (ie, triglycerides, 8-epi-PGF2{alpha}, CRP, malondialdehyde, and ox-LDL). For descriptive purposes, mean values are presented by using untransformed and unadjusted values. Results are expressed as mean ± SEs, and a 2-tailed value of P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Characteristics of cases and controls
General characteristics of 799 CAD cases and 925 controls are shown in Table 1Go. Cases were significantly older and heavier and had a larger waist-to-hip ratio than did controls, whereas there was no significant difference between the groups in sex distribution. Current smoking and alcohol consumption were more common in the controls than in the cases. Statins, antihypertensives, and antiplatelet agents were used more frequently in cases than in controls (47.8% and 0.9%, 69% and 4.8%, and 71% and 1.2%, respectively). Consequently, systolic and diastolic blood pressure and concentrations of total, LDL, and HDL cholesterol were lower in cases than in controls, although cases had higher concentrations of triglyceride. Oxidative stress and inflammation were evaluated by measurement of urinary 8-epi-PGF2{alpha}, plasma MDA, lymphocyte DNA damage, ox-LDL, and serum hs-CRP. Compared with controls, cases had significantly higher concentrations of urinary 8-epi-PGF2{alpha}, plasma MDA, and serum hs-CRP; significantly greater lymphocyte DNA damage; and significantly lower concentrations of ox-LDL. Lp-PLA2 activity was significantly higher in cases than in controls (32.9 ± 0.46 and 29.7 ± 0.42 nmol · mL–1 · min–1, respectively; P < 0.001) (Table 1Go).


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  		TABLE 1 Anthropometric and biochemical characteristics of controls and coronary artery disease (CAD) cases1

 
Correlation between lipoprotein-associated phospholipase A2 activity, lipoproteins, high-sensitivity C-reactive protein, and markers of oxidative stress
In controls, Lp-PLA2 activity was positively correlated with urinary excretion concentrations of 8-epi-PGF2{alpha} and lymphoctye DNA damage (tail DNA, tail length, and tail moment) after Bonferroni correction for multiple testing ({alpha} = 0.005) (Table 2Go). These associations remained significant after adjustment for age and medication use. In cases, Lp-PLA2 activity was positively correlated with urinary excretion concentrations of 8-epi-PGF2{alpha} after Bonferroni correction; however, Lp-PLA2 activity was not significantly correlated with age; BMI; total, HDL, or LDL cholesterol; triglyceride; MDA; ox-LDL; or CRP in either cases or controls (Table 2Go).


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  		TABLE 2 Correlation coefficients between lipoprotein-associated phospholipase A2 (Lp-PLA2) activity and lipid profiles, C-reactive protein, and oxidative stress markers in coronary artery disease (CAD) cases and controls1

 
Association of lipoprotein-associated phospholipase A2 activity and 8-epi-prostaglandin F2{alpha} concentration with coronary artery disease
Both Lp-PLA2 activity and urinary excretion concentrations of 8-epi-PGF2{alpha} showed a concentration-dependent association with CAD in the logistic regression model after control for the confounding factors in stages (Table 3Go). Lp-PLA2 activity in the highest quartile (≥41.0 nmol · mL–1 · min–1) was associated with a 2.14-fold risk of CAD (95% CI: 1.61, 2.84) compared with the lowest quartile of Lp-PLA2 activity (<20.4 nmol · mL–1 · min–1) in a model adjusted for sex, age, and BMI (model 1). After additional adjustment for blood pressure, smoking and alcohol consumption status, and LDL and HDL cholesterol, the OR increased to 2.47 (95% CI: 1.79, 3.40) (model 4). Further adjustment for statin therapy (model 5) attenuated the risk associated with high Lp-PLA2 activity, although the risk remained significantly elevated, with an OR of 1.92 (95% CI: 1.32, 2.79), in the highest quartile compared with the lowest quartile.


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  		TABLE 3 Odds ratios (OR) of coronary artery disease associated with lipoprotein-associated phospholipase A2 (Lp-PLA2) activity and urinary 8-epi-prostaglandin F2{alpha} (8-epi-PGF2{alpha}) excretion concentrations after various adjustments1

 
The urinary excretion concentration of 8-epi-PGF2{alpha} was associated with a higher OR for CAD in the third (≥1012.9–1381.8 pg/mg creatinine) and top (≥1381.8 pg/mg creatinine) quartiles, as compared with the lowest quartile (<744.6 pg/mg creatinine): the OR for 8-epi-PGF2{alpha} in the highest quartile was 2.19 (95% CI: 1.59, 3.0) after adjustment for sex, age, and BMI model (model 1) and 2.55 (95% CI: 1.60, 4.06) after further adjustment for blood pressure, smoking and alcohol consumption status, lipids, and statin therapy (model 5). However, when we also adjusted for Lp-PLA2 activity (model 6), the OR for the highest quartile was reduced to 1.80 (95% CI: 1.01, 2.94), and 8-epi-PGF2{alpha} concentrations in the highest quartile were significantly associated with a greater CAD risk only when compared with values in the lowest quartile.

Because the joint effect of the quartile of Lp-PLA2 activity and the quartile of urinary 8-epi-PGF2{alpha} concentration was significant (P < 0.001 for {chi}2 test with 15 df), the interactive effect of both markers with respect to CAD risk was evaluated (Figure 1Go). Compared with participants in the lowest categories of both variables, participants in almost all of the other categories had significantly greater risk of CAD; those in the lowest quartile of Lp-PLA2 activity and the second and third quartiles of 8-epi-PGF2{alpha} concentrations did not have greater risk. The combination of elevated Lp-PLA2 activity and elevated 8-epi-PGF2{alpha} concentrations was generally more significant than was the elevation of either one separately. Overall, the increase in CAD risk with increasing Lp-PLA2 activity was sustained with respect to increases in 8-epi-PGF2{alpha} concentrations; however, the relation between 8-epi-PGF2{alpha} and CAD risk was weakened above the second quartile of Lp-PLA2 activity.


Figure 1
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  		FIGURE 1. Association of lipoprotein-associated phospholipase A2 (Lp-PLA2) activity and urinary 8-epi-prostaglandin F2{alpha} (8-epi-PGF2{alpha}) concentrations with coronary artery disease. Odds ratios were adjusted for sex, age, BMI, systolic and diastolic blood pressure, and smoking and alcohol consumption status. Lp-PLA2 activity x 8-epi-PGF2{alpha} concentrations interactions were significant for coronary artery disease risk: *P < 0.05, **P < 0.01, and ***P < 0.001. Compared with persons in the lowest quartile of Lp-PLA2 activity (<20.4 nmol · mL–1 · min–1) and of 8-epi-PGF2{alpha} (<744.6 pg/mg creatinine), persons in most of the other categories had a significant association with a greater risk of coronary artery disease risk; those in the lowest quartile of Lp-PLA2 activity and the second and third quartiles of 8-epi-PGF2{alpha} did not have a significantly greater risk.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
To our knowledge, the present study is the first to focus on the relation between Lp-PLA2 activity and markers of oxidative stress and, in particular, on the association between urinary excretion of 8-epi-PGF2{alpha} and CAD. In this case-control study, Lp-PLA2 activity and urinary 8-epi-PGF2{alpha} concentration were higher in patients with CAD than in healthy controls, and both elevated Lp-PLA2 activity and urinary excretion concentration of 8-epi-PGF2{alpha} were significantly associated with the risk of CAD, even after control for potential confounders including HDL cholesterol, LDL cholesterol, and statin therapy. It is interesting that, when the combined effect of 8-epi-PGF2{alpha} concentration and Lp-PLA2 activity on CAD risk was examined, the association between 8-epi-PGF2{alpha} concentration and CAD risk was weakened by greater Lp-PLA2 activity. In addition, when CAD risk for the upper quartiles of 8-epi-PGF2{alpha} was compared with that for the lowest quartile, additional adjustment for Lp-PLA2 activity attenuated the risk associated with elevated 8-epi-PGF2{alpha}. Our data show the confirmatory association of elevated Lp-PLA2 activity with CAD risk, which is consistent with previous reports (2–5, 16, 17), and which sheds further light on a proatherogenic role of Lp-PLA2 in relation to oxidant stress.

Lp-PLA2 is known to catalyze hydrolysis of the sn-2 position of glycerophospholipids to liberate arachidonic acid, a precursor of eicosanoids such as prostaglandins and leukotrienes (18). Radical peroxidation of arachidonic acid results in a family of prostaglandin F2-isomers called F2-isoprostanes (13). One such F2-isoprostane is 8-epi-PGF2{alpha}, a sensitive and independent risk marker for coronary heart disease (19–21) that is probably released into biological fluids through a phospholipase-mediated pathway and consequently excreted in urine. Indeed, Stafforini et al (8) showed that the secreted form of Lp-PLA2 released F2-isoprostanes from the sn-2 position of phosphatidylcholine with high affinity, and Kono et al (22) recently reported that intracellular type II Lp-PLA2, which shares homology with the plasma enzyme Lp-PLA2, is involved in the metabolism of esterified 8-iso-PGF2{alpha}. Moreover, plasma samples from Lp-PLA2–deficient subjects do not release F2-isoprostanes from esterified precursors ex vivo (8). Our finding of a positive correlation between Lp-PLA2 activity and urinary excretion of 8-epi-PGF2{alpha} in both controls and cases adds evidence to support the possibility that this enzyme may modulate oxidant stress.

When the interactive effect between Lp-PLA2 activity and urinary excretion of 8-epi-PGF2{alpha} on CAD risk was evaluated, persons with Lp-PLA2 activity in the highest quartile (≥41.0 nmol · mL–1 · min–1) and 8-epi-PGF2{alpha} concentrations in the highest quartile (≥1381.8 pg/mg creatinine) had a CAD risk >8 times greater than that in persons with Lp-PLA2 activity (<20.4 nmol · mL–1 · min–1) and 8-epi-PGF2{alpha} (<744.6 pg/mg creatinine) in the lowest quartile (P < 0.001; 95% CI: 4.50, 16.0). Although this result has the limitation of reflecting only the relative risk in the present population, it may suggest that elevated Lp-PLA2 activity and urinary excretion concentrations of 8-epi-PGF2{alpha}, along with other factors that promote inflammation and oxidant stress, may drive the more susceptible status to atherosclerosis in persons. In addition, we show that the elevation of CAD risk by an increase in the urinary excretion of 8-epi-PGF2{alpha} was attenuated by greater Lp-PLA2 activity, which suggests that Lp-PLA2 may play an active role in atherogenesis, rather than simply being a risk marker. However, because the present study was designed as a case-control study, we are not currently able to explain the reason for such effects.

In the present study, Lp-PLA2 activity positively correlated with 8-epi-PGF2{alpha} and lymphocyte DNA damage in controls. These correlations persisted after Bonferroni correction for multiple testing ({alpha} = 0.005), even after adjustment for sex, age, BMI, smoking and alcohol consumption status, and medication therapy, all of which are known to affect concentrations of PGF2{alpha} (13, 23, 24) and degrees of DNA damage (13, 25–28) (data not shown). Semiquantitative analysis has shown that the isoprostane 8-epi-PGF2{alpha} is one of the most reliable markers of oxidative stress (19, 20, 29–31). Oxidative stress has been suggested as the major cause of DNA damage, and greater degrees of DNA damage in patients with CAD have been reported (32–34). In the present study, we measured lymphocyte DNA damage by using the comet assay, a sensitive and reliable determinant of oxidative stress and DNA damage (35). The positive correlation observed between Lp-PLA2 activity and both 8-epi-PGF2{alpha} and lymphocyte DNA damage suggests that persons with high Lp-PLA2 activity may have higher levels of lipid peroxidation and oxidative stress and may therefore have a greater risk of CAD than do persons with normal Lp-PLA2 activity. Such observations support the conclusion that Lp-PLA2 is proatherogenic (36).

Ox-LDL, another variable of lipid peroxidation, did not correlate with Lp-PLA2 activity in the present study. This lack of correlation may be partly explained by the low association between Lp-PLA2 and LDL cholesterol in the Korean population (37), which has relatively low concentrations of LDL cholesterol (115 mg/dL for middle-aged Koreans) and a high incidence of the Val279Phe variant in the Lp-PLA2 gene, which indicates loss of enzyme activity. The Val279Phe variant of Lp-PLA2 has been reported to be associated with a lower risk of cardiovascular disease in Korean men (37). However, this result may relate to previous findings that Lp-PLA2 does not affect the lipid peroxidation of LDL (38–40). In fact, this possibility is plausible, because the main role of Lp-PLA2 in atherogenesis is the hydrolysis of ox-LDL, which is generated when LDL becomes oxidized in the milieu of the artery wall (41).

MDA, another lipid peroxide, is widely used as an index of oxidative damage because of its ability to interact with lipoprotein (13); however, plasma MDA measurement was reported to be a less reliable assay than lymphocyte DNA damage for detecting the severity of vascular lesions in CAD patients (42). In the present study, the weak correlation observed between MDA and Lp-PLA2 activity may reflect differences in specificity and sensitivity for different lipid peroxides. Because the specificity of the MDA assay is low, MDA is regarded as a general indicator of oxidative stress rather than as a specific marker of lipid peroxidation (13, 43, 44).

We observed lower concentrations of total cholesterol, LDL cholesterol, and ox-LDL in CAD patients, which reflected their widespread use of statin therapy. Although statins can reduce Lp-PLA2 activity (45), CRP concentrations (46), and oxidative stress (47), we found that Lp-PLA2 activity, CRP, and urinary excretion of 8-epi-PGF2{alpha} were higher in CAD cases than in controls. Moreover, in these controls, CRP was positively correlated with urinary excretion concentrations of PGF2{alpha} (r = 0.134, P < 0.001) in the absence of correlation between Lp-PLA2 and CRP. This observation may indicate that inflammation is closely associated with oxidative stress. Schwedhelm et al (19) also found an association between CRP and urinary excretion concentrations of 8-epi-PGF2{alpha}, which was not confounded by lipid-lowering therapy or by total cholesterol, LDL-cholesterol, or triglyceride concentrations.

One limitation of the present study, as for all such case-control studies in which exposure and outcome are collected at one time-point, is the inability to assess the temporal sequence of the described associations. Despite this limitation, our results show an association of elevated Lp-PLA2 activity with a higher incidence of CAD in relation to greater oxidant stress and thus support the hypothesis that Lp-PLA2 represents a novel, viable marker for CAD risk.


   ACKNOWLEDGMENTS
 
We sincerely thank the research subjects who participated in the studies described in this report.

The authors' responsibilities were as follows—all authors: the development of the study protocol and the experimental design; JYK, YJ, BKL, JSC, SEK, HYY, TSJ, and DWJ: sample collection and conduct of the experiments; YJH and JYK: data analysis; JHL: wrote the draft of the manuscript with contributions from YJH; and all authors: contributions to and review and critique of the manuscript. None of the authors had a personal or financial conflict of interest.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Received for publication January 4, 2008. Accepted for publication May 29, 2008.





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