Context Myeloperoxidase (MPO), a leukocyte enzyme that promotes oxidation of
lipoproteins in atheroma, has been proposed as a possible mediator of atherosclerosis.
Objective To determine the association between MPO levels and prevalence of coronary
artery disease (CAD).
Design, Setting, and Patients Case-control study conducted from July to September 2000 in a US tertiary
care referral center, including 158 patients with established CAD (cases)
and 175 patients without angiographically significant CAD (controls).
Main Outcome Measures Association of MPO levels per milligram of neutrophil protein (leukocyte-MPO)
and MPO levels per milliliter of blood (blood-MPO) with CAD risk.
Results Leukocyte- and blood-MPO levels were both significantly greater in patients
with CAD than in controls (P<.001). In multivariable
models adjusting for traditional cardiovascular risk factors, Framingham risk
score, and white blood cell counts, MPO levels were significantly associated
with presence of CAD, with an OR of 11.9 (95% CI, 5.5-25.5) for the highest
vs lowest quartiles of leukocyte-MPO and an OR of 20.4 (95% CI, 8.9-47.2)
for the highest vs lowest quartiles of blood-MPO.
Conclusions Elevated levels of leukocyte- and blood-MPO are associated with the
presence of CAD. These findings support a potential role for MPO as an inflammatory
marker in CAD and may have implications for atherosclerosis diagnosis and
risk assessment.
Numerous epidemiologic studies have evaluated several inflammatory markers,
including C-reactive protein, various cytokines, adhesion molecules, and white
blood cell (WBC) count for their clinical usefulness in predicting risk of
cardiovascular disease.1-5
Recent investigations have suggested that myeloperoxidase (MPO), an abundant
enzyme secreted from activated neutrophils, monocytes, and certain tissue
macrophages (such as in atherosclerotic plaques), may be involved in the development
of coronary artery disease (CAD).
Myeloperoxidase synthesis occurs during myeloid differentiation in bone
marrow and is completed within granulocytes prior to their entry into the
circulation.6,7 The enzyme is
stored within primary granules of neutrophils and monocytes and is not released
until leukocyte activation and degranulation.6,7
Myeloperoxidase forms free radicals and diffusible oxidants with antimicrobial
activity. However, MPO also promotes oxidative damage of host tissues at sites
of inflammation, including atherosclerotic lesions.8,9
Immunohistochemical studies have demonstrated the presence of MPO in human
atherosclerotic lesions,10 and mass spectrometry
studies have shown that oxidation products generated by MPO are enriched in
human atheroma and in low-density lipoprotein (LDL) recovered from diseased
arterial tissue.8,9,11-13
Myeloperoxidase has been implicated as an enzymatic catalyst of LDL oxidation
in vivo, converting the lipoprotein into a high-uptake form for macrophages
leading to cholesterol deposition and foam cell formation.14,15
MPO also is capable of using nitric oxide as a physiologic substrate, thereby
serving as a catalytic sink for the nitric oxide, perhaps contributing to
endothelial dysfunction.16
Based on the links between MPO, oxidation of LDL, and the functional
deficiency of nitric oxide in atherosclerotic vessels, we hypothesized that
levels of MPO in leukocytes might serve to identify individuals at increased
risk for CAD. In this study we evaluated whether levels of MPO are associated
with the presence of angiographically documented CAD.
Based on logistic regression power calculations (assuming equal-size
groups), we estimated that 326 patients were needed to provide 80% power to
detect a statistically significant (α = .05) odds ratio (OR) of at least
2.0 for presence of CAD in the highest MPO quartile compared with the lowest
quartile. Patients (n = 333) were identified from 2 practices within the Cardiology
Department of the Cleveland Clinic Foundation. From July to September 2000,
85 consecutive patients were enrolled from the Preventive Cardiology Clinic
and simultaneously, 125 consecutive patients were enrolled from the catheterization
laboratory. Based on CAD prevalence in these patients, 116 additional control
subjects were needed. All patients who did not have significant CAD as determined
by coronary artery catheterization during the preceding 6 months were identified
from the catheterization database, and then 140 were randomly selected (based
on area code/telephone number) and invited to participate for MPO measurement.
Coronary artery disease was defined by a history of documented myocardial
infarction, prior coronary revascularization intervention (coronary artery
bypass graft surgery or percutaneous coronary intervention), or as the presence
of ≥50% stenosis in 1 or more coronary arteries identified during cardiac
catheterization. Exclusion criteria for the CAD group were an acute coronary
event within 3 months preceding enrollment, end-stage renal disease, or prior
bone marrow transplantation. The control group consisted of patients who had
undergone diagnostic coronary angiography that revealed no evidence of significant
CAD. Exclusion criteria for control subjects were 1 or more coronary vessels
with stenosis ≥50%, valvular heart disease, left ventricular dysfunction,
end-stage renal disease, bone marrow transplantation, or evidence of infection
or active inflammatory diseases as revealed by history and physical examination.
All patients were older than 45 years and afebrile.
Clinical history was assessed for diabetes mellitus, smoking (past and
present), hypertension, and whether any first-degree relatives had CAD (men
by age 50 years and women by age 60 years). Diabetes was ascertained with
a physician diagnosis of diabetes or fasting plasma glucose level higher than
126 mg/dL (7.0 mmol/L). Hypertension was defined as a physician diagnosis
of chronic hypertension or patient's blood pressure measured at higher than
140/90 mm Hg on examination. Current smoking was defined as smoking more than
5 cigarettes within the past 3 months. Study protocol and consent forms were
approved by the Cleveland Clinic Foundation Institutional Review Board and
written informed consent was obtained from all patients.
Samples were coded to ensure anonymity and all analyses were performed
in a blinded fashion. After an overnight fast, blood was drawn into tubes
containing EDTA and used to quantify WBC, LDL-C, high-density lipoprotein
cholesterol (HDL-C), total cholesterol (TC), and fasting triglycerides (TG).
Neutrophils were isolated by buoyant density centrifugation.17
Cell preparations were at least 98% homogeneous by visual inspection. Leukocyte
preparations were supplemented to 0.2% cetyltrimethylammonium bromide for
cellular lysis, incubated at room temperature for 10 minutes, snap frozen
in liquid nitrogen, and stored at −80°C until analysis.
Functional MPO was quantified by peroxidase activity assay of neutrophil
lysates. Briefly, detergent-lysed cells (104 per mL; triplicate
samples) were added to 20-mM phosphate buffer (pH 7.0) containing 14.4-mM
guaiacol, 0.34-mM H2O2, and 200-µM diethylene
triamine penta-acetic acid and the formation of guaiacol oxidation product
was monitored at A470 at 25°C.18,19
A millimolar absorbance coefficient of 26.6 mM−1·
cm−1for the diguaiacol oxidation product was used to calculate
peroxidase activity. One unit of MPO activity is defined as the amount that
consumes 1 µmol of H2O2 per minute at 25°C.
Myeloperoxidase activity reported is normalized either per milligram of neutrophil
protein (leukocyte-MPO) or per milliliter of blood (blood-MPO). The total
content of MPO in blood is dependent on MPO levels per leukocyte and the total
number of leukocytes. Since neutrophils possess more than 95% of the MPO content
in blood, blood-MPO (MPO units per milliliter of blood) was estimated by multiplying
the units of MPO activity per neutrophil by the absolute neutrophil count
(per microliter of blood, ×1000). Protein concentration was determined
as previously described.20
Levels of leukocyte-MPO in an individual were reproducible, demonstrating
less than ±7% variations in subjects over time (n = 6 men evaluated
once every 1-3 months for >2-year period). The coefficient of variance for
determination of leukocyte-MPO, as determined by analysis of samples multiple
times consecutively, was 4.2%. Leukocyte-MPO determination for 10 samples
analyzed on 3 separate days yielded a coefficient of variance of 4.6%. The
coefficient of variance for determination of blood-MPO, as determined by analysis
of samples multiple times consecutively, was 4.2%. Blood-MPO determination
for 10 samples analyzed on 3 separate days yielded a coefficient of variance
of 4.8%.
Myeloperoxidase mass per neutrophil was determined using an enzyme-linked
immunosorbent assay (ELISA). Capture plates were made by incubating 96-well
plates overnight with polyclonal antibody (Dako, Glostrup, Denmark) raised
against the heavy chain of human MPO (10 µg/mL in 10 mM of phosphate-buffered
saline, pH 7.2). Plates were washed and sandwich ELISAs were performed on
leukocyte lysates using alkaline phosphatase–labeled antibody to human
MPO. Myeloperoxidase mass was calculated based on standard curves generated
with known amounts of human MPO purified from leukocytes as described.17 Purity of isolated MPO was established by demonstrating
an RZ of 0.87 (A430/A280), sodium dodecyl sulfate–polyacrylamide
gel electrophoresis analysis, and in-gel tetramethylbenzidine peroxidase staining.14 Enzyme concentration was determined spectrophotometrically
using an extinction coefficient of 89 000 M−1·
cm−1/heme.
Patient characteristics are presented as either mean (SD) or median
(interquartile range) for continuous measures and as number (percentage) for
categorical measures. Differences between patients with CAD and controls were
evaluated with Wilcoxon rank sum or χ2 tests. Levels of MPO
were divided into quartiles for analyses because neither leukocyte-MPO nor
blood-MPO activity follows a Gaussian distribution. Unadjusted trends for
increasing CAD rates with increasing MPO activity were evaluated with the
Cochran-Armitage trend test. A modified Framingham Global Risk Score21 was determined using a documented history of hypertension
rather than the recorded blood pressure at time of catheterization.
Logistic regression models (SAS version 8.0, SAS Institute Inc, Cary
NC) were developed to calculate ORs associated with the combined second and
third quartiles of MPO activity and the highest quartile of MPO activity compared
with the lowest quartile. Adjustments were made for individual traditional
CAD risk factors (age, sex, diabetes, hypertension, smoking [ever or current],
family history, WBC, LDL-C, HDL-C, TC, and TG). Hosmer-Lemeshow goodness-of-fit
tests were used to evaluate appropriate model fit. Associations among continuous
variables were assessed with use of the Spearman rank-correlation coefficient.
Associations among categorical variables were assessed using Wilcoxon rank
sum tests.
The clinical and biochemical characteristics of study participants are
shown in Table 1. Patients with
CAD were older, more likely to be men, and more likely to have a history of
diabetes, hypertension, and smoking. Patients with CAD also had increased
fasting TG levels and were more likely to use lipid-lowering medications (predominantly
statins), aspirin, and other cardiovascular medications. The Framingham Global
Risk Score, absolute neutrophil count, and WBC count were significantly increased
in patients with CAD.
MPO Levels and Prevalence of CAD
Myeloperoxidase activity per milligram of neutrophil protein (leukocyte-MPO)
was significantly greater for patients with CAD than for controls (median
values, 18.1 U/mg vs 13.4 U/mg, respectively; P<.001
for trend and for difference) (Figure 1).
Individuals in the highest quartile of leukocyte-MPO levels had increased
risk of CAD compared with those in the lowest quartile (OR, 8.8; 95% CI, 4.4-17.5; P<.001 for trend) (Table 2). In an analysis that quantified MPO mass per neutrophil
using an ELISA in a random subset of subjects (n = 111), the results were
highly correlated (r = 0.95) with the activity measurements
(data not shown). Rates of CAD were also associated with increasing blood-MPO
quartiles (P<.001 for trend) (Figure 1, Table 2).
As shown in other studies, the Framingham Global Risk Score and WBC counts
were associated with rates of CAD (Table
2).
Leukocyte-MPO and CAD Risk Factors
Leukocyte-MPO levels were independent of age, sex, diabetes, hypertension,
smoking (ever or current), WBC count, LDL-C, TG, and Framingham Global Risk
Score. Weak negative correlations between leukocyte-MPO and both TC (r = −0.15; P = .005) and
HDL-C (r = −0.14; P
= .01) were observed. A positive correlation was observed between leukocyte-MPO
and absolute neutrophil count (r = 0.20; P<.001) and family history of CAD (median leukocyte-MPO: 15.9 [with
family history] vs 14.1 [without family history]; P
= .05). Similar correlations were noted for blood-MPO.
Multivariable Adjustments for Single and Multiple Risk Factors
Odds ratios for leukocyte-MPO and blood-MPO quartiles were adjusted
for individual traditional CAD risk factors. Since rates for CAD in the second
and third quartiles of leukocyte-MPO appeared comparable (Table 2), they were combined for all further analyses and are referred
to as the midrange levels in univariate and multivariable models. Odds ratios
for both the middle (second plus third) and highest (fourth), relative to
the lowest (first), quartiles of both leukocyte-MPO and blood-MPO remained
associated with CAD status following adjustments for individual traditional
CAD risk factors, WBC count, and Framingham Global Risk Score (data not shown),
with ORs ranging from 8.4 (95% CI, 4.2-16.9; P<.001)
after adjustment for HDL-C to 13.5 (95% CI, 6.3-29.1; P<.001) after adjustment for smoking. Diabetes, hypertension, smoking,
and to a lesser degree age, HDL-C, WBC count, and Framingham Global Risk Score,
also remained significant predictors for CAD status following single-factor
adjustments. Similar results were observed for blood-MPO following single-factor
adjustments for individual traditional CAD risk factors (data not shown).
In multivariable analyses with simultaneous adjustment for each of the
single risk factors that were significantly associated with CAD in the preceding
step, leukocyte-MPO remained the strongest predictor of CAD risk for both
the middle vs the low quartile (adjusted OR, 8.5; 95% CI, 3.7-19.7) and the
high vs the low quartile (adjusted OR, 20.3; 95% CI, 7.9-52.1). With further
adjustment for Framingham Global Risk Score and WBC count, the ORs for leukocyte-MPO
were 4.2 (middle vs low quartile) and 11.9 (high vs low quartile). Blood-MPO
also remained a strong predictor of CAD risk following multivariable adjustments
(Table 2). The adjusted ORs for
Framingham Global Risk Score and WBC count were also significant.
The results of this study suggest that MPO may serve as an independent
marker of CAD. Levels of functional MPO per leukocyte and per milliliter of
blood were associated with the risk of CAD in a study population that was
characterized angiographically for disease status, even following multivariable
adjustments for traditional risk factors and WBC count.
Several studies support potential links between MPO and the development
of CAD. Myeloperoxidase has been implicated as a participant in atherosclerosis
through mechanisms related to its role in inflammation,9,22
LDL oxidation,11-15,23,24
and nitric oxide consumption leading to endothelial dysfunction.16
Myeloperoxidase generates an array of diffusible oxidants9
and is capable of initiating lipid peroxidation25,26
and promoting protein nitration27,28
and crosslinking,29 processes known to occur
during the evolution of atherosclerosis.8,9,30-33
Myeloperoxidase also binds to LDL in plasma34
and promotes site-specific oxidation of the lipoprotein.35
Both immunohistochemical and mass spectrometry studies demonstrate that MPO
is present in, and promotes oxidative modification of, targets within human
atheroma at all stages of lesion development.8-10,22
Furthermore, LDL recovered from human atherosclerotic lesions is enriched
in multiple oxidation products formed specifically by MPO, such as chlorotyrosine11 and Schiff base adducts of p-hydroxyphenylacetaldehyde
(a tyrosine oxidation product17) with both
apolipoprotein B100 lysine residues12 and aminophospholipids.13
There are several clues to the potential functional consequences of
MPO-catalyzed oxidation in the artery wall. Isolated human monocytes use MPO
to oxidatively convert LDL into an atherogenic particle capable of promoting
cholesterol accumulation and foam cell formation.14
Uptake occurs via the scavenger receptor CD36,15
a receptor that appears to play a major role in foam cell formation in vivo.36 Myeloperoxidase may thus be involved in the atherosclerotic
process directly by promoting lesion development.
Myeloperoxidase also may play a role in the pathogenesis of acute coronary
syndromes through plaque destabilization.22
Circulating leukocytes release MPO during acute coronary syndromes.37 Macrophages containing MPO and MPO-dependent oxidation
products are selectively enriched in atheromas that have undergone plaque
rupture and ulceration.22 Moreover, hypochlorous
acid (HOCl), a primary oxidant generated by MPO,38
may promote extracellular matrix degradation in vivo.22
Myeloperoxidase-generated HOCl both activates latent matrix metalloproteinases
and inactivates their physiological inhibitors (eg, tissue inhibitor of metalloproteinase
1).39-41 Myeloperoxidase
thus may influence plaque stability and the propensity for provoking thrombosis.22
Myeloperoxidase also may contribute to CAD through promoting endothelial
dysfunction.16 Nitric oxide modulates MPO catalytic
activity42 and serves as a physiological substrate
for MPO.16 Myeloperoxidase attenuates nitric
oxide–dependent smooth muscle relaxation43
and preliminary studies with preconstricted vascular rings show that MPO attenuates
nitric oxide–mediated vasorelaxant responses.44
Thus, MPO may serve as a catalytic sink for nitric oxide, limiting its bioavailability
and function.9,16
Although multiple lines of evidence suggest potential mechanisms for
MPO in the development of cardiovascular disease, there are limited data in
humans or animals. A cross-sectional study of 92 MPO-deficient individuals
reported that MPO deficiency (a genetic disorder that occurs in 1:2000 to
1:5000 individuals6) is associated with a decreased
prevalence of cardiovascular events.45 A functional
polymorphism in the promoter region of the gene for MPO resulting in decreased
enzyme expression recently was reported to be associated with decreased risk
of CAD.46 Recent studies with MPO knockout
mice demonstrated increased atherosclerotic lesion development.47
However, further investigation demonstrated species-specific differences between
mouse and human, including the absence of MPO and its oxidation products within
lesions among wild-type mice.47
This study, to our knowledge, is the first direct attempt to correlate
levels of MPO in leukocytes and blood with angiographically documented CAD
status. While elevated MPO levels were associated with CAD, there was considerable
variation among individuals in the levels of MPO present within leukocytes.
Differences in leukocyte-MPO content, more than leukocyte counts, appear to
play a role in the association between MPO and CAD status. Heterogeneity in
MPO levels within circulating monocytes48,49
and atheromatous macrophages22 have also been
reported. While monocytes and certain subpopulations of macrophages are the
likely source of MPO in atheroma,10,22,28,50
we used neutrophils in our study as a surrogate because they are easier to
isolate, contain more than 95% of the circulating MPO, and because cell-specific
(ie, monocyte vs neutrophil) differences in MPO regulation have not been reported.
Indeed, little is known about the factors that regulate MPO expression in
vivo, either at the level of bone marrow or within the artery wall. While
a significant correlation was noted between leukocyte-MPO and the absolute
neutrophil count, the mechanism for this interaction is unknown. In our study,
leukocyte- and blood-MPO levels were stable within an individual over time,
with no significant differences in the specific activity (ie, units per milligram
of MPO protein) of MPO within leukocytes isolated from CAD patients and controls
(P = .92). Also, quantification of MPO through either
functional assays (eg, peroxidase activity) or by mass (eg, ELISA) yielded
consistent findings.
The association between MPO levels and CAD was apparent despite increased
use of lipid-lowering drugs, aspirin, and other cardiovascular agents (β-blockers,
calcium channel blockers, angiotensin-converting enzyme inhibitors) in the
CAD group. These medications did not appear to alter MPO levels as there were
no significant differences in leukocyte- or blood-MPO levels in controls taking
vs not taking each of these agents (P>.27 for all
comparisons). Moreover, leukocyte- and blood-MPO remained significant predictors
of CAD status for patients taking vs not taking each medication class. For
example, the adjusted OR for leukocyte-MPO (model 2) considering only patients
not taking lipid-lowering medications (middle vs low quartile: OR, 5.0; P = .006; high vs low quartile: OR, 15.4; P<.001) were similar to the overall ORs (Table 2). Similar results were seen for blood-MPO. In addition,
we examined the possible effects of aspirin intake on MPO levels by monitoring
MPO indices in 12 healthy men at baseline and following 2 weeks of aspirin
use (325 mg/d). We found no differences in leukocyte-MPO or blood-MPO levels
at baseline vs 2 weeks.
In summary, MPO levels are associated with the presence of angiographically
proven coronary atherosclerosis. If these findings are confirmed in other
populations and prove to be predictive of coronary events, MPO levels may
be helpful to identify patients with CAD who might otherwise not be identified
by routine screening methods. Further evaluation of MPO as a predictor of
future cardiac events in longitudinal studies and of MPO as a potential therapeutic
target for CAD appear warranted.
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