ORIGINAL CONTRIBUTION
Relationship of Paraoxonase 1 (PON1)
Gene Polymorphisms and Functional Activity
With Systemic Oxidative Stress
and Cardiovascular Risk
Tamali Bhattacharyya, MD, MS
Stephen J. Nicholls, MBBS, PhD
Eric J. Topol, MD
Renliang Zhang, MD, PhD
Xia Yang, PhD
David Schmitt, BA
Xiaoming Fu, MS
Mingyuan Shao, MS
Danielle M. Brennan, MS
Stephen G. Ellis, MD
Marie-Luise Brennan, PhD
Hooman Allayee, PhD
Aldons J. Lusis, PhD
Stanley L. Hazen, MD, PhD
S
YSTEMIC OXIDATIVE STRESS PRO-
motes a number of key events in
the development of atherosclerosis, and the search to identify
effective therapeutic antioxidant strategies is of potential clinical interest. For
example, in addition to promoting reverse cholesterol transport, highdensity lipoprotein (HDL) is also reported to promote systemic antiinflammatory and antioxidant effects.
The HDL associated esterase/lactonase
paraoxonase 1 (PON1) is implicated in
contributing to the anti-inflammatory
and antioxidant activities of the lipoprotein,1 although direct demonstration of
this in humans is lacking.
Several lines of evidence suggest that
PON1 promotes antioxidant and atheroprotective effects.2,3 PON1 inhibits
oxidation of low-density lipoprotein
(LDL) in vitro.2,4 Genetic deletion of
PON1 is associated with increased sus-
Context Paraoxonase 1 (PON1) is reported to have antioxidant and cardioprotective properties. The relationship between PON1 genotypes and functional activity with
systemic measures of oxidative stress and cardiovascular disease (CVD) risk in humans has not been systematically investigated.
Objective To investigate the relationship of genetic and biochemical determinants of
PON1 activity with systemic measures of oxidative stress and CVD risk in humans.
Design, Setting, and Participants The association between systemic PON1 activity measures and a functional polymorphism (Q192R) resulting in high PON1 activity with prevalent CVD and future major adverse cardiac events (myocardial infarction, stroke, or death) was evaluated in 1399 sequential consenting patients undergoing
diagnostic coronary angiography between September 2002 and November 2003 at
the Cleveland Clinic. Patients were followed up until December 2006. Systemic levels
of multiple structurally defined fatty acid oxidation products were also measured by
mass spectrometry in 150 age-, sex-, and race-matched patients and compared with
regard to PON1 genotype and activity.
Main Outcome Measures Relationship between a functional PON1 polymorphism
and PON1 activity with global indices of systemic oxidative stress and risk of CVD.
Results The PON1 genotype demonstrated significant dose-dependent associations
(QQ192⬎QR192⬎RR192) with decreased levels of serum PON1 activity and with increased levels of systemic indices of oxidative stress. Compared with participants with
either the PON1 RR192 or QR192 genotype, participants with the QQ192 genotype
demonstrated an increased risk of all-cause mortality (43/681 deaths [6.75%] in RR192
and QR192 and 62/584 deaths [11.1%] in QQ192; adjusted hazard ratio, 2.05; 95%
confidence interval [CI], 1.32-3.18) and of major adverse cardiac events (88/681 events
[13.6%] in RR192 and QR192 and 102/584 events [18.0%] in QQ192; adjusted hazard ratio, 1.48; 95% CI, 1.09-2.03; P=.01). The incidence of major adverse cardiac events
was significantly lower in participants in the highest PON1 activity quartile (23/315 [7.3%])
and 235/324 [7.7%] for paraoxonase and arylesterase, respectively) compared with those
in the lowest activity quartile (78/311 [25.1%] and 75/319 [23.5%]; P⬍.001 for paraoxonase and arylesterase, respectively). The adjusted hazard ratios for major adverse
cardiac events between the highest and lowest PON1 activity quartiles were, for paraoxonase, 3.4 (95% CI, 2.1-5.5; P⬍.001) and for arylesterase, 2.9 (95% CI, 1.8-4.7;
P⬍.001) and remained independent in multivariate analysis.
Conclusion This study provides direct evidence for a mechanistic link between genetic
determinants and activity of PON1 with systemic oxidative stress and prospective cardiovascular risk, indicating a potential mechanism for the atheroprotective function of PON1.
www.jama.com
JAMA. 2008;299(11):1265-1276
ceptibility of LDL to oxidation ex vivo,
increased measures of macrophage oxidative stress, and increased lesion size
in animal models of atherosclerosis;
©2008 American Medical Association. All rights reserved.
Author Affiliations are listed at the end of this article.
Corresponding Author: Stanley L. Hazen, MD, PhD,
Center for Cardiovascular Diagnostics and Prevention, Cleveland Clinic, 9500 Euclid Ave, NE-10, Cleveland, OH 44195 ([email protected]).
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RELATIONSHIP OF PARAOXONASE 1 WITH SYSTEMIC OXIDATIVE STRESS AND CVD RISK
conversely, overexpression of the human PON1 transgene in mice results in
reduced aortic lesion size and corresponding decreases in epitopes recognized by antibodies specific for oxidized lipid-protein adducts.5-8
Despite evidence that PON1 prevents atherosclerosis in animal models, it remains to be established whether
PON1 possesses atheroprotective and
antioxidant properties in humans. Several studies have suggested that PON1
may play an atheroprotective role but the
simultaneous associations between
PON1 polymorphisms and enzyme activity with cardiovascular disease (CVD)
risk have been reported in only some
population studies.1-3,9-13 Further confounding the relationship in humans has
been the discovery of multiple PON1
polymorphisms in both the coding region of the protein and the promoter,
some of which reportedly influence overall systemic activity levels.14
In a recent meta-analysis of 43 studies examining multiple single-nucleotide polymorphisms (SNPs) for PON1,
the most promising of the genotypes was
the Q192R variant, although this SNP
demonstrated only a weak overall association with coronary heart disease of uncertain relevance.15 However, each of the
contributing studies in the metaanalyses involved small cohorts of patients, with variable evaluation of functional activity and no quantitative
examination of systemic indices of oxidative stress. The major conclusions of
the meta-analyses therefore emphasized the need for both much larger and
mechanistic investigations of the role of
PON1 in human CVD, particularly since
no significant association for the Q192R
polymorphism was noted among the
larger studies in the meta-analyses, and
accompanying simultaneous functional and genetic studies were typically lacking.15 Thus, it remains to be established whether genetic and
biochemical determinants of PON1 are
linked to oxidative stress and CVD risk
in humans.
The Q192R polymorphism involves a
mutation from glutamine (Q, wild type)
to arginine (R, variant) at amino acid po-
sition 192 of the protein sequence. Functional PON1 activity can be measured by
its ability to hydrolyze exogenous substrates such as paraoxon and phenylacetate, reflecting so-called paraoxonase
and arylesterase activity, respectively.
Functional differences have been observed in hydrolysis rates of the Q192 vs
R192 alloenzymes using paraoxon as
substrate, although no difference has
been reported regarding their ability to
hydrolyze phenylacetate.16
In this present large, prospective
clinical study, we report a comprehensive in vivo investigation of a mechanistic link between the functional PON1
Q192R polymorphism, serum PON1
activity using dual enzyme activity measurements to account for the differential rate of hydrolysis of the alloenzymes, multiple indices of systemic
oxidative stress, and risk of both prevalent atherosclerotic CVD and nearterm incident major adverse cardiovascular events (myocardial infarction
[MI], stroke, and death).
METHODS
Study Design
and Sample Collection
PON1 activity and functional polymorphisms were determined in serum and
DNA samples of 1399 sequential consenting patients who participated in the
GeneBank study between September
2002 and November 2003. GeneBank
is a single-site (Cleveland Clinic, Cleveland, Ohio) sample repository generated from patients undergoing elective diagnostic coronary angiography
with extensive clinical and laboratory
characterization and longitudinal
observation.
For systemic measures of oxidative
stress, whole blood collected in EDTA
tubes was immediately spun, plasma
and buffy coat were isolated, and plasma
was stored under argon atmosphere
with antioxidant cocktail supplement,
as previously described.17 Serum for
PON1 activity measures was obtained
from serum separator tubes after 30 to
60 minutes of clotting time at room
temperature. All specimens were stored
at −80°C until time of analysis. Pa-
1266 JAMA, March 19, 2008—Vol 299, No. 11 (Reprinted)
tients were followed up on an annual
basis for adjudicated incident major adverse cardiac events and mortality until December 14, 2006. The GeneBank
study was approved by the institutional review board of the Cleveland
Clinic. All patients provided written informed consent prior to being enrolled in the study.
Clinical Diagnosis
and Definition of Outcomes
Information regarding demographics,
medical history, and medication use
was obtained by patient interview and
confirmed by chart review. Race information used in analyses was prespecified prior to the study and was based
on self-report. All clinical outcomes data
were verified by source documentation. Mortality was assessed using the
Social Security Death Index.18
Cardiovascular disease was defined
by the presence of coronary artery disease or peripheral arterial disease. Coronary artery disease included adjudicated diagnoses of stable or unstable
angina, MI (adjudicated definition
based on defined electrocardiographic
changes or elevated cardiac enzymes),
or angiographic evidence of at least 50%
stenosis of 1 or more epicardial vessels. Peripheral artery disease was defined as the presence of any extracoronary atherosclerosis and included
obstructive disease (including a history of intermittent claudication), amaurosis fugax, history of cerebrovascular accident, or evidence of either
arterial stenosis or aneurysmal disease
in the thoracic limbs or abdominal aorta
on Doppler ultrasound.
Prospective cardiovascular risk was
assessed by the incidence of major adverse cardiovascular events (MACE),
which included nonfatal and fatal MI,
nonfatal and fatal stroke, and allcause mortality. Nonfatal events were
defined as MI or stroke in patients who
survived at least for 48 hours following the onset of symptoms. We also assessed the risk of true incidence or the
first cardiovascular event in participants without a history of CVD at enrollment (baseline) and the risk for re-
©2008 American Medical Association. All rights reserved.
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RELATIONSHIP OF PARAOXONASE 1 WITH SYSTEMIC OXIDATIVE STRESS AND CVD RISK
current cardiovascular events in
participants with an established diagnosis of CVD at baseline according to
levels of PON1 activity.
Determination of PON1 Activity
Serum arylesterase and paraoxonase activities were independently measured by
UV spectrophotometry in a 96-well plate
format (Spectramax 384 Plus, Molecular Devices, Sunnyvale, California) using
phenyl acetate or paraoxon (SigmaAldrich, St Louis, Missouri) as substrates, respectively.19 Briefly, for arylesterase assays, initial hydrolysis rates were
determined at 270 nm in 50-fold diluted serum (final) in reactions mixtures composed of 3.4mM phenylacetate, 9mM Tris hydrocholoride, pH 8,
and 0.9mM calcium chloride at 24°C. An
extinction coefficient (at 270 nm) of
1310M−1 · cm−1 was used for calculating units of arylesterase activity, which
are expressed as the amount of phenyl
acetate hydrolyzed in micromoles per
minute per milliliter of serum.
For paraoxonase activity assays, rate
of generation of para-nitrophenol was
determined at 405 nm in 40-fold diluted serum (final) in reaction mixtures composed of 1.5mM paraoxon,
10mM Tris hydrocholoride, pH 8, 1M
sodium chloride, and 2mM calcium
chloride at 24°C. An extinction coefficient (at 405 nm) of 17 000 M−1 · cm−1
was used for calculating units of paraoxonase activity, which are expressed
as the amount of para-nitrophenol produced in nanomoles per minute per
milliliter of serum.
Paraoxonase and arylesterase assays
for each sample were performed in duplicates, with average measurements of
enzyme activity for each sample calculated. Each 96-well plate included blank
samples to monitor spontaneous hydrolysis of substrates and aliquots of serum
samples of 3 pooled calibrators (low, mid,
and high levels) with known activity levels to ensure assay quality of each plate
based on established acceptability criterion. The intra-assay and interassay coefficients of variance for performance of
arylesterase were 1.2% and 3.9%, respectively, and the intra-assay and interas-
say coefficients of variance for performance of paraoxonase activity assays
were 2.0% and 5.6%, respectively, on 20
replicates performed on 10 different days.
To establish normal ranges, serum arylesterase and paraoxonase activities
were also determined on 100 apparently healthy volunteers (50 men and
50 women) aged 55 years or older
(mean, 64 [SD, 4] years) responding to
local advertisements. Systemic arylesterase activity in this healthy, middleaged population ranged from 169.3 to
814.0 µmol/min/mL, with median levels of 605.8 µmol/min/mL (interquartile range [IQR], 517.6-666.8 µmol/
min/mL) of serum. Systemic
paraoxonase activity in this healthy,
middle-aged population ranged from
436.4 to 4025 nmol/min/mL, with median levels of 1264 nmol/min/mL (IQR,
647.0-1865 nmol/min/mL) of serum.
Genotyping
Of the 1399 participants, 1386 DNA
samples from the GeneBank cohort were
available for genotyping for the PON1
Q192R polymorphism (SNP rs662).
Primers for amplifying the sequences
containing the SNP were designed using
Primer 3 (http://www.genome.wi.mit
.edu/cgi-bin/primer/primer3_www
.cgi). Sense and antisense probes used for
fluorescence polarization–single base extension detection of the SNP were designed using Primer PREMIER (PREMIER Biosoft International, Palo Alto,
California). Each genotyping reaction
consisted of a single-plex polymerase
chain reaction followed by a single basepair extension reaction using either the
sense or antisense probe and dideoxynucleotides labeled with TAMRA or
R110 for the alternative alleles. The fluorescence signal was detected by fluorescence polarization using the analyst HT
(Molecular Devices) and genotypes were
determined based on the plot of TAMRA
vs R110 signal values.
Mass Spectrometry Assays
Plasma levels of structurally specific
species of hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), the 8-isopros-
©2008 American Medical Association. All rights reserved.
tane prostaglandin F2␣ (8-isoPGF2␣),
and their precursor fatty acids (arachidonic or linoleic acids) were quantified in samples of participants using
stable isotope dilution high-performance liquid chromatography with online electrospray ionization tandem
mass spectrometry. Prostaglandin F2␣-d4
(Cayman Chemicals, Ann Arbor, Michigan) was used as internal standard for
calibration of 8-isoPGF 2␣ and 15HETE-d8 (Cayman Chemicals) for other
oxidized fatty acids. Quantification of
total plasma levels of each analyte (free
plus esterified) were performed following addition of the appropriate isotopically labeled internal standards and
base-catalyzed hydrolysis, as previously described.17
Analyses were performed on plasma
specimens collected from 50 age-, sex-,
and race-matched participants of each
genotype (QQ192, QR192, and RR192)
who were randomly selected from
among the 1386 patients. To avoid bias,
50 participants with the genotype of the
lowest frequency in our cohort (RR192)
were initially randomly selected among
the 136 participants who carried that
genotype (50% male; 100% white; mean
age, 63 [SD, 2] years). An additional 50
participants from each of the QQ192
and QR192 cohorts, matched with the
QQ192 participants for age, sex, and
race, were also selected for mass
spectrometry analyses.
Statistical Analyses
Clinical diagnosis, outcome definition,
determination of PON1 activity, genotyping, and mass spectrometry analyses were each performed by investigators who were blinded to CVD status and
other measurements. Continuous variables are presented as mean (SD) or median (IQR) for non–normally distributed data and categorical variables as
numbers and percentages. Regression
analysis was used for calculating the variance (R2) explained by the PON1 polymorphism and activity measures. Quartile cut points were determined from the
enzyme activity levels of all 1399 study
participants at baseline. Analysis of variance or the Kruskal-Wallis test (for non–
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1267
RELATIONSHIP OF PARAOXONASE 1 WITH SYSTEMIC OXIDATIVE STRESS AND CVD RISK
normally distributed data) was used to
test the difference in mean oxidized fatty
acid levels according to PON1 genotype and tertiles of PON1 activity.
Kaplan-Meier methods were used to plot
time-to-event curves for PON1 activity
quartiles and genotypes and the logrank test was performed to assess differences between curves.
Logistic regression analysis was used
to calculate adjusted odds ratios (ORs)
for the association between activity levels and prevalent CVD status after adjusting for the traditional risk factors
and selected classes of medications
(statins and aspirin) as described herein.
Cox proportional hazard models
were performed to determine if PON1
is an independent predictor of future
cardiac events. Unadjusted hazard ratios (HRs) for clinical events were calculated with reference to the highest
quartile (corresponding to lowest risk).
The models were adjusted for all traditional cardiac risk factors, including
the Framingham ATP-III risk score (including diabetes status), log Creactive protein, body mass index, and
medication use (statins and aspirin).
Models were created separately for paraoxonase and arylesterase. All variables used in the models met the proportional hazards assumption by testing
them as time-dependent covariates in
the multivariate model.
Receiver operating characteristic
curves were plotted to estimate the C index for MACE with and without PON1
activity as predictors.20 The C index is
analogous to the area under the receiver operating characteristic curve but
takes into account right censoring.21 To
evaluate the contribution of PON1 activity as a predictive marker, we calculated the concordance indices22 with and
without each PON1 activity measurement in separate multivariate models that
included the variables described herein.
The improvement in predictability with
the addition of each of these wellestablished risk factors was assessed by
the difference in the concordance indices.23 The difference of the indices was
bias-corrected and bootstrapping was
used to generate 95% confidence inter-
vals (CIs). A 1-sample t test was performed to determine if the difference was
equal to zero.
Hazard ratios for clinical events were
calculated for participants with the
PON1 Q192R, polymorphism and were
adjusted after controlling for differences in the aforementioned traditional cardiac risk factors and medications. An additive model of inheritance
was also created to assess the change
in risk for having 1 and 2 copies of the
Q allele. The genotypes were given values of 0, 1, and 2 and entered in a Cox
proportional hazards model. All statistical analyses were performed using
SPSS, version 11 (SPSS Inc, Chicago, Illinois) and verified on SAS, version 8.2
(SAS Institute Inc, Cary, North Carolina). All P values are 2-sided, with
P⬍ .05 considered significant.
RESULTS
Clinical, Laboratory,
and Demographic Characteristics
The clinical characteristics of participants stratified according to a diagnosis of CVD at time of enrollment are
summarized in TABLE 1. Participants
with CVD were older, with a greater
prevalence of history of hyperlipidemia, diabetes, and hypertension and
greater use of aspirin and statins. While
66% of patients were taking statins, less
than 10% of the cohort were taking fenofibrates. Participants with CVD had
lower levels of HDL cholesterol
(HDL-C) and LDL cholesterol (LDL-C)
(the latter presumably because of increased statin use) and higher levels of
triglycerides and C-reactive protein.
In the entire cohort, 46.3% (642/1386)
had the QQ192 genotype, 43.9% (608/
1386)hadtheQR192genotype,and9.8%
(136/1386) had the RR192 genotype.
Whiteparticipantsdemonstratedagreater
frequency of the QQ192 genotype (621/
1254 [49.5%] vs 17/126 [13.5%];
P⬍.001) and lower frequency of the
RR192 genotype (92/1254 [7.4%] vs 44/
126 [34.9%]; P⬍.001) compared with
nonwhite participants.
Irrespective of race, paraoxonase and
arylesterase activity levels were significantly lower in participants with CVD in
1268 JAMA, March 19, 2008—Vol 299, No. 11 (Reprinted)
comparison with non-CVD patients: for
paraoxonase, the median was 860.6
(IQR, 442.6-1599) nmol/min/mL vs
1164 (IQR, 473.5-1825) nmol/min/mL
(P⬍.001), and for arylesterase, the median was 334.6 (IQR, 279.5-395.8) µmol/
min/mL vs 356.9 (IQR, 296-424.4) µmol/
min/mL (P⬍.001) (Table 1). Similarly,
systemic levels of serum paraoxonase and
arylesterase activity were lower in participants with CVD compared with
healthy middle-aged volunteers: for paraoxonase, median, 860.6 (IQR, 442.61599) nmol/min/mL vs 1264 (IQR,
647.0-1864.6) nmol/min/mL (P⬍.001),
and for arylesterase, 334.6 (IQR, 279.5395.8) µmol/min/mL vs 605.8 (IQR,
517.6-666.8) µmol/min/mL (P⬍.001).
During follow-up (mean, 44 [SD, 7]
months; minimum of 3 years), 13.8%
of participants (193/1399) experienced at least 1 MACE (death, 7.6%
[106/1399]; nonfatal MI, 5.7% [80/
1399]; and stroke, 1.1% [16/1399]).
PON1 Genotype
and Paraoxonase Activity
The relationships between PON1
Q192R genotypes and activity measures throughout the entire population are summarized in (TABLE 2). The
PON1 Q192R polymorphism genotypes were in Hardy-Weinberg equilibrium. Participants with the QQ192
genotype demonstrated significantly
less paraoxonase activity than participants with either the QR192 genotype
(median, 453.5 [IQR, 360.4-560.3]
nmol/min/mL vs 1436 [IQR, 11291800] nmol/min/mL; P ⬍.001) or the
RR192 genotype (median, 453.5 [IQR,
360.4-560.3] nmol/min/mL vs 2374
[IQR, 1978-2851] nmol/min/mL;
P ⬍.001) (Table 2).
While 49.4% of participants with the
wild-type QQ192 genotype (317/642)
were in the lowest quartile of PON1 activity, only 4.4% of participants with the
variant RR192 genotype (6/136) were
in the lowest activity quartile. In contrast, 3.9% of participants with the
QQ192 genotype (25/642) and 83.8%
with the RR192 genotype (114/136)
were in the highest activity quartile
(Table 2).
©2008 American Medical Association. All rights reserved.
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RELATIONSHIP OF PARAOXONASE 1 WITH SYSTEMIC OXIDATIVE STRESS AND CVD RISK
In the lowest activity quartile, 92.2%
of study participants (317/344) had the
QQ192 genotype and only 1.7% (6/
344) had the RR192 genotype. The reverse trend was observed in the highest activity quartile, with 7.2% of
participants (25/348) having the
QQ192 genotype and 32.8% (114/
348) having the RR192 genotype
(TABLE 3). Regression analysis confirmed that the PON1 Q192R polymorphism accounted for 58.5% (R2 =0.585;
P⬍.001) of the variation in serum paraoxonase activity levels throughout the
population.
PON1 Genotype, Paraoxonase
Activity, and Systemic
Oxidative Stress
Table 1. Baseline Characteristics and Prospective Events Among Participants Undergoing
Elective Diagnostic Cardiac Catheterization (N = 1399) a
With CVD
at Baseline
(n = 1116)
Characteristics
Race
White
P
Value
65.1 (10.9)
57.2 (11.8)
⬍.001
799/1116 (71.6)
135/283 (47.7)
⬍.001
1023/1111 (92.1)
243/282 (86.2)
.002
71/1111 (6.4)
38/282 (13.5)
⬍.001
Age, mean (SD), y
Male
Without CVD
at Baseline
(n = 283)
African American
Hyperlipidemia b
940/1093 (86.0)
168/277 (60.6)
⬍.001
Hypertension
843/1100 (76.6)
150/282 (53.2)
⬍.001
Diabetes mellitus c
433/1095 (39.5)
49/271 (18.1)
⬍.001
Current smoking
163/1116 (14.6)
33/283 (11.7)
.20
Statin use
717/1088 (65.9)
80/270 (29.6)
⬍.001
Aspirin use
873/1098 (79.5)
176/272 (64.7)
⬍.001
Body mass index d
Blood pressure, mean (SD), mm Hg
Systolic
Diastolic
29.7 (5.9)
29.7 (6.3)
.58
134.3 (21.2)
134.8 (21.3)
.96
74.0 (13.2)
75.5 (12.2)
.20
46.1 (13.0)
53.9 (15.9)
⬍.001
Plasma levels of multiple structurally
specific oxidized fatty acids were quantified in participants and analyzed for
their relationships with the PON1 genotype (TABLE 4) and paraoxonase activity (Table 4). Participants with the
RR192 genotype had lower levels of all
measured systemic indices of oxidative stress compared with age-, sex-, and
race-matched participants possessing
the QQ192 genotype (P ⬍.001 for all
comparisons). Consistent with these
findings, serum paraoxonase activity
levels were inversely correlated with
multiple direct systemic indices of oxidative stress in a dose-dependent fashion (Table 4). Collectively, these results provide both genetic and
biochemical support for the notion that
the PON1 Q192R variant strongly influences quantitative measures of systemic oxidative stress in humans.
Prospective event rates, No.
(Kaplan-Meier %) [95% CI]
MI
Association of the PON1 Q192R
Polymorphism With Prevalent CVD
and CVD Outcomes
Table 2. Distribution of Paraoxonase Activity Quartiles in Each PON1 Q192R Genotype a
An increased prevalence of coronary artery disease was observed in participants
withthePON1QQ192genotype(461/962
[47.9%] with vs 169/405 [41.7%] without coronary artery disease; P=.04). In
contrast, participants with the RR192
genotype showed the opposite tendency
with lower prevalence of coronary artery
disease (TABLE 5). Those with the PON1
QQ192genotypeweresimilarlyobserved
HDL-C, mean (SD), mg/dL
112.4 (36.6)
⬍.001
Triglycerides, median (IQR), mg/dL
138.0 (99.0-202.0)
115.5 (77.0-173.0)
⬍.001 e
Total cholesterol, mean (SD), mg/dL
178.4 (46.4)
193.9 (43.5)
⬍.001
LDL-C, mean (SD), mg/dL
99.0 (37.5)
C-reactive protein, median (IQR), mg/L
Framingham ATP-III score, mean (SD)
3.0 (1.5-7.0)
.005 e
2.4 (1.2-5.9)
13.3 (3.5)
⬍.001
11.4 (5.0)
Paraoxonase, median (IQR),
nmol/min/mL
860.6 (442.6-1599.0)
1164.0 (473.5-1825.0)
Arylesterase, median (IQR),
µmol/min/mL
334.6 (279.5-395.8)
356.9 (296.0-424.4)
.001 e
⬍.001
69 (7.2) [5.6-8.9]
11 (4.4) [1.8-6.9]
MI/CVA
84 (8.9) [7.1-10.7]
12 (4.7) [2.1-7.4]
.14
.05
Death
99 (10.3) [8.3-12.2]
7 (2.9) [0.8-5.0]
⬍.001
MACE
175 (17.9) [15.5-20.3]
18 (7.2) [4.0-10.4] ⬍.001
Abbreviations: CI, confidence interval; CVA, cerebrovascular accident; CVD, cardiovascular disease; HDL-C, highdensity lipoprotein cholesterol; IQR, interquartile range; MACE, major adverse cardiac events (ie, myocardial infarction, stroke, or death); MI, myocardial infarction; LDL-C, low-density lipoprotein cholesterol.
SI conversions: To convert HDL-C, LDL-C, and total cholesterol to mmol/L, multiply by 0.0259; to convert triglycerides
to mmol/L, multiply by 0.0113; to convert C-reactive protein to nmol/L, multiply by 9.524.
a Data are presented as No./total (%) unless otherwise indicated.
b Hyperlipidemia was defined as a total cholesterol level of higher than 200 mg/dL or treatment with an antihyperlipidemic
agent.
c Diabetes mellitus was defined as a fasting plasma glucose level of at least 125 mg/dL or treatment with hypoglycemic agents.
d Calculated as weight in kilograms divided by height in meters squared.
e By Wilcoxon rank-sum test.
PON1 Q192R Genotype
Paraoxonase Quartile,
nmol/min/mL
Median (IQR)
QQ192
(n = 642)
QR192
(n = 608)
RR192
(n = 136)
453.5 (360.4-560.3)
1436 (1129-1800)
2374 (1978-2851)
Quartile 4 (⬎1640)
25 (3.9)
209 (34.4)
114 (83.8)
Quartile 3 (1640-899.1)
14 (2.2)
321 (52.8)
14 (10.3)
Quartile 2 (899-450)
286 (44.5)
57 (9.4)
2 (1.5)
Quartile 1 (⬍450)
317 (49.4)
21 (3.5)
6 (4.4)
Abbreviations: CVD, cardiovascular disease; IQR, interquartile range; PON1, paraoxonase 1 gene.
a Data are presented as No. (%) unless otherwise indicated. Genotypes: QQ192, wild type; QR192, heterozygous; RR192,
mutant homozygous.
©2008 American Medical Association. All rights reserved.
(Reprinted) JAMA, March 19, 2008—Vol 299, No. 11
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1269
RELATIONSHIP OF PARAOXONASE 1 WITH SYSTEMIC OXIDATIVE STRESS AND CVD RISK
to have an increased likelihood of having
a history of coronary artery bypass graft
surgery (P=.03) and a tendency toward
increased history of percutaneous coronary intervention (P=.09) (data not
shown). In contrast, those with the PON1
RR192 genotype demonstrated opposite
tendencies (diminished history of revascularization;P=.09)(datanotshown).IndividualscarryingthePON1QR192genotype showed no observed differences in
prevalent peripheral artery disease within
the cohort (Table 5).
Of the 1386 participants with genotype information, follow-up data were
available in 1265 participants (QQ192
genotype, n = 584; QR192 genotype, n = 563; and RR192 genotype,
n = 118). The PON1 functional polymorphism Q192R was significantly
associated with CVD outcomes within
the cohort. For example, all adverse
cardiovascular outcomes monitored
were observed to a significantly lesser
degree in patients who carried 1 R
allele.
Kaplan-Meier estimates of all-cause
mortality and MACE revealed a significant trend in our study participants. Homozygous and heterozygous participants (RR192 and QR192 genotypes)
had significantly lower event rates for allcause mortality (43/681 [Kaplan-Meier
estimate, 6.75%]) compared with participants with the QQ192 genotype (62/
584 [Kaplan-Meier estimate, 11.10%];
P = .006). Similarly, the event rate for
MACE over the ensuing 3-year period
was lower with the RR192 and QR192
genotypes (88/681 [Kaplan-Meier estimate, 13.6%]) vs the QQ192 genotype
(102/584 [Kaplan-Meier estimate,
18.0%]; P=.03) (TABLE 6 and FIGURE).
In a multivariate model including
age, Framingham ATP-III risk score,
race, log C-reactive protein, body mass
index, and medication (statin and aspirin) use, the addition of the risk allele Q192 showed no association with
incident MI and stroke over the ensuing 3 years following enrollment but
was significantly associated with in-
creased likelihood of death and MACE
(Table 6). Compared with participants with either the RR192 or QR192
genotype, participants with the QQ192
genotype demonstrated an adjusted HR
of 2.05 (95% CI, 1.32-3.18) for allcause mortality (P = .001). Furthermore, MACE were also more likely to
occur over the ensuing 3-year period
in participants with the QQ192 genotype compared with participants with
either the RR192 or QR192 genotype
(adjusted HR, 1.48; 95% CI, 1.092.03; P=.01) (Table 6).
In separate analyses, an additive
model of inheritance was created to assess the change in risk for having a copy
of the Q allele. An adjusted HR for
3-year all-cause mortality of 1.58 (95%
CI, 1.12-2.24; P=.01) was observed for
having a Q allele. Furthermore, in this
model the presence of a Q allele was also
associated with an increased risk of having a MACE over the ensuing 3-year period (HR, 1.32; 95% CI, 1.04-1.69;
P =.03).
Table 3. Distribution of PON1 Q192R Genotypes in Each Paraoxonase Activity Quartile a
Paraoxonase Quartile, nmol/min/mL
PON1 Q192R Genotype
QQ192
QR192
RR192
Quartile 4 (⬎1640)
(n = 348)
Quartile 3
(1640-899.1)
(n = 349)
25 (7.2)
209 (60.1)
114 (32.8)
14 (4.0)
312 (92.0)
14 (4.1)
Quartile 2
(899-450)
(n = 345)
286 (82.9)
57 (16.5)
2 (0.6)
Quartile 1 (⬍450)
(n = 344)
317 (92.2)
21 (6.1)
6 (1.7)
Abbreviations: CVD, cardiovascular disease; IQR, interquartile range; PON1, paraoxonase 1 gene.
a Data are presented as No./total (%). Genotypes: QQ192, wild type; QR192, heterozygous; RR192, mutant homozygous.
Table 4. Total Plasma Oxidized Fatty Acid Levels According to PON1 Q192R Genotype and Tertile of Serum Paraoxonase Activity a
PON1 Q192R Genotype
Oxidized Fatty Acid
5-HETE
8-HETE
9-HETE
11-HETE
12-HETE
15-HETE
9-HODE
13-HODE
8-isoPGF2␣
QQ192
(n = 50)
QR192
(n = 50)
Paraoxonase Tertile
RR192
(n = 50)
20.1 (14.3-24.9) 15.5 (12.6-19.2) 9.1 (7.6-12.8)
3.0 (2.5-4.4)
2.5 (1.8-2.7)
1.5 (1.1-2.1)
42.7 (30.3-63.1) 30.1 (22.0-35.2) 17.2 (15.1-25.3)
5.8 (4.8-7.1)
7.6 (5.9-10.4)
24.3 (20.6-30.8)
35.5 (30.2-43.7)
38.7 (30.4-49.0)
11.1 (4.4-15.6)
4.5 (3.7-6.1)
6.1 (4.9-8.1)
3.7 (3.3-5.1)
4.4 (3.3-6.4)
20.2 (16.2-26.3) 13.1 (10.5-19.5)
28.8 (22.6-37.1) 20.3 (15.5-27.9)
29.6 (23.0-38.9) 22.2 (17.6-28.3)
11.5 (3.0-28.5)
2.8 (1.1-6.7)
P
Value
Tertile 1 (⬍630) Tertile 2 (630.1-1928) Tertile 3 (⬎1928)
(n = 50)
(n = 50)
(n = 50)
P
Value
⬍.001
⬍.001
⬍.001
21.8 (16.0-25.3)
3.3 (2.6-4.6)
45.2 (35.2-66.3)
16.1 (13.1-20.7)
2.5 (2.2-2.7)
30.4 (25.3-37.5)
8.4 (6.0-9.7)
1.2 (0.9-1.7)
15.8 (11.5-17.9)
⬍.001
⬍.001
⬍.001
⬍.001
⬍.001
5.9 (5.0-8.2)
7.8 (6.2-11.3)
4.6 (4.1-6.3)
6.4 (5.4-8.7)
3.6 (2.5-4.1)
3.8 (2.7-4.8)
⬍.001
⬍.001
⬍.001
⬍.001
⬍.001
⬍.001
24.7 (21.2-30.8)
36.3 (30.9-45.7)
39.8 (31.5-52.6)
12.1 (4.5-28.2)
22.0 (17.4-30.7)
33.0 (24.8-38.8)
30.5 (25.5-43.4)
14.2 (7.8-29.4)
11.3 (8.4-14.8)
18.3 (10.1-23.1)
19.3 (10.1-26.0)
1.6 (0.1-5.1)
⬍.001
⬍.001
⬍.001
⬍.001
Abbreviations: HETE, hydroxyeicosatetraenoic acid; HODE, hydroxyloctadecadienoic acid; PON1, paraoxonase 1 gene; QQ192, wild type subjects; QR192, heterozygous subjects;
RR192, mutant homozygous subjects; 8-iso PGF2␣, 8-isoprostane prostaglandin F2␣.
a Data are presented as median (interquartile range) of oxidized fatty acid in picomoles per milliliter.
1270 JAMA, March 19, 2008—Vol 299, No. 11 (Reprinted)
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RELATIONSHIP OF PARAOXONASE 1 WITH SYSTEMIC OXIDATIVE STRESS AND CVD RISK
Association of PON1 Activity
With Prevalent CVD
and CVD Outcomes
A higher prevalence of CVD was also
observed in patients with low serum activity levels of either paraoxonase or arylesterase. Following adjustment for differences in known CVD risk factors, this
association was not significant for paraoxonase (OR, 1.5; 95% CI, 0.97-2.2;
P =.07 for comparison between upper
and lower quartiles) but was significant for arylesterase (OR, 2.1; 95% CI,
1.4-3.1; P=.001). Low systemic levels
of enzyme activity were significantly associated with the presence of coronary artery disease (paraoxonase: OR,
1.5; 95% CI, 1.03-2.3; P = .03; arylesterase: OR, 2.0; 95% CI, 1.4-3.1;
P = .001) or in combination with peripheral artery disease (paraoxonase:
OR, 1.4; 95% CI, 0.9-2.0; P = .11; arylesterase: OR, 1.2; 95% CI, 1.0-2.11;
P =.05).
Event rates for all prospective cardiovascular events were significantly
lower in participants in the highest
PON1 activity quartile compared with
participants in the lowest quartile
(P⬍.001 for all prospective events comparisons) (TABLE 7 and Figure). The
frequency of incident cases of nonfatal MI or stroke among participants
within the highest quartiles of PON1
activity was 2.5% (8/315) based on
paraoxonase activity and 2.8% (9/
324) using arylesterase activity.
Higher rates of incident nonfatal MI
and stroke were observed in participants within the lowest quartile of paraoxonase activity (37/311 [11.9%]) and
arylesterase activity (40/319 [12.5%]).
Similarly, lower frequency of 3-year incident all-cause mortality was observed in participants within the highest quartiles of paraoxonase activity (17/
315 [5.4%]) and arylesterase activity
(16/324 [4.9%]) compared with participants within the lowest quartiles of
paraoxonase activity (37/311 [11.9%])
and arylesterase activity (41/319
[12.9%]). Lower event rates were also
noted for MACE among study participants in the highest quartiles of paraoxonase activity (23/315 [7.3%]) and
Table 5. Prevalent Disease by PON1 Q192R Genotype a
Prevalent Disease by
PON1 Q192R Genotype
Coronary artery disease
QQ192
QR192
RR192
Peripheral artery disease
QQ192
QR192
RR192
Disease Present, No. (%)
n = 962
461 (47.9)
410 (42.6)
91 (9.5)
n = 363
171 (47.1)
164 (45.2)
28 (7.7)
Disease Absent, No. (%)
n = 405
169 (41.7)
191 (47.2)
45 (11.1)
n = 1023
471 (46.0)
444 (43.3)
108 (10.6)
P Value
.03
.12
.35
.73
.56
.12
Abbreviation: PON1, paraoxonase 1 gene.
a QQ , wild type; QR , heterozygotes; RR , mutant homozygous. Of the 1399 participants, 1386 had genotype
192
192
192
information and 1367 had information on history of coronary artery disease.
arylesterase activity (25/324 [7.7%])
compared with event rates for MACE
among study participants in the lowest quartiles of paraoxonase activity (78/
311 [25.1%]) and arylesterase activity
(75/319 [23.5%]) (Table 7 and Figure).
Following multivariate analysis, serum PON1 activity measures remained independently associated with
prospective risk of cardiac events
(Table 7). The lowest quartiles of both
paraoxonase and arylesterase activity
were associated with a greater incident risk of nonfatal MI or stroke (paraoxonase: HR, 4.4; 95% CI, 2.0-9.6;
P⬍.001; arylesterase: HR, 4.5; 95% CI,
2.2-9.4; P⬍.001) during the 3-year follow-up interval. The risk of all-cause
mortality was also greatest in participants in the lowest quartiles of either
paraoxonase activity or arylesterase activity (paraoxonase: HR, 2.4; 95% CI,
1.3-4.4; P =.004; arylesterase: HR, 2.2;
95% CI, 1.2-4.2; P = .01). This translated to a greater incidence of MACE
in participants in the lowest quartiles
of paraoxonase and arylesterase activity (paraoxonase: HR, 3.4; 95% CI, 2.15.5; P⬍.001; arylesterase: HR, 2.9; 95%
CI, 1.8-4.7; P ⬍.001).
In further analyses, low systemic levels of arylesterase activity were associated with an increased risk of having a
first cardiovascular event (true incidence) among participants without
either a history of CVD or angiographic evidence of significant coronary artery disease (defined as ⱖ50%
stenosis) at baseline (adjusted HR, 5.8;
95% CI, 1.2-28.6; P=.03) (TABLE 8). In-
©2008 American Medical Association. All rights reserved.
creased risk of having a recurrent nonfatal MI or stroke, all-cause mortality,
and MACE was observed with low levels of paraoxonase and arylesterase activity measurements in participants with
an established diagnosis of CVD at enrollment (TABLE 9). The adjusted HR
for recurrent nonfatal MI or stroke was
4.5 (95% CI, 1.9-11.0; P=.001) for paraoxonase and 4.2 (95% CI, 1.9-9.6;
P = .001) for arylesterase between the
highest and lowest activity quartiles.
Participants in the lowest activity quartile for either paraoxonase or arylesterase were more likely to have a recurrent MACE compared with participants
in the highest activity quartile (paraoxonase: HR, 3.4; 95% CI, 2.0-5.9;
P⬍.001; arylesterase: HR, 2.4; 95% CI,
1.4-3.9; P ⬍.001).
Prognostic Value of PON1
The C index for the outcomes of nonfatal MI and stroke during a minimum
3-year follow-up period was 0.59 (with
traditional risk factors as predictors),
and significantly increased to either 0.66
(P=.007) or 0.69 (P=.001) in comparisons with traditional risk factors when
including systemic measures of either
paraoxonase or arylesterase activity, respectively. For the composite MACE
outcome, addition of either paraoxonase or arylesterase activity to traditional risk factors also significantly increased the predictive value of the
model during a mean of 44 (SD, 7)
months. For example, the C index of
0.67 with traditional risk factors alone
increased to 0.71 (P = .007) and 0.70
(Reprinted) JAMA, March 19, 2008—Vol 299, No. 11
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1271
RELATIONSHIP OF PARAOXONASE 1 WITH SYSTEMIC OXIDATIVE STRESS AND CVD RISK
Table 6. Relationship Between PON1 Q192R Genotype and CVD Outcomes
PON1 Q192R Genotypes
CVD Outcomes
RR192 ⫹ QR192
(n = 681)
Nonfatal MI/CVA
No./total (Kaplan-Meier %)
51/681 (7.97)
Adjusted HR (95% CI) a
1 [Reference]
All-cause mortality
No./total (Kaplan-Meier %)
43/681 (6.75)
Adjusted HR (95% CI) a
1 [Reference]
MACE
No./total (Kaplan-Meier %)
88/681 (13.59)
Adjusted HR (95% CI) a
1 [Reference]
QQ192
(n = 584)
43/584 (7.88)
1.01 (0.65-1.57)
62/584 (11.10)
2.05 (1.32-3.18)
102/584 (18.04)
1.48 (1.09-2.03)
P Value
.95
.96
.006
.001
.03
.01
Abbreviations: CI, confidence interval; CVA, cerebrovascular accident; CVD, cardiovascular disease; HR, hazard ratio;
MACE, major adverse cardiac events (ie, myocardial infarction, stroke, or death); MI, myocardial infarction; PON1,
paraoxonase 1 gene.
a Adjusted HRs were calculated for each clinical end point by adding the risk allele Q192 to the multivariate model
that included the Framingham ATP-III risk score of individual participants (including diabetes and smoking status),
race, log C-reactive protein, body mass index, and use of s2tatins and aspirin.
(P=.01) for risk factors plus paraoxonase or arylesterase, respectively.
COMMENT
Oxidative stress is thought to have a
pivotal role in the pathogenesis of a
number of chronic inflammatory disease processes, including atherosclerosis. The failure of ␣-tocopherol
supplementation studies with alleged
antioxidant properties to prevent cardiovascular events has brought the oxidation hypothesis of atherosclerosis into
question.24 Therefore, the search continues to identify effective strategies that
promote systemic antioxidant effects in
humans and to determine whether they
have a beneficial influence on the rate
of CVD.
The present genetic and biochemical studies demonstrate that the HDLassociated protein PON1 promotes pronounced systemic antioxidant effects in
humans, with coincident links to reduction in coronary artery disease
prevalence and prospective risks of
MACE. Our results demonstrate that
both the PON1 Q192R polymorphism
and serum PON1 activity are associated with both prevalent coronary artery disease and incident adverse cardiovascular events.
It has been speculated that PON1 contributes to the atheroprotective property of HDL via promotion of a systemic antioxidant effect.1,25 However,
there has been no definitive in vivo evi-
dence that PON1 promotes systemic antioxidant effects in humans. A recent
meta-analysis of 43 studies examining the
relationship between PON1 and clinical outcomes15 concluded that considerable uncertainty remained, since published studies to date primarily involve
small cohorts of participants. Moreover, they often failed to simultaneously examine PON1 genotype, systemic PON1 activity measures, and
cardiovascular outcomes, and none examined systemic quantitative indices of
oxidative stress.
In this study, we investigated the potential relationship between PON1 activity, PON1 genotype, systemic oxidative stress, and both coronary and
peripheral artery disease in a large, prospective cohort of patients to more fully
interrogate the aforementioned relationships. We demonstrate that the PON1
Q192R polymorphism is functional, resulting in increased enzymatic activity.
In parallel, we demonstrate that elevated systemic levels of multiple structurally distinct fatty acid oxidation products that are increased in both
atherosclerotic plaque and plasma of participants with CVD26,27 are associated
with low systemic levels of PON1 activity and the PON1 QQ192 genotype. Importantly, the plasma samples analyzed
in the present study were collected and
processed under conditions designed to
prevent artificial oxidation of lipids during both storage and analysis. The find-
1272 JAMA, March 19, 2008—Vol 299, No. 11 (Reprinted)
ing that levels of 9-HETE, an isomer of
arachidonic acid oxidation produced exclusively by free radical–mediated processes, are higher in participants with low
levels of paraoxonase activity suggests
that PON1 can influence oxidative events
beyond the cyclooxygenase and lipoxygenase pathways.
While the mechanism(s) for PON1mediated systemic antioxidant effects
remains to be determined, the present
findings strongly support a role for this
HDL-associated protein in modulating
systemic oxidative stress in humans.
Moreover, the present study suggests an
important mechanistic link among
PON1, systemic oxidative stress, and risk
of development of atherosclerotic heart
disease and its acute complications.
In a recent publication, amino acid
position 192 of PON1 was suggested to
participate in HDL binding.28 The Q192
alloenzyme was shown to bind to the
HDL particle with 3-fold lower affinity than the R192 alloenzyme and, consequently, exhibited lower stability, lipolactonase activity, and modulatory
effect on macrophage cholesterol efflux. The findings of the present clinical study complement these results,
demonstrating that individuals with the
arginine (R) mutation at position 192
have higher serum levels of PON1 activity, lower systemic indices of systemic oxidative stress, and corresponding reductions in both prevalent
coronary artery disease and prospective cardiac events.
This is, to our knowledge, the first
large, prospective study that comprehensively examines the genetics and biochemical activity of PON1 using dual enzyme measurements to predict prevalent
disease risks, as well as prospective risk
of MACE, while simultaneously also examining whether a potential mechanism for CVD associations is linked to
systemic oxidative stress measures in humans. Compared with prior studies that
often focused on lower-risk populations,15 the present cohort is also substantially enriched with patients with
both coronary and peripheral artery disease, enabling us to better study the association of the PON1 Q192R variant on
©2008 American Medical Association. All rights reserved.
Downloaded from jama.ama-assn.org at University of California - Los Angeles on January 6, 2012
RELATIONSHIP OF PARAOXONASE 1 WITH SYSTEMIC OXIDATIVE STRESS AND CVD RISK
Figure. Relationship Between Functional Genetic and Biochemical Indices of PON1 Activity and Incident Cardiovascular Risk
MI/CVA
Major Adverse Cardiac Events
Q192R Polymorphism
Q192R Polymorphism
30
Genotype
QQ
RQ
RR
10
25
Events, %
Events, %
15
5
20
15
10
5
Log-rank P = .95
Log-rank P = .02
0
0
0
183
365
549
732
915
1095
0
183
365
Days
No. at Risk
QQ
RQ
RR
584
563
118
544
522
112
527
517
110
501
492
101
732
915
1095
487
477
101
446
440
91
435
429
90
Days
487
477
101
446
440
91
435
429
90
584
563
118
544
522
112
Arylesterase Activity
527
517
110
501
492
101
Arylesterase Activity
30
15
Quartile
1
2
3
4
10
25
Events, %
Events, %
549
5
20
15
10
5
Log-rank P <.001
Log-rank P <.001
0
0
0
183
365
549
732
915
1095
0
183
365
Days
No. at Risk
Quartile 1
Quartile 2
Quartile 3
Quartile 4
319
317
318
324
289
290
300
310
278
284
296
307
263
267
277
297
549
732
915
1095
253
259
270
293
234
233
249
270
223
228
245
267
Days
253
259
270
293
234
233
249
270
223
228
245
267
319
317
318
324
289
290
300
310
Paraoxonase Activity
278
284
296
307
263
267
277
297
Paraoxonase Activity
15
30
10
Events, %
Events, %
25
5
20
15
10
5
Log-rank P <.001
0
0
183
365
549
732
915
1095
Log-rank P <.001
0
0
183
365
Days
No. at Risk
Quartile 1
Quartile 2
Quartile 3
Quartile 4
311
326
326
315
280
303
305
301
267
299
301
298
254
288
283
279
549
732
915
1095
288
290
275
277
283
259
253
257
279
256
247
252
Days
288
290
275
277
283
259
253
257
279
256
247
252
311
326
326
315
280
303
305
301
267
299
301
298
254
288
283
279
The paraoxonase 1 (PON1) Q192R genotypes are as follows: RR192 (mutant homozygous); QR192 (heterozygous); and QQ192 (wild type). In the top panels, log-rank P
values are shown for the at-risk genotype QQ192 vs RR192⫹QR192. PON1 activity (paraoxonase and arylesterase) were categorized into quartiles; Q1: lowest activity quartile; Q4: highest activity quartile. For paraoxonase, Q4⬎1640, Q3=1640-899.1, Q2=899-450, and Q1⬍450 nmol/min/mL. For arylesterase, Q4⬎403.9, Q3=403.9-338.5,
Q2=338.4-283, and Q1⬍283 µmol/min/mL. In the middle and bottom panels, log-rank P values across activity quartiles are shown. Days indicates number of days from
enrollment to first cardiac event. Event rates were calculated at 6-month intervals. Y-axis scales in blue indicate range from 0% to 15%. MI indicates myocardial infarction;
CVA, cerebrovascular accident.
©2008 American Medical Association. All rights reserved.
(Reprinted) JAMA, March 19, 2008—Vol 299, No. 11
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1273
RELATIONSHIP OF PARAOXONASE 1 WITH SYSTEMIC OXIDATIVE STRESS AND CVD RISK
Table 7. Risk of Prospective CVD Events in Relation to PON1 Activity Among Entire Cohort
Paraoxonase Activity Quartile, nmol/min/mL
CVD Events
Nonfatal MI/CVA
No./total (%)
Unadjusted HR (95% CI)
Adjusted HR (95% CI) a
All-cause mortality
No./total (%)
Unadjusted HR (95% CI)
Adjusted HR (95% CI) a
MACE
No./total (%)
Unadjusted HR (95% CI)
Adjusted HR (95% CI) a
Quartile 4
(⬎ 1640)
(n = 315)
Quartile 3
(1640-899.1)
(n = 325)
Quartile 2
(899-450)
(n = 326)
Quartile 1
(⬍ 450)
(n = 311)
8/315 (2.5)
1 [Reference]
1 [Reference]
23/325 (7.4)
27/326 (8.3)
37/311 (11.9)
3.0 (1.3-6.7)
3.3 (1.5-7.3)
5.0 (2.3-10.8)
2.9 (1.3-6.4)
3.1 (1.4-7.0)
4.4 (2.0-9.6)
17/315 (5.4)
1 [Reference]
1 [Reference]
21/325 (6.5)
27/326 (8.3)
37/311 (11.9)
1.2 (0.6-2.3)
1.3 (0.7-2.5)
2.7 (1.5-4.7)
1.1 (0.6-2.2)
1.3 (0.7-2.6)
2.4 (1.3-4.4)
23/315 (7.3)
1 [Reference]
1 [Reference]
44/325 (13.5)
48/326 (14.7) 78/311 (25.1)
1.9 (1.2-3.2)
2.1 (1.2-3.4)
3.7 (2.3-5.9)
1.9 (1.1-3.2)
2.0 (1.2-3.4)
3.4 (2.1-5.5)
Arylesterase Activity Quartile, µmol/min/mL
Quartile 4
(⬎ 403.9)
(n = 324)
Nonfatal MI/CVA
No./total (%)
Unadjusted HR (95% CI)
Adjusted HR (95% CI) a
All-cause mortality
No./total (%)
Unadjusted HR (95% CI)
Adjusted HR (95% CI) a
MACE
No./total (%)
Unadjusted HR (95% CI)
Adjusted HR (95% CI) a
9/324 (2.8)
1 [Reference]
1 [Reference]
Quartile 3
(403.9-338.5)
(n = 318)
Quartile 2
(338.4-283)
(n = 316)
Quartile 1
(⬍ 283)
(n = 319)
15/318 (4.7)
32/316 (10.1) 40/319 (12.5)
1.7 (0.8-4.0)
3.8 (1.8-8.0)
4.8 (2.3-9.9)
1.5 (0.7-3.6)
3.4 (1.6-7.2)
4.5 (2.2-9.4)
16/324 (4.9)
1 [Reference]
1 [Reference]
22/318 (6.9)
27/316 (8.5)
41/319 (12.9)
1.4 (0.8-2.7)
1.8 (1.0-3.3)
2.7 (1.5-4.8)
1.4 (0.7-2.8)
1.8 (0.9-3.4)
2.2 (1.2-4.2)
25/324 (7.7)
1 [Reference]
1 [Reference]
37/318 (11.6)
56/316 (17.7) 75/319 (23.5)
1.6 (0.9-2.6)
2.4 (1.5-3.9)
3.3 (2.1-5.2)
1.5 (0.9-2.5)
2.3 (1.4-3.7)
2.9 (1.8-4.7)
Abbreviations: CI, confidence interval; CVA, cerebrovascular accident; CVD, cardiovascular disease; HR, hazard ratio;
MACE, major adverse cardiac events (ie, myocardial infarction, stroke, or death); MI, myocardial infarction; PON1,
paraoxonase 1.
a Adjusted HRs were calculated including Framingham ATP-III risk score (including diabetes status), log C-reactive protein, body mass index, and use of statins and aspirin.
Table 8. Incidence of First CVD Event in Relation to PON1 Activity Among Patients With No
CVD at Baseline
Paraoxonase Activity Quartile, nmol/min/mL
First CVD Events
MACE
No./total (%)
Unadjusted HR (95% CI)
Adjusted HR (95% CI) a
Quartile 4
(⬎ 1640)
(n = 80)
Quartile 3
(1640-899.1)
(n = 61)
Quartile 2
(899-450)
(n = 57)
Quartile 1
(⬍ 450)
(n = 56)
4/80 (5.0)
1 [Reference]
1 [Reference]
3/61 (4.9)
1.0 (0.2-4.4)
0.6 (0.1-3.1)
3/57 (5.3)
1.1 (0.2-4.7)
1.2 (0.3-5.3)
8/56 (14.3)
3.0 (0.9-9.8)
1.7 (0.5-6.4)
Arylesterase Activity Quartile, µmol/min/mL
MACE
No./total (%)
Unadjusted HR (95% CI)
Adjusted HR (95% CI) a
Quartile 4
(⬎ 403.9)
(n = 91)
Quartile 3
(403.9-338.5)
(n = 61)
2/91 (2.2)
1 [Reference]
1 [Reference]
3/61 (4.9)
2.2 (0.4-13.4)
2.1 (0.3-12.8)
Quartile 2
(338.4-283)
(n = 51)
Quartile 1
(⬍ 283)
(n = 51)
3/51 (5.9)
10/51 (19.6)
2.7 (0.4-16.0)
9.7 (2.1-44.1)
2.4 (0.4-14.4)
5.8 (1.2-28.6)
Abbreviations: CI, confidence interval; CVD, cardiovascular disease; HR, hazard ratio; MACE, major adverse cardiac
events (ie, myocardial infarction, stroke, or death); PON1, paraoxonase 1.
a Adjusted HRs were calculated including the Framingham ATP-III risk score (including diabetes status), log C-reactive
protein, body mass index, and use of statins and aspirin.
1274 JAMA, March 19, 2008—Vol 299, No. 11 (Reprinted)
coronary and extracoronary atherosclerosis after controlling for established risk
factors. However, in the present study,
diminished systemic arylesterase activity levels remained significantly associated with 3-year incident MACE (composite of MI, stroke, or death) in the
subgroup of participants without history or clinical evidence of either coronary or peripheral artery disease and
without significant angiographic evidence of coronary artery disease. Furthermore, the present study also suggests a potential prognostic value of
PON1 activity measurement in patients. The addition of systemic PON1
activity measures to traditional risk factors and C-reactive protein provided a
significant incremental improvement in
the ability to predict clinical outcome
during a 3-year period.
Inrecentgenomewideassociationstudies,29,30 evidence for association of the
PON1 gene with coronary artery disease
or MI was not observed. One likely explanation is that genomewide association
studies are not ideal for assessing specific
candidate genes since the panel of SNPs
placed on the chip may not capture all of
the genetic variation for any particular
gene.31 Furthermore, genomewide association studies tend to identify genes with
the strongest genetic effect because of the
stringency in considering what is statistically significant. In this regard, a weak
associationwithPON1mayhavebeenobserved but not reported since it did not
exceedthethresholdforsignificancebased
on the thousands of statistical tests performed. Moreover, one of the phenotypes
wehavestudied,namely,mortalitywithin
thecohort,wasnottheexactsameasthose
studied in the genomewide association
studies,whichcouldalsoaccountforwhy
an association with PON1 was not reported.
ThePON1geneislocatedincloseproximity to 2 other members of the paraoxonasegenefamily,PON2andPON3,which
raises the possibility that the association
we have observed could be due to linkage disequilibrium with a variant in either
PON2orPON3.However,anexamination
of the HAPMAP database for the white
population(whichmatchestheGeneBank
©2008 American Medical Association. All rights reserved.
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RELATIONSHIP OF PARAOXONASE 1 WITH SYSTEMIC OXIDATIVE STRESS AND CVD RISK
cohort)showsthatthePON1Q192Rvariant is located within a haplotype block in
which the SNPs are in moderate linkage
disequilibriumwitheachother.Moreover,
this haplotype block only covers PON1
and does not extend to the PON2 and
PON3genes.Thus,whileitispossiblethat
variants of PON2 and PON3 could also
contribute to altered PON1 activity and
CVDrisk,theavailableHAPMAPdatasuggest that, in whites, the association observed with the PON1 Q192R variant is
not due to linkage disequilibrium with
other variants in the adjacent PON2 and
PON3genes,butlikelyresultsfromaltered
PON1function.Althoughitispossiblethat
other PON1 variants that are in linkage
disequilibrium with the Q192R SNP are
the causal alleles, recent studies using recombinantPON1mutantsatposition192
have shown that this residue is important
for HDL binding28 and suggest that the
Q192R SNP is the causal variant underlying the clinical associations that we observe. Further studies to address this issue are required.
A number of caveats to the present
study should be noted. All participants presented for elective diagnostic coronary angiography, which limits the generalization of the present
findings and raises the possibility of selection bias. However, measurements
of both paraoxonase and arylesterase activity in healthy volunteers are higher
than in both the CVD and non-CVD
participants evaluated in the present
study. Given that the majority of participants were white, it also remains to
be determined whether the same relationships are observed in large cohorts of other racial/ethnic groups. The
lower percentage of nonwhite participants in our cohort (7.9%) limited our
ability to analyze the mechanistic link
between PON1 and global indices of
systemic oxidative stress and thereby
its association with CVD in participants of other racial/ethnic backgrounds. It is also uncertain whether
concomitant medical problems influenced PON1 activity; however, inclusion of traditional cardiac risk factors including age, sex, race, diabetes,
hypertension, smoking, lipids, C-
reactive protein, and medication use
(statins and aspirin) in the multivariate analyses failed to alter the results.
It also is possible that the reduction
in PON1 activity may result from the
presence of vascular disease rather than
be the direct cause of future CVD
events. However, arylesterase activity
levels in participants with minimal angiographic evidence of coronary artery disease still remained independently associated with incident
cardiovascular events over the ensuing 3-year interval following participant enrollment, consistent with an
association of PON1 in early macrovascular atherosclerotic disease processes. Regardless, the current observation of a relationship among the
PON1 Q192R polymorphism and PON1
activity, oxidative stress, and CVD outcomes is consistent with findings in murine models and provides evidence that
the PON1 protein protects against the
development and propagation of CVD.
CONCLUSION
The current findings provide direct prospective evidence of an important
mechanistic link between the PON1
gene and PON1 systemic activity measures with both multiple quantitative
indices of oxidative stress and atherosclerotic heart disease development in
humans. Paraoxonase 1 is almost exclusively found to be associated with
HDL particles within the circulation and
has been argued to promote some of the
Table 9. Risk of Recurrent CVD Events in Relation to PON1 Activity Among Patients With
CVD at Baseline
Paraoxonase Activity Quartile, nmol/min/mL
Recurrent Events
Nonfatal MI/CVA
No./total (%)
Unadjusted HR (95% CI)
Adjusted HR (95% CI) a
All-cause mortality
No./total (%)
Unadjusted HR (95% CI)
Adjusted HR (95% CI) a
MACE
No./total (%)
Unadjusted HR (95% CI)
Adjusted HR (95% CI) a
Quartile 4
(⬎ 1640)
(n = 235)
Quartile 3
(1640-899.1)
(n = 265)
Quartile 2
(899-450)
(n = 269)
Quartile 1
(⬍ 450)
(n = 255)
6/235 (2.6)
1 [Reference]
1 [Reference]
22/265 (8.3)
24/269 (8.9)
40/255 (12.5)
3.4 (1.4-8.3)
3.6 (1.5-8.7)
5.3 (2.2-12.8)
3.2 (1.3-7.9)
3.3 (1.3-8.1)
4.5 (1.9-11.0)
15/235 (6.4)
1 [Reference]
1 [Reference]
20/265 (7.5)
24/269 (8.9)
40/255 (15.7)
1.2 (0.6-2.3)
1.4 (0.7-2.6)
2.6 (1.4-4.6)
1.1 (0.6-2.3)
1.4 (0.7-2.7)
2.4 (1.3-4.5)
19/235 (8.1)
1 [Reference]
1 [Reference]
41/265 (15.5)
45/269 (16.7) 70/255 (27.5)
2.0 (1.2-3.4)
2.1 (1.2-3.6)
3.7 (2.2-6.2)
2.0 (1.1-3.5)
2.1 (1.2-3.7)
3.4 (2.0-5.9)
Arylesterase Activity Quartile, µmol/min/mL
Quartile 4
(⬎ 403.9)
(n = 233)
Nonfatal MI/CVA
No./total (%)
Unadjusted HR (95% CI)
Adjusted HR (95% CI) a
All-cause mortality
No./total (%)
Unadjusted HR (95% CI)
Adjusted HR (95% CI) a
MACE
No./total (%)
Unadjusted HR (95% CI)
Adjusted HR (95% CI) a
Quartile 3
(403.9-338.5)
(n = 257)
Quartile 2
(338.4-283)
(n = 266)
Quartile 1
(⬍ 283)
(n = 268)
7/233 (3.0)
1 [Reference]
1 [Reference]
12/257 (4.7)
30/266 (11.3) 35/268 (13.1)
1.6 (0.6-4.1)
4.0 (1.8-9.1)
4.6 (2.0-10.4)
1.4 (0.5-3.6)
3.4 (1.5-7.9)
4.2 (1.9-9.6)
16/233 (6.9)
1 [Reference]
1 [Reference]
22/257 (8.6)
26/266 (9.8)
35/268 (13.1)
1.3 (0.7-2.4)
1.5 (0.8-2.8)
1.9 (1.1-3.5)
1.3 (0.6-2.5)
1.4 (0.7-2.8)
1.7 (0.9-3.2)
23/233 (9.9)
1 [Reference]
1 [Reference]
34/257 (13.2)
53/266 (19.9) 65/268 (24.3)
1.4 (0.8-2.4)
2.2 (1.3-3.5)
2.6 (1.6-4.2)
1.3 (0.7-2.3)
2.0 (1.2-3.3)
2.4 (1.4-3.9)
Abbreviations: CI, confidence interval; CVA, cerebrovascular accident; CVD, cardiovascular disease; HR, hazard ratio;
MACE, major adverse cardiac events (ie, myocardial infarction, stroke, or death); MI, myocardial infarction; PON1,
paraoxonase 1.
a Adjusted HRs were calculated including the Framingham ATP-III risk score (including diabetes status), log C-reactive
protein, body mass index, and use of statins and aspirin.
©2008 American Medical Association. All rights reserved.
(Reprinted) JAMA, March 19, 2008—Vol 299, No. 11
Downloaded from jama.ama-assn.org at University of California - Los Angeles on January 6, 2012
1275
RELATIONSHIP OF PARAOXONASE 1 WITH SYSTEMIC OXIDATIVE STRESS AND CVD RISK
anti-inflammatory and antioxidant effects attributed to HDL. Thus, the present studies also provide further support for the concept that functional
properties beyond the ability of HDL
and its associated proteins to promote
reverse cholesterol transport contribute to the overall ability of this lipoprotein to reduce or prevent development of atherosclerosis.
Author Affiliations: Department of Cell Biology (Drs
Bhattacharyya, Nicholls, Zhang, M.-L. Brennan, and
Hazen and Messrs Schmitt and Fu), Center for Cardiovascular Diagnostics and Prevention (Drs Bhattacharyya, Nicholls, Zhang, M.-L. Brennan, and Hazen and Messrs Schmitt and Fu), and Department of
Cardiovascular Medicine (Drs Nicholls, Ellis, and Hazen, Mr Shao, and Ms D. M. Brennan), Cleveland Clinic,
Cleveland, Ohio; Scripps Research Institute and Scripps
Clinic, La Jolla, California (Dr Topol); Departments of
Medicine (Drs Yang and Lusis), Human Genetics (Dr
Lusis), and Microbiology and Immunology (Dr Lusis),
University of California, and Department of Preventive
Medicine, University of Southern California (Dr
Allayee), Los Angeles.
Author Contributions: Dr Hazen had full access to all
of the data in the study and takes responsibility for
the integrity of the data and the accuracy of the data
analysis.
Study concept and design: Lusis, Hazen.
Acquisition of data: Bhattacharyya, Topol, Zhang,
Yang, Schmitt, Fu, M.-L. Brennan, Allayee, Lusis,
Hazen.
Analysis and interpretation of data: Bhattacharyya,
Nicholls, Topol, Yang, Shao, D. M. Brennan, Ellis, Lusis,
Hazen.
Drafting of the manuscript: Bhattacharyya, Nicholls,
Lusis, Hazen.
Critical revision of the manuscript for important intellectual content: Bhattacharyya, Zhang, Schmitt, Fu,
Shao, D. M. Brennan, M.-L. Brennan, Allayee, Lusis,
Hazen.
Statistical analysis: Bhattacharyya, Nicholls, Yang,
Shao, D. M. Brennan, Lusis, Hazen.
Obtained funding: Topol, Allayee, Lusis, Hazen.
Administrative, technical, or material support:
Bhattacharyya, Topol, Zhang, M.-L. Brennan, Allayee,
Lusis, Hazen.
Study supervision: Nicholls, Topol, Ellis, M.-L. Brennan,
Hazen.
Financial Disclosures: Dr Nicholls reports that he has
received speaking honoraria from Pfizer, AstraZeneca, Merck, Schering-Plough, and Takeda and consulting fees from Roche, AstraZeneca, Pfizer, and NovoNordisk. Dr Ellis reports that he is named as coinventor
on pending patents filed by the Cleveland Clinic referring to use of genetic markers in cardiovascular disease. Dr Ellis also reports that he has received research support from Lilly, Centocor, Boston Scientific,
and Cardlodx and has received consulting fees from
Celera, Cordis, Boston Scientific, and Abbott Vascular.
Dr Allayee reports that he is named as coinventor on
pending and approved patents filed by the Univer-
sity of California that are related to identification of
5-lipoxygenase as a gene involved in cardiovascularand diabetes-related traits. Dr Allayee also reports that
he has received honoraria/consulting fees from Reliant Pharmaceuticals and Corautus Genetics. Dr Lusis
reports that he has received research grant support
from Merck, Anthera, and Bristol-Myers Squibb. Dr
Hazen reports that he is named as coinventor on pending and approved patents filed by the Cleveland Clinic
that refer to the use of biomarkers to inflammatory
and cardiovascular diseases. Dr Hazen also reports that
he is the scientific founder of PrognostiX Inc; has received research grant support from Abbott Diagnostics, Pfizer, Merck, PrognostiX Inc, Hawaii Biotech,
ArgiNOx, Sanofi, and Takeda; and has received honoraria and consulting fees from Abbott Diagnostics,
BioSite, Merck, Lilly, Pfizer, PrognostiX Inc, Wyeth,
BioPhysical, and AstraZeneca. No other disclosures were
reported.
Funding/Support: This study was supported by National Institutes of Health grants P01 HL076491, P01
HL077107, P01 HL087018, P01 HL30568, and RO1
HL079353, the Case Western Reserve University/
Cleveland Clinic Clinical and Translational Science
Award (grant 1KL2RR024990), and AHA grant
0435223N. A portion of this work was conducted in
a facility constructed with support from Research Facilities Improvement Program grant C06 (RR1060001, CA62528-01, RR14514-01) from the National
Center for Research Resources.
Role of the Sponsors: The funding organizations played
no role in the design and conduct of the study; in the
collection, analysis, and interpretation of the data; or in
the preparation, review, or approval of the manuscript.
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Downloaded from jama.ama-assn.org at University of California - Los Angeles on January 6, 2012