Vitamin C and E Intake Is Associated With Increased
Paraoxonase Activity
Gail P. Jarvik, Nancey Trevanian Tsai, Laura A. McKinstry, Roohi Wani, Victoria H. Brophy, Rebecca J. Richter,
Gerard D. Schellenberg, Patrick J. Heagerty, Thomas S. Hatsukami, Clement E. Furlong
Downloaded from http://atvb.ahajournals.org/ by guest on June 17, 2017
Objective—Paraoxonase (PON1), an esterase physically associated with high density lipoprotein, has been shown to inhibit
atherogenic low density lipoprotein and high density lipoprotein oxidation. PON1 activity appears to be primarily under
genetic control with some environmental modification and is a predictor of vascular disease. Vitamins C and E, dietary
antioxidants, scavenge free-oxygen radical products that may depress PON1 activity. Therefore, we evaluated the
relationship between dietary vitamin C and E intake and PON1 activity.
Methods and Results—The vitamin C and E intakes of male white subjects (n⫽189) were estimated by using a
standardized food frequency survey. With covariates, vitamin C or E intakes were found to be significant positive
predictors of PON1 activity for the hydrolysis of paraoxon and diazoxon with the use of linear regression. Smoking and
use of statins were independent predictors of PON1 activity.
Conclusions—PON1 activity, which is primarily genotype dependent, varies with antioxidant vitamins, cigarette smoking,
and statin drug use. Because PON1 activity is a better predictor of vascular disease than is the currently described
genetic variation in PON1, further studies of the environmental influences on PON1 activity and additional PON1
genetic variants are warranted. (Arterioscler Thromb Vasc Biol. 2002;22:1329-1333.)
Key Words: antioxidants 䡲 genetics 䡲 vitamins 䡲 paraoxonase 䡲 smoking
P
araoxonase (PON1) genotype1–3 and activity4,5 have been
implicated in the pathogenesis of atherosclerosis. This
calcium-dependent esterase is physically associated with
HDL.6 – 8 Paraoxonase may protect against vascular disease
through its inhibition of LDL oxidation.9 Studies have demonstrated that PON1 activity is lower in patients with carotid
artery disease,5 coronary heart disease,10 and myocardial
infarction,4 even when no marginal effect of PON1 genotype
on disease is notable.
appears to have the largest effect on PON1 concentration,
accounting for 23% of the variance in PON1 arylesterase
activity.14
Environmental factors that alter PON1 activity include
tobacco consumption, which has been reported to depress
PON1 activity and concentration.15 PON1 POase activity has
been found to be increased by pharmacological therapy with
simvastatin16 and hormone replacement therapy.17 Fatty
meals may depress postprandial PON1 arylesterase activity.18
The concentration of PON1 mRNA has been shown to
increase or decrease when mice are challenged with an
atherogenic diet in a strain-dependent fashion.19 Pomegranate
juice has been reported to increase PON1 activity in mice.20
Exposure to organophosphate pesticides does not induce
PON1 activity.
The relationship between antioxidant intake and atherogenesis continues to be studied, with many conflicting results.21–26 These studies have been reviewed by Berliner27 and
Tribble,28 among others. Antioxidant supplements may be
expected to influence PON1 activity by altering oxidative
stress. The goal of the present study was to examine the effect
of the dietary antioxidants vitamin C (ascorbic acid) and
vitamin E (eg, ␣-tocopherol) on PON1 activities measured by
See page 1248
The activity of PON1 is under genetic and environmental
regulation. The PON1R192Q polymorphism, which results in
Arg instead of Gln at amino acid position 192, has been
shown to account for 73% to 76% of the variation in
hydrolysis activity of PON1 for paraoxon (POase activity)
and 12% to 25% of the variation in activity for the hydrolysis
of diazoxon (DZOase activity) in vitro with the use of an
assay in the presence of 2 mol/L NaCl.5,11 The PON1L55M
polymorphism seems to be correlated with the PON1 protein
concentration through linkage disequilibrium with promoter
polymorphisms12 and through increased protein turnover.13
Of the 5⬘ regulatory promoter region polymorphisms, PON1C-108T
Received May 31, 2002; accepted June 3, 2002.
From the Department of Medicine, Division of Medical Genetics (G.P.J., L.A.M., R.W., V.H.B., R.J.R., C.E.F.), and the Departments of Epidemiology
(G.P.J.), Neurology (G.D.S.), Genomic Sciences (G.P.J., C.E.F.), and Biostatistics (P.J.H.), University of Washington, and the Puget Sound Veterans
Affairs Health Care Systems (N.T.T., L.A.M., R.W., G.D.S., T.S.H.), Seattle, Wash.
Reprint requests to Gail P. Jarvik, MD, PhD, University of Washington Medical Center, Division of Medical Genetics, Box 357720, Seattle, WA 98195.
E-mail [email protected]
© 2002 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org
1329
DOI: 10.1161/01.ATV.0000027101.40323.3A
1330
Arterioscler Thromb Vasc Biol.
August 2002
the hydrolysis of paraoxon and diazoxon to determine
whether the role of the vitamins and PON1 antioxidant effects
might be interrelated.
Methods
Subjects
The subjects were white male veterans. The mean age was 65 years,
with a range of 48 to 88 years. There were 189 subjects: 94 men with
⬍15% carotid stenosis bilaterally, 23 men with 16% to 49% carotid
stenosis, 21 men with 50% to 79% carotid stenosis, 31 men with
⬎80% carotid stenosis and/or with carotid endarterectomy, and 20
men with lower extremity vascular disease without carotid stenosis.
Exclusionary criteria included familial hypercholesterolemia, total
fasting cholesterol ⬎400 mg/dL, hypercoagulable state, and/or use of
anticoagulant medication or inability to consent. Human Subjects
Oversight Committees at the University of Washington Medical
Center and the Veterans Affairs Puget Sound Health Care centers
approved the study, and written informed consent was obtained from
all participants.
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Survey Methods
Subjects were asked to complete a standardized food frequency
survey from The Health Professionals Follow-Up Study (1988,
Brigham and Women’s Hospital). The survey asked about (1) the
average frequency of intake over the previous year of specified
portions of 131 foods and (2) the use of vitamins and mineral
supplements, including dose and duration of multivitamins, vitamin
C, and vitamin E. Questions regarding brand of multivitamins and
breakfast cereals used were asked to clarify the quantities of specific
vitamin supplementation. Excluded from the analysis were those
subjects (1) whose caloric intake was not between 800 and 4200
kcal/d and (2) whose surveys had ⬎70 blank items of a total of 131
items. All vitamin usage was energy-adjusted to 2000 kcal/d. This
food frequency survey has been validated against two 1-week diet
records taken ⬇6 months apart.29 In that study, the adjusted
correlation coefficients between the food frequency questionnaire
and both 1-week diet records, which were adjusted for energy intake
and within-person variability in daily intake, were 0.92 for vitamins
C and E. The survey has predicted cardiovascular disease in relation
to fat intake,30 ␻-3 fatty acid intake,31 fiber,32,33 and vitamin E
consumption.34
Smoking status was ascertained by survey. Ten subjects who
reported that they did not currently smoke but reported 0 months
since the last cigarette were coded as current smokers. This code was
given because it seems likely that these subjects are current smokers
with the intention of quitting and because a prior study suggested that
the effects of smoking on PON1 remain for months after tobacco
cessation.35 The use of statins and other medications was ascertained
from Veterans Affairs Puget Sound Health Care pharmacy records.
PON1 Genotype and Activity Phenotype Methods
DNA was prepared from buffy coat preparations by a modification of
the procedure of Miller and Dykes,36 with the use of Puregene
reagents (Gentra). PON1192 genotypes were determined by using
polymerase chain reaction techniques and AlwI restriction enzyme
analysis.37 The PON1⫺108 promoter region polymorphism was typed
as recently described.12
PON1 paraoxon (POase activity) and diazoxon (DZOase activity)
hydrolysis rates were measured spectrophotometrically with lithium
heparin plasma, as previously described.38 All samples were run in
duplicate; the averaged value was used for analysis. PON1192
genotype can be predicted with high accuracy from examination of
the 2D plot of paraoxon and diazoxon hydrolysis rates.5,38 When
assignments did not match, genotyping and phenotyping studies
were repeated. One hundred eighty (95.2%) of the 189 subjects had
PON1192 genotype-phenotype agreement. These mismatches are
likely due to other PON1 variants that influence PON1 activity.12
Thus, the PON1 activity assays provide functional genomics for
TABLE 1.
Subject Characteristics
Subjects
Number
189
Men, %
100
White, %
100
Taking hypoglycemics, %
26.5
Taking lipid-lowering medication, %
33.3
Statin drug use, %
19.0
Taking antihypertensives, %
66.1
Current smokers, %
29.6
Pack years smoked, mean y
29.1
Total cholesterol, mean mg/dL
195.3
Calculated LDL-C, mean mg/dL
110.5
Triglycerides, mean mg/dL
183.9
VLDL-C, mean mg/dL
35.7
HDL-C, mean mg/dL
42.0
POase, U/L (sqrt-POase)
DZOase, U/L (sqrt-DZOase)
566.4 (23.8)
9920.2 (99.6)
variants that are not typed for and also reflect environmental
influences.
Lipid and HbAIc Measurements
Lipid measurements were performed on fasting whole plasma.
Standard enzymatic methods were used to determine the levels of
total cholesterol, triglycerides, and HDL cholesterol (HDL-C) on an
Abbott Spectrum analyzer.39 – 41 LDL cholesterol was calculated.42
Hemoglobin A1c (HbA1c) measurement used standard high-pressure
liquid chromatography.43 This assay does separate pre-HbA1c from
HbA1c, eliminating the false elevation that can result from high
serum glucose if pre-HbA1c is not removed.
Statistical Analysis
Marginal associations between vitamin intake and smoking status or
statin use were investigated by ANOVA. Linear regression was used
to determine whether vitamin C or vitamin E intake predicted PON1
POase and DZOase activities. The subjects’ ages and dummy
variables for their PON1192 and PON1⫺108 genotype, whether or not
they currently smoked, and pharmacological statin therapy for lipid
lowering were considered as covariates. PON1192QQ and PON1⫺108TT
genotypes were used as the reference genotype (coded 0,0) for the
dummy variable regression. All 4 quantitative variables significantly
deviated from a normal distribution. A natural logarithmic transformation was required for the vitamin C (ln-vitC) intake variable. A
square root transformation was used to adjust the vitamin E intake
(sq-vitE), DZOase (sq-DZOase), and POase (sq-POase) variables.
When total calories of dietary fat and grams of alcohol consumed
were considered as covariates, neither predicted PON1 activities, and
neither changed the relationship of vitamin C or E with PON1
activities (results not shown). HDL-C level was used as an additional
covariate. Multiplicative interaction for the prediction of PON1
activity between PON1192 and PON1⫺108 genotype dummy variables
and the antioxidant intakes were not significant and are not reported.
Power to detect interactions was expected to be low. The residuals of
the regression of age, current smoking, statin use, and PON1192 and
PON1⫺108 genotypes on sq-POase or sq-DZOase activity were
relocated to the activity means and resquared; they are described for
each vitamin intake tertile. Because of high correlations between the
antioxidant intakes and between the PON1 activities, adjustments for
multiple contrasts were not made. All analyses used SPSS for
Windows (version 10.0.5).
Jarvik et al
TABLE 2.
Antioxidant Intake and Paraoxonase Activity
1331
Dietary Antioxidant Effects on PON1 Activities
Predictor
B for sq-Poase
(SE)
P
B for sq-DZOase
(SE)
P
Considering vitamin C
ln-vitamin C
⬍0.0001*
0.54 (0.21)
0.011*
2.78 (0.74)
⫺1.15 (0.55)
0.039*
0.63 (1.93)
PON1192QR genotype
12.40 (0.52)
⬍0.0001*
⫺13.98 (1.81)
⬍0.0001*
PON1192RR genotype
22.15 (1.00)
⬍0.0001*
⫺30.58 (3.52)
⬍0.0001*
PON1⫺108CC genotype
4.80 (0.71)
⬍0.0001*
22.14 (2.49)
⬍0.0001*
PON1⫺108CT genotype
3.08 (0.60)
⬍0.0001*
12.17 (2.12)
⬍0.0001*
Current smokers
Statin drug use
Age
1.55 (0.62)
⫺0.10 (0.03)
0.014*
⬍0.0001*
2.98 (2.19)
⫺0.25 (0.09)
0.744
0.174
0.009*
Considering vitamin E
sq-vitamin E
0.08 (0.03)
0.005*
0.32 (0.10)
0.001*
⫺1.21 (0.55)
0.028*
0.17 (1.94)
0.931
PON1192QR genotype
12.35 (0.51)
⬍0.0001*
⫺14.24 (1.83)
PON1192RR genotype
22.04 (0.99)
⬍0.0001*
⫺31.39 (3.53)
⬍0.0001*
PON1⫺108CC genotype
4.80 (0.71)
⬍0.0001*
22.23 (2.51)
⬍0.0001*
PON1⫺108CT genotype
3.07 (0.60)
⬍0.0001*
12.30 (2.14)
⬍0.0001*
Statin drug use
1.67 (0.62)
0.008*
3.49 (2.21)
0.116
Current smokers
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Age
⫺0.10 (0.03)
⬍0.0001*
⫺0.26 (0.09)
⬍0.0001*
0.008*
*P⬍0.05; regressions include either ln-vitamin C or sq-vitamin E. PON1192QQ and PON1⫺108TT are
reference genotypes. See methods.
Results
As seen in Table 1, the correlation of ln-vitC with sq-vitE was
0.58 (P⬍0.01). sq-POase and sq-DZOase are highly correlated within PON1192 genotypes (0.77, 0.56, and 0.84 for the
PON1192 QQ, QR, and RR genotypes, respectively (all
P⬍0.001). There was a trend toward lower ln-vitC (P⫽0.06)
intake in current smokers. Decreased sq-vitE (P⫽0.25) intake
in smokers was not significant. The ln-vitC (P⫽0.9) and
sq-vitE (P⫽0.4) intakes did not differ significantly between
users and nonusers of statins.
Dietary intakes of vitamin C and of vitamin E were
significant predictors of PON1 activity with the use of POase
(P⫽0.011 and 0.005, respectively) and DZOase (P⫽0.001
for vitamins C and E) activities as measures for linear
regression with covariates (Table 2). Increased vitamin C and
E intakes were associated with an elevation of PON1 activities. Inclusion of either ln-vitC or sq-vitE into a regression
that included the other did not result in a significant improvement in sq-POase or sq-DZOase prediction. Although normalization is required for appropriate statistical testing, we
also computed the regression coefficients without normalizing transformations of PON1 activities or vitamin intake. The
multiple regression coefficients for vitC (milligrams per day)
and vitE (IU per day) prediction of POase activity (units per
liter) were 0.08 and 0.13, respectively. For the prediction of
DZOase activity, these were 1.72 and 1.81, respectively.
When all covariates were included in the regression, including
either vitamin C or E as predictors, PON1 POase and DZOase
activities declined with age (all P⬍0.009). Current smoking
predicted depressed POase activity (all P⬍0.04) but was not
predictive of DZOase activity (all P⬎0.7). When total calories
of dietary fat and grams of alcohol consumed were considered as
covariates, neither predicted PON1 activities, and neither
changed the relationship of vitamin C or E with PON1 activities
(results not shown). HDL-C level was uncorrelated with the
vitamin intakes but was a significant predictor of sq-POase and
sq-DZOase when added to the regression model containing age,
smoking, statin use, PON1⫺108 and PON1192 genotype, and either
vitamin C or E intake (all P⬍0.013). The effects of vitamin C or
E intake on PON1 activity remained significant when HDL-C
was added to the regression model (all P⬍0.012).
When sq-POase was adjusted for age and PON1⫺108 and
PON1192 genotype effects with the use of linear regression,
10.7% of the remaining POase variance was attributable to
sq-vitE, smoking status, and statin use, or 10.4% was from
ln-vitC, smoking status, and statin use. The marginal effect
attributable to either ln-vitC or sq-vitE was 3.6% or 4.0%,
respectively. The marginal effects of smoking and statins use
were 3.5% and 4.0%, respectively. When sq-DZOase was
adjusted for age and PON1⫺108 and PON1192 genotype effects,
7.8% of the remaining POase variance was attributable to
sq-vitE, smoking status, and statin use, or 10.2% was from
ln-vitC, smoking status, and statin use. The marginal effect
attributable to either ln-vitC or sq-vitE was 8.1% or 5.2%,
respectively. The marginal effects of smoking and statin use
were 0% and 2.2%, respectively. The portion of variance
attributable to these environments will vary with the prevalence of each factor. The mean POase activity, adjusted for
age, statin use, and PON1⫺108 and PON1192 genotype was
549.4 U/L for the lowest vitamin C intake tertile and 602.2
U/L for the highest vitamin C intake tertile. The adjusted
POase activity means for the lowest and highest vitamin E
intake tertiles were 549.0 and 602.2 U/L, respectively. The
mean adjusted DZOase activities for the lowest and highest
1332
Arterioscler Thromb Vasc Biol.
August 2002
vitamin C intake tertiles were 9298.7 and 10675.0 U/L,
respectively; for vitamin E, they were 9482.9 and 10453.0,
respectively.
Discussion
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In the present study, increased activity of PON1 (POase and
DZOase) was predicted by increased vitamin C and E intake.
The inhibition of LDL oxidation by HDL, which is due to the
hydrolysis of lipid peroxidases and the resulting inhibition of
lipid peroxides, appears to be, at least in part,6,44 – 46 a function
of the enzyme paraoxonase, which is a component of HDL.47
Watson et al45 showed that PON1 destroyed the multioxygenated molecules found in oxidized phosphatidylcholine.
Furthermore, they showed that inactivation of PON1 reduces
the ability of HDL to inhibit LDL modification and also
reduces the ability of HDL to inhibit monocytic-endothelial
interactions, both of which appear to be important in the
inflammatory response in arterial wall cells that promotes
atherogenesis. PON1 reduced mildly oxidized phospholipids
by eliminating oxidized derivatives of unsaturated fatty acids.6,45 Inactivation of PON1 by oxidized LDL can be
inhibited by antioxidants.48
The role of antioxidant vitamins C and E in the prevention
of vascular disease is still being clarified.27 Vitamin E may
inhibit vascular lesion progression.49 Although the effects of
PON1 on LDL oxidation appear to be independent of the
function of antioxidant vitamins,45 vitamins C and E have
been shown to inhibit LDL oxidation.50,51 Any reduction in
oxidative stress related to vitamin C and E intake may
preserve PON1 activity. The HDL effect on the protection of
LDL from oxidation, mainly attributable to PON1, is more
prolonged than the effects of antioxidant vitamins.52
Previous reports of PON1 depression by tobacco smoke in
ex vivo assays53 are consistent with the lower POase activity
associated with current smoking in the present study and a
prior study.35 In a Costa Rican sample, the PON1192 genotype
was associated with myocardial infarction only in nonsmokers,54 suggesting the importance of jointly considering environmental factors that modify PON1 activity. Additionally,
elevation of PON1 activity in statin drug users was consistent
with the reported effects of simvastatin on PON1 activity.16
The present study has several limitations. It relied on a survey
that asked subjects about their dietary habits on average over a
year coupled with objective laboratory results on a particular
day. This might be expected to underestimate true correlations if
the dietary effects are short term. Independent effects of vitamin
C and E intake were not evaluated because of their high
correlation. Importantly, it has been shown that those subjects
using vitamin supplements are more likely to engage in healthy
lifestyles, of which the factors are multivariate.55 Thus, it is
possible that some factor(s) correlated with vitamin intake is
actually influencing PON1 activity. The vitamin effects demonstrated in the present study are independent of any effect of
current smoking and statin drug use, both of which influence
PON1 activity. The vitamin effects on PON1 activity were also
independent of dietary fat (calories) and alcohol (grams), neither
of which predicted PON1 activities (results not shown). The
present study suggests the need for clinical intervention tests
evaluating causality and potential dosage effects, although the
results of short-term and long-term supplementation may differ.
Additionally, PON1 genotype distributions vary among ethnic
groups,56 –58 as may dietary effects on PON1 activity. Further
studies of women are also warranted.
In summary, PON1 activity is predicted by antioxidant
vitamin intake, smoking, and statin drug therapy, as well as
by variation in the PON1 gene. We cannot rule out the
possibility that the relationship between vitamin intake and
PON1 activity is secondary to a correlated unmeasured
healthy lifestyle, as discussed above. PON1 activity appears
to be a better predictor of vascular disease than genotype for
the PON1 coding region polymorphisms.4,5,10 For this reason,
the noncoding genetic and environmental determinants of
PON1 activity merit study as possible factors determining the
role of PON1 in vascular disease. Furthermore, the role of
PON1 and of dietary antioxidants in vascular disease may
both be best understood if jointly considered.
Acknowledgments
This work was funded by the Veteran Affairs Epidemiology Research and Information Center Program (award CSP 701S to G.P.J)
and an American Heart Association Physician Scientist Award (to
G.P.J.), with additional funding from National Institutes of Health
grants HL-67406-01A1, T32 AG-00057, and RO1 ES-09883. The
authors would like to thank the subjects for their participation and
thank Jeff Rodenbaugh and Tianji Yu for their technical assistance.
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Vitamin C and E Intake Is Associated With Increased Paraoxonase Activity
Gail P. Jarvik, Nancey Trevanian Tsai, Laura A. McKinstry, Roohi Wani, Victoria H. Brophy,
Rebecca J. Richter, Gerard D. Schellenberg, Patrick J. Heagerty, Thomas S. Hatsukami and
Clement E. Furlong
Arterioscler Thromb Vasc Biol. 2002;22:1329-1333; originally published online June 20, 2002;
doi: 10.1161/01.ATV.0000027101.40323.3A
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