wall of the mid and distal. CCA as the distance between the lumen-intima interface and the media- .... pack. = 20 cigarettes). Alcohol consumption was determined from the subject's esti- mate ...... Maggi E, Chiesa. R, Melissano. G, et al.
Combined effects of lipid peroxidation and antioxidant status on carotid atherosclerosis in a population aged 59-71 y: The EVA Study1’2 Claire Favier,
Bonithon-Kopp, and Pierre
ABSTRACT
Charles Coudray, Ducimeti#{232}re
There
are few
Claudine
epidemiologic
studies
fects of lipid peroxidation sis. The relation of lipid
and antioxidant status peroxidation evaluated
acid-reactive
(TBARS)
substances status
sclerosis
was
examined
on cognitive
from
and vascular
the EVA Study). and women aged
rid,
men
and
data
(Etude
Ultrasound thickness
of a longitudinal
study
examination (IMT) on
included the common
significantly
men
and
not.
and negatively
women
whereas
No association
either with
sex. carotid
found
than
in men
with
in
with
selenium
between
TBARS
plaques
strengthened plasma findings
was
However,
associated
plasma
and
TBARS
were
without.
concentrations
selenium, and carotenoids give some epidemiologic
adjustment vitamin E
CCA-IMT
in both
carotenoids and
significantly
those
Lipid
were
CCA-IMT higher
This
in
in men
association
of erythrocyte
was
vitamin
peroxidation,
antioxidants,
atherosclerosis,
pathogenesis
of atherosclerosis
E,
Oxidative
of
other
mechanisms
nicity of oxidized cutating monocytes phages.
In
immunogenic
Am
J Clin
LDL and
addition, and
and vasomotor Nutr
may
explain
cholesterol. inhibits the
it is cytotoxic can
properties 1997;65:l21-7.
adversely
increased
vitamin
affect
endothelial coagulation
Only in USA.
for cirmacrocells
and
pathways
a few clinical © 1997
E, and
because
a large
clinical
complications
sis per
American
studSociety
or carotid
atherosclerosis
/3-carotene
or carotenoids
majority
se. The
of them
have
regardsuch as are
considered
much
as endpoints
of atherosclerosis
and
hypothesis
of atherosclerosis
oxidation
not
protection
effective
may
lesions
be more
in the
atheroscleroemphain the early antioxidant
formation
of early
(16).
Thus,
the
Etude
present
sur
the
and and
associations
(plasma E) and
atherosclerosis
AND
METHODS
Study
is a 4-y
aging
(17).
volunteers aged City of Nantes information
The
was
When
sub-
selenium ultrasound
in a population
U258
INRA,
and
St Genes
Neurovasculaire,
study
population
on cognitive was
from the electoral and, to a lesser a subject
systematically
INSERM
et de Prevention
longitudinal study
59-71 y recruited (western France)
campaigns.
spouse
asked U360, Campanelle, Paris;
was
Laboratoire France;
Centre
Centre
d’ Examen
Received
May
Accepted
for Nutrition
16,
of
rolls of the extent, via
recruited,
to participate Paris;
and
composed
his
in the des
or
study
Maladies
de Diagnostic EVA,
France; and GREPO, Universit#{233}I Fourier, La Tronche, France. 2 Address reprint requests to C Bonithon-Kopp, INSERM U258, Broussais, 96 rue Didot, 75674 Paris Cedex, France.
for Clinical
to
biological
acid-reactive
defenses vitamin
carotid
of the
aimed
between
(thiobarbituric
antioxidant erythrocyte
data
Study)
y.
EVA
From
on baseline
Art#{233}riet(EVA
peroxidation
of early
59-71
The
performed
cross-sectional
of lipid
vascular
analysis
le Vieiltissement
measurements
I
(3,
evidence for an association atherosclerosis or related dis-
of oxidation damage and thus suggests that
her
of lipid
and myocardiat arterial disease
sizes the prime importance phases of atherosclerosis
M#{233}taboliques,
atheroge-
It is chemotactic motility of tissue for
of arteries. Printed
the
indexes
extensive as reviewed by several authors (12-15). Howthese studies have yielded conflicting results, possibly
aged
modification
some
On the other hand, epidemiologic data role of dietary or serum antioxidants
stances, TBARS) and carotenoids,
low-density lipoprotein (LDL) by tipoperoxide products leads to enhanced uptake by macrophages via the “scavenger” receptor and to cellular accumulation of cholesterol (foam cells) generating fatty streaks, generally considered the early step of plaque formation. As reviewed by Witztum and Steinberg (2), many
selenium,
more ever,
markers
evidence that oxidative may be involved in the
(1).
coronary,
eases is lacking. ing the preventive
in the early
vitamin
between
femoral,
6, 7) and
SUBJECTS
is a large body of experimental especially lipid peroxidation,
associations
(4,
examine
epidemiology
F#{232}ve, A lain
modification of LDL pectoris (5), peripheral
Our that
INTRODUCTION There processes,
reported
Marc
peroxidation or oxidative infarction (3, 4), angina
E,
below the lowest quartile. support to the hypothesis
lipid peroxidation and tow antioxidant status are involved phases of atherosclerosis. Am J C/in Nutr l997;65:l2l-7. KEY WORDS selenium, carotid
meaca-
have
Jean
8-1 1). Until now, epidemiologic between lipid peroxidation and
Art#{233}-
was composed of 1187 any history of coronary
rotid arteries (CCAs) and at the site of plaques. After for conventional cardiovascular risk factors, erythrocyte was
of anathero-
Touboul,
ies
ef-
carotid
sur le Vieitlisement
The study sample 59-7 1 y without
artery disease or stroke. surements of intima-media
markers
assessed
baseline
aging
the
on atheroscteroby thiobarbituric
biological
to ultrasonographicalty
of
Pierre-Jean
Nantes, H#{244}pital
1996.
publication
September
3, 1996.
121
Downloaded from www.ajcn.org by guest on July 17, 2011
tioxidant
Berr,
BONITHON-KOPP
122
if he or she was in the desirable
age range.
visit,
June
which
subjects
took
were
nations
of
place
between
recruited
their
During
1991
and
and high-resolution
carotid
arteries
the baseline
July
1993,
ultrasound
were
performed
them. The study protocol was approved thique du Centre Hospitalier Universitaire and written informed consent was
1389
exami-
in
1384
of
by the Comit#{233}d’dde Kremlin-Bic#{234}tre obtained from all
Ultrasonography Ultrasound
examinations
using
the
transducer age
were
Aloka of
of 0.30
of B-mode
performed
SSD-650,
frequency
resolution
axial
(Aloka,
were
four
Co.
MHz. This Acquisition,
7.5
mm.
images
by
measurements
were
it involved
Tokyo)
with
with
the
time
carotid
wall
of
the
(17).
mid
arteries
All
Optimal
mean
of the
defined
vessel
images
right
and
were
and
as localized
lumen
left
distal
wall
for
by
echo
which
the
the
on was
structures
IMT
sonographer
was
the
of
focused
the arterial
plaque
showing
a plaque
the
best
For all arterial were
shown
711
segments, stored
on
of
as
the
an
optical
used
CCA the
greatest
optimal an optical
or bifurcationwas
encroachment
into
the
an estimation
adjacent
that
both
the inter-
reduced as
with
plaque
could
of the IMT
after
software. absolute
using
and
intraobserver
the
repositioning
study
and were missing
those
who
(myocardial
(June-Octo-
excluded data
reported
infarction
from
in IMT any
or
history
angina
and/or
(n = 106) were also excluded. composed of 1 1 87 subjects (476
Thus, men
the and
Medical
were
question-
history participants that
gave
occupation, as smoking
administered
information
a standardized
about
demographic
medical history, drug use, and alcohol consumption. classified
background,
and personal With respect
as current
Life-long
habits such to smoking
smokers,
exposure
former
to smoking
was
estimated by the number of pack-years obtained by multiplying the average number of packs of cigarettes smoked per day the number of years Alcohol consumption
mate
of the
expressed systolic rest
average
amount
tensiometer the
was
of myocardial
in
infarction,
weekly
and
measurements of made with a digital
Spengler,
used
by
20 cigarettes). the subject’s esti=
ingested
Two independent pressure were
(SP9;
mean
pack from
of alcohol
as mL alcoholld. and diastolic blood
and
also
of smoking (1 was determined
Pans)
after
the
analysis.
angina
pectoris,
a tO-mm
Self-reported or stroke
was
recorded.
Laboratory
procedures
Blood 12-h
samples
fast.
lected
were
in evacuated
> 98%
drawn
Ten-millimeter
between
volumes
containers
with
heparin
of the participants.
Erythrocyte at 500 X g for
blood
ature. Plasma erythrocytes
and were
buffy washed
Plasma and erythrocyte until assayed. Standard formed daily and other
coats in
0800 and peripheral
of
were
obtained
for
pellets were obtained 15 mm at room temper-
were sterile
preparations laboratory determinations
0900 after a blood cot-
then removed 9 g NaCl/L
and the solution.
were stored at - 80 #{176}C determinations were perwithin 2 mo.
and
be
Assay
ofp/asma
interface.
Plasma
transversal
absorption
variabilities
procedure functions
were
selenium
was
disk.
the scanning
values ob(r = 0.71)
mm
sonographer
media-adventitia
longitudinal
were
in the
with
14) and
=
disease
stroke was
by centrifuging
profile
plaques
the
that
in
examination
mineralized, so
into
density
several
(n
0.07
women).
electronic
in the
of electronic
the
Subjects
respectively,
(18).
early
unreliable
77).
=
were
encroaching
(ie,
the
and
the
associated
EUREQUA
(n
smokers.
quality assessment, a rereading subsamples of images of both
stantially pressed
analysis
subjects
study was made on CCAs and bifurcationinternal carotid arteries as described elsewhere ( 17). Mean absolute differences and correlation coefficients between repeated readings were 0.06 mm and 0.82, respectively, for longitudinal CCA-IMT (n = 81), and 0.40 mm and 0.78 for plaque thickness (n = 52). In a previous study by our group, it was
the
were
respectively
considered
or never
were scanned occurrence of
placement
artery),
incidence
by extrapolating
CCA-IMT
were
smokers,
1 mm. The quanby measuring the IMT perpendicularly to the
When
was
on the far wall
images
and
segment
carotid
When
to obtain
For random
1991)
mm
performed
behavior,
be computer-assisted
inflections
internal
on the
vizualized made
interfaces
same
the
lumen.
had
could
at the site of plaque.
on
origin
of
examining
taken
present
the
and
identification
curve
ber
0.77),
=
exams
Briefly,
CCA
stored
CCA-IMT
tification of plaque thickness was made at the site of the maximal encroachment
calipers
(r
mm
Ultrasound
history
plaques
the
0.06
repositioning
(CCAs),
(first 2 cm) of the thickness (IMT) was
Both near and far walls of all arterial segments longitudinally and transversally to assess the
vessel
and
naire
analysis.
the
with
All
(EURE-
of examination.
common
and the origin The intima-media
far
plaques. The
procedures
a software
distance between the lumen-intima interface and the mediaadventitia interface by using an automated edge detection algorithm. Two longitudinal measurements of IMT were completed on both the right and left CCAs at a site free of any disk.
tamed
(r = 0.62), corresponding
0.10
whereas
solved in 0.1 mol plateform graphite
tion was obtained and
interassay
determined
by
(EAAS)
HNO3). furnace
by using CVs
Spectrophotometric
of
After
extracted
of the
was
spectrometry
electrothermal
(Perkin
Elmer
atomic 5100;
Elmer, Norwalk, CT) using Zeeman background (19). Plasma samples were diluted in 0.2% Triton
sub-
The aforementioned variabilities (cxdifferences and correlation coefficients)
selenium
was
precipitation
measured
nm (Uvikon tions
with
were
with calculated
standard
were
1 .4%
assay
of plasma
of proteins
hexane 860;
Nickel (10 as a modifier. and
with
a spectrophotometer on
Rotkrcuz, the
basis
.tg) was added in the Selenium concentra-
addition 1 .8%,
calibration.
Intra-
respectively.
carotenoids ethanol,
and the absorbance
Kontron,
Perkin
correction XlOO (dis-
carotenoids
were
of the hexane at 350,
Switzerland). of a molecular
450,
phase and
Concentraextinction
550
Downloaded from www.ajcn.org by guest on July 17, 2011
the carotid bifurcations, internal carotid arteries. on
at the
of the
a
system provides an processing, and stor-
computer-assisted
made
scanning
discrete
and
pectoris) or study sample
sonogra-
program specially designed for longitudinal studies QUA; TSI, Champs sur Mane, France) (18). Details of the protocol were described elsewhere
measured
were 0.10 mm (r = 0.58) with standard procedures
plaque assessment of coronary artery
participants.
phers
ET AL
TBARS, factor
at 450
350
and
550
at 450
nm
of
nm
were
CVs
HPLC
were
assay
saline
solution
cell
acetate
analysis, ble
for
of
assay
hexanc
8%
using
and
was
diluted
(20).
After
was
dried
react
59 subjects
plex. tanol.
The colored complex was The test tubes were stirred X g before the fluorescence
1000 emission
wavelengths
of 532
Tetraethoxypropanc and interassay CVs
was were
the
553
possi-
antioxidant
biochemical
(reference
corn-
reference
many)
and
the
Intra-
main
enzymatic
759350), respectively, supplied by cose concentrations were determined oxidase method (reference 61274; High-density-tipoprotein
(HDL)
zymatically
after
precipitation
tipoproteins
with
phosphotungstic
reference was
Statistical
Institute,
in Table
carotid
plaques
are
given
each
acid
Inc, Cary, analyses.
by
tested
sion
(carotid
NC)
Crude
and
of both
by linear
en-
women The oxidant
separately
were
Because
of
sex-related
differences
plaques
and
antioxidant
biomarkers
performed analyses
variables
were and
used data
Main
performed conventional
mean
(CCA-IMT)
presented
TBARS
or logistic
regres-
biomarkers
and and
the
by
and
variables.
relations
TBARS,
women
between analysis
separately.
adjusting
cardiovascular
as
with
as dependent in
and
and
antioxidant
in
higher
quartile
terms
between
were
was Mul-
Study and
higher
2. Significantly and
decrease plasma
this
vanand
also
tested.
higher
are of
concentrations
TBARS
were
was
inversely
found
was
between
with
in
both related
of
observed
in
of anti3. There
increasing
men
con-
and
women.
to erythrocyte
significant
only
CCA-IMT
and
vi-
in men. No both plasma
1
general
and in the
ultrasound EVA
characteristics
of the
59-7l-y-old
Study
Men
Women (n711)
(n476)
Age
population the prevalence
in men than in women
in CCA-IMT
carotenoids
association
was
(y)
65.0 plaques
(%)
index
blood
(mm)
(kg/m2)
pressure
blood cholesterol
Triacylglycerol (mmolIL)
Smoking
habits
(mm
pressure
64.9
(mm
Hg) Hg)
± 2.9
16.0
0.70
± 0.14
0.65
±
26.0
± 3.4
24.6
±
137.0
±
18.4
128.6
81.6
±
1 1.5
77.9
±
± 16.6
6.57
± 0.99
(mmollL) (mmollL)
1.46 4.07
±
1.78
± 0.42
± 0.88
4.27
± 0.92
1.41
± 0.99
1.15
± 0.53
5.75
±
1.01
5.31
± 0.78
0.37
(%) 24.6
83.0
Former
59.7
12.5
Current
15.8
4.5
0-40(mL/d)
13.3
35.6
40-80
57.9
59.7
use
(%)
(mUd)
>80(mL/d) Years
of education
l3y
risk
factors
‘1
y
±
SD.
10.5
6. 17 ± 0.94
Never
Alcohol
0.1 1 3.9
(mmoLIL)
(mmollL)
Glucose
± 3.0’
25.8
thickness
mass
HDL cholesterol LDL cholesterol
System
carotid and TBARS. between carotid
biomarkers
for
and distribution indexes of plasma erythrocyte vitamin E, and TBARS
biomarker
CCA-IMT
Diastolic
are
variable
CCA-IMT
significantly
The means carotenoids,
of
TABLE
Systolic
univariate
biomarkers
introduced
in men
for
associations
antioxidant where
were
E but
Body
cholesterol
Analysis
ratios
increase
as a dichotomous the first quartile)
of the EVA
1. The
a progressive
centrations
Intima-media
(precipitant,
LDL
Interaction
glucose. systemati-
than in men. mean concentrations of CCA-IMT by quartiles biomarkers and TBARS are shown in Table
Carotid
(22).
of subjects
antioxidant
TEARS
systematically
were
descriptive
regression
plaques)
quartile.
as a continuous
in Table
population
measured
Mg2
Statistical
and muttivariate and
were
graphic
(reference
B-containing
formula
or percentages
quartiles
univariate
atherosclerosis
tivariate
was
Mannheim).
the
first
odds
1 SD
or in each
biomarker considered first quartile or above
antioxidant
Total
of CCA-IMT
plaques
Linear
chotesterot
from
the
TBARS
Biom#{233}rieux (Paris). Gluby the enzymatic glucose Boehringer Mannheim).
Fnedewatd
procedures
multivaiate
means
the
Ger-
analysis
Standard
(SAS
with
kit
of apolipoprotein
Boehringer
543004;
computed
Mannheim,
triacylgtycerol
and
and
for
use
consump-
were
Adjusted
either
included
alcohol
triacylglycerols, variables
models.
calculated
characteristics
shown
Similarly,
were performed cholesterol kit
Mannheim,
regression
They pack-years,
of education,
cholesterol, confounding
considered
association
Boehringer PAP
to
antioxidant (below the
was
1,1,3,3
indexes
61227;
pressure,
years
were
(P < 0.001). selenium and
at pH 3
respectively.
assays enzymatic
blood
or biological
sex.
RESULTS
accordaldehyde
used as an external standard. 2. 1% and 3.2%, respectively.
Total cholesterol and triacylglycerol by enzymatic methods using the PAP
systolic
biomarkers
The
acid
in the
TBARS one
drugs,
plaques
tamin Standard
index,
with
in at least
and
a pink-colored
nm,
added
carotid
each able
then extracted by using 1-buvigorously and centrifuged at was read at excitation and
and
associated status
the
Intra-
of thiobarbituric #{176}C) to form
(95
mass
TBARS
from
revealed
catty
respectively.
two molecules
temperature
body
by
under
fluorometry
with
elevated
E
An external
E components.
9%,
significantly of antioxidant
29.1
2.7
(%) 12.4
18.7
65.8
66.9
21.8
14.3
Downloaded from www.ajcn.org by guest on July 17, 2011
and
red
of a-tocoph-
Plasma concentrations of TBARS were determined to the method of Richard et al (21). Briefly, the
groups
in
vitamin
standard
determination
123
tion, HDL and LDL Alt of these potential
detector.
vitamin
were
age,
at 290 nm was performed
excluded
that
markers
and inter-
ethanol,
phase
We
ATHEROSCLEROSIS
of lipid-lowering
with
an internal
quantity
were
Intra-
pyrogallol
and
artificial
CVs
TBARS
1%
The
used.
whom
presence
interassay
ing
with
to a spectrophotometer
were
at
absorbance
E (a-tocopherol)
precipitated
of a-tocopherol
Absorbances
‘.
obtained
.tL red blood cells
of 400
AND
respectively.
and quantification
coupled
cm
.
the
equation.
4.9%,
hexane.
stream
‘
vitamin
were
with
a nitrogen standard
and
combined
extracted
erol
5.4%
proteins
mol
an adequate
volume
blood
.
to correct
of erythrocyte
The sample
by HPLC
L
used
nm by applying
assay
was
134 000
ANTIOXIDANTS,
BONITHON-KOPP
124 TABLE 2 Means and distribution
indexes
of antioxidant
biomarkers
ET
and thiobarbituric
AL
acid-reactive
substances
(TBARS)
Men n
Women
SD
I ±
Quartile
values’
n
SD
I ±
Quartile
values’
pjnoLfL Plasma selenium Plasma carotenoids Erythrocyte vitamin TBARS ‘
Values
E
are the upper
Significantly
2-4
467
1.08
±
0.21
0.93,
1.07,
1.22
684
1.10
± 0.202
0.97,
1.10,
1.23
467
2.20
±
1 .50,
2.07,
2.80
687
3.07
±
2. 14, 2.90,
3.73
443
5.27
±
0.99 1.27
4.50,
5.26,
6.02
663
5.50
± l.27
447
2.91
± 0.39
2.62,
2.86,
3.14
665
2.97
±
cutpoints
different
of quartiles
from
men
t test):
0.05,
p
