Effect of Cardiorespiratory Fitness on Vascular Regulation
and Oxidative Stress in Postmenopausal Women
Vincent Pialoux, Allison D. Brown, Richard Leigh, Christine M. Friedenreich, Marc J. Poulin
Downloaded from http://hyper.ahajournals.org/ by guest on June 16, 2017
Abstract—Increasing evidence exists suggesting an important role for oxidative stress in the pathogenesis and progression
of hypertension in women via a decrease of NO production after menopause. Regular physical training has been shown
to upregulate antioxidant enzymatic systems, which may slow down the usual increase of oxidative stress in
postmenopausal women. The aims of this study were to determine the impact of fitness status on enzymatic antioxidant
efficiency, oxidative stress, and NO production and to determine the associations among oxidative stress, enzymatic
antioxidant and NO production, mean arterial blood pressure (MABP), and cerebrovascular conductance (CVC) in
postmenopausal women (n⫽40; 50 to 90 years old). Physical fitness, physical activity, resting MABP, and CVC were
measured. End product of NO, lipid peroxidation (malondialdehyde and 8-iso-prostaglandin F2␣), DNA oxidation
(8-hydroxy-2⬘-deoxyguanosine), protein nitration (nitrotyrosine), antioxidant glutathione peroxidase, and catalase
activities were measured in plasma. We identified significant negative associations between oxidative stress and indices
of physical fitness (malondialdehyde: r⫽⫺0.33, P⬍0.05; 8-iso-prostaglandin F2␣: r⫽⫺0.39, P⬍0.05; 8-hydroxy-2⬘deoxyguanosine: r⫽⫺0.35, P⬍0.05) and physical activity (malondialdehyde: r⫽⫺0.30, P⬍0.05; 8-iso-prostaglandin
F2␣: r⫽⫺0.41, P⬍0.01; 8-hydroxy-2⬘-deoxyguanosine: r⫽⫺0.39, P⬍0.05). Conversely, glutathione peroxidase was
positively correlated with fitness level (r⫽0.55; P⬍0.01). Finally, MABP and CVC were significantly associated with
8-hydroxy-2⬘-deoxyguanosine (MABP: r⫽0.36, P⬍0.05; CVC: r⫽⫺0.36, P⬍0.05), nitrotyrosine (MABP: r⫽0.39,
P⬍0.05; CVC: r⫽⫺0.32, P⬍0.05), and the end product of NO (MABP: r⫽⫺0.57, P⬍0.01; CVC: r⫽0.44, P⬍0.01).
These findings demonstrate that, after menopause, fitness level and regular physical activity mediate against oxidative
stress by maintaining antioxidant enzyme efficiency. Furthermore, these results suggest that oxidative stress and NO
production modulate MABP and CVC. (Hypertension. 2009;54:1014-1020.)
Key Words: oxidative stress 䡲 antioxidant enzymes 䡲 NO end products 䡲 menopause 䡲 fitness 䡲 blood pressure
T
radical scavengers prevents development of arterial hypertension in most hypertensive models in animals.1,5
Although the beneficial effect of hormone therapy on
cardiovascular risk after menopause is still controversial,6,7
the increased incidence of cardiovascular disease in postmenopausal women compared with premenopausal women8
may suggest that estrogen may have a protective effect on the
cardiovascular system. Oxidative stress is reported to increase
after menopause,9 suggesting that the decrease in sex hormones occurring at the time of menopause could predispose
women to higher levels of ROS.
Estradiol is thought to have antioxidant properties, because
it increases the expression of antioxidant enzymes and decreases NADPH oxidase enzyme activity and superoxide
production. Some studies have suggested that the increase in
oxidative stress in postmenopausal women could underlie, at
least in part, the mechanism whereby postmenopausal women
are at increased risk of cardiovascular disease.8 Indeed, it has
here is increasing evidence to implicate an important role
for reactive oxygen species (ROS) in the pathogenesis
and progression of arterial hypertension.1 Indeed, an increase
in ROS may decrease NO bioavailability in the vasculature
and further lead to endothelial dysfunction.2 This hypothesis
is supported by mechanistic evidence whereby superoxide
reacts with NO synthesized by endothelial NO synthase to
form the oxidant peroxynitrite, thereby inducing NO inactivation and an uncoupling of endothelial NO synthase.2
In this context, oxidative stress, defined as the imbalance in
favor of ROS generation over antioxidant defense, is usually
higher in the hypertensive population, as shown by accumulation of ROS end products from oxidized lipids and DNA in
patients with essential hypertension.3 Moreover, studies on
spontaneously hypertensive rats suggest that oxidative stress
involves enhanced NADPH oxidase activity, leading to ROS
overgeneration and dysfunctional endothelial NO synthase.1,4
Conversely, inhibition of ROS generation or treatment with
Received July 8, 2009; first decision July 27, 2009; revision accepted August 31, 2009.
From the Departments of Physiology and Pharmacology (V.P., A.D.B., R.L., M.J.P.), Medicine (R.L.), and Clinical Neurosciences (M.J.P.), Hotchkiss
Brain Institute (M.J.P.), and Libin Cardiovascular Institute of Alberta (M.J.P.), Faculty of Medicine, and Faculty of Kinesiology (V.P., M.J.P.), University
of Calgary, Calgary, Alberta, Canada; Alberta Health Services (C.M.F.), Calgary, Alberta, Canada.
Correspondence to Marc J. Poulin, Department of Physiology and Pharmacology, University of Calgary, HMRB-210, 3330 Hospital Dr NW, Calgary,
Alberta T2N 4N1, Canada. E-mail [email protected]
© 2009 American Heart Association, Inc.
Hypertension is available at http://hyper.ahajournals.org
DOI: 10.1161/HYPERTENSIONAHA.109.138917
1014
Pialoux et al
Downloaded from http://hyper.ahajournals.org/ by guest on June 16, 2017
been shown recently that reduced carotid artery compliance
of sedentary postmenopausal women was partially restored
by infusion of ascorbic acid.10 Interestingly, it also appears
that this compliance is not different between exercise-trained
postmenopausal and premenopausal women.11 Taken together, these data suggest that regular exercise might suppress
the reduction of arterial compliance via a decrease in oxidative stress. In this context, it has been demonstrated clearly
that, in a premenopausal population, regular physical training12,13 upregulates antioxidant enzymatic systems, which
may have implications for attenuating the usual increase in
postmenopausal-related oxidative stress. This paradigm is
supported by 2 recent studies14,15 that reported a decrease in
plasma thiobarbituric acid reactive substances after 8 and 24
weeks of exercise training in postmenopausal women. However, a recent study reported no significant difference in lipid
peroxidation between premenopausal and postmenopausal
women randomly matched for body mass index (BMI) and
waist circumference.16 The correlation reported between oxidative stress and body fat17 might suggest that the increase in
fat mass occurring at menopause18 may partially explain the
elevated levels of oxidative stress markers after menopause.
Finally, we demonstrated recently that cardiorespiratory
fitness and cerebral blood flow significantly impact cognitive
outcomes in older women.19 In addition, recent findings
demonstrating that exercise restored endothelium-dependent
dilation via improvement of antioxidant enzyme efficiency
and NO bioavailability in old mice.20 Here, we tested the
hypothesis that increases in antioxidant enzyme efficiency
and decreased oxidative stress, leading to NO bioavailability,
might be a potential mechanism to account for this observed
association between fitness and cardiovascular parameters. In
the current study, we investigated the impact of fitness status
on enzymatic antioxidant efficiency, oxidative stress, and NO
production in the same study population of postmenopausal
women who had participated in our original study.19 Secondary objectives were to determine the strength of the associations among oxidative stress, enzymatic antioxidant and NO
production, and arterial blood pressure and cerebrovascular
regulation.
Methods
Protocol
The study design and participant sample have been described
previously.19 In brief, a cross-sectional study was conducted of 42
white postmenopausal women aged 50 to 90 years identified in the
community through advertisements at the University of Calgary,
Calgary Health Region, and in the general community. The study
protocol was approved by the conjoint health research ethics board at
the University of Calgary. Additional details are included in an
online Data Supplement (please see http://hyper.ahajournals.org).
The subject’s menopausal status was determined by a self-report
of absence of menses for ⱖ12 months and by the assessment of
ovarian hormones levels. Additional details are included in the
online Data Supplement.
Subjects refrained from eating or drinking any caffeine-containing
beverages for ⱖ4 hours before each of the testing sessions. The first
visit was a screening session and involved collecting participant
demographic data and anthropometric measurements, completion of
a lifestyle questionnaire adapted from the Past Year Total Physical
Activity Questionnaire21,22 (from which a level of physical activity in
the past 12 months was estimated as the metabolic equivalent hours
Oxidative Stress and Blood Pressure in Women
1015
per week of activity), spirometry testing, and a carotid artery
ultrasound screening. The second visit involved a graded exercise
test on a modified recumbent cycle ergometer to determine maximal
oxygen consumption (V̇O2max), as described by Brown et al.19
During the third session, blood testing was preformed. Resting blood
pressure and transcranial Doppler ultrasound recordings from the
right middle cerebral artery were made while subjects sat quietly.
Systolic blood pressure, diastolic blood pressure, and mean arterial
blood pressure (MABP) were determined as the mean of 3 values
using an automated blood pressure monitoring device (Dinamap;
Johnson and Johnson Medical, Inc). MABP recorded was also
continuously recorded at the finger by photoplethysmography (Portapress, TPD Biomedical Instrumentation).
Measurements of middle cerebral artery peak velocity, as an index
of cerebral blood flow, were made at rest using transcranial Doppler
ultrasound.23,24 Additional details are included in an online Data
Supplement.
Biochemical Analyses
Blood was collected from the antecubital vein in two 7-mL EDTA
tubes for biochemical analysis at the beginning of the third session.
The plasma was obtained by centrifugation of the samples at 1000g
for 10 minutes at 4°C. Plasma was separated into aliquots and frozen
at ⫺80°C until assays could be performed.
Plasma levels of oxidative stress (ie, 8-hydroxy-2⬘deoxyguanosine [8-OHdG], malondialdehyde [MDA], and 8-isoprostaglandin F2␣ [8-iso-PGF2␣]), antioxidant enzymatic activities
(ie, plasma glutathione peroxidase [GPX] and catalase activities),
end product of NO (nitrites and nitrates), and ovarian hormone levels
were measured at rest. Additional details are included in an online
Data Supplement.
Statistical Analysis
To examine the effects of fitness on our primary outcome variables,
we divided the study population into 2 groups, on the basis of
aged-predicted V̇O2max values.25,26 Participants assigned to the fit
group had a V̇O2max ⬎100% of age-predicted values, whereas
participants assigned to the sedentary group had a V̇O2max ⬍100%
of age-predicted values.
Independent sample t tests were used to compare the fit and
sedentary groups. Pearson correlation coefficients between vascular
measures and biochemical markers (oxidative stress and enzymatic
antioxidant markers and end product of NO) were estimated.
Regression analyses were also controlled for BMI and fat mass using
partial correlations. Because of the exploratory nature of the study,
we did not perform multiple linear regressions. Statistical analyses
were performed with SPSS (version 15.0, SPSS, Inc). Differences
were considered significant at Pⱕ0.05.
Results
Of the 42 women who were tested, 2 subjects were excluded
from the statistical analyses; 1 woman did not complete the
V̇O2max test, and a blood sample was not collected on
another woman. Thus, of the 42 subjects tested, complete data
sets were available on 40 women (19 fit and 21 sedentary).
However, CVC data were calculated on only 39 subjects
because of the failure to locate a suitable transcranial Doppler
signal in 1 woman.
Anthropometric, fitness, and physical activity data of our
cohort are presented in Table 1. The study population
included older women who were, on average, moderately
active and fit, normal weight for height, and with a mean level
of education equivalent to a university undergraduate diploma. A difference between the fit and sedentary women
was found, particularly with respect to education, BMI, and
physical fitness.
1016
Hypertension
November 2009
Table 1.
Descriptive Characteristics of the Study Population
Fitness Groups
Variables
Age, y
Height, cm
Overall (n⫽40)
Fit (n⫽19)
Sedentary (n⫽21)
65.1⫾8.4
67.8⫾8.1
62.1⫾7.8
161.3⫾6.0
160.2⫾4.9
161.9⫾6.5
Weight, kg
66.7⫾10.0
61.9⫾7.2
71.1⫾10.5*
BMI, kg/m⫺2
25.6⫾3.7
24.2⫾2.7
28.2⫾3.9*
36.5⫾5.3
34.7⫾5.3
38.4⫾4.9*
25.6⫾5.6
28.9⫾4.8
22.3⫾4.3*
1.67⫾0.3
1.79⫾0.3
1.55⫾0.2*
101.2⫾22.6
119.6⫾12.8
82.9⫾13.5*
Physical activity, MET-h 䡠 wk⫺1 䡠 y⫺1
81.2⫾47.6
98.3⫾48.3
48.5⫾20.5*
Serum estradiol, pmol 䡠 L⫺1
25.7⫾37.4
13.8⫾35.1
34.8⫾37.3
Serum progesterone, nmol 䡠 L⫺1
1.13⫾0.54
1.19⫾0.47
1.44⫾0.57
Body fat, %
⫺1
VO2max relative, mL 䡠 kg
⫺1
䡠 min
VO2max absolute, L 䡠 min⫺1
VO2max percentage predicted, %
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Values represent mean⫾SD. Participants assigned to the fit group had a VO2max that was ⬎100%
of age-predicted values, whereas participants assigned to the sedentary group had a VO2max defined
as ⬍100% of age-predicted values. Vo2max indicates maximal oxygen consumption; MET, metabolic
equivalent.
*Data were significantly different from fit group by Pⱕ0.05.
Vascular Outcomes
MABP was higher in the sedentary group compared with the fit
group (77.3⫾12.6 versus 71.6⫾13.3 mm Hg; P⬍0.05). The
increase in MABP was attributable mainly to a higher systolic
blood pressure (113.0⫾20.7 versus 103.3⫾14.2 mm Hg;
P⫽0.045) in the sedentary group, although diastolic blood
pressure was also somewhat higher in this group (57.0⫾11.6
versus 52.0⫾11.3 mm Hg; P⫽0.08) as compared with the active
group. CVC was higher in the fit group compared with the
sedentary group (1.15⫾0.21 versus 1.01⫾0.17 cm.s⫺1.mm Hg⫺1;
P⫽0.04).
Ovarian Hormones
Group mean values of serum estradiol and progesterone are
included in Table 1. Estradiol and progesterone were not
significantly correlated with any antioxidant variables.
Biochemical Outcomes
End product of NO (NOx) was not statistically significantly
different in the fit group (40.2⫾21.5 mmol.L⫺1) compared
with the sedentary group (33.7⫾17.3 mmol.L⫺1; Table 2).
Oxidative stress was evaluated by analyzing blood oxidation
markers (Table 2). The sedentary group exhibited values that
were 270% and 93% higher than the fit group for 8-isoPGF2␣ (P⫽0.001) and 8-OHdG (P⫽0.02), respectively. The
antioxidant status response was evaluated by analyzing blood
antioxidant enzymatic activities (Table 2). GPX and catalase
activities were 26% (P⬍0.0001) and 36% (P⫽0.05) higher,
respectively, in the fit group (Table 2).
Physical Activity and Physical Fitness
Physical activity was positively correlated with absolute
V̇O2max (r⫽0.43; P⫽0.006) and V̇O2max expressed as a
percentage of aged-predicted values (r⫽0.48; P⫽0.003).
Correlation analyses between oxidative stress markers and
fitness parameters (with and without controlling for BMI and
fat mass) are presented in Table 3. The correlations between
V̇O2max (normalized for body weight) and oxidative stress
markers are presented in an online Data Supplement (Table
S1 and Figure S1, available at http://hyper.ahajournals.org).
Starting with markers of oxidative stress, 8-OHdG, 8-isoPGF2␣, and MDA were negatively correlated with V̇O2max
expressed as a percentage of age-predicted values and with total
physical activity over the past year (Figure 1 and Table 3). The
correlation with 8-OHdG and 8-iso-PGF2␣ remained statistically significant, even after controlling for BMI and fat mass.
Turning to markers of antioxidant enzyme activity, GPX
was positively correlated with V̇O2max expressed as a percentage of age-predicted values (Table 3). The correlations
between V̇O2max (normalized for body weight) and antioxidant enzyme activities are presented in an online Data
Supplement (Table S1 and Figure S2).
Looking at the relationships between oxidative stress and
antioxidant enzyme activity, GPX was negatively correlated
with 8-OHdG (r⫽⫺0.39; P⫽0.011) and with 8-iso-PGF2␣
Table 2. Mean Values of Plasma MDA, 8-OHdG, 8-iso-PGF2␣,
Nitrotyrosine, Catalase, and GPX Activities and NOx
Variables
Fit (n⫽19)
Sedentary (n⫽21)
8-OHdG, ␮g 䡠 L⫺1
51.3⫾54.0
98.9⫾70.5†
MDA, ␮mol 䡠 L
4.19⫾0.87
4.62⫾0.70
8-Iso-PGF2␣, ng 䡠 L⫺1
3.62⫾3.60
9.78⫾5.07†
Nitrotyrosine, nmol 䡠 L⫺1
157⫾19
163⫾18
GPX, ␮mol 䡠 L⫺1 䡠 min⫺1
14.1⫾2.7
11.1⫾1.5*
⫺1
Catalase, ␮mol 䡠 L⫺1 䡠 min⫺1
NOx, ␮mol 䡠 L⫺1
11.70⫾0.83
8.6⫾4.1*
40.2⫾21.5
33.7⫾17.3
Values represent mean⫾SD. Participants assigned to the fit group had a
VO2max that was ⬎100% of age-predicted values, whereas participants
assigned to the sedentary group had a VO2max defined as ⬍100% of
age-predicted values.
*Data are significantly different from the fit group at Pⱕ0.05.
†Data are significantly different from the fit group at Pⱕ0.01.
Pialoux et al
Oxidative Stress and Blood Pressure in Women
Table 3. Correlations Among Fitness Parameters and Plasma
Oxidative Stress and Antioxidant Enzyme Activity Controlled for
BMI and Fat Mass
Variables
P
Controlled for
BMI and Fat
Mass
P
8-OHdG, ␮g 䡠 L⫺1
vs predicted VO2max, %
⫺0.35
0.033
⫺0.35
0.04
vs physical activity,
MET-h 䡠 wk⫺1 䡠 y⫺1
⫺0.38
0.015
⫺0.33
0.05
MDA, ␮mol 䡠 L
vs physical activity,
MET-h 䡠 wk⫺1 䡠 y⫺1
⫺0.33
⫺0.30
0.03
0.05
⫺0.31
⫺0.30
0.06
0.07
8-iso-PGF2␣, ng 䡠 L⫺1
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vs physical activity,
MET-h 䡠 wk⫺1 䡠 y⫺1
⫺0.42
0.007
⫺0.37
0.03
vs predicted VO2max, %
⫺0.52
0.001
⫺0.35
0.04
0.55
0.001
0.58
GPX, ␮mol 䡠 L⫺1 䡠 min⫺1
vs predicted VO2max, %
14
12
10
8
6
4
2
⫺1
vs predicted VO2max, %
18
16
8-Iso-PGF2α (ng.l-1)
Pearson
Correlation
Coefficient
1017
0.001
VO2max indicates maximal oxygen consumption; MET, metabolic equivalent.
Correlation analyses were conducted on data from all of the volunteers (n⫽40).
(r⫽⫺0.29; P⫽0.05), whereas catalase was negatively correlated with 8-iso-PGF2␣ (r⫽⫺0.37; P⫽0.01).
Associations Among Oxidative Stress, NO Level,
and Vascular Parameters
Correlations among oxidative stress, NO levels, and vascular
parameters are presented in Table 4. Briefly, oxidative stress
markers 8-OHdG and nitrotyrosine were positively correlated
with MABP and negatively correlated with CVC and NOx.
NOx levels negatively correlated with MABP (Figures 2 and 3)
and positively correlated with CVC. The correlations among
oxidative stress, NO levels, and systolic blood pressure and
diastolic blood pressure are presented in an online Data
Supplement (Table S2).
Discussion
This is the first human study to examine the correlations
among fitness, oxidative stress, NO production, MABP, and
cerebrovascular resistance. We found greater oxidative stress
correlated with higher MABP values and lower values for
CVC and NO. Lower levels of NO production correlated with
higher MABP and lower CVC. Second, in support of the
hypotheses being tested, postmenopausal women with higher
fitness levels had higher antioxidant enzyme activity and
lower levels of oxidative stress. These results strengthen the
notion that the elevated MABP reported in women after
menopause is associated with an increase in oxidative stress
and can be positively modulated by maintaining antioxidant
enzyme efficiency through physical activity.
Effect of Fitness Level on Oxidative Stress
It has been suggested previously that the decrease in levels of
estrogen, and a loss of its natural antioxidant protective
effects, predisposes postmenopausal women to higher levels
of oxidative stress. The finding in this study of a positive
0
40
60
80
100
120
140
160
•
Predicted VO2 max (%)
Figure 1. Relationship between predicted V̇O2max and plasma
concentrations of 8-iso-PGF2␣ in postmenopausal women
(r⫽⫺0.52; P⫽0.001). E (n⫽21), Sedentary group (⬍100% agepredicted V̇O2max); F (n⫽19), fit group (⬎100% age-predicted
V̇O2max).
effect of fitness on oxidative stress in women is consistent
with previous reports.12,13 In the present study, oxidative
stresses assessed by plasma lipid oxidation, DNA oxidation,
and protein nitration markers were negatively correlated with
our 2 indices of physical fitness (ie, absolute V̇O2max, and
V̇O2max expressed as the percentage of aged-predicted
values25,26) and with the index of total physical activity over
the past year. These data strengthen the conclusion of recent
studies that reported decreases in oxidative stress (using
thiobarbituric acid reactive substances as an outcome marker)
after endurance training in postmenopausal women.14,15 In the
current study, we now provide evidence that fitness might
positively decrease oxidative stress and partially attenuate the
effects of aging on oxidative stress.27
The negative correlations between the antioxidant enzyme
activities and both 8-OHdG and 8-iso-PGF2␣ suggest that the
lower oxidative stress occurring with fitness is driven by an
improvement of the antioxidant enzymatic defenses, as demonstrated in the general population28,29 and the elderly.30 In
this context, the higher values of the antioxidant enzymes
GPX and catalase found in the fit group and the positive
relationship between GPX and the fitness variables support
the paradigm that regular exercise training upregulates antioxidant defenses in response to the acute increase of ROS
generation during a single bout of exercise.28
Although correlations between obesity and oxidative stress
have been reported in the literature, we did not find a
statistically significant correlation among BMI, fat mass, and
oxidative stress in this present study. This lack of correlation
is likely explained by the exclusion of obese subjects from the
present study (ie, BMI ⬎30).
Effect of Oxidative Stress on Blood Pressure and
Cerebrovascular Function
Although oxidative stress has been reported to contribute
to the increase in arterial blood pressure and the subsequent progression of hypertension,1 we are the first to
report a positive association between oxidative stress and
blood pressure in a normotensive population of postmeno-
1018
Hypertension
November 2009
Table 4. Correlations Among Cardiovascular Parameters and Plasma Oxidative
Stress and End Products of NO Controlled for BMI and Fat Mass
Pearson Correlation
Coefficient
Variables
P
Controlled for BMI
and Fat Mass
0.01
0.52
P
Nitrotyrosine, nmol 䡠 L⫺1
vs MABP, mm Hg
vs CVC, cm 䡠 s
⫺1
0.41
⫺1
䡠 mm Hg *
vs NOx, ␮mol 䡠 L⫺1
0.32
0.04
0.003
0.33
0.05
⫺0.62
⬍0.001
⫺0.62
⬍0.001
0.43
0.006
0.33
0.05
⫺0.33
0.05
8-OHdG, ␮g 䡠 L⫺1
vs MABP, mm Hg
vs CVC, cm 䡠 s
⫺1
⫺1
䡠 mm Hg *
vs NOx, ␮mol 䡠 L⫺1
⫺0.37
0.04
⫺0.25
NS
0.22
NS
0.10
NS
0.06
NS
⫺0.27
NS
⫺0.30
NS
0.18
NS
⫺0.12
NS
0.10
NS
0.29
NS
⫺0.18
NS
8-iso-PGF2␣, ng 䡠 L⫺1
vs MABP, mm Hg
vs CVC, cm 䡠 s
⫺1
⫺1
䡠 mm Hg *
vs NOx, ␮mol 䡠 L⫺1
vs MABP, mm Hg
vs CVC, cm 䡠 s
⫺1
⫺1
䡠 mm Hg *
vs NOx, ␮mol 䡠 L⫺1
⫺0.17
NS
⫺0.05
NS
⫺0.54
⬍0.001
0.009
NS
NOx, ␮mol 䡠 L⫺1
vs MABP, mm Hg
vs CVC, cm 䡠 s
⫺1
⫺1
䡠 mm Hg *
0.41
0.02
⫺0.49
0.005
0.37
0.03
Correlation analyses were conducted on data from all of the volunteers (n⫽40). NS indicates not
significant.
*Analyses were conducted on n⫽39.
pausal women. This finding corroborates previous data in
humans and animals in the context of hypertension. The
negative correlations between both 8-OHdG and nitrotyrosine and CVC suggest that the regulation of cerebrovascular tone may be determined, at least in part, by ROS and
peroxynitrite. These findings have broad clinical implications
in relation to mechanisms that regulate cerebral blood flow
and imply that oxidative stress–induced changes may contribute to the progression of cerebrovascular disease, such as
stroke.
Clinical trials of hypertension treatment with antioxidants
have been inconclusive and failed to demonstrate significant
beneficial effects of nonenzymatic antioxidants, such as
vitamins C and E, or ␤-carotene in the management of
MABP.31 The results of the present study suggest that the
improvement of enzymatic antioxidant defense by means of
greater physical activity may reduce MABP. On the basis of
our reported correlations between MAPB and both nitrotyrosine and 8-OHdG, a 25% reduction of oxidative stress
decreased MABP by 10 mm Hg. Although this result is not
necessarily generalizable to the clinical management of hypertensive patients, a decrease of blood pressure by
10 mm Hg (eg, 110 to 100 mm Hg) using medication reduces
the risk for a stroke event from 0.85 to 0.65.
100
Nitrotyrisine (nmol.l -1)
220
80
NOx (mmol.l -1)
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MDA, nmol 䡠 L⫺1
60
40
20
0
40
50
60
70 80 90 100 110 120
MABP (mmHg)
Figure 2. Relationship between MABP and NOx concentration
in serum in postmenopausal women (r⫽⫺0.54; P⬍0.001). E
(n⫽21), Sedentary group (⬍100% age-predicted V̇O2max); F
(n⫽19), fit group (⬎100% age-predicted V̇O2max).
200
180
160
140
120
100
40
50
60
70
80
90
100 110 120
MABP (mmHg)
Figure 3. Relationship between MABP and nitrotyrosine concentration in plasma in postmenopausal women (r⫽0.41;
P⫽0.01). E (n⫽21), Sedentary group (⬍100% age-predicted
V̇O2max); F (n⫽19), fit group (⬎100% age-predicted V̇O2max).
Pialoux et al
Effect of NO Production on Blood Pressure and
Cerebrovascular Function
Downloaded from http://hyper.ahajournals.org/ by guest on June 16, 2017
Exercise training in healthy individuals is known to elevate
NO bioavailability through a variety of mechanisms, including increased endothelial NO synthase enzyme expression
and activity.32 Moreover, in elderly women, Maeda et al33
reported that the plasma concentration of NOx was significantly increased by exercise training. In this present study,
despite a trend (⫹19%; P⫽0.10), NOx was not significantly
different in the fit group compared with the sedentary group,
most likely because of the relatively small number of subjects
and wide interindividual variability.
Nevertheless, NOx was negatively correlated with MABP,
systolic blood pressure, and diastolic blood pressure, thus
highlighting the role of NO in blood pressure regulation in a
postmenopausal population with a normal range of blood
pressure values. Our results also suggest that the negative
association found between plasma NO metabolites and the
incidence of hypertension in the general population34 might
also occur in hypertensive postmenopausal women. Finally,
NOx was positively correlated with cerebral vascular conductance, thereby supporting the hypothesis that the lower
vasodilator ability of cerebral vessels is likely attributable to
reduced bioavailability of NO.35
Interestingly, NOx was negatively correlated with nitrotyrosine, supporting emerging evidence for a role for improved
NO bioavailability after exercise training as a result of
reduced oxidative stress.36 However, other mechanisms activated by ROS are probably involved in endothelial dysfunction independent of the impairment of the NO pathway. By
mediating the increase in intracellular calcium concentration,
ROS plays a central role in the molecular cascade between
angiotensin II and the final vasoconstriction of vascular
smooth muscle.37 ROS is also known to induce atherosclerosis and subsequent endothelial dysfunction via an increase in
adhesion molecules, proinflammatory cytokines, and permeability of the endothelium.38
Perspectives
This research reveals that the modulation of oxidative stress
by regular physical exercise mediates blood pressure and
cerebral vascular conductance after menopause. This study
also shows how lifestyle might complement current medical
therapies in the prevention and management of cardiovascular disease. Nevertheless, this cross-sectional study needs to
be replicated in a larger randomized, controlled trial to further
verify our results. Moreover, direct causality among exercise,
oxidative stress, and blood pressure cannot be established
from these clinical studies of association. Therefore, future
intervention studies should determine whether enzymatic
antioxidant improvement resulting from physical training can
target and reverse the oxidative stress that occurs at the time
of the menopause and that likely contributes to elevated blood
pressure.
Acknowledgments
We are grateful to all of the volunteers for their cheerful and
dedicated participation to this study. We thank Dr Todd Anderson
and Andrew Beaudin for assistance with data collection and analysis
Oxidative Stress and Blood Pressure in Women
1019
and Linda Brigan for administrative support. We also thank Dr
David Hogan for helpful discussions.
Sources of Funding
Support was provided by the Alberta Heritage Foundation for
Medical Research (AHFMR; establishment grant to M.J.P.), the
Canadian Institutes of Health Research (operating grant to M.J.P.
and C.M.F.), and the Canadian Foundation for Innovation (new
opportunities fund to M.J.P.). V.P. is supported by postdoctoral
research awards from AHFMR, the Heart and Stroke Foundation of
Canada, and Canadian Institutes of Health Research Focus-on-Stroke
Program. A.D.B. was supported by studentship awards from AHFMR
and the Natural Research and Engineering Research Council of Canada.
R.L. is a Canadian Institutes of Health Research clinician scientist,
an AHFMR clinician investigator, and the GlaxoSmithKline Canadian Institutes of Health research professor of inflammatory lung
disease. C.M.F. is supported by an AHFMR Health Senior Scholar
award. M.J.P. is an AHFMR senior medical scholar.
Disclosures
None.
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Effect of Cardiorespiratory Fitness on Vascular Regulation and Oxidative Stress in
Postmenopausal Women
Vincent Pialoux, Allison D. Brown, Richard Leigh, Christine M. Friedenreich and Marc J.
Poulin
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Hypertension. 2009;54:1014-1020; originally published online September 28, 2009;
doi: 10.1161/HYPERTENSIONAHA.109.138917
Hypertension is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
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MANUSCRIPT NUMBER: HYPERTENSION/2009/138917
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ONLINE SUPPLEMENT
EFFECT OF CARDIORESPIRATORY FITNESS ON VASCULAR REGULATION AND
OXIDATIVE STRESS IN POSTMENOPAUSAL WOMEN
Vincent Pialoux1,2, Allison D. Brown1, Richard Leigh1,3, Christine M. Friedenreich4 and Marc J.
Poulin1,2,5,6,7
1
Department of Physiology & Pharmacology Faculty of Medicine, University of Calgary,
Canada
2
Faculty of Kinesiology, University of Calgary, Canada
3
Department of Medicine, Faculty of Medicine, University of Calgary, Canada
4
Alberta Health Services, Calgary, Canada
5
Department of Clinical Neurosciences, Faculty of Medicine, University of Calgary, Canada
6
Hotchkiss Brain Institute, Faculty of Medicine, University of Calgary, Canada
7
Libin Cardiovascular Institute of Alberta, Faculty of Medicine, University of Calgary, Canada
Corresponding author:
Dr. Marc J. Poulin, Department of Physiology & Pharmacology, University of Calgary, HMRB210, 3330 Hospital Drive NW, Calgary, Alberta, Canada T2N 4N1
Email: [email protected]
Phone: 403-220-8372
Fax: 403-210-8420
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EXPENDED MATERIALS AND METHODS
Protocol
Eligibility criteria included: non-smokers within the past 12 months, ability to perform moderate
exercise independently, body mass index (BMI) ≤30 kg/m2, normal spirometry, less than 20%
stenosis of either carotid arteries, and no evidence of significant co-morbid disease or
pharmacologic therapies, as determined by the study physician, that would interfere with their
ability to exercise or with study outcomes. The study requirements were fully explained to each
study participants, and written informed consent was obtained.
Estradiol was measured by electrochemiluminescent immunoassay using an Elecsys 2010 from
Roche Diagnostics. The measuring range was 18.4 – 15781 pmol/l, with an accuracy of 6.0%.
Progesterone was measured by chemiluminescent immunoassay using an Advia Centaur by
Siemens/Bayer Diagnostics. The measuring range was 0.48 – 190.8 nmol/l, with an accuracy of
6.0%.
Measurements of middle cerebral artery peak velocity, as an index of cerebral blood flow, were
made at rest using transcranial Doppler ultrasound (1;2). The right middle cerebral artery was
identified through the temporal window just above the zygomatic arch using search techniques
previously described (3;4). As there were no changes in P̄ throughout the experimental sessions V̄
P was used as the primary index of cerebral blood flow. The resting cerebral vascular tone was
calculated as cerebrovascular conductance (CVC), which is the ratio V̄P / MABP (5;6). All
cardiovascular parameters were acquired at 10 ms intervals and the values for each determined
beat-by-beat by specifically designed computer software (Chamber V2.43, University
Laboratory of Physiology, Oxford, UK).
Biochemical analyses
Concentrations of plasma 8-OHdG was determined using an ELISA kit from Cell BioLabs (Cell
Biolabs, Inc. San Diego, CA).The limits of detection for this assay are 1-200µg.l-1.
Concentrations of plasma MDA were determined as thiobarbituric reactive substances by a
modified method of Ohkawa et al (7), as previously described (8). Although MDA assay is often
associated with relevant methodological limitations (9), it was the most common lipid
peroxidation marker and it still widely used as marker of oxidative stress in the area of blood
pressure regulation.
Concentrations of plasma 8-iso-PGF2α were measured in the plasma using an ELISA kit (Cells
bioLabs, Inc. San Diego, CA). The limits of detection for this assay are 0.05-200 ng.l-1.
Concentrations of plasma Nitrotyrosine, as end product of protein nitration by ONOO-, were
measured using an ELISA kit from Cell BioLabs (Cell Biolabs, Inc. San Diego, CA). The limits
of detection for this assay are 1-8000 nmol.l-1.
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GPX in the plasma was determined by the modified method of Paglia and Valentine (10) using
hydroperoxide (H2O2) as a substrate. GPX was determined by the rate of oxidation of NADPH to
NADP+ after addition of glutathione reductase (GR), reduced glutathione (GSH), NADPH.
Catalase activity in the plasma was determined by the method of Johansson and Borg (11) using
hydroperoxide (H2O2) as substrate, and formaldehyde as standard. Catalase activity was
determined by the formation rate of formaldehyde induced by the reaction of methanol and H2O2
using catalase as enzyme.
The end-products of endothelium nitric oxide, nitrites and nitrates, were measured in the serum
using a commercially available kit (Cayman Chemical Company, Ann Arbor, MI, USA) based
on methods previously described (12). The sum of nitrite and nitrate in the plasma (NOx) is
considered an index of nitric oxide production (13).
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1. Ainslie PN, Ashmead JC, Ide K, Morgan BJ, Poulin MJ. Differential responses to CO2
and sympathetic stimulation in the cerebral and femoral circulations in humans. J
Physiol. 2005; 566:613-624.
91
92
93
2. Ide K, Worthley MI, Anderson TJ, Poulin MJ. Effects of the nitric oxide synthase
inhibitor L-NMMA on cerebrovascular and cardiovascular responses to hypoxia and
hypercapnia in humans. J Physiol. 2007; 584.1:321-332.
94
95
96
3. Poulin MJ, Liang P-J, Robbins PA. Fast and slow components of the cerebral blood flow
response to step decreases in end-tidal PCO2 in humans. J Appl Physiol 1998; 85:388397.
97
98
99
4. Ide K, Eliasziw M, Poulin MJ. The relationship between middle cerebral artery blood
velocity and end-tidal PCO2 in the hypocapnic-hypercapnic range in humans. J Appl
Physiol. 2003; 95:129-137.
REFERENCES
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101
5. Lautt WW. Resistance or Conductance for Expression of Arterial Vascular Tone.
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6. Claassen JA, Zhang R, Fu Q, Witkowski S, Levine BD. Transcranial Doppler estimation
of cerebral blood flow and cerebrovascular conductance during modified rebreathing. J
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7. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by
thiobarbituric acid reaction. Anal Biochem. 1979; 95:351-358.
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8. Pialoux V, Mounier R, Ponsot E, Rock E, Mazur A, Dufour S, Richard R, Richalet JP,
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9. Lefevre G, Beljean-Leymarie M, Beyerle F, Bonnefont-Rousselot D, Cristol JP, Therond
P, Torreilles J. [Evaluation of lipid peroxidation by measuring thiobarbituric acid reactive
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10. Paglia DE, Valentine WN. Studies on the quantitative and qualitative characterization of
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11. Johansson LH, Borg LA. A spectrophotometric method for determination of catalase
activity in small tissue samples. Anal Biochem. 1988; 174:331-336.
117
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12. Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR.
Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal Biochem. 1982;
126:131-138.
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13. Ohta M, Nanri H, Matsushima Y, Sato Y, Ikeda M. Blood pressure-lowering effects of
lifestyle modification: possible involvement of nitric oxide bioavailability. Hypertens
Res. 2005; 28:779-786.
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Table S1: Correlations between maximal oxygen consumption (normalized to body weight) and
plasma oxidative stress and antioxidant enzyme activity controlled or not for body mass index
and fat mass.
125
Pearson
Correlation
Coefficient
-0.52
0.001
Controlled for
BMI and Fat
mass
-0.30
0.07
8-iso-PGF2α (ng.l-1) vs.
VO2max (ml.kg-1.min-1)
-0.41
0.008
-0.17
ns
Nitrotyrosine (nmol.l-1) vs.
VO2max (ml.kg-1.min-1)
-0.31
0.04
0.18
ns
GPX (μmol.l-1.min-1) vs.
VO2max (ml.kg-1.min-1)
0.40
0.001
0.32
0.05
Variables
8-OHdG (μg.l-1) vs.
VO2max (ml.kg-1.min-1)
126
127
128
129
P
P
Abbreviations: VO2max, maximal oxygen consumption; 8-OHdG: 8-hydroxy-2'deoxyguanosine: 8-iso-PGF2α, 8-iso-Prostaglandin F2α, MDA: malondialdehydes: GPX:
glutathione peroxidase activity. Correlation analyses conducted on data from all volunteers
(n=40).
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Table S2: Correlations between cardiovascular parameters and plasma oxidative stress and endproducts of NO controlled or not for body mass index and fat mass.
Pearson
Correlation
Coefficient
P
Controlled for
BMI and Fat mass
0.24
0.13
0.23
0.16
vs. DBP (mmHg)
0.44
0.005
0.58
0.001
8-OHdG (μg.l-1)
vs. SBP (mmHg)
vs. DBP (mmHg)
0.48
0.33
0.002
0.04
0.43
0.32
0.01
0.05
8-iso-PGF2α (ng.l-1)
vs. SBP (mmHg)
vs. DBP (mmHg)
0.16
0.07
ns
ns
0.15
0.005
ns
ns
MDA (nmol.l-1)
vs. SBP (mmHg)
vs. DBP (mmHg)
0.36
0.06
0.02
ns
0.46
0.04
0.01
ns
NOx (μmol.l-1)
vs. DBP (mmHg)
vs. SBP (mmHg)
-0.50
-0.42
0.003
0.01
-0.45
-0.39
Variables
Nitrotyrosine (nmol.l-1)
vs. SBP (mmHg)
130
131
132
133
P
0.01
0.02
Abbreviations: 8-OHdG: 8-hydroxy-2'-deoxyguanosine, 8-iso-PGF2α: 8-iso-Prostaglandin F2α,
SBP: systolic blood pressure, DBP: diastolic blood pressure, MDA: malondialdehydes, NOx:
end-products of NO metabolism. Correlation analyses conducted on data from all volunteers
(n=40).
134
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SUPPLEMENTARY FIGURES
250
8-OHdG (µg.l-1)
135
200
150
100
50
0
10
15
20
25
30
35
40
VO 2max ( ml.min-1.kg-1)
Figure S1: Relationship between maximal oxygen consumption (VO2max) and 8-hydroxy-2'deoxyguanosine (8-OHdG) concentration in plasma in postmenopausal women.
Open circles (n=21) represent sedentary group (≤ 100% age predicted VO2max) and solid circles
(n=19) represent fit group (>100% age predicted VO2max).
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GPX (nmol.min-1.ml-1)
22
20
18
16
14
12
10
8
6
10
15
20
25
30
35
40
VO2max (ml.min-1.kg-1)
Figure S2: Relationship between maximal oxygen consumption (VO2max) and plasmatic
glutathione peroxidase activity (GPX) in postmenopausal women.
Open circles (n=21) represent sedentary group (≤ 100% age predicted VO2max) and solid circles
(n=19) represent fit group (>100% age predicted VO2max).
9