Clinical Science (2004) 106, 329–335 (Printed in Great Britain)
Effects of age and physical fitness on
microcirculatory function
Ferdinando FRANZONI∗ , Fabio GALETTA∗ , Carmela MORIZZO∗ , Valter LUBRANO†,
Carlo PALOMBO∗ , Gino SANTORO∗ , Eleuterio FERRANNINI∗
and Alfredo QUIÑONES-GALVAN†
∗
Department of Internal Medicine, University of Pisa School of Medicine, Via Roma 67, Pisa 56100, Italy, and †CNR Institute
of Clinical Physiology, Via Moruzzi 1, 56124 Pisa, Italy
A
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Sedentary aging is associated with endothelial dysfunction and nitric oxide (NO) impairment.
The aim of the present study was to assess the effects of regular physical exercise on nitrite/
nitrate (NOx) concentrations and microcirculatory function in older men compared with young
individuals. We measured NOx plasma concentrations and baseline and stimulated skin blood
flow (SBF) by laser Doppler flowmetry in 39 male athletes [range, 22–72 years; maximal oxygen
−1
−1
consumption (V̇ O2 max), 60.0 +
− 4.7 ml · min · kg of body weight (mean +
− S.D.)] and 45 age−1
7.1
ml
·
min
·
kg
of
body
weight−1 ). NOx
and sex-matched sedentary controls (V̇ O2 max, 38.0 +
−
concentrations were higher in athletes than in controls (50.4 +
− 16.3 compared with 39.0 +
−
15.4 µmol/l; P < 0.005), whereas baseline SBF was comparable. Hand SBF after heating and
ischaemia and foot SBF after heating were higher in athletes (P < 0.0001) than in controls. By
comparing the lowest and the highest tertile of age, sedentary young subjects had higher NOx
concentrations than sedentary older subjects (43.3 +
− 12.2 µmol/l
− 13.4 compared with 31.8 +
respectively; P < 0.05). Exercise abolished this difference (49.1 +
9.6
µmol/l
for
young
subjects and
−
52.1 +
11.5
µmol/l
for
older
subjects;
not
significant).
Resting
SBF
was
similar
in
all
the
subgroups,
−
but stimulated SBFs were lower in both subgroups of untrained compared with trained subjects.
NOx concentrations were positively correlated with V̇ O2 max (r = 0.46, P < 0.001). Stimulated
SBFs were correlated with NOx (r > 0.30, P < 0.05). These findings show that chronic exercise
may improve endothelial function in older (and young) men, probably by increasing NO availability.
INTRODUCTION
Aging represents a well-documented cardiovascular risk
factor [1]. One of the putative physiopathological mechanisms through which aging may lead to cardiovascular
damage is the promotion of changes in arterial structure
and function [2].
Endothelium plays an important role in the local
regulation of vascular tone and structure. This regulation
is mediated mainly by nitric oxide (NO) synthesis and
action [3]. A reduction in NO availability and action
would lead to endothelial dysfunction, and this derangement has been associated with other cardiovascular
risk factors, such as dyslipidaemia, diabetes mellitus,
essential hypertension and aging [4–7]. Thus endothelial
dysfunction represents a common feature and a crucial
event in cardiovascular disease [8].
Regular physical activity has long been considered
necessary for the achievement and the maintenance of
optimal health. Experimental and clinical studies have
provided strong evidence that physical training has beneficial effects on multiple cardiovascular risk factors,
such as dyslipidaemia, high blood pressure and glucose
tolerance [9,10]. By contrast, sedentary lifestyle has been
identified as a risk factor for the development of some
cardiovascular risk conditions and for coronary artery
Key words: aging, exercise, endothelial function, microcirculation, nitric oxide (NO).
Abbreviations: BMI, body mass index; NO, nitric oxide; eNOS, endothelial NO synthase; NOx, nitrate + nitrite; pu, perfusion
units; SBF, skin blood flow; V̇o2 max, maximal oxygen consumption.
Correspondence: Dr Fabio Galetta (e-mail [email protected]).
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disease. It has been reported that there is a strong relationship between physical inactivity and cardiovascular mortality [11]. Consequently, daily physical aerobic
activity is considered to be an effective component in
preventing cardiovascular events [1,9]. Recent evidence,
obtained from functional studies, suggests that regular
physical activity is associated with an increase in NO
availability and action in elderly [12–15] and young
people [13,16–18]. Thus it is possible that regular exercise
can counterbalance the effects of aging on endothelial
dysfunction.
In the present study, our aim was to assess the relationship between age, physical exercise, NO availability and endothelial function in both healthy older and
young subjects.
METHODS
Subjects
Thirty-nine competitive long-distance male runners [age,
42.4 +
− 15.3 years (mean +
− S.D.); range, 22–72 years] and
45 healthy age- and sex-matched sedentary volunteers
(range, 22–75 years) were studied. Athletes had been
training for 21 +
− 12 years. They were recruited from
various running clubs throughout the surrounding regions of Pisa and from the National Veterans Sport Club
of Pisa. They performed vigorous endurance exercise
more than five times/week and were active in national and
international road-running races. The sedentary subjects
were recruited through various forms of advertisements.
All subjects were free of cardiovascular disease or
other major medical disorders as assessed by clinical
history, physical examination, basal and stress electrocardiography, blood chemistry, haematology and urine
analysis. Subjects smoking more than five cigarettes/day
and/or consuming more than 60 g of ethanol/day (corresponding to half a litre of wine), or receiving any drug
treatment within the previous 2 months, were excluded
from the study.
Athletes were selected on the basis of maximal oxygen
consumption (V̇o2 max), as assessed by a graded exercise
test (cycle ergometer), of greater than 50 ml · min−1 · kg of
body weight−1 , whereas control subjects had a V̇o2 max
lower than 40 ml · min−1 · kg of body weight−1 [19]. The
time period between the last exercise and blood sampling
was at least 48 h.
All participants, who fasted for at least 12 h before the
start of the study, were requested to adhere to a lownitrate diet (e.g. exclusion of foodstuffs containing a
high concentration of nitrates, such as cured meat, fruits
and, in particular, leafy green vegetables) for 72 h before
collection of blood samples.
Fully informed consent was obtained from each subject
entering the study. The investigation conforms with the
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2004 The Biochemical Society
Declaration of Helsinki. The protocol was approved by
the Local University Hospital Ethics Committee.
Recording of blood pressure and
blood sampling
All subjects were examined at 09:00 hours in a quiet airconditioned room with the temperature maintained at
22–24 ◦ C. After introduction of an indwelling cannula
(20 gauge; Abbocath, Sling, Ireland) into the left antecubital vein, each subject was allowed to rest in a
supine position for at least 15 min before blood pressure
was recorded. Venous blood samples were collected in
tubes containing EDTA and processed immediately for
separation of plasma. Measurement of blood pressure was
carried out at least five times by means of a Dinamap
automatic recorder (Critikon, Tampa, FL, U.S.A.), with
an interval of 3 min between single readings.
Microcirculatory studies
The two groups underwent a laser Doppler study of
skin blood flow (SBF) by laser Doppler flowmetry
(Periflux PF4001, standard probe PF408; Perimed,
Jarfalla, Sweden) at upper and lower extremities. The laser
Doppler probe was fixed to the skin surface of the third
digit of the left hand and of the first digit of the left foot.
SBF was measured under basal conditions, after local
heating at 44 ◦ C and during reactive hyperaemia following
a 3-min brachial artery occlusion. The apparatus and technique have been described in detail previously [20]. The
output signal was linearly related to red cell flow as predicted by theoretical [21] and experimental [22] models.
The laser Doppler apparatus was connected to a PC
by RS232 interface. The program installed on the computer (Perisoft) allows the storage and analysis of the
recordings. The measurement of SBF is expressed in
arbitrary units [‘perfusion units’ (pu)]. Coefficient of
variation (three measurements) for baseline SBF was less
than 5 %.
Analytical methods
Serum concentrations of total cholesterol, high-density
lipoprotein-cholesterol and triacylglycerols (triglycerides) were determined by standard laboratory methods.
The concentration of low-density lipoprotein-cholesterol
was calculated using the Friedwald equation, and expressed as mmol/l.
We measured circulating levels of nitrite + nitrate
(NOx), which represent the final and stable end-product
of the NO metabolic pathway. Venous blood samples
were collected in the presence of EDTA and immediately
centrifuged at 2500 g for 15 min. Plasma samples were
stored at − 80 ◦ C for less than 2 weeks before analysis. At
the time of NOx assay, plasma samples were ultrafiltered
through 30 kDa cut-off filters and centrifuged at 1000 g
for 60 min in order to remove haemoglobin, which is
Nitric oxide and microcirculatory flow in older athletes
Table 1 Clinical characteristics of the study populations
Values are means +
− S.D. SBP, systolic blood pressure, DBP, diastolic blood
pressure, HDL, high-density lipoprotein; LDL, low-density lipoprotein. ∗ P < 0.05
and †P < 0.0001 compared with sedentary controls.
Characteristic
Sedentary controls
(n = 45)
Athletes
(n = 39)
Age (years)
BMI (kg/m2 )
SBP (mmHg)
DBP (mmHg)
Heart rate (beats/min)
Total cholesterol (mmol/l)
HDL-cholesterol (mmol/l)
LDL-cholesterol (mmol/l)
V̇ O2 max (ml · min−1 · kg−1 )
41.4 +
− 17.2
23.8 +
− 1.6
121.3 +
− 14.5
74.8 +
− 14.1
67.3 +
− 4.5
5.4 +
− 0.8
1.4 +
− 0.3
3.3 +
− 0.6
38 +
− 7.1
42.4 +
− 15.3
23.9 +
− 2.4
119.5 +
− 13.4
73.7 +
− 11.8
50.9 +
− 3.6†
4.7 +
− 0.4
∗
1.7 +
− 0.2
∗
2.6 +
− 0.3
60 +
− 4.7†
known to interfere with subsequent spectrophotometric
measurements. NOx concentrations in different dilutions
of plasma ultrafiltrate were determined by a colorimetric
assay kit (Cayman, Ann Arbor, MI, U.S.A.) based on a
three-step Griess reaction [23]. The results are expressed
as µmol/l. The lower limit of sensitivity for the evaluation
of plasma NOx concentration by the Griess colorimetric
assay was 1 µmol/l. The assay was found to be linear
over the range of NOx concentration 0–35 µmol/l, with
correlation coefficients greater than 0.99. Coefficients of
variation within or between days were lower than 5.5 %.
Statistical analysis
Results are expressed as means +
− S.D. ANOVA was used
to assess the mean differences between groups, followed
by Fisher’s post-hoc test. Linear correlation analysis
was used to assess relationships between variables. Differences were considered significant at a P value of < 0.05.
All statistical procedures and curve fitting for statistical
analysis were performed by means of StatView version
4.57 (Abacus Concepts Inc., Cary, NC, U.S.A.).
Figure 1 V̇ O2 max and NOx concentrations in the four
subgroups
V̇ O2 max (upper panel) and NOx (lower panel) were higher in both subgroups of
trained individuals. Results are expressed as means +
− S.D. Young sedentary,
n = 17; young athletes, n = 18; older sedentary, n = 12; older athletes, n = 15.
Upper panel, ∗ P < 0.0001 compared with sedentary subgroups; §P < 0.02
compared with older counterparts; ◦ P < 0.0002 compared with young athletes.
Lower panel, ∗∗ P < 0.01 and §§P < 0.002 compared with older sedentary. ns,
not significant.
270 +
− 30 pu), were significantly higher in athletes than in
sedentary individuals (P < 0.0001 for all measurements).
A positive statistical interaction between age and
V̇o2 max was found (P < 0.05). No age–endurance interaction was observed with respect to NOx concentrations
or SBF measurements.
Subgroup analysis
RESULTS
Demographic and clinical characteristics of the athletes
and sedentary groups are shown in Table 1. V̇o2 max was
significantly higher in athletes than in sedentary subjects
(P < 0.0001) as expected. NOx values were significantly higher in athletes compared with sedentary control
subjects (50.4 +
− 16.3 and 39.0 +
− 15.4 µmol/l respectively;
P < 0.005). Baseline SBF was similar in athletes and
controls (hand SBF, 206 +
− 36 compared with 190 +
− 29 pu
respectively, and foot SBF, 225 +
− 41 compared with 179 +
−
34 pu respectively; not significant). The hand reflow after
ischaemia (374 +
− 37 compared with 278 +
− 24 pu) and
after heating (332 +
− 29 compared with 271 +
− 27 pu), as
well as foot flow after heating (378 +
− 44 compared with
We selected the lowest (n = 35; age, 28 +
− 3 years) and the
highest (n = 27; age, 61 +
− 4 years) tertile of age to assess
the relationship between age, fitness, V̇o2 max, NOx and
SBF measurements at the extremes of the age distribution
(Figures 1 and 2).
The subgroups of older and young individuals, when
divided by trained status, were well-matched by age and
body mass index (BMI; results not shown). As expected,
V̇o2 max was higher in both subgroups of trained subjects when compared with sedentary individuals. Young
trained subjects exhibited the highest V̇o2 max (66.6 +
−
5.6 ml · min−1 · kg of body weight−1 ). The young sedentary subgroup had half the V̇o2 max observed in trained
−1
−1
counterparts (36.8 +
− 9.8 ml · min · kg of body weight ;
P < 0.0001), as expected. Older trained individuals
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F. Franzoni and others
Figure 3 Correlation analysis between V̇ O2 max and NOx in
the whole population
Baseline SBF measured at the hand and foot were
similar across the four subgroups (Figure 2). Stimulated
SBF (after heating or ischaemia) was significantly higher
in athletes than in sedentary people in both subgroups
(Figure 2).
Differences between groups were essentially the same
even after corrections for weight and BMI.
Correlation analysis
In the whole group, V̇o2 max was negatively correlated
with age (r = − 0.40, P < 0.002). In sedentary individuals,
this relationship was still significant (r = 0.37, P < 0.03),
whereas, in athletes, this relationship was more pronounced (r = 0.70, P < 0.0001).
NOx concentrations were positively correlated with
V̇o2 max (r = 0.46, P < 0.001; Figure 3). A direct relationship between NOx concentrations and stimulated
SBF was observed (Figure 4).
Figure 2 Hand basal SBF, hand and foot SBF after heating
and hand SBF after ischaemia in the four subgroups
DISCUSSION
Results are expressed as means +
− S.D. Young sedentary, n = 11; young athletes,
n = 13; older sedentary, n = 10; older athletes, n = 12. ∗∗ P < 0.004,
∗
P < 0.0001, §P < 0.001 and §§P < 0.05 compared with sedentary subgroups.
By studying a group of sedentary and trained subjects
with a large age range (22–75 years), we have tried to assess the relationship between age, exercise, NOx concentrations and endothelial function. Our findings have
shown that NOx concentrations were lower in older subjects, and that stimulated peripheral SBFs (e.g., after heating or ischaemia) were lower in sedentary people independent of age. Higher microcirculatory function was
observed in both groups of young and older individuals
who regularly perform physical exercise. This difference
was probably due to an increased action of NO and was
related to the higher V̇o2 max that characterizes regular
physical activity.
There is a large amount of experimental and clinical
data indicating that endothelial dysfunction is involved
not only in disease states such as atherosclerosis, hypertension, heart failure or diabetes, but also in the normal
physiological process of aging. Decreased endotheliumdependent vasodilation of the forearm and coronary
had a V̇o2 max 50 % higher than older controls
−1
(44.8 +
− 5.7 compared with 30.4 +
− 4.9 ml · min · kg of
−1
body weight ; P < 0.02; Figure 1).
Plasma NOx concentrations were higher in both
subgroups of trained subjects, which was independent of
age, and were slightly higher in sedentary young people
compared with sedentary older subgroup (P < 0.05;
Figure 1). Interestingly, physical exercise was not associated with a significantly higher NOx in young individuals; in contrast, endurance training was associated with
significantly higher NOx (P < 0.002) in older subjects.
Young athletes exhibited higher NOx concentrations
than older controls (P < 0.01). Training removed the difference in NOx concentrations observed between young
and older sedentary individuals (Figure 1).
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Nitric oxide and microcirculatory flow in older athletes
Figure 4 Correlation analysis between NOx and SBF in the
whole population
circulation has been associated with human aging [6,24–
26] but, to our knowledge, no information about microcirculatory function in elderly subjects and its relationship with exercise has been reported to date.
Previously reported experimental data have shown
[27–29] that chronic exercise training is associated with
enhanced endothelium-dependent vasodilation which is
accompanied by increased NO production in large
coronary vessels and microvessels in animals. Urinary
excretion of NO metabolites increases with increasing
levels of chronic physical activity [30] and, in patients
with cardiovascular disease, levels of urinary NO metabolites increase in direct relationship with the gain
in functional capacity, suggesting that increased NO
production may be a major adaptive mechanism by which
aerobic exercise training benefits the cardiovascular
system [30].
The mechanisms by which chronic exercise improves
vascular NO availability are not completely known.
Exercise induces a complex integrated physiological response that involves activation of circulating hormones and local autacoids, such as catecholamines, adenosine and ATP, which could exert an influence in the
long-term regulation of NO biosynthesis. In addition,
exercise increases myocardial oxygen consumption and
reduces vascular resistance at the coronary level. This
effect will elicit endothelial shear stress, which may increase endothelial NO synthase (eNOS) expression and
activity [31]. In line with this view, in vitro studies indicate that shear stress increases eNOS expression in endothelial cells [32,33]. Interestingly, shear-stress-responsive
elements have been found in the eNOS gene promoter,
indicating that shear stress can transcriptionally regulate
eNOS expression [34]. Other mechanisms implicated in
the increased availability of NO include the activation
of Ca2+ -activated K+ channels [29] and the increased expression of eNOS facilitated by exercise-induced expression of antioxidant enzymes [35,36].
In the present study, we observed that older trained
subjects have significantly higher NOx plasma concentrations than matched sedentary controls. Similar results
have been reported previously by us [12] and other groups
[13–15]. In the group of older trained individuals,
NOx concentrations were similar to that observed in
young people. This effect of exercise on NOx concentrations was not observed in young individuals, suggesting that eNOS in young people is expressed at a
higher level of activity independently of the trained
status. Accordingly, previous data [12] have shown that
endothelium-dependent vasodilation is preserved in a
young population and cannot be improved by chronic
aerobic exercise training. We have reported previously
[12] in elderly subjects that chronic physical activity
improves endothelial function (as measured by venous
plethysmography) by preventing oxidative stress and
restoring NO availability.
The use of the laser Doppler techniques to analyse
the microcirculatory SBF has been validated in previous
studies [37,38]. In fact, in patients with essential hypertension, SBF measured with laser Doppler flowmetry
was increased by acetylcholine infusion in parallel with
that observed with strain-gauge venous plethysmography
[37]. In patients with Raynaud disease, an NO-generating
system increased microcirculatory SBF as assessed by
photoplethysmography [38].
In the present study, NOx concentrations were significantly related to post-heating and post-ischaemia SBF,
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suggesting that NOx participates in the post-stimulus
regulation of blood flow, but the relatively low correlation coefficient (r ∼ 35) suggests that other factors are
implicated in the improvement of stimulated SBF observed in subjects performing chronic exercise. In this regard, other mediators that are modified by training, such
as prostaglandins [39], endothelin [40] and the oxidant/
antioxidant status [35], may be implicated.
It is possible that the beneficial effects of regular
physical training could be mediated by the increase in NO
availability and action, and this could be the basis for the
beneficial effects of exercise in some clinical conditions
associated with low fitness level, such as aging. It has
been reported previously [41] that endothelial NO action
could modulate sympathetic activity in humans. In this
context, it is possible that regular exercise may improve
endothelial function by reducing the chronic suppressive
influence exerted by sympathetic-adrenergic tone either
directly or by enhancing the sympathoinhibitory effect
of NO.
In conclusion, the present study suggests that chronic
regular aerobic training is associated with higher NO generation in both young and older subjects. These changes
are associated with higher microcirculatory endothelialdependent function.
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Received 9 July 2003/3 October 2003; accepted 30 October 2003
Published as Immediate Publication 30 October 2003, DOI 10.1042/CS20030229
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