The Ergogenic Effect of Recombinant Human Erythropoietin on V̇O2max Depends on the Severity of Arterial Hypoxemia
et al. (2008) The Ergogenic Effect of Recombinant Human Erythropoietin on V O2max Depends
on the Severity of Arterial Hypoxemia. PLoS ONE 3(8): e2996. doi:10.1371/journal.pone.0002996
The Ergogenic Effect of Recombinant Human Erythropoietin on V O2max Depends on the Severity of Arterial Hypoxemia
Paul Robach 0
Jose A. L. Calbet 0
Jonas J. Thomsen 0
Robert Boushel 0
Pascal Mollard 0
Carsten Lundby 0
Timothy Secomb, University of Arizona, United States of America
0 1 Ecole Nationale de Ski et d'Alpinisme, Chamonix, France, 2 The Copenhagen Muscle Research Centre , Rigshospitalet, Copenhagen , Denmark , 3 Department of Anaesthesia , Rigshospitalet, Copenhagen , Denmark , 4 Department of Physical Education, University of Las Palmas de Gran Canaria , Las Palmas , Spain , 5 Department of Sport Science, University of A rhus, A rhus, Denmark, 6 Department of Biomedical Sciences, University of Copenhagen , Copenhagen , Denmark , 7 Laboratoire ''Re ponses cellulaires et fonctionnelles a` l'hypoxie'' , EA 2363, A.R.P.E. , Universite Paris 13 , Bobigny , France
Treatment with recombinant human erythropoietin (rhEpo) induces a rise in blood oxygen-carrying capacity (CaO2) that unequivocally enhances maximal oxygen uptake (V O2max) during exercise in normoxia, but not when exercise is carried out in severe acute hypoxia. This implies that there should be a threshold altitude at which V O2max is less dependent on CaO2. To ascertain which are the mechanisms explaining the interactions between hypoxia, CaO2 and V O2max we measured systemic and leg O2 transport and utilization during incremental exercise to exhaustion in normoxia and with different degrees of acute hypoxia in eight rhEpo-treated subjects. Following prolonged rhEpo treatment, the gain in systemic V O2max observed in normoxia (6-7%) persisted during mild hypoxia (8% at inspired O2 fraction (FIO2) of 0.173) and was even larger during moderate hypoxia (14-17% at FIO2 = 0.153-0.134). When hypoxia was further augmented to FIO2 = 0.115, there was no rhEpo-induced enhancement of systemic V O2max or peak leg V O2. The mechanism highlighted by our data is that besides its strong influence on CaO2, rhEpo was found to enhance leg V O2max in normoxia through a preferential redistribution of cardiac output toward the exercising legs, whereas this advantageous effect disappeared during severe hypoxia, leaving augmented CaO2 alone insufficient for improving peak leg O2 delivery and V O2. Finally, that V O2max was largely dependent on CaO2 during moderate hypoxia but became abruptly CaO2-independent by slightly increasing the severity of hypoxia could be an indirect evidence of the appearance of central fatigue.
In a normoxic environment, recombinant human
erythropoietin (rhEpo) has long been known for its ergogenic properties.
Evidence of this is supported by numerous reports showing that
prolonged rhEpo treatment induces a consistent increase in
maximal oxygen uptake ( VO2max) . Furthermore, it has
been shown that endurance performance is dramatically
augmented after rhEpo treatment . The ergogenic effect of rhEpo is
primarily mediated by hematological changes, since rhEpo
stimulates - as endogenous renal Epo does - the proliferation of
erythroid progenitor cells that ultimately augments red blood cell
production, total hemoglobin mass and arterial oxygen content
[6,7], both factors being key determinants of oxygen transport
capacity and aerobic performance .
On the other hand, the ergogenic effect of rhEpo in a hypoxic
environment is much less documented. While exercise capacity is
consistently increased after rhEpo in normoxia, this is not the case
with exercise in hypoxia, since neither rhEpo injections  nor
autologous blood transfusion  were found to improve V O2max
at altitudes above 4000 m, in spite of enhanced arterial O2 content
(CaO2). Likewise, with altitude acclimatization, CaO2 and
systemic oxygen delivery are restored close to sea-level values,
however V O2max measured at high altitude is only barely
ameliorated [11,12]. The main mechanism of this dissociation
appears to be of cardiovascular origin, involving a reduction in
peak muscle blood flow with altitude acclimatization [11,13]
caused by a reduction in peak cardiac output and a redistribution
of blood flow toward non-exercising tissues in response to higher
CaO2 . Alternatively, it has been argued that in hypoxia
V O2max is not as tightly dependent on CaO2 as in normoxic
conditions, but is rather limited by the pressure gradient driving
diffusion from the capillaries to the muscle mitochondria, due to
the reduced arterial oxygen pressure (PaO2) [14,15]. Nevertheless,
since previous studies examining the influence of CaO2 on the
cardiovascular response to exercise have been carried out after
prolonged exposure to hypoxia, where accelerated erythropoiesis
and hypoxia coexist, it has not been possible to separate putative
effects linked to altitude acclimatization from those elicited by the
increase in CaO2.
The fact that recombinant Epo boosts maximal oxygen
transport at sea level but not at high altitude implies the existence
of an altitude threshold beyond which the ergogenic effect of
rhEpo on VO2max evanishes. Determining this threshold may
give further insights into the minimal level of hypoxia required to
initiate some mechanisms counteracting the advantageous
hematological adaptations conferred by rhEpo.
The first objective of this study was to determine how does the
rhEpo-induced rise in V O2max vary with the severity of hypoxia.
From the results obtained in response to this first question, our
second objective was to gain insights into the mechanisms involved
in the interactions between hypoxia, CaO2 and V O2max.
Eight Caucasian healthy male volunteers participated in the
study. Their characteristics (mean6SD) were age: 2767 yr,
height: 18064 cm, body mass: 8367 kg, and maximal oxygen
uptake: 4.160.2 l.min21. The subjects were active in recreational
sport activities such as jogging 12 times per week. During the
entire study period, the subjects were asked not to deviate from
their normal lifestyle, and this was well accepted by all. Written
informed consent to participate in the study was obtained from all
subjects. This study was carried out according to the Declaration
of Helsinki and was approved by the Ethical Committee of
Copenhagen and Frederiksberg Counties, Denmark. All the study
took place at sea level in Copenhagen, Denmark.
Treatment with Recombinant Human erythropoietin
After baseline measurements, treatment with rhEpo
(NeoRecormon, Roche, Mannheim, Germany) was started and lasted for
fifteen weeks. On each occasion, 5000 IU was injected as follows.
First two weeks: one injection every second day; third week: three
injections on three consecutive days; week four to fifteen: one
injection every week. All injections occurred between 08:00 and
10:00 a.m., and were preceded by 30 min of supine rest. Two
weeks prior to rhEpo treatment all subjects received 100 mg
iron.day21 orally, and this was maintained throughout the entire
study period. The present rhEpo treatment is an exploratory
model, which has been developed by the Copenhagen Muscle
Research Centre both for physiological studies and also for doping
testing purposes. Our rhEpo treatment, which aimed to mimic
what could be a procedure of rhEpo misuse, was shown to increase
red blood cell volume substantially (,10%) while maintaining the
hematocrit at around 50% (corresponding to the upper limit for
entering some competitions) throughout the study period .
Furthermore, our rhEpo treatment was associated with normal
circulating Epo levels during the maintenance period .
The overall experiment design of the study is summarized on
Non-invasive experiments. In order to gain insights into the
altitude threshold beyond which the effect of rhEpo on maximal
oxygen uptake becomes marginal, seven of the eight subjects
(subjects #1 to #7) underwent a non-invasive experiment on two
separate occasions, i.e. before and after five weeks of rhEpo
treatment. On each occasion, the subjects performed an
incremental cycling exercise until exhaustion in five conditions,
i.e. in normoxia and in four levels of acute normobaric hypoxia
equivalent to the altitudes of 1500 m (FIO2 = 0.174), 2500 m
(FIO2 = 0.153), 3500 m (FIO2 = 0.134) and 4500 m (FIO2 = 0.115).
In this study, the altitude of 1500 m refers to mild hypoxia, the
altitudes of 25003500 m to moderate hypoxia and the altitude
of 4500 m to severe hypoxia. The order between oxygenation
conditions was randomly balanced in a single-blind manner. Each
subject completed the five exercise tests over a period of 34
consecutive days with a maximum of two incremental tests per
day, and if so with a recovery period of at least 120 min between
the two tests. During baseline measurements, the non-invasive
experiment (five tests) took place one week before the invasive
experiment (two tests). On the experimental days the subjects
reported to the laboratory at 08:00 a.m., and a catheter was
inserted into an arm vein for lactate determination. Arterialized
blood was obtained from a capillary blood sample drawn from a
pre-warmed finger tip, by using an electrically heated pad (Heat
Pad, OBH). Respiratory variables were measured continuously, as
Cardiac output was measured continuously during cycling
exercise by a cardiac impedance technique (PhysioFlow PF-05,
Manatec biomedical, Paris, France). The cardiac impedance
device used in the present experiment has been previously
validated during exercise in healthy subjects [17,18]. This
technique, which measures the changes in thoracic impedance
during cardiac ejection in order to calculate stroke volume, has
been described elsewhere . Six thoracic impedance electrodes
(Ag/AgCl, Hewlett Packard 40493 E) were placed after skin
preparation as follow: four electrodes were placed at the base of
the neck and along the xiphoid for impedance signal and two
additional electrodes were placed for ECG signal. Resting arterial
blood pressure, measured by automatic sphygmomanometry while
the subject was sitting at rest on the ergometer, was used to
calibrate the thoracic impedance device. Heart rate and stroke
volume were continuously measured during the test. Cardiac
output was averaged over 15-s intervals.
Validity of cardiac impedance for cardiac output measurement. In order to
evaluate the validity of our cardiac impedance method for cardiac
output measurement during the non-invasive study, cardiac output
during incremental exercise in the invasive study was measured
simultaneously by the cardiac impedance technique and the
cardio-green dye dilution method. The agreement between the
two techniques was assessed by the method of Bland and Altman
. The Bland-Altman plot (Fig. 2), obtained from 55
simultaneous measurements, shows that the mean bias was
20.41 l.min21 with 95% confidence interval ranging between
24.83 and 4.01 l.min21.
Invasive experiments. In order to gain insights into the
control of maximal oxygen transport in hypoxia following
prolonged rhEpo treatment, seven of the eight subjects (subjects
#14 and #68) underwent an invasive experiment on two
separate occasions, i.e. before and after fourteen weeks of rhEpo
treatment. On each occasion, the subjects performed an
incremental cycling exercise until exhaustion in two conditions,
i.e. in normoxia and in normobaric hypoxia equivalent to an
altitude of 4500 m (FIO2 = 0.115). As for the non-invasive
experiment, here the altitude of 4500 m refers to severe
hypoxia. The order between oxygenation conditions was
randomly balanced in a single-blind manner and the recovery
time between the two exercise bouts was 120 min. On the
experimental days the subjects reported to the laboratory at 08:00
a.m., and catheters were placed under local anesthesia (2%
Lidocain) . Briefly, a 20 gauge catheter (Arrow, Ref.
ES14150, Reading, PA, USA) was inserted percutaneously using the
Seldinger technique into the right femoral artery, 25 cm below
the inguinal ligament and advanced 8 cm in the proximal
direction. This catheter was connected to a blood pressure
transducer positioned at the height of the fourth parasternal
intercostal space (T100209A, Baxter, Unterschleissheim,
Germany) and was also used to sample arterial blood. A similar
catheter was inserted in the same femoral artery 12 cm below the
previous catheter and advanced 510 cm in the proximal direction
for cardiac output measurement. Another 20 gauge catheter
(Arrow, Ref. ES-14150, Reading, PA, USA) was inserted in the
right femoral vein, 23 cm below the inguinal ligament and
advanced 8 cm in the distal direction, beyond the merger with the
saphenous vein for femoral venous blood sampling. This catheter
was also connected to a blood pressure transducer positioned at
the height of the fourth intercostal parasternal space (T100209A,
Baxter, Unterschleissheim, Germany) to measure femoral vein
pressure. In the same femoral vein, a venous catheter with side
holes (Radiopack TFE, Cook, Bjaerverskov, Denmark) was
inserted and advanced ,5 cm proximal to the inguinal ligament
for the injection of ice-cold physiological saline solution. A thin
polyethylene-coated thermistor (model 94-030-2.5F T.D. Probe,
Edwards Edslab, Baxter, Irvine, CA, USA) was inserted through
the venous catheter for blood flow measurements by the constant
infusion thermodilution technique . All these catheters were
connected to three-way stopcocks and, along with the thermistor,
sutured to the skin, under local anesthesia to minimize the risk of
movement during exercise. An additional venous catheter was
inserted into an antecubital vein to inject indocyanine green (ICG,
Akorn Inc, IL) for measuring cardiac output by the dye-dilution
method, as previously reported [22,23]. A three-lead
electrocardiogram (ECG) was displayed on a monitor during
catheterization and the rest of the experimental procedures
(Dialogue 2000, Danica, Copenhagen, Denmark). The ECG,
blood pressure and the temperatures registered by the thermistor,
as well as the infusate temperatures were recorded simultaneously
with the data acquisition system (MacLab 16/s ADInstruments,
Sydney, Australia). Cardiac output was measured continuously
during exercise by the cardiac impedance technique. Respiratory
variables were measured continuously, as explained below.
Incremental exercise. The experimental protocol consisted of
an incremental exercise bout during which the subjects cycled with a
cadence close to 80 revolutions per minute on an electrically-braked
ergometer (Excalibur, Lode, Groningen, The Netherlands), first at
100 watts (W) during 15 min, then by increasing workload by 40 W
every 90 seconds (s) until the subject could not sustain the pedaling
pace. The maximal workload (Wmax) was calculated with the
formula : Wmax = Wcompl+406(t/90), where Wcompl is the last
workload completed, t is the number of seconds that the final,
notcompleted workload was sustained and 40 (watts) is the workload
increment. During the invasive experiment, a blood sample was
obtained simultaneously from the femoral vein and artery, followed
by the measurement of leg blood flow, and cardiac output at the 6th
and 12th minutes at 100 W, within the last 45 s at each workload and
at maximal exercise, as close as possible to exhaustion. In this specific
study, only the measurements at the 12th minute at 100 W and
maximal exercise are reported. Also during normoxic trials the
values at 260 W were given as an estimate of the corresponding peak
workload attained in hypoxia (,280 W). During the non-invasive
experiment, a blood sample was obtained simultaneously from the
forearm vein site and the capillary sampling site at the 12th minute at
100 W and at maximal exercise. During all exercise tests, strong
verbal encouragement was given to the subjects. The subjects
practiced the incremental exercise until exhaustion twice before
performing baseline experiments.
Normobaric hypoxia. Normobaric hypoxia was simulated
by diluting ambient air with nitrogen via a mixing chamber, the
dilution being continuously regulated via an oxygen-pressure
probe (AltiTrainer 200, Sport and Medical Technology, Geneva,
Switzerland). This device allows the inspired partial pressure of O2
(PO2) to be set a pre-determined level of normobaric hypoxia, the
precision of the PO2 being 60.82 mmHg. At rest and during
exercise the subjects breathed through a face mask connected to
two-way respiratory valve. The inspiratory port of the three-way
valve was connected by a low resistance corrugated tube to a
three-way stopcock hidden from the subjects, delivering either
room air or normobaric hypoxia. During all the experiments in
hypoxia, the subjects breathed hypoxic mixture at rest for 10 min
Respiratory variables. Pulmonary V O2, CO2 production
( VCO2), and expired minute ventilation ( VE) were measured
continuously using an automated metabolic cart (Quark b2,
Cosmed Srl, Rome, Italy). Before each test ambient conditions
were measured, then gas analyzer and flowmeter were calibrated by
using high precision gases and a 3-litre syringe, respectively. The
ventilatory variables were recorded as averages of 15 seconds. The
highest 15-second measurement of V O2 was taken as representative
of VO2max. The reason for using this short interval is that leg blood
flow, leg V O2 and blood pressures were also assessed during a similar
Blood flow. Femoral venous blood flow was measured by
constant-infusion thermodilution, as described in detail elsewhere
. Briefly, ice-cold saline was infused through the femoral vein
simultaneously at flow rates sufficient to decrease blood
temperature at the thermistor level by 0.51uC. Infusate and
blood temperature were measured continuously during saline
infusion (Harvard pump, Harvard Apparatus, Millis, MA, USA)
via thermistors connected to the data acquisition system. Infusate
temperature was measured with a thermistor set in a flow-through
chamber (model 93-505, Edslab) connected to the venous catheter.
Blood flow was calculated on thermal balance principles, as
detailed by Andersen and Saltin .
Vascular Conductance. Systemic vascular conductance was
calculated as the cardiac output divided by the mean arterial
pressure. Leg vascular conductance was calculated as the quotient
between leg blood flow and the pressure difference between the
femoral artery and the femoral vein. All pressures were referred to
the fourth parasternal intercostal space.
Blood samples and calculations. Blood was sampled
anaerobically in heparinized syringes (or in capillary tubes
(Radiometer) for the non-invasive experiment, according to the
manufacturers instructions) and immediately analyzed for
hemoglobin concentration ([Hb]), hematocrit, oxygen saturation,
blood pH, CO2 and O2 tensions, lactate, and potassium (ABL700
Series, Radiometer, Copenhagen, Denmark). Capillary hematocrit
values obtained with the gas analyzer (ABL 700) were tested
against the micro-centrifugation method during a separate
procedure. A satisfactory agreement was found between the two
methods (results not shown). Blood gases were corrected for
measured femoral vein blood temperature (femoral venous and
arterial blood gases). Plasma norepinephrine concentration was
measured by ELISA (R&D Systems, Minneapolis, MN, USA).
Blood O2 content (ml.l21) in femoral artery and vein (CaO2 and
CfvO2 respectively) was computed from the saturation and [Hb],
i.e. (1.346[Hb]6SO2)+(0.0036PO2). Systemic oxygen delivery
was computed as the product of cardiac output and CaO2, while
leg O2 delivery was obtained multiplying leg blood flow by CaO2.
Leg V O2 was calculated using the direct Fick method, i.e. as the
product of leg blood flow and arterial-venous difference in oxygen
content. The systemic a-v O2 difference was calculated using the
measured whole body VO2 and cardiac output (indirect Fick
method). Leg lactate and potassium releases were calculated as the
product of leg blood flow and the venous-arterial difference of
lactate and potassium, respectively.
Red blood cell volume. Red blood cell volume was
determined by a carbon monoxide rebreathing method twice
before rhEpo treatment, and after 5 and 13 weeks of rhEpo
treatment. This dataset has been reported elsewhere .
The Kolmogorov-Smirnovs test was applied to examine the
normality in the distribution of data. Due to the small number of
participants in our study (n = 7), for each parameter, all the data
that were obtained at a given power output (i.e. at 100 W or at
peak exercise) were pooled before data distribution was examined
(n = 28). The Bartletts test was used to evaluate the uniformity of
variance between conditions. If variance was not found to be
homogeneous, ANOVA was replaced by the Welchs test. Once
normality and variance homogeneity were verified, the differences
in the measured variables among experimental conditions and
exercise levels were analyzed using two-way ANOVA with
repeated measures, with rhEpo and power output as
withinsubjects factors. Two separated ANOVA analyses were
performed, one for testing the effect of rhEpo in normoxia and the
other one for testing the effect of rhEpo in hypoxia. Another
twoway ANOVA (rhEpo6hypoxia) was applied to the measured
variables 1) at 100 W and 2) at peak exercise. When F was
significant in the ANOVA, planned pair-wise specific comparisons
were carried out using Students paired t test adjusted for multiple
comparisons with the Newman-Keuls procedure. Statistics were
done with the Statview software, version 5.0. (SAS Institute, Cary,
NC). The values are reported as arithmetic means6SE.
Differences were considered as significant for P,0.05.
rhEpo increases red blood cell volume and maximal
oxygen uptake in normoxia
The resting hematological changes observed among our
rhEpotreated subjects have been reported elsewhere . Briefly, rhEpo
was found to increase red blood cell volume by 9.4% after 5 weeks
and by 8.1% after 13 weeks. This was associated with an increase
in CaO2 of 15.7% after 5 weeks and of 14.5% after 13 weeks of
The ergogenic effect of rhEpo in normoxia is demonstrated
from the present data by the increase in maximal oxygen transport
1) at the systemic level with an augmentation of pulmonary
V O2max of 5.8% after 5 weeks (Fig. 3A and 3B) and of 7.2% after
14 weeks of treatment (Fig. 4A), and 2) at the level of exercising
muscles, as shown by the concomitant increase in peak leg VO2
after 14 weeks of rhEpo treatment (Fig. 4B).
rhEpo increases maximal oxygen uptake during acute
moderate hypoxia (15003500 m)
Our non-invasive study reveals that after 5 weeks of rhEpo
treatment the improvement in pulmonary VO2max observed in
normoxia, of 5.8%, did not decline with increasing levels of
inspired hypoxia up to 3500 m (Fig. 3A and 3B). Our results
instead suggest that the stimulating effect of rhEpo on maximal
oxygen transport was even more pronounced during moderate
hypoxia, as the gain in V O2max reached 14.0% at 2500 m
(P = 0.09 vs normoxia) and 17.5% at 3500 m (P,0.05 vs
normoxia) (Fig. 3B). Although the absence of any leg oxygen
transport measurement in this part of the study did not allow us to
unravel the underlying mechanisms, our results at least indicate
that this unexpectedly large gain in VO2max during moderate
hypoxia was related neither to a higher maximal cardiac output
nor to a larger rise in CaO2 following rhEpo in this condition
rhEpo does not increase maximal oxygen uptake during
acute severe hypoxia (4500 m)
Our results demonstrate that the potential for rhEpo to improve
maximal oxygen transport, evidenced both in normoxia and
during moderate hypoxia up to 3500 m, evanished during a more
severe hypoxic challenge equivalent to 4500 m. This finding
appears to be independent of the duration of rhEpo treatment, as
our data showed the same insignificant change in pulmonary
V O2max, of 1.9% after 5 weeks (Fig. 3A and 3B) and of 2.0% after
14 weeks of treatment (Fig. 3A). The demonstration that active
muscles failed to utilize more oxygen after rhEpo in severe hypoxia
of 4500 m comes from the observation of an unchanged peak leg
V O2 in this condition (Fig. 4B). Taken together, our data thus
suggest that the minimal level of inspired hypoxia required to
abolish the ergogenic properties of rhEpo is above 3500 m and
below 4500 m.
Why does rhEpo fail to improve maximal oxygen uptake
during acute severe hypoxia?
The fact that V O2max did not increase during hypoxia
(4500 m) following rhEpo treatment in spite of elevated CaO2
levels raises the question of the mechanisms involved. The results
here below, obtained from our invasive study, provide some
insights into these mechanisms.
Pulmonary gas exchange. Peak pulmonary ventilation
slightly increased after rhEpo in hypoxia (Table 3), without any
concomitant improvement in alveolar PO2 (Table 2).
Alveolar-toarterial O2 difference reached its highest value during maximal
exercise in hypoxia, regardless of rhEpo treatment. By contrast, in
normoxia, peak alveolar-to-arterial O2 difference tended to
decrease (P = 0.06) after rhEpo (Table 2). Finally, SaO2 was not
altered by rhEpo treatment, either during normoxia or during
hypoxia (Table 2).
Systemic oxygen transport. In normoxia, neither maximal
heart rate nor maximal stroke volume was affected by rhEpo and
this was also true in hypoxia (Fig. 5A and 5B). As a consequence,
maximal cardiac output was found similar in all conditions
(Fig. 6A). Whatever the oxygenation condition, CaO2 was
increased after rhEpo (Table 2) resulting in a higher maximal
systemic O2 delivery both in normoxia (P = 0.06) and in hypoxia
(P,0.05) (Fig. 6B). Systemic arterio-venous O2 difference,
although not wider following rhEpo, increased systematically as
power output rose, except during hypoxic exercise (Fig. 6C).
Similarly, systemic fractional O2 extraction failed to increase along
with power output in hypoxia after rhEpo, so that the calculated
value during hypoxic exercise was 16% lower than the
corresponding value obtained before rhEpo treatment (Fig. 6D).
Leg oxygen delivery. Peak leg blood flow was not altered,
either by rhEpo or by hypoxia (Fig. 6E). Peak leg O2 delivery
increased after rhEpo in normoxia only (Fig. 6F). Because CaO2
was augmented after rhEpo without any change in CfvO2 (Table 2),
leg arterio-venous O2 difference rose after rhEpo independently of
oxygenation (Fig. 6G). Finally, leg fractional O2 extraction was not
altered by rhEpo (Fig. 6H).
Distribution of cardiac output. Although neither maximal
cardiac output nor leg blood flow were significantly altered by
rhEpo, Fig. 7 reveals that the distribution of maximal cardiac
output was altered, as the fraction of peak cardiac output directed
to the exercising legs was 17% higher after rhEpo in normoxia.
Conversely, such a change in blood flow distribution did not occur
after rhEpo in hypoxia.
Blood pressure and vascular conductance. Mean arterial
blood pressure during maximal exercise was found to be higher
following rhEpo in normoxia, but not in hypoxia (Fig. 8A). The
same applied for systolic and diastolic blood pressures (results not
shown). Systemic vascular conductance during peak exercise
decreased after rhEpo in normoxia, while this parameter was not
Cardiac output, l.min21
Values are means6SE (n = 7). a reflects arterialized samples from capillary blood; v reflects arm venous samples. RER, respiratory exchange ratio. The subjects were
evaluated before (PRE) and after a 5-week treatment with recombinant human Epo (POST), in normoxia and in acute normobaric hypoxia equivalent to altitudes ranging
from 1500 m to 4500 m. *P,0.05 POST vs PRE. 1P,0.05 hypoxia vs normoxia.
Hypoxia (4500 m)
Hypoxia (4500 m)
Values are means6SE (n = 7). A, alveolar; a, arterial; fv, femoral vein; A-aDO2 alveolar-arterial O2 difference. The subjects were evaluated before (PRE) and after a 14-week
treatment with recombinant human Epo (POST), each time in normoxia and in acute normobaric hypoxia equivalent to 4500 m of altitude. *P,0.05 POST vs PRE.
1P,0.05 hypoxia vs normoxia.
altered by rhEpo in hypoxia (Fig. 8B). Following rhEpo, peak leg
vascular conductance was not affected, regardless of oxygenation
Acid-base balance, lactate and norepinephrine
concentrations in blood. Acid-base balance and blood
lactate concentrations are presented in Tables 1 and 3. The
levels of arm venous lactate (Table 1), as well as the levels of
arterial and femoral venous lactate (Table 3) and potassium (result
not shown) measured during peak exercise were similar in all the
experimental conditions. Furthermore, rhEpo did not induce any
change in leg lactate release (result not shown), regardless of
oxygenation. Finally, leg potassium release during peak exercise
was found to be similar in all the conditions (result not shown).
Of note is that rhEpo treatment was associated with a large
increase in arterial plasma norepinephrine during maximal
exercise, reaching 124% in normoxia and 286% in hypoxia
compared to the values observed before rhEpo treatment (Fig. 8D).
This study demonstrates that the well-known
VO2maxenhancing effect of rhEpo treatment during exercise in normoxia
is also present during mild to moderate hypoxia up to 3500 m, but
at 4500 m there is no benefit for V O2max from rhEpo treatment.
This finding implies that there is a threshold altitude between 3500
and 4500 m, from which little or no benefit should be expected
from an increased hemoglobin concentration. The main
mechanism responsible for this phenomenon resides on the distribution
of cardiac output between active muscles and the rest of the body.
During exercise in normoxia, the administration of rhEpo is
associated with higher CaO2 and systemic oxygen delivery
combined with a higher fraction of the cardiac output directed
to the legs, leading to higher peak leg O2 delivery and V O2.
During exercise in hypoxia CaO2 and systemic oxygen delivery
were also higher after rhEpo treatment, however the fraction of
the cardiac output directed to the legs was not increased
concurrently, leaving the increased CaO2 per se insufficient to
improve leg maximal oxygen delivery.
Because our study addresses the question of the interactions
between hypoxia, CaO2 and VO2max, a prerequisite for data
interpretation is that augmented CaO2 is the main, if not
unique factor by which rhEpo might alter V O2max. In support
of this hypothesis, we separately demonstrated in our
rhEpotreated subjects that if the rise in CaO2 associated with rhEpo
Hypoxia (4500 m)
Hypoxia (4500 m)
Femoral vein HCO32,
Values are means6SE (n = 7). a, arterial; fv, femoral vein; V E/V O2 and V E/V CO2, ventilatory equivalents for O2 and CO2, respectively. RER, respiratory exchange ratio. The
subjects were evaluated before (PRE) and after a 14-week treatment with recombinant human Epo (POST), each time in normoxia and in acute normobaric hypoxia
equivalent to 4500 m of altitude. *P,0.05 POST vs PRE. 1P,0.05 hypoxia vs normoxia.
treatment was acutely reversed by isovolemic hemodilution, the
gain in V O2max conferred by rhEpo was concurrently
rhEpo improves systemic maximal oxygen uptake during
Following rhEpo treatment VO2max was consistently improved
during moderate hypoxia, but not during severe hypoxia. To our
knowledge, only one research group has previously evaluated the
effect of an acute rise in CaO2 and red blood cell volume on
V O2max during moderate acute hypoxia. Their studies showed
that after autologous blood re-infusion VO2max was increased by
9.3% at 2278 m  and by 13% at 3566 m . It has also
recently been reported that at moderate altitude of 2340 m, the
15% increase in hemoglobin concentration induced by three
weeks of acclimatization in athletes resulted in a progressive rise in
CaO2 that was associated with a 8.9% increase in V O2max from
acute to chronic exposure to 2340 m . Although hemoglobin
concentration rose only by 11% after five weeks in our
rhEpotreated subjects, these previous data are nonetheless in line with
the present finding showing that increasing CaO2 is beneficial for
aerobic performance in moderate hypoxia.
A surprising observation was that the improvement in V O2max
was even larger during moderate hypoxia (,15%) than during
normoxia (,6%) following rhEpo. The observation of a similar
V O2max response at two consecutive levels of simulated altitudes
(2500 m and 3500 m) further supports the plausibility of our
observation. Our results indicate that in normoxia, the
improvement in V O2max was smaller than the concomitant rise in O2
delivery (,18%), while during moderate hypoxia (FIO2 = 0.134
0.153), the improvement in VO2max matched the increase in O2
delivery (,15%). Whatever the mechanism underlying this tighter
coupling might be, we suggest that O2 utilization responds more
efficiently to higher O2 supply when the O2 transport system
must cope with moderate arterial hypoxemia.
In severe hypoxia V O2max is not increased by rhEpo
treatment: potential mechanisms involved
The invasive and non-invasive experiments performed in this
study show that rhEpo treatment had no positive effect on
V O2max during severe acute hypoxia. This confirms recent data
from our laboratory, showing no increase in VO2max at 4100 m
following prolonged rhEpo treatment , as well as earlier
evidence demonstrating no augmentation in V O2max at 4300 m
after autologous blood re-infusion . The present finding is also
in agreement with high-altitude studies, where VO2max is
classically found barely changed with altitude acclimatization
(.4000 m), in spite of a large improvement in CaO2 [11,12,29].
Although in both situations (severe acute hypoxia after rhEpo or
chronic hypoxia) VO2max appears to be independent of the
enhancement of CaO2, the underlying mechanisms are different.
Indeed, prolonged rhEpo treatment stimulates erythropoiesis
without increasing hypoxia-inducible factor (HIF-1) levels, while
during prolonged hypoxia HIF-1 accumulation triggers Epo gene
activation therefore promoting erythropoiesis but also targets
many other genes involved in various physiological responses,
which may also have a negative effect on VO2max. In this regard
it has been shown that transgenic mice lacking prolyl hydroxylases
which otherwise always have HIF-1a downregulate aerobic
potential while increasing glycolytic capacity .
One major difference between the present data and
altitudeacclimatization studies relies on the responses of cardiac output
and leg blood flow. With chronic exposure to hypobaric hypoxia
(.4000 m), maximal cardiac output is blunted  and this is
thought to be a major cause of the failure to recover VO2max after
acclimatization . Peak leg blood flow is also decreased with
acclimatization [11,13], not only because cardiac output is lower,
but also because blood flow is preferentially directed toward
nonexercising tissues . By contrast, following rhEpo maximal
cardiac output and leg blood flow were found to be preserved in
acute severe hypoxia, in spite of elevated CaO2. Such cardiac
output response was in line with other data showing no change in
maximal cardiac output during acute hypoxia of similar severity
(40004500 m) without CaO2 manipulation [32,33]. Even so,
rhEpo failed to increase VO2max in severe hypoxia, thus raising
the question of the mechanism(s) abolishing the potential
advantage conferred by high O2 transport capacity.
Blood flow redistribution. One possible mechanism,
supported by our experimental data, is related to blood flow
regulation. In normoxia, rhEpo treatment allowed for a higher
fraction of maximal cardiac output to be directed toward
exercising muscles, resulting in higher peak leg O2 delivery and
leg V O2. Although this blood flow priority given to the active
tissue implied a flow limitation into the other vascular beds, it is
suggested that oxygenation in non-leg tissues - due to higher
CaO2- was nevertheless sufficient to allow for vital organs such as
heart, respiratory muscles or brain to ensure their homeostasis.
Since at maximal exercise there is a competition between tissues
for a limited amount of O2, a strong activation of the sympathetic
system is required to avoid hypotension  and maintain an
efficient match between O2 demand and blood flow distribution
. The higher norepinephrine concentrations in plasma during
normoxic exercise after rhEpo compared to the corresponding
value before rhEpo is compatible with increased sympathetic
activation  helping to direct a greater fraction of the cardiac
output to the legs.
However, the distribution of blood flow during exercise in
severe acute hypoxia was not altered by rhEpo, leaving peak leg
O2 delivery and leg VO2 unchanged. It is worth noting that at that
time, systemic fractional O2 extraction was reduced, unlike leg O2
delivery, therefore suggesting a limitation in O2 extraction into
non-leg tissues. Although we could not establish any causal
relationship between systemic O2 extraction and blood flow
redistribution, we speculate that the lack of blood flow
redistribution toward exercising muscles following rhEpo in hypoxia could
be related to the low O2 extraction observed in non-leg tissues.
Had blood flow decreased to non-leg tissue areas also in hypoxia,
where O2 extraction was already diminished in spite of arterial
hypoxemia, it is possible that O2 delivery/utilization in some
organs (i.e. brain, heart or respiratory muscles) and hence local O2
homeostasis would have been compromised. Beyond the causes of
this lower systemic O2 extraction, which remain unclear, our data
showed an increase in mixed venous O2 content during hypoxic
peak exercise, from 22 ml.l21 before rhEpo to 45 ml.l21 after
rhEpo, suggesting higher central venous oxygenation levels.
Pulmonary gas exchange was not altered by the increase in
hemoglobin concentration elicited by rhEpo treatment, and hence,
alveolar-to-arterial O2 difference and arterial O2 saturation were
similar to those observed before rhEpo. In agreement with this
finding isovolemic hemodilution at altitude did not alter
pulmonary gas exchange in altitude-acclimatized subjects .
Data on blood pressure and vascular conductance can also shed
light on leg versus systemic cardiovascular events that occurred
during maximal exercise following rhEpo. Indeed, we found that
blood pressure during normoxic maximal exercise increased after
rhEpo. Since such response was abolished after isovolemic
hemodilution , increased viscosity due to high hematocrit
was a likely candidate of this higher blood pressure, although a
direct rhEpo vasoconstrictor effect cannot be ruled out . In
association with higher blood pressure, we found that plasma
norepinephrine concentration at peak exercise reached higher
levels following rhEpo, independently of oxygenation. Although
we cannot deduce any causal link between circulating
norepinephrine and blood pressure from the present data, one possibility
is that the high levels of plasma norepinephrine reflected an overall
sympathetically-mediated vasoconstriction  which could be the
cause of elevated blood pressure. If so, the fact that blood pressure
did not increase similarly during hypoxic exercise in spite of
elevated norepinephrine could be related to hypoxia itself, which is
known to attenuate sympathetic vasoconstriction .
Nevertheless, the reason why norepinephrine was higher following rhEpo
remains unclear. A direct effect of rhEpo on norepinephrine can
be speculated, but has not been verified so far.
In summary, our data indicate that the reason why rhEpo does
not boost V O2max in severe acute hypoxia are: 1) a redistribution
of blood flow causing a lower peak leg blood flow following rhEpo
(see Fig. 6E and 7), such that O2 delivery to the legs in severe
hypoxia did not benefit from the increase in CaO2 brought up by
rhEpo treatment, and 2) a lower systemic O2 extraction.
Insights from the switch between moderate and severe
One major finding of the present study is that the gain in
V O2max following rhEpo evanished within 1000 m of altitude, i.e.
that rhEpo was able blunt V O2max decrement efficiently during
moderate hypoxia (FIO2 = 0.134) but failed to do so during slightly
more severe hypoxia (FIO2 = 0.115). One previous study is highly
relevant to the present one, because it showed a dominance of
central fatigue over peripheral muscle fatigue in influencing
exercise performance, only if arterial saturation was below 70
75% . Accordingly, we suggest that until the simulated altitude
of 3500 m, corresponding to SaO2 of ,78%, task failure in our
subjects was likely more dependent on peripheral than central
factors, thus enabling increased CaO2 to exert its maximal
influence on VO2max. By contrast, during exposure to a higher
altitude of 4500 m, SaO2 of ,70% was likely low enough to
trigger central nervous system (CNS) hypoxia and central fatigue
in our subjects, therefore accounting at least in part for our
results. Nevertheless, since this previous study  did not
manipulate CaO2 independently of PaO2 (as in the present one),
we cannot draw a definitive conclusion.
The timing difference for the non-invasive study (5 weeks) and
the invasive study (14 weeks) during the course of rhEpo
administration (see Fig. 1) raises the question of the comparability
of the two studies, because of a potential rhEpo time effect. Our
previously published data with the same subjects indicate that the
hematological changes induced by our rhEpo treatment were of
the same magnitude at weeks 5 and 13, as reflected by the similar
increases in red blood cell volume and CaO2 . Furthermore, the
similar gain in pulmonary V O2max seen either after 5 or after 14
weeks of rhEpo treatment, both in normoxia and hypoxia
(4500 m), suggests the attainment of a plateau in the V O2max
response within the first month of treatment, as previously shown
during 8 weeks of rhEpo administration .
Another point to be mentioned is the validity of the
noninvasive impedance technique we used in the present study. We
acknowledge that the measurement of cardiac output during
strenuous exercise with the thoracic cardiac impedance method is
a questionable technique , however a recent report testing the
same impedance device against the direct Fick method found that
cardiac impedance was reproducible (95% confidence interval
,400 ml.min21) and provided a clinically acceptable evaluation
of cardiac output in healthy subjects during an incremental
exercise . Our own comparison of the impedance method
against the cardio-green dye dilution technique showed values for
bias and 95% confidence interval (see Methods and Fig. 2) that were
very similar to those previously published .
What was already known in the field is that prolonged rhEpo
treatment boosts VO2max in normoxic conditions but not during
severe hypoxia. What the present study adds to the established
knowledge is that rhEpo efficiently blunted the decrement of
V O2max up to the altitude of 3500 m, supporting the idea that
CaO2 is a limiting factor of V O2max during moderate hypoxia.
Furthermore, our study demonstrates that rhEpo-stimulating effect
on V O2max was mediated not only by increased CaO2 but also by
preferential blood flow redistribution toward exercising muscle
tissue. We do not exclude the possibility that this advantageous
blood flow redistribution also occurred at moderate altitude,
therefore explaining why V O2max was substantially improved in
this condition. That such cardiovascular changes did not take
place during severe hypoxia would aim to protect some vital
organs from deleterious hypoxemia.
We are grateful to Pr. Bengt Saltin for his expert comments on this study.
Conceived and designed the experiments: PR JALC RB CL. Performed
the experiments: PR JALC JJT RB PM PR CL. Analyzed the data: PR
JALC JJT RB PM PR CL. Contributed reagents/materials/analysis tools:
JALC RB PM CL. Wrote the paper: PR JALC CL.
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