Key words
endurance performance - oxygen transport - exercise-induced desaturation - hypoxia
- altitude
Introduction
VO2max is a critical factor for endurance exercise performance because it sets the upper
limit for aerobic metabolism [1]
[19]
[20]. The primary limitation of VO2max is the rate of oxygen delivery to the working muscle [1]
[24]; many physiological parameters that influence oxygen delivery during exercise have
been described previously [1]
[20]
[23].
One parameter that influences VO2max is the total mass of hemoglobin in circulation (tHb) [35]. tHb influences VO2max both via its relationship with hemoglobin concentration ([Hb]) and arterial oxygen
content (CaO2) [35], and via its relationship with total blood volume, venous return and ventricular
filling, and maximal cardiac output [10]
[18]
[21]
[35]. However, tHb and [Hb] are not the only factors that influence CaO2; arterial oxygen partial pressure and arterial oxygen saturation (SaO2) also influence CaO2. During high-intensity exercise, SaO2 can drop significantly in a variety of athletes; this condition is known as exercise-induced
arterial desaturation (EIAD). When EIAD was initially described in the literature,
it was thought that decreased arterial oxyhemoglobin concentration during exercise
led to decreased CaO2. However, high-intensity exercise increases arterial blood temperature and can lead
to plasma volume shifts and hemoconcentration; in individuals that do not experience
EIAD, these changes can lead to an increase in CaO2 during high-intensity exercise [36], and therefore EIAD may simply prevent an increase in CaO2 during high intensity [15]. Regardless, it is clear that EIAD has a detrimental effect on VO2max, because ameliorating EIAD by increasing the fraction of inspired oxygen from
21% to 26% leads to an increase in VO2max only in individuals with EIAD [15].
At sea level, EIAD is uncommon in recreationally active subjects, but ~50% of elite
endurance athletes experience some degree of EIAD [25]. As altitude increases, EIAD is exacerbated in all individuals [22]. There is also significant interindividual variability in the severity of EIAD experienced
during exercise, even within groups of similarly trained athletes [12]
[26]. Therefore, assuming EIAD and tHb are independent, EIAD may influence the relationship
between tHb and VO2max.
Although tHb and EIAD both influence oxygen delivery during exercise, to date there
have been no studies looking at how these factors interact to influence VO2max. Therefore, the purpose of this study was to determine whether EIAD influences
the relationship between tHb and VO2max in moderately to highly trained competitive male and female endurance athletes
at moderate altitude (1,625 meters). Additionally, previous research has indicated
that females may be more likely to experience EIAD than males due to anatomical differences
[8]
[9]. However, direct comparisons of EIAD between competitive, endurance-trained male
and female athletes are lacking. Therefore, a secondary aim of this study was to compare
severity of EIAD at moderate altitude in men and women after taking into account aerobic
capacity.
Methods
Subjects
Seventeen female and sixteen male competitive endurance-trained cyclists and triathletes
residing at moderate altitude (1,500–2,000 meters) took part in this study. Subjects
were required to maintain moderate-altitude residence throughout participation in
the study, and testing occurred at 1,625 meters. Endurance-trained was defined as
cycling, running, and/or swimming for more than 10 h per week for men and more than
8 h per week for women over the month prior to inclusion in the study. All subjects
had participated in at least one discipline-specific competitive race in the previous
calendar year. At the time of the first visit, males were required to hold at a minimum
a USA Cycling Category 2 or USA triathlon license; females were required to hold at
a minimum a USA Cycling Category 3 or USA triathlon license. Subjects were screened
to ensure that they were between the ages of 18–42 years old, free from known cardio-respiratory
disease as assessed by the Physical Activity Readiness Questionnaire (PAR-Q), had
not donated blood in the previous 8 weeks, and were non-smokers. Subjects were not
excluded from the study if they had participated in short sojourns to sea level (<7
days) in the 8 weeks prior to participation in the study, because hemoglobin mass
has previously been shown to be stable for up to two weeks following descent to sea
level in endurance-trained moderate-altitude residents [28]. Females were screened to ensure they were not pregnant or breast feeding, and all
females undertook a urine hCG test prior to participating (AccuMed, USA).
Experimental design
Because duplicate measurements reduce the typical error of a measurement by √2 [17], measurements of all primary outcomes (tHb, VO2max, and SaO2) were performed twice. On the first of four visits to the lab, written informed consent
was obtained. In order to confirm that there were no changes in tHb throughout the
study, visits one and four consisted of identical measurements of tHb, whereas visits
two and three consisted of identical graded exercise tests (GXT) to measure maximal
oxygen uptake and SaO2 during exercise. All visits were separated by at least one day, and for each subject,
GXTs were performed at the same time of day, plus or minus one hour. This study was
conducted in accordance with the ethical standards of the International Journal of
Sports Medicine [16].
Total hemoglobin mass
Total hemoglobin mass was measured via the optimized carbon monoxide rebreathing procedure
[27]
[34] as described previously [32]
[33]. Subjects were instructed to refrain from exercise for two hours preceding these
visits due to the possible interactions between exercise and carboxyhemoglobin kinetics.
For this study, the coefficient of variability was 2.7% (95% confidence interval:
2.2%– 3.7%).
Graded exercise test
On visits two and three, subjects performed a maximal GXT on a cycle ergometer (Lode
Excalibur Sport, Groningen, Netherlands). For these visits, subjects were instructed
to arrive at the lab two hours postprandial, and were instructed to not consume alcohol
or perform vigorous activity for 24 h prior to either GXT. Prior to the GXT, body
mass was measured on a digital scale (Combics 1, Sartorius Weighing Technology, Göttingen,
Germany) in cycling clothes and without shoes. Oxygen consumption and other metabolic
parameters were measured via computerized open-circuit indirect calorimetry, which
was calibrated according to the manufacturer’s specifications (TrueOne 2400, Parvo
Medics, Sandy, UT, USA). Heart rate was measured using a heart rate monitor (Polar
Electro, Kempele, Finland), and peripheral oxygen saturation was measured continuously
via forehead pulse oximetry (Nellcor N-595, Medtronic, Minneapolis, MN, USA). Forehead
pulse oximetry was chosen due to its ability to determine SaO2 with relatively low bias and high precision compared to other non-invasive measurement
options [37]. After five minutes of rest on the ergometer, baseline SaO2 was measured for one minute. Each subject was then allowed a 10-min warm-up period
during which the subject rode at a self-selected power not exceeding the power output
of the first stage of the GXT. Following the warm-up period, the subject put on a
nose clip and began breathing through the open-circuit spirometry system. The GXT
began at an individualized power output of 4 W·kg−1 for males and 3 W·kg−1 for females, rounded down to the nearest 20 W increment, and increased 20 W every
minute until subjects reached volitional exhaustion or until cadence could not be
maintained above 60 RPM. This sex-specific protocol was used in order to elicit volitional
exhaustion in this subject population in about 10–12 min, which has been reported
to be an optimal duration for determining VO2max [4]. During each GXT, all subjects reached an RPE>17 and a HRmax within 10% of age-predicted HRmax. As previously described, VO2max was calculated as the highest 30-s average oxygen consumption; SaO2 at VO2max was calculated as the average SaO2 during the same 30 s used to determine VO2max [3]
[32]. Peak power output was calculated as described previously using the following equation
[3]
[32]:
Mild, moderate, and severe EIAD were classified as a SaO2 at VO2max between 93–95%, 88–93%, and<88%, respectively [7]. The coefficients of variation for VO2max and SaO2 at VO2max using these duplicate measurements were 3.5% (95% confidence interval: 2.8–4.7%)
and 1.4% (95% confidence interval: 1.2–1.8%), respectively.
Saturation-adjusted tHb
To determine how saturation influences the relationship between tHb and VO2max, saturation-adjusted tHb was calculated as sat-adj tHb=tHb · (S
a
O
2
at VO
2
max).
Statistics
To determine if there were differences between duplicate tests, dependent t-tests
were used to compare measurements. In order to determine the degree of agreement between
duplicate measurements, intraclass correlation coefficients were calculated; to assess
the typical error of duplicate measurements, the coefficient of variation was calculated
as a percent change [17]. To determine the relationship between tHb and VO2max, as well as the relationship between saturation-adjusted tHb and VO2max, simple linear regressions were performed. Linear regression models were compared
using Williams t-test. To assess differences in SaO2 at VO2max between sexes, multiple linear regression was performed with sex and VO2max as independent variables and SaO2 at VO2max as the dependent variable. For all regression analyses, bivariate normality was
assessed using Q-Q plots and Shapiro-Wilk tests on the residuals, homoscedasticity
and linearity were assessed using scatter plots of normalized residuals, and autocorrelation
was assessed using the Box-Pierce test. Unstandardized regression coefficients from
linear models are reported using the symbol β. All analyses were performed in R, version
3.3.2 (R Core Team, Vienna, Austria), and alpha was set to 0.05. Trends were noted
if 0.05<p<0.1. Results are represented as the mean±SD.
Results
Subject characteristics and measures of reliability between duplicate measures are
given in [Table 1]. Of the 33 subjects who participated, 32 completed all experimental procedures;
one female subject was withdrawn due to illness after completing the first two visits,
so her results represent only a single measurement of each parameter.
Table 1 Subject Characteristics and Measurement Variability.
|
Male n=16
|
Female n=17
|
Coefficient of variation (%)
|
Intra-class correlation coefficient
|
|
Age
|
25.6±4.6
|
28.6±6.0
|
–
|
–
|
|
Weekly Training Duration (hours)
|
16.3±5.4 (Range: 10–30)
|
13.5±4.1 (Range: 8.2–25)
|
–
|
–
|
|
Body Mass (kg)
|
69.7±4.8
|
58.4±5.1
|
0.8
|
0.99
|
|
Height (cm)
|
182.1±4.1
|
165.5±5.1
|
–
|
–
|
|
Absolute VO2max (L·min−1)
|
5.12±0.45
|
3.22±0.43
|
3.5
|
0.99
|
|
Normalized VO2max (mL·kg−1·min−1)
|
73.4±5.4
|
55.2±5.9
|
3.5
|
0.96
|
|
RER at VO2max
|
1.05±0.05
|
1.07±0.06
|
1.4
|
0.86
|
|
HR max
|
184±9
|
185±11
|
2.0
|
0.85
|
|
Peak Power Output (W)
|
429±26
|
292±31
|
2.4
|
0.99
|
|
SaO2 at Rest (%)
|
98.18±0.94
|
98.86±0.87
|
0.8
|
0.49
|
|
SaO2 at VO2max (%)
|
88.8±3.1
|
92.0±2.8
|
1.4
|
0.86
|
|
Desaturation from Rest (%)
|
9.1±3.5
|
6.9±2.6
|
16.1
|
0.84
|
|
Absolute tHb (g)
|
977±102
|
647±100
|
2.7
|
0.99
|
|
Normalized tHb (g·kg−1)
|
14.0±1.3
|
11.1±1.3
|
2.7
|
0.99
|
Values are mean±SD. VO2max: Maximal oxygen uptake during graded exercise test; RER: Respiratory exchange
ratio; HR max: maximum heart rate during graded exercise test; Peak Power Output:
peak power output during graded exercise test; SaO2: arterial oxyhemoglobin saturation; tHb: total hemoglobin mass. Weekly training duration
was calculated from self-reported hours of endurance training per week over the month
preceding inclusion in study. tHb parameters are the average of two measurements,
whereas all graded exercise test parameters (VO2max, RER at VO2max, HR max, Peak Power Output, SaO2 at VO2max, and Desaturation from Rest) are taken from the 2nd graded exercise test, because VO2max was significantly higher for this test.
There were no significant differences between tests for the following measures: tHb,
body mass, SaO2 at rest, SaO2 at VO2max, desaturation from rest, maximum heart rate, maximum RER, peak power output, and
maximum RPE (all p=N.S.). VO2max was significantly higher for the second GXT, both when expressed as L·min−1 (delta=0.064 L·min−1, p=0.048) and when expressed as mL·min−1·kg−1 (delta=1.21 mL·min−1·kg−1, p=0.02). The change in VO2max between trials was not different between males and females (p=N.S.). Although
there was no significant difference in peak power output between tests, the difference
in VO2max between tests was related to the difference in peak power output (p<0.001, r2=0.44), which indicates that there may have been a learning effect that took place
between GXTs. The magnitude of the difference in VO2max between tests was small (<2%). To examine whether this difference influenced our
conclusions, all further analyses were performed twice, once using results from the
average of both GXTs, and once using results from only the second GXT. There were
no differences in any conclusion regardless of which variables were used, and therefore
all results are presented from the second GXT, includingVO2max, SaO2 at VO2max, peak RER, and peak power output.
Exercise-induced arterial desaturation
At VO2max, the average desaturation from rest was 9.1±3.5% for males and 6.9±2.6% for females.
In males, SaO2 at VO2max ranged from 81.7% to 94.0%; in females SaO2 at VO2max ranged from 85.7% to 95%. Overall, 94% of subjects experienced greater than 4%
desaturation from rest (95% confidence interval: 80%–99%), with no statistical difference
between men and women ([Table 2]). SaO2 at VO2max was negatively related to VO2max, both when expressed as an absolute (r=–0.58, p<0.001) and when normalized to
body mass (r=–0.55, p<0.001). When split by sex, this relationship was observed only
in the female cohort (for females, absolute: r=–0.59, p<0.05; normalized: r=–0.62,
p<0.05). There was no significant difference in the severity of EIAD between males
and females after accounting for VO2max (p=N.S.).
Table 2 Prevalence of Exercise-induced Arterial Desaturation.
|
EIAD Severity
|
Male (%) n=16
|
Female (%) n=17
|
|
Mild
|
12.5 [1–38]
|
24 [7–50]
|
|
Moderate
|
50 [25–75]
|
53 [28–77]
|
|
Severe
|
38 [15–65]
|
12 [1–36]
|
|
Overall
|
100 [79–100]
|
88 [64–99]
|
Values are presented as percent [95% confidence interval]. EIAD: exercise-induced
arterial desaturation. Mild, moderate, and severe EIAD were classified as an SaO2 at VO2max between 93–95%, 88–93%, and<88%, respectively [3].
Relationship between tHb and VO2max
tHb was positively related to VO2max when both parameters were expressed as absolute values (βtHb=5.07; r2=0.88, p<0.001; [Fig. 1a]) and when both parameters were normalized to body mass (βtHb=4.70; r2=0.73, p<0.001; [Fig. 2a]). When split by sex, the magnitude of the correlation decreased, but similar relationships
were observed in both males (absolute: r2=0.44, p<0.01; normalized: r2=0.32, p=0.02) and in females (absolute: r2=0.67, p<0.01; normalized: r2=0.42, p<0.01).
Fig. 1 Relationship between VO2max and a absolute total hemoglobin mass (tHb); b total hemoglobin mass adjusted by arterial oxygen saturation during maximal aerobic
exercise, without taking into account body mass. No significant difference was found
between regressions (p=N.S.).
Fig. 2 Relationship between VO2max normalized by body mass and a body mass-normalized total hemoglobin mass (tHb); b total hemoglobin mass adjusted by arterial oxygen saturation during maximal aerobic
exercise, after normalizing by body mass. No significant difference was found between
regressions (p=N.S.).
Saturation-adjusted tHb
When tHb was adjusted by SaO2 at VO2max, this parameter was positively related to VO2max, both when expressed as absolute values (β=6.05; r2=0.87, p<0.001; [Fig. 1b]) and when normalized to body mass (β=5.65; r2=0.68, p<0.01; [Fig. 2b]). When compared to the model between tHb and VO2max, there was no significant difference between the amounts of explained variability,
either for the absolute (p=N.S.; [Fig. 1]) or for the body mass normalized models (p=N.S.; [Fig. 2]).
tHb and SaO2 at VO2max
Across all subjects, when tHb was normalized by body mass, it was negatively related
to SaO2 at maximal exercise (r2=0.32, p<0.001; [Fig. 3]). When split by sex, there was a trend for this relationship to show up in females
(r2=0.22, p=0.06) but not males (r2=0.06, p=N.S.). After diagnostic testing of this model, this analysis was re-run after
removing one male subject who exerted a high degree of influence on the original model.
In the reduced data set, there was still a significant relationship between tHb and
SaO2 at VO2max (r2=0.202, p=0.01). A secondary analysis of this data revealed that when only subjects
who experienced an SaO2 at VO2max less than 91% were analyzed (which included 15 subjects), r2 increased from 0.32 to 0.48 (p<0.01 for this model).
Fig. 3 Relationship between SaO2 at VO2max and total hemoglobin mass (r2=0.324, p<0.001).
Discussion
The primary findings from this study are that 1) adjusting tHb by SaO2 at VO2max did not improve the amount of explained variability in VO2max, and 2) after correcting for aerobic capacity, there was no difference in severity
of EIAD between males and females. Additionally, we found that tHb was negatively
related to SaO2 at VO2max.
The finding that SaO2 at VO2max did not explain additional variability in the relationship between tHb and VO2max was contrary to our hypothesis. This was despite a high prevalence of EIAD and
a large range of observed desaturation values in this cohort. If tHb and SaO2 at VO2max were both independent predictors of VO2max, it would be expected that the relationship between tHb and VO2max would improve after taking into account SaO2 at VO2max. However, accounting for SaO2 at VO2max resulted in no significant changes in the relationship between these variables,
which indicates that tHb and EIAD may not be independent. This concept is supported
by our finding that SaO2 at VO2max was negatively correlated to tHb ([Fig. 3]). Although we do not have direct evidence to explain these results, there are two
possible physiological explanations. One possibility is that individuals with high
tHb may experience more severe desaturation during exercise due to the presence of
high blood volume. High blood volume enables greater venous return and ventricular
filling, and is therefore a prerequisite for high cardiac output; however, as cardiac
output increases, pulmonary capillary transit time decreases, which is believed to
be one of the predominate factors leading to exercise-induced hypoxemia and desaturation
in athletes [6]
[31].
Another possible explanation for this finding is that desaturation during exercise
may influence the regulation of tHb. It is well documented that hypoxia can augment
erythropoiesis and increase tHb [13]. Although very little research has been performed to directly study this issue,
previous research indicates that hypoxemia during high-intensity exercise may interact
with environmental hypoxia to create a larger erythropoietic stimulus in individuals
who experience more severe EIAD. For example, in athletes with SaO2 at VO2max below 91%, three minutes of maximal aerobic exercise at sea level was found to
elevate serum erythropoietin (EPO) for at least 24 h following exercise, and were
found to increase reticulocyte count 96 h following exercise, whereas individuals
with SaO2 at VO2max above 91% had no significant change in EPO or reticulocytes over time [29]. Additional research by the same group found that the increase in circulating EPO
24 h following high-intensity exercise was ~50% higher when the exercise was performed
in simulated normobaric hypoxia (2,100 meters compared to 1,000 meters) [30]. This finding was hypothesized to be a function of the significantly lower SaO2 during exercise in the simulated hypoxia compared to the control condition. To our
knowledge, only one study has followed up on these results to examine whether high-intensity
exercise performed in hypoxia can augment increases in hemoglobin mass over time compared
to the equivalent training in normoxia. Brocherie et al. reported that 6 bouts of
sprint interval training performed in hypoxia throughout a two-week live-high/train-low
training camp increased tHb by 4% on average, compared to a control group that lived
high but performed sprint interval training in normoxia, which increased tHb only
by 2.8% (although these were not statistically different, in part due to low sample
size) [2]. Taken together, these results raise the possibility that individual variability
in EIAD severity during exercise may be one factor that influences tHb in endurance
athletes, especially over long durations of time or in athletes residing at moderate
altitude. However, we do not have direct evidence to support this hypothesis, and
therefore further experimental research is required to fully elucidate the relationship
between EIAD and tHb.
Our finding that there was no difference in severity or prevalence of EIAD at moderate
altitude between endurance-trained males and females is in contrast to our hypothesis.
Previous work has identified that females experience more severe mechanical respiratory
constraints during exercise compared to males [8]
[14], which can exacerbate EIAD and has led to the hypothesis that females may be more
likely than males to exhibit EIAD [5]. Our findings do not support this hypothesis. Our lab has previously published similar
findings in endurance-trained male and female cyclists, where we reported no significant
difference between males and females in SaO2 at VO2max at moderate altitude [3]. Interestingly, this study did find a statistically significant difference in the
degree of desaturation in males versus females, but it was males, not females, that
exhibited larger desaturation. One important caveat to this previous finding is that
the statistical tests used for these comparisons did not take into account aerobic
capacity, which is known to be a confounding variable for EIAD [5]
[7]
[8]. Taken together with previous work in this area, our findings seem to indicate that
although females may generally have respiratory anatomy that increases susceptibility
to EIAD, the severity of this phenomenon depends upon a complex interplay of several
physiological, anatomical, and environmental (i. e., altitude) factors, some of which
seem to be of different importance in males versus females.
One strength of this study was that we were able to examine a relatively large sample
of both male and female endurance athletes across a range of abilities. This can help
to explain the finding that VO2max increased between GXTs. This finding is not unprecedented; for example, Edgett
et al. recently analyzed the reliability of VO2max measured over three GXTs performed on a cycle ergometer in 45 recreationally active
adults, using a similar exercise protocol to this study [11]. This group found an increase of 0.066 L O2 * min−1 between the first and second GXT, and an increase of 0.030 LO2 * min−1 between the second and third GXT (although only the first and third were statistically
different). Interestingly, the magnitude of the change in VO2max between the first and second GXT is almost identical to the statistically significant
increase of 0.064 LO2 * min−1 that we found in the current work. Although this increase in VO2max was statistically significant, the small magnitude of this change (<2%) is likely
not of practical significance and did not have an effect on our conclusions. However,
this finding is important because it helps to quantify the magnitude of the learning
effect during graded exercise tests on VO2max.
One limitation of this study is that despite the relatively large sample size, it
is still possible that SaO2 explains a small but significant portion of the variability in the relationship between
tHb and VO2max that we were underpowered to detect. Another limitation of this study is that
we did not directly measure arterial oxygen saturation or oxygen content. Although
pulse oximetry has previously been shown to reliably measure SaO2 during exercise [37], direct measurements of arterial blood gas, pH, and temperature during exercise
could lead to further insights on this topic. Finally, it is difficult to determine
causal relationships when looking at cross-sectional data. An additional factor that
may influence the relationship between tHb and VO2max is maximal cardiac output, and future research is therefore required on this subject.
Conclusions
At moderate altitude, over 90% of endurance-trained males and females experienced
EIAD. Despite a wide range of exercise-induced desaturation values, taking into account
SaO2 at VO2max did not improve the relationship between tHb and VO2max at moderate altitude. This finding may be in part due to a relationship between
oxyhemoglobin desaturation during exercise and tHb, which warrants further investigation.
Future research is required determine how other physiological parameters, such as
cardiac output, influence the relationship between tHb and VO2max.
