1
Two protocols to measure mitochondrial capacity in women and adolescent girls: A
31P-MRS
preliminary study
2
ABSTRACT
The phosphocreatine (PCr) recovery time constant (τ) following exercise provides a
measure of mitochondrial oxidative capacity. The purpose of this investigation was to use
two different protocols to determine τ in adolescent females.
31
P-MR spectra were
collected during two exercise tests in 6 adolescent girls (13.8 ± 0.3 y) and 7 women (23.2
± 3.4 y). The first test consisted of 23 repeated 4 s maximal isometric calf contractions
separated by 12 s recovery; PCr recovery between the final 18 contractions was used to
calculate τ. The second test was a sustained 20 s maximal contraction; recovery was fitted
with an exponential function to measure τ. PCr τ did not significantly differ between
groups: (gated exercise: 4 girls: 16 ± 5 s, 7 women: 17 ± 5 s, p; sustained exercise: 6
girls: 19 ± 6 s, 7 women: 19± 4 s). Bland-Altman analysis demonstrated a close
agreement between sustained and gated exercise. Both gated and sustained exercise
appear feasible in a pediatric population, and offer a non-invasive evaluation of
mitochondrial oxidative capacity.
3
INTRODUCTION
Following exercise, the time constant (τ) for the monoexponential recovery of muscle
phosphocreatine (PCr) serves as a valid measure of mitochondrial capacity (23, 26), as
dictated by the resynthesis of PCr via the creatine kinase equilibrium (equation 1).
ATP + Cr ↔ PCr + ADP + H+
(1)
The speed of the monoexponential recovery of PCr is determined by the mitochondrial
capacity of the muscle (9, 22, 26), since glycolytic metabolism is considered to be
negligible within a few seconds of the end of exercise (11). PCr recovery has been used
to study the effects of age (4, 9, 21, 29), training (33), and disease (1, 27) on
mitochondrial capacity.
Mitochondrial capacity in children is an important physiological property, which could be
used to investigate the efficacy of pharmacological and therapeutic interventions on
muscle function, as well as to develop a greater understanding of age and maturation
related changes in skeletal muscle function. A measure of mitochondrial capacity in
health and disease is clearly desirable in young people. For example, a recent study
suggested that PCr recovery might reveal a predisposition to insulin resistance in children
and adolescents (13).
P-MRS allows the noninvasive measurement of PCr τ, but its application in a clinical
31
setting has been limited. To measure PCr τ in young people, several repeat recovery
transitions must be averaged in order to estimate the parameters of the kinetic response
4
with confidence when using low intensity exercise models (5, 40), or a sustained muscle
contraction for ~ 15-30 s (10, 32), is needed to perform the measurement over a single
test. Clearly these tests are not suitable for clinical work, either due to the cost and time
demands by multiple sustained tests, or due to the maximal exertion required to perform
sustained muscle contraction. A study published by Slade and colleagues (32) used a
gated exercise model to determine the PCr recovery τ in adults in a single visit and
without strenuous exercise. This study reported a significant correlation (r=0.82) between
the gated PCr τ and that measured after 30 seconds of intense repetitive exercise. Given
the potential application of gated exercise to enable the assessment of the PCr τ within
the clinical setting, the purpose of the current study was to establish the suitability of
gated exercise for use within a pediatric population.
PARTICIPANTS AND METHODS
Participants and protocol
Seven women (23.2  3.4 y, 63.2 ± 5.5 kg, 1.65 ± 0.04 m) and six adolescent girls (13.8 
0.3 y, 51.3 ± 9.0 kg, 1.61 ± 0.05 m, 1.4 ± 0.5 y from age at peak height velocity (17))
were recruited to participate in the study, which was approved by the institutional ethics
committee. All participants were healthy and recreationally active. Informed consent was
obtained from all adult participants, and the consent and assent of a parent or guardian
and each adolescent, respectively, was obtained. Participants completed 3 exercise tests
on a calf ergometer whilst lying supine within a 1.5 T magnetic resonance scanner
(Philips Gyroscan Intera) with a 6 cm 31P transmit-receive surface coil positioned beneath
the belly of the right calf muscle. Participants completed a familiarization session in a
5
mock MR scanner, during which they practiced sustaining a consistent force using the
ergometer and initiating contractions when cued by a visual display. On a subsequent
day, all tests were completed in a single data acquisition session. Tests were completed in
a standardized order, chosen to ensure that fatigue or metabolic changes resulting from
the sustained contraction did not affect the results of the gated test. 10 minutes of rest was
provided between each test. Participants completed: 1) a set of 5 calf muscle maximal
voluntary contractions (MVC), 2) a gated exercise test lasting 6 minutes; and 3) a 20 s
sustained calf muscle MVC. Muscle PCr and pH were determined by 31P-MRS in tests 2
and 3.
Ergometer and measurement of MVC (Test 1)
A custom-made calf ergometer was used for all exercise protocols. The participant lay
supine with the right foot secured to the ergometer with the ankle plantar-flexed at ~ 30
degrees and Velcro straps securing the participant’s hips and legs. The knee was slightly
flexed for the participant’s comfort. The ergometer consisted of a foot pedal through
which force was transmitted to a static bar perpendicular to the foot. An MR compatible
force transducer (Interface, Scottsdale, AZ, USA) measured the force applied. The heel
cradle of the ergometer was adjusted for each participant to ensure that the ball of the foot
was positioned over the force transducer. The ergometer was calibrated for force before
each test, and corrected for the weight of the participant’s foot on the heel cradle when in
place and resting.
6
Each participant completed 5 MVCs, each lasting 4 s and separated by 1 minute of rest.
The three trials with the highest force were averaged to determine a ‘target’ force for
exercise tests 2 and 3. A visual display projected on the scanner provided force feedback
to the participants.
Gated exercise (Test 2)
The gated exercise test consisted of 23 MVCs, each lasting 4 s, with 12 s of recovery
allowed following each contraction. PCr was measured every 4 s, immediately prior to
and following contraction, as well as twice during recovery. PCr data from the first 5
contractions were discarded to eliminate contractions where depletion during exercise
and resynthesis during recovery were not balanced. PCr values corresponding to the end
of recovery and the end of contraction for the remaining 18 contraction cycles were
averaged (Figure 1). These values were used, along with the time between contractions,
to calculate the PCr recovery τ (16).
PCr τ = -Δt/(ln[D/(D+Q)])
(2)
where τ is the PCr recovery time constant, Δt is the recovery interval between
contractions, D is the steady-state decrease in PCr (the average PCr at the end of
recovery) and Q is the PCr cost of each contraction. Recovery was assumed to be
monoexponential, and PCr recovery between contractions was assumed to balance PCr
depletion due to contraction. Intracellular pH was determined each minute during
exercise and recovery.
7
Sustained exercise (Test 3)
Following 5 minutes of rest from the gated protocol, each participant completed a
sustained 20 s MVC, and PCr recovery from this contraction was monitored for 6
minutes. PCr recovery over the 6 minutes was fitted to an exponential equation of the
form:
PCr(t) = PCrend - PCr(0)(1-e(-t/))
(3)
Where PCrend is the fully recovered PCr, PCr(0) is the PCr at the end of exercise, t is time,
and τ is the time constant for the exponential recovery of PCr. Intracellular pH was
measured over 20 s of exercise and each minute during recovery.
31
P-MR spectroscopy
Prior to exercise testing, gradient echo images were initially acquired to ascertain the
position of the muscle relative to the coil. Matching and tuning of the coil was then
carried out, followed by an automatic shimming protocol on the signal volume that
defined the calf muscle, so as to optimize the signal from the muscle under investigation.
During the test protocol
31
P spectra were obtained using an adiabatic pulse every 1 s,
with a spectral width of 1500 Hz. Phase cycling with 4 phase cycles was employed,
leading the acquisition of spectra every 4 s. The areas of the spectra acquired were then
quantified using a non-linear least squares peak-fitting software package (jMRUI
Software, version 3.0) (25) employing the AMARES fitting algorithm (38). Spectral
8
areas were fitted assuming presence of the following peaks: inorganic phosphate (Pi),
phophodiester, PCr, α-ATP (2 peaks, amplitude ratio 1:1), γ-ATP (2 peaks, amplitude
ratio 1:1) and β-ATP (3 peaks, amplitude ratio 1:2:1). T1 is typically assumed to remain
constant during exercise (4, 29, 18). Intracellular pH was calculated from the shift in the
Pi peak during exercise (34).
Data analyses
Potential mean differences in physiological parameters between adolescent and adult
females were analysed using independent t-tests. The agreement between the PCr τ
determined from the gated and sustained exercise tests were compared using BlandAltman analysis (22) and dependent t-tests. The change in pH during the gated test was
analysed using repeated measures ANOVA, and post-hoc testing was carried out using
planned t-tests with the Bonferroni correction. All analyses were performed using
GraphPad Prism 4 for Windows (GraphPad Software Inc.) with rejection of the null
hypothesis accepted at an alpha level of 0.05.
RESULTS
Table 1 shows the parameters of the PCr recovery during gated exercise, and after a
sustained contraction. The force and PCr responses for both the gated and sustained
exercise tests for a representative participant are shown in figure 2. MVC force was
significantly lower in girls than women (p=0.01). Force was maintained throughout the
gated test; the force on the last contraction was 99 ± 9% of the force on the first
contraction. There were no significant differences between the gated and sustained
9
exercise tests in PCr τ (p=0.40) or end-exercise PCr (p=0.06). However, it was not
possible to measure PCr τ using the gated method in two girls due to low signal-to-noise
ratio. Mean 95% confidence intervals for the fit of the exponential curve to PCr following
recovery were ~ ± 1 s in both girls and women.
Intracellular pH was measured each minute throughout the two exercise tests (Figure 3).
Repeated measures ANOVA revealed that pH changed significantly in the gated test.
Post-hoc testing identified a significant increase from baseline to the first minute
(p=0.005) and then did not change through the rest of the test (p>0.1 for all comparisons).
However, following the 20 s contraction, pH decreased. The average nadir was 6.88 ±
0.04, a significant decrease from baseline (6.96 ± 0.06, p<0.001). The time of this nadir
was midway (2-4 minutes) through recovery.
Figure 4 illustrates the agreement between the PCr recovery τ measured following
sustained exercise and the PCr recovery τ measured using the gated protocol. BlandAltman analysis revealed a mean bias of 2 s (p=0.37), with 95% limits of agreement
(LOA) from -14 to 18 s.
DISCUSSION
The purpose of this study was to examine the feasibility of gated exercise in a pediatric
population to determine PCr τ. PCr recovery τ is an important measure of mitochondrial
capacity in young people, but measurement of this variable often requires that
participants complete repeated exercise tests, which are averaged together (4, 40, 30), or
10
a sustained exercise bout, which can be challenging. By reducing the number or intensity
of tests required from participants, gated exercise might be an efficient method to
measure mitochondrial capacity in young people at minimal cost, time commitment, and
strain from participants. To the best of our knowledge this is the first time gated exercise
has been measured in young people and presents exciting opportunities for advancing
methodological protocols. In the current study, gated exercise was used to measure PCr
recovery τ in 67% of girls and 100% of women. In these participants, the Bland-Altman
analysis revealed a mean bias of 2 s, with 95% LOA of -14 to 18 s, suggesting that there
is no directional bias in the tests, and that some cases, the tests differ in their estimates of
recovery τ. However, in 9/11 subjects, the two estimates of τ were within ±6 s of each
other. PCr recovery kinetics in children and adolescents compared with adults differ by
12-40 seconds where age differences are reported (14, 36). Metabolic recovery following
exercise has been reported to be slowed by 47% in Duchenne muscular dystrophy carriers
compared with healthy controls (20), by 67% in female athletes with cystic fibrosis
compared with matched controls (31), and by 48% in patients with mitochondrial
myopathy compared with healthy controls (35). This would lead to a 9-13 second slowing
compared with the healthy subjects in the current study. Thus, we believe that either of
these tests would be able to detect differences with age or disease in pediatric subjects.
Importantly, there are many potential applications of the gated protocol in a pediatric
population, particularly in the investigation of the effects of disease and treatment.
Diseases such as cystic fibrosis (12), muscular dystrophy (3), and mitochondrial
myopathy (39) are thought to affect mitochondrial capacity and exercise tolerance in
11
children – changes in mitochondrial capacity with therapy or treatment could be noninvasively monitored using this protocol. As well, this type of exercise might be useful in
the prediction of insulin sensitivity in children. A recent study has suggested that PCr
recovery differs in children with impaired insulin response compared with healthy
children, regardless of other risk factors such as body weight (13). Finally, gated exercise
might be used to elucidate the development of mitochondrial capacity in young people
and to investigate the effects of habitual physical activity and intensive sports training in
this population. For example, this technique could be applied longitudinally to measure
changes in mitochondrial capacity with high intensity swimming or gymnastics training
in young people.
The use of gated exercise to calculate PCr recovery  is based on the assumption that
recovery from brief exercise follows a predictable monoexponential time course. It is
widely accepted that an exponential function describes PCr recovery kinetics when pH
does not appreciably decrease from resting values (17, 23), although more intense
exercise might result in more complex patterns of recovery (16). Assuming
monoexponentiality of PCr recovery, the time for recovery and the amplitude of PCr
recovery can be measured and the parameters (specifically the τ) of the exponential
response can be calculated. Use of this method depends on the selection of an exercise
intensity which results in incomplete PCr recovery between contractions. Gated exercise
has been used in animals (7, 15) and in several muscle groups in adults (8, 17, 19, 32) to
determine the rate of PCr recovery.
12
Anaerobic metabolism might affect the results of this investigation in two ways. First,
intracellular pH is known to affect the speed of PCr recovery (18, 37). Specifically, even
a slight decrease in pH slows the PCr τ (37). In the current study, pH remained slightly
elevated above baseline throughout the gated exercise test. However, there was a
significant, though small, decrease in pH during recovery from the sustained exercise
test. This decrease is attributable to proton generation during phosphocreatine
resynthesis, and represents a limitation of using the sustained exercise test to measure
PCr τ. Second, glycolysis has been demonstrated to continue for several seconds
following the cessation of exercise (11, 16). The duration of prolonged glycolysis varies
from study to study, likely reflecting differences in the preceding exercise duration and
intensity. It is possible that glycolysis continued over the early stages of recovery during
the gated protocol, following sustained exercise, or both. A prolonged glycolytic
contribution to energy metabolism could lead to errors in the calculation of the PCr τ in
the gated or sustained analysis.
In two of the adolescent participants in the current study, it was impossible to discern the
parameters of the PCr recovery curve. Due to children’s inherently smaller muscle mass,
the signal-to-noise ratio of the PCr and Pi peaks in
31
P-MR spectra is often poor
compared with spectra collected in adults. Previous investigators have addressed this by
averaging repeated recovery transitions together to increase confidence in the parameters
of the PCr response in children (4, 40). Gated exercise, which depends on averaging
recovery parameters over a number of repeated contractions within a single testing
session, offers another method of improving confidence. However, a low signal-to-noise
13
ratio can also cause problems in the collection and interpretation of data during gated
exercise, as in this study. The signal-to-noise ratio can be improved by increasing the size
of the muscle being interrogated (for instance, the quadriceps rather than the calf muscle)
and/or by using a magnet with a higher field strength, and signal-to-noise ratio should be
a primary consideration in planning a
31
P-MRS study in a pediatric population. Further
studies using this protocol should also consider using a larger sample size and broader
range of ages in the pediatric subjects.
There was good agreement between the PCr τ derived from the gated exercise and that
measured following sustained exercise in 11 adolescent girls and women. The mean bias
was 2 seconds, and in 4 girls and 5 women, the difference was ± 6 s or less. The mean
difference between the two estimates was 24% of the average PCr τ. Metabolic recovery
following exercise has been reported to be slowed by 47% in Duchenne muscular
dystrophy carriers compared with healthy controls (20), by 67% in female athletes with
cystic fibrosis compared with matched controls (31), and by 48% in patients with
mitochondrial myopathy compared with healthy controls (35). Thus, we believe that this
protocol may have acceptable clinical precision. Although PCr τ measured following
sustained exercise is generally regarded as the gold standard measurement of oxidative
capacity, the potential for acidosis to affect this measure must be taken into account (28).
In the sample published by Slade et al. (32), the mean difference between PCr τ measured
using gated and burst exercise was found to be 6 s. These authors report that the
correlation between the two estimates of τ was high (r=0.82). However, the use of
14
correlations to assess agreement has been criticised on the grounds that it measures
association rather than agreement (6). Slade et al. report a slight decrease in pH following
burst exercise similar to the decrease in pH found during recovery from sustained
exercise in the current study. As discussed above, the assumption of monoexponential
recovery is only valid where there is no significant acidosis. It is possible that the
sustained exercise protocol risks underestimating mitochondrial capacity as a result of the
effect of acidosis on PCr resynthesis.
In conclusion, this study has demonstrated that both gated and sustained exercise can be
used to determine PCr recovery  in most adolescent girls and adult women. Both
exercise protocols were tolerated well by the participants, which is important in pediatric
groups and makes both methods prospectively applicable to patient groups. The brief
exercise bouts of the gated protocol are more similar to children’s naturally sporadic
activity patterns (2). In most participants, the gated estimate of PCr  corresponds well
with PCr τ measured following sustained exercise. However, in two participants, poor
signal-to-noise ratio prevented determination of PCr τ using the gated protocol, a
limitation of this technique. Given the clinical implications of mitochondrial capacity for
exercise, recovery, and fatigue, PCr recovery kinetics are important to understand in
adolescents and children in health and disease. This study encourages further exploration
of gated exercise as a cost-effective and well-tolerated method to estimate PCr τ in young
people.
15
Acknowledgements
We thank the students and staff at St. James School for their involvement in this project;
we are also grateful to Bob Wiseman, Ron Meyer, Ted Towse, and David Childs for their
involvement in developing the ergometer used in this project.
The authors do not report any conflict of interest
16
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21
FIGURE LEGENDS
Figure 1. Average PCr during a gated exercise test. The beginning and end of the test are
indicated with vertical lines, while the horizontal lines show the mean depletion of PCr at
the end of each contraction and the end of each recovery period, which were used to
calculate PCr τ.
Figure 2. PCr (top) and force (middle) for a 13 year old girl during a gated exercise test
(left) and during 20 s of sustained exercise and 6 minutes of recovery (right). Dashed
vertical lines indicate the beginning and end of the exercise test.
Figure 3. Intracellular pH (mean and SD) averaged from all participants during the gated
(left) and sustained (right) tests.
Figure 4. Bland-Altman plot for gated τ and τ measured following sustained isometric
contraction for four adolescent girls (●) and seven adult women (○). Dashed lines
indicate the mean bias and limits of agreement. The points for two women (4 and 6,
Table 1) overlap – this point is marked (*).