J Appl Physiol 113: 175–183, 2012.
First published May 10, 2012; doi:10.1152/japplphysiol.00319.2012.
Noninvasive evaluation of skeletal muscle mitochondrial capacity with near-infrared
spectroscopy: correcting for blood volume changes
Terence E. Ryan, Melissa L. Erickson, Jared T. Brizendine, Hui-Ju Young, and Kevin K. McCully
Department of Kinesiology, University of Georgia, Athens, Georgia
Submitted 12 March 2012; accepted in final form 3 May 2012
mitochondrial capacity; electrical stimulation; oxidative metabolism
MEASURING SKELETAL MUSCLE oxidative metabolism has been important in understanding muscle function in health and disease
(19, 23). Noninvasive methodologies have enhanced the study of
mitochondrial function, particularly in human participants. The
primary noninvasive method of measuring mitochondrial function
has been magnetic resonance spectroscopy (MRS), and in particular the use of the kinetics of phosphocreatine (PCr) resynthesis
after exercise as a direct assessment of mitochondrial capacity (30,
35). However, MRS is a costly technique that requires large,
expensive equipment and a high level of technical expertise,
making measurement difficult. Near-infrared spectroscopy
(NIRS) provides a noninvasive measure of muscle oxygenation
(9, 10, 20, 31). Commercially available NIRS devices typically
provide information about the relative changes in oxygenated
Address for reprint requests and other correspondence: T. E. Ryan, 330
River Rd., Univ. of Georgia, Athens, GA 30602 (e-mail: [email protected]).
http://www.jappl.org
hemoglobin/myoglobin (O2Hb), deoxygenated hemoglobin/
myoglobin (HHb), and total hemoglobin or blood volume (tHb).
Compared with MRS, these NIRS devices are much smaller, less
expensive, portable, and easier to use, making them more practically useful for a clinical setting.
NIRS measurement of skeletal muscle oxygen consumption has been done previously using both venous and arterial
occlusions (2, 18, 28, 36). However, a number of researchers have suggested that during occlusions there is a blood
volume change that could confound the slope measurements
for oxygen consumption (4, 38). Blood volume changes
during occlusions can mask changes in the NIRS signals due
to oxygen consumption. Some researchers have suggested
that the deoxygenated hemoglobin/myoglobin (HHb) signal
is less sensitive to blood volume changes (5, 17). Motobe
and colleagues (36) first described a method for measuring
mitochondrial capacity using transient arterial occlusions
and NIRS. In this study, participants performed shortduration handgrip exercise followed by repeated transient
arterial occlusions to measure muscle oxygen consumption
(mV̇O2) (see Figure 1A of Ref. 36). The repeated arterial
occlusions were fit to a monoexponential curve and the time
constant was calculated as a measure of muscle mitochondrial function (see Figure 1B of Ref. 36). After a closer look
at Figure 1A of Ref. 36, there is an obvious blood volume
(tHb) change during the arterial occlusions, which has an
influence on the measurement of mV̇O2. In order for the
arterial occlusion method of measuring mV̇O2 to be valid,
the NIRS signal should reflect a symmetrical change in
O2Hb and HHb, and therefore no change in tHb (tHb ⫽
O2Hb ⫹ HHb). The dissociation of oxygen molecules from
oxyhemoglobin/myoglobin, which supplies the oxygen required for oxidative phosphorylation, should be reflected in
equal and opposite changes in the NIRS signals for O2Hb
and HHb.
In this paper, we provide detailed methods for blood volume
corrections, which allow for the reliable assessment of skeletal
muscle mitochondrial function using NIRS. We have applied a
blood volume correction to data from a variety of subjects
including healthy college-aged participants (both recreationally active and sedentary) and clinical populations such as
individuals with spinal cord injury. We also assessed the
reliability and test-retest reproducibility of our measurements
in young, healthy individuals.
MATERIALS AND METHODS
Subjects
Twenty-four subjects (14 male, 10 female) were tested in this
study. Subjects were chosen to represent a wide variety of muscle
oxidative capacity and included college-aged endurance-trained and
8750-7587/12 Copyright © 2012 the American Physiological Society
175
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Ryan TE, Erickson ML, Brizendine JT, Young HJ, McCully KK.
Noninvasive evaluation of skeletal muscle mitochondrial capacity with
near-infrared spectroscopy: correcting for blood volume changes. J Appl
Physiol 113: 175–183, 2012. First published May 10, 2012;
doi:10.1152/japplphysiol.00319.2012.—Near-infrared spectroscopy
(NIRS) is a well-known method used to measure muscle oxygenation
and hemodynamics in vivo. The application of arterial occlusions
allows for the assessment of muscle oxygen consumption (mV̇O2)
using NIRS. The aim of this study was to measure skeletal muscle
mitochondrial capacity using blood volume-corrected NIRS signals
that represent oxygenated hemoglobin/myoglobin (O2Hb) and deoxygenated hemoglobin/myoglobin (HHb). We also assessed the reliability and reproducibility of NIRS measurements of resting oxygen
consumption and mitochondrial capacity. Twenty-four subjects, including four with chronic spinal cord injury, were tested using either
the vastus lateralis or gastrocnemius muscles. Ten healthy, ablebodied subjects were tested on two occasions within a period of 7 days
to assess the reliability and reproducibility. NIRS signals were corrected for blood volume changes using three different methods.
Resting oxygen consumption had a mean coefficient of variation (CV)
of 2.4% (range 1–32%). The recovery of oxygen consumption (mV̇O2)
after electrical stimulation at 4 Hz was fit to an exponential curve,
which represents mitochondrial capacity. The time constant for the
recovery of mV̇O2 was reproducible with a mean CV of 10% (range
1–22%) only when correcting for blood volume changes. We also
examined the effects of adipose tissue thickness on measurements of
mV̇O2. We found the mV̇O2 measurements using absolute units to be
influenced by adipose tissue thickness (ATT), and this relationship
was removed when an ischemic calibration was performed, supporting
its use to compare mV̇O2 between individuals of varying ATT. In
conclusion, in vivo oxidative capacity can be assessed using blood
volume-corrected NIRS signals with a high degree of reliability and
reproducibility.
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•
Ryan TE et al.
sedentary, as well as four individuals with chronic spinal cord injury
(SCI). Ten healthy, young subjects (6 male, 4 female) were tested on
two separate days over a period of 1 wk to assess the reliability and
reproducibility of our measurements. The study was conducted with
the approval of the Institutional Review Board at the University of
Georgia (Athens, GA), and all subjects gave written, informed consent
before testing.
Experimental Protocol
Fig. 2. Muscle oxygenated hemoglobin/myoglobin (O2Hb; as a percentage of
the ischemic calibration) during rest, resting arterial occlusions, and a 15-s
electrical stimulation exercise followed by a series of transient arterial occlusions after exercise. The final 3–5 min are an ischemic calibration used to
determine a relative concentration.
was performed prior to this 3- to 5-min arterial occlusion to increase
metabolic rate, thereby minimizing the duration of the arterial occlusion and the discomfort imposed on the participants.
NIRS Device
NIRS signals were obtained using a continuous-wave NIRS
device (Oxymon MK III, Artinis Medical Systems), which consists
of two channels (2 equivalent pulsed light sources, 2 avalanche
photodiode detectors, shielding from ambient light), and uses
intensity-modulated light at a frequency of 1 MHz, and laser
diodes at three wavelengths (905, 850, and 770 nm) corresponding
to the absorption wavelengths of oxyhemoglobin (O2Hb) and
deoxyhemoglobin (HHb), with an autosensing power supply (⬃40
W at 110 –240 V). The probe was set for two source-detector
separation distances after measurement of adipose tissue thickness.
The NIRS data were collected at 10 Hz.
Calculation of Muscle Oxygen Consumption
mV̇O2 was calculated as the slope of change in O2Hb and HHb
during the arterial occlusion using simple linear regression. mV̇O2 was
calculated in absolute units using a differential pathlength factor
(DPF) of 4, as suggested by the manufacturer. mV̇O2 was also
expressed as a percentage of the ischemic calibration per unit time.
This measurement was made at rest and repeated a number of times
after exercise. The postexercise repeated measurements of mV̇O2 were
fit to a monoexponential curve according to the formula below:
y ⫽ End ⫺ Delta ⫻ e⫺1⁄Tc
(1)
For this equation, y represents relative mV̇O2 during the arterial
occlusion, End is the mV̇O2 immediately after the cessation of exercise, Delta is the change in mV̇O2 from rest to end exercise, and Tc is
the fitting time constant.
Correction for Blood Volume
Fig. 1. Experimental setup for measurements on the gastrocnemius muscle. A
similar set up was used for measurements on the vastus lateralis muscle. BP,
blood pressure; NIRS, near-infrared spectroscopy.
NIRS data were analyzed using custom-written routines for Matlab
v. 7.13.0.564 (The Mathworks, Natick, MA). The calculation of a
blood volume correction factor (␤) is based on the assumption that
during an arterial occlusion, changes in O2Hb and HHb occur with a
1:1 ratio that represents mitochondrial oxygen consumption only (no
arterial oxygen delivery or venous return of deoxygenated blood), thus
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Each subject was placed on a padded table, supine, with both legs
extended (0° of flexion). The right foot was placed into a home-built
isometric exercise device to limit motion artifact in the NIRS signal.
The foot was strapped firmly to the exercise device using nonelastic
Velcro straps proximal to the base of the fifth digit, and the knee was
supported. The NIRS optode was placed at the level of the largest
circumference of the muscle of interest (medial gastrocnemius or
vastus lateralis) and secured with Velcro straps and biadhesive tape.
Four aluminum foil electrodes attached to a Theratouch 4.7 stimulator
(Rich-mar, Inola, OK) were placed on the skin, two proximal and two
distal to the NIRS optode (Fig. 1). A blood pressure cuff (Hokanson
E20 cuff inflator; Bellevue, WA) was placed proximal to the NIRS
optode.
Testing occurred on one visit to the laboratory. Subjects who
participated in the reproducibility study were tested on a second
occasion 3–7 days after the first session. Adipose tissue thickness
(ATT) was measured at the site of the NIRS optode using B-mode
ultrasound (LOGIQ e; GE HealthCare). Subjects were instructed not
to consume caffeine or tobacco on the day of the test or to use alcohol
or perform moderate or heavy physical activity for at least 24 h before
the test.
The test protocol consisted of a baseline measurement of muscle
oxygenation, followed by inflation of a blood pressure cuff (250 –300
mmHg) for the measurement of resting mV̇O2. To increase mV̇O2, 15
s of continuous electrical stimulation at 4 Hz was applied to the
muscle. Previous studies have shown short-duration exercise produces
similar initial rates of phosphocreatine resynthesis (40). The current
intensity was adjusted for each individual to produce twitch contractions at the maximal tolerable level. Pilot experiments suggested that
small differences in stimulation level did not influence measurements
of metabolic rate (data not reported). Immediately following the
electrical stimulation, a series of 10 –18 brief (5–10 s) arterial occlusions were applied to measure the rate of recovery of mV̇O2 back to
resting levels. To maximize our ability to measure the recovery of
mV̇O2 while minimizing the discomfort to participants, the duration
between arterial occlusions begins at 5 s and extends to 20 s by the
end of the repeated occlusions (i.e., 5 s for cuffs 1–5, 10 s for cuffs
6 –10, 15–20 s for cuffs 11–15). Finally, to normalize the NIRS signal,
a 3- to 5-min arterial occlusion was applied to completely deoxygenate the tissue under the optode (i.e., 0% oxygenation), and the peak
hyperemic response upon release of the cuff was used to indicate
100% oxygenation (Fig. 2). A brief 5-s electrical stimulation period
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Table 1. Physical characteristics of participants
Muscle Tested
Height, cm
Weight, kg
Age, yr
Sex, M/F
ATT, mm
Medial gastrocnemius (n ⫽ 10)
Vastus lateralis (n ⫽ 14)
172.8 ⫾ 6.9
175.2 ⫾ 12.8
67.5 ⫾ 7.7
71.2 ⫾ 16.0
23.3 ⫾ 3.0
35.5 ⫾ 15.4
6 M/4 F
8 M/6 F
6.0 ⫾ 1.6
8.0 ⫾ 2.8
Values are expressed as means ⫾ SD. M, men; F, women; ATT, adipose tissue thickness.
making the area under the NIRS probe a closed system. The following
equation can be used to conceptualize the blood volume change:
˙
O2 ⫹ ⌬blood volume
⌬NIRSSignal ⫽ mV
(2)
␤(t) ⫽
共ⱍ
ⱍO Hb(t)ⱍ
ⱍ ⱍ
2
O2Hb(t) ⫹ HHb(t)
ⱍ兲
(3)
For this equation, ␤ is the blood volume correction factor, t represents
time, O2Hb is the oxygenated hemoglobin/myoglobin signal, and
HHb is the deoxygenated hemoglobin/myoglobin signal. ␤ is a nondirectional factor that represents the proportionality of the blood
volume change (values range from 0 to 1).
We used three approaches to determine ␤.
Method 1. This method calculates a single ␤ for each arterial
occlusion. The proportion of blood volume change attributed to the
O2Hb signal (␤) is calculated for each data point during an arterial
occlusion. Next, the average ␤ is calculated for these data points.
Once ␤ is obtained, it is applied to each individual data point using
Eqs. 4 and 5 below. A single correction factor (␤) is applied to each
individual data point over the course of the arterial occlusion.
Method 2. This method calculates a ␤ for each data point and can
account for small changes in the proportionality of the blood volume
change throughout a cuff. Each data point is then corrected using its
corresponding ␤ according to Eqs. 4 and 5 below.
Method 3. This method calculates a global ␤ and assumes this value
does not change throughout the test. This global ␤ is then applied to
each arterial occlusion. To calculate ␤, a postprocessing correction
was performed using an iterative least-squares error method for
obtaining ␤ (using Nelder-Mead algorithm from Matlab’s fminsearch
function). This routine searches for the ␤ that minimized the sum of
squared residuals (a residual being the difference between O2Hb and
HHb signals).
Once ␤ was defined, its application to the raw data is shown below:
O2Hbc ⫽ O2Hb ⫺ [tHb ⫻ (1 ⫺ ␤)]
(4)
HHbc ⫽ HHb ⫺ (tHb ⫻ ␤)
(5)
where O2Hbc and HHbc are the corrected oxygenated and deoxygenated hemoglobin/myoglobin signals, respectively; tHb if the arithmetic sum of the uncorrected O2Hb and HHb (the blood volume signal
from the NIRS device); and ␤ is the blood volume correction factor.
In Eq. 4, the raw O2Hb signal is corrected by subtracting the
proportion of the blood volume [tHb ⫻ (1 ⫺ ␤)] change attributed to
O2Hb, thus producing a corrected O2Hb signal that represents the
O2Hb signal minus the oxygenated blood volume change. Similarly,
in Eq. 5, the raw HHb signal is corrected by subtracting the proportion
of blood volume [tHb ⫻ ␤] change attributed to HHb, thus producing
Statistical Analysis
Data are presented as means ⫾ SD. Test-retest reliability was
analyzed using coefficient of variation (CV) and intraclass correlation
coefficients (ICC). Relative reliability is related to an individual
maintaining his/her position within a sample for repeated measurements (1). We assess this type of reliability with the ICC, which
indicates error in measurements as a proportion of the total variance
in scores. ICC analysis was done using a downloadable spreadsheet
(22). The absolute reliability can be described as the degree to which
repeated measurements vary for individuals. This was performed by
calculating the CV for each subject, which is the SD of measurements
recorded during both tests divided by the mean of the two tests. CV
was expressed as a percentage. Statistical analyses were performed
using SPSS 19.0 (IBM, Armonk, NY). The relationship between two
variables was analyzed by least-squares regression analysis. Significance was accepted when P ⬍ 0.05.
RESULTS
All subjects were able to complete testing with no adverse
events. The physical characteristics of participants are shown
in Table 1.
Blood Volume Correction
All data were analyzed using the three approaches described
above for correction of the blood volume influence on the NIRS
signal. There were no differences in resting, postexercise mV̇O2,
or the recovery time constant using the three methods (P ⫽ 0.39).
All three methods produced similar results (R2Method1.Method2 ⫽
0.99; R2Method1.Method3 ⫽ 0.98; R2Method2.Method3 ⫽ 0.96). While
all three approaches produced similar results, correcting each data
point (method 2) for its corresponding blood volume change was
the most robust method and produced the most reliable results.
Therefore, the remainder of the results was presented using
method 2 (individualized ␤’s for each arterial occlusion). The
application of the blood volume correction can be seen in Fig. 3.
Without correcting for blood volume, there is an unequal
change in the O2Hb and HHb signals during arterial occlusions
in nearly all of the tests. We found no relationship between the
recovery time constants for the O2Hb and HHb signals (P ⫽
0.78) without the blood volume correction (Fig. 4A). When
correcting for blood volume, there is a strong relationship
between the O2Hb and HHb signals (P ⬍ 0.001) as shown in
Fig. 4B. We did find the HHb signal was less susceptible to
blood volume changes than the O2Hb signal in most cases.
Because blood volume changes mask changes in oxygen consumption, the raw data, uncorrected for blood volume, were
excluded from the remainder of the analysis. The average ␤ for
all subjects and all test days was 0.52 ⫾ 0.21 (mean ⫾ SD).
The blood volume correction (␤) was variable between individuals (CV ⫽ 40.1%). However, ␤ was consistent between
cuffs, and is not influenced by the metabolic rate of the tissue
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The change in NIRS signals (both HHb and O2Hb) during an arterial
occlusion occurs due to a metabolic consumption of oxygen and a
blood volume flux from the redistribution of heme between highpressure arteries/arterioles and low-pressure veins/venules. Therefore,
accurate quantification of mV̇O2 requires either 1) no change in blood
volume, or 2) the removal of blood volume change from the NIRS
signal (i.e., mV̇O2 ⫽ ⌬NIRSSignal ⫺ ⌬blood volume).
To correct NIRS signals for changes in blood volume, the blood
change must be proportioned into oxygenated and deoxygenated
sources. The equation below describes the calculation for this correction factor:
a corrected HHb signal that represents the HHb signal minus the
deoxygenated blood volume change.
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Fig. 3. Postexercise arterial occlusion data for the oxygenated hemoglobin/myoglobin (O2Hb), deoxygenated hemoglobin/myoglobin (HHb), and the blood
volume (tHb) signals for one individual uncorrected (A) and with a blood volume correction (B). Magnification of the final two cuffs for uncorrected (C) and
corrected (D) signals are also shown. Monoexponential recovery curves are also shown for uncorrected (E) and corrected (F) data. The correction used was the
individualized ␤i approach (see MATERIALS AND METHODS for description).
(Fig. 5). The mean CV between cuffs within a single test was
7.5% (range ⫽ 0.3–26%). ␤ (within subjects) was more variable between test days 1 and 2 (CV ⫽ 34%). Figure 5 shows
the group average ␤ over the time for a resting arterial
occlusion and the first end-exercise arterial occlusion. Although ␤ was variable between subjects, all individuals presented similar responses: no change in ␤ over the first 3 s
followed by a gradual rise in ␤ during the final 1 s of occlusion
(Fig. 5). Within-subject ␤ from the resting occlusions was not
statistically different from end-exercise ␤ for both test 1 and
test 2 (P ⫽ 0.414 and P ⫽ 0.486, respectively). Resting ␤ was
not statistically different for participants between test 1 and test
2 (P ⫽ 0.823). Similarly, end-exercise ␤ was not statistically
different for participants between test 1 and test 2 (P ⫽ 0.805).
Reproducibility
The reproducibility of the NIRS measurements for resting
mV̇O2 and the end-exercise recovery of mV̇O2 was investigated
by testing 10 individuals on two separate occasions. All values
for resting mV̇O2 and the recovery of mV̇O2 as well as the CV
and ICC are shown in Table 2.
Resting oxygen consumption. Resting oxygen consumption
was measured by NIRS during an arterial occlusion (⬃270
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179
Influence of Depth
Fig. 4. Comparisons between the recovery time constants for the oxygenated
hemoglobin/myoglobin (O2Hb) and deoxygenated hemoglobin/myoglobin
(HHb) signals without (A) and with (B) the blood volume correction.
mmHg). Resting mV̇O2 of all participants from day 1 and day
2 is shown in Fig. 6A. The mean within-subject CV for
comparison between days was 2.4% (range 1–32%). There
were no differences in resting oxygen consumption between
All NIRS testing was performed using two interoptode
distances ranging from 3 to 4.5 cm. We compared resting
mV̇O2 measured at the shallow vs. deep NIRS signals and
found good agreement between channels (r ⫽ 0.91, P ⬍ 0.001)
only when values were expressed as a percentage of the
ischemic calibration. Using a differential pathlength factor
(DPF) of 4, as recommended by the manufacturer, the relationship between shallow and deep penetration depths is
weaker but still significant (r ⫽ 0.68, P ⬍ 0.001). Figure 8
illustrates the agreement between penetration depths for the
corrected recovery time constants. There were no significant
differences in mV̇O2 between penetration depths (P ⫽ 0.21).
Effects of ATT on mV̇O2
ATT was 6.0 ⫾ 1.6 mm on top of the medial gastrocnemius
and 8.0 ⫾ 2.8 mm on top of the vastus lateralis muscle. ATT
ranged from 3.3 to 12.0 mm in our participants. Resting mV̇O2
was expressed two ways: using the recommended DPF of 4 to
Fig. 5. Blood volume correction factor (␤) calculated for each individual data point over the course a resting arterial occlusion (A) and the first end-exercise
arterial occlusion (B). Slope measurements of oxygen consumption were made over the first 3 s. Data presented are the mean changes in ␤ for all individuals
and all tests. Error bars were omitted for visual presentation, as ␤ was variable between individuals. Despite individual differences the change in ␤ over time
was similar between individuals for resting cuffs (low metabolic rate) and end-exercise cuffs (high metabolic rate).
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the vastus lateralis and gastrocnemius muscles. We also compared the reliability and reproducibility of resting mV̇O2 from
a single arterial occlusion (⬃30 s) with the average of three
10-s arterial occlusions (see Fig. 2). Averaging the three short
resting cuffs decreased the within-subjects CV compared with
the single cuff (CV ⫽ 16% vs. 20%, for the average of 3
measurements and single measurement, respectively). This
suggests that averaging three short arterial occlusions for the
measurement of resting oxygen consumption produces a modest reduction in the variability. We saw no evidence for short
repeated cuffs influencing resting metabolic rate.
End-exercise recovery of mV̇O2. End-exercise recovery of
mV̇O2 was measured by NIRS using brief (5–10 s) repeated
arterial occlusions (⬃270 mmHg). The time constant for the
recovery of mV̇O2 following electrical stimulation was 24.5 ⫾
4.0 s (CV ⫽ 16%) for day 1 and 22.1 ⫾ 2.1 s (CV ⫽ 10%) for
day 2. Recovery mV̇O2 time constants of all participants from
day 1 and day 2 are shown in Fig. 6B. Representative mV̇O2
recovery curves from single subject for day 1 and day 2 are
shown in Fig. 7. The mean coefficient of variation between day
1 and day 2 was 10.6% (Table 2).
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Table 2. Resting and end-exercise oxygen consumption from NIRS
Resting mV̇O2, %/s
O2Hb
Uncorrected
Corrected
HHb
Uncorrected
Corrected
End-exercise recovery
O2Hb Tc, s
Uncorrected
Corrected
HHb Tc, s
Uncorrected
Corrected
Day 1 (n ⫽ 10)
Day 2 (n ⫽ 10)
Mean CV, %
ICC (95% CI)
0.25 ⫾ 0.11
0.28 ⫾ 0.08
0.24 ⫾ 0.17
0.28 ⫾ 0.11
31.4
2.4
⫺0.44
0.19
0.32 ⫾ 0.11
0.28 ⫾ 0.08
0.33 ⫾ 0.12
0.28 ⫾ 0.11
10.7
2.4
0.08
0.19
24.3 ⫾ 8.2
24.5 ⫾ 4.0
23.7 ⫾ 6.1
22.1 ⫾ 2.1
21.0
10.6
0.14
0.68
28.3 ⫾ 7.6
24.5 ⫾ 4.0
23.3 ⫾ 7.6
22.1 ⫾ 2.1
20.6
10.6
0.57
0.67
Values are expressed as means ⫾ SD. Resting muscle oxygen consumption (mV̇O2) is expressed as a percentage of the ischemic calibration per second. The
end-exercise recovery of mV̇O2 is expressed as the time constant (Tc) of the exponential fit. NIRS, near-infrared spectroscopy; CV, coefficient of variation; ICC,
intraclass correlation coefficient; 95% CI, 95% lower and upper confidence intervals.
Fig. 6. Reproducibility results for resting muscle oxygen consumption (mV̇O2)
(A) and the postexercise recovery time constant of muscle oxygen consumption
(B) for day 1 and day 2. Data are presented for each individual that completed
two test sessions.
DISCUSSION
The primary finding of this study was the demonstration of
methods to correct NIRS signals to account for changes in
blood volume. The three different methods of blood volume
correction resulted in consistent and reliable NIRS-based signals of skeletal muscle O2Hb and HHb during arterial occlusion measurements of oxidative metabolism. The appearance
of blood volume changes in the NIRS signal, even with arterial
and venous occlusion, has been reported by others (5, 8, 13, 17,
25). The data from our raw, uncorrected signals support these
volume effects. Our data also support the idea that the HHb
signal is less susceptible to changes in blood volume (17).
While less influenced by blood volume shifts with occlusion,
the uncorrected HHb signal resulted in recovery time constants
that were twice as variable when compared with the blood
volume-corrected HHb time constants. This suggests that the
blood volume correction is needed to accurately detect oxygen
consumption measurements for all the signals coming from the
NIRS devices.
The exact cause of blood volume changes during arterial
occlusions is currently unknown. Blood volume changes could
be attributed to movement of heme concentrations from larger
vessels (undetectable by NIRS) to microvascular beds detected
Fig. 7. Sample recovery curves for a healthy, able-bodied subject from day 1
and day 2. Raw, blood volume-corrected data are represented by solid squares
(day 1) and open triangles (day 2).
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calculate absolute concentration changes and as a percentage of
the ischemic calibration. The influence of ATT on mV̇O2 for both
methods is shown in Fig. 9. There was a significant relationship
between ATT and mV̇O2 (r ⫽ 0.41, P ⫽ 0.026) calculated using
the DPF method (Fig. 9A). This relationship was no longer
existent (r ⫽ 0.006, P ⫽ 0.668) when expressing resting mV̇O2 as
a percentage of the ischemic calibration (Fig. 9B).
NIRS Evaluation of Skeletal Muscle Mitochondrial Capacity
Fig. 8. Comparisons between the NIRS interoptode distances for blood volume-corrected recovery time constants (Tc).
Ryan TE et al.
181
which is slightly higher than our average resting mV̇O2 of
0.28%/s.
In this study, we provide evidence that blood volumecorrected NIRS measurements for the recovery rate of mV̇O2
after electrical stimulation exhibit good reliability. We reported
a mean CV of 10% (range 1–22%) between day 1 and day 2.
Our results are comparable to coefficients of variation reported
for phosphocreatine recovery kinetics (27, 33, 40). The time
constant for recovery of mV̇O2 measured in the gastrocnemius
muscle also agrees with some reported values for phosphocreatine recovery (16, 21), but is faster than other reports (15, 32,
33, 37). One possible explanation for this difference is the
individuals included in the reproducibility portion of this study
were aerobically conditioned. Endurance-trained individuals
have faster phosphocreatine recovery rates compared with their
sedentary or less aerobically fit counterparts (26, 34).
There are a number of potentially confounding factors for
the quantification of continuous-wave NIRS signals including
unknown optical pathlength, absorption, and scattering coefficients, as well as the influence of ATT. The development of
frequency- and time-domain NIRS devices has enabled the
continuous measurement of the pathlength and absorption/
scattering coefficients (3, 10 –12, 14, 41). However, the influence of ATT on NIRS signals still exists even with more
advanced NIRS devices (24). As in the case of this study, and
others using continuous-wave NIRS, ATT will influence the
Fig. 9. Relationship between resting mV̇O2 and adipose tissue thickness (ATT).
A: calculation of resting mV̇O2 in absolute units using a differential pathlength
factor (DPF) ⫽ 4. B: calculation of mV̇O2 as a percentage of the ischemic
calibration. Data represent resting metabolic rate from all subjects tested and
all test days.
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by NIRS (31). Since the NIRS probe is measuring distally to
the arterial occlusion in the vascular tree, there is still a
redistribution of heme under the NIRS probe between the
high-pressure arterial and low-pressure venous systems. The
blood volume correction factor (␤) was consistent between
cuffs and was not influenced by the metabolic rate of the tissue.
While the correction factor value was variable between subjects, all subjects had similar changes in ␤ over time. The ␤ did
not change over the first 3 s of arterial occlusion (where the
slope measurements for oxygen consumption were made),
followed by a slight increase during the final 1–2 s of the
occlusion. In most cases seen in this study the proportion of
heme was primarily oxygenated heme, suggesting the influx of
detectable signal was coming from the arterial side of the
vascular tree. The effect of correcting NIRS signals for blood
volume changes on measurements of oxygen consumption
varies depending on both the magnitude and composition of the
blood volume change. In most cases, the uncorrected data have
large blood volume changes, primarily oxygenated heme, compared with the change due to metabolism. Therefore removing
the blood volume change reverses the direction of the O2Hb
signal during the arterial occlusions, thus allowing for the
measurement of oxygen consumption. In some cases, the blood
volume change is smaller than seen in Fig. 3, and as a result
correcting for blood volume has a small effect on the measured
slopes. Because the correction factor to account for blood
volume was more variable between participants and test sessions, we suggest that the correction method needs to be
applied to each set of experiments. Furthermore, because the
proportion of blood volume change can occur over time, we
feel that correcting each data point in the O2Hb and HHb NIRS
signals (method 2) produces the most accurate results.
In this study, resting oxygen consumption was more variable
than the postexercise recovery of oxygen consumption. The
variability of resting measurements of muscle metabolism in
this study was consistent with previous studies. Van Beekvelt
et al. (38) measured resting mV̇O2 using venous and arterial
occlusion methods, and reported similar between-subjects reliability (CV ⫽ 33%) to our results (CV ⫽ 30% and 39% for
day 1 and day 2, respectively). Hamaoka et al. (18) reported
resting muscle oxygen consumption as a percentage change in
the O2Hb signal of five health male subjects to be 0.39%/s,
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NIRS Evaluation of Skeletal Muscle Mitochondrial Capacity
Ryan TE et al.
method with those types of devices. In addition, more studies
need to be conducted to explore differences in blood volume
changes between individuals, and to accurately quantify NIRS
measurements of oxygen consumption. Furthermore, comparisons also need to be made to established methods such as
magnetic resonance spectroscopy.
GRANTS
This study was funded in part by National Institutes of Health Grant
HD-039676.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: T.E.R., M.L.E., and K.K.M. conception and design of
research; T.E.R., M.L.E., J.T.B., and H.-J.Y. performed experiments; T.E.R.
analyzed data; T.E.R., M.L.E., J.T.B., H.-J.Y., and K.K.M. interpreted results
of experiments; T.E.R. prepared figures; T.E.R. drafted manuscript; T.E.R.,
M.L.E., J.T.B., H.-J.Y., and K.K.M. edited and revised manuscript; T.E.R.,
M.L.E., J.T.B., H.-J.Y., and K.K.M. approved final version of manuscript.
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This study used two interoptode separation distances. This
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potential effect of penetration depth on the metabolic measurements. The depth of muscle activation with electrical stimulation was unknown in this study, which could mean that some
inactive tissue is contributing to NIRS signals. We did not find
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people with different ATT thicknesses and intramuscular fat
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In this study, the calculation of mV̇O2 in absolute units was
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our measurements of mV̇O2 as a percentage change per unit
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In conclusion, we measured mitochondrial capacity using
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muscle oxygen consumption and the recovery rate of muscle
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volume changes, the metabolic exchange between O2Hb and
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changes. This study was performed using a continuous-wave
NIRS system. The need to account for heme signals with
different oxygen saturations moving into the NIRS-detectable
tissue area should apply to time and frequency domain NIRS
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