J Appl Physiol 102: 358-367, 2007.
First published October 5, 2006; doi:10.1152/japplphysiol.00920.2005
8750-7587/07 $8.00
Effects of assuming constant optical scattering on measurements of muscle oxygenation by near-infrared spectroscopy during exercise
Leonardo F. Ferreira,1
Dennis M. Hueber,2 and
Thomas J. Barstow1
1Departments of Kinesiology and Anatomy and Physiology, Kansas State University, Manhattan, Kansas; and 2ISS Inc., Champaign, Illinois
Submitted 28 July 2005
; accepted in final form 25 September 2006
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ABSTRACT
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The aim of this study was to examine the effects of assuming constant reduced scattering coefficient (µ
) on the muscle oxygenation response to incremental exercise and its recovery kinetics. Fifteen subjects (age: 24 ± 5 yr) underwent incremental cycling exercise. Frequency domain near-infrared spectroscopy (NIRS) was used to estimate deoxyhemoglobin concentration {[deoxy(Hb+Mb)]} (where Mb is myoglobin), oxyhemoglobin concentration {[oxy(Hb+Mb)]}, total Hb concentration (Total[Hb+Mb]), and tissue O2 saturation (Sti
), incorporating both continuous measurements of µ and assuming constant µ. When measuring µ, we observed significant changes in NIRS variables at peak work rate 
[deoxy(Hb+Mb)] (15.0 ± 7.8 µM), [oxy(Hb+Mb)] (–4.8 ± 5.8 µM), Total[Hb+Mb] (10.9 ± 8.4 µM), and Sti(–11.8 ± 4.1%). Assuming constant µ resulted in greater (P < 0.01 vs. measured µ) changes in the NIRS variables at peak work rate, where [deoxy(Hb+Mb)] = 24.5 ± 15.6 µM, [oxy(Hb+Mb)] = –9.7 ± 8.2 µM, Total[Hb+Mb] = 14.8 ± 8.7 µM, and Sti= –18.7 ± 8.4%. Regarding the recovery kinetics, the large 95% confidence intervals (CI) for the difference between those determine measuring µ and assuming constant µ suggested poor agreement between methods. For the mean response time (MRT), which describes the overall kinetics, the 95% confidence intervals were MRT – [deoxy(Hb+Mb)] = 26.7 s; MRT – [oxy(Hb+Mb)] = 11.8 s, and MRT – Sti= 11.8 s. In conclusion, µ changed from light to peak exercise. Furthermore, assuming a constant µ led to an overestimation of the changes in NIRS variables during exercise and distortion of the recovery kinetics.
tissue oxygen saturation; incremental exercise; kinetics; recovery
NEAR-INFRARED SPECTROSCOPY (NIRS) has been used to determine the concentration of light-absorbing chromophores. In the near-infrared region (650–900 nm), the main absorbing chromophores are hemoglobin (Hb) and myoglobin (Mb), both of which demonstrate an oxygenation-dependent absorption (12). It has been almost three decades since Jöbsis (25) first described the application of NIRS to monitor cerebral and muscle oxygenation in vivo. Although much advance has been made in the field since then (review Refs. 17, 37), one of the early problems faced by Jöbsis still remains in NIRS, i.e., light attenuation in tissues is determined not only by absorption, but also by loss due to scattering. According to the Beer-Lambert law, the attenuation of light in a nonscattering medium is given by
 | (1) |
where A is light attenuation; Io is incident light; I is emergent light; 
is the specific extinction coefficient; [C] is the chromophore concentration (e.g., [Hb] + [Mb]); and L is the light pathlength (distance between points where light enters and leaves the medium). However, tissues are generally highly scattering media, and scattering will increase the pathlength of light, augmenting both the probability of light absorption and loss of light. The Beer-Lambert law was modified to account for these effects on light attenuation [13, 37, but see discussion by Sassaroli and Fantini (38)].
 | (2) |
where the differential pathlength factor (DPF) is included to account for the extended pathlength due to scattering, and G represents the losses due to scattering. Consequently, quantitative measurements of [C] {i.e., oxyhemoglobin concentration [oxy(Hb+Mb)] and deoxyhemoglobin concentration [deoxy(Hb+Mb)]} cannot be made without knowledge of DPF and G. In continuous-wave NIRS, the assumption is made that DPF and G are always constant, and changes in [C] from an arbitrary baseline are then calculated from changes in light attenuation. The belief that assuming constant scattering provides accurate estimates of muscle oxygenation during dynamic exercise has been based on good agreement between oxygenation determined by NIRS and direct measurements in vitro (e.g., Refs. 2, 7). Due to its relative ease of use and noninvasive nature, NIRS has gained wide acceptance in the investigation of muscle oxygenation in exercising humans (3), without any direct examination of the potential confounding effects of the underlying assumption that scattering is constant during exercise.
Light scattering is a complex process occurring when photons cross membrane boundaries due to the changes in the refractive index (37). It has been shown in vitro (35) that increases in total Hb concentration (Total[Hb+Mb]) measured by NIRS were associated with elevations in the "reduced-scattering" coefficient (µ). Previous studies have shown an increase in Total[Hb+Mb] during exercise (7, 11, 16, 20, 21), which suggests that µ could increase concurrently with Total[Hb+Mb] during exercise. If so, the assumption of constant µ could distort the changes in muscle oxygenation, depending on the effects on each wavelength studied (see Eqs. 4 and 5 below).
The tissue optical properties can be determined quantitatively by time-resolved spectroscopy (12, 17). One of these techniques, frequency-domain multidistance (FDMD) spectroscopy, uses intensity-modulated light and applies concepts from diffusion theory to determine both light absorption, due to alterations in [oxy(Hb+Mb)] and [deoxy(Hb+Mb)], and scattering characteristics, based on a two-layer tissue model (12, 14, 22, 37). The two measurements derived by FDMD spectroscopy are the absorption coefficient (µa, cm–1) and µ(cm–1) (14). The theory underlying the measurements of µa and µ by frequency-domain spectroscopy is different from that presented above for continuous-wave spectroscopy (12, 22), but the frequency domain measurements can be related to attenuation of light in biological tissues described in Eqs. 1 and 2 as follows
 | (3) |
and µ= (1 – g)·µs, where µs is the scattering coefficient and g is the mean cosine of the scattering angle (for more details see Refs. 24, 37). From these relationships, it is important to keep in mind that µa is directly proportional to [C] and µ is the optical parameter directly related to light attenuation due to scattering, and DPF is a function of µa and µ(see Eq. 6).
In addition to changes in tissue oxygen saturation (Sti) for steady-state exercise, the kinetics of muscle (re)oxygenation during recovery from exercise have also been extensively studied (1, 7, 23, 32, 33). These kinetics reflect the dynamic interaction between O2 delivery and uptake during recovery and could reveal important alterations associated with, for example, disease states, such as evidenced by microvascular PO2 kinetics in rats (34). However, following cessation of exercise, Total[Hb+Mb] and other metabolic processes with potential to influence µ return to preexercise values, such that changes in µ likely exist during this time period. Under these circumstances, the recovery kinetics of muscle oxygenation might be altered in an unpredictable manner when determined under the assumption of constant µ in healthy subjects (7, 33) and patients (1, 23, 32). It is as yet unclear what effects, if any, the assumption of constant µ has on the kinetic parameters of muscle oxygenation compared with results determined by continuous measurement of µ following the cessation of exercise.
Although µ may be directly related to tissue Total[Hb+Mb], several other physiological responses and biochemical processes associated with exercise might contribute to increase, decrease, or maintain µ constant (see DISCUSSION). Therefore, the purposes of the present study were twofold. The first was to determine the actual pattern of µ during incremental exercise and recovery. The second purpose was to compare the effects of assuming constant µ vs. measuring µ on muscle oxygenation during exercise and its recovery kinetics following cessation of exercise. We hypothesized that µ would increase during exercise, in association with changes in Total[Hb+Mb]. However, we could not anticipate what would be the results for each NIRS variable {i.e., [oxy(Hb+Mb)], [deoxy(Hb+Mb)], Total[Hb+Mb], and Sti}, because these would depend on the relative effect on µ for each wavelength used to determine [oxy(Hb+Mb)] and [deoxy(Hb+Mb)] (see Eqs. 4 and 5 below). Moreover, if µ changes during recovery from exercise, we hypothesized that the assumption of constant µ would likely distort the recovery kinetics of muscle oxygenation compared with those determined by dynamically measuring µ.
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METHODS
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The study involved 15 healthy subjects (13 men, 2 women) with mean ± SD age, weight, and height as follows: 24 ± 5 yr, 71 ± 13 kg, and 177 ± 12 cm. We selected apparently lean subjects (body mass index = 23 ± 2 kg/m2) to minimize the confounding effects of adipose tissue thickness (42). After being informed of all procedures and potential risks of participation, each subject signed a written consent form. The experimental protocol was approved by the Institutional Review Board for Research Involving Human Subjects at Kansas State University.
The subjects performed an incremental exercise test on an electronically braked cycle ergometer (Corival 400, Lode, The Netherlands). The test involved a 4-min period of baseline cycling (20 W) at 60 rpm followed by a progressive (ramp) increase in exercise intensity (15–30 W/min) to volitional exhaustion, with a subsequent 6-min recovery period that consisted of cycling at 20 W (60 rpm).
Muscle oxygenation was evaluated by a frequency-domain multidistance NIRS system (OxiplexTS model 96208, ISS, Champaign, IL). In this study, we used a single probe consisting of eight laser diodes operating at two wavelengths (690 and 830 nm, four at each wavelength) and a photomultiplier tube. The laser diodes and photomultiplier tube were connected to a lightweight plastic probe by optical fibers consisting of two parallel rows of emitter fibers and one detector fiber bundle comprising source-detector separations of 2.0, 2.5, 3.0, and 3.5 cm for both wavelengths. The frequency modulation of laser intensity was 110 MHz, and the heterodyne detection was performed at a 5-kHz cross-correlation frequency. The output frequency was >25 Hz. The probe was positioned longitudinally on the belly of the vastus lateralis muscle 
15 cm above the patella. After the area was carefully shaved and dried, the margins of the probe were bound to the thigh (Skin-Bond, Smith & Nephew, Largo, FL) and secured with Velcro straps around the thigh. No movement (sliding) was observed in any exercise test. The near-infrared spectrometer was calibrated on each test day after a warm-up period of at least 30 min. The calibration was done with the optical probe placed on a calibration phantom with optical properties previously measured, and correction factors were determined and automatically implemented by the equipment's software for the calculation of variables of interest during the data collection (24). The calibration routine was initiated after a warm-up period of 30 min and lasted 15–30 s.
The multiple-distance frequency domain tissue spectrometer provides the average value (dc), amplitude (ac), and phase (
) of the modulated light intensity, which, by using equations derived from diffusion theory (14, 22, 24), allows for continuous measurement of µa and µ for each wavelength (
= 690 and 830 nm). Thus absolute measurements of [oxy(Hb+Mb)] and [deoxy(Hb+Mb)] (expressed in µM) could be made using the Beer-Lambert law (Eq. 1) directly. Assuming only water and Hb as absorbing species, µa() = 
· [oxy(Hb+Mb)] + HHb·[deoxy(Hb+Mb)] + 
· [H2O], and water content equal to 70% and constant, [deoxy(Hb+Mb)] and [oxy(Hb+Mb)] can be determined as
 | (4) |
and
 | (5) |
where the variables of the equations are defined above; is specific extinction coefficient; [HHb] is [deoxy(Hb+Mb)]; [HbO2] is [oxy(Hb+Mb)]; and 830 and 690 are 830- and 690-nm wavelengths, respectively. In this study, we used the (ac, ) pair for the quantitative determination of µa and µ, which minimizes the effects of background light. Further details on the theory and algorithms involved in the technology used herein have been presented elsewhere (12, 14, 15, 22, 37). Total[Hb+Mb] and Sti were calculated as follows: Total[Hb+Mb] (µM) = [deoxy(Hb+Mb)] + [oxy(Hb+Mb)] and Sti(%) = [oxy(Hb+Mb)] 100/{[deoxy(Hb+Mb)] + [oxy(Hb+Mb)]}.
Continuous-wave spectroscopy systems require the assumption that µ is constant throughout the measurement period. The dc signal from the frequency domain spectrometer is similar to the continuous-wave intensity data, allowing for the investigation of the effects of assuming constant µ on the NIRS variables during incremental exercise and recovery. We determined the mean µ at 690 nm (µ
) and 830 nm (µ
) during the last 120 s of baseline pedaling, when µ was relatively constant in all subjects. The data were then reanalyzed off-line with µ assumed constant throughout the protocol, with a value equal to the mean of the baseline period for each subject. In this instance, [deoxy(Hb+Mb)] and [oxy(Hb+Mb)] were calculated by the "DPF method" (13, 15) using software developed by one of the authors (D. M. Hueber). The formula used to determine DPF (15) was
 | (6) |
This formula provides a distance-independent and more simplistic approximation of DPF values, compared with the distance-dependent expression for the "semi-infinite" model (10).
When µ is assumed constant, the changes in the NIRS signal induced by interventions are considered the relevant responses, because absolute values are not available (12). Therefore, changes in [deoxy(Hb+Mb)], [oxy(Hb+Mb)], Total[Hb+Mb], and Sti for assumed constant µ and dynamically measured µ during incremental exercise were determined by subtracting the mean baseline value from the exercise data. For further analysis, the data were averaged in bins corresponding to 10% of the peak work rate, which represents an average of 45–90 s each, depending on the duration of the incremental test.
Kinetics analysis.
The NIRS data were converted to second-by-second values, and the kinetics of [deoxy(Hb+Mb)], [oxy(Hb+Mb)], and Sti during recovery from exercise were determined by nonlinear regression using a least squares technique (Marquadt-Levenberg, SigmaPlot 2001, Systat Software). The model used for fitting the responses consisted of a two-exponential term with a time delay
 | (7) |
 | (8) |
where f(t) is [oxy(Hb+Mb)], Sti, or [deoxy(Hb+Mb)]; EE is end of exercise; AF and AS are the amplitudes, TDF and TDS are the time delays, and 
F and S are the time constants of the exponential responses for the fast and slow components, respectively, for each variable. The relevant amplitude of the slow component (A
) was calculated as AS [1 – e
], and the total amplitude of the response (AT) was defined as AF + A. In some subjects, overshoots and/or undershoots were observed. In these cases, the data were fitted up to the plateau region of the fast portion of the response, and a similar period of time was included in the curve-fitting for measured µ and assumed constant µ. At least 10 s of data before the end of exercise were included in all curve fittings to serve as the baseline before recovery. The overall kinetics were determined by the mean response time (MRT) where the parameters are from Eq. 2 and subsequent text.
Statistical analysis.
A repeated-measures analysis of variance (two within-factors: exercise intensity and scattering) was performed to compare the changes of NIRS variables during incremental exercise, while the Fisher's least significant difference post hoc test was used for pairwise comparisons (NCSS 2000, NCSS Statistical Software, Kaysville, UT). One-way ANOVA was used to analyze the µ response to exercise and recovery. Pearson's product-moment correlation was used to determine association between variables. To evaluate the agreement of the kinetic parameters for measured µ and assumed constant µ, the method of Bland and Altman (5, 30) was used. One sample t-test (or Wilcoxon signed-rank test) and linear regression were used to analyze the results of the Bland-Altman plots of recovery kinetic parameters (30). Values were reported as means ± SD, unless otherwise specified. The 95% confidence intervals (CIs) were calculated using a conservative estimate for small sample sizes (n < 100), as suggested by Ludbrook (30). Significance was declared when P 
0.05.
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RESULTS
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The peak work rate achieved during the incremental exercise was 287 ± 63 W. DPF decreased slightly, but significantly from baseline [690 nm DPF (DPF690) = 5.22 ± 0.88; 830 nm DPF (DPF830) = 4.49 ± 0.83] to peak exercise (DPF690 = 4.88 ± 0.86, DPF830 = 4.27 ± 0.80, P 0.002 vs. baseline).
The values of µ for both wavelengths during baseline cycling and incremental exercise are shown in Fig. 1. During baseline cycling, µ ranged from 5.60 to 6.54 cm–1, and µ ranged from 4.10 to 6.16 cm–1, indicating substantial variability of µ across subjects. We observed that µ increased significantly during incremental exercise, while on average µ did not change significantly (Fig. 1).
A representative response of NIRS variables during the incremental exercise test is shown in Fig. 2, while the absolute values for NIRS variables are presented in Table 1. The effects of assuming constant µ on the changes in NIRS variables during incremental exercise are depicted in Fig. 3. Assuming constant µ resulted in overestimated [deoxy(Hb+Mb)], [oxy(Hb+Mb)], and Sti(where is change) for exercise intensities 
30–40% peak work rate. Assuming constant µ led to overestimation of changes in Total[Hb+Mb] for intensities 81–90% peak work rate.
A representative response of recovery kinetics of [deoxy(Hb+Mb)], [oxy(Hb+Mb)], and Sti is shown in Fig. 4. The Bland-Altman plots for the kinetic parameters of [deoxy(Hb+Mb)], Sti, and [oxy(Hb+Mb)] during recovery are shown in Figs. 5–7. In general, assuming constant µ resulted in an overestimation of the total amplitude of change for [deoxy(Hb+Mb)], Sti, and [oxy(Hb+Mb)]. The differences between F determined using measured µ vs. constant µ for Sti and [oxy(Hb+Mb)] did not differ significantly from zero; however, F of [deoxy(Hb+Mb)] was slower when assuming constant µ's. Importantly, the range of differences between F for measured vs. constant µ's (Fig. 5) was greater than the 95% CI for the parameter estimate (data not shown). In most subjects, TDF was slightly longer for Sti(Fig. 6) and [oxy(Hb+Mb)] (Fig. 7) when µ was continuously measured vs. assumed constant. The MRT of [deoxy(Hb+Mb)] for constant µ tended to be longer (P = 0.06) than that determined by measuring µ. The difference between MRT of measured vs. constant µ for Sti(P = 0.20) and [oxy(Hb+Mb)] (P = 0.29) did not reach significance. Overall, the data were widely scattered with large 95% CIs, suggesting that in some subjects there was poor agreement between kinetic responses determined when measuring µ compared with assuming constant µ.
Surprisingly, there was no significant correlation between baseline Total[Hb+Mb] and µ(P = 0.98) or µ(P = 0.09) (data not shown), given that a previous study reported a positive correlation for measurements in vitro (35). During exercise, some subjects showed a significant correlation between µ and Total[Hb+Mb], while others demonstrated an increase in Total[Hb+Mb] with only minor changes in µ(Fig. 8), suggesting that the profile of µ during exercise could not be explained simply by the pattern of Total[Hb+Mb] response.
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DISCUSSION
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The principal new finding of our study was that µ increased significantly during incremental exercise, while in general µ did not change from baseline to peak exercise. Furthermore, assuming constant µ led to an overestimation of changes in [deoxy(Hb+Mb)], [oxy(Hb+Mb)], and Sti during incremental exercise performed above low-to-moderate intensities. Similarly, for the kinetics of [deoxy(Hb+Mb)], [oxy(Hb+Mb)], and Sti during recovery from incremental exercise, there was poor agreement between the parameters yielded by measuring µ compared with those found by assuming constant µ. Based on these findings, the assumption that µ remains constant from rest to maximal exercise, and during recovery thereafter, appears to be invalid.
Light scattering during incremental exercise.
Near-infrared light traveling through tissue is scattered primarily when crossing membrane boundaries as a consequence of changes in the refractive index among media enclosed by the membranes (cell and intracellular organelles) (37). During incremental exercise, there was a significant increase in µ's of the skin-fat-muscle compartment for 690 nm (Fig. 1); however, µ did not increase significantly, suggesting that, on average, µ of near-infrared light with longer wavelengths are less affected or might actually decrease during exercise. If we assume a linear trend for the relationship between NIRS wavelength and changes in µ's during exercise, we predict that, for 760–776 nm (shortest wavelengths used in some continuous-wave NIRS systems), the changes in µ would be 0.50–0.40 cm–1, respectively. In this context, when assuming constant µ, the changes in light absorption at peak exercise were overestimated by 70% at 690 nm (data not shown). From this, we predict that, for = 760 nm, the changes in light absorption (or optical density) would be overestimated by 50%, if µ is assumed constant.
Even though blood volume comprises 3–5% of total tissue volume, the blood compartment can have a measurable effect on µ(6, 35). Previous studies have shown an association between Total[Hb+Mb] under the area sampled by the probe and µ[venous occlusion, cited by Paunescu et al. (35)]. In the present study, we found that the relationship between Total[Hb+Mb] and µ was variable across subjects and wavelengths (Fig. 8). Thus it appears that the increase in capillary hematocrit associated with arteriolar vasodilation (27), assessed here by Total[Hb+Mb], is not the main factor determining the profile of µ during exercise. If so, we would expect qualitatively similar relationships between Total[Hb+Mb] and µ for both wavelengths studied, as seen in vitro (35).
In addition to blood volume, a frequently neglected factor that will likely contribute to increase µ during exercise is the increase in red blood cell (RBC) flux (cells/s) (26), reflecting a greater number of RBCs exposed to the incident light over a given period of time. Moreover, changes in a cell's osmolarity, such as those produced by alterations in the concentration of anions and cations during muscle contraction, will affect its refractive index and, therefore, the tissue scattering properties. Consistent with this notion, neuronal depolarization has been suggested to decrease µ(8), whereas an increase in intracellular Ca2+ (15) and swelling (39) is thought to increase µ. Larger molecules could also affect µ, such as the µ reducing effects of blood glucose in resting humans (6). In this context, we speculate that accumulation of metabolic by-products (e.g., muscle creatine, inorganic phosphate, and lactate) and blood-borne hormones (e.g., catecholamines) might also affect µ. In contrast, reduced Hb might decrease near-infrared light scattering (8, 9). Therefore, Hb deoxygenation during exercise may be one of the factors counteracting the increase in µ associated with elevation of Total[Hb+Mb] (and RBC flux).
Although the mechanisms mentioned above are potential explanations for our observations, the different patterns of change in µ of both wavelengths (Fig. 1) makes difficult the association with specific physiological responses. In fact, the divergent results for µ at 690 nm and 830 nm are in disagreement with Mie theory of light scattering (µs), which predicts that an increase in scattering would be evident in all wavelengths, although to a lower extent with increasing wavelengths (28). However, Mie theory is a rough approximation primarily because it considers a single size of scatterers and single-scattering events. Moreover, we measured µ, where µ= µs·(1 – g), and g can be a factor of wavelength and particle size. Therefore, multiple scattering events and changes in the "effective" particle size, which is influenced by orientation and index of refraction, might account for the results observed in the present study. However, we acknowledge that factors besides scattering could be responsible for the outcomes of our study. For example, under the assumption of macroscopic tissue homogeneity (see Assumptions and limitations), changes in the structure of the tissue might be measured as changes in µ.
Effects of assuming constant µ
on NIRS variables.
Previous studies have examined the response of NIRS variables during incremental exercise, assuming constant µ through the DPF method (2, 4, 7, 21, 41). In the present study, minor but significant changes were observed for DPF690 (7%) and DPF830 (5%) and assuming constant µ led to a substantial overestimation of [deoxy(Hb+Mb)], [oxy(Hb+Mb)], and Sti during incremental exercise compared with results from dynamically measuring µ with FDMD spectroscopy (Table 1 and Fig. 3). In general, the responses of [deoxy(Hb+Mb)], but not [oxy(Hb+Mb)] (Fig. 3) or Sti(e.g., Fig. 4), were qualitatively similar when comparing data from measured and assumed constant µ. Based on our estimates for changes in µ across wavelengths (see above), systems using longer wavelengths (750 nm) might overestimate the changes in NIRS variables during exercise to a lesser extent than shown in our study. Another important aspect to consider is that light absorption from longer wavelengths (830 nm) predominantly originates from [oxy(Hb+Mb)] due to its greater specific extinction coefficient compared with [deoxy(Hb+Mb)] (43), although both wavelengths contribute to determine [deoxy(Hb+Mb)] and [oxy(Hb+Mb)] (see Eqs. 4 and 5). Thus the effects of assuming constant µ might be less pronounced in measurements of [oxy(Hb+Mb)].
A central problem with the application of NIRS to physiology is the overlapping spectrum of Hb and Mb over the range of wavelengths studied (600–850 nm) (16). The contribution of Hb vs. Mb has been investigated in two studies that came to disparate conclusions (31, 40). A shortcoming of these studies was that both used continuous-wave spectroscopy with µ assumed constant. Our results clearly show that assuming constant µ can distort the characteristics of light absorption stemming from Hb/Mb, suggesting that much caution is needed when interpreting data collected under this assumption.
Recovery kinetics of muscle oxygenation.
The kinetics of muscle oxygenation assuming constant µ during recovery from exercise have been used to evaluate the dynamic balance between muscle blood flow and O2 uptake in health (7) and disease (1, 32). Our investigation of the effects of assuming constant µ on recovery kinetics of NIRS variables demonstrated that, for some parameters, there was a constant bias when comparing the results determined by measuring µ and assuming constant µ(Figs. 4–7). In general, the large 95% CI for the difference between parameters from measured µ and constant µ(Bland-Altman plots, Figs. 5–7) indicates that there was poor agreement between the two methods. This suggests that assuming constant µ might lead to erroneous parameter estimates describing the kinetics of muscle oxygenation, which will depend on the dynamics, and directional changes, of µ following the end of exercise. A detailed description of the time course of µ was outside the scope of our study; however, we noticed that the majority of the change in µ was fast, followed by a minor slower response (data not shown). This might at least partially explain why the MRT, which describes the overall kinetics, was affected to a greater extent than the TDF and F of muscle oxygenation, when µ was assumed constant.
The effects on the TDF and F, albeit small, have implications for the investigation of, for example, exercise training (7) or disease states, such as peripheral vascular disease (32) and congestive heart failure (1). Alterations in each kinetic parameter (or phase) of muscle oxygenation recovery could be related to different mechanisms mediating the adjustment of muscle blood flow and muscle oxygen uptake. Therefore, a more accurate description of the dynamics of muscle oxygenation during recovery from exercise requires continuous measurement of µ, since this optical parameter was not constant following the transition from peak exercise to baseline "unloaded" cycling.
Assumptions and limitations.
The limitations of our study are related to the assumptions made in NIRS in general. A central assumption is that Hb is the only absorbing compound in the tissue volume interrogated. In addition to Mb (see above), other absorbing species (e.g., lipids, melanin) will attenuate the near-infrared light; nonetheless, these are expected to be constant throughout the measurement period. A constant tissue water content equivalent to 70% of the total volume was assumed for each NIRS measurements. The effect of this assumption on the NIRS signal is unknown; however, changes in the water content will modify the tissue refractive index and, consequently, the µ. In addition, the increase in muscle temperature during exercise will provoke changes of the water and lipid spectra; however, it is unclear how the 1–2°C increase in muscle temperature (29) will affect the results of NIRS measurements of muscle oxygenation. Another assumption, which remains unsubstantiated in skeletal muscle during exercise, is that the relative contributions of arterial and venous blood to the NIRS signals remain constant from rest to peak exercise and recovery. Although this may have a minor effect since the microvascular volume is predominantly (85%) composed of capillaries (36). Finally, it is possible that a normalization procedure, such as scaling changes during exercise to the deflection induced by limb ischemia at peak exercise (2), might minimize the error in absolute changes incurred by assuming constant µ. However, due to discomfort, this procedure is of limited utility and would not correct the dynamic disparities demonstrated herein.
The accuracy of frequency-domain NIRS relies on the validity of assumptions about the tissue optical characteristics made to derive µa and µ. These assumptions are that the tissue is macroscopically homogeneous, scatters isotropically, and µ>> µa. The tissue macroscopic homogeneity is certainly not true for in vivo measurements due to the skin-fat layer separating the NIRS probe from the muscle (main source of changes in µa). However, the multidistance method strongly reduces the effects of a superficial layer on the determination of µa and µ(19). The superficial layer has major effects on the light intensity (used in single-distance methods), whereas the slopes of dc, ac, and used to determine µa and µ in the FDMD technique are less affected in a two-layer model (19).
The approximations used in the equations of FDMD spectroscopy can cause "cross talk" between µa and µ, where changes in µ could lead to apparent variations in µa that are "unreal" (i.e., not caused by absorbing chromophores) (24). The changes in µa were consistent across subjects, increasing during incremental exercise and decreasing with similar overall temporal characteristics following the end of exercise (not shown). Conversely, µ was more variable with an increase, decrease, or no change from baseline to peak exercise and recovery (as discussed above). This suggests that absorption-scattering cross talk does not fully explain the increase in µ. However, we must acknowledge that the ability to measure µa and µ comes at the expense of a lower signal-to-noise ratio compared with assuming constant µ(for example see Figs. 2 and 5). Further assumptions and possible limitations of the frequency-domain method have been discussed in previous publications (14, 19, 24, 37).
In summary, we have demonstrated that µ increased during exercise whereas, on average, µ did not change significantly. Moreover, these changes in µ could not be explained by alterations in Total[Hb+Mb] measured by NIRS. The effect of assuming a constant µ, using the DPF method of continuous-wave spectroscopy, was an overestimation of changes in [deoxy(Hb+Mb)], [oxy(Hb+Mb)], and Sti during incremental exercise and recovery. Assuming constant µ also affected the kinetic parameters describing the recovery of muscle oxygenation {[deoxy(Hb+Mb)], [oxy(Hb+Mb)], and Sti} compared with the results yielded by continuously measuring µ. Since it is not possible to determine a priori which subjects will demonstrate changes in µ during exercise, the assumption of constant µ appears to be invalid for a range of wavelengths. Therefore, if mechanistic insights are to be gained from investigation of physiologically relevant variables of NIRS, µ cannot be assumed constant during dynamic exercise. However, the appropriate validation of NIRS should rely on a direct comparison between measurements of venous oxygen saturation in the blood draining the muscle and NIRS, especially the [deoxy(Hb+Mb)] (18) and preferably with continuous measurements of µ(11, 20).
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GRANTS
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Financial support for this study was provided by a Fellowship from the Ministry of Education/CAPES-Brazil to L. F. Ferreira, ISS Inc. to D. M. Hueber, and American Heart Association (0151183Z) to T. J. Barstow.
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ACKNOWLEDGMENTS
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The authors thank Barbara J. Lutjemeier, Dana K. Townsend, and Allison J. Harper for invaluable technical assistance and ISS Inc. for loaning the instrument used in this study.
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FOOTNOTES
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Address for reprint requests and other correspondence: T. J. Barstow, Dept. of Kinesiology, 1A Natatorium, Kansas State Univ., Manhattan, KS, 66506–0302 (e-mail: tbarsto{at}ksu.edu)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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ABSTRACT
METHODS
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, group mean; error bars, 95% confidence interval (CI). The mean difference between kinetic parameters for measured µ
