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Departments of Anatomy and Physiology and Kinesiology, Kansas State University, Manhattan, Kansas
Submitted 20 August 2004 ; accepted in final form 4 January 2005
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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cap) and muscle O2 uptake (
O2m) in the microcirculation. The purposes of the present study were to estimate the time course of cap from the kinetics of the primary component of pulmonary O2 uptake (O2p) and [HHb] throughout exercise, and compare the cap kinetics with the O2p kinetics. Nine subjects performed moderate- (M; below lactate threshold) and heavy-intensity (H, above lactate threshold) constant-work-rate tests. O2p (l/min) was measured breath by breath, and [HHb] (µM) was measured by NIRS during the tests. The time course of cap was estimated from the rearrangement of the Fick equation [cap = O2m/(a-v)O2, where (a-v)O2 is arteriovenous O2 difference] using O2p (primary component) and [HHb] as proxies of O2m and (a-v)O2, respectively. The kinetics of [HHb] [time constant (
) + time delay [HHb]; M = 17.8 ± 2.3 s and H = 13.7 ± 1.4 s] were significantly (P < 0.001) faster than the kinetics of O2 [ of primary component (P); M = 25.5 ± 8.8 s and H = 25.6 ± 7.2 s] and cap [mean response time (MRT); M = 25.4 ± 9.1 s and H = 25.7 ± 7.7 s]. However, there was no significant difference between MRT of cap and P-O2 for both intensities (P = 0.99), and these parameters were significantly correlated (M and H; r = 0.99; P < 0.001). In conclusion, we have proposed a new method to noninvasively approximate cap kinetics in humans during exercise. The resulting overall cap kinetics appeared to be tightly coupled to the temporal profile of O2m. exercise; skeletal muscle; oxygenation
m) can be gained from the investigation of its response in the transitional phase (i.e., kinetics) (21, 27), but because of methodological constraints the kinetics of m in humans have been studied primarily in larger vessels (1, 16, 22, 31, 37, 42, 44).
The difficulty in obtaining measurements with a time resolution that allows reliable kinetic analysis during large muscle mass exercise (e.g., cycling or running) has led to a predominant use of knee extensor or forearm exercise with measurements of blood flow made by Doppler ultrasound (11, 22, 31, 37, 42, 44). These investigations have shown that the m response is biphasic with an initial fast phase determined by the combined effects of muscle contraction (muscle pump) (41) and possibly rapid vasodilation (48) followed by a second slower phase that appears to match O2 delivery and utilization (43).
Several studies have addressed the relationship between m and muscle O2 uptake (O2m) kinetic response after the onset of exercise (5, 12, 14, 16, 22, 31). Although Grassi et al. (16) and Koga et al. (25) demonstrated a similar time course for O2 uptake (O2) and m during moderate exercise, other human (22, 31) and animal studies (5, 12, 13) have shown that m reaches a steady-state level sooner than O2m [i.e., 5–10 s faster time constant ()]. However, it is possible that the adjustment of m in the microcirculation of active skeletal muscles may differ from that measured in larger conduit arteries (27). To date, for technical and ethical reasons, assessing the kinetics of muscle capillary blood flow (cap) in humans has been problematic. Resolution of this discrepancy in m kinetics relative to those of O2m is crucial to advancing our understanding of the mechanisms that govern the control of both m and O2m in health and disease.
Near-infrared spectroscopy (NIRS) provides a noninvasive measure of muscle oxygenation (or O2 extraction) in the microcirculation. Although distinction between hemoglobin (Hb) and myoglobin (Mb) with regard to absorption of the near-infrared light cannot be made, the deoxygenated Hb/Mb (deoxy-Hb/Mb) signal obtained by NIRS has been used as an index of local O2 extraction reflecting the O2m-to-m ratio in the capillaries (9, 15). The time course of deoxy-Hb/Mb after the onset of exercise resembles qualitatively and quantitatively the arteriovenous O2 difference [(a-v)O2] observed in separate investigations (12, 14, 16). Although these studies have not directly compared the kinetics of deoxy-Hb/Mb and (a-v)O2, collectively, these observations suggest that the contribution of Mb to the NIRS signal does not distort the similarity between deoxy-Hb/Mb and (a-v)O2 kinetics. In fact, inferences about m kinetics have been made on the basis of the deoxy-Hb/Mb profile after the onset of exercise (8, 9, 15).
On the basis of the above review, we propose that the kinetics of cap could be estimated noninvasively by solving the Fick equation for blood flow [cap = O2m/(a-v)O2], using the primary component of O2p and deoxyhemoglobin ([HHb]) kinetics as surrogates of O2m and (a-v)O2 kinetics, respectively. Results from computer simulations suggest that the amplitude of the O2m and (a-v)O2 responses have little influence on the calculated m kinetics (10a). Thus, assuming that the relative contributions of arterial and venous blood to the [HHb] signal remain constant, so that the proportionality of [HHb](t) to (a-v)O2 over time also remains relatively constant, the temporal (kinetic) characteristics of cap should be preserved. Therefore, the aims of the present study were to estimate the kinetics of cap from the time course of O2p (primary component) and [HHb] after the onset of exercise and compare the overall kinetics of the estimated cap with the estimated O2m kinetics. Because the preponderance of studies investigating the temporal association between m and O2m have shown that blood flow adjusted at a faster rate than O2 uptake (5, 12, 22, 31), we hypothesized that the estimated cap kinetics would also be faster than the O2m kinetics.
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METHODS |
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Protocol. Subjects performed the exercise protocol on 3 separate days within a period of 2 wk and were instructed to avoid strenuous exercise for at least 24 h preceding each visit to the laboratory. On the first visit, seat height and handlebar position on the cycle ergometer were recorded and these were reproduced on subsequent tests.
The first visit was used to familiarize the subjects with testing procedures and to determine the peak O2 uptake (O2 peak), estimated lactate threshold (LT), and work rates for the constant-work-rate tests. All exercise tests were performed on an electronically braked cycle ergometer (Corival 400, Lode, The Netherlands). The incremental exercise test involved 4 min of baseline cycling at 60 rpm followed by a progressive (ramp) increase in exercise intensity (15–30 W/min) to volitional exhaustion. O2 peak was defined as the highest O2 achieved during the test averaged over a 15-s interval. The LT was estimated from gas-exchange measurements by using the V-slope method, ventilatory equivalents, and end-tidal gas tensions (4, 50). The work rates calculated to elicit 90% of the LT O2 (90% LT) and a O2 halfway between the LT and O2 peak [50% 
= O2 LT + 0.5·(O2 peak – O2 LT)] were determined. Over the next two visits, the subjects performed a total of four to six bouts of constant-work-rate exercise at 90% LT (6 min each) interspersed by 6 min at 20 W, and 2 bouts at 50% (8-min duration). The first bout was preceded by 4 min of baseline pedaling at 20 W. O2p and muscle oxygenation were measured continuously in all subjects and transitions.
Pulmonary gas exchange (O2 and CO2 production) and minute expired ventilation were measured breath by breath by an "open-circuit" method (CardiO2, Medical Graphics, St. Paul, MN). Before each exercise test, the volume signal was calibrated by pumping a 3-liter syringe at flow rates spanning the range expected during the exercise studies, while the O2 and CO2 analyzers were calibrated with gases of known concentration. Heart rate was recorded from the electrocardiogram using a modified lead I configuration and stored in the breath-by-breath file.
Muscle oxygenation was evaluated by a frequency-domain multidistance (FDMD) NIRS system (OxiplexTS, ISS, Champaign, IL). The principles of operation and algorithms utilized by the equipment have been described in detail elsewhere (18). In this study, we used a single channel 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 are 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 selected as 31.25 Hz. The probe was positioned longitudinally on the belly of the vastus lateralis muscle 
15 cm above the patella. To minimize motion artifacts and contamination of the signal by ambient light, the margins of the probe were bound to the thigh with a skin cement (Skin-Bond, Smith & Nephew, Largo, FL) after careful shaving and drying of the area, and secured with Velcro straps around the thigh. The probe position was marked to check for any sliding and for accurate repositioning on subsequent test days. 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 block (phantom) with absorption and reduced scattering coefficients previously measured, and correction factors were determined and automatically implemented by the equipment's software for the calculation of the absorption coefficient (µA) and reduced scattering coefficient (µs) for each wavelength during the data collection (20).
The FDMD method provides continuous measurement of absolute concentration of oxyhemoglobin ([HbO2]), [HHb] (expressed in µM), and µs (1/cm). The [HHb] reported in the present study was calculated incorporating the continuous measurement of µs made throughout the exercise test, i.e., without assuming a constant value for scattering.
Kinetics analysis.
The breath-by-breath O2 and NIRS-oxygenation data were converted to second-by-second values. For each subject, the O2p and NIRS-oxygenation were time aligned to the start of exercise and ensemble averaged to generate a single data set for each variable, at each exercise intensity, to improve the signal-to-noise ratio and confidence of fitting procedures. The kinetics of O2p, [HHb], and cap were determined by nonlinear regression using a least squares technique (Marquardt-Levenberg, SigmaPlot 2001, Jandel Scientific). The model used for fitting the [HHb] response consisted of a single exponential with a time delay (primary component of Eq. 1) or a two-exponential term (primary and slow component of Eq. 1) when an additional "slow component" was observed
 | (1) |
O2p response was fitted by conventional equations (Eq. 2) with two- (phases 1–2) and three- (phases 1–3) exponential terms for exercise below and above the LT, respectively
 | (2) |
I, P, and S are the time constants of the exponential responses of interest for each variable. The initial (cardiodynamic) component of O2p was described up to TDP and the amplitude of the response at TDP (AI) calculated as AI = AI·(1 – e–TDP/I). The relevant amplitude of the primary component was calculated as AP = AI + AP. The 95% confidence limits for p of O2 kinetics were on average p ± 17% (moderate exercise) and p ± 35% (heavy exercise).
Muscle capillary blood flow.
The design used to investigate the kinetics of cap in this study is conceptually similar to that applied in modeling studies (2) and, more recently, in rat muscle preparations (5). The cap response to exercise was derived from the kinetics of O2p and [HHb]. The kinetics of the primary component of O2p during constant-work-rate exercise has been shown to approximate the O2m kinetics (2, 3, 16, 39), whereas [HHb] measured by NIRS is thought to be a function of the muscle O2 uptake-to-blood flow ratio (O2m/m) (8, 15). The [HHb] signal has been shown to be less sensitive than [HbO2] to changes in blood volume under the field of interrogation, thus better representing the muscle oxygenation status during steady- and nonsteady-state situations (10, 15). Hence, assuming that arterial O2 saturation did not change appreciably during moderate and heavy exercise in the present subjects, the [HHb] response was considered to be proportional to O2 extraction (a-v)O2. Therefore, by rearranging the Fick equation, the temporal characteristic of cap could be estimated from the ratio of O2m to [HHb], specifically,
 | (3) |
cap is quantitatively uncertain because the precise proportional contribution of arterial and venous blood to the [HHb] signal are unknown for skeletal muscle. However, if this distribution remains constant from baseline to exercise in a given subject, the temporal (kinetic) characteristic of [HHb], and thus cap, should be preserved. Consistent with this, the results from computer simulations have shown that the amplitude of O2 and (a-v)O2 responses have little influence on the calculated blood flow kinetics (10a). The O2m kinetics were estimated by using the kinetic parameters of O2p obtained from the curve fitting, i.e., by assuming that O2m rose exponentially at time zero with the time constant and amplitude determined for the primary component of the O2p response (e.g., Fig. 1). The resulting estimated O2m kinetics were used to estimate the cap kinetics (see Figs. 2–3). The time course of cap was analyzed with exponential equations as described above (Eq. 2), where cap is substituted for O2. At present, we do not know whether simple exponential equations provide the best mathematical description of the cap response; however, we used methods similar to those previously employed to investigate the kinetics of m (22, 31, 37, 44). The mean response time (MRT) for cap, which approximates the time to reach 63% of the response, was calculated as
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RESULTS |
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O2 peak was 48.8 ± 7.0 ml·kg–1·min–1, and the estimated LT occurred at 56.3 ± 8.5% O2 peak. The work rates for the constant-work-rate tests were 115.0 ± 35.6 W (90% LT) and 205.7 ± 55.7 W (50% ).
Preliminary analysis showed no main effect for the order of the moderate exercise bout averaged for visits 2 and 3 on cap kinetics (MRT cap = 25.9 ± 6.4 s, 25.1 ± 6.9 s and 25.6 ± 7.2 s for bouts 1, 2, and 3, respectively; P = 0.73). Therefore, cap kinetics during exercise at 90% LT was estimated on the basis of [HHb] (and O2p) response from the four to six transitions ensemble averaged to yield a single data set for each subject.
The estimated O2m, [HHb], and estimated cap response of a representative subject are shown in Fig. 2 (90% LT) and 3 (50% ), and Fig. 4 depicts the best regression fit through the cap presented in Figs. 2 and 3. The kinetic parameters of cap and [HHb] during moderate- and heavy-intensity exercise are presented in Table 1. As previously shown (15), the TDI of [HHb] for heavy exercise was shorter than for moderate exercise (P < 0.001), whereas no significant difference was observed for I of [HHb].
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O2, [HHb], and cap are shown in Fig. 5. The MRT of [HHb] (TDP + P) was significantly faster than cap and O2 kinetics for moderate and heavy exercise (Fig. 5). However, the overall kinetics of cap was similar to the estimated O2 kinetics for both exercise intensities, i.e., MRT of cap (MRT-cap) was not significantly different from P-O2 (Fig. 5). No exercise intensity effect was observed for either the MRT-cap or P-O2. Finally, there were significant correlations between MRT-cap and the estimated O2m (P-O2) for moderate (r = 0.99; P < 0.001) and heavy exercise (r = 0.99; P < 0.001) (Fig. 6).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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O2p and [HHb], and to test the hypothesis that the resulting cap kinetics were faster than O2m kinetics. The main novel finding was that the estimated temporal profile of m in the microcirculation (cap) was tightly coupled to O2m kinetics. To the best of our knowledge, this is the first study to noninvasively estimate, in humans, the time course of on-transient m in the microcirculation. Although the [HHb] signal from NIRS has been used to investigate the balance between blood flow and O2 uptake in the microcirculation under various experimental conditions (8, 9, 15), the kinetics of cap itself have not previously been estimated as in the present study.
Validity of assumptions.
Two primary assumptions were made to estimate the temporal profile of cap from the Fick principle. These involved the use of O2p (primary component) and [HHb] kinetics as surrogates of O2m and O2 extraction kinetics, respectively. Therefore, the validity of these assumptions should be addressed before discussing the cap profile observed herein.
O2p as O2m.
The O2m was determined on the basis of the kinetics of the primary component of O2p. The O2p on-transient is characterized by two (moderate intensity) or three phases (heavy intensity) (51), where the initial component has a cardiodynamic origin ("cardiodynamic hyperpnea") (51). The time course of the primary component of O2p has generally been shown or predicted to closely reflect (within 10%) the O2m response (2, 3, 16, 39). Grassi et al. (16) directly measured leg O2 and O2p during cycling exercise and found that kinetics of the primary component of O2p were similar to, and consequently a good approximation of, the leg O2 kinetics. In a different modality (knee-extension exercise), it has been demonstrated that phosphocreatine breakdown (from 31P-NMR) followed a similar dynamic profile to that of the primary component of O2p during moderate-intensity (below LT) exercise (39). Regarding the heavy exercise intensity domain (50% , present study), 86% of the O2p slow component comes from the exercising legs (36). Therefore, on the basis of experimental and theoretical studies (2), it is reasonable to assume that the primary and slow component of O2p approximates the time course of the muscle O2 response during moderate and heavy exercise.
Deoxy-Hb as (a-v)O2. Hb and Mb have similar absorption spectra that at present cannot be distinguished by NIRS devices incorporating two to four wavelengths. There is ongoing controversy as to what extent the NIRS signal contains qualitatively significant information from Mb (33, 40, 46). It has been generally accepted that the NIRS signal evolves predominantly from changes in oxy- or deoxy-Hb (34); however, for its relevance to the present study, this issue deserves further consideration.
In rat thigh muscles perfused with perfluorocarbon to eliminate the NIRS signal arising from Hb, an interference from Mb of less than 10% was observed (40). These results were confirmed in human muscle by use of 1H-NMR (33). In contrast, Tran et al. (46), also using 1H-NMR, showed that in the human gastrocnemius the deoxy-Mb kinetics matched the NIRS oxygenation profile during cuff occlusion, suggesting that the latter signal originated from Mb. It is noteworthy that the 1H-NMR studies (33, 46) analyzed the tissue O2 saturation (% St
= [HbO2]/total[Hb]), whereas recent studies (9, 15) have emphasized the use of [HHb] as an index of O2 extraction (see below). The deoxy-Mb signal from the quadriceps muscle did not change from 50–60% to 100% maximum work rate (38) whereas [HHb] continued to demonstrate a work rate dependency above 50–65% maximal O2 (17). Collectively, these observations suggest that NIRS light absorption in the quadriceps muscle is mainly associated with Hb during exercise.
The NIRS output has often been used to describe a global % St, and comparisons with direct measurements of venous O2 saturation during exercise have been made (7, 32). There are two shortcomings to these studies that are relevant to the present one. First, as discussed by DeLorey et al. (9), femoral venous O2 saturation includes blood flow through both active and inactive muscles, whereas the St was obtained from the vastus lateralis (active) only. To this point, Wilson et al. (53) found good agreement between St from NIRS of the contracting dog gracilis muscle and direct measurements of the isolated venous effluent O2 saturation from the same muscle. Second, changes in blood volume under the area of tissue sampled by the probe (cf. Fig. 4 in Ref. 32) will affect the [HbO2] and St signals, independent of any changes in O2 extraction. On the other hand, the [HHb] is insensitive to blood volume changes (10) and has been used to assess variations in muscle O2 extraction (8, 9). Indeed, Grassi et al. (15) pointed out the striking similarity of [HHb] kinetics during moderate and heavy exercise with the time course of (a-v)O2. Specifically, in the dog gastrocnemius (13), where venous outflow is isolated and, thus, errors due to multiple vessels draining the exercising muscle and muscle-to-sampling site transit delay are minimized, the O2 extraction kinetics (TD 7.5 s and 8 s, Ref. 13) were similar to those found for [HHb] in the present (Table 1) and other studies in exercising humans (9, 15). These findings provide a framework supporting the assumption of [HHb] as a noninvasive surrogate of (a-v)O2 to estimate the temporal profile of muscle capillary blood flow by the Fick principle. However, to our knowledge, no study has directly compared the kinetics of [HHb] with those of (a-v)O2 for a single muscle and its entire venous outflow in exercising humans. Thus this assumption must await direct validation.
Because the cap kinetics were estimated from indirect measures of O2m and (a-v)O2 kinetics, the estimated cap kinetics will have some error when compared with the "true" cap kinetics. Inasmuch as the error introduced by the assumption that [HHb](t) 
(a-v)O2(t) is not known, we cannot predict the extent of error that will be present in the estimated cap kinetics. However, because the kinetics of O2p (primary component) reflects the O2m kinetics with a ±10% error (see above), we might predict that the estimated cap kinetics will represent the true cap kinetics within 
10% (or 2.5 s for the MRT-cap observed herein). Nevertheless, on the basis of the relationship between m vs. O2m kinetics resulting from computer simulations of O2m and (a-v)O2 response to exercise (10a), and the estimated MRT-cap vs. P-O2 (Fig. 6), it appears that the error due to the assumptions made are not much greater than 10%.
Kinetics of m.
The muscle hemodynamic response after the onset of moderate exercise is characterized by two phases (for review, see Ref. 43). In the present investigation, the estimated muscle cap response was also better described by a two-exponential model (three-exponential for heavy exercise) with the initial phase (phase 1) lasting 15–20 s. These results are in concert with the kinetics of bulk blood flow determined in larger human vessels (31, 42) and those of red blood cells in capillaries of contracting rat spinotrapezius muscle (24).
The rapid phase 1 response of m is thought to be due to a mechanical effect of muscle contraction (i.e., muscle pump) and possibly rapid vasodilation (49). Pharmacological interventions (55) and theoretical predictions (24) suggest a lack of vasodilation during phase 1; however, recent studies have indirectly pointed to the presence of a rapid vasodilation sufficient to elevate blood flow in conduit arteries (19, 48). It is noteworthy that the kinetics of phase 1 appear to be faster, with a greater contribution to the overall response, in rest-to-exercise transitions (24, 31, 42) compared with exercise-to-exercise transitions (present study, Ref. 16). This response may reflect a greater muscle pump effect because contraction frequency is an important determinant of the muscle pump (41). Our results, within the constraints of the assumptions made to estimate cap kinetics (see above), suggest that a biphasic capillary blood flow response (24) is also present in the human muscle microcirculation.
The second, slower phase of blood flow adjustment has been associated with feedback metabolic control, and several vasodilators have been proposed to mediate the response (21, 27, 43). The time constant of the primary component (or phase 2) of cap in the present study (26 s, Table 1) was faster than those reported for the femoral artery (40 s, Ref. 31; and 59 s, Ref. 25). The cause of this disparity is not clear, but it is important to emphasize that a different exercise modality (knee extension) was utilized in these studies compared with cycling in the present study. Also, in the present study, cap kinetics were estimated on the basis of assumptions that would introduce some error in the estimated vs. "true" time constant of cap (discussed above). However, it is unlikely that the potentially random error introduced by our assumptions would account for the 14- to 25-s systematic difference with these previous studies. The overall blood flow response estimated in the present study by the mean response time was slower than some (22, 31) but similar to other (16, 25) studies on larger human vessels, when the relationship between m and O2m kinetics is considered (Fig. 4A). In general, our results differ from those studies that investigated rest-to-exercise transitions (22, 31) but agree with results from studies using exercise-to-exercise protocols (16, 25). Therefore, the inconsistency of results could be related to differences in the phase 1 profile of m and its relative contribution to the overall response (see above).
For heavy exercise, comparison of MRT or P of blood flow with previous investigations in humans is not straightforward because we chose to truncate the MRT so as to reflect the primary component of the response, whereas others have included the blood flow increase associated with the O2 slow component (1, 11, 37). The rationale for limiting the calculation of the heavy exercise MRT was that we were interested in determining the relationship between the primary components of the estimated cap and O2m kinetics. Furthermore, it is not clear whether the time course of [HHb] approximates the dynamics of (a-v)O2 during the slow component phase of O2 (36), which technically limited us from determining the MRT for the total response (primary and slow component). On the basis of the relationship presented in Fig. 4B, our results for cap kinetics are in agreement with the primary component of m kinetics in the dog gastrocnemius muscle preparation contracting at O2 peak (14).
Dynamic coupling of m to O2m.
In the present study, the estimated temporal profile of cap was directly related to the O2m kinetics. As seen in Fig. 4, the association between blood flow and O2 kinetics has been demonstrated by a number of investigators (13, 16, 22, 25, 31). Some studies have shown that the time constants for adjustment of blood flow were 5–10 s faster than those of O2 during moderate exercise (13, 22, 31), whereas others have found similar kinetics for m and O2 (16, 25) for moderate exercise. Our results are in agreement with the latter studies and suggest that in the human muscle microcirculation, under the experimental conditions and assumptions of this investigation, the dynamic adjustment of blood flow is intimately coupled to the time course of O2m. As noted above, rest-to-exercise transitions were associated with overall m kinetics faster than O2m kinetics (22, 31), whereas exercise-to-exercise transitions ("unloaded" to exercise) demonstrated m kinetics similar to O2 kinetics (16, 25). This could be the result of differences in the initial phase of the m response, possibly because of a greater muscle-pump effect during rest-to-exercise transitions (see above). Collectively, this raises an interesting question: Does the muscle-pump-induced increase in blood flow alter the coupling between m and O2m during rest-to-exercise transitions? The mechanisms underlying the close association between m and O2m kinetics are not presently clear because studies of m in the microcirculation during the transitional phase of exercise are scarce. Pharmacological blockade of vasodilator pathways did not change the overall time course of bulk blood flow adjustment (e.g., Ref. 42), but the possibility of different effects on the microcirculation cannot be excluded (27).
In this context, it has been suggested that the time constant of phase 2 should be used to investigate the control of m because phase 1 may have a mechanical origin that increases blood flow indiscriminately to active and nonactive muscle fibers (21). In the present model, we chose to evaluate the temporal association between the MRT for cap and P of O2p. As discussed above, P-O2 is a reasonable approximation of muscle O2 kinetics, whereas the MRT was selected to reflect the overall kinetics of blood flow due to the present uncertainty about the mechanism(s) of cap phase 1. In fact, recent studies have indicated the presence of a rapid vasodilation that is related to muscle metabolism (48). Therefore, on the basis of current knowledge of the blood flow response to exercise, it is our contention that the mean response time gives a better representation of the overall temporal profile of blood flow. Using this analysis, the kinetics of cap were found to be similar to O2 kinetics during moderate and heavy exercise. From these results alone, it is difficult to make inferences about the potential role of O2 delivery to determine the kinetics of O2m during upright cycling exercise (present study). O2m kinetics reflect the interaction between O2 delivery and metabolic inertia (47). If cap (and presumably O2 delivery) kinetics had been clearly faster than O2 kinetics, it would suggest that O2 delivery was not the limiting factor to O2m kinetics. Conversely, if the kinetics of cap were slower than O2 uptake kinetics, it might suggest an O2 delivery limitation to O2m kinetics. However, our results lie between these two extremes. It is important to note that similar kinetics for cap and O2m does not necessarily mean that cap (and by inference O2 delivery) limits O2m kinetics. To this point, augmented O2 delivery in the transitional phase of moderate exercise did not change significantly the kinetics of O2 (12, 30). In contrast, during heavy exercise, enhanced O2 delivery resulted in faster O2 kinetics in some (pump-perfused muscle, Ref. 14; prior exercise, Ref. 45), but not all studies (prior exercise, Ref. 52). In our study, estimated O2m and cap kinetics were similar for both exercise intensities (Fig. 6). Therefore, it cannot be ascertained, from our data only, how O2 delivery and metabolic inertia interact to determine O2m kinetics (for further discussion on this topic, see Refs. 45, 47, and 52).
Methodological considerations.
The basic assumptions made to estimate the temporal profile of cap were discussed in detail above. It is important to recognize, however, that possible subtle changes in the exponential characteristic of O2m response occurring in the transitional phase will be masked by the breath-by-breath noise of O2p, which prevents statistical justification of higher order models, but has important physiological implications (23); however, resolution of this limitation is not possible at present. In addition, we have assumed that O2m and (a-v)O2, estimated locally from NIRS, were proportionately distributed among the exercising muscles. However, the skeletal muscles recruited are not homogeneous, either with respect to their relative contribution to the work of cycling or with regard to muscle fiber type, recruitment pattern, and distribution of blood flow (26). At present, resolution of both the intra- and intermuscular heterogeneity of blood flow and O2 during cycling is not possible and must await further advances in methods such as those recently reported (35).
Two inherent limitations of NIRS are the small tissue volume sampled by the probe and the relatively shallow light penetration depth. In this study, we used the vastus lateralis on the basis of electromyography activity, which indicates that the vastus lateralis provides a good representation of muscle recruitment during cycling (29). Contribution from other muscles (by electromyography) during heavy exercise may become important during the period corresponding to the slow component of O2p (6), but this is expected to have minor effects on our results because we restricted our analysis to the primary component of O2p, [HHb], and estimated cap. Regarding the light penetration depth, the predominance of type II fibers in superficial muscle areas (28) could result in slower estimated cap kinetics, if the lower endothelium-dependent vasodilator response of type II fibers demonstrated in animal muscles (54) is observed in human muscles. The potential influence of fiber- type regionalization on the NIRS estimated cap kinetics must await further studies. Finally, we assumed that the relative contribution of arterioles, capillaries, and venules to the [HHb] signal remained constant during the exercise period. This is a technical limitation of NIRS in general that cannot be solved at present. However, on the basis of the similarity between the temporal profile of [HHb] observed in the present study and recently reported by others (9, 15), and the dynamics of O2 extraction measured in other studies (12, 14, 16), a possible shift in the vessels contributing to the [HHb] signal would not invalidate our assumption.
In summary, this study introduced a new method to noninvasively estimate the time course of cap from the kinetics of O2p and [HHb] from NIRS. The resulting estimated cap kinetics were similar to O2p (phase 2) kinetics, indicating that in the microcirculation m is tightly coupled to muscle O2 uptake after the onset of exercise. Moreover, the temporal profile of the estimated cap response suggests that in human muscles the kinetics of capillary blood flow is biphasic, as previously shown in the rat muscle (24).
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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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) for moderate exercise (A, from 
, present study; 
, Koga et al. (knee extension, Ref. 
, Grassi et al. (dog gastrocnemius, Ref. 
, MacDonald et al. (knee extension, Ref. 
, Behnke et al. (rat spinotrapezius, Ref.