INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

DURING EXERCISE, the ability of the working muscle in healthy individuals to increase its O₂ uptake (O₂) is limited by either a central or a peripheral mechanism (37). The central mechanism represents the systemic increase in O₂ delivery to the muscle, which includes enhanced blood flow through the capillaries. The peripheral mechanism focuses on the diffusion of O₂ from hemoglobin (Hb) to the mitochondria and the metabolic regulation of oxidative phosphorylation. Proponents of a central mechanism point out a tight correlation between O₂ and blood flow () while others have countered that such a mechanism cannot account for the large residual venous PO₂ at maximal O₂ uptake (O_{2 max}; see Refs. 13-15, 40). In the latter group's view, convective control is only secondary to diffusion control. Despite numerous studies, the limiting mechanism for O₂ is still under debate.

Even though both models entail an O₂ gradient role, the diffusion model emphasizes the gradient from the capillary to the mitochondria, which is a driving force of O₂ transport. Analysis of the role of gradients, however, requires a measurement of the mean end-capillary and the cellular partial pressure of O₂ (PO₂). For the cellular PO₂, studies have assumed that the mitochondrial PO₂ is approximately zero. Such an assumption is dictated largely by the current limitation in observing quantitatively the cellular PO₂ in muscle during exercise. Honig et al. (16) have already demonstrated in cryosection experiments that, even though the intracellular PO₂ is quite low, it does decrease with exercise intensity (16). The observation implies that the cell can modulate its O₂ gradient and suggests that myoglobin (Mb)-facilitated diffusion, which becomes increasingly pronounced as the cellular PO₂ falls, contributes significantly to O₂ transport to the mitochondria (11, 46).

The intracellular PO₂ during exercise is then a key variable in helping to determine the regulatory mechanism of O₂ in exercising muscle. In addition, the intracellular PO₂ response during exercise can help clarify the hypothesis that O₂ supply limits O_{2 max}. If the critical PO₂ in the cell is less than ~2.9 mmHg (Mb p50 at 39°C), then the PO₂ at the mitochondria is estimated to be at the Michaelis constant of the cytrochrome oxidase activity (3, 43). The cellular O₂ supply can then limit the aerobic capacity of working muscles without metabolic limitation, which can modulate the phosphorylation potential and redox state (8, 45). However, if the critical intracellular PO₂ is well above 2.9 mmHg, then cytochrome oxidase is saturated even at O_{2 max}, suggesting that O₂ alone is not modulating mitochondrial respiration (25). Unfortunately, measurement of intracellular PO₂ in human skeletal muscle during intense exercise poses a formidable technical challenge.

In recent years, ¹H NMR has presented an approach to measure the intracellular PO₂ with the Mb signals, first in myocardium and subsequently in skeletal muscle (19, 21, 42). In fact, one study has now shown that Mb desaturates rapidly to 51 and 60% of control under normoxic and hypoxic exercise conditions, respectively; yet, surprisingly, Mb desaturation is not proportional to increased work output (32). The results are provocative and have significant impact on the current view of respiratory control in muscle. They compel us to reexamine the relationship between work output and Mb desaturation in a plantar flexion exercise protocol, which is less prone to motional artifact than the reported quadricep exercise. As a result, the NMR data are much improved and show that both the ¹H-deoxy-Mb and ³¹P high-energy phosphate signal intensities change in proportion to power output or O₂. At O_{2 max}, Mb is 48% desaturated. The present study's results indicate that the cellular PO₂ can modulate the O₂ gradient and that Mb-facilitated diffusion may play a significant role in regulating O₂. Moreover, O₂ availability does not appear to limit O₂ during exercise.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Experimental design. The protocol for this study was reviewed and approved by the Human Subjects Welfare Committee of the University of California, Davis. Four young adult men (Table 1) were recruited from the student body of the university. All were untrained volunteers, who gave written consent to participate in this study. They were informed of the procedures, requirements, and risks. Each subject reported to the laboratory several times to practice plantar flexion exercise and to become familiar with breathing through the respiratory apparatus. During this time, the exercise intensity was adjusted so that the subject could reach peak O₂ uptake (O_{2 peak}).

Two sessions were required to complete this study. Session I was conducted in the Human Performance Laboratory and involved characterizing each subject's body composition, steady-state O₂ at several intensities of plantar flexion exercise, and O_{2 peak} for plantar flexion exercise. A fiberglass (ScotchCast; 3M) cast was made from the subject's lower leg from below the knee to the ankle. The cast was used as another means to calibrate the percent desaturation of the deoxy-Mb signal and to estimate the lower leg volume, which was then used to normalize the net change in O₂. Session II was performed at the nuclear magnetic resonance (NMR) Research Facility at General Electric (GE) Medical Systems (Fremont, CA) and involved duplicate protocols for plantar flexion exercise for determination of metabolic phosphates by ³¹P NMR and deoxy-Mb by ¹H NMR. Both protocols corresponded to the protocol performed in session I.

Body composition. Air displacement plethysmography was employed to determine body composition with the BOD POD instrument (LMI, Concord, CA). The method has been described in detail elsewhere (26). Each subject was weighed to the nearest gram, and the height was measured to the nearest centimeter. The raw body volume was determined in duplicate and averaged. Next, thoracic gas volume was then measured in the BOD POD. Body density was calculated as the ratio of body mass to corrected body volume. Percent body fat was then calculated from body density using the Siri (35) formula.

Plantar flexion ergometer. The same ergometer was employed to assess the mechanical power, energy transfer rate by whole body indirect calorimetry, the muscle concentrations of P_i, phosphocreatine (PCr), ATP, and intracellular pH by ³¹P NMR, and deoxy-Mb by ¹H NMR during plantar flexion exercise of the calf muscles. The ergometer consists of a three-sided box with dimensions of 25.4 cm wide × 25.4 cm high × 91.4 cm long with a foot pedal on an axle at one end and a moveable back plate at the other end of the box. An aluminum bar served as an end stop for the pedal arc during plantar flexion exercise. Latex rubber tubing (1.3 cm diameter and 34.3 cm length) with a Hooke's constant of 31.12 N/cm length change was attached to the back plate and the axle of the foot pedal. Resistance to plantar flexion can be varied by the number of tubes used and/or by changing the stretch of tubing between the axle and back plate. Mechanical work of plantar flexion involved moving the pedal against a specified resistance through an arc of 3.8 cm. The pedal movement was controlled by stops for forward and reverse movements with plantar flexion and relaxation. In this study, power was incremented by varying the contraction frequency from 30 to 70 contractions/min (0.5-1.17 Hz) with the resistance and plantar flexion arc held constant. Contraction frequency was not measured but was controlled by requiring the subject to follow a metronome beat, with verbal assistance from a technician for added control. The technician monitored the exercise protocol, which required the subject to push the pedal until it stops at the aluminum bar. Any learning effect was minimized by having the subject practice the exercise procedures several times before the experiments.

Steady-state O₂ and O_{2 peak} for plantar flexion exercise. Each subject performed three to five intensities of plantar flexion exercise of the dominant leg, each for 3 min with a 5-min recovery period after each bout. Energy expenditure was determined continuously by indirect calorimetry. The sequence was as follows: after resting for 10 min in a supine position, the subject breathed for 5 min through a mouthpiece and tubing connected to a SensorMedics metabolic cart (model 2900) for breath-by-breath measurements of resting O₂ and CO₂ production (CO₂). Next, the subject performed a series of three to five exercise bouts at progressively higher intensities by varying the frequency from 30 to 70 contractions/min (0.5-1.17 Hz) on the foot ergometer, with the resistance held constant. Each bout lasted 3 min and was followed by 5 min of resting recovery. The calculated mean value during the last 30 s of each bout was used to characterize the O₂, CO₂, and respiratory exchange ratio for the bout.

After a rest period of 10-15 min, the subject's O_{2 peak} was determined by holding the resistance constant and progressively increasing the contraction frequency each minute until the subject could no longer maintain the required cadence. O₂ and CO₂ were determined throughout the test as described above. O₂ was averaged over each 15-s interval. The highest O₂ value was designated as the subject's O_{2 peak}.

NMR. NMR measurements were performed on a 1-m bore diameter GE Signa scanner at 1.5 T. ¹H (63.86 MHz) NMR signal acquisition utilized a body coil transmit/surface coil (5-in. diameter)-received configuration. Magnetic field shimming used a three-point Dixon method to improve the field homogeneity, yielding a water line width of ~40 Hz (34). A selective excitation pulse sequence was optimized to excite the deoxy-Mb and deoxy-Hb histidyl-F8 NH signals, ~4.6 kHz from the water resonance (28). Numerical simulation and experimental data verified that the experimental pulse length of 800 µs had a full width at half-maximum excitation of 2 kHz. At an offset of 800 Hz or 13 ppm from the excitation maximum, the pulse power dropped by 25%. Each data block was comprised of 200 transients or 45 s signal averaging time. The repetition time was 160 ms. The spectral width was 16 kHz, and the data block size was 512. All spectra were referenced to the water signal as 4.65 ppm at 35°C, which in turn was calibrated against sodium 3-(trimethyl)propionate as 0 ppm.

³¹P (25.85 MHz) NMR signal acquisition utilized a conforming flexible coil that wrapped around the leg. A 50-mm slice was selected and then excited with a self-refocused 45° radio frequency pulse. The effective echo time was set at 2.5 ms (24). The other acquisition parameters were as follows: spectral width, 2.5 kHz; data points, 2,048; acquisition time, 820 ms; recycle time, 2 s. Each ³¹P NMR spectrum consisted of 50 transients and required a total acquisition time of 140 s. All spectra were apodized with a 15-Hz exponential function and referenced to PCr as 0 ppm.

The percent Mb desaturation was obtained in two different ways. After the final exercise bout, a pressure cuff above the knee was inflated to 240 mmHg to occlude the blood flow. Within 5 min, the deoxy-Mb signal reached a steady-state level, which was then considered as 100% desaturated. A soft cast was also made for each subject's leg, from the knee down to the ankle, and was used to calibrate the area intensity of the observed Mb signal. The cast was prepared with Scotchast Plus (3M) and was filled with 0.2 mM met-Hb solution, which approximated the physiological Mb concentration in tissue. ¹H-met-Hb spectra were then acquired with acquisition parameters, which were identical to the ones used in the leg exercise experiment. The peak intensity at 85.5 ppm was then utilized as a basis to quantitate the observed Mb intensity in exercising gastrocnemius muscle (23). No longitudinal relaxation time (T₁)-based saturation factor correction was necessary, since the T₁ values of both the Mb and Hb signals are sufficiently rapid to permit full recovery within the recycle time (38).

Data were imported from the Signa system to a Sun Sparc2 workstation and were processed using the GE Omega 6.0 software package. All spectra were zero filled to 2,000 and apodized using a 50 Hz Gaussian-exponential function. All spectra were baseline corrected and referenced to water at 4.65 ppm at 35°C.

Statistical analysis. Values reported are given as the means ± SE. Least-squares regression and correlational analyses were performed for net O₂, deoxy-Mb, PCr, P_i, ATP, and pH relationships with plantar flexion power output for each individual using SigmaStat (version 1; Jandel Scientific, San Rafael, CA). The mean slope coefficient and pooled SE estimate of regression for each relationship were used for power analysis. Statistical significance was accepted at P  0.05 with power   0.80. The power of these tests was 0.9 or greater for n = 4 tests.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Selected characteristics of the subjects are presented in Table 1 and show that these young, untrained adult men are relatively lean for their age and gender.

A representative example of a stack plot of ¹H NMR deoxy-Mb for subject A is illustrated in Fig. 1. Figure 1A shows no detectable deoxy-Mb at rest where Mb presumably is fully saturated with O₂. Figure 1, B-D, shows that the magnitude of the deoxy-Mb peak grows with power outputs of 9.4, 11.5, and 14.7 watts, respectively. Within 5 min of recovery from exercise at 14.7 watts, the deoxy-Mb signal disappears into the noise (Fig. 1E), indicating that Mb has become saturated with O₂.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   1H nuclear magnetic resonance (NMR) spectra from gastrocnemius muscle at rest (A) and during plantar flexion at power outputs of 9.4 (B), 11.5 (C), and 14.7 (D) watts. E: spectrum after 5 min of rest recovery. Signal arises from the proximal histidyl-NH of deoxymyoglobin, and its intensity reflects increasing cellular deoxygenation.

The steady-state ATP, PCr, P_i, and intracellular pH results at rest and with variations in exercise intensity are presented in Table 2 and Fig. 2. Muscle ATP does not change relative to rest when power output is varied from 7.8 to 15.1 watts for plantar flexion exercise. In contrast, muscle PCr decreases, whereas P_i increases linearly with power output (Fig. 2, B-D). PCr falls to 33%, and P_i rises to 81% of the resting PCr level at the highest intensity of exercise, which elicits O_{2 peak}.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   31P NMR spectra from gastrocnemius muscle at rest (A) and during plantar flexion at power outputs of 9.4 (B), 11.5 (C), and 14.7 (D) watts. E: spectrum after 5 min of rest recovery. Phosphocreatine (PCr) declines with increasing work output, whereas ATP remains constant and is set at 8.2 mM.

Figure 3 shows the graph of ATP and PCr level as a function of plantar flexion work output. ATP is set at 8.2 mM and is the normalizing value for the PCr and P_i. Clearly, the ATP level remains constant throughout the exercise protocol (Fig. 3A). PCr falls 4.5%/watt output. The corresponding graphs for P_i and pH are shown in Fig. 4. P_i increases with exercise at a rate of 4.1%/work output.

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Graphs of ATP (A) and PCr (B) as a function of plantar flexion power output in 4 untrained men. PCr level declines with increasing work output, whereas ATP remains constant. All signals are expressed as percent PCr. Resting PCr signal is normalized to 100%, and the resting ATP concentration is set at 8.2 mM (12). The regression line for PCr is based on the mean values (n = 4). Repeated-measures ANOVA shows significance (P = 0.00856), with 45 vs. 70 rpm significance at P < 0.05. Regression of PCr vs. power output is also significant for each subject. The slopes, SE estimate, and P values for subjects A, B, C, and D are (5.48, 0.791, 0.0202), (5.41, 0.737, 0.0180), (3.90, 0.531, 0.0181), and (3.17, 0.126, 0.0016), respectively. Using the mean of the slopes (4.49) as a measure of the change in PCr per unit change in power and the pooled SE estimate (0.606), the power is 0.99. A sample size, n = 3, is needed to detect a difference at P = 0.05.

View larger version (12K): [in this window] [in a new window]   Fig. 4.   Graph of Pi (A) and pH (B) as a function of plantar flexion power output in 4 untrained men. Pi rises with increasing power output. Intracellular pH, calculated from the chemical shift of Pi relative to PCr, decreases with power output. Signal intensity is represented as percent resting PCr.

The relationship between normalized net O₂ and plantar flexion power is shown in Fig. 5. As exercise intensity increases, the O₂ also increases linearly. Because the net O₂ of 13.2 ± 2.1 and 12.8 ± 1.2 ml · min¹ · 100 ml leg volume¹ for the last two power outputs of 15.1 ± 0.3 and 17.6 ± 1.0 watts are not significantly different (P > 0.05, paired t-test), these values are averaged to yield a net O_{2 peak} of 13.0 ± 1.6 ml · min¹ · 100 ml¹. This O_{2 peak} satisfies the criterion for O_{2 max} for leg muscles performing plantar flexion exercise.

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Net O2 uptake (O2) as a function of plantar flexion power output in 4 untrained men. As plantar flexion power increases, so does O2. The values reflect the difference between the average O2 of the last 30 s of exercise and 5 min of rest and are expressed as means ± SE. Values are normalized for each individual's lower leg volume, which was obtained from the cast of the lower leg. O2 values for the last two exercise intensities were not significantly different (paired t-test, P < 0.05), indicating that O2 peaked at 15.1 watts, the highest intensity employed for the NMR experiments. The regression line is based on the mean values (n = 4). Differences between O2 for the 3 exercise intensities are significant (P = 0.000883), with Bonferroni's post hoc test showing that the results from the 45 vs. 70 rpm experiments are significant. Regression of O2 vs. power (without the 0,0 points) for each subject shows that the slopes for each subject are significant. Slopes, SE estimate, and P values for subjects A, B, C, and D are (1.17, 0.387, 0.05), (0.692, 0.0329, 0.0002), (0.629, 0.130, 0.017), and (0.885, 0.129, 0.0064), respectively. Using the mean of the slopes as a measure of the change in O2 per unit change in power (3.37) and the pooled SE estimate (0.931), the power is 0.994. A sample size , n = 4, is needed to detect a difference at P = 0.05.

The relationship between deoxy-Mb signal and exercise power and net O₂ is presented in Fig. 6. A distinct linear relationship is found between the percent Mb desaturation and power output (Fig. 6A) and between percent Mb desaturation and net O₂ (Fig. 6B). Mb desaturates progressively as work output or O₂ increases. At O_{2 peak}, Mb desaturates to 48.4%. A similar relationship is apparent with PCr. As Mb desaturates, the PCr level falls linearly as does pH (data not shown).

View larger version (12K): [in this window] [in a new window]   Fig. 6.   Graph of the percent deoxymyoglobin (deoxy-Mb) intensity as a function of power output (A) and net O2 (B). As the O2 increases with muscle power output, the intracellular PO2, as reflected in the deoxy-Mb proximal histidyl NH signal, declines linearly. The regression lines are based on mean values (n = 4). Repeated-measures ANOVA on deoxy-Mb also shows significance (P = 0.00632), with 45 vs. 70 and 55 vs. 70 (P < 0.05). Regression analysis of deoxy-Mb vs. power (without the 0,0 points) for each subject shows that the slopes are significant for each subject. The slope, SE estimate, and P values for subjects A, B, C, and D are (5.55, 0.864, 0.010), (2.702, 0.199, 0.0054), (3.02, 0.248, 0.0067), and (2.81, 0.464, 0.0261), respectively. Using the mean of the slopes (3.53) as a measure of the change in deoxy-Mb per unit change in power and the pooled SE estimate (0.516), the power is 0.99. A sample size, n = 3, is needed to detect a difference at P = 0.05. Dotted line corresponds to peak O2 at 15.1-W output.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Resting state intracellular PO₂. The proximal histidyl NH signal of deoxy-Mb reflects the degree of tissue oxygenation during rest and exercise. At rest, no signal is detected, whereas during exercise the signal appears. Given the excellent signal to noise of the deoxy-Mb peak under the normalization condition of the cuffed leg spectra, a 10-20% deoxy-Mb saturation would certainly reveal an observable signal above the noise. Because none is detected, even after the addition of several reference spectra (data not shown), the resting PO₂ of skeletal muscle must be sufficient to saturate MbO₂ >80%, which reflects a resting cellular PO₂ >10 Torr (given a Mb p50 of 2.9 at 39°C), and is consistent with muscle cryosection data (10, 11).

Mb desaturation with work output. As muscle work output increases, O₂ flux must also increase to match the enhanced O₂. Such an enhanced O₂ flux is governed phenomenologically by Fick's law of diffusion where DO₂ is the lumped conductance for O₂ diffusion transport in tissue and (PO₂)_cap and (PO₂)_mito are the partial pressures of O₂ at the capillary and at the mitochondria, respectively.

A driving force for O₂ flux involves then a gradient from the capillary to the mitochondria. Because the mitochondrial PO₂ is assumed to be approximately zero, only the capillary PO₂ is often used to determine the interaction between the O₂ gradient and O₂. However, according to Honig et al. (16), the cellular PO₂ can also modulate the O₂ flux. Intracellular PO₂ is low but not zero. As work intensity increases, the cellular PO₂ can decline, as reflected by Mb desaturation, and therefore enhances the gradient driving force for O₂ flux (11). Even though O₂ has increased, the intracellular PO₂ has fallen. A rise in O₂ in face of a drop in intracellular PO₂ is consistent with an enhanced role of Mb in facilitating O₂ diffusion to the mitochondria during exercise (44, 46).

Validation of the Honig hypothesis in a dynamic model has presented a formidable challenge, since experimental measurements must assess the intracellular PO₂ during exercise. With ¹H NMR, a strategy has emerged to observe the intracellular PO₂ with the signals of Mb (22). Indeed, the deoxy-Mb proximal histidyl NH signal in our study demonstrates clearly that intracellular PO₂ in exercising skeletal muscle falls progressively with work output. As the work output varies from 7.8 ± 0.32 to 15.1 ± 0.3 watts, Mb desaturates from 30.2 ± 2.4 to 48.4 ± 5.1%. Both the O₂ and work output form linear relationships with Mb desaturation.

These results are in contrast with a previous NMR report, which shows that Mb desaturates rapidly to a constant 51% under exercise intensity >50% of O_{2 max} (32). Below 50% of O_{2 max}, a dubious experimental point is slightly <50% and suggests the presence of a linear response region at low work output. In the present study, the exercise protocol also elicits O₂ that spans the range above 50% of O_{2 max}. At the highest level of exercise performance, the subjects reach O_{2 peak}.

The observed pH and P_i/PCr change also supports the interpretation that our subjects are exercising above 50% O_{2 max}. At the highest work output of 15 watts, the cellular pH is 6.87 ± 0.16, which is consistent with the value of 6.554 ± 0.325 observed by Richardson et al. (32) during exercise at 95% of O_{2 max}. Because calf muscle has heterogeneous fibers, the pH values during exercise can range from 6.2 to 7.1 (27, 39). The pH observation during exercise is then consistent with a high work output.

Moreover, the P_i-to-PCr ratio also supports a high O₂ exercise. Studies have reported values ranging from 1.3 to 4.1 during maximal exercise (4, 31, 39). At the highest work output, the P_i-to-PCr ratio is 2.4. Although the study by Richardson et al. (32) reports P_i-to-PCr ratios approaching 10, the unacceptably large SE precludes any comparative analysis. In that study, the reported PCr-to-P_i ratio is 0.1 ± 0.2 during exercise at 95% of O_{2 max}.

Clearly, the discrepancy in the two reports requires further study and may originate from differences in the interrogated muscle groups (quadriceps vs. gastrocnemius), the subject's athletic training, and NMR acquisition/processing methodology.

Diffusional conductance. Because the Mb is desaturating as exercise intensity increases, the diffusion equation (Eq. 1) would imply that O₂ gradient from the capillary to the cell is indeed modulating O₂ delivery to match the O₂. To determine the specific relationship requires the assessment of the O₂ as a function of exercise intensity. Even though the present study has utilized whole body O₂, both theoretical and empirical studies have shown that the kinetics of O₂ of working muscle is the same as pulmonary O₂ in phases 2 (metabolic) and 3 (steady state; see Refs. 5 and 6). A number of other studies have demonstrated that the increase in leg O₂ accounts for >57% (ranging from 57 to 93%) of the increment in whole body O₂ during leg exercise (1, 2, 18, 20, 30, 36, 41).

The experimentally determined change in () O₂ from exercise level 1 to 3 is 7 ml · min¹ · 100 ml leg volume¹, whereas the [(PO₂)_cap  (PO₂)_mito] rises from 31.4 (40-8.6) to 36.1 (40-3.9) Torr, assuming a constant mean end-capillary PO₂ of 40 Torr. Even though the O₂ has increased by a factor of 2, the PO₂ has only increased by a factor of 1.15. If the mean end-capillary PO₂ is 13 Torr, then the PO₂ is now altered to 4.4 (13-8.6) and 8.1 (13-3.9), respectively. The change would imply that the O₂ gradient is sufficient to match the enhanced O₂ and that DO₂ is relatively constant. Because DO₂ is a lumped constant, which includes aggregate capillary surface area and capillary to cell distance, a relatively constant DO₂ would diminish the contribution of diffusion-controlled regulation of O₂ flux during exercise.

Nevertheless, DO₂ is multifactorial and nonlinear. Relatively small changes can enhance O₂ flux (9, 17). The extent of DO₂ modulation is unclear from the present experimental data, since, in part, the NMR observes only a spatially averaged signal and cannot discriminate any heterogeneity, which can complicate the interpretation (29). The specific heterogeneity in question, O₂/, is difficult to assess, since at present no definitive measurements can resolve the O₂/ heterogeneity contribution in exercising muscle. Researchers have argued reasonably against any significant contribution, which is an underlying assumption in the above analysis (32).

Determinant of respiration during exercise. Even with the modulation of O₂ delivery, the O₂ supply does not appear to be sufficient to meet the O₂ demand during exercise without metabolic adaptation. Because oxidative phosphorylation depends on the phosphorylation potential or charge, redox state, ADP, and carbon substrate availability, the associated metabolite levels can also regulate respiration. Indeed, as O₂ increases, the ³¹P-PCr signal declines, whereas the P_i signal increases. The P_i-to-PCr ratio, which reflects the ADP concentration, shifts from 0.19 to 2.7 and is consistent with the linear relationship between percent peak power vs. ADP (4).

Although the O₂ gradient increases with exercise intensity and therefore enhances the driving force for O₂ transport, the intracellular PO₂ nevertheless drops and suggests that an additional O₂ diffusion route to the mitochondria becomes increasingly significant. Such a role is postulated for Mb. Even the enhanced driving force for O₂ flux, however, does not preclude an apparent ADP-dependent stimulation of respiration or any modulation from the lumped O₂ conductance factor, which includes O₂ unloading and aggregate capillary surface area. Respiratory control does not appear to depend solely on the regulation of O₂ delivery or supply. Nevertheless, the study has demonstrated that the O₂ gradient does shift dramatically during exercise along with metabolic adaptation. Further study will employ the NMR strategy to define the relationship between O₂ supply and metabolism as a coordinate set of respiration controls during exercise in skeletal muscle.

In exercising skeletal muscle, the enhanced O₂ with increased work output correlates linearly with MbO₂ desaturation. As O₂ increases, Mb desaturation also increases, reflecting a fall in the cellular PO₂. At O_{2 peak}, Mb is desaturated by 48%. The linear relationship between O₂ and Mb desaturation supports the notion that the O₂ gradient from the vasculature to the cell is enhancing the O₂ flux. Yet, despite the increased O₂ flux, PCr level still falls, reflecting a rise in ADP. The results indicate that the regulation of O₂ involves both an O₂ gradient and metabolic control.

Perspectives