Skeletal muscle intracellular PO₂ assessed by myoglobin desaturation: response to graded exercise

¹ Department of Medicine, University of California San Diego, La Jolla, California 92093; and ² Department of Radiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6021

	ABSTRACT

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The relationship between skeletal muscle intracellular PO₂ (iPO₂) and progressive muscular work has important implications for the understanding of O₂ transport and utilization. Presently there is debate as to whether iPO₂ falls progressively with increasing O₂ demand or reaches a plateau from moderate to maximal metabolic demand. Thus, using ¹H magnetic resonance spectroscopy of myoglobin (Mb), we studied cellular oxygenation during progressive single-leg knee extensor exercise from unweighted to 100% of maximal work rate in six active human subjects. In all subjects, the Mb peak at 73 ppm was not visible at rest, whereas the peak was small or indistinguishable from the noise in the majority of subjects during progressive exercise from unweighted to 50-60% of maximum work rate. In contrast, beyond this exercise intensity, a Mb peak of consistent magnitude was discernible in all subjects. When a Mb half saturation of 3.2 Torr was used, the calculated skeletal muscle PO₂ was variable before 60% of maximum work rate but in general was relatively high (>18 Torr, the measurable PO₂ with the poorest signal-to-noise ratio, in the majority of cases), whereas beyond this exercise intensity iPO₂ fell to a relatively uniform and invariant level of 3.8 ± 0.5 Torr across all subjects. These results do not support the concept of a progressive linear fall in iPO₂ across increasing work rates. Instead, this study documents variable but relatively high iPO₂ from rest to moderate exercise and again confirms that from 50-60% of maximum work rate iPO₂ reaches a plateau that is then invariant with increasing work rate.

oxygen; work rate; diffusion; oxygen transport

	INTRODUCTION

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PRESENTLY THERE IS DEBATE as to whether intracellular PO₂ (iPO₂) falls progressively with increasing O₂ demand or reaches a plateau from moderate to maximal metabolic demand. For many years, the general consensus was that iPO₂ declines as exercise intensity increases (8, 12). This has been fueled by the concept of the "anaerobic threshold" in which iPO₂ falls, leading to anaerobiosis during incremental exercise and resulting in a large increase in lactate efflux from the exercising skeletal muscle (44). In 1995, our laboratory utilized ¹H magnetic resonance spectroscopy (MRS) to calculate iPO₂ during a progression from moderate to intense exercise with the quadriceps muscles and revealed a constant iPO₂ but rapidly increasing lactate efflux (34, 36). Unfortunately, this research was limited to the study of 50-100% of maximum work rate by a prototype ergometer.

Added fuel to this controversial issue was recently provided by the recently published findings of Mole et al. (27), who also utilized ¹H MRS to study intracellular oxygenation during progressive plantar flexion exercise and found that myoglobin (Mb) became increasingly desaturated with increasing mitochondrial respiration rate. These contradictory findings and the recent work examining the role of myoglobin (7, 11) have rekindled the interest in the relationship between iPO₂ and changing metabolic demand. There remains uncertainty as to the relationship between these two variables across the full scope of exercise capacity (29). The characterization of the relationship between work rate and iPO₂ has important ramifications for the understanding of O₂ transport and utilization in skeletal muscle. Specifically, this relationship has direct impact on issues such as the lactate efflux, the role of Mb, and the role of diffusion gradients and O₂ conductance (D_O2) in the transport of O₂ from blood to muscle cell.

Thus the purpose of this study was to test the hypothesis that iPO₂ in skeletal muscle falls from a relatively high resting value to a low iPO₂ plateau at a moderate work rate that continues until the attainment of maximal exercise capacity. To achieve this, ¹H MRS of Mb desaturation was again utilized as an indicator of iPO₂ in the human quadriceps at rest and throughout a graded exercise test to maximum performed on a single-leg knee extensor ergometer.

	METHODS

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Subjects

Seven recreationally active males (age: 30.5 ± 4.5 yr, weight: 78.4 ± 10.2 kg, and height: 178.4 ± 4.4 cm; means ± SD) volunteered to participate in this study after informed, written consent was obtained according to the University of Pennsylvania Human Subjects Committee requirements. All of the subjects performed endurance exercise two to five times a week, but none was actively competing or training for a specific sporting event.

Exercise Modality and Protocol

Single-leg knee extensor exercise, designed to limit work to the quadriceps muscles of the left leg (32), was employed. Before the exercise protocol, all subjects were familiarized with the testing environment and ergometer. Briefly, subjects were semirecumbent in a chair within a 2.0-Tesla Oxford imaging magnet. A special ankle boot placed on the left leg connected them by a bar to the ergometer placed at the end of the magnet (34). Contractions of the quadriceps muscles caused the lower part of the leg to extend from an angle of 90° to 170°. Therefore, the lower leg traveled with an arc-shaped trajectory of ~80°. The momentum of the ergometer passively returned the relaxed leg to the start position, and, as a result, the quadriceps muscle was functionally isolated (30). One half hour before the exercise test, ischemia in the left leg was induced by inflation of a suprasystolic cuff to allow the calibration of the Mb signal. The graded exercise test required subjects to maintain each work rate for 120 s, after which the work rate was incremented. The subjects continued until they were unable to maintain a cadence of 60 rpm for the entire 2 min.

Determination of iPO₂

Spectra were collected from the muscle region below the 7-cm-diameter surface coil double-tuned to proton (85.45 MHz) and phosphorus (34.59 MHz) placed over the rectus femoris portion of the quadriceps group (40), ~20-25 cm proximal to the knee. Recently, at a similar anatomic location, Rolf et al. (38) determined the fiber type composition in the rectus femoris of physically active subjects to be 60% (45-71) type I, 36% type IIa (24-45), and 7% (0-7) type IIx. In an autopsy study on young accidental death victims, Johnson et al. (20) reported that the rectus femoris is made up of 42% type I fibers and 58% type II. These data illustrate the effect of activity on this assessment and are in line with the multitude of data more commonly collected from the vastus lateralis portion of the quadriceps femoris (3, 19, 20). However, the "sensitive region" for the MRS data collection is limited to semispherical volume under the coil, with its deepest portion equal to approximately half the coil width (3.5 cm). Thus, although there is a potential for a combination of data to be collected from the vastus lateralis, vastus medialis, and vastus intermedius, the <100 cm³ of muscle sampled was most likely predominantly from the rectus femoris (1).

Details of the theory behind oxygen-sensitive Mb signals have been published previously (5, 34). Briefly, the heme iron exhibits oxygen-dependent spin states that in turn influence nearby protons. The N- proton on proximal histidine F8, one of the ligands coordinated to the iron, is particularly sensitive these changes. When oxygen is bound to the active site, the resonance of this proton is hidden beneath the dominant water signal. However, when Mb becomes deoxygenated, changes in the iron spin state shift this peak to a temperature-dependent position that is clearly distinct from all other resonances.

Under conditions of cuff ischemia, intramuscular O₂ depletes within ~6-8 min of occlusion (43). Therefore, the plateaued signals obtained during the 10th and 12th min of cuff occlusion (270 Torr) represent complete deoxygenation of Mb and were used to estimate total Mb content within the muscle. During exercise, the fractional deoxy-Mb (^fdeoxy-Mb) was determined by the normalizing signal areas to the average signal obtained during the last minutes of cuff ischemia. The conversion from ^fdeoxy-Mb to PO₂ values was then calculated from the oxygen-binding curve for Mb
where ^fMb_O2 is the fraction of Mb that is oxygenated and P₅₀ is the O₂ pressure where 50% of the Mb binding sites are bound with O₂. The temperature-dependent Mb half saturation (P₅₀) of 3.2 Torr was used (39). The assessment of all Mb spectra was performed by a proprietary peak-finding program that removed the potentially subjective component of this process. Although detailed reliability studies utilizing these MRS methods are sparse, in five subjects we performed the cuff deoxy-Mb assessment on 2 separate days in the same anatomic location and recorded deoxy-Mb signals of 2,240 ± 350 and 2,100 ± 445 arbitrary units on days 1 and 2, respectively. This 6% variation is supportive of reliable measurement. Because the main goal of this study was to optimize the signal-to-noise ratio for the Mb signal, the normal interleaved data collection of ³¹P was not performed, because this process significantly diminishes this ratio.

Statistical and Mathematical Analyses

Linear regression analysis was utilized on both the individual and averaged data to assess the relationship between both deoxy-Mb levels and iPO₂. Each data set was analyzed for signal-to-noise ratio, and the poorest ratio was documented.

	RESULTS

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Deoxy-Mb Signals During Cuff Ischemia

In all subjects, there was no discernible Mb desaturation at rest, both before cuff inflation and before exercise (Figs. 1 and 2). After several minutes of cuff ischemia, the Mb peak developed from within the noise from cuff inflation to minutes 6-8 and plateaued in minutes 8-12 of cuffing (Fig. 1). Loss of deoxygenated Mb signal was apparent rapidly (20 s) after cuff deflation (Fig. 1).

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Stack plot showing spectra for an individual subject during rest, 12 min of cuff ischemia, and recovery (last spectrum).

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Stack plot showing spectra for an individual subject during rest and progressive exercise to maximum work rate.

Deoxy-Mb Signals During Exercise

Although the signal-to-noise ratio in all exercise bouts was good (poorest = 6-7:1), there was little measurable deoxy-Mb desaturation during low-intensity exercise (<50-60% of maximum work rate) (Fig. 2). A single notable exception was a single subject who exhibited a large deoxy-Mb peak (indicative of 4-5 Torr iPO₂) throughout the unweighted and low-intensity exercise levels (Fig. 3). With the poorest signal-to-noise ratio achieved and a Mb P₅₀ of 3.2 Torr, an iPO₂ of >18 Torr was often immeasurable. Consequently, although all subjects were successfully studied before 50% of maximum work rate, only four subjects demonstrated a measurable and typically rather inconsistent iPO₂ before this level of exercise intensity. Beyond 60% of maximum work rate, the deoxy-Mb signal rose consistently to 47 ± 0.03% of the cuff signal (3.8 ± 0.3 mmHg). The regression analysis between work rate and both iPO₂ and deoxy-Mb signal with work rate from 60% on revealed no relationship indicating that intracellular oxygenation remained relatively constant at this 50% level until the attainment of maximal exercise (Figs. 2 and 3). It is important to recognize that all subjects (n = 7) were studied successfully both below and above 60% of maximum work. Thus the lack of deoxy-Mb signal in the less intense exercise period is clearly indicative of relatively high iPO₂ values, whereas measurable data were attainable in all seven subjects at all work rates from 60% of maximum work rate onwards (Fig. 3). Similar to the cuff data, the exercise deoxy-Mb signal again disappeared within 20 s of the cessation of exercise.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   A: individual relationships between intracellular PO2 and work rate during increasingly intense dynamic knee extensor exercise. B: averaged deoxy-myoglobin (Mb) signal and work rate during increasingly intense knee extensor exercise. Responses of 7 subjects are illustrated. It should be noted that incomplete data are the result of minimal myoglobin desaturation and therefore relatively high cellular PO2 values. As indicated by the n = 7 across the complete spectrum of work intensities; however, data were only attainable on the fraction of subjects indicated.

	DISCUSSION

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The major findings of this study are that 1) at the onset of a graded exercise test iPO₂ is clearly reduced in some subjects but not all, and 2) beyond 50-60% of maximum work the variability in iPO₂ between subjects is greatly reduced and falls to a relatively uniform low level (~4.0 Torr) and is relatively constant despite the continued large increase in work rate and therefore O₂ consumption (Fig. 3). Thus these data continue to support the concept that intracellular oxygenation state does not fall linearly with increasing metabolic rate and for the first time provide an indication of oxygen availability from submaximal to maximal single-leg knee extensor exercise. Although the findings are straightforward in nature, there are many far-reaching implications of these data that are addressed specifically below.

Oxygen Transport From Capillary to Cell

Because oxygen uptake (O₂) = D_O2 (mean capillary PO₂  cellular PO₂), a doubling of O₂ from submaximal to maximal exercise (27, 34) can be achieved by a twofold increase in either D_O2 or the PO₂. However the PO₂ is greatly influenced by the somewhat illusive value of mean capillary PO₂. For example, if mean capillary PO₂ is, as we have previously calculated using both arterial and muscle effluent PO₂ measurements (33, 34), in the region of 40 Torr during submaximal exercise and falls to only 35 Torr at maximal exercise, even reasonably large changes in iPO₂ have little or no impact on the gradient from blood to cell: Submaximal gradient = (40  8.6) = 31.4; whereas maximal gradient = (35  3.9) = 31.1 [intracellular PO₂ from Mole et al. (27)]. However, if one specifically "selects" a much lower mean capillary PO₂, as Mole et al. did, the effect of a falling iPO₂ is much greater: Submaximal gradient = (13  8.6) = 4.4; whereas maximal gradient = (13  3.9) = 8.1 (again iPO₂ from Mole et al.). Use of this latter scenario, with little basis for the mean capillary value used, may lead to the conclusion that the change in O₂ gradient from submaximal to maximal exercise is sufficient to match the increased O₂ and that D_O2 may be considered constant.

The present data reveal once again that during knee extensor exercise iPO₂ is invariant from 50 to 60% of work rate maximum and, when viewed in conjunction with our calculations of mean capillary PO₂, clearly indicate that there is very little benefit to be gained by a significant reduction in iPO₂. In fact, the increasing O₂ must be facilitated by a linear increase in D_O2 with progressively intense exercise (34). This is not a unique observation as the diffusing capacity of the lung is recognized to increase at least threefold from rest to high-intensity exercise (45).

Oxygen Supply and Demand as Determinants of Maximal O₂ Uptake

Our laboratory (15, 16, 33) and others (46, 47) have documented the important role that O₂ plays in modulating cellular bioenergetics. However, the present finding that iPO₂ remains constant (much above the level during cuff ischemia) may appear difficult to reconcile with the observation that variations in inspired O₂ availability alters iPO₂, which in turn affects maximal metabolic rate (15, 33). At first assessment the latter, but not the former, observation appears to support the concept that O₂ supply is an important determinant of maximal metabolic capacity (34). However, a reconciliation of these data is possible by approaching them with similar logic to that employed to explain the observation that there is a large gradient from blood to cell, and venous PO₂ (representative of end capillary PO₂) does not fall to zero even at maximal O₂ uptake (O_2 max) (34, 42). The linking concept here is one of a finite D_O2 at maximal exercise that may limit O₂ transport (41). There are certainly similarities between the profile for venous PO₂ (a hyperbolic function) and iPO₂ (Fig. 3) with increasing work rate. Thus a mitochondrial D_O2, which limits O₂ conductance from the cytosol to mitochondria, may explain the inability for Mb PO₂ to fall to a greater extent before the cessation of high-intensity exercise. In this scenario, a gradient exists from capillary to cytosol (~30 Torr) and from cytosol to mitochondria (~4 Torr). It should be noted that although the gradient is vastly different in each case, the physiological significance of this apparently abundant O₂ that is potentially limited in terms of availability might well be equal. Hence, in both locations, the detection of adequate O₂ for mitochondrial respiration does not clearly indicate that O₂ is in excess because there is still the potential for limited O₂ transport to the site of use, to the cytochromes themselves.

Anaerobic Threshold

From 50% of O_2 max to O_2 max, it has previously been recognized and published (36) that iPO₂ failed to progressively fall despite large increases in lactate production. The present and more complete intracellular profile data (Fig. 3) indicate that iPO₂ tends to fall from rest to low-intensity exercise (<50-60% of work rate maximum), falls dramatically to a constant level during higher intensity graded exercise (50-60% to 100% of muscle O_2 max), and thus appears unrelated to the documented rise in net muscle lactate efflux with increasing skeletal muscle work (36). Consequently, these data again demonstrate that, as assessed by cytosolic oxygenation state (deoxy-Mb) during incremental exercise, skeletal muscle cells do not become "anaerobic" as lactate levels suddenly rise, because intracellular PO₂ is well preserved at a low but constant level even at maximal exercise. Given that arterial epinephrine levels stimulate glycolysis and are closely related to skeletal muscle lactate efflux in both normoxia and hypoxia, the catecholamine-mediated rise in pyruvate may be responsible for the observed rise in muscle lactate efflux during progressively intense exercise and for the elevated lactate efflux in hypoxia (25, 36).

Role of Mb in Skeletal Muscle

The initial perception that Mb plays an important role in O₂ flux (48, 49) has been widely supported by the modeling literature (6, 7, 26, 28). Additionally, there are clear variations in Mb concentration that support the role of Mb in the flux of O₂ from blood to aerobically active muscle: breast muscle of the nonflying chicken is white and lacks this protein, whereas the breast muscle of ducks and geese (which can fly) is red and rich in Mb (23). However, there is mounting evidence against a primary role of Mb-mediated O₂ diffusion. Specifically, in terrestrial mammals, variations in Mb concentration within fibers and between fibers has been found to be inconsistent with the varied aerobic capacity of the tissue (4). Additionally, although Mb has been documented to diffuse in vivo (perhaps an important criterion for a significant contribution to O₂ flux), the comparatively low diffusivity recorded has been suggested to indicate only a minor role in intracellular O₂ transport (22).

Most recently and perhaps most significantly, the successful development of mice without Mb, generated by gene knockout, but with normal exercise capacity initially implied that Mb is not essential to meet the requirements of exercise in this terrestrial mammal (7, 11). However, follow-up studies and reports have clearly indicated that there are many compensatory changes to the O₂ transport system that must accompany this loss of Mb to sustain life, let alone facilitate normal exercise capacity (10, 13). These findings are highly supportive of an important role of Mb in O₂ transport.

The present data indicate that in human skeletal muscle Mb desaturates in heavily exercising muscle, a criterion necessary for the models that support the role of Mb in the transport of O₂ (6, 7, 26, 28). Additionally, beyond 50-60% of maximum work rate, we have documented a relatively constant percentage of deoxy-Mb despite large changes in work rate. This suggests that, in a fashion similar to that by which creatine phosphokinase buffers ATP concentrations during increasing metabolic demand, Mb serves to buffer O₂ concentrations with increasing work (Fig. 4). Both intracellular PO₂ and ATP can be considered perfectly homeostatic in terms of concentration, whereas their flux can change by orders of magnitude with progressively more intense exercise (18). The significant desaturation of Mb (~50% of the desaturation during cuff ischemia) is additionally supportive of the facilitative diffusive role that Mb may play in O₂ transport because Mb needs to become desaturated if it is to facilitate O₂ movement from blood to cell (14). At the lower intensity work rates (<50%), the majority of subjects revealed very little deoxy-Mb, which may be interpreted as minimal need for either the facilitation of O₂ diffusion or the buffering of O₂ concentration by Mb.

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Illustration of the similar buffering role of myoglobin and creatine phosphokinase in respiration: both maintain the concentration (indicated by square brackets) of essential cellular substances (O2 and ATP, respectively) despite vast changes in flux rate. CPK, creatine phosphokinase; Cr, creatine; PCr, phosphocreatine.

In these physically active individuals, the metabolic system appears to clearly capable of transporting and maintaining cellular PO₂ without the assistance of myoglobin until 50-60% of maximum work rate. The one clear exception to this is the single subject who demonstrated significant Mb desaturation throughout the progressive exercise, even during the initial warm-up period (0%, zero-resistance knee extensor exercise). It is not clear why this difference exists, but the somewhat anecdotal observation that this subject was one of the least physically active subjects may suggest a different relationship between O₂ supply and demand for this less active subject. On the basis of this observation, it will be important to study the relationship between Mb desaturation and skeletal muscle work in sedentary subjects whose exercise capacity appears to be governed more by O₂ demand than O₂ supply, unlike their active and exercise-trained counterparts studied in the majority here (31, 35).

Limitations

Assessment of deoxy-Mb. As is clear from the present data (Fig. 3A), this method of assessing deoxy-Mb levels results in limited quantifiable data when the muscle is well oxygenated. It is also important to recognize that Mb exists only within the cytosol; thus this MRS technique accesses cytosolic oxygenation, one step removed from the mitochondria, where oxidative phosphorylation occurs. Additionally, although the cytochromes might contribute to visible muscle pigment, it is generally accepted that there is a positive correlation between muscle redness and Mb content (24).

As noted earlier, the human quadriceps femoris muscles are not structurally homogenous; hence, the present MRS technique offers an average signal that is weighted toward the intracellular oxygenation state of type I fibers. This is of importance during the type of progressive exercise utilized in the present study because both type II fiber types are relatively quiescent at lighter work rates but become increasingly involved in performing the work as the exercise intensity moves from moderate to high levels (9). Interestingly, this limitation adds validity to the recorded low or immeasurable levels of deoxy-Mb during light work because here the fibers recruited are mostly type I fibers that are rich in Mb. However, as the work intensity increases and additional fibers containing little or no Mb are recruited, the ability to determine the Mb signal will not be influenced, but no new information from these newly recruited fibers will be collected. Along similar lines, the use of a large MR coil that facilitates the collection of data across several muscles with varying fiber types and varying involvement in the exercise may lead to misleading results (2). This may have been the case in the work of Mole et al. (27), whose MRS assessment undoubtedly involved both the gastrocnemius and the soleus (17). However, this is less likely to be the case in the present study because of the use of a small surface coil on a large relatively homogeneous muscle group, in terms of both fiber type and recruitment during the exercise (30).

Conversion from deoxy-MB signal to PO₂. The Mb-oxygen binding curve is nonlinear, and hence the hyperbolic shape of the Mb oxygen-binding pattern is drastically influenced by P₅₀. Reported values for the P₅₀ of Mb vary in the literature from 1.5 to 5.5 Torr (49). Alone, this choice has a profound effect on the estimation of Mb associated PO₂. Thus Mb desaturation should always be reported in addition to calculated PO₂.

Recently, Jurgens et al. (21) questioned the validity of this conversion process because of the measurement of an average deoxy-Mb signal and then subsequent conversion to a PO₂ (using the nonlinear Mb desaturation curve) rather than the direct measurement and subsequent averaging of iPO₂. They illustrate this point with theoretical Mb saturation values of 97 and 50% that average to 74%, with equivalent PO₂ values of 77.6 and 2.4 Torr that average to 40 Torr. When the average Mb saturation is converted to a PO₂, it results in a much lower PO₂ of 6.7 Torr (P₅₀ = 2.4). This suggests a sixfold error in the estimation of PO₂.

This simplistic illustration, although worthy of discussion, is flawed. Initially, it should be recognized that both the high and low levels of saturation used in this illustration are equally weighted. Hence, for this large underestimation of PO₂ to, in fact, be representative of the studies using Mb and MRS, 50% of the sample volume measured by proton MRS (~100 cm³) (33, 34, 36) would have to exhibit this high level of Mb saturation (97%). On the basis of the simultaneous assessment of intracellular bioenergetics in the same muscle volume using ³¹P MRS, this is most unlikely to be the case [mean intramuscular pH = 6.55, >95% PCr depletion, and 50% ATP depletion (33, 34, 36)]. These values are clearly values associated with a system that has, as a whole, been greatly perturbed. In addition, the existence of any region (especially half the coil volume) with a PO₂ of 77 Torr in heavily working muscle with an inflowing PO₂ of ~120 Torr, an outflowing PO₂ of ~18 Torr, and a calculated mean capillary PO₂ of ~40 Torr is highly unlikely (33, 34, 36). Thus the mathematical illustration made by Jurgens et al. (21) is misleading. If, as is probably more likely to be the case, 19/20 units of the muscle volume demonstrate a 50% Mb saturation and 1/20 units demonstrates a 97% Mb saturation, the mean Mb saturation is now 52%. The individual PO₂ values are unchanged (2.4 and 77.6 Torr), but the mean PO₂ calculated directly is 6.2 Torr, whereas the PO₂ calculated from Mb saturation is 2.2 Torr. Thus high levels of iPO₂ do have the potential to be underrepresented, but under more physiologically representative conditions this limitation may be much less important than previously portrayed (21).

Perspectives

Clearly the present data support the concept that intracellular oxygenation does not fall progressively with increasing muscular work. However, it should be emphasized that, even with inclusion of other laboratories' findings, the present consensus would suggest that at moderate levels of work the iPO₂ is already low (~5 mmHg) and venous PO₂/mean capillary PO₂ is greater than 30 Torr. Thus, as work increases, there is minimal scope for a decrease in iPO₂, and therefore the explanation for the majority of the increase in O₂ with increasing work must be the result of a markedly greater increase in D_O2. These findings once again emphasize the important interplay between both the convective and diffusive components of the O₂ transport system in determining skeletal muscle O_2 max.

	ACKNOWLEDGEMENTS

As always, the investigators are thankful for the participation of the subjects in this research. Special thanks must go to Ivan Dimitrov for invaluable assistance with this project.

	FOOTNOTES

This study was concurrently supported by the National Heart, Lung, and Blood Institute Grant HL-17731 and Regional Resource Grant RR-02305.

Address for reprint requests and other correspondence: R. S. Richardson, Dept. of Medicine, Univ. of California San Diego, La Jolla, CA 92093-0623 (E-mail: rrichardson{at}ucsd.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.

Received 10 April 2001; accepted in final form 15 August 2001.

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