INTRODUCTION

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BECAUSE MAXIMAL OXYGEN consumption (O_{2 max}) = (Ca_O2  Cv_O2), where is cardiac output and Ca_O2 and Cv_O2 are arterial and venous O₂ content, respectively, it can be expected that breathing a hyperoxic gas may raise body O_{2 max} by 5-10% in normal fit healthy human subjects through an increase in Ca_O2 (16, 32). This has been experimentally confirmed at the muscular level in normal fit healthy subjects during conventional cycle exercise by Knight et al. (14), who reported an increase in leg O_{2 max} of 8% and a corresponding 9% increase in maximal work rate due to elevated Ca_O2 with an unchanged leg blood flow. These data directly support the concept that, in fit healthy human subjects, muscle O_{2 max} is limited by O₂ supply: when provided with more O₂, skeletal muscle utilizes more O₂ and produces more work. However, perhaps the most compelling evidence in support of the argument that O_{2 max} is set by O₂ supply rather than biochemical limitation was first documented through the introduction of the knee-extensor (KE) model by Andersen et al. in 1985 (2, 3) and is now illustrated with more recent data in Fig. 1 (14, 24). Here, it is apparent that mitochondrial O₂ uptake (O₂) can more than double when central limitations to O₂ delivery are not present. These differences in mitochondrial metabolic rate, elucidated by the study of a single leg during whole body cycle exercise (14) and in the functionally isolated human quadriceps during single-leg KE exercise (24), raise two specific questions: 1) Can muscle O_{2 max} during KE exercise increase even further with increased O₂ delivery? 2) Is there a constant ability to extract O₂ in human skeletal muscle that constrains the relationship between O₂ delivery and O_{2 max} regardless of the exercise paradigm?

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Large increase in mitochondrial O2 uptake facilitated by changing exercise paradigm from cycling (14) to knee extension (24), where cardiac output and muscle O2 delivery are not limiting. Inasmuch as both sets of data were collected in endurance-trained subjects, mitochondrial O2 uptake was calculated on the basis of an assumed mitochondrial fiber volume of 7.5%, a myofibril volume of 80%, and a muscle density of 1.06 g/cm (9, 10, 20). Active muscle mass used in normalization was 7.5 kg for cycle exercise and 2.5 kg for knee extension.

To answer these questions, trained cyclists [similar in characteristics to subjects studied previously during cycle ergometry (14)] performed maximum KE exercise in hypoxia [0.12 inspired O₂ fraction (FI_O2)], normoxia (0.21 FI_O2), and hyperoxia (1.0 FI_O2). Thus the primary objective of this study was to test the hypothesis that muscle O_{2 max} during human KE exercise would not only be reduced in hypoxia as a result of a fall in O₂ delivery but, more importantly, would be elevated in hyperoxia as the result of an increased O₂ supply and that both would be governed by a constant muscle O₂ transport conductance from blood to muscle. In addition, the combination of the present results and previous data demonstrating that conventional cycle O_{2 max} is O₂ supply dependent (14) afforded a unique opportunity to examine the relationship between O₂ supply and O_{2 max} across a wide range of specific muscle activity.

	METHODS

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Subjects. Seven healthy nonsmoking male competitive bicycle racers who regularly rode 200-400 miles/wk volunteered to participate in the study, which had been approved by the University of California San Diego Human Subjects Committee. Health histories and physical examinations were completed and written informed consent was obtained according to requirements of the University of California San Diego Human Subjects Committee. The physical characteristics of the subjects were as follows: 26.1 ± 0.7 (SE) yr of age, 185.8 ± 0.7 cm height, and 77.1 ± 1.3 kg body wt. Subjects performed an incremental cycle ergometry test (100 W initial work rate increased by 25 W/min until fatigue), and their O_{2 max} averaged 5.03 ± 0.09 l/min or 65.2 ± 1.1 ml · min¹ · kg¹.

Preliminary tests. Subjects performed two to three training bouts on the dynamic KE apparatus to ensure familiarity with this exercise modality. The final two practice periods involved two graded maximal KE exercise tests (20 W initial work rate increased by 5 W/min) and a simulated final experiment adhering to the planned protocol but without any vascular catheterization. Indirect open-circuit calorimetry described in detail previously (26) was used to measure pulmonary ventilation and expired gases with use of a commercially available software package (Consentius Technologies, Salt Lake City, UT). In hyperoxic conditions the technical limitations of measuring ventilatory O₂ exchange at very high inspired O₂ concentrations ([O₂]) precluded collection of pulmonary gas exchange data (32).

Exercise model. The subject lay supine on a padded bed with the KE ergometer placed in front of him (25). The resistance to KE was provided via a fiberglass bar attached to the crank of the ergometer and to a specially designed shin brace worn by the subject. This ergometer was a prototype, and as such work rates could not be measured in conventional units of work, but work rate was prescribed and measured as a percentage of the maximum resistance (weight in g, resisting the flywheel turning) at the end of the preliminary graded maximal test. Dynamic contractions of the KE muscles were performed at 60/min. Contractions of the quadriceps femoris muscle caused the lower part of the leg to extend from ~90 to 170° flexion. The momentum of the flywheel returned the relaxed leg to the start position. Anecdotal subject reports, force tracings, electromyography, and T2-weighted magnetic resonance imaging (2, 23, 26) support the conclusion that active contractions are limited to the quadriceps muscles during this exercise modality.

Experimental protocol. After the catheterization procedures three bouts of exercise were performed: 1) left leg KE during room air breathing, 2) left leg KE at 0.12 FI_O2, and 3) left leg KE during 1.0 FI_O2. The order of these exercise bouts across subjects was arranged to create a balanced design. For each exercise bout, the work rate was increased from an unweighted warm-up to the previously determined maximum work rate followed by progressive 5% increases in work rate to ensure that a true maximum was achieved. Data were obtained at each level. Each incremental exercise bout was completed in 8-12 min. The sequence of events at each work rate was as follows: 1) 3-ml blood samples were taken [for measurement of PO₂, PCO₂, pH, and arterial O₂ saturation (Sa_O2)], and 2) femoral venous blood flow was measured. Duplicate measurements of all variables were then taken.

Exercise study with femoral blood flow and blood-gas measurements. Within 1 wk of the preliminary studies, subjects returned to the laboratory in the morning when two catheters (radial artery and left femoral vein) and a thermocouple (left femoral vein) were placed using sterile technique, as previously reported (19, 26). Briefly, a 20-gauge, 3.2-cm-long arterial catheter was inserted percutaneously under local anesthesia (1% lidocaine) in the radial artery of the nondominant hand for arterial blood sampling. Subsequently, a 1.25-mm-OD catheter (model DSA 400L, Cook, Bloomington, IN) was introduced percutaneously into the left femoral vein 2 cm below the inguinal ligament and advanced 7 cm distally. This catheter has an open end and also 10 pinhole side ports in the distal 2.5 cm oriented in all directions around the catheter. This ensures that, during injection of cold saline, thin streams are ejected at all orientations into the vein, facilitating mixing across the vein lumen. The second catheter consisted of a thin (0.64-mm-diameter) polyethylene-coated thermocouple (model IT-18, Physitemp Instruments, Clifton, NJ) that was advanced from approximately the same location proximally 10 cm into the left femoral vein toward the heart. Each catheter was attached to the skin by means of adhesive tape and positioned to minimize the risk of movement or creasing. During exercise, iced saline was infused through the Cook catheter at flow rates sufficient to decrease blood temperature at the thermocouple by >1°C. Infusions were continued for 10-15 s until femoral vein temperature had stabilized at its new lower value. Saline injection rate was measured by weight change in a reservoir bag suspended from a force transducer, which was calibrated before and after each experiment. Blood flow was calculated on thermal balance principles, as detailed by Andersen and Saltin (3). Quadriceps O₂ was calculated as the product of arteriovenous [O₂] difference (see below) and blood flow.

Blood analyses. Samples (3-4 ml) of arterial and femoral venous blood were withdrawn from the catheters anaerobically to measure PO₂, PCO₂, pH, O₂ saturation, and Hb concentration ([Hb]). All measurements were made on an IL 1306 blood-gas analyzer and an IL 482 CO-oximeter (Instrumentation Laboratories, Lexington, MA). Between each sample, electrodes were calibrated and demonstrated acceptable reproducibility (SD of repeated determinations 1.5 Torr for PO₂ and PCO₂ and 0.003 for pH). [O₂] was calculated as 1.39 × [Hb] × measured O₂ saturation  0.003 × measured PO₂. Arteriovenous [O₂] difference was calculated from the difference in radial arterial and femoral venous [O₂]. This difference was then divided by arterial concentration to give O₂ extraction.

Validity and reliability of the thermodilution blood flow technique. This method of measuring blood flow has now been used on many occasions in research from this and other laboratories (3, 4, 13, 19, 29). In each of these experiments the ultimate criterion of validity (which initially should be whether the measurements of blood flow and arteriovenous differences yield proper values for O₂) was achieved on the basis of the slopes of the O₂-work rate relationship (26). However, in addition to a direct validation of the thermodilution technique with dye dilution (12), recent agreement between the thermodilution technique and ultrasound Doppler estimates of femoral arterial blood flow during KE exercise adds considerable validity to both methodologies (22).

Thigh volume measurement. With use of thigh length, circumference, and skinfold measurements, thigh volume was calculated to allow an estimate of quadriceps femoris muscle mass, as suggested by Andersen and Saltin (3) and others (11). This calculation of muscle mass and the consequent normalization of and O₂ assume that this is the only muscle mass involved in the KE exercise, an assumption recently verified in the KE human exercise model (23).

Muscle O₂ transport conductance and mean capillary PO₂ () calculations. Muscle O₂ transport conductance (D_O2) and were calculated as described previously (31) but only at 100% of maximum work rate. Briefly, a numerical integration procedure is used to determine the value of D_O2, assumed constant along the capillary, that produces the measured femoral venous PO₂, given the measured arterial PO₂. Additional explicit assumptions of this calculation are as follows: 1) mitochondrial PO₂ is negligibly small at O_{2 max}, and 2) the only explanation of O₂ remaining in the femoral venous blood is diffusion limitation of O₂ efflux from the muscle microcirculation. Perfusion-O₂ heterogeneity and perfusional or diffusional shunt are considered negligible. To the extent that these phenomena contribute O₂ to femoral venous blood, the parameter D_O2 is a conductance coefficient that expresses the diffusing capacity that would be required to achieve the measured O_{2 max}, with the assumption of only diffusion limitation. This assumption cannot be avoided for the lack of specific means for detecting perfusiono₂ heterogeneity and shunt. is the numerical average of all PO₂ values computed, equally spaced in time, along the capillary from the arterial to the venous end. Although is useful for graphical purposes (see RESULTS), our conclusions come from statistical comparisons of D_O2 among the three experimental conditions.

Statistical analyses. At maximal exercise, variables were tested for a significant difference among the three different inspired O₂ conditions by repeated-measures ANOVA and a Tukey post hoc analysis. All statistics were computed using a commercially available software package (Graph Pad, San Diego, CA). Variables were considered significantly different when P  0.05.

	RESULTS

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Work rate, quadriceps blood flow, quadriceps O₂, and pulmonary O₂. On the basis of data attained in previous KE studies (24), the competitive nature of the subjects, and the reproducibility of maximal work rates in the preliminary and experimental study days, it was evident that these subjects were at or extremely close to O_{2 max} in each condition; however, because of the experimental design, a plateau in the O₂-work rate relationship was not attainable. From other trials with these subjects on another KE ergometer (the present ergometer did not allow a classic work rate calculation), the maximum work rate could be equated to 80-100 W, depending on the subject. Pulmonary O₂ at maximal KE rose to only 45% of that previously recorded during maximal conventional cycle ergometry, indicating that whole body maximal effort was not invoked. Quadriceps O₂ varied with inspired O₂, being lower in hypoxia than in normoxia and greater in hyperoxia than in normoxia (Table 1, Figs. 2-4). Maximal muscle blood flow was not different between hyperoxia and normoxia but was significantly lower in hypoxia (Table 1).

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Relationship between maximal O2 consumption (O2 max) and mean capillary and femoral venous PO2 in hypoxia, normoxia, and hyperoxia [inspired O2 fraction (FIO2) = 0.12, 0.21, and 1.0, respectively] during knee extension.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Relationship between O2 max and mean capillary PO2 during knee extension and cycle exercise in hypoxia, normoxia, and hyperoxia. In each case, O2 transport conductance is represented by lines connecting data collected in same exercise paradigm. O2 transport conductance is significantly elevated in knee-extensor compared with cycle exercise: # P < 0.05 (14).

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Similar relationship between O2 max and O2 delivery during conventional cycle exercise (14) and knee-extensor data collected in hypoxia, normoxia, and hyperoxia. Maximal O2 extraction in each knee extension is significantly reduced compared with maximal cycle exercise O2 extraction: # P < 0.05.

Blood O₂ content, O₂ delivery, and O₂ extraction. At maximal exercise, Ca_O2 was significantly reduced in hypoxia and significantly increased in hyperoxia, whereas Cv_O2 was unaffected by alterations in inspired O₂ (Table 1). O₂ delivery reflected the changes in Ca_O2 and blood flow and consequently was significantly reduced in hypoxia and significantly elevated in hyperoxia in comparison to normoxia (Table 1). O₂ extraction, at maximal exercise, was not significantly influenced by the inspired [O₂].

Hb O₂ saturation, blood O₂ partial pressures, and muscle D_O2. At maximal exercise, Sa_O2 was significantly reduced in hypoxia and significantly elevated in hyperoxia (Table 1). As expected, the magnitude of Sa_O2 reduction in hypoxia was much greater than the elevation in hyperoxia because of the 100% ceiling for Hb O₂ saturation. Sv_O2 was significantly lower in hypoxia than in normoxia but was not different from the normoxic value in hyperoxia. With the exception of Pv_O2 in hyperoxia compared with normoxia (which was elevated but did not achieve statistical significance), Pa_O2, Pv_O2, and were significantly decreased in hypoxia and significantly elevated in hyperoxia in comparison to normoxia (Table 1). Additionally, in the three FI_O2 levels, Pv_O2 and each varied proportionately with O_{2 max} (Fig. 2). D_O2 did not vary with FI_O2 during maximal exercise (Table 1, Fig. 2).

Cycle vs. KE O₂ extraction. O₂ extractions previously reported in similar subjects during cycle exercise in hypoxia, normoxia, and hyperoxia were 93.3 ± 1.0, 91.8 ± 1.4, and 89.7 ± 4.4%, respectively (14). Each of these O₂ extractions was significantly higher than the equivalent extraction recorded during the present KE study (Table 1, Fig. 4).

	DISCUSSION

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The major observation in this study is that even in an exercise paradigm where O₂ delivery per unit of muscle mass is very high, an elevated O₂ delivery afforded by breathing 100% O₂ results in an increase in O_{2 max} in trained human skeletal muscle. This provides evidence that in trained human subjects normoxic KE, which has demonstrated the highest mass-specific skeletal muscle O₂ in humans (Fig. 1), is limited by O₂ supply and not O₂ demand. Additionally, factors that determine O₂ supply and D_O2 from blood to skeletal muscle play a key role in determining O_{2 max} (Figs. 1, 3, and 4). Specifically, during maximal single-leg KE the proportional relationship between both Pv_O2 and and O_{2 max} accompanying elevations and reductions in FI_O2 are consistent with the concept of tissue diffusion limitation of O_{2 max} in normal humans (Fig. 2) (31). Additionally, the observed similarity in the slope of the relationship between O₂ delivery and muscle O_{2 max} in KE and cycle exercise (14) suggests that skeletal muscle O_{2 max} is responsive to variations in O₂ delivery but is bound by the ability of skeletal muscle to extract O₂, which is itself dependent on the exercise paradigm (Fig. 4).

Although it is likely that there will always be disagreement on the topic of what limits muscle O_{2 max}, in addition to the present findings, several recent studies have provided evidence supporting the concept that O₂ supply, rather than biochemical limitation (8, 30), sets O_{2 max}. Specifically, despite using an optical technique similar to that used by Stainsby et al. (30), Duhaylongsod et al. (7) reported contrasting results in the canine gracilis muscle, where maximal exercise resulted in near-complete reduction of cytochrome aa₃. This was interpreted to reflect deficient O₂ provision to this muscle (7). In humans, Richardson et al. (25) measured in vivo myoglobin desaturation at maximal exercise as an endogenous probe of intracellular PO₂ and found a proportional fall in muscle O_{2 max} with a hypoxically induced reduction in intracellular PO₂, thus providing support for the concept that maximal respiratory rate (O_{2 max}) is limited by O₂ supply (25). During whole body exercise, indirect pulmonary gas-exchange measurements in humans have continued to support the importance of O₂ supply in determining muscle O_{2 max} (1, 17), whereas more direct evidence attained by blood-gas and blood flow measurements in humans during cycle exercise have also recently been provided by Knight et al. (14). Here normoxic leg O_{2 max} was increased by 8% in hyperoxia (1.0 FI_O2) and reduced by 30% in hypoxia (0.12 FI_O2). Additionally, in the dog gastrocnemius, Richardson et al. (27) increased the diffusive component of O₂ supply by a rightward shift in the Hb-O₂ dissociation curve while maintaining a constant convective O₂ delivery (1.0 FI_O2) and found a concomitant increase in O_{2 max}. These data demonstrated not only the O₂ supply dependence of canine skeletal muscle but also the importance of diffusive O₂ transport at maximal exercise. On the other hand, in support of demand limitation, the human KE model has recently revealed that, although the average flux through the tricarboxylic acid cycle is much lower than the maximal activity of several traditional marker enzymes (e.g., citrate synthase) (6), oxyglutarate dehydrogenase, another tricarboxylic acid cycle enzyme, is fully activated during the high O₂ recorded under these conditions and may be a factor in limiting maximal metabolic rate (6).

As illustrated in Fig. 1, a clear indication that O₂ supply governs muscle O_{2 max} became apparent with the introduction of the functionally isolated KE model by Andersen and Saltin (3). The comparison that we make here between data collected from human quadriceps acting as part of whole body (cycle) exercise (14) and the KE in isolation confirms much higher specific mitochondrial O₂ when central limitations to O₂ delivery are not present (Fig. 1). The present data extend this observation and illustrate that, within either exercise paradigm, increased or decreased O₂ delivery results in a similar change in muscle O_{2 max} (dictated by the interaction of O₂ delivery with D_O2, Fig. 2). When this same analysis is used to compare the D_O2 in KE and cycle exercise, it is apparent that KE results in a significantly elevated D_O2 (Fig. 3). However, between exercise paradigms the change in O_{2 max} is restrained by the ability of human skeletal muscle to extract O₂, which is significantly reduced in KE (Fig. 4). Thus the increased maximal D_O2 in KE does not directly translate to a similar increase in maximal O₂ extraction, despite the fact that the increase in O₂ extraction with increasing O₂ during KE has previously been shown to be similar in nature to that seen in cycle exercise (26). This again illustrates that "peripheral" limits to O₂ flux by diffusion from capillary to muscle interact with O₂ delivery to determine O₂ extraction and ultimately O_{2 max}. Here it should be recognized that O₂ extraction (e) is not a pure reflection of peripheral factors (i.e., D_O2), inasmuch as it incorporates other "central" factors {i.e., blood flow () and the shape of the Hb-O₂ dissociation curve (): O₂ extraction = 1  exp[D_O2/()]} (18). However, it is evident that the interaction of the variables that constitute O₂ extraction appears to be a limitation that constrains changes in muscle O_{2 max} with changes in muscle O₂ delivery. Hence, in KE, despite an increased D_O2 and a similar relationship between O₂ extraction and O₂ (26), O₂ extraction at O_{2 max} appears to be attenuated by high muscle blood flows.

Finally, Rowell et al. (29) reported that, during maximal KE exercise, blood flow increased to compensate for the reduced Ca_O2 and thus maintain muscle O_{2 max}. Recently, two independent groups replicated the study of Rowell et al. and found that maximal blood flows were actually reduced in hypoxia. It was concluded that the previous data were probably submaximal and not maximal, as suggested previously (15, 24). These recent studies disagreed as to the method by which submaximal O₂ was maintained constant in hypoxia compared with normoxia: Richardson et al. (24) found that extraction was increased and muscle blood flow remained unaltered, whereas Koskolou et al. (15) reported an elevation in blood flow with constant extraction. However, at maximal exercise, both research groups were in agreement that blood flow did not increase (in fact, it was reduced) to compensate for the reduced Ca_O2, and thus O₂ delivery and muscle O₂ were reduced (15, 24). Evidently, a greater muscle blood flow could have been achieved (as seen in normoxia and hyperoxia); however, in hypoxia this higher level was not attained. When examined in relation to work rate (15, 24), the maximal blood flow in hypoxia exhibits the same relationship as in normoxia but was reduced in proportion to the lowered work rate. The mechanism for this apparent coupling between absolute work rate, O₂, and blood flow at maximal exercise, but not during submaximal exercise, is unclear and deserves further investigation.

Summary. It has now been repeatedly demonstrated that an increase in O₂ delivery can increase O_{2 max} (1, 5, 7, 14, 17, 21, 25, 27, 32), which suggests that O₂ supply limitation exists. As isolated human quadriceps exercise does not approach the upper limits of , this exercise paradigm has previously unveiled a skeletal muscle metabolic reserve and results in the highest mass-specific O₂ and work rates recorded in humans (24, 26, 28). This observation is evidence of O₂ supply limitation of muscle O_{2 max}. The present study supports these findings and by increasing O₂ delivery demonstrates that, in normoxic conditions of isolated KE, muscle O_{2 max} is not limited by mitochondrial metabolic rate but, rather, by O₂ supply.