Journal of Applied Physiology Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

J Appl Physiol 84: 995-1002, 1998;
8750-7587/98 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Richardson, R. S.
Right arrow Articles by Wagner, P. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Richardson, R. S.
Right arrow Articles by Wagner, P. D.

Vol. 84, Issue 3, 995-1002, March 1998

Increased VO2 max with right-shifted Hb-O2 dissociation curve at a constant O2 delivery in dog muscle in situ

Russell S. Richardson, Kuldeep Tagore, Luke J. Haseler, Maria Jordan, and Peter D. Wagner

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

If the diffusive component of O2 transport in muscle is important in determining exercise capacity, an increased capillary-to-tissue PO2 difference should enhance gas exchange from blood to skeletal muscle during exercise. Thus a rightward shift in the O2 dissociation curve should theoretically increase O2 extraction and improve maximal O2 uptake (VO2 max). To test this hypothesis, we used the canine gastrocnemius muscle to study maximal exercise in eight dogs at a normal P50 (33.1 ± 0.4 Torr) and with the O2 dissociation curve shifted to the right by an allosteric modifier of hemoglobin (Hb) (methylpropionic acid, RSR-13; P50 = 53.2 ± 5.0 Torr). Four control dogs were also studied before and after infusion of vehicle. O2 (100%) was inspired during exercise to maintain arterial saturation in both conditions. The muscle was surgically isolated and electrically stimulated (tetanic train: 0.2-ms stimuli for 200-ms duration at 50 Hz, once per s). To maintain O2 delivery (pre-RSR-13 = 19.1 ± 2.9; RSR-13 = 19.6 ± 2.5 ml · 100 g-1 · min-1), the muscle was pump perfused. At a constant O2 delivery, RSR-13 significantly increased percent O2 extraction (pre-RSR-13 = 61 ± 4.0; RSR-13 = 75.5 ± 4.7) and muscle VO2 max (pre-RSR-13 = 11.8 ± 2.1; RSR-13 = 14.2 ± 1.5 ml · 100 g-1 · min-1). This improvement in VO2 max with increased P50 demonstrates its O2 supply dependence when P50 is normal and the importance of O2 diffusive transport to muscle at maximal exercise.

oxygen affinity; exercise; diffusion; partial pressure of oxygen at 50% hemoglobin saturation; skeletal muscle; oxyhemoglobin; maximal oxygen uptake

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

OXYGEN DELIVERY to skeletal muscle is dependent on the interaction between the convective transport of O2 in blood and, once released from the hemoglobin (Hb) carrier molecule, its subsequent diffusion down an O2 tension (PO2) gradient to the mitochondria. Convective O2 delivery is set by muscle blood flow (Q) and arterial O2 content (CaO2), whereas the diffusive component is determined by the magnitude of the PO2 gradient from blood to the O2-consuming mitochondria and the physical conductance for O2 of the pathway between them. The PO2 gradient itself is the consequence of O2 delivery, the affinity of Hb for O2, the muscle metabolic rate, and the O2 conductance from blood to muscle (24, 30).

Thus the position of the O2 dissociation curve (ODC), conveniently described by P50 (PO2 at which 50% Hb is saturated), has an important role in O2 transport. Of course, variations in P50 have ramifications not only in the periphery but also during O2 loading in the lungs. A high P50 opposes the association of O2 in the lungs but favors its release to the tissues and vice versa. Thus, if arterial PO2 is maintained to permit normal O2 saturation, a right-shifted ODC may be advantageous during exercise conditions, since it allows Hb desaturation to occur at higher levels of mean capillary PO2 (PcO2), increasing the PO2 gradient and thus promoting a greater O2 flux from capillary blood to skeletal muscle (25). There is now considerable experimental evidence, collected in animal and human skeletal muscle, that indicates that O2 conductance (DmO2), from blood to mitochondria, is an important determinant of maximal muscle O2 uptake (VO2 max ) (3, 18, 21, 29). These data have recently been supported by evidence of a large difference in PO2 between blood and the intracellular compartment in normal human muscle, as measured by myoglobin-associated PO2, across a wide range of exercise intensities (19).

In the ongoing search to find methods to increase O2 delivery to ischemic tissue, recent pharmaceutical developments have lead to the discovery of allosteric effectors of Hb-O2 affinity, which are active in whole blood (17). These allosteric effectors result in a similar right shift of the ODC to the naturally occurring erythrocyte allosteric effector 2,3-diphosphoglyceric acid (2,3-DPG), but they bind at a different site and so may produce an additive right shift in the ODC in the presence of 2,3-DPG. One compound of this series, 2-(4-{[(3,5-dimethylanilino)carbonyl]methyl}phenoxy)-2-methylpropionic acid (RSR-13), significantly increases the P50 in vivo but has a lower affinity for serum albumin than the other compounds from this series and therefore may be the preferred allosteric effector to study clinically (1).

It has previously been illustrated that in the presence of a right shift in the ODC produced by allosteric Hb-O2 modifiers [with constant convective O2 delivery and O2 uptake (VO2)], venous PO2 and tissue PO2 are both increased (9, 32). Thus manipulations of the P50 offer the opportunity to vary O2 delivery to the tissues without altering blood flow and CaO2 and therefore isolating the specific effects of diffusive O2 movement into the tissue. Using this approach, Hogan et al. (4) demonstrated that an increased Hb affinity (low P50) resulted in a diminished VO2 max in the isolated canine gastrocnemius muscle. Because the estimated DmO2 was not different in the normal and left-shifted conditions and the decline in VO2 max was proportional to the fall in calculated PcO2, it was concluded that peripheral DmO2 (blood to muscle) played a role in determining VO2 max. However, these data illustrate the O2 supply dependence of VO2 max only under conditions in which the PO2 gradient from blood to tissue is reduced and do not address the issue of whether VO2 max is O2 supply dependent under normal conditions and thus could be increased by producing a larger PO2 gradient. This would provide a better understanding of the relationship between maximal mitochondrial respiration rate and O2 supply under normal physiological conditions. Consequently, the purpose of this study was to determine the effect of a decreased Hb affinity produced by an infusion of RSR-13 on skeletal muscle VO2 max, while ensuring constant O2 delivery by controlling Q and CaO2.

    METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Twelve adult mongrel dogs with a weight range of 14-26 kg were anesthetized with pentobarbital sodium (30 mg/kg) and kept under a suitable level of anesthesia by maintenance doses as required. The dogs were intubated with a cuffed endotracheal tube and were ventilated (Harvard 613) to maintain arterial PO2, PCO2, and pH in the normal range. Esophageal temperature was monitored by a thermistor and maintained at 36-38°C by heating pads.

Surgical preparation. The functional and vascular isolation of the left gastrocnemius-flexor digitorum superficialis muscle complex (referred to as gastrocnemius) was achieved as described previously (23). In brief, a medial incision was made through the skin of the left hindlimb from the ankle to midthigh. The muscles that overlie the gastrocnemius (sartorius, gracilis, semitendinosus, and semimembranosus) were retracted from the field of view by doubly ligating and cutting between the ties. The arterial and venous circulation of the gastrocnemius was isolated by ligating all vessels from both the popliteal artery and vein and femoral vein, which were not directly connected to the gastrocnemius. The left popliteal vein, distal to the gastrocnemius, was cannulated, and the venous return from the muscle was diverted to the cannulated jugular vein. The right femoral artery was cannulated and connected to the left femoral artery, allowing the now isolated muscle to be perfused by blood from this contralateral artery. Perfusion of the isolated muscle was achieved by systemic pressure at rest and controlled by a roller pump (Cole-Palmer, Chicago, IL) during exercise periods. Muscle perfusion pressure was constantly monitored through a pressure transducer in this line, close to the head of the muscle. Systemic arterial blood pressure was also monitored continuously from a catheterized carotid artery. Heparin (150 U/kg) was given to the animals at the completion of the surgery. The left sciatic nerve, which innervates the gastrocnemius, was ligated and cut. To reduce the potential for cooling and drying, all exposed tissues were covered with saline-soaked gauze.

When surgically isolated, the Achilles tendon was attached to an isometric myograph (Interface Manufacturing, Scottsdale, AZ) to measure tension development. The hindlimb was fixed at the knee and ankle and attached to the myograph with struts to minimize movement. At the end of each experiment, weights were used to calibrate the tension myograph.

Isometric muscle contractions were elicited by electrical stimulation of the sciatic nerve (tetanic train: 6-8 V, 0.2-ms stimuli for 200-ms duration at 50 Hz, once per s). The muscle was stimulated in this fashion for 3-3.5 min. Before each contraction period, the resting muscle length was adjusted to achieve the greatest contractile response to a single stimulation. This ensured that the initial tension development was not affected by slippage in the system that may have occurred in the previous contraction period. Before each exercise period the blood supply to the isolated muscle was switched from self-perfusion to pump perfusion. Before the beginning of the contraction period, adequate time (2-3 min) was allowed for conditions to stabilize at a blood flow which matched those at rest with self-perfusion.

Experimental protocol. We studied the gastrocnemius electrically stimulated to elicit maximal exercise in eight dogs at a normal P50 and then again with the ODC shifted to the right by the allosteric modifier of Hb (RSR-13, Allos Therapeutics). Before each exercise period, arterial and venous blood samples were analyzed at varied levels of inspired O2 (12, 16, 21, 30, and 100% O2), and the data were used to calculate the P50 and Hill coefficient (n) with the Hill equation. A preliminary series of studies in which we monitored the change in P50 over time (to assess effect of half-life of RSR-13) indicated the validity of producing a single ODC from a series of blood samples when all samples were bracketed within a 10- to 15-min period. The dose of RSR-13 (100 mg/kg) was diluted in 100 ml of 0.9% NaCl and was infused over a 20-min period after the first exercise bout. Due to the reduction in arterial O2 saturation produced by the rightward shift in the ODC, all dogs breathed 30% O2 between contraction periods and 100% O2 during each contraction sequence. This successfully maintained arterial O2 saturation at >= 99.6% during exercise both before and after the P50 change. O2 delivery during each bout of exercise was kept constant by alterations in the blood flow to the maximally exercising isolated muscle as necessary. Due to the 1.5- to 2-h half-life of RSR-13, we were unable to produce the preferred balanced experimental design in which one-half the animals received the RSR-13 treatment first and normal Hb in the second exercise period. To address this potential ordering effect, we evaluated the effect of time and repeated exercise bouts on four control dogs that did not receive the RSR-13 but did experience exactly the same protocol (including a sham infusion of saline) and performed two exercise bouts over the same period of time as the RSR-13-treated animals.

Measurements. Duplicate arterial blood samples were taken from the carotid artery, and the venous samples were taken from the catheter extension of the popliteal vein, which ran to the jugular vein. Arterial and venous samples were drawn anaerobically during the last 20 s of each contraction. Arterial samples were analyzed immediately for PO2, PCO2, pH, O2 saturation, and Hb concentration ([Hb]), to minimize the rapid fall in PO2 commonly seen with a high PO2 when breathing 100% O2. Venous samples, not prone to this problem, were placed on ice and analyzed after the arterial samples. Between taking of blood samples blood flow measurements of the venous outflow were made by both timed blood collections into a graduated cylinder and by an in-line ultrasonic flow probe (Transonic Systems). Both methods of measurement were in agreement within 1-4 ml/min; thus only the flow probe values are reported.

Blood PO2, PCO2, and pH were measured with a blood gas analyzer (IL-1306, Instrumentation Laboratories, Lexington, MA) and then corrected for measured body temperature while O2 saturation and [Hb] were measured on a CO-oximeter (IL-482, Instrumentation Laboratories). Plasma bicarbonate concentration was calculated from the measured pH and PCO2 values by using the Henderson-Hasselbalch equation. Blood lactate concentrations were determined by using a blood lactate analyzer (model 1500, Yellow Springs Instrument, Yellow Springs, OH). Blood O2 concentration was calculated as 1.39 ml/O2 × [Hb] g/100 ml × measured O2 saturation + 0.003 ml · O2-1 · 100 ml-1 × measured PO2. Arteriovenous O2 concentration difference was calculated from the difference in carotid artery and popliteal venous O2 concentration. This difference was then divided by arterial concentration to give O2 extraction. Gastrocnemius VO2 was calculated as the product of arteriovenous O2 concentration difference and blood flow. The standard P50 of the blood was calculated before each exercise bout by varying the inspired O2 concentration (see Experimental protocol; Fig. 1). The Hill equation was then used to calculate the P50 and n for each ODC in both the normal and right-shifted conditions.


View larger version (20K):
[in this window]
[in a new window]
  		Fig. 1.   Typical effect, on a single dog, of RSR-13 infusion on hemoglobin (Hb)-O2 dissociation curve. P50, PO2 at 50% Hb saturation; Hill n, Hill coefficient.

Before RSR-13 infusion, PcO2 was calculated by using a Bohr integration technique as described previously (26). Briefly, with the recently substantiated assumption that, under normal P50 conditions, mitochondrial PO2 is close to zero at maximal exercise (19), a numerical integration procedure was used to calculate the value of DmO2, assumed constant along the capillary, that produces the measured femoral venous PO2, given the measured arterial PO2. This calculation also allowed an estimate of PcO2 as the average of all PO2 values computed, at equal time intervals along the capillary from the arterial to the venous end, assuming the only explanation for O2 remaining in the femoral venous blood is diffusion limitation of O2 efflux from the muscle microcirculation. On the basis of previous studies that have recorded a substantially elevated intracellular PO2 (although at rest) after the infusion of RSR-13 (9, 32), it is probably inappropriate to attempt to calculate DmO2 or PcO2 after RSR-13, since the essential assumption of a low intracellular PO2 may be no longer valid. However, for interpretation purposes, we have reported these calculated variables with the assumption that intracellular PO2 is zero.

At the end of the experiment, both the exercised and contralateral gastrocnemius muscles were removed and weighed. The contralateral muscle weight was used to normalize variables to muscle mass, and the exercised muscle was weighed to indicate the amount of any edema that had resulted from the protocol.

Statistical analyses were performed on both the results in the RSR-13 and control data pre- and postinfusion by using paired t-tests and linear regression. The control animals were not compared statistically with the RSR-13 treatment group by experimental design, since this would have undermined the power of the paired statistical analyses and increased the number of animals required for the study. For the treatment group, the degrees of freedom were 7 and the controls 3 in all variables. A probability level of P < 0.05 was considered significant. Data are reported as means ± SE.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

The principal variables related to O2 transport, gas exchange, and acid-base balance in the blood perfusing the muscles during the RSR-13 condition and the controls are presented in Tables 1 and 2. The P50 in the RSR-13-treated group increased from 33.1 ± 0.4 Torr (similar to that seen in control animals) to 53.2 ± 5.0 Torr. Despite this large right shift in the ODC, the elevated inspired O2 concentration of 100% was sufficient to maintain the arterial Hb saturation level at >= 99.6 in the RSR-13-treated group. In both the control and RSR-13-treated animals, [Hb] was somewhat reduced in the second exercise bout. This was the combined result of bleeding due to the surgical intervention, blood sampling, and the volume of saline added through flushing sample lines and infusions. As a result of this fall in [Hb], CaO2 was reduced in the second exercise bout in all animals. However, by experimental design, O2 delivery was matched in all pre- and postinfusion exercise bouts by increasing the pump controlled Q (Table 2, Fig. 2). Under these conditions of constant O2 delivery, the control group showed no change in O2 extraction or VO2 max between the first and second exercise bout, whereas the RSR-13-treated group showed a significant increase in O2 extraction (Table 2, Fig. 3), resulting in a significant (20%) increase in VO2 max (Table 2, Fig. 2). The administration of the RSR-13 produced a significant reduction in arterial blood pressure measured in the carotid artery, apparent in the second exercise bout in this group and not in the sham saline-infused control group (Table 2).

                              
View this table:
[in this window]
[in a new window]
  		Table 1.   Principal variables related to O2 transport and acid-base balance at VO2 max

                              
View this table:
[in this window]
[in a new window]
  		Table 2.   Principal variables related to O2 delivery, utilization, and gas exchange at VO2 max


View larger version (22K):
[in this window]
[in a new window]
  		Fig. 2.   Effect of RSR-13 infusion (P50 = 53.2 Torr; B) and sham saline infusion (P50 = 34 Torr; A) on O2 delivery and O2 uptake. VO2 max, maximal O2 uptake. * Significantly different from preinfusion value (P <=  0.05).


View larger version (20K):
[in this window]
[in a new window]
  		Fig. 3.   Significantly increased O2 extraction after an increase in P50 with RSR-13 infusion in comparison to unchanged saline-infused controls. * Significantly different from preinfusion value (P <=  0.05).

The average weight of the nonexercised gastrocnemius was 82 ± 7 g and that of the surgically isolated and exercised muscle group was 95 ± 6 g because of the increased filtration pressures in the vascular tree of this muscle during exercise. There was no difference in the average increase in muscle weight between the control and RSR-13-treated animals.

Because not all animals exhibited similar degrees of O2 extraction at VO2 max before the administration of RSR-13, we examined the relationship between the increase O2 extraction at VO2 max after RSR-13 and the percent O2 extraction at VO2 max before RSR-13 infusion (Fig. 4). Onto this analysis, we placed the control animals that received the saline infusion. From this analysis, which indicates the effect of the infusion on extraction (limited by bounds of possible improvement: an animal with an initially high extraction has little room for improvement), we divided the group of eight RSR-13-treated dogs into five responders and three nonresponders. Among the five animals that did respond, we found a strong correlation (r2 = 0.72) between initial O2 extraction and the effect that RSR-13 had on O2 extraction. Thus, in animals with initially poor extraction, the right-shifted ODC greatly increased O2 extraction, whereas in initially high extractors, there was little effect. For the remaining three of the RSR-13-treated animals, the response was not measurable and fell among the results of the sham saline-infused animals. In fact, the 95% confidence intervals constructed for each regression line support the grouping of the data in this manner, since the confidence intervals do not overlap and the nonresponders fall in the region of the control animals (Fig. 4).


View larger version (30K):
[in this window]
[in a new window]
  		Fig. 4.   Variable responses to a rightward shift in O2 dissociation curve. Note a strong inverse relationship between change in O2 extraction after RSR-13 infusion and O2 extraction pre-RSR-13 for 5 dogs, whereas 3 dogs responded no differently than saline-infused controls. Note that 1) 95% confidence intervals for regression lines through RSR-13-responsive animals and controls contain respective data (responders and controls + nonresponders) and do not overlap, suggesting a legitimate division of data, and 2) this graph embodies certain mathematical restraints because it has a component of preinfusion values on both axes. However, it does illustrate concept of a maximal achievable O2 extraction and that not all dogs moved toward this upper limit even when treated with RSR-13.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

The principal observation in this study is that, under conditions of constant convective arterial O2 delivery, an increase in P50 allowed exercising skeletal muscle to achieve a greater VO2 max. This provides evidence that VO2 max at a normal P50 is not determined by mitochondrial metabolic limits but, rather, by O2 supply: an increase in P50 produces a steeper O2 gradient (driving force) from capillary-to-tissue, providing more O2 and allowing tissue VO2 max to increase. This is of importance because it has been controversial as to whether VO2 max during normal delivery conditions is limited by inadequate oxygenation of the mitochondria (8, 19).

Role of O2 supply and O2 diffusion in determining VO2 max. It has been demonstrated that an increase in O2 delivery can increase VO2 max (2, 10, 16, 31), suggesting that O2 supply limitation does exist. However, it has also been shown that this is not the unique determinant of VO2 max (26). The present experimental findings support the concept that, for a given O2 delivery, the amount of O2 that can be extracted and used by the working muscle is determined by the DmO2 and the PO2 gradient from the erythrocyte to the mitochondria (Fick's law of diffusion). Theoretically, if the O2 conductance is held constant and DmO2 does not change, a right-shifted ODC should decrease the rate at which the capillary PO2 declines as O2 is removed by the working muscle, thereby increasing the capillary-to-tissue PO2 driving gradient along the capillary length. This rightward shift in the ODC should then increase VO2 max, if DmO2 is an important determinant of VO2 max. Previously, this theoretical analysis was tested directly by the calculation of PcO2 and DmO2 and the determination of their relationship to VO2. In the present scenario, it may not be appropriate to attempt to estimate either the PcO2 or DmO2 after the infusion of RSR-13 because previous findings have determined that intracellular PO2 can rise to levels in excess of 32 Torr (at rest) after this Hb modification (9) and an inherent assumption of these calculated variables is that intracellular PO2 is close to zero at maximal exercise (7, 26). It is interesting to note that, after RSR-13 infusion, an estimate of intracellular PO2 by using the Bohr integration technique (assuming same DmO2 as before RSR-13 infusion) indicated that tissue PO2 may be as high as 24 Torr. However, if these variables are calculated by using the assumption that intracellular PO2 is zero, they suggest that PcO2 was greatly elevated and DmO2 was significantly reduced. The unexpected change in DmO2 could be the result of either an incorrect assumption of the intracellular PO2 or a vascular effect of the infusion of the RSR-13. The former is the most probable explanation. Due to the inability to calculate these variables with confidence after RSR-13, we have utilized venous PO2 as our best approximation of end-capillary PO2 (6) (Fig. 5). Such a substitution of venous PO2 for calculated PcO2 has previously produced qualitatively interchangeable conclusions about DmO2 (19). This analysis suggests that there was an increase in VO2 max and no substantial change in DmO2 (Fig. 5).


View larger version (17K):
[in this window]
[in a new window]
  		Fig. 5.   Assuming venous PO2 represents end-capillary PO2 (6), unchanged relationship between VO2 max and venous PO2 after RSR-13 administration illustrates that diffusional O2 conductance (DmO2) has remained unchanged.

Variable responses to right-shifted ODC. Although we have no information to explain why some animals demonstrated no response to the increase in P50 while others did (Fig. 4), we hypothesize that the dichotomy may be due to a differing dependence on diffusion of O2 and offer two possible interpretations. First, if in the nonresponders, muscle O2 shunts and heterogeneity (between local VO2 and blood flow; rather than diffusion limitation) accounted for the residual O2 in muscle venous blood at VO2 max (15), an increase in PcO2 would have no effect on VO2 max or O2 extraction (at a constant O2 delivery). Second, the animals that did not respond to the increased P50 may have already reached their mitochondrial metabolic limits of O2 utilization before the administration of RSR-13. In this scenario, the increased intracellular availability of O2, due to the elevated blood to tissue PO2 gradient, would not increase VO2 max. An additional explanation for this varied response could be that the electrical stimulation was not sufficient in some animals to promote an increased VO2 max despite the elevated O2 availability. However, this is not probable, since this level of electrical stimulation has previously been demonstrated to elicit maximal work in many conditions (5). It should be reiterated that, despite this differential response to RSR-13, statistical analyses of the data indicated increased O2 extraction and VO2 max in the RSR-13-treated group as a whole, not just in those we identify as responders.

Magnitude of VO2 max increase. VO2 max rose by 20% with an increase in P50 from 33 to 53 Torr (Tables 1 and 2, Fig. 2), similar in magnitude to the reduction reported by Hogan et al. (4) with a decrease in P50 from 32 to 23 Torr (-17%). The increase in VO2 max in the present study is predicted by a simple model of O2 transport from the mouth to muscle mitochondria (28) (Fig. 6). To calculate the estimated effect on VO2 max of a change in P50, we utilized the present O2 transport data and assumed no mitochondrial metabolic limit (thus intracellular PO2 would be sufficiently close to zero to be ignored) and a constant muscle diffusing capacity. That the effect on VO2 max per Torr change in P50 was greater in the study by Hogan et al. may be explained by the hyperbolic nature of the increase in VO2 max with increased P50. Although Fig. 6 shows the measured increase in VO2 max after RSR-13 was predictable on the basis of diffusive transport, several other factors may be involved. One possibility is that the increase in O2 availability with the increase in P50 was relatively greater than the remaining mitochondrial metabolic reserve to consume O2. Another factor that may have played a role was that, due to blood loss, blood sampling, and saline administrations, [Hb] fell after RSR-13 to match O2 delivery and Q had to be increased in the second exercise bout (Tables 1 and 2). The increased Q and consequent decrease in capillary transit time (20) may have attenuated the increase in VO2 max with the elevated P50. However, this need to increase Q occurred in both RSR-13-treated and control animals, and the latter group did not demonstrate a significant decrease in VO2 max. Finally, RSR-13, while producing a rapid and reproducible increase in the P50, achieved some of this effect by altering the cooperative binding properties of the Hb with O2 (Fig. 1). This was evidenced by a change in n from 2.8 ± 0.03 in the preinfusion and control animals to 1.4 ± 0.06 after the RSR-13 infusion, as reported previously (9, 14). This change in cooperativity results in a flattening of the physiologically important steep section of the ODC, lessening the effect of the rightward shift. This too may have influenced the magnitude of change in VO2 max for the increase in P50.


View larger version (28K):
[in this window]
[in a new window]
  		Fig. 6.   Change in VO2 max with an increase in P50, compared with predicted response modeled with all factors that influence O2 transport, except P50, held constant (27). Assumes no mitochondrial limit to O2 uptake, 100% O2 inspired, and a constant DmO2. * Significantly different from preinfusion value (P <=  0.05).

Here, it is also pertinent to note that, in addition to the work of Hogan et al. (4), Schumacker et al. (22) assessed the effect of a reduced P50 (by sodium cyanate infusions) on exercise performance in dogs on a treadmill and found no effect on O2 extraction and exercise performance. In this study (22), because the animals were only minimally instrumented, it was not possible to control O2 delivery to the exercising muscles at maximal exercise. However, Schumacker et al. (22) did demonstrate that, for the same O2 delivery, less O2 was extracted during exercise when the ODC was shifted to the left, consistent with the findings of Hogan et al. (4) and the present study, thus suggesting an important role of P50 in determining O2 extraction.

Hemodynamic changes due to right-shifted ODC. Mean arterial blood pressure was significantly reduced after the RSR-13 infusion, whereas it remained constant in the control animals (Table 2). These findings are in agreement with the report of Kunert et al. (11) with RSR-13 infusion in the rat but are in contrast to the findings of Liard and Kunert (14), who reported that, in dogs, RSR-13 caused an increase in total peripheral resistance modulated by a modest increase in mean arterial pressure and a significant decrease in cardiac output. It is not clear why systemic pressure was reduced in the present study, and since, cardiac output was not measured, it is not possible to calculate any changes in peripheral resistance. However, by using the present data, we can evaluate the effect of the right shift in the ODC on the hemodynamics in the exercising gastrocnemius muscle itself (Table 2). Perfusion pressure was not different between contraction periods in RSR-13-treated or control groups, but vascular resistance was significantly decreased and conductance increased during the exercise after the RSR-13 infusion. It has previously been suggested that elevated levels of O2, as produced by a right-shifted ODC, would result in excess tissue PO2, which in turn would result in a vasoconstrictor response (13). This has been experimentally documented in several studies at rest utilizing Hb modifiers (11, 12, 14) and supports the role of O2 in the autoregulatory process. The present data illustrate the opposite response, and this may be because this study examined skeletal muscle during exercise, whereas previous studies have all been performed at rest. During exercise, the elevated mitochondrial metabolic demand may offset the effect of a high PO2 on vascular conductance, diminishing the local autoregulatory signal to vasoconstrict.

In summary, RSR-13 infusion significantly increased P50 and, at a constant arterial O2 delivery, resulted in an increase in O2 extraction and a consequent increase in muscle VO2 max. This indicates, for the first time, that the canine gastrocnemius muscle is normally O2 supply limited, even when the animal is breathing 100% O2. In addition, the increase in VO2 max was proportional to the increase in venous PO2. Taken together, these findings support the concept that diffusion of O2 between the erythrocyte and mitochondria plays a role in determining VO2 max. In contrast to previous hemodynamic studies, which were performed at rest, RSR-13, despite increasing intravascular PO2, produced a fall in vascular resistance within the exercising muscle.

    ACKNOWLEDGEMENTS

We are as usual indebted for the expert technical assistance of Harrieth Wagner, Nick Busan, and Jeffrey Struthers.

    FOOTNOTES

This study was supported by National Heart, Lung, and Blood Institute Grant HL-17731. R. S. Richardson was a Parker B. Francis Fellow in Pulmonary Research during this research.

The RSR-13 used in this research was kindly provided by Allos Therapeutics, Denver, CO.

Address for reprint requests: R. S. Richardson, Dept. of Medicine 0623, University of California, La Jolla, CA 92093-0623.

Received 17 April 1997; accepted in final form 31 October 1997.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Abraham, D. J., F. C. Wireko, and R. S. Randad. Allosteric modifiers of hemoglobin: 2-(4-{[(3,5-disubstituted anilino)carbonyl]methyl}phenoxy)-2-methylpropionic acid derivatives that lower the oxygen affinity of hemoglobin in red cell suspensions, whole blood, and in vivo in rats. Biochemistry 31: 9141-9149, 1992[Medline].

2.   Barclay, J. K., and W. N. Stainsby. The role of blood flow in limiting maximal metabolic rate in muscle. Med. Sci. Sports Exerc. 7: 116-119, 1975.

3.   Hogan, M. C., D. E. Bebout, and P. D. Wagner. Effect of hemoglobin concentration on maximal O2 uptake in canine gastrocnemius muscle in situ. J. Appl. Physiol. 70: 1105-1112, 1991[Abstract/Free Full Text].

4.   Hogan, M. C., D. E. Bebout, and P. D. Wagner. Effect of increased HbO2 affinity on VO2 max at a constant O2 delivery in dog muscle in situ. J. Appl. Physiol. 70: 2656-2662, 1991[Abstract/Free Full Text].

5.   Hogan, M. C., J. Roca, J. B. West, and P. D. Wagner. Dissociation of maximal O2 uptake from O2 delivery in canine gastrocnemius in situ. J. Appl. Physiol. 66: 1219-1226, 1989[Abstract/Free Full Text].

6.   Honig, C. R., T. E. Gayeski, A. Clark, and P. A. A. Clark. Arteriovenous oxygen shunt is negligible in resting and working gracilis muscles. Am. J. Physiol. 261 (Heart Circ. Physiol. 30): H2031-H2043, 1991[Abstract/Free Full Text].

7.   Jeneson, J. A. L., J. S. Taylor, D. B. Vigneron, T. S. Willard, L. Carvajal, S. J. Nelson, J. Murphy-Boesch, and T. R. Brown. 1H MR imaging of anatomical compartments within finger flexor muscles of the human forearm. Magn. Reson. Med. 15: 491-496, 1990[Medline].

8.   Jobsis, F. F., and W. N. Stainsby. Oxidation of NADH during contractions of circulated skeletal muscle. Respir. Physiol. 4: 292-300, 1968[Medline].

9.   Khandelwal, S. R., R. S. Randid, P. Lin, H. Meng, R. N. Pittman, H. A. Kontos, S. C. Choi, D. J. Abraham, and R. Schmidt-Ullrich. Enhanced oxygenation in vivo by allosteric inhibitors of hemoglobin saturation. Am. J. Physiol. 265 (Heart Circ. Physiol. 34): H1450-H1453, 1993[Abstract/Free Full Text].

10.   Knight, D. R., W. Schaffartzik, D. C. Poole, M. C. Hogan, D. E. Bebout, and P. D. Wagner. Hyperoxia increases leg maximal oxygen uptake. J. Appl. Physiol. 75: 2586-2594, 1993[Abstract/Free Full Text].

11.   Kunert, M. P., J. F. Liard, and D. J. Abraham. RSR-13, an allosteric effector of hemoglobin, increases systemic and illiac vascular resistance in rats. Am. J. Physiol. 271 (Heart Circ. Physiol. 40): H602-H613, 1996[Abstract/Free Full Text].

12.   Kunert, M. P., J. F. Liard, D. J. Abraham, and J. H. Lombard. Low-affininty hemoglobin increases tissue PO2 and decreases arteriolar diameter and flow in the rat cremaster muscle. Microvasc. Res. 52: 58-68, 1996[Medline].

13.   Ledinham, J. M. Autoregulation in hypertension: a review. J. Hypertens. 7: S97-S104, 1989.

14.   Liard, J. F., and M. P. Kunert. Hemodynamic changes induced by low blood oxygen affinity in dogs. Am. J. Physiol. 264 (Regulatory Integrative Comp. Physiol. 33): R396-R401, 1993[Abstract/Free Full Text].

15.   Piiper, J. Unequal distribution of blood flow in exercising muscle of dog. Respir. Physiol. 80: 129-136, 1990[Medline].

16.   Powers, S. K., J. Lawler, J. A. Dempsey, S. Dodd, and G. Landry. Effects of incomplete pulmonary gas exchange on VO2 max. J. Appl. Physiol. 66: 2491-2495, 1989[Abstract/Free Full Text].

17.   Randad, R. S., M. A. Mahran, A. S. Mehanna, and D. J. Abraham. Allosteric modifiers of hemoglobin: design, synthesis, testing and structure-allosteric activity relationship of novel hemoglobin oxygen affinity decreasing agents. J. Med. Chem. 34: 752-757, 1991[Medline].

18.   Richardson, R. S., D. R. Knight, D. C. Poole, S. S. Kurdak, M. C. Hogan, B. Grassi, and P. D. Wagner. Determinants of maximal exercise VO2 during single leg knee extensor exercise in man. Am. J. Physiol. 268 (Heart Circ. Physiol. 37): H1453-H1461, 1995[Abstract/Free Full Text].

19.   Richardson, R. S., E. A. Noyszeski, K. F. Kendrick, J. S. Leigh, and P. D. Wagner. Myoglobin O2 desaturation during exercise: evidence of limited O2 transport. J. Clin. Invest. 96: 1916-1926, 1995.

20.   Richardson, R. S., D. C. Poole, D. R. Knight, and P. D. Wagner. Red blood cell transit time in man: theoretical effects of capillary density. In: Advances in Experimental Medicine and Biology, edited by M. C. Hogan, O. Mathieu-Costello, D. C. Poole, and P. D. Wagner. New York: Plenum, 1995, vol. XVII, p. 517-528.

21.   Roca, J., M. C. Hogan, D. Story, D. E. Bebout, P. Haab, R. Gonzalez, O. Ueno, and P. D. Wagner. Evidence for tissue diffusion limitation of VO2 max in normal humans. J. Appl. Physiol. 67: 291-299, 1989[Abstract/Free Full Text].

22.   Schumacker, P. T., A. J. Sugget, P. D. Wagner, and J. B. West. Role of hemoglobin P50 in O2 transport during normoxic and hypoxic exercise in the dog. J. Appl. Physiol. 59: 749-757, 1985[Abstract/Free Full Text].

23.   Stainsby, W. N., and A. B. Otis. Blood flow, blood oxygen tension, oxygen uptake, and oxygen transport in skeletal muscle. Am. J. Physiol. 206: 858-866, 1964.

24.   Stein, J. C., and M. L. Ellsworth. Microvascular oxygen transport: impact of a left-shifted dissociation curve. Am. J. Physiol. 262 (Heart Circ. Physiol. 31): H517-H522, 1992[Abstract/Free Full Text].

25.   Stringer, W., K. Wasserman, R. Casaburi, J. Porszasz, K. Maehara, and W. French. Lactic acidosis as a facilitator of oxyhemoglobin dissociation during exercise. J. Appl. Physiol. 76: 1462-1467, 1994[Abstract/Free Full Text].

26.   Wagner, P. D. Gas exchange and peripheral diffusion limitation. Med. Sci. Sports Exerc. 24: 54-58, 1992[Medline].

27.   Wagner, P. D. Algebraic analysis of the determinants of VO2 max. Respir. Physiol. 93: 221-237, 1993[Medline].

28.   Wagner, P. D. A theoretical analysis of factors determing VO2 max at sea level and altitude. Respir. Physiol. 106: 329-343, 1996[Medline].

29.   Wagner, P. D., J. Roca, M. C. Hogan, D. C. Poole, D. E. Debout, and P. Haab. Experimental support of the theory of diffusion limitation of maximum oxygen uptake. In: Oxygen Transport to Tissue, edited by J. Piiper, T. K. Goldstick, and M. Meyer. New York: Plenum, 1990, vol. XII, p. 825-833.

30.   Weibel, E. R. The pathway for oxygen. In: Structure and Function in the Mammalian Respiratory System. London: Harvard Univ. Press, 1984.

31.   Welch, H. G. Hyperoxia and human performance: a brief review. Med. Sci. Sports Exerc. 14: 253-262, 1982[Medline].

32.   Woodson, R. D. Functional consequences of altered blood oxygen affinity. Acta Biol. Med. Ger. 40: 733-736, 1981[Medline].

JAP 84(3):995-1002
0161-7567/98 $5.00 Copyright © 1998 the American Physiological Society


This article has been cited by other articles:


Home page
 Eur Respir JHome page
P. D. Wagner
The biology of oxygen
Eur. Respir. J., April 1, 2008; 31(4): 887 - 890.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
L. J. Haseler, A. Lin, J. Hoff, and R. S. Richardson
Oxygen availability and PCr recovery rate in untrained human calf muscle: evidence of metabolic limitation in normoxia
Am J Physiol Regulatory Integrative Comp Physiol, November 1, 2007; 293(5): R2046 - R2051.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
N. Lai, G. M. Saidel, B. Grassi, L. B. Gladden, and M. E. Cabrera
Model of oxygen transport and metabolism predicts effect of hyperoxia on canine muscle oxygen uptake dynamics
J Appl Physiol, October 1, 2007; 103(4): 1366 - 1378.
[Abstract] [Full Text] [PDF]

Home page
 Canadian J. AnesthesiaHome page
G. M.T. Hare, A. Harrington, E. Liu, J. L. Wang, A. J. Baker, and C. D. Mazer
Effect of oxygen affinity and molecular weight of HBOCs on cerebral oxygenation and blood pressure in rats: [L'effet de l'affinite pour l'oxygene et du poids moleculaire des TOBH sur l'oxygenation et la tension arterielle chez les rats].
Can J Anesth, October 1, 2006; 53(10): 1030 - 1038.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
C. Lundby, M. Sander, G. van Hall, B. Saltin, and J. A. L. Calbet
Maximal exercise and muscle oxygen extraction in acclimatizing lowlanders and high altitude natives
J. Physiol., June 1, 2006; 573(2): 535 - 547.
[Abstract] [Full Text] [PDF]

Home page
 Exp. Biol. Med.Home page
E. T. Donnelly, Y. Liu, and S. Rockwell
Efaproxiral (RSR13) Plus Oxygen Breathing Increases the Therapeutic Ratio of Carboplatin in EMT6 Mouse Mammary Tumors.
Experimental Biology and Medicine, March 1, 2006; 231(3): 317 - 321.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
B. Saltin, J. A. L. Calbet, and P. D. Wagner
Point: In health and in a normoxic environment, VO2 max is limited primarily by cardiac output and locomotor muscle blood flow
J Appl Physiol, February 1, 2006; 100(2): 744 - 748.
[Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
J. A. L. Calbet, H.-C. Holmberg, H. Rosdahl, G. van Hall, M. Jensen-Urstad, and B. Saltin
Why do arms extract less oxygen than legs during exercise?
Am J Physiol Regulatory Integrative Comp Physiol, November 1, 2005; 289(5): R1448 - R1458.
[Abstract] [Full Text] [PDF]

Home page
 JCOHome page
H. Choy, A. Nabid, B. Stea, C. Scott, W. Roa, L. Kleinberg, J. Ayoub, C. Smith, L. Souhami, S. Hamburg, et al.
Phase II Multicenter Study of Induction Chemotherapy Followed by Concurrent Efaproxiral (RSR13) and Thoracic Radiotherapy for Patients With Locally Advanced Non-Small-Cell Lung Cancer
J. Clin. Oncol., September 1, 2005; 23(25): 5918 - 5928.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
L. J. Haseler, A. P. Lin, and R. S. Richardson
Skeletal muscle oxidative metabolism in sedentary humans: 31P-MRS assessment of O2 supply and demand limitations
J Appl Physiol, September 1, 2004; 97(3): 1077 - 1081.
[Abstract] [Full Text] [PDF]

Home page
 Adv. Physiol. Educ.Home page
R. S. Richardson
OXYGEN TRANSPORT AND UTILIZATION: AN INTEGRATION OF THE MUSCLE SYSTEMS
Advan Physiol Educ, December 1, 2003; 27(4): 183 - 191.
[Abstract] [Full Text] [PDF]

Home page
 JCOHome page
E. Shaw, C. Scott, J. Suh, S. Kadish, B. Stea, J. Hackman, A. Pearlman, K. Murray, L. Gaspar, M. Mehta, et al.
RSR13 Plus Cranial Radiation Therapy in Patients With Brain Metastases: Comparison With the Radiation Therapy Oncology Group Recursive Partitioning Analysis Brain Metastases Database
J. Clin. Oncol., June 15, 2003; 21(12): 2364 - 2371.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
J. A. L. Calbet, R. Boushel, G. Radegran, H. Sondergaard, P. D. Wagner, and B. Saltin
Why is VO2 max after altitude acclimatization still reduced despite normalization of arterial O2 content?
Am J Physiol Regulatory Integrative Comp Physiol, February 1, 2003; 284(2): R304 - R316.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
R. S. Richardson, E. A. Noyszewski, B. Saltin, and J. Gonzalez-Alonso
Effect of mild carboxy-hemoglobin on exercising skeletal muscle: intravascular and intracellular evidence
Am J Physiol Regulatory Integrative Comp Physiol, November 1, 2002; 283(5): R1131 - R1139.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
K. K. Henderson, H. Wagner, F. Favret, S. L. Britton, L. G. Koch, P. D. Wagner, and N. C. Gonzalez
Determinants of maximal O2 uptake in rats selectively bred for endurance running capacity
J Appl Physiol, October 1, 2002; 93(4): 1265 - 1274.