Increased plasma 0, solubility improves 0, uptake of in situ dog muscle working maximally MICHAEL C. HOGAN, DAVID AND PETER D. WAGNER




Divisiun of Physiology, Department of Medicine, University of California, and Alliance Pharmaceutical Corporation, San Diego, California 92122 HOGAN, MICHAEL C., DAVID C. WILLFORD, PETER E. KEIPERT, N. SIMON FAITHFULL, AND PETER D. WAGNER. Increased plasma muscle ulorking

0, solubility improues U2 uptake of in situ dog maximally. J. Appl. Physiol. 73(6): 2470-2475,

1992.-A perfluorocarbon emulsion [formulation containing 90%wt/vol perflubron (perfluorooctylbromide); Alliance Pharmaceutical] was used to increase 0, solubility in the plasma compartment during hyperoxic low hemoglobin concentration ([Hb]) perfusion of a maximally working dog muscle in situ. Our hypothesis was that the increased plasma 0, solubility would increasethe muscle0, diffusing capacity (Do,) by augmenting the capillary surface area in contact with high [O,]. Oxygen uptake (VO,) was measuredin isolated in situ canine gastrocnemius(n = 4) while working for 6 min at a maximal stimulation rate of 1 Hz (isometric tetanic contractions) on three to four separateoccasionsfor eachmuscle.On eachoccasion, the last 4 min of the 6-min work period was split into 2 min of a control treatment (only emulsifying agent mixed into blood) and 2 min of perflubron treatment (6 g/kg body wt), reversing the order for each subsequentwork bout. Before contractions, the [Hb] of the dog was decreasedto 8-9 g/100 ml and arterial PO, was increasedto 500-600 Torr by having the dog breathe 100%0, to maximize the effect of the perflubron. Muscle blood flow was held constant between the two experimental conditions. Plasma0, solubility was aImost doubledto 0.005 ml 0, a100ml blood-l t TOrr-l by the addition of the perflubron. Muscle O2delivery and maximal i’o, were significantly improved (at the sameblood flow and [Hb]) by 11 and 12.6%, respectively (P -C0.05), during the perflubron treatment compared with the control. 0, extraction by the muscleremained the samebetween the two treatments, as did the estimate of Do,. These resultsindicate that improving plasma0, solubility to the degreeobservedincreasedmusclevu2 only in proportion to the amount that convective 0, transport was enhancedand that under the conditions of this study, the increased0, solubility in the plasmacompartment did not improve the capacity for diffusional conductanceof 0, into the muscle. skeletal muscle; perflubron; perfluorooctylbromide; uxygen consumption; gasexchange; exercise; blood perfusate; lactate, diffusion limitation

RECENT STUDIEShave suggested that the interaction between the perfusive conductance of 0, to the working tissue and the capacity for diffusive movement of 0,

from hemoglobin (Hb) to mitochondria sets the maximal 0, uptake (VO 2 mag)for the particular conditions (15, 16, 19). For a given 0, delivery (flow X arterial 0, content), the amount of 0, that can be extracted and used by the 2470



San Diego, La Jolla 92093-0623;

working tissue, according

to Fick’s law of diffusion, is determined by the diffusing capacity of the muscle (Do2) and the PO, gradient from the red blood cell to the mitochondria l


2 = Do,( Pco,-Pmito,,)


where VO, is the 0, uptake, PZo, is an average capillary PO,, and Pmitoo, is an average mitochondrial PO,. The DO, is a lumped conductance parameter that takes into account all of the variables that determine the resistance to 0, diffusion from the red blood cell to the mitochondria (e.g., 0, off-loading kinetics from Hb, membrane resistances, diffusional distances, erythrocyte transit time, 0, solubilities, capillary surface area). The estimated Do2 of the tissue may change in value as these factors are altered. Recently, experimental studies (5,8, 9) and theoretical modeling (4, 7, 10, 13, 18) have suggested that the major portion of the PO, gradient between Hb and cytochromes in skeletal muscle occurs outside the myocyte so that the major resistance to 0, diffusion lies between Hb and the myocyte cytoplasm. We have demonstrated previously that when [Hb] is decreased in blood perfusing maximally working muscle, while muscle blood flow is kept constant, the estimated Do, decreases considerably (14). When the lower [Hb] conditions were compared with similar 0, deliveries induced by either hypoxemia (15) or ischemia (unpublished observations), the amount of 0, that was extracted by the maximally working muscle was significantly less during the lower [Hb] conditions compared with either hypoxemia or ischemia, as was the estimated Do,. The decreased capacity to extract 0, suggests that reductions in [Hb] increase the resistance to the diffusion of 0, into the tissue. This could have been a result of changes in the convective distribution of blood flow in the microcirculation (even though total muscle blood flow at the different concentrations of Hb was kept the same during these experiments; Ref. 14), the slowness of the kinetic offloading of 0, from Hb becoming more of a resistance factor during reduced [Hb] (ll), or possibly an increase in the red blood cell spacing causing less of the capillary surface area to be available at any instant for 0, diffu-

sion. The latter hypothesis comes from Federspiel and Pope1 (7), who have suggested, using theoretical modeling, that the flux of 0, from a particle (erythrocyte) through the capillary wall is limited to the near vicinity of

0161-7567&E $2.00 Copyright 0 1992the AmericanPhysiological Society

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the particle and that 0, flux becomes negligible in the plasma spaces between erythrocytes. They suggested that not only will the convective delivery of 0, be diminished during low erythrocyte conditions but also the mass transfer coefficient (DO,) for 0, would be reduced with increased erythrocyte spacing in the capillary. This is because the 0, diffusion coefficient for plasma is low compared with the facilitated transport within the erythrocyte by Hb and within the muscle cell by myoglobin. It was the purpose of this study to increase the 0, solubility of plasma, using a perfluorocarbon emulsion [AF0104, containing 90% wtfvol perflubron (perfluorooctylbromide); Alliance Pharmaceutical] in an attempt to improve the 0, diffusion coefficient and 0, concentration of the intererythrocyte spaces and thereby increase the capillary area available for 0, diffusion. Our hypothesis was that if the intererythrocyte spacing is in fact an important determinant of the Do,, then increasing the plasma 0, solubility should result in an increased VO, MBX that is greater than that expected just from the convective increase in 0, delivery (i.e., further augmented by an increased 0, extraction caused by a higher Do,). METHODS

All experimental protocols were approved by the animal use committee in the Medical School and adhered to American Association of Laboratory Animal Care guidelines. Four adult mongrel dogs of either sex with a weight range of 15-20 kg were anesthetized with pentobarbital sodium (30 mg/kg). The dogs were intubated with cuffed endotracheal tubes. Maintenance doses of pentobarbital sodium were given as required. Heating pads were used to maintain esophageal temperature near 37OC. The animals were given heparin at a dosage of 1,500 U/kg atier the surgery. Ventilation was maintained with a Harvard 613 ventilator. Surgical preparation. The left gastrocnemius-flexor digitorum superficialis muscle complex (for convenience referred to as the gastrocnemius) was isolated in each dog as described previously (16). Briefly, a medial incision was made through the skin of the left hindlimb from midthigh to the ankle. The sartorius, gracilis, semitendinosus, and semimembranosus muscles, which overlie the gastrocnemius, were doubly ligated and cut between the ties. All vessels draining into the popliteal vein except for those from the gastrocnemius were ligated to isolate the venous outflow from the gastrocnemius. The arterial circulation to the gastrocnemius was isolated by ligating all vessels from the femoral and popliteal artery that did not enter the gastrocnemius. The left popliteal vein was cannulated, and the venous outflow was returned to the animal via a jugular catheter. The right femoral artery was catheterized for arterial blood sampling. This catheter was connected to the left femoral artery so that the isolated muscle was perfused by blood from this contralateral artery. Perfusion was accomplished either directly from the contralateral (systemic pressure ---self perfused) or via a Sigmamotor pump to control flow. All experimental periods were conducted using pump perfusion so that muscle blood flow could be controlled. A pressure transducer in this line at




the head of the muscle constantly monitored perfusion pressure. A carotid artery was also catheterized to monitor systemic blood pressure. The left sciatic nerve, which innervates the gastrocnemius, was doubly ligated and cut between ties. To prevent cooling and drying, all exposed tissues were covered with saline-soaked gauze and with a sheet of plastic wrap. After the muscle was surgically isolated, the Achilles tendon was attached to an isometric myograph (Statham 1360 transducer) to measure tension development. The hindlimb was fixed at the knee and ankle and attached to the myograph with struts to minimize movement. Weights were used at the end of each experiment to calibrate the tension myograph. An outflow line from a second perfusion pump was inserted, through a three-way stopcock, into the line that connected the first perfusion pump to the head of the muscle. At the juncture of the lines from the two perfusion pumps was a mixing device (several sections of screw threads) that ensured complete mixing of the solutions from both pumps. As stated, the first perfusion pump carried arterial blood to the muscle from the contralateral femoral artery, whereas the second perfusion pump was used to mix either the perflubron solution (PFB) or the control (C) solution (the emulsion vehicle solution containing all excipients except the PFB) directly into the blood just before the blood entered the muscle. In this way, only the blood perfusing the muscle, and not the whole animal, carried the experimental perfusate. Isometric muscle contractions (tetanic) were elicited by stimulation of the sciatic nerve with square-wave impulses (4-6 V) of 0.2-ms duration for 200 ms at 50 Hz. The muscle was stimulated at 1 contraction/s for 6 min, which results in the VO 2maxthat can be achieved by this preparation (2). Before each contraction period, the resting muscle was passively stretched until a tension setting of -10 g force/g muscle mass (estimated before the experiment) was recorded. This ensured that the initial tension development was not affected by slippage in the system that might have occurred during the prior contraction period. This resting muscle length was slightly less than the length at which the contractile response was greatest. Experimental protocol. On each experimental day after the muscle had been isolated and the catheters inserted, blood was slowly removed from the dog and replaced with saline (blood pressure was maintained) until the [Hb] level was down to 8-9 g/100 ml. At this time, the dog was also placed on 100% 0, to fully saturate arterial Hb. The blood removed from the animal was also diluted to the same approximate hematocrit, was kept well stirred and at 37”C, and was given back to the animal when venous blood from the muscle was not returned to the animal. Before the first contraction period, the blood supply to the isolated muscle was switched from self-perfusion to pump-perfused, and enough time was allowed for conditions to stabilize at a blood flow similar to the self-perfused one. Each experiment (n = 4) consisted of three to four separate 6-min contraction periods for the isolated muscle. Each 6-min contraction period (described above) was

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separated by 20-25 min of rest. Each 6-min contraction period was the same in that the first 2 min were a warmup period, followed by a 2min experimental condition, and then another 2-min experimental condition. The two 2-min experimental conditions consisted of the PFB or the C condition in both orders (sequence assigned randomly). Blood flow to the muscle (perfusion pressure at -100 mmHg) was set in the first 2-min warm-up period, and flow was kept constant for the subsequent two experimental conditions (the next 4 min of the 6-min contraction period). The second perfusion pump could be adjusted to achieve the proper mixing of the PFB or vehicle emulsion with the blood flowing into the muscle (6 g PFBikg body wt). The flow of PFB that was mixed into the inflowing blood was matched by the same flow of vehicle emulsion alone (C). Both the PFB and vehicle emulsion were bubbled with 100% 0, before being mixed with the inflowing blood. During the PFB condition, the venous effluent was collected and not returned to the animal. This amount of blood was replaced by the blood removed from the animal previously. Measurements. Arterial blood samples from the arterial line just before entering the muscle (after mixing of the PFB or vehicle emulsion with the arterial blood) and venous samples from the left popliteal vein as close to the gastrocnemius as possible were drawn anaerobically during the last 20 s of each of the 2-min experimental time periods and were kept on ice. Venous blood flow measurements were then made by timed blood collections into a graduated cylinder. A previously published study by Barbee et al. (I), using a maximal stimulation pattern, had determined that a near steady-state flow and 90, were achieved by the end of 2 min. Blood lactate concentrations were determined from the arterial and venous samples using a Yellow Springs Instruments 23L blood lactate analyzer. Blood PO,, Pco~, and pH were measured within 5-8 min with a blood gas analyzer (Instrumentation Laboratory model 813) at 37OC while [Hb], percent 0, saturation, and 0, content were measured with an Instrumentation Laboratory 282 CO-oximeter. Because the turbidity of the perflubron emulsion in blood interfered with the spectrophotometric readings of the Instrumentation Laboratory 282 COoximeter, all 0, contents were also measured with a LexO,-Con (Lexington Instruments), and these 0, contents were used fur subsequent calculations. These instruments were calibrated before each experiment and often throughout each experiment. Plasma bicarbonate concentration was calculated from the measured pH and PCO, values using the Henderson-Hasselbalch equation. Blood hematocrits were measured for each sample. Plasma 0, solubility was determined by assuming that at the high arterial PO, (Pa,,; -500 Torr) all of the Hb within the erythrocytes was fully saturated with 0, (1.39 ml 0,/g Hb) and that the remainder of the 0, carried in the blood was dissolved in the plasma. A Bohr integration technique was used to calculate muscle 0, diffusing capacity using Fick’s law of diffusion as a simple model of capillary gas exchange, as outlined previously (15, 19). Briefly, it is assumed that the Pmito,, is zero and constant along the length of the capillary, which has been demonstrated to be approximately



true by Honig and co-workers (5, 8, 9), so that the 0, uptake at any point along the capillary is a product of the muscle DO, and the PcoZ (the driving force) at that point. In this way, a value of DO, can be calculated to account for the measured fall in PO,, and 0, contents, from the arterial to venous ends of the muscle. From the associated PO, profile, PCoZ can be calculated as the average of all PO, values from arterial to venous end of the capillary. We take this as an estimate of the mean diffusional driving pressure for 0, for those conditions. This analysis, which assumes that the incomplete extraction of 0, by the maximally working muscle due to perfusional or diffusional shunts and blood flowNo, heterogeneity is negligible, is useful only as a simple means of comparing similar conditions. The Fick principle was used to calculate muscle VO, and lactate release. The muscle was removed and weighed at the end of each experiment. Statistics. Two-way analysis of variance was used for the statistical analysis, and the 0.05 level of significance was used. To minimize the total number of animals used, the C and PFB treatments that were compared were repeated three to four times for each isolated muscle (n = 4) for a total of 15 matched comparisons. It should be noted, however, that if the measurements obtained are averaged for each muscle (so that three to four comparisuns are averaged within each muscle), all values that were found to be significantly different with the 15 matched comparisons remain significantly different with n = 4 (repeated measures analysis of variance). RESULTS

Mean weight of the exercised gastrocnemius muscles (n = 4) removed after the end of the experiments was 76 t 6 (SE) g. The addition of 6 g/kg body wt of PFB (-6 g/70 ml blood) to the arterial blood perfusing the muscle increased the 0, solubility of the plasma from 0.003 to 0.005 ml 100 ml-l Torr? Although measurements were not obtained at the end of the initial 2-min warm-up period preceding the C or PFB infusion conditions, it should be noted that when the infusion of either PFB or vehicle began at the end of the 2-min warm-up period, no changes were noted in developed tension, perfusion pressure, or muscle resistance. This would indicate that the infusion of either PFB or vehicle (C) at the concentrations given had no effect on the muscle hemodynamics. The principal variables describing 0, transport and acid-base balance in the blood perfusing the muscles during the C and the PFB conditions are presented in Table 1. The high Pao, reflects the inspired 0, concentration of 100% that was used to ensure that arterial 0, saturation of Hb was maximal. Although the Pa,, was statistically significantly higher during the PFB condition (likely due to the perflubron being bubbled with 100% 0, before mixing with the arterial blood), this difference is physiologically insignificant because the slope of the O2 dissociation curve is so small at such high PaOz values. Because the PFB and the vehicle emulsion were mixed into the inflowing arterial blood at the same dilution (-1 part l


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1. Blood parameters corresponding to control vehicle und perflubrun emulsion conditions


Hct, % Pa,, , Torr Pacoz, Torr PH, [L a 1a? mM

[HCOJ,, mM BPm, Torr

26k-1 557tl6* 37&l 7.41t0.02 5.5t0.8 2422

26&l 496_+21 37tl 7.4Ot,O.O2 5.8t0.9 2422 101+2

Values are means + SE for I5 matched treatments. C, control vehicle; PFB, perflubron emulsion; Hct, blood hematocrit; Pa?,, arterial PO,; pace, 9 arterial PCO~; pH, , arterial pH; [La],, arterial lactate BPm, muscle arterial blood concn; CHWI,~ arterial [HCO& pressure. * Significantly different from control (P < 0.05).

2. Summary of principal variables related tu 0, transport and gas exchunge for control vehicle and perflubron emulsion conditions








ml min-l 100 g-’ Pvoz, Torr C& extraction, % PEo,, Torr Do,, ml. 100 g-’ 9mine1 0Torr-’ La, pm01 mine1 100 g+ Fatigue index, %



33+1 66t3

35*1 67-t2 62t2" 0.17-1-0.01 43+10 86k2

QhX3X~ l




58tl O.l7+0.02 2528 86t2




e =PFB












mmin -I)

FIG. 1. Relationship between 0, delivery and oxygen uptake (TO,) for perflubron emulsion (PFB) and vehicle control (C) conditions during hyperoxemic (n = 15) treatment. Values are means t SE. Line drawn .--_. is line of constant 0, extraction passing through 2 points and - __ongm.



Cao,, ml/100 ml Muscle blood flow, ml min-l -100 g-’ U2 delivery, ml. min-’ -100 g-l l



A cv 4--9 2 --






IE lo--I cT 8-g




Values are means t SE for 15 matched treatments. Caoz, arterial O2 content; O2 delivery, Cao, X muscle blood flow; vogmax, mqximal O2 uptake; Pvoz , muscle effluent venous POP; 0, extraction, VO,,,/O, delivery; PEoz, calculated mean capillary PO,; Du,, calculated muscle O2 diffusing capacity; La, muscle lactate output (flow X venoarterial difference); fatigue index, final tension development/initial tension development. * Significantly different from control (P < 0.05).



40 PO2




FIG. 2. Relationship between VO, and both effluent venous PO, (Pq,,) and calculated mean capillary PO, (Peon) for PFB and C condition; during hyperoxemic (n = 15) treatments.-Values are means t SE. Line of positive slope is line of best fit between VCI, and PE,, that passes through origin, representing constant muscle-diffusing capacity.

sure to the muscle between the two conditions, there was no difference in muscular vascular resistance. The principal observation (independent of computaPFB or vehicle solution to 10 parts arterial blood), the hematocrit of the blood entering the muscle was not dif- tions such as PC, and Do,) was that vogmax was increased significantly by 11% during the PFF condition. ferent between the two treatments. The other arterial between O2 delivery and VO, is preblood acid-base parameters, along with the muscfe perfu- The relationship sion pressure, were also not different. Arterial blood sented in Fig. 1. The increase in maximal VU, during the same as the increase in 0, lactates were elevated in both conditions because of the PFB was proportionally delivery, so that the 0, extraction ratio [Ca,, - venous low [Hb]. remained the same between the two The data relevant to 0, transport, utilization, and gas 0, content/CaO,l exchange are presented in Table 2. Because of the higher conditions. In 13 of the 15 matched treatments, even with 0, solubility in the plasma compartment during the PFB the reverse ordering, the VO, was greater during the PFB treatment, the arterial 0, content (Ca,,) of the blood condition. The PO, of the muscle effluent venous blood different between PFB and entering the muscle was significantly greater during this (Pvo,) was not significantly condition. Although the amount of 0, carried by the C. The estimates of muscle Do, and PC,, indicate that erythrocytes was the same (because of 100% Hb saturathe PEoS during PFB was significantly greater without tion and equal hematocrits), more 0, could be carried by there being any change in the calculated Doz. Figure 2 the plasma during perfusion with PFB. Because muscle illustrates the relationship between VO, m8xand both calblood flow was deliberately kept constant between the culated PC,, and measured PvoZ for the means of the 15 two conditions as planned and because Ca,, was sig- matched conditions. The line drawn for PC,, is the line of nificantly greater during the PFB treatment, this re- best fit (by least squares) for the points th& also passes through the origin; such a line indicates the best estimate sulted in 0, delivery being significantly higher during PFB perfusion. Also, with muscle blood flows being iden- of Do2 (as estimated by the slope of the relationship OO,/ tical and no significant difference in arterial blood pres- PC,,) for the two conditions. Downloaded from by ${individualUser.givenNames} ${individualUser.surname} ( on December 13, 2018.






The important result of this study was that at similar Pa hematocrit, and muscle blood flow, the higher O$&ubility of the plasma compartment during the PFB condition resulted in a significantly greater Cao,, 0, L delivery, and Vo, m8X.Under the conditions of this study, the increase in VO, maxduring the PFB condition appears to be strictly related to the increase in convective 0, delivery, with no apparent increase in . the muscle Do,. Although it is possible that the increase in plasma O,-solubility achieved in this study was not of the magnitude necessary to measure small changes in Do,, the consistency of the 0, extraction ratios between C and PFB conditions indicate no change in the ability of the muscle to extract 0, even with the higher convective 0, delivery. Muscle Do,. It is well known that maximally working muscle is not capable of extracting all of the 0, delivered. This incomplete extraction can be due to several causes: perfusional shunts, diffusional shunts, bo,/perfusion heterogeneity, diffusion limitation, or non-OJimited . VO 2max* In recent publications (15, 16, 19), we have presented evidence suggesting that the rate at which 0, can diffuse from Hb to mitochondria in the peripheral tissue has an important role in determining

Increased plasma O2 solubility improves O2 uptake of in situ dog muscle working maximally.

A perfluorocarbon emulsion [formulation containing 90% wt/vol perflubron (perfluorooctylbromide); Alliance Pharmaceutical] was used to increase O2 sol...
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