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Am J Physiol Heart Circ Physiol 277: H290-H298, 1999;
0363-6135/99 $5.00
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Vol. 277, Issue 1, H290-H298, July 1999

Effects of FIO2 on hemodynamic responses and O2 transport during RSR13-induced reduction in P50

Otto Eichelbrönner, Andreas Sielenkämper, Mark D'Almeida, Christopher G. Ellis, William J. Sibbald, and Ian H. Chin-Yee

A. C. Burton Vascular Biology Laboratory, University of Western Ontario, London, Ontario, Canada N6A 4G5


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Reduced Hb-O2 affinity facilitates O2 release to tissue but may impair pulmonary O2 uptake, affecting cardiac output and systemic vascular resistance (SVR). We studied the effects of shifting the O2-dissociation curve (ODC) to the right with a continuous infusion of RSR13, an allosteric modifier of Hb, and of different inspired O2 fractions (FIO2) on arterial O2 saturations (SaO2) in Hb and on hemodynamics in nonanesthetized rats. At an FIO2 of 0.21, SaO2 fell during RSR13 from 95 to 81%. Elevation of FIO2 to 0.30 returned SaO2 to baseline in the RSR13 group. The decrease in mean arterial pressure (MAP) was significantly greater in the control than in the RSR13 group at 30% O2. Cardiac index (CI) increased only during RSR13 at 21% O2 and returned to baseline at 30% O2. In contrast, SVR decreased after RSR13 was infused at 21% O2 but returned to baseline at 30%O2, whereas controls showed the opposite, a sustained SVR. In the follow-up period, when 21 O2% was reestablished and mild anemia was present, MAP and SVR fell significantly more in controls, whereas CI only increased in controls. Lactate was significantly lower in the RSR13 than in the control group during RSR13 and the follow-up period. These results demonstrate that 1) continuous infusion of RSR13 produces a constant shift in the O2 tension at which Hb is 50% saturated (P50), 2) FIO2 of 0.30 compensates for the effects of increased P50 on pulmonary O2 loading, and 3) right-shifted ODC combined with supplemental O2 may improve tissue O2 availability.

oxygen affinity; oxygen transport; oxygen tension at half-saturation of hemoglobin; tissue oxygenation availability


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE MAIN PRINCIPLES of O2 transport and tissue oxygenation are convection and diffusion. Either of these may set significant limits to O2 availability to the tissues (32). The former is determined by cardiac output (CO), arterial O2 content (CaO2), and the distribution of blood flow between and within organs. Diffusive O2 transport plays a key role in both O2 uptake in the lungs and O2 transport from erythrocytes to tissue. Its determinants in the lungs are the inspired O2 fraction (FIO2), tissue O2 conductance, and Hb-O2 affinity. In the periphery, however, O2 diffusion is governed by the O2 tension (PO2) gradient, the O2 conductance of the tissue, and the Hb-O2 affinity. Because the PO2 gradient, the driving force for O2 diffusion, is generated by convective O2 delivery (DO2) and Hb-O2 affinity, the position of the Hb O2-dissociation curve (ODC) therefore represents a crucial factor for tissue oxygenation.

When Hb-O2 affinity is increased, a lower tissue PO2 is necessary to release O2 from Hb. As a consequence, the O2 gradient between red blood cells and the mitochondria is reduced and less of the O2 transported is available for the tissues (12), whereas O2 loading in the lungs is increased. In contrast, a right-shifted ODC favors release of O2 from Hb at a higher tissue PO2, thus increasing the O2 gradient between red blood cells and mitochondria (2). The reduced Hb-O2 affinity, however, impairs pulmonary O2 uptake, and supplemental O2 may therefore be required to maintain arterial O2 saturation (SaO2), convective DO2, and, thereby, the PO2 gradient from the blood to the tissue.

The synthetic allosteric effector of the Hb molecule, RSR13 [2-(4-{[(3,5-dimethylanilino)carbonyl]methyl}phenoxy)-2-methyl propionic acid] reduces the Hb-O2 affinity. As RSR13 binds at a different site (near the top of the alpha -subunits) than 2,3-diphospho-D-glycerate (DPG), the naturally occurring allosteric modifier of the Hb (in the cleft between the two beta -subunits), it generates an additive rightward shift of the ODC in the presence of DPG that should facilitate added O2 release to the tissue (1). Wei et al. (31) reported that, when superfused on brain tissue, RSR13 decreased venous Hb saturations (SvO2) and reduced hypoxia-induced vasodilation, thereby suggesting improved tissue oxygenation. Khandelwal et al. (13) measured a 66% increase in tissue PO2 after a bolus injection of RSR13. Recently, Kunert et al. (15) reported that a bolus injection of RSR13 shifted the O2 tension at which Hb was 50% saturated (P50) to 58 mmHg, followed by a decrease in CO and an increase in systemic vascular resistance (SVR). Because such a large shift in P50, induced at room air, substantially reduces convective DO2, we assumed that these hemodynamic changes were more likely due to inadequate rather than improved tissue oxygenation.

We speculated that infusion of RSR13 at ambient air would reduce SaO2, thereby reducing CaO2 and convective DO2. This reduction on convective DO2 would then result in a lower PO2 gradient in the periphery that could impair tissue O2 availability. As a consequence, SVR would fall and CO would increase to maintain tissue O2 availability. We also hypothesized that increasing FIO2 to restore SaO2 and CaO2 would ameliorate such adaptive circulatory responses. In the present experiment, we continuously infused RSR13 and varied the FIO2 to measure the effects of this approach on the circulatory determinants of O2 transport. We specifically asked the following questions. 1) Does a continuous infusion of RSR13 produce a consistent shift in P50? 2) Does a moderate increase in FIO2 compensate for the effects of an increased P50 on pulmonary O2 uptake and convective O2 transport? 3) What effects does a continuous infusion of RSR13 have on CO, SVR, and mean arterial pressure (MAP) at different FIO2? The findings of this study could contribute to a better understanding of the complex effects of a right-shifted ODC and help to optimize the experimental and clinical applications of RSR13.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The study protocol was reviewed and approved by the Council of Animal Care of the University of Western Ontario. The animals were acclimatized for 1 wk in our laboratory and housed in cages with food and water ad libitum.

Animal Model

Fourteen male Sprague-Dawley rats (Charles River, Quebec, Canada) weighing 375-440 g were used in this study. Under pentobarbital sodium anesthesia (65 mg/kg ip), all rats were instrumented with an arterial line advanced into the aorta via the left femoral artery and with venous catheters inserted into the left femoral vein and the right jugular vein. A thermodilution CO probe (IT-21 thermocouple, Physiotemp Instruments, Clifton, NJ) was positioned in the aortic arch via the right carotid artery. Both the cannulas and the thermocouple were tunneled subcutaneously, exteriorized at the interscapular region, and guided into a swivel device. After surgery, rats were allowed to recover for 24 h. The lines were continuously flushed to maintain patency. Analgesia was provided by a constant infusion of fentanyl (3 µg/h) that was terminated the next day, 2 h before the experiment.

Experimental Protocol

After the 24-h recovery period following catheter insertion, animals were placed in a chamber in which different FIO2 could be generated and maintained constant during the study period. The FIO2 was produced by mixing room air with pure O2 and was monitored continuously with a Miniox-1 O2 analyzer (Catalyst Research) by placing its sensor within the chamber. Atmospheric pressure in the chamber was maintained by outlets to compensate for the continuous inflow of the air-O2 mixture. To provide steady-state conditions, we allowed animals to adapt to the chamber environment for ~30 min before baseline measurements and after each change in FIO2. After acclimatization and equilibration, baseline measurements of hemodynamics [MAP, CO, and central venous pressure (CVP)], temperature, lactate, Hb concentration, and saturation as well as blood gas analyses were performed twice every 20 min between each data set. RSR13 was then administered as a bolus short-term infusion (95 mg/kg for 30 min) followed by a continuous infusion of RSR13 (54 mg · kg-1 · h-1) to maintain a plateau of the effect on P50 estimated by a consistent reduction of Hb SaO2. Thirty minutes after the maintenance infusion of RSR13 was started, all measurements were repeated. The FIO2 was increased from 0.21 to 0.25 and then to 0.30 with data collection at each FIO2. Subsequently, RSR13 administration was turned off while the increased FIO2 of 0.30 was continued for another 100 min (Fig. 1). To detect a possible carryover effect of RSR13, we reduced the FIO2 of 0.30 to room air halfway through the follow-up period (50 min after RSR13 was stopped) accompanied by acquisition of the standard data set (M9) (Fig. 1). At the end of the experiment (120 min after RSR13 was terminated) the final data set (M10) was collected at room air conditions (Fig. 1). In the control group (Con) a corresponding volume of saline instead of the RSR13 solution was infused over the same time period. After the experiments were completed, animals were euthanized with an overdose of pentobarbital, and a postmortem examination was conducted to verify the positions of all the catheters and to inspect the internal organs.


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  		Fig. 1.   Experimental design. RSR13, synthetic allosteric modifier of Hb; FIO2, inspired O2 fraction; M1-M10, measurements 1-10.

Measurements and Calculations

Hemodynamics. For assessment of MAP and CVP, arterial and jugular lines were connected to pressure transducers (Uniflow, Baxter, Toronto, Canada) joined to a multichannel amplifier recording system (Hewlett-Packard, Mississauga, Ontario, Canada). CO was measured using the thermodilution technique by injecting 0.3 ml of saline at room temperature via the jugular vein. The thermocouple signal was converted by a Cardiotherm 500 AC-R CO computer (Columbus Instruments, Columbus, OH).

O2 transport. Hb concentration ([Hb]) and Hb SaO2 were assessed using a cooximeter (OSM-II Hemoximeter, Radiometer, Copenhagen, Denmark). Blood gas analyses were measured from arterial and venous blood samples using a blood gas analyzer (ABL 520, Radiometer) linked with a hemoximeter (OSM3, Radiometer). Blood samples were placed on ice immediately after they were withdrawn. DO2, O2 consumption (VO2), O2 extraction (O2 Ex), and CaO2 as well as venous O2 content (CvO2) were calculated using the following equations: 1) DO2 = CaO2 × CO, 2) VO2 = (CaO2 - CvO2) × CO, 3) O2 Ex = (CaO2 - CvO2)/CaO2, 4) CaO2 = [(1.34 × [Hb] × SaO2)/100] - (PaO2 × 0.0031), and 5) CvO2 = [(1.34 × [Hb] × SvO2)/100] - (PvO2 × 0.0031), where CaO2 - CvO2 is arteriovenous O2 content difference Hb, PaO2 is arterial PO2, and PvO2 is venous PO2.

P50 measurements. The P50 values were obtained from venous blood samples using the ABL 520. In a previous study we evaluated the validity and reliability of the these readings by comparing the results from the ABL 520 to values obtained by multipoint tonometry technique (IL 237 Tonometer, Instrumentation Laboratories, Lexington, MA) as a reference. These data demonstrate that the ABL 520 reliably reproduces the P50, when analyzed from venous blood, but consistently underestimates it by 4.6 mmHg. Therefore, we adjusted our measurements using this correction factor (3).

Lactate. Lactate concentrations were determined by means of a quantitative, enzymatic method (Paramax Analytical System, Baxter, Mississauga, Ontario, Canada).

Statistics

For statistical analysis of the data, SigmaStat 1.0 software (Jandel, San Raphael, CA) was used. A two-way ANOVA for repeated measurements was applied to the data, completed by a post hoc analysis (Student-Newman-Keuls method) or t-tests followed by the Bonferroni procedure where applicable. For all comparisons, differences were considered significant at a P value of <0.05. Data are presented as means ± SE if not indicated differently.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Twenty-four hours after the catheters were inserted, none of the animals showed signs of infection such as reduced activity, piloerection, or exudations around the eyes and nose. Postmortem examination exhibited normal thoracic and intestinal organs without signs of infection, ischemia, or necrosis.

P50

The continuous infusion of RSR13 (loading dose combined with a maintenance dose) induced a consistent and significant (P < 0.05) increase in P50 throughout the experiment (Fig. 2). In controls, baseline P50 started at 36.7 ± 0.9 mmHg and remained unchanged at all time points. In RSR13-treated animals, baseline P50 was elevated from 36.8 ± 0.8 to 47 ± 0.7 mmHg after the loading infusion of drug and was maintained at this level for the entire infusion period by a maintenance infusion (54 mg · kg-1 · h-1). One hour after the RSR13 infusion was discontinued, P50 declined to 39 ± 1.2 mmHg and was close to pretreatment values after 120 min (38.1 ± 0.9 mmHg) (Fig. 2). The average difference in P50 between the control and RSR13 groups during the continuous infusion of RSR13 was 10 ± 1 mmHg.


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  		Fig. 2.   Comparison of O2 tension at which Hb is 50% saturated with O2 (P50) from rats that received an intravenous infusion of vehicle only (Con; n = 5) and from rats that received a loading dose of RSR13 (95 mg/kg) followed by a maintenance dose (54 mg · kg-1 · h-1) (n = 8). Values are means ± SE. * Significantly different from control (P < 0.05).

O2 Transport

Systemic O2 Ex (Fig. 3A) was comparable between the groups at baseline and during the treatment period. In the follow-up period, however, O2 Ex significantly increased above baseline only in the RSR13 group, whereas systemic VO2 (Table 1) remained unchanged in both groups throughout the experiment. Convective DO2 (Table 1) was also not significantly different between groups but showed a continuous decline over time. In the RSR13-treated animals, DO2 fell below baseline at room air but returned to the DO2 level of controls with supplemental O2. CaO2 (Fig. 3B) was comparable in both groups at baseline (control: 18 ± 0.5 ml/100 ml; RSR13: 18 ± 0.3 ml/100 ml). Infusion of RSR13 significantly lowered CaO2 at room air (control: 17 ± 0.3 ml/100 ml; RSR13: 14 ± 0.4 ml/100 ml) and at 25% O2 (control: 16 ± 0.8 ml/100 ml; RSR13: 13 ± 0.3 ml/100 ml). With 30% O2, CaO2 of RSR13-infused rats returned to values comparable to those of control animals (control: 14 ± 0.6 ml/100 ml; RSR13: 13 ± 0.3 ml/100 ml). Over time, CaO2 declined similarly in both groups due to reduction of [Hb] from repeated blood sampling (M10; control: 8.7 ± 0.8 g/dl; RSR13: 8.4 ± 0.3 g/dl). For both groups, similar SaO2 were measured at baseline (control: 95 ± 1 mmHg; RSR13: 94 ± 1 mmHg) (Fig. 3C). After the infusion of RSR13, SaO2 fell to 81 ± 0.7 mmHg at room air (P < 0.05), whereas it remained at baseline level in the control animals. Stepwise elevations of FIO2 increased SaO2 in the RSR13-treated rats to 90% at 0.25 and to 95% at 0.30, whereas SaO2 increased to supranormal values in the control group (98% at 0.25, 99% at 0.30). When FIO2 was reduced to room air in the follow-up period, SaO2 fell again in both groups with a tendency toward lower values in the RSR13-infused animals (control: 95 ± 1 mmHg; RSR13: 91 ± 1 mmHg). The comparison of the first-hour values in the follow-up period (M9) with baseline values showed a significantly lower SaO2 only in the RSR13 group (P < 0.05), whereas SaO2 in the controls matched the baseline value. Two hours after infusion of RSR13 was terminated (M10), SaO2 were similar between the groups and corresponded to the baseline measurements in each group.

                              
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  		Table 1.   O2 transport



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  		Fig. 3.   A: comparison of systemic O2 extraction (O2 Ex) between saline-infused (Con) and RSR13-infused animals. B: comparison of arterial O2 content (CaO2) between saline- and RSR13-infused animals. C: time course of arterial O2 saturation (SaO2) between saline- and RSR13-infused animals. Values are means ± SE for n = 5 control and 8 RSR13-infused animals. * Significant difference for treatment effect between groups at same time point (P < 0.05); # significantly different from baseline within control animals (P < 0.05); § significantly different from baseline within RSR13 group (P < 0.05).

Hemoglobin

Both groups started the experiment with similar arterial [Hb] (control: 14 ± 0.5 g/dl; RSR13: 14 ± 0.4 g/dl). Because of blood sampling for the different measurements, [Hb] decreased to 8.7 ± 0.8 g/dl in controls and 8.4 ± 0.3 g/dl in RSR13-treated rats at the end of the study (Fig. 4).


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  		Fig. 4.   Time course of arterial Hb concentration in control and RSR13-treated animals during experiment. Decline of Hb concentration is caused by repeated blood sampling. Values are means ± SE. # Significantly different from baseline within control animals (P < 0.05); § significantly different from baseline within RSR13 group (P < 0.05).



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  		Fig. 5.   Changes of hemodynamic parameters from baseline values. A: comparison of differences in cardiac index from baseline measurements (Delta CI) between control and RSR13-treated group at different FIO2. B: effect of infusion of saline or RSR13 on changes of systemic vascular resistance index from baseline (Delta SVRI) during experiment. C: comparison of changes in mean arterial pressure from baseline (Delta MAP) between control and RSR13-treated animals. Values are means ± SE. * Significant difference for treatment effects between groups at corresponding time points (P < 0.05); # significantly different from baseline within control animals (P < 0.05); § significantly different from baseline within RSR13 group (P < 0.05).

Hemodynamics

Both groups commenced the experiment with comparable blood pressure values (Table 2). At an FIO2 of 0.25, MAP fell below baseline in control animals and remained at the lower value until the end of the experiment. The RSR13 group maintained blood pressure during the increased FIO2 period. The blood pressure of the RSR13-treated animals was lower than baseline only at 60 min (M9) and 120 min (M10) after RSR13 was discontinued (Table 2). The changes in blood pressure from baseline (Delta MAP; Fig. 5C) were significantly different between groups. At M9 and M10 the fall in blood pressure from baseline was greater in control than in RSR13-infused animals (Fig. 5C).

                              
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  		Table 2.   Hemodynamic parameters

Cardiac index (CI = CO/100 g body wt) (Table 2) was equivalent between groups at baseline. After RSR13 or saline was infused, CI was stable in the control but significantly increased in the RSR13 group at room air and at an FIO2 of 0.25. Further increments of supplemental O2 to an FIO2 of 0.30 combined with RSR13 returned CI to baseline. The increments of FIO2 up to 0.30 did not affect CI in control animals. In the follow-up period, when FIO2 was reduced to room air, CI significantly rose above previous and baseline values in control rats, whereas RSR13-treated rats kept their CI comparable to baseline values. At both measurements in the follow-up period, the change of CI from baseline (Delta CI) was significantly higher in the control than in the RSR13 group (Fig. 5A).

In comparison to CI, the SVR index (SVRI = SVR/100 g body wt) progressed in the opposite direction and showed a decline over time in both groups (Table 2). Infusion of saline and supplemental O2 had no influence on SVRI, whereas reexposure to ambient air in the follow-up period led to a fall of SVRI below baseline. In the RSR13 group, SVRI was lower than in the control after the loading dose of RSR13 at FIO2 of 0.21 and 0.25. Over time, SVRI decreased in the RSR13 group at 25% O2 but returned to baseline level with 30% O2 (Table 2). The analysis of the changes in SVRI from baseline (Delta SVRI; Fig. 5B) demonstrates a fall below baseline in both groups at 25% O2 and a restoration of Delta SVRI at 30% O2 back to a level not significantly different from baseline in the RSR13, whereas the control animals further declined. The intergroup comparison of Delta SVRI revealed significant differences in the follow-up period with a more pronounced fall in the control group.

Lactate

At baseline, lactate concentration values were comparable in both groups (control: 0.9 ± 0.1 mmol/l; RSR13: 0.8 ± 0.1 mmol/l) and remained similar during infusion of RSR13 or saline combined with 25% O2 (control: 1.1 ± 0.1 mmol/l; RSR13: 0.9 ± 0.2 mmol/l) (Fig. 6). At 30% O2, still combined with RSR13 or saline (M8) and at the end of the study (M10), lactate was reduced in the RSR13 (M8: 0.37 ± 0.1 mmol/l; M10: 0.5 ± 0.1 mmol/l) but sustained in the saline infused group (M8: 1.1 ± 0.2 mmol/l; M10: 1.0 ± 0.1 mmol/l).


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  		Fig. 6.   Effects of infusion of saline and RSR13 on arterial lactate concentrations at different FIO2 throughout experiment. Values are means ± SE. * Significant difference between groups at corresponding same time points (P < 0.05).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The binding of RSR13 to Hb changes the allosteric configuration of Hb and thereby reduces its affinity for O2. This reduction in Hb-O2 affinity enhances O2 availability to the tissues by facilitating O2 release from the Hb in the periphery (16, 31). The opposite effects occur in the lungs, rendering it more difficult for the Hb to combine with O2. The alveolar O2 tension necessary to compensate for the impact of reduced Hb-O2 affinity on pulmonary O2 uptake has not been previously reported. There are also no data about the effects of continuous administration of RSR13 combined with elevated inspiratory O2 tension on hemodynamics and O2 transport. We therefore designed an experiment to determine the FIO2 that restores O2 loading in the lungs and to evaluate the effects of right-shifted ODC produced by a continuous infusion of RSR13 on hemodynamics and O2 transport at different FIO2.

In the present study, we demonstrated for the first time that in awake and healthy rats a continuous infusion of RSR13 produces a consistent increase in P50. This increase in P50 significantly reduced SaO2 at room air. An elevation of FIO2 to 0.30 was sufficient to restore SaO2 to baseline values. In addition, we found that a continuous infusion of RSR13 combined with supplemental O2 did not significantly alter MAP, CO, or SVR during the infusion period. In the postinfusion period, however, when supplemental O2 was abandoned and RSR13 was still present in the plasma as evidenced by reduced SaO2, MAP, CO, and SVR exhibited more stability in the RSR13-treated than in the control animals. This suggests a better compensation in terms of tissue O2 availability in the RSR13-treated animals in the anemic state caused by repeated blood sampling.

RSR13 is a synthetic allosteric modifier of the Hb molecule. The binding of RSR13 with Hb induces a conformational change in the molecule. In contrast to DPG, the naturally occurring allosteric effector, which binds in the cleft between the two beta -subunits, RSR13 binds near the top of the alpha -subunits extending to the alpha ,beta -subunit interfaces of the Hb molecule (1, 22). Because of the different binding site, RSR13 produces an additive rightward shift of the Hb O2-dissociation curve in the presence of DPG.

The position of the Hb O2-dissociation curve represents a key element in the multifactorial process of O2 uptake in the lungs, O2 transport by the blood, and the release of O2 from the Hb molecule in the tissues. A shift of the curve either to the left or the right, indicated by a decrease or an increase of the P50, respectively, may have both beneficial and disadvantageous effects, exerting considerable impact on both pulmonary O2 uptake and peripheral O2 release. In a recent experimental study, Kunert et al. (15) administered Hb effectors to rats as bolus infusions at room air. They observed a drop of SaO2 to 60% at the most, expressing a serious impairment of O2 delivery at room air.

Furthermore, as a consequence of altered Hb-O2 affinity, the function of Hb as a tissue O2-buffer system is affected and the Hb, which is mainly responsible for stabilizing the O2 pressure in the tissues, is no longer able to maintain physiological tissue O2 tensions (10). For these reasons, a leftward shift is expected to generate lower, and a rightward shift to generate higher, tissue O2 tensions than normal. The latter was investigated in studies performed by Khandelwal et al. (13) and Wei et al. (31) after superfusion or intraperitoneal application of RSR13 and congeners measuring tissue O2 tensions in skeletal muscle of mice or Hb SvO2 in feline brain tissue. Khandelwal et al. (13) found significantly increased tissue O2 tensions in the skeletal muscle, whereas Wei et al.(31) measured significantly reduced Hb SvO2 in the vein draining the RSR13 superfused tissue unit. These studies support the hypothesis that a right-shifted curve improves tissue O2 availability. Both the experiments performed by Kunert et al. (16) and the latter studies, however, also demonstrated that changes in Hb SaO2 and/or in tissue O2 tensions cause hemodynamic and microvascular adaptations due to changed O2 availability, such as increased SVR, reduced CO, and alterations in vessel diameters.

Our study, in which a different infusion and dosing regimen was used, accompanied by stepwise elevated FIO2, demonstrated the feasibility of generating and maintaining a significant rightward shift of the O2-dissociation curve with the possible benefit of eased O2 release while preserving Hb SaO2 and convective DO2. In contrast to previous studies using single bolus regimen injections, we used a continuous administration of RSR13, which did not cause adverse effects on hemodynamics. An explanation for this different observation may be that supplemental O2 restored CaO2 and thereby maintained convective DO2.

A high-dose bolus infusion of a drug induces a characteristic plasma concentration and response profile that is described by a rapid increase in plasma concentration to a peak value followed by an immediate decline of the plasma concentration, lacking a stable plateau phase of the drug effect, i.e., with no steady state (17). It has been shown that, after a bolus infusion of 200 mg/kg of RSR13, P50 was increased by 60% but started to fall 30 min after administration (15). For consistent effects of the drug and limited side effects, a plateau phase is required at which infusion and elimination are balanced (21). We applied a loading dose of 95 mg/kg followed by a maintenance dose of 54 mg · kg-1 · h-1. This regimen produced an average increase in P50 of 10 ± 1 mmHg and maintained the P50 at that elevated level throughout the infusion period. Two hours after RSR13 infusion was discontinued, P50 returned to values that were not statistically different from baseline. These data point out that the RSR13-induced shift of P50 can be regulated and stabilized at a plateau to provide a consistent effect.

CO and SVR are active modules of the cardiovascular system of the body that control acute and long-term blood flow to organs to supply the required nutrients according to the metabolic demands of the tissue (9). As a consequence of these mechanisms, MAP can be maintained or reestablished by increasing either CO or SVR. A body of evidence suggests that tissue O2 availability plays a crucial role in controlling blood flow, resulting in adaptations of CO, SVR, and MAP (4, 6, 9). Accordingly, altering the Hb-O2 affinity in favor of a facilitated O2 release should interact with these mechanisms. A bolus injection of RSR13 (300 mg/kg ip) increased tissue PO2 up to 100% over baseline 20-80 min after administration, associated with an increase in P50 to 60-70 mmHg (31). Other studies, using either RSR13 (15) or inositol hexaphosphate (27), provided direct evidence that a shift of P50 to ~60-70 mmHg increased SVR and reduced CO. These data suggest that an acute and pronounced shift of P50 to the right elevates tissue O2 tensions probably far above physiological limits and triggers pronounced adaptive responses to restore lower tissue O2 tensions to avoid excessive tissue hyperoxia. Consequently, blood flow is reduced by constricting or closing the precapillary arterioles to reduce excess O2 availability, resulting in either an increase of SVR and/or a fall in CO as observed in previous studies (16, 18). In the present study, MAP was not significantly different between groups throughout the infusion of RSR13 when combined with different FIO2. Over time, blood pressure decreased little but significantly in both groups. Because the fall in blood pressure happened in both groups, a drug-associated decline in blood pressure is unlikely. A loss of intravascular volume by blood sampling is a possible but unlikely explanation, because the volume withdrawn by sampling was replaced by an adjusted saline volume so that CVP was comparable between the groups and was maintained during the study. Thus it is unlikely that an altered volume status would account for the observed time course of the blood pressure. Another and more likely hypothesis explaining this phenomenon is a decrease in blood pressure induced by a fall in SVR due to a restricted O2 availability in the periphery and an increase in venous return due to decreased blood viscosity because of anemia (11, 14, 20). Indeed, [Hb] was reduced to as low as 60-70% of the baseline value, a situation that may reduce O2 availability in the tissue (7) and that reduces whole blood viscosity (9, 19).

SVR progressively declined in both groups and was lower than baseline in the RSR13 animals at an FIO2 of 0.25, whereas the controls still showed a normal SVR. After termination of the drug and the supplemental O2, SVR in the control animals dropped 30% below baseline, whereas the RSR13-treated animals were able to sustain SVR. CI responded the opposite way; it was elevated at FIO2 of 0.25 in the RSR13-treated group but normal in the control animals. However, at room air during the follow-up period, CI increased in the control while remaining normal in the RSR13 group, indicating a greater hemodynamic tolerance likely due to an improved O2 availability. These observations are also supported by hemodynamic responses to changes in SaO2 after RSR13 infusion. Infusion of RSR13 shifted P50 to the right and thereby impaired pulmonary O2 loading, which resulted in a reduced SaO2 as low as 80% at room air. This decline in saturation diminished CaO2 with less O2 available to be set free in the microcirculation for tissue oxygenation. Consequently, compensatory mechanisms aimed to maintain tissue O2 availability, such as a decrease in SVR and an increase in CO, are induced. Further elevation of the FIO2 reestablished pulmonary O2 loading and CaO2. This restored convective DO2 provides the O2 to rebuild a capillary PO2 gradient necessary to normalize or reinstall adequate tissue PO2, thereby counteracting the demand of hemodynamic regulatory responses.

The different hemodynamic performance between the two groups in the follow-up period at room air appears to be caused by a sustained shift in P50. Despite discontinuation of the drug earlier, RSR13 still seemed to be present to a relevant extent. Sixty minutes after RSR13 was discontinued, the mean difference in P50 was still 4.6 mmHg, which just failed to reach statistical significance. The arterial Hb saturations, which at this point were significantly lower than baseline only in the RSR13-treated group, confirm a sustained effect of RSR13 on the Hb-O2 affinity. Although the difference in P50 values was small, we hypothesize that it was biologically significant and effective to affect and favor O2 release and to optimize tissue O2 availability. Similar hemodynamic responses were observed after the transfusion of erythrocytes with DPG levels 1.5 times normal in hypoxic baboons (25). After the PaO2 was restored to normal, CO was significantly lower in the baboons transfused with high-DPG erythrocytes. In that study the P50 values were 3-5 mmHg higher in the high-DPG group (25). Stuecker et al. (26) reported that, in isolated rat heart, reduced Hb affinity decreased coronary blood flow, increased coronary PO2, and slightly elevated myocardial VO2. These findings suggest that even small changes in P50 can trigger physiologically relevant changes in O2 availability and adaptive hemodynamic responses.

Evidence for the improved tissue O2 availability is also supported by the lower lactate concentrations and the increase in O2 Ex above baseline that we measured in the RSR13-treated animals. Acute changes in arterial lactate concentration have been shown to represent changes in the balance between tissue O2 need and availability during infectious and noninfectious conditions (5, 24, 30). The presence of hypoxic tissue areas and areas that are on the verge of dysoxia have been visualized in healthy resting skeletal muscle using the NADH fluorescence technique. Toth et al. (28) reported that in <= 5% of the resting skeletal muscle O2 supply appeared insufficient to support oxidative metabolism. In addition, they found that there are sites with low but still sufficient O2 tensions that might become hypoxic relatively rapidly (28). It is also possible that this pattern of inadequate tissue oxygenation may occur to an even greater extent in selected organs with microvascular architecture that predisposes them to oxygenation heterogeneities. Organs with poor capillary reserves or those with countercurrent arteriovenous microvascular arrangements such as the gut and the kidney are most sensitive to these oxygenation disturbances (8). An elevated P50 allows Hb desaturation at higher mean capillary PO2, increasing the PO2 gradient and thereby promoting higher O2 flux from capillaries to tissue mitochondria or enabling O2 to diffuse over longer distances in the tissue to poorly oxygenated regions. Given a constant or, as in this study, a comparable convective DO2 in both groups, the improved diffusive O2 transport may have favored a more homogenous and adequate tissue oxygenation in the treatment group. Supporting evidence for this concept was recently provided by Richardson et al. (24), who demonstrated in a model of constant convective DO2 that a RSR13-induced increase in diffusive DO2 enhanced maximal VO2 in exercising skeletal muscle (24). In line with these findings, Valeri et al. (29) reported beneficial effects of an increased P50 on arterial and coronary sinus lactate concentrations after infusion of red blood cells enriched with DPG, the natural allosteric effector of Hb.

In contrast to previous studies, we induced a more moderate and constant reduction in Hb-O2 affinity. We also applied stepwise increasing FIO2 until the rightward shift-induced drop of SaO2 was compensated, thereby restoring CaO2 and convective DO2 in the RSR13 group to physiological conditions. This may explain the contrasting effects found in this study compared with previous reports.

In summary, these results indicate that a continuous administration of RSR13 generates a consistent rightward shift of the Hb O2-dissociation curve. In contrast to a bolus injection applied in preceding studies, continuous infusion of RSR13, combined with supplemental O2, has no detrimental effects on systemic hemodynamics. These findings suggest a stabilizing impact of RSR13 on MAP and a mitigating effect on the compensatory hemodynamic responses to anemia-induced limitation in tissue O2 availability. Thus an increase in P50, in conjunction with moderately elevated FIO2, may be beneficial in situations in which O2 availability does not meet tissue O2 needs and in which an increase in diffusive O2 transport may be useful.


    ACKNOWLEDGEMENTS

We gratefully thank Allos Therapeutics, Denver, CO, for providing RSR13 for the experiments. We also acknowledge the blood gas laboratory of the London Health Sciences Centre, South Street Campus, for analyzing the numerous blood samples.


    FOOTNOTES

This work was supported by a grant from Allos Therapeutics, Denver, CO.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: I. H. Chin-Yee, London Health Sciences Centre, Westminster Campus, 800 Commissioners Rd. East, London, Ontario, Canada N6A 4G5 (E-mail: ian.chinyee{at}lhsc.on.ca).

Received 25 June 1998; accepted in final form 1 March 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 277(1):H290-H298
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society


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