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) in hamster window chamber
1Department of Bioengineering, University of California-San Diego, La Jolla, California 92093; and 2Departments of Medicine and of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461
Submitted 17 February 2004 ; accepted in final form 3 June 2004
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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surface-modified hemoglobin; functional capillary density; oxygen-carrying capacity; blood substitutes; tissue oxygen delivery; hemoglobin cooperativity
A widely accepted premise for designing a blood substitute is that its Hb should have an oxygen dissociation curve similar to that of RBCs right shifted, i.e., having a high P50 to facilitate oxygen unloading. Because most of previously proposed oxygen-carrying formulations have been found to induce vasoconstriction, presumably due to nitric oxide scavenging, some of the current efforts are aimed at devising Hb molecules that do not cause vasoconstriction (15). The study of Winslow et al. (32) supports the hypothesis that high P50 and oxygen delivery are intertwined because facilitated oxygen release from Hb causes an oxygen overload of the arteriolar wall, leading to vasoconstriction as the microcirculation strives to regulate blood flow to lower oxygen delivery to the arterioles (8).
Previous studies by our group have shown that extreme hemodilution to a hematocrit (Hct) level of 
11% is a practical model for testing the efficacy of OCPEs in maintaining microvascular function and tissue metabolism. In the hamster window chamber model, this level of Hct defines the threshold at which the organism becomes oxygen supply limited (26), which magnifies the ability of a given material to sustain normal conditions upon introduction into the circulation. Using this model, we previously investigated microvascular oxygen delivery by different OCPEs. In these experiments, animals underwent hemodilution by progressive exchange-transfusion with Dextran 70 until their Hct reached 40% of baseline (level 2 hemodilution). Subsequent exchange-transfusion was continued with the test materials to a final Hct of 11% (20).
A nonhypertensive polyethylene glycol (PEG)-Hb conjugate, MP4 (29), with a significantly left-shifted oxygen dissociation curve (P50 = 5.4 mmHg) and low cooperativity, is presently in phase II clinical trials. MP4 was studied by us (27), using the extreme hemodilution experimental protocol, to determine how efficiently oxygen is delivered by this high-oxygen affinity Hb molecule to the tissue at the microcirculation level. In this study, MP4, at a plasma concentration of 1 g/dl and Hct of 11%, was superior in maintaining functional capillary density (FCD), blood pressure, and a positive base excess (BE) compared with RBCs in identical protocols using Dextran 70, a non-OCPE, and Oxyglobin (Biopure; Boston, MA), a bovine glutaraldehyde polymerized molecular Hb-based blood substitute with 50-mmHg P50 and no cooperativity (29).
MP4 is a PEG 5K-Hb conjugate consisting of an average of six copies of PEG-5K chains conjugated to Hb at its Cys-93(
) and thiolated 
-amino groups through succinimido propyl linkage (SPr-PEG5K)6-Hb. Application of this material as a blood substitute leads to the introduction of half a gram of PEG for each gram of Hb into the circulation. Although all evidence shows that PEG is fully biocompatible when introduced into the circulation, even in the quantities associated with the potential clinical use of MP4, decreasing the eventual biological load of clearing this material from the organism appears to be a desirable goal. Furthermore, the cost of PEG reagents and the ultimate cost of the material to be deployed as a blood substitute is strongly dependent on the amount of PEG reagent needed to attach the desired number of PEG chains onto the Hb tetramer. These considerations led us to further explore the design of different forms of PEG-Hb conjugates to determine whether similar efficacy can be obtained using a lesser number of PEG copies (and a lesser amount of material).
In the present study, we investigated the microvascular effects of an OCPE where Hb was conjugated with two copies (i.e., attaching 1 PEG polymer to each of 2 specific binding sites) of either 5- or 10-kDa PEG chains at the sulfhydryl group of Cys-93() using maleimide phenyl PEG. These two site specifically modified PEGylated Hb are referred to as (SP-PEG5K)2-Hb (PK52-Hb) and (SP-PEG10K)2-Hb (PK102-Hb), respectively (15). To compare the efficacy with previous results using maleidophenyl PEG-Hb (MP4), we investigated how the newly designed samples of PEG-Hb support microvascular function in the extreme hemodilution protocol already used in the analysis of the previously tested materials (13).
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Preparation of PK52-Hb and PK102-Hb.
Purified human Hb molecules were modified by a process that precisely controls the number of PEG molecules reacting with the Hb molecule as well as the site of conjugation, leading to a biochemically homogeneous molecular preparation (a process termed "site-specific Hb PEGylation"). Samples were prepared by reacting Hb with a maleidophenyl PEG reagent. Chromatographically purified Hb was dialyzed extensively against PBS. The extremely pure Hb was reacted with maleimide phenyl PEG reagents at a concentration of 0.25 mM protein (tetramer) and PEG 5.0 mM maleimide. The reaction was maintained at pH 7.4 and at 4°C for 16 to 20 h. After the reaction, samples were tangential flow filtrated to remove excess PEG reagent using a 70-kDa membrane. They were monitored by size exclusion chromatography of the solution with Pharmacia FPLC unit using two Superose 12 columns connected in series. The effluent was monitored at 540 nm (for Hb), and its refractive index was measured to insure that there were no PEG reagents present. The solution free from PEG reagent contains the site specifically PEGylated Hb and was further purified by ion exchange chromatography on Q-sepharose as described previously (13). The purity of the products was established by reverse-phase HPLC, FPLC, and tryptic mapping of the PEGylated -chains of these samples. The amino acid sequencing of the PEG-modified tryptic petide confirmed the modification of Cys-93(). The resistance of Lys-95() of the PEGylated Hb samples for tryptic digestion appears to be a consequence of modification of Cys-93() by PEG maleimide (1).
Hypervolemic infusion. Animals were infused with a bolus of P5K2-Hb or P10K2-Hb equivalent to 10% of their blood volume via the jugular vein at a rate of 0.2 ml/min (top load). Mean arterial blood pressure (MAP), heart rate (HR), and microvessel diameters were studied 10 and 30 min after the start of infusion. These inclusion criteria represent the minimum acceptable parameters rather than the range of normal values used in our previous studies.
Acute isovolemic exchange transfusion (hemodilution) protocol. Progressive hemodilution to a final systemic Hct level of 11% was accomplished with three isovolemic exchange steps. This protocol is described in detail in our previous reports (20). Briefly, the volume of each exchange-transfusion step was calculated as a percentage of the blood volume, estimated as 7% of body weight. An acute anemic state was induced by lowering the systemic Hct by 60% with two steps of progressive isovolemic hemodilution using 6% Dextran 70 (molecular mass = 70 kDa), exchange levels 1 and 2. Level 1 exchange was 40% of blood volume and level 2 exchange was 35% of blood volume, respectively.
After the level 2 exchange, the animals were randomly divided into three experimental groups by assigning each animal to an experimental group according to a sorting scheme based on a list of random numbers (2). Level 2 exchange was followed by level 3, exchanging 35% of blood volume using a nonoxygen carrier and the test materials. The experimental group L3-D70 was hemodiluted with Dextran 70 to a Hct of 11%. The test materials were studied in two groups of animals labeled L3-P5K2 and L3-P10K2; both groups had a reduced Hct of 11%. The Hb concentration in the test materials, P5K2 and P10K2, was 4 g/dl (23).
Because mixed blood and dilution material is withdrawn during the exchanges, a 110% blood volume exchange was needed to reduce the Hct to 11% (23). The transfusion solutions were filtered (in-line, 0.2-µm filter, at a rate of 100 µl/min, CMA100 Microinjection Pump, CMA) and infused into the jugular vein catheter. Blood was simultaneously withdrawn by hand at the same rate from the carotid artery catheter according to a previously established protocol (23). Data were taken after a 5-min stabilization period.
Blood chemistry and biophysical properties. Arterial blood was collected in heparinized glass capillaries (0.05 ml) and immediately analyzed for arterial PO2 (PaO2), arterial PCO2 (PaCO2), BE, and pH (Blood Chemistry Analyzer 248, Bayer; Norwood, MA). The comparatively low PaO2 and high PaCO2 of these animals is a consequence of their adaptation to a fossorial environment. Blood samples for viscosity and colloid osmotic pressure (COP) measurements were quickly withdrawn from the animal with a heparinized 3-ml syringe at the end of the experiment for immediate analysis. Viscosity was measured in a Brookfield DV-III cone/plate Rheometer with a CPE-40 cone spindle at a shear rate of 160 s–1. COP was measured using a Wescor 4420 Colloid Osmometer (31). Oxygen equilibration curves were measured at 37°C using a Hem-O-Scan Aminco instrument (SLM Aminco Instruments; Urbana, IL). P5K2 or P10K2 was diluted in 100 mM phosphate buffer (pH 7.4) to a Hb concentration of 1 mM. P50 was obtained by noting the PO2 corresponding to 50% saturation under the assumption that hemoglobin was 100% saturated at ambient oxygen (21% O2) (28). P50 values were 8.6 mmHg for P5K2 and 8.3 mmHg for P10K2 versus 33.9 mmHg for hamster RBCs. Cooperativity for these molecules was n = 2.2 (Fig. 1).
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Functional capillary density. Functional capillary density (FCD; in cm–1) is the total length of RBC-perfused capillaries divided by the area of the microscopic field of view (23). Capillary segments are considered functional if RBCs are observed to transit over a 30-s period. FCD was tabulated from the capillary lengths with RBC flow in an area comprosed of 10 successive microscopic fields (420 x 320 µm2). Detailed mappings were made of the chamber vasculature to study the same microvessels throughout the experiment.
Microhemodynamic parameters.
Arteriolar and venular blood flow velocity were measured on-line using the photodiode cross-correlation technique (6) (Fiber-Optic Photo Diode and Velocity Tracker model 102B, Vista Electronics; San Diego, CA). The centerline velocity (V) was corrected according to vessel size to obtain the mean RBC velocity (12). The video image shearing technique was used to measure vessel diameter (D) on-line. Blood flow (Q) was calculated from the measured parameters as Q = V x 
(D/2)2.
Microvascular PO2 distribution. High-resolution noninvasive microvascular PO2 measurements were made using phosphorescence quenching microscopy (PQM) (19). PQM is based on the oxygen-dependent quenching of phosphorescence emitted by the albumin-bound metalloporphyrin complex after pulsed light excitation. PQM is independent of the dye concentration within the tissue and is well suited for detecting hypoxia because its decay time is inversely proportional to the PO2 level, causing the method to be more precise at low PO2s. This technique is used to measure both intravascular and extravascular PO2 because the albumin-dye complex continuously extravasates the circulation into the interstitial tissue (5). Interstitial PO2 measurements have been found to be identical to simultaneous measurements made with recessed oxygen electrodes (2). Tissue PO2 was measured in tissue regions in between functional capillaries. The reported values are the average of several determinations in several animals. PQM allows for precise localization of the PO2 measurements without subjecting the tissue to injury. These measurements provide a detailed understanding of microvascular oxygen distribution and show whether oxygen is delivered to the interstitial areas.
The system setup has been described in previous references (10). Animals received a slow intravenous injection of 15 mg/kg body wt at a concentration of 10.1 mg/ml palladium-meso-tetra(4-carboxyphenyl)porphyrin (Porphyrin Products; Logan, UT), which was allowed to circulate for 20 min before PO2 measurements. The phosphorescence was excited by pulsed light (30 Hz, 4-µs duration) for a period of <5 s; the measurement site was microscopically vignetted by an adjustable slit. For intravascular measurements, an optical rectangular slit 5 x 35 µm was positioned longitudinally within the vessel of interest. For interstitial tissue measurements, a 6 x 6-µm slit was placed in intercapillary spaces in regions void of large vessels. The phosphorescence decay curves were analyzed off-line using a standard single-exponential least-squares numerical fitting technique, and the resultant time constants were applied to the Stern-Volmer equation to calculate PO2 using predetermined parameters of lifetime in the absence of oxygen and quenching constant corrected for this animal model (19).
Experimental procedures. Baseline systemic, microvascular, and hemodynamic characterization were performed before the start of the exchange protocol (and palladium porphyrin injection). After each exchange and a stabilization period of 5 min, systemic and/or microvascular measurements were performed. Exchanges began every hour. After the exchange transfusion, the same measurements were repeated, and the PO2 distribution was then determined using PQM (9). The duration of the experiment was 3–4 h.
Oxygen delivery and extraction.
The rate of oxygen delivered and used by the circulation at both the systemic and microvascular level in the tissue of the hamster window preparation was calculated according to the formulation of Sakai et al. (17)
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A-V %saturation is the arteriolar-venular difference in oxygen saturation of RBCs and plasma Hb, respectively, and Q is the venular flow. In all cases, the blood oxygen content was determined by using the oxygen dissociation curves corresponding to each of the oxygen-carrying blood components to calculate the oxygen content at the measured blood PO2. Data on oxygen saturation were obtained as previously described (30). In this analysis, microvascular Hct was corrected according to the findings of Lipowsky and Firrell (11). Data analysis. Results are presented as means ± SD unless otherwise noted. All data are presented as absolute values and ratios relative to baseline values. A ratio of 1.0 signifies no change from baseline, whereas lower and higher ratios are indicative of changes proportionally higher and lower than baseline. The same vessels and functional capillary fields were followed so that direct comparisons to their baseline levels could be performed allowing for more robust statistics for small sample populations. For repeated measurements, time-related changes were assessed by ANOVA.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Exchange transfusion. Fifteen animals (55–65 g body wt) entered the hemodilution, exchange-transfusion protocol, and all tolerated the experiment without any visible discomfort.
Hematological changes. The blood exchange transfusion resulted in a final Hct of 11.0 ± 0.9. The P5K2-Hb and P10K2-Hb exchange transfusions resulted in a plasma Hb level of 0.8 ± 0.1 g/dl, which increased the total Hb in the blood (RBCs + Hb dissolved in plasma) to 4.8 ± 0.4 g/dl after completion of level 3 exchange. Thus oxygen-carrying capacity at this level was significantly lower than that found at level 2, after the last Dextran 70 transfusion, which was 6.2 ± 0.4 g/dl (Hct of 17.0 ± 1.0).
Colligative properties.
Blood viscosities and COP after the level 3 exchange were sampled at 15 min after completion of the exchange. Table 3 shows that viscosity ranged from 2.1 ± 0.2 for Dextran 70 to 2.0 ± 0.3 for P5K2-Hb and P10K2-Hb. Blood viscosity at level 3 exchange using MP4 was 1.8 ± 0.1 cP. These results are somewhat unexpected because P10K2-Hb had a significantly higher solution viscosity than P5K2-Hb (2.7 vs. 1.4 cP; Table 1). The highest blood viscosity after the level 3 exchange was obtained with Dextran 70-only exchanges (2.1 ± 0.2 cP); however, this value was not significantly different from the other values.
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Systemic and blood gas parameters. Changes in the systemic and blood gas parameters from baseline are presented in Table 2. HR after hemodilution and then the exchange-transfusion with the PEGylated materials was in the range of 1.10–1.14 of baseline. The slight increase in HR was not statistically different from one another but was significantly different from baseline (P < 0.05). MAP was statistically lower for all extreme hemodilution tests but was not different from baseline in the previous study using MP4.
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Microhemodynamics.
Level 3 exchange arteriolar and venular diameters were not statistically different from control with P5K2-Hb and P10K2-Hb, which paralleled previous findings with MP4. Arteriolar diameter did not change significantly with Dextran 70, but venular diameter constricted at level 3 exchange. For the same groups, arteriolar flow velocities were 83% of control and venular flow velocities were not statistically significant from control. By comparison, arteriolar and venular flow velocities for MP4 were 60% (arteriolar) and 65% (venular) of control, respectively, whereas Dextran 70 caused flow velocity to rise to 120% of control. Combining the diameter and flow velocity data allowed us to calculate arteriolar and venular flows (as shown in Fig. 3) showing that in the P5K2-Hb and P10K2-Hb exchanges, arteriolar flow was 85% of baseline and not statistically different from baseline for the venules. In the level 3 exchange with Dextran 70, the corresponding values of flow were 70% (arteriolar) and 90% (venular) of baseline, respectively. For MP4, we previously found 62% (arteriolar) and 75% (venular) of baseline, respectively. The results of calculating shear stress in arterioles and venules are also shown (Fig. 4). It should be noted that the latter level of shear stresses were essentially lower than normal.
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Microvascular oxygen distribution. PO2 measured using phosphorescence microscopy after the exchange-transfusion with P5K2 and P10K2 showed that materials produced virtually identical distributions of microvascular PO2 (arterioles averaged 36 mmHg and venules averaged 13 mmHg) (Fig. 5). By comparison, our previous study with MP4 presented arterioles with 39 ± 5 mmHg, whereas venular PO2 was 2 ± 1 mmHg (27). Exchange with Dextran 70 yielded an arteriolar PO2 of 33 mmHg, whereas the venules had a PO2 of 3 ± 2 mmHg, which was statistically significantly lower from the exchanges with the test materials.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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11%, a condition of extreme hemodilution near the critical oxygen supply limitation. Furthermore, because the Hb solutions were formulated at 4% g/dl, the increase of total oxygen-carrying capacity due to their infusion was minimal; thus changes in microvascular and systemic parameters should provide a clear indication of the efficacy of these materials. The principal finding of this study is that P5K2-Hb and P10K2-Hb administered in an extreme hemodilution protocol yielded a tissue PO2 of 7.0 ± 2.5 mmHg. In a previous study using the same protocol, MP4, a PEG-conjugated Hb molecule with a greater number of PEG copies attached to its surface, and Dextran 70 resulted in tissue PO2 of 1.7 ± 0.5 and 1.3 ± 1.2 mmHg, respectively (27). The increased tissue PO2 obtained with the newly designed materials appears to be due to a series of incremental changes in microvascular and macrovascular hemodynamics comprising the increase of arteriolar and venular blood flow, increased FCD, and increased cardiac index. Although none of the individual changes were statistically different among one another, they all showed a trend of being somewhat improved when P5K2-Hb and P10K2-Hb were compared with MP4. It would seem that the cumulative effect of this trend leads to the statistically different FCD and tissue PO2 found in our study (24).
Oxygen delivery and extraction shows a trend of being somewhat greater for P5K2-Hb and P10K2-Hb than MP4 (Fig. 6). This is mostly due to the cardiac index being increased with the new Hb formulation relative to that obtained with MP4. This increase may be in part due to the lowered peripheral resistance as suggested by the significantly increased venular flow. An additional factor may be that improved tissue oxygen and FCD seen in the window chamber may be the reflection of a more general systemic phenomenon, which includes improved oxygen delivery to the heart and therefore improved cardiac function.
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20%. The intrinsically low P50 of these formulations ensure that oxygen delivery is targeted to anoxic regions, making the most efficient use of the additional oxygen-carrying capacity, which is not used for oxygenating regions with high oxygen delivery and PO2 (14).
The increased tissue PO2 caused by the present compounds relative to MP4 in conditions of severe anemia may be beneficial in reducing the incidence of localized anoxia. Tissue PO2 in the case of MP4 was 1.7 ± 0.5, indicating a combined variability due to instrumentation accuracy and inherent biological variability of ±0.5 mmHg. Therefore, 16% of the tissue may be in the anaerobic condition (1.2 mmHg or lower, 1 SD from the average), depending on the relative partition between measurement variability and inherent tissue variability. Tissue PO2 attained with the present compounds is 7.0 ± 2.5 mmHg; therefore, <2% of the tissue (2 mmHg or lower, 2 SD from the mean) may be in anaerobic conditions, thus significantly decreasing the potential for developing damage due to focal ischemia.
The partition of oxygen PO2 between arterioles and venules shown in Fig. 5 indicates that MP4 causes blood to release more oxygen than the new compounds, a finding that appears to contradict the data that the tissue PO2 is higher after the administration of the new compounds. This discrepancy is reconciled by noting that in these extreme hemodilution experiments RBCs still carry 80% of oxygen and undergo a similar change in saturation as they traverse the microcirculation in the presence of either of the PEG-Hb molecules. The principal difference, however, is that the new compounds cause an 40% increase in microvascular flow. Averaging oxygen delivery and extraction calculated on a vessel to vessel basis for each group shows that a greater amount of oxygen is delivered by the new compounds (Fig. 6 and Table 4). Although the results on oxygen delivery and extraction are not statistically different, tissue PO2 is increased, which is statistically significant.
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In these experiments, final total Hb in the circulation was the same in all tests, or 4.8 g/dl total Hb, with the same partition between RBC Hb and molecular Hb, 4.0 ± 0.4 and 0.8 ± 0.1 g/dl, respectively. Therefore, the results found are independent of total oxygen-carrying capacity and Hb present and should reflect the differences in transport properties of the modified Hb molecules determined by the differences in chemistry used to conjugate PEG to the molecular surface of Hb. The primary factors that may influence the physiological effects of these PEG-Hb conjugates are 1) the amount of PEG mass per Hb tetramer; 2) differences in oxygen affinity between PK52-Hb, PK102-Hb, and MP4; and 3) the level of cooperativity of the two site-specific PEGylated Hb, n = 2.2, compared with MP4, n = 1.2.
The differences in the amount of PEG conjugated with the Hb molecule may produce differences in the water bound to the Hb compound and therefore affect the hydrodynamic radius and volume of the molecule and, therefore, its viscosity. However, there was no significant difference in physiological responses between formulation with two copies of PEG of 5 kDa each and two copies of 10 kDa each, although the viscosity of the administered solutions was significantly different (1.3 and 2.7 cP, respectively; Table 1). The solution viscosity of MP4, which has the largest amount of PEG (5–6 copies of 5 kDa each) was intermediate, or 2.3 cP (all viscosities measured at 4% concentration). Once these materials, including MP4, were introduced into the circulation, they were diluted and resulted in plasma viscosities that were essentially identical, in the range of 1.3–1.4 cP, with blood viscosities also being similar and in the range of 1.8–2.0 cP, a value that is affected by slight differences in RBC concentration (Table 3). In a previous study by Tsai et al. (23), it was shown that to significantly increase FCD in extreme hemodilution with a viscogenic non-OCPE (Dextran 500), plasma viscosity had to increase to 2.2 cP, a value that is significantly above the values of plasma viscosity obtained in extreme hemodilution with the compounds tested.
Dilution and possibly autotransfusion due to the initially elevated COP of the exchanged solutions caused all COP plasma measurements (including the Dextran 70 group) to be in the range of 16.4–16.8 mmHg, which is close to the normal blood values of 17.6 ± 0.7 mmHg in this species, whereas in the previous study with MP4, plasma COP was 22.8 ± 2.4 mmHg (27). Consideration of the lack in correlation between viscosity and COP data of the bulk solutions, as well as the similarity of COP once the materials were introduced into the circulation, leads to the conclusion that the resulting plasma and blood viscosities attained, as well as the final COP, are not determining factors in explaining the difference in tissue PO2 found in this study.
Low oxygen affinity has been proposed to be a major factor in making these modified Hbs effective and economic as oxygen carriers, because this property insures that oxygen is delivered solely to regions of low PO2, bypassing normal oxygenated tissue domains. However, the difference between molecules is not significant, with P5K2-Hb and P10K2-Hb with P50s of 8.6 and 8.4 mmHg, respectively, being somewhat higher than MP4 with P50 of 5.4 mmHg. These high-oxygen affinities appear to act as a reservoir of oxygen for tissue compartments with low PO2 ensuring that oxygen is only released where PO2 is low, a function requiring only a small amount of Hb. The hypothesis that these comparatively small amounts of low-oxygen affinity species of Hb in the circulation are effective in eliminating hypoxic pockets in the tissues is supported by the finding that the small amount of Hb leads to a positive acid-base balance, which cannot be achieved solely with Dextran 70. The somewhat higher P50 of P5K2-Hb and P10K2-Hb may improve oxygen release relative to MP4. This parameter is still considerably below that of normal hamster blood, which is 33.9 mmHg; however, a contribution from the higher P50 cannot be excluded.
An important difference between the newly developed PEGylated Hb and MP4 is that these molecules exhibit a greater cooperativity than MP4 causing the molecule’s bound oxygen to be released at low tissue PO2s. Conversely, a molecule like MP4 will retain some oxygen even when exposed to a low PO2 environment.
The data on cardiac index allows the calculation of arterial oxygen delivery (normalized to body weight). This parameter is slightly lower than baseline but not significantly different from the normal control for the PEGylated Hbs, because of the increased extraction due to the low venular PO2 (Fig. 6). Arterial oxygen delivery was significantly reduced for Dextran 70 only in extreme hemodilution, and oxygen delivery to the microcirculation was significantly reduced relative to baseline due to the combination of reduced FCD, reduced microvascular flow, and low blood oxygen-carrying capacity (25).
All the PEGylated Hbs in these conditions of extreme hemodilution appear to supply the tissue with somewhat more oxygen than blood in control conditions because oxygen consumption at the microcirculation shows the tendency to be higher than baseline. Considering that tissue PO2 is significantly lower than baseline [21 ± 4 mmHg (21)], it appears this is a direct function of venular PO2.
In conditions of impaired oxygen delivery usually associated with lowered cardiac output such as shock and extreme hemodilution, PaO2 becomes elevated above normal as blood flow becomes insufficient to extract all the available oxygen from the lungs. This phenomenon is particularly evident in hamsters, which, being adapted to a fossorial environment, have low PaO2s. In these studies, the presence of these high-affinity Hbs may increase the amount of oxygen captured by the lung by binding residual low PO2 oxygen that otherwise would be expired, leading to increased arterial blood PO2.
In summary, the results presented here show the use of either of the two site-specific PEGylated Hbs achieve the same results as with MP4 in terms of oxygen delivery to the tissue. FCD, cardiac index, and acid-base balance were also maintained to near-normal values by introducing a comparatively small amount of PEGylated Hb into the circulation. Although oxygen delivery and consumption by the microcirculation was not statistically different between molecules, P5K2-Hb and P10K2-Hb showed the tendency to deliver more oxygen than MP4 in the same protocol, leading to a significantly higher tissue PO2. This difference appears to be due to the greater cooperativity of these molecules. This result indicates that tissue PO2 in the present experiments is in part a function of the shape of the oxygen dissociation curve of Hb and not indicative of the level of tissue oxygen supply carried to the tissue from the lungs. The present results also show that the type of linker (between PEG and maleimide) and the number and size of PEG chains are important determinants of the physical functional properties of PEG-Hb molecules conjugates that affect their P50, cooperativity, and colligative properties (16). Because these new materials are formulated using comparatively lower amounts of PEG, they may present some practical advantages relative to PEGylated Hbs with greater amounts of PEG.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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P < 0.05 relative to baseline; 
P < 0.05 compared with Dextran 70; 
P < 0.05 compared with MP4. Cardiac output (CO) and calculated cardiac index (CI) (means ± SD) in each animal group were as follows: baseline (CO: 17.5 ± 1.3 ml/min, CI: 210 ± 24 ml·kg–1·min–1, n = 6); level 1 exchange (CO: 24.8 ± 3.6 ml/min, CI: 290 ± 28 ml·kg–1·min–1); level 2 exchange (CO: 28.1 ± 4.2 ml/min, CI: 345 ± 33 ml·kg–1·min–1); level 3 (L3) Dextran 70 (CO: 14.2 ± 1.9 ml/min, CI: 170 ± 27 ml·kg–1·min–1, n = 6); level 3 P5K2 (CO: 20.2 ± 2.1 ml/min, CI: 310 ± 29 ml·kg–1·min–1, n = 5); and level 3 P10K2 (CO: 22.6 ± 2.6 ml/min, CI: 330 ± 36 ml·kg–1·min–1, n = 6); n, no. of vessels studied. MP4 data were reported in Ref. 
