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Departments of 1Anesthesiology and 2Surgery, Karolinska Institute at Söder Hospital, S-118 83 Stockholm; and 3Swedish Defense Research Agency (FOI), SE-172 90 Stockholm, Sweden; 4Sangart, Inc., San Diego 92121; and 5Department of Bioengineering, University of California San Diego, San Diego, California 92093
Submitted 16 May 2003 ; accepted in final form 22 December 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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polyethylene glycol; 4% maleimide polyethylene glycol hemoglobin; hemoglobin; shock; blood substitutes
chains (-Hb) by the US Army (10, 11) showed these effects to such a degree that further development was abandoned by the Army and subsequently also by Baxter Healthcare (27, 28). Polymerized bovine Hb demonstrated similar effects in humans (12, 13). Whereas several of these cross-linked and/or polymerized Hb products are still under development (31), our hypothesis is that the lack of clinical success is predictable, based on animal studies in a variety of models that demonstrate vasoconstriction (38).
Nitric oxide (NO) scavenging by cell-free Hb would appear to be an obvious explanation for the vasoactive effects; however, several modified Hb with different hypertensive effects have nearly identical NO-binding properties (24). We have proposed an alternative hypothesis. In an in vitro system, McCarthy et al. (16) found that, by decreasing the diffusivity of cell-free Hb by conjugation with polyethylene glycol (PEG), the diffusive transfer of oxygen in a low-PO2 environment could be reduced or eliminated, compared with HbA0 or -Hb. We further suggested that this modified Hb could preserve oxygen delivery (
O2) to capillaries in vivo and limit vasoconstriction in the microcirculation, resulting from premature unloading of oxygen at the level of small arterioles. Consistent with this hypothesis, we found that PEG-modified Hb did not lead to elevated systemic blood pressure and resistance in rats, compared with -Hb or polymerized Hb, and further demonstrated that PEG-Hb could preserve O2, oxygen consumption (
O2), and acid-base status (39).
In view of these findings, a new surface-modified human Hb [maleimide PEG Hb (MalPEG-Hb)] was designed by using novel attachment of six strands of maleimide-activated PEG (5 kDa) to human Hb (32). Based on the previous demonstration that the pig circulation was very sensitive to the hypertensive effects of cell-free Hb (11), the present experiments were performed to determine whether MalPEG-Hb solutions caused the characteristic hemodynamic effects observed previously with other Hb solutions. In addition, we examined O2 and O2 in response to hemorrhage and resuscitation to determine whether the concentrations of plasma Hb tested support oxygen requirements to offset the effects of hemodilution after resuscitation. To accomplish these goals, three different formulations of MalPEG-Hb were compared with 10% pentastarch (PS) and 4% stroma-free Hb (SFH) as resuscitation solutions in anesthetized pigs following controlled hemorrhage of 50% of estimated blood volume. The results confirm the lack of hemodynamic effects previously reported and further demonstrate that MalPEG-Hb preserves O2 after severe hemorrhage and resuscitation in pigs.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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-amino groups (lysine), which were then conjugated to six strands per tetramer of maleimide-activated PEG, 5-kDa molecular mass (NOF, Tokyo, Japan) (1, 32). One formulation of pentastarch, three formulations of MalPEG-Hb, and one formulation of SFH were prepared before infusion: 1) PS, 10 g/dl pentastarch (Pentaspan, a gift from B. Braun Medical, Irvine, CA); 2) MP4, containing 4.2 g/dl MalPEG-Hb in lactated Ringer solution; 3) HS4, containing 4.2 g/dl MalPEG-Hb and 5 g/dl pentastarch; 4) HS2, containing 2 g/dl MalPEG-Hb in 5 g/dl pentastarch; and 5) SFH, containing 4.3 g/dl SFH in lactated Ringer solution. Although the molecular mass of MalPEG-Hb is 
95 kDa, compared with 64 kDa for unmodified Hb, concentrations are expressed on heme basis. That is, concentrations are estimated from optical density and extinction coefficients for Hb, so that, gram for gram, MalPEG-Hb has the same O2 capacity as native Hb. The physical properties of the solutions are summarized in Table 1. Oxygen equilibrium curves were measured by using the method described previously (33), and other physical characterizations were as previously reported (32).
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Animal preparation. All animal experiments were performed at the Department of Experimental Traumatology, Defense Research Agency (FOI), Stockholm, Sweden, at the Söder Hospital. The local institutional review committee approved the protocols. Animals (n = 38) were female or castrated male Swedish landrace pigs, weighing an average of 20.8 kg (range, 17.0-27.5 kg). The PS, HS2, HS4, and MP4 animals were randomized as to test article on the day of experiment without identification of the animal or knowledge of its weight, condition, or any other parameters. The SFH studies were performed as a separate cohort and were not randomized as to treatment group.
Before anesthesia, pigs were given 10-15 ml of ketamine-HCl (50 mg/ml) by intramuscular injection in the neck, followed by intravenous injection of 3-4 ml of pentobarbital sodium (50 mg/ml) and 0.25 ml atropine (0.5 mg/ml) in a catheterized ear vein. Anesthesia was maintained by continuous infusion of ketamine with the use of an Atom syringe infusion pump, model 235 (Atom Medical, Tokyo, Japan), at 50 mg·kg-1·h-1. The level of anesthesia was checked regularly by withdrawal reflex. A tracheostomy was performed, and, after insertion of an endotracheal tube, the animals were mechanically ventilated with room air (Siemens 900C servo ventilator) at 3.5-4.5 l/min, with a frequency of 20 breaths/min to maintain arterial PCO2 of 34-41 Torr.
The left external jugular vein was cannulated for administration of anesthetic and resuscitation solutions. The left common carotid artery was cannulated by cut-down for withdrawal of blood samples. The pulmonary artery was accessed by using a Swan-Ganz thermodilution catheter (Baxter, model 132F, 5 Fr) via the right external jugular vein for measurement of central venous pressure (CVP), pulmonary arterial pressure (Ppa), and cardiac output by thermodilution. The right femoral artery was cannulated for continuous measurement of arterial pressure. Arterial pressure, Ppa, and CVPs were monitored by using a BioPac MP100 data-acquisition unit (BIOPAC Systems, Goleta, CA) equipped with UFI model 1050BPR and TSD104A pressure transducers, connected through a UIM 100A Universal interface to a Compaq Presario 1810 laptop computer with PCMCIA interface card. Data were collected at 100 Hz. Cardiac output was measured by using an Edwards 9420A Laboratory Cardiac Output computer (Edwards Laboratories, Santa Ana, CA) and the thermodilution catheter, by injection of 5 ml of saline at ambient temperature into the pulmonary artery catheter. The average of two measurements that agreed to within 10% of each other was used.
Experimental groups and hemorrhage protocol. After anesthesia and surgical instrumentation, animals were assigned to one of five groups: PS (n = 8), HS2 (n = 8), HS4 (n = 8), MP4 (n = 8), and SFH (n = 6). After a 30-min stabilization period, a 50% hemorrhage (50% of blood volume) was begun (blood volume calculated as 65 ml/kg) at constant rate (1.08 ml·kg-1·min-1) by using the arterial catheter. Because all animals were of comparable weight, the duration of hemorrhage was predetermined to be 30 min in all pigs. Fifteen minutes after completion of the blood withdrawal, resuscitation fluid was initiated at a rate of 1.14 ml·kg-1·min-1, the entire volume to be infused over the course of 20 min. The replacement volume, arbitrarily 70% of the shed volume, was chosen because the solutions are hyperoncotic (see Table 1) and thus would lead to overexpansion of the blood volume if volumes equal to the shed volume were replaced. Consequently, 450 ml of each product were infused into each animal, corresponding to 0, 900, 900, 450, and 900 mg/kg of Hb in the PS, MP4, HS4, HS2, and SFH groups, respectively.
Hemodynamic measurements. Arterial pressure, Ppa, and CVP signals were recorded continuously and sampled at 100 Hz by the BIOPAC system. Mean pressures were calculated as diastolic pressure + 
(systolic-diastolic pressure). Heart rate for each beat was calculated as the reciprocal of the interval between successive pressure peaks. Minute averages for mean arterial pressure (MAP), mean pulmonary arterial pressure (MPAP), CVP, and heart rate were calculated from the continuously recorded data. Cardiac output was recorded at baseline, after hemorrhage, and thereafter every 15 min.
Systemic vascular resistance index (SVRI) was calculated as
 | (1) |
where MAP is in mmHg, CVP is in mmHg, and 
is cardiac index (ml·min-1·kg body wt-1) (units for SVRI are dyn·kg·s·cm-5). An approximation of pulmonary vascular resistance (PVRI*) was calculated, without the measurement of pulmonary capillary wedge pressure, by
 | (2) |
where MPAP is in mmHg (units for PVRI* are dyn·kg·s·cm-5). Cardiac output, stroke volume, systemic vascular resistance, and pulmonary vascular resistance are expressed as "index" values, normalized to body weight.
Hematology. Arterial and venous blood samples were drawn into plastic syringes and immediately capped and measured by using a GEM Premier Plus blood-gas analyzer (Instrumentation Laboratories, Lexington, MA) for PO2, PCO2, pH, and base excess (BE). Hct was measured by using centrifuged arterial blood samples in microhematocrit tubes. Total Hb was measured by using a HemoCue B-Hemoglobin (HemoCue, Ängelholm, Sweden), and plasma Hb was measured, after centrifugation of whole blood, by using a HemoCue Plasma/Low Hb system, also from HemoCue. Plasma methemoglobin was not measured routinely. However, it was shown in control experiments that met-MalPEG-Hb does not increase over the period of these experiments (data not shown).
Although red cell Hb is confined to the red cell space, it is measured in whole blood, and the value is used as if Hb were evenly distributed in blood. Plasma Hb, however, is measured only in plasma. Therefore, to compare the two Hb compartments, it is necessary to correct plasma Hb to the same volume of distribution as total Hb. This quantity, blood-free Hb (BFH) is
 | (3) |
where Hct is expressed as a fraction. Red blood cell Hb concentration is then calculated as the difference between total Hb and BFH. All values are expressed as grams per decaliter of blood.
Red cell Hb saturation was calculated by using the Rovida formulas for pig blood (25) using a 2,3-diphospho-D-glycerate-to-Hb molar ratio of 1.9 measured in one of our animals (unpublished observation). The saturation of the plasma Hb was calculated by using the PO2 as measured and the Adair parameters according to the generalized Adair equation
 | (4) |
where Y is fractional saturation, p is PO2, and a values are the parameters derived from oxygen equilibrium curve measurements (40).
Calculation of O2, O2, and oxygen extraction ratio (OER) were
 | (5) |
 | (6) |
 | (7) |
where HbRBC is red blood cell Hb, Hbpl is plasma Hb, SaO2 is arterial O2 saturation, SaO2 RBC is arterial RBC O2 saturation, SaO2 pl is arterial plasma O2 saturation, SplO2 is plasma O2 saturation, SvO2 is venous O2 saturation, SvO2 RBC is venous RBC O2 saturation, SvO2 pl is venous plasma O2 saturation, a-vCo2 is arteriovenous oxygen content, and CaO2 is arterial O2 content. The OER could be calculated as the fractional release of O2 from red blood cells, plasma Hb, or total blood Hb. In all calculations, O2 content (ml/dl) is assumed to be 1.34 x Hb concentration (g/dl).
Relative blood volume. The total blood volume cannot be calculated accurately because the initial blood volume is not known. However, knowing the volume of blood removed and the Hct of that blood and with the assumption that the initial blood volume is 65 ml/kg and body weight in kilograms, a dilution factor can be calculated. These factors can then be compared for the different solutions studied. For example, if the Hct before and after resuscitation are Hct1 and Hct2, the red cell mass RCM1 and RCM2, and the total blood volumes TBV1 and TBV2, respectively, and if the volume of the removed blood is Vb and its Hct is Hctb, then
 | (8) |
which reduces to
 | (9) |
assuming the starting blood volume is 65 ml/kg. If the Hct of the removed blood is Hct1, then
 | (10) |
Statistics. Data are presented as means ± SE. Data were tested for normal distribution by using the Brown-Forsythe test. All data with equal variance were analyzed by using a two-way repeated-measures ANOVA, with treatment group and time as the two factors. Differences within a given treatment group from the baseline value (over time) were tested with Dunnett's test. Differences between treatment groups at a specific time point were tested by using Student's t-test, with comparisons made only to the PS group. Data with unequal variance were rank-transformed, and the tests described above were applied to the transformed data. All data were analyzed by using JMP Statistical Software (version 4.0.4, SAS Institute, Cary, NC). Differences were considered significant if P < 0.05.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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4.0 cP. Thus PS and HS4 are well matched to each other and to blood, whereas the viscosity of HS2 and MP4 is less than that of blood but greater than the viscosity of SFH. Although oncotic pressures [colloid oncotic pressure (COP)] generally parallel viscosity, the relationship is not linear. Thus PS, whose viscosity is 4.0 cP, has a COP of 90 mmHg, but HS4, also with a viscosity of 4 cP, has a significantly higher COP (129 mmHg). The O2 affinities of the MalPEG-Hb solutions are all very high, and we cannot distinguish between the values given in Table 1 for MP4, HS2, and HS4. The O2 half saturation pressure of SFH is significantly higher than those of the MalPEG-Hb solutions, and its cooperativity (Hill coeffi-cient) is greater. Experimental groups and overall survival. A total of 38 pigs were studied. The range of body weights was 17.0-27.5 kg (95% confidence interval, 20.6-22.3 kg). The mean weight of the SFH group (24.33 ± 0.72 kg) was significantly greater than that of the HS2 (20.25 ± 0.73 kg) and the PS (20.06 ± 0.72 kg) groups. There were no other significant differences between mean body weights. Three animals died before completion of the experimental protocol and are not included in the data analysis. One animal had received PS, and one had received HS4. The third animal had been randomized to receive MP4, but died before the beginning of resuscitation.
Hematological response to hemorrhage and resuscitation. Designations of statistical differences are described in the text, but are omitted from Figs. 1, 2, 3, 4, 5, 6, 7, 8 to avoid confusion from the number of groups and time points. There were some small but significant differences in Hct and total Hb values at baseline, resulting from variability inherent in pigs of this size and age (Table 2). The Hct changed slightly, but not significantly, at the completion of hemorrhage and fell dramatically during resuscitation, remaining at 50% of baseline level for the duration of the study. After resuscitation, Hct was significantly higher than PS in the MP4 group and SFH group, and this difference persisted only in the SFH group for the duration of the observation period, presumably because SFH has the lowest COP and, therefore, leads to less hemodilution. As expected, the total Hb and plasma Hb were significantly higher in all animals that received Hb-containing solutions (Fig. 1, Table 2). Plasma Hb was measurable only in the four groups of animals treated with Hb, contributing 20% of the total Hb in animals treated with MP4, HS4, and SFH, and 10% in animals treated with HS2.
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The degree of hemodilution in each of the experimental groups is given by the ratio TBV1/TBV2 (Table 2). The calculated values are only approximations, but indicate that the degree of plasma expansion is related to the oncotic pressure of the test solutions (Table 1). In particular, it should be noted that the animals that received SFH were underresuscitated relative to the other groups. The degree of blood volume expansion for SFH is significantly less than that for the PS group immediately postresuscitation and 60 min later.
Blood-gas and acid-base status. Arterial PO2 fell slightly after resuscitation in the MP4, HS2, and HS4 animals (Table 3), and this was significant in the HS4 group (P < 0.05 from baseline). These values did not reach statistical significance compared with those of the PS group. The mixed-venous PO2 fell in all groups after hemorrhage and remained below baseline in each of the groups administered Hb solutions. Mixed-venous PO2 was significantly lower in the SFH animals compared with the PS animals immediately postresuscitation, but there were no other statistically significant differences between groups. Arterial pH values fell significantly from baseline in all groups after resuscitation, and these recovered in all groups except the SFH-treated animals. Arterial pH was significantly less in SFH animals compared with PS-treated animals at 120 min postresuscitation. Finally, arterial BE fell in all groups but failed to recover only in the SFH animals. At 120 min postresuscitation, BE was significantly lower in this group (-1.43 ± 2.09 meq/l) compared with the PS animals (6.76 ± 1.78 meq/l).
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Systemic hemodynamic response. Baseline values for hemodynamic parameters are reported in Table 4. There were no differences in the baseline values for any of these variables, and there were no differences immediately after hemorrhage in any of the groups. After resuscitation, cardiac index rose above baseline in the PS, HS2, and HS4 animals. Cardiac index increased less in the MP4 and SFH animals, and the cardiac index was significantly lower in MP4 animals (resuscitation to 60 min) and in SFH animals (resuscitation to 120 min), compared with PS (Fig. 2). Cardiac index was maintained slightly above baseline in MP4 animals and actually fell below baseline in SFH-treated animals, reflecting the difference in COP.
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MAP rose to values greater than baseline in all groups that received Hb-containing solutions, regardless of their formulation, but not in PS animals (Fig. 3A). MAP was significantly greater in all groups, compared with PS, from immediately after resuscitation to 75 min postresuscitation, but had returned to baseline in all groups at 120 min. Similar results were found for CVP. CVP was significantly lower after infusion of SFH, compared with PS, from resuscitation to 30 min postresuscitation, consistent with its lower COP.
SVRI, calculated according to Eq. 1, is best examined as differences between measured value and baseline (Fig. 3B) because of individual differences in baseline values among animals. SVRI was unchanged immediately after hemorrhage in all groups, but fell significantly below the baseline value after administration of PS and for 90 min thereafter. SVRI fell immediately after resuscitation with HS2, but was unchanged at all other time points after administration of MalPEG-Hb solutions. With SFH, SVRI rose significantly above baseline at 30-60 min postresuscitation and was significantly greater than PS at all time points (resuscitation to 120 min).
Pulmonary hemodynamic response. MPAP fell during hemorrhage and increased above baseline on resuscitation in all groups (Fig. 4A). The pattern of response of the MPAP was identical to the response in systemic pressure, except that the sensitivity in the pulmonary circulation appears to have been greater in all groups. Compared with infusion of PS, MPAP values were significantly higher in all groups immediately after resuscitation through 45 min postresuscitation. MPAP recovered toward baseline in each of the groups but remained significantly elevated from baseline values in the MP4 and SFH groups at the completion of the protocol (120 min postresuscitation).
We did not measure left arterial pressure or pulmonary capillary wedge pressure in these experiments, so calculation of PVRI* according to Eq. 2 is an approximation and, therefore, an overestimation. As with SVRI, because of variability between animals, the PVRI* is best appreciated by examining the differences at each time point relative to baseline (Fig. 4B). This analysis shows that SFH produces the greatest degree of pulmonary vascular resistance but that some increase is also seen for MP4 and, to a still lesser degree, HS4. PVRI* was elevated significantly above baseline in SFH (resuscitation through 120 min) and MP4 (resuscitation through 60 min). The formulations HS2 and HS4 did not result in elevated resistance from baseline at any point. Thus, with the caution that absolute resistance cannot be calculated from our data, the analysis of PVRI* suggests that the effect on the pulmonary circulation is in the order SFH > MP4 > HS4 > HS2, and that PS causes a slight, but not significant, reduction in pulmonary resistance from baseline.
Heart rate and stroke volume index. The heart rate response to hemorrhage was quite variable (Fig. 5A), and there were no statistically significant differences among the groups at any point in the experiment. However, as significant differences did occur in the cardiac output (Fig. 2), the computed stroke volume index (SVI = cardiac index/heart rate) is different for the various groups (Fig. 5B). The SVI fell equally in all groups during hemorrhage. After resuscitation, SVI increased signifi-cantly above baseline in PS, HS2, and HS4 groups but did not change in the MP4 and SFH groups. SVI actually tended to fall below baseline after resuscitation with SFH, although the decline did not reach statistical significance from baseline. Compared with the PS group, SVI was significantly lower in SFH (resuscitation through 120 min) and in the MP4 group (resuscitation through 45 min).
Blood-gas and oxygen transport. Arterial O2 content fell with hemorrhage and resuscitation due to loss of red cell mass and subsequent hemodilution after infusion of the resuscitation fluids. However, perfusion and oxygenation of tissue appeared to be adequate in all of the groups of animals, except those that received SFH, as indicated by BE (Fig. 6A) and lactic acid (Fig. 6B) measurements. BE fell significantly from baseline in all groups during hemorrhage, resuscitation, and 15 min postresuscitation. This began to normalize in all groups at 30 min after resuscitation, but continued to deteriorate in SFH animals, and BE was significantly different from baseline and compared with the PS group (P < 0.05) between 75 and 120 postresuscitation. Although lactic acid did not normalize completely in any of the groups, the trend was in the direction of normalization, except in the SFH animals, in which the trend reversed and lactic acid appeared to be rising by the end of the observation period.
O2 was not significantly different among the various groups, as shown by a comparison of relative changes (Fig. 7A). O2 was maintained in all groups except the PS animals, in which O2 fell significantly from baseline values and did not recover (Fig. 7B). Statistical significance was achieved for differences between Hb-resuscitated and PS-treated animals from 45 min to the end of the experiment at 120 min postresuscitation.
The amount of O2 delivered by plasma Hb is small in all cases because of the low-plasma Hb concentration (Table 2). Thus the maintenance of O2 in these severely anemic animals after resuscitation and hemodilution is a result, in part, of elevated OER both from red blood cells and from plasma Hb (Fig. 8). Oxygen extraction from red blood cells (Fig. 8A) increased significantly during hemorrhage in all groups, normalized after resuscitation, and increased subsequently in all groups except PS (P < 0.05 at 45-120 min). Interestingly, the OER from red blood cells was significantly lower in the PS animals compared with animals that received Hb, regardless of the formulation, suggesting that plasma Hb facilitates O2 from red blood cells. Oxygen extraction from plasma Hb was greatest in SFH animals and intermediate in animals resuscitated with MalPEG-Hb solutions (Fig. 8B), reflecting the lower oxygen affinity of SFH compared with MalPEG-Hb.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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-cross-linked Hb (38). The present study was carried out to determine whether a new Hb derivative, based on our current understanding of the mechanism of vasoconstriction (16), would demonstrate vasoconstriction in swine, considered to be a sensitive animal model. Because -cross-linked Hb is no longer available, due to the shutdown of the US Army's pilot plant, we used SFH as a reference material. The primary result of these studies demonstrates that formulations of MalPEG-Hb did not promote an increase in systemic vascular resistance, a direct measure of vasoconstriction, in contrast to numerous reports with other formulations of cell-free Hb (4, 6, 19, 35). SFH, used as a control solution, produced a greater rise in systemic and pulmonary vascular resistance and was, therefore, less effective in satisfying tissue O2 demands as demonstrated by significantly less correction in BE and lactic acid after resuscitation. These studies with PEG-modified human Hb extend, in a large animal model, our previous observations with PEG-modified bovine Hb in rats (39) and underscore the importance of eliminating vasoconstriction in achieving adequate resuscitation from hemorrhagic shock.
The hemodynamic consequences of administration of unmodified cell-free Hb have been known for many years, and the studies demonstrating hypertension, vasoconstriction, reduced cardiac output, and bradycardia in animal models and humans have been reviewed recently (38). Hess and coworkers (10) reported sustained (>3 h) increases in MAP and MPAP in pigs after hemorrhage and resuscitation with unmodified Hb and -Hb. They also showed that cardiac output failed to rise after resuscitation, and systemic vascular and pulmonary resistance paralleled the increased pressure. Moreover, despite the increased arterial oxygen content contributed from the plasma Hb, mixed-venous oxygen content was also elevated, and oxygen utilization was not improved in the animals receiving Hb compared with those receiving human serum albumin or lactated Ringer solution. The authors concluded that the potential benefit from improved oxygen-carrying capacity was offset by the systemic vasoconstriction. This conclusion was supported by a recent study in which exchange transfusion with Hb solutions was followed by a 60% hemorrhage in conscious rats (39). In that study (39), exchange transfusion with -Hb initially increased MAP and systemic vascular resistance and decreased heart rate. The vasoconstriction in rats transfused with -Hb was exacerbated during subsequent hemorrhage to such an extent that O2 and O2 were limited, acid-base disturbances were evident, and survival was compromised. That report concluded that the pressor effect of -Hb did not correlate positively with survival but, instead, caused the greater mortality seen in those animals. In the same study, animals transfused with PEG-modified bovine Hb exhibited no vasoconstriction, no acid-base disturbance, no decline in cardiac output, and demonstrated the greatest survival. Thus, in addition to the obvious requirement for oxygen-carrying capacity, the need for the lack of vasopressor activity in Hb-based oxygen carriers is clear from the foregoing studies.
Whereas a number of modifications to the Hb molecule have attempted to reduce the hypertensive actions of cell-free Hb, the studies published to date have not demonstrated the elimination of these effects (4, 6, 19, 35). Data from clinical studies of polymerized human Hb report efficacy without hypertension in cases of acute trauma (8, 17), but published data from preclinical studies are lacking. In contrast, the cumulative evidence with PEG-modified Hb (2, 3, 36, 39) demonstrates the lack of hemodynamic actions in a number of animal models. The primary observation in the present study, i.e., lack of hypertension or rise in systemic vascular resistance, represents the first publication, to our knowledge, of this finding with PEG-modified Hb in a large-animal model of hemorrhage and resuscitation. This is an important fundamental observation regarding Hb-based oxygen carriers, especially in view of the current lack of consensus on several issues. First, the mechanism of vasoconstriction and hypertension observed with Hb-based solutions is unclear, and potential candidates include NO-scavenging (5, 20) endothelin release (14), potentiation of catecholamines (9), and alteration of autoregulation (16). Second, the number of different Hb formulations and animal models has hampered comparison between laboratories and published observations. Third, despite the fact that vasoconstriction with most Hb-based solutions in preclinical models is well recognized, this agreement does not extend to clinical development, and programs are still in place with a number of different solutions shown to induce hypertension and vasoconstriction in animals (7, 31). Finally, depending on the specific clinical indication being considered, differing arguments exist as to whether the hypertension is, in fact, an undesirable property of Hb-based oxygen carriers (22). Whereas the current results cannot address most of the above issues, the demonstrated lack of vasoactivity in an animal model known to be sensitive to the vascular actions of Hb provides the first evidence that MalPEG-Hb eliminates the dramatic rise in systemic vascular resistance observed previously with resuscitation using Hb solutions after hemorrhage in pigs (4, 11, 15) or sheep (34).
A secondary observation from the present study relates to the metabolic consequences of resuscitation with the different solutions. Each of the MalPEG-Hb solutions maintained O2 for the duration of the resuscitation period and induced near-complete recovery of both the BE and the lactate concentrations. In contrast, BE and lactate concentrations began to deteriorate in SFH-treated animals 90 min after resuscitation, despite the fact that calculated O2 was similar to that in MalPEG-Hb-treated animals. This difference suggests that O2 during resuscitation is not uniform with different Hb solutions and that maintenance of O2 in SFH-treated animals does not necessarily signal an improvement in regional metabolic status as reflected by the surrogate markers of tissue oxygenation, i.e., BE and lactate. The deterioration in BE and lactate in SFH-treated animals indicates that significant portions of tissues remained ischemic in those animals, most likely as a consequence of regional vasoconstriction and increased peripheral resistance. The poor metabolic recovery in SFH-treated animals, despite equivalent O2 (Fig. 7) and enhanced oxygen extraction (Fig. 8), documents, in our model, the inefficiency of oxygen transport by unmodified Hb (10) and -cross-linked Hb (11, 39) demonstrated previously. Animals resuscitated with PS exhibited a different profile, i.e., BE and lactate recovered similar to that in MalPEG-Hb animals, despite the fact that O2 was significantly lower during the 120-min recovery period. The recovery of BE and lactate suggests the absence of any regional hypoperfusion, an observation supported by the increased cardiac output and decreased peripheral vascular resistance with PS. The observation of reduced O2 after resuscitation with PS is interesting and might reflect a less efficient repayment of the O2 debt incurred during the brief shock period, compared with that observed in animals resuscitated with Hb solutions. Other studies have demonstrated that resuscitation with crystalloid or non-Hb colloidal solutions provides adequate resuscitation from shock, reflected by improvement in hemodynamic and acid-base status (4, 10, 15), and the present results support this view. Whereas our results do not allow clear distinction of the metabolic effects of MalPEG-Hb from those of PS, an improvement in O2 with MalPEG-Hb has been demonstrated previously in the microcirculation of the hamster skinfold preparation (30). That study compared hemodilution with dextran, MalPEG-Hb, and polymerized bovine Hb (Oxyglobin) and demonstrated improvement in perfused capillary density, local tissue O2 and O2, and systemic acid-base status with MalPEG-Hb. The observed improvement with MalPEG-Hb in the hamster model was demonstrated to result from enhanced utilization of oxygen transported by red blood cells (30) to the capillary bed. The present study results suggest that similar effects might occur in the swine hemorrhage model, although the relationship between O2, O2, and markers of metabolic status with Mal-PEG-Hb solutions warrants further experimentation.
The animal model and hemorrhage protocol in the present study were chosen due to prior experience of reproducible hemodynamic and metabolic changes in our laboratory (41) and to facilitate comparison with previously tested Hb-based solutions (4, 15). Three animals (10%) died immediately before or during the resuscitation, confirming the severity of the hemorrhage protocol. In the remaining animals, hemorrhage elicited a reproducible fall in MAP between 35 and 57% and in MPAP between 33 and 43% in the five groups of animals. In addition, the fall in BE (-4.4 to -6.0 meq/l) and rise in lactate concentration (+2.4 to +4.5 meq/l) were similar in each of the five groups of animals, indicating that the hemodynamic and metabolic responses to hemorrhage were similar across the groups. In response to resuscitation, MAP and cardiac output returned to values higher than baseline, yet systemic vascular resistance rose above baseline only in the SFH animals. Pulmonary vascular resistance increased in each of the groups receiving Hb solutions, whereas pulmonary resistance fell in PS-treated animals. These data are difficult to interpret adequately in the absence of left arterial or pulmonary capillary wedge pressure because estimation of resistance requires both pressure and volume data. In the case of the systemic circulation, CVP is available, but, in the case of the pulmonary circulation, we can only speculate about the volume status. If, as seems reasonable, left atrial pressure increased with increased cardiac return (elevated CVP), then the pulmonary resistances that we calculate would be an overestimation. Resolution of this issue must await subsequent measurements of left atrial and/or pulmonary capillary wedge pressures. This shortcoming notwithstanding, the results of this study agree with the observations using unmodified Hb and -Hb reported by Hess et al. (10). In that study, systemic vascular resistance doubled immediately after resuscitation and did not decline with either solution for 4 h. Pulmonary vascular resistance increased two- to threefold over baseline and remained elevated for the same duration. Heart rate declined immediately on resuscitation and remained depressed for the duration of monitoring. In contrast, our present observations with MalPEG-Hb solutions more closely resemble the results with human serum albumin and Ringer lactate in the study by Hess et al. (10), where systemic and pulmonary vascular resistance were unchanged after resuscitation.
The overall comparison of these Hb solutions is confounded somewhat by their differing COP. Thus we estimate that, after restoration of 70% of the shed blood volume, the animals that received SFH were incompletely resuscitated, with blood volumes estimated to be 65% of baseline, compared with 100% for the MalPEG-Hb and PS solutions. This may explain, in part, the less satisfactory response in the SFH animals in regard to acid-base restoration. However, as the primary purpose of the study was to evaluate systemic and pulmonary hemodynamics, we elected to match the dose of Hb, on heme basis, rather than their volume effects. Hence the solutions were matched for volume administered and Hb concentration. The optimal oncotic pressure of a Hb-based O2 carrier has been somewhat controversial (37). One fear has been that oncotic pressure could oppose hydrostatic pressure, which is necessary for glomerular filtration (26). However, recent studies indicate that reduced endothelial swelling and improved microvascular perfusion more than offset any detriment due to increased oncotic pressure in patients undergoing major abdominal surgery (21). Our results support the concept that hypertonic and/or hyperoncotic solutions are beneficial in resuscitation from hemorrhagic shock (6, 23, 29, 36).
The present results indicate that hemodynamic and metabolic resuscitation after hemorrhage can be effectively accomplished with MalPEG-Hb solutions. We did not observe a difference in the hemodynamic actions with the four resuscitation solutions (excluding SFH). In fact, it appeared that the high oncotic pressures of PS, MP4, HS2, and HS4 enabled normalization of hemodynamic parameters before complete transfusion with 70% of shed volume, and adequate resuscitation might have been realized with smaller volumes than those chosen for the present study. This is an important finding in view of the need for resuscitation fluid volumes to be minimized for military use (18, 21). The fact that we observed similar improvement in O2 in the three groups of animals receiving MalPEG-Hb suggests that their ability to oxygenate tissue is not linear with total Hb concentration. The development of an ideal resuscitation solution will ultimately take into consideration the O2 transport characteristics and concentration of modified Hb and the oncotic pressure and viscosity of the solution. However, there appears to be good agreement that vasoconstriction and pressor activity are undesirable, and probably unsafe, qualities for Hb-based resuscitation fluids (7, 17, 22, 27, 28, 38), especially when the mechanism responsible remains poorly understood. The present study lends further evidence to the concept that MalPEG-Hb solutions can be administered without the adverse effects of vasoconstriction and elevated vascular resistance observed by basic and clinical researchers for more than 50 years.
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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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-cross-linked hemoglobin and pyridoxalated hemoglobin polyoxyethylene conjugate solutions on gastrointestinal regional perfusion in hemorrhagic shock. J Trauma 45: 457-469, 1998.[Web of Science][Medline]
-Crosslinked hemoglobin: was failure predicted by preclinical testing? Vox Sang 79: 1-20, 2000.[CrossRef][Web of Science][Medline]
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