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Department of Medicine, School of Medicine, University of California, San Diego, and Department of Veterans Affairs Medical Center, San Diego, California 92161
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES
ABSTRACT 
Migita, Russell, Armando Gonzales, Maria L. Gonzales, Kim D. Vandegriff, and Robert M. Winslow. Blood volume and cardiac index
in rats after exchange transfusion with hemoglobin-based oxygen
carriers. J. Appl. Physiol. 82(6):
1995-2002, 1997.
We have measured plasma volume and cardiac index
in rats after 50% isovolemic exchange transfusion with human
hemoglobin cross-linked between the 
-chains with
bis(3,5-dibromosalicyl)fumarate (Hb) and with bovine hemoglobin
modified with polyethylene glycol (PEGHb). Hb and PEGHb differ in
colloid osmotic pressure (23.4 and 118.0 Torr, respectively), oxygen
affinity (oxygen half-saturation pressure of hemoglobin = 30.0 and 10.2 Torr, respectively), viscosity (1.00 and 3.39 cP, respectively), and
molecular weight (64,400 and 105,000, respectively). Plasma volume was
measured by Evans blue dye dilution modified for interference by plasma
hemoglobin. Blood volumes in PEGHb-treated animals were significantly
elevated (74.0 ± 3.5 ml/kg) compared with animals treated with
Hb (49.0 ± 1.2 ml/kg) or Ringer lactate (48.0 ± 2.0 ml/kg) or with controls (58.2 ± 1.9 ml/kg). Heart rate reduction
after Hb exchange is opposite to that expected with blood volume
contraction, suggesting that Hb may have a direct myocardial
depressant action. The apparently slow elimination of PEGHb during the
2 h after its injection is a consequence of plasma volume expansion:
when absolute hemoglobin (concentration × plasma volume) is
compared for PEGHb and Hb, no difference in their elimination
rates is found. These studies emphasize the need to understand blood
volume regulation when the effects of cell-free hemoglobin on
hemodynamic measurements are evaluated.
INTRODUCTION HEMOGLOBIN-BASED OXYGEN carriers are not yet available
for routine blood replacement, because the many biological consequences of high-concentration cell-free hemoglobin are not completely understood. Among these is a propensity to raise systemic blood pressure in humans and animals (28). Impurities were initially implicated as a cause (6, 23), but extensive purification has not
eliminated this unwanted property. Several factors can potentially
contribute to vasoconstriction; among them are vasoactivity due to NO
scavenging (19), local autoregulation in the microcirculation (15, 26),
and adrenergic stimulation. At least part of the cause for the
vasoactivity is believed to be the high affinity of hemoglobin for NO
(8), but to what extent the expansion of circulating blood volume due
to oncotic effects of hemoglobin plays an additional role has not been
established.
Oncotic pressure is only one of a number of properties of cell-free
hemoglobin that will affect its physiological effects and clinical
utility. For example, modified hemoglobins leave the plasma at
different rates, presumably as a consequence of the specific type of
modification and dose; polymerized and conjugated hemoglobins have the
longest intravascular retention times, and intramolecular cross-linked
hemoglobin appears to have the shortest plasma retention time (13).
Hemoglobin solutions may also affect vascular permeability (2, 22). It
is important to determine the most appropriate combination of oncotic
pressure and vascular retention for a clinical application: higher
oncotic pressure may be useful in shock, but in other situations,
hyperoncotic hemoglobin solutions have been suggested to cause tissue
damage in the heart and liver (4) or volume overload. Quantitative data
to describe the effect of hemoglobin solutions on blood volume are not
available in the literature. The purpose of the present study is to
examine such effects with two representative solutions with markedly
different oncotic properties.
Numerous methods have been used to determine plasma volume, usually by
radiolabeled albumin (16, 21, 27) or by dye-dilution techniques (1, 9,
25). Evans blue dye (EBD, T-1824), an inert material used for >50
years for plasma volume determination (7), is attractive in blood
substitute research, because measurement is simple, it is
nonradioactive, and it has a long history of use in rats and other
small animals.
The use of EBD in the presence of cell-free plasma hemoglobin raises
several special problems: 1) the
optical spectra of EBD and hemoglobin overlap;
2) EBD binds to albumin and leaves
the circulation at a rate that reflects the transudation of albumin through the vessel wall (7), and after a few hours EBD begins to
reenter the bloodstream via the lymphatics; and
3) EBD, a diazo compound, could bind
significantly to hemoglobin, as it does to albumin, and could alter the
optical spectrum of hemoglobin and/or albumin. One objective of
the present study is to validate the EBD method in the presence of
cell-free hemoglobin.
METHODS All experiments and procedures were approved by the San Diego Veterans
Affairs Medical Center Animal Studies Subcommittee.
hemoglobin; cardiac output; polyethylene glycol-modified
hemoglobin; blood substitutes; colloid osmotic pressure; plasma volume; Evans blue dye; bovine hemoglobin
-chains with bis(3,5-dibromosalicyl)fumarate (Hb) was
prepared at the Letterman Army Institute of Research as previously
described (29). It was formulated in Ringer lactate and supplied at
~15 or 7.9 g/dl. The latter solutions were used for the in
vivo studies. Polyethylene glycol-modified hemoglobin (PEGHb) was a
gift from Enzon (Piscataway, NJ) and was infused at a concentration of
5.5 g/dl. Human serum albumin (25 g/dl stock) was obtained from Baxter Health Care.
Table 1.
Characteristics of hemoglobin solutions
Hb PEGHb
Source
US Army
Enzon
[Hb],
g/dl
7.9
5.5
P50, Torr
30.0
10.2
COP, mmHg
23.4
118.0
Viscosity, CP
1.0
3.39
Mol mass, Da
68,849 (64,400)
123,380 (105,000)
Excluded volume,
nm3
1,034
93,273
Hb, human hemoglobin cross-linked between -chains with
bis(3,5-dibromosalicyl)fumarate; PEGHb, polyethylene-modified
hemoglobin; [Hb], hemoglobin concn; P50, oxygen
half-saturation pressure of hemoglobin; COP, colloid osmotic pressure.
Weight 
average molecular weight was determined by oncotic pressure
measurements; values based on known structure are given in
parentheses.
Hb, PEGHb, and albumin were calculated from the oncotic
pressure measurements (3). Oxygen equilibrium curves were calculated
from absorption spectra at various
PO2 values at 37°C in 0.1 M
bis-tris(hydroxymethyl)aminomethane buffer, 0.1 M
Cl
.
Surgical preparation of animals.
Male Sprague-Dawley rats (210-350 g body wt; Charles River) were
fed ad libitum before all experiments. Rats were anesthetized with 250 µl of a mixture of ketamine (71 mg/ml; Aveco, Fort Dodge IA),
acepromazine (2.85 mg/ml; Fermenta, Kansas City, MO), and xylazine
(2.85 mg/ml; Lloyd Laboratories, Shenandoah, IA). The area of the left
femoral artery and vein was exposed by blunt dissection, and a
polyethylene catheter (PE-50) was placed into the abdominal aorta via
the femoral artery to allow rapid withdrawal of arterial blood. An
identical catheter was placed in the inferior vena cava via the femoral
vein to allow injection of the EBD. Catheters were tunneled
subcutaneously, exteriorized through the tail, and flushed with ~100
µl of normal saline.
Hemodynamic monitoring.
The femoral artery catheter was connected through a stopcock to a
pressure transducer (model 1050, UFI, Morro, CA), and arterial pressure
was recorded continuously using a data-collection system (model
MP100WSW, BIOPAC Systems, Goleta, CA). The data were stored in digital
form for subsequent analysis. Cardiac output was measured by injection
of cold saline via the jugular vein. Mixing occurs in the pulmonary
capillary beds, and cardiac output was measured by a thermodilution
catheter and cardiac output computer (Columbia Instruments, Columbus,
OH). Systolic, diastolic, and mean pressures were also calculated for
each heartbeat. Mean values were averaged for each minute of data
collected at a rate of 100 Hz.
Exchange transfusion.
After anesthesia, rats were allowed to recover for 90 min and placed in
Plexiglas restrainers. The arterial and venous cannulas were flushed
with 200 and 100 µl, respectively, of heparinized saline (100 U/ml).
Test solutions were filtered through a 0.22-µm filter immediately
before infusion. The rats were connected to an infusion pump (model
4262000, Labconco, Kansas City, MO) so that blood was removed from the
femoral artery and exchange fluid was replaced simultaneously via the
femoral vein. Exchange transfusions were done at a rate of ~0.5
ml/min to a total volume of solution that equaled 50% of estimated
total blood volume (60 ml/kg). In the case of Ringer lactate
experiments, a 1:1 exchange led to hemodynamic instability. Thus the
exchange ratio was 2:1, in which the volume of Ringer lactate returned
to the animal was twice that removed.
Several groups of animals were used for these studies. EBD cannot be
given repeatedly to the same animal, so each animal represents a single
time point after exchange transfusion. Five animals were used for each
EBD blood volume determination. Serial cardiac output could be measured
repeatedly in the same animal, however. Thus five rats each were used
for Hb and PEGHb and three for Ringer lactate to measure cardiac
output after exchange transfusion.
Plasma and blood volume determination.
After exchange transfusion, rats were allowed to rest in their
restrainers for 0, 30, 60, or 120 min. Blood volume was measured in
five Hb- and PEGHb-transfused animals at each time point and in
three animals transfused with Ringer lactate at each time point. A
preinjection sample of 150 µl of blood was collected in three
premeasured, heparinized capillary tubes immediately before EBD
injection. This preinjection sample was centrifuged in an IEC Micro MB
centrifuge for 5 min to obtain the preinjection hematocrit. Each tube
was then scored just above the red cell-plasma interface, and 50 µl
of this plasma were pipetted into a 1-ml spectrophotometer cuvette (VWR
Scientific) and diluted 20:1 in 0.1 M
tris(hydroxymethyl)aminomethane · HCl buffer, pH 9.0 (this pH was chosen to minimize the effect of methemoglobin on the
measured spectra). Plasma samples were also used to measure hemoglobin concentration using the Hemocue instrument. Optical spectra of the
EBD-containing plasma samples were measured between 450 and 750 nm
using a diode array spectrophotometer (model 3000, Milton Roy) and
stored. All post-EBD injection samples were handled in a similar
fashion. Immediately after collection of the preinjection sample, EBD
(5 mg/ml solution; New World Trading, DeBary, FL) was drawn into a
Hamilton syringe to a volume that would deliver a dose of 0.05 mg/ml of
predicted plasma volume.
Blood samples were collected immediately before injection of EBD and
then at 5-min intervals for a total of five samples. The absorbance at
620 nm (A620) was corrected
first for the presence of hemoglobin
(A620 blank), then for turbidity
(A740). Measurements at 5, 10, 15, and 20 min after EBD injection were analyzed by linear regression
to determine the extrapolated time 0 EBD A620.
Plasma volume (PLV) was determined as follows
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Hb and PEGHb.
EBD-hemoglobin binding.
To determine the extent to which EBD binding to hemoglobin affects the
measured optical spectrum, solutions of EBD, hemoglobin, and EBD + hemoglobin were compared. Test solutions consisted of rat plasma and
hemoglobin mixtures. Rat plasma was collected in capillary tubes from
cannulated arteries and was subsequently centrifuged and pooled. The
concentration of EBD in the test mixtures was 2.5 µg/ml, and the
hemoglobin concentration was 0.125 mM. Both of these concentrations
approximate the in vivo conditions of subsequent experiments.
Statistical methods.
Differences between all group means were evaluated using a one-way
analysis of variance. Individual differences between groups were
assessed using a post hoc Bonferroni multiple-comparisons test.
Comparisons were performed only when P < 0.05. P < 0.05 was considered
significant.
RESULTS
Hb is taken from our previous studies
(29). The value for PEGHb, 12.3 Torr, was obtained in our laboratory.
COP is significantly higher for PEGHb than for Hb or albumin
(Fig. 1). The molecular weights reported in
Table 1 were calculated from the oncotic pressure measurements (3) and
are therefore number-average values. The values in parentheses in Table
1 were calculated from the known structure of the molecules in the case
of Hb and the value reported previously from PEGHb (20). The
number-average molecular weight of PEGHb is significantly higher than
its calculated molecular weight or the molecular weight of Hb and
is consistent with the high excluded volume. Thus, although the protein
mass of PEGHb is less than twice that of Hb, the molecule
occupies ~100 times the volume.
,
Polyethylene glycol-modified hemoglobin (PEGHb); 
, albumin; 
,
human hemoglobin cross-linked between -chains with
bis(3,5-dibromosalicyl)fumarate (Hb). Stock solutions were
diluted with normal saline.
Hematocrit. Hematocrit was measured before EBD injection in the same animals used for measurements of plasma and blood volume (Fig. 2). Hematocrits were significantly lower in animals that received PEGHb than in those that received
Hb or Ringer lactate at all time points (0, 30, 60, and 120 min). There was no significant difference between hematocrits
at the various time points within the group that received PEGHb.
Hematocrits in the PEGHb-transfused animals were 16.1% at 0 min,
18.1% at 30 min, 18.9% at 60 min, and 18.0% at 120 min.
), PEGHb (), or Hb (). Largest drop is seen in
PEGHb-treated animals, in which expansion of blood volume is greatest
(see Fig. 4). Values are means ± SE;
n = 5 for each group.
* Significant differences (P < 0.05) between groups treated with Hb and PEGHb.
When animals treated with
Hb were compared with those treated
with Ringer lactate alone, there were significant differences at only
30 and 60 min, with Hb-transfused animals having slightly lower
hematocrits at both time points. Hematocrits in the Hb-transfused group were 21.3% at 0 min, 20.3% at 30 min, 21.8% at 60 min, and 24.4% at 120 min. Hematocrits in the group transfused with Ringer lactate were 22.6% at 0 min posttransfusion, 23.6% at 30 min, 25.0%
at 60 min, and 24.6% at 120 min.
EBD-hemoglobin binding.
Figure 3 shows the spectra of EBD,
hemoglobin, and EBD + hemoglobin in plasma. Differences between the
spectra of the mixture and the sum of both components measured
independently were not significant. Figure 3 also
demonstrates a small but significant overlap of the hemoglobin and EBD
spectra at 620 nm. Thus measurement of
A620 in the plasma just before
injection of EBD is essential to obtain the corrected
A620 due to EBD alone in the
plasma volume experiments (12).
EBD disappearance. Figure 4 shows the disappearance rates for EBD in the plasma in rats after injection. The rate of disappearance of EBD after exchange transfusion with
Hb or hemodilution with
Ringer lactate increases with time (more negative slope), but the rate
in the PEGHb animals remains constant. The difference in rate between Hb- and PEGHb-transfused animals is significant
(P < 0.05) at 60 and 120 min after
exchange, indicating that EBD escapes at approximately twice the rate
of PEGHb.
), PEGHb (), or
Hb (). Values are means ± SE;
n = 5 for each group. A630, absorbance at 630 nm.
Negative slopes indicate a faster rate of Evans blue dye disappearance
from plasma. * Significantly faster
(P < 0.05) disappearance rate for
Hb than for PEGHb 60 and 120 min after exchange transfusion.
Rates for Hb-treated animals are never significantly different
from rates for Ringer lactate-treated animals.
Plasma and blood volume. In control rats not subjected to hemodilution, we found a plasma volume of 39.6 ± 1.3 ml/kg and a blood volume of 58.2 ± 1.9 ml/kg. These values are in excellent agreement with those reported in the literature using 125I-labeled albumin (27), indocyanine green (65.7 ml/kg) (27), and EBD (60.8 ml/kg) (5). Total blood volume adjusted for body weight for the three solutions is shown in Fig. 5. Plasma and blood volumes were significantly higher in animals exchange transfused with PEGHb than in animals exchange transfused with
Hb or Ringer lactate and were significantly higher than in controls at all time
points. Average blood volumes were 50% greater in rats transfused with
PEGHb than in controls immediately after completion of the exchange
transfusion. Mean blood volume in PEGHb-transfused rats was 74 ± 3.5 ml/kg immediately after transfusion, 66 ± 11.5 ml/kg at 30 min, 62 ± 3.9 ml/kg at 60 min, and 61 ± 3.8 ml/kg at 120 min. There were no significant differences between blood volumes when
PEGHb-treated groups were compared at different time points.
), PEGHb (), or Hb (). Blood volume rises after exchange transfusion in PEGHb-treated animals but drops in Hb- and Ringer lactate-treated animals. Values are means ± SE;
n = 5 for each group.
* Significant differences between Hb- and PEGHb-treated
animals (P < 0.05).
When animals treated with
Hb were compared, total blood volumes
were significantly lower than control only at 60 and 120 min. Whereas
blood volumes were consistently higher in the Hb- than in the
Ringer lactate-transfused group, there were no significant differences
between the two groups when post hoc comparisons were made. Mean blood
volumes in Hb-treated animals were 49 ± 1.2 ml/kg at 0 min,
48 ml/kg at 30 min, 45 ± 2.9 ml/kg at 60 min, and 42 ± 2.8 ml/kg at 120 min. When Hb-transfused animals were compared at
different time points, animals had significantly lower blood volumes at
120 min than at 0 and 30 min posttransfusion.
In animals receiving only Ringer lactate solution, blood volumes were
significantly lower than in controls at 0, 60, and 120 min after
transfusion. Mean blood volumes in these animals were 48 ± 2.0 ml/kg immediately after exchange transfusion, 47 ± 2.6 ml/kg at 30 min, 43 ± 0.2 ml/kg at 60 min, and 42 ± 5.8 ml/kg at 120 min.
There were no significant differences in blood volume among animals
receiving Ringer lactate at different time points.
Hemoglobin.
The hemoglobin concentrations ([Hb]) are shown in Fig.
6. Figure
6A shows the measured plasma
concentrations in grams per deciliter. The data were fit to the
expression
![[Hb] = <IT>A</IT>(exp<SUP>−<IT>kt</IT></SUP>)](/content/vol82/issue6/fulltext/1995/img003.gif)
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Hb and PEGHb: 3.53 and >200 h, respectively. Expansion of the
blood volume prevents an accurate picture of the actual clearance rate
of the two hemoglobins, however, because of the dynamic changes in the
blood volume (Fig. 5). The plasma hemoglobin concentration calculated
on a gram per kilogram body weight basis (hemoglobin concentration × plasma volume) is shown in Fig.
6B. Analysis of these curves now
yields identical
t1/2 values for
the two hemoglobins: 2.96 h.
, solid line) and Hb (, dashed line).
A: plasma concentration.
B: absolute hemoglobin. Absolute
values were calculated using hemoglobin concentrations
(A) and plasma volume of
distribution (Fig. 5). Values are means ± SE;
n = 5 for each group.
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Hb and PEGHb, respectively. It is not possible to
calculate absolute disappearance rates over 10 h, because we have blood volume data only for the first 2 h after exchange transfusion. However,
Fig. 5 shows that there was little change in blood volume beyond 2 h
and that blood volume has essentially returned to the control level in
our model by that time. Thus, taken together, these data suggest that
the absolute clearance rate for PEGHb may be biphasic: relative to
Hb, it is initially removed more slowly. This could be a result
of greater molecular size or, possibly, a reduced rate of metabolism.
Blood pressure and heart rate.
In a different series of experiments, continuous mean arterial pressure
readings were obtained from exchange-transfused rats. Five rats
received PEGHb, six received Hb, and six received Ringer lactate
(Fig. 7). The exchange transfusion was
carried out from 30 to ~55 min in these experiments. As shown in Fig. 7, Hb produces a significant
(P < 0.05) rise in mean arterial blood pressure compared with animals treated with Ringer lactate or
PEGHb. Exchange with Hb also produces a significant decrease in
heart rate compared with PEGHb or Ringer lactate
(P < 0.05). The heart rate rises in
the animals treated with Ringer lactate compared with baseline or
PEGHb-treated animals (P < 0.05).
),
Hb (), or Ringer lactate (solid line). Exchange of 50% of
estimated blood volume (0.5 ml/min) was begun at time
0. A: blood pressure
rises (* P < 0.05) in
Hb-treated animals compared with animals treated with PEGHb or
Ringer lactate. B: heart rate rises
(+ P < 0.05) in animals treated with Ringer lactate compared with PEGHb-
and HB-treated animals. * Heart rate after exchange
transfusion is significantly lower in Hb-treated animals than in
animals treated with Ringer lactate or PEGHb
(P < 0.05). Values are means ± SE; n = 5 for each group.
Cardiac output. Cardiac output was measured in a separate series of experiments at 0, 30, 60, and 120 min after 50% exchange transfusion. Results were corrected for body weight and are expressed as cardiac index in Fig. 8. In animals receiving PEGHb, cardiac outputs were markedly elevated over the 120 min after transfusion. There was little change in cardiac output in animals that received
Hb, and cardiac output was decreased in animals receiving only
Ringer lactate in a 50% exchange transfusion. Figure
9 shows that cardiac index is directly
correlated to blood volume.
, n = 3),
PEGHb (, n = 5), or Hb (,
n = 5). Values are means ± SE.
Exchange of 50% of estimated blood volume (0.5 ml/min) is complete at
time 0.
DISCUSSION
Red blood cells exert very little COP. However, cell-free hemoglobin, like other plasma proteins, does exert COP, and, in general, the magnitude of the COP is a colligative property, i.e., related to the concentration of molecules in the plasma. Thus traditional thinking has been to strive to develop hemoglobin solutions as red cell substitutes that minimize COP while maximizing oxygen capacity (10). In contrast, many non-oxygen-carrying plasma expanders, such as the starches, are effective precisely because they do exert COP and thereby maintain the vascular volume and cardiac output (24). Furthermore, considerable success has been achieved in the experimental resuscitation of animals in shock with hypertonic saline dextran (17), again because of its high COP. There has been little attention to the effects of various cell-free hemoglobin solutions on plasma and blood volume. This study compared two hemoglobin solutions with very different oncotic pressures with regard to their effects on blood volume and cardiac output using EBD dilution and thermodilution, respectively.
EBD has been used for many years to measure plasma and blood volumes. It is bound to albumin, and therefore the circulating albumin exchanges with interstitial albumin. The use of EBD to measure plasma volume in the presence of cell-free hemoglobin has not been described in the literature. Therefore, we were concerned 1) because of possible interference of the A620 measurement in plasma, 2) because of possible interaction of hemoglobin and EBD, and 3) because hemoglobin could alter the disappearance volume of albumin from the plasma.
Spectral analysis of mixtures of EBD and hemoglobin solutions compared with spectra of each individually indicate that the EBD or hemoglobin spectra are not fundamentally altered at the wavelengths used in the calculation of plasma volume in our experiments. Furthermore, in preliminary experiments, we established that, by subtracting plasma A620 obtained after exchange transfusion and before EBD injection, we were able to accurately estimate premeasured volumes of blood and hemoglobin solutions. Our determination of normalized blood volume in normal rats agreed closely with published observations (5).
In regard to the disappearance of EBD from the plasma, we found that
the rates were significantly different for PEGHb- and Hb-treated
animals, the latter being significantly faster after 60 min and
approximately twice as fast 120 min after exchange transfusion. This
unexpected result raises the interesting possibility that greater
vascular leakiness is induced by Hb than by PEGHb. In fact, the
disappearance rate for the PEGHb animals did not change significantly
over the 120 min of the experiments. This observation may provide a
clue in understanding the interaction of cell-free hemoglobin with
endothelium (18) and is worthy of further experiments.
We found significantly higher blood volumes, lower hematocrits, and
higher cardiac outputs in animals transfused with PEGHb than in those
transfused with Hb or Ringer lactate. These observations are all
physiologically consistent with the oncotic pressures of these
solutions. Influx of extravascular fluid in response to an oncotic
gradient would increase blood volume and dilute the circulating red
cell mass, causing a decreased hematocrit. Expanded blood volume should
increase output, according to Starling's law.
The heart rate response to exchange transfusion is worthy of special
comment. Tachycardia is expected when blood volume is depleted. Indeed,
this is the case with exchange transfusion with Ringer lactate.
However, Hb and Ringer lactate produce the same contraction of
the blood volume (Fig. 5), but Hb depresses the heart rate (Fig.
7B). Thus it appears that the
relative bradycardia produced by Hb is not volume mediated, and
our results suggest a direct depressive effect on the heart.
The lack of a significant change in blood pressure in PEGHb-treated
animals is not easily understood. However, it does raise the question,
Can the vasoactive property of hemoglobin be generalized to all
molecules? Moreover, until more detail is known about the ability of
PEGHb to bind and release NO, whether this apparent difference between
PEGHb and Hb is due to different reactivities with NO or to some
other property will remain unknown. There are at least two alternative
explanations for the different vasoactivities. First, PEGHb is a much
larger molecule (Table 1), and therefore its entry into the
interstitial space might be restricted. Indeed, this is suggested by
the difference in EBD disappearance slopes shown in Fig. 4. Second, the
oxygen binding curves of the two solutions are markedly different
(unpublished data): PEGHb displays a lower oxygen half-saturation
pressure of hemoglobin and Hill coefficient, whereas these two
properties of Hb are closer to the values for blood.
Hb-transfused animals show a slight but significant decrease in
blood volume compared with controls but a large increase in blood
pressure, in agreement with data reported by Hess and co-workers (14)
in a dehydrated swine shock model. Thus, whereas Hb may be less
oncotically active than PEGHb, it appears to be more vasoactive. In
experiments carried out by Gulati and others (11), increases in cardiac
output after infusion of commercially prepared Hb (DCLHb)
resulted in an increase, rather than a decrease, in cardiac output. It
is not clear whether this discrepancy is due to some difference in the
solutions or in the protocols studied. Resolution of the difference
would be important as the solutions near clinical use. As expected, our
animals that received Ringer lactate had lower blood volumes, higher
hematocrits, and lower cardiac outputs than animals in the Hb or
the PEGHb group.
The oncotic effect on blood volume has important implications for the
estimation of plasma retention times of modified hemoglobins. In our
experiments, we were able to calculate the absolute plasma retention
(hemoglobin concentration × plasma volume) in addition to
hemoglobin concentration alone. This analysis (Fig. 6) led to a
surprising result: whereas the plasma hemoglobin concentration t1/2 values for
Hb and PEGHb are markedly different, the absolute rate of
disappearance from the circulation is approximately the same. This
phenomenon should be taken into account when pharmacokinetic studies of
any protein with oncotic activity are performed.
The model we have chosen for the present studies is not intended to be a clinical model. Rather, it was selected to permit study of fluid shifts after administration of hemoglobin solutions. In an elective surgical setting, hemodilution would probably be less aggressive, and the effects on blood volume and hemodynamics would not be as severe as we report here. In shock-resuscitation, where larger volumes might be used, oncotic effects could be much more significant. However, the importance of the model and of these experiments is to demonstrate that each modified hemoglobin may have different physiological effects and that, to use cell-free hemoglobin solutions wisely and safely in clinical settings, these effects must first be understood.
ACKNOWLEDGEMENTS
The authors thank Renee Schad for expert assistance in preparation of the manuscript.
FOOTNOTES
Address for reprint requests: R. M. Winslow, VA Medical Center, 3350 La Jolla Village Dr., San Diego, CA 92161.
Received 30 July 1996; accepted in final form 29 January 1997.
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