Oxygen Gradients In Vivo Seen by a
High Oxygen Affinity HB Polymer
Enrico Bucci1 and Raymond C. Koehler2
Summary. Cell-free hemoglobin based oxygen carriers (HBOC) wet the
endothelial surfaces and deliver oxygen directly to tissues, bypassing plasma.
Simulations show that carriers with oxygen affinity higher than blood would
best deliver oxygen to tissues, although good delivery is produced within a
large range of affinities. We tested this hypothesis using a solution of either
a high oxygen affinity polymer (ZL-HbBv, P50 = 4 mmHg) or of sebacoyl
crosslinked hemoglobin, DECA, with P50 = 30 mmHg. The polymer does not
extravasate and does not produce a pressor response in infused animals. ZLHbBv decreased the volume of cerebral infarct by 40% in mice, while in the
cat the lower affinity DECA failed to reduce the infarct volume. At reduced
plasma viscosity ZL-HbBv produced a cerebral vasoconstriction due to excessive oxygen delivery, while at high plasma viscosity it produced a compensating vasodilation. In rabbit jejunum membranes, superfused under hypoxic
conditions, the presence of the DECA allowed metabolites transport across
the mucosa. Equivalent suspensions of red cells failed to allow transport.
It is suggested that non-extravasating HBOC with high oxygen affinity can
still deliver oxygen to ischemic tissues. Under nonischemic conditions with
reduced blood viscosity cerebral vasoconstriction appears to occur in
response to hyperoxygenation of tissues.
Key words. Blood substitutes, Oxygen gradients, Stroke, Microcirculation,
Zero-link polymers
Department of Biochemistry and Molecular Biology, University of Maryland, Baltimore
Medical School, 108 N. Greene St., Baltimore, MD 21201, USA
2
Department of Anesthesiology and Critical Care, Johns Hopkins Medical Institutions, 600
N. Wolfe St., Baltimore, MD 21205, USA
1
62
Oxygen Gradients and a High Affinity Hb Polymer
63
Introduction
The design of hemoglobin based blood substitutes should satisfy two main
parameters: oxygen affinity and size. The prevailing opinion is that the ideal
cell-free oxygen carrier should have oxygen affinity, and binding cooperativity similar to those of blood, namely P50 = 27 mmHg and a binding cooperativity with the index “n” near 2.7.
This opinion has been challenged by Vandegriff and Winslow [1], who claim
that the oxygen carriers should have affinities higher than that of blood.
Regarding size, the old concept that stabilized tetramers were big enough to
prevent extravasation because they did not appear in the urine of infused
animal, was incorrect. In fact extravasation still was detectable in the
lymphatics [2], and was associated with large increase of mean arterial
pressure.
Recently we obtained data using a high affinity, nonextravasating polymer
of bovine hemoglobin and a crosslinked hemoglobin with affinity similar to
that of blood. Data on brain microcirculation and focal ischemia were consistent with numerical simulations anticipating oxygen delivery as function
of oxygen affinity in vivo.
Gradients of Oxygen Pressure
As shown in Fig. 1, blood plasma is an interface which regulates the transport
of oxygen from the lungs to the tissues. A gradient is formed from a partial
pressure of oxygen near 100 mmHg at the lungs, to the partial pressure at the
mitochondrial level, where oxygen pressure is very low. In the absence of a
carrier, the dissolved oxygen would be gradually released in amounts paral-
Fig. 1. Blood as an interface between lungs and mitochondria
64
E. Bucci and R.C. Koehler
lel to the decreasing partial pressure gradient and not much is left when it
reaches the tissues. Instead when oxygen is transported by a carrier, the transported amount is released only when the gradient becomes compatible with
its oxygen affinity, assuring a large amount of oxygen even at low partial pressure of oxygen.
There is a fundamental difference between the transport produced by a cell
free carrier and a carrier segregated inside a membrane as in the red cells. A
stringent regulation is provided by the poor solubility of oxygen in plasma.
Oxygen released by the red cells cannot exceed oxygen solubility, therefore it
is allowed only as replacement of consumption. The result is that red cells are
an excellent buffer of plasma’s oxygen tension. The end point of oxygen delivery by the red cells is plasma, not the tissues. Instead, as anticipated by the
facilitated diffusion across liquid interfaces described by Wittenberg et al. [3],
cell-free carriers chelate oxygen molecules at one end of the interface (in the
lungs) and physically transport them to the other end (the endothelial walls
of the capillaries) bypassing the fluid (plasma) and delivering oxygen directly
across the interface (to tissues).
It is instructive to simulate, using the classical Hill equation, the fractional
release of oxygen by cell free carrier as function of their P50 and binding
cooperativity, when exposed to these gradients. For simulation purposes we
assumed a partial pressure of oxygen at the mitochondria of 2.0 and
Fig. 2. Dependence of fractional delivery of oxygen (DY) on the P50 values of oxygen carriers exposed to the gradients of partial pressure shown in parenthesis. Square and circle
are for cooperativity with n = 3 and n = 1 respectively
Oxygen Gradients and a High Affinity Hb Polymer
65
0.1 mmHg respectively. As shown in Fig. 2, for a gradient between 100 and
2 mmHg, with a cooperativity index n = 3 the delivery is practically the same
for P50 values between 4 and 50 mmHg, where the delivery is more than 90%
of the oxygen content. When the cooperativity decreases to n = 1.0. The delivery is still near 60 % between P50 of 4 and 50 mmHg. More dramatic are simulations assuming a gradient between 100 and 0.1 mmHg, For n = 3 the
delivery is close to 100% at P50’s between 1.0 and 20.0 mmHg, declining only
slightly to 90% at higher P50 values. When n = 1 the delivery is still 90% at
P50 between 2 and 5 mmHg, declining to about 60 % at higher P50 values. In
essence the simulations suggest that best delivery is obtained with low, or very
low P50 values. Also, the curves are flat, suggesting an ample tolerance of
oxygen affinities. Cooperativity increases the delivery.
Brain Focal Ischemia and Microcirculation Evidences
We used a polymer of bovine hemoglobin (ZL-HbBv) [4], and hemoglobin A
intramolecularly crosslinked with sebacic acid (DECA) [5]. Suffice here to say
that ZL-HbBv is a large molecule with hydrodynamic radius Rh = 240 nm,
P50 = 4.0 mmHg and no oxygen binding cooperativity. It does not extravasate
and does not produce a “pressor response” in infused animals. DECA is a
stabilized tetramer which does not dissociate into dimers [5]. It has P50 =
30 mmHg and binding cooperativity with n = 2.0.
Stroke Response to Infusions of ZL-HbBv
Cerebral infarct in mice was produced by occlusion of the middle cerebral
artery [6]. Infusion of ZL-HbBv, with P50 near 4 mmHg, decreased by 40% the
volume of the infarct, probably because of the oxygen delivered to ischemic
tissues by the carrier (Fig. 3). Instead in the cat the size of cerebral stroke was
not reduced by infusions of DECA [7] (Fig. 3). These data suggest that the
high affinity with no binding cooperativity ZL-HbBv was more efficient than
the lower affinity, high cooperativity DECA.
Microvascular Response to Infusions of ZL-HbBv
In the cat, when the viscosity of circulating blood was decreased by anemia,
as produced by exchange transfusions, the oxygen carried by ZL-HbBv produced a moderate decrease in the diameter of the pial arteries, as opposed to
the vasodilation produced by albumin infusion. In the absence of a pressor
response, the reduced arterioles diameter was interpreted as a regulation to
prevent excessive oxygen delivery [4,8]. Conversely, when plasma viscosity
was increased 2.7 times by infusion of PVP, and the vasodilation produced by
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E. Bucci and R.C. Koehler
Fig. 3. Upper panel, A 40% exchange transfusion with ZL-HbBv polymer reduces the
infarct size of mice brain, 1 day after a 2 h period of middle cerebral occlusion. Lower panel,
In the cat a 40% exhange transfusion of DECA failed to reduce the size of the infarct after
6 h of middle cerebral artery occlusion
albumin solutions was not sufficient to maintain a normal oxygen supply, the
oxygen carried by ZL-HbBv produced an extra vasodilation probably as if to
further compensate for the diminished oxygen supply [8] (Fig. 4). These
observations are consistent with simulations showing that carriers with high
oxygen affinity and no oxygen binding cooperativity still transport and
deliver oxygen in vivo. Actually, the oxygen delivery by ZL-HbBv elicited a
regulatory response of either vasoconstriction or vasodilation to compensate
for change in viscosity.
Oxygen Gradients and a High Affinity Hb Polymer
67
Fig. 4. Percent change in diameter of pial cerebral arteries
(<50 mm) in anesthetized cats in
a time control group, and 1 h after
exchange transfusion with solutions of either albumin, albumin
+ PVP, ZL-HbBv, and Zl-HbBv +
PVP. (n = 5 in all groups)
(Adapted from Rebel et al. [8])
Fig. 5. Comparison of metabolite transport across rabbit jejunum membranes superfused
in Ussin chambers with equivalent 3% hemoglobin in perfusates containing either cell-free
DECA or bovine red cells. (Adapted from Bucci et al. [10]). Transport is proportional to
the mA of the short circuit current elicited by the transport
Red Cells Deliver Oxygen only to Surrounding Fluid
Superfusion in Ussin Chambers
Rabbit jejunum membranes transport glucose and amino acids from the
mucosa to the serosa side when superfused with salines in Ussin chambers
[9]. The transport is oxygen sensitive. Ringer perfusates must be equilibrated
with 95% oxygen and 5% CO2. We have shown that with 3% w/v DECA in
Ringer it was possible to equilibrate the perfusate with only 30% oxygen.
Under these conditions Ringer alone would not allow transport. When we
compared the transport obtained with equivalent 3% hemoglobin content in
either DECA solutions or in bovine red cells suspensions, no transport was
produced by the red cells [10] (Fig. 5). It should be stressed that the oxygen
affinity of DECA and bovine red cells are very similar with P50 of 30 and
27 mmHg and a cooperativity index of 2.0 and 2.5, respectively [5,11].
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E. Bucci and R.C. Koehler
As anticipated by the model, the red cells could not deliver oxygen above
the amount dissolved in perfusates equilibrated with 30% oxygen. They only
buffered a partial pressure of oxygen insufficient to allow metabolite transport. Instead the cell-free carrier, with a similar oxygen affinity, bypassed the
Ringer and directly delivered sufficient amounts of oxygen.
Discussion
Although the proposed model is only a gross oversimplification, it is still consistent with the experimental data. The main difference between the delivery
of oxygen by red cells and by cell-free HBOC’s is that, due to the facilitated
diffusion where oxygen molecules are physically transported by the carriers
through the blood interface, cell-free hemoglobins “bypass” plasma.
These considerations strongly suggest that the oxygen affinity characteristics of cell free carriers are not a limiting factor for their physiologic competence. Our data on cerebral infarcts would suggest that high oxygen affinity
carriers are more efficient than the low affinity ones in reducing the injury
size. Also, at reduced viscosity ZL-HbBv seemed to deliver an excessive
amount of oxygen which elicited vasoconstriction of cerebral pial arterioles.
Conversely at high plasma viscosity it produced a compensatory vasodilation.
These opposite effects confirm that both the vasoconstriction and vasodilation were regulatory phenomena stimulated by oxygen delivery.
It should be stressed that the effects of ZL-HbBv on brain arterioles, and in
particular the vasodilation, could be interpreted as due to oxygen delivery
only because ZL-HbBv did not elicit a pressor response. Also, it is very important to recognize the potentially excessive delivery of oxygen of a high affinity carrier. This property of cell free oxygen carriers should be investigated,
so as either to avoid the risk of hyperoxygenation, or to take advantage of it,
according to needs.
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