Oxygen Gradients In Vivo Seen by a High Oxygen Affinity HB Polymer

Oxygen Gradients In Vivo Seen by a High Oxygen Affinity HB Polymer

Enrico Bucci

and Raymond C. Koehler

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. ZL- HbBv 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 exces- sive oxygen delivery, while at high plasma viscosity it produced a compen- sating 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

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1Department of Biochemistry and Molecular Biology, University of Maryland, Baltimore Medical School, 108 N. Greene St., Baltimore, MD 21201, USA

2Department of Anesthesiology and Critical Care, Johns Hopkins Medical Institutions, 600 N. Wolfe St., Baltimore, MD 21205, USA

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 cooperativ- ity similar to those of blood, namely P50 = 27 mmHg and a binding cooper- ativity 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 con- sistent 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

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 trans- ported amount is released only when the gradient becomes compatible with its oxygen affinity, assuring a large amount of oxygen even at low partial pres- sure 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 deliv- ery 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 car- riers exposed to the gradients of partial pressure shown in parenthesis. Square and circle are for cooperativity with n = 3 and n = 1 respectively

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 deliv- ery is still near 60 % between P50 of 4 and 50 mmHg. More dramatic are sim- ulations 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 pro-

duced 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

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.

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 6h of middle cerebral artery occlusion

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% CO

. 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].

Fig. 4. Percent change in dia- meter of pial cerebral arteries (<50 mm) in anesthetized cats in a time control group, and 1 h after exchange transfusion with solu- tions 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

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 trans- port. 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 con- sistent 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 characteris- tics of cell free carriers are not a limiting factor for their physiologic compe- tence. 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 vasodila- tion 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 impor- tant to recognize the potentially excessive delivery of oxygen of a high affin- ity 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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