Cell-free Hemoglobin, Oxygen Off-Load and Vasoconstriction

Different Efficacy In Vitro of Hemoglobin-based Oxygen Carriers and Red Cells

A vast literature exists which either theoretically explore the mechanism of oxygen delivery from blood to the surrounding tissues, or simulates oxygen delivery and absorption by red cells suspension and cell-free hemoglobin solutions using artificial capillary systems. Very little is known on the physiologic efficiency of cell-free hemoglobin solutions to sustain life of isolated organs and tissues.

The oxygen dependent, active transport of metabolites across ileal mucosa, superfused in Ussing chambers, offers a means for testing the oxygen transport efficacy of hemoglobin-based oxygen carriers. The Ussing system was modified by substituting the original oxygenation chambers with membrane oxygenators.

Rabbit intestinal tissues were mounted in Ussing cuvettes perfused with Ringer solutions containing either cell-free sebacoyl crosslinked hemoglobin A (DECA) or red cells. DECA is human hemoglobin intramolecularly crosslinked with a sebacyl residue between the two β 82 lysines of the partner subunits across the β cleft. It does not dissociate into dimers and does not appear in the urines. In 0.1 M Tris-HCl buffer, at pH 7.4, and 37 ° C, its oxygen affinity has a P 50 near 30 mm Hg and an oxygen binding cooperativity with the Hill parameter near 2.0.

Classically in order to maintain tissue metabolism, as monitored by metabolites transport, the perfusates are equilibrated with 95 % oxygen, 5 % carbon dioxide. However when 3 g/dl of DECA were added to the Ringer, upon lowering the amount of oxygen in the equilibrating gas, amino acid and glucose transports needed only 30 % oxygen. At this low oxygen tension either Ringer alone or perfusates containing a suspension of red cells, diluted to a hemoglobin concentration of 3 g/dl, failed to maintain transport.

These experiments show that hemoglobin-based oxygen carriers deliver oxygen to tissue under simulated physiological conditions, with an efficacy higher than that of an equivalent suspension of red cells.

It appears that the mechanism of oxygen transport by cell-free hemoglobin is different from that of red cells. Oxygen transport by red cells is regulated, as classically proposed, by oxygen partial pressure, oxygen affinity, rate of oxygen release from hemoglobin, rate of diffusion of molecular oxygen and rate of oxygen consumption. In contrast oxygen transport by cell-free HBOC is a phenomenon practically unregulated, subject only to the rate of metabolic consumption. This encourages a revision of the design of red cell substitutes, so far based solely on the attempt of mimicking the oxygen affinity of blood.