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Stroma-free hemoglobin solutions have been the subject of extensive studies as potential acellular oxygen carriers. Oncotic pressure considerations limit the hemoglobin concentration of the solutions (6-8 g/dl) to one-half the normal whole blood values. Furthermore, the free hemoglobin has a short plasma half-life (2-4 hours). In principle, polymerization offers a means of normalizing the oxygen-carrying capacity as well as extending the plasma half-life. Pyridoxylation of hemoglobin prior to polymerization provides an acceptable P50. Pyridoxylated hemoglobin (14-16 g/dl) was polymerized with a 12.5 percent glutaraldehyde solution. Since the goal was to obtain a 15 g per dl solution iso-oncotic with plasma, the polymerization reaction was monitored by the drop in colloid osmotic pressure. The reaction was quenched with 1.3 M lysine when the colloid osmotic pressure reached normal values (20-25 torr). The polymerization yield was 80 percent, with molecular weights ranging from 120,000 to 600,000 Dalton. The polymerized hemoglobin had a binding coefficient of 1.32, a P50 of 16 torr, a Bohr coefficient of -0.12, and a Hill coefficient of 1.7. The viscosity of the solution was 4.5 centipoise. The methemoglobin levels were comparable to that of unpolymerized stroma-free hemoglobin. Polymerized hemoglobin solutions provide a normal oxygen-carrying capacity with a P50 comparable to that of unpolymerized stroma-free hemoglobin solutions.

Stroma-free hemoglobin (SFH) solution is an acellular oxygen carrier under development as a temporary red blood cell (rbc) substitute. SFH will be of value if it contributes to oxygen loading (delivery) and unloading (consumption). If there is no interaction between red cell hemoglobin and SFH, then the composite oxygen dissociation curve for whole blood (WB) can be written as: SWB = rSSFH + (1 - r) Srbc, where S is the fractional saturation, and r is the SFH/WB hemoglobin ratio. This composite whole-blood curve was compared with the calculated curve, and the root mean square difference was 0.9%. Direct and indirect methods of measuring oxygen concentration ([O2]) of whole blood or plasma were compared. The regression line was [O2]indirect = 0.995 [O2]direct + 0.07 (r = 0.995, N = 16, p less than .01). The adequacy of anaerobic separation of plasma was evaluated by measuring the changes in PO2 and PCO2. The PO2 increased by less than 2 torr and the PCO2 decreased by less than 1 torr during in vitro tests. Both the direct and indirect methodology yielded consistent [O2] values for each carrier.

Polymerized pyridoxylated hemoglobin provides a means of obtaining an acellular oxygen carrier that can be infused at a normal hemoglobin concentration, with a normal COP and is stable in vivo. We have also shown that rats can survive a total exchange transfusion with poly SFH-P (Sehgal et al, 1980). It thus become a prime candidate for further in vivo assessment of its oxygen transport characteristics.

Polymerization of pyridoxylated stroma free hemoglobin (poly SFH-P) yields a solution with a normal hemoglobin concentration, normal colloid osmotic pressure (COP), and a suitable P50. In the present study we compared its in vivo O2-carrying capacity and intravascular persistence with those of pyridoxylated hemoglobin (SFH-P), following a partial exchange transfusion (900 ml) in adult baboons (n = 4 in each group). Poly SFH-P in plasma had an O2-carrying capacity that was 77% greater than that achieved with SFH-P (8.0 versus 4.5 vol%). Furthermore, poly SFH-P provided 5.0 vol% at the end of 24 hours and 2.5 vol% at the end of 48 hours; in contrast SFH-P provided only 2.2 vol% at the end of 6 hours and none within 24 hours. A normal COP was maintained throughout the postexchange period with poly SFH-P despite a 68% increase in plasma protein concentration (from 7.1 to 11.9 gm/dl). The plasma P50 of poly SFH-P decreased from 16 torr immediately after exchange to 12 torr at the end of 48 hours. The in vivo rate of conversion to methemoglobin was similar with both solutions. Polymerized pyridoxylated hemoglobin is currently the only modification of hemoglobin solution that approximates the O2-carrying capacity of whole blood and can be infused without altering the plasma COP.

Stroma-free hemoglobin (SFH) and Fluosol-DA (FL-DA) are the two acellular oxygen carriers that represent potential red cell substitutes. Although both can support life at zero hematocrit, their effectiveness as O2 carriers at intermediate hematocrits has been unclear. In the presence of red cells, the efficacy of an acellular O2 carrier can be assessed by its contribution to O2 delivery and O2 consumption. This study evaluated the efficacy of SFH and FL-DA in the presence of red cells at hematocrits less than or equal to 10%. The results illustrate that SFH and FL-DA do contribute to O2 delivery and O2 consumption in the presence of red cells. The data indicate that SFH and FL-DA are both effective acellular O2 carriers.

Risks inherent in the administration of blood products have increased efforts to avoid homologous transfusion. Although this has increased interest in autologous transfusion and intraoperative salvage, little attention has been focused on efforts to enhance endogenous erythropoiesis as a method of minimizing exposure to homologous blood. Recombinant human erythropoietin (rHuEPO) has been shown to enhance erythropoiesis. The purpose of this study is to evaluate the effect of rHuEPO, administered postoperatively, on a model of acute blood loss. Eleven adult male baboons were randomized into two groups. All animals underwent a laparotomy and an exchange transfusion, with 6% hetastarch, to a final hematocrit of 15%. Group I (N = 6) received 1,000 units/kg of recombinant human erythropoietin daily for the first 14 postoperative days. Group II (N = 5) received an equivalent volume of placebo. All animals were given supplemental vitamin B12, folate and 200% of shed iron, as iron dextran IV, after exchange transfusion. Response was observed for a period of 35 days. All animals survived the protocol. There were no adverse reactions to rHuEPO or surgical complications. The hematocrits were similar between groups at baseline and after exchange transfusion. The maximal rate of erythropoiesis was significantly faster in the rHuEPO group (2.1 vs. 1.3%/day; p less than 0.01). The time required to return to hematocrits of 30% (9.9 vs. 17.4 days, p less than 0.001) and to baseline hematocrits (11.9 vs. 32.1 days, p less than 0.01) were both significantly shorter in the rHuEPO group. The data show that rHuEPO accelerates the recovery from anemia in the postoperative setting. Acceleration of erythropoiesis represents another alternative to homologous transfusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Polymerization of SFH-P currently provides the only approach that leads to a hemoglobin solution that approximates the O2 carrying capacity of whole blood and can be infused without altering the COP of plasma. It appears to have an adequate O2 loading and unloading characteristic and a greatly improved intravascular half-life. In addition, stable shelf-life at 4 degrees C of greater than five months, makes it a prime candidate for future pre-clinical and clinical investigations.

In summary, we remain optimistic about the potential value of perfluorochemicals as acellular oxygen carriers. The results of our first patient show that at an FiO2 of 1.0, Fluosol-DA was not necessary. The oxygen content of the plasma alone was adequate, although the FiO2 was at an unsafe level. At the safe FiO2 of 0.6, however, the Fluosol-DA was needed, since the oxygen content of the plasma was inadequate. Fluosol-DA was thus necessary and effective. At an FiO2 = 0.6, the total of the oxygen content of plasma and the oxygen content of Fluosol-DA was adequate. Although these results are encouraging, the temporary nature of this benefit is an important limitation. We hope that the additional data from both our clinical study and the multicenter study will help resolve some of these questions in the future.

The left anterior descending coronary artery was cannulated in anesthetized, open-chest dogs for use as a coronary test bed to assess the coronary vascular actions of oxygenated, unmodified and pyridoxylated, partially cross-linked (polymerized), stroma-free hemoglobin solutions (SFHS). The actions of these SFHSs were assessed in this test bed, by comparison with perfusion with whole blood. Unmodified SFHS caused significant vasoconstriction, whereas pyridoxylated, partially polymerized SFHS did not do so. The existing coronary flow during perfusion with either SFHS preparation, under basal conditions, did not increase during intracoronary infusion of adenosine. Coronary flow-autoregulation was also altered during perfusion with either SFHS, because the "normal" reactive hyperemia response, observed in the control experiments, was not seen. These findings suggest that both unmodified and pyridoxylated cross-linked (70% polymers) hemoglobin preparations possess some vascular activity when tested in the canine coronary circulation.