Alternatives to Transfusion Therapy

Abstract

Summary and Conclusion Attempts to develop a hemoglobin-based red-cell substitute have spanned many decades12,13 but have not yet resulted in a clinically useful product. The issues that have prevented clinical application have been primarily ones of safety and not efficacy.27 Numerous animal studies have documented the efficacy of stroma free hemoglobin.3,12 Though effective, there were limitations that were of concern. Oncotic considerations limit the concentration of the infusate SFH to 6 to 8 g/dL, or half-normal. Owing to the loss of organic phosphate modulators of P50 such as 2,3-DPG, the P50 of SFH is typically between 12 to 14 torr, which again is half the normal value. And finally, the intravascular half-life of SFH is too short, ranging from 2 to 6 hours.3 Polymerization provides a means of correcting these limitations of SFH. The high O2 affinity can be greatly diminished by covalent binding of pyridoxal-5′-phosphate to the N-terminal of the β-chains. COP exerted by a protein solution is proportional to the number of discrete colloid particles. By polymerization, the number of colloid particles are reduced, leading to a decrease in COP. Our data show that this can indeed be achieved in a reproducible fashion. The rate at which this diminution of COP is accomplished determines the yield of polymeric species, as well as their molecular weight distribution. We demonstrate that the polymerization can be reproducibly controlled to result in a yield of 75% to 85% polymers, with a molecular weight distribution of 128,000 to 400,000 daltons. The number average and the weight average molecular weights indicate that the large proportion of the polymers represent the crosslinking of two tetramers. The data that reflect the interaction of O2 with Poly SFH-P indicate that the O2-carrying function of hemoglobin has not been significantly altered by the chemical modifications. The binding coefficient of O2 is unchanged. As one would have anticipated, there is a loss of cooperativity (diminished Hill coefficient) between the hemoglobin chains, suggesting structural restrictions in the polymeric species owing to the crosslinking. One would expect to see a reduced alkaline Bohr effect, and our data confirm that. And finally, one would expect to see some increase in O2 affinity with polymerization. This is indeed the case, though the P50 of Poly SFH-P is comparable to banked blood (18 to 22 torr). A modified hemoglobin solution, to be clinically useful, would need to have a reasonable shelf-life. Our data demonstrate that polymerized hemoglobin can be stored in the cold (4°C to 8°C) for several months with minimum change in methemoglobin concentration. Furthermore, our HPLC and viscosity data clearly indicate that no significant alterations in the polymeric species occur during storage.