Preparation and Characterization of Crosslinked and Polymerized Hemoglobin Solutions

Abstract

In 1982 we synthesized 2-Nor-2-formylpyridoxal 5′-phosphate (NFPLP) and subsequently showed that coupling of the ß chains of hemoglobin (Hb) by this organic phosphate compound according to Benesch et al. (1) lowers the oxygen affinity and prolongs the retention time in the circulation of rats and rabbits with a factor 3 by prevention of excretion via the kidneys. Optimal conditions for the purification of HbNFPLP either by ion-exchange chromatography or by heat treatment were established with recoveries of 70% and 85%, respectively. By extrapolation from the data in rats and rabbits a half life of about 8 hours can be expected in the circulation of humans. However, under some conditions a further prolongation is required.

The aim of further modification of HbNFPLP was to achieve a retention time of about 24 hours. Polymerization with glutaraldehyde to polyHbNFPLP resulted in a mixture of polymers of different size. We determined the optimal degree of polymerization with respect to the effects on vascular retention time, oncotic activity, viscosity and oxygen affinity. Depending on the degree of polymerization we found in rats a 5-to 7-fold increase in vascular half-life compared to native Hb. The change in oxygen affinity was found to be independent of the polymer size (P₅₀ = 18–22 mmHg). A limiting factor for polymerization is the increase in viscosity, which was dramatic when large polymers (>300 kD) were present in the preparation. Polymerization to an average molecular mass of 200 kD was considered optimal: a vascular half-life of 15 hours in rats, an iso-oncotic concentration of 10 g Hb/100 ml and a viscosity of 1.5 cp, close to plasma viscosity (1.2 cp). We conclude that polyHbNFPLP can be produced reproducibly and is a potential oxygen-carrying plasma expander. Finally, the properties of the crosslinked and polymerized Hb solutions are compared.