Preparation and in vitro characteristics of polymerized pyridoxylated hemoglobin

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Abstract
Stroma-free Hb solutions were the subject of extensive studies as potential acellular O2 carriers. Oncotic pressure considerations limit the Hb concentration of the solutions (6-8 g/dl) to 1/2 the normal whole blood values. The free Hb has a short plasma half-life (2-4 h). In principle, polymerization offers a means of normalizing the O2-carrying capacity as well as extending the plasma half-life. Pyridoxylation of Hb prior to polymerization provides an acceptable P50 [O2 partial pressure at 50% saturation of Hb]. Pyridoxylated Hb (14-16 g/dl) was polymerized with a 12.5% glutaraldehyde solution. Since the goal was to obtain a 15 g/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%, with MW ranging from 120,000-600,000 daltons. The polymerized Hb 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 Hb. Polymerized Hb solutions provide a normal O2-carrying capacity with a P50 comparable to that of unpolymerized stroma-free Hb solutions.