Preventing the oxidation of hemoglobin in solution is one of the major requirements for the successful production and long-term storage of hemoglobin-based blood substitutes. To this end we have studied the effects of antioxidants on the rate of methemoglobin formation and disappearance in solutions of human and bovine hemoglobin at 4 degrees C and 37 degrees C. Ascorbate and desferal (5 mM) were observed to act as prooxidants, increasing the rate of methemoglobin formation at 37 degrees C. Trehalose, mannitol, glucose, and EDTA (5 mM) had no significant effect. Glutathione and NADH (10 mM) were the most effective antioxidants tested, causing a significant decrease in the rate of methemoglobin formation at 37 degrees C for periods of up to 50 hours. The combination of these antioxidants in bovine hemoglobin at 4 degrees C resulted in the reduction of methemoglobin levels to nearly undetectable levels in approximately 150 hours. In addition, NADH and glutathione were found to reduce methemoglobin levels to 10% over a period of 100 hours in a sample of human hemoglobin that had been stored at 4 degrees C for one year and had 60% methemoglobin. These results suggest that the prevention and reversal of methemoglobin formation during the long-term storage of hemoglobin solutions and hemoglobin-based blood substitutes may now be possible.

For any viable blood substitute, questions of long-term storage and shelf-life must be addressed. Recently, we have made great progress in improving the stability of the blood substitute, liposome encapsulated hemoglobin (LEH). We have concentrated our efforts on protecting LEH in solution and in the long-term preservation of LEH by lyophilization. In particular, we have been able to retard and in some cases, reverse the oxidative process of metHb formation in solution by the addition of antioxidants such as NADH and glutathione. We have been able to regenerate Hb preparations with 60% metHb by the addition of 10 mM NADH and glutathione. In these preparations addition of these antioxidants results in a decrease of metHb levels from 62% to 15% over the course of 12.5 days at 4°C. We have also explored the use of protective solutes such as the disaccharide trehalose in the preservation of LEH in the freeze-dried state. Addition of increasing amounts of trehalose and other disaccharides results in the inhibition of lyophilization-induced fusion events and in the retention of hemoglobin within the unilamellar liposomal vesicles following rehydration.