Abstract
ABSTRACT Hemoglobin has been used as raw materials for manufacturing blood substitutes. However, because of its high oxygen affinity and short vascular retention time, limitations on hemoglobin as a blood substitute in clinical therapy have been reported in the literature. To decrease its oxygen affinity, hemoglobin has been modified by pyridoxylation (PLP-Hb) and followed by polymerization with glutaraldehyde. It was reported that the polymerized hemoglobin showed a P50 value of 19 to 22 mm Hg. Nevertheless, the reaction rate of hemoglobin with glutaraldehyde is too fast to control its molecular weight distribution. Additionally, the glutaraldehyde-polymerized hemoglobin is relatively unstable and may release glutaraldehyde residues during storage or sterilization. It was reported that glutaraldehyde is cytotoxic even at low doses. This may impair the biocompatibility of the polymerized products. In an attempt to overcome the aforementioned problems, two naturally occurring crosslinking agents, genipin and reuterin, were used by our group to polymerize hemoglobin. The first study was to investigate the feasibility of using genipin to polymerize hemoglobin as a blood substitute. The results indicated that the rate of hemoglobin polymerization by glutaraldehyde was significantly faster than that by genipin and it readily produced polymers with molecular masses greater than 500,000 daltons. It was found that the maximum degree of hemoglobin polymerization by genipin was approximately 40% if over-polymerization is to be prevented. With increasing the reaction temperature, hemoglobin concentration, and genipin-to-hemoglobin molar ratio, the duration taken to achieve the maximum degree of hemoglobin polymerization by genipin became significantly shorter. The P50 value of the unmodified hemoglobin was 9 mm Hg, while that of the genipin-polymerized PLP-hemoglobin increased to 21 mm Hg. It was found in a rat model that the genipin-polymerized PLP-hemoglobin increased the survive ratio of rats in a 50% blood exchange. The half-life of the unpolymerized hemoglobin in circulation was about 1.5 h, while that of the genipin-polymerized PLP-hemoglobin was approximately 12.5 h. In the second study, we used another naturally occurring crosslinking agent, reuterin, to polymerize hemoglobin. The results indicated that the rate of hemoglobin polymerization by reuterin was significantly slower than that by glutaraldehyde. In an animal study, it was found that animals transfused with the reuterin-polymerized PLP-hemoglobin up to a 50% blood exchange all survived (n = 6), while animals transfused with allograft plasma died in 3~5 h after transfusion (n = 6) and those transfused with phosphate buffered saline (pH 7.4, n = 6) and unpolymerized hemoglobin (n = 6) survived one out of six. The half-life of the unpolymerized hemoglobin in circulation was about 1.5 h, while that of the reuterin-polymerized hemoglobin was approximately 12 h. In conclusion, genipin and reuterin are promising agents to polymerize hemoglobin as blood substitutes.