Polyethylene glycol conjugated bovine hemoglobin containing 15% MetHb plays approving effect in exchange transfusion rabbit model.

PEG-bHb was developed by Kaizheng Biotech (Beijing, China), and pre-clinical research was completed. The objective of this study was to investigate the safe concentration of MetHb in PEG-bHb. The study was accomplished by examining the effects of PEG-bHb containing 5%, 8%, 15%, and 25% methemoglobin (MetHb), respectively, on cardiovascular system, blood chemistry, pathology of liver and kidney in rabbits following a 50% exchange transfusion. The results showed that PEG-bHb containing 5%, 8%, 15%, and 25% MetHb could keep four groups of experimental rabbits (5/5) alive until the 8th day after 50% exchange infusion as autologous whole blood did, and were superior to dextran 40 (2/5). MetHb concentration in PEG-bHb, no more than 25%, did not affect the PEG-bHb function on resuscitation of hemorrhaged rabbits by physiological measurements and blood chemistry assays. Histology study using optic and electron microscopy showed that there were slight pathological changes in hepatocytes and renal tubule epithelia in rabbits, which were infused by PEG-bHb containing 5%, 8%, and 15% MetHb. Partial organelles collapse was observed in rabbits resuscitated by PEG-bHb containing 25% MetHb. In conclusion, PEG-bHb is safe and effective when the MetHb concentration is at or below 15%.

B to O erythrocyte conversion by the recombinant alpha-galactosidase.

BACKGROUND: Human group O red blood cells have great benefit in specialized transfusion areas such as armed conflict and natural calamity. The group B antigen differs structurally from group O antigen only by the addition of one terminal alpha-linked galactose residue. In this study we aimed to remove the terminal galactose from group B red blood cell to get group O red blood cell. METHODS: alpha-galactosidase cDNA was cloned by RT-PCR from Catimor coffee beans grown on Hainan Island of China. The vector for alpha-galactosidase cDNA expression was constructed and transferred into Pichia pastoris cells by electroporation. The transgenic cells were cloned by fermentation and the recombinant alpha-galactosidase was purified by ion exchange chromatography. After studying the biochemical characters of alpha-galactosidase, we have used it in converting human erythrocytes from group B to group O. RESULTS: The purity of recombinant alpha-galactosidase was higher than 96%, which was thought to be suitable for the use of blood conversion. Enzymatically converted human group O red blood cells (ECHORBC) exhibited membrane integrity, metabolic integrity, normal cell deformation and morphology. There were no coagulation between ECHORBC and any group of human blood. The ECHORBC will keep normal structure and function for a period of 21 days at 4 degrees C in monoammoniumphosphate nutrient solution. Experiments with Rhesus monkeys and gibbons showed that transfusion of enzymatically converted erythrocytes was safe. CONCLUSION: ECHORBC can be easily obtained from group B red blood cell by alpha-galactosidase digestion. This study suggests that ECHORBC could be transfused to patients safely and efficiently.

[Study on Polyethylene Glycol Modified Porcine Hemoglobin Using Computer Modeling]

The structure analysis of porcine hemoglobin alphabeta dimer and the calculation of solvent accessible surface of the amino acids showed the epsilon-amino groups of the lysine are suitable for modification by polyethylene glycol (PEG). The modification of the lysine residues will not affect the carring oxygen capacity of Hb. Three types of linker have been designed to connect PEG and porcine hemoglobin. The lysines between porcine and bovine hemoglobin (pHb and bHb) are highly conserved, but the solvent accessible surface of conserved lysines are different. These suggested that the properties of homologous proteins are similar in pHb and bHb, but the characteristic derived from the homology analysis will be deviated from the actual status. The results of molecular dynamics simulation suggested that the chemical modified porcine hemoglobin would be no immunogenicity.