Preparation Of Hemoglobin Oxygen Carrier And Its Effect On Blood Pressure In Mice

Matrix-free Hemoglobin needs to be modified to increase its molecular weight or molecular radius before it can be used in clinical transfusion.Among them,increasing molecular weight by cross-linking agent polymerization is the most concerned method for the preparation of Hemoglobin oxygen carrier.However,the unpolymerized hemoglobin(64kD)in the polymerized product has been shown to be highly toxic,except for the nephrotoxicity of the dimer itself,which depolymerizes to 32 kD in physiological solution,other studies have shown that Hemoglobin(64kD)causes vasoconstriction and hypertension,so all commercial hemoglobin oxygen carriers are trying to remove the hemoglobin(64kD)from the polymer products.In order to ensure that the hemoglobin oxygen carrier in clinical application does not contain hemoglobin molecule(64kD),this paper reduced the content of Hemoglobin(64kD)in the polymerization reaction to a certain extent by increasing the viscosity of the polymerization reaction and using the method of polymerization,the cause of hypertension induced by hemoglobin oxygen carrier was studied by animal experiment.During the preparation of Hemoglobin in the liquid phase,80% glycerol was used to increase the viscosity of the polymerization,the concentration of hemoglobin was 5 mg/ml,the molar ratio of hemoglobin to Glutaraldehyde was 1:240,and the time of polymerization was 50 min,after the end of the polymerization,DMAB should be used to stop the reduction,and the content of Hemoglobin(64kD)in the product can be reduced to about 21% by the method of increasing viscosity.In the process of preparing hemoglobin in solid phase,the hemoglobin is first fixed by electrostatic adsorption using anion resin,at the same time according to the molar ratio of Glutaraldehyde and glycol 1:20 to prepare ACETAL,then acetals were added into the saturated adsorbed hemoglobin medium,the molar ratio of Hemoglobin to Glutaraldehyde was controlled to 1:300,the reaction time was 60 min,the polymerization was terminated by DMAB,and the product was eluted by 1 M Nacl solution,the content of Hemoglobin Molecule(64kD)in polymerized hemoglobin prepared by the method of solid phase polymerization is only 1%.In animal experiments,four samples were injected into four groups of mice by the method of "Top-load" animal model: direct intravenous injection,using polymerized hemoglobin with different oxygen affinity and intramolecular cross-linked Hemoglobin,the changes of blood pressure of mice were monitored in real-time.The blood pressure of mice injected with deoxygen-polymerized Hemoglobin was almost unchanged.The blood pressure of mice injected with oxy-polymerized hemoglobin increased slightly during injection,but the fluctuation range was less than 10%,the increase of blood pressure in the group of intra-molecular cross-linked hemoglobin with deoxygenated and oxygenated lasted from the beginning of injection to 60 Min,but the whole fluctuation range was about 10%.The preliminary conclusion drawn from the above experiments is that there is no significant correlation between the increase of blood pressure and the content of oxygen affinity and Hemoglobin(64kD),but this may be related to the low concentration of the sample and other experimental interference factors,the clinical cause of increased blood pressure needs to be further studied.