The Effect Of Nitric Oxide On The Quality Of Red Blood Cell During Preservation

The effect of red blood cell (RBC) transfusion is closely related to the quality of RBC during storage. The latest clinical research proved that supplement of nitric oxide (NO) to stored RBC significantly improved RBC deformability and attenuate adverse effects of transfusion. According to the preliminary achievements in our lab, we speculate NO improves the whole RBC quality by affecting certain cells.Objective:To investigate the effect of SNP or L-arg on the quality of preservation of RBC, and to clarify the part mechanism of NO acting on RBC by exploring the relationship of NO and glycolysis metabolic.Method:The RBC with different concentrations of SNP or L-arg incubated for 1 hour were separated by density-gradient-separation, and then we optimized concentration of SNP and L-arg by detecting RBC deformability and osmotic fragility. RBC unites stored up to 35 days were sampled at selected storage times (day 1, day 14, day 35). Samples were separated according to their desity using Percoll after incubating with SNP or L-arg at room temperature for 1 hour, and RBC with different density was measured with RBC number ratio, age, deformability, osmotic fragility, ATP content, PS and CD47 positive expression rate. DL-GA has been currently considered as the most ideal glycolysis inhibitor. DL-GA was shown to inhibi the PK and HK activity to resist the glycolysis. In the storage telophase, the mixed RBC and the density fractionated RBC were divided into 6 aliquots:a glycolysis inhibitior, DL-glyceraldehydes (DL-GA) was added to one aliquot, SNP and L-arg were added separately to the second and the third aliquot, SNP and DL-GA were added together to the forth one, L-arg and DL-GA were added to the fifth one, and the sixth adding the same volume of PBS served as control. The six aliquots were then incubated at room temperature for 2 hours to examine the RBC deformability and the activity of two critical rate-limiting enzymes (pyruvate kinase, PK and hexokinase, HK) in the glycolysis path.Results:In the preliminary and metaphase, the 10μM SNP group and the 10μM L-arg group showed more significant than other groups on the deformability and osmotic fragility, while the terminal stage SNP and L-arg groups were both 100μM. At the storaged timepoint, the storage quality of the forth layer (older) RBC was improved significantly after adding SNP or L-arg. Besides, the RBC adding SNP or L-arg with 35-day storage was also found to be improved obviously when measuring the related indexes (deformability, osmotic fragility, ATP, etc.). In the storage telophase, SNP or L-arg was shown to increase RBC deformability and the PK and HK activity, but if SNP or L-arg was added with DL-GA, the above indexes were inhibited the NO improving effect.Conclusion:The best concentration of the two drugs (SNP, L-arg) was 10 μM in the preliminary stage and metaphase; in the terminal stage the best concentration both was 100 μM. This study demonstrated that NO was only capable to improve the storage quality of the old RBC and injured RBC. Also, NO was found to promote the cellular metabolism through the glycolysis path, resulting in improving the RBC storage quality.