| Blood transfusion has been playing an important role in treating anemia and blood loss. However, studies show that in some cases, blood transfusion poses a significant risk to survival and health of patients. For example, it can cause ischemic events and Reperfusion injuries in some severe patients, especially those suffering from serious cardiac dysfunction. These adverse reactions used to be attributed to several time-dependent changes including the loss of RBC shape and flexibility, increase in RBC adhesiveness and affinity for oxygen, decrease in the concentration of molecular modulators of oxygen, 2,3 - DPG and ATP. Moreover, even fresh processed blood has been observed to decrease tissue oxygenation, an effect that predates many of these injury mechanisms. It was not until recently that the causes were identified. Studies demonstrate that nitric oxide in banked blood has been reduced during storage and consumed after 24h. NO plays a key role in keeping the tension of blood vessels stable and regulating the stability of blood pressure. With the participation of a variety of factors, it is generated by catalysis of nitric oxide synthase (NOS) and L- arginine (L-arg), and then transported into the surrounding tissues after combination with hemoglobin(Hb-NO), to play the role in vasodilation. The reduction of Hb - NO is strongly correlated with decrease of banked blood vasodilation. In this study, we attempted to improve the Hb-NO level in the banked blood using the NOS of red blood cells by adding NO synthetic substrate-L - arg and NOS agonists–ATP in order to keep the NO content of banked blood, restore vasodilation of banked blood, and to enhance the safety and effectiveness of clinical transfusion after evaluating blood additives. Through an analysis of indexes affecting the ability to transport oxygen in banked blood and by using the biochemical analyzer and blood gas analyzers, laser diffraction erythrocyte deformation apparatus, we detected the ion level, blood gas, deformation and collected indexes of RBC in banked blood. Using Griss and the uv photodissociation - chemiluminescence method, the SNO - Hb contents of blood were analyzed. The results showed that levels of K+ in banked blood rose compared with fresh blood(P<0.01), Na+ gradually declined(P<0.01) while Cl- and Ca2+ remained unchanged (P > 0.01). The average pH values and bicarbonate (HCO3 -) concentration in plasma were gradually reduced (P < 0.01), metabolites increased (P < 0.01), partial pressure of oxygen and oxygen saturation gradually increased (P < 0.05), and the deformation of RBC declined significantly (P < 0.01). Total Hb - NO content (ppb) in the banked blood at 0d, 3d, 14d, 21d 7d, 28d, 35d was 152.00±32.58, 48.25±18.79, 34.00±21.15, 35.75±25.47, 40.35±13.02, 39.75±20.61, 33.75±9.78(P<0.01) respectively. In summary, some indexes of the banked blood changed in the course of storage, especially so as evidenced by the decrease of total Hb– NO, which led to the decline of vasodilation.Based on studies on nitric oxide synthase (eNOS) of the endothelial type in banked erythrocytes, eNOS in RBC was identified by Western blotting and immunofluorescence technology. eNOS can catalyze L - Arg and molecular oxygen to form NO. NO and nucleophilic substance can generate colored compounds. Accordingly, we tested the activity of eNOS. The type of NOS playing a role in RBC was identified using nitric oxide fluorescence probe (DAF - FM DA) and a single type of NOS inhibitors. The level of eNOS was represented by gray analysis, the result of which at 0d, 7d, 14d, 21d 28d, 35d was 0.2175, 0.1935, 0.197, 0.2015, 0.203, 0.202 respectively. The eNOS content in RBC remained stable (P > 0.01). The blood was preserved at 4℃for 0d 20d, 35d 10d, and the eNOS activity (U/gHb) was respectively 31.28±8.97, 36.95±9.44, 36.13±3.9, 37.93±5.14. The eNOS activitiy of each groups was of no statistic significance (P > 0.05). Using L - NAME as the inhibitor, the generation of NO was detected as 306.83±14.98, 315.98±55.75, 328.57±14.65, 342.62±56.44 in control group when blood was saved for 0d, 10d, 20d, 35d in 4℃, and was 116.51±44.02, 124.90±39.57, 127.75±29.42, 156.64±9.65 in inhibitor group, so the inhibitor proved to be highly effective (P < 0.05). Test results showed that active eNOS existed in RBC, and its content and activity were stable during storage without apparent change.Effects of L - Arg and ATP on the NO content in banked blood were studied. L - Arg is a substrate to synthesize nitric oxide (NO). ATP provides high-energy compounds for metabolism activities of red blood cells stored at 4℃. ATP is also an agonist foreNOS, able to promote activation and phosphorylation on the 83th threonine. In order to maintain the levels of NO in banked blood, L - Arg at different concentrations was added to improve the content of total Hb - NO. The results showed that the content of Hb - NO of RBC was 152.0±36.6,48.3±18.8,34.0±21.2,35.8±21.5,40.3±13.0,39.8±20.6 and 33.8±9.8 in the control group stored for 0d,3d,7d,14d,21d,28d,35d. The content was 167.8±39.3,104.3±21.8,98.5±12.6,85.5±15.5,95.0±23.8,89.3±18.3 and 77.3±15.0 in L-Arg group of 300μmol/, 199.8±23.5,143.0±34.2,148.3±40.0,134.00±30.6,124.0±25.8,116.8±20.1 and 104.5±24.3 in L - Arg group of 3000μmol/. In two L-arg groups, the content was higher than in the control group in the same period (P < 0.01), suggesting that L - arginine can increase the content of total Hb - NO in red blood cells detected after adding ATP at 0d,7d,14d,21d. Compared with the control group, the content of total Hb - NO in RBC was not significantly different between the groups (P > 0.05). The content of total Hb - NO was not impacted by ATP. Therefore, it was concluded that L -Arg played an important role in keeping the the content of total Hb - NO in banked blood. ATP had no effect on the content of total Hb - NO.Studies on blood function after restored vasodilation were also conducted. Firstly, the vasodilation function was evaluated after adding L - arg in banked blood. The vasodilation of banked blood was tested and analyzed by rat aortic rings experiment. The results showéd that the vasodilation of banked blood was 44.2±3.47,37.02±12.70 and 24.36±6.12 in the control group stored for 7d,16d,35d and was 76.23±5.46,63.97±24.62 and 55.17±12.1 in L-arg group, which was remarkably different from the control group (P<0.01). Secondly, the quality of blood to which L-arg was added was evaluated. The concentration of K+, Na+, Cl-, SO2, PO2, PCO2, pH, HCO3-, ME, deformability and aggregation of RBC were tested at 0d, 7d, 14d, 21d, 28d, 35d. The results were not significantly different from the control group(P>0.05). Finally, the free hemoglobin of banked blood was detected at different time points. The concentration (mg/L) of the free hemoglobin of banked blood was 200.53±42.72, 364.07±71.72 and 455.32±65.58 in L-arg group at 21d, 28d and 35d. which was lower than that in the control group (283.51±90.72, 486.53±103.44 and 595.74±112.53) for same stored time(P<0.01). Test results showed that L-arg could improve vasodilation of banked blood without impacting the quality of banked blood by inhibiting the free hemoglobin during subsequent storageTo sum up, in this study we found that NO content was decreased significantly when blood was stored at 4℃, which might cause vasodilation down. The content and activity of RBC eNOS did not change significantly after preservation. This suggested that the decrease of NO synthesized by eNOS was correlated positively with consumption of L - arg as metabolic substrates, which was of theoretical value for synthesizing NO by eNOS of RBCs themselves. The research found that L-arg played an important role in increasing NO in banked blood. The concentration-response relation was determined between L-arg and NO. And L-arg could obviously improve vasodilation of banked blood without adverse effects on the quality of blood. These results provided a theoretical basis for the development of a new blood protectant and the improvement of the quality of banked blood. |
