The Association Of Red Blood Cell Damage With Free Fatty Acids

BackgroundBlood loss is a common complication typically occurs following total hip arthroplasty (THA) and total knee arthroplasty (TKA). However, a kind of anemia which is followed after arthroplasty due to hidden blood loss (HBL). The quantity of hidden blood loss can be calculated based on Gross equation:total blood loss volume subtracts visible blood loss volume. Studies have shown that, there is about 30% of total blood loss caused by hidden blood loss perioperation of total knee arthroplasty. It means that the result of hidden blood loss is up to 547-1473 ml, but the mechanism is not clear yet.Because of the use of tourniquet, blood loss in total knee arthroplasty (TKA) is so little that can be ignored. The amount of visible blood loss in perioperative of TKA could be estimated by the total amount of bleeding during the surgery with the addition of the amount drawn by drainage tube after the operation. In theory, recycling drainage and reinfusion of blood is sufficient to correct the patient perioperative blood loss. However, study found that autologous blood transfusion appears insufficient to correct the postoperative anemia, and autologous transfusion amount only equivalent to 50% of the total amount of blood loss. The unexplained fall of hemoglobin is so-called hidden blood loss, which often results in receiving blood transfusion.There are multiple theories concerning the causes and underlying mechanisms of hidden blood loss. Sehat et al speculated that the recessive bleeding, at least part of the loss, is the result of hemolysis; however, the cause of the hemolysis is not clear. Haien’s study found that autologous blood transfusion can cause hemolysis. Specifically, when 1.3L was autotransfused, plasma hemoglobin levels reached 50 mg/L. At this concentration, the hemoglobin level was sufficient to cause hemolysis and may also result in insufficient autologous blood transfusion. Sehat et al reported similar findings. They reported that because of the existence of haptoglobin, the level of hemoglobin is not sufficient to cause hemoglobinuria; instead, it may cause the blood to be insufficient even after autologous blood transfusion. Shen et al reported no significant differences between the autologous blood transfusion group and the allogeneic transfusion group in terms of hidden blood loss. However, autologous blood transfusion is a safe and effective way to decrease the use of allogeneic blood and avoid complications associated with its transfusion. Tetro et al reported that it is important to use a tourniquet, which may cause the HBL. Their study showed that patients who did not receive a tourniquet during surgery were 16% lower than the user, but still reached the absolute value of the HBL(600ml). The "third mesooecium" theory has also tried to explain this phenomenon. Using labeled red blood cells, Sehat et al showed that the unexplained blood loss was a result of a large amount of acute fluid storage in the interstitial space. Hou et al observed a large number of labeled red blood cells in tissue clearance by using radiolabeled red blood cells after the operation, instead of using them in systemic circulation. This led to further reduced levels of hemoglobin. This may be associated with bone marrow fat, bone cement, and bone debris, which may leak into the bloodstream during operation. This can cause an abnormal capillary bed to open. However, there is no hard evidence to fully support this claim. Despite these findings, additional work is needed to fully understand the underlying mechanisms governing hidden blood loss.Free fatty acids, metabolites of fatty emboli, stimulate neutrophils to produce reactive oxygen species (ROS), such as superoxide anion radical (·O2-), hydroxyl radicals(·OH), and hydrogen peroxide. These reactive oxygen species can damage the cell membrane via the oxidation of unsaturated fatty acids. For example, hydrogen peroxide (H2O2) has been shown to penetrate the blood cell membrane and directly oxidize hemoglobin (Hb) to ferryl hemoglobin (ferryl Hb), which is incapable of carrying oxygen. Thus, this enhances anoxic damage to red blood cells. On the other hand, according to the diagnostic criteria of fatty embolism syndrome (FES) raised by Guard, the unexplained decrease of HCT is one of minor criteria. And the unclear anemia offen come out ahead of the typical symptoms in lung and brain. It is worth noting that the common characteristic of FES and hip and knee joints replacement is rising level of fatty droplets in blood. Pressure of the medullary cavity increased during the process of artificial arthroplasty installation. The later would play an important role in the pathological process when fatty droplets enter heart, brain, lung and other organs through blood circulation which has been confirmed by TEE. Clinical evidence also suggests that there is a close correlation between the heart and lung dysfunction and thromboembolic events. Apart from the mechanical obstruction in these organs capillaries, free fatty acids, the metabolites of fat droplets of can stimulate neutrophils to generate reactive oxygen species ROS, and finally damage the red blood cells. ROS generated commonly in two ways:First, the mitochondrial pathway, ROS is mainly produced in the complex Ⅰ and Ⅲ of mitochondrial respiratory chain. The a-glycerophosphate dehydrogenase mGPH and citric acid cycle and a-ketoglutarate dehydrogenase of Complex Ⅱ also play a role in ROS producting processes. Second, non-mitochondrial pathway, PUFA metabolic transformation that occurred in effect of endoplasmic reticulum and cytochrome P450 monooxygenase is the primary means of generating ROS. Further, the oxidation of NADPH also contribute to the generation of ROS. Mitochondrial pathway of ROS generation rate mainly depends on the coupling portions for electrons, as well as the efficiency of complex Ⅰ and Ⅲ proton pump. FMN, Fe-S cluster and Q binding domain and the level of transmembrane potential determine the velocity of electron across mitochondrial membrane in complexes, and then control the production of ROS. The forward and reverse electron transfers also influence the generation rate of ROS. Long-chain FFA and it metabolite can prompts the generation of ROS in mitochondria pathway. FFA can accelerate the generation of ROS by decreasing stream of electrons when combining with subunits of complex Ⅰ and Ⅲ. FFA can also stimulate the production of ROS through following approaches:a. As a carrier of photonic, FFA reduce the dependency of ROS production on reversed electron transfer; b. FFA constrains GSSG transferring to reduced glutathione (GSH) through adapting combined enzyme agent, so as to slower the cleaning rate of glutathione peroxidase to hydrogen peroxide; c. FFA could do harm to the mobility of high affinity membrane on mitochondrial inner membrane.On the other hand, FFA is the irritant produced by superoxide that relied by NADPH oxidase in neutrophils, which belongs to non-mitochondrial pathway. O2- generated in mitochondria in the matrix area and cytoplasm transfer to H2O2 by affected with Mn-superoxide dismutase Mn-SOD and ZnCu-SOD. The latter effected by catalase would break down to H2O and 02, or converted into H2O by the action of glutathione. Apart from this, H2O2 can transfer to HOCl in neuteophile granulocyte affected by myeloperoxidase as well. Neutrophils can eliminate causative agent and foreign body by producing ROS. In physiological conditions, it is important to the survival of cells and organs that the equilibrium state between oxidation and reduction process. However, the over-production of ROS can damage the cell membrane that contains large quantity of unsaturated fattyacid PUFA. Peroxiredoxin 2 exists extensively on red cell membrane, protects red cells from oxidation damage, but research show that 5 uM H2O2 is enough to oxidize this enzyme. The oxidative damage of hemoglobin is mainly in its structure and function change, resulting in the degeneration and sediment and production of hemoglobin. Hydrophilic H2O2 can penetrate membrane of red blood cell and oxidize hemoglobin directly to ferry1 Hb, which loss its oxygen-carrying capacity, the process of spontaneous reduction to ferrohemoglobin is also very slowly, further more cannot recover to the normal condition. Hypochlorous acid oxidized glutathione and membrane protein SH in red blood cells can increase the osmotic fragility of cells and damage cellular deformability. There was a research proving that oxidation of hypochlorous acid to red cells make it more sensitive to hemolysis induced effect of Stichodactyla heliantus Ⅱ.All above indicate us when line in the process of the artificial joint replacement the marrow cavity pressure increase abnormally, and the fatty droplets enters into blood circulation (which had been proved via endoscopic esophageal ultrasound during the surgery), free fatty acid, metabolite of fatty droplets, would stimulate neutrophils to produce H2O2 and HOCL, and the H2O2 and HOCL can oxidize and depletion of the superoxide dismutase (SOD), glutathione peroxidase, and total antioxidant capacity (TDC) on the cell membrane surface of red blood cell, resulting in oxidation and degradation of hemoglobin. Damaged red cell membrane, increased osmotic fragility of cells and the feryyl hemoglobin loss oxygen carrying capacity were main causes of anemia. At the same time, we proposed that the unexplained anemia during the process of FES is due to the oxidative stress inducing activity, which in accordance with the mechanism of hidden blood loss in hip and knee replacement surgery.Objective To study the effects of free fatty acids on red blood cells and hemoglobinin of rats, and explore the pathogenesis of hidden blood loss.Methods The experiment was divided into two parts, different concentrations of free fatty acids alternatives which inciude linoleic, arachidonic acid were injected by the tail vein to rats, structuring a high level free fatty acid model in vivo model. Further analysis and research were undergone in the experimental group for which had significant changes. Mainly detecting the condition of changes of red cells and oxidation-reluctant. Blood samples were collected before treatment,24 h,48 h, and 72 h after administration, and examined the red blood cell count (RBC), levels of hemoglobin (Hb), glutathione peroxidase (GSH-PX) activity, total superoxide dismutase (T-SOD) activity, hydrogen peroxide (H2O2) in the blood, and as well as ferryl hemoglobin, which is generated by the oxidation of Hb. Moreover, red blood cell morphology was examined under a polarizing microscope to observe the dynamic changes.Results The second parts of the experiment result was consistent with the tendency of first part, and both successed in constructing hidden blood loss in animal. RBC and Hb content decreased in all the experimental group and the control groups 24 hours after administration, and the changes in the experimental groups were more significant (p<0.05); At the same time, the GSH-PX vitality, T-SOD vitality, H2O2 content in the experimental group is also significantly reduced compared with the control group (p<0.05).48 hours after administration, Hb and RBC had no significant changes in the control group, but the linoleic acid group, arachidonic acid group was still significantly decreased (p<0.01), and the same tendency reflected in the GSH-PX vigor, T-SOD vigor, H2O2 content (p<0.01). Pathologic changes of red blood cell morphology occurred in both experimental group 24,48 hours after administration. Shown as pleomorphism, shrinkage, even rupture and necrosis under the microscope. Meanwhile, it is found that the absorption value of experimental groups were significantly increased after treatment, especially in the 24 hours after the administration,and peaking value appeared at 425nm which is consistent with ferryl hemoglobin characteristic; 72 hours later, RBC and Hb content in blood is relatively stable, GSH-PX vigor, T-SOD vigor, H2O2 content also had the tendency to recover to equilibrium status.Conclusion High level of linoleic acid and arachidonic acid can both lead to acute damage of red blood cells. And the mechanism of its pathological is free fatty acid-induced redox reactions, which can cause oxidative damage to red blood cells and hemoglobin, and giverise to hidden blood loss.