In this study, we confirmed the existence of HBL by examining and analyzing the perioperative blood indexes of patients with knee arthroplasty, and the results showed that the total perioperative blood loss was 1036 ml and significantly lower than the earlier reported 1474 ml, suggesting that the improvement of perioperative management could reduce the total blood loss and visible blood loss. But the proportion of HBL is 54.5%, which is higher than the 50% reported previously [4], indicating that the control and management of HBL is crucial.
There are many hypotheses about HBL. Some studies had suggested that HBL may be due to partial hemolysis caused by blood transfusion. However, clinical studies had found that there was no difference in HBL between the transfusion group and non-transfusion group [13, 14]. However, in order to eliminate potential interference, perioperative blood transfusion patients were not included in the study. It is reported that the use of tourniquet during TKA can increase the risk of HBL. The study divided the patients into the tourniquet group and non- tourniquet group, and the results showed that there was no significant difference in operation time between the groups, and the visible blood loss in the non-tourniquet group increased about 180 ml compared with the tourniquet group. Instead, the HBL was about 16% lower than that in the tourniquet group. However, there was still significant HBL in the non- tourniquet group that could not be explained [15, 16]. In addition, the " third mesooecium" theory also partially explained the HBL. Boswell et al. believe that the HBL occurs after TKA due to tissue blood oozing and fluid oozing into the surrounding tissue clearance (i.e., the third space) [17, 18]. Therefore, after the surgery, we routinely use elastic bandages to bandage the affected limb from the ankle to the middle of the thigh and pressurize appropriately to reduce bleeding, and applied a negative pressure drainage ball to drain the hematocele or hydrops in the joint cavity. With these measures, it was difficult for the blood to penetrate into the surrounding tissue space, and the amount of HBL triggered by this way was limited.
In the previous study, we successfully constructed the animal model of HBL by injecting linoleic acid and arachidonic acid into rats. Linoleic acid and arachidonic acid are important components of FFA. Our study found that when the blood linoleic acid or linolenic acid reached a certain concentration, it would induce oxidative stress response, resulting in the destruction of red blood cells and HBL [19–21].
HBL often occurs after joint replacement and traumatic fracture surgery, their common feature is that a large amount of fat droplets leak out of the medullary cavity, and penetrate deep into the circulation, tissues and cells; Especially during joint replacement, the manipulation of the femur and tibia can increase the pressure of the medullary cavity and force the fat particles into the blood circulation [6, 7, 22, 23]. The effects of its metabolites on tissue cells need further study. Our results showed that the concentration of FFA in the blood increased significantly 48 h postoperatively, indicating that large amounts of small fat droplets into the bloodstream during the operation, and the RBC and Hb were significantly reduced; At the same time, HBL was also the most obvious, and a quantity of damaged red blood cells can be found under the microscope. 72 h after the operation, with the catabolic metabolism of fat particles, FFA concentration decreased significantly, and erythrocyte and hemoglobin level gradually increased. Meanwhile, the morphology of red blood cells began to improve under the microscope, and the number of erythrocytes with atypia decreased gradually. This indicates that FFA, a metabolite of small fat droplets, is closely related to the reduction and destruction of red blood cells after surgery.
Studies have pointed out that the FFA can help to induce the generation of ROS in endothelial and vascular smooth muscle cells [24]. High concentration of FFA stimulation can increase the production of highly reactive molecular oxygen clusters and reactive nitrogen clusters, which triggers oxidative stress, prolonged imbalance between the production of highly reactive molecules and anti-oxidant effects resulting in tissue damage. These active molecules can directly oxidize and damage DNA, proteins and lipids, and can also act as functional molecular signals, activating a variety of stress-sensitive signaling pathways in cells, which are closely related to insulin resistance and impaired β-cell function [25, 26]. Studies have shown that high levels of FFA lead to a large amount of ROS production and oxidative stress, which can also activate stress-sensitive signaling pathways. As fatty acid synthase (FAS) genes activate Akt and sterol regulatory element binding protein-1 under hypoxic conditions Up-regulated, so FAS expression level is associated with hypoxia in the body. It is worth noting that Akt is also up-regulated by H2O2; Under hypoxic conditions, the level of FAS protein is significantly increased as well as the production of ROS in cells [27, 28]. It suggests that the expression of FAS gene is actively controlled by hypoxia, which is also related to the amount of ROS in cells. During knee arthroplasty, the use of tourniquets can lead to poor blood supply and tissue hypoxia in the affected limb over a period of time, and local tissue swelling and reduced activity after operation also exacerbate blood and tissue hypoxia. As the concentration of fat droplets in blood and tissues increased after surgery, the FAS required for metabolism increases. Therefore, blood and tissue hypoxia will accelerate the catabolic metabolism of H2O2 in the blood. As a consequence, the level of H2O2 in the blood decreased significantly 24 h and 48 h after surgery. High concentration of FFA will induce the production of ROS in vascular smooth muscle cells, the results of flow cytometry showed that ROS levels increased significantly after 48 hours, which may lead to an imbalance of redox reaction. Free radicals can damage cells by acting on polyunsaturated fatty acids on cell membranes, exacerbating oxygen free radical reactions, and lipid peroxidation, the intermediates of lipid peroxidation can react with membrane proteins to polymerize and cross-link proteins. In addition, the carbonyl products of lipid peroxidation (such as malondialdehyde) can also attack the amino groups of membrane protein molecules, resulting in intramolecular and intermolecular cross-linking of proteins. On the other hand, free radicals can also covalently bind directly to enzymes or receptors on the membrane [29–32]. These oxidations damage the spatial configuration of enzymes, receptors and ion channels embedded in the membrane system, destroying the integrity of the membrane and affecting the function of the membrane and antigen specificity, which eventually leads to the cell damage and lesions. Therefore, the oxidative stress induced by FFA can destroy red blood cells and result in erythropenia and HBL.
SOD is an antioxidant metal enzyme in body. It can catalyze the disproportionation of superoxide anion radicals to generate oxygen and H2O2,which plays a crucial role in the balance between oxidation and antioxidant activity in the body. So, SOD is closely related to the occurrence and development of many diseases [32]. The activity of total superoxide dismutase (T-SOD) can reflect the ability of the body to remove oxygen free radicals [33]. Glutathione peroxidase (GSH-Px) is an important peroxidase widely found in the body. The active center of GSH-Px is selenocysteine, and its activity can reflect the selenium level of the body. Selenium, as a component of the GSH-Px enzyme system, can catalyze the transformation of GSH into GSSG, and reduce toxic peroxides to non-toxic hydroxyl compounds so as to protect the structure and function of cell membranes from the interference and damage of oxides [34–35]. The decrease of NADPH was linearly related to the activity of GSH-Px. The physiological function of GSH-Px in plasma is mainly to catalyze GSH, participate in the peroxidation reaction, remove peroxides and hydroxyl radicals generated during cellular respiratory metabolism, thereby reducing the peroxidation of polyunsaturated fatty acids in the cell membrane [19, 36]. Studies have shown that FFA can stimulate neutrophils to produce H2O2 and hypochlorous acid, which can oxidize and deplete SOD and GSH-Px on the surface of the cell membrane [37, 38]. In our study, with the increase of FFA concentration in blood, the activities of T-SOD and GSH-Px decreased significantly, and the morphology of red blood cells also showed obvious cytomembrane destruction, suggesting that the lipid droplet metabolite FFA in the blood induced a series of oxidation reactions in the body. The imbalance of the redox reaction leads to the destruction and significant reduction of red blood cells. Accompanied by the metabolism of FFA and the disappearance of oxidative stress stimuli, the Hb and RBC increased by self-compensation, the activities of T-SOD and GSH-Px return to equilibrium.