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Arachidonic acid causes hidden blood loss–like red blood cell damage through oxidative stress reactions
j o u r n a l o f s u r g i c a l r e s e a r c h m a y 2 0 1 7 ( 2 1 1 ) 1 4 e2 0
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Arachidonic acid causes hidden blood losselike red blood cell damage through oxidative stress reactions Tao Yuan, MD,a,1 Yu Cong, MD, PhD,a,1 Jia Meng, MD, PhD,a Hong Qian, MD,b Wei Ye, MD,c Wen-Shuang Sun, MD,b Jian-Ning Zhao, MD, PhD,a,* and Ni-Rong Bao, MD, PhDa,* a
Department of Orthopedic, Nanjing Jinling Hospital, Nanjing, China Department of Orthopedic of Jinling Hospital (Nanjing), Southeast University, School of Medicine, Nanjing, China c Department of Orthopedic of Jinling Hospital (Nanjing), Southern Medical University, School of Medicine, Nanjing, China b
article info
abstract
Article history:
Background: Hidden blood loss (HBL) often occurs in the prosthetic replacement for joint,
Received 12 July 2016
but the mechanism is still not clear.
Received in revised form
Materials and methods: This study tried to establish an animal model of HBL by injecting
12 October 2016
arachidonic acid (AA) into the Sprague-Dawley rats. Different concentrations of AA were
Accepted 30 November 2016
injected into the tail veins of the rats, and blood samples were collected before and after
Available online 14 December 2016
administration at 24, 48, and 72 h. A complete blood count was obtained by to find the hemoglobin (Hb) and red blood cell (RBC) count changes. The glutathione peroxidase (GSH-PX) and
Keywords:
total superoxide dismutase (T-SOD) activities and hydrogen peroxide (H2O2) levels were detec-
Arachidonic acid
ted. The morphological changes of erythrocyte were observed under a polarizing microscope.
Erythrocyte injury
The absorbance values of the blood samples were tested to determine the presence of ferryl Hb.
Oxidative stress
Results: HBL occurred in the experimental groups when the concentration of AA reached
Hidden blood loss
10 mmol/L; Hb and RBC values decreased sharply at 24- and 48-h postinjection. This was followed by reduced activities of GSH-PX and T-SOD and decreased levels of H2O2. Moreover, the pathologic changes of red cell morphology mainly presented as pleomorphic RBC morphology, including cell rupture. The absorbance values of the blood samples were in accordance with ferryl Hb features. RBC and Hb values were relatively stable at 72 h. The GSH-PX and T-SOD activities and H2O2 levels gradually increased up to a balanced state. Conclusions: The study concluded that high concentrations of AA can induce oxidative stress reactions in the body, causing acute injury of RBCs, which is closely related to HBL. ª 2016 Elsevier Inc. All rights reserved.
Introduction Hidden blood loss (HBL) is one of the most common complications of arthroplasty,1,2 which causes blood loss in spite of autologous or equivalent allogeneic blood transfusion in
accordance with the dominant blood loss. Numerous hypotheses have addressed this topic1,3-7; however, the pathogenesis of HBL is still unknown. Pressure of the intramedullary increased rapidly, during the process of installing prosthesis in an artificial joint replacement surgery,
This work has been conducted in compliance with ethical standards. * Corresponding authors. Department of Orthopedic, Nanjing Jinling Hospital, 305 Zhongshan East Road, Nanjing 210002, China. Tel./fax: þ25 80860015. E-mail addresses: [email protected] (J.-N. Zhao), [email protected] (N.-R. Bao). 1 Co-first authors. 0022-4804/$ e see front matter ª 2016 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2016.11.060
yuan et al arachidonic acid causes hbl-like red blood cell injury
causing fat droplets in the bone marrow cavity enter into the blood circulation, which plays an important role in the process of pathology of some diseases and has been verified by transesophageal echocardiography.8 Free fatty acids (FFAs) are metabolites of fat. Studies have shown that it can stimulate neutrophils to promote the generation of reactive oxygen species (ROS).9 ROS can attack macromolecular substances and cytoderm, damage the microstructure and membrane proteins of cells, and increase the cell membrane permeability, resulting in tissue injury and pathologic change in the body.10-12 In a previous research, the effect of linoleic acid on Sprague-Dawley (SD) rats was studied.13 The hemoglobin (Hb) and red blood cell (RBC) values in that study were found to decrease. This was followed by an increase in the linoleic acid concentration and glutathione peroxidase (GSH-PX) and total superoxide dismutase (T-SOD) activities, and an obvious decrease in hydrogen peroxide (H2O2) levels. The study further observed the morphology of RBCs and absorbance change of the blood samples and found a large deformation of RBCs in the experimental group. The absorbance peak production reflected the existence of ferryl Hb. Thus, the study confirmed that the linoleic acideinduced oxidative stress can cause acute damage of RBCs. Arachidonic acid (AA) is an important part of the FFA and metabolites of linoleic acid.14 In the present study, by injecting different concentrations of AA to SD rats, the changes in RBCs and the associated redox reactions were observed. The relationship between AA and HBL was also explored and the mechanism of HBL was explained, so as to obtain a useful HBL model.
Materials and methods Animal model preparation Adult male SD rats (weight: 225 15 g) purchased from the Chinese Academy of Science, Nanjing University Animal Center, China, were used for all experiments. All animals received humane care in compliance with “The Principles of Laboratory Animal Care” formulated by the National Society of Medical Research and “The Guide for the Care and Use of Laboratory Animals” published by the U.S. National Institutes of Health (National Research Council, 1996). The animals were housed at a set temperature (24 C) in a humidity-controlled room with a 12-h light/dark photoperiod. All experimental procedures were carried out strictly in accordance with the care and use of laboratory animals, which were approved by the National Institute of Health’s Guide for the Care and Use of Laboratory Animals and the Institutional Animal Care and Use Committee at Nanjing University, China. All animals were anesthetized with ether inhalation before operation.
15
microscope (Nikon ECLIPSE 50i, Japan), and centrifuge (Hermle Universal Centrifuge Z323, Germany). The H2O2 concentration and GSH-PX and SOD activities were determined with commercially available assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). AA was obtained from Sigma-Aldrich (MO).
Experimental protocol and drug A total of 50 SD rats were assigned randomly into five groups: one control group and four experimental groups, with 10 rats in each group. First of all, AA was dissolved in a 20% ethanol solution. Different concentrations of AA were made up by diluting AA in 20% ethanol solution. The control group was treated with 20% ethanol solution alone. The experimental groups were further divided into four groups, AA-A, AA-B, AAC, and AA-D, with concentrations of 5, 10, 20, and 40 mmol/L, respectively. The mode of administration was intravenous injections of 0.5 m per dosage through the tail veins. The rate was slowed down, and the concentration of administration was minimized to avoid unnecessary deaths. Before formal experiments, preliminary experiments were conducted to explore the best-dosage range notation. The present study showed that half of the mortality rates occurred at a dosing concentration of 40 mmol/L, and the effective concentration ranged between 5-40 mmol/L. All the rats were monitored continuously until they recovered from anesthesia. The rats were then sacrificed by spinal dislocation method. The study found that they suffered from several symptoms, including dyspnea, cyanosis, hemoptysis labiali, and convulsions. Throughout the process, the rats had no special discomfort. Blood samples were collected from the caudal vein before injection and at 24-, 48-, and 72-h postinjection (0.5 mL each time). Blood smears were prepared and stained with Wright’s stain to observe morphologic changes. RBC and Hb values were obtained through the automatic blood analysis system. A spectrophotometer was used to measure the concentration of H2O2 and the activities of GSH-PX and T-SOD15; for each of these measurements, absorbance values were determined at 405, 412, and 550 nm, respectively, according to the manufacturer’s instructions. All samples were processed within 2 h of collecting the blood samples. Spectral changes of Hb in the AAC group were typically measured with a spectrophotometer. The Hb (10 mmol/L) was mixed with buffer (0.1 mol/L sodium phosphate) containing 100-mmol/L diethylenetriaminepentaacetic acid. All procedures were performed at 25 C.
Statistical analysis All calculations and statistical analyses were performed using the SPSS version 19.0 software. Values were expressed as mean standard deviation and analyzed by one-way analysis of variance, followed by Dunnett’s t-test. In all cases, P < 0.05 was regarded as significant.
Instruments and reagents The following instruments were used in this study: microplate reader (Bio-Rad680, USA), hematology analyzer (Sysmex XE-5000, Japan), spectrophotometer (Hewlett Packard 8453 UV-visible diode array spectrophotometer, USA), polarizing
Results This study showed that administering AA at a concentration of 20 mmol/L caused RBC and Hb values to decrease
16
j o u r n a l o f s u r g i c a l r e s e a r c h m a y 2 0 1 7 ( 2 1 1 ) 1 4 e2 0
significantly, indicating that HBL occurred in the experimental group (Fig. 1A and B). By increasing the concentration of AA, the level/amount of RBC and Hb decreased obviously. However, at a concentration of 40 mmol/L, half of the AA-D group rats died within 5 min of administration. Pulmonary embolism was considered to be the main cause of death in this case16; the rats suffered from dyspnea, cyanosis, hemoptysis labiali, and convulsions prior to death. Further research was carried out among AA-B, AA-C, and control groups. No significant differences were obtained between them in blood tests and other indicators before the experiment. Based on the phenomenon observed, it was inferred that after 24 h, the blood cell got injured. First, the RBC and Hb values dropped to various degrees (Fig. 1). Hb and RBC values in the control group decreased by 8.50 2.35 g/L and 0.18 0.07 1012/L, respectively, whereas those in the AA-B group decreased by 18.25 3.30 g/L and 0.95 0.12 1012/L, respectively, and those in the AA-C group decreased by 24.52 2.55 g/L and 1.31 0.22 1012/L, respectively. However, changes among the AA-B, AA-C, and the control groups were statistically significant (P < 0.05). Activities of GSH-PX, and T-SOD and the concentration of H2O2 had a similar tendency (Fig. 2A-C). Changes in Hb and RBC values were apparent among the AA-B, AA-C, and control groups after 48 h of administration. The changes in GSH-PX and T-SOD activities and the H2O2
Fig. 2 e Comparison of GSH-PX, (A) T-SOD (B), and H2O2 (C) between control and experimental groups. Data are shown as means ± SE; *P < 0.05, #P < 0.01.
Fig. 1 e Comparison of Hb (A) and RBC (B) changes between control and experimental groups. Data are shown as means ± standard error (SE); *P < 0.05, #P < 0.01.
concentration were consistent with the changes in Hb and RBC values (P < 0.01). At 72 h, RBC and Hb values were relatively stable, and GSH-PX and T-SOD activities and the H2O2 concentration gradually increased to the rebalancing state (Fig. 2). The presence of ferryl Hb, formed by reacting with H2O2, was detected by its reaction with sulfide ions producing a characteristic absorbance band around 620 nm.17 The effect of AA on the hemolysis of RBCs, either on its own or in conjunction with ROS, can be used to evaluate the degree of oxidation injury of erythrocytes. Blood samples were collected from the AA-C group before administration and then every 24 h thereafter. The absorbance peaks were detected at approximately 425 nm corresponding to the ferryl Hb Soret peak18 (Fig. 3). The morphology of erythrocyte was observed under a polarizing microscope. The cell-wall structure, morphology, and metabolic features were normal in all groups before the AA treatment. After 24 h of administration, apparent morphologic changes were observed in the AA-B and AA-C groups, and the changes continued when observed after 48 h. A polarized light microscope showed 20% of erythrocyte
yuan et al arachidonic acid causes hbl-like red blood cell injury
Fig. 3 e (A) Changes in AA-C absorbance values as determined using a spectrophotometer. Measurements were taken every 24 h. For clarity, only a small subset of the spectra collected is shown, and the peak at 425 nm corresponds to a ferryl Hb Soret peak. (B) Statistical analysis of absorbance changes between control and AA-C groups, and the spectral changes at 425 nm. Values are shown as mean ± SE; *P < 0.05, #P < 0.001. deformation, mainly exhibited as pleomorphism, shrinkage, rupture, and breaking (Fig. 4F and G).
Discussion Previous studies have confirmed that the injury of blood cell caused by linoleic acid is close to the pathology of HBL.13 AA is an important component of FFA, which is also associated with linoleic acid metabolism. The presence of unsaturated double bonds in the AA structure makes it vulnerable to free radical attack.19 Due to the special oxidation nature of the unsaturated fatty acids, numerous free radicals and active substances are induced.20 If ROS generated exceeds the oxygen radical absorbance capacity, the active oxygen will release into the extracellular space and attack the cells and the surrounding tissue. The present study demonstrated that AA is closely related to red blood cell damage, indicating that FFA can induce oxidative stress and cause HBL. Hb and RBC values are good indicators that reflect the extent to which erythrocyte is damaged. In the present study, Hb and RBC values reduced in all the experimental and control groups, but the changes in the AA-B and AA-C groups were more remarkable, showing obvious damage to erythrocyte, and hence indicating that HBL occurred in these groups. Since the blood samples accounted for 2.8%-4.2% of the blood capacity of rats, this directly resulted in a decrease of Hb and
17
RBC values. Moreover, the intravenous injection of ethanol diluent may have also induced an increased volume, cell lyses, and rupture of erythrocyte.21,22 All the aforementioned reasons may have caused the decrease of Hb and RBC values. Although same amount of blood samples were collected for both experimental and control groups, Hb and RBC values in the experimental groups decreased more significantly, indicating that HBL occurred in the experimental groups, and that AA was responsible for the change in Hb and RBC values. AA can stimulate the production of ROS through the following ways: (1) AA can irritate the nicotinamide adenine dinucleotide phosphate oxidase by activating calcium ion channels on the cell membrane to produce large amounts of ROS1,23; (2) an increase in AA concentration in the blood can raise lipid peroxidation and oxidized glutathione content, which may promote the formation of ROS24; (3) AA can also induce neutrophils to release oxygen radicals, and activate the receptor-interacting protein-1 to stimulate the generation of ROS25,26; and (4) AA can also inhibit the activity of the mitochondrial respiratory chain enzyme complexes I and III and increase the mobility of the mitochondrial membrane.27 This suggests that ROS can be generated through cyclooxygenase and lipoxygenase metabolic pathways, which include lipid peroxides and superoxide anion. GSH-PX is an important H2O2-removing enzyme in human cells, which couples the reduction of H2O2 with the oxidization of glutathione, thus removing H2O2 and protecting the integrity of the cell membrane function.28,29 The vitality of GSH-PX inversely correlates with the severity of cell damage caused by oxygen-free radicals. High levels of FAA inhibit the oxidization of glutathione, slowing the activity of GSH-PX and hence the rate of removal of H2O2.30 It was likely that the stress response caused by the blood collection process was accountable for the partial decrease of the GSH-PX and T-SOD activities and H2O2 levels in the first 24 h. Changes between the experimental and control groups were significantly different, showing AA had an influence on the pathologic role of RBCs. T-SOD plays a vital role in keeping the balance of oxidation and antioxidation in our body, thus removing superoxide-free radical from aerobic organisms and protecting against the toxic effect of the oxygen-free radicals.31 The SOD in blood has a specific inhibitory effect on superoxide anion; therefore, the T-SOD vitality reflects the ability of organisms in removing oxygen-free radicals. Studies have shown that FFA can stimulate neutrophils to produce H2O2 and hypochlorous acid; deplete SOD, GSH-PX, and total antioxidant capacity on the surface of red cell membrane; and create additional oxidative denaturation of Hb.9,32 The present study showed that GSH-PX and T-SOD depleted and decreased obviously in the AA-B and AA-C groups 48 h after administration. Moreover, H2O2 was decomposed into H2O and O2 by Mn superoxide dismutase and Zn superoxide dismutase, or translated into H2O directly by the action of glutathione, resulting in the decline of H2O2 levels.33,34 The oxidative stress response diminished as AA gradually metabolized, and the enzymes associated with oxidative stress returned to equilibrium. Hb is an important component of RBCs. Ferrous Hb [Fe (II) Hb] is responsible for delivering oxygen to tissues and can be
18
j o u r n a l o f s u r g i c a l r e s e a r c h m a y 2 0 1 7 ( 2 1 1 ) 1 4 e2 0
Fig. 4 e Changes in red blood cell morphology. Blood smears were observed under polarized light microscope (400 3 magnification). Blood samples were collected before injection and at 24-, 48-, and 72-h postinjection. Cell morphology did not obviously change in the control group (A-D). However, it showed 20% of erythrocyte deformation, mainly exhibited as pleomorphism, shrinkage, rupture, and breaking cells (black arrow) in the AA-C group (E-H) treated with AA (20 mmol/L). (Color version of figure is available online.)
slowly oxidized to ferric Hb [Fe (III) Hb].18,35 Ferryl Hb (Fe4þ¼O2), which is produced by the reaction of methemoglobin with strong oxidants, loses the function of carrying and transporting oxygen.36,37 Studies have shown that lipid peroxidation increases in the process of ferryl Hb formation and aggravates lipid membrane damage.38,39 H2O2 is one of the active substances involved in the severe damage of in vivo cells, while its reaction with Fe(II) Hb and Fe (III)Hb results in the formation of ferryl Hb.40 These physiological responses are closely related to ischemia in vivo. Ferryl Hb can oxidize nucleic acids and lipids, and its globulin radical is unstable, which rapidly shifts to a stable and long-lived ferryl radical.41,42 Ferryl Hb will automatically revert to the ferric (met-) Hb form under acidic conditions.43 The reduced met-Hb is different from the initial ferric Hb, ferrihemochrome, and Hb subunit formed in the process of reduction. The presence of ferryl Hb can be judged by the extinction value of the blood samples. Previous studies have found that when Hb reacts with H2O2 at pH 7-8, the peak value of absorbance appears at 408 and 425 nm, which represent the met-Hb and ferryl Hb.17,18,38 The experimental groups in the present study also
showed an absorbance peak at 425 nm, whereas the control group had no significant change, which proved the generation of ferryl Hb. This result indicates that strong oxidative stress in the blood system of the experimental group leads to the intense oxidation of Hb. Tests on the blood samples indicated that the levels of H2O2 consistently declined at 24 and 48 h after the treatment. These results clearly showed that a large number of H2O2 was consumed after the injection of AA. Part of the H2O2 is involved in the destruction process of RBCs by oxidation, which can be verified through morphologic changes of RBCs. Groups of crenated, heteromorphic, and even exploded erythrocytes appeared at 24 h after the administration of AA, and the situation improved after 72 h, indicating that the oxidative action on erythrocyte induced by AA gradually weakened and disappeared. The pathologic changes of erythrocyte morphology can be considered as the result of protein oxidation and lipid peroxidation, and permeability of red cell membrane changes. However, strong oxidation can also directly lead to the destruction of RBCs.
yuan et al arachidonic acid causes hbl-like red blood cell injury
In conclusion, AA and linoleic acid are both important components of FFAs. The results are consistent with the previous studies on linoleic acid. In conclusion, AA can induce oxidative stress reaction and stimulate the production of ROS. Oxidative stress induced by AA can act on erythrocyte and cause HBL. The limitation of this study was that the change of ROS was not measured, which could have made the results more intuitive and credible. More research remains to be perfected to explain the pathogenesis of HBL, for example, the use of antioxidant intervention experiments on rats and clinical observation. These will eventually be beneficial to explain the mechanism of HBL and provide evidence base for clinical prevention and treatment of HBL.
Acknowledgment This study was supported by the Clinical Science and Technology Foundation of Jingling hospital (No. 2016016), and the Scientific Innovation Research of the Chinese People’s Liberation Army (PLA) (14QNP034) and Natural Science Foundation of Jiangsu Province (BK20161385) Authors’ contributions: B.N.R. and Z.J.N. contributed to the ideas and final version for this commentary, Y.T, C.Y, M.J, Q.H, Y.W, and S.W.S contributed to the literature search, animal experiment, data analysis, article drafting, and editing.
Disclosure The authors reported no proprietary or commercial interest in any product mentioned or concept discussed in the article.
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ScienceDirect journal homepage: www.JournalofSurgicalResearch.com
Arachidonic acid causes hidden blood losselike red blood cell damage through oxidative stress reactions Tao Yuan, MD,a,1 Yu Cong, MD, PhD,a,1 Jia Meng, MD, PhD,a Hong Qian, MD,b Wei Ye, MD,c Wen-Shuang Sun, MD,b Jian-Ning Zhao, MD, PhD,a,* and Ni-Rong Bao, MD, PhDa,* a
Department of Orthopedic, Nanjing Jinling Hospital, Nanjing, China Department of Orthopedic of Jinling Hospital (Nanjing), Southeast University, School of Medicine, Nanjing, China c Department of Orthopedic of Jinling Hospital (Nanjing), Southern Medical University, School of Medicine, Nanjing, China b
article info
abstract
Article history:
Background: Hidden blood loss (HBL) often occurs in the prosthetic replacement for joint,
Received 12 July 2016
but the mechanism is still not clear.
Received in revised form
Materials and methods: This study tried to establish an animal model of HBL by injecting
12 October 2016
arachidonic acid (AA) into the Sprague-Dawley rats. Different concentrations of AA were
Accepted 30 November 2016
injected into the tail veins of the rats, and blood samples were collected before and after
Available online 14 December 2016
administration at 24, 48, and 72 h. A complete blood count was obtained by to find the hemoglobin (Hb) and red blood cell (RBC) count changes. The glutathione peroxidase (GSH-PX) and
Keywords:
total superoxide dismutase (T-SOD) activities and hydrogen peroxide (H2O2) levels were detec-
Arachidonic acid
ted. The morphological changes of erythrocyte were observed under a polarizing microscope.
Erythrocyte injury
The absorbance values of the blood samples were tested to determine the presence of ferryl Hb.
Oxidative stress
Results: HBL occurred in the experimental groups when the concentration of AA reached
Hidden blood loss
10 mmol/L; Hb and RBC values decreased sharply at 24- and 48-h postinjection. This was followed by reduced activities of GSH-PX and T-SOD and decreased levels of H2O2. Moreover, the pathologic changes of red cell morphology mainly presented as pleomorphic RBC morphology, including cell rupture. The absorbance values of the blood samples were in accordance with ferryl Hb features. RBC and Hb values were relatively stable at 72 h. The GSH-PX and T-SOD activities and H2O2 levels gradually increased up to a balanced state. Conclusions: The study concluded that high concentrations of AA can induce oxidative stress reactions in the body, causing acute injury of RBCs, which is closely related to HBL. ª 2016 Elsevier Inc. All rights reserved.
Introduction Hidden blood loss (HBL) is one of the most common complications of arthroplasty,1,2 which causes blood loss in spite of autologous or equivalent allogeneic blood transfusion in
accordance with the dominant blood loss. Numerous hypotheses have addressed this topic1,3-7; however, the pathogenesis of HBL is still unknown. Pressure of the intramedullary increased rapidly, during the process of installing prosthesis in an artificial joint replacement surgery,
This work has been conducted in compliance with ethical standards. * Corresponding authors. Department of Orthopedic, Nanjing Jinling Hospital, 305 Zhongshan East Road, Nanjing 210002, China. Tel./fax: þ25 80860015. E-mail addresses: [email protected] (J.-N. Zhao), [email protected] (N.-R. Bao). 1 Co-first authors. 0022-4804/$ e see front matter ª 2016 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2016.11.060
yuan et al arachidonic acid causes hbl-like red blood cell injury
causing fat droplets in the bone marrow cavity enter into the blood circulation, which plays an important role in the process of pathology of some diseases and has been verified by transesophageal echocardiography.8 Free fatty acids (FFAs) are metabolites of fat. Studies have shown that it can stimulate neutrophils to promote the generation of reactive oxygen species (ROS).9 ROS can attack macromolecular substances and cytoderm, damage the microstructure and membrane proteins of cells, and increase the cell membrane permeability, resulting in tissue injury and pathologic change in the body.10-12 In a previous research, the effect of linoleic acid on Sprague-Dawley (SD) rats was studied.13 The hemoglobin (Hb) and red blood cell (RBC) values in that study were found to decrease. This was followed by an increase in the linoleic acid concentration and glutathione peroxidase (GSH-PX) and total superoxide dismutase (T-SOD) activities, and an obvious decrease in hydrogen peroxide (H2O2) levels. The study further observed the morphology of RBCs and absorbance change of the blood samples and found a large deformation of RBCs in the experimental group. The absorbance peak production reflected the existence of ferryl Hb. Thus, the study confirmed that the linoleic acideinduced oxidative stress can cause acute damage of RBCs. Arachidonic acid (AA) is an important part of the FFA and metabolites of linoleic acid.14 In the present study, by injecting different concentrations of AA to SD rats, the changes in RBCs and the associated redox reactions were observed. The relationship between AA and HBL was also explored and the mechanism of HBL was explained, so as to obtain a useful HBL model.
Materials and methods Animal model preparation Adult male SD rats (weight: 225 15 g) purchased from the Chinese Academy of Science, Nanjing University Animal Center, China, were used for all experiments. All animals received humane care in compliance with “The Principles of Laboratory Animal Care” formulated by the National Society of Medical Research and “The Guide for the Care and Use of Laboratory Animals” published by the U.S. National Institutes of Health (National Research Council, 1996). The animals were housed at a set temperature (24 C) in a humidity-controlled room with a 12-h light/dark photoperiod. All experimental procedures were carried out strictly in accordance with the care and use of laboratory animals, which were approved by the National Institute of Health’s Guide for the Care and Use of Laboratory Animals and the Institutional Animal Care and Use Committee at Nanjing University, China. All animals were anesthetized with ether inhalation before operation.
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microscope (Nikon ECLIPSE 50i, Japan), and centrifuge (Hermle Universal Centrifuge Z323, Germany). The H2O2 concentration and GSH-PX and SOD activities were determined with commercially available assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). AA was obtained from Sigma-Aldrich (MO).
Experimental protocol and drug A total of 50 SD rats were assigned randomly into five groups: one control group and four experimental groups, with 10 rats in each group. First of all, AA was dissolved in a 20% ethanol solution. Different concentrations of AA were made up by diluting AA in 20% ethanol solution. The control group was treated with 20% ethanol solution alone. The experimental groups were further divided into four groups, AA-A, AA-B, AAC, and AA-D, with concentrations of 5, 10, 20, and 40 mmol/L, respectively. The mode of administration was intravenous injections of 0.5 m per dosage through the tail veins. The rate was slowed down, and the concentration of administration was minimized to avoid unnecessary deaths. Before formal experiments, preliminary experiments were conducted to explore the best-dosage range notation. The present study showed that half of the mortality rates occurred at a dosing concentration of 40 mmol/L, and the effective concentration ranged between 5-40 mmol/L. All the rats were monitored continuously until they recovered from anesthesia. The rats were then sacrificed by spinal dislocation method. The study found that they suffered from several symptoms, including dyspnea, cyanosis, hemoptysis labiali, and convulsions. Throughout the process, the rats had no special discomfort. Blood samples were collected from the caudal vein before injection and at 24-, 48-, and 72-h postinjection (0.5 mL each time). Blood smears were prepared and stained with Wright’s stain to observe morphologic changes. RBC and Hb values were obtained through the automatic blood analysis system. A spectrophotometer was used to measure the concentration of H2O2 and the activities of GSH-PX and T-SOD15; for each of these measurements, absorbance values were determined at 405, 412, and 550 nm, respectively, according to the manufacturer’s instructions. All samples were processed within 2 h of collecting the blood samples. Spectral changes of Hb in the AAC group were typically measured with a spectrophotometer. The Hb (10 mmol/L) was mixed with buffer (0.1 mol/L sodium phosphate) containing 100-mmol/L diethylenetriaminepentaacetic acid. All procedures were performed at 25 C.
Statistical analysis All calculations and statistical analyses were performed using the SPSS version 19.0 software. Values were expressed as mean standard deviation and analyzed by one-way analysis of variance, followed by Dunnett’s t-test. In all cases, P < 0.05 was regarded as significant.
Instruments and reagents The following instruments were used in this study: microplate reader (Bio-Rad680, USA), hematology analyzer (Sysmex XE-5000, Japan), spectrophotometer (Hewlett Packard 8453 UV-visible diode array spectrophotometer, USA), polarizing
Results This study showed that administering AA at a concentration of 20 mmol/L caused RBC and Hb values to decrease
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significantly, indicating that HBL occurred in the experimental group (Fig. 1A and B). By increasing the concentration of AA, the level/amount of RBC and Hb decreased obviously. However, at a concentration of 40 mmol/L, half of the AA-D group rats died within 5 min of administration. Pulmonary embolism was considered to be the main cause of death in this case16; the rats suffered from dyspnea, cyanosis, hemoptysis labiali, and convulsions prior to death. Further research was carried out among AA-B, AA-C, and control groups. No significant differences were obtained between them in blood tests and other indicators before the experiment. Based on the phenomenon observed, it was inferred that after 24 h, the blood cell got injured. First, the RBC and Hb values dropped to various degrees (Fig. 1). Hb and RBC values in the control group decreased by 8.50 2.35 g/L and 0.18 0.07 1012/L, respectively, whereas those in the AA-B group decreased by 18.25 3.30 g/L and 0.95 0.12 1012/L, respectively, and those in the AA-C group decreased by 24.52 2.55 g/L and 1.31 0.22 1012/L, respectively. However, changes among the AA-B, AA-C, and the control groups were statistically significant (P < 0.05). Activities of GSH-PX, and T-SOD and the concentration of H2O2 had a similar tendency (Fig. 2A-C). Changes in Hb and RBC values were apparent among the AA-B, AA-C, and control groups after 48 h of administration. The changes in GSH-PX and T-SOD activities and the H2O2
Fig. 2 e Comparison of GSH-PX, (A) T-SOD (B), and H2O2 (C) between control and experimental groups. Data are shown as means ± SE; *P < 0.05, #P < 0.01.
Fig. 1 e Comparison of Hb (A) and RBC (B) changes between control and experimental groups. Data are shown as means ± standard error (SE); *P < 0.05, #P < 0.01.
concentration were consistent with the changes in Hb and RBC values (P < 0.01). At 72 h, RBC and Hb values were relatively stable, and GSH-PX and T-SOD activities and the H2O2 concentration gradually increased to the rebalancing state (Fig. 2). The presence of ferryl Hb, formed by reacting with H2O2, was detected by its reaction with sulfide ions producing a characteristic absorbance band around 620 nm.17 The effect of AA on the hemolysis of RBCs, either on its own or in conjunction with ROS, can be used to evaluate the degree of oxidation injury of erythrocytes. Blood samples were collected from the AA-C group before administration and then every 24 h thereafter. The absorbance peaks were detected at approximately 425 nm corresponding to the ferryl Hb Soret peak18 (Fig. 3). The morphology of erythrocyte was observed under a polarizing microscope. The cell-wall structure, morphology, and metabolic features were normal in all groups before the AA treatment. After 24 h of administration, apparent morphologic changes were observed in the AA-B and AA-C groups, and the changes continued when observed after 48 h. A polarized light microscope showed 20% of erythrocyte
yuan et al arachidonic acid causes hbl-like red blood cell injury
Fig. 3 e (A) Changes in AA-C absorbance values as determined using a spectrophotometer. Measurements were taken every 24 h. For clarity, only a small subset of the spectra collected is shown, and the peak at 425 nm corresponds to a ferryl Hb Soret peak. (B) Statistical analysis of absorbance changes between control and AA-C groups, and the spectral changes at 425 nm. Values are shown as mean ± SE; *P < 0.05, #P < 0.001. deformation, mainly exhibited as pleomorphism, shrinkage, rupture, and breaking (Fig. 4F and G).
Discussion Previous studies have confirmed that the injury of blood cell caused by linoleic acid is close to the pathology of HBL.13 AA is an important component of FFA, which is also associated with linoleic acid metabolism. The presence of unsaturated double bonds in the AA structure makes it vulnerable to free radical attack.19 Due to the special oxidation nature of the unsaturated fatty acids, numerous free radicals and active substances are induced.20 If ROS generated exceeds the oxygen radical absorbance capacity, the active oxygen will release into the extracellular space and attack the cells and the surrounding tissue. The present study demonstrated that AA is closely related to red blood cell damage, indicating that FFA can induce oxidative stress and cause HBL. Hb and RBC values are good indicators that reflect the extent to which erythrocyte is damaged. In the present study, Hb and RBC values reduced in all the experimental and control groups, but the changes in the AA-B and AA-C groups were more remarkable, showing obvious damage to erythrocyte, and hence indicating that HBL occurred in these groups. Since the blood samples accounted for 2.8%-4.2% of the blood capacity of rats, this directly resulted in a decrease of Hb and
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RBC values. Moreover, the intravenous injection of ethanol diluent may have also induced an increased volume, cell lyses, and rupture of erythrocyte.21,22 All the aforementioned reasons may have caused the decrease of Hb and RBC values. Although same amount of blood samples were collected for both experimental and control groups, Hb and RBC values in the experimental groups decreased more significantly, indicating that HBL occurred in the experimental groups, and that AA was responsible for the change in Hb and RBC values. AA can stimulate the production of ROS through the following ways: (1) AA can irritate the nicotinamide adenine dinucleotide phosphate oxidase by activating calcium ion channels on the cell membrane to produce large amounts of ROS1,23; (2) an increase in AA concentration in the blood can raise lipid peroxidation and oxidized glutathione content, which may promote the formation of ROS24; (3) AA can also induce neutrophils to release oxygen radicals, and activate the receptor-interacting protein-1 to stimulate the generation of ROS25,26; and (4) AA can also inhibit the activity of the mitochondrial respiratory chain enzyme complexes I and III and increase the mobility of the mitochondrial membrane.27 This suggests that ROS can be generated through cyclooxygenase and lipoxygenase metabolic pathways, which include lipid peroxides and superoxide anion. GSH-PX is an important H2O2-removing enzyme in human cells, which couples the reduction of H2O2 with the oxidization of glutathione, thus removing H2O2 and protecting the integrity of the cell membrane function.28,29 The vitality of GSH-PX inversely correlates with the severity of cell damage caused by oxygen-free radicals. High levels of FAA inhibit the oxidization of glutathione, slowing the activity of GSH-PX and hence the rate of removal of H2O2.30 It was likely that the stress response caused by the blood collection process was accountable for the partial decrease of the GSH-PX and T-SOD activities and H2O2 levels in the first 24 h. Changes between the experimental and control groups were significantly different, showing AA had an influence on the pathologic role of RBCs. T-SOD plays a vital role in keeping the balance of oxidation and antioxidation in our body, thus removing superoxide-free radical from aerobic organisms and protecting against the toxic effect of the oxygen-free radicals.31 The SOD in blood has a specific inhibitory effect on superoxide anion; therefore, the T-SOD vitality reflects the ability of organisms in removing oxygen-free radicals. Studies have shown that FFA can stimulate neutrophils to produce H2O2 and hypochlorous acid; deplete SOD, GSH-PX, and total antioxidant capacity on the surface of red cell membrane; and create additional oxidative denaturation of Hb.9,32 The present study showed that GSH-PX and T-SOD depleted and decreased obviously in the AA-B and AA-C groups 48 h after administration. Moreover, H2O2 was decomposed into H2O and O2 by Mn superoxide dismutase and Zn superoxide dismutase, or translated into H2O directly by the action of glutathione, resulting in the decline of H2O2 levels.33,34 The oxidative stress response diminished as AA gradually metabolized, and the enzymes associated with oxidative stress returned to equilibrium. Hb is an important component of RBCs. Ferrous Hb [Fe (II) Hb] is responsible for delivering oxygen to tissues and can be
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Fig. 4 e Changes in red blood cell morphology. Blood smears were observed under polarized light microscope (400 3 magnification). Blood samples were collected before injection and at 24-, 48-, and 72-h postinjection. Cell morphology did not obviously change in the control group (A-D). However, it showed 20% of erythrocyte deformation, mainly exhibited as pleomorphism, shrinkage, rupture, and breaking cells (black arrow) in the AA-C group (E-H) treated with AA (20 mmol/L). (Color version of figure is available online.)
slowly oxidized to ferric Hb [Fe (III) Hb].18,35 Ferryl Hb (Fe4þ¼O2), which is produced by the reaction of methemoglobin with strong oxidants, loses the function of carrying and transporting oxygen.36,37 Studies have shown that lipid peroxidation increases in the process of ferryl Hb formation and aggravates lipid membrane damage.38,39 H2O2 is one of the active substances involved in the severe damage of in vivo cells, while its reaction with Fe(II) Hb and Fe (III)Hb results in the formation of ferryl Hb.40 These physiological responses are closely related to ischemia in vivo. Ferryl Hb can oxidize nucleic acids and lipids, and its globulin radical is unstable, which rapidly shifts to a stable and long-lived ferryl radical.41,42 Ferryl Hb will automatically revert to the ferric (met-) Hb form under acidic conditions.43 The reduced met-Hb is different from the initial ferric Hb, ferrihemochrome, and Hb subunit formed in the process of reduction. The presence of ferryl Hb can be judged by the extinction value of the blood samples. Previous studies have found that when Hb reacts with H2O2 at pH 7-8, the peak value of absorbance appears at 408 and 425 nm, which represent the met-Hb and ferryl Hb.17,18,38 The experimental groups in the present study also
showed an absorbance peak at 425 nm, whereas the control group had no significant change, which proved the generation of ferryl Hb. This result indicates that strong oxidative stress in the blood system of the experimental group leads to the intense oxidation of Hb. Tests on the blood samples indicated that the levels of H2O2 consistently declined at 24 and 48 h after the treatment. These results clearly showed that a large number of H2O2 was consumed after the injection of AA. Part of the H2O2 is involved in the destruction process of RBCs by oxidation, which can be verified through morphologic changes of RBCs. Groups of crenated, heteromorphic, and even exploded erythrocytes appeared at 24 h after the administration of AA, and the situation improved after 72 h, indicating that the oxidative action on erythrocyte induced by AA gradually weakened and disappeared. The pathologic changes of erythrocyte morphology can be considered as the result of protein oxidation and lipid peroxidation, and permeability of red cell membrane changes. However, strong oxidation can also directly lead to the destruction of RBCs.
yuan et al arachidonic acid causes hbl-like red blood cell injury
In conclusion, AA and linoleic acid are both important components of FFAs. The results are consistent with the previous studies on linoleic acid. In conclusion, AA can induce oxidative stress reaction and stimulate the production of ROS. Oxidative stress induced by AA can act on erythrocyte and cause HBL. The limitation of this study was that the change of ROS was not measured, which could have made the results more intuitive and credible. More research remains to be perfected to explain the pathogenesis of HBL, for example, the use of antioxidant intervention experiments on rats and clinical observation. These will eventually be beneficial to explain the mechanism of HBL and provide evidence base for clinical prevention and treatment of HBL.
Acknowledgment This study was supported by the Clinical Science and Technology Foundation of Jingling hospital (No. 2016016), and the Scientific Innovation Research of the Chinese People’s Liberation Army (PLA) (14QNP034) and Natural Science Foundation of Jiangsu Province (BK20161385) Authors’ contributions: B.N.R. and Z.J.N. contributed to the ideas and final version for this commentary, Y.T, C.Y, M.J, Q.H, Y.W, and S.W.S contributed to the literature search, animal experiment, data analysis, article drafting, and editing.
Disclosure The authors reported no proprietary or commercial interest in any product mentioned or concept discussed in the article.
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