Emergency. 2018; 6 (1): e54 OR I G I N A L RE S E A RC H Burn-induced Oxidative Stress and Serum Glutathione Depletion; a Cross Sectional Study Arash Beiraghi-Toosi1,2, Roya Askarian3, Faezeh Sadrabadi Haghighi3, Mohammad Safarian4, Fereshteh Kalantari1, Seyed Isaac Hashemy1∗ 1. Surgical Oncology Research Centre, Mashhad University of Medical Sciences, Mashhad, Iran. 2. Department of Plastic Surgery, Imam Reza Hospital, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran. 3. Department of Clinical Biochemistry, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran. 4. Department of Nutrition, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran. Received: April 2018; Accepted: August 2018; Published online: 31 August 2018 Abstract: Introduction: Several studies have shown the role of oxidative stress in pathophysiology of burn injuries. This study aimed to evaluate the changes of oxidant-antioxidant levels during the week following burn injuries and its correlation with grade of burn. Methods: In this prospective cross-sectional study, changes of total glutathione, reduced glutathione (GSH), oxidized GSH (GSSG), GSH/GSSG ratio, as well as Pro-oxidant-antioxidant balance (PAB) were investigated on the 1st, 2nd and 7th days of admission in patients with > 15% burns. Results: 40 patients with the mean age of 21.1 ± 14.5 were studied (47.5% male). More than 50% of patients were in the 18 – 55 years age range and over 70% had 20% – 60% grade of burn. Total serum glutathione level and GSH had significant decreasing trends (P < 0.001) and GSSG and GSH/GSSG ratio had increasing trends (p < 0.001). No significant correlation was observed between serum GSH level and the total body surface area (TBSA) of burn injury (r = 0.047; p = 0.779). The evaluation of PAB and its correlation with TBSA showed a significant and direct association between them on the 1st (coefficient = 0.516; p = 0.001), 2nd (coefficient = 0.62; p <0.001), and 3rd (coefficient = 0.471; p = 0.002) day of follow up. Conclusion: According to this study, the redox perturbation oc- curred in burn injury which was measured and proved by decreased GSH/GSSG ratio as well as the shift of PAB in favour of oxidants. Besides, since PAB positively correlated with the severity of dermal damage, it might suggest the application of antioxidants as a part of therapeutic protocol for which the dosage should be proportionate to the surface area of the damaged skin. Keywords: Oxidative stress; oxidants; antioxidants; Glutathione; Burns © Copyright (2018) Shahid Beheshti University of Medical Sciences Cite this article as: Beiraghi-Toosi A, Askarian R, Sadrabadi Haghighi F, Safarian M, Kalantari F, Hashemy Seyed I. Burn-induced Oxidative Stress and Serum Glutathione Depletion; a Cross Sectional Study. Emergency. 2018; 6(1): e54. 1. Introduction Burn injury is a kind of inflammatory disease in which, be- sides the local dermal damages, systemic complications such as systemic inflammatory response syndrome, multiple or- gan failure and sepsis, which are the leading causes of mor- bidity and mortality, may occur (1, 2). Different factors such as inflammatory responses are involved in the pathophys- iology of these systemic complications. Oxidative stress is ∗Corresponding Author: Seyed Isaac Hashemy; Surgical Oncology Research Centre, Mashhad University of Medical Sciences, Mashhad, Iran. Email: hashemyi@mums.ac.ir Tel: +98-9158112664 one of these factors, which happens due to the formation of free radicals at the area of injury due to intravascular stim- ulation of neutrophils and increased xanthine oxidase activ- ity (3, 4). The subsequent transportation of these free radi- cals to distant parts through blood flow may explain one of the mechanisms by which other organs such as liver, heart and lungs can get damaged in patients with burn injury (5). LaLonde et al. even suggested a causal relationship between increased redox perturbation and inflammation (6). These damages are because of the high reactivity of free radicals, including both reactive oxygen species (ROS) and reactive nitrogen species (RNS), which react with and oxidize cel- lular molecules such as proteins, lipids and nucleic acids This open-access article distributed under the terms of the Creative Commons Attribution NonCommercial 3.0 License (CC BY-NC 3.0). Downloaded from: www.jemerg.com A. Beiraghi-Toosi et al. 2 (7, 8) leading to a wide variety of diseases including can- cers, autoimmune diseases, cardiovascular diseases, etc (9- 14). On the other hand, the body is equipped with enzymatic as well as non-enzymatic antioxidants to recuperate the re- dox homeostasis (15-18). Glutathione, a tripeptide, is such a molecule, which plays an important role in providing and maintaining a reduced intracellular environment, which is necessary for cell survival (19). During oxidative stress, when pro-oxidant-antioxidant balance (PAB) is disrupted in favour of the former, reduced glutathione (GSH) is oxidized to the form of GSSG, and GSH/GSSG ratio, which is normally about 100/1 in cells, decreases to values of about 10:1 and even 1:1 (20-23). This study aimed to investigate if oxidative stress happens during burn injury, how it proceeds during the first week of injury and whether it correlates with the severity of dermal damage. For this purpose, two markers of oxidative stress were selected: the GSH/GSSG ratio and PAB. The latter is a newly-established method through which the whole redox status can be investigated using a simple method (24). 2. Methods 2.1. Study design and setting This prospective cross-sectional study was conducted on burn patients who were hospitalized in Imam Reza Hospi- tal, Mashhad, Iran, during a one-month period from Febru- ary 2016 to March 2016. The changes of total glutathione, GSH, GSSG, as well as GSH/GSSG ratio were investigated on the 1st, 2nd and 7th days after a thermal burn in- jury. This project was ethically approved by the Research Council of Mashhad University of Medical Sciences (code: IR.MUMS.REC.1388.102), and an informed consent was pro- vided by each patient. 2.2. Participants Patients with over 15% burn of total body surface area (TBSA) of the second-degree were enrolled using non probability convenience sampling method. Those with known systemic diseases such as cardiovascular diseases, diabetes mellitus, smoking, opium addiction, etc. as well as patients with a recent history of consuming antioxidants such as Vitamin C and multivitamins in which the redox status could be af- fected were excluded from this study. 2.3. Measurements After careful history taking and clinical examination, the pro- tocol of study was explained to patients or relatives and an informed consent was obtained. Blood samples from each patient were collected three times: on the 1st day (during the first 6 hours after the injury), 2nd day (24 hours after the injury), and 7th day of the injury to study serum GSH in both oxidized and reduced forms as well as PAB. Blood sam- ples were collected free of any anticoagulant, kept at room temperature for 30 minutes to clot. Thereafter, the sam- ples were centrifuged at 2000×g for 15 min at 4 ◦C to sep- arate the serum, which was subsequently deproteinated by adding an equal volume of metaphosphoric acid 1 M in dis- tilled water to the sample. After 5 minutes of incubation at 25◦C, the mixture was centrifuged at 3000×g for 5 minutes and the supernatant was collected carefully. The concentra- tion of serum GSH was measured by the method described before by Ellman, which is based on the reduction of 5,5’- Dithiobis-(2-Nitrobenzoic Acid) (DTNB) by free thiols to 2- nitrobenzoic acid anion (TNB–), which is a yellowish com- pound (22, 27, 28). The absorbance of samples was mea- sured spectrophotometrically at 412 nm, and the concentra- tion of glutathione in each sample was calculated using a standard curve, which had been provided with different con- centrations of glutathione (0-15 µM in MES buffer). In order to measure the concentration of GSSG, excluding GSH, 10 µl of 2-vinylpyridine 1 M in ethanol was added to 1 ml of de- proteinated sample, incubated at 25◦C for 60 min to block free thiols of GSH. Subtraction of GSSG level from total glu- tathione concentration equals to GSH level. Serum PAB was measured before deproteination as described before (24). In brief, it was performed by adding a cation to the TMB (3, 3 ’, 5, 5’-Tetramethylbenzidine) solution by peroxidase en- zyme. The cationized TMB solution (blue colored) was sub- sequently reduced by antioxidants present in the sample or standards, and converted to the reduced TMB (colorless). The higher the level of antioxidants in the sample was, the lower the density of blue color would be. For this purpose, the standard curve was prepared by mixing varying percent- ages of hydrogen peroxide 250 µM and uric acid 3 mM in 10 mM of NaOH. The resulting absorption was read at a wave- length of 450 nm. The percentage of burnt TBSA was calcu- lated using the law of Lound & Browder (25, 26). All materials and reagents were purchased from Sigma-Aldrich, Germany. 2.4. Data gathering Demographic information (age, sex), vital signs (tempera- ture, heart rate, respiratory rate), and the percentage of TBSA were recorded for all participants using a pre-design check list. There were no missing data. All data were collected prospectively by a trained nurse. Laboratory tests were per- formed by MSc students in Clinical Biochemistry. 2.5. Statistical Analysis We have performed the Kolmogorov-Smirnov test to check the normality of data. Repeated measures ANOVA, paired sample t-test, and correlation coefficient tests were used to analyse the data. The level of significance was considered as p-value ≤0.05. SPSS 11.0 software was used for statistical This open-access article distributed under the terms of the Creative Commons Attribution NonCommercial 3.0 License (CC BY-NC 3.0). Downloaded from: www.jemerg.com 3 Emergency. 2018; 6 (1): e54 Figure 1: Serum glutathione changes following burn injury on the 1st, 2nd, and 7th day of admission. analysis and data are presented as Mean ± standard devia- tion (SD). 3. Results 3.1. Baseline characteristics of studied patients 40 patients with the mean age of 21.1 ± 14.5 (3 – 56) were studied (47.5% male). Table 1 shows the baseline character- istics of studied patients. More than 50% of patients were in 18 – 55 years age range and > 70% had 20% – 60% burn. 3.2. GSH changes following burn injury Figure 1 and table 2 summarize the changes of total serum glutathione level, GSH, GSSG, and GSH/GSSG ratio during the one week follow-up. Total serum glutathione level and GSH had significant decreasing trends (P < 0.001) and GSSG and GSH/GSSG ratio had increasing trends (p < 0.001). No significant correlation was observed between serum GSH level and the TBSA of burn injury (r = 0.047; p = 0.779). 3.3. PAB changes following burn injury The evaluation of PAB and its correlation with TBSA showed a significant and direct association between them on the 1st (coefficient = 0.516; p = 0.001), 2nd (coefficient = 0.62; p <0.001), and 3rd (coefficient = 0.471; p = 0.002) day of follow up (figure 2). This open-access article distributed under the terms of the Creative Commons Attribution NonCommercial 3.0 License (CC BY-NC 3.0). Downloaded from: www.jemerg.com A. Beiraghi-Toosi et al. 4 Table 1: Baseline characteristics of studied patients Variables Number (%) Sex Male 19 (47.5) Female 31 (52.5) Age (years) < 18 16 (40.0) 18 – 35 12 (30.0) 35 – 55 11 (27.5) ≥ 55 1 (2.5) Burn grade* (%) <20 3 (7.5) 20 – 40 26 (65.0) 40 – 60 3 (7.5) ≥ 60 8 (20.0) *: Calculated using the law of Lound & Browder. Table 2: Changes of total serum glutathione level, reduced glu- tathione (GSH), oxidized glutathione (GSSG), and GSH/GSSG ratio during the one week follow-up after burn injury among the studied patients Variables Values P Total glutathione (µM) 1s t 10.52 ± 1.38 2n d 4.13 ± 0.96 < 0.001 7t h 3.91 ± 1.18 GSH 1s t 8.05 ± 1.46 2n d 1.32 ± 0.95 < 0.001 7t h 0.55 ± 0.51 GSSG 1st 2.46 ± 0.81 2n d 2.81 ± 0.89 < 0.001 7t h 3.37 ± 1.01 GSH/GSSG ratio 1st 3.66 ± 1.44 2n d 0.55 ± 0.44 < 0.001 7t h 0.17 ± 0.15 Data are presented as mean ± standard deviation. 4. Discussion The aim of this study was to determine how the formation of reactive oxygen species and its subsequent oxidative stress proceed during the first week of burn injury; for this pur- pose, GSH/GSSG ratio and PAB were chosen as the mark- ers of the redox status. Based on this study, we observed time-dependent decreasing trends for both the serum total glutathione and GSH during the first week of burn trauma. However, an increasing trend was recorded for the serum oxidized glutathione within the first week of injury, which was time-dependent as well. Moreover, since the damaged body surface area is one of the factors involved in both the indication of hospitalization and prognosis, we planned to Figure 2: Correlation of pro-oxidant-Antioxidant Balance (PAB) with total body surface area (TBSA) burnt. investigate the possible correlation between this factor and the above-mentioned redox markers. No significant cor- relation between serum glutathione level and the TBSA of burn injury was observed in our study. However, PAB val- ues showed a significant and direct association with burnt TBSA. The perturbation of redox homeostasis during burn injury has been addressed in a number of studies (4-6, 29, 30). In this study, we could also demonstrate a significant degree of redox perturbation, which was documented as the decreased level of serum total and reduced glutathione as well as GSH/GSSG ratio, which were accompanied with in- creased level of oxidized glutathione. The observed changes were time-dependent, which is supported by the results of Szczesny, B. et al (31). However, as observed in this study, the magnitude of increase in serum oxidized glutathione (GSSG) was not as big as the decrease of serum glutathione (GSH) concentration. Con- sidering this fact, along with decreased level of total serum glutathione, it might be suggested that GSH is probably re- cruited during burn injury to protect cellular and extracellu- lar proteins from irreversible oxidations such as the forma- tion of sulfinic and sulfonic acid (32) through glutathiony- lation of these proteins (figure 3) (33, 34). Since this post- translational modification is reversible, proteins can retain their activities after controlling the acute phase of injury (35- 38). On the other hand, since the administration of ascorbic acid as a part of the routine therapeutic protocol in our hos- pital was started from the second day of hospitalization, the extent of oxidative damage as a consequence of burn injury could be much more significant if there were not any antiox- idants in the drug regimen. The more significant decrease of total glutathione and GSH, This open-access article distributed under the terms of the Creative Commons Attribution NonCommercial 3.0 License (CC BY-NC 3.0). Downloaded from: www.jemerg.com 5 Emergency. 2018; 6 (1): e54 Figure 3: Possible mechanism and consequences of oxidative stress in burn injury. This schematic figure suggests two pathways of GSH consumption during burn injury including its oxidation to GSSG and its application in glutathionylation. as well as the more dramatic decrease in GSH/GSSG ratio from the 1st day to 2nd day in comparison to changes from the 2nd day to 7th day could also be explained by the pro- tective effects of ascorbic acid against oxidative damages. In this study, the first sample was taken during the first 6 hours of hospitalization, and the second sample was taken 24 hours later and before the administration of ascorbic acid, which is a part of the routine therapeutic protocol for burn injury in Imam Reza hospital, Mashhad, Iran. The third sample was taken on day 7, which means the serum redox status of pa- tients was studied after the administration of a distinguished antioxidant for 6 days and daily administration of ascorbic acid. Since the administration of ascorbic acid could not be avoided due to ethical reasons, we could not study the changes of glutathione as well as PAB without use of any an- tioxidant in this group of patients. Performing the same study in animal models can be recommended to solve this limita- tion. There are several studies showing the advantages of an- tioxidant therapy in management of burn injuries (39). N- acetylcysteine is an antioxidant and its effects on oxidative stress have been studied (8, 29). Ascorbic acid is another ex- ample of antioxidants that is shown to attenuate the oxida- tive stress (40). In a study by LaLonde et a.l, using rats as ani- mal models, oral administration of a mixture containing glu- tathione, N-acetylcysteine and ascorbic acid showed a pro- tective effect against burn-induced altered cell energetic (6). However, the severity of burn injury was not introduced as a factor for determination of the required dosage of antioxi- dants in any of these studies. 5. Limitation The oxidative stress after burn is supposed to start quickly. Therefore, the first sample had to be taken as soon as possi- ble. However, considering the time interval between injury and hospitalization and since our hospital is the referral cen- ter for a large area of Iran, more we than 6 hours might have passed at from the trauma at the time of admission but de- cided to take the samples from the patients during the first 6 hours of admission anyway. For other samples, we should have ideally checked our patients on all days of hospitaliza- tion; but financially we had limitations, which made us be restricted to the 2nd and 7th days. 6. Conclusion According to this study, the redox perturbation in burn in- jury, which was measured as the PAB value positively corre- lated with the severity of dermal damage, which may suggest that the dosage of antioxidants should be proportionate to the surface area of skin that is damaged. 7. Appendix 7.1. Acknowledgements We are very grateful to all patients who participated in this study. This work was based on the Research Project No. 88482, as the MSc dissertation of Roya Askarian, financed by Research Council of Mashhad University of Medical Sciences. 7.2. Author contribution ABT, MS and SIH contributed to the conception and design of the study. RA, FSH and SIH contributed to the acquisition, analysis and interpretation of data. RA, FSH and SIH drafted the manuscript; ABT, MS and SIH revised it critically for con- tent. All authors read and approved the final manuscript sub- mitted for publication. 7.3. 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Action of ascorbic acid as a scavenger of active and stable oxygen radicals. The American Journal of Clin- ical Nutrition. 1991;54(6):1119S-24S. This open-access article distributed under the terms of the Creative Commons Attribution NonCommercial 3.0 License (CC BY-NC 3.0). Downloaded from: www.jemerg.com Introduction Methods Results Discussion Limitation Conclusion Appendix References