key: cord-0767415-dlwy2r3e
authors: Karkhanei, Behrouz; Ghane, Elaheh Talebi; Mehri, Fereshteh
title: Evaluation of oxidative stress level: total antioxidant capacity, total oxidant status and glutathione activity in patients with Covid-19
date: 2021-05-17
journal: New Microbes New Infect
DOI: 10.1016/j.nmni.2021.100897
sha: c7eef3bb0679397404f0f03ce41856351bfe73b0
doc_id: 767415
cord_uid: dlwy2r3e

Covid-19 disease, as a dangerous global pandemic, has led to high morbidity and mortality in all countries. There is a lot of evidence for the possible role of oxidative stress in coronavirus disease 2019 (Covid-19). In the current study, we aimed to measure the levels of glutathione (GSH), total antioxidant capacity (TAC), and total oxidant status (TOS) in the serum of patients with Covid-19. A total of 96 individuals with and without Covid-19 were enrolled and divided into four groups, including hospitalized group in non-intensive care units (non-ICU) (n=35), hospitalized group in intensive care units with endotracheal intubation (EI) (ICU with EI) (n=19), hospitalized group in intensive care units without endotracheal intubation (ICU without EI) (n=24), and healthy people without Covid-19 disease as our control group (n=18).The present study revealed that the TOS level was significantly lower in the group of control (P = 0.001), and level of (GSH) remarkably increased in the patients' groups (P < 0.001). TAC activity in non-ICU group of patients had no significant difference in comparison with control group. However, in hospitalized patients' groups in ICU with and without EI this activity was significantly different from control group (P<0.001). Moreover, there was a significant relationship between the levels of TOS, GSH, and TAC with blood Oxygen saturation (SpO2), fever, duration of hospitalization, and the prognosis of this disease (P<0.001). Area under the curve (AUC) (CI, 95%) of TOS, TAC and GSH-C to predict death among patients were respectively 0.907 (0.841, 0.973), 0.735 (0.626, 0.843) and 0.820 (0.725, 0.914). ROC curve analysis showed that TOS, TAC and GSH-C have the potential specificity and sensitivity to distinguish between alive and dead patients. We found that elevated levels of oxidative stress and reduction of antioxidant indices can aggravate disease’s severity in hospitalized patients with Covid-19. Therefore, it can be suggested to apply antioxidant agents as one of the effective therapeutic strategies in these groups.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) emerged in late December 2019 and was responsible for the COVID-19 pandemic [1] . COVID-19 has several unique features compared with other coronavirus infections [2] . In the most vulnerable individuals (for example, older, obese, or diabetic individuals), the virus sometimes triggers a cascade of acute biological events due to excessive levels of reactive oxygen species (ROS), which can, unfortunately, lead to ventilation of patients and even dying [3] . Critical characteristics of this pandemic are high rate of spread, lack of knowledge, lack of effective treatment, and high mortality. Symptoms can vary drastically; they include fever (99%), chills, dry cough (59%), sputum production (27%), fatigue (70%), lethargy, arthralgias, myalgias (35%), headache, dyspnea (31%), nausea, vomiting, anorexia (40%), and diarrhea; Some carriers may be asymptomatic, whereas others can experience acute respiratory distress syndrome (ARDS) and death [4] . Therefore, it is required to identify complicated pathogenic mechanisms of this virus in order to decrease the time of hospitalization and mortality rate. It is known that oxidative stress is associated with the severity of the disease.Oxidative stress is involved in aging and found in certain chronic pathologies, such as diabetes mellitus, cancers, hypertension, coronary heart disease, etc., and certain infections, especially by RNA viruses, belonging to coronaviruse family [5] . Some authors have postulated that oxidative stress might be as an important player in the activation of the inflammasome during SARS-CoV-2 infection [6] . Over the past months, the COVID-19 crisis has seriously jeopardized the capabilities of most healthcare systems worldwide. Given to recent studies, oxidative stress is an essential factor in increasing the severity of COVID-19 in some patients, and it is associated with pulmonary dysfunction, cytokine storm, and viral sepsis derived from SARS-CoV-2 infection [7] . The deleterious effects of ROS on the functions of both pulmonary cells and red blood cells (RBCs) can be seen as a major contributor of hypoxic respiratory failure in the most severe cases of COVID-19 [3] . Since oxidative stress seems to play an important role in the pathogenesis of respiratory syncytial virus (RSV) and possibly other viral-associated lung diseases, antioxidant intervention would represent a rational approach for the treatment of lower respiratory tract infections, caused by, various respiratory viral infections [8] . The pathogenesis of many diseases can be as a result of apoptosis cascade induced by oxidative stress induced apoptotic signalingoxidative stress, leading to ROS increases and/or antioxidant decreases, J o u r n a l P r e -p r o o f disruption of intracellular redox homeostasis, and irreversible oxidative modifications of lipid, protein, or DNA [9] . In general, respiratory viral infections, such as SARS-CoV-2 infection cause cytokine production, inflammation, cell death, and other pathophysiological processes, which can be linked with redox imbalance or oxidative stress [10] . All these processes are associated with the development of oxidative stress, which makes an important contribution to the pathogenesis of viral infections [11] . Strengthening immune system and reducing inflammation and oxidative stress through diet and nutrition, such as consuming sufficient protein, vitamin C, vitamin E, vitamin A, zinc, carotenoids, and polyphenols play an essential role in fighting against COVID-19 [12, 13] . In general, changes in redox homeostasis in infected cells are one of the key events that is linked to infection with respiratory viruses, inflammation, and subsequent tissue damage [14] . It appears that oxidative stress play a critical role in the pathogenesis of COVID-19, as perpetuating the cytokine storm cycle, blood clotting mechanism, and exacerbating hypoxia [15] . It is known that oxidative stress is associated with the severity of the disease, but its status of this biomarker is unclear in patients hospitalized in the hospital with various conditions, in terms smoking and drug use, place of residence, occupation, fever, the length of hospitalization, and SpO2. Therefore, the present study was conducted to evaluate oxidative stress biomarkers' levels in hospitalized Covid-19 patients and control individuals in public hospitals of Hamadan City, in Iran in regarding factors mentioned.

This case-control study was conducted at the public hospitals of Hamadan City, located in the west of Iran. Ninety-six Covid-19 patients were selected in four groups, including hospitalized group in non-intensive care units (non-ICU Group) (n=35), hospitalized group in intensive care units with endotracheal intubation (EI) (ICU with EI Group) (n=19), hospitalized group in intensive care units without endotracheal intubation (ICU without EI group) (n=24), and healthy people without Covid-19 disease as control group (n=18), between May and September in 2020.

The inclusion criteria for selecting healthy people as our control group were the people who had no Covid-19 symptoms, or a history of visiting a doctor or being hospitalized due to Covid-19, and for patient groups were patients admitted in hospital due to infection with Covid-19, with positive Covid-19 RT-PCR test, and identified based on World Health Organization interim guidelines J o u r n a l P r e -p r o o f [16] . Subjects with a history of diabetes, hypertension, cancers, and autoimmune disorders were excluded from control and case groups. Also, subjects were excluded if they had a specific regimen or took antioxidant supplements such as vitamin C, vitamin E, Coenzyme Q10, Nacetylcysteine, and selenium. All patients or their surrogates had completed the consent form before being involved in this investigation.

We used a researcher-made questionnaire that included some factors, such as age, sex, education, smoking and drug use, place of residence, occupation, fever, the length of hospitalization, and SpO 2 for selected individuals in the patient and control groups. After that, venous blood samples were collected within a minimum of 24 hours after admission. The samples were centrifuged at 3000g for 10 minutes; serums were separated, liquated, and stored at -20 C until analyzed.

The serum levels of glutathione (GSH), total antioxidant capacity (TAC) and total oxidant status (TOS) were measured by using the commercially available ELISA kits according to the instructions of the company (ZellBio GmbH, Veltlinerweg 42, and 89072 Ulm, Germany).

The distribution of qualitative data was described by frequencies and percentages and compared between four groups (control group, non-ICU group, ICU without EI group, and ICU with EI group) by chi square and Fisher exact test. The quantitative data was described as the mean ± standard deviation (SD), median and interquartile range (IQR) and their Normal distribution was evaluated by Shapiro-Wilk test. In case of normal distribution, the mean of quantitative data was compared in two groups using independent t-test, and in four groups using one-way Analysis of Variance (ANOVA), otherwise Mann-Whitney and Kruskal-Wallis tests were done. Moreover, the pairwise comparisons were done, using Tuckey post hoc test. The receiver operating characteristic (ROC) curve and the area under the curve (AUC) were used to determine the feasibility of using oxidative status as a classifier to predict death among patients. All analyses were performed at 0.05 significance levels using SPSS version 23 (SPSS Inc., USA) and GraphPad Prism version 6 for Windows.

The main demographic characteristics of the study population are shown in Table 1 . No significant difference in age, sex, smoking, and opium use was observed between different groups (P>0.05).

There was a significant difference between these four different groups according to their education level, job position, and residential area. In this regard, 73.68%, 73.68%, and 63.16% of infected patients in ICU with EI were illiterate, unemployed, and lived-in rural areas, respectively.

According to the results, body temperature (fever) of patient groups was higher than those in control group (P<0.001), which indicated that 63.16% of patients in ICU with EI, 70.83% of patients in ICU without EI, and 20.00% of patients (non-ICU), had fever. Our findings illustrated that the duration of hospitalization of patients was variable among different groups. Over 80% of patients in ICU without EI were hospitalized in hospital for less than one week, while 89.47% of patients in ICU with EI were hospitalized for more than one week. Based on the results, SpO2 significantly decreased in patient groups according to the severity of the disease (P<0.001). In addition, a significant difference between case groups was observed in the outcome of the disease (P<0.001). 

Results for oxidative stress markers, including TOS, GSH, and TAC, were described by mean and 95% confidence intervals in different patient groups compared with the control group, presented in Figure 1(a-c) . According to the presented results in Figure 1(a-c) , the TOS levels in different case groups were significantly higher than the control group. The mean ± SD for this biomarker was Table 2 represents the relationship between oxidative stress biomarkers and the level of other parameters in all patients. The results in this Table indicate that those patients with fever, SpO2 lower than 88 percent, the length of hospitalization higher than one week and dead patients had significantly higher mean (SD) and median (IQR) of TOS and lower mean (SD) and median (IQR) of GSH and TAC. On the other hand, the TOS/GSH and TOS/TAC ratios in both control and the patient group are shown in Table   3 . In our study the TOS/GSH ratio was 0.003 ±0.002, 0.070 ±0.018, 0.224 ±0.069, 0.907 ±0.331 in control group, Non-ICU group, ICU without EI group and ICU with ET group, respectively, and TOS/TAC ratio was 0.019 ±0.011, 0.323±0.106, 0.061 ±0.025, 0.306±0.129in control group, Non-ICU group, ICU without EI group and ICU with ET group, respectively. Our study showed the TOS/GSH ratio was 0.590±0.025 and 0.129±0.031 in expired patients and discharged patients, respectively; and the TOS/TAC ratio was 0.318±0.115 and 0.063±0.015 in expired patients and discharged patients, respectively. Also, we found that 78.95% and 25% of patients in ICU with and without ET deceased, while all patients were alive in Non ICU group. ROC curve analysis showed that a) TOS and TAC, b) GSH-C have the potential specificity and sensitivity to distinguish between alive and dead patients.

In the present study, we investigated the role of oxidative stress biomarkers in different groups of patients hospitalized in all public hospitals of Hamadan city in Iran. According to previous studies, an imbalance in the production of reactive species and the body's inability to detoxify these reactive species is referred to oxidative stress [17, 18] . It is known that oxidative stress is triggered by a wide variety of viral infections, including HIV 1, viral hepatitis B,C, and D viruses, herpes viruses, respiratory viruses ,such as corona viruses. [5] and is associated with severity and predictive of outcome. Although, it has been clearly understood that many of viral, bacterial, and parasitic infections trigger the production of reactive oxygen (ROS) and nitrogen (RNS) species implicated in lung tissue injury and epithelial barrier dysfunction , but understanding the molecular inflammation mechanisms of stress oxidative contributing to Covid-19 progression is a current clinical need to improve therapies in patients [19] . As mentioned in previous studies, hypoxia induced by lung injury can be due to mitochondrial dysfunction. Mitochondrial dysfunction leads to a relative decrease in oxygen and energy production and increase in ROS production. In this regard, superoxide, H2O2 and other reactive species are mainly produced by the mitochondrial respiratory chain. Hydrogen peroxide causes the expression of many genes that activate proinflammatory cytokines in macrophages, neutrophils, and endothelial cells through NADPH oxidase (NOx) to produce more superoxide and H2O2 [15, 20] . Also, published researches showed covid-19 microbiota dysbiosis disturbs mitochondrial homeostasis via the production of toxic as gases hydrogen sulfide (H2S) and nitrogen oxide (NO) [20] . These processes ultimately, lead to oxidative damage in COVID-19 patients. Survey data describes that oxidative damage occurs in humans and the demographic, physical, or nutritional factors may be associated with it [21] . Our results demonstrated that oxidative stress profile in Covid-19 patients is closely related to patients' health level and demographic characteristics. Owing to our results, the serum level of TOS, as one of the oxidative stress biomarkers, was higher in patients with acute disease conditions, like those in ICU with and without endotracheal intubation, than in control group (Fig 1a) . In general, factors such as medication, environmental pollutants, and dietary components highlight the importance of an optimal nutrient status to reduce inflammation and oxidative stress: thereby, they strengthen the immune system during the Covid-19 crisis [12] . Our study showed that there is a direct [25] . High levels of ROS with deprived antioxidant mechanisms are of great importance for viruses to replicate, and cause disease [26] .

Although reactive species are frequently formed after viral infections and antioxidant defenses, such as enzymatic and non-enzymatic components, protect against reactive species, but sometimes these defenses are not completely adequate [17] .The major antioxidant enzymes, directly involved in the neutralization of ROS and RNS are superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GRx) [27] . The non-enzymatic antioxidants are also divided into metabolic antioxidants and nutrient antioxidants. Metabolic antioxidants belonging to endogenous antioxidants are produced by metabolism in the body, such as lipoid acid, glutathione, L-ariginine, coenzyme Q10, melatonin, uric acid, bilirubin, metalchelating proteins, transferrin, etc. [28, 29] . However, nutrient antioxidants belonging to exogenous antioxidants are compounds and cannot be produced in the body and must be provided through foods or supplements, such as vitamin E, vitamin C, carotenoids, trace metals (selenium, manganese, and zinc), flavonoids, omega-3, and omega-6 fatty acids, etc. [18, 27] . The practice of physical exercises acts as a modulator of the immune system. During and after physical exercise, pro-and anti-infammatory cytokines are released, lymphocyte circulation increases, as well as cell J o u r n a l P r e -p r o o f recruitment. Such practice has an effect on the lower incidence, intensity of symptoms and mortality in viral infections as covid-19 [30] . As shown in Figure 2b, disease [19, 22, 36, 37] . Glutathione (GSH) as a thiol compound is one of the most important small molecular weight antioxidants, produced in the cell [38] . Based on the results we obtained, glutathione level (GSH) as an antioxidant was significantly lower in patients with Covid-19

hospitalized in different hospital wards compared with the control group (Fig 1c) . Our study also showed GSH levels in infected patients with Covid-19 have an indirect relationship with fever, duration of hospitalization and direct relationship with SpO2. Also there was a direct relation between TOS/GSH ratio and TOS/TAC ratio and the severity of covid-19 disease. Glutathione is a peptide composed of three amino acids and a free radical scavenger, preventing damage induced by ROS in oxidative stress conditions. The thiol function of GSH gives it the role of reducing J o u r n a l P r e -p r o o f agents related to ROS. Many studies have emphasized the advantages of glutathione in the body, acting as an anti-viral factor and managing Covid-19 patients [34, [39] [40] [41] [42] . The higher levels of glutathione may improve an individual's responsiveness to viral infections [43] . In particular, glutathione is known to protect host immune cells through its antioxidant mechanism, and it is also responsible for the optimal function of various cells, such as those in the immune system [43] [44] [45] .

According to several studies, Covid-19 patients with moderate and severe illness had lower levels of glutathione, higher ROS levels, and greater redox status (ROS/GSH ratio) than those patients with a mild illness [38, 44, 46, 47] . Taken together, applying antioxidants therapy can be useful as a promising approach for lowering oxidative stress and accompanying complications of viral infections. However, further experiments in vivo and clinical trials are necessary to reveal the potential effects of these therapeutic approaches on viral diseases with unknown mechanisms, like

Covid-19.

Our studies showed a significant association between oxidative stress biomarkers and disease severity in different groups of hospitalized patients. In addition, our results illustrated that lifestyle and education level has played an important role in increasing stress levels and disease progression. In fact, there was a significant relationship between oxidative stress levels and patients' condition in terms of SpO2, fever, the median duration of hospitalization as well as the outcome of this disease. It seems that strategies for reducing or preventing oxidative stress may help in Covid-19 management. According to many studies on the role of oxidative stress in the pathophysiology of Covid-19 disease, it seems that further investigations should be conducted to determine the time of onset of antioxidants and their required dose to treat this disease.

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The authors would appreciate the Deputy of Research and Technology, Hamadan University of Medical Sciences for financial support of this research.

All authors contributed to the work presented in the manuscript.

All authors declared no conflict of interest regarding this paper. 

J o u r n a l P r e -p r o o f

We the undersigned declare that this manuscript is original, has not been published before and is not currently being considered for publication elsewhere.We would like to draw the attention of the Editor to the following publications of one or more of us that refer to aspects of the manuscript presently being submitted. Where relevant, copies of such publications are attached.We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us.All the authors have contributed significantly in different parts of the method of working, reading and writing the manuscript. All authors has read and approved the final version of the manuscript. All authors have signed author agreement form and have agreed to submit to archives of medical research.We understand that the Corresponding Author is the sole contact for the Editorial process. He/she is responsible for communicating with the other authors about progress, submissions of revisions and final approval of proofs.Signed by all authors as follows: