Oxidative stress index can be a new marker related to disease severity in COVID-19

Introduction

Coronavirus Disease 2019 (COVID-19) is a global pandemic and caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) affecting the whole world emerges in a broad spectrum. It can cause serious complications such as respiratory failure, sepsis, acute respiratory distress syndrome (ARDS) and organ damage especially in hospitalized patients with COVID-19 pneumonia [1].

Previous studies revealed that severely ill patients prone to have a high concentration of various inflammatory cytokines, including interleukin-6 (IL-6) and tumor necrosis factor (TNF-α). The overproduction of these pro-inflammatory cytokines has been described as a cytokine storm. It causes an increased systemic inflammatory response and multiorgan failure due to vascular hyperpermeability and may contribute to the higher mortality rates in patients with COVID-19 [2, 3].

Pathophysiological processes such as increase in cytokine release and inflammation are frequently encountered in respiratory viral infections, including COVID-19 and accordingly, may be related to oxidative stress. Recent studies described that increased reactive oxygen species (ROS) and antioxidant defense mechanisms deprivation have a very important role in the pathogenesis of SARS-CoV infection [4-6]. It has been suggested that ARDS in patients with COVID-19 depends on activation of the oxidative stress that is coupled with inflammatory machinery and resulting in an increased pro-inflammatory host response [4]. Various mechanisms have been suggested to explain increased ROS generation, including increased angiotensin II, a prooxidant and pro-inflammatory substance, due to angiotensin-converting enzyme II (ACE) II inhibition (I), respiratory burst generating superoxide radicals and H₂O₂ by activated macrophages and neutrophils (II), ROS production by heme and iron released by damaged erythrocytes (III), hypoxemia caused by alveolar edema (IV) [7]. Although previous studies have revealed a relationship between systemic oxidative stress and viral diseases such as HCV [8], there is not enough data in the literature for SARS-CoV-2 infection.

This study aimed to determine the relationship between systemic oxidative balance and inflammation and their roles in the severity of the disease at different stages of patients with COVID-19.

Materials and methods

Results

The demographic characteristics of 64 patients and 24 healthy individuals participating in the study are given in Table 1. The mean age of the patients with the severe group was significantly higher than the other groups (p<0.001, Table 1).

The clinical characteristics of COVID-19 patients are given in Table 2. Comorbidities including DM (17%), hypertension (12.5%), CHD (14%), CKD (4.6%) and CPD (7.8%) were observed in 22 patients (34.4%) participating in the study.

Biochemical data of the patients and the healthy subjects are shown in Table 3. Serum levels of lymphocytes in the severe group significantly decreased and LDH, CRP, D-dimer and fibrinogen levels were significantly increased compared to mild, moderate and control groups.

Oxidative stress parameters (TAS, TOS and OSI) and pro-inflammatory cytokines (IL-6 and TNF-α) are shown in Figures 1 and 2, respectively. TAS levels were significantly decreased in all study groups compared to controls (mild p=0.026, moderate p=0.027 and severe p=0.001). On the other hand IL-6, TNF-α, TOS and OSI levels were significantly elevated in the severe group compared to other groups (p<0.001). However, the significant difference of IL-6, TOS and OSI levels between severe and other groups still existed after adjusting for the age (Figures 1D–F and 2CD–FC Figures 1D–F and 2C Figures 1D–F and 2C Figures 1D–F and 2C, D).

Figure 1:

One way ANOVA test (Post hoc: LSD) was applied for figure 1a, 1b and 1c. Data were expressed as mean ± SD. a) TAS, total antioxidant status. *p=0.026, **p=0.027, ***p=0.001. b) TOS, total oxidant status. *p<0.001 c) OSI, oxidative stress index. *p<0.001, **p=0.007, ***p=0.014. The levels of TAS, TOS and OSI adjusted for the age were presented in figure 1d, 1e and 1f. Data were expressed as mean ± SEM. d) *p=0.004, e) *p<0.0001, **p=0.001, ***p=0.005, ****p=0.031, f) *p>0.0001, p=0.002, p=0.025.

Figure 2:

One way ANOVA test (Post hoc: LSD) was applied for figure 1a and 1b. Data were expressed as mean ± SD. a) IL-6: Interleukin-6. *p=0.014, **p=0.016, ***p<0.001. b) TNF-α, tumor necrosis factor-α. *p<0.001, **p=0.018, ***p=0.010, ****p=0.025 The levels of IL-6 and TNF-α adjusted for the age were presented in figure 1c and 1d. Data were expressed as mean ± SEM. d) *p<0.0001, **p=0.003.

The correlation of pro-inflammatory and oxidative stress parameters were given in Table 4. Serum levels of TOS and OSI showed a positive moderate correlation with IL-6, CRP, ferritin, fibrinogen and D-dimer. And IL-6 levels were correlated with TNF-α, CRP, ferritin, fibrinogen and D-dimer.

We performed ROC analyses to evaluate the diagnostic potential for the disease severity and detect the optimal cut-off values of laboratory, inflammatory and oxidative stress parameters in COVID-19 patients (Table 5 and Figure 3). We detected that areas under the ROC curve for TAS, TOS, OSI, ferritin, fibrinogen, D-dimer, TNF-α, IL-6 and CRP were 0.7077, 0.9557, 0.8890, 0.8210, 0.9443, 0.9925, 0.6852, 0.8730 and 0.8812, respectively. Also 95% confidence intervals for TOS, OSI, ferritin, fibrinogen, D-dimer, IL-6 and CRP were 0.9169–0.9946, 0.8236–0.9544, 0.7343–0.9076, 0.8983–0.9904, 0.9805–1.005, 0.7981–0.9480 and 0.8125–0.9499, respectively (p<0.0001).

Figure 3:

AUC, area under the receiver operating characteristic curve; TAS, total antioxidant status; TOS, total oxidan status; OSI, oxidative stress index; IL-6, interleukin-6; TNF-α, tumor necrosis factor-alfa; CRP, C-reactive protein.

Multiple regression analyses indicating CRP, D-dimer, fibrinogen, IL-6, LDH and ferritin with OSI in patients with COVID-19 are shown in Table 6. These results revealed that CRP and IL-6 might explain and the regression model was statistically significant (p<0.001 and p=0.022, respectively).

Discussion

This is the first study evaluating the systemic oxidative stress parameters in COVID-19 patients and their relationship with inflammation and disease severity. We found a significant deprivation in antioxidant defense mechanism and an increase in total oxidative stress in COVID-19 patients. TOS levels were also significantly different between study groups and related to disease severity. We also found a significant correlation in TOS and calculated OSI levels among IL-6, ferritin, D-dimer, CRP and fibrinogen. Serum pro-inflammatory cytokine levels were significantly different in mild, moderate and severe groups and IL-6 were significantly correlated with TNF-α, CRP, ferritin, fibrinogen and D-dimer.

There is strong evidence that deterioration of the antioxidant defense mechanism leads to increased oxidative stress [13]. Previous studies have suggested that the imbalance in oxidant and antioxidant systems plays an important role in the pathogenesis of COVID-19 [4, 14]. There is also a strong relationship between oxidative stress and inflammation caused by innate and adaptive immune systems in the disease progress [3, 15]. In the current study a significant increase was observed in TOS and OSI levels of study groups compared to controls (Figure 1). We also found that TOS and OSI levels were significantly different between severe moderate and mild groups (Figure 1). TOS levels measurement is based on the oxidation of Fe²⁺ to Fe³⁺ in acidic medium due to the presence of oxidant species [10]. And it is used to evaluate total oxidative stress. However OSI level is a better indicator for systemic balance because it is calculated as TOS and TAS ratio [11]. Up to now, there is no study in literature evaluating systemic oxidative balance using OSI in COVID-19. There are several mechanisms explaning increased oxidative stress in COVID-19. It has been shown that SARS-CoV-2 virus enters into alveolar epithelial cells by binding to ACE II receptors and blocks its activity. Diminished ACE II activity leads to increased angiotensin II levels, which has oxidant and pro-inflammatory properties [16]. The lungs are the primary affected organs in COVID-19. Virus infection also causes alveolar edema together with inflammation and leads to decreased oxygen saturation, which is another well-known contributor factor in ROS production [17]. Our results indicate that increased systemic oxidative stress is related to disease severity in patients with COVID-19. We also found that TOS and calculated OSI levels were correlated with ferritin, CRP, IL-6, fibrinogen and D-dimer levels. Additionally, area under the ROC curve for TOS and OSI were 0.9169–0.9946 (at cut-off value of 14.68 µmol H₂O₂ Eq/L) and 0.8236–0.9544 (at cut-off value of 9.15 arbitrary unit), respectively (p<0.0001). The area under the ROC curve for TOS and OSI were similar to D-dimer, fibrinogen and CRP (Table 5) and also the changes in calculated OSI levels were due to TOS levels. Therefore, TOS and OSI levels can be a new predictor marker for the disease severity and used in the management of the disease.

We also evaluated inflammatory cytokine levels. Serum IL-6 and CRP levels were significantly increased in the patient groups compared to the controls (Figure 2 and Table 3). IL-6 were also significantly different between severe, moderate and mild groups. Serum IL-6 levels seem related to disease severity. There is a moderate correlation between IL-6 and CRP (Table 4) and IL-6 were correlated with OSI values (r=0.447, p<0.001). Area under the ROC curve for IL-6 and CRP were 0.7981–0.9480 and 0.8125–0.9499, respectively (p<0.0001). Moreover, multiple regression analyses showed that 23% of the alterations in OSI values might be explained by CRP and IL-6, and the regression model was statistically significant (p<0.001 and 0.022, Table 6). These results implicate that IL-6 and CRP significantly contributed to the changes in the OSI levels. Besides, increased pro-inflammatory cytokines are related with systemic oxidative balance and play a role together in the progression of the disease. These findings also support the previous studies suggesting a positive feedback loop between inflammation and oxidative stress in COVID-19.

Ferritin is another inflammatory marker and several studies have shown that ferritin levels were increased in COVID-19 [18]. In the current study we found higher ferritin levels in all study groups compared to controls (Table 3). Ferritin is an acute phase reactant and plays an important role in iron metabolism. Free iron is highly toxic for tissues because it causes oxidative damage through fenton reaction. In a recent study, Liu W et al. suggested that viral proteins attack hemoglobin beta chains and lead to iron relase, which make contribution to the tissue damage [19]. Ferritin is used to storage free iron in tissues to prevent oxidative stress. Therefore, increased ferritin levels in COVID-19 is an expected result and can be related to prevention of iron toxicity [18]. However, in our study, ferritin levels were more prominent in patients with severe disease (p<0.001) and correlated with systemic oxidative (TOS and OSI) and inflammatory (IL-6, CRP) parameters (Table 4).

Another contributing factor responsible for disturbed oxidative balance is excessive consumption of antioxidant substrates. It has been shown in many disorders including pulmoner diseases, HIV infections, and age related disorders [20, 21]. The excessive consumption of antioxidants can contribute to the disease progression in SARS CoV-2 infection [14]. Previous studies showed that SARS-CoV-2 virus reduce glutathione production [14, 22, 23]. But there is no study evaluating TAS levels in COVID-19. In this study, we evaluated serum TAS levels and found significant decrease in mild, moderate and severe groups compared to healthy controls (Figure 1). TAS assay shows total antioxidant capacity and briefly based on the principle of initiating colored dianisidyl radicals due to hydroxyl radicals produced by fenton reaction. Potential antioxidant substrates in serum such as vitamin C, reduced glutathione, bilirubin and uric acid are suppressed the color formation depending on their concentrations as an indicator of serum total antioxidant capacity [9]. It can be suggested that decreased TAS levels may be due to the consumption of serum antioxidants including glutathione in COVID-19 patients. And this consumption is initiated from the early stage of the disease (Figure 1).

It is well known that fibrin/fibrinogen degradation products and D-dimer increase in COVID-19 [3, 15]. We found significant elevation in levels of fibrinogen and D-dimer in patient groups and confirmed the previous studies (Table 3). However, we found significant correlations between fibrinogen and D-dimer levels with TOS, OSI, IL-6, CRP and ferritin. These results implicate that fibrinogen and D-dimer levels are related to the increased oxidative stress and inflammation in COVID-19.

COVID-19 patients with comorbidities have a poor prognosis and underlying diseases such as hypertension, cardiac disease etc. have been reported as risk factors for severe disease and death. Of the 64 patients included in our study, 22 (34.4%, Table 2) patients had comorbidities including DM, hypertension, CHD, CKD, and CPD. Consistent with the literature, the rate of comorbidities in patients in the severe group was higher than other groups [24, 25]. Serum AST, CK, LDH activites and creatinine levels were significantly higher only in severe group compared to controls. But only CK and LDH levels were above the reference ranges. LDH enzyme levels indicate heart, lung, kidney, muscle tissue and erythrocyte damage. It increases due to multipl organ damage in COVID-19 patients [3, 26]. In this study, LDH enzyme activities were correlated with oxidative stress and inflammatory parameters (Table 4). Our study results support that the increasing LDH levels were associated with disease severity in COVID-19.

The small sample size due to single-center design is one of the limitations of this study. Another limitation is that critically ill patients were not included in the study group.

Conclusions

In the present study, TOS and OSI levels were increased due to the disease severity and showed a positive correlation with other prognostic markers, including inflammatory and coagulation markers. The measurement of TOS levels and calculated OSI values, an inexpensive and automated method, can be an important marker for evaluating the disease severity and used in the management of COVID-19 patients.