Evaluation of oxidative stress level: reactive oxygen species, reduced glutathione, and D-dimer in patients hospitalized due to COVID-19

ABSTRACT

Elevated D-dimer levels at hospital admission may also indicate a higher likelihood of progressing to a severe or critical state. This study aimed to assess reactive oxygen species (ROS), non-enzymatic antioxidant reduced glutathione (GSH), and D-dimer levels in COVID-19 patients upon admission, examining their association with mortality outcomes. Data was collected from the medical records of 170 patients hospitalized in a referral hospital unit between March 2020 and December 2021. Patients were divided into two groups: the ward bed group (n = 87), comprising 51% with moderate clinical conditions, and the intensive care unit (ICU) group (n = 83), comprising 49% with severe conditions. The mean age was 59.4 years, with a male predominance of 52.4%. The overall death rate was 43%, with 30.6% in the moderate group and 69.4% in the severe group. The average time from symptom onset to hospitalization was 6.42 days. Results showed that non-survivors had high D-dimer and ROS counts, longer ICU stays, and worse saturation levels at admission. In conclusion, elevated ROS and D-dimer levels may contribute to worse outcomes in critically ill patients, potentially serving as specific and sensitive predictors of poor outcomes upon admission.

KEYWORDS:

• SARS-CoV-2
• pandemic
• oxidative stress
• reduced glutathione
• d-dimer
• reactive oxygen species
• critical illness
• intensive care unit (ICU)

1. Introduction

The challenge in terms of containing the COVID-19 pandemic is being compounded by SARS-CoV-2 subvariants that demonstrate greater potential for transmissibility and resistance to existing vaccines and therapies [1]. These facts may be associated with adaptive mutations in the viral genome that alter the pathogenic potential of the virus [2]. Although the progression of the disease is still not fully understood, there are strong indications that oxidative stress, caused by an increase in reactive oxygen species (ROS) and/or a reduction in the antioxidant defense system, is one of the crucial factors for the evolution of the clinical picture [3]. Thus, patients with mild symptoms of the disease have a rapid recovery, while those with moderate to severe disease signs have a high mortality and poor prognosis [4].

Patients with COVID-19 and in the severe condition have increased levels of reactive oxygen species, free radicals and often have decreased levels of glutathione, triggering a cytokine storm [5]. In addition to inducing a cytokine storm, SARS-CoV-2 triggers redox imbalance, causing lung inflammation, acute respiratory distress syndrome (ARDS), multiple organ failure, resulting in patient death [6,7].

Although the progression of the disease is still not fully understood, there are strong indications that oxidative stress, caused by an increase in reactive oxygen species (ROS) and/or a reduction in the antioxidant defense system, is one of the crucial factors for the evolution of the clinical picture [3]. Oxidative stress has been observed in other inflammatory and infectious diseases, and may be the point of connection with the cytokine storm caused by COVID-19, aggravating tissue damage and inducing hypoxia and organ failure [8]. However, clinical recovery may be associated with antioxidant components through enzymatic systems that help prevent oxidative damage to cells and regulate metabolic pathways essential for homeostasis [9].

Still in this context, a host defense response is expected through the respiratory epithelial lining (GSH, beta-defensins and immunoglobulin A (IgA)), which positively regulate the innate and adaptive immune responses and help to prevent the progression of the infection to more serious stages [6]. Evidence indicates that the decrease in antioxidant levels [10–12], especially reduced glutathione (GSH), in the presence of pre-existing comorbidities, can add individual fragility and aggravate the viral infection picture [13]. These factors shift the redox homeostasis to oxidative stress, thus exacerbating lung inflammation and leading to ARDS, multiple organ failure and patient death [7].

Information on oxidative status through redox biomarkers can help to better understand the evolution of COVID-19 and provide support for clinical decision-making, avoiding the mortality outcome [8,14,15]. In this context, the need to evaluate the levels of biomarkers such as ROS, GSH, D-dimer and their association with prognosis and mortality outcome is justified at the time of admission of patients with COVID-19.

2 Material and methods

2.1 Study design, sample characterization and ethical procedures

This is a quantitative, descriptive and cross-sectional study, involving 170 patients diagnosed with COVID-19, from March 2020 to December 2021, who were admitted to a reference hospital. Data were collected from electronic medical records through the Hospital and Outpatient Management System (GSUS) program of patients of both genders and aged over 18 years. As inclusion criteria, hospitalized individuals participated and had reactive results for SARS-CoV-2 through nasopharyngeal swab collection, using the gold standard test – real-time polymerase chain reaction (qRT-PCR). As exclusion criteria, individuals with a non-reagent or undetermined diagnosis were adopted, as well as in cases of impossibility of carrying out the collection of biological material (blood) and data from the patients’ medical records.

The project was approved by the Ethics Committee in Research involving Human Beings (CEP) and by the Ethics Committee in Research (CEP), Opinion n° 4.224.011 and Certificate of Presentation for Ethical Appreciation (CAAE) n° 31837720.5.0000.0107.

2.2 Study design

During hospitalization, patient data were recorded in a mixed way, using printed forms standardized by the hospital institution and electronic medical records, through the Hospital and Outpatient Management System program (GSUS).

At the time of hospitalization of patients, a sample of 9 mL of venous blood was collected via venipuncture of the forearm, by the nursing team. Part of the biological material (4 mL) was placed in a tube containing EDTA anticoagulant, which was immediately stored in an ultra-freezer (−80°C) at the Laboratory of the State University of Western Paraná (UNIOESTE) until the moment of carrying out the analyzes of the oxidative status biomarkers. The rest of the blood (5 mL) was placed in a tube containing citrate anticoagulant, refrigerated and immediately delivered to an outsourced laboratory for sample processing, with plasma separation and subsequent D-dimer analysis. This was processed at the time of patient admission by the Laboratory that serves the hospital unit.

2.3 Oxidative stress biomarker assays

In order to determine ROS levels, the whole blood sample was diluted (1:10) in 10 mM Tris HCl (pH7.4) and incubated with 10 μL of 2’,7'-dichlorofluorescein diacetate (DCHF-DA; 1 mM), at 37°C for 60 min. ROS levels were determined by a spectrofluorimetric method, using the assay, where DCHF-DA, which is enzymatically hydrolyzed by intracellular esterase enzymes to form non-fluorescent DCFH, and it is rapidly oxidized to form 2’,7'-dichlorofluorescein (DCF), highly fluorescent in the presence of ROS. The fluorescence intensity of DCF is proportional to the amount of ROS present in the sample. The emission of DCF fluorescence intensity was recorded at 520 nm, with excitation of 488 nm, for excitation, 520 nm for emission and beam 1.5. ROS levels were expressed in arbitrary units (AU) [16]. This analysis was performed at the Federal University of Pampa (UNIPAMPA), in Itaqui, Rio Grande do Sul, Brazil.

Reduced glutathione (GSH) levels were evaluated using the procedure described by [17]. First, in order to measure the GSH levels, the whole blood sample was deproteinized with 10% trichloroacetic acid and after previous hemolysis with 1% triton. Next, the supernatant was used to react the levels of non-protein thiol groups (SH-NP) which were determined after the reaction with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and the color developed was measured spectrophotometrically at 412 nm. SH-NP levels indicate an indirect measure of GSH and results are expressed as µmol GSH/mL whole blood. This analysis was carried out at the Federal University of Fronteira Sul (UFFS), in Realeza, Paraná, Brazil.

2.4 D-dimer

Analyzes of D-dimer were performed on the Semiautomatic Fluorescence Analyzer DETECH (Guangzhou, China). Blood was collected in a sodium citrate tube, centrifuged for 5 min at 4.000 rpm, and then a 100 µL aliquot of whole blood was incubated in the cassette for 15 min before reading. Results with a value > 0.5 mg/L indicate stable coagulation and fibrinolysis. The results are expressed in ng/mL of whole blood.

2.5 Statistical analysis

The distribution of qualitative data was described by frequencies and percentages and compared between two groups (patients hospitalized in ward beds and ICU beds) using the chi-square test and Fisher's exact test. Quantitative data were described as mean ± standard deviation (SD), median and interquartile range (IQR), and their normal distribution was evaluated using the Shapiro–Wilk test. In case of normal distribution, the means of the quantitative data were compared in two groups by the independent t test; and in the case in which the distribution was not normal, the Mann–Whitney test was performed and for the SpO2 variable, the Kruskal–Wallis test. The receiver operating characteristic (ROC) curve and the area under the curve were used to determine the feasibility of using oxidative status and D-dimer biomarkers as classifiers to predict death among patients. All analyzes were performed at significance levels of 0.05, using SPSS version 23 (SPSS Inc., USA) and GraphPad Prism version 3 for Windows.

3 Results

3.1 General and clinical characteristics of patients with Covid-19

Regarding data collection, the predominance of the age group of patients over 60 years old, male, hospitalizations for more than 7 days and in the year 2021 was evidenced. In total, 170 patients participated, whose main demographic characteristics are presented in Table 1. No significant differences regarding gender and age were observed between groups (p > 0.05). Nonetheless, there was a significant difference in relation to the non-survivors group, in which admissions to the ICU sector prevailed (69.4%). As for the number of days of hospitalization, there was a predominance of hospitalizations >1 week, with 73.6%, and even patients with SpO2 < 88 at the time of hospitalization represented 31.9%.

Table 1. General characteristics of the total sample and stratified by non-survivors and survivors.

	Total (n = 170) n (%)	Non-survivors (n = 72) n (%)	Survivors (n = 98) n (%)	P-value
Gender				0.148
Male	87 (51.2%)	42 (58.3%)	45 (45.9%)
Female	83 (48.8%)	30(41.7%)	53(54.1%)
Age				0.359
18–59 years	81 (47.6%)	28 (38.9%)	53 (54,1%)
60–94 years	89 (52.4%)	44 (61.1%)	45 (45,9%)
Hospitalization sector				<0.000
Clinics	87 (51.2%)	22 (30.6%)	65 (66.3%)
ICU	83 (48.8%)	50 (69.4%)	33 (33.7%)
Hospitalization length				0.005
≤1 week	67 (39.4%)	19 (26.4%)	48 (49%)
>1 week	103 (60.6%)	53 (73.6%)	50 (51%)
SpO2				0.016
<88	37 (21.8%)	23 (31.9%)	14 (14.3%)
88–90	21 (12.4%)	06 (8.3%)	15 (15.3%)
>90	112 (65.9%)	43 (59.7%)	69 (70.4%)

Note: ICU = intensive care unit; SAH = systemic arterial hypertension; OPD = obstructive pulmonary disease.

The results for D-dimer markers, ROS and GSH were described by means and 95% confidence intervals in the groups of non-survivors and survivors, and then presented in Figure 1. D-dimer levels were significantly higher in the non-survivors group, as well as ROS, p < 0.009, p < 0.05, respectively. GSH, on the other hand, but without statistical significance (p < 0.79) (Wilcoxon Signed Rank Test).

Figure 1. Comparison of D-dimer, ROS and GSH levels between non-survivor patients (n = 72) and group of survivors (n = 98) hospitalized with COVID-19.

Table 2 represents the relationship between D-dimer level and other parameters in all patients. The results of this table indicate that patients hospitalized in the ICU had significantly higher mean (SD) and median (IQR) D-dimer.

Table 2. Relationship between D-dimer (ng/mL) and other demographic characteristics of the studied population.

Result	N	Mean	SD	Median	IQR	P-value
Gender						0.712
Male	50	1,644.75	2,228.16	913.44	1,315.43
Female	53	2,383.14	3,263.02	790	1,865.43
Age						0.113
19–59 years	52	1,631.61	2,421.98	650	1,149.04
60–86 years	51	2,425.49	3,148.21	960.52	1,857.18
Hospitalization sector						0.024
Clinics	53	1,424.14	2,223.50	731.07	955.70
ICU	50	2,661.29	3,240.53	1,058.60	3,205.31
Hospitalization length						0.851
≤1 week	35	1,889.07	2,583.43	903,00	1.374,24
>1 week	68	2,094.51	2,949.98	890,08	1.732,91
SpO2						0.502
<88	23	1,446.81	2,020.73	731.07	1,293.89
88–90	12	2,714.29	3,598.05	1,073.61	3,368.30
>90	68	2,098.47	2,903.83	912.91	1,720.32

Wilcoxon test.

Table 3 represents the relationship between GSH level and the other parameters in all patients. The results of this table indicate that there was no significant difference between any parameter.

Table 3. Relationship between reduced glutathione (GSH) (µmol/mL) and other demographic characteristics of the studied population.

Result	N	Mean	SD	Median	IQR	P-value
Gender						0.238
Male	68	110.63	30.47	107.61	41.64
Female	58	106.33	41.50	98.12	38.86
Age						0.126
19–59 years	63	114.30	38.30	110.68	46.41
60–86 years	63	102.99	32.62	96.87	33.26
Hospitalization sector						0.511
Clinics	61	109.34	32.99	104.37	41.30
ICU	65	108.00	38.64	104.96	37.83
Hospitalization length						0.741
≤1 week	49	107.94	30.45	104.37	40.85
>1 week	77	109.10	39.13	104.96	39.96
SpO2						0.882
<88	26	105.46	29.09	114.69	35.97
88–90	16	113.90	39.86	106.20	42.10
>90	84	108.64	37.26	98.30	43.26

Wilcoxon test.

Table 4 represents the relationship between ROS level and the other parameters in all patients. The results of this table indicate that patients hospitalized in the ICU had significantly higher mean (SD) and median (IQR) ROS level.

Table 4. Relationship between reactive oxygen species (ROS) (AU) and other demographic characteristics of the studied population.

Result	N	Mean	SD	Median	IQR	P-value
Gender						0.403
Male	87	6,402.19	7,006.70	5,081.00	5,089.00
Female	83	6,579.48	5,828.76	5,247.00	5,751.00
Idade						0.685
19–59 years	81	6,355.43	6,753.37	4,585.00	5,316.00
60–86 years	89	6,610.08	6,177.23	5,247.00	5,812.00
Hospitalization sector						0.038
Clinics	87	5,882.49	6,444.25	4,254.00	4,964.00
ICU	83	7,124.22	6,413.00	5,412.00	5,544.00
Hospitalization length						0.285
≤1 week	67	5.459,52	4.025,27	4.419,00	4.303,00
>1 week	103	7.158,25	7.555,54	5.412,00	6.330,00
SpO2						0.240
<88	37	8,743.16	9,963.54	5,867.00	7,136.50
88–90	12	4,650.80	3,283.92	4,171.00	5,667.00
>90	112	6,088.60	5,149.05	4,854.00	5,180.75

Wilcoxon test.

In Figure 2, the analysis of the ROC (Receiver Operating Characteristic) curve showed that D-dimer and ROS have potential specificity and sensitivity to distinguish between living and dead patients. D-dimer: 75.44% sensitivity and 58.70% specificity (p < 0.009, cutoff value of 1059 ug/L); GSH: 61.11% sensitivity and 50% specificity (p:0.79); ROS: 56.12% sensitivity and 63.89% specificity (p:0.05, cutoff value of 4,647 AUµM), by Younden Index (J) [18].

Figure 2. Analysis through the ROC curve (Receiver Operating Characteristic) demonstrating the sensitivity and specificity of D-dimer, GSH and ROS between survivors and non-survivors.

4 Discussion

It was shown that, upon hospital admission, tests such as elevated ROS and D-dimer are associated with an increased risk of needing to be hospitalized in the ICU. The same biomarkers showed potential specificity and sensitivity as predictors of poor outcome upon admission. In this sense, the novelty of this study is the use of the ROS and D-dimer biomarkers to predict prognosis of COVID-19 patients [10–12].

The unregulated production of ROS generates oxidative stress and contributes to the pathophysiology of several diseases [19]. Similar results were evidenced in a study carried out with 15 patients with COVID-19 hospitalized in the ICU, 12 patients with sepsis and non-COVID-19 and 18 healthy volunteers, in which the production of ROS was markedly elevated and with median values 09 times higher than in healthy controls and showed an increase in mechanically ventilated and ICU admissions [20].

ROS are signaling molecules that regulate a wide variety of physiological functions. They are part of the mechanisms that lead to the elimination of virus-infected cells and the recovery of the patient [21]. ROS are generated endogenously especially through the nicotinamide adenine dinucleotide phosphate oxidase (NADPH) complex [13]. Nonetheless, the increase in ROS may be associated with an imbalance in redox homeostasis, significantly contributing to the worsening of COVID-19 and with varying symptoms from mild to critical [13,22,23].

It was also observed that critically ill patients hospitalized the ICU had significantly higher values for D-dimer markers. Nonetheless, changes in relation to D-dimer can be predictors of severity and reflect in poor prognosis, mainly associated with thrombotic events [24]. A prospective cohort study demonstrated a 10% increase in mortality risk for every 10% increase in interleukin (IL)−6 or D-dimer levels [22].

In recent years, some biomarkers of inflammation and systemic coagulation have become available in the main analyzers of laboratory tests and can help in terms of tracking and detecting the severity of the disease [25]. It was found that there were high D-dimer levels in critically ill patients, just as the Chinese retrospective study showed that D-dimer levels showed higher values at the time of hospital admission in those patients who evolved to a severe or critical illness and required intensive treatment [26]. During the pandemic, other markers indicating the severity of COVID-19 deserve to be highlighted: procalcitonin, serum ferritin, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) and IL-6. Measuring inflammatory markers can help in terms of monitoring and evaluating severity and prognosis [27].

GSH is the most abundant non-enzymatic antioxidant found in airway epithelial lining fluid and acts as a vital intra and extracellular antioxidant, protecting against oxidative/nitrosative stress and interceding in pro-inflammatory processes in the lungs [8,28]. However, this study did not show relevance of values between gender, hospitalization sector and days before hospitalization. There was a slight discharge only in relation to hospitalized aged between 18 and 59 years. Scientific evidence indicates that the increase in bodily GSH could also contribute to reducing the number of critically ill patients, while low levels of GSH could contribute to increasing the clinical severity of patients affected by SARS-COV-2 due to the increase of free radicals (ROS) [29,30].

It was observed a mean age of 59.4 years, a predominance of males in 52.4%, a frequency of death of 43% and a mean time of 6.42 days from the onset of symptoms to hospitalization. Viral infection stimulates the generation of elevated levels of ROS that disrupt redox homeostasis and elicit oxidative stress and inflammation, biological responses closely related to disease aggravation [30]. A study carried out with 710 patients in China with SARS-CoV-2 pneumonia found 52 patients in critical condition. Of these, 32 (61.5%) died at 28 days, and the median duration from ICU admission to death was 7 days for non-survivors. Compared to survivors, non-survivors were older, with a mean age of 64.6 years [21].

Finally, ROS and D-dimer presented a relationship, since they were associated with an increased risk of needing to be hospitalized in the ICU. This relationship appear, once, the immune response leads to excessive production and accumulation of ROS, that cause clinical signs characteristic of COVID-19, such as decreased oxygen saturation, vasoconstriction, elevated cytokines, alteration of hemoglobin properties, cardiac and/or renal injury and enhanced D-dimer [32], that reflects poor prognosis, mainly associated with thrombotic events [24].

Biomarkers may collaborate in terms of detecting and monitoring parameters linked to oxidative stress in SARS-CoV-2 infected patients. It was sought to find evidence correlating ROS, but further investigations and future studies are of extreme relevance. It is concluded that elevated ROS and D-dimer may contribute to worse outcomes in critically ill patients, demonstrating potential specificity and sensitivity as predictors of poor outcome upon admission.

Study limitations

Our results should not be extrapolated to the general population, because the patients who entered this hospital unit were probably admitted at different stages of the disease, and it is not possible to establish a causal relationship due to the study design.

Furthermore, the patient samples obtained to perform the analysis were only whole blood and not plasma and PBMCs, samples usually used to apply the methodology here performed.

Acknowledgements

All authors contributed equally to the writing and preparation of the manuscript. All authors approved the final version of the manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

The author(s) reported there is no funding associated with the work featured in this article.