Aminothiols exchange in coronavirus disease 2019

1 Material and methods

This study was approved by the Medical Ethics Commission of Chita State Medical Academy (No.104, 2020-11-11), and informed consent in writing was provided by the legal guardians.

The experimental group was composed of 48 patients with newly diagnosed mild to severe COVID-19 according to the Ministry of Health's temporary guidelines “Prevention, diagnosis, and treatment of novel coronavirus infection, version 8.1, 01.10.2020” and received inpatient treatment in Chita city hospital No.1^[10]. Including criterion was laboratory-confirmed severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. Excluding criteria were HIV, hepatitis B, hepatitis C, exacerbations of chronic cardiovascular diseases, terminal stages of malignancy, decompensated liver, and renal failure. 20 healthy volunteers were included in the control group.

The participants were divided into 4 subgroups according to the serum levels of IL-6. The first subgroup was composed of healthy subjects with IL-6 levels of 0–7 pg/mL; the second > 7–15 pg/mL; the third > 15–25 pg/mL; and the fourth greater than 25 pg/mL. According to Chest computer tomography severity score, patients of the third and fourth subgroups suffered from moderate to severe pneumonia: 1–2 and 3–4, respectively.

Total serum levels of reduced and oxidized aminothiols were assessed with a highly effective liquid chromatography method. Concentrations of MDA-oxLDL and IL-6 were measured using ELISA (Biomedica Gruppe, Germany; Vector Best, Russia).

Statistical analysis was performed using Mann-Whitney U-test and the Kruskal-Wallis test. Data obtained are presented as median and 25th percentile to 75th percentile; P < 0.05 was considered as significant.

2 Results

Baseline characteristics of the patients are presented in Table 1.

Among the patients, the prevalence of male subjects was uncovered for all subgroups. Moderate COVID-19 pneumonia with elevation of IL-6 plasma level from 13 to 25 pg/mL was diagnosed in more than a half of patients (54.6%). Severe pneumonia was associated with more than 25 pg/mL concentration of IL-6. Normal body temperature was detected in 20% of patients, low-grade fever in 34%, moderate fever in 38%, and high-grade fever in 8% (P = 0.000 1). 57% of patients developed arterial hypertension, predominantly in the 4^th subgroup (P = 0.000 3). Patients with IL-6 levels of > 15–25 pg/mL developed low-grade fever, moderate lung injury (CTS 1–2), and decreased arterial blood oxygen saturation to 96%–97%. Patients with IL-6 levels over 25 pg/mL developed moderate fever, severe lung injury (CTS 3–4), decreased arterial blood saturation down to < 96%, and increased systolic blood pressure over 150 mmHg and diastolic blood pressure over 90 mmHg.

There were no accompanying diseases in patients with mild COVID-19 and in controls. Patients with lung damage demonstrated approximately equal percentage of comorbidity (Table 1).

IL-6 level was within the normal range in controls. Patients who had no clinical manifestations or had been diagnosed with mild COVID-19 (nasopharyngitis symptoms) were assigned to the 2^nd subgroup according to the IL-6 levels of > 7–15 pg/mL. Patients with IL-6 levels of > 15–25 pg/mL and higher had moderate to severe COVID-19 pneumonia and were composed of the 3^rd and 4^th subgroups.

Assessment of total aminothiols revealed decreases in Cys, CysGly, and Glu-Cys levels coupled with an elevation of plasma glutathione, regardless of the severity of COVID-19 (Table 2, Fig.1).

Fig. 1

Aminothyols in mild COVID-19

Cys, cysteine; CysGly, cysteine-glycine; GSH, glutathione; Hcy, homocysteine; Glu-Cys, glutamate-cysteine; COVID-19, coronavirus disease 2019.

In patients with IL-6 levels ranging from > 15–25 pg/mL to > 25 pg/mL, glutathione was increased by 1.8 (P = 0.035) and 2.5 (P = 0.000 4) times, respectively. There was decrease of GSH/GSSG ratio in patients with IL-6 level more than 7.2 pg/mL. Rise in MDA-oxidized LDLP was detected in all examinated patients: the highest level was revealed in the 2^nd subgroup (P < 0.05), the lowest – in the 4^th subgroup (P = 0.002) (Table 2).

3 Discussion

Decreases in Cys and CysGly could be considered a sign of enhanced synthesis of glutathione and an indirect marker of intensive ROS production.

The development of oxidative stress in COVID-19 could be explained by the following mechanisms:

Respiratory viruses induce mitochondrial dysfunction and ROS generation via activation of NADPH oxidase, xanthine oxidase, and iNO-synthase^{[11,12,13,14]}; SARS-CoV-2 activates ATP, which causes an elevation of AT-II level and then activates NADPH oxidase and peroxynitrite thereby exaggerated ROS production^[15]; SARS-CoV-2 particles bind to CD147 on erythrocytes surface and enter cells, leading to the destruction of hemoglobin and release of iron, which can induce oxidative damage^[16]; Activated phagocytes produce ROS and initiate an inflammatory process^[17].

The lowest precursors of glutathione have been associated with an increased concentration of IL-6 to more than 25 pg/mL, an indicator of exacerbated oxidative stress in patients with more severe lung injury.

Increasing serum levels of glutathione and homocysteine could be caused by their leakage during exocytosis of virus-containing vesicles, which does not depend on inflammation intensity.

Significantly reduced GSH/GSSG ratios in COVID-19 patients compared to controls had no correlation with disease severity, which could presumably be a sign of antioxidants insufficiency. Decreased CySH/CySS ratio was, in contrast, significantly different in all investigated subgroups. The value of the ratio reflects redox balance in biological fluids and can be considered as one of the important vital signs^[18]. Cysteine and methionine oxidation end with the formation of disulfides^[19]. It is particularly dangerous if such a process involves proteins enriched with cysteine residues such as mitogen-activated protein kinases, receptors of insulin and insulin-like growth factor, and ion channels. Hence, dimerization or autoactivation of these proteins without specific signaling molecules can cause cell dysfunction. It has been suggested that thiol and disulfide groups at cell-virus interphase could act as donors or acceptors of electrons, which are required for conformation changes promoting a fusion of viruses and host cells^[20].

Free radicals, especially ROS, cannot be eliminated from the cells, due to their signaling function. However, the human body has intrinsic ROS-buffering capacities to maintain the functionalities of proteins under moderate oxidative stress conditions. Nonetheless, some proteins, for example, low-density lipoproteins, are much less resistant to oxidation damage and subsequent degradation^[21]. Our data demonstrated an elevation of MDA-oxLDL in all COVID-19 patients with the highest values in the 2^nd subgroup (P = 0.000 01), which could be related to higher saturation of the ROS-buffering mechanism and greater impairment of the cellular respiration, thereby enhanced ROS production.

4 Conclusions

Inflammatory process caused by SARS-CoV-2 alters aminothiols metabolism along with rise in serum level of IL-6 which is associated with development of more severe lung injury, declining of arterial blood O₂-saturation and increase in blood pressure. Reduced antioxidant capacity could worsen disease course due to propagated inflammatory process, expanded tissue damage and, thus provide unfavorable outcome.