INTRODUCTION
Coronavirus disease 2019 (COVID-19), a contagious virus highly contagious illness caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), this virus has significantly affected the global population, resulting in more than 6 million deaths worldwide and has emerged as Since the 1918 influenza pandemic, this is the most consequential worldwide health emergency. Clinical research has significantly improved understanding of SARS-CoV 2 and the management of COVID-19, but limiting the spread of the virus and its variants has become a major concern. Mutant variants of SARS-CoV-2 continue to cause outbreaks in a number of countries.[1]
There are several symptoms of the disease that appear in its early stages, including fever, weakness, headache, cough, and myalgia. However, severe symptoms may include systemic inflammatory response syndrome, acute respiratory distress syndrome that involves multiple organs, diffuse intravascular coagulation, and shock. COVID-19 can cause thrombosis in both the venous and arterial systems due to a number of risk factors, such as inflammation, thrombocyte activation, endothelial dysfunction, and blood flow stasis caused by immobility.[2,3] Patients with severe disease have been shown to have a different pattern of hematologic, biochemical, inflammatory, and immunological biomarker abnormalities than those with moderate systemic disease; in addition, the authors found the discovery of many novel biomarkers.[4]
Homocysteine (Hcy) is synthesized from the essential amino acid methionine. Many studies found Hcy is a factor that promotes thrombosis and is involved in the production of reactive oxygen species (ROS), which in turn leads to oxidative stress, endothelial disruption, and the irreversible inactivation of protein C and thrombomodulin.[5] High levels of Hcy in the blood significantly raise the risk of damage to both small and large blood vessels.[6,7] Multiple pathways have been proposed by which Hcy might either cause severe COVID-19 infection or prevent it from being controlled.[8,9]
COVID-19 infects host cells by binding virus-specific “S” spikes to human angiotensin-converting enzyme 2 (ACE-2). In contrast to Ang (1–7), which works through MAS1 protooncogene G-protein-coupled receptors (GPCRs) receptors, Ang-II operates through the AT1 and AT2 receptors.[10] Humans not only possess ACE2 other angiotensinogen involved in Ang II to Ang1–7 processing may impact Spike proteins of SARS-CoV-2 and ACE2 receptor interactions.[11,12] New studies show that deregulation of the renin-angiotensin system (RAS) causes COVID-19 complications. As a result of the interaction between SARS-CoV-2 (mediated by the Spike protein of the virus) and ACE2 receptors, angiotensin II imbalances, and angiotensin1–7 imbalances play a key role in COVID-19’s clinical characteristics and severity.[13–16] In ACE2-deficient hearts, higher myocardial Ang II and reduced Ang1–7 were associated with enhanced ROS generation.[17]
Plasminogen activator inhibitor-1 (PAI-1), a serine protease inhibitor 1, is involved in blood coagulation. Multiple diseases have been linked to thrombosis by it elevation.[18] PAI-1, a serine protease inhibitor, regulates clot breakdown in the body by reducing the action of tPA and the enzyme urokinase (uPA).[19,20] Using both active and total PAI-1 levels from 19 studies, a meta-analysis found a link between PAI-1 levels and mortality due to sepsis.[21] Acute viral sepsis results from a direct organ attack by disseminated SARS-CoV-2, an immunological pathogenesis brought on by a systemic cytokine storm, and dysfunctions in the microcirculation.[22]
Accordingly, a number of studies that investigated the association between risk and severity of Covid-19 and Hcy, Ang (1–7), and PAI-1 levels were either debated or insufficient. As a result, it was important to conduct this study to clarify the relationship between serum Hcy, Ang (1–7), and levels, respectively, and the early diagnosis of patients.
METHODS
Research design and participants
The study included 88 positive COVID-19 patients (48 women, 40 men) and 40 (23 women, 17 men) healthy adults of various ages. All patients who participated in this study had positive viral nucleic acid assays (reverse transcription polymerase chain reaction) performed on respiratory samples from nasal/oropharyngeal swabs. The exclusion criteria include: (1) Individuals under 16 years of age, (2) patients with respiratory diseases, and (3) those who have taken chemotherapy in the past 3 months. Hcy, Ang 1–7, and PAI-1 levels in the blood were determined using the enzyme-linked immunosorbent assay (ELISA) technique and an ELISA kit assay. The study was carried out in three hospitals (Al Diwaniyah teaching hospital, Al-Hussein Teaching Hospital, and Marjan medical city) in the provinces of Iraq included (Al Diwaniyah government, Al Muthanna government, and Babil government) respectively during the period from February/2022 to June/2022.
Data collection
The patients received the same course of treatment depending on the severity of infection, and all blood were collected from participants under identical circumstances; patients receiving continuous positive airway pressure therapy were in severe condition, while nonsevere patients had symptoms of simple-medium such as fever and upper respiratory symptoms but did not require to stay in hospitals. In addition to demographic information, such as age, vaccination, and gender, as well as information about any previous medical conditions and medications, were collected from all patients.
Laboratory technique
For this study, a 5 ml sample of peripheral blood was collected in gel tubes (the sample was allowed to clot at room temperature under sterile conditions for an appropriate amount of time and centrifugated for 10-15 minutes at 1500 rounds per minute (rpm) to determine the biomarkers included in study. Serum samples were prepared to measure the levels of Hcy, Ang 1–7, and PAI-1 using ELISA assay kits from the bioassay technology laboratory. According to the manufacturer’s instructions, all chemicals and standards were used. ELISA experiment’s methodology in the supplementary methods [Tables S1 and S2].
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Table S1: Components of kit enzyme-linked immunosorbent assay
Table S2: The standard concentrations in kits of enzyme-linked immunosorbent assay
Ethical consideration
The study was conducted in accordance with patients verbal and analytical approval before the sample was taken. A local ethics committee at the College of Science, University of Babylon, reviewed and approved the study protocol and subject information, and consent form in accordance with the regulations of the Ministry of Health in Iraq.
Study sample size
Using Raosoft online software, the sample size was calculated based on common reports and the acceptable margin of error (4%–8%) (the margin of error = 8% at 128 samples).
Statistical analysis
Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS) version of SPSS 27.0 software (IBM company). For analyzing the data included in the present study, descriptive analysis was provided using mean, SD, median, and interquartile range (IQR). The receiver operation characteristic (ROC) curve was used to calculate cutoff values for parameters to determine which biomarker aid in predicted risk of infection and severity diagnosis, we performed binary logistic regression analysis to identify independent predictors of disease severity
RESULTS
In the present study, Table 1 shows the mean ± standard deviation (SD) age of controls and patients was 34 ± 11 and 53.4 ± 17.8, respectively, and the median (IQR) was 31 (24.7–43.5) and 56 (39–68), the table also shows the gender (female and male) and vaccination status. when investigated the levels of Hcy by ELISA found there was a significant increase in patients serum compared with a healthy group (P = 0.001), the median (IQR) was 14.88 (IQR 5.45–23.4) for Covid-19 patients while 3.62 (IQR 2.75–5.27) for healthy and the mean ± SD for patients and control were (16.17 ± 9.33 vs. 4.46 ± 2.73) nmol/mL, respectively, as shown in Table 2 and Figure 1.
Table 1: The demographic characteristics of coronavirus disease 2019 patients and control subjects
Table 2: Evaluation of homocysteine, angiotensin 1–7, and plasminogen activator inhibitor-1 protein levels in studied groups
Figure 1: Homocysteine levels in patients and control groups
Moreover, the results showed the Ang 1–7 levels in this study, there was a decrease in measured levels in significant difference (P = 0.01) for the COVID-19 patients; on the contrary, healthy was higher. The mean ± SD was 70.77 ± 25.75 ng/mL in the serum patients in comparison to healthy controls 91.38 ± 96 ng/mL and the median (IQR) in patients and healthy were (67.95 [IQR 55.83–90.21 vs. 83.7] [IQR 68.8–122.8]) as in Table 2 and Figure 2. At the same time, the P value for PAI-1 was (0.01) which refers to a significant increase in PAI-1 level in COVID-19 patients than in healthy patient’s median (IQR) was 4.39 (3.87–5.77), whereas controls median was 3.38 (2.72–3.9), as shown in Table 2 and Figure 3.
Figure 2: Angiotensin 1–7 levels in studied groups
Figure 3: PAI-1 levels in studied groups. PAI-1: Plasminogen activator inhibitor-1
As shown in Table 3, the correlation between serum levels of Ang 1–7 and age in patients was only significant (r = 0.63; P 0.03), but not in controls, while Hcy and PAI-1 correlations were not significant in either group.
Table 3: The correlation between age and the parameters tested in patients and control group
Based on the graph ROC analysis, the cutoff level for homocysteine (Hcy) >10.3 nmol/mL discriminated severe cases from non-severe cases in the patients’ group with an area under the ROC curve (AUC) of 0.79, 95%CI 0.62–0.96, P = 0.005, as shown in Table 4.
Table 4: The area under the curve of homocysteine, angiotensin 1–7, and plasminogen activator inhibitor-1 protein levels in patients with coronavirus disease 2019 infection were measured using a receiver operating characteristic curve
The serum Ang 1–7 levels of the cutoff value = 89.6 ng/L and the AUC (0.84, 95% CI 0.54–0.9, P = 0.02) in maximal 81% specificity and 62.5% sensitivity, regarding PAI-1 levels had an AUC of 0.81, 95% CI (0.78–1) and the optimal cutoff value was 4.89 (ng/ml) at the highest sensitivity and specificity (93% and 81.3%), respectively [as shown in Table 4 and Figure 4].
Figure 4: Receiver operating characteristic curve for measuring Hcy, Ang 1–7, and PAI-1 to calculate the area under curve. PAI-1: Plasminogen activator inhibitor-1, Hcy: Homocysteine
The binary logistic regression model analyzed parameters (Hcy, Ang 1–7, and PAI-1) based on the severity of COVID-19 at 87.5%, it was statistically significant at P < 0.001 with Χ2 = 28.6, and this mean model fit with the predictor. In addition, the HL test predicted goodness fit to model P > 0.05 and the results of logistic regression found the three parameters decrease and indicated (Hcy and PAI-1) dependent factors on other biomarkers in the present study of the prediction of COVID-19 to contribute of severity, while Ang 1–7 was an independent factor to contribute of increase severity and In other words, each decrease in the level of Ang 1–7 has no relationship with the level of Hcy or PAI-1 Table 5.
Table 5: Logistic regression analysis showed the impact of homocysteine, angiotensin 1–7, and plasminogen activator inhibitor-1protein parameters
DISCUSSION
Biomarkers are needed to predict COVID-19 death, and to identify high-risk individuals more quickly and effectively to improve close monitoring and intensive treatment. A study was conducted to investigate the association between serum levels of Hcy, Ang 1-7, and PAI 1 levels on admission to the hospital, and the risk of COVID-19 infection. We found Hcy levels were higher in COVID-19 patients than in the control group, suggesting a greater risk of respiratory complications in COVID-19 patients with elevated Hcy. Hcy has been proposed as a critical biomarker for cardiovascular complications among patients with COVID-19 infection.[23] Previous studies showed that infections with numerous viruses, such as the human immunodeficiency virus, the hepatitis virus, and the human papillomavirus, were associated with higher Hcy concentrations.[24–26]
A study of COVID-19 patients diagnosed based on chest CT data discovered that Hcy has a predictive level and concluded that Hcy level is a significant parameter in the follow-up of COVID-19 disease.[27] In the second study, the median blood Hcy concentration in COVID-19 patients was 27.5 mol/L, while it was 1.8 mol/L in the control group, when patients were compared to controls, there was a statistically significant rise in the Hcy level at (P = 0.001).[28] In local study confirmed Hcy levels significantly increase in COVID-19 patients compared to healthy was lower.[29] Similar to the findings of the current study, serum Hcy levels were tested in 304 hospitalized COVID-19 patients, and it was discovered that nonsurvivors had significantly higher levels than survivors.[23] A study by Yang etal. 2020 confirmed Hcy may be a useful biomarker for the severity of COVID-19.[30]
Loss of ACE-2 receptor function caused by SARS-CoV-2 inhibits the production of Ang II, reduces its inactivation, and reduces the synthesis of Ang-(1–7).[31] In the current study found decreased Ang 1–7 in COVID-19 patients while higher in control when measured the levels in ELISA and this may be because of ACE2 is receptor to spike protein of SARS-CoV-2 and this lead to reduce the activity ACE2 with higher levels of Ang II together with limited production of Ang-(1 − 7).[32] In addition to logistic regression model confirmed that Ang 1–7 in this study independent factor to increase risk of infection and severity compared with Hcy and PAI-1were dependent parameters. The results of our study are consistent with those of Henry etal.,[33] who reported that Ang 1–7 levels were significantly lower in COVID-19 patients compared to controls and in those admitted to the ICU versus those who did not require critical care. In addition, in the second study, Ang 1–7 was repressed in positive patients compared to the control. The RAS correlates negatively with pro-inflammatory cytokines and D-dimer, suggesting that increased activity in this counter-regulatory arm is responsible for the hyper-immune responses and diffuse coagulation activation found in COVID-19.[34]
When compared to the severe COVID-19 cases in Henry et al. study, the level of Ang (1-7) found in donor plasmas that did not undergo COVID-19 was 2.5–10 times greater and discovered that patients who required hospitalization in the intensive care unit had even lower Ang (1-7) levels at admission.[33]
PAI-1 is one of the most essential and quickest fibrinolytic system inhibitors in plasma. PAI-1’s major function is to block plasminogen activators at the protease site by cleaving a particular arginine-valine peptide link.[35] We analyzed serum PAI-1 and found elevation in COVID19 patients, whereas the control group had lower levels, and these results need to be investigated to determine if the plasminogen system plays a role in disease progression. There are few studies on PAI-1, the primary molecule in the plasminogen cascade, which is necessary for fibrinolytic activity in COVID-19 patients, and prospective studies are required. In results of a study similar to the current study found PAI-1 were significantly higher in severe COVID-19 patients (P < compared to nonsevere and healthy control.[36] In addition, it has been shown that PAI-1 plasma concentrations may serve as a biomarker for ARDS disease development and as a good indicator of mortality.[37] Some investigators found an association between higher PAI-1 levels and the COVID-19 cytokine storm, particularly IL-6 levels.[38] In another study, authors analyzed PAI-1 in two diseases COVID-19 and sepsis and found that covid-19 patients had elevated levels of PAI-1 than sepsis disease patients.[39]
CONCLUSION
In our study, we investigated Hcy, Ang 1–7, and PAI-1 levels, which is an important parameter in patients to be diagnosed early with COVID-19 and prevent the progression it. Clinicians may use Hcy to identify patients at risk of severe COVID-19 infections by assessing their Hcy levels according to ROC curves, it is a good parameter, and in thrombosis and cardiovascular disease patients, homocysteine is an important parameter. Angiotensin 1–7 in the present study showed decrease in COVID-19 than control, in addition to ROC curve model results confirmed it was good biomarker, and by logistic model found an independent factor to decrease in patients without the two others. The levels of PAI-1 were significantly higher in COVID-19 patients than in controls, which is an important parameter in the fibrinolytic step and acts as a good marker-based ROC curve for predicting COVID-19 severity. We believe that our study is one of the few prospective studies that involves three biomarkers’ measures to assess COVID-19 participants. More studies are needed to assess Hcy, Ang1–7, and PAI-1 for follow-up. This can reduce mortality and stop the disease from progressing.
Limitations of study
There are some limitations to our study, including the following: the demographics and clinical features of the participants (medical history and laboratory tests) were not evaluated in this study; second, the sample size was not remarkable for this study, which limited the accuracy of the evaluation.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
Acknowledgments
The authors would like to express deep thanks to the University of Babylon, College of Science, Department of biology for their support in access for articles.
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