Is Homocysteine Associated with the Prognosis of Covid-19 Pneumonia

Background/Aim. Coronavirus disease 2019 (COVID-19) is a life-threatening disease characterized by a prothrombotic state. Because homocysteine (Hcy) is a potential biomarker in thrombotic disease, this article aims to highlight the role of Hcy in the prognosis of COVID-19 pneumonia. Methods. This prospective study was conducted between April 2021 and December 2021 at the University of Health Sciences, Antalya Training and Research Hospital. 162 patients admitted to the emergency department for COVID-19 pneumonia and scheduled for hospitalization in the intensive care unit (ICU) or COVID-19 ward of the chest disease department were included in the study. Hcy levels and other necessary laboratory parameters were analyzed. Results. 134 patients were admitted to the COVID-19 ward and 28 to the ICU. Hcy levels were significantly higher in ICU patients than in ward patients (p : 0.001). Of the 134 patients, 55 later required ICU treatment for various reasons and were transferred to the ICU. Hcy (p : 0.010), ferritin (p : 0.041), and LDH (p : 0.010) were significantly higher in patients who were transferred to the ICU than in patients who remained in the ward. The Hcy level was associated with a poor prognosis. It was found that each unit increase in the Hcy level approximately doubled the risk of death in patients with COVID-19 (odds ratio: 1.753). Discussion. There are few studies examining the association between high Hcy levels and disease severity in COVID-19. Our study supports previous studies and shows the association between the need for intensive care and high Hcy levels. Conclusion. A high Hcy value is a helpful marker in determining the need for critical care on admission to the emergency department and a marker of poor prognosis in COVID-19 pneumonia.


Introduction
Since the 2019 coronavirus (COVID-19) pandemic, reliable biomarkers associated with the disease prognosis are needed for early identifcation of high-risk patients. Identifcation of new biomarkers helps to categorize patients immediately after diagnosis, plan hospitalization, and improve clinical management of the disease. COVID-19 infection is associated with coagulopathy, and there is an increase in procoagulant factors such as fbrinogen and D-dimer [1,2]. Te incidence of pulmonary embolism is increased [3], and microvascular thrombosis in the lungs is demonstrated in the autopsy series and in COVID-19 acute respiratory distress syndrome [4][5][6]. Leukocytes activated during the infammatory storm involving IL-6 may damage the endothelium in capillaries and impair the thromboprotective mechanism of the endothelial cells. On the other hand, the virus can infect cells via the angiotensin-converting enzyme-2 receptor, which is mainly expressed on endothelial cells and cause vascular damage [7][8][9][10]. Tis endotheliitis may be the cause of this microthrombotic disorder, which particularly afects the lungs and kidneys [8,11]. In addition, recent studies have shown that homocysteine (Hcy) may be associated with this microvascular thrombosis and the severity of COVID-19 disease [12][13][14]. Hcy is associated with pulmonary and venous thrombosis, and high plasma Hcy levels signifcantly increase the incidence of vascular injury in both small and large vessels [15][16][17]. Concentrations above the 90th percentile are associated with an increased risk of degenerative and atherosclerotic processes in the coronary, cerebral, and peripheral circulatory systems [18]. Although Hcy is an efective cardiovascular risk biomarker and cardiovascular complications are considered crucial in hospitalized COVID-19 patients, there are few detailed studies on this parameter. Te aim of our study was to compare Hcy levels in patients admitted to the emergency department for COVID-19 pneumonia and scheduled for inpatient admission to the intensive care unit (ICU) or COVID-19 ward and to demonstrate the association between Hcy levels and disease prognosis.

Methods
Our study was designed as a prospective cross-sectional study. Te study was approved by the Ethics Committee of the University of Health Sciences, Antalya Training and Research Hospital (date: 18/03/2021, number: 3/22), and written informed consent was obtained from all patients/ guardians. Te report was prepared in accordance with the Declaration of Helsinki. Tis study was conducted between April 2021 and December 2021 at the University of Health Sciences, Antalya Training and Research Hospital, COVID-19 ward in the chest disease unit and the ICU. A total of 162 patients who were admitted to the emergency department and had an indication for hospitalization in the ICU or COVID-19 ward due to COVID-19 pneumonia were included. Patients were excluded if they were younger than 18 years and older than 75 years; had a history of malignancy, coronary artery disease or valvular heart disease, liver or kidney failure, chronic infammatory disease, and thromboembolic disease; were taking antiplatelet or anticoagulant medications; and were pregnant or breastfeeding. Te diagnosis of COVID-19 infection was made from nasopharyngeal swabs that were positive in the reverse transcription-polymerase chain reaction. Te diagnosis of pneumonia was made by thoracic computed tomography (CT), laboratory tests, and clinical evaluation. Te indications for hospitalization in the ICU and COVID-19 ward of the patients were determined according to the treatment guidelines of the Turkish Ministry of Health regarding COVID-19 for adult patients [19]. Within the frst 2 hours after hospitalization, blood samples were taken from the patients for necessary biochemical and hematological tests and were studied in our laboratory. Te biochemical tests, including BUN (blood urea nitrogen), creatinine, albumin, ALT (alanine aminotransferase), AST (aspartate aminotransferase), LDH (Lactate dehydrogenase enzyme), ferritin, total cholesterol (TC), triglycerides (TG), HDL-C (high-density lipoprotein-cholesterol), and CRP (C-reactive protein), were analyzed by the spectrophotometric method using Beckman Coulter commercial kits in the Beckman Coulter AU5800 (Beckman Coulter Inc. CA, USA) autoanalyzer. LDL-C (lowdensity lipoprotein-cholesterol) was calculated according to the Friedewald formula [20]. Hemogram (whole blood analysis) was analyzed on Sysmex XT-2000i (Sysmex, Kobe, Japan). Fibrinogen and D-dimer assays were performed on the ACL-TOP 700 (Instrumentation Laboratory, Bedford, Massachusetts, USA) coagulation autoanalyzer using ACL Synthesis commercial kits. After performing these tests, blood samples were centrifuged at 3500 rpm for 10 minutes. Te separated serums were stored in sealed 1.5 ml Eppendorf tubes at −80°C for later assay to measure the serum Hcy level. Te separated serums were thawed at room temperature on the study day to determine the Hcy levels. Human Hcy was analyzed using ELISA commercial kits (AFG Bioscience, Illinois, United States, intraassay reproducibility (precision within an assay) <8%, interassay reproducibility (precision between assays) <10%, detection range: 0.75-15 µmol/L, sensitivity: 0.3 µmol/L). Treatment of patients was started in accordance with the COVID-19 treatment guidelines of the Turkish Ministry of Health when patients were enrolled in the study. Patients received 10 days of favipiravir therapy, low-molecularweight heparin, and prednisolone according to the degree of pneumonia. Patients were followed up during hospitalization.

Statistical Analysis
Te SPSS 23.0 program was used for the analysis. Conformity of variables to the normal distribution was examined using visual (histogram and probability graphs) and analytical methods (Kolmogorov-Smirnov/Shapiro-Wilk tests). Descriptive analyzes are performed using the mean and standard deviations for normally distributed variables. Means and standard deviations of patients' age and biochemical data were reported, and the diference between groups was analyzed with Student's t-test. Diferences between categorical variables were evaluated with the chi-square test. Whether the parameters were normally distributed or not, Student's t-test and the Mann-Whitney U test were used to evaluate the biochemical data of the patients according to the poor prognosis status. Te statistical signifcance level was set as p value <0.05. Te efects of some biochemical fndings of patients on the disease prognosis and mortality risk were evaluated by the logistic regression analysis. Decision-making of Hcy levels in predicting the probability of exitus was analyzed by the receiver operating characteristics (ROCs) curve. Sensitivity and specifcity values of the signifcant cut-of value were calculated. In the evaluation of the area under the curve, it was considered to be statistically signifcant in cases where the type-1 error level was below 5%.

Results
According to the current clinical status of 162 patients enrolled in the study, 134 (82.7%) were admitted from the emergency department to the COVID-19 ward and 28 (17.3%) were admitted to the ICU. Patients in the ICU and in the COVID-19 ward were compared (Table 1). Regarding admission symptoms, nausea/vomiting occurred more frequently in patients in the COVID-19 ward (p: 0.005). SpO 2 (oxygen saturation) levels at the time of admission were comparable between the 2 groups (p: 0.073). Te length of hospital stay was longer in ICU patients than in ward patients (p: 0.0001). Laboratory results of patients in the ICU and in the COVID-19 ward were compared (Table 2). It was found that albumin levels were lower in ICU patients than in ward patients (p: 0.011 ). Hcy levels were signifcantly higher in ICU patients than in ward patients (p: 0.001). Of the 134 patients admitted to the COVID-19 ward on admission, 55 of them later developed a need for ICU care for various reasons and were transferred to the ICU. Hcy (p: 0.010), ferritin (p: 0.041), and LDH (p: 0.010) were signifcantly higher in patients who were transferred to the ICU than in patients who remained in the ward. Tere were 61 patients with a hospital stay of less than 10 days and 101 patients with a hospital stay of more than 10 days. When comparing patients with hospitalization of more than 10 days and less than 10 days, fbrinogen (p: 0.019), ferritin (p: 0.021), CRP (p: 0.016), and LDH (p: 0.004) were higher in patients with hospitalization of more than 10 days while lymphocyte count (p: 0.002) was lower. It was found that 112 patients were discharged and 50 were exitus. Te values of Hcy (p: 0.0001), D-dimer (p: 0.003), N/L ratio (p: 0.019), CRP (p: 0.0001), and LDH (p: 0.016) were signifcantly higher in exitus patients compared to discharged patients. Te comparison of laboratory values between groups according to the prognosis status is shown in Table 3. We evaluated the efects of some biochemical markers known to be important for the prognosis of COVID-19 and Hcy on the prognosis of the disease (depending on whether they are exitus or not) by the logistic regression analysis. It was found that each unit by which Hcy increased approximately doubled the risk of death in patients with COVID-19 (odds ratio: 1.753) ( Table 4). When the cut-of value of Hcy in predicting ICU    International Journal of Clinical Practice admission was taken as 5.85, the sensitivity was found to be 66% and the specifcity as 67% (Table 5). Te ROC curve is given in Figure 1. Considering the correlation between Hcy and other parameters, Hcy was positively related with D-dimer and platelets count (p: 0.01 and p: 0.19, respectively) ( Table 6).

Discussion
It is known that the severity and prognosis of COVID-19 disease are diferent in each patient. It has been shown that 10-15% of mild cases have a severe course and 15-20% of severe cases may require intensive treatment [21]. In our study, 17% (28 patients) of 162 patients admitted to the emergency department had an indication for admission to the ICU. During the follow-up of 134 patients in the ward, 44% (55 individuals) of them required intensive care for various reasons. Numerous biomarkers continue to be studied to predict the disease prognosis and identify patients who may require intensive care. Studies have shown that lymphopenia, low platelet counts, and a high N/L ratio are related to disease severity [22]. Liver tests indicative of liver injury, particularly ALT, ferritin, CRP levels, and D-dimer, have been shown to be associated with a poor prognosis and Alb, albumin; LDH, lactate dehydrogenase enzyme; CRP, C-reactive protein; N/L, neutrophil-to-lymphocyte ratio; Hcy, homocysteine; OR, odds ratio * p < 0.05.     mortality, and a decrease in albumin levels also indicates a poor prognosis [22]. Our study shows that low albumin and high Hcy levels may be indicators of which patients should be transferred to the ICU on admission to emergency department. It is also evident from our study that these two parameters are related to the severity of the disease. Te parameters associated with survival in our study were Hcy, D-dimer, N/L ratio, CRP, and LDH. It was found that each unit by which Hcy increased approximately doubled the risk of death in patients with COVID-19. Hcy was associated with poor prognostic parameters, including exitus, and need for intensive care. According to recent studies, people with COVID-19 have a signifcantly higher risk of developing coagulopathy and thrombosis, two conditions in which Ddimer levels rise, especially in severe courses. It is thought that the amino acid Hcy, which is essential for coagulation, may potentially be a factor in these conditions [14]. As an indicator of the likelihood of unfavorable disease progression in COVID-19 patients, elevated Hcy levels appear to be benefcial. In the study by Yang et al. involving 273 COVID-19 patients, Hcy levels were measured and chest CT was performed on hospital admission. Tey performed CT on day 7 ± 2 of admission to assess disease progression during the frst week. Hcy levels were signifcantly higher on imaging in patients with disease progression than in patients without progression [12]. Ponti et al. measured plasma Hcy levels in 304 hospitalized COVID-19 patients and found that serum Hcy levels were signifcantly higher in survivors than in exitus patients [13]. Keskin et al. treated 117 hospitalized COVID-19 patients, and Hcy levels were higher in the severe disease group (respiratory rate above 30/min, blood oxygen saturation below 93%, respiratory failure, shock, or other organ failure requiring intensive care) than in the mild disease group (respiratory rate below 30/min and blood oxygen saturation above 90% on room air) [23]. In the study by Fouda et al. of 40 pediatric patients with COVID-19, the Hcy levels were higher in patients than in controls and were associated with disease severity and need for intensive care [24]. Tere are few studies investigating the association between high Hcy levels and disease severity in COVID-19 [25]. Our study supports these studies and demonstrates the association between the need for intensive treatment and high Hcy levels. A limitation was the small number of patients receiving intensive treatment at the time of hospitalization. Patients in the study received similar treatments. Before starting the treatment, the blood drawn was centrifuged and stored, and Hcy measurements were obtained from these serums. Terefore, the treatment had no efect on Hcy levels.

Conclusion
A high Hcy level is a helpful marker in determining the need for intensive care on admission to the emergency department and a marker of poor prognosis in COVID-19 pneumonia. Measurement of Hcy in patients admitted to the hospital with COVID-19 pneumonia is benefcial for early identifcation of patients with a poor prognosis and for predicting the need for intensive care. Identifying patients who have a poor prognosis at the time of admission and appropriately predicting the need for hospitalization in the ICU may help to improve early management of these patients in the ICU and reduce mortality.

Data Availability
Data analyzed during the current study are available from the corresponding author upon request.

Additional Points
What is Known? What is New?. A limited number of previous studies have found that homocysteine may be associated with poor prognosis in COVID-19 and that it is higher in severe disease than in mild disease. However, in our study, we found that the measurement of homocysteine is a guide for early decision-making about which patients need intensive care on initial admission to the emergency department, and we also showed that it is directly related to mortality and is a useful tool for early identifcation of patients who may need intensive care.

Ethical Approval
Ethics committee approval was obtained from the Clinical Research Ethics Committee of the University of Health Sciences, Antalya Training and Research Hospital, No: 3/22.

Consent
Te informed consent of the patient or guardian was taken prior to the writing of the manuscript.

Conflicts of Interest
Te authors declare that they have no conficts of interest.