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Original article

Homocysteine as a predictor of clinical outcomes in patients hospitalized for COVID-19

Sanja Aleksić1, Miloš Ratinac2
  • University Clinical Center of Kragujevac, Clinic for Hematology, Kragujevac, Serbia
  • University Clinical Center of Kragujevac, Clinic for Pulmonology, Kragujevac, Serbia

ABSTRACT

Introduction: COVID-19 is still a global health, social and economic problem. Treating this systemic infection remains a challenge. Numerous biomarkers have been studied so far as potential prognostic parameters, hematological and inflammation parameters most of all.

Aim: Our main goal is to examine the association of homocysteine level with the clinical outcome, as well as whether homocysteine level changes through the course of the disease.

Materials and methods: In our research, blood samples were collected from 131 patients who were SARS-CoV-2 positive and were treated at the University Clinical Center of Kragujevac. The inclusion criteria were as follows: men and women above 18 years of age, a signed informed consent for participation in the study, patients SARS-CoV-2 positive for the first time (which was confirmed by a rapid antigen test or a PCR test), treated in hospital and belonging to any of the three COVID-19 severity categories. Blood samples were taken on the first, fifth and tenth day of the hospitalization period and the course of the disease was monitored. Statistical data processing was done using the IBM SPSS Statistics v.21 program.

Results: Based on our research, we concluded that homocysteine level changed during the hospitalization period and that it had a growing trend. There was a statistical significance between the levels measured on the first, fifth and tenth day. We showed that the patients suffering from hypertension (HTN) had higher levels of the examined parameter compared to the patients who were not hypertensive. It was also observed that homocysteine levels increased with age. There was a statistical significance between homocysteine levels and the outcome of the disease.

Conclusion: Based on our study, we can say that patients with high homocysteine levels (on the fifth day of the hospitalization period) have a worse clinical outcome than patients with lower homocysteine levels. Patients with lower homocysteine levels have a better chance of recovery.


INTRODUCTION

At the end of 2019, a new virus called SARS-CoV-2 was identified as the cause of pneumonia in the city of Wuhan, China, and in March 2020 the World Health Organization (WHO) declared a pandemic and named the disease coronavirus disease 2019 [1]. According to WHO criteria from September 2022, the disease is classified into three categories according to its severity: critical COVID-19, severe COVID-19, and mild COVID-19 [2]. The infection is associated with the risk of thromboembolic events based on inflammation, endotheliitis, hyperviscosity and hypercoagulability [3]. Numerous studies have reported a high incidence of venous thromboembolism in patients with severe clinical picture of COVID-19 despite the use of thromboprophylaxis therapy [4],[5]. Such patients can develop myocardial infarction, cerebrovascular events, arterial thrombosis or microthrombosis affecting different organs, including the lungs [6]. Coagulopathy is associated with poor outcomes, so thromboprophylaxis is suggested in all patients with no clear contraindication [3]. Numerous studies have examined many markers that predict or could predict the prognosis, the course and the outcome of this disease [4]. Many inflammatory markers have been examined, as well as numerous immunological, hematological, and biochemical parameters [7]. One of the examined biomarkers is the amino acid homocysteine which has attracted great attention and been widely researched ever since it was discovered [8],[9]. Homocysteine is a semi essential sulfur-containing amino acid which is derived from an essential amino acid methionine, is not present in peptides and proteins and is not ingested through food [7],[8]. Homocysteine metabolism involves the process of methylation and transsulfuration. The methylation process requires vitamin B12, folic acid and the enzyme methylenetetrahydrofolate (MTHFR), whereas the transsulfuration process requires vitamin B6 and the enzyme cystathionine- β-synthase [6],[10]. Impaired homocysteine metabolism usually results in hyperhomocysteinemia which is defined as homocysteine level above 15µmol/l [6]. Refsum et al. were among the first to conduct a large-scale study on a population of over 18,000 subjects and they concluded hyperhomocysteinemia was associated with an increased risk of hospitalization in cardiovascular patients, the risk being higher in patients over 65 years of age. Van der Meer et al. found that hyperhomocysteinemia was associated with an increased risk of thrombosis [11]. In addition to this, the literature describes the risk of some other diseases including cerebrovascular diseases, neurodegenerative diseases, complications in pregnancy, osteoporosis, malignant diseases, but also a more severe clinical picture and a poor outcome in patients suffering from COVID-19 [8],[9]. The three largest studies on the correlation between COVID-19 and homocysteine levels were conducted in Italy, China and Turkey and their results were used for a meta-analysis in June 2022, whereby in a total of 694 hospitalized patients an elevated serum homocysteine level was a poor prognostic sign [6]. Yang et al. included 273 patients in their research where they measured homocysteine level on the first day of the hospitalization period followed by accompanying diagnostics that included CT of the chest, and then after 5-7 days the disease progression was assessed by repeating CT. The patients with higher homocysteine levels on hospital admission also had progression on CT scan [12]. Ponti et al. conducted a study that included 304 patients whose plasma homocysteine level was measured on hospital admission, and they showed that the level of this substance was higher in patients with lethal outcome [10]. Keskin et al. also measured plasma homocysteine level in 117 patients on hospital admission classifying them into two groups (mild and severe disease) and the results showed that the patients with severe disease had higher homocysteine levels compared to the patients who had mild disease [13].

Aim: Hypercoagulability and a tendency to thrombosis in COVID-19 patients have attracted great attention. Studies examining numerous markers of homeostasis have been conducted. In our research we examined a parameter that is not routinely analyzed except in patients suspected to have acquired thrombophilia. We decided to analyze this parameter over time, i.e. several times during the hospitalization period of COVID-19 patients. Our main aim was to examine the association of homocysteine levels with the outcome of the disease, as well as to find out if the levels changed during the course of the disease.

MATERIALS AND METHODS

Sampling

In our research, blood samples were collected from 131 patients hospitalized due to SARS-CoV-2 infection at the University Clinical Centre of Kragujevac. The inclusion criteria were as follows: men and women above 18 years of age, a signed informed consent for participation in the study, patients SARS-CoV-2 positive for the first time (which was confirmed by a rapid antigen test or a PCR test), treated in hospital and belonging to any of the three severity categories according to WHO criteria. Patients’ blood was sampled on the first, fifth, and tenth day of the hospitalization period and the course of the disease was monitored. One test tube of blood (3 ml) was sampled in each case. The analysis was performed in the hematology laboratory of the Clinic for hematology of the University Clinical Centre of Kragujevac using a standardized machine. Out of 131 participants, there were 53 women (40.5%) and 78 men (59.5%). The participants were 19 to 90 years old, the average age being 62.3±16.3 years.

Statistical analysis

Statistical data processing was done using the IBM SPSS Statistics v.21 program. Normal distribution of data was analyzed using Kolmogorov-Smirnov test for normality. The Friedman test was used to analyze homocysteine levels measured on the first, fifth and tenth day. To analyze homocysteine levels measured on the first, fifth and tenth day in relation to categorical variables with two responses t-test was used for independent samples if the data by category followed a normal distribution, whereas the Mann-Whitney U test was applied if the data by category did not follow a normal distribution. For the analysis of homocysteine levels measured on the first, fifth, and tenth day in relation to categorical variables with more than two responses one-factor ANOVA was used for different groups if the data by category followed a normal distribution, whereas the Kruskal-Wallis test was chosen if the data by category did not follow a normal distribution. Correlation and regression were used for the analysis of homocysteine levels measured on the first, fifth, and tenth day in relation to a quantitative variable (participants’ age), i.e. Spearman’s Rank correlation coefficient (Spearman’s rho) was interpreted. The results were considered statistically significant if the significance (p) was less than or equal to 0.05.

RESULTS

In descriptive statistical analysis of continuous variables, the lowest and the highest values were taken into consideration, as well as the mean and standard deviation. The results of this analysis are presented in Table 1.

Table 1. Descriptive homocysteine levels during the course of the disease

Table 1. Descriptive homocysteine levels during the course of the disease

In descriptive statistical analysis of categorical variables, absolute frequency and relative frequency were considered. The results of this analysis are shown in Table 2.

Table 2. Descriptive statistical analysis of patients’ samples

Table 2. Descriptive statistical analysis of patients’ samples

The Friedman test was used to analyze homocysteine levels measured on the first, fifth, and tenth day. Applying the Friedman test, we found that there was a statistically significant difference (chisquared statistics=45.319, df=2, p < 0.001) between homocysteine levels measured at three different times. For subsequent measurements we used the Wilcoxon test in order to determine the exact moments between which there is a statistically significant difference. Using the Wilcoxon test, we concluded that there was a statistically significant difference between homocysteine levels at all three measurements. A graphical presentation of this result can be seen in Chart 1.

Figure 1. Homocysteine levels during the hospitalization period

Figure 1. Homocysteine levels during the hospitalization period

The Mann-Whitney U test was used to analyze homocysteine levels in relation to gender. Using this test, we concluded that there was no statistically significant difference between homocysteine levels measured on the first, fifth, and tenth day in relation to gender. To analyze the relationship between homocysteine levels measured on the first, fifth, and tenth day and participants’ age, correlation and regression method was used, i.e. Spearman’s Rank correlation coefficient was interpreted. Using the correlation method, i.e. interpreting Spearman’s Rank correlation coefficient, we concluded that there was a moderately weak, positive and statistically significant correlation between homocysteine levels measured on the fifth day and the participants’ age. The results of this analysis are shown in Table 3.

Table 3. The significance of homocysteine levels measured on the fifth day of the hospitalization period in relation to age, clinical outcome, and the presence of hypertension

Table 3. The significance of homocysteine levels measured on the fifth day of the hospitalization period in relation to age, clinical outcome, and the presence of hypertension

To analyze homocysteine levels in relation to the clinical outcome the independent samples t-test was used. Applying the independent samples t-test we found that there was no statistically significant difference between homocysteine levels measured on the first and tenth day in relation to the clinical outcome, but that there was a statistically significant difference in relation to homocysteine levels measured on the fifth day. The results are shown in Table 3 and Chart 2.

Figure 2. The average homocysteine level on the 5th day in relation to the clinical outcome

Figure 2. The average homocysteine level on the 5th day in relation to the clinical outcome

To analyze homocysteine levels in relation to disease severity, the Kruskal-Wallis test was applied. Using the Kruskal-Wallis test we found that there was a statistically significant difference between homocysteine levels measured on the first, fifth, and tenth day in relation to disease severity. To analyze homocysteine levels in relation to the presence of hypertension in participants, the Mann-Whitney U test was applied. We concluded that there was a statistically significant difference between homocysteine levels measured on the fifth day in relation to the presence of hypertension in participants. The results of this analysis are shown in Table 3. Significantly higher homocysteine levels measured on the fifth day were found in participants suffering from hypertension. A graphical presentation of homocysteine levels measured on the fifth day in relation to the presence of hypertension is shown in Chart 3.

Figure 3. Homocysteine level on the 5th day in relation to the presence of hypertension

Figure 3. Homocysteine level on the 5th day in relation to the presence of hypertension

DISCUSSION

So far, homocysteine levels have been analyzed on hospital admission in order to determine their correlation with the outcome of the disease and the clinical picture [6]. In our study, we checked homocysteine levels through the hospitalization period, i.e. through the course of the disease. Patients’ blood was sampled on the first day of the hospitalization period and then on the fifth and tenth day. We included 131 patients of both sexes (53 women and 78 men) suffering from all three disease severity categories of COVID-19. The participants were 19 to 90 years old, the average age being 62.3±16.3 years. Mild COVID-19 was found in 29 patients (22.1%), 77 patients (58.8%) suffered from severe COVID-19 and 25 patients (19.1%) had critical COVID-19. 111 patients (84.7%) were discharged to home for home health care, whereas 20 patients (15.3%) had a lethal outcome. Due to the large number of studies on the association between homocysteine and cardiovascular diseases, we also classified patients according to the presence of hypertension (HTN) [13],[14]. 104 patients suffered from HTN (79.4%), whereas 27 patients did not have HTN (20.6%). Previous studies showed a correlation between elevated homocysteine levels on the first day of the hospitalization period and the severity of the clinical picture, as well as the final outcome. Higher levels of homocysteine were found in patients with severe COVID-19 compared to patients with mild disease, as well as in patients with lethal outcome in comparison with those who recovered [10],[11]. Moreover, higher homocysteine levels are associated with disease progression on CT [10]. Our study presented a statistical significance of homocysteine levels analyzed through time; more precisely, during the course of the disease the levels increased. The lowest homocysteine level measured on the first day was 2.82 and the highest level was 29.4 (the mean 7.49; standard deviation 4.80). On the fifth day, the lowest measured level was 4.86 and the highest was 29.52 (the mean 8.07; standard deviation 3.45). We concluded there was a statistically significant correlation between homocysteine levels measured on the fifth day of the hospitalization period and the clinical outcome (p < 0.05). The patients with a lethal outcome had significantly higher levels of this biomarker on the fifth day of the hospitalization period in comparison with the patients who recovered. We also noticed that the patients who suffered from HTN had higher homocysteine levels than the patients who were not hypertensive. There is a statistically significant difference between homocysteine levels measured on the fifth day in relation to the presence of hypertension in participants. Apart from this, we noticed that the level of the examined parameter increased with age. There is a statistically significant correlation between homocysteine levels measured on the fifth day and participants’ age (p < 0.05). Homocysteine level measured on the fifth day was the most significant finding being statistically significantly correlated with the clinical outcome, the presence of hypertension and age.

CONCLUSION

Based on the results of our study, we can conclude that homocysteine levels in patients hospitalized for COVID-19 changed through the course of the disease, i.e. that they increased. We showed that homocysteine levels measured on the fifth day significantly affected the outcome of the treatment. COVID-19 patients who had higher homocysteine levels had worse clinical outcomes, i.e. a greater chance of a lethal outcome. Patients with lower homocysteine levels had a better prognosis and recovered more quickly.

  • Conflict of interest:
    None declared.

Informations

Volume 3 No 4

December 2023

Pages 395-402
  • Keywords:
    homocysteine, COVID-19, clinical outcome
  • Received:
    03 November 2022
  • Revised:
    06 December 2022
  • Accepted:
    09 December 2022
  • Online first:
    25 December 2022
  • DOI:
  • Cite this article:
    Aleksić S, Ratinac M. Homocysteine as a predictor of clinical outcomes in patients hospitalized for Covid-19. Serbian Journal of the Medical Chamber. 2022;3(4):395-402. doi: 10.5937/smclk3-40984
Corresponding author

Miloš Ratinac
Clinic for Pulmonology, University Clinical Centre of Kragujevac
30 Zmaj Jovina Street, 34000 Kragujevac, Serbia
E-mail: This email address is being protected from spambots. You need JavaScript enabled to view it.



REFERENCES

1. Nikolopoulou GB, Maltezou HC. COVID-19 in Children: Where do we Stand? Arch Med Res. 2022 Jan;53(1):1-8. doi: 10.1016/j.arcmed.2021.07.002. [CROSSREF]

2. World Health Organization. Therapeutics and COVID-19: living guideline. 2022 September [HTTP]

3. Sarkar M, Madabhavi IV, Quy PN, Govindagoudar MB. COVID-19 and coagulopathy. Clin Respir J. 2021 Dec;15(12):1259-74. doi: 10.1111/crj.13438 [CROSSREF]

4. Llitjos JF, Leclerc M, Chochois C, Monsallier JM, Ramakers M, Auvray M et al. High incidence of venous thromboembolic events in anticoagulated severe COVID-19 patients. J Thromb Haemost. 2020 Jul;18(7):1743-46. doi: 10.1111/ jth.14869 [CROSSREF]

5. Ali MAM, Spinler SA. COVID-19 and thrombosis: From bench to bedside. Trends Cardiovasc Med. 2021 Apr;31(3):143-60. doi: 10.1016/j.tcm.2020.12.004 [CROSSREF]

6. Carpenè G, Negrini D, Henry BM, Montagnana M, Lippi G. Homocysteine in coronavirus disease (COVID-19): a systematic literature review. Diagnosis (Berl). 2022 Jun 16;9(3):306-10. doi: 10.1515/dx-2022-0042 [CROSSREF]

7. Koklesova L, Mazurakova A, Samec M, Biringer K, Samuel SM, Büsselberg D et al. Homocysteine metabolism as the target for predictive medical approach, disease prevention, prognosis, and treatments tailored to the person. EPMA J. 2021 Nov 11;12(4):477-505. doi: 10.1007/s13167-021-00263-0. [CROSSREF]

8. Jakubowski H. Homocysteine Modification in Protein Structure/Function and Human Disease. Physiol Rev. 2019 Jan 1;99(1):555-604. doi: 10.1152/ physrev.00003.2018 [CROSSREF]

9. Hermann A, Sitdikova G. Homocysteine: Biochemistry, Molecular Biology and Role in Disease. Biomolecules. 2021 May 15;11(5):737. doi: 10.3390/ biom11050737. [CROSSREF]

10. Ponti G, Maccaferri M, Ruini C, Tomasi A, Ozben T. Biomarkers associated with COVID-19 disease progression. Crit Rev Clin Lab Sci. 2020 Sep;57(6):389- 99. doi: 10.1080/10408363.2020.1770685. [CROSSREF]

11. Refsum H, Nurk E, Smith AD, Ueland PM, Gjesdal CG, Bjelland I et al. The Hordaland Homocysteine Study: a community-based study of homocysteine, its determinants, and associations with disease. J Nutr. 2006 Jun;136(6 Suppl):1731S-1740S. doi: 10.1093/jn/136.6.1731S. [CROSSREF]

12. Yang Z, Shi J, He Z, Lü Y, Xu Q, Ye C et al. Predictors for imaging progression on chest CT from coronavirus disease 2019 (COVID-19) patients. Aging (Albany NY). 2020 Apr 10;12(7):6037-48. doi: 10.18632/aging.102999. [CROSSREF]

1. Nikolopoulou GB, Maltezou HC. COVID-19 in Children: Where do we Stand? Arch Med Res. 2022 Jan;53(1):1-8. doi: 10.1016/j.arcmed.2021.07.002. [CROSSREF]

2. World Health Organization. Therapeutics and COVID-19: living guideline. 2022 September [HTTP]

3. Sarkar M, Madabhavi IV, Quy PN, Govindagoudar MB. COVID-19 and coagulopathy. Clin Respir J. 2021 Dec;15(12):1259-74. doi: 10.1111/crj.13438 [CROSSREF]

4. Llitjos JF, Leclerc M, Chochois C, Monsallier JM, Ramakers M, Auvray M et al. High incidence of venous thromboembolic events in anticoagulated severe COVID-19 patients. J Thromb Haemost. 2020 Jul;18(7):1743-46. doi: 10.1111/ jth.14869 [CROSSREF]

5. Ali MAM, Spinler SA. COVID-19 and thrombosis: From bench to bedside. Trends Cardiovasc Med. 2021 Apr;31(3):143-60. doi: 10.1016/j.tcm.2020.12.004 [CROSSREF]

6. Carpenè G, Negrini D, Henry BM, Montagnana M, Lippi G. Homocysteine in coronavirus disease (COVID-19): a systematic literature review. Diagnosis (Berl). 2022 Jun 16;9(3):306-10. doi: 10.1515/dx-2022-0042 [CROSSREF]

7. Koklesova L, Mazurakova A, Samec M, Biringer K, Samuel SM, Büsselberg D et al. Homocysteine metabolism as the target for predictive medical approach, disease prevention, prognosis, and treatments tailored to the person. EPMA J. 2021 Nov 11;12(4):477-505. doi: 10.1007/s13167-021-00263-0. [CROSSREF]

8. Jakubowski H. Homocysteine Modification in Protein Structure/Function and Human Disease. Physiol Rev. 2019 Jan 1;99(1):555-604. doi: 10.1152/ physrev.00003.2018 [CROSSREF]

9. Hermann A, Sitdikova G. Homocysteine: Biochemistry, Molecular Biology and Role in Disease. Biomolecules. 2021 May 15;11(5):737. doi: 10.3390/ biom11050737. [CROSSREF]

10. Ponti G, Maccaferri M, Ruini C, Tomasi A, Ozben T. Biomarkers associated with COVID-19 disease progression. Crit Rev Clin Lab Sci. 2020 Sep;57(6):389- 99. doi: 10.1080/10408363.2020.1770685. [CROSSREF]

11. Refsum H, Nurk E, Smith AD, Ueland PM, Gjesdal CG, Bjelland I et al. The Hordaland Homocysteine Study: a community-based study of homocysteine, its determinants, and associations with disease. J Nutr. 2006 Jun;136(6 Suppl):1731S-1740S. doi: 10.1093/jn/136.6.1731S. [CROSSREF]

12. Yang Z, Shi J, He Z, Lü Y, Xu Q, Ye C et al. Predictors for imaging progression on chest CT from coronavirus disease 2019 (COVID-19) patients. Aging (Albany NY). 2020 Apr 10;12(7):6037-48. doi: 10.18632/aging.102999. [CROSSREF]


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