COVID-19: The accumulation of oxysterols in the erythrocytes is a biomarker for case severity

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A new study by Saudi scientist from King Abdulaziz University and King Abdulaziz University Hospital has found that SARS-CoV-2 infections lead to the buildup of oxysterols in erythrocytes and also the accumulation of acylcarnitines, both of which can be toxic and damaging to the host cells, contributing to disease severity.

The study findings were published on a preprints server and is currently being peer reviewed for publication in the journal: BMC Respiratory Research.
https://www.researchsquare.com/article/rs-2473292/v1

In this work, blood samples were collected from thirty patients clinically diagnosed with COVID-19 infection of moderate to severe stage, and 30 matched healthy controls. LC-IT-MS was used to analyze the extract of the erythrocyte samples collected from moderate to severe COVID-19 patients.

The IT-MS scan confirmed five oxidation products derived from cholesterol and six acylcarnitines (Fig. 1). A modified LC-MS analytical procedure was applied to quantitatively estimate the targeted analytes [44]. Special precautions were applied during sample preparation to avoid the analyte’s oxidation during sample handling.

The extracted ion-chromatogram showed relatively intense MS peaks corresponding to the precursor positive ions of oxysterols and acylcarnitines (Fig. 2&3).

Cell death, Inflammation, N-glycosylation, thrombosis have been studied and among the critical COVID-19 biomarkers that illustrate the case severity [45].

The characterized analytes were quantitatively determined since these entities of metabolites have been reported as erythrocytic pathogenic biomarkers released due to the harmful impact on the biological cells [9, 21, 24, 46]. The summary of the results we obtained is consistent with the report published by Reva et al.

It has been described that COVID-19 virus considered the human erythrocytes as a preferred target’s host for multiplication and cell damage [47].

Table 1 shows the calibration parameters of the assayed targeted analytes. The quantitation results showed a significant increase in oxidized cholesterol and acylcarnitines (Table 2). 7-Ketocholesterol and 4-cholestenone corresponded to the highest level of oxysterols associated with two hydroxylated-cholesterol metabolites. 4-Cholestenone and 7-ketocholesterol were increased in the erythrocyte of COVID-19 patients by ten and five folds, respectively, compared with the amount found in the healthy subjects.

The concentration of acylcarnitines and oxysterols in healthy versus COVID-19 erythrocytes was statistically analyzed using principal component analysis (PCA).

The found concentrations of the targeted metabolites were statistically used as input data variables to get the multivariate statistical plot. The principal component analysis showed an apparent aggregation of each group separately (Fig. 5). The Association of the data of both oxysterols with acylcarnitines in one PCA figure showed better segregation based on the values of the percentage of PC1 and PC2 axis.

Also, the significantly increased oleoyl-L-carnitine, 4.96 ± 2.67 µg/mL, and palmitoyl-L-carnitine, 3.88 ± 2.01 µg/mL, were relatively the highest level among the six acylcarnitine found in COVID-19 erythrocytes. COVID-19 patients were sorted according to the severity of the case based on the clinical diagnosis and the occurrence of complications.

The first ten cases were defined as critical cases, and the rest were considered moderate cases according to the WHO guidelines [37]. The concentration of 7-KCh, 4-Ch, and C18:1_CARN was plotted versus patient numbers, as shown in Fig. 6. This figure showed that the more severe the COVID-19 infection, the higher concentration of the accumulated oxysterol and C18:1_CARN.

The concentration range of 7-ketocholesterol and 4-cholestenone in the erythrocytes of COVID-19 patients defined as severe was 52.0 to 16.6 and 4.5 to 1.5 µg/mL, respectively. However, the moderate COVID-19 cases showed a concentration range of 16.2 to 5.7 and 1.4 to 0.3 µg/mL for 7-KCh and 4-Ch, respectively.

The lowest concentration of both 7-KCh and 4-Ch in the COVID-19 patients was higher than the average values obtained from healthy volunteers, as shown in Table 2. The level of 7-KCh in the erythrocyte of moderate and sever cases was increased by 2.0–4.5, and 4.5–12.5 fold, respectively (Fig. 6).

Analysis of 7-KCh in erythrocytes was preferred over serum due to its presence at a relatively higher concentration, which facilitates measurement and early detection in mild cases of COVID-19. In this study the average amount of 7-KCh in the erythrocytes of healthy volunteers was 3.9 µg/mL.

However, it has been reported that the serum concentration of 7-KCh was 20 ng/mL and increased in COVID-19 patients [21, 24]. Marcello et al found that the serum level of 7-KCh was increased 2–3.5, and 2–5 fold in moderate and severe COVID-19 cases, respectively [24]. Based on these data, it could be concluded that 7-KCh, specifically, increased significantly in both erythrocyte homogenate and serum.

Also, the severe COVID-19 cases showed a higher level of C18:1_CARN. Covid-19 patients showed a significantly higher concentration level of oxysterol which expresses the oxidative stress and, subsequently, the risk of apoptosis [16, 21]. Although the mechanism of hypercoagulation has not been understood [11], it has been reported that oxysterols are toxic and pathogenic [13, 15, 24].

Conclusions

Based on the data obtained and the previous reports, it could be concluded that COVID-19 patients are at a very high risk of thrombosis in the circulating blood, atherosclerosis, myocardial infarction, and pulmonary failure due to the lipidomic dysregulation of the erythrocytes-lipid composition.

Also, patients with COVID-19 infection showed that the erythrocyte’s homogenate contains a significantly high concentration of 7-ketocholesterol, 4-cholestenone, and six acylcarnitines.

These lipidomes are very harmful to biological cells, namely red blood cells, and lead to thrombosis formation. It is highly advised to urgent prescribe antiplatelet drugs (aspirin, clopidogrel) to COVID-19 patients.

The concentration range of 7-ketocholesterol and 4-cholestenone in the erythrocytes of COVID-19 patients defined as severe was 52.0 to 16.6 and 4.5 to 1.5 µg/mL, respectively. However, the moderate COVID-19 cases showed a concentration range of 16.2 to 5.7 and 1.4 to 0.3 µg/mL for 7-KCh and 4-Ch, respectively.

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