Accumulation of oxysterols in the erythrocytes of COVID-19 patients as a biomarker for case severity

doi:10.21203/rs.3.rs-2473292/v1

Background: Due to the high risk of COVID-19 patients to the formation of thrombosis in the circulating blood, atherosclerosis, and myocardial infarction, it was necessary to study the lipidomic of the erythrocytes. The aim of this work was to analyze the pathogenic oxysterols and acylcarnitines in the erythrocyte’s homogenate of COVID-19 patients and to estimate the case severity from the level of oxysterols.

Methods: A linear ion trap mass spectrometry coupled with high-performance liquid chromatography was used to investigate the extract of erythrocytes homogenate. The toxic biomarkers that primarily induce the generation of dead red blood cells, were characterized, and quantified in the erythrocytes of COVID-19 patients and matched with healthy volunteers.

Results: A total of 30 patients and 30 healthy volunteers were enrolled. The concentration of five oxysterols and six acylcarnitines in the erythrocyte’s homogenate of COVID-19 patients was significantly upregulated matching with healthy subjects at p <0.05. The average total concentration of oxysterols was 23.36 ± 13.47 μg/mL in the erythrocytes of COVID-19 patients, while samples of healthy volunteers showed a total concentration of 4.92 ± 1.61 μg/mL. The average concentration level of 7-ketocholesterol and 4-cholestenone in the COVID-19 patients was higher by five and ten-fold compared to the healthy subjects. Also, the average concentration of acylcarnitines in the erythrocyte's homogenate of COVID-19 patients was high by 2-to-4-fold in comparison with the healthy volunteers.

Conclusions: The abnormally high levels of oxysterols and acylcarnitines found in the erythrocytes of COVID-19 patients were associated with the severity of the case's complications and substantial risk of thrombosis. The concentration of oxysterols in the erythrocyte homogenate could be useful as a diagnostic biomarker to stand on the COVID-19 case severity.

COVID-19

Thrombosis

7-Ketocholesterol

4-Cholestenone

Acylcarnitines

Erythrocytes

Ion-trap-mass spectrometry

Oxysterols

Six acylcarnitines and five oxysterols were confirmed in COVID-19 patients
The levels of 4-cholestenone and 7-ketocholesterol in the erythrocytes of COVID-19 patients were abnormally high
Both, 7-KCh and 4-Chn potentially induce apoptosis of RBCs and thrombus formation
Particularly, the two pathogenic oxysterols, 4-Chn and 7-KCh, were increased in the erythrocytes by a factor of 10 and 5 folds, respectively
Principal component analysis showed that oxysterols could be used as a prognostic biomarker of COVID-19
Disease severity is directly proportion to the level of oxysterols and acylcarnitines in the erythrocytes
The LC-IT-MS method was capable to detect, characterize and quantify the abnormal oxysterols

The pandemic coronavirus outbreak started in the Chinese city of Wuhan and was defined by WHO on February 11, 2020, as COVID-19. The COVID-19 infection and complications are well described by the World Health Organization [1, 2]. The COVID-19 disease is caused by a virus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The general clinical symptoms of this viral infection are fever, myalgia, general fatigue, and dry cough [3]. Some cases suffer more aggressive illnesses leading to pneumonia, breathing dysfunction [4], and cardiac injury, which might end with death [5]. The most life-threatening complications of COVID-19 infection include pneumonia, acute respiratory distress syndrome, and severe respiratory failure, which hamper the oxygen reaching the bloodstream.

Lefrançais et al. reported that the bone marrow produces platelets, and the lung generates 50% of total platelets [6]. However, Wool et al. reported that thrombocytopenia might negatively impact the prognosis in COVID-19 patients [7], which is not entirely accurate, as recently published [8]. Researchers could not conclude that COVID-19 exhibit a particular effect on the hemostatic system or cause hemostatic system activation [7, 9]. The confirmed COVID-19-infected cases are varied in severity. The severe COVID-19 infection can be life-threatening and lead to serious complications affecting multiorgan such as the lung, heart, and kidneys which are believed to be mediated by a condition known as cytokine release syndrome [10].

COVID-19 patients are very prone to blood hypercoagulation [8]. To date, the pathophysiology and the mechanism of hypercoagulation have not been understood [11]. However, many hypercoagulation mechanisms have been postulated and attributed to the impact of endothelial cell injury, activation of the coagulation factor, inflammation and inactivation of fibrinolysis [9]. Lucijanic et al. found that a smaller proportion of COVID-19 patients present with thrombocytosis than with thrombocytopenia [8]. However, COVID-19 patients with a higher level of platelets are at high risk of venous thromboembolism. Recent reports showed that the COVID-19 autopsy has evidence of pulmonary endothelial viral inclusions, increased angiogenesis, apoptosis, and increased alveolus-microthrombi [12].

Oxysterols are the oxidation products of cholesterol. Oxysterols have an important role in the biological process of cholesterol homeostasis, apoptosis, and autophagy lipid metabolism [13]. The blood level of oxysterol may be considered a diagnostic biomarker for specific diseases or prediction of the incidence of certain diseases, such as multiple sclerosis, osteoporosis, Alzheimer's disease, psychomotor diseases, and cancer [13, 14]. The accumulation of oxysterols as an end product of cholesterol results in chronic pathogenesis accompanied by cellular damage, as in neurodegenerative disease, congestive heart failure, and acute viral infections [15]. Some oxysterols exert potent oxidation damage to cells and acute inflammatory effects at low concentrations detected in the lesions of atherosclerosis, acute coronary syndrome, and neurodegenerative diseases [16]. This atherosclerosis is due to oxidized lipids and cholesterol [17–19].

Ohashi et al. tested the antiviral effect of some oxysterols against COVID-19 activity using a cell-culture assay module [20]. They reported that 7-ketocholesterol (7-KCh) inhibits the propagation of SARS-CoV-2 in culture cells. However, 7-KCh exerts a toxic effect and induces cell damage [16]. Because of these different toxic activities, 7-KCh has been claimed to contribute to the pathogenesis of COVID-19. It has been postulated that the assay level of 7-KCh in plasma could be used as a prognostic biomarker for identifying the severity of COVID-19. This information could help identify patients at risk of worse clinical outcomes and optimize their medical care, thus perhaps decreasing the number of patients needing mechanical ventilation [16]. In severe cases of COVID-19 infection, it has been hypothesized that 7-KCh could be a biomarker predicting the disease severity and its potential complications [21]. Tang et al reported that the lipidomic analysis of erythrocytes collected from heart failure patients revealed oxysterols accumulation [22]. Also, patients with high 7-KCh in plasma have shown extraordinarily more giant dead cells that make the plaques harder [23].

It has been reported that both 7-ketocholesterol and 7β-cholesterol are moderately increased in the serum of COVID-19 patients using gas chromatography-mass spectrometry as an assay method [24].

The literature review showed that the most suitable method of analysis of oxysterols in tissues and plasma is the utilization of isotope dilution-mass spectrometry, with the use of individual deuterium-labeled analytes as internal standards [25, 26]. Samples intended to analyze cholesterol and oxysterol should be carefully handled since these compounds are oxidized by air within 30 min [27]. Pataj et al. described an LC-MS method for quantifying eight oxysterols in plasma and red blood cells of healthy humans using N,N-dimethylglycine as pre-column derivatization reagents to improve the ionization efficiency at ESI [28].

Acylcarnitine translocase existed in extracellular and auto-activated to transport the long-chain fatty acids into mitochondria for β-oxidation used by the cells as an energy source [29]. Abnormal expression of some acyl-Co enzyme-A leads to the accumulation of some acyl-carnitines, which results in a toxic effect on the cell [29]. Important changes in acylcarnitine metabolism have been found in diseases such as dementia [30], heart failure [31], and coronary artery disease [32].

Barberis et al 2020 demonstrated that COVID-19-infected patients had high concentrations of eighteen acylcarnitines in the plasma [33]. They showed that mitochondria secreted more elevated levels of acylcarnitines, characterized by long-chain fatty acids, which could exhibit a high-risk factor for lung injury with uncontrolled oxidation of the fatty acid. The accumulated lipids in the air fluid could increase the infection severity [34]. It has been described that the viruses that infect the respiratory system, such as the flu virus, induce the accumulation of acylcarnitine, and a subsequent increase of acylcarnitine levels in the plasma of COVID-19 patients might be connected to this mechanism. As previously suggested, acylcarnitine accumulation may lead to toxicity due to the amphiphilic long-chain acylcarnitine, which has been demonstrated to inhibit ion channels, disrupt calcium signaling, and impair adenosine-triphosphate [35].

Herein, we investigated the correlation between COVID-19 and abnormal biogenic materials. The lipidomic profile of cholesterol, some characteristic oxysterols, and acylcarnitines were studied to explain and justify the pathogenesis, respiratory dysfunction, and atherosclerosis.

Because of the key role of acylcarnitines in cellular metabolism in different diseases, they could be expressive diagnostic or prognostic biomarkers. Therefore, we studied the Lipidomics of acylcarnitines in COVID-19 patients because it can help enhance the evolution of disease diagnosis and treatment technology.

Standard lipids were obtained from Avanti Polar Lipids, Inc. (AL, USA), and the purity of each was more than 99.0%; cholesterol, 4β-hydroxycholesterol-4-acetate, 4β-hydroxycholesterol, 7-ketocholesterol (7-KCh), 4-cholestenone (4-Chn), 4,6-cholestadien-3-one, O-palmitoleoylcarnitine (C16:1_CA), palmitoyl-L-carnitine (C16:0_CA), linoleoyl-L-carnitine (C18:2_CA), oleoyl-L-carnitine (C18:1_CA), stearoyl-L-carnitine (C18:0_CA), and 3-hydroxyoleoyl-carnitine (3OH-C18:1_CA). Three deuterated compounds were purchased and used as internal standards, including oleoyl-L-carnitine-d9 (IS1), cholesterol-d6 (IS2), and 7-KCh-d7 (IS3).

Instruments and conditions

Thermo Scientific LTQ-XL linear ion trap mass spectrometer coupled with Accela autosampler and Accela pump (San Jose, CA, USA). The ion source; is the electrospray ionization (ESI) compartment. The system was controlled with Xcalibur® Thermo Fisher Scientific Inc, version 2.07 SP1. Spray voltage, 5.0 kv, sheath gas flow rate, 42 mL/min, auxiliary gas, 10 mL/min, capillary voltage, 60v, capillary temperature, 325 °C. The collision energy was 35 v. Column, Eclipse Plus C18, 3.5 μm, 4.6 x 100 mm (Agilent, Palo Alto, USA), column oven, 40±3 °C. Tray temperature, 20 °C. The mobile system was composed of (A) water: methanol: ammonium hydroxide solution 25% (75: 25: 0.4, v/v), (B) methanol: chloroform: ammonium hydroxide solution 25% (95: 5: 0.4, v/v) and (C) methanol: chloroform: ammonium hydroxide solution 25% (75: 25: 0.4, v/v). The flow rate was 400 μL/min. The pump was programmed at 0 – 2 min to deliver 65%A, decreased to 35%A at 9 min, decreased to 15%A at 30 min, decreases to 5%A at 40, and decreased to 1%A at 49 – 70 min.

The ion trap-mass spectrometer (IT-MS) detector was programmed to monitor ions by applying positive scan mode, 100 - 1200 m/z, and dependent auto-fragmentation mode. MSⁿ spectra generation for ions exceeds the mass count of 1000. Data were saved as raw files for further investigation by Processing Setup, Thermo Xcalibur 4.5.474.0, 1/14/2022., and FreeStyle™ 1.8 SP2, Modern Data Visualization Software, Version 1.8.63.0, Build Date: Friday, July 30, 2021, Copyright © 2021 Thermo Fisher Scientific Inc. The NIST Mass Spectral Search Program, Version 2.4, build Mar 25, 2020. The NIST database was enriched by the online MassBank of North America (MoNA) and is metadata-centric (https://mona.fiehnlab.ucdavis.edu).

Solutions and calibration levels

A mixture of chloroform and methanol (2: 1, v/v) was used as a solvent matrix to prepare standard stock solutions and all other dilutions. A 1 liter of this solvent was bubbled with nitrogen gas, 99.999%, for 1 min to expel any dissolved oxygen. The internal standard (IS) solution mixture was prepared in this solvent to contain 50 ng/μL of each: oleoyl-L-carnitine-d9, cholesterol-d6, and 7-ketocholesterol-d7. The stock calibrant solution mixture containing 500 μg/μL of each acylcarnitine, cholesterol, and oxysterol was prepared and kept at 4 °C in a glass vial sealed under nitrogen gas. A serial dilution mixture of this stock calibrant mixture was prepared to get a concentration range of 2 to 120 ng/μL for each analyte. A volume of 5 μL was injected for LC-MS analysis. Seven calibration solution levels were prepared by mixing 25 μL of the internal standard solution mixture with 25 μL of the calibrant mixture. The final concentration of the calibrant has spanned a range of 1 to 60 ng/μL with a constant concentration of each IS, 25 ng/μL.

Blood sample collection

Patients suffering from the general symptoms of COVID-19 infection, including fever, chest pain with respiratory complaints, and general fatigue, were immediately subjected to the polymerase chain reaction (PCR) test. The nasopharyngeal swab sample was collected from the COVID-19 candidate and exposed to the PCR testing method because of its high specificity and sensitivity [36]. This study included only the hospitalized patients clinically diagnosed and categorized as having moderate and severe COVID-19 infection in compliance with the international guidelines of COVID-19 case severity [37]. Patients diagnosed as asymptomatic were excluded.

The ethical committee approved the blood sample collection at King Abdulaziz University Hospital, reference number 408-20, and the National Committee of Biology and Medical Ethics registration number is “HA-02-J-008”. The study exclusion criteria included pregnant women, volunteers receiving any hypolipidemic drugs, diabetic patients, cases who received streptokinase injections, and acute cases under mechanical ventilation. Each group involved 30 volunteers involving, male and female, equally. Included volunteers aged 40 to 75 years old, admitted to the hospital and under medical supervision. Informed consent was read, understood, and signed by the patients. Healthy volunteers were also selected carefully and had recent medical investigation data at King Abdulaziz University Hospital, Jeddah, Saudi Arabia.

A volume of 5 mL of blood sample was collected from the arm vein over sodium-EDTA, swirled, and immediately sent to the analysis lab. The blood samples were centrifuged at 3000 rpm for 15 min. The supernatant plasma was aspirated with a Pasteur pipette, transferred to a 7-mL brown-glass vial, and kept at -80 °C. The red residue was mixed with 5 mL saline (0.9% sodium chloride in water, w/v), mixed by gently swirling, and centrifuged at 3000 rpm/10 min. The upper aqueous layer was aspirated and rejected. The remaining erythrocytes were then vortexed for 1 min and left in a sonication bath for 15 min to homogenize the ruptured erythrocytes. The samples of the homogenate erythrocytes were extracted immediately or otherwise kept at -80 °C until analysis. The samples were stored in 7-mL brown-glass vials, flushed with nitrogen gas, labeled, and tightly screw-capped before storage or analysis.

Calibration curve and assay method

Oleoyl-L-carnitine-d9 (+m/z 435.4), was used as an internal standard for the assay of the targeted acylcarnitines, but cholesterol-d6 (+m/z 375.3) was used as an internal standard for the assay of cholesterol. At the same time, 7-ketocholesterol-d7 (+m/z 408.3) was used as an internal standard for the assay of oxysterols, including 7-KCh, 4-Chn, 4,6-cholestadien-3-one, 4β-hydroxycholesterol-4-acetate, and 4β-hydroxycholesterol. The calibration parameters are shown in Table 1. The calibration curves were constructed by plotting the MS-relative response of the analyte to the IS (Y-axis) and the concentration of the targeted analyte (X-axis). The squared regression coefficient was close to unity, and the response factor was close to 0.04. The ESI source was maintained and tuned daily at +m/z 524.33 to minimize the ionization suppression due to the contamination of the ion source. Samples that showed an analyte concentration outside the calibration range were diluted or concentrated by nitrogen gas to get an MS response within the valid range.

Sample preparation for the analysis

The glass vial containing the homogenate of erythrocytes was vortexed for 10 sec. A volume of 100 μL was transferred to a screw-capped 15-mL glass test tube, mixed with 6 mL of the extraction solvent (chloroform: methanol, 2: 1, v/v), 25 μL of the IS solution mixture, and left under a gentle stream of nitrogen gas for 30 sec before capping. This mixture was vortexed for 2 minutes, sonicated for 15 minutes, vortexed for 30 seconds, and centrifuged at 5000 rpm for 15 minutes. The organic layer was separated by decantation to a 25-mL syringe fitted with a 0.22 μm PTFE filtration membrane, filtered into a 15-mL glass test tube, and dried with nitrogen gas over a water bath adjusted at 40 °C. The remaining residue was quantitatively transferred to a total recovery vial 1-mL using 200 μL of the same extraction solvent and gently bubbled with nitrogen gas to dryness. The residue was reconstituted in 50 μL chloroform and methanol; 2: 1, v/v, capped under nitrogen gas, vortexed, and a volume of 5 μL was injected for LC-IT-MS analysis.

Statistical analyses

The quantitative results collected from the analysis of the targeted analytes were statistically evaluated by using principal component analysis (PCA) to determine the score profile using Multivariate Statistical Package software (MVSP, version 3.22, Kovach Computing Services, Pentraeth, Isle of Anglesey, UK, 2013).

Special precautions

Patients who participated in this study were described clinically as moderate to severe COVID-19 patients [37]. The blood samples collected from COVID-19 cases were centrifuged at a speed of not more than 3000 rpm. The COVID-19 samples that were separated at a faster centrifugal rate, >3400 rpm/5 min, showed the fragility of red blood cells as evidenced by the appearance of red hemoglobin in the supernatant-plasma fluid. Nitrogen gas was used along the erythrocyte’s separation, extraction, and storage process to avoid the formation of oxysterols due to air oxidation. Also, COVID-19 cases exposed to mechanical ventilation were excluded for the possible formation of oxidizing biomaterials due to exposure to oxygen in the inhaled air.

Extraction of erythrocytes

Several extraction trials have been reported to recover the highest percentage of lipids and cholesterols from the erythrocytes [22, 38]. All reported extraction trials have been conducted to obtain an extract with the highest yield, purity, and low red pigmentation. In this work a modified Bligh and Dyer [39] extraction method was applied. The applied extraction procedure considered not only the extraction yield and purity but also the nature and stability of the extracted biogenic material. Auto-oxidation of the biomolecules was avoided throughout the sample preparation procedure. Our laboratory immediately received the collected blood samples to separate the plasma from the erythrocytes. The erythrocytes homogenate was kept at -80 °C in a culture tube sealed under nitrogen gas until analysis. The extraction solvent was also freshly prepared and bubbled with nitrogen gas before use. All these precautions were taken to avoid any oxidation through sample preparation. The final sample extract collected in the autosampler vial showed a faded red pigmentation.

The erythrocytes homogenate was used as the targeted sample for investigation in COVID-19 patients to monitor the oxidized cholesterol derivatives that might generate a multi-thrombotic particle and the possible formation of plaque [40, 41].

Homogeneous erythrocytes collected from healthy volunteers were used as a matrix for preparing quality control (QC) samples. Four QC samples spiked with the oleoyl-L-carnitine-d9, cholesterol-d6, and 7-KCh-d7, at high, median, low, and limit of quantification (LOQ) concentration levels (60, 20, 2, 0.2 ng/μL, respectively), were prepared and extracted. The analysis data of the QC samples, based on the MS-peak area, have shown an average percentage recovery of 95 ±3.7 % with a % error of not more than 4.0% of the three deuterated IS. The IT-MS chromatograms were examined for any expected oxidation products of the spiked cholesterol-d6 due to bad sample handling. This procedure showed no oxidation products derived from the spiked cholesterol-d6. The coextracted unidentified biogenic materials were omitted by removing the unwanted +m/z ions and subtracted as LCMS bases peak value from the extracted ion chromatogram.

Characterization of oxysterols and acylcarnitines

The average IT-MS² spectra of the oxysterols and acylcarnitines were retrieved after a dependent IT-MS scan subjected to a collision energy of 35v and re-analyzed by assigning the peak of interest at the corresponding retention time with the zooming option. The IT-MS² spectra of the detected precursor ions were investigated by NIST 2020 supported. The NIST database was updated to a release of 2022 using a retrievable NSIT-format online database of the Mass Bank of North America (MoNA) at https://mona.fiehnlab.ucdavis.edu. The identified precursor ions showed a delta-mass difference of ±0.002 m/z applying targeted-zooming fragmentation mode.

The ionization efficiency and, subsequently, the detectability of cholesterol, oxysterols, and acylcarnitines at the ESI-MS was improved by investigating the effect of pH of the mobile system. The mobile phase containing ammonium hydroxide (0.4%, w/v) showed intense precursor ions of cholesterol, oxysterol, and acylcarnitine, while formic acid (0.1%, w/v) showed a very low ESI-MS response or little peaks of the targeted analytes. . The pH variation of the mobile phase does not affect the peak retention time; however, an intense and precise peak response was observed in the presence of ammonia solution.

The IT-MSⁿ spectra of the confirmed lipidomes in the extract of erythrocytes obtained from Covid-19 patients were; O-palmitoleoyl-L-carnitine (a), palmitoyl-L-carnitine (b), linoleoyl-L-carnitine (c), oleoyl-L-carnitine (d), stearoyl-L-carnitine (e), 4,6-cholestadien-3-one (f), 4β-hydroxycholesterol 4-acetate (g), 4β-hydroxycholesterol (h), 4-cholestenone (i), 7-ketocholesterol (j) as shown in Fig. 1. The NIST similarity index of the characterized analytes was close to 95%. These identified biogenic materials were analyzed quantitatively in the erythrocytes extract collected from healthy and COVID-19 patients.

Quantitative results of oxysterols and acylcarnitines

The concentration of the identified major acylcarnitines and oxysterols was calculated from the corresponding calibration curve (Table 1). The extracted ion chromatogram of each analyte was first identified, assigned, and integrated, then included within the list of peaks to be processed to calculate the concentration using Thermo-Xcalibur software (version 4.5.474.0, January 2022). The concentration of each analyte was calculated based on the relative peak area of the targeted compound to the assigned IS. Table 2 shows the average concentration of acylcarnitines, cholesterol, and oxysterols in erythrocytes of healthy volunteers and COVID-19 cases. The statistical student t-test was calculated, and the significant change in the values was computed at p-values of 0.05, n = 30 for each group. Data revealed that acylcarnitines, cholesterol, and oxysterols significantly increased in COVID-19 cases. The extracted ion chromatogram of all targeted lipidomes was relatively more intense in COVID-19 cases, matching the healthy volunteers (Fig. 2&3).

The erythrocyte homogenate collected from the 30 patients showed three ketonic and two hydroxylated cholesterol derivatives. The significantly increased 7-ketocholesterol (also known as 7-oxocholesterol), 18.56±10.69 μg/mL, and 4-cholestenone, 1.50±0.85 μg/mL, are relatively the highest levels and the most pathogenic biomarker found in the erythrocyte homogenate of COVID-19 cases. Fig. 4 shows the fold change of the found oxysterols. The average concentration of 4-cholestenone and 7-ketocholesterol was increased by ten and five folds, respectively, compared with the amount found in the erythrocyte of healthy subjects. 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.

The quantitative data of six acylcarnitines and six oxysterols (including cholesterol among the variables) in the erythrocyte of healthy versus COVID-19 cases were statistically analyzed using principal component analysis (PCA). As seen in Fig. 5, the PCA score plot showed a separate aggregate between the healthy and COVID-19 groups. The PCA plot was processed based on the abundance of significant different (p <0.05) between the two groups considering oxysterols (Fig. 5a), acylcarnitines (Fig. 5b), and combined concentration values of both oxysterols and acylcarnitines (Fig. 5c). These results suggest the possibility of using the total concentrations of oxysterols alone or with acylcarnitines in the erythrocyte as a diagnostic biomarker for COVID-19 disease. The PCA scores of the healthy volunteers are more aggregated than those of COVID-19 patients. The PCA scores of COVID-19 patients were found to wider range along the PC1 and PC2 axis due to the difference in the case severity and, subsequently, the quantitative results. This score distribution was also expressed as a higher PC1% (59.1 to 70.9%) than PC2% (8.6 to 13.5%) (Fig. 5).

4-Cholestenone (4-cholesten-3-one), is one of the intermediate oxidation products of cholesterol. It has been reported that 4-Chn is highly mobile in the erythrocytes’ membranes, affecting the cholesterol flip-flop and efflux. It has been reported that cholestenone formation may cause long-term functional defects in red blood cells [42]. Chandra et al 2022 claimed that cholestenone is a unique oxidation product of cholesterol that exists extra and intra-cellular in patients suffering from Mycobacterium tuberculosis infection [43]. This study confirmed that cholestenone, namely 4-cholestenone, was accumulated in the erythrocyte’s membrane. Cholestenone could be detected at concentrations below 0.08 μg/mL in the plasma of COVID-19 patients classified as severe. The literature review showed no data that identify or quantify 4-cholestenone in the blood of COVID-19 patients. Moreover, the LC-MS analysis data confirmed that COVID-19 patients showed an accumulation of three ketonic cholesterols in the erythrocyte's membrane, including 7-ketocholesterol, 4-cholestenone, and 4,6-cholestadien-3-one. This higher level of oxysterol found in the erythrocytes homogenate of COVID-19 patients is not only a diagnostic biomarker or case severity but also could be considered as an alarm to expect aggregation of dead cells and subsequent high risk to form multiple sclerosis in the respiratory system or circulating blood [16, 21].

Moreover, the analysis of erythrocytes homogenate in COVID-19 patients also showed a significant increase of six acylcarnitines, as shown in Table 2. The concentration of the quantified acylcarnitines showed a 2-to-4-fold increase, matching with the data obtained from the healthy volunteers (Fig. 4). Acylcarnitine accumulation has been reported to have a toxic effect on biological cells [29]. Significant changes in acylcarnitine metabolism have been observed in diseases such as dementia [30], heart failure [31], and coronary artery disease [32]. The moderate to severe cases of COVID-19-infected humans clinically suffer difficulty breathing, dementia, and heart problems. All these symptoms could be attributed to the accumulated acylcarnitines.

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].

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.

4-Chn, 4-cholestenone; 7-kCh, 7-ketocholesterol; COVID-19, coronavirus disease of 2019; ESI, electrospray ionization; HPLC, high performance-liquid chromatography; IT-MS, ion trap mass spectrometry; PCA, principal component analysis

Author contributions

Alaa Khedr: conceived the study, designed the study, obtained ethics approval, analyzed samples, wrote the manuscript, statistical calculation, and data interpretation. Maan T. Khayat, Ahdab N. Khayyat, and Hany Z. Asfour:participate in the data interpretation and manuscript writing. Rahmah A. Alsilmi: collected the blood samples, estimated the case severity, collected the signed patient consent, and participated in the clinical data interpretation. Ahmed K. Kammoun: participated in the sample analysis, manuscript writing, statistical calculations, and data interpretation.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Funding

This research work was funded by Institutional Fund Projects under grant no (IFPRC-166-2020). Therefore, the authors gratefully acknowledge technical and financial support from the Ministry of Education and King Abdulaziz University, Jeddah, Saudi Arabia.

Availability of data and materials

The data that support the findings of this study are available from the corresponding author, A Khedr, upon reasonable request.

Patient consent

The patient consent of all participants was kept confidentially at the King Abdulaziz University Hospital.

Ethics approval and consent to participate

The ethical committee approved the blood sample collection at King Abdulaziz University Hospital, reference number 408-20, and the National Committee of Biology and Medical Ethics registration number is “HA-02-J-008”.

Consent for publication

Not applicable.

Competing interests

The authors have no conflicts of interest to declare.

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Table 1 Calibration parameters of the acylcarnitines and oxysterols in the homogenate of erythrocytes

t_R, min	Name	Squared Regression coefficient, r²	Slope*	Intercept	Range, ng/μL	LOD, ng/μL
25.82	O-Palmitoleoyl-L-carnitine	0.9991	0.0425	0.0355	2.0 - 60	0.50
30.94	Palmitoyl-L-carnitine	0.9988	0.0399	0.0415	2.0 - 60	0.50
28.71	Linoleoyl-L-carnitine	0.9995	0.0517	0.0222	2.0 - 60	0.50
32.52	Oleoyl-L-carnitine	0.9999	0.0401	0.0066	2.0 - 60	0.50
37.64	Stearoyl-L-carnitine	0.9992	0.0452	0.0299	2.0 - 60	0.50
16.72	3-Hydroxyoleoylcarnitine	0.9987	0.0407	0.0425	2.0 - 60	0.50
32.55	Oleoyl-L-carnitine-d9, IS1	N/A	N/A	N/A	N/A	N/A
60.61	Cholesterol	0.9999	0.0417	0.0101	2.0 - 60	0.50
49.11	4,6-Cholestadien-3-one	0.9995	0.0398	0.0211	2.0 - 60	0.50
38.52	4β-Hydroxycholesterol-4-acetate	0.9997	0.0335	0.0198	2.0 - 60	0.50
38.82	4β-Hydroxycholesterol	0.9991	0.0392	0.0324	2.0 - 60	0.50
50.28	4-Cholestenone	0.9998	0.0378	0.0060	2.0 - 60	0.50
40.19	7-Ketocholesterol	0.9999	0.0414	0.0022	2.0 - 60	0.50
60.82	Cholesterol-d6, IS2	N/A	N/A	N/A	N/A	N/A
40.22	7-ketocholesterol-d7, IS3	N/A	N/A	N/A	N/A	N/A

* The regression line was drawn by plotting the concentration (X independent variable), ng/μL, versus the peak area ratio of the analyte to the internal standard (Y dependent variable).

Table 2 The concentration of acylcarnitines, cholesterol, and oxysterols in erythrocytes of healthy volunteers and hospitalized COVID-19 cases. p-Values calculated at probability ± 0.05, n = 30 of each group.

t_R, min	Name	Exact mass	Precursor ion, +m/z	Healthy, concentration μg/mL ±SD		COVID-19, concentration, μg/mL ±SD		p-value	**
25.82	O-Palmitoleoyl-L-carnitine	397.32	398.32	0.19	±0.22	0.35	±0.20	0.01	↑
30.94	Palmitoyl-L-carnitine	399.33	400.33	1.96	±0.69	3.88	±2.01	0.00	↑
28.71	Linoleoyl-L-carnitine	423.33	424.34	0.91	±0.44	2.20	±1.24	0.00	↑
32.52	Oleoyl-L-carnitine	425.35	426.36	2.00	±0.65	4.96	±2.67	0.00	↑
37.64	Stearoyl-L-carnitine	427.36	428.38	1.07	±0.36	1.64	±0.69	0.00	↑
16.72	3-Hydroxyoleoylcarnitine	441.35	442.36	0.01	±0.00	0.06	±0.03	0.00	↑
32.55	Oleoyl-L-carnitine-d9, IS1	434.35	435.37	-		-		-	-
60.61	Cholesterol	386.35	369.37	0.06	±0.01	0.19	±0.09	0.00	↑
49.11	4,6-Cholestadien-3-one	382.32	383.34	0.19	±0.05	0.95	±0.43	0.00	↑
38.52	4β-Hydroxycholesterol-4-acetate	444.36	367.38	0.47	±0.16	1.83	±1.13	0.00	↑
38.82	4β-Hydroxycholesterol	402.35	385.37	0.17	±0.13	0.52	±0.37	0.00	↑
50.28	4-Cholestenone	384.34	385.36	0.15	±0.13	1.50	±0.85	0.00	↑
40.19	7-Ketocholesterol	400.33	401.33	3.94	±1.14	18.56	±10.69	0.00	↑
60.82	Cholesterol-d6, IS2	392.35	375.35	-		-		-	-
40.22	7-Ketocholesterol-d7, IS3	407.33	408.34	-		-		-	-

* Average m/z value obtained with zooming-dependent fragmentation mode

** Statistical significance, ↑ significant increase.

No competing interests reported.

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Graphical Abstract

Accumulation of oxysterols in the erythrocytes of COVID-19 patients as a biomarker for case severity

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