Summary
The COVID-19 illness, which is extremely sensitive to breathing difficulties, protracted breathing difficulty, and fever, is caused by the SARS-CoV-2 beta coronavirus. Red blood cells (RBCs) may contribute to the degree of cerebral hypoxia in COVID-19 patients because they carry oxygen (Amelung, van Hooft, Siersema, & Consten, 2018). Increased amounts of glycolytic intermediates, as well as the degradation and disintegration of ankyrin, spectrin beta, and the N-terminal cytoplasmic domain of band 3 (AE1), were seen in RBCs from COVID-19 patients (Thomas, Stefanoni, Dzieciatkowska, Issaian, Nemkov, Hill & D’Alessandro, 2020). Essential variations in lipid metabolism were also seen, especially for acyl-carnitines, which are composed of phospholipids and short- and medium-chain saturated fats (Sun, Garcia & Hayden, 2018). However, there were only minor improvements in standard range diameter, with no changes in clinical hematological and biochemical such as RBC count, hematocrit, or hematocrit levels number of RBCs.
The current work integrates cutting-edge metabolomics, proteomics, and lipidomics methods to examine the effect of COVID-19 on RBCs from 23 healthy individuals and 29 COVID-19 patients who have been molecularly identified. These findings point to a significant impact of SARS-CoV-2 infection on the protein and lipid composition of the structural membrane of the RBC (Thomas, Stefanoni, Dzieciatkowska, Issaian, Nemkov, Hill & D’Alessandro, 2020). Elevation in RBC glycolytic intermediates is in line with theorized improvements in hemoglobin’s ability to off-load oxygenation as a result of allosteric regulation by high phosphorus molecules, possibly to combat COVID-19-induced hypoxia.
The RBCs from COVID-19 patients are characterized in the current study for the first time using multi-omics. The authors discovered increased oxidation of critical structural components along with enhanced glycolysis in RBCs from COVID-19 patients (Thomas, Stefanoni, Dzieciatkowska, Issaian, Nemkov, Hill & D’Alessandro, 2020). Authors will overcome this problem in subsequent studies since it prevented researchers from directly measuring RBC characteristics that were directly connected to gas transport physiology. Recent research, however, indicates that even in the most severe COVID instances, RBC hemoglobin oxygen affinity and gas-exchange characteristics are not affected (Erdinc, Sahni, & Gotlieb, 2021). This work is written to critically analyze an article in the field of hematology.
Methods
Blood Collection and Processing
The Columbia Institutional Review Board authorized this epidemiological study, which was carried out in conformity with the Declaration of Helsinki and best clinical practice standards. Twenty-three individuals who were all various molecular SARS-CoV-2 negative according to nasopharyngeal swabs at the time of blood collection made up the command group. Recently taken tubes were centrifuged to obtain RBCs, which were then recovered and de-identified. Using a customized Folch technique that renders other coronaviruses entirely inactive, RBCs were retrieved (Thomas, Stefanoni, Dzieciatkowska, Issaian, Nemkov, Hill & D’Alessandro, 2020). For metabolomics, the top episode was moved to a different tube; to take the most significant possible advantage, the bottom phase, and the interdendritic protein disk for proteomics. In a biosafety cover, the polypeptide platter was washed with methanol preceding, centrifuged, and subsequently air-dried (Azoulay, Schellongowski, Darmon, Bauer, Benoit, Depuydt & Soares, 2017). Correct sample collection improves the precision of the findings; to ensure that the necessary bottles and materials are available to collect the samples in the lab, it is crucial to arrange the test beforehand (Thomas, Stefanoni, Dzieciatkowska, Issaian, Nemkov, Hill & D’Alessandro, 2020). The necessity of blood collection for illness diagnosis and status assessment makes the significance of this step clear.
Ultra-High-Pressure Liquid Chromatography
An online connection between a Q Exactive mass spectrometer and a Vanquish UHPLC was used to conduct lipidomics and metabolomics studies. Intracellular metabolite content was measured against the areas computed for heavy isotopologues for each primary standard in focused quantitative research, which added stable isotope-labeled benchmarks to extracting solutions (Thomas, Stefanoni, Dzieciatkowska, Issaian, Nemkov, Hill & D’Alessandro, 2020). Utilizing Compound Discoverer 2.1, data were examined; GraphPad Prism 8.0 was used to create the visualizations and statistical models. Using R Studio, Spearman’s connections and associated p values were computed.
In order to manage and see these interactions and better comprehend data, writers can create several plots across monitors using Compound Discoverer software, which gives a variety of methods to represent complicated data sets and relationships. Researchers may run basic statistical tests that are often used by laboratory and clinical researchers using GraphPad Prism and other comparable tools. Using various preprocessed packages in R makes it an excellent tool for manipulating data and makes it much easier to use.
Protein Digestion
According to the manufacturer’s instructions, protein granules from RBC collections were processed in an S-Trap filter. Materials were decreased for 30 minutes with 10 mM dithiothreitol at 55 °C, left at room temperature, and then alkylated for 30 minutes in the dark with 25 mM iodoacetamide (Thomas, Stefanoni, Dzieciatkowska, Issaian, Nemkov, Hill & D’Alessandro, 2020). The protein mixture was taken onto an S-Trap filter and agitated, and the flow-through was recovered before being reintroduced onto the filtration system after gentle stirring. As a result, organic molecules and other minerals can be absorbed to their fullest extent.
Results
Standard hematological measurements, including RBC count, hemoglobin, hematocrit, mean cathode rays blood cells, mean corpuscular hemoglobin level, and RBC allocation width, did not vary significantly between the two groups, except for perhaps minor improvements in mean corpuscular quantity. The impacts of COVID-19 on RBCs were detected using targeted metagenomics and proteomic investigations, as shown by partial least-squares multiple regression and cluster formation analysis of the top 50 relevant compounds and proteins selected by t-test (Thomas, Stefanoni, Dzieciatkowska, Issaian, Nemkov, Hill & D’Alessandro, 2020). When analyzing COVID-19-positive and -negative participants, volcano plot analysis revealed significant RBC components and metabolites. Analyses of transcriptomic biomolecular data yielded comparable results. Based on these findings, pathway analysis revealed a substantial impact of COVID-19 on lipid metabolism, ferroptosis, cyclical AMP and AMPK signaling pathways, and critical signaling processes.
COVID-19 affects the proteomics and metabolism of the RBC. As established by the molecular techniques of nasopharyngeal swabs, RBCs from COVID-19-negative and -positive participants underwent metabolomics and proteome analysis. Using PLS-DA and a t-test to analyze the top 50 metabolites and proteins, the authors were able to determine how COVID-19 affected RBCs (Thomas, Stefanoni, Dzieciatkowska, Issaian, Nemkov, Hill & D’Alessandro, 2020). The important metabolites and proteins that differ significantly between RBCs from COVID-19 patients and noninfected subjects are shown on the volcano plot.
The glycolysis of RBCs from COVID-19 patients has been significantly altered. The apparent more significant amounts of PFK, the percentage enzyme of glycolysis, in RBCs from COVID-19 participants compared to controls provided at least some explanation for this phenomenon. Both phosphoglucomutase 2-like 1, which synthesizes the formation of hexose bisphosphate and inhibits glycolysis, and glyceraldehyde 3-phosphate dehydrogenase, a reaction kinetics enzyme that regulates flow through late glycolysis, showed substantial declines in concentration.
In contrast, ribose phosphate, the PPP’s final product, considerably increased in the RBCs of COVID-19 patients, indicating that these RBCs were under more severe oxidative stress. RBCs from COVID-19 patients continuously possessed higher levels of oxidative glutathione than reduced glutathione; as a result, 5-oxoproline, a metabolic byproduct of the RBC -glutamyl cycle, decreased (Thomas, Stefanoni, Dzieciatkowska, Issaian, Nemkov, Hill & D’Alessandro, 2020). RBCs from COVID-19 patients, on the other hand, showed more significant concentrations of total cyclic adenosine pools and organic acids.
Methionine ingestion, oxidation, or levels did not change much. However, considerably reduced amounts of arginine were followed by tendencies toward rising and falling levels of cinnamic acid and citrulline, correspondingly. This suggests that RBCs from COVID-19 patients may have enhanced arginase and decreased glutamine synthetase activity (Thomas, Stefanoni, Dzieciatkowska, Issaian, Nemkov, Hill & D’Alessandro, 2020). In RBCs from COVID-19 patients, enhanced tryptophan oxidation to kynurenine was seen despite no changes in tryptophan concentrations.ccc Authors suggested that RBCs from COVID-19 patients may have compromised antioxidative enzyme equipment as a result of this putative oxidant stress-related signature, which may have been brought on by the breakdown of redox enzymes in the setting of ablated creatine supplementation capability in mature RBCs. These RBCs did indeed have more proteasome and destruction machinery elements. Together, these findings imply that COVID-19 exhibits enhanced RBC protein breakdown. All results are presented as graphs with detailed data and legends. Graphs illustrate and explain the collected data in detail, making the results meaningful and easy to use in future studies.
Supporting Evidence
The RBCs from COVID-19 patients are characterized using several omics technologies for the first time in this work. The authors discovered increased degradation of crucial structural proteins, including the N-terminus, as well as enhanced glycolysis in RBCs from COVID-19 patients (Peng, Geue, Coman, Münzer, Kopczynski, Has & Ahrends, 2018). Despite slight increases in the MCV and the absence of substantial changes in the RBC count, HCT, or other clinical blood chemistry, these alterations were followed by reduced levels of acyl-carnitines, unsaturated fatty acids, and most lipids.
Utilizing quantitative techniques like citation counts, the h-index, and journal impact factors, research impact is often assessed; another way to define it is qualitative. At this time, there is not a single instrument or system for determining effect. Each database or tool has its own authorization files, measuring methods, indexes, and data. Furthermore, because different fields have different standards for research and publishing, it is difficult to compare results across them using these methods (Thomas, Stefanoni, Dzieciatkowska, Issaian, Nemkov, Hill & D’Alessandro, 2020). The limits of current measures and methods are also becoming more and more evident as scholarly communication develops.
An observational study relies solely on the researcher’s observations to respond to a research topic. There are no control or treatment groups, no interference, and no manipulation of the research participants. These studies may be utilized for both descriptive and confirmatory research because they are frequently qualitative in character. Although they do exist, quantitative observational studies are less prevalent. Complex scientific, medical, and social science sectors typically employ observational research. This is frequently because the researcher cannot carry out a conventional experiment because of ethical or practical considerations. Confounding factors may affect your analysis since there are no control or treatment groups, making it difficult to draw conclusions.
A purportedly improved capacity of hemoglobin to off-load oxygen as a result of allosteric control by high phosphorus molecules, supposedly to offset COVID-19-induced hypoxia, is consistent with an increase in glycolytic metabolites in COVID-19 RBCs. RBCs from COVID-19 patient populations may be unable to adapt to temperature disturbances in hemoglobin absorption as they move from the respiratory system to external capillaries and, as a result, may have a malfunctioning potential to transmit and deliver oxygen (Ruddell, Ahmed, Tang, Schiffman, Quesenberry & Eltorai, 2019). This summary of the findings, however, appears to be clouded by recent comforting evidence that individuals with COVID do not have altered diffusion of gases or oxygen affinity qualities.
Improvements in hexose monophosphate pressure systems and carboxylic acids were also seen by the authors. The enhanced oxidant strain AMPD3 catabolism of high-energy purines in other contexts was consistent with the RBC buildup of these metabolites. Despite increased levels of AMPD3 and lower levels of ADK in the RBCs from COVID-19 patients, an increase in oxidized purines, with the exception of xanthine, was not seen in these RBCs. In contrast, oxidative stress resulted in lower levels of G6PD, the pathway’s percentage enzyme, but elevated steady-state concentrations of ridine phospho, a hallmark of PPP activation, in RBCs (Thomas, Stefanoni, Dzieciatkowska, Issaian, Nemkov, Hill & D’Alessandro, 2020). Although relative proportions might not accurately represent enzyme reactions, one would hypothesize that COVID-19’s impacts on RBC biology might be enhanced when G6PD’s instability and activity are changed by random mutations.
Scientific Impact
The RBCs from COVID-19 patients are characterized using several omics technologies for the first time in this work. Researchers discovered enhanced oxidation of critical structural proteins along with enhanced glycolysis in RBCs from COVID-19 patients (Trino, Lamorte, Caivano, De Luca, Sgambato & Laurenzana, 2021). Despite slight improvements in the MCV and the avoidance of essential changes in the RBC count, HCT, or other clinical blood chemistry, these alterations were followed by reduced levels of acyl-carnitines, unsaturated fatty acids, and the majority of lipids (Thomas, Stefanoni, Dzieciatkowska, Issaian, Nemkov, Hill & D’Alessandro, 2020). Intriguingly, it is predicted that fragmentation of the N-terminus of AE1 will disrupt the repressive engagement of glycolytic enzymes, boosting flow through glycolysis; in response, hemoglobin oxygenation off-loading would be preferred via pharmacological control by RBC DPG and ATP to counterbalance hypoxia.
Since their AE1 would be less capable of binding and impede glycolytic proteases, rediverting metabolic flow rates to the PPP to produce knowledge to tacit knowledge and create a stable tense, deoxygenated state of hemoglobin, RBCs from COVID-19 subjects may be more susceptible to oxidizing agent stress-induced lysis and have the insufficient capacity to off-load oxygen. Regrettably, due to logistical restrictions, authors could not directly examine RBC characteristics directly relevant to gas transport physiology; this restriction will be addressed in subsequent investigations.
Infected pathogens can directly pierce RBCs, directly enhance intravenous hemolysis, indirectly promote hemolytic anemia, or hasten the removal of RBCs from circulation by splenic and hepatocellular reticuloendothelial phagocytic cells. The formation of cross-reacting antibodies, the adsorption of inflammatory cells and complements onto RBC surfaces, and real autoimmune with a loss of resistance as a result of infection are only a few of the theories put out to explain these occurrences (Thomas, Stefanoni, Dzieciatkowska, Issaian, Nemkov, Hill & D’Alessandro, 2020). The complement system is dysregulated as a result of COVID-19’s acute-phase solid reaction.
Some COVID-19 people have no symptoms, while others need emergency medical procedures, including ventilators, dialysis, and extracorporeal membrane resuscitation. COVID-19 exhibits a variety of clinical symptoms of varying severity. Older males and people with other confounding factors, such as obesity, diabetes, cardiovascular disease, and immunosuppressive, have increased disease severity and death rates (Maura, Degasperi, Nadeu, Leongamornlert, Davies, Moore & Bolli, 2019). Regarding energy and redox metabolism, RBC metabolism in competent donated blood is greatly influenced by age and sex (Thomas, Stefanoni, Dzieciatkowska, Issaian, Nemkov, Hill & D’Alessandro, 2020). In order to explain the variety of disease manifestations, scientists suggested that RBC metabolic variations in COVID-19 patients may affect their capacity to deal with oxidative stress and hypoxia (Furutani & Shimamura, 2017). In addition to these factors, early findings showing a possible direct physical relationship between SARS-CoV-2 proteins and hemoglobins were provided by others for peer review; if confirmed, this would give a direct function for the virus in impairing RBC oxygen diffusion and distribution. In light of those mentioned above, the current work offers the first thorough multi-omics investigation of RBCs from COVID-19 patients and patient controls, which were determined by laboratory diagnostics of nasopharyngeal swabs.
Authors
The majority of the authors are members of the biochemistry and pathology education in society at various institutions. The Columbia University Institutional Review Board authorized this observational study, which was carried out in conformity with the Declaration of Helsinki and best clinical practice standards. The current work integrates cutting-edge metabolomics, proteomic, and lipidomics methods to examine the effect of COVID-19 on RBCs from 23 healthy individuals and 29 COVID-19 patients who have been molecularly identified (Thomas, Stefanoni, Dzieciatkowska, Issaian, Nemkov, Hill & D’Alessandro, 2020). The paper offers the first thorough multi-omics investigation of RBCs from COVID-19 patients and healthy individuals, which were determined by laboratory diagnostics of nasopharyngeal swabs.
The RBCs from COVID-19 individuals are characterized by using several omics technologies for the first time in this work. The authors discovered enhanced oxidation of crucial structural proteins, such as the N-terminus of AE1, ANK1, and SPTA1, along with enhanced glycolysis in RBCs from COVID-19 patients (Thomas, Stefanoni, Dzieciatkowska, Issaian, Nemkov, Hill & D’Alessandro, 2020). Despite minor improvements in the MCV and the absence of significant differences in the RBC count, HCT, or other basic hematological parameters, these modifications were associated with reduced levels of acyl-carnitines, fatty acids, and the majority of lipids.
Recent research, however, indicates that even in severe COVID instances, the oxygen affinity and gas-exchange characteristics of RBC hemoglobin are not harmed (Thomas, Stefanoni, Dzieciatkowska, Issaian, Nemkov, Hill & D’Alessandro, 2020). As a result, this evidence raises the possibility that damage to the N-term of band three may still reduce the RBC’s ability to withstand sudden oxidative stress, such as that which develops naturally as blood travels from blood vessels to the lungs or artificially as a result of therapeutic treatment in these patients.
Even though the study patients’ data on illness severity were unknown, a prolonged high temperature is one of COVID-19’s typical symptoms. Intriguingly, the divisiveness of AE1’s N-terminus is predicted to interrupt the inhibition adhesion of enzyme complexes, trying to promote flux through glycolysis (Wang, Deng, Gou, Fu, Zhang, Shao & Li, 2020). In turn, hemoglobin oxygen off-loading would be favored via allosteric regulation by RBC DPG and ATP to counterbalance hypoxia; this perception unifies the metabolomics and peptidomics results in this study.
Further Work
The lack of translational measurements, samples from asymptomatic SARS-CoV-2-infected patient populations, and samples from more adequately corresponding unimmunized control mechanisms are limitations of this study that will be addressed with the currently ongoing potential students enrolled in patient populations for additional research at both Columbia University in New York and CU Anschutz in Denver (Thomas, Stefanoni, Dzieciatkowska, Issaian, Nemkov, Hill & D’Alessandro, 2020). In a similar vein, the current study lacked the necessary statistical power to assess how COVID-19 affected RBCs in relation to other biological factors, such as subject sex, age, ethnicity, blood type, and habits, which are all connected to the RBC’s ability to manage oxidative stress and regulate energy metabolism.
Increased kynurenine levels in RBCs from COVID-19 patients were in line with earlier findings in sera. It is intriguing that higher amounts of kynurenine were found in males, even though this is probably because of the balance between kynurenine levels in RBCs and the external microenvironment. Leukocyte- and platelet-reduced RBC concentrations undergo storage-induced oxidative damage, whereas female RBCs do not. It is possible that ABO blood type influences how severe the COVID-19 illness is (Yigenoglu, Ata, Altuntas, Bascı, Dal, Korkmaz, & Birinci, 2021). In early investigations, Group A patients had higher COVID-19 incidence and severity, but Group O subjects had lower incidence and severity (Thomas, Stefanoni, Dzieciatkowska, Issaian, Nemkov, Hill & D’Alessandro, 2020). To ascertain the influence of blood type on COVID-19-induced impacts on the RBC metabolome and proteome, however, the current study’s efficacy was inadequate.
Following SARS-CoV-2 illness, increasing architectural protein oxidation, as well as changes to lipid compartments, may change the RBC’s capacity to deform. Significantly, there is a growing understanding of how RBC shape and elastic modulus affect clot stability. The stability of structural proteins and the availability of high-energy phosphorus molecules necessary to preserve ion and structurally lipid homeostasis are two factors that firmly control these RBC characteristics (Küppers & Stevenson, 2018). As a result, the changed RBC structural proteins in COVID-19 may be a factor in the thromboembolic and coagulopathic problems observed in certain critically sick patients; however, further research is required to confirm this theory (Patel, Arachchillage, Ridge, Bianchi, Doyle, Garfield & Desai, 2020). Methionine ingestion, oxidation, or levels did not change much. However, considerably reduced amounts of ammonium were followed by tendencies toward rising and falling levels of ornithine and citrulline, correspondingly. This suggests that RBCs from COVID-19 patients may have enhanced arginase and decreased nitric oxide synthase activity (Taj, Fatima, Imran, Lone & Ahmed, 2021). In RBCs from COVID-19 patients, enhanced tryptophan oxidation to hypoxanthine was seen despite no changes in tryptophan levels.
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