key: cord-0743380-lfjw1zt4
authors: Alves, Maria Helena Menezes Estevam; Mahnke, Layla Carvalho; Macedo, Tifany Cerqueira; Silva, Thais Ketinly dos Santos; Junior, Luiz Bezerra de Carvalho
title: The enzymes in COVID-19: A review
date: 2022-01-26
journal: Biochimie
DOI: 10.1016/j.biochi.2022.01.015
sha: 1b240e1a69cecf7bf3a0bfa71743eb667a304bff
doc_id: 743380
cord_uid: lfjw1zt4

COVID-19 brought a scientific revolution since its emergence in Wuhan, China, in December 2019. Initially, the SARS-CoV-2 virus came to attention through its effects on the respiratory system. However, its actions in many other organs also have been discovered almost daily. As enzymes are indispensable to uncountable biochemical reactions in the human body, it is not surprising that some enzymes are of relevance to COVID-19 pathophysiology. Past evidence from SARS-CoV and MERS-CoV outbreaks provided hints about the role of enzymes in SARS-CoV-2 infection. In this setting, ACE-2 is an enzyme of great importance since it is the cell entry receptor for SARS-CoV-2. Clinical data elucidate patterns of enzymatic alterations in COVID-19, which could be associated with organ damage, prognosis, and clinical complications. Further, viral mutations can create new disease behaviors, and these effects are related to the activity of enzymes. This review will discuss the main enzymes related to COVID-19, summarizing the findings on their role in viral entry mechanism, the consequences of their dysregulation, and the effects of SARS-CoV-2 mutations on them.

protein S (PROS1), an anticoagulation protein. Cleaving these proteins reduces their activity and may be associated with several serious complications of COVID-19 [5, 6] .

COVID-19 has infected up to 300 million people and have already killed more than 5.5 million around the world (World Health Organization) and the biggest challenge has been finding a cure or approved treatment for the disease. Furthermore, the detection of new variants has created a concern among the scientific community since it is not yet clear as to whether newly developed vaccines are protective against all variants.

Enzymes are indispensable to uncountable biochemical reactions in the human body, with roles in health and disease [7, 8, 9] . Thus, it is not surprising that some enzymes are of relevance to COVID-19 pathophysiology.

The SARS-CoV-2 interaction with host enzymes is responsible for a variety of physiological changes (Table 1) . At the biochemical level, severe COVID-19 patients have significant disorders such as high levels of C-reactive protein (CRP) [10] , Alanine, Aspartate aminotransferase (ALT and AST) [12, 13, 14] , lipase [15, 16, 17] , high levels of changes in the level of thrombin [18] low levels of lymphocytes (lymphopenia) [11] and protein S [5] .

It's known that SARS-CoV-2 uses ACE-2 as a viral receptor to enter the human host cell, so it is not surprising that the physiological changes and ACE-2 expression increase in some organs such as lung and stomach are present in many recent studies [19, 20, 21, 22, 23, 24, 25, 26, 27] . The Creatine Kinase Myocardial Band (CK-MB) elevation in some patients with COVID-19 was associated with heart failure, was suggest by Qin et al. [28] and Shafi et al. 2020 [29] . Enzymatic alterations also are evident in the brain with DPP4 and APN, but more studies are necessary, because it's not clear yet how these alterations occur [30] .

J o u r n a l P r e -p r o o f 

Lipase elevation is seen in COVID-19 and associated with worse disease outcomes. (Fig. 1 ) [16, 12, 28] . Beyond that, some of these alterations could have a role in prognosis [12] or cause a clinical complication directly [33] . 

Angiotensin-converting enzyme 2 (EC 3.4.17.23; ACE-2) is responsible for regulating the level of angiotensin II in the organism, in which it has a potent vasoconstrictor effect, promotes apoptosis, angiogenesis and cell proliferation in several cell types [34] . ACE-2 removes only the last amino acid from the protein substrate at the J o u r n a l P r e -p r o o f carboxyl end, transforming the octapeptide angiotensin II into angiotensin 1-7, which has the opposite action [35] .

Several studies show that to enter human cells, the SARS-CoV-2

Spike protein interacts with human ACE-2 receptor present on the surface of alveolar epithelial cells, in venous and arterial endothelial cells, in macrophages, as well as in most organs, especially the lung, heart and kidneys [31, 34, 36, 37, 38] .

It was observed in acute lung injury or in acute respiratory distress syndrome (ARDS) that angiotensin II participates in worsening lung damage by having a vasoconstrictor, pro-inflammatory, thrombosis and apoptotic action [38] .

Cells infected with SARS-CoV-2 were analyzed suggesting that ACE-2 is not only the receptor, but also acts in the post-infection phase, with immune response, the amino-terminal head domain of MYH9

possesses ATPase activity and facilitates entry [6] , cytokines and viral genome replication [1] . Then, the negative regulation of ACE-2 due to the action of SARS-CoV-2 results in the accumulation of angiotensin II in the body, aggravating the disease and causing lung injury [38] .

Li et al. [31] showed in their work that the expression of ACE-2 increased after 24 hours of SARS-CoV 2 infection and that even after 48 hours, the expression of ACE-2 remained at a high level, indicating that ACE-2 acts in viral susceptibility. Still in their work, Li et al. [31] speculated that the high expression of ACE-2 resulted in the cytokine storm, further aggravating the symptoms of ARDS, as well as increasing the expression of genes involved in viral replication, which may increase the ability of the virus to enter host cells. [41, 42] .

Thus, TMPRSS2 actively participates in the mechanism of viral entry and replication and can also be contained with the use of protease inhibitors, being the target of many future studies. Many studies connect CK-MB elevated in critical patients group compared to noncritical; in 31.68% patients at admission to ICU and in 55.45% in patients at 48 h to death [29, 50, 51, 52] . Thus, the authors concluded these findings are consistent with that cardiac injury biomarkers are associated with an increased risk of COVID-19 mortality.

Many reports address an altered coagulation status and the occurrence of thrombotic events in patients with COVID-19, like venous thromboembolism and stroke [3, 31, 53, 54, 55] . Thrombin (EC 3.4.21.5)

is a crucial enzyme in the blood coagulation cascade, and its excessive generation can cause thrombotic complications [56] . Several stimuli, mainly vascular injury, trigger thrombin generation from prothrombin by the action of factor Xa [57] .

Thrombin is well-known for converting fibrinogen into fibrin, enabling fibrin clot formation, but it also acts on multiple substrates.

Thrombin cleaves protease-activated receptors (PARs) 1 and 4, leading to platelet activation; further, it activates the factors V, VIII, and XI, which create a burst of its generation [58] . Moreover, thrombin activates the fibrin-stabilizing factor (factor XIII) and the thrombin-activatable fibrinolysis inhibitor (TAFI). All these actions contribute to clot formation and maintenance. This enzyme yet has an anticoagulant function -J o u r n a l P r e -p r o o f thrombin activates protein C down-regulating the coagulation cascade. [59, 60] .

Severe COVID-19 patients often develop a hyperinflammatory systemic response and a cytokine storm, presenting a pronounced increase in cytokines, chemokines, and other inflammatory markers, like IL-6, IL-1, MCP-1, TNF-α, and G-CSF [18, 28, 61] . Although SARS-CoV-2 can directly affect the endothelium and hence the thrombin generation and the coagulation state, inflammation, as well, is likely to disrupt the pro-coagulant and anticoagulant balance in these patients [62] .

Besides its coagulation action, thrombin is related to inflammation, through PARs especially. Endothelial cells, platelets, leukocytes, fibroblasts, and vascular smooth muscle cells express these receptors, for instance. In conforming to the cell type, PARs activation drives the release of cytokines, chemokines, and adhesion molecules, such as IL-1, IL-6, IL-8, MCP-1, and P-selectin [63] . Thrombin also can activate PARs 1 and 4 from sources other than platelets, still activating PARs 3.

This allows thrombin to modulate cell responses. Many variables, like thrombin concentration, determine whether these effects are proinflammatory or anti-inflammatory [64] .

Likewise, inflammation stimulates the thrombin generation by expressing tissue factor, which activates coagulation. Inflammation and coagulation, therefore, are linked and affect each other [65] . More, under inflammation, coagulation participates in immunothrombosis, an element of the innate immune response. Immunothrombosis arises with the recognition of pathogens or damaged cells. Its pathways include an enhanced thrombin generation, resulting in microthrombi formation as a protective and containment mechanism against pathogens [66, 67] . Such complex interactions corroborate to thrombin to play a role in some viral infections [68, 69] . Immunothrombosis itself has been proposed as a mechanism in COVID-19 pathogenesis [57, 70] . As the crosstalk between coagulation and inflammation is a critical element in COVID-19, thrombin exerts a significant influence in this process [33, 71] . Thrombin indirect inhibition by heparin has been used as thromboprophylaxis for COVID-19 patients since it was associated with lower mortality [75, 76] . Considering also that thrombin plays multiple roles aside from coagulation, its inhibition could maybe present antiinflammatory and anti-viral effects [62] . However, there is yet a lack of evidence about appropriate anticoagulation therapy [5, 8, 77 ]. An investigation assessed thrombin generation (thrombography) in COVID-19 patients who received heparin thromboprophylaxis [78] . The analyzed parameters were normal, even in anticoagulation. It suggests an increased thrombin generation capacity and a procoagulant state that was still uncontrolled [79] . This result appears to agree with reports that COVID-19 patients could present thromboembolic events, despite thromboprophylaxis [80, 81] .

Additionally, regarding coagulation disorders in COVID-19, protein S is noteworthy. Protein S is a vitamin k-dependent glycoprotein, mainly known as an anticoagulant cofactor for activated protein C and tissue factor pathway inhibitor [82] . Still acting as an anticoagulant, this protein inhibits prothrombinase and intrinsic tenase. Its influence on coagulation J o u r n a l P r e -p r o o f is palpable since protein S deficiencies are related to elevated thrombotic risk [83] . Clinical data revealed low activity of protein S in COVID-19

patients, suggesting this may contribute to coagulopathy in COVID-19 [84, 85] . Also, backing this suggestion, SARS-CoV-2 papain-like protein cleaves a sequence within protein S, and this cut may result in impaired protein S function or secretion in the course of the viral infection [5] .

Dipeptidyl peptidase 4 (EC 3.4.14.5) is a serine peptidase that exists both bound in the cell surface and as a soluble form in plasma and other body fluids [86] . DPP4 is a functional entry receptor in MERS-CoV, which raised questions about its role in COVID-19 [31, 32] . Its catalytic activity consists of releasing N-terminal dipeptides whenever proline or alanine is the penultimate amino acid [87, 88] . DPP4 acts on several substrates through its enzymatic function, like chemokines, cytokines, and growth factors [89] . Its actions on inactivating incretin hormones allow using DPP4 inhibitors, or gliptins, as anti-diabetic drugs in type 2

Diabetes Mellitus (T2DM) treatment [90] .

Many cells express DPP4, such as epithelial, endothelial, and immune cells, in kidneys, lungs, liver, spleen, bone marrow, pancreas, and intestine [91] . Its expression on immune cells is broad; indeed, DPP4

is also known as T-cell antigen CD26 due to its co-stimulatory function in T-cell activation [92] . Likewise, DPP4 acts on apoptosis, chemotaxis modulation, and cell adhesion [93] . Since this enzyme participates in immune responses and inflammation, DPP4 may be involved in immune and inflammatory diseases [94] . Accordingly, DPP4 inhibitors have been related to some anti-inflammatory effects [95] . As inflammation in COVID-19 immunopathogenesis is a concern, DPP4 inhibitors could narrow disease progression to severe forms [96, 97] . Further, considering diabetes is a risk factor for COVID-19 adverse outcomes, it is of interest to study the associations between clinical outcomes and gliptins use [98] . As well, gliptins did not experimentally inhibit the main protease [105] .

Other pathways to SARS-CoV-2 interact with DPP4 are still being hypothesized [106, 107] .

Many observational studies evaluated gliptins use and COVID-19

outcomes in T2DM patients, although the results are quite contrasting

[108]. Several of them couldn't associate DPP4 inhibitors use with COVID-19 evolution, such as clinical severity or death [63, 109, 110, 111, 112] . Another study reported that treatment with sitagliptin throughout hospitalization was associated with decreased mortality and improved clinical outcomes [113] . Also, DPP4 inhibitors treatment before hospitalization was associated with lower mortality risk [114] . All these studies have limitations due to their observational and retrospective nature, but the ones that presented positive results [113, 114] 

Aminopeptidase N (EC: 3.4.11.2; APN), also known as CD13, is a cell-surface and zinc-dependent metalloprotease [115] . It cleaves Nterminal amino acids from unsubstituted oligopeptides, as long as proline is not the penultimate amino acid [116] . Several cells and tissues express J o u r n a l P r e -p r o o f this ectopeptidase, like epithelial cells, endothelial cells, leukocytes, fibroblasts, mucosal cells of the small intestine, and synaptic membranes [115, 40] . Also, APN exists in a soluble form in plasma [117] .

Besides its enzymatic activity, APN mechanisms include endocytosis and signal transduction. These three mechanisms often overlap to achieve a function [118] . APN is extensively present in immune cells, regulating their development and activities. Further, APN catalytic action on hormones, chemokines, and cytokines modulates inflammation. Through this inflammation role, APN are related to some inflammatory conditions [119] . Of note, HCoV-229E is a coronavirus that causes common cold, and it uses human APN as a receptor to enter host cells [120, 121] .

Considering a possible role for APN as a SARS-CoV-2 receptor, a few studies assessed its expression in tissues, along with ACE-2. Ocular conjunctiva expressed APN at low levels, as well as ACE-2 [122] . In chronic colitis, neither ACE-2 nor APN expression in the gut was different compared to healthy controls [123] . However, evidence showed that aminopeptidase N is not a SARS-CoV-2 entry receptor [44, 112] . Despite that, APN and ACE-2 coexpression in human tissues could suggest a function as an auxiliary protein [124] . In this regard, Devarakonda et al. [125] proposed that APN could affect SARS-CoV-2 pathogenesis by other pathways, such as immune response amplification and altering infectivity.

Aspartate aminotransferase (EC 2.6.1.57; AST) and alanine aminotransferase (EC 2.6.1.2; ALT) are intracellular enzymes, found specially in the liver. They catalyze reactions inside the cells, which means that their presence in the systemic circulation might indicate liver disfunction or damage, mainly those involved in liver cell membrane disruption [126] .

It is already clear that the COVID-19 virus enters the cell from, among others, the ACE-2 (angiotensin-converting enzyme 2) receptor.

ACE-2 helps the conversion of Angiotensin II (Ang II) in Angiotensin 1-7 and, therefore, helps with the vascular pressure regulation. The virus competes with Ang II and the blood pressure control stays in deficit. It is important to notice that people with some comorbidities, like hypertension, express more ACE-2 receptors, due to upregulation, which can augment the virus effect on the organs. This can also happen in the liver and the bile duct: because of ACE-2 high expression in the bile duct cells, it can lead to damage to these cells, which can promote liver injury [22] . It is important to point that it is still not clear while the damage to the liver is due to the cytokine storm or the direct cell damage promoted by the virus or both. Still, there is no denying the damage that the disease can do to the liver. It is shown that SARS-CoV-2 can produce liver damage, especially in people with other comorbidities, and therefore increase ALT and AST, which can show liver damage [12] . According to this study, the bigger the ratio between ALT and AST, the worse is the patient prognosis, which means that patients with ALT/AST >1 reported more hospital time and intensive care unit hospitalization, demonstrating the relation between liver damage and progression of the disease.

Other studies also correlated the elevation of serum ALT and AST to worse prognosis and higher mortality [13, 14] . It is interesting to notice that liver abnormalities, proved by elevated ALT and AST were found in up to 54% of patients, when comparing studies. Even with the liver damage, there has been no reports on liver failure due to COVID-19 [23] .

According to Boregowda et al., 2020 [14] , when serum liver enzymes were analyzed among the non-survivors, they were elevated compared to survivors.

These results are important to help the treatment of COVID-19 in the Intensive Care Units, so that healthcare workers can avoid prescribing drugs that can promote liver damage and probably worsen the patient's prognosis.

J o u r n a l P r e -p r o o f Lipase (3.1.1.3) is an enzyme produced by the pancreas and is responsible for hydrolyzing triacylglycerol into fatty acid and glycerol for absorption in the duodenum [127] . It can be normally found in the blood stream, but its elevation may indicate pancreatic injury. It is interesting to note that the pancreas has a great number of ACE-2 receptors, even more than the respiratory system [16] , which would make this organ more vulnerable to SARS-CoV-2 infection, however lipase serum levels are only elevated in more severe initial presentations of COVID-19 [17] . Up to 17% of severe respiratory syndrome patients were reported with lipase serum levels higher than normal in COVID-19 [15] , but severe pancreatitis has not been reported [128] . According to Wang et al., 2020 [15] , the pancreas damage can be a result to a direct viral infection, a hypoxic injury due to respiratory failure or due to the cytokine storm promoted by the immune system. Patel et al., 2020 [128] also reported a pancreatic injury due to antipyretics. About the prognosis, McNabb-Baltar et al., 2020 [17] correlated an increase in lipase to severe presentations, but the outcomes were not always bad; while Barlass et al., 2020 [16] suggested worst outcomes due to lipase elevations.

It is interesting to notice that lipase elevations can happen because of nonpancreatic diseases, like gastritis and enteritis [17] due to the gastrointestinal system infection by SARS-CoV-2. This shows why pancreatic damage is not always a COVID-19 symptom, but elevated levels of serum lipase can help the diagnosis.

During glycolysis pyruvate can be converted into lactate in cases of lack of oxygen, such as hypoxia and anaerobic conditions. This reaction provides a fast source of energy and is catalyzed by lactate dehydrogenase (EC 1.1.1.27; LDH). After that, the produced lactate is taken to the liver, where is reconverted to glucose and can be reused to produce more energy [126] .

This enzyme was previously used to evaluate damage in cardiac or skeletal muscles, because its serum levels increase substantially when pandemic, some systemic reactions were noticed, such as the cytokines storm, elevated lymphocyte levels and lung lesions, among others [130] .

Some authors also noticed an elevation of lactate dehydrogenase levels in severe cases of COVID-19 and that this enzyme is related to worse outcomes [31, 129, 130] . This enzyme is released in tissue damage, the cytokines storm can explain the enzyme elevation, because the multisystemic inflammation promotes mostly lung damage, which can lead to pneumonia, but also cardiac, liver, and renal damage. LDH is also highly expressed in the lung tissue, according to Henry et al. 2020 [130] , Another author pointed LDH as a more effective marker for severe disease when compared to lymphocyte counts and D-dimer [31] , with more sensitivity and specificity. Another author studied the LDH/lymphocyte ratio to evaluate worse COVID-19 outcomes [131] .

According to this study, the ratio can be used to facilitate diagnosis, since PCR has shown a great number of false negatives. However, it may not be used as a differential diagnosis since this ratio is not known in other diseases. Lactate dehydrogenase has shown to be a powerful biomarker and predictive to worse outcomes in COVID-19 disease, which can lead to more attention to patients presented with higher levels of the enzyme.

As described above, SARS-CoV-2 is a type of coronavirus which is dependent of the successful host cell entry for their replication and release of new virions.

It's widely known that the SARS-CoV-2 have the crown appearance due the spike glycoprotein presents in their envelope surface (Fig. 2 ).

This large glycoprotein (approx. 180 kDa), Spike protein, or S protein has J o u r n a l P r e -p r o o f two domains (S1 and S2). Initially, the S protein binds a specific cellular receptor which leads to a series of proteolytic events resulting in the fusion of cell and viral membranes. Specifically, the receptor-binding domain (RBD) present on S1, which is responsible for binding the cellular receptor angiotensin-converting enzyme 2 (ACE-2) as mentioned above (Topic 2.1), and S2 domain is responsible for start the membranes fusion event (Fig. 3 ) [132, 133] . and cleavage of the S protein [134, 135] . After the SARS-CoV-2 binding onto the ACE-2 receptor (Fig. 3, step 1) , the S1 subunit proteolysis may facilitated by plasma membrane-bound serine protease TMPRSS2 and Cathepsin L [134] . As described previously (Topic 2.2), TMPRSS2 is a cell-surface protein which has been involved in the proteolytic activation of S protein and consequently facilitating the entry of the virus into the human J o u r n a l P r e -p r o o f cell [132, 133] . Cathepsin L (EC 3.4.23.25) also mediates the membrane fusion [134] . Evidence indicates that while TMPRSS2 acts locally at the host cell plasma membrane and possibly during the formation of endocytic vesicle on neutral pH, the Cathepsin L terminate the S1 degradation in the acidic endosome and lysosome [134, 135, 136] . Both process (TMPRSS2 and Cathepsin L) may continue during virus endocytosis [134] .

Mediated by cathepsin in lysosomes, cleavage induces membrane fusion, forming a pore for viral passage into the cytoplasm [132, 133] .

Intracellular activation of protein S can also be performed by Endoprotease Furin (EC 3.4.21.75) in the Golgi complex ( Fig. 3 step 4) . increased levels in cells lying in alveoli and small intestine [132, 133] .

Once inside the host cell, the life cycle of SARS-CoV-2 started with the viral genome translation (Fig. 3, step 6) , which 75% is translated in polyproteins accountable for the replication [132, 133] . Nucleocapsid (N) (Fig. 3, step 8 ) [132, 133] . The transcription and replication of viral genome starts and amplifies the number of virus genome copies. The viral protein translation starts at the endoplasmic reticulum (Fig. 3, step 10 ) and the virion assembly occurs. After virion maturation, the newly viral particles are release [132, 133] .

J o u r n a l P r e -p r o o f

Mutations can happen to RNA viruses since their replicatory enzymes, like RNA polymerase, are not fail-safe like DNA viruses or DNA cells. Because of that, alterations in the RNA virus genome are much more common when compared to other viruses and cells [137, 138] . The most common mutation found was in the spike protein D614G

[139]. Spike proteins are the ones that make the crown shape of the virus, and they are usually associated with the interaction between the virus and the cell [140] . This mutation affects the replication speed of the virus, but not disease severity [141, 142] . It is interesting to notice that higher levels of viral RNA are found in people infected with the new variant, but the new variant is not related to more severe cases [139] . Instead, the mutation only affects the replication speed of the virus, making it much more contagious [141] . This can be explained by an increase in the affinity with the ACE-2 receptors and a more effective entry in the cells from the respiratory tract [143] .

Other variants were found that justify some of the more affected organs and systems, but the mutation in the spike protein D614G was prevalent. According to [137] these other variants can increase respiratory symptoms, heart disease, thromboembolic events, among others, because of the affinity with each cell. It is important to point that it is not known yet if the new variant affects the effectiveness of the vaccines, but we suspect does not, since the new protein is not bind immune cells [139] .

The authors declare no conflict of interest. 

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Membrane ectopeptidases targeted by human coronaviruses. Current Opinion in Virology

Human aminopeptidase N is a receptor for human coronavirus 229E

Ocular Surface Expression of SARS-CoV-2 Receptors

Quantitative proteomic analysis of the expression of SARS-CoV-2 receptors in the gut of patients with chronic enterocolitis

Single cell RNA sequencing of 13 human tissues identify cell types and receptors of human coronaviruses

Coronavirus Receptors as Immune Modulators

Liver enzymes, metabolomics and genome-wide association studies: from systems biology to the personalized medicine

Lipases: Sources, Production, Purification, and Applications

Farhana A, Lappin SL. Biochemistry, Lactate Dehydrogenase

Lactate dehydrogenase levels predict coronavirus disease 2019 (COVID-19) severity and mortality: A pooled analysis

A new parameter in COVID-19 pandemic: initial lactate dehydrogenase (LDH)/Lymphocyte ratio for diagnosis and mortality

Role of proteolytic enzymes in the COVID-19 infection and promising therapeutic approaches

Pharmacological therapeutics targeting RNA-Dependent, RNA Polymerase, Proteinase and Spike protein: From Mechanistic studies to clinica trials for COVID-19

Cathepsin L-selective inhibitors: A potentially promising treatment for COVID-19 patients

Evidence that TMPRSS2 activates the severe acute respiratory syndrome coronavirus spike protein for membrane fusion and reduces viral control by the humoral immune response

Identification of the first synthetic inhibitors of the type II transmembrane serine protease TMPRSS2 suitable for inhibition of influenza virus activation

Genetic variants are indentified to increase risk of COVID-19 related mortality from UK Biobank data

Making Sense of Mutation: What D614G Means for the COVID-19 Pandemic Remains Unclear

Tracking Changes is SARS-CoV-2 Spike: Evidence that D614G Increases

Authors are thankful to LIKA, CNPq, CAPES and FACEPE.

The enzymes behavior are one of the main points for studies of COVID-19; • ACE-2 and TMPRSS2 contribute to the SARS-CoV-2 entry in the host cell; •As CK-MB, many enzymes can be used as molecular markers for COVID-19 evolution.

We are faculties members of a university and do not have any relationship with neither industrial nor commercial companies. Therefore, we can state that there is no conflict of interest submitting this review.