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Citation: Soto, M.E.;
Guarner-Lans, V.; Díaz-Díaz, E.;
Manzano-Pech, L.;
Palacios-Chavarría, A.;
Valdez-Vázquez, R.R.;
Aisa-Álvarez, A.;
Saucedo-Orozco, H.; Pérez-Torres, I.
Hyperglycemia and Loss of Redox
Homeostasis in COVID-19 Patients.
Cells 2022,11, 932. https://doi.org/
10.3390/cells11060932
Academic Editor: Zhixiang Wang
Received: 10 February 2022
Accepted: 7 March 2022
Published: 9 March 2022
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4.0/).
cells
Article
Hyperglycemia and Loss of Redox Homeostasis in
COVID-19 Patients
María Elena Soto 1, †, Verónica Guarner-Lans 2,† , Eulises Díaz-Díaz 3, Linaloe Manzano-Pech 4,
Adrían Palacios-Chavarría5, Rafael Ricardo Valdez-Vázquez 5, Alfredo Aisa-Álvarez 1,
Huitzilihuitl Saucedo-Orozco 1and Israel Pérez-Torres 4,*
1
Department of Immunology, Instituto Nacional de Cardiología Ignacio Chávez, Juan Badiano 1, Sección XVI,
Tlalpan, Mexico City 14080, Mexico; elena.soto@cardiologia.org.mx (M.E.S.);
alfredoaisaa@gmail.com (A.A.-Á.); huitzilihuitls@hotmail.com (H.S.-O.)
2Department of Physiology, Instituto Nacional de Cardiología Ignacio Chávez, Juan Badiano 1, Sección XVI,
Tlalpan, Mexico City 14080, Mexico; veronica.guarner@cardiologia.org.mx
3Department of Reproductive Biology, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán,
Vasco de Quiroga 15, Sección XVI, Tlalpan, Mexico City 14000, Mexico; eulisesd@yahoo.com
4Department of Cardiovascular Biomedicine, Instituto Nacional de Cardiología Ignacio Chávez,
Mexico City 14080, Mexico; loe_mana@hotmail.com
5Critical Care Unit of the Temporal COVID-19 Unit, Citibanamex Center, Mexico City 11200, Mexico;
a2novi@hotmail.com (A.P.-C.); rrvaldezvazquez@gmail.com (R.R.V.-V.)
*Correspondence: israel.perez@cardiologia.org.mx; Tel.: +52-5573-2911 (ext. 25203); Fax: +52-5573-0926
† These authors contributed equally to this work.
Abstract:
The infection with SARS-CoV-2 impairs the glucose–insulin axis and this contributes to
oxidative (OS) and nitrosative (NSS) stress. Here, we evaluated changes in glucose metabolism that
could promote the loss of redox homeostasis in COVID-19 patients. This was comparative cohort
and analytical study that compared COVID-19 patients and healthy subjects. The study population
consisted of 61 COVID-19 patients with and without comorbidities and 25 healthy subjects (HS).
In all subjects the plasma glucose, insulin, 8-isoprostane, Vitamin D, H
2
S and 3-nitrotyrosine were
determined by ELISA. The nitrites (NO
2−
), lipid-peroxidation (LPO), total-antioxidant-capacity
(TAC), thiols, glutathione (GSH) and selenium (Se) were determined by spectrophotometry. The
glucose, insulin and HOMA-IR (p< 0.001), 8-isoprostanes, 3-nitrotyrosine (p< 0.001) and LPO were
increased (p= 0.02) while Vitamin D (p= 0.01), H
2
S, thiols, TAC, GSH and Se (p< 0.001) decreased
in COVID-19 patients in comparison to HS. The SARS-CoV-2 infection resulted in alterations in the
glucose–insulin axis that led to hyperglycemia, hyperinsulinemia and IR in patients with and without
comorbidities. These alterations increase OS and NSS reflected in increases or decreases in some
oxidative markers in plasma with major impact or fatal consequences in patients that course with
metabolic syndrome. Moreover, subjects without comorbidities could have long-term alterations in
the redox homeostasis after infection.
Keywords:
SARS-CoV-2; homeostasis redox; hyperglycemia; lipid peroxidation; selenium; thiols;
nitrotyrosine; H2S
1. Introduction
Coronavirus disease 2019 (COVID-19) is caused by the type 2
β
-coronavirus and it
induces a severe acute respiratory syndrome (SARS-CoV-2). This disease causes multiple
organ failures that results from an exacerbated cytokine storm by the immune system.
The cytokine storm is initially aimed at bringing down the infection and may have as a
consequence, a possible fatal outcome for the patient [1].
COVID-19 impairs glucose homeostasis and metabolism in non- and diabetes mellitus
(DM) and metabolic syndrome (MS) patients due to the cytokine storm, inflammation,
angiotensin II-converting enzyme (ACE2) down-regulation and the direct injury to the
Cells 2022,11, 932. https://doi.org/10.3390/cells11060932 https://www.mdpi.com/journal/cells
Cells 2022,11, 932 2 of 23
pancreatic
β
-cells [
2
]. It also deteriorates the already impaired glucose homeostasis in
patients with DM and in patients with other comorbidities such as obesity, insulin resistance
(IR), hyperinsulinemia and hypertension in whom alterations in insulin secretion are
already present and contribute to hyperglycemia [
3
]. Furthermore, increased inflammation
and massive production of cytokines generate IR which adds to the deterioration of the
insulin secretion by β-cells [4].
In addition, high glucose levels also contribute to the virulence and replication of
SARS-CoV-2 since it is associated with a decrease in the number of phagocytic and nuclear
polymorphic leukocytes [
4
]. Moreover, the virus sequesters the host’s mitochondrial
function and shifts it from aerobic to anaerobic [
5
]. In this state, the pyruvate produced
from glucose during glycolysis is oxidized to lactate, thereby increasing glucose levels in
the cytosol and leading to the generation of limited amounts of adenosine-5-triphosphate
(ATP) [
5
,
6
]. The viral replication also consumes large amounts of ATP and therefore, its
concentration is depleted. In this condition, lactate is not metabolized by gluconeogenesis
and accumulates in the blood, leading to a loss of the balance in glucose metabolism [
7
].
The increase in hyperglycemia contributes a high production of inflammatory cytokines
and cellular mediators implied in pro-thrombotic process present in COVID-19 patients. [
8
].
All these alterations favor oxidative stress (OS) but also, the viral infection induces the
cytokine storm by the immune system that contribute to OS [
9
]. This alteration leads to
positive feedback systems for each element and/or metabolic pathway and may contribute
to severe pneumonia with possible multiple organ failure (MOF) in the COVID-19 patient.
On the other hand, an overproduction of reactive oxygen species (ROS) leads to a
deprivation of the antioxidant mechanisms which are crucial to decrease or abolish viral
replication and the level of infection present in the patient [
10
]. Therefore, SARS-CoV-2
favors an increased threshold in the production of ROS and reactive nitrogen species (RNS).
The overproduction of these molecules is associated with elevated activity and/or ex-
pression of the inducible nitric oxide synthase (iNOS), nicotinamide-adenine dinucleotide
phosphate (NADP) oxidases, cyclooxygenase 2, xanthine oxidase and with alterations in
the mitochondrial functions that activate transcription factors such as nuclear factor kappa
B subunit (NFkB) resulting in an exacerbated proinflammatory state and the production
of interleukins [
5
,
11
]. Therefore, Interleukin (IL)-6, IL-8 and tumor necrosis factor alpha
(TNF-
α
) are increased in bronchial epithelial cells and alveolar macrophages and these
cytokines can then activate macrophages and neutrophils, resulting in the destruction of the
alveolar wall, the collapse of small airways, hyper-permeability of pulmonary capillaries
and pulmonary edema, that result in the deterioration of pulmonary gas exchange [
10
].
These changes contribute to acute respiratory distress syndrome (ARDS), chronic obstruc-
tive pulmonary disease (COPD) and acute lung injury (ALI) [
1
]. However, the complex
combination of the alterations in glucose metabolism and the association with the loss of
redox homeostasis in COVID-19 patients have not been completely analyzed. Therefore, the
aim of this study was to evaluate changes in glucose metabolism that could contribute to the
loss of redox homeostasis in the plasma of patients with moderate and severe pneumonia
by SARS-CoV-2 with and without comorbidities. In this study, we also tested whether the
use of the presence of the anti-N protein and anti-S protein antibodies of the SARS-CoV-2
virus at the time of admission of the patients can be used as a diagnostic tool for COVID-19.
2. Materials and Methods
2.1. Study Type
This was a comparative cohort and analytical study that compared COVID-19 patients
and healthy subjects (HS). The study was run between 20 August and 20 September 2020.
The study population consisted of 61 patients over 18 years of age who were admitted to the
intensive care unit (ICU) of the CITIBANAMEX Center and that had not developed septic
shock, secondary to moderate or severe pneumonia due to COVID-19. Diagnostic criteria
for septic shock were based on the Sepsis-3 consensus [
12
]. Exclusion from this study
occurred when patients were younger than 18 years of age, when they were not able to
Cells 2022,11, 932 3 of 23
grant an informed consent or when they refused to be included. Patients were also excluded
if pregnant or breastfeeding or if they were under chronic use (last 6 months) or recent use
of steroids, statins or antioxidants. The hospitalized patients included were considered
to have moderate or severe symptoms considering their ventilatory status. Moderate
COVID-19 patients were classified according to the Horowitz index which is defined as the
ratio of partial pressure of oxygen (PaO
2
in mmHg) in blood to the fraction of oxygen in
inhaled air (FiO
2
), (PaO
2
/FiO
2
) with value index percentage of fraction
>200 mmHg [13]
.
Patients with the severe condition required invasive mechanical intubation according to
the criteria of Berlin for ARDS. The Berlin definition proposes 3 categories of ARDS based
on the severity of hypoxemia: mild (200 mmHg < PaO
2
/Fio2
≤
300 mmHg), moderate
(100 mmHg < PaO
2
/FiO
2
200 mmHg) and severe (PaO
2
/FiO
2≤
100 mmHg), along with
explicit criteria related to timing of the syndrome’s onset, origin of edema and the chest
radiographic findings. The ARDS definition task force was considered [14].
Twenty-five HS were matched by age and gender. HS were negative for SARS-CoV-2.
The collection of peripheral blood samples was carried out by venipuncture. In these sub-
jects, there was no suspicion of inflammatory disease or presence of degenerative disorders
such as thyroid and autoimmune diseases, DM, dyslipidemia, arterial hypertension and MS.
The intake of some medications that could interfere with the results of the study such as an-
tioxidant drugs and non-steroidal anti-inflammatory drugs was considered, and the drugs
were suspended 48 h before the obtainment of the sample. Ethical approval to perform the
study was obtained from the local ethics committee on 19 August 2020 (Control-9867/2020,
register REG. CONBIOETICA-09-CEI-011-20160627). A written informed consent for en-
rollment or consent to use data from the patients was obtained directly from them or their
legal surrogates. The protocol was registered (TRIAL REGISTRATION: ClinicalTrials.gov
(accessed on 6 February 2022) Identifier: NCT 04570254). Results related to this study
were previously reported by Chavarría et al. during the pre-treatment and post-treatment
evaluation with antioxidants [
11
]. We observed that the glucose levels were increased since
the admission of the patients. It was therefore considered that, in addition to the main
objective, the evaluation if this increase needed to be explored since it could be related to
the oxidative background present in these patients.
The collection of peripheral blood samples was carried out by venipuncture when
patients entered the ICU and they tested positive by the qRT-PCR test. In total, 34 moderate
and 27 severe COVID-19 patients were included and 25 HS. The blood samples were
centrifuged for 20 min at 936 g and 4
◦
C. The plasma of the samples was placed in 3 or
4 aliquots and stored at −30 ◦C.
2.2. Collection of Samples to Verify Infection by SARS-CoV-2 upon Admission to the Hospital
Paired saliva and nasopharyngeal swab samples were collected from all patients who
were suspected to be infected by SARS-CoV-2. Samples were classified as positive for SARS-
CoV-2 when both the N1 and N2 primer-probe sets were detected. The presence of the SARS-
CoV-2 virus was evaluated using specific probes for the detection of the virus in conjunction
with the real-time reverse transcriptase polymerase chain reaction technique (qRT-PCR).
To evaluate organ dysfunction, the SOFA score (neurologic, respiratory, hemodynamic,
hepatic and hematologic) was calculated at admission [12].
2.3. Detection in Plasma of the N and S Protein Antibodies of the SARS-CoV-2 Virus
The anti-N and anti-S protein antibodies of the SARS-CoV-2 virus were detected using
two ELISA-type immunoassays. The plates were coated with 100
µ
L/well of 5
µ
g/mL of
recombinant SARS-CoV-2 nucleocapsid protein derived from E. coli (CODE: 230-01104)
or 2.5
µ
g/mL of recombinant SARS-CoV-2 Spike protein, S1 subunit derived from E. coli
(CODE: 230-01101), supplied by RayBiotech Company (RayBiotech Inc., Georgia, GA,
USA) and 100
µ
L per well of diluted plasma of the patients and added to the plates. The
absorbance in the plates was measured on an ELISA reader Chromate 4300 (Awareness
Technology, Inc., Palm City, FL, USA), at 450 nm. The percentages of positivity of each
Cells 2022,11, 932 4 of 23
patient, for each type of anti-viral protein antibodies were evaluated and calculated using
the following mathematical model: [(Absorbance at 450 nm of the patient/absorbance
at 450 nm of the negative control)
−
1]
×
100, for both anti-N protein and anti-S protein
antibodies [15].
2.4. Laboratory Tests
Laboratory tests were made for the COVID-19 patients to determine acute-phase reac-
tants, hemoglobin, leukocytes, lymphocytes, platelets, creatinine, urea nitrogen, glucose,
C-reactive protein (CRP), albumin, D-dimer, ferritin, IL-6 and oxygen saturation. Data from
the patient’s medical history including demographic data, prior illnesses to infection by
SARS-CoV-2, test result for COVID-19 and whether mechanical ventilation were used for
the analysis of the results.
2.5. Glucose, Insulin, and HOMA-IR Concentrations
Glucose concentrations were determined using the enzymatic commercial kit from
Pointe Scientific (Pointe Scientific Inc., Michigan, MO, USA), and insulin was determined
by radioimmunoassay as previously described by Jiménez et al. [
12
]. (The HOMA index of
IR was calculated by HOMA−IR = (insulin µU/mL ×glucose mmol/L)/22.5 [16].
2.6. 8-Isoprostane, Vitamin D, H2S, and 3-Nitrotyrosine Concentrations
The kits for the determination of 8-isoprostane and Vitamin D were provided by
Cayman Chemical Company, Michigan MO, USA. (8-isoprostane ELISA kit Item No. 516351
and vitamin D ELISA kit Item No. 501050). This assay is based on the competition between
8-isoprostane and an 8-isoprostane–acetycholinesterase conjugate for a limited number
of 8-isoprostane-specific rabbit antiserum binding sites. The product of this enzymatic
reaction has a distinct yellow color and adsorbs strongly at 412 nm. The assay for Vitamin
D is based on the competition between vitamin D and a conjugated vitamin D-acetyl-
cholinesterase. The product of this enzymatic reaction has a distinctive yellow color and
absorbs strongly at 412 nm. Hydrogen sulfide (H
2
S) was quantified by a commercial
colorimetric assay kit obtained from Elab science Biotechnology Co., Ltd., Houston, TX,
USA. (Cat No. E-BC-K355-M). H
2
S reacts with an acetate solution to form ZnS which can be
dissolved in an alkaline solution. Methylene blue is formed in the presence of Fe
3+
and can
absorb at 665 nm. 3-nitrotyrosine (3-NT) was determined with a kit provided by LifeSpan
BioSciencies, Seattle, WA, USA) (3-nitrotyrosine ELISA kit No. LS-40120). This assay is
based on the competitive ELISA principle and is measured at a wavelength of 450 nm. The
measurements were made using a visible light micro plate reader (Stat Fax 3200 Awareness
Technology Palm City, FL, USA).
2.7. Oxidative Stress Markers
2.7.1. Nitrites (NO2–)
The NO
2–
levels in plasma were determined by the Griess reaction. In total, 100
µ
L of
plasma previously deproteinated with 0.5 N, NaOH and 10%, ZnSO
4
were centrifuged at
1789
×
gfor10 min. The supernatant was recovered and 200
µ
L of 1% sulfanilamide and
200
µ
L of 0.1% N-naphthyl-ethyl diamine were added. The total volume was adjusted to
1 mL. The calibration curve was obtained with a solution of KNO
2
5–0.156 nM and the
absorbance was measured at 540 nm [17].
2.7.2. Lipid Peroxidation Levels (LPO)
In total, 50
µ
L CH
3
-OH with 4% BHT plus phosphate buffer pH 7.4 were added to
100
µ
L of plasma. The reaction tube was incubated to 100
◦
C for 1 hour and centrifuged
at 936 g at room temperature for 2 min. Then, the n-butanol phase was extracted, and the
absorbance was measured at 532 nm [
18
]. This test is based on the reaction of malondialde-
hyde, a secondary product of the oxidation of fatty acids with three or more bonds, with
Cells 2022,11, 932 5 of 23
thiobarbituric acid in an acid medium and at high temperature, generating a pink-colored
product, the value is expressed in nM of malondialdehyde (MDA) per 1 mL of plasma.
2.7.3. Evaluation of Total Antioxidant Capacity (TAC)
In total, 100
µ
L of plasma were suspended in 1.5 mL of a reaction mixture prepared
as follows: 300 mM acetate buffer pH 3.6, 20 mM FeCl
3
6H
2
O and 10 mM of 2,4,6-Tris-2-
pyridyl-s-triazine dissolved in 40 mM HCl. These reactants were added in a relation of
10:1:1 v/v, respectively. After mixing, the samples were incubated at 37
◦
C for 15 min in
the dark. The absorbance was measured at 593 nm [19].
2.7.4. Thiol Concentrations
The technique used was previously described by Erel and Neselioglu [
20
], with some
adaptations and modifications carried out in our laboratory. In total, 50
µ
L of plasma were
suspended reduced with 100
µ
L of KBH
4
10 mM dissolved in CH
3
OH-bidistilled H
2
O,
(1:1 vol/vol) for 3 min, then, 700
µ
L of buffer (6.7 mM formaldehyde, 10 mM EDTA and
Tris 100 mM, pH 8.2) was added for 3 min. Then, 100
µ
L of DTNB 10 mM in CH
3
OH was
added for 4 min. The calibration curve was obtained with solution GSSG 1 mg/1 mL and
the absorbance was measured at 415 nm.
2.7.5. Glutathione Levels (GSH)
In total, 100
µ
L of plasma previously deproteinized with 20% trichloroacetic acid
(vol/vol) and centrifugated to 10,000
×
gfor 5 min plasma was added to 800
µ
L of phosphate
buffer 50 mM, pH 7.3, plus 100
µ
L of 5, 5
0
-dithiobis-2-nitrobenzoic acid 1 M. The mixture
was incubated at room temperature for 5 min and absorbance was read at 412 nm [18].
2.7.6. Selenium
The technique used was previously described by Soto et al. [
21
]. All solutions were
made with tridistilled H
2
O and were only used for the assay and discarded. In brief, in
new Corning sterile polypropylene centrifuge tubes, 200
µ
L of plasma and 500
µ
L of acid
mixture (4:1 vol/vol of HNO
3
+ HCl) plus 500
µ
L of 10% H
2
O
2
were added and incubated
at 120
◦
C for 4 h. After incubation, 100
µ
L of tridistilled H
2
O, 150
µ
L of 0.5 N NaOH,
200
µ
L of 30% formaldehyde, 200
µ
L of a mixture containing 0.5N of N
2
S and 0.5 N of
Na
2
SO
3
, plus 250
µ
L of 0.01 M of EDTA (pH 10.2), and 300
µ
L of 4 mM of toluidine blue
were added. Samples were incubated for 15min at 25
◦
C. At the end of the incubation,
they were centrifuged at 448rcf for 2 min and the absorbance was read at 600 nm. The
calibration curve was performed using 100 ng/mL Na
2
SeO
3,
and the samples were treated
under conditions similar to those of the experimental samples.
2.8. Statistical Analysis
The Sigma Plot 14 program (Jendel Corporation, San José, CA, USA, 1986–2017) was
used to generate the analysis and graphs. Statistical significance was determined by the
Mann–Whitney rank sum test followed by the normality test (Shapiro–Wilk). Differences
were considered statistically significant when p
≤
0.05. The calculation of the antibodies
against N and S proteins of the SARS-CoV-2 virus was carried out by the analysis of
frequency performed using Fisher’s exact test.
3. Results
3.1. Demographic Characteristics
A total of 61 COVID-19 patients were examined, out of which 44 (72%) were men
and 17 (28%) were women. Patients had an age range of 56
±
13 years. The average body
mass index was 29
±
4 kg/m
2
. Normal weight was found in 13 (21%) and overweight
in 24 (39%). Comorbid conditions prior to SARS-CoV-2 infection were dyslipidemia in
11 (18%), systemic arterial hypertension (SAH) in 7 (8%), DM in 6 (10%), DM plus dys-
lipidemia in 5 (8%), DM plus SAH in 5 (8%), SAH plus dyslipidemia in 3 (5%), SM in 9
Cells 2022,11, 932 6 of 23
(15%), chronic obstructive lung disease in 1 (1.6%) and chronic kidney disease in 2 (3.3%).
Temperature was 36.6
±
0.46
◦
C. Other variables expressed as median and minimum–
maximum ranges, respectively, are included such as arterial blood oxygen pressure (PaO
2
,
66.9,
34–223 mmHg
), partial pressure of carbon dioxide (PCO
2
, 31.7,
12.2–81.2 mmHg
),
Kirby’s index which is PaCO
2
/inspired fraction of oxygen (FiO
2
128, 26.8–299 mmHg),
oxygen saturation (SpO
2
/FiO
2
138, 50–280 mmHg), urea (16,
5.6–106.7 mg/dL
), ureic
nitrogen (16,
5.6–196.7 mg/dL
), total cholesterol (136,
69–217 mg/dL
), triglycerides (133,
62–726 mg/dL
), high-density lipoprotein (31,
14–60 mg/dL
), low-density lipoprotein (70,
28–40 mg/dL
), total bilirubin (0.60,
0.12–4.10 mg/dL
), direct bilirubin (0.20,
10–1.20 mg/dL
),
leukocytes (8.8, 2–25 10
3
/
µ
L), lymphocytes 0.8, 0.14–9.6 10
3
/
µ
L), platelets median (244,
16–576 103/µL
), ferritin 541, 147–2592 ng/mL, CRP 146, 20–2450 mg/L), IL-6 67,
7.8–638.5 pg/mL
) and D-dimer 700, 136–16,400
µ
g/mL). The demographic characteris-
tics of the COVID-19 patients are shown in the Table 1.
Table 1. Demographic characteristics at admission of patients infected with COVID-19.
Total
n= 61 (100) Number
and Percentage
Moderate
n= 34 (56) Number
and Percentage
Severe
n= 27 (44) Number
and Percentage
p
Women 17 (28) 11 (32) 6 (22) NS
Men 44 (72) 23 (68) 21 (78) NS
Age 56 ±13 54 ±12 59 ±14 NS
BMI (kg/m2)29 ±4 29 ±4 29 ±4 NS
Temperature (◦C) 36.6 ±0.46 36.5 ±0.43 36.7 ±0.49 NS
Laboratory at the admission Median (Min–Max) range
PAO2(58.5–67.1 mmHg) 66.9 (34–223) 67 (34–223) 66.4 (62–93) NS
PCO2(30.4–40 mmHg) 31.7 (12.2–81.2) 32 (12.2–81.2) 31.6 (22–71) NS
PAO2/FIO2(>164) 128 (26.8–299) 145 (26.8–281) 113 (30–299) NS
SpO2/FIO2(>300) 138 (50–280) 157 (88–240) 128 (50–280) 0.007
Urea (<40 mg/dL) 16 (5.6–106.7) 29.2 (16–60) 30 (13–224) NS
Creatinine (mg/dL) 0.90 (0.5–5.3) 1 (0.5–2.5) 0.8 (0.5–5.3) NS
Ureic Nitrogen (7–25 mg/L) 16 (5.6–196-7) 15.7 (7.5–36) 17 (5.6–106.7) NS
TC (<200 mg/dL) 136 (69–217) 140 (69–217) 132 (86–190) NS
HDL (mg/dL) 31 (14–60) 32 (14–60) 31 (14–45) NS
LDL (mg/dL) 70 (28–140) 64 (35–135) 79 (28–140) NS
DHL (mg/dL) 253 (124–515) 233 (128–412) 255 (124–515) NS
TB (mg/dL) 0.60 (0.12–4.10) 0.50 (0.12–1.3) 0.70 (0.33–4.10) 0.02
DB (mg/dL) 0.20 (0.10–1.20) 0.20 (0.10–1.2) 0.20 (0.10–0.8) NS
TG (<150 mg/dL) 133 (62–726) 137 (62–726) 133 (77–328) NS
Leukocytes (3.5–10.3 ×103/µL)8.8 (2–25) 8.2 (2–14) 11.5 (3–25) 0.003
Lymphocytes (0.99–3.2 ×103/µL)0.8 (0.14–9.6) 0.92 (0.42–9.6) 0.69 (0.14–8.2) 0.01
Platelets (150,000–500,000 ×103/µL)244 (16–576) 226 (16–576) 254 (122–412) 0.057
Ferritin (11–307 ng/mL) 541 (147–2592) 513 (147–2100) 592 (175–2592) NS
IL-6 (pg/mL9) 67 (7.8–638.5) 30.2 (7.8–304) 94 (7.8–639) 0.003
Index N/L 11.5 (1–89) 10 (3–89) 13 (1–83) NS
D-Dimer (0–24 µg/mL) 700 (136–16440) 615 (136–5130) 810 (210–16,640) 0.08
CRP (1–3 mg/L) 146 (20–2450) 280 (20–1380) 146 (32–2450) NS
Comorbidities (%)
DM 6 (10) 4 (7) 2 (3) NS
SAH 5 (8) 3 (5) 2 (3) NS
Dyslipidemia 11 (18) 8 (13) 3 (5) NS
DM + Dyslipidemia 5 (8) 2 (3) 3 (5) NS
DM + SAH 5 (8) 2 (3) 3 (5) NS
Cells 2022,11, 932 7 of 23
Table 1. Cont.
Total
n= 61 (100) Number
and Percentage
Moderate
n= 34 (56) Number
and Percentage
Severe
n= 27 (44) Number
and Percentage
p
SAH + Dyslipidemia 3 (5) 0 2 (5) NS
SM 9 (15) 6 (10) 3 (5) NS
Healthy Subjects without comorbidities 17 (28) 8 (13) 9 (15) NS
Normal weight 13 (21) 6 (18) 7 (26) NS
Overweight 24 (39) 17 (50) 7 (26) 0.06
Obesity 24 (39) 11 (33) 13 (48) NS
COPD 1 (1.6) 0 1 (4) NS
ECKD 2 (3.3) 0 2 (8) NS
Norepinephrine 18 (29) 1 (3) 17 (63)
0.0001
Enteral nutrition 26 (43) 22 (65) 4 (15) 0.001
Deaths 1 (1.6) 0 1 (4) NS
Abbreviations: BMI = Body mass index, HR = Heart rate, MAP = Mean arterial pressure,
HDL = High-density lipoproteins
, LDL = Low-density lipoproteins, TB = Total bilirubin, DB = Direct bilirubin,
IL = Interleukin
,
N/L = neutrophil&/lymphocyte
, DM = Diabetes mellitus, SAH = Systemic arterial hyperten-
sion,
CD = Cardiovascular disease
, COPD = Chronic obstructive pulmonary disease, ECKD = End-stage chronic
kidney disease. FiO
2
= inspired fraction of oxygen, PAO
2
= Blood pressure oxygen, PCO
2
= Partial pressure carbon
dioxide, SpO
2
= Arterial oxygen saturation, ECKD = End-stage chronic kidney disease, TC = Total cholesterol,
TG = Triglycerides, MS = Metabolic syndrome.
3.2. Detection of Antibodies against the N and S Proteins of the SARS-CoV-2 Virus
The presence of anti-N protein and anti-S protein antibodies of the SARS-CoV-2 virus
was determined in the present study at the time of admission. In the baseline measurement,
53 of the 61 patients tested positive for the presence of anti-N protein antibodies (86.9%),
while 8 patients, 4 corresponding to the group who had a moderate respiratory disease,
and 4 corresponding to the group of patients who progressed poorly and required the
use of an artificial respirator, showed an absence of anti-N protein antibodies. A week
later, seven of the eight patients who did not have detectable anti-N protein antibodies
at the beginning, already tested positive for the presence of this type of antibody. The
frequencies of positivity of the antibodies against the N and S proteins were of 31/34 with
91.2% frequency and 7/34 with 20.6% frequency, respectively, in patients with a moderate
illness and of 26/27 with 96.3% frequency and 7/27 with 25.9% frequency, respectively,
in patients with severe COVID-19. There was no statistical difference between the groups
with moderate or severe acute respiratory disease in the analyses of frequency performed
using Fisher’s exact test. Therefore, the results suggest that the serological response to the
SARS-CoV-2 virus does not appear to be the condition responsible for progression to a
more severe stage requiring artificial ventilation.
3.3. Glucose, Insulin and HOMA-IR Concentrations
The moderate and severe COVID-19 patients showed a significant increase (p< 0.001)
in glucose, insulin concentrations and HOMA-IR in plasma in comparison with the HS,
(Figure 1a–c, respectively).
Cells 2022,11, 932 8 of 23
Cells 2022, 11, x FOR PEER REVIEW 8 of 22
Figure 1. (a) The values of glucose significantly increased in the moderate and severe COVID-19
patients in comparison with HS. (b) Insulin had a significant increase in the moderate and severe
COVID-19 patients in comparison to HS. (c) The HOMA index significantly increased in the mod-
erate and severe COVID-19 patients in comparison with HS. Abbreviations: HS = healthy subjects,
HOMA index = marker of insulin resistance which if the values are greater than 2.5 is an indication
of insulin resistance.
HS
0
100
200
300
400
500
600
Glucose (mg / dL)
COVID-19
Moderate COVID-19
Severe
p<0.001
p<0.001
HS
0
1
2
3
4
5
COVID-19
Moderate COVID-19
Severe
Insulin (ng / mL)
p<0.001
p<0.001
HS
0
20
40
60
80
HOMA-index
COVID-19
Moderate COVID-19
Severe
p<0.001
p<0.001
A
B
C
a
b
c
Figure 1.
(
a
) The values of glucose significantly increased in the moderate and severe COVID-19
patients in comparison with HS. (
b
) Insulin had a significant increase in the moderate and severe
COVID-19 patients in comparison to HS. (
c
) The HOMA index significantly increased in the moderate
and severe COVID-19 patients in comparison with HS. Abbreviations: HS = healthy subjects, HOMA
index = marker of insulin resistance which if the values are greater than 2.5 is an indication of
insulin resistance.
Cells 2022,11, 932 9 of 23
3.4. 8-Isoprostane, Vitamin D, H2S, and 3-Nitrotyrosine Concentrations
The 8-isoprostane and 3-NT concentrations showed a significant increase (p< 0.001,
Figure 2a,b). However, Vitamin D (p= 0.03 and p= 0.01, Figure 3a) and H
2
S (p< 0.001 and
p= 0.007, Figure 3b) levels showed a decrease in moderate and severe COVID-19 patients
in comparison to HS.
Cells 2022, 11, x FOR PEER REVIEW 9 of 22
3.4. 8-Isoprostane, Vitamin D, H2S, and 3-Nitrotyrosine Concentrations
The 8-isoprostane and 3-NT concentrations showed a significant increase (p < 0.001,
Figure 2a,b). However, Vitamin D (p = 0.03 and p = 0.01, Figure 3a) and H2S (p < 0.001 and
p = 0.007, Figure 3b) levels showed a decrease in moderate and severe COVID-19 patients
in comparison to HS.
Figure 2. (a) The 8-isoprostane concentrations statistically increased in both the moderate and severe
COVID-19 patients in comparison with HS. (b) 3-NT statistically increased in the moderate and
severe COVID-19 patients in comparison to HS. Abbreviations: HS = healthy subjects, 3-NT = 3-
nitrotyrosine.
HS
0
200
400
600
800
1000
8-isoprostane (pg/ml of plasma)
COVID-19
Moderate COVID-19
Severe
p<0.001
p<0.001
HS
-0.01
0.00
0.01
0.02
0.03
0.04
0.05
3-NT
(nM / ml plasma)
COVID-19
Moderate COVID-19
Severe
p<0.001
p<0.001
A
B
a
b
Figure 2.
(
a
) The 8-isoprostane concentrations statistically increased in both the moderate and severe
COVID-19 patients in comparison with HS. (
b
) 3-NT statistically increased in the moderate and severe
COVID-19 patients in comparison to HS. Abbreviations: HS = healthy subjects, 3-NT = 3-nitrotyrosine.
Cells 2022,11, 932 10 of 23
Cells 2022, 11, x FOR PEER REVIEW 10 of 22
Figure 3. (a) Changes in the values of Vit D and (b) changes in the concentration of the H2S, there
was a significant decrease in the moderate and severe COVID-19 patients in comparison with HS.
Abbreviations: Vit D = Vitamin D, H2S = hydrogen sulfide, HS = healthy subjects.
3.5. Oxidative Stress Markers
The LPO index showed a significant increase (p = 0.02 and p = 0.004, Figure 4a). How-
ever, the TAC (p < 0.001, Figure 4b), NO2– (p < 0.001, Figure 4c), the thiols concentrations
(p = 0.02 and p = 0.006, Figure 5a) and GSH levels (p < 0.001, Figure 5b) showed a decrease
in moderate and severe COVID-19 patients in comparison with HS.
HS
-20
-10
0
10
20
30
40
50
60
Vit D (ng/ml of plasma)
COVID-19
Moderate COVID-19
Severe
p=0.03
p=0.01
HS
H2S (mmol/L of plasma)
20
25
30
35
40
45
COVID-19
Moderate
p<0.001
p=0.007
COVID-19
Severe
A
B
a
b
Figure 3.
(
a
) Changes in the values of Vit D and (
b
) changes in the concentration of the H
2
S, there
was a significant decrease in the moderate and severe COVID-19 patients in comparison with HS.
Abbreviations: Vit D = Vitamin D, H2S = hydrogen sulfide, HS = healthy subjects.
3.5. Oxidative Stress Markers
The LPO index showed a significant increase (p= 0.02 and p= 0.004, Figure 4a). How-
ever, the TAC (p< 0.001, Figure 4b), NO
2–
(p< 0.001, Figure 4c), the thiols concentrations
(
p= 0.02
and p= 0.006, Figure 5a) and GSH levels (p< 0.001, Figure 5b) showed a decrease
in moderate and severe COVID-19 patients in comparison with HS.
Cells 2022,11, 932 11 of 23
Cells 2022, 11, x FOR PEER REVIEW 11 of 22
Figure 4. (a) The LPO index significantly increased in the moderate and severe COVID-19 patients
in comparison with HS. (b) The TAC significantly decreased in the moderate and severe COVID-19
patients in comparison to HS. (c) The NO2– concentration significantly decreased in the moderate
and severe COVID-19 patients in comparison with HS. Abbreviations: LPO = Lipid peroxidation,
TAC = Total antioxidant capacity, NO2– = Nitrites.
0
2
4
6
8
LPO (nM MDA / ml of plasma)
HS COVID-19
Moderate COVID-19
Severe
p=0.02
p=0.004
0
5000
10000
15000
20000
25000
30000
35000
TAC (nM Trolox/ml of plasma)
p<0.001
p<0.001
HS COVID-19
Moderate COVID-19
Severe
A
B
p<0.001
0
20
40
60
80
HS COVID-19
Moderate COVID-19
Severe
NO2- (nM / ml of plasma)
p<0.001
C
a
b
c
Figure 4.
(
a
) The LPO index significantly increased in the moderate and severe COVID-19 patients
in comparison with HS. (
b
) The TAC significantly decreased in the moderate and severe COVID-19
patients in comparison to HS. (
c
) The NO
2–
concentration significantly decreased in the moderate
and severe COVID-19 patients in comparison with HS. Abbreviations: LPO = Lipid peroxidation,
TAC = Total antioxidant capacity, NO2–= Nitrites.
Cells 2022,11, 932 12 of 23
Cells 2022, 11, x FOR PEER REVIEW 12 of 22
Figure 5. (a) The thiol concentrations significantly decreased in the moderate and severe COVID-19
patients in comparison to HS. (b) GSH concentration statistically decreased in the moderate and
severe COVID-19 patients in comparison with HS. (c) The Se concentration significantly decreased
in both the moderate and severe COVID-19 patients in comparison with HS. Abbreviations: HS =
healthy subjects, GSH = glutathione, Se = selenium.
HS
GSH (mM/ ml of plasma)
0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
COVID-19
Moderate COVID-19
Severe
p<0.001
p<0.001
HS
-0.002
0.000
0.002
0.004
0.006
0.008
0.010
0.012
0.014
0.016
Se (nM / ml of plasma)
COVID-19
Moderate COVID-19
Severe
p<0.001
a
b
c
0
5
10
15
20
25
30
35
Thiols (mM /ml of plasma)
p=0.02
p=0.006
HS COVID-19
Moderate COVID-19
Severe
Figure 5.
(
a
) The thiol concentrations significantly decreased in the moderate and severe COVID-
19 patients in comparison to HS. (
b
) GSH concentration statistically decreased in the moderate
and severe COVID-19 patients in comparison with HS. (
c
) The Se concentration significantly de-
creased in both the moderate and severe COVID-19 patients in comparison with HS. Abbreviations:
HS = healthy subjects, GSH = glutathione, Se = selenium.
Cells 2022,11, 932 13 of 23
3.6. Selenium
The Selenium concentration only decreased in severe COVID-19 patients in compari-
son to HS (p< 0.001, Figure 5c).
4. Discussion
In the present study, the presence of anti-N protein, and anti-S protein antibodies of the
SARS-CoV-2 virus was determined at the time of admission of the patients. In most of the
patients, antibodies against these proteins were present at the time of admission and in the
rest of them, the antibodies appeared a few days later. During the analytical validation of
the ELISA to measure anti-N protein antibodies, it was found that up to 11% of subjects may
not have measurable amounts of anti-N protein antibodies at the time of the manifestation
of clinical symptoms. This was because these subjects are slow to produce antibodies and
require a longer time to respond. However, between 4 and 10 days after the onset of the
manifestations of clinical symptoms, patients already have measurable concentrations of
anti-N protein antibodies. These results confirm the known fact that the measurement of
anti-N protein antibodies is the most effective serological measurement for diagnosing
the SARS-CoV-2 virus infection [
15
,
22
]. This is due to the fact that the N protein of the
SARS-CoV-2 virus is a highly immunogenic protein that is overexpressed and released into
the bloodstream simultaneously with the viral particles. Therefore, its measurement is a
highly useful analytical tool for the epidemiological management of the pandemic [
22
].
The low positivity values for anti-S protein antibodies, both in the frequency of positive
patients, and in the percentage of positivity, suggests that these patients had difficulty in
producing neutralizing antibodies, necessary to counteract the infectious capacity of the
SARS-CoV-2 virus [
15
,
22
]. For this reason, these subjects required hospitalization and were
in need of specialized clinical management and even the use of artificial respirators.
On the other hand, several studies have shown that COVID-19 triggers a transient
hyperglycemia and impairs pancreatic
β
-cell function. This has been associated with
inflammation and the cytokine storm that may lead to IR. These changes contribute to a
positive feedback cycle in the development and progression of hyperglycemia in COVID-
19 patients. Hyperglycemia may also induce OS and glucolipotoxicity. However, in
subjects with MS these changes are more aggressive, and comorbidities may lead to fatal
outcomes [
23
]. Our results show that the hyperglycemia, hyperinsulinemia and IR were
present in our series of 61 moderate and severe COVID-19 patients.
The association of the COVID-19 infection and altered glucose metabolism has previ-
ously been reported [
24
], and explained by the capacity of the virus to hijack mitochondrial
function leading to an anaerobic function [
5
]. In this condition, there is an increase in lactate
levels that provokes an increase in the dependency on glycolysis in hepatocytes, which is
reflected in an increase in glucose and lactate levels in the blood. This state favors viral
replication since the virus requires large amounts of energy for biosynthetical process [
5
].
Moreover, the increase in glucose levels elevates the pool of free fatty acids (FFA) which are
necessary for the formation of viral membranes. It also increases nucleotides needed for
RNA synthesis for viral replication [5].
As a consequence of glucose alterations, DM and MS patients have an increased risk of
severe pneumonia by COVID-19 with fatal outcomes. This is reflected in a higher mortality
in subjects with these comorbidities [
25
]. In addition, prolonged hyperglycemia could
worsen the course of COVID-19 via glycation of pancreatic ACE2 and the transmembrane
serine protease 2 (TMPRSS2), thus facilitating the SARS-CoV-2 binding and entrance to
pancreatic
β
-cells [
2
]. However, SARS-CoV-2 may infect the pancreatic
β
-cells through
other proteinases such as neuroplin-1 and transferrin receptor [
26
,
27
]. Studies in human
islets infected
in vitro
and studies in postmortem autopsied tissue found a phenotypic alter-
ation or trans-differentiation of the
β
-cells with decreased insulin and increased glucagon
secretion that could lead to a fatal outcome [
26
,
27
]. However, these studies had great
limitations such as the lack of a determination of circulating insulin concentrations in
COVID-19 patients.
Cells 2022,11, 932 14 of 23
Our results showed that hyperglycemia and hyperinsulinemia were present in plasma
of patients with and without comorbidities upon admission to the ICU. These results agree
with those reported by Montefusco et al. who showed that in 551 patients hospitalized for
COVID-19, the SARS-CoV-2 induces IR and disrupts
β
-cells function which can result in
clinically evident hyperglycemia detectable even in the post-acute phase [
28
]. This suggests
that both the hyperglycemia and hyperinsulinemia depend on the SARS-CoV-2 infection,
the viral replication cycle and the viral load. As the disease progresses, there is an increase
in glucose levels elicited by the demand of the virus for its replication, which favors a
continuous overstimulation of the
β
-cell [
24
]. This results in an increase in insulin levels
that eventually leads to depletion of insulin stores. This worsens hyperglycemia, and
finally deteriorates
β
-cell function.
In vitro
studies have demonstrated that the hepatitis
virus (HCV) infection of human
β
-cells also leads to hypersecretion of insulin in the initial
stages of the infection and that it later causes a reduction in insulin in the
β
-cells [
29
].
Another study in pancreatic cells infected with HCV demonstrated that hyperinsulinemia
and pancreatic
β
-cell hyperfunctionality is aimed to maintain glucose homeostasis [
30
].
Furthermore, hyperinsulinemia may disturb fibrinolysis by elevating the plasminogen
activator inhibitor type 1 and increase thrombosis in COVID-19 patients [31].
An inadequate insulin secretion by decompensated
β
-cells may cause increasingly
higher blood glucose levels that bathe the islet. This leads to a spectrum of consequences
for the
β
-cell, including glucose desensitization,
β
-cell exhaustion, and eventually glu-
cose toxicity [
32
]. Furthermore, this sustained response of the pancreas aimed towards
maintaining glucose homeostasis, leads to the activation of pathways of apoptosis and trans-
differentiation if it does not decrease the infection. These result, with time, in a decreased
secretion of insulin by the
β
-cells and lower glucose-stimulated insulin secretion [
33
]. In
addition, subjects without comorbidities may develop hyperglycemia after three days of
the COVID-19 infection. This elevation in glucose levels can be reversed within 2 weeks.
However, 10% of patients could develop DM later in time. This is important because these
findings are not observed in other viral forms of pneumonia, suggesting the involvement of
the pancreatic axis in the coronavirus infection [
34
]. Furthermore, subjects with DM show
pre- and post-prandial hyperglycemia as well as diabetic ketoacidosis when infected by
SARS-CoV-2 [
34
]. In our experimental series, out of 61 patients infected with SARS-CoV-2,
45 had comorbidities while 17 did not report comorbidities and, nevertheless, all presented
alterations in glucose metabolism [
34
]. Hyperglycemia inactivates the glucose transporters
(GLUT) which are normally triggered by insulin. In fibroblasts infected with the human
cytomegalovirus, the IE72 trans activator of viral promoters decreases the mRNA of GLUT1
and increases the mRNA of GLUT-3, -4 and -8 from the early stage of infection that are
3 times more effective in transporting glucose. This increase is independent from the
Akt-mediated metabolic pathway that depends on insulin concentrations. This suggests
that the SARS-CoV-2 could probably have a similar metabolic pathway in glucose transport
independent of hyperglycemia and hyperinsulinemia present in COVID-19 patients [35].
In addition, our results showed that the IR state in COVID-19 patients is present. In
this sense, it has been described that binding of insulin to the insulin receptor or insulin-like
growth factor 1 receptor (IGF1R) results in the autophosphorylation of insulin receptor
substrate 1/2 (IRS1/2) at its tyrosine residues and in the subsequent activation of two main
pathways, the phosphoinositide-3-kinase (PI3K-Akt) pathway and the mitogen activated
protein kinase (MAPK) pathway. Conversely, serine phosphorylation of the IRS1/2 attenu-
ates insulin signaling by decreasing insulin-stimulated tyrosine phosphorylation and this
can produce IR [
36
]. A study using liver biopsy specimens obtained from non-diabetic
HCV-infected patients showed that HCV impairs the insulin-stimulated tyrosine phospho-
rylation of hepatic IRS1, resulting in reduced PI3K-Akt activation without any alteration in
the MAPK pathway [
37
]. Another study in a transgenic mouse model expressing the HCV
genotype 1 core protein indicated that the core protein was responsible for inducing IR
in the liver via suppression of tyrosine phosphorylation in IRS1, which is associated with
DM [38].
Cells 2022,11, 932 15 of 23
During IR, the action of insulin at the cellular level is reduced in several tissues, which
increases the secretion of this hormone by the pancreas. The HOMA-IR index is used
as an indicator of IR [
39
]. IR is defined by an augmented production of glucose by the
liver, reduced glucose uptake by muscle and elevated lipolysis. In this condition, muscles,
adipose tissue and the liver do not respond to insulin. Moreover, IR is related to a range
of risk factors for cardiovascular disease, including MS, hypertension, OS, dyslipidemia,
inflammation and glucose intolerance [
40
,
41
]. Different studies have demonstrated that
SARS-CoV-2 induces IR [
42
]. Our results show that COVID-19 patients presented an
increase in the HOMA index. This result implies that during the progression of the
infection, IR forces
β
-cells to elevate the synthesis of insulin trying to restore the normal
blood glucose level [
43
]. Furthermore, IR leads to an increased pancreatic expression of
the ACE2 receptors, increasing the binding affinity for the S protein of the SARS-CoV-2.
Therefore, there is an increased vulnerability for the COVID-19 infection in subjects with
IR [44].
The alterations in the glucose–insulin axis in COVID-19 patients with or without
comorbidities, suggest that it is very important to control the glycemic state using dif-
ferent drugs such as the metformin, which improves IR and peripheral glucose uptake
through the activation of AMP-dependent protein kinase [
45
]. Metformin also exerts
pleiotropic effects through the AMP-independent pathway including anti-inflammatory
and immunomodulatory effects and it also inhibits the synthesis and release of CRP, IL-1
β
-
induced IL-6, and ferritin from macrophages, endothelial cells, smooth muscle vascular
cells and hepatocytes [
46
]. Metformin also reduces the binding of SARS-CoV-2 to ACE2 by
inducing functional changes in the transmembrane enzyme by AMP-dependent phospho-
rylation [47].
Hyperglycemia, hyperinsulinemia and IR contribute to increase ROS that attack free
fatty acid (FFA) or esterified arachidonic acid in the phospholipids of the membrane and
constitute conditions that are frequently found in COVID-19 and in patients with MS.
Hyperglycemia, hyperinsulinemia and IR increase the rate of lipolysis which is accompa-
nied with liberation of FFA to the circulation. An increased fatty acid (FA) metabolism is
essential for the formation of the viral membranes including those of SARS-CoV-2 [
48
].
Furthermore, the increase in FFA and the alteration in FA metabolism increase gluconeo-
genesis, reduce
β
-cells mass and provide substrates for the synthesis of triglycerides that
are elevated in COVID-19 patients [
48
]. The attack of the membrane phospholipids and
arachidonic acid by ROS result in the formation of isoprostanes that are molecules that are
considered as markers of LPO and oxidative injury. In this sense, 8-isoprostane has been
considered an ideal marker for pathophysiology such as bronchoalveolar lavage fluid of
patients with interstitial lung disease [
49
]. Our results show that 8-isoprostanes and IL-6
were elevated in both moderate and severe COVID-19 patients. A study in rats with EPOC
also showed an increase in 8-isoprostanes and IL-6 in lung lysates associated with ACE2
overexpression and another study demonstrated an increase in urinary 8-isoprostanes
levels in patients with chronic obstructive pulmonary disease [
50
,
51
]. This suggests that
the 8-isoprostanes are associated in pneumonia by SASR-CoV-2 and comorbidities present
in MS and that their synthesis is a consequence of the alteration of the FA metabolism
and the oxidative background. These alterations contribute to ACE2 over-expression that
facilitates the entrance of the virus into the host cells. However, more investigations are
necessary to verify this hypothesis. In addition, the alterations in the glucose–insulin axis
and FA metabolism [
48
] may lead to an increase in OS, increased inflammation, interleukin
storm, and decreased mobilization of leukocytes, phagocytic activity, TAC and impairment
of endothelial function [
52
]. In this sense, it has been reported that the TAC levels were
considerably lower in serum of mild and severe COVID-19 patients in comparison with
control subjects [
53
]. Our results showed alterations in some markers of OS such as LPO,
TAC, thiols, 8-isoprotanes, GSH, 3-NT and NO
2–
concentrations. In ARDS and COPD
associated with COVID-19, the hypoxic conditions result in inflammation that may lead to
OS. Increased OS leads, as a consequence, to a decrease in nitric oxide (NO) release mainly
Cells 2022,11, 932 16 of 23
because ROS interfere with the activity of the NO synthases and cause an inadequate
production of tetrahydrobiopterin [
54
]. Optimal concentrations of O
2
are required for the
synthesis of NO by the nitric oxide synthases.
Changes in the activity of these synthases are reflected in a decrease in their metabo-
lites including NO
2–
. This metabolite was decreased in moderate and severe patients in
our study. Furthermore, the newly synthetized NO might be oxidized by ROS to peroxini-
trite (ONOO
–
) which is the most aggressive RNS and may contribute to the inflammatory
process associated with the interleukin storm in the COVID-19 patients. Low levels of NO
also induce proliferation of vascular smooth muscle cells, platelet aggregation, elevated
pro-inflammatory cytokines such as IL-6, chemokine expression, low-density lipoprotein
oxidation, and expression of vascular cell adhesion molecule-1, stimulation of thrombolysis
and monocyte chemotactic protein-1 through the inhibition of the NF-
κ
B signaling pathway.
Moreover, decreased NO following an oxidative burst can stimulate the production of
metalloproteinases-2 and -9 that contribute to pulmonary damage [
55
]. Hypoxia also in-
creases the presence of protons, in a similar way as in a neutral or acidic pH, compromising
the stability of ONOO
−
and decomposing peroxynitrous acid to form nitrates (NO
3−
) and
NO
2−
[
54
]. ONOO
−
causes the irreversible process of nitration of tyrosines in proteins,
it oxidizes the thiol groups in them [
56
] and it also lowers the H
2
S concentration. In this
sense, H
2
S is a potent gasotransmitter that is decreased in COVID-19 patients [
57
]. Recent
studies provide evidence of the beneficial role of H
2
S in COVID-19 patients. In 10 patients
with severe COVID-19 intravenous N-acetylcysteine a potential H
2
S releaser decreased
the inflammation [
58
]. H
2
S is able to positively modulate concentrations of cytokines by
reducing pro-inflammatory IL-6 and TNF-
α
. This suggests that the decrease in the H
2
S
levels contributes in part to a pro-inflammatory state in SARS-CoV-2 infection. In addition,
moderate or high IL-6 levels have been associated with the decrease in the cysteine and
taurine concentration which are essential amino acids for H
2
S synthesis [
59
]. Our results
show that 3-NT in plasma proteins was increased both in moderate and severe patients.
This result suggests that COVID-19 courses with nitrosative stress (NSS) [
5
,
60
], which is
associated with ferroptosis [5].
Ferroptosis results from mitochondrial sequestration by the viruses and contributes to
an oxidative environment by increasing the Fenton–Haber–Weiss reaction which results
in OS and NSS production [
5
], and in the ferroptosis present in COVID-19 patients [
61
].
The NSS and OS damage disulphide bonds (thiols), which are necessary to stabilize the
architecture of proteins. Viruses and pathogens rely on the proper redox state for their
–S–S– bonds or sulfhydryl groups that are needed for their entrance to the host cells. The S1
subunit is responsible for receptor binding and it contains the receptor-binding domain
which contains disulphide bridges that are indispensable for the union with the ACE2.
Moreover, the ACE2 receptor and the antioxidants enzymes also contain disulphide bridges,
and therefore, dynamic disulphide bridge homeostasis is very important for both viral
replication and for the antioxidant defense in the patient [
3
]. Our results show that the level
of thiols in plasma from patients with COVID-19 was decreased in moderate and severe
cases. This result is similar to the one found in another study where it was demonstrated
that COVID-19 patients’ course with low levels of thiols and that their level can even
be a marker that reflects the gradual increase in the severity of the infection [
62
]. This
result is very important because different investigations have reported that the treatment
with agents capable of elevating the reducing thiol groups such as GSH, allicin, garlic and
N-acetylcysteine, can restore the homeostasis of thiols and decrease the degree of viral
infection increasing the TAC and decreasing the LPO in COVID-19 patients [63].
On another hand, the low levels of H
2
S favor a decrease in the thiol concentration
because this gasotransmitter reduces the –S–S– in the proteins and enzymes and increases
the expression of TMPRSS2 which facilities the SARS-CoV-2 entrance into cells [
64
]. Low
levels of H
2
S have been reported in ARDS and COPD associated with the SARS-CoV-2
infection [
65
]. In this sense, exogenously applied H
2
S donors such as NAC protect from
lung damage, including that produced by ARDS, COPD, ALI, asthma pulmonary fibrosis
Cells 2022,11, 932 17 of 23
and hypoxia-induced pulmonary hypertension in animal models and in humans. This
therapy has therefore been proposed as an alternative strategy in this pandemic [66].
Moreover, levels of Vitamin D are low in diseases that course with hyperinsulinemia
such as COVID-19 and in the pathologies that comprise the MS [
67
,
68
]. Vitamin D exerts its
effects by binding to a nuclear Vitamin D receptor, which is expressed in various immune
cells, with particularly high levels in dendritic cells, macrophages, T and B lymphocytes that
are increased when the innate immune response is elicited [
69
]. During hyperinsulinaemic
states, Vitamin D3 is sequestered into adipocytes, and inactivated in the kidney [
42
]. Our
results show that Vitamin D levels were decreased in moderate and severe COVID-19
patients. This suggests that hyperinsulinemia may decrease the concentration of this
vitamin having an unfavorable impact upon the innate immune response. Moreover,
this deficiency may increase the interaction of the S protein with ACE2, because its low
levels favor an increase in the TMPRSS2 protease which is essential for the entrance of the
virus [
70
]. Vitamin D deficiency also decreases innate cellular immunity by lowering the
expression of defensins which maintain the gap junctions in the endothelial cells of the lung.
Rupture of these junctions is caused by the SARS-CoV-2 leading to ARDS and pulmonary
edema [
71
]. High concentrations of Vitamin D may also have benefic effects such as the
induction of the vasorelaxant ACE2/Ang-(1-7)/Mas receptor axis, which protects against
acute lung injury and ARDS [
72
]. In addition, low levels of Vitamin D are associated with Se
deficiency and are related to COVID-19 severity, old age, obesity, diabetes and dyslipidemia.
However, in COVID-19 patients without comorbidities, there is also a decrease in Vitamin D
and Se in comparison with the healthy subjects [
68
]. The participation of Se is also important
in COVID-19 because it has synergic effects with Vitamin D and E acting as antioxidants.
Furthermore, Se is indispensable for the presence of 25 seleno-enzymes such glutathione
peroxidase, thioredoxin reductases (TrxR), deiodinases and methionine sulfoxide reductases
which play an important role in maintaining the redox homeostasis [
73
]. Our results show
that the concentrations of Se and GSH were decreased in severe COVID-19 patients. This
suggests that the redox homeostasis which is dependent on the 25 seleno-enzymes is altered
and this condition is associated with severe COVID-19. Moreover, this impacts on the
presence of low thiols GSH and H
2
S concentrations in plasma, which contribute to an
increase in OS and NSS present in these patients. This is reflected in the rise of the LPO
index accompanied with depletion of the TAC. In this sense, Se deficiency in mice was
associated with enhanced virulence of enteroviruses and the development of myocardial
lesions [
73
]. The homeostasis of thiols and GSH depend on TrxR and glutathione reductase
which are seleno-enzymes. Low levels of the GSH in COVID-19 patients are associated
with ferroptosis and with a down-regulation of GPX4 and TrxR [
5
,
61
]. A deficiency in
GSH is linked to HIV progression and poor survival of HIV-infected individuals [
74
], and
high concentrations of GSH in blood decrease the virulence of the infection by dengue
and chikungunya [
75
]. Our results suggest that a reduction in the GSH concentration may
contribute to an increase in OS and NSS and favor the decrease and increase, respectively,
in the TAC and LPO in COVID-19 patients. Figure 6summarizes the alterations in the
glucose–insulin axis by SARS-CoV-2.
Cells 2022,11, 932 18 of 23
Cells 2022, 11, x FOR PEER REVIEW 18 of 22
Figure 6. Alteration of the glucose–insulin axis by SARS-CoV-2, and the impact on some antioxidant
markers in patients. (1) Entry of COVID-19 through various receptors in the pancreas. (2) COVID-
19 increases lactate levels, favoring viral replication. (3) Increased free fatty acids are used for viral
membrane formation. (4) Increased glucose overstimulates pancreatic β-cells and subsequently im-
pairs their function. (5) Chronically increased insulin concentrations will lead to depletion of insulin
reserves. (6) Hyperinsulinemia may favor the development of thrombosis. (7) Hyperglycemia inac-
tivates GLUT 1 but SARS-CoV-2 could increase -3, -4 and -8, transporters. (8) Increased lipolysis
blocks the response to insulin by the liver, adipose tissue and muscles. (9) Metformin prevents the
interaction of the ACE2 receptor and COVID-19. (10) Increased free fatty acid synthesis favors the
generation of 8-isoprostanes, which favor ACE2 receptor overexpression. (11) ONOO– favors inter-
leukin storm. (12) ONOO– decreases H2S concentration. (13) Nitrosative stress and depletion of GSH
concentrations is associated with ferroptosis and mitochondrial sequestration by the virus. (14) Un-
der conditions of hyperinsulinemia, Vitamin D is sequestered in adipose tissue and inactivated in
the kidney, which favors the interaction of virus S protein with the ACE2 receptor. (15) Vitamin D
deficiency generates ruptures in the gap junctions of lung endothelial cells. (16) Vitamin D defi-
ciency is associated with Se deficiency which affects selenoenzymes. Abbreviations: ARDS = acute
respiratory distress syndrome, H2O2 = hydrogen peroxide, H2S = sulfhydryl acid, G-6-P = glucose 6
phosphate, GR = glutathione reductase, GSH = glutathione, GSSG = oxidized glutathione, NO = ni-
tric oxide, NSS = nitrosative stress, O2– = superoxide, ONOO– = peroxynitrite, ROS = reactive oxygen
species, SOD = Superoxide dismutase.
5. Conclusions
The results suggest that infection with SARS-CoV-2 in patients with and without
comorbidities results in alterations in the glucose–insulin axis which leads to hyperglyce-
mia, hyperinsulinemia and IR. These alterations increase OS and NSS that is reflected in
increases or decreases in some oxidative markers in plasma with major impacts or fatal
consequences in patients that course with MS. Moreover, subjects without comorbidities
could have long-term alterations in the redox homeostasis after infection.
Figure 6.
Alteration of the glucose–insulin axis by SARS-CoV-2, and the impact on some an-
tioxidant markers in patients. (
1
) Entry of COVID-19 through various receptors in the pancreas.
(
2
) COVID-19 increases lactate levels, favoring viral replication. (
3
) Increased free fatty acids are
used for viral membrane formation. (
4
) Increased glucose overstimulates pancreatic
β
-cells and
subsequently impairs their function. (
5
) Chronically increased insulin concentrations will lead
to depletion of insulin reserves. (
6
) Hyperinsulinemia may favor the development of thrombo-
sis. (
7
) Hyperglycemia inactivates GLUT 1 but SARS-CoV-2 could increase -3, -4 and -8, trans-
porters. (
8
) Increased lipolysis blocks the response to insulin by the liver, adipose tissue and muscles.
(
9
) Metformin prevents the interaction of the ACE2 receptor and COVID-19. (
10
) Increased free
fatty acid synthesis favors the generation of 8-isoprostanes, which favor ACE2 receptor over-
expression. (
11
) ONOO
–
favors interleukin storm.
(12) ONOO–
decreases H
2
S concentration.
(
13
) Nitrosative stress and depletion of GSH concentrations is associated with ferroptosis and mi-
tochondrial sequestration by the virus. (
14
) Under conditions of hyperinsulinemia, Vitamin D is
sequestered in adipose tissue and inactivated in the kidney, which favors the interaction of virus S pro-
tein with the ACE2 receptor. (
15
) Vitamin D deficiency generates ruptures in the gap junctions of lung
endothelial cells.
(16) Vitamin D
deficiency is associated with Se deficiency which affects selenoen-
zymes. Abbreviations:
ARDS = acute respiratory distress syndrome
,
H2O2= hydrogen peroxide
,
H
2
S = sulfhydryl acid, G-6-P = glucose 6 phosphate, GR = glutathione reductase,
GSH = glutathione
,
GSSG = oxidized glutathione
, NO = nitric oxide,
NSS = nitrosative stress
,
O2–= superoxide
,
ONOO–= peroxynitrite, ROS = reactive oxygen species, SOD = Superoxide dismutase.
5. Conclusions
The results suggest that infection with SARS-CoV-2 in patients with and without
comorbidities results in alterations in the glucose–insulin axis which leads to hyperglycemia,
hyperinsulinemia and IR. These alterations increase OS and NSS that is reflected in increases
or decreases in some oxidative markers in plasma with major impacts or fatal consequences
Cells 2022,11, 932 19 of 23
in patients that course with MS. Moreover, subjects without comorbidities could have
long-term alterations in the redox homeostasis after infection.
5.1. Study Limitations
One of the limitations of this study is that we could not study SARS-CoV-2-infected
subjects with mild symptoms or asymptomatic COVID-19. The center in which this work
was carried out was a reference center and received patients with moderate to severe
symptoms sent from other hospitals. Another limitation is that in the evaluation of certain
parameters such as D-Dimer, Ferritin, N/L index, there is a tendency to an elevation in
moderate and critical patients; however, this cannot be defined in a concrete way because a
larger sample size would be required for this specific evaluation. However, it is known that
these parameters increase, and our results show this same tendency. It is important to state
that the sample for this study was adjusted for the specific question of the investigation.
One of the limitations of this study is also that we were not able to measure markers with
thromboelastography to define the co-participation of a hyperglycemic state with changes
in PAI-1 and fibrinolysis. It is currently known that a subset of patients with COVID-19
have a higher risk of bleeding, and high levels of tissue-type plasminogen activator (tPA)
and plasminogen activator inhibitor 1 (PAI- 1) have been associated with a worse lung
function. An elevated tPA is independently correlated with mortality. The levels of any of
these molecules can increase independently of the other; however, a change in one can have
consequences on another. The interaction of plasminogen activators, both tissue-type (tPA)
and urokinase-type, and their main inhibitor, PAI-1, play a key role in the regulation of
fibrinolytic activity. Impaired fibrinolysis has been suggested in COVID-19 patients, which
could further increase thrombotic risk. This has been evidenced by markedly reduced clot
lysis at 30 min by thromboelastography in critically ill COVID-19 patients [
76
]. Another
limitation of the study is that the oxidative markers can be altered by the age, gender
and pathological comorbidities which was not contemplated in this work but would be
interesting to address in a future investigation.
5.2. Perspectives
According to the results of our study, the use of a combined therapy with antioxidants
such as NAC and metformin could attenuate SARS-CoV-2 infection in patients with COVID-
19, as has already been shown in various studies [11].
Author Contributions:
I.P.-T., M.E.S. and V.G.-L. designed the study and wrote the manuscript.
V.G.-L
. revised and structured the manuscript. I.P.-T. and M.E.S. designed the laboratory determina-
tions, the tables, performed, and planned the statistical analysis, contributed to the conceptualization
of the project, methodology and statistical analysis. E.D.-D. designed and made glucose, insulin,
HOMA index and detection of N and S proteins antibodies. L.M.-P. designed and made the graphical
abstract and Selenium determination. A.P.-C., R.R.V.-V., A.A.-Á. and H.S.-O. treated and recruited
the patients in the intensive care unit and collected the biochemical results. All authors have read
and agreed to the published version of the manuscript.
Funding:
This work was supported by Consejo Nacional de Ciencia y Tecnologia (CONACYT)
México, Project Number 312167.
Institutional Review Board Statement:
The studies involving human participants were reviewed
and approved by ethical approval from the local ethics committee on 19 August 2020 (Control-
9867/2020, register REG. CONBIOETICA-09CEI-011-20160627). A written informed consent for
enrollment or consent to use patient data was obtained from each patient or their legal surrogate. The
protocol was registered (TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT 04570254).
Informed Consent Statement:
The patients/participants provided their written informed consent
to participate in this study. Written informed consent was obtained from the individual(s) for the
publication of any potentially identifiable images or data included in this article.
Data Availability Statement:
The datasets generated and analyzed during the current study are
available from the corresponding author on reasonable request.
Cells 2022,11, 932 20 of 23
Acknowledgments:
Thank to Instituto Nacional de Cardiología “Ignacio Chávez” for the payment
for the open access to this paper.
Conflicts of Interest:
The authors declare that they have no known competing financial interest or
personal relationships that could have appeared to influence the work reported in this paper.
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