Preliminary Findings on the Association of the Lipid Peroxidation Product 4-Hydroxynonenal with the Lethal Outcome of Aggressive COVID-19

Abstract

Major findings of the pilot study involving 21 critically ill patients during the week after admission to the critical care unit specialized for COVID-19 are presented. Fourteen patients have recovered, while seven passed away. There were no differences between them in respect to clinical or laboratory parameters monitored. However, protein adducts of the lipid peroxidation product 4-hydroxynonenal (HNE) were higher in the plasma of the deceased patients, while total antioxidant capacity was below the detection limit for the majority of sera samples in both groups. Moreover, levels of the HNE-protein adducts were constant in the plasma of the deceased patients, while in survivors, they have shown prominent and dynamic variations, suggesting that survivors had active oxidative stress response mechanisms reacting to COVID-19 aggression, which were not efficient in patients who died. Immunohistochemistry revealed the abundant presence of HNE-protein adducts in the lungs of deceased patients indicating that HNE is associated with the lethal outcome. It seems that HNE was spreading from the blood vessels more than being a consequence of pneumonia. Due to the limitations of the relatively small number of patients involved in this study, further research on HNE and antioxidants is needed. This might allow a better understanding of COVID-19 and options for utilizing antioxidants by personalized, integrative biomedicine approach to prevent the onset of HNE-mediated vitious circle of lipid peroxidation in patients with aggressive inflammatory diseases.

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antioxidants
Article
Preliminary Findings on the Association of the Lipid
Peroxidation Product 4-Hydroxynonenal with the Lethal
Outcome of Aggressive COVID-19
Neven Žarkovi´c 1,* , Biserka Orehovec 2, Lidija Milkovi´c 1, Bruno Barši´c 2, Franz Tatzber 3,
Willibald Wonisch 3, Marko Tarle 2, Marta Kmet 2, Ana Matai´c 4, Antonia Jakovˇcevi´c 4, Tea Vukovi´c 1,
Danijela Tali´c 1, Georg Waeg 5, Ivica Lukši´c 2,6, Elzbieta Skrzydlewska 7and Kamelija Žarkovi´c 4,6


Citation: Žarkovi´c, N.; Orehovec, B.;
Milkovi´c, L.; Barši´c, B.; Tatzber, F.;
Wonisch, W.; Tarle, M.; Kmet, M.;
Matai´c, A.; Jakovˇcevi´c, A.; et al.
Preliminary Findings on the
Association of the Lipid Peroxidation
Product 4-Hydroxynonenal with the
Lethal Outcome of Aggressive
COVID-19. Antioxidants 2021,10,
1341. https://doi.org/10.3390/
antiox10091341
Academic Editors:
Dimitrios Kouretas,
Konstantinos Poulas,
Konstantinos Farsalinos and
Joanna Floros
Received: 15 July 2021
Accepted: 24 August 2021
Published: 25 August 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
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iations.
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1Laboratory for Oxidative Stress (LabOS), Ru ¯
der Boškovi´c Institute, 10000 Zagreb, Croatia;
Lidija.Milkovic@irb.hr (L.M.); Tea.Vukovic@irb.hr (T.V.); Danijela.Talic@irb.hr (D.T.)
2Clinical Hospital Dubrava, 10000 Zagreb, Croatia; biserka.orehovec@gmail.com (B.O.);
barsicbruno@gmail.com (B.B.); tarlemarko1@gmail.com (M.T.); zmarta.km@gmail.com (M.K.);
luksic.ivica@gmail.com (I.L.)
3
Omnignostica Ltd., 3421 Höflein an der Donau, Austria; franz@tatzber.at (F.T.); willi.wonisch@aon.at (W.W.)
4Clinical Hospital Centre Zagreb, 10000 Zagreb, Croatia; ana.mataic@gmail.com (A.M.);
antonia.jakovcevic@gmail.com (A.J.); kamelijazarkovic@gmail.com (K.Ž.)
5Institute of Molecular Biosciences, Karl Franzens University, 8010 Graz, Austria; georg.waeg@uni-graz.at
6University of Zagreb School of Medicine, 10000 Zagreb, Croatia
7
Department of Inorganic and Analytical Chemistry, Medical University of Bialystok, 15-089 Bialystok, Poland;
elzbieta.skrzydlewska@umb.edu.pl
*Correspondence: zarkovic@irb.hr; Tel.: +385-1-4571212
Abstract:
Major findings of the pilot study involving 21 critically ill patients during the week after
admission to the critical care unit specialized for COVID-19 are presented. Fourteen patients have
recovered, while seven passed away. There were no differences between them in respect to clinical
or laboratory parameters monitored. However, protein adducts of the lipid peroxidation product
4-hydroxynonenal (HNE) were higher in the plasma of the deceased patients, while total antioxidant
capacity was below the detection limit for the majority of sera samples in both groups. Moreover,
levels of the HNE-protein adducts were constant in the plasma of the deceased patients, while in
survivors, they have shown prominent and dynamic variations, suggesting that survivors had active
oxidative stress response mechanisms reacting to COVID-19 aggression, which were not efficient in
patients who died. Immunohistochemistry revealed the abundant presence of HNE-protein adducts
in the lungs of deceased patients indicating that HNE is associated with the lethal outcome. It seems
that HNE was spreading from the blood vessels more than being a consequence of pneumonia. Due
to the limitations of the relatively small number of patients involved in this study, further research on
HNE and antioxidants is needed. This might allow a better understanding of COVID-19 and options
for utilizing antioxidants by personalized, integrative biomedicine approach to prevent the onset of
HNE-mediated vitious circle of lipid peroxidation in patients with aggressive inflammatory diseases.
Keywords:
COVID-19; oxidative stress; lipid peroxidation; 4-hydroxynonenal (HNE); inflamma-
tion; antioxidants; peroxides; free radicals; reactive oxygen species (ROS); blood vessels; lungs;
immunohistochemistry; HNE-ELISA
1. Introduction
First cases of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory
syndrome coronavirus 2 (SARS-CoV-2) infection, were reported at the very end of the year
2019. In 2020, the virus has spread worldwide, causing pandemics as never seen before.
Since the COVID-19 outbreak, scientists are trying to understand the pathology of the
disease in order to improve the patient’s outcome. While only a few drugs were approved
Antioxidants 2021,10, 1341. https://doi.org/10.3390/antiox10091341 https://www.mdpi.com/journal/antioxidants

Antioxidants 2021,10, 1341 2 of 10
by the Food and Drug Administration as appropriate to treat patients with COVID-19, the
first vaccines are implemented worldwide according to the urgent criteria, occasionally
causing inexplicable side effects. Major complications of severe COVID-19 infection include
acute respiratory distress syndrome (ARDS), sepsis, and multiple organ dysfunction or
failure (MOF) [
1
]. Each of these severe disorders is associated with inflammation and
excessive generation of reactive oxygen species (ROS), affecting body redox balance, thus
causing oxidative damage of macromolecules under the pathophysiological process of
oxidative stress (OS). Therefore, the potential use of antioxidant treatments to prevent
COVID-19 progression have been already suggested [2–4].
One of the underlying mechanisms of organ damage in COVID-19 could be mitochon-
drial dysfunctions resulting in excessive production of ROS, triggered by inflammatory
response to SARS-CoV-2 infection, hence resembling other types of sepsis characterized by
severe inflammation [
5
]. There are various pathophysiological sources of OS, causing either
acute or chronic disbalance of pro- and anti-oxidants, many of which do not need to result
in severe health disorders unless they induce lipid peroxidation (LPO). Namely, under such
aggressive OS, polyunsaturated fatty acids (PUFAs, notably
ω
-6 fatty acids or n-6 fatty
acids, especially arachidonic and linoleic acid) in cellular biomembranes become targets for
ROS-induced damage, triggering a self-catalyzed, non-enzymatic chain reaction of LPO.
Thus, damaged cells in the affected tissues, together with erythrocytes in blood, generate
ROS, and release of free iron, which can consequently amplify LPO through Fenton reac-
tions and promote LPO and ferroptosis [
6
], resulting in membrane dysfunction/destruction
and MOF in COVID-19 patients [
7
]. Indeed, the autopsy findings in COVID-19 patients
revealed a ferroptosis signature in the epicardium and myocardium in the area of severe
SARS-CoV-2 myocarditis associated with generalized lipid peroxidation [
8
]. Similarly, a
pilot study on oxidative stress in COVID-19 patients hospitalized in an intensive care unit
(ICU) for severe pneumonia has indicated systemic OS in critically ill COVID-19 patients
manifested by increased LPO and deficits of antioxidants [9].
Final products of LPO are reactive aldehydes that have a longer lifetime (minutes
to hours) than is the lifetime of ROS (nano to milliseconds) and might accordingly rep-
resent better biomarkers of SARS-CoV-2 induced OS. Among the LPO-derived reactive
aldehydes, the most bioactive seems to be 4-hydroxynonenal (HNE). Namely, HNE acts
as “second messenger of free radicals”, spreading OS and LPO even in the absence of
ROS, accumulating in blood vessels in an age-dependent manner [
10
,
11
]. The affinity of
HNE to bind to proteins, modifying their structure and function and acting dynamically
in a concentration-dependent manner not only as a cytotoxic product of LPO, but also
as a signaling, regulatory molecule, makes HNE a valuable bioactive marker for various
diseases associated with OS [
12
,
13
] and their respective therapies [
14
], suggesting it could
also be a predictive biomarker of SARS-CoV-2 induced OS. In favor of our assumption
are recent autopsy findings of HNE in COVID-19 patient [
8
] and a positive correlation
of HNE expression with the functional receptor of SARS-CoV-2, angiotensin-converting
enzyme 2 (ACE2), also suggesting significant involvement of this particular LPO product
in COVID-19 pathogenesis [15].
Therefore, to test our hypothesis that the onset of the vicious circle of LPO mediated by
HNE might be relevant for the pathogenesis of COVID-19 and its outcome, we used genuine
enzyme-linked immunosorbent assay (ELISA) specific for the HNE-protein adducts in
the blood of critically ill patients who survived and those how were killed by aggressive
COVID-19. The same monoclonal antibody used for the HNE-ELISA was eventually used
for immunohistochemistry of lungs of some deceased COVID-19 patients.
2. Materials and Methods
2.1. Patients
This study was done upon the ethical approval 2020-1012-13 of the Clinical Hospital
Dubrava in Zagreb, serving as the national center for COVID-19, thus, providing medical
care for patients suffering from the most aggressive COVID-19. Only patients who signed

Antioxidants 2021,10, 1341 3 of 10
the informed written consent and who were hospitalized at the ICU for the period long
enough to collect three samples of blood starting on the first day after admission, followed
by the blood sampling every second day afterward.
Of the 21 patients involved (11 men and 10 women of average age 65.3
±
14.6 years),
fourteen patients have recovered, while seven died. Although survivors were on the
average ten years younger than patients who died (62.0
±
14.7 years vs. 71.9
±
12.4),
there was no significant difference between them in respect to age (p> 0.1), comorbidities
(3 patients had diabetes mellitus type 2, one passed away, while two survived), clinical
treatment or laboratory parameters monitored.
2.2. Laboratory Analysis
For the analysis of biochemical and immunochemical parameters, samples of blood
were centrifuged at 1370
×
gfor 10 min and sera were analysed immediately after centrifu-
gation or were stored at −80 ◦C to be analysed afterwards.
Leukocytes were counted in EDTA-anticoagulated blood on the DxH 800 Hematology
Analyzer (Beckman Coulter, Tokyo, Japan). Concentrations of C-reactive protein (CRP)
and ferritin were measured using immunoturbidimetric assays on the AU 5800 analyzer
(Beckman Coulter, Tokyo, Japan). Lactate dehydrogenase, EC 1.1.1.27 (LDH) activity was
measured using a kinetic UV test on AU 5800 analyzer (Beckman Coulter, Tokyo, Japan).
Levels of interleukin 6 (IL-6) and Procalcitonin (PCT) were measured using chemilumines-
cent immunoassays on the UniCel DxI 800 Access Immunoassay System (Beckman Coulter,
Tokyo, Japan).
An enzymatic assay was used to test ROS scavenging of sera using uric acid standards
as described before [
16
]. In parallel, a complementary assay for total serum peroxides was
done [
16
] using hydrogen peroxide standards. However, only results for serum peroxide
levels were obtained, because for the majority of sera samples, total antioxidant capacity
was below the detection limit equivalent to 11.7
µ
M of the uric acid standard. Sera samples
were also used to determine the titer of the autoantibodies against oxidized low-density
lipoproteins (LDL) using the oLAB ELISA as described before [17].
Parallel to the sera sampling, the EDTA plasma samples were prepared for the HNE-
ELISA analysis done as described before [
18
,
19
] using a genuine monoclonal antibody
specific for the HNE-histidine (HNE-His) epitopes.
2.3. Immunohistochemistry
Finally, immunohistochemical analysis using the same monoclonal antibody, as in the
case of the HNE-ELISA, was done for the tissue specimens obtained by autopsy of patients
that eventually passed away at the University Hospital for Infectious Diseases, Zagreb.
Formalin-fixed, paraffin-embedded tissue specimens of lungs were analyzed by pathol-
ogists with expertise in the field, as described before [
13
,
20
]. The 3,3
0
-Diaminobenzidine
tetrahydrochloride (DAB) was used as chromophore giving brown colored reaction in case
of positive immunohistochemical reaction for the HNE-His, with blue-colored contrast
staining of hematoxylin.
2.4. Statistics
Comparison of the mean values of the HNE-protein adducts in plasma between
the two groups of patients was carried out using the t-test, the differences between the
incidence of patients with static levels and those with dynamic changes of the HNE levels
were compared between survivors and the deceased patients by chi-square test, while
correlations between biomarkers analysed in comparison to the HNE levels in plasma were
done by the Pearson Rtest.
3. Results
The analysis of HNE levels in the EDTA-plasma of patients analyzed by the gen-
uine HNE-ELISA using a non-commercial monoclonal antibody specific for the HNE-His

Antioxidants 2021,10, 1341 4 of 10
epitopes revealed significantly higher (p< 0.05) average plasma values of HNE-protein
adducts in the blood of the deceased patients than in the blood of survivors for the first
days (1–3) in hospital (Figure 1).
Antioxidants 2021, 10, x FOR PEER REVIEW 4 of 10
correlations between biomarkers analysed in comparison to the HNE levels in plasma
were done by the Pearson R test.
3. Results
The analysis of HNE levels in the EDTA-plasma of patients analyzed by the genuine
HNE-ELISA using a non-commercial monoclonal antibody specific for the HNE-His
epitopes revealed significantly higher (p < 0.05) average plasma values of HNE-protein
adducts in the blood of the deceased patients than in the blood of survivors for the first
days (1–3) in hospital (Figure 1).
Figure 1. Values of the HNE-protein adducts determined in the EDTA-plasma of COVID-19 patients by the genuine HNE-
ELISA specific for the HNE-His adducts. The amounts of the HNE are presented in pmol/mg protein values, while the
asterisk indicates a significant difference (p < 0.05) between the average values determined for the plasma of survivors and
for the deceased COVID-19 patients.
However, values of HNE-His detected in the plasma of survivors slightly increased
on the fifth day, reaching the levels that were not significantly different from the values
of the deceased patients indicating that the high levels of the HNE-protein adducts in
plasma were not the only HNE-related parameter predicting the lethal outcome of aggres-
sive COVID-19. Namely, the individual levels of HNE determined at different time points
in the blood of patients have shown that the levels of HNE were stable in the blood of
patients who died, but have shown dynamic variations in the blood of survivors revealing
significantly different patterns between the two groups (chi-square p < 0.01). Namely, 6/7
patients who passed away did not show prominent variations of the HNE levels in the
Figure 1.
Values of the HNE-protein adducts determined in the EDTA-plasma of COVID-19 patients by the genuine
HNE-ELISA specific for the HNE-His adducts. The amounts of the HNE are presented in pmol/mg protein values, while
the asterisk indicates a significant difference (p< 0.05) between the average values determined for the plasma of survivors
and for the deceased COVID-19 patients.
However, values of HNE-His detected in the plasma of survivors slightly increased
on the fifth day, reaching the levels that were not significantly different from the values of
the deceased patients indicating that the high levels of the HNE-protein adducts in plasma
were not the only HNE-related parameter predicting the lethal outcome of aggressive
COVID-19. Namely, the individual levels of HNE determined at different time points
in the blood of patients have shown that the levels of HNE were stable in the blood of
patients who died, but have shown dynamic variations in the blood of survivors revealing
significantly different patterns between the two groups (chi-square p< 0.01). Namely,
6/7 patients who passed away did not show prominent variations of the HNE levels in the
blood collected at different time points (as an exemption, we consider the 3rd patient from
the left side of the respective upper graph, who had a transient decrease of HNE levels in
the blood on the third day), while relatively stable, although gradually decreasing levels of
HNE were observed only in 1/14 survivors (the 5th patient from the left side on the bottom
graph). Hence, these findings suggest that survivors had active oxidative stress response
mechanisms reacting to the COVID-19 aggression, which were not efficient in the patients
who died.

Antioxidants 2021,10, 1341 5 of 10
The individual variations of the HNE levels in the blood were compared using Pearson
correlation with all available physical and laboratory parameters of the patients, but due
to the relatively small number of patients per group, the R values were significant for the
deceased patients only for HNE and IL-6 (R-0.83, p< 0.05) on the first day after admission to
the ICU. Although trends of differentially positive vs. negative correlations were observed
at different time points for other parameters between survivors and the deceased patients,
due to the lack of statistical significance, these trends are not presented.
However, because of possible high importance, we wish to say that the persistent
negative correlation trend between the HNE levels in the blood and the levels of the au-
toantibodies against oxidized LDL (oLAB) was observed only in the blood of the deceased
patients, while for survivors significantly positive correlation (R 0.54, p< 0.05) was ob-
served on the last day of evaluation, followed by the recovery from COVID-9. Similarly,
a trend of negative correlation was also observed between the HNE levels and the total
peroxide levels in sera of the patients who died but were gradually lost in the terminal
stage (day 5), while such a trend was not observed for survivors.
It should be mentioned here that the complementary assay for the total antioxidant
capacity was also carried out, but the levels of antioxidants were too low to be determined
(bellow detection limit) for the majority of samples in both groups, suggesting again that
OS is one of the major pathogenic factors in aggressive COVID-19, for which patients
needed additional oxygen support at ICU.
Microphotographs presented in Figure 2show the immunohistochemical appearance
of HNE-His adducts in the lungs of the deceased COVID-19 patient.
Antioxidants 2021, 10, x FOR PEER REVIEW 5 of 10
blood collected at different time points (as an exemption, we consider the 3rd patient from
the left side of the respective upper graph, who had a transient decrease of HNE levels in
the blood on the third day), while relatively stable, although gradually decreasing levels
of HNE were observed only in 1/14 survivors (the 5th patient from the left side on the
bottom graph). Hence, these findings suggest that survivors had active oxidative stress
response mechanisms reacting to the COVID-19 aggression, which were not efficient in
the patients who died.
The individual variations of the HNE levels in the blood were compared using Pear-
son correlation with all available physical and laboratory parameters of the patients, but
due to the relatively small number of patients per group, the R values were significant for
the deceased patients only for HNE and IL-6 (R-0.83, p < 0.05) on the first day after admis-
sion to the ICU. Although trends of differentially positive vs. negative correlations were
observed at different time points for other parameters between survivors and the de-
ceased patients, due to the lack of statistical significance, these trends are not presented.
However, because of possible high importance, we wish to say that the persistent
negative correlation trend between the HNE levels in the blood and the levels of the au-
toantibodies against oxidized LDL (oLAB) was observed only in the blood of the deceased
patients, while for survivors significantly positive correlation (R 0.54, p < 0.05) was ob-
served on the last day of evaluation, followed by the recovery from COVID-9. Similarly,
a trend of negative correlation was also observed between the HNE levels and the total
peroxide levels in sera of the patients who died but were gradually lost in the terminal
stage (day 5), while such a trend was not observed for survivors.
It should be mentioned here that the complementary assay for the total antioxidant
capacity was also carried out, but the levels of antioxidants were too low to be determined
(bellow detection limit) for the majority of samples in both groups, suggesting again that
OS is one of the major pathogenic factors in aggressive COVID-19, for which patients
needed additional oxygen support at ICU.
Microphotographs presented in Figure 2 show the immunohistochemical appearance
of HNE-His adducts in the lungs of the deceased COVID-19 patient.
Figure 2. The immunohistochemical appearance of HNE in the lungs of the deceased COVID-19
patient. Eosinophilic (HE) liquid of edema in the alveoli (asterisks) shows abundant HNE content
(HNE-His giving brown-colored immunopositive reaction of DAB staining). However, inflamma-
tory mononuclear cells in the alveoli and in their septa (black arrows) are mostly negative for HNE
(only blue-colored hemotoxylin contrast staining is present), with some macrophages are positive
for HNE (yellow arrow). On the contrary, blood vessels (both their walls and the blood content,
indicated by the black stars) are HNE-positive, as are the hyaline membranes on the surface of the
alveolar septa (red arrows). Conclusion—HNE might be responsible for the fatal outcome, but more
Figure 2.
The immunohistochemical appearance of HNE in the lungs of the deceased COVID-19 patient. Eosinophilic (HE)
liquid of edema in the alveoli (asterisks) shows abundant HNE content (HNE-His giving brown-colored immunopositive
reaction of DAB staining). However, inflammatory mononuclear cells in the alveoli and in their septa (black arrows) are
mostly negative for HNE (only blue-colored hemotoxylin contrast staining is present), with some macrophages are positive
for HNE (yellow arrow). On the contrary, blood vessels (both their walls and the blood content, indicated by the black stars)
are HNE-positive, as are the hyaline membranes on the surface of the alveolar septa (red arrows). Conclusion—HNE might
be responsible for the fatal outcome, but more likely due to the systemic, vascular oxidative stress (sepsis-like, originating
from the blood), not due to the pulmonary inflammatory cells, which are mostly negative for HNE.

Antioxidants 2021,10, 1341 6 of 10
Immunohistochemistry was done by the same monoclonal antibody used for the
HNE-His ELISA thus detecting the same epitopes on the proteins modified by HNE due to
the onset of OS in the blood and in the lungs. As can be seen, the cell-free inflammatory
edema in the lungs contained abundant amounts of HNE-protein adducts. Similarly, blood
vessels were loaded with HNE, as were the hyaline membranes suggesting that abundant
HNE might be responsible for the vicious circle of OS in COVID-19, leading to the fatal
outcome. However, pulmonary OS was probably not caused by an oxidative burst of
inflammatory cells in the lungs, which were mostly negative for HNE, but was a crucial
component of systemic OS spreading HNE by blood through the entire organism.
4. Discussion
While excessive inflammation plays an important role in aggressive COVID-19, the
relevance of this generated oxidative stress is uncertain, mostly due to the short lifetime of
free radicals and the complexity of the disease itself [
21
–
23
]. On the other hand, reactive
aldehyde HNE, the particular LPO product of PUFAs, which regulates the sensitivity of cells
to OS and their adaptation acting as the second messenger of free radicals and a signaling
molecule, has a longer lifetime mostly because of high affinity for binding to proteins [
24
,
25
].
This pilot study revealed the abundant presence of the HNE-protein adducts in the lungs
of patients who died from aggressive COVID-19. The HNE-protein adducts were also
higher in the blood of these patients than in the blood of the equally-treated critically
ill patients who survived COVID-19 even five days before the death/recovery and were
correlated with the levels of the oxygen saturation, inflammatory parameters, and the
other parameters of oxidative stress analyzed. Due to the relatively small size of the group
of deceased patients (n= 7), the only significant correlation in the group of the deceased
patients was observed between HNE and IL-6 levels upon admission to the ICU and was
strongly negative (R-0.83). This is not surprising because HNE can act as a signaling
molecule suppressing IL-6 production [
26
], but it is worth mentioning that it was only
transient and was only observed for the patients who eventually died from COVID-19.
In spite of high levels of R, which were mostly not significant due to the relatively small
number of patients in the pilot study, obvious trends were observed showing a lot of
differences between patients who survived and patients who died, which deserve further
studies involving more patients.
Among other aspects of COVID-19 related OS and LPO further, studies should evalu-
ate the immune response to oxidatively modified proteins, as we have observed a trend of
negative correlation between HNE-protein adducts and the titers of autoimmune antibod-
ies generated against oxidized LDL, only in the patients who died. Although HNE-ELISA
determines mostly HNE bound to histidine residues of serum albumin, the assay also
detects HNE-His present on the other sera proteins [
19
,
27
]. Since HNE-His is also a major
epitope of the oxidized LDL [
27
], our results suggest the involvement of the immune sys-
tem in the fight with toxic mediators of OS, notably HNE in COVID-19 patients. Moreover,
the observed constantly negative trend of correlation between HNE-ELISA and oLAB
assays in non-survivors might be interpreted as exhaustion of the immune system, which
cannot produce specific anti-OS-IgG in sufficient amounts and should be further studied.
On the other hand, HNE is also binding to the histidine moiety of angiotensin [
27
], which,
together with the fact that HNE interferes with bioactivities of angiotensin [
28
,
29
] and is
positively correlated with the ACE2 [
15
], increase the need for further research on HNE in
the pathogenesis of COVID-19.
Immunohistochemistry revealed abundant HNE-protein adducts, especially in the
blood vessels and inflammatory edema of the lungs affected by COVID-19. Surprisingly,
inflammatory, mononuclear cells in the alveoli and in their septa were HNE-negative
in spite of the fact that for inflammatory cells, HNE acts as a signaling molecule and
pleiotropic regulatory factor, while macrophages, which were only exceptionally HNE-
positive, are otherwise known to generate ROS and abundant HNE [
30
,
31
]. These findings
suggest systemic, vascular oxidative stress in COVID-19 patients and the spread of HNE in

Antioxidants 2021,10, 1341 7 of 10
the form of protein adducts by blood. That is in agreement with findings of toxic HNE-
protein adducts accumulation in atherosclerotic blood vessels, proportional to the age, but
in potentially reversible form of histidine adducts [
11
,
32
,
33
]. Although in our pilot study
there was no significant difference between the age or patients who survived and those
who died, it should be mentioned that survivors were, on average, ten years younger. Our
previous research on the atherosclerotic aorta revealed that the accumulation of the HNE-
protein adducts reaches its maximum around the age of 65 [
11
], which was the average
age of our patients taken together. Hence, we can hypothesize that the age of patients
might play an important role in the survival of COVID-19 also due to the age-dependent
accumulation of HNE in the wall of blood vessels, while the possible release of toxic HNE
from the blood vessels into other tissues should be further studied, especially because
the similar accumulation of the blood-originating HNE was also revealed in abdominal
adipose tissue of obese people with metabolic syndrome [
34
]. This assumption deserves
further research, which could increase our knowledge on the oxidative stress response not
only to COVID-19 aggression but also about other stress- and age-associated diseases for
which HNE is known to be an important factor of pathogenesis or hormesis [35–38].
Finally, it should be mentioned that trend of negative correlation of HNE in the
blood and the total serum peroxides observed in our study associated with undetectable
serum antioxidant capacity (according to the TAC assay) of COVID-19 patients resembles
findings of other studies and points to the high relevance of antioxidants in defense against
aggressive COVID-19. Among potential antioxidants present in tissues, including blood,
uric acid is often neglected, although it contributes a lot to the TAC analysis of sera, for
which we even used uric acid as standard. Although we did not evaluate urate levels
in the blood of our patients, further studies should do that with respect to the possible
onset of ischemic/reperfusion injury that might cause systemic oxidative stress generating
uric acid.
5. Conclusions
If further studies confirm our preliminary findings, novel options for the introduction
of an integrative biomedicine approach to COVID-19 and other severe inflammatory
diseases could be developed based on personalized medicine and the careful use of the most
promising antioxidants that could regulate HNE metabolism and its bioactivities. It is likely
that such options will include the use of lipid-soluble antioxidants to prevent the onset
of LPO, such as tocopherol and lycopene, but together with water-soluble antioxidants,
such as vitamin C, micronutrients needed to maintain physiological functions of enzymatic
antioxidants (such as zinc and selenium) and eventually HNE-scavengers resembling GSH—
the dominant physiological scavenger of HNE, such as those based on N-acetylcysteine,
together with other credible natural or synthetic regulators of OS and LPO in particular.
Author Contributions:
Conceptualization, N.Ž., K.Ž., B.O. and B.B.; methodology, L.M., T.V., D.T.,
M.K., M.T., G.W., A.J. and A.M.; software, L.M.; validation, F.T., W.W. and L.M.; formal analysis, N.Ž.,
E.S., I.L. and K.Ž.; investigation, B.B., M.T., B.O., M.K. and L.M.; resources, E.S., I.L., N.Ž., F.T. and
W.W.; data curation, N.Ž., B.O. and L.M.; writing—original draft preparation, N.Ž.; writing—review
and editing, N.Ž. L.M., F.T., B.B., B.O., W.W. and K.Ž.; visualization, N.Ž., K.Ž. and L.M.; supervision,
N.Ž. and K.Ž.; project administration, N.Ž., E.S., I.L. and K.Ž.; funding acquisition, E.S., N.Ž., I.L., F.T.
and W.W. All authors have read and agreed to the published version of the manuscript.
Funding:
This research was supported by the Polish National Agency for Academic Exchange NAWA
as a part of the International Academic Partnerships (PPI/APM/2018/00015/U/001) and by core
funding of the Rudjer Boskovic Institute.
Institutional Review Board Statement:
The study was conducted according to the guidelines of the
Declaration of Helsinki and approved by the Institutional Review Board of the Clinical Hospital
Dubrava in Zagreb by ethical approval 2020-1012-13.
Informed Consent Statement:
Informed consent was obtained from all subjects involved in the study.
Data Availability Statement: Data is contained within the article.

Antioxidants 2021,10, 1341 8 of 10

Antioxidants 2021,10, 1341 9 of 10
Acknowledgments:
Kind support in materials by Omnignostica is acknowledged. The panelist of
the HrZZ call IP-CORONA-2020-12 denied financial support for this research imposing its realization
by the pilot study presented. The authors are grateful to the reviewers for supportive, constructive,
and encouraging comments on this paper.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
Coronavirus disease 2019 (COVID-19); 4-Hydroxynonenal (HNE); severe acute respi-
ratory syndrome coronavirus 2 (SARS-CoV-2); acute respiratory distress syndrome (ARDS);
multiple organ dysfunction or failure (MOF); oxidative stress (OS); reactive oxygen species
(ROS); lipid peroxidation LPO); polyunsaturated fatty acids (PUFAs); low-density lipopro-
teins (LDL); enzyme-linked immunosorbent assay (ELISA); angiotensin-converting en-
zyme 2 (ACE2); HNE-histidine (HNE-His); intensive care unit (ICU); interleukin 6 (IL-6);
immunoglobulin G (IgG), hematoxylin/eosin staining (HE); 3,3
0
-Diaminobenzidine stain-
ing (DAB).
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