Abstract and Figures

The airway epithelial glycocalyx plays an important role in preventing severe acute respiratory syndrome coronavirus 2 entry into the epithelial cells, while the endothelial glycocalyx contributes to vascular permeability and tone, as well as modulating immune, inflammatory, and coagulation responses. With ample evidence in the scientific literature that coronavirus disease 2019 (COVID19) is related to epithelial and endothelial dysfunction, preserving the glycocalyx should be the main focus of any COVID-19 treatment protocol. The most studied functional unit of the glycocalyx is the glycosaminoglycan heparan sulfate, where the degree and position of the sulfate groups determine the biological activity. N-acetylcysteine (NAC) and other sulfur donors contribute to the inorganic sulfate pool, the rate-limiting molecule in sulfation. NAC is not only a precursor to glutathione but also converts to hydrogen sulfide, inorganic sulfate, taurine, Coenzyme A, and albumin. By optimising inorganic sulfate availability, and therefore sulfation, it is proposed that COVID-19 can be prevented or at least most of the symptoms attenuated. A comprehensive COVID19 treatment protocol is needed to preserve the glycocalyx in both the prevention and treatment of COVID-19. The use of NAC at a dosage of 600 mg bid for the prevention of COVID-19 is proposed, but a higher dosage of NAC (1200 mg bid) should be administered upon the first onset of symptoms. In the severe to critically ill, it is advised that IV NAC should be administered immediately upon hospital admission, and in the late stage of the disease, IV sodium thiosulfate should be considered. Doxycycline as a protease inhibitor will prevent shedding and further degradation of the glycocalyx.
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Review Article
N-Acetylcysteine and Other Sulfur-Donors as a Preventative and
Adjunct Therapy for COVID-19
Heidi N du Preez ,
1
Colleen Aldous ,
2
Hendrik G Kruger ,
1
and Lin Johnson
3
1
Catalysis and Peptide Research Unit, University of KwaZulu-Natal, Westville Campus, Durban, South Africa
2
College of Health Sciences, University of KwaZulu-Natal, Durban, South Africa
3
School of Life Sciences, University of KwaZulu-Natal, Durban, South Africa
Correspondence should be addressed to Heidi N du Preez; hdp@heididupreez.com
Received 7 April 2022; Accepted 7 July 2022; Published 10 August 2022
Academic Editor: Benedetto Natalini
Copyright ©2022 Heidi N du Preez et al. is is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
e airway epithelial glycocalyx plays an important role in preventing severe acute respiratory syndrome coronavirus 2 entry into
the epithelial cells, while the endothelial glycocalyx contributes to vascular permeability and tone, as well as modulating immune,
inflammatory, and coagulation responses. With ample evidence in the scientific literature that coronavirus disease 2019 (COVID-
19) is related to epithelial and endothelial dysfunction, preserving the glycocalyx should be the main focus of any COVID-19
treatment protocol. e most studied functional unit of the glycocalyx is the glycosaminoglycan heparan sulfate, where the degree
and position of the sulfate groups determine the biological activity. N-acetylcysteine (NAC) and other sulfur donors contribute to
the inorganic sulfate pool, the rate-limiting molecule in sulfation. NAC is not only a precursor to glutathione but also converts to
hydrogen sulfide, inorganic sulfate, taurine, Coenzyme A, and albumin. By optimising inorganic sulfate availability, and therefore
sulfation, it is proposed that COVID-19 can be prevented or at least most of the symptoms attenuated. A comprehensive COVID-
19 treatment protocol is needed to preserve the glycocalyx in both the prevention and treatment of COVID-19. e use of NAC at
a dosage of 600 mg bid for the prevention of COVID-19 is proposed, but a higher dosage of NAC (1200mg bid) should be
administered upon the first onset of symptoms. In the severe to critically ill, it is advised that IV NAC should be administered
immediately upon hospital admission, and in the late stage of the disease, IV sodium thiosulfate should be considered.
Doxycycline as a protease inhibitor will prevent shedding and further degradation of the glycocalyx.
1. Introduction
e pandemic surrounding coronavirus disease 2019
(COVID-19), caused by severe acute respiratory syndrome
coronavirus 2 (SARS-CoV-2) infection, has been associated
with immense economic and social disruption around the
globe, apart from high mortality rates. More than two years
later, there is still a need to find effective therapeutic solutions
to prevent and treat established disease. Because of their
various physiological functions in viral infection and in-
flammation, the use of N-acetylcysteine (NAC) and sodium
thiosulfate (STS) [1, 2] was recommended in the very early
stages of the pandemic, by the authors and various other
scientists, for both prevention and treatment of COVID-19. It
is important to understand the science underlying these
recommendations, which will be explained in this review.
We have recently reviewed the pathogenesis of COVID-
19 [3] that provides, inter alia, a comprehensive overview of
sulfation of the glycocalyx (GL), as well as highlighting the
importance of an intact sulfated GL for natural protection
against SARS-CoV-2 infection [3, 4]. We provide a short
summary here of our undersulfation hypothesis, so that the
remainder of the manuscript (treatment of COVID-19) may
be understood better. GL will refer to both the epithelial
glycocalyx (EpGL) and endothelial glycocalyx (EnGL), un-
less specified.
Since the GL surrounds all eukaryotic cells, the EpGL
serves as the interface between our internal cells and the
Hindawi
Advances in Pharmacological and Pharmaceutical Sciences
Volume 2022, Article ID 4555490, 21 pages
https://doi.org/10.1155/2022/4555490
external world. It therefore serves as the first defence of the
innate immune system, and if compromised, we are vul-
nerable to the onslaught of various pathogens, viruses, and
environmental toxins [3]. e GL is a dense layer of proteins
and carbohydrate chains that forms a mesh that extends into
the extracellular matrix and is involved in many critical bi-
ological processes. Composed of membrane-bound glyco-
proteins, proteoglycans (PGs), and highly sulfated
glycosaminoglycan (GAG) side-chains (Figure 1), the GL is
particularly essential for barrier functions [5], as well as
modulating immunity [3], inflammation [6, 7], and coagu-
lation [811]. As a consequence of their interaction with
multiple proteins, cell surface heparan sulfate (HS) PGs
(HSPGs) are involved in developmental, regeneratory, in-
fectious, and inflammatory processes [12]. It is important to
note that the density and position of sulfate groups on the HS
structure will mainly determine these HS-protein interactions
[3, 13], and therefore the biological activity of sulfation.
Sulfation occurs in all tissues and involves the transfer of
a sulfate group to various substrates, such as proteins, GAGs,
lipids, hormones, various drugs, and toxins [14–18], [3, 19].
e availability of inorganic sulfate in the host was identified
as the rate-limiting factor to sulfation [18, 20, 21]. e degree
of sulfation and the trans-sulfuration pathway [22–24] is
involved in many aspects of health, since it controls such a
wide range of substances. e intact GL layer consists of
many highly sulfated GAG chains, providing a negative
charge density for the epithelial (Ep) and endothelial (En)
surface layer [3, 25], and binding capacity for a variety of
positively charged protein ligands [26]. e negative charge,
mainly due to the presence of sulfate groups, is critical to
biological and barrier function, especially in preventing the
binding of negatively charged pathogens [3]. Changes in the
charge density, and thus the degree of GAG sulfation, are
often associated with various disorders [3, 27]. While dif-
ferent tissues and cell types vary in HS structure, the
structure of HS can vary between individuals and with age. It
seems evident that these differences in HS composition may
contribute to tissue tropism and/or host susceptibility to
infection by viruses and various other pathogens [3, 27–29].
Pre-COVID-19 vaccination, the elderly were most sus-
ceptible to SARS-CoV-2, where the typical profile of the
critically ill patient was 65 years and older, presenting with
comorbidities and ARDS [30, 31]. e main comorbidities
related to COVID-19 were preexisting lung, heart and
kidney disease, diabetes mellitus (DM) [32], obesity [33, 34],
cancer, and cerebrovascular pathologies [35]. Dietary fac-
tors, such as low vitamin D and glutathione status, also
increase susceptibility to COVID-19 [36, 37]. Today, more
than two years later, immunosuppression in a younger
population group seems to be a major predisposing factor
for SARS-CoV-2 infection [38, 39]. Accumulating research
relates dysfunction of the GL to ageing and these COVID-19
comorbid conditions [40, 41], which are also associated with
chronic inflammation [3, 7, 34] and oxidative stress [42].
is highlights the probability that a disrupted GL is the
main predisposing factor to COVID-19 susceptibility [3, 43].
As evident in COVID-19-related comorbid conditions,
oxidative stress and chronic inflammation result in
attenuation of the GL [7, 34, 42]. Moreover, En dysfunction
increases systemic inflammation and oxidative stress, deg-
radation of GL, and induces a procoagulant and anti-
fibrinolytic state [35].
Since we are all exposed to SARS-CoV-2, the main
question is why more than 80% of the world population did
not become infected before the introduction of the COVID-
19 vaccine, were asymptomatic, or showed only very mild
symptoms [44–46], compared to the small percentage of
people that became severely ill [22, 47]. e variable factor
does not seem so much the expression of angiotensin
converting enzyme 2 (ACE2) receptors, but rather the de-
gree of GAG sulfation, which will affect the integrity of the
GL, increasing susceptibility to SARS-CoV-2 infection and
attenuating the ability to modulate inflammatory and
coagulatory responses when the GL is undersulfated [3].
ACE2 expression is low in a well-preserved sulfated GL,
which acts as a structural barrier [4]. erefore, it is evident
that the GL’s structural and functional properties play a
critical role in the pathogenesis of COVID-19. In this review,
we will highlight the treatment strategies to best preserve the
GL, with an emphasis on NAC and other sulfur donors as
precursors to inorganic sulfate to support sulfation.
2. Glycocalyx
2.1. Structural Properties of the Glycocalyx (GL). e extra-
cellular matrix that surrounds all cells is a complex external
layer of PGs, glycoproteins bound to sialic acid, GAGs, gly-
colipids, and proteins such as collagen [5, 48]. is glyco-
protein polysaccharide covering is referred to as the GL.
Plasma proteins such as antithrombin (AT) and albumin are
also bound within the EnGL [49]. PGs are core proteins an-
chored to the apical membrane of Ep- and EnCs, with several
GAG side-chains attached covalently to the PGs (Figure 1).
Glycosaminoglycan chains that bind to PGs are long
linear, hydrophilic [50], negatively charged polysaccharides
(PSs), namely, HS, chondroitin sulfate, dermatan sulfate,
keratan sulfate, and nonsulfated hyaluronan [34, 48, 51, 52].
HS is the most studied and predominant GAG and acts as a
receptor and reservoir for cytokines [53], enzymes, growth
factor proteins, such as AT [12], and cell adhesion proteins,
thereby modulating their distribution, concentration, and
biological activity [54–56]. HSPG plays a critical role in
maintaining and regulating various functions, including
viral entry into target cells, vascular permeability [57], in-
flammatory and immune responses, and coagulation activity
[3]. e GAG chain length and the number and location of
sulfate groups in the various domains determine the binding
affinity to the specific proteins [51, 58–60] and, therefore,
biological function [12, 61, 62]. e degree of sulfation exerts
the main influence on biological activity, therefore the GL’s
ability to modulate inflammation and coagulation, apart
from barrier functions [3].
2.2. Sulfation. In mammalian cells, the sulfation process
begins with the uptake of inorganic sulfate from the ex-
tracellular milieu [15], where the inorganic sulfate must be
2Advances in Pharmacological and Pharmaceutical Sciences
activated before reacting with the acceptor molecule. In
mammals, this activated form of sulfate is sulfonucleotide 3-
phosphoadenosine 5-phosphosulfate (PAPS), which is the
universal sulfate donor for sulfotransferase (ST or SULT)
reactions [11, 15]. STs transfer sulfate from PAPS to a
specific position on the GAG structure [15], and therefore
contribute significantly to the diversity of GAGs [3, 27].
2.3. Physiological Role of the Glycocalyx. Under normal
healthy physiological conditions, HSPGs interact with and
modulate the activity of several molecules [63], including
protective enzymes such as superoxide dismutase (SOD) [64],
cytokines [65], growth factors [25, 66], anticoagulant factors,
albumin [57], Hedgehogs, Wingless, and protease inhibitors
[67]. erefore, apart from its essential role in barrier function
and pathogen evasion [3], the GL will contribute to the
regulation of coagulation, leukocyte adhesion [1, 65, 68], and
protection against inflammation and oxidative stress [3, 49].
Any change in these functions is correlated with a wide range
of pathophysiological consequences such as accelerated in-
flammation, capillary leak syndrome, consequent oedema
formation [6], hypercoagulation, platelet hyperaggregation,
and loss of vascular responsiveness [3, 25, 69]. e GL and HS
do not only regulate physiological processes but are also
implicated in many pathologies, including cancer, infectious
and vascular diseases, and neurodegenerative disorders
[1, 3, 5, 27, 56, 63, 7073].
Of relevance to COVID-19, the primary role of the EpGL
is the ability of a highly sulfated intact, negatively charged
GL, to repel SARS-CoV-2 through electrostatic forces,
thereby preventing cell invasion [3]. Most viral particles
have a net negative charge at neutral pH, and SARS-CoV-2 is
no exception. Various research reports confirmed the SARS-
CoV-2 spike protein (SP) and net GRAVY score to be highly
negative [74–77]. Nonetheless, if the GL is undersulfated and
3-O-sulfotransferase 3B (3OST-3B) is overexpressed, as is
the case during chronic inflammatory conditions, the GL
may facilitate SARS-CoV-2 entry [3]. Moreover, an
undersulfated and degraded GL will not only increase
susceptibility to SARS-CoV-2 infection, but will also hamper
the ability to modulate the immune and inflammatory re-
sponse and coagulation.
3. Sepsis Model, Degradation of the Glycocalyx
In COVID-19 patients that present with severe symptoms,
sepsis has set in, which is a complicated condition that can be
defined as severe organ dysfunction caused by an uncon-
trollable host response to infection [78]. After exposure to an
infectious pathogen or inflammatory insult, alteration in the
composition of the GL is one of the earliest features during
sepsis [65, 68]. Following infection, acute injury, or in-
flammatory conditions, glucuronidases, such as heparanases
(HPSEs) [58, 66, 79], reactive oxygen species (ROS) [65], and
other proteases [5, 80, 81], cause shedding of HS and PGs
[25, 57, 82]. Subsequently, adhesion molecules such as
E-selectin and intercellular adhesion molecules are exposed
on the denuded endothelium [6, 40, 80, 83], which result in
accelerated inflammation [84], vascular permeability
Soluble proteoglycan
(shedding)
Syndecan
(core protein)
Heparan
sulfate
Proteoglycan Carbohydrate
Sialic acid
Hyaluronic acid
Epithelial cells
CD44
Epithelial glycocalyx layer
CAG-chain
Chondroitin
sulfate
Glycoprotein
Sulfate (SO42-)
Figure 1: Schematic presentation of the epithelial glycocalyx (GL) in mammalian cells, forming a mesh-like structure that projects into the
extracellular matrix (ECM). e proteoglycan presented is a syndecan with heparan sulfate and chondroitin sulfate glycosaminoglycan
(GAG) side chains. e sulfate groups (SO
42
) are indicated in red dots, attached to the GAG and glycoprotein side chains. Hyaluronic acid
is unsulfated and anchored to the receptor CD44 on the cell membrane. e glycoprotein consists of a protein base with carbohydrate chains
(orange circles) extending into the extracellular space with sialic acid (orange square) bound at the terminal position of the oligosaccharide
chains. Soluble proteoglycans and free GAG chains can appear in the extracellular space.
Advances in Pharmacological and Pharmaceutical Sciences 3
[58, 85], oedema, platelet aggregation, hypercoagulation,
and a loss of vascular responsiveness [3, 40, 65, 68, 86, 87].
Pulmonary EnGL degradation increases the availability
of En surface adhesion molecules to circulating micro-
spheres. It contributes to neutrophil adhesion, leading to
diffuse alveolar damage, interstitial lung oedema, and clot
formation during acute lung injury in sepsis and ARDS
[61, 68, 78]. ere exists a clear association between alveolar
epithelial and endothelial GL shedding and the establish-
ment of ARDS [81, 82] and COVID-19 [4]. Moreover,
circulating HS fragments are capable of influencing growth
factors and other signaling pathways distant to the site of GL
injury, which explains the systemic (i.e. para- or endocrine)
consequences of GL degradation [51, 88], and supports the
relationship between renal damage and the systemic
proinflammatory state observed in sepsis [58] and COVID-
19. Sepsis-associated induction of HPSE triggers degrada-
tion of vascular HS, leading to the collapse of the pulmonary
and renal EnGL [51, 58]. While pulmonary EnGL loss
contributes to lung injury via promotion of lung oedema and
neutrophil adhesion, it was found that HPSE-mediated
glomerular HS degradation could induce an early loss of
glomerular filtration in the absence of kidney oedema or
inflammation [51].
Apart from viral infections [82], multiple factors can
cause degradation of the GL, including major surgery, is-
chemia/reperfusion, hypoxia/reoxygenation, mechanical
ventilation [3, 48, 69], sodium overload [64, 80], ox-LDLs
[5, 40, 86], sepsis, haemorrhagic shock, excessive shear
stress, hypervolemia [48, 49], hyperglycaemia [68, 80], in-
flammatory cytokines such as tumour necrosis factor α
(TNFα) [5, 85], interleukin (IL)-1β, IL-6, and IL-10
[48, 57, 58], matrix metalloproteinases (MMPs) [64, 65, 87],
and reactive oxygen/nitrogen species [6].
Preserving the integrity of the GL is paramount both in
the prevention and treatment of infectious diseases. Since
most of the physiological properties of the GL are deter-
mined by the degree of GAG sulfation, ways to optimise
inorganic sulfate, the rate-limiting substrate for sulfation,
should be the primary focus of treatment. e intact Ep- and
EnGL, with adequate sulfated GAGs, prevent viral infection
and replication [3], determine vascular permeability, anti-
coagulant, anti-thrombotic, and anti-adhesive effects
[11, 89], attenuate blood cell–vessel wall interactions [1],
mediate shear stress sensing [40], enable balanced signaling,
antioxidant properties [49], modulate inflammation [5], and
fulfil a vasculoprotective role [25, 26, 48, 73]. In this review,
we will expand on the role of NAC as a precursor to in-
organic sulfate and its potential role as a preventive and
therapeutic agent for combatting COVID-19.
4. NAC as a Physiological Precursor
Most of the literature to date focuses on NAC as a precursor
to glutathione (GSH) [30, 42, 90–93]. However, it is im-
portant to realise that after free NAC enters a cell, it is
rapidly hydrolysed to release cysteine (Cys), the rate-limiting
substrate for intracellular GSH [94, 95], as well as hydrogen
sulfide (H
2
S) [22, 30, 96, 97], inorganic sulfate [18], taurine
[24, 98], Coenzyme A [99] and albumin synthesis [100]
(Figure 2). NAC, therefore, has many physiological func-
tions, apart from being an antioxidant precursor to GSH.
Since SARS-CoV-2 infection has been demonstrated to
cause rapid depletion of sulfur amino acids (SAAs) due to
oxidative stress or inflammation-induced proteolysis [22],
amino acid replenishment becomes even more critical [18].
NAC treatment has been demonstrated to successfully re-
plenish SAA levels, where a rapid increase in circulating Cys
levels has been observed within hours following NAC
supplementation [22]. During a rat study, the pharmaco-
kinetics of NAC revealed that after oral administration, 77%
NAC was maintained in the body and only 3% was excreted
in faeces, while another human study indicated that after
oral supplementation of NAC at 400 mg per single dose, the
level of Cys increased to 4 mg/L in plasma [101].
4.1. Glutathione. Since Cys is the rate-limiting substrate in
GSH and inorganic sulfate intracellular synthesis, low Cys
levels will therefore result in lower GSH and inorganic
sulfate levels. rough a series of redox reactions in the
plasma, NAC directly affects the amino acid pool of ex-
tracellular cystine and intracellular Cys [18, 31]. NAC will
favour GSH synthesis during oxidative stress. If there is a
higher demand for GSH because of oxidative stress, such as
during viral infection [102], extreme or endurance exercise
[103], and hypoxic conditions [22, 94], it will have an in-
hibitory effect on inorganic sulfate synthesis. Lowered redox
potential, common in high-risk COVID-19 patients, in-
cluding older adults and those with uncontrolled DM, causes
alterations in the TNFαreceptor activity towards a proin-
flammatory state [22, 31, 95]. It is known that infection by
RNA-viruses induces oxidative stress in host cells, and
growing evidence indicates that viral replication is regulated
by the redox state of the host cell [30, 94, 104, 105]. e aged
patients with moderate and severe COVID-19 illness, and
men had lower plasma levels of reduced GSH [30, 37], higher
ROS levels, and greater redox status (ROS/GSH ratio) than
COVID-19 patients with mild illness [37, 94, 106]. Erel et al.
found that thiol levels decreased as the severity of the disease
increased [107], while du Preez et al. hypothesised that low
thiol levels/sulfur deficiency is an underlying cause of
COVID-19 [3]. If these patients were low in GSH [108, 109],
one can assume that Cys levels were low [102] and conse-
quently inorganic sulfate, or it could be ascribed to a higher
demand for Cys or inorganic sulfate during severe illness
[106, 107, 110]. Bartonlini et al. demonstrated in vitro that
SARS-CoV-2 infection impairs the metabolism of cellular
GSH and its role in the redox homeostasis of cellular
proteins, and results in changes in the composition of ex-
tracellular thiols [102, 111]. Even though it was found that
COVID-19 patients with glucose-6-phosphate dehydroge-
nase (G6PD) deficiency [112, 113] and those with the glu-
tathione S-transferase (GST) theta 1 (GSTT1)
/
genotype or
Ile 105Val glutathione S-transferase P1 (GSTP1) polymor-
phism, were more susceptible to infection and had higher
mortality rates [114, 115], the availability of Cys will have a
greater influence on redox status and outcome. e age-
4Advances in Pharmacological and Pharmaceutical Sciences
dependent decline of GSH and Cys in extracellular fluids has
been hypothesised to not only be the actual causative factor
[3], but also a molecular marker of increased risk of infection
and development of serious COVID-19 [106, 107, 109, 116].
4.2. Hydrogen Sulfide (H
2
S). Endogenous H
2
S supports
basal, physiological, cellular bioenergetic functions, while
the activity of this metabolic support decreases with phys-
iological ageing [117]. Multiple biological regulatory roles
for H
2
S as an endogenous gaseous transmitter have been
established over the last decade [22, 97, 118]. H
2
S is pro-
duced, respectively, by cystathionine-β-synthase (CBS),
CSE, and 3-mercaptopyruvate sulfurtransferase (3-MST)
[22, 96, 118] (Figure 2). CBS and CSE serve as endogenous
stimulators of cellular bioenergetics, while 3-MST serves as a
regulator of cellular bioenergetics [118]. Moreover, during
physiological oxygen reduction, H
2
S serves as a stimulator of
electron transport in mammalian cells, by acting as a mi-
tochondrial electron donor [117, 118]. It seems probable that
stress and hypoxic states [97, 117] favour H
2
S as an
“emergency” substrate that balances and complements the
electron-donating effect of Krebs cycle-derived electron
donors [119]. Of note is that the cell’s free H
2
S concentration
is likely to be constantly in dynamic equilibrium with a
much larger pool of bound forms of sulfur, including thiol
groups of proteins. Mitochondrial production of H
2
S starts
with Cys [97, 106, 117], and the assumption can therefore be
made that under hypoxic conditions, there would be an
increased demand for H
2
S as electron donor [22], with a
consequent inhibitory effect on inorganic sulfate and GSH
synthesis, with Cys as the rate-limiting compound [119].
Even though one of the main H
2
S catabolic pathways in the
mitochondria leads to the formation of thiosulfate, which is
ultimately converted into sulfate [96], H
2
S would be fav-
oured as an electron donor under hypoxic and stress con-
ditions. is will affect the degree of HS sulfation [54].
Moreover, increases in intracellular H
2
S and ROS levels,
mediated through hypoxic conditions [22], may synergis-
tically induce membrane depolarisation [117]. is will
result in increased levels of cytosolic calcium ions, which will
lead to the activation of the endoplasmic reticulum (ER)
stress response involved in the initiation of apoptosis [120].
Indeed, corona-derived viral protein deposits were found to
result in ER stress and mitochondrial dysfunction in the
affected EpCs [121].
Intrinsically synthesised H
2
S is extensively studied be-
cause of the role it plays as a proinflammatory mediator at
high concentrations. Inhibition of endogenous H
2
S allevi-
ates the diseased inflammatory condition, whereas
Methionine
Homocysteine
Cystathionine
Cystine Cysteine
Albumin
Cysteine sulfinic acid
Taurine Sulfite (SO32-)
SUOX
3-MST
CBS
CSE
Coenzyme A
Glutathione
H2S
PAPS
Sulfate
(SO42-)
Sulfite
(SO32-)
Thiosulfate
(S2O32-)
CDO
CBS
Figure 2: Sulfur metabolism diagram. e essential sulfur amino acid methionine converts to cysteine, a precursor to albumin, cystine,
coenzyme A, glutathione, hydrogen sulfite (H
2
S), taurine, and sulfate (SO
42
). H
2
S can convert to thiosulfate (S
2
O
32
) and sulfite (SO
32
),
which are oxidised to sulfate. e enzyme cystathionine-β-synthase (CBS) converts homocysteine to cystathionine, while cysteine
dioxygenase (CDO) is responsible for the conversion of cysteine to cysteine sulfinic acid. Sulfite oxidase (SUOX) oxidises sulfite to sulfate.
H
2
S is generated from cysteine by three different pathways through either CBS, cystathionine-c-lyase (CSE), or 3-mercaptopyruvate
sulfurtransferase (3-MST). Adapted with permission by CC by 4.0 [3].
Advances in Pharmacological and Pharmaceutical Sciences 5
exogenous H
2
S, released from natural sulfur compounds
and SAAs, has shown protective effects in biological systems
[117, 119]. H
2
S is stimulatory at lower concentrations, while
it has an inhibitory effect on cellular bioenergetic functions
at higher concentrations [118]. Inflammation, hypoxia, and
calcium overload upregulate CSE gene expression and a
simultaneous increase in H
2
S concentration [118, 119].
Conversely, H
2
S at low physiological concentrations
[22, 97, 122] has been demonstrated to exhibit potent an-
tiviral and anti-inflammatory properties [106, 123], muco-
lytic activity [96], as well as ameliorating various
manifestations of inflammation, including ROS, nitric oxide,
TNFα, and IL-6 [22, 122], and prevents endothelial dys-
function in cardiovascular-related pathologies [96]. More-
over, H
2
S attenuates pulmonary tissue injury by inducing
ACE2 upregulation, while H
2
S may exhibit its antiviral
activity against SARS-CoV-2 by interfering with both ACE2
and transmembrane protease serine 2 [22, 106]. COVID-19
patients with a more favourable outcome displayed higher
circulating H
2
S levels than those found in patients with
severe COVID-19 pneumonia [43, 96]. It seems evident that
the H
2
S concentration must remain in a homeostatic balance
to exert its protective effects. Even though Cys is the rate-
limiting substrate for H
2
S production, pyridoxal 5-phos-
phate (vitamin B6) is a very important cofactor, not only
supporting CBS and CSE, but also the conversion of Cys to
inorganic sulfate via CDO, along with iron. When supple-
menting with NAC to increase Cys levels, it is imperative to
ensure a deficiency of the important cofactor nutrients does
not impair the metabolic conversions in the sulfur meta-
bolism pathway.
4.3. Inorganic Sulfate. Even though a small percentage of
inorganic sulfate is found in foods and various sources of
drinking water, the major source of inorganic sulfate for
humans is from the biodegradation of dietary proteins. First,
the essential sulfur amino acid methionine (Met) is con-
verted to Cys [18]. ereafter, the cytosolic enzyme, CDO,
catalyses the conversion of Cys to cysteine sulfinic acid, the
initial step in the transsulfuration pathway that leads to the
formation of inorganic sulfate and taurine [124] (Figure 2).
CDO is found primarily in the liver and brain, with some
heart, kidney, and thyroid activity. It is known that the
CDO-catalysed step is rate-limiting and that this metabolic
route is responsible for the production of the majority of
inorganic sulfate in vivo, since the absorption of inorganic
sulfate across the gastrointestinal tract is relatively inefficient
in humans [124]. e primary regulatory mechanism for
CDO enzymatic activity functions at the posttranslational
level. Under normal physiological conditions, high post-
prandial levels of Met or Cys will enhance the activity of
CDO through inhibition of its degradation via the ubiquitin
proteasome pathway. In the case of liver cirrhosis, however,
CDO activity is significantly decreased despite high Met
levels. It seems prudent that CDO expression is regulated at
the mRNA level rather than the protein level. It was found
that the proinflammatory cytokines, IL-1β, TNFα, and
transforming growth factor-beta (TGF-β), downregulate
CDO at mRNA level [98, 124]. is is evident in various
health conditions with an autoimmune or inflammatory
component, where high plasma levels of Cys with low
plasma concentrations of inorganic sulfate are generally
observed [124]. One can consequently expect reduced levels
of HS sulfation during inflammatory conditions [3].
Most hospitalised COVID-19 patients in ICU are being
placed on enteral nutrition [125]. Met is an essential amino
acid, while Cys is conditionally essential since it is not always
synthesised sufficiently, and preterm neonates have a relative
inability to convert Met into Cys [126]. Most enteral and
parenteral amino acid mixtures lack Cys since it is unstable in
solution [126, 127]. A short-bowel syndrome rat model found
that when enteral diets were supplemented with Cys and Met,
improved gut mucosal and plasma cysteine/cystine redox
potential and enhanced adaptive ileal mucosal growth were
observed [126]. It is important to note that Met’s contribution
to the PAPS pool is 610 times lower than that of Cys [21].
Patients on enteral nutrition most likely do not get sufficient
amounts of Cys [128], and during the cytokine storm seen in
COVID-19, the conversion of Cys or NAC to inorganic
sulfate appears to be reduced or inhibited due to the inhib-
itory effect of cytokines on CDO [124]. While adding Cys to
enteral feed and intravenous (IV) NAC for the moderate to
severely ill should be considered, IV STS should be explored
in late-stage COVID-19, since it is more readily converted to
inorganic sulfate. An easily digestible protein is recommended
for mild to moderately ill patients, such as whey protein, high
in Cys and albumin [129]. Another aspect to consider with
enteral and parenteral nutrition is the very high calcium
content in comparison to low levels of Cys and magnesium
[126, 130, 131]. Both SARS-CoV and high levels of intra-
cellular calcium ions would induce ER stress [120, 132]. Since
high calcium levels also upregulate CSE gene expression [119],
thereby increasing H
2
S levels and consequently membrane
depolarisation [117], even more calcium ions will be released,
creating a vicious cycle exacerbating inflammatory stress
signaling [133] and apoptosis [120].
Cys is one of the amino acids in the hepatic precursor
amino acid pool involved in both albumin and inorganic
sulfate synthesis. e close arrangement between albumin
synthesis and catabolic rates [134] suggests that a state of rapid
equilibrium exists between the amino acid pools concerned
with albumin and inorganic sulfate synthesis in the liver. It
should be noted that whereas human serum albumin (HSA) is
exclusively manufactured in the liver, hepatic inorganic
sulfate also mixes with inorganic sulfate formed in other
tissues in the extracellular fluid space. Inorganic sulfate
synthesis in a healthy person mostly depends on the extent of
reusing the sulfur from sulfate to synthesise new sulfur
compounds. ese conversions can occur through the in-
tervention of microorganisms in the intestinal tract, although
similar conversions can also occur in tissue cells [135].
erefore, it seems probable that competitive inhibition exists
in the synthesis between sulfate and albumin [134]. Pecora
et al. indicated that the pathway of sulfate recruitment in the
catabolism of SAAs was active in vivo [21]. ey discovered
that GAGs are undersulfated due to reduced extracellular
sulfate uptake [134], caused by a mutation in diastrophic
6Advances in Pharmacological and Pharmaceutical Sciences
dysplasia sulfate transporter (Dtdst), which was reversed with
the application of hypodermic NAC. is confirms that
amino acid catabolism will contribute to the intracellular
sulfate pool when extracellular sulfate availability is low.
erefore, the contribution of thiol compounds, such as NAC
and HSA, to GAG sulfation, becomes significant by increasing
the inorganic sulfate plasma concentration [21, 134]. Con-
versely, low levels of Cys or HSA will result in undersulfated
GAGs. e dietary contribution to GAG sulfation has been
reviewed extensively elsewhere [3].
Apart from NAC and HSA being sulfur donors, STS,
methylsulfonylmethane (MSM) [136], and sulfated PSs
[35, 137–140] will increase intracellular inorganic sulfate
levels and thus exhibit antiviral and immune modulating
properties. STS provides protection against ischemic brain
injury and acute lung injury through inhibition of nuclear
transcription factor kappa B (NF-κB) activation and TNFα-
induced production of cytokines and ROS, thereby pre-
venting upregulation of IL-6. It is well established that STS is
a potent antioxidant and anti-inflammatory agent, and it has
been used to treat cyanide poisoning [141, 142] and calci-
phylaxis [143] with a remarkably safe track record. STS also
acts as an H
2
S donor [122]. Taken together, these obser-
vations suggest a therapeutic potential for STS in COVID-
19, taken orally, inhaled or intravenously [2, 122].
As a source of organic sulfur, MSM will increase the
synthesis of GSH [144] and inorganic sulfate. Amir-
shahrokhi showed that MSM attenuates paraquat-induced
pulmonary and hepatic injury in mice, demonstrated by the
reduction of TNFα, malondialdehyde (MDA), and myelo-
peroxidase (MPO) levels, and an increase in SOD, GSH, and
catalase (CAT) levels in lung and liver tissues [144]. Several
studies demonstrated that MSM inhibits lipopolysaccharide-
induced release of oxidative stress biomarkers, such as nitric
oxide and prostaglandin E2 in macrophages, through
downregulation of NF-κB signaling [145, 146]. Kalman et al.
reported that MSM potentially inhibits the translocation of
the p65 subunit of NF-κB to the nucleus, therefore mini-
mising downstream events associated with local and sys-
temic inflammation [147–149]. Indeed, supplementation
with MSM will minimise the expression of many proin-
flammatory cytokines [144, 147, 148, 150]. is is confirmed
in a study where MSM attenuated experimental colitis by
reducing IL-1βlevels and protected against hepatic liver
injury by decreasing TNFαand IL-6 levels [144, 150]. In
another study, MSM significantly mitigated lung and pan-
creatic histopathological changes, decreased serum amylase
and MPO activity, and inhibited caerulein-induced IL-1β
expression. Moreover, MSM reduced caerulein-induced H
2
S
levels by decreasing CSE expression in the lungs and pan-
creas, and increased CD34+ expression [149]. Many of these
beneficial properties of MSM could be attributed to in-
creased intracellular levels of inorganic sulfate and therefore
enhanced GAG sulfation.
4.4. Albumin. Under normal physiological conditions,
oncotic pressure in the vascular system is maintained by
HSA [40]. e shedding of HSPGs promotes albumin
leakage and therefore reduces tissue turgor [134]. Hypo-
albuminemia is frequently observed in patients with DM,
hypertension, and chronic heart failure, and they are sta-
tistically most vulnerable to SARS-CoV-2 infection.
Hypoalbuminemia is a known factor in sepsis, ARDS, and
COVID-19 [59, 151]. It has been reported that low albumin
levels are seen in almost 81% of COVID-19 deaths [151].
Hypoalbuminemia, vascular disease, and coagulopathy
have all been linked to COVID-19 and have been shown to
predict outcomes independent of age and morbidity [151]. All
these conditions can be related to the degree of GAG sulfation
[3]. HSA also plays a vital role in fat metabolism by binding
fatty acids and maintaining them in a soluble form in the
plasma. Hyperlipemia, therefore, occurs in clinical situations
of hypoalbuminemia [152], which has been associated with
COVID-19 [151]. Important research questions are: does
hypoalbuminemia predispose patients to COVID-19, or is it a
consequence of the diseaseor both?
ere is no doubt that the binding of SARS-CoV-2 to
ACE2 receptors influences various processes, such as vaso-
constriction, kidney injury, cardiovascular disease, apoptosis,
and oxidative processes [153], but the effect of the degraded
GL and shedding of HS is mostly overlooked in COVID-19
research. It has been well established that proteinuria occurs
when the En barrier function is compromised [154], resulting
in changes in plasma protein concentration, particularly HSA
[151]. In a rat model, removal of HS by HPSE led to increased
permeability for both albumin and ferritin, and injection of
antibodies to HS led to acute selective proteinuria [155].
Studies showed that 59% of COVID-19 patients already
presented with proteinuria upon hospital admission, where
22% of nonventilated patients and 90% of ventilated patients
developed acute kidney injury [156], which confirms EnGL
degradation in COVID-19 patients.
Plasma proteins, such as albumin [40, 57, 64, 68, 80] and
fresh frozen plasma [48, 49], may protect the GL. After cold
ischemia, it was observed that albumin supplementation
significantly attenuated pronounced shedding of the GL [5],
and consequently adhesion of leukocytes [25], with reduced
interstitial oedema [49]. Meli showed that additional al-
bumin in the perfusion medium positively affects the for-
mation, support, and preservation of the EnGL [40]. Since
albumin is degraded into amino acids [152], it will increase
Cys levels and indirectly act as a precursor to inorganic
sulfate. Moreover, albumin has immunomodulatory and
anti-inflammatory [49], antioxidant, anticoagulant, and
antiplatelet-aggregational properties [40].
NAC and Cys play an essential role as the rate-limiting
substrate in many vital physiological processes, and it is
crucial to understand the homeostatic balance between the
sulfur substrates to maintain physiological function [43]. It
seems evident that a low level of Cys or inorganic sulfate
predisposes to COVID-19 [3], and paradoxically, there is a
higher demand for these sulfur substances during severe
illness [3, 18]. Moreover, since NAC, H
2
S, GSH, and al-
bumin demonstrate many similar physiological functions,
their donation of sulfur [43, 157] for inorganic sulfate
synthesis, could probably be credited for their various
physiological actions.
Advances in Pharmacological and Pharmaceutical Sciences 7
5. NAC as a Therapeutic Agent
Various research studies indicated that NAC modulates the
immune system [158], inhibits viral binding and suppresses
replication [123, 159], plus reduces inflammation
[22, 30, 91, 94, 105, 160], apart from its antibiofilm [123] and
antioxidant properties [22, 92, 161–163]. NAC also has
clinical benefits such as in cough and dry eyes, and as
mucolytic [94, 106, 164, 165]. Since NAC increases GSH and
inorganic sulfate production, it can modulate various
smoking-related end-points. It was shown that NAC at-
tenuated several biomarker alterations, such as inflamma-
tion and lung airspace enlargement, in heavy smokers who
received NAC (600 mg twice daily) for six months [30]. It
was demonstrated that NAC increased GSH levels in plasma
[102] and bronchoalveolar lavage fluid in humans [166],
which should subsequently lead to an increase in inorganic
sulfate levels. Various studies demonstrate the beneficial role
of NAC in recovery facilitation after cerebral ischemia and
traumatic brain injury and in the treatment of cerebro-
vascular vasospasm after subarachnoid haemorrhage [42].
NAC could therefore potentially be promising in amelio-
rating the long-term residual neuropsychiatric and neuro-
cognitive conditions seen in long COVID
[3, 153, 159, 167, 168].
Apart from being a precursor to GSH, various other
mechanisms have been ascribed to NAC’s antioxidant and
anti-inflammatory properties. NAC downregulates inflam-
masome NLRP3 mRNA expression, therefore decreasing
proinflammatory cytokine expression and release from ac-
tivated mononuclear phagocytes; NAC inhibits the endo-
toxin-induced release of IL-1β, IL-8, and TNFα; prevents
systemic endotoxemia and inflammatory response by im-
proving gut barrier dysfunction [121], where previous
studies have shown that COVID-19 has been associated with
gut barrier dysfunction and systemic endotoxemia; and
NAC downregulates programmed cell death protein 1 ex-
pression in CD4+ and CD8+ lymphocytes, therefore in-
creasing their counts and longevity [158]. NAC also
increases IL-2 production and proliferation, thereby pro-
moting the activation and modulation of T and
B lymphocytes [169, 170]. ese immunostimulatory
properties of NAC further justify its use for COVID-19,
since reduced levels of CD4+ and CD8+ T lymphocytes have
been observed in critically ill COVID-19 patients [171]. Its
immunostimulant properties could also be beneficial to
prevent subsequent opportunistic bacterial infections. Un-
like glucocorticoids, NAC would seem to have a more
favourable effect on modulating both the inflammatory and
immune responses against infection [172, 173]. Many of
these properties exhibited by NAC could likely be ascribed to
increased intracellular inorganic sulfate levels.
Oxidative processes enhance viral infection by pro-
moting replication in infected cells, inhibition of cell pro-
liferation, and cell apoptosis induction [35, 95]. Studies
found that NAC treatment significantly reduced the fre-
quency of influenza in the elderly and the severity and
duration of most symptoms [31, 95, 160, 162]. To replicate,
RNA-viruses need active NF-κB pathway support within
host cells [158]. In a recent study, it was found that NF-κB is
a mediator of SARS-CoV-2 pulmonary pathology, since it
triggers the production of numerous proinflammatory cy-
tokines [162]. It was demonstrated that the suppression of
hypoxia-inducible NF-κB [92] significantly reduced the
replication rate of mammalian coronaviruses. NAC has been
shown to inhibit NF-κB [30, 95, 105] and replication of
influenza A viruses in human lung EpCs in a dose-depen-
dent manner. NAC also reduced the production of proin-
flammatory cytokines, such as IL-6, IL-8, CXCL10, and
CCL5 [106, 123, 165], hence reducing chemotactic migration
of monocytes [160]. In addition, it has also been demon-
strated that NAC inhibits replication of other viruses [165],
such as HIV and respiratory syncytial virus [30, 160]. is
means that NAC and other thiol donors should potentially
inhibit SARS-CoV-2 as well [91], because of its ability to
downregulate NF-κB [35, 96, 160, 162]. e activity of
monocyte chemotactic protein-1 (MCP-1) is upregulated by
NF-kB, where MCP-1 amplifies inflammation and has been
associated with the pathophysiological processes observed in
COVID-19 patients [174]. Since NAC decreases both NF-κB
and MCP-1 levels [175], it demonstrates another mechanism
for reducing inflammation in COVID-19 patients. More-
over, Debnath et al. showed that NAC bound to SARS-CoV-
2 SP and resulted in a threefold weakening of SP binding
affinity with ACE2 receptors [90]. Furthermore, an inter-
esting interrelationship exists between vitamin D deficiency
and sulfur metabolism [3, 113]. In animal studies, co-sup-
plementation of vitamin D and NAC showed a greater
benefit in increasing 25(OH) vitamin D levels, and in re-
ducing oxidative stress and inflammatory biomarkers [36].
NAC was also shown to protect GL shedding, since HS is
expected to restore the GL when it is shed from EnCs [68].
Pecora et al. indicated an increase in GAG sulfation due to
NAC catabolism. erefore, the contribution of thiols to
sulfation becomes significant when their plasma concen-
tration is increased [21]. An infusion of NAC attenuated
hyperglycemia-induced degradation of the EnGL and co-
agulation [80], indicating the ability to restore the EnGL
[43]. Many experiments with various sulfur-containing
compounds confirmed the inhibiting effect of Cys on co-
agulation [3, 176]. De Flora et al. proposed the adminis-
tration of NAC as a possible strategy to preserve En function
and limit micro-thrombosis in severe forms of COVID-19
[30, 168]. NAC reduces thrombotic complications by
inhibiting the activity of plasminogen activator inhibitor-1,
which is a procoagulant positively correlated with severe
cases of COVID-19 [177, 178]. Furthermore, due to NAC’s
ability to break disulfide bonds, it disrupts platelet aggre-
gation and breaks the bond between blood cells and the
clotting factor, thereby maintaining blood fluidity and ox-
ygen flow in the specific area [106, 107]. NAC could
therefore reduce the activation of the characteristic coagu-
lation cascade of severe COVID-19 [3, 168]. Apart from the
effect of sulfation in coagulopathy [3], NAC may also exert
its anti-coagulopathy role by interrupting the vitamin K
reducing electron transfer pathway, which otherwise could
result in cerebral haemorrhage if co-administered with other
anticoagulants and acetaminophen. Regular monitoring of
8Advances in Pharmacological and Pharmaceutical Sciences
the international normalised Ratio (INR) and prothrombin
time (PT) is therefore recommended for patients taking
anticoagulants and NAC simultaneously [179]. However,
administration of NAC alone did not worsen haemorrhagic
stroke outcome, suggesting that NAC exerts thrombolytic
effects without significantly impairing normal hemostasis
[168]. Daid et al. successfully treated a COVID-19 patient
with intrahepatic haemorrhage with the application of IV
NAC [180].
Various case reports using NAC to treat COVID-19
patients successfully have recently appeared in the literature
[22, 30, 91, 123, 159, 160]. In a larger cohort study, it was
found that IV NAC significantly improved disease condi-
tions in 10 severely respirator-dependent COVID-19 pa-
tients, aged between 38 and 71 years, including one with
G6PD deficiency. Apart from improved lung function, IV
NAC administration significantly reduced inflammatory
markers, such as C-reactive protein and ferritin [91, 94, 160].
NAC administration has also been successful as a prophy-
lactic intervention for ventilator-associated pneumonia [31].
De Flora also describes various cases where IV NAC suc-
cessfully treated ARDS and increased extracellular total
antioxidant power and total thiol molecules [30]. Puyo et al.
also described a successful case study where IV NAC and
oral hydroxychloroquine (HCQ) were used in combination.
Previous work has shown that HCQ and NAC modulate the
innate immune system, reduce hypercoagulability, and in-
hibit thrombosis [181]. NAC also potentiates the vasodilator
and antiaggregatory effects of nitric oxide, which is valuable
in the context of acute heart failure, myocardial ischemia,
and infarction [30], while it improved renal oxygenation in
acute kidney injury in a rat model by decreasing free radicals
[161]. Chavarr´
ıa et al. compared the effect of the antioxidants
vitamin E, vitamin C, NAC, and melatonin with pentox-
ifylline as adjuvant therapy in COVID-19 patients with
moderate to severe pneumonia, where the simultaneous use
of NAC (600 mg twice daily every 12 h) and pentoxifylline
demonstrated the best effect in patients with severe symp-
toms [104].
Even though various clinical studies reported contra-
dicting evidence [159], NAC could serve as a first-line drug
for COVID-19 due to its structural and functional char-
acteristics [3, 35, 106, 160, 162]. NAC is widely available,
inexpensive, tolerable, and safe, and has been FDA approved
for many years. It could be used in an “off-label” manner to
improve therapeutic strategies for COVID-19 [37, 160].
When used for acetaminophen overdose, NAC is safe at
doses of up to 980 mg/kg over 48 hours [181]. It is evident
that NAC administered intravenously, orally, or inhaled,
should suppress SARS-CoV-2 replication and may reduce
symptoms if used timely. NAC’s potential therapeutic
benefits include scavenging ROS radicals extracellularly,
replenishing intracellular GSH and inorganic sulfate, and
suppressing cytokine storm and T cell protection, thus
mitigating inflammation, coagulation, and tissue injury
[30, 31, 160, 181, 182]. What is more, NAC inhibits the
downstream activities post TNFɑreceptor activation and
gene expression of TNFɑand IL-6 while under oxidative
stress [31, 182]. In a placebo-controlled study with
peritoneal dialysis patients, administration of NAC (600 mg
bid for eight weeks) has been shown to reduce the plasma
levels of inflammatory markers, including complement C3
[30]. Furthermore, in vivo NAC modifies the function of the
renin/angiotensin system, which is probably mediated by
inhibition of ACE2 activity. By blocking ACE2, NAC will
potentially protect patients from the deleterious effects of
angiotensin-2, which seems to be a potentially useful strategy
in SARS-CoV-2 infection [30, 123, 162]. NAC administra-
tion as first-line therapy, or as an adjunct therapy combined
with other antiviral agents [30], may dramatically reduce
hospital admission rates, the need for ventilation and
mortality [35, 94, 160]. Altay et al. had great success with the
application of a mixture of combined metabolic activators,
such as NAC and nicotinamide adenine dinucleotide pre-
cursors, to facilitate a more rapid symptom-free recovery in
COVID-19 patients [111].
Furthermore, since proteases, such as thrombin, have
been reported to support the cleavage of syndecan 1 PG
ectodomains, thus facilitating shedding of the GL, the
therapeutic use of protease inhibitors should be considered
[57]. Among the tested inhibitors is doxycycline [25, 57, 87],
an appealing candidate as a repurposed drug in the treat-
ment of COVID-19, with an established safety track record
and solid preclinical rationale [183]. In several studies, re-
searchers demonstrated that doxycycline inhibited matrix
MMP activity, which significantly reduced GL shedding and,
therefore, leukocyte adhesion to EnCs in response to in-
flammatory and ischemic stimuli [5, 25, 183]. Apart from
NAC, the early administration of doxycycline will also play
an essential role in preserving the GL. It is, therefore, ap-
parent that a comprehensive integrative protocol is needed
to treat COVID-19.
6. Treatment Recommendations for COVID-19
e main goal of any COVID-19 treatment regimen should
be the preservation of an intact well-sulfated GL
[3, 25, 49, 82]. erefore, for the prevention and treatment of
COVID-19, we propose the treatment options and dosages
summarised in Table 1, which are based on clinical obser-
vation, the science [3] and evidence outlined in this review.
Please note that the proposed treatments and dosages are
based on what is currently cited in the literature and could
only be adopted either under accepted hospital protocols or
as part of clinical trials.
Even though we propose that NAC and STS can be used
as first-line therapy, their application would complement
any other treatment protocol as a safe adjunct intervention.
COVID-19 is a complex disease that cannot be treated with a
single drug approach. Various strategies to preserve the
intact sulfated GL need to be applied. It is, however, essential
to prescribe a sulfur donor such as NAC or STS when drugs
that are administered need sulfation for their metabolism,
such as steroids, aspirin, colchicine, acetaminophen, and
nonsteroidal anti-inflammatory drugs [16, 186, 187];
[19, 165, 188, 189]. Apart from preventing the depletion of
inorganic sulfate when these drugs are given, sulfur-donor
supplements would complement the anti-inflammatory
Advances in Pharmacological and Pharmaceutical Sciences 9
drugs through their synergistic immune-modulatory action
[3]. Even though quercetin and melatonin have been shown
to be very beneficial in the early treatment of COVID-19
[190–192], they also require sulfation to be metabolised.
NAC should therefore also be considered as an adjunct to
these early treatment protocols.
We propose that for the prevention of COVID-19 in the
general population, especially in health care workers,
frontline personnel, and those at high risk with comor-
bidities, a cysteine derivative such as NAC [30], carbocis-
teine, or erdosteine [106, 184] should be taken daily. ese
cysteine derivatives, and MSM as sulfur-donor [136], will
favourably contribute to the Cys and inorganic sulfate pool.
Rogliani et al. did a meta-analysis on the effect of NAC
1200 mg/day, carbocisteine 1500 mg/day, and erdosteine
600 mg/day on chronic obstructive pulmonary disease and
found erdosteine to be superior in efficacy, compared to
NAC and carbocisteine [184]. Even though NAC has been
labelled as “low bioavailability” for decades, oral adminis-
tration of NAC given within 8 to 10 hours of acetaminophen
overdose displays the same detoxification capacity compared
to the IV route. It was reported that 600 mg NAC in capsule
form was able to reach a level of 16 μM NAC in the pe-
ripheral blood within half an hour after administration
[160]. Oral NAC at a dosage of 600 mg bid significantly
decreased the frequency and severity of influenza [94, 95]. It
is, therefore, expected that high dose oral NAC (1200 mg,
bid) [94] can improve innate immunity through sulfation of
the GL, and adaptive immunity by elevating GSH levels
in lymphocytes, in addition to modulating neutrophil
functions during the development of COVID-19 [3, 160]. As
a preventive measure, oral NAC (600 mg bid) should be an
effective and economical strategy to prevent SARS-CoV-2
infection and modulate the immune response [193]. Upon
the first onset of symptoms, such as fever, sore throat, or dry
cough, a higher dosage of oral NAC (1200 mg bid) should be
considered to alleviate symptoms and accelerate recovery
from viral infection [105, 160]. Note that high doses of oral
NAC could result in intolerable gastrointestinal adverse
effects such as nausea, vomiting, and diarrhoea [159]. Other
micronutrients, such as zinc [194], selenium [106, 195, 196],
magnesium, vitamins A, C, and D [113, 116, 197], should
form part of a successful integrative protocol [110], while
sulfur-donors such as MSM, allicin [157], or marine-derived
sulfated PSs can also be considered. MSM is entirely safe and
effective, taken at daily dosages of up to 4 g to prevent in-
fection and modulate the immune response [136]. ese self-
treatment strategies to prevent viral infection with oral or
inhalable NAC [22], or the use of other sulfur-donors,
should help many SARS-CoV-2-infected patients to safely
and cost-effectively recover at home. Since molybdenum,
iron, and various B-vitamins are also important cofactor
nutrients in sulfur metabolism, a balanced wholefood nu-
trient-dense diet should, therefore, form the cornerstone of
any prevention and treatment regimen, supplying all macro-
and micro-nutrients needed to prevent infection and
maintain redox balance [3, 43].
In patients experiencing moderate to severe COVID-19
symptoms, IV NAC administration upon hospital admission
should be considered as a standard practice of care, if
allowed within the safety protocols of the hospital. e idea
is to preserve and repair the GL to prevent a cytokine storm,
and the sooner proper measures can be applied, the better
the outcome that could be expected [22]. Shi & Puyo re-
ported that patients with mild-to-moderate acute lung injury
had significantly improved systemic oxygenation when in-
travenous (IV) NAC treatment (40 mg/kg/day) was given for
three days, as well as reduced need for ventilatory support
and lower mortality rate [160]. In another study, a 1200mg/
day oral dose of NAC was given for two weeks, which was
Table 1: Summary of a proposed therapeutic strategy to prevent and combat COVID-19.
Disease state Sulfur-donor Protease inhibitor Comments
Prevention
Either NAC 1200 mg/day, carbocisteine 1500 mg/
day, erdosteine 600 mg/day [184], or MSM 2 g/day
[136].
Health care workers, frontline
personnel, and those at high risk with
comorbidities.
Mild disease Double up the dosages indicated above.
Adequate dietary protein intake is
important; they can add whey protein to
the diet [21, 129, 134].
Moderate to
severe symptoms
IV NAC upon hospital admission (100 mg/kg/day)
for 7 to 10 days [160]
Doxycycline 100 mg
qid 5 to 7 days
[25, 57, 87]
Add L-cysteine to enteral feed
[126, 127].
Severe to
critically ill
Sodium thiosulfate—for 5 to 7 days and when
symptoms subside, every 2
nd
or 3
rd
day.
Adults: 100mL (25 g) of STS (rate of 5 mL/minute).
Paediatric 0–18 years: 1 mL/kg of body weight
(250 mg/kg or approximately 30–40 mL/m
2
of
BSA) (rate of 2.5 to 5 mL/minute) not to exceed
50 mL total dose of STS [185]
or IV NAC (150 mg/kg/day) for 7 to 10 days [160]
Doxycycline 100 mg
bid 7 to 10 days
[25, 57, 87]
STS might be a better option than NAC
to modulate the cytokine storm in the
critically ill.
Add L-cysteine to enteral feed
[126, 127].
Give albumin [40, 57] or fresh frozen
plasma [48, 49].
Avoid high tidal volume ventilation
[3, 48, 69, 123].
Avoid both hypervolemia and
hypernatremia [65, 68, 69].
10 Advances in Pharmacological and Pharmaceutical Sciences
sufficient to prevent deterioration due to severe respiratory
failure requiring invasive or noninvasive mechanical ven-
tilation in hospitalised patients with moderate or severe
COVID-19 pneumonia, and reduced 14- and 28-day mor-
tality [158]. Nonetheless, Taher et al. saw no clinical benefit
with the application of NAC at a dose of 40 mg/kg/day
diluted in 5% dextrose, given as a continuous intravenous
infusion for 3 consecutive days, in patients with mild to
moderate COVID-19-associated ARDS [159]. It should be
noted that NAC was given at low dosage and in addition to
standard-of-care treatment. A significant percentage of
patients in both the NAC and control group received
dexamethasone and other medications, which could reduce
the degree of HS sulfation and therefore attenuated the GL
[3, 198]. It is known that glucocorticoids may have intrinsic
immunosuppressive drawbacks when applied at the wrong
time, high dosages, and long term [3, 172, 173, 199, 200],
while NAC is not immunosuppressive [159]. When higher
concentration of IV NAC was given, better clinical outcomes
could be expected. Shi & Puyo noted that high dosages of
NAC will effectively reduce viral replication and significantly
alleviate pneumocyte damage, as well as modulate immune
responses and therefore prevent a cytokine storm [160].
NAC