International Journal of

Molecular Sciences

Review
Oxidative Stress Markers in Multiple Sclerosis
Félix Javier Jiménez-Jiménez 1, * , Hortensia Alonso-Navarro 1 , Paula Salgado-Cámara 1 , Elena García-Martín 2 and
José A. G. Agúndez 2

1 Section of Neurology, Hospital Universitario del Sureste, Arganda del Rey, E-28500 Madrid, Spain;
hortalon@yahoo.es (H.A.-N.); paula.salgado.camara@gmail.com (P.S.-C.)
2 University Institute of Molecular Pathology Biomarkers, Universidad de Extremadura, E-10071 Cáceres,
Spain; elenag@unex.es (E.G.-M.); jagundez@unex.es (J.A.G.A.)
* Correspondence: fjavier.jimenez@salud.madrid.org

Abstract: The pathogenesis of multiple sclerosis (MS) is not completely understood, but genetic fac-
tors, autoimmunity, inflammation, demyelination, and neurodegeneration seem to play a significant
role. Data from analyses of central nervous system autopsy material from patients diagnosed with
multiple sclerosis, as well as from studies in the main experimental model of multiple sclerosis, ex-
perimental autoimmune encephalomyelitis (EAE), suggest the possibility of a role of oxidative stress
as well. In this narrative review, we summarize the main data from studies reported on oxidative
stress markers in patients diagnosed with MS and in experimental models of MS (mainly EAE), and
case–control association studies on the possible association of candidate genes related to oxidative
stress with risk for MS. Most studies have shown an increase in markers of oxidative stress, a decrease
in antioxidant substances, or both, with cerebrospinal fluid and serum/plasma malonyl-dialdehyde
being the most reliable markers. This topic requires further prospective, multicenter studies with a
long-term follow-up period involving a large number of patients with MS and controls.

Keywords: multiple sclerosis; pathogenesis; risk factors; oxidative stress; biological markers; animal
models

Citation: Jiménez-Jiménez, F.J.; 1. Introduction

Alonso-Navarro, H.; Salgado-Cámara,
Multiple sclerosis (MS), which is characterized mainly by inflammation, demyelination,
P.; García-Martín, E.; Agúndez, J.A.G.
and neuronal degeneration, is considered to be a chronic autoimmune disease with a
Oxidative Stress Markers in Multiple
Sclerosis. Int. J. Mol. Sci. 2024, 25,
genetic predisposition affecting the central nervous system. To date, at least 200 loci with
6289. https://doi.org/10.3390/
genome-wide significance have been associated with the risk for MS through genome-wide
ijms25126289 association studies (GWAS) [1,2]. Most of the described associations, however, show a
modest odds ratio (OR) and explain close to half of its heritability [1,2], HLA (in particular,
Academic Editor: Irmgard Tegeder
the HLA-DRB1*15:01 haplotype) being the only one that has shown a strong association
Received: 29 January 2024 with MS risk [1]. It has been suggested that, together with genetic predisposition, some
Revised: 10 March 2024 environmental factors, gene–environment, and environment–environment interactions,
Accepted: 3 June 2024 including smoking, infections (mainly Epstein–Barr virus seropositivity or exposure), low
Published: 7 June 2024 sun exposure/low vitamin D levels, and obesity may be related to the etiopathogenesis of
MS and with MS onset and progression [3–5]. Since it has been suggested that oxidative
stress is closely related to inflammation (for example, in inflammatory conditions, immune
cells can liberate reactive oxidant substances leading to oxidative stress and, on the other
Copyright: © 2024 by the authors.
hand, the oxidative damage produced by free radicals can induce an inflammatory response
Licensee MDPI, Basel, Switzerland.
through the Toll-like receptors and inflammasomes) [6,7], with MS being a prototype of
This article is an open access article
inflammatory diseases, oxidative stress could also play a role in the etiopathogenesis of MS.
distributed under the terms and
Figure 1 depicts the possible interaction between the different mechanisms proposed in the
conditions of the Creative Commons
etiopathogenesis of MS, including oxidative stress.
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).

Int. J. Mol. Sci. 2024, 25, 6289. https://doi.org/10.3390/ijms25126289 https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 2 of 20
Int. J. Mol. Sci. 2024, 25, 6289 2 of 20

Possibleinteractions
Figure1.1.Possible
Figure interactionsbetween
betweenthe
thedifferent
differentpathogenetic
pathogeneticmechanisms
mechanismsin
inmultiple
multiplesclerosis.
sclerosis.

The term “oxidative stress” designates the imbalance between the production of reac-
The term “oxidative stress” designates the imbalance between the production of re-
tive oxygen species (ROS) and the ability of a biological system to neutralize intermediate
active oxygen species (ROS) and the ability of a biological system to neutralize intermedi-
reagents or to repair the resulting damage. Biomarkers of oxidative stress can be divided
ate reagents or to repair the resulting damage. Biomarkers of oxidative stress can be di-
into molecules modified by their interaction with ROS or free radicals derived from nitrogen
vided into molecules modified by their interaction with ROS or free radicals derived from
(RNS) and into molecules of the antioxidant system in response to an increase in redox stress.
nitrogen (RNS) and into molecules of the antioxidant system in response to an increase in
These include lipid peroxidation, protein oxidation, DNA oxidation markers, enzymes or
redox stress. These include lipid peroxidation, protein oxidation, DNA oxidation markers,
protein with antioxidant actions, other prooxidant and antioxidant substances, and global
enzymes or protein with antioxidant actions, other prooxidant and antioxidant sub-
markers of oxidative processes, such as the total oxidant status/capacity (TOS/TOC), total
stances, and global markers of oxidative processes, such as the total oxidant status/capac-
antioxidant status/capacity (TAS/TAC), and oxidative stress index (OSI).
ity (TOS/TOC),
This narrativetotalreview
antioxidant
aims tostatus/capacity (TAS/TAC),
analyze the results and oxidative
of published studies onstress index
the possible
(OSI).
role of oxidative stress in multiple sclerosis, mainly those related to oxidative stress markers
This narrative
in different tissuesreview aims to analyze
from patients the with
diagnosed results of published
MS, studies on the
but also case–control possible
association
role of oxidative
studies stress inassociation
on the possible multiple sclerosis, mainly
of candidate those
genes related
related to to oxidative
oxidative stress
stress mark-
with risk
ers in different tissues from patients diagnosed with MS, but also case–control
for MS, and studies showing the presence of oxidative stress in experimental models of association
studies
multiple onsclerosis.
the possible association
To this end, we of candidateagenes
performed PubMed related to oxidative
Database search stress with to
from 1966 risk
28
for MS, and studies showing the presence of oxidative stress in experimental
December 2023, crossing the terms “multiple sclerosis” and “oxidative stress”. The search models of
multiple
retrievedsclerosis. To this that
1672 references end,were
we performed a PubMed
manually selected Database
to include search
only from
those 1966related
strictly to 28
December
to the topic 2023, crossing
(a total of 201the terms “multiple sclerosis” and “oxidative stress”. The search
references).
retrieved 1672 references that were manually selected to include only those strictly related
2. the
to Oxidative
topic (a Stress
total ofMarkers in Patients with Multiple Sclerosis
201 references).
2.1. Oxidative Stress Markers in the Brain and Spinal Cord
2. Oxidative Stress
The results of Markers
studies on in oxidative
Patients with Multiple
markers in theSclerosis
brains or spinal cord of patients
2.1.
with Oxidative
MS, with Stress
most Markers
of themin the Brain and
compared toSpinal Cord[8–31], are summarized in Table S1.
controls
Many autopsy studies describe an increase in
The results of studies on oxidative markers in the various markers
brainsoforlipid peroxidation
spinal [8–15],
cord of patients
an increase in carbonylated proteins [16], markers of DNA damage
with MS, with most of them compared to controls [8–31], are summarized in Table S1. [9,11,12,17], and ni-
trotyrosine
Many (a marker
autopsy studies of nitrosative
describe stress) [8,11]
an increase in themarkers
in various brains ofofpatients diagnosed with
lipid peroxidation [8–
MS, especially in active MS plaques. The enzymatic activity of superoxide-dismutase
15], an increase in carbonylated proteins [16], markers of DNA damage [9,11,12,17], and
1 and 2 is upregulated
nitrotyrosine (a marker inof active demyelinating
nitrosative stress) [8,11]lesions [10]
in the and of
brains in patients
cerebellar gray matter
diagnosed withof
patients with MS [11], and catalase activity is increased in active demyelinating
MS, especially in active MS plaques. The enzymatic activity of superoxide-dismutase 1 lesions [11].
In contrast,
and glutathione
2 is upregulated peroxidase
in active (GPx) [10]
demyelinating and [10]
lesions catalase
and [10] activitiesgray
in cerebellar are similar
ma er of to
those of controls in cerebellar gray matter. Iron content has been found
patients with MS [11], and catalase activity is increased in active demyelinating lesions to decrease in
Int. J. Mol. Sci. 2024, 25, 6289 3 of 20

MS inactive lesions [12], mitochondrial protein expression is increased, and mitochondrial

complex IV activity is upregulated in MS lesions [18]. Studies with proton magnetic reso-
nance spectroscopy (1 HMRS) found decreased glutathione in some brain regions of MS
patients [19–21].
Several studies have also reported the upregulation of multiple enzymes and proteins
involved in oxidative processes, such as NAD(P)H:quinone oxidoreductase 1 (NQO1) [22],
some subunits of NADPH oxidase 2 [24], nicotinamide adenine dinucleotide phosphate ox-
idase 1 (NOX1) [24], nicotinamide adenine dinucleotide phosphate oxidase organizer [24],
heme oxygenase 1 (HO-1) [25], myeloperoxidase (MPO) [26], metallothionein I + II [11],
peroxiredoxins (PRX) 2 [23] and 5 [27], endoplasmic reticulum stress-related signaling path-
way molecules [28], transcription factor NF-E2-related factor 2 (Nrf2) [29], DJ-1 protein [29],
genes involved in mitochondrial protein synthesis (MRPL18, 14, 23; MRPS15, 22) [24],
genes involved in adenine nucleotide translocation (SLC25A4) [24], and genes induced by
oxidative stress and involved in the oxidative stress defense (UCP3, GRPEL1, TXNRD2,
ISCU, AASS, ACADL, DMGDH, and CADS) [24], in brain MS lesions in comparison to
control brains. On the other hand, peptidases of the 20S and 26 proteasomes [31], regulatory
caps 11S α and 19S [31], and nuclear-encoded genes of the respiratory chain [24], mitochon-
drial DNA-encoded gene rays (ND1, ND2, ND3, ND5, ND6, COX1, and CYTB) [24], were
found to be decreased or downregulated when compared to controls. Finally, brain concen-
trations of 20S proteasome α, β1, β2, and β5 subunits [31], calpain [31], cathepsin B [31],
and mitochondrial LonP [31] have been reported to be similar in MS patients compared
to controls.

2.2. Oxidative Stress Markers in Cerebrospinal Fluid (CSF)

Table S1 summarizes the results of studies related to markers of oxidative stress in the
CSF from MS patients compared to controls [32–67]. Most of these studies found increased
CSF levels of markers of lipid peroxidation, such as malonyl-dialdehyde, hydroxyalkenals,
diene conjugates, 4-hydroxy-nonenal, oxidized phosphatidylcholine, and isoprostanes
in patients diagnosed with MS compared to controls [13,32–35,37–40,45–47], with some
exceptions [36]. CSF levels of certain prostaglandins were increased in patients with MS in
most studies [35,41–44], but were similar to controls in others [36–38].
CSF protein carbonyl concentrations were increased in MS patients compared to
controls in two studies [40,47], and were similar to controls in another [35]. Protein-linked
neuroketals were found to be increased [35], and advanced glycoxidation end products
were similar to controls [48], respectively, in two studies. Advanced oxidation protein
products were increased in the CSF of MS patients compared to controls [49] and were
similar for the different clinical subtypes of MS [50]. CSF levels of markers of DNA damage
were increased in MS patients compared to controls [38,50]. Nitrotyrosine (a marker of
nitrosative stress) was also increased in MS patients compared to controls [36].
CSF iron levels were reported to be similar in MS patients and controls [51], and
transferrin was found to be decreased in MS patients with a shorter MS duration [52].
CSF concentrations of copper [51,53], ceruloplasmin [53], and matrix-metalloproteinase
9 (MMP9) [57] were increased, while ferric-reducing antioxidant power [50] and ferrox-
idase activity [54] were described as being similar in MS patients compared with con-
trols. The total antioxidant status or capacity (TAS or TAC) were decreased in MS (es-
pecially in RRMS) patients compared to controls in three studies [37,43,56], increased in
one study [34], and similar in another [37]. The CSF total thiol (SH) groups were de-
creased in MS patients [33,49]. GPx [32] and glutathione-reductase (GSSG-R) were found
to be increased and aryl esterase activity was similar [58] in comparison with controls in
isolated studies.
The CSF concentrations of several antioxidants, such as ascorbate [51] and the an-
tiaging antioxidant protein Klotho [55], were decreased in MS patients. CSF levels of
alpha-tocopherol were similar in MS patients and controls [59]. Two studies measuring CSF
uric acid concentrations found increased levels of this antioxidant and its precursors hy-
Int. J. Mol. Sci. 2024, 25, 6289 4 of 20

poxanthine and xanthine in MS patients compared to controls [63,64], while another study
described similar values of uric acid and its metabolite allantoine in MS and controls [62].
CSF levels of the excitatory amino acid L-glutamate were decreased in MS patients
compared to controls [60]. CSF levels of nitric oxide (NO) metabolites were reported to
be increased in MS patients compared to controls in three studies [36,42,57], and similar
to those of controls in another [61]. CSF levels of trace metals involved in oxidative stress
processes were the subject of a single study, which described increased lead, decreased
magnesium, and similar calcium, manganese, and zinc levels in patients with primary
progressive MS (PPMS) compared to those with secondary progressive MS (SPMS) and
controls [51]. The CSF human serum albumin (HAS), mercaptoalbumin (HMA), and non-
mercaptoalbumins 1 and 2 (HNA1 and HNA2) [65] levels were reported to be similar
in MS patients and controls. CSF neutrophil gelatinase-associated lipocalin (NGAL) was
increased [39] or similar [38] compared to controls in two studies by the same group. Finally,
DJ-1 [66], periredoxins 2 (PRX2) [38,39], and β-site amyloid precursor protein-cleaving
enzyme 1 (BACE1) [67] were increased in the CSF of patients with MS compared to controls.

2.3. Oxidative Stress Markers in Blood Cells

The results of studies addressing concentrations of oxidative stress markers in blood
cells (erythrocytes, leukocytes, peripheral blood mononuclear cells/lymphocytes, and
platelets) [68–84] are summarized in Table S1. MDA/TBA/TBARS levels were described
as increased in erythrocytes [68] and leukocytes [72] from patients diagnosed with MS
compared with controls, and were higher in patients with a more severe disease [72].
Radical oxygen species production was increased as well in platelets from patients with
SPMS compared to controls [83]. However, other lipid peroxidation markers, such as
diene conjugate and fatty acid patterns of phospholipids, were reported as being similar
in MS patients and controls in erythrocytes [69] and leukocytes [69,73,74]. Advanced
oxidation protein products were increased in erythrocytes from patients with CIS and
RRSS (being even higher in RRSS and patients with a more severe disease) [68], and
in platelets from SPMS patients (higher in patients with a more severe disease) [83] in
comparison with controls, 3-nitrotyrosine was found to be increased in platelets from SPMS
patients compared to controls [83], and DNA damage was increased in leukocytes from
patients with RRMS [75] and in PBMC from patients with MS compared to controls [81].
Global mitochondrial activity PBMC levels were similar in MS patients and controls in one
study [80], while another study found a significant increase in mitochondrial respiratory
chain complexes I, II, III, IV, and V [78], and another found a decreased complex IV in MS
patients compared to controls [82].
Regarding antioxidant enzyme activities or levels of antioxidant substances, in patients
with MS compared to controls:
a. SOD activity was decreased in erythrocytes [68,70], leukocytes [70], and peripheral
blood mononuclear cells (PBMC) [77]. Related to this finding, superoxide anion
(O2 − ) production in MS patients was increased in PBMC [78] and in platelets [73],
but was similar in leukocytes [76] from MS patients compared to controls.
b. GPx activity was decreased in erythrocytes [69] and decreased [72] or similar [69]
in leukocytes.
c. Catalase activity was decreased [71] or similar [69] in erythrocytes, similar in leuko-
cytes [69], and increased in PBMC [77].
d. Myeloperoxidase activity was similar [76].
e. Reduced (GSH) and oxidized glutathione (GSSG) concentrations were reported as
being similar in erythrocytes [69] and leukocytes [69,72].
f. Coenzyme Q10 concentrations were similar in erythrocytes [69] and decreased in
leukocytes [69], and alpha-tocopherol levels were similar in erythrocytes [69] and
leukocytes [69].
g. The TAS was similar [73,74] and total antiradical activity was increased [73,74]
in leukocytes.
Int. J. Mol. Sci. 2024, 25, 6289 5 of 20

h. Thiol group concentrations were decreased [83], and NADPH oxidase (NOX-1),
cytochrome c oxidase subunit 1 expression and glyceraldehyde 3-phosphate dehy-
drogenase activity (GADPH) were increased in platelets [83].
Finally, HO-1 [40], HsC70 [40], Hsp72 [40], and Trx concentrations [40], polyADP
ribose (PAR) synthesis [81], and polyADP ribose polymerase-1 (PARP1) expression [81]
were increased, HO-2 [40] and Hsp70-2 concentrations [80] were similar, and sirtuin 1 con-
centrations were increased [40] or similar [77] in PBMC from patients with MS compared
with controls.

2.4. Oxidative Stress Markers in Serum/Plasma

The results of studies addressing serum and/or plasmatic levels of oxidative stress
markers in patients diagnosed with MS compared to controls [33,34,36,40,44,48–54,56,58,
59,62–66,69,72,73,75,85–161] are summarized in Table S1.
Regarding lipid peroxidation markers, serum/plasma concentrations of MDA/TBA/
TBARS were increased in MS patients compared to controls in 16 studies [33,64,72,75,85–
88,90–94,96–98], but were similar in 4 studies [36,89,95,100] and decreased in 2 other stud-
ies [34,99]. Serum levels of MDA + hydroxyalkenals [101], “lipid oxidizability” [102], lipid
peroxides [103], and fluorescent lipid peroxidation products [104], and lipid hydroperox-
ides [105–108] were increased in MS patients in most studies, and similar in others [89,103].
Serum tert-butyl hydroperoxide and hydroperoxide concentrations were reported to be
similar [110–112,115] or increased in MS patients [109,114]. Serum HNE [40] and fatty acid
patterns of phospholipids [69] levels were increased in MS patients compared to controls,
while prostaglandin-F2alpha and F2-isoprostane were reported to be increased [36,116] or
decreased [44].
Serum/plasma levels of protein carbonyls were described as increased in MS or RRMS
patients compared to controls in nine studies [40,75,92,96,109,117–120], and similar between
two groups in three other studies [108,110,115]. Advanced glycoxidation end products
were normal [48,91] or increased [120], and advanced oxidation protein products were
increased in MS according to eight studies [49,106,112,115,120–122,124], with this increase
being more marked in patients with a more severe disease [115], higher ferritin levels [110],
and with a trend towards a decrease during follow-up [123]. Other authors reported similar
serum/plasma levels of advanced oxidation protein products in MS patients and controls
(with a trend towards lower values in patients with low vitamin D levels) [111] and a lack
in differences among the different evolutive types of MS [50]. Serum fructosamine levels
were reported as increased in MS patients in a single study [91]. Serum/plasma levels
of markers of oxidative stress/damage of DNA, such as 8-OHdG [105,118,125] and DNA
single-strand breaks [99], were increased in MS patients.
Serum/plasma ferric-reducing antioxidant power (FRAP) and total-reducing antioxi-
dant power (TRAP) were decreased [93,108,110,111,122,127] or similar [50,91,124] in MS
patients compared to controls, and were similar in MS patients with different degrees of
severity [107]. In comparison to controls, MS patients showed decreased [129,132,133] or
similar [51,110,114,128,131,134] serum/plasma iron levels, increased ferritin levels [107,108,
110,133] or those similar to controls [110], decreased [52,97] or similar [114,128] transferrin
levels, higher soluble transferrin receptor levels [118,131], and similar lactoferrin levels to
controls [135]. The serum/plasma total ferroxidase activity was decreased [56,136], cerulo-
plasmin increased [114,119,137], and copper increased [53,114,132] or similar [51,129,134]
in MS compared to controls.
Serum/plasma TAS/TAC or total antiradical activity were reported to be decreased [34,
77,78,87,91,94,96,102,109,114,118,130,132,138,139,141] or similar [56,73,85,89,95,97,100,126,
142], TOS increased [87,97,103,118,126,129,130,132,139,140,143] or similar [103,129,140], and
oxidative stress index (OSI) increased [87,97,126,139] or similar [103] in patients with MS
compared to controls.
Serum/plasma total thiol group concentrations were decreased in MS patients com-
pared to controls in most studies [33,49,75,96,109,112,122,143], with one exception [144],
Int. J. Mol. Sci. 2024, 25, 6289 6 of 20

and were similar in patients with MS with low vs. those with high ferritin levels. Native
thiol levels were found to be decreased in one study [143] and similar to those of controls
in another [144]. In comparison to controls, serum plasma SOD activity from MS patients
was reported to be similar [90,100,103,146], increased in MS patients [86,97,118,145], or
decreased in MS patients [75,143]; total glutathione was similar [89] or increased in MS
patients [118]; GSH was similar [69,89,100] or increased in MS patients [118]: GSSG was
similar [68] or decreased in MS patients [118]; GSSG-reductase activity was decreased in
MS patients [118,146]; GPx activity was similar [69,100,146], increased in MS patients [101],
or decreased in MS patients [103,118,135]; catalase activity was increased [75,97,103] or
similar [69,100]; and GST was similar [146] or decreased in MS patients [118].
Serum/plasma paraoxonase (PON1) activity was decreased in most studies in pa-
tients with MS [58,105,127,146], while others reported an increase [148] or similarity to
controls [93]. Arylesterase activity in MS patients was similar to controls in three stud-
ies [123,139,146] and decreased in another [58].
Serum/plasma concentrations of coenzyme Q10 were reported as being decreased in
MS patients compared to controls [64,69,89] or similar in MS patients and controls [151].
Similarly, four studies showed lower serum/plasma alpha-tocopherol levels in MS pa-
tients [59,69,72,75] and another showed non-significant differences when compared to
controls [69]. In comparison with controls, serum/plasma gamma-tocopherol levels were
decreased in MS patients in a single study [64], beta-carotene was decreased in two stud-
ies [72,152] and similar in one [64], and ascorbic acid was decreased in two studies [75,86]
and similar in another two [51,72].
Many studies have addressed serum/plasma levels of NO metabolites (nitrates + ni-
trites) in MS patients and controls; seven showed a significant decrease [36,87,107–111], one
showed a non-significant trend towards a decrease [61], and five others reported a signifi-
cant increase [86,99,101,103,112] of these parameters in MS patients. Serum nitrotyrosine
levels were increased in MS patients compared to controls [36,92,117].
Several studies have addressed serum/plasma trace metal concentrations in MS pa-
tients and controls, described in detail in Table S1, with varying results. The most consistent
findings were increased cadmium [129,132,154,155], aluminum [129,132,134], molybde-
num [129,132,134], tin [129,132], zirconium [129,132], and arsenic levels in MS patients [88,
93,134,155], and a lack in differences in serum/plasma levels of lead [129,132,154,155], mer-
cury [129,132], strontium [129,130,132], vanadium [129,132,134], and wolframium [129,132].
Serum/plasma levels of uric acid were decreased in patients with MS compared to con-
trols in three studies [97,158,159], increased in another two [63,86], and similar to controls in
two more [109,156], while the serum levels of the related substances hypoxanthine, xanthine,
and uridine were found to be increased [158] and allantoine was similar in MS patients
compared to controls. Serum/plasma uric acid levels from MS patients were decreased
in current smokers compared with non-smokers and ex-smokers [160]. Serum/plasma
bilirubin levels were decreased in MS patients [97,157]. Ischemia-modified albumin was
increased [140] and irisin and nesfatin-1 were decreased in MS patients compared to con-
trols [161]. Finally, serum/plasma levels of HAS [65], HMA [65], HNA1 [65], HNA2 [65],
and DJ-1 [66] from MS patients did not differ significantly from those of controls.

2.5. Oxidative Stress Markers in Other Fluids

Several studies found increased levels of lipid peroxidation markers [85,162], increased
levels of aluminum [162], decreased levels of silicon [162], increased 8-iso-prostaglandin
(PG-)F2alpha levels [163], neopterin/creatinine ratio [164], and prolyl oligopeptidase lev-
els [165] and decreased levels of alpha2-macroglobulin [165] in urine from MS patients
compared to controls (Table S1).
Karlík et al. [91] described an increase in salivary levels of TBA/TBARS and advanced
glycation end-products, decreased FRAP, and similar advanced oxidation protein products
and TAS in patients with MS compared to controls (Table S1).
Int. J. Mol. Sci. 2024, 25, 6289 7 of 20

3. Genetic Variants of Genes Related to Oxidative Stress in Patients with

Multiple Sclerosis
The possible association between single nucleotide polymorphisms (SNPs) or deletions
in genes related to oxidative stress and the risk of developing MS has been the subject of sev-
eral case–control association studies, which are summarized in Table S2 [113,155,166–180].
Most of these studies found a lack of a direct association between these SNPs and mul-
tiple sclerosis, including the most common SNPs in CYP2D6 [166], GSTP1 [167–169],
GSTM1 [113,155,167,170], GSST1 [167,170], GSTM3 [167], PON1 rs662 [173,174], PON1
rs854560 [173], NQO1 rs1800556 [176], HMOX1 2071747 [177], HMOX2 rs270363 [177],
HMOX2 rs1051308 [177], NCF1 D7S2518 [178], NCF2 [178], NCF4 [178], CYBA [178],
CYBB rs9330580 [178], NOS1 rs1879417 [179], NOS3 rs2070744 [180], and HNF1A-AS1
rs7953249 [181] genes.
Other authors reported a significant association for GSTM1 null polymorphism [171],
GSTT1 null polymorphism [171], MPO rs2333227 [172], PON1 rs854560 [174], GLO1
rs1049346 [173], NQO1 rs1800566 [169,175], OGG1 rs1052133 [113], NCF1 D7S1870 [178],
CYBB rs5963310 [178], NOS2 rs2297518 [179], CAT rs7943316 [179], and TRPP2-AS
rs933151 [181] with the risk of developing MS, and a decreased risk related with SOD2
rs187947 [179] and GPX4 rs713041 [179]. Mann et al. [167] described an association between
the combination of GSTM1 null polymorphism and GSTP1 rs1695 alleles and the presence
of GSTM3 rs1799735 with severe disability in patients with an MS duration longer than
10 years [167]. Alexoudi et al. [169] described an interaction between GSTP1 rs1695 and
NQO1 rs1800665 and the risk for MS. Finally, Agúndez et al. [178] described an association
of the HMOX2 rs1051308AA genotype and rs1051308 with risk for MS in males.

4. Data from Experimental Models of Multiple Sclerosis

4.1. Lipid Peroxidation Markers
Perianes-Cachero et al. [182] described an increase in lipid peroxidation, and in SOD,
GPx, GSSG-reductase activities (assessed using spectrophotometry), and a decrease in
catalase activity (assessed using spectrophotometry) and GSH concentrations (assessed
using a fluorometric method) in the hippocampus of 6-week-old female Lewis rats with
chronic relapsing experimental autoimmune encephalomyelitis (EAE), the most important
animal model of MS.
Dimitrijević et al. [183] reported an increased MDA (assessed using a colorimetric
method) and superoxide anion levels, decreased GSH concentrations, decreased SOD
activity (assessed using spectrophotometry), increased NOS3 and xanthine oxidase (an
enzyme responsible for the synthesis of uric acid) expression (assessed using quantitative
real time-polymerase chain reaction—qRT-PCR), and increased AOPP in the spinal cord
from Dark Agouti rats with EAE, and increased plasma AOPP levels (assessed using
high-performance size-exclusion matrix chromatography) in the same MS model.
Jhelum et al. [184] reported increased peroxidation, increased mRNA levels of several
ferroptosis genes, increased nuclear receptor coactivator 4 (NCOA4) expression (assessed
using qRT-PCR), decreased GPx4 activity (assessed using Western blot analysis), and
decreased total glutathione (assessed using spectrophotometry) in the brain of female mice
with EAE of C57BL/6. C57BL/6 OlaHSD [15] and C57BL/6 female mice with EAE [185]
showed increased levels of acrolein or their metabolites (assessed using Western blot [15] or
liquid chromatography/tandem mass spectrometry [185]) in the spinal cord and urine. A
decrease in mRNA expression was described for the cytoplasmic isoform of GPx4 (assessed
using RT-PCR) in the spinal cord from female C57BL/6 mice with EAE [186]. Smerjac and
Bizzozzero [187] described increased lipid peroxidation markers (assessed by a colorimetric
method) before the appearance of neurological symptoms, in the spinal cord of seven-week-
old male Lewis rats with acute EAE.
Int. J. Mol. Sci. 2024, 25, 6289 8 of 20

4.2. Protein Oxidation Markers

Smerjac and Bizzozzero [187] described increased protein carbonylation and degra-
dation (assessed using Western blotting) at the time of maximal clinical disability and a
decreased glutathione concentration (assessed using spectrophotometry) in the spinal cord
of seven-week-old male Lewis rats with acute EAE. The same group described an increased
protein carbonylation (using the OxyBlot™ protein oxidation detection kit) within cerebel-
lar astrocytes, which was maximal in the acute phase and decreased in the chronic phase
of the disease [188], and in the spinal cord [189] of eight-week-old female C57BL/6 mice
with EAE.
Castegna et al. [190] described an increased protein oxidation (assessed with OxyBlot
and mass spectrometry analyses), increased glutamate/glutamine, and decreased natural
antioxidant levels (assessed with liquid chromatography-tandem mass spectrometry analy-
sis, LC-MS/MS), which paralleled disease activity in the brain of female 10–11-week-old
PLSJL mice with EAE.

4.3. Heme Oxygenase 1 (HO-1)

Several authors have reported the increased expression of heme oxygenase 1 (HO-1)
by using the Western blot analysis [184,191–193] and decreased expression of NADPH
cytochrome P450 reductase (which is required for the catalytic activity of HO-1) expres-
sion [191] in the brain of female C57BL/6 mice [184], female SJL mice [191], pregnant
Sprague-Dawley rats [192], and adult male Lewis rats [193] with EAE. An increase in
HO-1 expression in the EAE brain was inhibited and exacerbated, respectively, through the
coadministration of inducers of inhibitors of HO-1 [193]. HO-1 was increased in the spinal
cord of the EAE rodents [192]. In addition, a significant upregulation in HO-1 and the iron
storage protein ferritin (assessed with RT-PCR) in a demyelination model of mutant rats
(dmv rats) was described in comparison with a hypomyelination model (mv rats) [194].

4.4. NAD(P)H Oxidases (NOX)

NAD(P)H oxidase (NOX) enzymes (assessed using a fluorescence lifetime imaging
method with a two-photon laser-scanning microscope) were also found to be overactivated
in inflammatory monocytes, activated microglia, and astrocytes of the brain and peripheral
CD11b(+) cells of CerTN L15 x LysM tdRFP mice with EAE [195]. In addition, NOX2 deletion
or deficiency could prevent EAE induction in female C57BL/6 gp91phox−/− [Nox2 KO
mice (gp91Cybbtm1Din/J)] mice (assessment performed using qRT-PCR) [196,197].

4.5. Other Markers

Hasseldam et al. [198] reported a loss in mitochondrial membrane potential and ox-
idative changes (assessed using a spectrophotometric method) in the brains of female Dark
Agouti rats with EAE, present approximately 10 days before clinical onset. Other authors,
using two-photon imaging, described increased mitochondrial oxidation in oligodendro-
cytes from MOG-cre mice, CCR2-RFP x CX3CR1-GFP mice, mito-roGFP2-Orp1 mice, and
Ai14 reporter mice with EAE [199].
Aheng et al. [200] described a higher severity of EAE in a model of mice lacking the
inducible nitric oxide synthase (iNOS) gene, while mice lacking simultaneously uncoupling
protein 2 (UCP2) and iNOS genes developed milder EAE.
Johnson et al. [201] described a relationship between the deficiency of nuclear-factor-
erythroid-2-related factor 2 (Nrf2) (assessed using qPCR) and a more severe clinical course,
a more rapid onset, and a greater percentage of Biozzi ABH mice back-crossed onto Nrf2-
KO mice developing EAE after the administration of myelin oligodendrocyte glycoprotein
(MOG 35-55), suggesting a role of Nrf2 in modulating the neuroinflammatory response.
Honorat et al. [202] reported an increased expression of xanthine oxidase (assessed
using a fluorometric assay) in infiltrating macrophages and microglia of the spinal cord
from 8-week-old female SJL/J mice with EAE, which decreased with the preadministration
Int. J. Mol. Sci. 2024, 25, 6289 9 of 20

of a potent xanthine oxidase inhibitor, thereby, suggesting a role of xanthine oxidase in the
pathogenesis of EAE.
Metabolomic studies in plasma (using ultra-high-performance liquid chromatography-
orbitrap-mass spectrometry—UHPLC-Orbitrap-MS) from C57BL/6J EAE mice showed a
downregulation in glycerophospholipids and fatty acyls and upregulation in glycolipids,
taurine-conjugated bile acids, and sphingolipids, and an increase in NOX activity and
MMP9 during disease progression [203]. Increased superoxide anion concentrations and
upregulation in NOS3 in the pituitary and adrenal glands were reported, as well as an
increase in MDA and GSH levels and in catalase activity (assessed using electron paramag-
netic resonance spectroscopy) in adrenal glands of 2-month-old female rats of Dark Agouti
with EAE [204]. Plasma concentrations of IgG antibodies with peroxidase [205], oxidoreduc-
tase [205], and catalase [206] activities (assessed using spectrophotometry) were increased
at different stages of EAE in C57BL/6, Th, 2D2 mice [205], and C57BL/6 mice [206].

4.6. Effects of Exposure of Neuronal Cultures to Pathological Products of MS Patients

Vidaurre et al. [207], in a study comparing 13 MS patients with 10 HC, showed that
acute exposure of culture neurons to the CSF from MS patients induced oxidative stress
and decreased the expression of neuroprotective genes (assessed using RT-PCR and a quan-
titative lipidomic analysis), which was attributed to an increased content of the ceramides
C16:0 and C24:0. Finally, the injection of cultured neurons and oligodendrocytes killed
by oxidized phosphatidylcholines obtained from MS lesions induced focal demyelinating
lesions with prominent axonal loss in the spinal cord of mice [14].

5. Discussion and Conclusions

Many findings point to the possible role of oxidative stress in the pathogenesis of MS.
This is supported by a demonstrated increase in markers of oxidation of lipids, proteins, and
DNA, and markers of nitrosative stress, together with changes in the activity of enzymes
and proteins involved in oxidative stress, both in the brain and/or the spinal cord of MS
patients (in samples from autopsies) and in experimental models of MS, mainly in different
strains of rodents with EAE (although data from these experimental models could have a
low predictive value). Most studies on CSF and blood cells have also shown an increase in
several markers of oxidative stress and a decrease in several antioxidant substances in MS
patients compared to controls. However, a recent meta-analysis of oxidative stress markers
in CSF confirmed only a significant association of CSF MDA levels, but not of other potential
markers, with MS [208]. Even though studies of concentrations of markers of oxidative
stress in serum/plasma, urine, and other tissues have not shown conclusive results, most
studies and the results of a meta-analysis showed an increase in serum/plasma MDA
and albumin concentrations in patients diagnosed with MS compared to controls [208].
Similarly, the majority of studies showed a decrease in serum/plasma TAS/TAC and
serum/plasma levels of SH groups and an increase in TOS and OSI in patients with MS.
In summary, the main alterations found in studies addressing oxidative stress markers
in patients with MS included the following;
a. Increased markers for lipid peroxidation, protein oxidation, and DNA oxidation.
b. Increased mitochondrial activity.
c. Increased NO nitrotyrosine (therefore, increased nitrosative stress).
d. Increased TOS and OSI and decreased TAS.
e. Decreased iron, copper, and ceruloplasmin.
f. Increased SOD and catalase activities.
g. Decreased GSH levels and normal or decreased GPx activity.
h. Increased NQO1, NOX1, NOX2, and HO-1 activities.
i. Increased myeloperoxidase and peroxiredoxins 1 and 2 activities.
j. Increased endoplasmic reticulum stress proteins.
k. Decreased concentrations of uric acid and related substances.
l. Decreased ascorbate concentrations.
Int. J. Mol. Sci. 2024, 25, 6289 10 of 20

To date, none of the studied variants in genes related to oxidative stress have shown
an unequivocal association with MS. Potential reasons for the controversies seen between
the results of various studies, both in those addressing biochemical parameters and studies
on genes related to oxidative stress, include sample size, differences in sample collection
and the methods used, and possibly factors associated with the participants (for example,
some studies were not matched by age and/or sex or treatment with disease-modifying
therapies). Moreover, in the case of genetic case–control association studies, there was a
lack of replication studies.

6. Future Directions
We suggest that future studies aiming to establish the possible role of oxidative stress
in the pathogenesis of MS should fulfill, at least, the following conditions:
a. Design prospective and multicenter studies with a long-term follow-up period (1 year).
b. The recruitment of a large number of patients diagnosed with MS according to
standardized criteria [209], not exposed to any therapy for this disease, and a similar
number of age- and sex-matched healthy controls who do not fulfill clinical criteria
for the diagnosis of MS and without a family history of MS.
c. Both MS patients and controls involved in such studies should not have obesity or
undernutrition, should not suffer from oncologic, acute infectious diseases, kidney,
liver, thyroid, or parathyroid disease, have no recent history of traumatism or surgery,
and no atypical dietary habits (i.e., diets consisting exclusively of one type of food-
stuffs, such as vegetables, and others). They should not use therapy with steroids,
diuretics, diphosphonates vitamins, calcium or mineral supplements, or drugs that
could affect oxidative stress. In addition, pregnant women should be excluded.
d. It would be desirable to collect plasma/serum and blood cells for the analysis of
multiple oxidative stress biomarkers and to obtain blood DNA for genetic studies of
genes related to oxidative stress, both in MS patients and controls, at baseline.
e. Patients with MS should undergo periodic clinical evaluations every 3–4 months to
evaluate the evolutive type and severity of the disease according to standardized
scales such as the EDSS [210].
f. A new collection of plasma/serum and blood cells should be performed for the
analysis of multiple oxidative stress biomarkers at the end of the follow-up to evaluate
the changes induced by the different treatments used for MS.

Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/ijms25126289/s1.
Author Contributions: F.J.J.-J.: Conceptualization, Methodology, Investigation, Validation, Formal
Analysis, Investigation; Writing—Original Draft; Writing—Review and Editing; Project Admin-
istration. H.A.-N.: Conceptualization, Methodology, Investigation, Validation, Formal Analysis,
Investigation; Writing—Original Draft; Writing—Review and Editing; Project Administration. P.S.-C.:
Conceptualization, Methodology, Investigation, Validation, Formal Analysis, Investigation; Writing—
Original Draft; Writing—Review and Editing; Project Administration. E.G.-M.: Conceptualization,
Methodology, Investigation, Validation, Formal Analysis, Investigation; Writing—Original Draft;
Writing—Review and Editing, Project Administration, Obtaining Funding. J.A.G.A.: Conceptual-
ization, Methodology, Investigation, Validation, Formal Analysis, Investigation; Writing—Original
Draft; Writing—Review and Editing, Project Administration, Obtaining Funding. All authors have
read and agreed to the published version of the manuscript.
Funding: The work at the authors’ laboratory was supported in part by grants PI18/00540 and
PI21/01683 from Fondo de Investigación Sanitaria, Instituto de Salud Carlos III, Madrid, Spain, and
IB20134 and GR21073 from Junta de Extremadura, Mérida, Spain. Partially funded with FEDER funds.
Acknowledgments: We recognize the effort of the personnel of the Library of Hospital Universitario
del Sureste, Arganda del Rey, who retrieved an important number of papers for us. James McCue, a
native English speaker with expertise in editing scientific articles, extensively revised the English text.
Int. J. Mol. Sci. 2024, 25, 6289 11 of 20

Conflicts of Interest: The authors declare that the research was conducted in the absence of any
commercial or financial relationships that could be construed as potential conflicts of interest.

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