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Citation: Jakovljevi´c, D.; Nikoli´c, M.;
Jovanovi´c, V.; Vidonja Uzelac, T.;
Nikoli´c-Koki´c, A.; Novakovi´c, E.;
Miljevi´c, ˇ
C.; Milovanovi´c, M.;
Blagojevi´c, D. Influence of Long-Term
Anti-Seizure Medications on Redox
Parameters in Human Blood.
Pharmaceuticals 2024,17, 130.
https://doi.org/10.3390/ph17010130
Academic Editor: Abdeslam
Chagraoui
Received: 17 December 2023
Revised: 9 January 2024
Accepted: 12 January 2024
Published: 18 January 2024
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
pharmaceuticals
Article
Influence of Long-Term Anti-Seizure Medications on Redox
Parameters in Human Blood
Danijel Jakovljevi´c 1,2 , Milan Nikoli´c 1, Vesna Jovanovi´c 1, Teodora Vidonja Uzelac 2,
Aleksandra Nikoli´c-Koki´c 2, * , Emilija Novakovi´c 3,4 ,ˇ
Cedo Miljevi´c 5, Maja Milovanovi´c 6
and Duško Blagojevi´c 2
1Department of Biochemistry, Faculty of Chemistry, University of Belgrade, 11158 Belgrade, Serbia;
jakovljevicdanijel96@gmail.com (D.J.); mnikolic.chem@gmail.com (M.N.); vjovanovic@chem.bg.ac.rs (V.J.)
2
Department of Physiology, Institute for Biological Research “Siniša Stankovi´c”, National Institute of Republic
of Serbia, University of Belgrade, 11108 Belgrade, Serbia; teodora.vidonja@ibiss.bg.ac.rs (T.V.U.);
dblagoje@ibiss.bg.ac.rs (D.B.)
3Clinic for Mental Disorders “Dr. Laza Lazarevi´c”, 11000 Belgrade, Serbia; emilija.novakovic@med.pr.ac.rs
4Faculty of Medicine, University of Priština, 38220 Kosovska Mitrovica, Serbia
5Outpatient Department, Institute of Mental Health, School of Medicine, University of Belgrade,
11000 Belgrade, Serbia; cedo.miljevic35@gmail.com
6Department for Epilepsy and Clinical Neurophysiology, Institute of Mental Health, School of Medicine,
University of Belgrade, 11000 Belgrade, Serbia; jmtmilov@gmail.com
*Correspondence: san@ibiss.bg.ac.rs
Abstract: Background: Epilepsy is a chronic brain disease affecting millions of people worldwide,
but little is known about the impact of anti-seizure medications on redox homeostasis. Methods:
This study aimed to compare the effects of the long-term use of oral anti-seizure medications in
monotherapy (lamotrigine, carbamazepine, and valproate) on antioxidant enzymes: superoxide dis-
mutase, catalase, glutathione peroxidase, glutathione reductase, haemoglobin, and methaemoglobin
content in erythrocytes, and concentrations of total proteins and thiols, nitrites, lipid peroxides and
total glutathione in the plasma of epilepsy patients and drug-naïve patients. Results: The results
showed that lamotrigine therapy led to lower superoxide dismutase activity (p< 0.005) and lower
concentrations of total thiols (p< 0.01) and lipid peroxides (p< 0.01) compared to controls. On the
other hand, therapy with carbamazepine increased nitrite levels (p< 0.01) but reduced superoxide
dismutase activity (p< 0.005). In the valproate group, only a decrease in catalase activity was observed
(p< 0.005). Canonical discriminant analysis showed that the composition of antioxidant enzymes in
erythrocytes was different for both the lamotrigine and carbamazepine groups, while the controls
were separated from all others. Conclusions: Monotherapy with anti-seizure medications discretely
alters redox homeostasis, followed by distinct relationships between antioxidant components.
Keywords: anti-seizure medications; redox homeostasis; antioxidant enzymes; erythrocytes; plasma
1. Introduction
Epilepsy is one of the most common neurological disorders in children and adults. It
is described as a group of disorders characterised by chronic, recurrent, and paroxysmal
changes in motor and sensory neurological functions due to a disturbance in the electrical
activity of a population of neurons [
1
]. In epileptogenesis, many different factors cause
or support the progression of epilepsy at different levels [
2
]. The epileptic seizure itself
can generate an excessive amount of reactive oxidative species and reactive nitrogen
species in the region of abnormal neuronal activity, and reactive oxidative species-induced
changes in the structure of glutamate receptors increase neuronal excitability and seizure
susceptibility [
3
]. In addition, oxidative stress appears to contribute to the development
of neuroinflammation, neurodegeneration, and impaired neurogenesis, which in turn
Pharmaceuticals 2024,17, 130. https://doi.org/10.3390/ph17010130 https://www.mdpi.com/journal/pharmaceuticals
Pharmaceuticals 2024,17, 130 2 of 12
may promote epileptogenesis [
4
]; this has been confirmed in various models of epileptic
rats [5,6].
Oxidative stress is defined as an imbalance between the production of cellular oxidants
(reactive oxygen species such as superoxide, hydrogen peroxide, and hydroxyl radicals
and lipid peroxides, as well as reactive nitrogen species such as peroxynitrite) and their
removal by the following antioxidant enzymes: superoxide dismutase, catalase, glutathione
peroxidase, and glutathione reductase, and antioxidants (e.g., glutathione) in the cell [
7
].
The antioxidant enzymes and antioxidants coordinate their activity and form an antioxidant
system that reduces the oxidising agents that cause cell damage and death [
8
]. Peroxidation
of membrane lipids, caused by an increase in free radical formation or a decrease in the
activity of the antioxidant defence system, is thought to play a crucial role in controlling
seizures [9].
Epilepsy is treated with oral anti-seizure medications, a chemically heterogeneous
group of drugs that have been categorised into generations according to the date of their
introduction into clinical use. Carbamazepine and valproate belong to the old anti-seizure
medications, while lamotrigine is one of the newer drugs on the market [
10
]. Carba-
mazepine and valproate are the drugs of first choice for epilepsy in Europe and the USA.
They are used to treat generalised seizures (generalised tonic-clonic seizures, absence
seizures, and myoclonic seizures) and epileptic selection seizures—valproate and carba-
mazepine are the treatment of choice for partial seizures (simple partial, complex partial,
and secondary generalized tonic-clonic seizures) and epilepsy syndromes. Valproate is
the drug of choice for generalized epilepsies, and carbamazepine is the preferred drug
for partial epilepsies [
11
]. Carbamazepine is a sodium channel blocker that affects the
serotonin system [
12
]. The mechanisms of action of valproate are unclear, but they appear
to affect GABA levels and block voltage-gated sodium channels [
13
]. Lamotrigine is a
sodium channel-blocking antiepileptic drug that also suppresses the release of glutamate
and aspartate [
14
]. Although they are chemically different, they all prevent seizures. How-
ever, many anti-seizure medications are metabolised to produce reactive metabolites that
can bind macromolecules and thus both impair function on a molecular level and elicit
systemic toxicity [
15
]. The role of anti-seizure medications in oxidative/antioxidative
processes is controversial [
4
] and ranges from a moderate antioxidant to a prooxidant
effect. Although the main root of its oxidant mechanisms is not scientifically established, an
exacerbation of oxidative stress during antiepileptic drug therapy could also be one of the
causes of pharmacotherapy-resistant epilepsy. These prooxidant effects could exacerbate
seizure activity by increasing hyperexcitability and/or causing neuronal damage, which
may lead to the loss of efficacy of seizure medications or apparent functional tolerance
and adverse side effects. Experimental evidence suggests that almost all first-, second-,
and third-generation anti-seizure medications lose their anti-seizure effect with prolonged
therapy, albeit in varying degrees [16].
Human erythrocytes have effective antioxidant protection consisting of enzymatic
and non-enzymatic pathways that counteract reactive oxygen species to maintain redox
regulation in the body [
7
]. On the other hand, changes in the structure and activity of
antioxidant defence enzymes under the direct influence of chronic (long-term) drug intake
as biomarkers of oxidative stress in erythrocytes could be valuable parameters for the
organism’s susceptibility to chronic therapy with anti-seizure medications. Interestingly,
there is not much data in the literature on the effects of selected anti-seizure medications on
antioxidant defence activity in the blood of treated patients. Therefore, the aim of this study
was to investigate the effects of long-term monotherapy with these drugs on the antioxidant
defence enzymes in the erythrocytes and oxidative (haemoglobin/methaemoglobin ratio
of erythrocytes and lipid peroxide levels in plasma) and antioxidative (total glutathione
and thiol groups in plasma) parameters.
Pharmaceuticals 2024,17, 130 3 of 12
2. Results
Before comparing the different anti-seizure therapies, the influence of the daily dosage
regime on the level of selected antioxidant parameters was first measured for each of
the three anti-seizure medications. Daily dosage was divided into three ranges for each
anti-seizure separately. The results showed no significant differences between the daily
anti-seizure medication doses in the individual groups. Therefore, our further analyses
were performed on the entire group sample, regardless of the daily dosing regimen.
Compared to the control, the activity of copper-zinc superoxide dismutase was signifi-
cantly reduced in the patient groups treated with lamotrigine (p< 0.005) and carbamazepine
(p< 0.005). The mean value in the carbamazepine group was lower than in patients treated
with lamotrigine (p< 0.005) and valproate (for both: p< 0.005), and the lamotrigine group
was significantly lower compared to the valproate group (p< 0.01) (Figure 1).
Figure 1. The activity of antioxidant enzymes and concentration of haemoglobin and methaemoglobin
in erythrocytes of patients treated with anti-seizure medications and the control group. Statistically
significant differences between the experimental and control groups: * p< 0.05, ** p< 0.01, and
*** p< 0.001.
All three anti-seizure medication therapy groups showed lower catalase activity than
the control group, but there is only a statistically significant difference between the con-
trol and valproate groups (p< 0.005). Regarding the anti-seizure medication groups, a
significant decrease in catalase activity was found between valproate and lamotrigine
(p< 0.05)
and between valproate and carbamazepine (p< 0.005). There were no differences
in glutathione peroxidase activity in any of the four groups studied (Figure 1).
Compared to controls, there were no significant differences between glutathione
reductase activities in the erythrocytes of patients treated with anti-seizure medications.
However, among the groups of patients, lamotrigine had higher glutathione reductase
activity than carbamazepine (p< 0.005), and the activity in the carbamazepine group was
lower than in the group of patients treated with valproate (p< 0.005) (Figure 1).
No changes in haemoglobin concentration existed between the controls and the three
groups of patients treated with anti-seizure medications. The average percentage concen-
tration of oxidised haemoglobin in the erythrocytes of the participants in all groups was
also not significantly different. In addition, they were very uniform (approx. 1.4% of total
haemoglobin) and slightly lower in the carbamazepine patient group (Figure 1).
The concentration of total thiol groups in plasma was significantly lower in the lamot-
rigine group compared to the control (p< 0.01). There were no significant changes in total
proteins or glutathione concentrations (Figure 2).
Pharmaceuticals 2024,17, 130 4 of 12
Figure 2. The levels of total proteins, lipid peroxides, sulfhydryl groups, glutathione, and nitric
oxide (as nitrite) in the blood plasma of patients treated with anti-seizure medications and the control
group. Statistically significant differences between the experimental and control groups: ** p< 0.01,
and *** p< 0.001.
The estimated nitric oxide concentration was higher in the carbamazepine group than
in the control group (p< 0.01). Regarding the anti-seizure medication groups, significant
differences were found between lamotrigine and carbamazepine (p< 0.005) and between
lamotrigine and valproate (p< 0.005) (Figure 2).
Lipid peroxides were lower in the lamotrigine group compared to the control group
(
p< 0.01
) and in the carbamazepine (p< 0.001) and valproate (p< 0.001) groups (Figure 2).
For canonical discriminant analysis, we used 4 variables (antioxidant enzymes):
copper-zinc superoxide dismutase, catalase, glutathione peroxidase, and glutathione reduc-
tase for each group (anti-seizure medications). Statistical analysis showed that our model
was very significant (Wilks’ Lambda F (12,180) = 11.511; p< 0.0001) and that copper-zinc
superoxide dismutase (p< 0.001), catalase (p< 0.001), and glutathione reductase (p< 0.001)
had the largest significant differences. Chi-square tests with successive roots showed that
there were two significant roots (canonical discriminant functions), D1 (p< 0.001) and D2
(p< 0.001), with their standardized coefficients for canonical variables.
D1 = −0.82 SOD1* + 0.4 CAT* + 0.14 GPx* −0.36 GR* (p< 0.001)
D2 = −0.56 SOD1* −0.72 CAT* −0.2 GPx* + 0.55 GR* (p< 0.001)
* SOD1-cooper-zinc superoxide dismutase, CAT-catalase, GPx-glutathione peroxidase, and
GR-glutathione reductase.
Means of canonical variables for each group (a) and the canonical scores for each
case (b) are presented in Figure 3and plotted in two-dimensional discriminant canonical
space (corresponding to functions D1 and D2). The canonical discriminant analysis showed
that the anti-seizure therapy led to a different composition of the antioxidant defence in
the erythrocytes.
Pharmaceuticals 2024,17, 130 5 of 12
Figure 3. Two-dimensional discriminant analysis of patients treated with anti-seizure and the control
group (a) and the canonical score for each case (b).
Function D1 showed that copper-zinc superoxide dismutase activity in erythrocytes
contributes most to the difference. Function D2 showed that this contribution corresponds
to catalase and glutathione reductase. By using the canonical variables, we were able to
distinguish the control group from all the other groups and separate the lamotrigine and
valproate groups from the carbamazepine group.
3. Discussion
In this paper, we analysed the effects of three anti-seizure monotherapies on the level
of oxidative/antioxidative components in patients’ blood. Our results showed that chronic
therapy changes the activity and composition of antioxidant enzymes. Long-term anti-
seizure monotherapy attenuated the activity of antioxidant enzymes activity in the analysed
parameters, erythrocytes, and blood plasma, but in slightly different ways. Lamotrigine
therapy decreased lipid peroxides, followed by lower levels of superoxide dismutase and
sulfhydryl groups in blood plasma. On the other hand, carbamazepine increased circulating
nitric oxide concentration (estimated by nitrite concentrations), followed by a decrease
in copper-zinc superoxide dismutase. In these two groups, the mechanisms for reducing
copper-zinc superoxide dismutase appeared to differ. Although the lowest methaemoglobin
levels were found in the carbamazepine group, the lower copper-zinc superoxide dismutase
activity may be related to the lower pro-oxidative conditions. Methemoglobin levels in
all treated groups were within the limits expected for healthy controls (1.5%), indicating
that haemoglobin oxidation, as the main site for primary superoxide formation, was
not increased and was even slightly reduced in the carbamazepine group. This result
implies lower oxidative pressure in patients receiving long-term anti-seizure medications.
This finding was even more significant in the group receiving lamotrigine, where overall
lipid peroxidation decreased, as did sulfhydryl groups, indicating less oxidative stress. In
addition, catalase activity was significantly reduced in the valproate group, indicating lower
hydrogen peroxide levels. Since the daily dose (and thus the dosage) did not influence the
patients, the changes observed were due to the long-lasting use of these medications.
From an arbitrary look, the statistical changes observed coincide with changes in other
components that follow the general pattern of reduction in activity, but not uniformly. These
minor changes, which were not significantly different from controls, resulted in significant
differences between the groups of treated patients, which were most pronounced when
glutathione reductase activity was considered; glutathione reductase activity was signifi-
Pharmaceuticals 2024,17, 130 6 of 12
cantly lower in the carbamazepine group compared to valproate and lamotrigine, but not
compared to controls. Therefore, the parameters indicating the composition of antioxidant
enzymes considered in this study (activities of antioxidant enzymes of those considered as
an antioxidant system: copper-zinc superoxide dismutase, catalase, glutathione peroxidase,
and glutathione reductase) were highly significant (expressed as D1 and D2) in canonical
discriminant analysis, which indicates a different composition of the antioxidant system as
a whole and not as a single component. Small, non-significant changes during therapy led
to a shift in antioxidant composition after a long treatment period. Thus, carbamazepine
appeared to differ substantially from both valproate and lamotrigine, while all groups
differed from the controls (Figure 3).
However, since anti-seizure medications are primarily considered sodium chan-
nel blockers, the difference could be due to some additional mechanisms and/or drug
metabolism. Valproate is bound to albumin and affects the GABA-ergic and endocrine
systems, whereas carbamazepine needs to be converted to epoxide via cytochrome P450
to be bioactive, increasing reactive oxygen species. All three substances are metabolised
and excreted by the formation of glucuronide conjugates. Valproate has been reported to
reduce glutathione and superoxide dismutase levels in the brain of control rats after 45 days,
demonstrating that anti-seizure medications alter the levels of oxidants/antioxidants per
se [
17
]. The same study showed that lamotrigine did not induce significant changes in
oxidative/antioxidative balance parameters in the brains of control rats. Considering all
these processes, long-term therapy with anti-seizure medications is a factor that affects the
balance between oxidants and antioxidants and, thus, the antioxidant defence in different
ways, which in turn is reflected in the blood oxidative/antioxidative balance of the blood.
This phenomenon was shown in a study on untreated epileptic children and children
receiving monotherapy with valproic acid and carbamazepine for up to 7 and 12 months,
respectively [
9
]. However, some previous results on the effects of anti-seizure medica-
tions on antioxidant activity during therapy are contradictory, ranging from a moderate
antioxidant effect to an enhancement of the oxidative process [
4
]. Our results showed that
anti-seizure medications reduce antioxidant enzymes, suggesting lower oxidative stress,
as shown in previous studies, but various antioxidant parameters were lower [
3
,
18
]. The
protective effect likely depends on the therapy duration and sampling time. Differences
in the design of similar studies should be taken into account, mainly regarding the age
of the subjects, the period (a few years or more than ten years) during which they have
been taking prescribed medication, the daily doses of these drugs, and whether the control
group consisted of healthy subjects or naïve neurological patients.
In one study that analysed five enzymes (including glutathione-S-transferase), the
activity of glutathione peroxidase was significantly lower, and the activity of glutathione
reductase was considerably higher in patients treated with valproate [
19
]. Our results
also showed an involvement of glutathione reductase activity, which was not significantly
different compared to controls, but was significantly different between patients on anti-
seizure medications. Consistent with our results, some other authors also found that
valproate therapy did not change glutathione peroxidase activity [
20
]. Prolonged valproate
therapy (7–14 years) significantly increased copper-zinc superoxide dismutase activity,
contradicting our results. Still, the control group in this study consisted of clinically
healthy individuals and not patients [
21
]. Peker et al. (2009) found that valproate therapy
increased serum levels of nitric oxide in epileptic children by about 10% compared to the
control group [
22
]. Karabiber et al. (2004) also showed that nitrite and nitrate levels were
significantly higher in epileptic children treated with valproate and carbamazepine [
23
].
Geronzi et al. (2018) showed that carbamazepine increased the release of ATP and nitric
oxide-derived metabolites from erythrocytes into the lumen, leading to an increased nitric
oxide pool in the vasculature [
24
]. Our results confirmed this increase of elevated nitric
oxide in the blood serum of patients from the carbamazepine group, which appeared to be
one of the common carbamazepine effects.
Pharmaceuticals 2024,17, 130 7 of 12
Cengiz et al. (2000) found a significant decrease in total glutathione concentration in
the valproate and carbamazepine groups [
25
]. No significant differences were found in our
studies. However, a notable difference in our study design is that the subjects were children
aged 1–15 years with epilepsy and healthy controls (without epilepsy). This finding also
suggests that the oxidative/antioxidative effects of anti-seizure medications depend on
the age of the patients and cannot be generalised, with important considerations for the
interaction(s) with other medications, age, and morbidity.
With regard to lamotrigine, Huang et al. (2014) discovered a significant decrease in
copper-zinc superoxide dismutase activity, similar to our results, and observed a decrease
in glutathione peroxidase after at least 36 months of monotherapy with this drug [
26
]. They
also found that total glutathione concentrations in blood plasma were much higher in
treated patients than the control subjects, with no changes in non-glutathione sulfhydryl
compounds (thiols) and no differences in lipid peroxide concentrations between groups.
Other studies investigating lamotrigine have shown lower lipid peroxidation than untreated
epilepsy patients or healthy controls, e.g., in [
27
]. The same result was also obtained in
our study.
Our results showed that the mechanisms of antioxidant action of the studied anti-
seizure medications differ, leading to a shift in antioxidant composition reflecting the
oxidant-antioxidant balance, which is considered an essential process in epileptogenesis.
Although our study has some limitations (e.g., the comparison between drug-naive patients
and patients receiving long-term monotherapy, excluding a group of healthy individuals
chronically treated with these drugs and the comparison between healthy individuals
without medication and healthy individuals with medication and epileptics with medi-
cation), we have shown that long-term therapy (at least six months) with the anti-seizure
medications studied alters the balance between oxidants and antioxidants through different
mechanisms and components. The exact impact of the observed changes on pathophysiol-
ogy cannot be determined without parallel studies of clinical manifestations in patients.
However, our study was conducted when the epileptogenic period was over, therapy
was underway, clinical manifestations were in the foreground, and the antioxidant system
was tracking not only the reactive oxygen species involved in epilepsy but also the effects
of medications at different levels. Studying antioxidant systems can help assess the role,
importance, and extent of particular anti-seizure medications in regulating the balance
between oxidants and antioxidants. This approach may help to establish antioxidant proto-
cols for complementary effects, including oxidative stress in epileptogenesis and epilepsy.
Several antioxidants are considered protective in epilepsy [
5
], such as
α
-tocopherol [
28
],
coenzyme Q
10
[
6
], melatonin [
29
], resveratrol [
30
],
α
-lipoic acid [
31
], curcumin [
32
], ascor-
bic acid [
33
], and vitamin D [
34
], but none of them showed a large and significant effect.
Therefore, the combination of anti-seizure medications and antioxidants could be helpful.
Still, the combination must be carefully considered since each anti-seizure medication
affects the balance between oxidants and antioxidants through its specific mechanism.
4. Materials and Methods
4.1. Subjects
This study was conducted at the Institute of Mental Health Belgrade, Serbia, in 2019
and 2020 in accordance with the Declaration of Helsinki and the Good Clinical Practice
guidelines. The study protocol was approved by the Ethics Committee of the Institute
of Mental Health School of Medicine, University of Belgrade, Belgrade, Serbia (licence
No. 30/26), and patients gave informed written consent.
Patients with epilepsy aged 18–70 years were recruited consecutively during their
regular appointments at the Department of Epilepsy and Clinical Neurophysiology of the
Institute of Mental Health in Belgrade. The diagnosis and type of epilepsy were established
by a neurologist (M.M.) [
35
,
36
] based on seizure semiology, neurological examination, and
video-electroencephalography (V-EEG). Of the final sample of 67 patients with epilepsy,
24 had idiopathic generalised epilepsy, and 43 had focal epilepsy. Based on more than
Pharmaceuticals 2024,17, 130 8 of 12
six months of monotherapy with anti-seizure medications (lamotrigine, carbamazepine,
and valproate), three groups were formed with 22 and 23 epilepsy patients of both sexes
and different ages (Table 1). In the semi-structured interview, each patient’s demographic
and clinical characteristics, the dosage and administration regimen of the seizure med-
ication, concomitant diseases, and drug therapy were recorded. Patients were initially
categorised into three ranges for each antiepileptic drug according to the daily dosage
(lamotrigine group: <100 mg, 100–200 mg, and >200 mg per day; valproate group:
<500 mg
,
500–1000 mg
, and >1000 mg per day; carbamazepine <400 mg, 400–800 mg, and >800 mg
per day). As there were no significant differences in the measured parameters due to daily
dosing, they were further summarised as one group for each anti-seizure medication. An
exclusion criterion was the presence of a severe concomitant disease that required the con-
comitant use of other medications. The concomitant diseases of the patients studied ranged
from hypovitaminosis D, hypertension, and hyperlipidaemia to fibrillatio atriorum, chori-
oretinitis focalis, and posthysterectomy. Other medications besides anti-seizure medications
included vitamin D, captopril, propafenone, acetylsalicylic acid, and hydrochlorothiazide.
The control group consisted of drug-free epilepsy patients before the start of seizure therapy
(to compare the results obtained). Comorbidities in the control group mainly included
headaches and, in one case, diabetes.
Table 1. Anamnestic data for patients who participated in this study.
Group Gender Average Years
(Mean ±SD) Smokers Other
Medications Comorbidity
M F
Controls 4 6 44.9 ±18.4 1/10 3/10 4/10
Lamotrigine 7 15 64.2 ±17.9 0/22 15/22 18/22
Carbamazepine 12 11 42.1 ±16.5 9/23 15/23 19/23
Valproate 19 3 33.6 ±19.7 5/22 5/22 6/22
4.2. Blood Sampling Collection, Isolation and Haemolysis of Erythrocytes
Venous blood (approximately 5 mL) was obtained after overnight fasting (
10–12 h
)
and stored directly in vacutainers coated with an anticoagulant film (EDTA, 1 g/L). Within
48 h after sampling, the whole blood sample was processed. The erythrocytes were settled
from the blood via centrifugation at 3000
×
gfor 10 min. The plasma in the supernatant
was separated and stored in the freezer. The intermediate layer was discarded, and the ery-
throcytes were washed three times with five volumes of saline (0.9% NaCl) and separated
via centrifugation, as described above. Erythrocyte hemolysis was performed by adding
3 mL of ice-cold MiliQ water to 0.5 mL of washed erythrocytes, followed by mixing. The
obtained haemolysates were stored in the freezer until use (maximum seven days).
4.3. Preparation of Haemolyses for Determination of Antioxidant Enzyme Activity
To determine the activity of copper-zinc superoxide dismutase, it was necessary to first
precipitate the haemoglobin from the analysed haemolyses [
37
]. Briefly, 0.4 mL of ice-cold
distilled water was added to 0.4 mL of haemolysate (in 2 mL Eppendorf tube), followed by
0.4 mL of ice-cold ethanol. After stirring the samples briefly on a Vortex, 0.3 mL of ice-cold
chloroform was added dropwise to the resulting suspension. After vigorous stirring on
a vortex (about 30 s per sample), the contents were centrifuged (5000
×
g, 5 min). The
resultant clear (and colourless) supernatants were separated from the precipitated proteins
(haemoglobin). These samples were left in the freezer until determination. The adrenaline
method was used to determine total copper-zinc superoxide dismutase activity [
38
]. One
copper-zinc superoxide dismutase unit was defined as the amount of the enzyme necessary
to decrease the rate of adrenaline auto-oxidation by 50% at pH 10.2. Copper-zinc superoxide
dismutase activity was expressed in activity units per gram of haemoglobin (U/g Hb).
Pharmaceuticals 2024,17, 130 9 of 12
Aliquots of haemolysate were used to determine the activity of catalase, glutathione
peroxidase, and glutathione reductase using a Shimadzu UV-160 spectrophotometer
(Tokyo, Japan).
This haemolysate was further diluted to determine catalase activity: 10
µ
L of haemolysate,
10
µ
L of 96% ethanol, and 980
µ
L of distilled water were added, and the resulting solution
was mixed well on a Vortex. Catalase activity in dilute haemolysate was determined
according to [
39
]. One unit of catalase activity was defined as the amount of the enzyme
that decomposes 1 mmol H
2
O
2
per minute at 25
◦
C and pH 8.0; catalase activity was
expressed in activity units per gram of haemoglobin (U/g Hb).
Selenium-dependent glutathione peroxidase activity was determined by the glu-
tathione reduction of t-butyl hydroperoxide using a modification of the assay described
by Paglia and Valentine (1967) [
40
]. Glutathione peroxidase activity is expressed as the
amount of the enzyme needed to oxidise 1
µ
mol NADPH per minute at 25
◦
C and pH 7.0
per gram of haemoglobin; the unit of activity is
µ
M NADPH min
−1
g Hb
−1
. Glutathione
reductase activity was determined using the method of Glatzle and colleagues (1974) [41].
One unit of glutathione reductase activity was defined as the amount of the enzyme needed
to oxidise 1 µmol NADPH per minute at 25 ◦C and pH 7.4 per gram of haemoglobin.
4.4. Determination of Total and Methaemoglobin in Haemolyses
The total haemoglobin content in the analysed haemolyses was determined by the
standard Drabkin cyano-methaemoglobin method [
42
]. The absorbance was measured at
545 nm (Shimadzu UV-1800 UV/Visible Scanning Spectrophotometer 1800;
Kyoto, Japan)
.
The haemoglobin concentration was expressed in mg/mL. The content of oxidised
methaemoglobin was determined based on the unique spectral characteristics of this
haemoglobin derivative: a small peak at 630 nm [
43
]. To record the spectra, we used a
Thermo Scientific NanoDrop 2000c spectrophotometer, Waltham, USA. Levels of
methaemoglobin are expressed in percentages of total haemoglobin.
4.5. Determination of Plasma Parameters
Total protein concentration, concentration of total plasma thiols, nitrite concentration,
lipid peroxides level, and total glutathione level were determined in the stored plasma
(after centrifugation of whole blood). Total protein concentration was measured with an
accurate and rapid method with Biuret reagent [
44
]. The addition of a cupric salt to an alka-
line protein solution produces a reddish-violet colour, measuring 546 nm. The total protein
concentration was expressed in grams per litre (g/L). A spectrophotometric assay based on
5,5
′
-dithiobis(2-nitrobenzoic acid) acid (DTNB or Ellman’s reagent) was used to determine
total plasma thiols [
45
]. Concentrations were expressed in mM. Nitric oxide concentra-
tion was indirectly measured by reconversion of nitrate/nitrite to nitric oxide by Griess
reagent [
46
]. The method was based on the formation of a coloured azo compound with
maximum absorption at 548 nm in the reaction between sulfanilic acid (or sulfanilamide)
and N-(1-naphthyl)ethylenediamine in an acidic environment in the presence of nitrite.
Nitrite concentration was expressed in
µ
M. Total protein concentration, total plasma thiols,
and nitrite concentration were measured using a Shimadzu UV-1800 spectrophotometer
(Tokyo, Japan).
The level of lipid peroxides was determined using a Biotek Synergy H1 microplate
reader, according to the method described by [
47
]. In this method, we used a thiobarbituric
acid assay to measure lipid peroxide levels in the plasma. Lipids and proteins were
precipitated using a phosphotungstic acid-sulfuric acid system to eliminate disturbing
substances such as glucose and water-soluble aldehydes. Also, the reaction of thiobarbituric
acid with these lipids was carried out in an acetic acid solution to avoid its reaction with
other substances, such as sialic acid. After the thiobarbituric acid reaction, the product was
measured via fluorometry because the thiobarbituric acid reaction product is a fluorescent
substance. Levels of lipid peroxides are expressed in nmol per ml of blood. The total
Pharmaceuticals 2024,17, 130 10 of 12
glutathione concentration was determined on the same reader, according to the method
described by [48]; the concentration was expressed in g/L.
4.6. Statistical Analyses
Statistical analyses were performed according to the protocols described by Hinkle
et al. (2002), Manley (1986), and Blagojevi´c et al. (1998) [
49
–
52
], which have been subse-
quently applied to study neurological conditions [
53
–
55
]. The results are expressed as the
mean value followed by the standard error of the mean (SEM). The data were analyzed us-
ing analysis of variance (ANOVA) and Tukey post-hoc Honest Significant Difference (HSD)
test for unequal sample size was used to determine significant differences between groups.
The canonical discriminant analysis measured the effects of anti-seizures on overall con-
nections between individual antioxidant enzyme components at the total group level and
the significance of these changes. Correlation analysis was used to calculate the relations
between individual components, but an alternative statistical test, canonical discriminant
analysis, was used to calculate differences between groups, considering the complete corre-
lation matrix of a particular group (i.e., the composition of antioxidant defence enzymes) to
others. A probability level of p< 0.05 was considered statistically significant.
5. Conclusions
Our results suggest that long-term monotherapy with these anti-seizure medications
can affect oxidant/antioxidant balance, where copper-zinc superoxide dismutase (lamot-
rigine and carbamazepine) and catalase (valproate) were primarily affected (decreased).
Lamotrigine significantly reduced the level of lipid peroxides and the total plasma con-
centration of the sulfhydryl group, suggesting lower oxidative stress and protectivity. In
addition, carbamazepine increased the formation of nitric oxide, which seems to be a
common effect. We demonstrated that long-term therapy with anti-seizure medications can
change the oxidant/antioxidant balance through different mechanisms and components.
Author Contributions: Conceptualisation, M.N., ˇ
C.M. and M.M.; methodology, A.N.-K.; software,
T.V.U.; validation, A.N.-K. and E.N.; formal analysis, D.J. and V.J.; investigation, D.J.; resources, M.M.;
data curation, T.V.U.; writing—original draft preparation, D.J.; writing—review and editing, D.B.,
M.N. and A.N.-K.; supervision, ˇ
C.M. and M.M.; project administration, D.B. and M.N.; funding
acquisition, D.B. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the Ministry of Education, Science and Technological De-
velopment of the Republic of Serbia (grant numbers: 451-03-47/2023-01/200007 and 451-03-47/
2023-01/200168).
Institutional Review Board Statement: The study protocol was approved by the Ethics Committee
of the Institute of Mental Health School of Medicine, University of Belgrade, Belgrade, Serbia (licence
No. 30/26).
Informed Consent Statement: Informed consent was obtained from all the subjects involved in
this study.
Data Availability Statement: Data is contained within the paper.
Conflicts of Interest: The authors declare no conflicts of interest.
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