Multiple sclerosis (MS) is a multifactorial disease of the central nervous system (CNS) characterized by inflammation and demyelination [
Hyperactivity OS enzyme is responsible for the production of free radicals [
Multiple sclerosis may occur in several forms, that is, relapsing-remitting (RRMS), secondary progressive (SPMS), and primary progressive (PPMS). Currently, MS treatment is based on immunomodulatory therapy. The I-line of treatment includes interferon (IFN) beta, glatiramer acetate, dimethyl fumarate, and teriflunomide, whereas the II-line includes fingolimod (FG), natalizumab (NT), and alemtuzumab.
The most widely used I-line drugs are interferon (IFN) beta and glatiramer acetate. Their effectiveness is comparable in the treatment of the RRMS [
Due to the fact that the effectiveness of therapy is still limited, new therapeutic strategies are constantly under investigation [
It is believed that new immunomodulatory drugs may have an influence on OS level in patients with MS [
We assessed OS parameters in the blood serum of RRMS patients in the Clinic of Neurology in Zabrze, Poland. The parameters were assessed in patients treated with II-line immunomodulatory therapy compared to newly diagnosed RRMS patients (de novo group), RRMS patients treated with IFN beta, and healthy subjects. We assessed the relationship between OS parameters and gender, age, disease duration, degree of disability in the Expanded Disability Status Scale (EDSS), ARR, and MRI Gd + lesions.
One hundred and twenty-one patients diagnosed with RRMS according to the McDonald criteria of 2010 were enrolled in the study. In addition, the study involved 41 volunteers (controls) that did not suffer from MS.
The inclusion criteria (study group) are the following:
RRMS patients diagnosed according to the 2010 McDonald criteria. The immunomodulatory treatment with interferon 10 ml of venous blood Age ≥ 18 years Female or male Written informed consent for participation in the study Patient free from relapse Blood samples were not taken immediately after drug administration.
The exclusion criterion (study group) is the following:
Lack of consent to participate in the study.
The inclusion criteria (control group) are the following:
Generally healthy people (diagnosis of MS was excluded; treatment of hypertension was not a contraindication) Nonsmokers.
The exclusion criteria (control group) are the following:
Smokers Lack of consent to participate in the study.
Patients were divided into the following groups, according to the type of immunomodulatory therapy:
De novo group—patients newly diagnosed with RRMS without immunomodulatory therapy (24 patients) IFN—RRMS patients treated with IFN beta-1a (30 FG—RRMS patients treated with FG (0.5 mg/daily p.o.) (39 patients) NT—RRMS patients treated with NT (300 mg once a month IV) (26 patients) Control—a control group of nonsmoking healthy volunteers (41 persons).
A sample of 10 ml venous blood were obtained and placed in chilled tubes with 1 mg/ml EDTA-K3 as anticoagulant. After centrifugation of blood samples, the obtained serum was frozen at −80°C. The following OS parameters were determined:
concentration of lipid hydroperoxides (LHP), malondialdehyde (MDA), lipofuscin (LPS), total oxidative status (TOS).
The determination of the level of the parameters was conducted using standard methods.
The concentration of LHP in serum was determined by the method of Sodergren et al. Xylene xangan was used. The procedure was based on the oxidation of iron (II) ions to iron (III) ions in acidic medium. Then, iron (III) ions with xylene orange form a colorful complex, up to a blue-purple coloration. The reading was made with a 560 nm filter using the Perkin Elmer VICTOR-X3 reader. The concentration was read from the calibration curve prepared with the aid of appropriate H2O2 concentrations. Values are expressed in
The MDA concentration was determined in serum using the MDA reaction with thiobarbituric acid according to Ohkawa. For reading, the LS45 spectrometer from Perkin Elmer was used at 515 nm (absorbance) and 552 nm (emission) spectrophotometer. Spectrofluorometric reading, unlike spectrophotometry (at 532 nm), is more specific, and it does not interfere with hemoglobin; no bile duct interference is observed. The method was modified by adding sodium sulphate and BHT, which further increased the specificity of the method. MDA concentrations were read from the standard curve using standard 1,1,3,3-tetraethoxypropane and expressed in micromoles per liter of serum (
Serum LPS concentration was determined according to the method of Tsuchida et al. Ethanol-ether mixtures 3 : 1 (
The values are expressed in relative lipid extract fluorescence (RF), where the value of 100 RF corresponds to the fluorescence of the solution of 0.1
The TOS assay is based on the oxidation of iron (II) ions to iron (III) ions in acidic medium. Then, iron (III) ions with xylene orange form a colorful complex, up to a blue-purple coloration.
The absorbance readings were made with a 560 nm filter on the PerkinElmer VICTOR-X3.
The concentration was calculated from the calibration curve using H2O2 as the standard.
Values are expressed in
Demographic data, clinical disease onset, disease duration, clinical form of MS, the type of treatment, ARR, the degree of disability in the EDSS scale, and lesions on MRI were obtained from medical databases and the Department of Neurology. The results were stored in the database prepared specifically for this purpose in Microsoft Excel.
STATISTICA 9.1 was used for the statistical analysis.
Homogeneity of continuous variables between groups was analyzed using the parametric ANOVA test (for normally distributed variables) or the nonparametric test Kruskal-Wallis ANOVA (for variables whose distribution was not normal).
Post hoc analysis using Tukey’s test with the Bonferroni correction was conducted in the case of statistically significant differences. The Student
The study was approved by the Bioethics Committee of the Medical University of Silesia in Katowice, Poland. This study was conducted in accordance with the Helsinki criteria for patient trials. The approval number of The Bioethical Commission was KNW/0022/KB1/37/16 of 19th April 2016.
A group of patients with RRMS and the control group proved to be homogeneous in terms of gender and age. Women accounted for 66.94% of patients and 78.05% of the control (Table
General characteristics of the study groups divided into MS patients (RRMS) and healthy people (control).
Group | RRMS | Control |
|
---|---|---|---|
|
121 | 41 | 0.157 |
Age (years) | 37.5 ± 14 | 36 ± 21 | 0.588 |
Gender (% of females) | 66.94 | 78.05 | 0.181 |
RRMS: relapsing-remitting multiple sclerosis; statistical significance for
Table
The demographic and clinical characteristics of the study group.
Group | De novo RRMS | INF | FG | NT | Control |
---|---|---|---|---|---|
|
24 | 32 | 39 | 26 | 41 |
Age (years) | 43.05 ± 12.73 | 40.50 ± 9.45 | 36.49 ± 11.67 | 33.96 ± 8.45 | 39.46 ± 12.30 |
Gender (% of females) | 66.67 | 71.88 | 56.41 | 76.92 | 78.05 |
Disease duration (years) | NA | 4.39 ± 4.51 | 6.66 ± 4.27 | 5.96 ± 3.35 | NA |
EDSS (score) | 2.52 ± 1.65 | 1.98 ± 0.86 | 3.08 ± 1.10 | 3.08 ± 1.14 | NA |
ARR ( |
0.84 ± 0.83 | 0.25 ± 0.44 | 0.37 ± 0.64 | 0.12 ± 0.33 | NA |
Gd + MRI lesions ( |
0.76 ± 0.83 | 0.43 ± 1.13 | 0.09 ± 0.39 | 0.12 ± 0.61 | NA |
T2 MRI lesions ( |
19.05 ± 3.99 | 18.53 ± 4.38 | 20.25 ± 1.86 | 18.50 ± 4.90 | NA |
De novo RRMS: patients with a newly diagnosed relapsing-remitting multiple sclerosis; RRMS INF: RRMS patients treated with interferon beta; FG: RRMS patients treated with fingolimod; NT: RRMS patients treated with natalizumab; EDSS: Expanded Disability Status Scale; ARR: annualized relapse rate; NA: nonapplicable.
Table
The detailed clinical characteristics of RRMS patients prior to inclusion in the II-line treatment
Group | FG | NT |
---|---|---|
Time of I-line treatment (years) | 2 ± 2 | 2 ± 2.5 |
Type of treatment in I-line: | ||
INF beta-1a (%) | 35.9 | 40 |
INF beta-1b (%) | 46.15 | 45 |
OG (%) | 17.95 | 15 |
EDSS (pkt) | 3.5 ± 1 | 3.5 ± 1 |
ARR ( |
2.02 ± 0.77 | 2.31 ± 0.73 |
Gd + MRI lesions ( |
2 ± 4 | 3 ± 3 |
T2 MRI lesions ( |
18.39 ± 5.70 | 14.56 ± 8.19 |
The detailed clinical characteristics of RRMS patients after inclusion in the II-line treatment
Group | FG | NT |
|
---|---|---|---|
Time of II-line treatment (years) | 2.14 ± 1.39 | 2.09 ± 1.27 | 0.841 |
% of patients without relapses | 71.43 | 87.50 | 0.143 |
% of patients without clinical progression | 28.57 | 33.33 | 0.721 |
% of patients without radiological progression | 93.75 | 95.83 | 0.732 |
T2 MRI lesions ( |
20.25 ± 1.86 | 18.50 ± 4.90 | 0.380 |
RRMS INF: RRMS patients treated with interferon beta; FG: RRMS patients treated with fingolimod; NT: RRMS patients treated with natalizumab; OG: RRMS patients treated with octan glatiramer; EDSS: Expanded Disability Status Scale; ARR: annualized relapse rate; statistical significance for
The treatment effects are presented as the percentage of patients free from relapse (FG group 71.43% versus NT group 87.5%), free of clinical disease progression at the end of the observation (FG group 28.57% versus NT group 33.33%), and radiological disease progression at the end of the observation (FG group 93.75% versus NT group 95.83%). The number of T2 MRI lesions was represented as N number (FG group 20.25 ± 1.86 versus NT group 18.50 ± 4.90). The results did not differ significantly between the two groups (Table
Patients with RRMS were divided into four groups. The fifth group was the control group.
The comparison of the parameters of oxidative stress in serum in the study groups
Group | De novo RRMS | INF | FG | NT | Control |
|
---|---|---|---|---|---|---|
|
24 | 33 | 39 | 26 | 41 | |
LHP ( |
24.64 ± 40.64 | 3.37 ± 16.32 | 3.07 ± 7.72 | 3.99 ± 7.05 | 0.84 ± 0.62 |
|
MDA ( |
6.31 ± 3.18 | 3.11 ± 3.83 | 3.39 ± 1.75 | 3.63 ± 2.15 | 2.56 ± 0.51 |
|
TOS ( |
39.11 ± 64.51 | 5.35 ± 35.47 | 8.48 ± 11.59 | 6.87 ± 15.4 | 2.39 ± 1.08 |
|
LPS (RF) | 950.47 | 869.3 ± 293.9 | 808.52 ± 247.59 | 745.71 ± 260.59 | 764 ± 167.77 |
|
Post hoc analysis in the study groups
Parameter | LHP | MDA | TOS | LPS | ||||
---|---|---|---|---|---|---|---|---|
Group | FG | NT | FG | NT | FG | NT | FG | NT |
De novo RRMS |
|
|
|
|
|
|
NS |
|
INF | NS | NS | NS | NS | NS | NS | NS | NS |
Control |
|
|
NS | NS | NS | NS | NS | NS |
Post hoc analysis in the study groups
Parameter | LHP | MDA | TOS | LPS | ||||
---|---|---|---|---|---|---|---|---|
Group | INF | Control | INF | Control | INF | Control | INF | Control |
De novo RRMS |
|
|
|
|
|
|
NS |
|
Control |
|
|
|
|
|
|
NS |
|
De novo RRMS: patients with a new diagnosed relapsing-remitting multiple sclerosis; RRMS INF: RRMS patients treated with interferon beta; FG: RRMS patients treated with fingolimod; NT: RRMS patients treated with natalizumab; LHP: lipid hydroxyperoxides; MDA: malondialdehyde; TOS: total oxidative status; LPS: lipofuscin; NA: nonapplicable; statistical significance for
All groups were compared with respect to the selected parameters of OS in serum. The study groups showed the difference in the concentrations of LHP, MDA, LPS and TOS. The post hoc analysis was conducted for FG and NT groups only for statistically significant
Significantly lower concentrations of LHP, MDA and TOS were observed in NT and FG groups compared to the de novo group. In addition, a lower concentration of LPS was observed in the NT group compared to the de novo group. Both NT and FG groups were not different from the IFN group in terms of OS parameters. Significantly higher MDA concentrations were noted in the groups treated with II-line immunomodulatory therapy as compared to the control group. The IFN group also had significantly lower all parameters of OS compared to the de novo group, but they were higher in the IFN group compared to the control group. It should be stressed that in the FG and NT groups only the MDA concentration was higher than that in the control. Other parameters were not different from the control group.
Women were selected from all of the groups. The post hoc analysis revealed that all women treated with II-line drugs had lower levels of OS parameters such as MDA, LPH, and TOS compared to women who had not been previously treated. Women in the IFN group had lower levels of MDA, LHP, and TOS compared to women diagnosed de novo, but MDA and LHP levels were higher in the IFN group compared to healthy women. However, in the FG and NT groups, this difference was not observed.
The comparison of the selected parameters of oxidative stress in serum in women
Group | De novo RRMS | INF | FG | NT | Control |
|
---|---|---|---|---|---|---|
|
16 | 23 | 22 | 20 | 32 | |
LHP ( |
35.65 ± 28.07 | 12.62 ± 16.86 | 8.02 ± 11.09 | 7.11 ± 11.04 | 0.86 ± 0.48 |
|
MDA ( |
6.33 ± 2.57 | 4.36 ± 2.52 | 4.02 ± 1.94 | 4.10 ± 2.07 | 2.61 ± 0.44 |
|
TOS ( |
21.86 ± 30.08 | 21.86 ± 30.08 | 18.04 ± 27.84 | 14.58 ± 17.53 | 2.46 ± 0.71 |
|
Post hoc analysis for women
Parameter | LHP | MDA | TOS | |||
---|---|---|---|---|---|---|
Group | FG | NT | FG | NT | FG | NT |
De novo RRMS |
|
|
|
|
|
|
INF | NS | NS | NS | NS | NS | NS |
Control | NS | NS | NS | NS | NS | NS |
Post hoc analysis for women
Parameter | LHP | MDA | TOS | |||
---|---|---|---|---|---|---|
Group | INF | Control | INF | Control | INF | Control |
De novo RRMS |
|
|
|
|
|
|
Control |
|
|
|
|
NS |
|
De novo RRMS: patients with a newly diagnosed relapsing-remitting multiple sclerosis; RRMS INF: RRMS patients treated with interferon beta; FG: RRMS patients treated with fingolimod; NT: RRMS patients treated with natalizumab; LHP: lipid hydroxyperoxides; MDA: malondialdehyde; TOS: total oxidative status; NA: nonapplicable; statistical significance for
Then, men were selected from all of the groups. The post hoc analysis revealed that all the men treated with II-line drugs had a lower level of LHP than men who had not been previously treated. In men, OS parameters from the IFN group were not different from the OS parameters in the de novo group. Healthy men had lower most of these parameters compared to men diagnosed de novo and the IFN group.
The comparison of the selected parameters of oxidative stress in serum in men
Group | De novo RRMS | INF | FG | NT | Control |
|
---|---|---|---|---|---|---|
|
8 | 9 | 17 | 6 | 9 | |
LHP ( |
20.59 ± 13.87 | 18.57 ± 25.81 | 4.10 ± 3.72 | 9.29 ± 7.28 | 0.70 ± 0.51 |
|
MDA ( |
5.73 ± 1.92 | 5.12 ± 3.43 | 3.64 ± 0.99 | 4.60 ± 1.79 | 2.43 ± 0.24 |
|
TOS ( |
32.99 ± 21.51 | 31.24 ± 41.71 | 8.92 ± 7.05 | 21.48 ± 18.95 | 2.27 ± 0.98 |
|
Post hoc analysis for men
Parameter | LHP | |
---|---|---|
Group | FG | NT |
De novo RRMS |
|
NS |
INF | NS | NS |
Control | NS | NS |
Post hoc analysis for men
Parameter | LHP | MDA | TOS | |||
---|---|---|---|---|---|---|
Group | INF | Control | INF | Control | INF | Control |
De novo RRMS | NS |
|
NS |
|
NS |
|
Control |
|
|
|
|
NS |
|
De novo RRMS: patients with a newly diagnosed relapsing-remitting multiple sclerosis; RRMS INF: RRMS patients treated with interferon beta; FG: RRMS patients treated with fingolimod; NT: RRMS patients treated with natalizumab; LHP: lipid hydroxyperoxides; NA: nonapplicable; statistical significance for
Moderately positive correlations were found in the NT group between the concentrations of MDA, LHP, TOS, and ARR. No other significant correlation was found.
The most important correlations of the selected parameters of oxidative stress in serum for RRMS patients treated with II-line drugs.
Parameter | Age (years) | Disease duration (years) | ARR | EDSS | Gd + MRI lesions ( |
Group |
---|---|---|---|---|---|---|
TOS | NS | NS | NS | NS | NS | FG |
MDA | NS | NS | NS | NS | NS | |
LHP | NS | NS | NS | NS | NS | |
TOS | NS | NS |
|
NS | NS | NT |
MDA | NS | NS |
|
NS | NS | |
LHP | NS | NS |
|
NS | NS | |
TOS | NS | NS | NS | NS |
|
IFN |
MDA | NS | NS | NS | NS |
|
|
LHP | NS | NS | NS | NS |
|
FG: RRMS patients treated with fingolimod; NT: RRMS patients treated with natalizumab; EDSS: Expanded Disability Status Scale; ARR: annualized relapse rate; LHP: lipid hydroxyperoxides; MDA: malondialdehyde; TOS: total oxidative status; NA: nonapplicable; R: Pearson linear correlation coefficient.
In the IFN group, only a positive correlation was found between Gd + MRI lesions and LHP and MDA and TOS concentrations.
The contribution of OS to MS is very complex and linked to many mechanisms (Figure
Possible significance of oxidative stress in the pathomechanism of multiple sclerosis. CNS: central nervous system; ROS: reactive oxygen species; RNS: reactive nitrogen species; OS: oxidative stress; ICAM: intercellular adhesion molecule.
This study showed an impact on OS parameters in MS patients treated with II-line immunomodulatory therapy. Better understanding of the effects of II-line drugs may help explain the mechanism of OS in the pathogenesis of MS. It is possible that this direction of research may allow in the future to introduce new therapies based on the oxidative/antioxidant system. What is more, there may be some possibility of using new markers to evaluate treatment response [
Unfortunately, there are some limitations to these markers, due to the fact that the level of OS may depend on many factors such as age, gender, activity level, diet, smoking, and exposure to toxic substances. Our study took into account the gender and age of patients with MS and the control group.
Both in NT and FG groups, a reduction was observed in the level of parameters such as TOS, LHP, and MDA compared to the newly diagnosed patients. Similar results were also obtained in the IFN and the control group.
Some study suggested that FG might even change MDA levels in the hippocampus in the rat model of autism [
Researchers suggested a multidirectional and very complex mechanism of FG action, also listing immunosuppression and antitumor activity [
Early studies suggested that FG can induce apotheosis of cells as well as protect cells from OS. One study demonstrated that FG could induce apoptosis of tumor cells, lymphocytes, and atypical neutrophils by rapid translocation of heat shock protein 27 to the cell surface. The authors of that study suggested that FG acted through the necrosome signalling complex and the OS machinery [
Studies on FG in other diseases showed its positive effect in acute stroke [
In our study, lipid peroxidation (LPH) in patients treated with II-line was more severe compared to controls, and in the IFN group all examined parameters were higher than those in the control group. However, the level of the selected OS parameters in patients treated with IFN beta and II-line drugs was not different.
The data on the impact of IFN beta on the reduction of OS are still unclear. It is supposed that IFN beta treatment in combination with other agents or antioxidants could decrease the level of oxidative parameters. For instance, the treatment with IFN beta and glatiramer was reported to have reduced tumor necrosis factor alpha (TNF-
It is difficult to assess the participation of IFN beta in oxidative processes. The suppression of OS by IFN beta cannot be ruled out as compared to patients treated with II-line therapy.
Patients treated with NT had a lower TOS and lower lipid peroxidation levels than untreated patients. Several authors suggested the impact of the monoclonal antibody on the whole OS level in MS patients [
Scientific studies confirmed that certain genotypes of detoxification enzymes such as NQO1 and the GSTP gene possibly showed a better clinical outcome after NT therapy [
In one of the studies, 22 MS patients were treated with NT. It was observed that NT prompted a decrease in oxidative-damage biomarker levels after 14 months and induced nuclear translocation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) which was responsible for the activation of the antioxidant pathway [
The level of LPS was higher in patients treated with NT compared to healthy controls in our results. Konig et al. suggested that antioxidants could reduce the level of accumulation of LPS in the mitochondria of senescent cells [
Generally, patients with RRMS had higher lipid oxidation (MDA, LHP) and TOS compared to the control group. Therefore, MS may be associated with higher peripheral OS in RRMS patients. Interestingly, similar findings were reported in other studies [
Tasset et al. pointed out the limitations of the studies related to peripheral samples. The authors listed such limitations as patient-specific features, the nature of the sample (tissue, cerebrospinal fluid, plasma, or erythrocytes), and differences in the study situation (experimental models and clinical studies with different characteristics). In this study, they observed an increased level of TOS, 8-OHdG, and carbonylated proteins in MS patients qualified for the treatment with NT. On the other hand, the samples were taken before administration of the drug (no information about the previous treatment) [
However, it can be seen that patients treated with II-line have more advanced disease than patients treated with I-line. In our results, the levels of MDA and TOS in the FG and the NT groups were not different from the control group, which may suggest a potential influence of these drugs on OS.
On the other hand, our study was limited to a small number of patients and the control group. Therefore, it was difficult to properly evaluate the effects of FG, NT, and IFN on OS in the MS patients.
The present results concerning OS level in RRMS patients in relation to gender, age, disease duration, EDSS, ARR, and Gd (+) MRI lesions are consistent with some reports of other authors.
In RRMS women treated with FG, NT, and IFN, most of the OS parameters were lowered compared to the RRMS women with the newly diagnosed disease, whereas in men only LHP was reduced in the FG group. These results are not representative enough due to the fact that the group of women was larger compared to the male group. The comparison between women and men with MS did not reveal differences (data not shown). It is known that women are more likely to have MS, which may be related to genetic predisposition [
Mifflin et al. demonstrated that male mice with EAE given daily access to running wheels had significantly less OS compared to females with EAE. This may suggest that there are sex-specific effects on disease-related outcomes connected with exercise [
The data on the level of OS and gender of MS patients are insufficient to make it clear which gender predisposes to greater exposure to oxidative processes.
The level of OS in patients treated with NT and FG was not associated with new lesions on MRI. This correlation was found only for the IFN group.
In the AFFIRM study, NT was associated with a 76% reduction in new T1-hypointense lesions (the development of black holes) at 2 years (
As the study showed, FG reduced brain volume loss (BVL) and promoted no evidence of disease activity (NEDA-4) in MS patients [
No correlations between the selected OS parameters and age, disease duration, or EDSS were observed in the RRMS group treated with FG and NT.
These data are—at least in part—in agreement with other results [
However, one study showed an increase in OS parameters (IL-10, TNF-
Our results did not reveal EDSS differences in EDSS < 2, 2–4, and >4 groups (results not shown) and OS. In this division, however, the type of treatment was not included. No differences were observed in the whole MS group. Only patients with RRMS were included. It seems that RRMS, secondary progressive MS, and primary progressive MS patients and the control group should be compared in order to better assess the severity of the disease and OS. For example, Lam et al. observed that plasma concentrations of F2-isoprostanes and prostaglandin F2alpha (PGF2
In our study, no correlation was found between the duration of the disease and the oxidative stress parameters of patients in I-line and II-line patients. One recent study demonstrated that NOx decreased with MS duration, which was significant for patients treated with II-line drugs [
In our study, patients undergoing immunomodulatory treatment presented generally lower OS parameters than the untreated patients. There is a chance that the new biomarkers may be used in the future to evaluate treatment response. The major limitation of using peripheral samples is connected with the fact that we do not know exactly how the OS parameters in serum reflect the processes occurring in the CNS during MS. Previously conducted studies were—at least in part—in agreement with our results, especially due to the fact that patients with MS have a disturbed oxidative system which results in higher OS parameters. It appears that OS parameters in serum of patients with MS did not correlate with the disease severity. It is possible that understanding the contribution of OS in MS will enable the implementation of new therapies based on the oxidative/antioxidative system. The study revealed only some differences in the oxidative system of patients treated with IFN and patients treated with II-line drugs. These differences included higher lipid peroxidation parameters in patients treated with IFN compared to the control group. Additionally, our study attempted to clarify whether II-line drugs may influence the level of OS in MS patients.
The authors declare that there is no conflict of interests regarding the publication of this paper.