Introduction

Multiple sclerosis (MS) is a demyelinating neurodegenerative disease and is considered to be one of the main inflammatory conditions that affects the central nervous system (CNS) (Reynolds et al. 2011; Ljubisavljevic et al. 2013). MS is among the most common causes of non-traumatic chronic neurological disability in young adults that affects approximately 2.5 million people worldwide (Trapp and Nave 2008; Reinhardt et al. 2014). The majority of individuals, accounting for 80–85 % of MS patients, exhibit the form of relapsing-remitting MS (RRMS), which is characterized by periods of exacerbation and remission of neurological symptoms (Ransohoff et al. 2015).

The etiological aspects of MS are still the major target of extensive studies; nonetheless, vitamin D deficiency currently appears to be the most robust modifiable risk factor for MS development, possibly because of the importance of this vitamin in neuroimmune functions (Pierrot-Deseilligny and Souberbielle 2013). Vitamin D receptors are presented in immune cells, and therefore, vitamin D is considered to be an immunomodulatory molecule (Correale et al. 2009; Gröber et al. 2013). It has been demonstrated that this vitamin is able to reduce the production of pro-inflammatory cytokines and increase the secretion of anti-inflammatory cytokines (Hanwell and Banwell 2011). The hypothesis that adequate vitamin D supplementation can contribute to the prevention of MS has been proposed (Ascherio et al. 2012); however, there are many questions involving the protective effect of this vitamin in the course of MS that are not completely understood yet.

In recent years, evidence has also suggested that reactive oxygen species (ROS) and oxidative stress contribute to several mechanisms underlying the pathogenesis of MS lesions (Lassmann and van Horssen 2011). ROS in MS are generally derived from inflammatory cells and play a role in tissue injury, demyelization, and axonal degeneration (van Horssen et al. 2011). Moreover, ROS are known to mediate the migration of monocytes, leading to a dysfunction in the blood–brain barrier (Mirshafiey and Mohsenzadegan 2009).

The ROS are produced continuously by cells as part of their metabolic processes (Fialkow et al. 2007). However, an imbalance in the oxidative/antioxidative status leads to a pro-oxidant process called oxidative stress that has been revealed to be the main contributor to neuroinflammation and neurodegeneration in diseases, such as MS (Gonsette 2008; Ljubisavljevic et al. 2014). In this sense, this process can cause irreversible damage to important cell structures, resulting in modifications of lipids, proteins, and DNA (Djordjevic et al. 2008), leading to cell dysfunction.

The susceptibility of cells to oxidative damage depends on the state of their antioxidant defense, which is composed of endogenous enzymatic and non-enzymatic systems (Mirshafiey and Mohsenzadegan 2009). The enzymatic system is composed of a variety of antioxidant enzymes, such as superoxide dismutase (SOD) and catalase (CAT) (Miller et al. 2010). SOD is mainly responsible for the dismutation of superoxide radicals to hydrogen peroxide, which can be degraded subsequently by the enzyme CAT (Ortiz et al. 2013).

Ascorbic acid (vitamin C), α-tocopherol (vitamin E), and organic compounds containing sulfhydryl groups (SH), called thiols, should be highlighted among the non-enzymatic antioxidants (Halliwell 2012). Vitamin C has antioxidant properties and an immunomodulatory action that protects various molecules of damage caused by ROS (Villacorta et al. 2007), while vitamin E also has important antioxidative and immunomodulatory properties (Løken-Amsrud et al. 2013). It is considered to be the first line of defense against lipid peroxidation (Pekmezci 2011). In addition, non-protein thiols (NPSH) are extremely important because they are involved in bioreduction and detoxification processes, thereby regulating intracellular redox homeostasis (Van Laer et al. 2013).

Furthermore, the enzyme delta-aminolevulinic acid dehydratase (δ-ALA-D, EC 4.2.1.24) has been suggested to be a marker of oxidative stress in human pathologies (Rocha et al. 2012), wherein alterations in the activity of this enzyme are closely related to process oxidative stress. δ-ALA-D participates in heme biosynthesis and is essential for all aerobic organisms (Sassa 1998). It is a thiol-dependent enzyme that can represent the circulating oxidative status better because it is highly sensitive to –SH oxidation by pro-oxidant elements (Paniz et al. 2007).

In this context, although many studies have demonstrated alterations in the oxidative stress parameters in MS, many of these data are controversial and the relation with disease progression is uncertain (Koch et al. 2006; van Horssen et al. 2011). In addition, considering that δ-ALA-D can be an important marker of oxidative stress and that there are no studies in the literature evaluating the role of this enzyme in MS, the aim of this study was to evaluate δ-ALA-D, CAT and SOD activities, as well as the lipid peroxidation, carbonyl protein, vitamin D, vitamin C, vitamin E, and NPSH levels, and DNA damage in samples from patients with RRMS. These parameters were analyzed to obtain a better understanding of the role of δ-ALA-D and the oxidative process in the RRMS patients.

Materials and Methods

Study Population

This study included 58 volunteers who live in the city of Santa Maria, located in the central region of the state of the Rio Grande Sul (Brazil). The subjects were divided into two groups: MS patients (n = 29) and healthy individuals (n = 29) as a control group. The diagnosis of MS was based on the McDonald criteria (McDonald et al. 2001), and all patients had RRMS. Most patients were treated with a current immunosuppressive therapy such as interferon β and glatiramer acetate according to medical guidelines. The general patient characteristics are shown in Table 1. All of the subjects provided written informed consent to participate in this study, and the Human Ethics Committee of the Health Science Center from the Federal University of the Santa Maria approved the protocol under number 26757114.4.0000.5346.

Table 1 General characteristics of subjects
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Blood Serum Collect

Twelve milliliters of blood was obtained from each patient and used for biochemical determinations. The same procedure was carried out for the control group. Blood samples were collected after an overnight fast by the venous puncture technique in Vacutainer® (BD Diagnostics, Plymouth, UK) tubes with heparin and EDTA and without an anticoagulant system. For blood serum separation, the blood was centrifuged at 3500 rpm for 15 min at room temperature, and the clot was discarded.

The serum was used to determine the vitamin D, vitamin C, vitamin E, NPSH, and TBARS levels as well as the protein carbonyl content. CAT and SOD activities were determined using whole blood collected in citrated tubes and diluted to a ratio of 1:10 in a saline solution, whereas heparin was used as an anticoagulant agent for the determination of δ-ALA-D activity and the comet alkaline assay in leukocytes.

Determination of the Hematological Parameters

The erythrocyte quantification, hemoglobin, hematocrit, mean corpuscular volume (VCM), mean corpuscular hemoglobin (HCM), mean corpuscular hemoglobin concentration (CHCM), and red cell distribution width (RDW) were determined in whole blood and analyzed with MEK-4100 automated equipment (Nihon Kohden, Japan).

δ-Aminolevulinic Dehydratase (δ-ALA-D) Enzyme Assay

δ-ALA-D activity was assayed in whole blood according to the method of Sassa (1982) by measuring the rate of porphobilinogen (PBG) formation. The reaction was started by adding δ-ALA to a final concentration of 2.4 mM in a phosphate-buffered solution. A total of 200 μL of sample were incubated for 2 h at 37 °C. The reaction was stopped by the addition of 250 μL of trichloroacetic acid (TCA). The reaction product was determined using modified Ehrlich’s reagent at 555 nm, with a molar absorption coefficient of 6.1 × 104 M−1for the Ehrlich-porphobilinogen salt. ALA-D activity was expressed as nmol porphobilinogen (PBG) mg−1 protein h−1.

Determination of Lipid Peroxidation

The index of lipid peroxidation was estimated colorimetrically by measuring thiobarbituric acid reactive substances (TBARS) in serum as previously described (Jentzsch et al. 1996) with some modifications. In short, 200 µL of serum or standard (malondialdehyde [MDA] 0.03 mM) was added to the reaction mixture containing 1 mL of 1 % ortho-phosphoric acid and 0.25 mL of an alkaline solution of thiobarbituric acid-TBA (final volume 2.0 mL) followed by 45 min of heating at 95 °C. After cooling, the serum and standard samples of MDA were read at 532 nm against the blank of the standard curve. The results are expressed as nmol MDA/mL of serum.

Quantification of the Protein Carbonyl Content

Protein oxidation was measured by an estimation of the carbonyl groups in serum performed according to the method of Levine et al. (1990), with slight modifications. This method is based on the quantification of the protein carbonyl by reaction with 2,4-dinitrophenylhydrazine (DNPH) in acidic medium. First, serum proteins were precipitated using 0.5 mL of 10 % trichloroacetic acid (TCA) from 1 mL of serum and were centrifuged at 3500 rpm for 15 min, discarding the supernatant. One-half milliliter of 10 mmol/L DNPH in 2 mol/L HCl was added to precipitate the protein and was incubated at room temperature for 30 min. After incubation, 0.5 mL of 10 % TCA was added to the protein precipitate, and the tubes were centrifuged at 3500 rpm for 15 min. After discarding the supernatant, the precipitate was washed twice with 1 mL of ethanol/ethylacetate (1:1), centrifuging out the supernatant to remove the free DNPH. The precipitate was dissolved in 1.5 mL of a protein dissolving solution (2 g sodium dodecyl sulfate and 50 mg ethylenediamine tetraacetic acid in 100 mL 80 mmol/L phosphate buffer, pH 8.0) and incubated at 37 °C for 10 min. The color intensity of the supernatant was measured using a spectrophotometer at 370 nm against 2 mol/L HCl. The carbonyl content was calculated by using the molar extinction coefficient (21 × 103 L/mol cm), and the results were expressed as nanomoles of carbonyl per milligram of protein.

The Comet Alkaline Assay (Single Cell Gel Electrophoresis)

The comet assay has been developed as a method of detecting cellular DNA damage, and it is broadly used in a variety of fields, such as biological monitoring and genetic toxicology (McKelvey-Martin et al. 1993). The distance migrated by cellular DNA during electrophoresis directly reflects the extent of the DNA damage that is present. Therefore, we used this method to measure DNA damage in leukocytes isolated from RRMS patients and healthy subjects. The alkaline comet assay was performed as described by Singh et al. (1988) in accordance with the general guidelines for the use of the comet assay (Hartmann et al. 2003). Isolated human lymphocytes were suspended in 0.7 % low-melting-point agarose and phosphate-buffered saline (PBS) at 37 °C and placed on microscopic slides with a layer of 1 % agarose. Slides were incubated in an ice-cold lysis solution at 4 °C for 1 h to remove cell proteins, leaving DNA as “nucleoids.” After the lysis procedure, slides were placed on a horizontal electrophoresis unit and covered with a fresh buffer (300 mM NaOH and 1 mM EDTA, pH > 13) for 20 min at 4 °C to allow DNA unwinding and the expression of alkali-labile-sites. This was followed by electrophoresis at 25 V, 300 mA, for 40 min at a constant temperature. The slides were then silver-stained, as described by Nadin et al. (2001). All of the steps, from sample collection to electrophoresis, were conducted under yellow light to minimize the possibility of cellular DNA damage. One-hundred cells (50 cells from each of the two replicate slides) were selected and analyzed. Cells were visually scored according to tail length and received scores from 0 (no migration) to 4 (maximal migration). Therefore, the damage index for cells ranged from 0 (all cells with no migration) to 400 (all cells with maximal migration). The comet parameters were analyzed as a percentage of DNA in the comet tail and tail moment. The slides were analyzed under blind conditions by at least two different individuals.

SOD and CAT Activities

The SOD activity in the whole blood measurement is based on the inhibition of the radical superoxide reaction with adrenalin as described by Misra and Fridovich (1972). Briefly, the SOD activity was determined in a reaction medium containing glycine-NaOH (50 mM, pH 10) and adrenalin (1 mM) by measuring the speed of adrenochrome formation, observed at 480 nm. In this method, the SOD present in the sample competes with the detection system for radical superoxide, and the oxidation of adrenalin leads to the formation of adrenochrome, a colored product that is detected by the spectrophotometric reaction. One SOD unit was defined as the enzyme amount that inhibits the speed of oxidation of adrenalin by 50 %. The results are expressed in units of SOD/mg of protein.

CAT enzyme activity in whole blood was measured with the method of Aebi (1984) with slight modifications. For the determination of the CAT activity, a 20 µL aliquot of blood was added to a cuvette, and spectrophotometric determination was started by the addition of 70 µL of freshly prepared 0.3 mol/L H2O2 in potassium phosphate buffer (50 mM, pH 7.0) to give a final volume of 1 mL. The rate of the H2O2 reaction was monitored at 240 nm for 2 min at room temperature. The CAT activity was calculated using the molar extinction coefficient (0.0436 cm2/μmol), and the results were expressed as nmol/mg of protein.

Vitamin D Quantification

Vitamin D or 25-hydroxyvitamin D was measured in serum by radioimmunoassay (ADVIA Centaur kit commercial; Siemens Medical Solutions Diagnostic, Los Angeles, CA, USA). Serum 25(OH)D concentrations of <20 ng/mL were defined as 25(OH)D deficient, as well as being defined as insufficient when <30 ng/mL, and 25(OH)D > 30 ng/mL was considered sufficient (Holick et al. 2011; Wacker and Holick 2013).

Vitamin C Levels

Vitamin C was estimated in serum as described by Jacques-Silva et al. (2001). The samples were deproteinized with trichloroacetic acid (TCA) 10 %, using 0.5 mL of sample and 0.5 mL of TCA that was vortex-mixed for 15 s and submitted to centrifugation once more. An aliquot of 300 µL of supernatant was removed and incubated at 37 °C in medium containing 4.5 mg/mL dinitrophenylhydrazine hydrazine (DNPH), 0.6 mg/mL thiourea, 0.075 mg/mL CuSO4, and 0.675 mol/L H2SO4. After 3 h, 1 mL of 65 % H2SO4 was added, and the samples were read at 520 nm. The content of ascorbic acid was calculated using a standard curve, following the same procedure used for the samples.

Vitamin E Content

Serum vitamin E was measured by a modified method of Hansen and Warwick (1969). A total of 140 µL of sample, 20 µL of butylated hydroxytoluene 10 mM (BHT), and 2.1 mL of ethanol solution (66 %) were added to 140 µL of Milli-Q water (Millipore, Bedford, MA, USA) in a covered tube. This mixture was vortex-mixed for 10 s, and 3.5 mL of n-hexane was added and mixed for 1 min, followed by centrifugation at 3500 rpm for 10 min. Next, 3 mL of the superior phase was transferred to fluorimeter cuvettes, and vitamin E was measured in the fluorimeter (excitation: 295 nm; emission: 340 nm). The concentration was determined by the calibration curves with α-tocopherol, following the same procedure used for the samples.

NPSH Determination

NPSH was determined in serum as previously described by Ellman (1959), with minor modifications. Aliquots of 100 µL of serum were added to a phosphate buffer 0.3 mol/L (0.85 mL), pH 7.4. Then, the 10 mM 5-5′-dithio-bis (2-nitrobenzoic acid) (DTNB) (0.05 mL) was added, and the reaction was read spectrophotometrically at 412 nm. A standard curve using cysteine was added to calculate the content of thiol groups in samples. The results are expressed as µmol NPSH/mL of serum.

Protein Determination

Protein was measured by the Coomassie blue method according to Bradford (1976) using serum albumin as a standard.

Statistical Analysis

Data were analyzed statistically with Student’s t test for independent samples. Correlations between parameters were performed by Pearson’s correlation. Differences were considered significant when P < 0.05. All data were expressed as the mean ± standard error of the mean (SEM).

Results

This study consisted of 29 subjects with RRMS (19 women, aged 18–60 years; 10 men, aged 13–58 years) and 29 controls (20 women, aged 22–58 years; 9 men, aged 23–52 years). The general characteristics of the study participants are shown in Table 1. All of the patients involved in this study had the RRMS form, but none of them was a smoker or had a family history of MS. The treatment used for the majority of the female patients was glatiramer acetate, and they were diagnosed for the first time with MS approximately 10 years previously.

The hematological parameters of RRMS patients and control group are shown in Table 2. All of the individuals showed a hemogram compatible with their age and gender in accordance with reference values. In the present study, we analyzed the number of erythrocytes, hemoglobin, hematocrit, and hematological indices (i.e., VCM, HCM, CHCM, and RDW). No significant differences were found in any parameter between RRMS patients and healthy individuals.

Table 2 Blood parameter of MS patients and healthy subjects
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The results of our study indicated, for the first time in the literature, that RRMS patients show alterations in δ-ALA-D activity. As can be observed in Fig. 1, statistical analysis revealed a significant increase in the δ-ALA-D activity in the whole blood of the RRMS patients (96.7 %) compared with healthy individuals (P < 0.0001).

Fig. 1
figure 1

δ-ALA-D activity in the whole blood of healthy subjects and MS patients. The results are expressed as the mean ± SEM. ***Indicates a significant difference from the control group (Student’s t test, ***P < 0.0001, n = 29)

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Figures 2, 3, and 4 show the results obtained for the oxidative stress parameters, such as the lipid peroxidation, protein carbonyl, and comet assay, respectively. As can be observed, the lipid peroxidation estimated by the TBARS levels was significantly increased in the serum of RRMS patients (49.9 %) compared with the healthy group (Fig. 2, P < 0.0001). Similar results were obtained in relation to the protein oxidation determined by the protein carbonyl content in serum. The protein carbonyl content was also significantly increased in serum from RRMS patients (19.3 %) in relation to the control group (Fig. 3, P < 0.05). The comet image of blood cells showed different damage levels (Fig. 4a). In addition, as seen in Fig. 4b, the RRMS patients presented significantly higher DNA damage (33.1 %) compared to the control group (P < 0.001).

Fig. 2
figure 2

Levels of thiobarbituric acid reactive substances (TBARS) in the serum of healthy subjects and MS patients. The results are expressed as the mean ± SEM. ***Indicates a significant difference from the control group (Student’s t test, ***P < 0.0001, n = 29)

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Fig. 3
figure 3

Protein carbonyl content in the serum of healthy subjects and MS patients. The results are expressed as the mean ± SEM. *Represents a significant difference from the control group (Student’s t test, *P < 0.05, n = 20)

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Fig. 4
figure 4

The comet assay on the leukocytes of healthy subjects and MS patients. a Comet image of blood cells showing a nucleus without damage and nuclei with different damage levels. b DNA damage comparison between healthy subjects and MS patients. DNA index damage calculation Σ (1,2,3,4 comet class)/100. **Represents a significant difference from the control group (Student’s t test, **P < 0.001, n = 22)

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Table 3 describes the activity of δ-ALA-D and its correlation with oxidative stress parameters in RRMS patients. As can be observed, the δ-ALA-D activity was reversely correlated with the TBARS levels in RRMS patients (Pearson r: −0.420, P < 0.05). No statistically significant difference was found for the correlation between δ-ALA-D activity and carbonyl protein levels (Pearson r: −0.381, P = 0.107). However, the activity of the ALA-D enzyme demonstrated a significant positive correlation with SOD (Pearson r: 0.535, P < 0.05) and CAT activities (Pearson r: 0.560, P < 0.05). These findings show a correlation between the increase in the activity of this enzyme and the decrease in markers of oxidative damage in RRMS patients.

Table 3 Activity of δ-ALA-D and its correlation with oxidative stress parameters in RRMS patients
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The next set of experiments was performed to analyze the antioxidant enzyme status in whole blood in RRMS patients. Figure 5 shows that SOD activity was significantly decreased in whole blood from MS patients (25.7 %) compared with the healthy subjects (Fig. 5a, P < 0.001). On the other hand, we found a significant increase in CAT activity in the whole blood of RRMS patients (10.7 %) in relation to the control group (Fig. 5b, P < 0.05).

Fig. 5
figure 5

Enzymatic antioxidant: superoxide dismutase (a) and catalase (b) activities in the whole blood of healthy subjects and MS patients. The results are expressed as the mean ± SEM. **Represents a significant difference from the control group (Student’s t test, **P < 0.001, n = 29)

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In addition, our results also showed alterations in the non-enzymatic antioxidant parameters in serum from MS patients. The levels of vitamin C and vitamin E in serum are shown in Fig. 6 for vitamin C (6a) and E (6b). We found an inhibition of 54.4 % for vitamin C and 51 % for vitamin E in the serum of RRMS patients compared with the control group (P < 0.0001). Moreover, the NPSH levels were significantly decreased in the serum from RRMS patients (9.9 %) compared with the healthy subjects (Fig. 6c, P < 0.001).

Fig. 6
figure 6

Non-enzymatic antioxidant: vitamin C (a), vitamin E (b), and non-protein thiol (NPSH) (c) levels in the serum of healthy subjects and MS patients. The results are expressed as the mean ± SEM. ***Represents a significant difference from the control group (Student’s t test, ***P < 0.0001, n = 29 and **P < 0.001, n = 20, respectively)

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In the present study, the vitamin D content was significantly decreased in the serum from RRMS patients (37.4 %) compared with the control group (Fig. 7, P < 0.05).

Fig. 7
figure 7

Vitamin D quantification in the serum of healthy subjects and MS patients. The results are expressed as the mean ± SEM. *Represents significant difference from the control group (Student’s t test, *P < 0.05, n = 29)

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Discussion

In recent years, several studies concerned with the relation between oxidative stress and MS have been reported, and this topic is considered to be of great clinical relevance for MS (Ljubisavljevic et al. 2013; Friese et al. 2014). In line with this, researchers have suggested that, in MS, excess ROS are responsible for leukocyte migration and cause mitochondrial dysfunction (Lee et al. 2012). Increasing evidence links the onset of new outbreaks in MS to the ROS increase, with subsequent oxidative damage (van Horssen et al. 2008). Apart from the possible involvement of stress in MS, another important point is the activity of the enzyme δ-ALA-D in this disease. δ-ALA-D, together with other oxidative stress biomarkers, can perform a key role as a marker of oxidative stress and faulty metabolic processes (Valentini et al. 2008).

Previous investigations, including those of our research group, have reported a reduction in δ-ALA-D activity accompanied by changes in the hemoglobin levels in individuals diagnosed with chronic inflammatory diseases, such as type 2 diabetes mellitus (Souza et al. 2007) and lung cancer (Zanini et al. 2014). Nonetheless, it is important to note that, in the present study, we verified a significant increase in δ-ALA-D activity in whole blood from RRMS patients (Fig. 1). This increase may be due to the high demand of heme groups in an attempt to maintain erythropoiesis under normal conditions, since in the presence of chronic inflammatory processes, there is an increase of pro-inflammatory cytokines with the consequent reduction of erythropoietin (Han et al. 2011). In fact, we observed that the patients involved in this study did not present significant differences in the blood parameter levels compared with the control group (Table 2).

Corroborating this hypothesis, we recently demonstrated that the pro-inflammatory cytokine levels, such as IL-1, IL-6, TNF-α and IFN-γ, are increased in the serum of RRMS patients (Polachini et al. 2014). Furthermore, Johansson and Strandberg (1972) reported higher δ-ALA-D activity in rheumatoid arthritis patients. Taken together, these findings demonstrated that the increase in the activity of this enzyme also may be related to the autoimmune process, which is present in both diseases. Reinforcing this line of reasoning, there was no change in δ-ALA-D activity in whole blood when tested in vitro in the presence of potential plasma concentrations of the drugs that are used to treat RRMS patients, i.e., glatiramer acetate and interferon β (data not shown). In this context, these findings support the argument that it is the pathological condition that generates the alterations in δ-ALA-D activity.

Another important aspect to be discussed is that the increase in δ-ALA-D activity found in this study could be contributing to the reduced oxidative damage by the accumulation of substrate 5-aminolevulinic acid (ALA) since the accumulation of ALA has been associated with the overproduction of ROS (Rocha et al. 2003). Thus, we can speculate that the increase in δ-ALA-D activity can be due to excess ALA because it is a precursor for the synthesis of heme, cytochrome, and vitamin B12 (Kang et al. 2012). Evidence has suggested that the demand for vitamin B12 might be an increase in recurrent myelin repair processes or chronic immune reactions in MS (McCaddon 2013). In addition, the myelin expresses the pathway of heme synthesis (Morelli et al. 2012). Therefore, we suggest that enhanced δ-ALA-D activity can be beneficial in this disease, contributing to the balance of metabolic processes and reducing oxidative stress.

Thus, to investigate the existence of damage, TBARS and carbonyl protein levels were evaluated in serum from RRMS patients. We observed a significant increase in both parameters compared with the control group (Figs. 2, 3). Similar results were obtained in previous studies that also confirmed significantly increased lipid peroxidation and protein carbonyls in patients with MS (Sadowska-Bartosz et al. 2013; Yousefi et al. 2014). Researchers have suggested that the lipid peroxidation products were increased in MS patients and could possibly be valid predictors of disease progression, and these products could result from ROS overproduction and the generation of metabolites due to alterations in energy metabolism (Tavazzi et al. 2011). Furthermore, Gonzalo et al. (2012) reported that MS-associated lipid peroxidation would include increased protein modification that would serve as driving force for enhanced autoimmune response and progression of the disease, thereby lipid peroxidation could be a significant pathogenic factor in MS. In addition, protein carbonyl formation seems to be a common phenomenon during oxidation, and its quantification can be used as biomarker protein damage (Dalle-Donne et al. 2003). Reinforcing this line of reasoning, Rommer et al. (2014) have described that carbonyl proteins may also serve as marker for oxidative stress in the cerebrospinal fluid (CFS) of relapsing and progressive MS patients. Considering these statements, we may infer that the lipid and protein damage observed in our study may contribute to neurological dysfunction in RRMS patients.

In this study, we also showed DNA damage in the leucocytes of MS patients compared with the control group (Fig. 4). It is important to observe that lipid peroxidation, protein oxidation, and DNA damage generally lead to cell dysfunction and death by apoptosis or necrosis due to overproduced superoxide radicals (McCord and Edeas 2005). This increase in DNA damage may suggest that patients with RRMS have a higher number of apoptotic cells, and therefore, an apoptosis mechanism may be responsible for the effect on the lymphocyte function in MS (Grecchi et al. 2012).

In addition, hydrogen peroxide (H2O2), one of the main ROS, is a typical inducing agent in oxidative DNA damage (Benhusein et al. 2010). In line with this, in our study, we observed that SOD activity is decreased in whole blood from RRMS patients compared with the control group (Fig. 5a). SOD is the first line of cellular defense against oxidative injury, which involves the removal of superoxide anions and H2O2 and has a crucial antioxidant role in human health (Johnson and Giulivi 2005). A possible explanation for this reduction in SOD activity may be the excess of free radicals that may result in the inactivation of this enzyme (Schmatz et al. 2012). Furthermore, the decrease in the activity of this antioxidant enzyme might be responsible for the oxidative alterations observed in our study in RRMS patients (Miller et al. 2013). Corroborating these results, other studies have reported that activity of SOD is reduced in erythrocytes from MS patients (Kopff et al. 1993; Miller et al. 2010).

Another important enzymatic antioxidant defense is CAT, which acts in the detoxification of H2O2 and plays an important role in the adaptive response of cells in pathological conditions (Matés and Sánchez-Jiménez 1999). It operates when the clearance of high concentrations of H2O2 is required (Mirshafiey and Mohsenzadegan 2009). In the present study, the activity of CAT was significantly increased in whole blood from RRMS patients compared with healthy subjects (Fig. 5b). This finding may reflect an effort to partially compensate for the increased oxidative stress or may be the result of an increase in the concentration of H2O2 in the circulation. In addition, an increase in CAT activity and TBARS levels may be explained by the hypothesis proposed by Ljubisavljevic et al. (2013), which supposed that H2O2 can diffuse from the sites of generation in the perivascular space and induce the peroxidation of lipids in all CNS cells, axonal membranes, and myelin, inducing CAT activity due to its dependence on the H2O2 concentration.

The antioxidants, synthesized endogenously or exogenously administered, are reducing agents that neutralize ROS before they damage different biomolecules (Miller et al. 2013). Thus, we can speculate that the increased levels of oxidative stress biomarkers and decreased levels of important antioxidant enzymes found in this study may indicate a probable increase in ROS levels resulting in the depletion of cellular antioxidants.

To reinforce the previously mentioned findings, in the next set of experiments, we verified the non-enzymatic antioxidant levels, such as vitamin C, vitamin E, and NPSH. The levels of all of these non-enzymatic antioxidant defenses were significantly decreased in serum of RRMS patients compared with the control group (Fig. 6a–c). Studies have demonstrated that the reduction of antioxidants is an important contributor to the progression of degenerative neurological diseases due to oxidative damage to cell components (Mazza et al. 2007). In this sense, it has been shown that the treatment with antioxidants combined with the conventional therapies used for treatment of MS might be beneficial (Gilgun-Sherki et al. 2004; Løken-Amsrud et al. 2013).

Evidence suggests that vitamin E, which is found in high concentrations in the membranes of immune cells, can promote beneficial effects in neurodegenerative diseases through its antioxidant properties and mainly by reducing the degree of oxidative stress (Li-Weber et al. 2002; Ricciarelli et al. 2007). A possible mechanism for this condition could be higher a consumption of this vitamin to counterbalance MS chronic neurodegeneration (Salemi et al. 2010), thereby helping to decrease inflammation and immune-mediated tissue damage (Mora et al. 2008).

With this line of reasoning, the regeneration of vitamin E (α-tocopherol) is performed by vitamin C (Villacorta et al. 2007). Thus, the low levels of vitamin C in patients with RRMS observed in our study suggest that protection against lipid peroxidation will not occur due to reduction in the normal concentrations of these vitamins, which are essential to prevent lipid peroxidation (Padh 2005). Furthermore, vitamin C has a protective role against the oxidation of –SH groups, which may not occur due to the reduction in its levels (Paniz et al. 2007). Additionally, it is important to note that the significant decrease in NPSH and vitamin C levels, together with the increase in TBARS levels found in this study, can create a strategic position against ROS-mediated brain damage (Mitosek-Szewczyk et al. 2010). Our results are in accordance with others studies that show cellular oxidative damage together with reduced antioxidant capability in patients with MS compared to healthy controls (Salemi et al. 2010; Pasquali et al. 2015).

From these findings, we can postulate that this significant reduction in non-enzymatic antioxidant defenses found in MS patients is due to the scarcity of these non-enzymatic antioxidants or by depletion of the antioxidants, which when consumed may make up for the increase in oxidative stress. Oxidative stress leads to systematic, peripheral, and CNS changes, which probably induce each other (Haider et al. 2011).

Another important aspect to be discussed is that the vitamin D levels were also decreased in the serum of MS patients compared with health individuals (Fig. 7). These results are in agreement with studies that reported that the serum levels of 25(OH)D, a modulator of immune function, are lower in MS patients in comparison with healthy controls (Ozgocmen et al. 2005). One possible mechanism for this reduction is a decrease in synthesis or an increase in 25(OH)vitamin D degradation, which was observed in MS patients (Correale et al. 2009). The low concentration of vitamin D could contribute to the pro-inflammatory response in RRMS patients because this vitamin induces IL-10 synthesis, which is considered to be the main anti-inflammatory cytokine (Allen et al. 2012). In line with this, in previous studies, we found a significant decrease in IL-10 in RRMS patients (Polachini et al. 2014).

Preliminary evidence suggested that a poor vitamin D status is associated with an unfavorable course of MS and appears to influence the incidence of subsequent relapse and/or disability in MS patients (Smolders et al. 2008; Simpson et al. 2010). In addition, the high circulating levels of 25(OH)D have been related to a lower risk of MS (Munger et al. 2004). According to Gianforcaro and Hamadeh (2014), it has been demonstrated that vitamin D can reduce the expression of the biomarkers of oxidative stress and inflammation in MS patients. In line with this, researchers have suggested that the expression of the MHC class II allele HLA-DRB1*15, which is a major MS genetic risk factor, is directly stimulated by vitamin D (Ramagopalan et al. 2009).

It is also important to emphasize that oxidative stress, DNA defects, and insufficient vitamin D levels may contribute to the development and progression of MS (Mao and Reddy 2010). Based on the findings of this study, we suggest that RRMS patients have significant increases in cell damage associated with a reduction in non-enzymatic and enzymatic antioxidant defenses, which may contribute to enhancement of pro-inflammatory processes and the consequent oxidative stress. On the other hand, it is important to note that the increase in ALA activity can represent an important cell defense mechanism.

In conclusion, for the first time, we reported that δ-ALA-D activity is altered in RRMS. We also showed a relationship between the biomarkers of oxidative stress and vitamin D levels in RRMS patients. Taken together, these results may contribute to a better understanding of the complex pathogenic mechanisms involved in this disease and thus potential novel strategies for the treatment of patients with MS.