Markers of oxidative stress in plasma and saliva in ...

Clinical Biochemistry 48 (2015) 24–28

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Markers of oxidative stress in plasma and saliva in patients with multiple sclerosis☆ Martin Karlík a, Peter Valkovič a, Viera Hančinová b, Lucia Krížová b, Ľubomíra Tóthová c,d,⁎, Peter Celec c,d,e,f a

2nd Department of Neurology, Faculty of Medicine, Comenius University, Bratislava, Slovakia Department of Neurology, Slovak Medical University, Bratislava, Slovakia Institute of Molecular Biomedicine, Faculty of Medicine, Comenius University, Bratislava, Slovakia d Center for Molecular Medicine, Slovak Academy of Sciences, Bratislava, Slovakia e Institute of Pathophysiology, Faculty of Medicine, Comenius University, Bratislava, Slovakia f Department of Molecular Biology, Faculty of Natural Sciences, Comenius University, Bratislava, Slovakia b c

a r t i c l e

i n f o

Article history: Received 10 June 2014 Received in revised form 4 September 2014 Accepted 30 September 2014 Available online 7 October 2014 Keywords: Multiple sclerosis Reactive oxygen species Carbonyl stress Saliva EDSS

a b s t r a c t Backround: Oxidative stress plays a role in multiple sclerosis. Saliva can be potentially used to study the disease progression or treatment, because of its non-invasiveness and easy collection. But studies on saliva and multiple sclerosis are missing. The aim of this study was to compare the concentrations of salivary oxidative stress markers in patients and healthy controls. Objective: Whole saliva and blood samples were collected from 29 patients and 29 healthy controls. Samples were collected during relapse, after corticosteroid therapy, and after three months. Markers of oxidative, carbonyl stress and antioxidant status were measured. Results: In plasma, thiobarbituric acid reacting substances, advanced oxidation protein products and fructosamine were significantly higher in patients compared to controls (by 271%, 46% and 24%, respectively; p b 0.01). Total antioxidant capacity in plasma was lower by 20% (p b 0.01) in patients versus controls. In saliva, higher levels of thiobarbituric acid reacting substances and advanced glycation end-products were observed in patients when compared to controls (by 51% and 49% respectively; p b 0.01). Ferric reducing ability was reduced by 38% (p b 0.05) in patients with multiple sclerosis. Conclusion: According to our knowledge, this is the first report showing higher markers of oxidative stress and lower antioxidant status in patients with multiple sclerosis in saliva. © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Introduction Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system and the most common cause of non-traumatic disability in young adults [1]. The development of multiple sclerosis is considered to be an interaction of genetic predisposition, environmental factors and aberrant immune response, but the precise etiology of the disease remains unknown [2]. Various pathophysiological mechanisms such as inflammation, axonal damage, oxidative injury, excitotoxicity, demyelination and remyelinaton are implicated in pathogenesis of multiple sclerosis [3]. In recent studies, increased focus has been placed on oxidative stress in pathogenesis of multiple sclerosis [4,5]. Oxidative stress occurs, when excessive amount of reactive oxygen species (ROS) is formed or when the antioxidant capacity is decreased. ☆ Disclosure: No conflict of interest to declare. ⁎ Corresponding author at: Institute of Molecular Biomedicine, Faculty of Medicine, Comenius University, Sasinkova 4, 811 08 Bratislava, Slovakia. Fax: +421 2 59357631. E-mail address: [email protected] (Ľ Tóthová).

Such imbalance in favor of ROS leads to loss of blood–brain barrier integrity, myelin destruction and nerve tissue degeneration [6]. Oxidative stress might be also a consequence of inflammation. The central nervous system is particularly vulnerable to free radicals because of high oxygen utilization, low concentration of antioxidants and antioxidant enzymes and high content of polyunsaturated fatty acids, which are highly susceptible to peroxidation [7]. Many regions of the brain are rich in iron, which can catalyze oxidative damage and associated cellular injury via the Fenton reaction [8]. Oxidative stress impairs the function of proteins, lipids, saccharides and nucleic acids. Currently, variables, which are used to evaluate the disease activity or to monitor treatment effectivity depend on either relapses rate, outcomes of MRI and changes in scores. Effective stratification and prognosis markers for individual patients with MS are not available. This leads to development of new assessment tools with better accuracy and predictability and it represents a major challenge in MS management [9]. Generally, these biomarkers can be found in blood, cerebrospinal fluid or are based on imaging modalities of which improving MRI techniques belongs to gold standard. Plasmatic or cerebrospinal fluid biomarkers belong to most evolving, including inflammatory cytokine measurement

http://dx.doi.org/10.1016/j.clinbiochem.2014.09.023 0009-9120/© 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

M. Karlík et al. / Clinical Biochemistry 48 (2015) 24–28

[10], specific microRNA determination [11] or transcriptome signature analysis [12] and some others. All of these are either non-specific or has limited use caused by missing reproducibility. Price, complicated equipment needed to measure the molecular markers or transcriptome as well as relative invasiveness resulting from blood or cerebrospinal fluid collection are another cause for a need of a new simple, costeffective and non-invasive biomarkers. Saliva can be collected noninvasively, with an easy access and the possibility of repeated sampling. Hence, saliva represents an attractive diagnostic fluid for detecting biomarkers of various pathological conditions. Increased concentrations of salivary markers of oxidative stress were reported in oral diseases [13,14], as well as in systemic diseases [15]. In MS patients, only little is known about oxidative stress markers in other biofluids apart from plasma and cerebrospinal fluid. Indeed, similarly to other markers, markers of oxidative stress are also nonspecific. However, to measure them, only little and relatively cheap equipment is needed. Non-specificity can be addressed by measuring several markers of oxidative stress at once without additional costs. In addition, the possibility of measuring these in saliva brings the noninvasive techniques and therefore more compliance from patients, potentially from home over period of time. Additionally, only little amount of saliva is needed to measure all of these markers and they can be measured quickly in laboratory settings. The aim of this study was to analyze markers of oxidative stress in plasma and saliva samples of patients with multiple sclerosis as well as to describe the dynamics of oxidative stress markers in saliva to verify the potential of these markers to monitor the therapeutic response during the course of the disease. Subjects and methods The study was approved by the local medical ethics committee. All patients and healthy individuals gave written informed consent before inclusion into the study. Twenty nine patients with clinically diagnosed multiple sclerosis according to 2010 revised McDonald criteria [16] were included in the study. A relapse was defined as patient-reported symptoms or objectively observed signs typical of an acute inflammatory demyelinating event in the central nervous system (CNS) with a duration of at least 24 h in the absence of fever or infection [16]. Exclusion criteria included duration of symptoms more than 7 days and corticotherapy within last 30 days. The neurologic deficit was scored with the Kurtzke expanded disability status scale during the time of disease relapse [17]. The control group consisted of 29 healthy individuals, matched with the patients according to age, gender and smoking status. The healthy individuals did not show any clinical or laboratory characteristics of autoimmune, renal, cardiovascular or liver disease (Table 1). Blood and saliva samples were collected between 8:00 and 10:00 am, after overnight fasting. The samples from patients were collected 3 times—[1] on the first day of admission to hospital, [2] after taking the high dose of intravenous methylprednisolone therapy (MP therapy, total dose 3000–5000 mg), and [3] after 2–3 months of disease remission. Whole unstimulated saliva samples were collected into sterile tubes (Sarstedt, Germany). Saliva samples with visible blood contamination were not included in the study. Blood samples were obtained from antecubital vein into 2 tubes with K3EDTA and subsequently centrifuged (3000 ×g for 10 min). Plasma and saliva samples were stored at −20 °C until further analysis. Biochemical analysis The marker of protein oxidation—advanced oxidation protein products (AOPP)—was determined using spectrophotometry after addition of phosphate buffered saline (PBS) into the samples and exposure to glacial acetic acid for 2 minutes. The mixture of chloramine T and potassium iodine was used for construction of the calibration curve. Specific absorbance was taken at 340 nm [18].

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Table 1 Clinical characteristic of patients and controls. Characteristic

Male gender, n (%) Age (mean ± SD) Age range Disease duration (years, mean ± SD) Disease course Relapse remitting Secondary progressive Primary progressive C-reactive protein (mg/L, mean ± SD) WBC (×109 cells/L, mean ± SD) Smokers Non-smokers EDSS (mean ± SD) EDSS range Newly diagnosed patients Without therapy Interferon therapy Glatirmer acetate therapy Natalizumab therapy Fingolimod therapy

Control

Patients with MS

n = 29

n = 29

9 (31.03%) 38.29 ± 14.69 23–78 N/A N/A

9 (31.03%) 37.48 ± 14.54 21–76 8.14 ± 10.56 24 (82.75%) 4 (16.67%) 1 (3.44%) 2.27 ± 2.23 7.12 ± 1.91 5 24 3.32 ± 1.88 1.0–8.5 8 15 8 3 2 1

N/A N/A 8 21 N/A N/A N/A N/A N/A N/A N/A N/A

Thiobarbituric acid reacting substances (TBARS), as markers of lipoperoxidation, were measured after addition of water, thiobarbituric and acetic acids, which formed colored complexes with TBARS after incubation at 94 °C for 45 min [19]. The samples were then cooled to 4 °C. Afterwards, 100 μl of n-butanol was added. Phase separation was performed by centrifugation at 2000 ×g for 10 min. Upper phase was then carefully removed and measured at 553 nm emission and 515 nm excitation wavelengths. TBARS contents were quantified based on calibration curve made using 1,1,3,3-tetramethoxypropane. To assess carbonyl stress in the samples, advanced glycation endproducts (AGEs) and fructosamine were measured. For AGE determination, samples were diluted in PBS (pH = 7.2, 1:10) and calibrated with AGE-modified bovine serum albumin. The absorbance of the prepared reaction mixture was immediately measured at 440 nm emission and 370 nm excitation wavelength, respectively [20]. Fructosamine was assessed by measuring the absorbance at 530 nm after addition of nitro blue tetrazolium solution (pH = 10.35) into the samples and incubation for 15 min at 37 °C [21]. To prepare a calibration curve, the mixture of 1deoxy-morpholino-D-fructose, sodium chloride and albumin was used. The marker of antioxidant status, ferric reducing ability of saliva/ plasma (FRAS/FRAP) was measured according to Benzie [22]. An initial absorbance of freshly prepared FRAP reagent (a mixture of acetate buffer, TPTZ (tripyridyl-s-triazine), FeCl3·6H2O and water, heated to 37 °C) was measured at a wavelength of 593 nm. Then samples and standards (prepared using FeSO4·7H2O) were added, incubated for 4 minutes at room temperature, and specific absorbance was measured again. Total antioxidant capacity (TAC) of samples was measured in the presence of acetate buffer (pH = 5.8) and the initial absorbance was recorded, as described by Erel [23], following the addition of 2,2′-azino-bis(3ethylbenzthiazoline-6-sulphonic acid) (ABTS) in acetate buffer with hydrogen peroxide. After 5 minute incubation at room temperature, the absorbance at 660 nm was recorded again. The difference to the initial values was calculated. To construct the calibration curve a watersoluble derivative of tocopherol was used. All measurements were performed using Saphire II multi-mode plate reader (Tecan, Austria). All chemicals and reagents used were purchased from Sigma-Aldrich (Germany). Statistical analysis Data were analyzed using GraphPad Prism 6.0 (California, USA). For comparison of oxidative stress markers between the control group and MS patients, the unpaired nonparametric Kruskal–Wallis test with subsequent Dunn's multiple comparison test were used. For the dynamics

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of the markers, Friedman test with Dunn's multiple comparison test were used. P values less than 0.05 were considered significant. Data are presented as mean + standard error of the mean (SEM). Results Plasma concentrations of TBARS and AOPP were significantly higher by 271% and 46%, respectively in MS patients when compared to the control group (p b 0.001 and p b 0.01, respectively, Fig. 1). Similarly, fructosamine was higher in MS patients when compared to controls by 24% (p b 0.01). Another marker of carbonyl stress—AGEs seem to be higher in MS patients compared to control group without reaching statistical significance (p = n.s.). Total antioxidant capacity of plasma was lower in MS patients when compared to controls by 20% (p b 0.01). No differences in FRAP concentrations between the MS patients and the control group were observed. Similar results were obtained from saliva (Fig. 2). Significantly higher TBARS concentrations were observed in MS patients when compared to the control group (by 51%; p b 0.001). Patients with MS showed also higher concentrations of AGEs (by 49%; p b 0.01). The

ability of saliva to reduce ferric ions was lower in MS patients vs control by 38% (p b 0.05). TAC seems to be lower without reaching statistical significance (p = n.s.). MP therapy did not cause a significant change in markers of oxidative stress, neither in plasma nor in saliva samples (Figs. 1, 2). No significant correlation was observed between the expanded disability status scale (EDSS) and the oxidative stress markers (data not shown). Discussion CNS contains high concentrations of polyunsaturated fats and has high oxygen consumption, which can lead to excessive lipid peroxidation impairing the brain tissue in patients with MS [24]. In line with previous studies, increased concentrations of oxidative stress markers were found in plasma samples in our patients with MS [6,25–27]. Plasma concentrations of TBARS were significantly higher in MS patients when compared to healthy controls. On contrary, in another recent study, no differences in serum concetrations of malondialdehyde in 20 newly diagnosed patients with relapse–remitting MS in comparison to healthy controls were reported [28]. Other markers, such as conjugated dienes

Fig. 1. Plasma markers of oxidative, carbonyl and antioxidative status in patients with multiple sclerosis and controls and the dynamics of measured plasma markers in patients with multiple sclerosis during relapse, after the treatment and during remission. Data are presented as mean + SEM, * represents p b 0.05, ** represents p b 0.01 and *** represents p b 0.001. AGEs—advanced glycation end-products, AOPP—advanced oxidation protein products, TBARS—thiobarbituric acid reacting substances, FRAS - ferric reducing ability of saliva, TAC—total antioxidant capacity.


were higher in these MS patients. In our study, most of the patients were not newly diagnosed (Table 1). This difference may suggest that CNS lipid peroxidation is lower in early stage of the disease. Additionally, higher plasma concentrations of AOPP and fructosamine in MS patients were found in our study. These results are in agreement with the results obtained by Sadowska-Bartosz et al. (2013), who observed higher AOPP concentrations in patients with relapse–remitting multiple sclerosis (the most common type of MS, where relapses are followed by remission of symptoms) without treatment and in patients with clinical relapse [27]. In the present study, lower TAC of plasma was found in MS patients. No difference in FRAP concentrations between MS patients and the control group was observed. Conflicting results regarding the markers of antioxidant status were reported by Koch et al. (2008) [29] who found increased concentration in MS patients, while in other studies decreased antioxidant status in MS patients was reported [30–32]. To reduce oxidative injury, cells have defense mechanisms consisting mainly of antioxidant enzymes [33]. Ongoing oxidative stress causes

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depletion of antioxidants in the CNS, but increases the amount of endogenous antioxidant enzymes. Increase in antioxidant enzymes is considered to be the up-regulated response to oxidative stress in order to minimize the free radical-caused damage [6]. To our knowledge, this is the first study evaluating oxidative stress markers in saliva in MS. In parallel to plasma values, significantly higher salivary levels of TBARS and AGEs in MS patients in comparison to controls were observed. In antioxidant stress markers, FRAS was significantly lower in patients group. However, no significant difference in salivary TAC between patients and controls was observed. The concentration of oxidative stress markers in saliva depends on the circadian rhythm, and the presence or absence of oral diseases. Significantly lower concentrations of FRAS in comparison to TAC in saliva might have occurred due to the fact, that the FRAS assay measures primarily the non-protein antioxidant capacity while TAC assay measures the antioxidative effect of proteins as well, which have an important contribution to the antioxidant status of saliva [23]. The increase in FRAS levels could be the consequence of fluctuations in the amount of low

Fig. 2. Salivary markers of oxidative, carbonyl and antioxidative status in patients with multiple sclerosis and the dynamics of measured salivary markers in patients with multiple sclerosis during relapse, after the treatment and during remission. Data are presented as mean + SEM, * represents p b 0.05, ** represents p b 0.01 and *** represents p b 0.001. AGEs—advanced glycation end-products, AOPP—advanced oxidation protein products, TBARS—thiobarbituric acid reacting substances, FRAP—ferric reducing ability of plasma, TAC—total antioxidant capacity.

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molecular antioxidants [13]. Protracted release of high molecular weight AOPP in saliva might have caused similar concentrations of this biomarker in patient and control group. In our study, MP therapy did not cause a significant change in oxidative stress markers, neither in plasma nor in saliva. The dynamics was highly variable and complex. In the literature, controversial findings are reported on the effect of MP therapy on oxidative stress markers in CNS. Greco et al. (2004) observed decreased lipid peroxidation in plasma after MP therapy in MS patients, who had the lowest values of isoprostanes during the remission [34]. Keles et al. (2001) reported, that TBARS were significantly increased in plasma and, particularly, in the cerebrospinal fluid compared to control group before MP therapy, which caused a significant decrease of plasma levels of TBARS [35]. In the study of Seven et al. (2013) MP treatment did not change TBARS and conjugate diene levels, but significantly decreased 8-epi-PGFα levels in serum of MS patients [28]. Actually, partial glucocorticoid resistance in MS patients was described [36]. The inter-individual variability of MP therapy effects may be present in different subjective or objective clinical response and different changes in expression of cytokines, chemokines, oxidative stress markers and other molecules. There does not appear to be a relation between oxidative stress markers and EDSS. The effect of MP therapy on peripheral oxidative stress markers is highly complex due to strong inter-individual variability. The main limitation of the presented study is the variability of the clinical status and different treatment of patients, described in Table 1. Analysis of subgroups of patients is not possible due to the low number of patients included. This study had strict exclusion criteria, especially in terms of concurrent diseases. However, patients did not undergo a dental examination, and oral diseases could also affect the oxidative stress markers. All other known factors influencing salivary markers of oxidative stress such as food intake, chewing gums, antioxidant vitamin intake, sampling daytime, or physical activity were excluded or reduced as much as possible. Also, the number of patients was reduced because of low relapse rate. The low relapse rate was probably due to the positive effect of modern immunomodulatory therapy as was reported previously [37]. Another source of variation was the variable dosage and length of the treatment. This was mainly because of high heterogeneity of the patients and their treatment response. More patients and samples could help us to cope with this variability in the future. In summary, higher concentrations of oxidative stress markers in MS patients vs control in plasma were found, supporting previous research showing that oxidative stress is involved in the pathogenesis of multiple sclerosis. Additionally, our study proved that these markers are higher also in saliva, making saliva a potentially useful biofluid for monitoring of MS. Further studies are needed to support these results. Interventional studies might test the possibility of an adjuvant antioxidant therapy in patients with MS. Acknowledgments This study is the result of the implementation of the project University Science Park for Biomedicine in Bratislava supported by the Research and Development Operational Programme funded by the European Regional Development Fund (ITMS 26240220087). References [1] Sellner J, Kraus J, Awad A, Milo R, Hemmer B, Stuve O. The increasing incidence and prevalence of female multiple sclerosis—a critical analysis of potential environmental factors. Autoimmun Rev 2011;10:495–502. [2] Chastain EM, Miller SD. Molecular mimicry as an inducing trigger for CNS autoimmune demyelinating disease. Immunol Rev 2012;245:227–38. [3] Tully M, Shi R. New insights in the pathogenesis of multiple sclerosis—role of acrolein in neuronal and myelin damage. Int J Mol Sci 2013;14:20037–47. [4] van Horssen J, Witte ME, Schreibelt G, de Vries HE. Radical changes in multiple sclerosis pathogenesis. Biochim Biophys Acta 1812;2011:141–50.

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