Original article
Free radical peroxidation products in cerebrospinal fluid and serum of patients with multiple sclerosis after glucocorticoid therapy

Introduction

Multiple sclerosis (MS) is a disabling, inflammatory and demyelinating disease of the central nervous system (CNS) of unknown aetiology. MS is characte-rised by recurrent events of autoimmune-mediated demyelination and axonal loss. The disease presents different immunological pictures, clinical courses and radiological images [6,34]. Oxidative stress, which is usually defined as a preponderance of the production of free radicals over their elimination, is commonly implicated in the development of brain damage and reactive oxygen species (ROS) contribute to several mechanisms underlying the pathogenesis of MS lesions [7]. Because free radical peroxidation alters the structure of biological membranes, and thereby affects their physical and chemical properties such as permeability, resorption or potential, it can be expected to play an important role in the pathomechanism of MS [14]. Under normal conditions, free radicals are involved in a chain of various reactions necessary for regular cell functioning. Among biochemical mechanisms responsible for the toxic effects of free radicals the major one is lipoxygenation. Lipid endoperoxides resulting from lipoxygenation are not durable, and during their degradation subsequent free radicals and malondialdehyde (MDA) are produced [12,33]. Lipoxygenation and protein alterations are involved in biomembrane configuration abnormalities and the ensuing modification of its flow and permeability. These activated oxygen species are believed to be detoxified by cellular antioxidative enzymes such as superoxide dismutase (SOD) [10,11,31]. Non-protein thiols are important factors of the cellular defense against free radicals [44], whereas protein thiols are determined by the structure and function of many proteins [29]. Non-protein brain thiols consist mostly of reduced glutathione (GSH) under normal circumstances [35,39]. In MS, free radical peroxidation can be included as a possible pathogenic factor.
In the past few years, the role of glucocorticoids (GC) in MS relapse treatment has strengthened [23]. Their strong anti-inflammatory action makes their use the most effective method for overcoming and shortening the duration of neurological symptoms during relapses. GC activity consists mainly of the induction of specific, inflammation suppressing genes and repression of genes coding proteins which are engaged in immune and inflammatory reactions [1,22,32]. It comes to rapid inhibition of inflammatory reactions, sealing the blood-brain barrier and decreasing the magnetic resonance imaging (MRI) number of gadolinium up-taking foci [41]. Moreover, an attempt was made to determine the relationship between these biochemical characteristics of MS patients and remission of disease after methylprednisolone (MP) treatment.
The aims of this study were to verify the hypothesis that free radical peroxidation might be one of the factors implicated in the pathophysiology of MS and find out whether these biochemical parameters change after GC therapy.

Material and methods
Sample studied
Participants in the study were patients admitted to the Neurology Department of the Medical University of Lublin. Thirty seven patients (20 female and 17 male) with a mean age of 35.2 ± 5.3 with RRMS (according to the criteria in McDonalds’ et al. [24]) were consecutively studied during relapse. Relapse was defined as a worsening on the Expanded Disability Status Scale (EDSS) by 1.0 point, new clinical symptoms of subjective character or objectively existing, lasting at least 24 hours, in the absence of infection or fever, after a period not shorter than 30 days of neurological status stability. The patients were evaluated at three different time-points, at relapse, after five days of IVMP following a dose of 1.0 g/day and in the remitting period. All 37 patients had white matter lesions on brain MRI scans. Clinical disease severity was scored by Kurtzke’s EDSS [18]. The mean time for duration of clinical symptoms was 6.4 years (± 5.1). The mean number of relapses during the course of the disease in patients was 3.3 (± 1.3) (Table I).
Patients with kidney, liver, endocrine, immunolo-gical, inflammatory or infectious disorders were excluded on the basis of history, physical examinations and laboratory evaluations. No patients had received anti-inflammatory, immunosuppressive, immunomodulatory, steroid or hormonal treatment for at least three months before this study-point.
Cerebrospinal fluid (CSF) samples were collected from patients with active MS relapse before the initiation of IVMP therapy (A0). Venus blood samples were collected from patients in active relapse - before IVMP therapy (B0), after five days of IVMP therapy (B5) and in remitting period (BR). The control group consisted of 10 age-adjusted healthy volunteers whose blood and CSF were being collected for clinical indications.
The study was approved by the scientific ethics committee of the Medical University in Lublin, Poland. All persons gave their informed consent prior to their inclusion in the study.
CSF was concentrated by vacuum centrifugation (JW Electronic, Poland).

Biochemical investigation

1. To estimate the thiobarbituric acid-reactive material (TBAR) content in CSF or serum of blood we used a spectrophotometric technique of absorption determination modified from Slater and Sawyer [38]. CSF or serum of blood samples were extracted with 20% trichloroacetic acid, and centrifuged at 3000 × γ for 10 min. 0.67% thiobarbituric acid (TBA) diluted in buffer (26 mM Tris-HCl, pH 7.0) was added to supernatant. The mixture was heated in a water bath at 100^oC for 10 min. After cooling the sample, the absorption level was read spectrophotometrically at the light wave length of 532 nm against a reagent assay. TBAR content was quantified as the malondialdehyde (MDA) extinction molar coefficient E = 1.56 × 105 M-1 cm-1, and the result was expressed in MDA nmoles per mg protein.
2. The amount of sulfhydryl (SH) groups, including the estimation of both of total SH groups (t-SH) and of the so-called non-protein soluble SH groups (s-SH) was estimated in CSF and serum. The term non-protein SH groups denotes those originating from SH compounds present in the supernatants obtained after precipitation of proteins with trichloroacetic acid. SH group content was determined by a modified method of Sedlak and Lindsay [36], based on the Ellman spectrophotometric technique using dithionitrobenzoic acid (DTNB). In computations the molar coefficient E = 13 983 M-1 cm-1 was used. Its value was determined by investigation of cysteine and reduced glutathione standard solutions. Total protein was measured by the method of Lowry et al. [20] using bovine serum albumin as standard.
3. Superoxide dismutase (SOD-1) activity were assayed in serum using the methods of Misra and Fridovich [27]. SOD-1 activity was determined at 37^oC by recording the increase in absorbance at 480 nm following the autooxidations of adrenaline, inhibited by SOD-1. One unit (U) of this activity is defined as the amount of enzyme inhibiting the adrenaline auto- oxidation in 50%. Haemoglobin concentration in the haemolysate was estimated after conversion into the cyanmethaemoglobin form using a commercial reagent (Biomed, Lublin, Poland) at 540 nm.

Data analysis

The results were expressed as the mean ± SD. The statistical significance of the differences was determined by analysis of variance (ANOVA) followed by Student’s t test for paired samples. A value of p < 0.001 and p < 0.05 was regarded as significant.

Results

In the biochemical investigation of patients with MS, selected parameters (i.e., free radical peroxidation products TBAR, SOD and SH groups content) turned out to significantly differentiate between MS patients and controls. The obtained results are presented separately for each patient group (cf. methods).
The results of TBAR (Table II) suggested, that peroxidation is taking place in MS, as observed in all examined groups, especially the relapse group. We noted that the level of TBAR increased by 81.85% in CSF during relapse compared with controls (p < 0.05). In serum of patients in all examined groups, we observed significantly 3 times higher levels of TBAR than in CSF. In serum during relapse, this level was higher by 104.87% (p < 0.001) compared with the control values. After five days of IVMP administration, the levels of TBAR decreased by 30.93% compared with the relapse group, but was still higher by 74.94% than in controls (p < 0.001). During the remitting period the level of TBAR decreased in comparison with the relapse and B5 groups, but was still higher by 39.48% compared with controls’ (p < 0.05).
In CSF and serum (Table III) we noticed statistically insignificant slight increases in the levels of t-SH groups in MS relapse in comparison with control group levels. After IVMP treatment, the level of t-SH groups in serum decreased compared with controls’ levels (p < 0.05). During the remitting period the level in serum also decreased in comparison with the level in the control group. The level of s-SH in CSF was higher by 65.71% than in the control group (p < 0.05). In serum, the level of s-SH group also increased by 30.66% compared with controls’ levels (p < 0.05). The treatment prompted an increase in the level of s-SH by 42.79% (p < 0.001). In the remitting period the level of s-SH increased by 8.20% compared with the control group (p < 0.05), but decreased by 22.46% compared with the level in MS relapse (p < 0.05) and by 34.59% compared with the level after five days of treatment (p < 0.05). Considering the above described groups, the level of p-SH groups was as follows. In CSF and serum it decreased respectively by 31.72% (p < 0.001) and 28.97% (p < 0.001) in comparison with the levels in controls. After five days of treatment the level in serum decreased by 58.27% compared with controls’ levels (p < 0.001). In the remitting period, a slight increase in the level was shown compared with the level in MS relapse and after five days of treatment, but it was still lower by 18.83% in comparison with the level in controls (p < 0.05).
The level of SOD-1 (Table IV) in serum during relapse decreased slightly by 4.48% compared with the controls’ levels (p < 0.05); however, after five days of treatment it increased by 11.34% (p < 0.05) and during remission by 9.7% (p < 0.05) compared with the controls.

Discussion

MP is used as a standard drug in treating MS relapses. Studies in the literature on the effects of MP on the immune system in MS are controversial. Some authors [4,9,23,26] support the view that MP treatment induces a decrease in CD4+ cells. However, there are other reports stating that corticosteroids have no effect on the cell subpopulation [5,8,15]. Reactive oxygen species (ROS) are produced upon interaction of monocytes with brain endothelium, which leads to tight-junction alterations, cytoskeleton rearrangements, loss of blood-brain barrier integrity, and subsequent extravasation of leucocytes into the CNS [44]. Some publications support the view that the generation of ROS enhances in demyelinating diseases [3,21,40,42]. When free radicals concentration exceeds the level that can be handled by defence system, they can cause tissue destruction by disrupting biomembrane structure and functions, SH-containing enzymes, polysaccharide structure and nuc-leic acid activity [40,43]. The nature and degree of CNS damage can be inferred from alterations in CSF and serum levels of these products [37].
In this study we observed significantly higher CSF and serum levels of TBAR in MS patients during relapse compared with controls, which indicated an increase of peroxidation products in the relapse phase of MS. Our findings were in accordance with results described by Koch et al. [16] and Ortiz et al. [30]. It was found that TBAR level in the serum decreases after MP treatment and had the lowest value during the remission phase of the disease compared with controls. This indicated an enhanced process of lipid peroxidation in the CNS and probable damage of biomembranes in the brain [17,43]. The lipid peroxidation process intensity was differentiated across the MP treatment; the lowest TBAR level was noted in groups of patients in remission phase and the highest (significantly higher than that in the controls) in patients with relapse of MS.
Protein thiols are important for structure and function of many proteins [29], whereas non-protein thiols are important for the cellular defence system against free radicals [44].
In relapse phase, patient s-SH groups were elevated in CSF and the serum compared with the controls, whereas patient p-SH groups were decreased. After MP treatment, patient s-SH groups significantly increased in serum compared with the controls and patient p-SH groups significantly decreased. SH compounds such as reduced GSH, submit to peroxidation (dehydrogenation) and are transformed into the disulphide compounds (RSSR). Changes in the mutual transformation of SH and RSSR result in a modification of the oxidative processes involving lipid acid dehydrogenase, GSH reductase and thioredoxin systems. In all groups of patients, differences in the decrease of the number of p-SH groups were correlated with an increase in TBAR levels, which suggested that non-protein thiols were in a strategic position against ROS-mediated brain damage [2,19,25,44]. The level of SOD-1 superoxide dismutase increased after MP treatment and this rise was retained during remission.
Our study partially confirmed the results of Naidoo and Knapp [28], who showed higher concentrations of MDA in the serum but did not observed changes of MDA in CSF of patients during remission (we estimated MDA in CSF during relapse). On the other hand Hunter et al. [13] demonstrated significant increases in the level of MDA in CSF of patients with MS.
In conclusion, we noticed significant difference between SM patients and control group in peroxidation products. The changes were better seen during relapses of the disease. Following MP treatment the levels of these indicators approached control values especially in the remission phase of the disease. One of oxidative stress exponents is an increase of MDA level in serum – a marker of lipids’ peroxidation. This happens both in CSF and the serum during the relapse phase. Also, the changes in thiol groups testify the beginning of oxidative-reductive reaction in the relapse phase of the disease and the inhibition of free radicals activity after the treatment and in the remission phase. The biggest antioxidative importance is arrogated to SOD-1 which catalyzes dismutation of superoxide anion and, together with catalase and glutathione peroxydase, they perform protective activities against intracellular accumulation of free radicals. Decrease of activity of SOD-1 leads to cell exposure to harmful effect of free radicals. Increase of SOD-1 level after treatment and during remission period testify the protection against free radicals activity. The changes suggest that glucocorticoids protect against free radicals’ attacks and cause the stabilization of investigated indicators in the remission period.

References

 1. Airla N, Luomala M, Elovaara I, Kettunen E, Knuutila S, Lehtimäki T. Suppresion of immune system genes by methylprednisolone in exacerbations of multiple sclerosis. Preliminary results. J Neurol 2004; 251: 215-219.  
2. Aizenman E, Hartnett KA, Reynolds IJ. Oxigen free radicals regulate NMDA receptor function via a redox modulatory site. Neuron 1990; 5: 841-846.  
3. Arundhati J, Kalipada P Oxidative stress kills human primary oligodendrcytes via neural sphingomyelinase: implication for multiple sclerosis. J Neuroimmune Pharmacol 2007; 2: 184-193.  
4. Dufour A, Salmaggi A, La Mantia L, Eoli M, Nepolo A, Milanese C. High-dose methylprednisolone treatment-induced changes in immunological parameters in progressive MS patients. Int J Neurosci 1994; 75: 119-128.  
5. Frequin ST, Lamers KJ, Borm GF, Barkhof F, Jongen PJ, Holm- mes OR. T-cell subsets in the cerebrospinal fluid and peripheral blood of multiple sclerosis patients treatment with high-dose intravenous methylprednisolone. Acta Neurol Scand 1993; 88: 80-86.  
6. Forman HJ, Fisher AB. Role of glutathione peroxidase in tole-rance and adaptation of rats to hyperoxia. In: Bors W, Saran M, Tait D (eds.). Oxygen Radicals in Chemistry and Biology. Walter de Gruyter, Berlin, New York 1984; pp. 699-704.  
7. Frohman EM, Racke MK, Raine CS. Multiple sclerosis – the plaque and its pathogenesis. N Engl J Med 2006; 354: 942-955.
 8. Gallo P, Chiusole M, Sanzari M, Sivieri S, Piccinno MG, Argentiero V, Rizzotti P, Tavolato B. Effect of high-dose steroid therapy on T-cell subpopulations. A longitudinal study in MS patients. Acta Neurol Scand 1994; 89: 95-101.
 9. Gelati M, Corsini E, Durfour A, Ciusani E, Massa G, Frigerio S, Milanese C, Nespolo A, Salmaggi A. Reduced adhesion of PBMNCs to endothelium in methylprednisolone-treated MS patients: preliminary results. Acta Neurol Scand 1997; 96: 283-292.
10. Gsell W, Conrad R, Hickethier M, Sofic E, Frölich L, Wichart I, Jellinger K, Moll G, Ransmayr G, Beckmann H, Riederer P. Decrease catalase activity but unchanged superoxide dismutase activity in brains of patients with dementia of Alzheimer type. J Neurochem 1995; 64: 1216-1223.
11. Han MH, Hwang SI, Roy DB, Lundgren DH, Price JV, Ousman SS, Fernald GH, Gerlitz B, Robinson WH, Baranzini SE, Grinnell BW, RaineCS, Sobel RA, Han DK, Steinman L. Proteomic analysis of active multiple sclerosis lesions reveals therapeutic targets. Nature 2008; 451: 1076-1081.
12. Hossmann KA. Pathophysiological basis of translational stroke research. Folia Neuropathol 2009; 47: 213-227.
13. Hunter MIS, Nlemadim BC, Davidson DLW. Lipid peroxidation products and antioxidant proteins in plasma and cerebrospinal fluids from multiple sclerosis patients. Neurochem Res 1985; 10: 1645-1652.
14. Jana A, Pahan K. Oxidative stress kills human primary oligodendrocytes via neutral sphingomyelinase: implications for multiple sclerosis. J Neuroimmune Pharmacol 2007; 2: 184-193.
15. Kirk P, Compston A. The effect of methylprednisolone on lymphocyte phenotype and function in patients with multiple sclerosis. J Neuroimmunol 1990; 26: 1-8.
16. Koch M, Mostert J, Arutjunyan AV, Stepanov M, Teelken A, Heersema D, De Keyser J. Plasma lipid peroxidation and progression of disability in multiple sclerosis. Eur J Neurol 2007; 14: 529-533.
17. Kramer JH, Misik V, Weglicki WB. Lipid peroxidation-derived free radical production and postischemic myocardial reperfusion injury. Ann NY Acad Sc 1994; 723: 180-196.
18. Kurtzke JE. Rating neurologic impairment in multiple sclerosis: an expanded disability status scale (EDSS). Neurology 1983; 33: 1444-1452.
19. Lei SZ, Pan ZH, Aggarwal SK, Chen HSV, Hartman J, Sucher N, Lipton SA. Effects of nitrate oxide production on the redox modulatory site of the NMDA receptor-channel complex. Neuron 1992; 8: 1087-1099.
20. Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ. Protein measu-rement with the Folin phenol reagent. J Biol Chem 1951; 193: 265-275.
21. Love S, Jenner P. Oxidative stress in neurological disease. Brain Pathol 1999; 9: 55-56.
22. Lung DY, Bloom JW. Update on glucocorticoid action and resis-tance. J Allergy Clin Immunol 2003; 111: 3-22.
23. Martínez-Cáceres EM, Barrau MA, Brieva L, Espejo C, Barberá N, Montalban X. Treatment with methylprednisolone in relapse of multiple sclerosis patients: immunological evidence of imme-diate and short-term but not long-lasting effects. Clin Exp Immunol 2002; 127: 165-171.
24. McDonald WI, Compston A, Edan G, Goodkin D, Hartung HP, Lublin FD, MaFarland HF, Paty DW, Polman CH, Reingold SC, Sandberg-Wollheim M, Sibley W, Thompson A, von den Noot S, Weinshenker BY, Wolinsky JS. Recommended diagnostic criteria for multiple sclerosis: guidelines from the International Panel on the diagnosis of multiple sclerosis. Ann Neurol 2001; 50: 121-127.
25. Meister A, Anderson ME. Glutathione. Annu Rev Biochem 1983; 52: 711-760.
26. Michałowska-Wender G, Wender M. Peripheral blood cell immu-nomarkers in the course of methylprednisolone treatment of multiple sclerosis relapses. Folia Neuropathologica 2008; 46: 134-138.
27. Misra HP, Fridovich J. The role of superoxide anion in the autooxidation of epinephrine and simple assay for superoxide dismutase. J Biol Chem 1972; 247: 3170-3175.
28. Naidoo R, Knapp ML. Studies of lipid peroxidation products in cerebral fluid and serum in multiple sclerosis and other conditions. Clin Chem 1992; 38: 2449-2454.
29. Orrenius S, Burkitt MJ, Kass GEN, Dypbukt J, Nicotera P. Calcium ions and oxidative cell injury. Ann Neurol 1992; 32: S33-42.
30. Ortiz GG, Macías-Islas MA, Pacheco-Moisés FP, Cruz-Ramos JA, Sustersik S, Barba EA, Aguayo A. Oxidative stress is increased in serum from Mexican patients with relapsing-remitting multiple sclerosis. Dis Markers 2009; 26: 35-39.
31. Percy ME, Dalton AJ, Markovic VD, Crapper McLachlan DR, Hummel JT, Rusk ACM, Andrews DF. Red cell superoxide dismutase, glutathione peroxidase and catalase in Down Syndrome patients with and without manifestations of Alzheimer disease. Am J Med Gen 1990; 35: 459-467.
32. Perumal JS, Caon C, Hreha S, Zabad R, Tselis A, Lisak R, Khan O. Oral prednisone taper following intravenous steroids fails to improve disability or recovery from relapses in multiple sclerosis. Eur J Neurol 2008; 15: 677-680.
33. Placer Z, Cushman L, Johnson B. Estimation of product of lipid peroxidation malondialdehyde in biochemical systems. Anal Bioch 1966; 16: 359-364.
34. Raine CS. Multiple sclerosis: classification revisited reveals homogeneity and recapitulation. Ann Neurol 2008; 63: 1-3.
35. Rehncrona S, Folbergrova J, Smith DS, Siesjo BK. Influence of complete and pronounced incomplete cerebral ischemia and subsequent recirculation on cortical concentracions of oxidized and reduced glutathione in the rat. Neurochem 1980; 34: 477-486.
36. Sedlak J, Lindsay RH. Estimation of total protein bound and non-protein sulfhydryl groups in tissue with Ellman’s reagent. Anal Biochem 1968; 25: 192-205.
37. Selmaj K, Jurewicz A, Matysiak M, Raine CS. Soluble Nogo-A in CSF is not a usefule biomarker for multiple sclerosis. Neurology 2009; 72: 1708-1709.
38. Slater TF, Sawyer BC. The stimulatory effects of carbon tetrachloride and other halogenoalkanes on peroxidative reactions in rat liver fractions in vitro. Biochem J 1971; 123: 805-814.
39. Slivka A, Mytilineou C, Cohen G. Histochemical evaluation of glutathione in brain. Brain Res 1987; 409: 275-284.
40. Smith J, Kapoor R, Felts PA. Demyelination: the role of reactive oxygen and nitrogen species. Brain Pathol 1999; 9: 69-92.
41. Southorn PA, Powis G. Free radicals in medicine. Chemical nature and biologic reactions. Mayo Clin Proc 1998; 63a: 381-389.
42. Sulkowski G, Dąbrowska-Bouta B, Kwiatkowska-Patzer B, Strużyńska L. Alterations in glutamate transport and group I metabotropic glutamate receptors in the rat brain during acute phase of experimental autoimmune encephalomyelitis. Folia Neuropathol 2009; 47: 329-337.
43. Van Horssen J, Schreibelt G, Drexhage J, Hazes T, Dijkstra CD, van der Valk P, de Vries HE. Severe oxidative damage in multiple sclerosis lesions coincidences with enhanced antioxidant enzyme expression. Free Radic Biol Med 2008; 45: 1729-1737.
44. Wilson JX. Antioxidant defense of the brain: a role for astrocytes. Can J Physiol Pharmacol 1997; 75: 1149-1163.