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Serum uric acid and multiple sclerosis
Clinical Neurology and Neurosurgery 108 (2006) 527–531
Serum uric acid and multiple sclerosis M. Rentzos a,∗ , C. Nikolaou b , M. Anagnostouli a , A. Rombos a , K. Tsakanikas a , M. Economou b , A. Dimitrakopoulos a , M. Karouli a , D. Vassilopoulos a a
Department of Neurology, Aeginition Hospital-Athens Medical School, 72-74 Vas.Sophias Av, Greece b Department of Biopathology-Immunology, Aeginition Hospital-Athens Medical School, Greece Received 22 April 2005; received in revised form 2 August 2005; accepted 8 August 2005
Abstract Peroxynitrite (PN) has been implicated in multiple sclerosis (MS) and its animal model experimental allergic encephalomyelitis. Uric acid (UA) serum levels of MS patients, a natural scavenger of PN, were found lowered in some recent studies. Objective/purpose: The objective of our study was to correlate UA serum levels and several clinical parameters of MS. We also tried to investigate serum UA changes during treatment with immunomodulating or immunosuppressing drugs in the last 6 months. Patients and methods: We measured UA serum levels in 190 patients with MS and 58 age and gender matched patients with inflammatory (IND) and non-inflammatory diseases (NIND) studied as control groups. UA levels were correlated with clinical parameters as type of the disease, duration, disability, magnetic resonance imaging (MRI) activity and female gender. Results: In the overall MS group, patients were found to have significantly lower mean serum uric acid levels compared with the IND (p = 0.0029) and the NIND group (p < 0.0001). UA serum concentrations were not inversely correlated with duration of the disease (p = 0.87), with disability as assessed by Expanded Disability Status Scale (EDSS) score (p = 0.67) and MRI activity (p = 0.36). Treatment with immunomodulating or immunosuppressing drugs had no influence in UA levels (p = 0.85). Patients with Clinically Isolated Syndromes (CIS) were found to have significantly lower UA concentrations compared with IND and NIND patients (p = 0.009 and <0.001, respectively). Conclusions: Our findings suggest that lower serum UA levels in MS patients may represent a primary, constitutive loss of protection against nitric oxide and the development of CNS inflammation and tissue damage may not have a direct effect to UA serum levels. They also provide support that the earlier increase of UA serum levels might be beneficial in the future treatment of MS. © 2005 Elsevier B.V. All rights reserved. Keywords: Uric acid; Multiple sclerosis; Treatment; Neuroprotection
1. Introduction Nitric oxide and its oxidizing congeners, such as peroxynitrite (PN), have been implicated in the immunopathogenesis of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Elevated levels of nitric oxide synthase have been found in macrophages, microglia and astrocytes in demyelinating plaques of MS patients [1]. The presence of increased levels of reactive nitrogen oxide species (RNOS) in the central nervous system (CNS) and cerebrospinal fluid of MS patients may sug∗
Corresponding author. Tel.: +30 2 10 7289200. E-mail addresses: [email protected], [email protected] (M. Rentzos). 0303-8467/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.clineuro.2005.08.004
gest that RNOS are involved in the pathogenesis of MS [2,3]. MS is a chronic inflammatory demyelinating disease of the CNS. It is well established that activated T-cells and monocytes play an important pathogenetic role [4]. Reactive oxygen and nitrogen species may be involved in inflammation, demyelination and axonal injury occurring independently in MS [5]. Peroxynitrite, one of the most damaging oxidants, exert a toxic effect on neurons, axons and glia cells and may enhance apoptosis [6]. Uric acid (UA), the naturally occurring end product of purine metabolism, is a strong scavenger of PN [7]. Its administration inhibited the production of free radicals and suppressed EAE [7,8]. Patients suffering from MS have lower circulating UA levels than healthy control patients or patients
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M. Rentzos et al. / Clinical Neurology and Neurosurgery 108 (2006) 527–531
with other neurological diseases [9–12]. In some of these studies, UA levels did significantly correlated with disease activity but in others did not. This is very important because correlation between UA serum levels and clinical measures of MS may suggest a primary, constitutive loss of protection against PN or a secondary effect related to its oxidation by RNOS and free radicals. Two recent studies have shown that high-dose methylprednisolone and glatiramer acetate therapy increased serum UA levels [11,12]. In another study, the authors suggested that uric acid may serve as an easily detectable marker of disease activity as well as response to therapy [13].
2. Materials and methods
Fifteen patients with MS were treated with steroids, 9 with interferon-, 2 with steroids and interferon- and 2 with azathioprine within last 6 months previous to examination. Ethical consent has been obtained by all patients and controls for studying UA serum levels. Exclusion criteria were treatment with acetylsalicylic acid, thiazide diuretics, steroids, ibuprophen and other drugs that could increase or reduce UA levels as well as subjects with diabetus mellitus or renal failure. Blood samples were collected after all patients had received the same diet for at least 5 days. UA serum levels were determined by using a commercially available enzymatic assay (Uricase-PAP) according to the manufacturer’s protocol. The normal range of serum UA values is 2.3–6.1 mg/dl in women and 3.6–8.2 mg/dl in men.
2.1. Patients 3. Statistical analysis Serum samples were studied in 261 individuals comprising the following groups: (1) 190 patients with MS (78 men and 112 women, aged 16–69 years, mean 38.6); (2) 28 patients with inflammatory neurological diseases (IND) (12 men and 16 women, aged 19–74 years, mean 42), 15 out of these patients had Guillain–Barre Syndrome (GBS), 8 had Chronic Inflammatory Demyelinating Polyradiculoneuropathy (CIDP) and 5 had meningitis; (3) 30 patients with non-inflammatory neurological diseases (NIND) (12 men and 18 women, aged 23–71 years, mean 41.5), 4 out of these patients had movement disorder, 3 had motor neuron disease, 6 were demented, 5 had stroke, 6 had non-inflammatory neuropathies, 2 had tension headache and 4 had migraine. The second and third group of patients was studied as a control group. All of MS patients had definite MS according to the criteria of McDonald et al. [14] except of 29 patients that had Clinically Isolated Syndrome (CIS) and were at high risk for the development of multiple sclerosis. In CIS group patients with clinical features such as optic neuritis, diplopia, hemiparesis or cerebellar ataxia were included. These patients presented the first demyelinating event and had two or more clinically silent brain lesions on magnetic resonance imaging (MRI) [15]. All the MS patients were scored by the Expanded Disability Status Scale (EDSS) [16]. Mean EDSS score was 2.8 ± 1.4, range 0–7.5. The mean duration of the disease was 7 ± 7.5, range 0.08–43 years. Of the 161 patients with MS 113 had the relapsing–remitting (RR) type of the disease, 32 had the secondary progressive (SP), and 16 the primary progressive (PP) type of the disease. Patients with MS were divided into two groups: 35 with brain or spinal cord (MRI) activity and 155 without. Thirty patients with radiological activity had a clinical relapse. Patients with MS were considered to have MRI activity if they had one or more enhancing lesions in T1-weighted spin-echo images after gadopentate dimeglumine (Gd-DTPA) injection. Gd-DTPA was given intravenously at a dose 0.1 mmol/kg and about 15 min after contrast injection a T1-weighted sequence was repeated.
Results were expressed as mean ± standard deviation. The Student’s t-test and the Mann–Whitney U-test were used, as appropriate, to compare titres of UA between the examined study groups. Data are expressed as the mean ± S.D. All comparisons were two-sided, with a p-value of less than 0.05 used to indicate statistical significance. The statistical software used for this analysis was Statistica 6.0.
4. Results Women in each group were found to have significantly lower mean serum uric acid values (3.04 ± 0.82 mg/ml) when compared with men (4.06 ± 0.93 mg/dl), especially in MS group (p < 0.00001) (Table 1). In the overall MS group, patients were found to have significantly lower mean serum UA levels (3.48 ± 0.99 mg/dl) compared with the IND (4.34 ± 1.27 mg/dl), p = 0.0029 and the NIND patients Table 1 Correlation of UA levels with clinical and demographic characteristics Variables
Mean ± S.D. (mg/dl)
Range (mg/dl)
Disease duration <10 years, n = 80 >10 years, n = 110
3.55 ± 1 3.47 ± 0.97
1.2–6.7 1.7–6.2
MRI activity Active, n = 35 Inactive, n = 155
3.65 ± 1 3.45 ± 0.94
2.2–6.7 1.2–6.3
EDSS <3.5, n = 130 >3.5, n = 60
3.49 ± 1 3.42 ± 1
1.2–6.3 1.7–5.9
Sex Female, n = 112 Male, n = 78
3.04 ± 0.82 4.06 ± 0.93
1.2–6 2.0–6.7
Correlation of serum UA levels with disease duration, MRI activity, disability and sex. Values represent the mean ± S.D. Values in parenthesis represent the minimum and the maximum value. MRI: magnetic resonance imaging, EDSS: Expanded Disability Status Scale.
M. Rentzos et al. / Clinical Neurology and Neurosurgery 108 (2006) 527–531
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Table 2 Doses and duration of treatment with immunomodulatory or immunosuppressive drugs
Fig. 1. Distribution of UA levels in serum samples of patients with MS, IND and NIND. MS: multiple sclerosis, IND: inflammatory neurological diseases, NIND: non-inflammatory neurological diseases.
(4.5 ± 1.2 mg/dl), p < 0.0001 (Fig. 1). Serum UA levels were significantly lower in patients with RRMS (3.4 ± 1 mg/dl) compared with patients with IND (p = 0.0015) and NIND (p = 0.0006) (Fig. 2). Serum UA levels were also decreased in SPMS (3.5 ± 0.95 mg/dl) and CIS patients (3.5 ± 0.97 mg/dl) compared with IND (p = 0.0067, 0.0004) and NIND patients (p = 0.009, <0.001) (Fig. 2). Patients with PPMS (3.7 ± 0.95 mg/dl) had not significantly lower UA levels compared with IND patients (p = 0.08). They had mildly lower UA levels compared with NIND patients (p = 0.03) (Fig. 2). Among MS patients, patients with MRI activity had not significantly lower serum UA levels (3.65 ± 1 mg/dl) when compared with patients with no MRI activity (3.45 ± 0.94 mg/dl; p = 0.36) (Table 1). We divided patients with MRI-active and MRI-inactive MS in groups with
Fig. 2. Distribution of UA levels in serum samples of patients with RR-MS, SP-MS, PP-MS, CIS, IND and NIND. RR-MS: relapsing–remitting multiple sclerosis, SP-MS: secondary progressive multiple sclerosis, PP-MS: primary progressive multiple sclerosis, CIS: Clinically Isolated Syndromes, IND: inflammatory neurological diseases, NIND: non-inflammatory neurological diseases.
Patients
Drugs
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
Inf -1␣ for 10 months MP 2.5 g IV in 5 days, 1 month ago Inf -1a for 6 months MP 1.5 g IV in 3 days, 3 months ago Inf -1a for 1 year Inf -1b for 8 months Inf -1b for 1.5 years Inf -1a for 4 months AZA for 8 months MP 1.5 g IV for 3 days, 2 months ago MP 2 g IV for 4 days, 6 months ago MP 2.5 g IV for 5 days, 6 months ago Inf -1a for 10 months, MP 1.5 g IV 10 months ago MP 1.5 g IV, 2 months ago Inf -1b for 5 months MP 2 g IV for 4 days after admission Inf -1a for 10 months MP 2 g IV for 4 days 3 months ago MP 2.5 g IV for 5 days 1 month ago MP 1.5 g IV for 3 days 6 months ago Inf -1a for 7 months Inf -1a for 5 months, stop 2 months before admission, MP 1.5 g IV for 3 days 5 months ago AZA for 6 months, MP 2 g IV for 4 days 5 months ago MP 1.5 g IV for 3 days 3 months ago MP 1.5 g IV for 3 days 4 months ago Inf -1b 1 month ago MP 2 g IV for 4 days 3 months ago MP 2 g IV for 4 days 15 days ago
23 24 25 26 27 28
Abbreviations: MP, methylprednisolone; Inf, interferon; AZA, azathioprine; IV, intravenous; . . . ago, days or months before admission.
different clinical patterns of MS. RR-MS-active patients did not show significant differences in serum UA levels (n = 21, 3.98 ± 0.85) compared with RR-MS-inactive patients (n = 92, 3.2 ± 1.02), p < 0.088. Comparison between SP-active (n = 7, 3.9 ± 0.35) and SP-inactive MS patients (n = 25, 3.75 ± 1.45) did not show differences in UA levels (p < 0.89). We did not compare UA concentrations between PP-active and PP-non-active MS patients since only 2 PPMS patients had active MRI. Uric acid levels were not lower in patients with longer duration of the disease (>10 years) or patients with more disabling disease (EDSS > 3.5) (p = 0.87 and 0.67, respectively) (Table 1). We compared UA levels between patients treated with immunomodulating or immunosuppressing drugs within last 6 months and untreated patients. Previous treatment had no influence in UA levels (p = 0.85). Doses and duration of treatment are included in Table 2.
5. Discussion Peroxynitrite acts on CNS tissue by lipid peroxidation, release of oxygen species, tyrosine nitration and DNA strand breakage or mutation [17–21]. High levels of NO and its
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metabolites were found in the CSF of MS patients during relapse [22,23]. There are some recent studies correlating UA levels and clinical parameters of MS [5,9,10]. UA levels were found reduced either in the overall MS group or in the group with active disease or increased MRI activity compared with OND or healthy subjects. In our study, UA levels in MS patients were found significantly lower than control patients. Neuroinflammation or neurodegeneration should not be considered potential causal factors of this phenomenon since they form basic pathogenetic characteristics not only of MS but of IND and NIND as well. Comparison of UA levels in MS patients according to MRI activity, disability and duration of the disease showed no significant difference. No trend toward lower UA levels between different MS subtypes according to MRI activity (RR-active versus RR-inactive, SP-active versus SP-inactive) or according to clinical activity have been found. The above results of our study may favor the view that MS patients might have a primary reduced antioxidant reserve. However, it remains uncertain whether the low values of UA in patients with MS are a cause or a consequence of the disease activity. Hooper et al. have recently shown in a mouse model of multiple sclerosis that a primary reduced antioxidant reserve may coexist with the increased consumption of UA as a scavenger [24]. In a recent study, UA serum levels in patients with optic neuritis were found reduced compared to control group patients [25]. In our study, UA levels in the subgroup of patients with CIS were found significantly different compared to IND and NIND patients but did not show significant difference compared to other clinical subtypes of definite MS. This may suggest that UA levels are low from the beginning, years before the definite diagnosis of the disease and the development of CNS inflammation, and that tissue damage may not have a direct effect to UA serum levels. A significant difference between serum UA levels in RRMS and SP-MS compared with IND and NIND, respectively, has also been found. In the contrary, in the subgroup of patients with the PP form of the disease UA levels were not found significantly different compared to IND patients (p = 0.08) and were mildly different compared to NIND group patients (p = 0.03). This is probably due to the unequal distribution of gender between patients with various subtypes of MS and patients with PP-MS. These patients present male gender preponderance. However, it might be the result of the different immunogenetic profile of the RR-MS regarding to the other clinical forms of the disease. Neurodegeneration rather than inflammation has been implied as a more relevant pathogenetic mechanism in PP-MS. This is supported by the lower IgG index compared to RR-MS, the lower number of lesions on MRI and the absence of efficacy of immunomodulating or immunosuppressing treatment [26,27]. All studied groups and subgroups were age and gender matched. Female MS patients had significantly lower UA levels compared to male patients (p < 0.000001) as also reported in previous studies [5,10,25]. There is not any satisfactory explanation for these results. Recent studies try to partially
explain the female predominance in MS with the inverse correlation between serum UA concentrations and female gender [5,10,25]. UA suppresses the inflammatory processes, the CNS tissue damage and death in animal models of MS [24]. Interferon-␥ is a major contributor to NOS induction [28] while interferon was shown both to induce and to inhibit the NOS induction of human glial cells [12,29]. This action is potentially doserelated. This might explain why non-significant UA changes were found after treatment with interferon- 1a [11,12]. However, the results of clinical studies suggest the increase of serum uric acid levels by high-dose methylprednisolone and glatiramer acetate in patients with MS. In the present study, we studied retrospectively the result of previous treatment with interferon-, high-dose methylprednizolone or azathioprine in UA serum levels. There was not significant correlation of UA levels in treated patients compared with not treated. Our findings do not exclude the possible effect of immunomodulating and immunosuppressing treatment in UA levels of MS patients. To determine whether immunomodulatory treatments may increase or not serum UA levels, double-blind prospective placebo controlled studies in greater number of patients will be needed. Irrespective of whether lower serum UA levels in MS patients represent a primary, constitutive loss of protection against nitric oxide and its oxidizing congeners or a secondary effect as a result of UA scavenging activity, the results of our study support the hypothesis that higher serum uric acid might be beneficial in the treatment of MS. There is growing evidence that axonal loss rather than inflammation and demyelination underlies the disability progression in MS (1). The neuroprotective properties of antioxidants like UA make these agents promising tools in future treatment protocols in MS (3). In one recent study, inosine, a precursor of UA, has been used in order to avoid degradation by gastrointestinal bacteria of UA administrated orally [30]. Three of 11 patients given inosine presented clinical improvement, while the rest had no sign of relapse. Two of the patients with gadolinium enhanced lesions in brain MRI before inosine, had no any lesion after 10 months of inosine therapy. Given the small number of patients no definite conclusions can be drawn about inosine efficacy. To estimate the benefit or not of higher UA serum levels to individuals with MS a greater number of patients should be studied in the future. In blood of both monozygotic and dizygotic twins the uric acid levels were lower in the twin with the disease than in the healthy twin [31]. This might suggest the use of antioxidant treatment in individuals at high risk of developing MS (e.g. unaffected monozygotic or dizygotic twins) in order to prevent MS clinical manifestations [9]. The absence of any relationship between uric acid levels and MRI activity in our study may suggest that even if uric acid is involved in MS as a scavenger of peroxynitrite, its role in the modification of disease activity is far from being established. Future therapies targeting to increase UA levels in MS may start early in the course of the disease in order
M. Rentzos et al. / Clinical Neurology and Neurosurgery 108 (2006) 527–531
to promote neuroprotection and to prevent axonal injury and irreversible tissue damage.
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[17] Radi R, Beckman JS, Bush KM, Freeman BA. Peroxynitriteinduced membrane lipid peroxydation: the cytotoxic potential of superoxide and nitric oxide. Arch Biochem Biophys 1991;288:481– 7. [18] Vladimirova O, O’Connor J, Cahill A, Alder H, Butunoi C, Kalman B. Oxidative damage to DNA in plaques of MS brains. Mult Scler 1998;4:413–8. [19] Whiteman M, Halliwell B. Protection against peroxynitritedependent tyrosine nitration and alpha 1-proteinase inactivation by ascorbic acid. A comparison with other antioxidants. Free Radic Res 1996;25:275–83. [20] Ischiropoulos H, Zhu L, Chen J, et al. Peroxynitrite-mediated tyrosinase nitration catalyzed by superoxide dismutase. Arch Biochem Biophys 1992;298:431–7. [21] Hooper DC, Ohnishi ST, Kean R, Numagami Y, Dietzschold B, Koprowski H. Local nitric oxide production in viral and autoimmune diseases of the central nervous system. Proc Natl Acad Sci USA 1995;92:5312–6. [22] Brundin L, Morcos E, Olsson T, Wiklund NP, Andersson M. Increased intrathecal nitric oxide formation in multiple sclerosis; cerebrospinal fluid nitrite as activity marker. Eur J Neurol 1999;6: 585–90. [23] Yamashita T, Ando Y, Obayashi K, Uchino M, Ando M. Changes in nitrite and nitrate (NO2 − /NO3 − ) levels in cerebrospinal fluid of patients with multiple sclerosis. J Neurol Sci 1997;153:32–4. [24] Hooper DC, Scott GS, Zborek A, et al. Uric acid, a peroxynitrite scavenger, inhibits CNS inflammation, blood–CNS barrier permeability changes, and tissue damage in a mouse model of multiple sclerosis. FASEB J 2000;14:691–8. [25] Knapp CM, Constantinescu CS, Tan JH, McLean R, Cherryman GR, Gottlob I. Serum uric acid levels in optic neuritis. Mult Scler 2004;10:278–80. [26] Thompson AJ, Polman CH, Miller DH, et al. Primary progressive multiple sclerosis. Brain 1997;120:1085–96. [27] Michalopoulou M, Nikolaou C, Tavernarakis A, Alexandri NM, Rentzos M, Chatzipanagiotou S, et al. Soluble interleukin-6 receptor (sIL-6R) in cerebrospinal fluid of patients with inflammatory and non inflammatory neurological diseases. Immunol Lett 2004;94:183– 9. [28] Lorsbach RB, Murphy WJ, Lowenstein CJ, Snyder SH, Russell SW. Expression of the nitric-oxide synthase gene in mouse macrophages activated for tumor cell killing. Molecular basis for the synergy between interferon-␥ and lipopolysaccharide. J Biol Chem 1993;268:1908–13. [29] Hua LL, Liu JS, Brosnan CF, Lee SC. Selective inhibition of human glial inducible nitric oxide synthase by interferon-beta: implication for multiple sclerosis. Ann Neurol 1998;43:384–7. [30] Spitsin S, Hooper DC, Leist T, Streletz LJ, Mikheeva T, Koprowski H. Inactivation of peroxynitrite in multiple sclerosis patients after oral administration of inosine may suggest possible approaches to therapy of the disease. Mult Scler 2001;7:313–9. [31] Spitsin S, Hooper DC, Mikheeva T, Koprowski H. Uric acid levels in patients with multiple sclerosis: analysis in mono-and dizygotic twins. Mult Scler 2001;7:165–6.
Serum uric acid and multiple sclerosis M. Rentzos a,∗ , C. Nikolaou b , M. Anagnostouli a , A. Rombos a , K. Tsakanikas a , M. Economou b , A. Dimitrakopoulos a , M. Karouli a , D. Vassilopoulos a a
Department of Neurology, Aeginition Hospital-Athens Medical School, 72-74 Vas.Sophias Av, Greece b Department of Biopathology-Immunology, Aeginition Hospital-Athens Medical School, Greece Received 22 April 2005; received in revised form 2 August 2005; accepted 8 August 2005
Abstract Peroxynitrite (PN) has been implicated in multiple sclerosis (MS) and its animal model experimental allergic encephalomyelitis. Uric acid (UA) serum levels of MS patients, a natural scavenger of PN, were found lowered in some recent studies. Objective/purpose: The objective of our study was to correlate UA serum levels and several clinical parameters of MS. We also tried to investigate serum UA changes during treatment with immunomodulating or immunosuppressing drugs in the last 6 months. Patients and methods: We measured UA serum levels in 190 patients with MS and 58 age and gender matched patients with inflammatory (IND) and non-inflammatory diseases (NIND) studied as control groups. UA levels were correlated with clinical parameters as type of the disease, duration, disability, magnetic resonance imaging (MRI) activity and female gender. Results: In the overall MS group, patients were found to have significantly lower mean serum uric acid levels compared with the IND (p = 0.0029) and the NIND group (p < 0.0001). UA serum concentrations were not inversely correlated with duration of the disease (p = 0.87), with disability as assessed by Expanded Disability Status Scale (EDSS) score (p = 0.67) and MRI activity (p = 0.36). Treatment with immunomodulating or immunosuppressing drugs had no influence in UA levels (p = 0.85). Patients with Clinically Isolated Syndromes (CIS) were found to have significantly lower UA concentrations compared with IND and NIND patients (p = 0.009 and <0.001, respectively). Conclusions: Our findings suggest that lower serum UA levels in MS patients may represent a primary, constitutive loss of protection against nitric oxide and the development of CNS inflammation and tissue damage may not have a direct effect to UA serum levels. They also provide support that the earlier increase of UA serum levels might be beneficial in the future treatment of MS. © 2005 Elsevier B.V. All rights reserved. Keywords: Uric acid; Multiple sclerosis; Treatment; Neuroprotection
1. Introduction Nitric oxide and its oxidizing congeners, such as peroxynitrite (PN), have been implicated in the immunopathogenesis of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Elevated levels of nitric oxide synthase have been found in macrophages, microglia and astrocytes in demyelinating plaques of MS patients [1]. The presence of increased levels of reactive nitrogen oxide species (RNOS) in the central nervous system (CNS) and cerebrospinal fluid of MS patients may sug∗
Corresponding author. Tel.: +30 2 10 7289200. E-mail addresses: [email protected], [email protected] (M. Rentzos). 0303-8467/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.clineuro.2005.08.004
gest that RNOS are involved in the pathogenesis of MS [2,3]. MS is a chronic inflammatory demyelinating disease of the CNS. It is well established that activated T-cells and monocytes play an important pathogenetic role [4]. Reactive oxygen and nitrogen species may be involved in inflammation, demyelination and axonal injury occurring independently in MS [5]. Peroxynitrite, one of the most damaging oxidants, exert a toxic effect on neurons, axons and glia cells and may enhance apoptosis [6]. Uric acid (UA), the naturally occurring end product of purine metabolism, is a strong scavenger of PN [7]. Its administration inhibited the production of free radicals and suppressed EAE [7,8]. Patients suffering from MS have lower circulating UA levels than healthy control patients or patients
528
M. Rentzos et al. / Clinical Neurology and Neurosurgery 108 (2006) 527–531
with other neurological diseases [9–12]. In some of these studies, UA levels did significantly correlated with disease activity but in others did not. This is very important because correlation between UA serum levels and clinical measures of MS may suggest a primary, constitutive loss of protection against PN or a secondary effect related to its oxidation by RNOS and free radicals. Two recent studies have shown that high-dose methylprednisolone and glatiramer acetate therapy increased serum UA levels [11,12]. In another study, the authors suggested that uric acid may serve as an easily detectable marker of disease activity as well as response to therapy [13].
2. Materials and methods
Fifteen patients with MS were treated with steroids, 9 with interferon-, 2 with steroids and interferon- and 2 with azathioprine within last 6 months previous to examination. Ethical consent has been obtained by all patients and controls for studying UA serum levels. Exclusion criteria were treatment with acetylsalicylic acid, thiazide diuretics, steroids, ibuprophen and other drugs that could increase or reduce UA levels as well as subjects with diabetus mellitus or renal failure. Blood samples were collected after all patients had received the same diet for at least 5 days. UA serum levels were determined by using a commercially available enzymatic assay (Uricase-PAP) according to the manufacturer’s protocol. The normal range of serum UA values is 2.3–6.1 mg/dl in women and 3.6–8.2 mg/dl in men.
2.1. Patients 3. Statistical analysis Serum samples were studied in 261 individuals comprising the following groups: (1) 190 patients with MS (78 men and 112 women, aged 16–69 years, mean 38.6); (2) 28 patients with inflammatory neurological diseases (IND) (12 men and 16 women, aged 19–74 years, mean 42), 15 out of these patients had Guillain–Barre Syndrome (GBS), 8 had Chronic Inflammatory Demyelinating Polyradiculoneuropathy (CIDP) and 5 had meningitis; (3) 30 patients with non-inflammatory neurological diseases (NIND) (12 men and 18 women, aged 23–71 years, mean 41.5), 4 out of these patients had movement disorder, 3 had motor neuron disease, 6 were demented, 5 had stroke, 6 had non-inflammatory neuropathies, 2 had tension headache and 4 had migraine. The second and third group of patients was studied as a control group. All of MS patients had definite MS according to the criteria of McDonald et al. [14] except of 29 patients that had Clinically Isolated Syndrome (CIS) and were at high risk for the development of multiple sclerosis. In CIS group patients with clinical features such as optic neuritis, diplopia, hemiparesis or cerebellar ataxia were included. These patients presented the first demyelinating event and had two or more clinically silent brain lesions on magnetic resonance imaging (MRI) [15]. All the MS patients were scored by the Expanded Disability Status Scale (EDSS) [16]. Mean EDSS score was 2.8 ± 1.4, range 0–7.5. The mean duration of the disease was 7 ± 7.5, range 0.08–43 years. Of the 161 patients with MS 113 had the relapsing–remitting (RR) type of the disease, 32 had the secondary progressive (SP), and 16 the primary progressive (PP) type of the disease. Patients with MS were divided into two groups: 35 with brain or spinal cord (MRI) activity and 155 without. Thirty patients with radiological activity had a clinical relapse. Patients with MS were considered to have MRI activity if they had one or more enhancing lesions in T1-weighted spin-echo images after gadopentate dimeglumine (Gd-DTPA) injection. Gd-DTPA was given intravenously at a dose 0.1 mmol/kg and about 15 min after contrast injection a T1-weighted sequence was repeated.
Results were expressed as mean ± standard deviation. The Student’s t-test and the Mann–Whitney U-test were used, as appropriate, to compare titres of UA between the examined study groups. Data are expressed as the mean ± S.D. All comparisons were two-sided, with a p-value of less than 0.05 used to indicate statistical significance. The statistical software used for this analysis was Statistica 6.0.
4. Results Women in each group were found to have significantly lower mean serum uric acid values (3.04 ± 0.82 mg/ml) when compared with men (4.06 ± 0.93 mg/dl), especially in MS group (p < 0.00001) (Table 1). In the overall MS group, patients were found to have significantly lower mean serum UA levels (3.48 ± 0.99 mg/dl) compared with the IND (4.34 ± 1.27 mg/dl), p = 0.0029 and the NIND patients Table 1 Correlation of UA levels with clinical and demographic characteristics Variables
Mean ± S.D. (mg/dl)
Range (mg/dl)
Disease duration <10 years, n = 80 >10 years, n = 110
3.55 ± 1 3.47 ± 0.97
1.2–6.7 1.7–6.2
MRI activity Active, n = 35 Inactive, n = 155
3.65 ± 1 3.45 ± 0.94
2.2–6.7 1.2–6.3
EDSS <3.5, n = 130 >3.5, n = 60
3.49 ± 1 3.42 ± 1
1.2–6.3 1.7–5.9
Sex Female, n = 112 Male, n = 78
3.04 ± 0.82 4.06 ± 0.93
1.2–6 2.0–6.7
Correlation of serum UA levels with disease duration, MRI activity, disability and sex. Values represent the mean ± S.D. Values in parenthesis represent the minimum and the maximum value. MRI: magnetic resonance imaging, EDSS: Expanded Disability Status Scale.
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Table 2 Doses and duration of treatment with immunomodulatory or immunosuppressive drugs
Fig. 1. Distribution of UA levels in serum samples of patients with MS, IND and NIND. MS: multiple sclerosis, IND: inflammatory neurological diseases, NIND: non-inflammatory neurological diseases.
(4.5 ± 1.2 mg/dl), p < 0.0001 (Fig. 1). Serum UA levels were significantly lower in patients with RRMS (3.4 ± 1 mg/dl) compared with patients with IND (p = 0.0015) and NIND (p = 0.0006) (Fig. 2). Serum UA levels were also decreased in SPMS (3.5 ± 0.95 mg/dl) and CIS patients (3.5 ± 0.97 mg/dl) compared with IND (p = 0.0067, 0.0004) and NIND patients (p = 0.009, <0.001) (Fig. 2). Patients with PPMS (3.7 ± 0.95 mg/dl) had not significantly lower UA levels compared with IND patients (p = 0.08). They had mildly lower UA levels compared with NIND patients (p = 0.03) (Fig. 2). Among MS patients, patients with MRI activity had not significantly lower serum UA levels (3.65 ± 1 mg/dl) when compared with patients with no MRI activity (3.45 ± 0.94 mg/dl; p = 0.36) (Table 1). We divided patients with MRI-active and MRI-inactive MS in groups with
Fig. 2. Distribution of UA levels in serum samples of patients with RR-MS, SP-MS, PP-MS, CIS, IND and NIND. RR-MS: relapsing–remitting multiple sclerosis, SP-MS: secondary progressive multiple sclerosis, PP-MS: primary progressive multiple sclerosis, CIS: Clinically Isolated Syndromes, IND: inflammatory neurological diseases, NIND: non-inflammatory neurological diseases.
Patients
Drugs
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
Inf -1␣ for 10 months MP 2.5 g IV in 5 days, 1 month ago Inf -1a for 6 months MP 1.5 g IV in 3 days, 3 months ago Inf -1a for 1 year Inf -1b for 8 months Inf -1b for 1.5 years Inf -1a for 4 months AZA for 8 months MP 1.5 g IV for 3 days, 2 months ago MP 2 g IV for 4 days, 6 months ago MP 2.5 g IV for 5 days, 6 months ago Inf -1a for 10 months, MP 1.5 g IV 10 months ago MP 1.5 g IV, 2 months ago Inf -1b for 5 months MP 2 g IV for 4 days after admission Inf -1a for 10 months MP 2 g IV for 4 days 3 months ago MP 2.5 g IV for 5 days 1 month ago MP 1.5 g IV for 3 days 6 months ago Inf -1a for 7 months Inf -1a for 5 months, stop 2 months before admission, MP 1.5 g IV for 3 days 5 months ago AZA for 6 months, MP 2 g IV for 4 days 5 months ago MP 1.5 g IV for 3 days 3 months ago MP 1.5 g IV for 3 days 4 months ago Inf -1b 1 month ago MP 2 g IV for 4 days 3 months ago MP 2 g IV for 4 days 15 days ago
23 24 25 26 27 28
Abbreviations: MP, methylprednisolone; Inf, interferon; AZA, azathioprine; IV, intravenous; . . . ago, days or months before admission.
different clinical patterns of MS. RR-MS-active patients did not show significant differences in serum UA levels (n = 21, 3.98 ± 0.85) compared with RR-MS-inactive patients (n = 92, 3.2 ± 1.02), p < 0.088. Comparison between SP-active (n = 7, 3.9 ± 0.35) and SP-inactive MS patients (n = 25, 3.75 ± 1.45) did not show differences in UA levels (p < 0.89). We did not compare UA concentrations between PP-active and PP-non-active MS patients since only 2 PPMS patients had active MRI. Uric acid levels were not lower in patients with longer duration of the disease (>10 years) or patients with more disabling disease (EDSS > 3.5) (p = 0.87 and 0.67, respectively) (Table 1). We compared UA levels between patients treated with immunomodulating or immunosuppressing drugs within last 6 months and untreated patients. Previous treatment had no influence in UA levels (p = 0.85). Doses and duration of treatment are included in Table 2.
5. Discussion Peroxynitrite acts on CNS tissue by lipid peroxidation, release of oxygen species, tyrosine nitration and DNA strand breakage or mutation [17–21]. High levels of NO and its
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metabolites were found in the CSF of MS patients during relapse [22,23]. There are some recent studies correlating UA levels and clinical parameters of MS [5,9,10]. UA levels were found reduced either in the overall MS group or in the group with active disease or increased MRI activity compared with OND or healthy subjects. In our study, UA levels in MS patients were found significantly lower than control patients. Neuroinflammation or neurodegeneration should not be considered potential causal factors of this phenomenon since they form basic pathogenetic characteristics not only of MS but of IND and NIND as well. Comparison of UA levels in MS patients according to MRI activity, disability and duration of the disease showed no significant difference. No trend toward lower UA levels between different MS subtypes according to MRI activity (RR-active versus RR-inactive, SP-active versus SP-inactive) or according to clinical activity have been found. The above results of our study may favor the view that MS patients might have a primary reduced antioxidant reserve. However, it remains uncertain whether the low values of UA in patients with MS are a cause or a consequence of the disease activity. Hooper et al. have recently shown in a mouse model of multiple sclerosis that a primary reduced antioxidant reserve may coexist with the increased consumption of UA as a scavenger [24]. In a recent study, UA serum levels in patients with optic neuritis were found reduced compared to control group patients [25]. In our study, UA levels in the subgroup of patients with CIS were found significantly different compared to IND and NIND patients but did not show significant difference compared to other clinical subtypes of definite MS. This may suggest that UA levels are low from the beginning, years before the definite diagnosis of the disease and the development of CNS inflammation, and that tissue damage may not have a direct effect to UA serum levels. A significant difference between serum UA levels in RRMS and SP-MS compared with IND and NIND, respectively, has also been found. In the contrary, in the subgroup of patients with the PP form of the disease UA levels were not found significantly different compared to IND patients (p = 0.08) and were mildly different compared to NIND group patients (p = 0.03). This is probably due to the unequal distribution of gender between patients with various subtypes of MS and patients with PP-MS. These patients present male gender preponderance. However, it might be the result of the different immunogenetic profile of the RR-MS regarding to the other clinical forms of the disease. Neurodegeneration rather than inflammation has been implied as a more relevant pathogenetic mechanism in PP-MS. This is supported by the lower IgG index compared to RR-MS, the lower number of lesions on MRI and the absence of efficacy of immunomodulating or immunosuppressing treatment [26,27]. All studied groups and subgroups were age and gender matched. Female MS patients had significantly lower UA levels compared to male patients (p < 0.000001) as also reported in previous studies [5,10,25]. There is not any satisfactory explanation for these results. Recent studies try to partially
explain the female predominance in MS with the inverse correlation between serum UA concentrations and female gender [5,10,25]. UA suppresses the inflammatory processes, the CNS tissue damage and death in animal models of MS [24]. Interferon-␥ is a major contributor to NOS induction [28] while interferon was shown both to induce and to inhibit the NOS induction of human glial cells [12,29]. This action is potentially doserelated. This might explain why non-significant UA changes were found after treatment with interferon- 1a [11,12]. However, the results of clinical studies suggest the increase of serum uric acid levels by high-dose methylprednisolone and glatiramer acetate in patients with MS. In the present study, we studied retrospectively the result of previous treatment with interferon-, high-dose methylprednizolone or azathioprine in UA serum levels. There was not significant correlation of UA levels in treated patients compared with not treated. Our findings do not exclude the possible effect of immunomodulating and immunosuppressing treatment in UA levels of MS patients. To determine whether immunomodulatory treatments may increase or not serum UA levels, double-blind prospective placebo controlled studies in greater number of patients will be needed. Irrespective of whether lower serum UA levels in MS patients represent a primary, constitutive loss of protection against nitric oxide and its oxidizing congeners or a secondary effect as a result of UA scavenging activity, the results of our study support the hypothesis that higher serum uric acid might be beneficial in the treatment of MS. There is growing evidence that axonal loss rather than inflammation and demyelination underlies the disability progression in MS (1). The neuroprotective properties of antioxidants like UA make these agents promising tools in future treatment protocols in MS (3). In one recent study, inosine, a precursor of UA, has been used in order to avoid degradation by gastrointestinal bacteria of UA administrated orally [30]. Three of 11 patients given inosine presented clinical improvement, while the rest had no sign of relapse. Two of the patients with gadolinium enhanced lesions in brain MRI before inosine, had no any lesion after 10 months of inosine therapy. Given the small number of patients no definite conclusions can be drawn about inosine efficacy. To estimate the benefit or not of higher UA serum levels to individuals with MS a greater number of patients should be studied in the future. In blood of both monozygotic and dizygotic twins the uric acid levels were lower in the twin with the disease than in the healthy twin [31]. This might suggest the use of antioxidant treatment in individuals at high risk of developing MS (e.g. unaffected monozygotic or dizygotic twins) in order to prevent MS clinical manifestations [9]. The absence of any relationship between uric acid levels and MRI activity in our study may suggest that even if uric acid is involved in MS as a scavenger of peroxynitrite, its role in the modification of disease activity is far from being established. Future therapies targeting to increase UA levels in MS may start early in the course of the disease in order
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to promote neuroprotection and to prevent axonal injury and irreversible tissue damage.
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