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Homocysteine, vitamin B12 and folate levels in Iranian patients with Multiple Sclerosis: A case control study
Clinical Neurology and Neurosurgery 115 (2013) 1802–1805
Contents lists available at SciVerse ScienceDirect
Clinical Neurology and Neurosurgery journal homepage: www.elsevier.com/locate/clineuro
Homocysteine, vitamin B12 and folate levels in Iranian patients with Multiple Sclerosis: A case control study Mehdi Moghaddasi a,b , Mansoureh Mamarabadi a,b,∗ , Nafiseh Mohebi a,b , Hadie Razjouyan c , Mahbubeh Aghaei a,b a
Department of Neurology, Rasool Akram Hospital, Tehran University of Medical Sciences, Tehran, Iran Iranian Center of Neurological Research (ICNR), Tehran, Iran c Department of Medicine, Drexel University, Monmouth Medical Center, Long Branch, 07744, USA b
a r t i c l e
i n f o
Article history: Received 30 March 2012 Received in revised form 19 February 2013 Accepted 2 May 2013 Available online 10 June 2013 Keywords: Homocysteine Vitamin B12 Folate Multiple Sclerosis
a b s t r a c t Background: Recently, homocysteine (Hcy), folate, and vitamin B12 have been proposed to have several roles on MS pathogenesis. Objective: We performed this study to determine the role of serum levels of Hcy, vitamin B12, and folate in patients with relapsing remitting MS (RRMS) and compared them with healthy controls. Methods: We recruited 75 RRMS patients and 75 subjects as controls with the same age and sex. Homocysteine was measured using fluorimetric high-performance liquid chromatography. Plasma folate and vitamin B12 levels were measured through ion-capture method. Results: Mean plasma levels of vitamin B12, folate, and Hcy in cases were 342.64 ± 210.66 pg/ml, 9.74 ± 4.77 ng/ml, and 22.73 ± 11.63 M/L, respectively, which showed significant difference in comparison with the controls. In addition, there were significant correlations between mean serum Hcy levels and duration of disease (r = 0.2, p = 0.05) and treatment with interferon (r = 0.21, p = 0.01). In cases, Hcy level was higher among those on  interferon (24.56 ± 11.87 vs. 19.71 ± 10.75, p = 0.01). Conclusions: We concluded that serum levels of vitamin B12 and folate decreased in RRMS patients, but Hcy levels increased significantly. It seems necessary to conduct prospective trials to determine whether the treatment with supplements and correct biomarker levels in the early stage of the disease can change the course of the disease. We recommend regular checking of the serum level of Hcy in patients who use disease-modifying drugs. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Multiple sclerosis (MS) is one of the most common neurological disorders, which is more prevalent in women and causes major morbidity in young adults [1]. It imposes an enormous economic burden on individual and society. Its prevalence differs based on geographic latitude of countries and increased in recent years [2]. The definite mechanism of MS is still unknown, but overt reaction of the immune system to environmental factors is a well-known causing factor [2–4]. Among these factors, recent studies are focused on the role of vitamin B12, folate, and homocysteine (Hcy). Folate and vitamin B12 have fundamental roles in CNS function especially methionine synthase-mediated conversion of homocysteine to methionine, which is essential for synthesis of DNA and RNA (Fig. 1) [5,6]. Therefore, B12 and folate deficiency can lead to an increased level of Hcy [6].
On the other hand, Hcy may have a neurotoxin effect that activates aspartate receptor, which leads to cell death [7,8]. Some studies showed the relation between high level of serum Hcy and low levels of serum B12 and folate in the MS groups [9–12]. By contrast, some reports found no significant difference in Hcy level between MS patients and controls [12,13]. Therefore, there are some controversies on the role of Hcy, folate, and vitamin B12 on MS pathogenesis. It seems necessary to elucidate the potential mechanisms of evolving MS and correct them for preventing more disability. It may lead to decrease the burden of disease on the society and young patients. We carried this study to determine the role of serum levels of Hcy, vitamin B12, and folate in Iranian patients with relapsing-remitting MS and compared them with healthy controls. 2. Material and methods 2.1. Study population
∗ Corresponding author at: Tehran University of Medical Sciences, Tehran, Iran. Tel.: +98 21 64352496; fax: +98 21 66517118. E-mail address: [email protected] (M. Mamarabadi). 0303-8467/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.clineuro.2013.05.007
This case control study was conducted at Rasool Akram Hospital affiliated to Tehran University of Medical Sciences, Tehran,
M. Moghaddasi et al. / Clinical Neurology and Neurosurgery 115 (2013) 1802–1805
1803
Fig. 1. Role of folate, B12 and B6 in metabolism of homocysteine [6]. THF: tetrahydrofolate.
Iran. The institutional medical ethics committee approved the study, and all patients and controls enrolled after signing their written informed consent. Seventy-five patients with diagnosis of relapsing-remitting MS (RRMS) were enrolled as cases (n = 75). All of them were in remission. The diagnosis of RRMS was based on revised McDonald Criteria diagnostic scheme [14]. Age- and sex-matched controls were selected sequentially from healthy volunteers. All patients and controls had normal blood pressure, lipid profile, and liver and renal function tests and had no risk factor for atherosclerosis. None of the cases and controls had received vitamin B12 and folate supplementary products, drugs that increase serum homocysteine level, and corticosteroids in the past 2 months. All participants were interviewed by one neurologist. The demographic, past medical history, family history, smoking, and medication history were questioned. Patients’ disability score was estimated according to extended disability status scale (EDSS).
2.2. Blood collection and laboratory analysis After overnight fasting, a total of 10 ml of venous blood was collected and centrifuged immediately. All samples were transferred to the laboratory in the cold box. Homocysteine was measured using flourimetric high-performance liquid chromatography (M/L). Serum B12 and folate levels were measured through the ion-captured method (pg/ml and ng/ml). All samples were assayed with the same kit (Bosch, Stuttgart, Germany). 2.3. Statistic The data were expressed as mean ± standard deviation (SD). Chi-square test and t-test were used for qualitative and quantitative data, respectively. We used Pearson correlation analysis for assessing correlation between age and folate, B12, and Hcy levels. We consider p < 0.05 as statistically significant. All statistical
Fig. 2. Box and Whisker plot of homocysteine (Hcy), vitamin B12, and folate in MS patients and controls (all were significantly different between two groups).
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M. Moghaddasi et al. / Clinical Neurology and Neurosurgery 115 (2013) 1802–1805
Table 1 Baseline characteristics of the study population. Characteristics
Patients (RRMS)
Control
Number Age (years), mean (SD) Sex (F/M) Duration of disease (min–max) in years EDSS, mean (SD) Biomarkers, mean (SD) Plasma vitamin B12 (pg/ml) Plasma folate (ng/ml) Plasma homocysteine(M/L)
75 31.97 (9.05) 57/18 4.3 (0.25–17) 2.54 (1.90)
75 32.33 (8.86) 46/29 N/A N/A
342.64 ± 210.66 9.74 ± 4.77 22.73 ± 11.63
426.48 ± 284.65 11.64 ± 5.76 11.04 ± 4.71
analyses were performed with SPSS version 16 for Windows (SPSS Inc., Chicago, IL, USA). 3. Results A total of 75 RRMS patients with a mean age of 31.97 ± 9.05 years entered the study; 57 of whom were female subjects (76%). We included 75 healthy controls with the same age and sex (p = NS). Baseline characteristics of the subjects are listed in Table 1. The mean EDSS in the MS group was 2.54 (range, 2.00–8.00). There was a significant association between the type of first symptom of disease and EDSS (p < 0.001). EDSS was lower in patients with optic neuritis and pyramidal symptoms than in patients with brainstem lesions and myelitis. Forty-four patients were taking -interferon. In the present study, mean plasma levels of vitamin B12, folate, and Hcy in patients were 342.64 ± 210.66 pg/ml, 9.74 ± 4.77 ng/ml, and 22.73 ± 11.63 M/L, respectively (Fig. 2), which showed significant difference in comparison with controls. The mean level of Hcy was higher in male subjects, and the mean level of vitamin B12 was lower in female subjects; however, these differences were not significant. There were no significant associations between level of serum markers (Hcy, vitamin B12, and folate) and age at disease onset in RRMS, but patients with higher level of Hcy and lower levels of vitamin B12 and folate were younger at first attack of demyelination. Patients were divided into three groups based on EDSS scores (Table 2). EDSS scores were higher in cases with low levels of folate and vitamin B12 and high level of Hcy, but these associations were not significant. It was also shown that there were significant correlations between mean serum Hcy level and duration of disease (r = 0.2, p = 0.05) and treatment with interferon (r = 0.21, p = 0.01), although this association was not seen for folate and vitamin B12 (r = 0.03, p = 0.7, and r = 0.2, p = 0.06, respectively). 4. Discussion The purpose of this case–control study was to elucidate the role of serum Hcy, folate, and vitamin B12 levels in RRMS. We concluded
p-value 0.8 0.07
0.01 0.04 0.00
that serum levels of vitamin B12 and folate decreased in RRMS patients; however, Hcy level increased. Our findings are consistent with the most previous studies, in which low folate and vitamin B12 levels were seen in MS patients. On the other hand, there are some controversies about the role of Hcy metabolism in MS [11,15–17]. This study is in line with some [9,10,18] but not all [2,11,19] previous studies regarding Hcy levels in MS. Ramsaransing et al. [9] showed that mean plasma levels of Hcy was higher in MS patients than in controls. Baig and Ali Qureishi [17] found that elevated levels of Hcy were associated with significantly decreased levels of vitamin B12 in serum and CSF. Besler et al. [11] showed that low levels of serum folate and B12 coincide with high levels of serum Hcy, but it was not significant in comparison with normal cases. Hyperhomocysteinemia may have different effects in MS. For example, high levels of Hcy can reduce the S-adenosylmethionine, which is important in methylation reactions and neuronal homeostasis [20]. Hypomethylation of myelin basic protein may lead to destabilize myelin structures and interrupt remyelination. Moreover, Hcy can induce neuronal toxicity because of reactive oxidative species [6]; thus, mild increase in Hcy levels can have important consequences for central nervous system myelination. We found that in patients who were receiving  interferon, serum levels of Hcy were higher. It can be explained in different ways. First, these patients might have a higher baseline Hcy level. Second, interferon could increase the serum level of Hcy without any effect on B12 and folate. This could happen with probable effect of interferon on downstream metabolic pathway of homocysteine (Fig. 1), such as its possible interaction with vitamin B6. Vitamin B6 is a cofactor of cystathione synthase, a necessary enzyme for downstream metabolism of homocysteine. Third, these patients might have low vitamin B6 levels at baseline, which could affect the consequent interactions beyond the homocysteine leading to hyperhomocysteinemia. We need a welldesigned prospective study, such as a before and after treatment study, with a larger number of population along with measuring vitamin B6 level to scrutinize the association between Hcy and  interferon.
Table 2 Interpretation of plasma biomarkers levels in different contexts of multiple sclerosis patients. Vitamin B12 (pg/ml), p-value
Folate (ng/ml), p-value
Homocysteine (M/L), p-value
Sex Female Male
321.09 ± 147 414.88 ± 345.90
0.7
10.08 ± 4.89 8.59 ± 4.30
0.21
23.26 ± 11.57 21.06 ± 11.98
0.29
EDSS EDSS ≤ 3 3 < EDSS < 6 EDSS ≥ 6
316.77 ± 150.07 425.36 ± 318.67 309.5 ± 182.8
0.1
10.10 ± 5.07 9.12 ± 3.91 9.56 ± 5.41
0.7
22.27 ± 11.87 23.35 ± 10.72 20.85 ± 11.27
0.6
0.66
10.07 ± 4.86 9.25 ± 4.68
0.34
24.56 ± 11.87 19.71 ± 10.75
0.01
Receiving  interferon 349.93 ± 227.25 Yes No 342.20 ± 187.46
M. Moghaddasi et al. / Clinical Neurology and Neurosurgery 115 (2013) 1802–1805
Our results indicate that there were association between high levels of Hcy and low levels of folate/vitamin B12 and higher EDSS scores. It seems that vitamin B12 requirements increase in patients with chronic immune disease and myelin repair. Therefore, we could expect changes in Hcy levels because of decrease in vitamin B12 and folate that influence myelin stability. There are some limitations in our study. First, it would be better to recruit patients that were not under treatment with immunomodulators to investigate Hcy role on MS pathogenesis without any confounders. Second, we have a relatively small population in our study. In conclusion, our results suggest the possible role of Hcy, folate, and vitamin B12 on MS pathogenesis and disability scores of patients. Considering the burden of the disease, it seems necessary to conduct prospective trials to determine whether supplement therapy and correcting biomarker levels in early stage of disease can change the disease course. We recommend regular checking of the Hcy serum level in patients who use  interferon as diseasemodifying drugs. References [1] Mamarabadi M, Razjouyan H, Mohammadi F, Moghaddasi M. Assessment of outcomes predictors after first attack of optic neuritis. The Canadian Journal of Neurological Sciences 2011;38(6):887–95. [2] Ashtari F, Salehi Abari SH, ShayganNejad V. Serum Homocysteine level in patients with Multiple Sclerosis. Journal of Research in Medical Sciences 2005;10(5):302–4. [3] Lublin FD, Miller AE. Demyelinating diseases of the central nervous system. In: Bradley WG, Daroff RB, Fenichel GM, Jankovic J, editors. Neurology in clinical practice. Philadelphia: Elsevier; 2008. p. 1583–4. [4] Riley CS, Tullman MJ. Multiple sclerosis. In: Rowland LP, Pedley TA, editors. Merritt’s neurology. Philadelphia: Lippincott Williams and Wilkins; 2010. p. 903. [5] Reynolds E. Vitamin B12, folic acid and the nervous system. Lancet Neurology 2006;5(11):949–60.
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[6] Moghaddasi M, Mamarabadi M, Mirzadeh S, Freydoonnejad AA, Razjouyan H. Homocysteine, vitamin B12 and folate levels in Iranian patients with ischemic stroke. Neurological Research 2010;32(9):953–6. [7] Diaz-Arrastia R. Homocysteine and neurologic disease. Archives of Neurology 2000;57:1422–8. [8] Lipton SA, Kim WK, Choi YB. Neurotoxicity associated with dual action of homocysteine at the N-methyle–Daspartat receptor. Proceedings of the National Academy of Sciences of the United States of America 1994;11:5923–8. [9] Ramsaransing GSM, fokkema Teelken A, Arutjunyan AV, Koch M, Dekeyser J. Plasma homocysteine level in multiple sclerosis. Journal of Neurology, Neurosurgery, and Psychiatry 2006;77:189–92. [10] Tajouri L, Martin V, Gasparini C, Ovcaric M, Curtain R, Lea RA, et al. Genetic investigation of methylenetetrahydrofolate reductase (MTHFR) and catecholO-methyl transferase (COMT) in multiple sclerosis. Brain Research Bulletin 2006;69(3):327–31. [11] Besler HT, Comoglu S. Lipoprotein oxidation, plasma total antioxidant capacity and homocystein level in patients with multiple sclerosis. Nutritional Neuroscience 2003;6(3):189–96. [12] Masoud SA, Fakharian E. Relationship between homocystein blood level and multiple sclerosis. International Journal of Medicine and Medical Sciences 2009;1(5):230–2. [13] Triantafyllou N, Evangelopoulos ME, Kimiskidis VK, Kararizou E, Boufidou F, Fountoulakis KN, et al. Increased plasma homocysteine levels in patients with multiple sclerosis and depression. Annals of General Psychiatry 2008;7:17. [14] Polman CH, Reingold SC, Edan G, Filippi M, Hartung HP, Kappos L, et al. Diagnostic criteria for multiple sclerosis: revisions to the “McDonald Criteria”. Annals of Neurology 2005;58(6):840–6. [15] Reyold EH. Multiple sclerosis and vitamin b12 metabolism. Journal of Neurology, Neurosurgery, and Psychiatry 1992;55:339–40. [16] Russo C, Morabito F, Luise F, Piromalli A, Battaglia L, Vinci A, et al. Hyperhomocysteinemia is associated with cognitive impairment in multiple sclerosis. Journal of Neurology 2008;255:64–9. [17] Baig SM, Ali Qureishi G. Homocystein and vitamin B12 in multiple sclerosis. Biogenic Amines 1995;11(6):479–85. [18] Vrethem M, Mattsson E, Hebelka H, Leerbeck K, Osterberg A, Landtblom AM, et al. Increased plasma homocysteine levels without signs of vitamin B12 deficiency in patients with multiple sclerosis assessed by blood and cerebrospinal fluid homocysteine and methylmalonic acid. Multiple Sclerosis 2003;9:239–45. [19] Rio J, Montalban J, Tintore M, Codina A, Malinow MR. Serum homocysteine levels in multiple sclerosis. Archives of Neurology 1994;51:1181. [20] Kim S, Lim IK, Park GH, Paik WK. Biological methylation of myelin basic protein: enzymology and biological significance. The International Journal of Biochemistry & Cell Biology 1997;29:743–51.
Contents lists available at SciVerse ScienceDirect
Clinical Neurology and Neurosurgery journal homepage: www.elsevier.com/locate/clineuro
Homocysteine, vitamin B12 and folate levels in Iranian patients with Multiple Sclerosis: A case control study Mehdi Moghaddasi a,b , Mansoureh Mamarabadi a,b,∗ , Nafiseh Mohebi a,b , Hadie Razjouyan c , Mahbubeh Aghaei a,b a
Department of Neurology, Rasool Akram Hospital, Tehran University of Medical Sciences, Tehran, Iran Iranian Center of Neurological Research (ICNR), Tehran, Iran c Department of Medicine, Drexel University, Monmouth Medical Center, Long Branch, 07744, USA b
a r t i c l e
i n f o
Article history: Received 30 March 2012 Received in revised form 19 February 2013 Accepted 2 May 2013 Available online 10 June 2013 Keywords: Homocysteine Vitamin B12 Folate Multiple Sclerosis
a b s t r a c t Background: Recently, homocysteine (Hcy), folate, and vitamin B12 have been proposed to have several roles on MS pathogenesis. Objective: We performed this study to determine the role of serum levels of Hcy, vitamin B12, and folate in patients with relapsing remitting MS (RRMS) and compared them with healthy controls. Methods: We recruited 75 RRMS patients and 75 subjects as controls with the same age and sex. Homocysteine was measured using fluorimetric high-performance liquid chromatography. Plasma folate and vitamin B12 levels were measured through ion-capture method. Results: Mean plasma levels of vitamin B12, folate, and Hcy in cases were 342.64 ± 210.66 pg/ml, 9.74 ± 4.77 ng/ml, and 22.73 ± 11.63 M/L, respectively, which showed significant difference in comparison with the controls. In addition, there were significant correlations between mean serum Hcy levels and duration of disease (r = 0.2, p = 0.05) and treatment with interferon (r = 0.21, p = 0.01). In cases, Hcy level was higher among those on  interferon (24.56 ± 11.87 vs. 19.71 ± 10.75, p = 0.01). Conclusions: We concluded that serum levels of vitamin B12 and folate decreased in RRMS patients, but Hcy levels increased significantly. It seems necessary to conduct prospective trials to determine whether the treatment with supplements and correct biomarker levels in the early stage of the disease can change the course of the disease. We recommend regular checking of the serum level of Hcy in patients who use disease-modifying drugs. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Multiple sclerosis (MS) is one of the most common neurological disorders, which is more prevalent in women and causes major morbidity in young adults [1]. It imposes an enormous economic burden on individual and society. Its prevalence differs based on geographic latitude of countries and increased in recent years [2]. The definite mechanism of MS is still unknown, but overt reaction of the immune system to environmental factors is a well-known causing factor [2–4]. Among these factors, recent studies are focused on the role of vitamin B12, folate, and homocysteine (Hcy). Folate and vitamin B12 have fundamental roles in CNS function especially methionine synthase-mediated conversion of homocysteine to methionine, which is essential for synthesis of DNA and RNA (Fig. 1) [5,6]. Therefore, B12 and folate deficiency can lead to an increased level of Hcy [6].
On the other hand, Hcy may have a neurotoxin effect that activates aspartate receptor, which leads to cell death [7,8]. Some studies showed the relation between high level of serum Hcy and low levels of serum B12 and folate in the MS groups [9–12]. By contrast, some reports found no significant difference in Hcy level between MS patients and controls [12,13]. Therefore, there are some controversies on the role of Hcy, folate, and vitamin B12 on MS pathogenesis. It seems necessary to elucidate the potential mechanisms of evolving MS and correct them for preventing more disability. It may lead to decrease the burden of disease on the society and young patients. We carried this study to determine the role of serum levels of Hcy, vitamin B12, and folate in Iranian patients with relapsing-remitting MS and compared them with healthy controls. 2. Material and methods 2.1. Study population
∗ Corresponding author at: Tehran University of Medical Sciences, Tehran, Iran. Tel.: +98 21 64352496; fax: +98 21 66517118. E-mail address: [email protected] (M. Mamarabadi). 0303-8467/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.clineuro.2013.05.007
This case control study was conducted at Rasool Akram Hospital affiliated to Tehran University of Medical Sciences, Tehran,
M. Moghaddasi et al. / Clinical Neurology and Neurosurgery 115 (2013) 1802–1805
1803
Fig. 1. Role of folate, B12 and B6 in metabolism of homocysteine [6]. THF: tetrahydrofolate.
Iran. The institutional medical ethics committee approved the study, and all patients and controls enrolled after signing their written informed consent. Seventy-five patients with diagnosis of relapsing-remitting MS (RRMS) were enrolled as cases (n = 75). All of them were in remission. The diagnosis of RRMS was based on revised McDonald Criteria diagnostic scheme [14]. Age- and sex-matched controls were selected sequentially from healthy volunteers. All patients and controls had normal blood pressure, lipid profile, and liver and renal function tests and had no risk factor for atherosclerosis. None of the cases and controls had received vitamin B12 and folate supplementary products, drugs that increase serum homocysteine level, and corticosteroids in the past 2 months. All participants were interviewed by one neurologist. The demographic, past medical history, family history, smoking, and medication history were questioned. Patients’ disability score was estimated according to extended disability status scale (EDSS).
2.2. Blood collection and laboratory analysis After overnight fasting, a total of 10 ml of venous blood was collected and centrifuged immediately. All samples were transferred to the laboratory in the cold box. Homocysteine was measured using flourimetric high-performance liquid chromatography (M/L). Serum B12 and folate levels were measured through the ion-captured method (pg/ml and ng/ml). All samples were assayed with the same kit (Bosch, Stuttgart, Germany). 2.3. Statistic The data were expressed as mean ± standard deviation (SD). Chi-square test and t-test were used for qualitative and quantitative data, respectively. We used Pearson correlation analysis for assessing correlation between age and folate, B12, and Hcy levels. We consider p < 0.05 as statistically significant. All statistical
Fig. 2. Box and Whisker plot of homocysteine (Hcy), vitamin B12, and folate in MS patients and controls (all were significantly different between two groups).
1804
M. Moghaddasi et al. / Clinical Neurology and Neurosurgery 115 (2013) 1802–1805
Table 1 Baseline characteristics of the study population. Characteristics
Patients (RRMS)
Control
Number Age (years), mean (SD) Sex (F/M) Duration of disease (min–max) in years EDSS, mean (SD) Biomarkers, mean (SD) Plasma vitamin B12 (pg/ml) Plasma folate (ng/ml) Plasma homocysteine(M/L)
75 31.97 (9.05) 57/18 4.3 (0.25–17) 2.54 (1.90)
75 32.33 (8.86) 46/29 N/A N/A
342.64 ± 210.66 9.74 ± 4.77 22.73 ± 11.63
426.48 ± 284.65 11.64 ± 5.76 11.04 ± 4.71
analyses were performed with SPSS version 16 for Windows (SPSS Inc., Chicago, IL, USA). 3. Results A total of 75 RRMS patients with a mean age of 31.97 ± 9.05 years entered the study; 57 of whom were female subjects (76%). We included 75 healthy controls with the same age and sex (p = NS). Baseline characteristics of the subjects are listed in Table 1. The mean EDSS in the MS group was 2.54 (range, 2.00–8.00). There was a significant association between the type of first symptom of disease and EDSS (p < 0.001). EDSS was lower in patients with optic neuritis and pyramidal symptoms than in patients with brainstem lesions and myelitis. Forty-four patients were taking -interferon. In the present study, mean plasma levels of vitamin B12, folate, and Hcy in patients were 342.64 ± 210.66 pg/ml, 9.74 ± 4.77 ng/ml, and 22.73 ± 11.63 M/L, respectively (Fig. 2), which showed significant difference in comparison with controls. The mean level of Hcy was higher in male subjects, and the mean level of vitamin B12 was lower in female subjects; however, these differences were not significant. There were no significant associations between level of serum markers (Hcy, vitamin B12, and folate) and age at disease onset in RRMS, but patients with higher level of Hcy and lower levels of vitamin B12 and folate were younger at first attack of demyelination. Patients were divided into three groups based on EDSS scores (Table 2). EDSS scores were higher in cases with low levels of folate and vitamin B12 and high level of Hcy, but these associations were not significant. It was also shown that there were significant correlations between mean serum Hcy level and duration of disease (r = 0.2, p = 0.05) and treatment with interferon (r = 0.21, p = 0.01), although this association was not seen for folate and vitamin B12 (r = 0.03, p = 0.7, and r = 0.2, p = 0.06, respectively). 4. Discussion The purpose of this case–control study was to elucidate the role of serum Hcy, folate, and vitamin B12 levels in RRMS. We concluded
p-value 0.8 0.07
0.01 0.04 0.00
that serum levels of vitamin B12 and folate decreased in RRMS patients; however, Hcy level increased. Our findings are consistent with the most previous studies, in which low folate and vitamin B12 levels were seen in MS patients. On the other hand, there are some controversies about the role of Hcy metabolism in MS [11,15–17]. This study is in line with some [9,10,18] but not all [2,11,19] previous studies regarding Hcy levels in MS. Ramsaransing et al. [9] showed that mean plasma levels of Hcy was higher in MS patients than in controls. Baig and Ali Qureishi [17] found that elevated levels of Hcy were associated with significantly decreased levels of vitamin B12 in serum and CSF. Besler et al. [11] showed that low levels of serum folate and B12 coincide with high levels of serum Hcy, but it was not significant in comparison with normal cases. Hyperhomocysteinemia may have different effects in MS. For example, high levels of Hcy can reduce the S-adenosylmethionine, which is important in methylation reactions and neuronal homeostasis [20]. Hypomethylation of myelin basic protein may lead to destabilize myelin structures and interrupt remyelination. Moreover, Hcy can induce neuronal toxicity because of reactive oxidative species [6]; thus, mild increase in Hcy levels can have important consequences for central nervous system myelination. We found that in patients who were receiving  interferon, serum levels of Hcy were higher. It can be explained in different ways. First, these patients might have a higher baseline Hcy level. Second, interferon could increase the serum level of Hcy without any effect on B12 and folate. This could happen with probable effect of interferon on downstream metabolic pathway of homocysteine (Fig. 1), such as its possible interaction with vitamin B6. Vitamin B6 is a cofactor of cystathione synthase, a necessary enzyme for downstream metabolism of homocysteine. Third, these patients might have low vitamin B6 levels at baseline, which could affect the consequent interactions beyond the homocysteine leading to hyperhomocysteinemia. We need a welldesigned prospective study, such as a before and after treatment study, with a larger number of population along with measuring vitamin B6 level to scrutinize the association between Hcy and  interferon.
Table 2 Interpretation of plasma biomarkers levels in different contexts of multiple sclerosis patients. Vitamin B12 (pg/ml), p-value
Folate (ng/ml), p-value
Homocysteine (M/L), p-value
Sex Female Male
321.09 ± 147 414.88 ± 345.90
0.7
10.08 ± 4.89 8.59 ± 4.30
0.21
23.26 ± 11.57 21.06 ± 11.98
0.29
EDSS EDSS ≤ 3 3 < EDSS < 6 EDSS ≥ 6
316.77 ± 150.07 425.36 ± 318.67 309.5 ± 182.8
0.1
10.10 ± 5.07 9.12 ± 3.91 9.56 ± 5.41
0.7
22.27 ± 11.87 23.35 ± 10.72 20.85 ± 11.27
0.6
0.66
10.07 ± 4.86 9.25 ± 4.68
0.34
24.56 ± 11.87 19.71 ± 10.75
0.01
Receiving  interferon 349.93 ± 227.25 Yes No 342.20 ± 187.46
M. Moghaddasi et al. / Clinical Neurology and Neurosurgery 115 (2013) 1802–1805
Our results indicate that there were association between high levels of Hcy and low levels of folate/vitamin B12 and higher EDSS scores. It seems that vitamin B12 requirements increase in patients with chronic immune disease and myelin repair. Therefore, we could expect changes in Hcy levels because of decrease in vitamin B12 and folate that influence myelin stability. There are some limitations in our study. First, it would be better to recruit patients that were not under treatment with immunomodulators to investigate Hcy role on MS pathogenesis without any confounders. Second, we have a relatively small population in our study. In conclusion, our results suggest the possible role of Hcy, folate, and vitamin B12 on MS pathogenesis and disability scores of patients. Considering the burden of the disease, it seems necessary to conduct prospective trials to determine whether supplement therapy and correcting biomarker levels in early stage of disease can change the disease course. We recommend regular checking of the Hcy serum level in patients who use  interferon as diseasemodifying drugs. References [1] Mamarabadi M, Razjouyan H, Mohammadi F, Moghaddasi M. Assessment of outcomes predictors after first attack of optic neuritis. The Canadian Journal of Neurological Sciences 2011;38(6):887–95. [2] Ashtari F, Salehi Abari SH, ShayganNejad V. Serum Homocysteine level in patients with Multiple Sclerosis. Journal of Research in Medical Sciences 2005;10(5):302–4. [3] Lublin FD, Miller AE. Demyelinating diseases of the central nervous system. In: Bradley WG, Daroff RB, Fenichel GM, Jankovic J, editors. Neurology in clinical practice. Philadelphia: Elsevier; 2008. p. 1583–4. [4] Riley CS, Tullman MJ. Multiple sclerosis. In: Rowland LP, Pedley TA, editors. Merritt’s neurology. Philadelphia: Lippincott Williams and Wilkins; 2010. p. 903. [5] Reynolds E. Vitamin B12, folic acid and the nervous system. Lancet Neurology 2006;5(11):949–60.
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[6] Moghaddasi M, Mamarabadi M, Mirzadeh S, Freydoonnejad AA, Razjouyan H. Homocysteine, vitamin B12 and folate levels in Iranian patients with ischemic stroke. Neurological Research 2010;32(9):953–6. [7] Diaz-Arrastia R. Homocysteine and neurologic disease. Archives of Neurology 2000;57:1422–8. [8] Lipton SA, Kim WK, Choi YB. Neurotoxicity associated with dual action of homocysteine at the N-methyle–Daspartat receptor. Proceedings of the National Academy of Sciences of the United States of America 1994;11:5923–8. [9] Ramsaransing GSM, fokkema Teelken A, Arutjunyan AV, Koch M, Dekeyser J. Plasma homocysteine level in multiple sclerosis. Journal of Neurology, Neurosurgery, and Psychiatry 2006;77:189–92. [10] Tajouri L, Martin V, Gasparini C, Ovcaric M, Curtain R, Lea RA, et al. Genetic investigation of methylenetetrahydrofolate reductase (MTHFR) and catecholO-methyl transferase (COMT) in multiple sclerosis. Brain Research Bulletin 2006;69(3):327–31. [11] Besler HT, Comoglu S. Lipoprotein oxidation, plasma total antioxidant capacity and homocystein level in patients with multiple sclerosis. Nutritional Neuroscience 2003;6(3):189–96. [12] Masoud SA, Fakharian E. Relationship between homocystein blood level and multiple sclerosis. International Journal of Medicine and Medical Sciences 2009;1(5):230–2. [13] Triantafyllou N, Evangelopoulos ME, Kimiskidis VK, Kararizou E, Boufidou F, Fountoulakis KN, et al. Increased plasma homocysteine levels in patients with multiple sclerosis and depression. Annals of General Psychiatry 2008;7:17. [14] Polman CH, Reingold SC, Edan G, Filippi M, Hartung HP, Kappos L, et al. Diagnostic criteria for multiple sclerosis: revisions to the “McDonald Criteria”. Annals of Neurology 2005;58(6):840–6. [15] Reyold EH. Multiple sclerosis and vitamin b12 metabolism. Journal of Neurology, Neurosurgery, and Psychiatry 1992;55:339–40. [16] Russo C, Morabito F, Luise F, Piromalli A, Battaglia L, Vinci A, et al. Hyperhomocysteinemia is associated with cognitive impairment in multiple sclerosis. Journal of Neurology 2008;255:64–9. [17] Baig SM, Ali Qureishi G. Homocystein and vitamin B12 in multiple sclerosis. Biogenic Amines 1995;11(6):479–85. [18] Vrethem M, Mattsson E, Hebelka H, Leerbeck K, Osterberg A, Landtblom AM, et al. Increased plasma homocysteine levels without signs of vitamin B12 deficiency in patients with multiple sclerosis assessed by blood and cerebrospinal fluid homocysteine and methylmalonic acid. Multiple Sclerosis 2003;9:239–45. [19] Rio J, Montalban J, Tintore M, Codina A, Malinow MR. Serum homocysteine levels in multiple sclerosis. Archives of Neurology 1994;51:1181. [20] Kim S, Lim IK, Park GH, Paik WK. Biological methylation of myelin basic protein: enzymology and biological significance. The International Journal of Biochemistry & Cell Biology 1997;29:743–51.