Plasma homocysteine levels in patients with multiple sclerosis in the Greek population

Available online at www.sciencedirect.com ScienceDirect Journal of the Chinese Medical Association 76 (2013) 611e614 www.jcma-online.com Original Article Plasma homocysteine levels in patients with multiple sclerosis in the Greek population Evangelia Kararizou*, George Paraskevas, Nikolaos Triantafyllou, George Koutsis, Maria E. Evangelopoulos, Dimitrios Mandellos, Constantinos Sfagos, Elisabeth Kapaki Department of Neurology, Athens National University, Aiginition Hospital, Athens, Greece Received September 19, 2012; accepted January 14, 2013 Abstract Background: In recent years, there has been increasing interest in the role of plasma homocysteine (Hcy) as a possible risk factor for several diseases of the central nervous system. The aim of this study was to determine the plasma levels of Hcy in a group of multiple sclerosis (MS) patients from a Greek population and the possible correlation with age, disability status, activity or duration of disease, sex, and treatment. Methods: The MS group that was studied consisted of 46 patients and a total of 42 healthy individuals served as a control group. Plasma Hcy levels were determined by means of high-performance liquid chromatography coupled with fluorescence detection, after precolumn derivatization with 4-Fluoro-7-aminosulfonylbenzofurazan (ABD-F). Results: Statistical analysis revealed that, in the MS patients, Hcy levels were not significantly different as compared to those in the controls. Men presented with higher Hcy levels than women in the MS group; however, age, disease subtype, disease duration, relapse rate, and Expanded Disability Status Scale score/Multiple Sclerosis Severity Score did not significantly affect Hcy levels in MS patients. Conclusion: The preliminary data suggest that Hcy levels were not elevated in our sample of Greek MS patients, which does not support previous findings of a significant correlation between elevated serum Hcy levels and MS. Further studies to establish a possible association between MS and Hcy levels in the context of different ethnic groups with different habits are needed. Copyright Ó 2013 Elsevier Taiwan LLC and the Chinese Medical Association. All rights reserved. Keywords: homocysteine; multiple sclerosis; vitamin B12 1. Introduction Homocysteine (Hcy) is a sulfhydryl-containing amino acid. Methionine in food is demethylated to Hcy, which is further processed to cysteine, or remethylated to methionine. However abnormalities in this pathway may cause increased Hcy levels. It has been suggested that increased plasma Hcy levels may be an important yet potentially treatable risk factor, or a contributor to the mechanisms of several neurological diseases including multiple sclerosis (MS).1,2 Several recent studies found elevated plasma Hcy levels in patients with MS, but another found no significant difference in plasma Hcy concentrations between patients with MS and controls.3e5 According to the 2003 study of Vrethem et al, the increased plasma Hcy levels in MS patients are not generally associated with vitamin B12 deficiency.6 In the present study we determined the plasma levels of Hcy in a group of MS patients from the Greek population and investigated a possible correlation with age, disability status, disease activity, disease duration, sex, and treatment. 2. Methods 2.1. Patients * Corresponding author. Dr. Evangelia Kararizou, Neurologic Clinic, Aiginition Hospital, 72e74, Vassilisis Sofias Avenue, 11528 Athens, Greece. E-mail address: ekarariz@med.uoa.gr (E. Kararizou). This study was approved by the local ethics committee of our hospital and performed according to the ethical standards 1726-4901/$ - see front matter Copyright Ó 2013 Elsevier Taiwan LLC and the Chinese Medical Association. All rights reserved. http://dx.doi.org/10.1016/j.jcma.2013.07.002 612 E. Kararizou et al. / Journal of the Chinese Medical Association 76 (2013) 611e614 of the Helsinki Declaration. The MS group comprised 46 patients. Patients with depression or other psychiatric or pathological conditions were excluded. All patients had MS according to the revised McDonald criteria.7 Twenty-one (45.7%) patients had the relapsingeremitting form of MS, five (11%) the secondary progressive form, five (11%) the primary progressive form, and the remaining 15 (32.6%) had a clinically isolated syndrome with multiple lesions on magnetic resonance imaging and abnormal cerebrospinal immunoglobulin G index and/or oligoclonal bands (all of them developed MS within the next 2 years). The mean (standard deviation) age onset was 30 ( 8) years and mean (standard deviation) disease duration was 5.5 ( 4.7) years (range 0.1e22 years). Median (25the75th percentile) Expanded Disability Status Scale (EDSS) and Multiple Sclerosis Severity Score (MSSS) were 2 (1.5e3.5) and 4.2 (2.6e7.3), respectively. In patients experiencing relapse, the median relapse rate was 0.8 (range: 0.2e2). Twenty (43.5%) patients were at relapse when blood samples were obtained. It was our purpose to study as many treatment-naı̈ve patients as possible. Thus, in 36 patients (78%), blood sampling was performed prior to treatment was initiated, and only nine patients were already on interferon or other immunomodulatory treatment. The control group consisted of 42 healthy individuals of comparable age from the Greek population. None of the patients and controls had abnormal B12 or folate levels, and none reported using vitamin supplements in the past 2 years. Plasma Hcy level were measured by high-performance liquid chromatography with precolumn derivatization and fluorescence detection (excitation 385 nm, emission 515 nm) by the use of a commercial kit (Homocysteine in serum/ plasma; Chromsystems GmbH, München, Germany). 2.2. Statistical analysis All variables were checked for normality and equality of variances by the ShapiroeWilk’s and Levene’s tests, respectively. Hcy levels did not follow the normal distribution and data are presented in terms of median values and quartiles. However, logarithmic transformation restored the violation and permitted the use of analysis of covariance (ANCOVA). Age and sex have been observed to affect Hcy levels in normal individuals, therefore, a general factorial two-way ANCOVA model with diagnostic group and sex as factors and age as a covariate was used.8 Effects of sex, age, disease subtype, relapse rate, EDSS/MSSS and the presence or absence of relapse or treatment were also tested in the MS group by ANCOVA models and with multiple regression separately in the control and MS groups. The t test, c2 test, and Spearman’s rank correlation coefficient were also used as appropriate. 3. Results The results are summarized in Tables 1 and 2 and Figs. 1 and 2. For comparison between the MS and control groups, ANCOVA revealed no significant effect of the diagnostic Table 1 Demographic and biochemical data of studied groups. n (M/F) Age (y)a Hcy (mM)b Control MS p 42 (21/21) 35.8  10.3 10.8 (8.1e13.7) 46 (14/32) 34.2  9.2 11 (9e13) NSc NSd NSe F ¼ female; Hcy ¼ homocysteine; M ¼ male; MS ¼ multiple sclerosis; NS ¼ not significant. a Mean  standard deviation. b Median (25the75th percentile). c Yates’ continuity corrected c2 test. d t test. e Analysis of covariance with diagnostic group and sex as factors and age as covariate. group (Fig. 1A) or age; however, sex did affect the model significantly with men showing significantly higher Hcy levels than women (F ¼ 31.78, df ¼ 1, p < 0.00001). Men presented with higher Hcy levels than women in both the MS (F ¼ 7.45, df ¼ 1, p ¼ 0.01 and control groups (F ¼ 13.75, df ¼ 1, p ¼ 0.00065; Fig. 1B and C). Age, disease subtype, presence or absence of relapse or treatment at the time of blood sampling, disease duration, age at disease onset, relapse rate, and EDSS/MSSS did not affect significantly Hcy levels in MS. In the control group, male age did not affect Hcy levels significantly (Table 2, Fig. 2). 4. Discussion In recent years, there has been an increase in interest in the role of plasma Hcy as a possible risk factor for several diseases of the central nervous system. Hyperhomocysteinemia is common in Alzheimer’s disease, vascular dementia, and Parkinson’s disease, particularly after L-dopa treatment.1,2,9,10 Increased Hcy results in increased levels of proinflammatory cytokines such as tumor necrosis factor-a, interleukin (IL)-1b and IL-6, prostaglandin E2 and chemokine CC ligand 2 (monocyte chemoattractant protein-1) in both the central nervous system and serum and nuclear factor-kB/p65 subunit in the central nervous system.11 Conversely, inflammation and activation of astrocytes by IL-1b and tumor necrosis factor-a resulted in increased levels of Hcy, whereas Hcy-induced endoplasmic reticulum protein may be involved in inflammatory mechanisms and autoimmunity.12,13 Several studies have found increased plasma Hcy levels in MS patients.4e6 Besler and Comoglou14 reported elevated Table 2 Relation between Hcy level and clinical parameters in MS patients. b Age (y) Disease duration (y) EDSS score Age of onset (y) Annual relapse rate p 0.113 0.017 0.003 0.084 0.119 0.267 0.489 0.986 0.556 0.516 EDSS ¼ Expanded Disability Status Scale; Hcy ¼ homocysteine; MS ¼ multiple sclerosis. 613 E. Kararizou et al. / Journal of the Chinese Medical Association 76 (2013) 611e614 A B C Ctrl MS 10 100 Hcy (µM) 100 Hcy (µM) Hcy (µM) 100 10 10 p = 0.01 p = 0.00065 1 CTRL 1 MS M 1 F M F Ctrl = control; F = female; Hcy = homocysteine; M = male; MS = multiple sclerosis. Fig. 1. Homocysteine levels in the multiple sclerosis and control groups (horizontal bars indicate medians). plasma Hcy levels in patients with secondary progressive MS compared to healthy controls. According to Ramsaransing et al,4 elevated plasma Hcy occurs in both the benign and progressive disease course of MS. They noticed that there were no differences in plasma Hcy concentrations between the three MS subgroups. The purpose of the recently published study by Triantafyllou et al15 was to investigate plasma Hcy levels in patients with MS and depression. They found significantly increased plasma Hcy levels in MS patients with depression compared to the controls. In conclusion, the authors suggested that the moderately disabled MS patients with increased Hcy are particularly prone to develop depressive symptoms. Contrary to the above findings, the results of the present study indicate that, in MS patients without depression or other psychiatric disorders, plasma Hcy levels are not elevated, leading to speculation that Hcy may not participate in the pathogenetic mechanisms of MS; at least not in the population studied. There is also another study by Rı́o et al3 reporting no significant difference in plasma Hcy concentrations between patients with MS and controls. Teunissen et al16 investigated serum Hcy levels in patients with MS and A compared the results between MS subtypes, but the levels were not elevated in patients with MS compared with controls. The authors discussed the question of whether Hcy has a direct impact on MS, or reflects a more general neurodegenerative process. Hcy has been considered as a marker for vitamin B deficiency,17 and vitamin B12 is important for myelination of the central nervous system. An association between vitamin B12 deficiency and MS has been reported.18 However, in their study, Vrethem et al6 concluded that B12 deficiency, in general, is not associated with MS. Plasma Hcy levels may show some variation according to the nutritional habits of the studied population, especially B12 and folate intake, therefore, it is possible that different results may be found in different geographic areas and different ethnic groups.19,20 The studies that found elevated Hcy levels appear to have originated from northern countries including Sweden, The Netherlands, and the United Kingdom, whereas our data are in accordance with the results from Spain. The report of Cappuccio et al21 indicates that South Asian Hindus have B 100 Hcy (µM) Hcy (µM) 100 10 10 1 1 No treatment Treatment RR PP/SP Hcy = homocysteine; PP = primary progressive; RR = relapsing-remitting; SP = secondary progressive. Fig. 2. (A) Homocysteine levels in the no treatment and treatment groups of patients. (B) Homocysteine levels in different disease subtypes (horizontal bars indicate medians). 614 E. Kararizou et al. / Journal of the Chinese Medical Association 76 (2013) 611e614 higher levels of Hcy than other ethnic groups, in part (though not exclusively) due to their vegetarianism. By contrast, people of African origin tend to have lower levels of tHcy than whites. Czajkowska et al, 22 correlating their results to the dietary habits in young healthy Polish men, concluded that habitual daily intake of protein and consequently methionine has a beneficial effect on plasma Hcy levels. In addition, it seems feasible that the protein effect on plasma Hcy is due to the action of methionine and protein-originating vitamins. All of the patients enrolled in the present study were of Greek origin, who generally followed the Mediterranean diet, thus, it is possible that a well-preserved nutritional status prevented a rise in plasma Hcy.23,24 As mentioned earlier, men presented with higher Hcy levels than women in our study, which is contrary to the predominance of female sex in MS. However, the phenomenon of higher Hcy levels in men is well established and may play a role in the increased cardiovascular risk found in men.25 However, the increased levels of Hcy in men cannot be explained by differences in folate and B12 levels alone. Other factors may be involved, including increased demethylation of methionine associated with higher creatinine production in men, which may be influenced by sex hormones.26 In our study, clinical variables such as disease subtype, presence or absence of relapse or treatment at the time of blood sampling, disease duration, age at disease onset, relapse rate and EDSS/MSSS did not significantly affect Hcy levels in MS. This is in agreement with the findings of Ramsaransing et al,4 who reported no difference in Hcy levels between MS patients with a benign and those with a progressive disease course. In conclusion, the findings in our sample of the Greek population do not support previous studies that showed a significant correlation between elevated serum Hcy levels and MS. Further studies to establish the possible association between MS and Hcy levels in the context of different ethnic groups with different habits are needed. 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