Hyperhomocysteinemia and methylenetetrahydrofolate reductase polymorphism in patients with Parkinson's disease

Neuroscience Letters 404 (2006) 56–60

Hyperhomocysteinemia and methylenetetrahydrofolate reductase polymorphism in patients with Parkinson’s disease D. Religa a,b,∗ , K. Czyzewski c , M. Styczynska a , B. Peplonska a , J. Lokk b , M. Chodakowska-Zebrowska c , K. Stepien d , B. Winblad b , M. Barcikowska a a

Department of Neurodegenerative Disorders, Medical Research Center, Polish Academy of Sciences, Warsaw, Poland Neurotec, Section of Experimental Geriatrics, Karolinska Institutet, Novum, Level 5, SE-141 86 Stockholm, Sweden c Department of Neurology, MSWiA Hospital, Warsaw, Poland d Central Laboratory, MSWiA Hospital, Warsaw, Poland

b

Received 23 January 2006; received in revised form 9 May 2006; accepted 17 May 2006

Abstract Elevated levels of homocysteine have been observed in Parkinson’s disease (PD) patients treated with levodopa. However, it is not studied if duration of PD or PD per se is associated with hyperhomocysteinemia. In the present study, the levels of homocysteine in 99 levodopa-treated PD patients, 15 untreated PD patients and 100 controls were examined. We focused on the influence of levodopa dose, duration of therapy and disease as well as genetic (C677T methylenetetrahydrofolate reductase (MTHFR) polymorphism) and environmental factors. We found that levodopa-treated PD patients had elevated homocysteine plasma levels as compared to controls (p < 0.05), but the levels did not depend on levodopa doses. Another factor influencing homocysteine level was the duration of PD (p < 0.001). The frequency of allele C677T of MTHFR gene did not differ between PD and controls. In conclusion, hyperhomocysteinemia is associated with the duration of PD and levodopa treatment and possibly also with PD per se. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Homocysteine; Parkinson’s disease; Methylenetetrahydrofolate reductase polymorphism; Levodopa

Parkinson’s disease (PD) is one of the most studied and best understood neurological disorders. However, there is still a need to define modifiable risk factors to prevent the disease. Although the exact cause of PD is unknown, experimental data suggest that factors such as homocysteine may sensitize dopaminergic neurons to age-related dysfunction and death [8]. The discovery of levodopa opened a new era in PD treatment, but this therapy has troublesome side effects in the form of motor fluctuations and drug-related dyskinesias. According to the most recent studies it is proposed that levodopa treatment also may lead to hyperhomocysteinemia. Epidemiological studies have shown elevated levels of homocysteine in stroke, coronary heart disease [5], Alzheimer’s disease (AD) [35], cerebral white matter changes [13], brain atrophy [34], Huntington disease (HD) [3] and mild cognitive Abbreviations: PD, Parkinson’s disease; tHcy, total fasting plasma homocysteine; MTHFR, methylenetetrahydrofolate reductase; MMSE, Mini mental state examination ∗ Corresponding author. Tel.: +46 8 736141060; fax: +46 8 58583880. E-mail address: [email protected] (D. Religa). 0304-3940/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2006.05.040

impairment [4]. Increased plasma homocysteine levels were also observed in levodopa-treated PD subjects [19,23]. These findings were confirmed by other larger studies and it has been proposed that hyperhomocysteinemia is caused by levodopa treatment rather than by PD per se [24,33,37]. Additionally, it would be worth testing the hypothesis that the duration of disease could be connected with higher levels of homocysteine. The longer is duration of disease, the more progression and the more frequent use of levodopa. The aim of this study was to test the relationship between homocysteine levels and PD with regard to duration of disease and levodopa treatment. We focused on the influence of dose, the duration of levodopa therapy, as well as both genetic (C677T MTHFR polymorphism) and environmental factors (Vitamin B12 and folic acid) on homocysteine levels. We also studied the association between high homocysteine levels and cognitive impairment in PD patients, as the development of dementia is the principal risk factor for shortened life in PD [21]. PD patients were recruited from and diagnosed at a Day Clinic of the Department of Neurodegenerative Disorders of

D. Religa et al. / Neuroscience Letters 404 (2006) 56–60

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Table 1 Descriptive statistics for the study groups

N Age Years since PD diagnosis Levodopa treatment Duration of treatment Levodopa doses MMSE Folic acid Homocysteine Vitamin B12 MTHFR CC genotype MTHFR CT genotype MTHFR TT genotype

Levodopa-treated PD patients

Controls

Levodopa-non-treated PD patients

99 70.5 ± 7.57 6.06 ± 4.05*** – 4.8 ± 10.35 681.2 ± 328.7 27.1 ± 2.3 9.21 ± 4.23 17.25 ± 5.96* 326.7 ± 174.9* 41.5% 51% 7.5%

100 71.2 ± 6.0 – – – – 29.2 ± 0.5 7.56 ± 5.39 14.43 ± 4.48 413.5 ± 241.3 55% 38% 7%

15 66.0 ± 7.11 1.97 ± 1.02 – – – 26.2 ± 6.3 7.45 ± 2.82 16.37 ± 5.53 273.0 ± 86.5** 46.7% 40% 13.3%

The laboratory reference range for homocysteine is 4–12 ␮mol/l, for folate 5.3–14.4 ng/ml and for Vitamin B12 157–1059 pg/ml. Results are expressed in mean and S.D. MMSE, Mini mental state examination; MTHFR, methylenetetrahydrofolate reductase. * p < 0.05. ** p < 0.01. *** p < 0.001.

Medical Research Centre at the Polish Academy of Sciences in Warsaw. PD patients were at Hoehn and Yahr stage 3.0 or less [14]. The diagnosis of PD was made by a movement disorders specialist (K.C.) based on the diagnostic criteria for PD [11]. Those required presence of at least two of the three cardinal signs of PD (resting tremor, rigidity, or bradykinesia) without a known or suspected alternative cause of parkinsonism. We excluded the subjects with conditions that could affect homocysteine levels, such as cancer, renal insufficiency, vascular disease, diabetes mellitus, hypertension, malnutrition, use of anticonvulsants or antifolate drugs and neurological and psychiatric disorders other than PD. Blood samples were collected from newly diagnosed patients or patients coming for follow-up visits. The control group was recruited from cognitively intact elderly patients from surgery and internal medicine (coming for a control visit) as well as from University of Third Age and we used the same exclusion criteria as for the PD group. Global cognitive functions were assessed using mini mental state examination (MMSE) [9]. Mean value of MMSE was 29.2 ± 0.5 for the control group. All patients and control group subjects were Caucasians. Written informed consent to participate in the study was obtained from all participants or their relatives. The local Ethics Committee for Medical Research at MSWiA Hospital in Warsaw (Poland) and at Karolinska Institutet in Stockholm (Sweden) have approved the study. Blood samples were collected from each participant after overnight fasting (12–14 h). DNA was isolated from blood leukocytes. All plasma specimens were stored at −80 ◦ C before homocysteine level determination. Total fasting homocysteine (tHcy) concentrations in blood plasma were measured with a fluorescence polarization assay (Abbott IMx Homocysteine Assay). Serum folate levels were determined using Abbott Laboratories AxSYM Folate Reagent assay, and plasma Vitamin B12 by immunoassay. Normal ranges are from 4 to 12 ␮mol/l for homocysteine, 5.3–14.4 ng/ml for folate, and 157–1059 pg/ml for Vitamin B12 . The MTHFR genotype was determined using

polymerase chain reaction performed in Biometra UNO II Thermocycler using the protocol described previously [10,31]. Analysis of regression and ANOVA followed by post-hoc Newman–Keuls tests were used to analyze the results of these studies. Statistical analyses were performed with Statistica (release 6.0, Statsoft Inc., Tulsa OK, USA). The demographic characteristics of the analyzed groups are shown in Table 1. Numbers of patients in the groups were 99 PD patients treated with levodopa, 15 PD patients not treated with levodopa and 100 controls. The patients in the groups had similar age: 70.5, 66 and 71.2 years, respectively. The following factors were considered in the study: duration of the disease (expressed in years since PD diagnosis), fact of levodopa treatment, duration of treatment, levodopa doses, creatinine, MMSE, levels of folic acid, homocysteine and Vitamin B12 . Multivariate analysis of regression was performed. The final model included age, duration of the disease, MMSE, doses of levodopa, levels of B12 and folic acid (R = 0.54; adjusted R2 = 0.25; F(6,99) = 6.8; standard error of estimates 5.2). Results of the analyses are presented in Table 1. The analyses were followed by ANOVA for homocysteine, duration of disease, MMSE, B12 , since these factors indicated statistical significance, followed by post-hoc Newman–Keuls tests. The mean plasma tHcy levels were significantly higher in PD patients treated with levodopa (17.25 ± 5.96) than in controls (14.43 ± 4.48) (p < 0.05). There was no significant difference in tHcy levels between PD non-treated patients (16.37 ± 5.53) and controls; however, a trend towards higher levels in PD patients was observed. The levels of tHcy did not show dependence on levodopa doses. In the presence of low folic acid levels tHcy increases in all three study groups (Fig. 1). The distribution of MTHFR C677T alleles was not significantly different in levodopa-treated PD, non-treated PD and controls (Table 1). There was no Hcy-disturbing impairment in kidney function as measured by creatinine in the groups. The PD population had lower Vitamin B12 levels than controls (p < 0.05). Additionally,

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Fig. 1. Relation between serum folic acid levels and plasma tHcy levels in all study subjects from the three studied groups: levodopa-treated PD, levodopanon-treated PD and control groups. The solid lines show the mean value.

Fig. 3. Relation between the duration of PD and plasma tHcy levels in two PD groups: levodopa-treated PD and levodopa-non-treated groups. The solid lines show the mean value.

in the presence of low Vitamin B12 levels, homocysteine levels rose significantly in PD patients (Fig. 2). Hyperhomocysteinemia correlated with cognitive impairment evaluated by MMSE (p < 0.05). Since homozygous TT polymorphism, deficiency of Vitamin B12 and/or folic acid can affect level of homocysteine, we performed similar additional analysis of regression followed with ANOVA excluding such patients. In this case we also found significant difference between patients with PD treated with levodopa and healthy control p < 0.05. One important factor that differentiated our study groups is the duration of the disease. As is presented by the regression analysis model, the duration of disease correlates stronger with concentration of homocysteine (p < 0.05) than doses of levodopa (p = 0.3). The levodopa-treated group had longer disease duration than non-treated subjects; however, with no significant age difference. In both groups the levels of homocysteine rose with duration of the disease (p < 0.001) (Fig. 3) and the increase was higher than that observed with age (Fig. 4). Our study clearly shows an association between levodopa therapy and levels of plasma homocysteine, which is in accordance with previous findings [2,18,24,37]. Furthermore, we have found that homo-

cysteine levels depend not only on levodopa treatment, but are influenced even more by the duration of the disease. Increased plasma tHcy levels have been reported to be associated with AD [7,31], psychogeriatric disorders other than dementia [26] and worse cognitive performance in normal aging [32]. In some studies on specific populations, such as centenarians or very old people, these associations are not seen [30]. Hyperhomocysteinemia is associated with both cognitive and affective symptoms in the PD population [28]. In the present study we found a significant, independent association between increased plasma tHcy levels and cognitive impairment defined by MMSE. It has earlier been shown that development of dementia is the strongest risk factor for shortened life in the PD population [21]. The hyperhomocysteinemia seen in our levodopa-treated patients is not a unique causal factor for dementia, but since it may be treatable, it should be avoided. It has been observed that elevated plasma tHcy have an independent, graded association with concurrent cognitive impairment as measured with the MMSE in healthy elderly community dwellers [29]. Presence of hyperhomocysteinemia can be a partial explanation to the common cognitive impairment seen in the PD population. It has also been shown that elevated homocysteine levels

Fig. 2. Relation between serum Vitamin B12 levels and plasma tHcy levels in all study subjects from the three studied groups: levodopa-treated PD, levodopanon-treated PD and control groups. The solid lines show the mean value.

Fig. 4. Relation between the age of the patients and plasma tHcy levels in two PD groups: levodopa-treated PD and levodopa-non-treated groups. The solid lines show the mean value.

D. Religa et al. / Neuroscience Letters 404 (2006) 56–60

in levodopa-treated PD patients are associated with an increased risk for cardiovascular disorders [33]. In a recent study with Huntington disease patients, higher tHcy levels was observed in the group with twice as long duration of the disease [3]. In a study on PD patients by O’Suilleabhain et al. a modest increase of homocysteine after the initiation of levodopa treatment was observed and the magnitude of the levodopa-associated tHcy elevation was lower than previously reported [27]. These results may give an additional argument for looking closer at a correlation between the duration of neurodegenerative disorders and hyperhomocysteinemia. Hyperhomocysteinemia can be caused by genetic mutations or/and environmental factors. The most prevalent mutation leading to elevated plasma tHcy is the MTHFR C677T mutation, which makes the MTHFR enzyme thermolabile. We found that the prevalence of MTHFR C677T mutation is not significantly different in controls and PD patients in our study population. We did not observe a protective effect of the C allele against hyperhomocysteinemia in PD subjects. However, in a Japanese population, the homozygosity for the T allele of MTHFR 677 was shown to be a risk factor for development of PD [37]. It was also observed that PD patients with the CT or TT genotype of MTHFR had significantly higher tHcy levels than controls or PD patients with the CC homozygosity [36]. Experimental studies have shown that dietary folate deficiency causes hyperhomocysteinemia and sensitizes dopaminergic neurons to dysfunction and death in an animal model of PD [8]. Additionally, it was not observed that folate deficiency alone cause clinical signs of motor disturbances in mice. In humans homocysteine can be rapidly taken up by neurons via a specific membrane transporter [12] and the intracellular increase of homocysteine promotes DNA damage [17]. These data suggest a mechanism whereby dietary folate might reduce a risk of developing PD, especially in a subgroup of people having a mutation in enzymes that regulate the homocysteine metabolism. From a mechanistic perspective it has been reported electrophysiological sign of peripheral neuronal dysfunction suggesting, to a certain extent, sensory nerve action potential could be a surrogate marker for the levodopa metabolism—induced elevation of Hcy levels and the aggravation of the ongoing central neurodegenerative process [22]. Moreover, in an experimental model in rodents Hcy has been shown to be toxic to the dopaminergic systems possibly accelerating the progression of PD [20]. It was also seen that the levels of tHc are elevated in the CSF of patients with AD and PD [15]. As CSF better reflects the brain changes, these findings give additional argument that hyperhomocysteiemia in PD may be related to the pathogenesis of the disease. From the meta-analysis data of clinical trials it was concluded that in Western populations daily supplementation with a combination of 0.5–5 mg folic acid and 0.5 mg Vitamin B12 could be expected to reduce blood homocysteine concentrations by 25–30% (for example, from about 12 to 8–9 ␮mol/l) [1]. An alternative method to reduce increased homocysteine levels is therapy with betaine, successfully used in homocystinuria. One pilot study was performed in AD patients providing a basis for pursuing larger, controlled trials of betaine in AD [16]. Betaine

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treatment could also be of interest in prevention of hyperhomocysteinemia seen in PD. Chen et al. investigated whether higher intake of folate, Vitamin B6, or Vitamin B12 was related to a lower risk of PD in the Health Professionals Follow-up Study (1986–2000) and the Nurses’ Health Study (1980–1998) [6]. Their findings do not support the hypothesis that higher intake of folate or Vitamins B6 and B12 lowers the risk for a development of PD. However, a prospective intervention trial is necessary to give the full answer to the question of the necessity of high folic acid supplementation in order to decrease cognitive and psychiatric symptoms associated with hyperhomocysteinemia in PD. In such a trial potential side effects should also be evaluated as vitamin supplementation to different mechanisms of homocysteine degradation, i.e. to cysteine, that could be toxic. To conclude, our study confirms the results of previous investigations indicating that levodopa treatment leads to hyperhomocysteinemia. However, we observed that the duration of PD also is associated with increased homocysteine levels, as well as, tentatively, the disease per se. Keeping normal or higher folic acid and Vitamin B12 levels through intake of food rich in these vitamins or direct supplementation in the PD population may prevent increase in blood homocysteine levels. This is a simple, cheap intervention free of side effects. It could hypothetically reduce the progress of PD and decrease the hazard ratios for both ischaemic heart and cerebrovascular diseases as well as cognitive disturbances in PD patients [25]. However, the evaluation of such anti-hyperhomocysteinemia strategies in prevention of dementia, affective symptoms and vascular episodes in PD needs further and larger studies. Acknowledgments The study was supported by a grant from Polish Academy of Sciences (Grant KBN), Swedish Institute (New Visby Programme) and Dementia Foundation. M.S. is a recipient of the L’Oreal-UNESCO for Women in Science Fellowship and the Foundation for Polish Science Fellowship. References [1] Lowering blood homocysteine with folic acid based supplements: metaanalysis of randomised trials. Homocysteine Lowering Trialists’ Collaboration, BMJ 316 (1998) 894–898. [2] P. Allain, A. Le Bouil, E. Cordillet, L. Le Quay, H. Bagheri, J.L. Montastruc, Sulfate and cysteine levels in the plasma of patients with Parkinson’s disease, Neurotoxicology 16 (1995) 527–529. [3] J. Andrich, C. Saft, A. Arz, B. Schneider, M.W. Agelink, P.H. Kraus, W. Kuhn, T. Muller, Hyperhomocysteinaemia in treated patients with Huntington’s disease homocysteine in HD, Mov. Disord. 19 (2004) 226–228. [4] S. Annerbo, L.O. Wahlund, J. Lokk, The relation between homocysteine levels and development of Alzheimer’s disease in mild cognitive impairment patients, Demen. Geriatr. Cogn. Disord. 20 (2005) 209–214. [5] J.C. Chambers, O.A. Obeid, H. Refsum, P. Ueland, D. Hackett, J. Hooper, R.M. Turner, S.G. Thompson, J.S. Kooner, Plasma homocysteine concentrations and risk of coronary heart disease in UK Indian Asian and European men, Lancet 355 (2000) 523–527. [6] H. Chen, S.M. Zhang, M.A. Schwarzschild, M.A. Hernan, G. Logroscino, W.C. Willett, A. Ascherio, Folate intake and risk of Parkinson’s disease, Am. J. Epidemiol. 160 (2004) 368–375.

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