Plasma homocysteine and MTHFR C677T genotype in levodopa-treated patients with PD

Subjects and methods.

We analyzed 90 patients with PD and 50 control subjects (table). Patients with PD received conventional pharmacotherapy, including levodopa/carbidopa and other antiparkinsonian drugs. All subjects were Japanese and gave informed consent before participation in our study. Blood samples were drawn in the morning from a peripheral vein. Plasma homocysteine and cysteine levels were determined using high-performance liquid chromatography with fluorescent detection (HPLC-FD) by Hyland’s6 method, with some modifications. A PCR was performed on leukocyte genomic DNA samples with previously reported primers.5 Serum folate and vitamin B₁₂ levels were measured by chemiluminescent immunoassay at the Special Reference Laboratory (Tokyo, Japan). Comparisons of plasma homocysteine and cysteine levels between patients with PD and control subjects were performed by two-tailed Mann–Whitney U test. Comparisons of homocysteine and cysteine in C677T MTHFR genotype groups of patients with PD and control subjects were performed using one-way ANOVA followed by Fisher’s Protected Least Significant Difference. Spearman rank correlation test was used for correlation analyses of homocysteine, cysteine, age, gender, serum folate, vitamin B₁₂, and patients’ other information.

Results.

Plasma homocysteine levels in patients with PD were 16.3 ± 14.2 nmol/mL, and they were higher than those of control subjects (10.2 ± 5.3 nmol/mL, p < 0.001) (figure 1A). Plasma cysteine levels in patients with PD were similar to levels in control subjects (patients with PD: 281.5 ± 102.1 nmol/mL; control subjects: 283.6 ± 101.8 nmol/mL) (see figure 1B). There was no difference in cysteine levels between the PD and control groups.

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Figure 1. (A) Plasma levels of homocysteine in control and PD groups. Significantly higher homocysteine levels were observed in patients with PD (16.3 ± 14.2 nmol/mL) than in control subjects (10.2 ± 5.3 nmol/mL) *p < 0.001 (Mann-Whitney test). (B) Plasma levels of cysteine in controls and PD. No difference is demonstrated between control subjects (283.6 ± 101.8 nmol/mL) and patients with PD (281.5 ± 102.1 nmol/mL). Open circles = control subjects; dark circles = patients with PD.

Patients with PD and control subjects were separated into three groups on the basis of C677T MTHFR genotype. Plasma homocysteine levels in the patients with PD were 13.6 ± 8.1 nmol/mL in the C/C genotype group, 12.6 ± 7.3 nmol/mL in the C/T genotype group, and 27.2 ± 23.3 nmol/mL in the T/T genotype group. In patients with PD, homocysteine levels in the T/T genotype group were remarkably higher than in those of the other genotype groups. In control subjects, however, homocysteine levels were 11.0 ± 6.5 nmol/mL in the C/C genotype group, 8.3 ± 3.4 nmol/mL in the C/T genotype group, and 11.7 ± 4.6 nmol/mL in the T/T genotype group. There were no statistical differences between the three control genotype groups (figure 2A). In each genotype group, homocysteine levels in PD had a tendency to be higher than those in control subjects. Plasma cysteine levels were not different within each genotype group in PD and control groups (data not shown).

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Figure 2. A) Plasma homocysteine levels in MTHFR genotype groups in control subjects and patients with PD. Homocysteine levels of the T/T genotype in PD were higher than those of the other 5 groups. *p < 0.0001 (ANOVA followed by Fisher’s PLSD). Open circles = control subjects; dark circles = patients with PD. B) Correlation between homocysteine levels and serum folate levels in PD. In the MTHFR T/T genotype with PD, homocysteine levels are inversely correlated with serum folate. The coefficient curve indicates a correlation between homocysteine levels and serum folate levels in the T/T genotype group (p < 0.01, r = −0.804 [Spearman rank correlation test]). Open square = C/C genotype (n = 16); open triangle = C/T genotype (n = 13); dark circles = T/T genotype (n = 12).

Only 41 patients with PD agreed to the measurement of serum folate and vitamin B₁₂. Serum folate levels of 40 patients with PD were within the normal range (>2.4 ng/mL). One patient had a very mild folate deficiency (2.3 ng/mL). Levels of serum folate were 7.4 ± 2.8 ng/mL in 41 patients with PD. Serum folate levels were 7.9 ± 3.0 ng/mL in the C/C genotype group (n = 16), 7.6 ± 2.6 ng/mL in the C/T genotype group (n = 13), and 6.6 ± 2.9 ng/mL in the T/T genotype group (n = 12). There were no statistical differences in folate levels between the three genotype groups. However, in the T/T genotype of patients with PD, folate levels were inversely correlated with homocysteine levels (p < 0.01, r = −0.804). No correlation was found between folate levels and homocysteine levels in the C/C and C/T genotype groups of PD (see figure 2B).

Serum vitamin B₁₂ levels of 41 patients with PD were 803.8 ± 403.9 pg/mL, and all were within the normal range (>249 pg/mL). There were no significant differences in vitamin B₁₂ levels between the three genotype groups, and there was no correlation between vitamin B₁₂ levels and homocysteine levels in any of the genotype groups (data not shown).

Age, gender, Hoehn–Yahr Stage, duration of PD, daily levodopa dose, and other antiparkinsonian drugs had no correlation with homocysteine (data not shown).

Discussion.

We investigated plasma homocysteine, cysteine, serum folate, vitamin B₁₂, and the MTHFR genotype of patients with PD. The homocysteine levels in 90 patients with PD were higher than those in 50 control subjects. Our large study confirms previous smaller reports.2,3,4 We also demonstrated that the T/T genotype patients with PD had extreme hyperhomocysteinemia and that homocysteine levels were higher if serum folate levels were low and vice versa.

Homocysteine is a metabolite of S-adenosylmethionine via S-adenosylhomocysteine. S-adenosylmethionine plays an important role as a methyl donor in transmethylation reactions. Transmethylation reactions occur in the methylation of not only nucleic acid, proteins, and phospholipids, but also of catecholamine. Concentrations of S-adenosylmethionine are decreased in PD, suggesting that transmethylation reactions in dopa and catecholamine metabolism increase in PD.7 Increased methylation should cause an increase in homocysteine and a decrease in S-adenosylmethionine. The results of this study may reflect such metabolic disorders associated with methylation in PD.

Homocysteine levels in the PD group tended to be higher than those of control subjects in all genotypes, and homocysteine levels were not elevated in the T/T genotype in the control group. According to Müller,4 plasma homocysteine levels of levodopa-treated patients with PD increase compared with nondrug-treated patients and with control subjects. Levodopa causes increased plasma homocysteine levels in rats.8 Regrettably, we failed to confirm levodopa-induced hyperhomocysteinemia because all the patients in this study received phamacotherapy including levodopa. Judging from our results, the combination of T/T genotype and low folate level might also cause further hyperhomocysteinemia in PD. This evidence suggests that differences in homocysteine levels among C/C, C/T, and T/T genotypes may result not only from the MTHFR enzyme activity itself but also from the difference in capacity for levodopa-associated metabolism in each genotype.

Folate deficiency has been identified as one of the causes of hyperhomocysteinemia.9 In a previous report,9 homocysteine levels were significantly elevated in cases whose folate levels were under 2.0 ng/mL. In our study, all patients investigated except one showed normal folate levels. Folate levels had no relationship with homocysteine levels in the C/C and C/T genotype groups. By contrast, in the T/T genotype group, folate levels had a significant inverse correlation with homocysteine levels. According to the correlation curve, the homocysteine level of the T/T genotype patient, whose folate level is under approximately 5.0 ng/mL, might elevate to more than 20 nmol/mL. Therefore, T/T genotype patients with PD might be advised to keep folate levels over 5.0 ng/mL. The T/T genotype may cause not only an increase in thermolability of the enzyme5 but also a decrease in tolerance to folate deficiency.

Homocysteine injures both neuronal and endothelial cells.10 This study suggests that low folate levels (including the lower part of the normal range) may cause hyperhomocysteinemia in T/T genotype cases of PD. This might be an important risk factor for vascular disease as a complication in PD, and it might be involved in the pathogenesis of nigrostriatal degeneration because of its putative effect on dopaminergic neurons.10 As low folate level enhances hyperhomocysteinemia, folate supplementation might be warranted in levodopa-treated patients with PD, especially T/T genotype patients.