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Keywords:

  • Brain atrophy;
  • Hyper-total-homocysteinemia;
  • C677T MTHFR polymorphism;
  • Enzyme-inducing antiepileptic drugs

Summary

  1. Top of page
  2. Summary
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

Purpose: Brain atrophy (BA) is observed in 20–50% of patients with epilepsy. Hyper-total-homocysteinemia (hyper-tHcy), which occurs in 10–40% of patients, is considered to be a risk factor for cardiovascular diseases and BA. The present study was aimed at investigating the possible association of hyper-tHcy with BA in a population of patients with epilepsy.

Methods: Fifty-eight patients (33 M/25 F, 43.5 ± 13.1 years of age) chronically treated with antiepileptic drugs (AEDs) and 60 controls matched for age and sex were enrolled. All participants underwent determination of plasma tHcy, folate, vitamin B12, and C677T methylene-tetrahydrofolate-reductase (MTHFR) polymorphism genotyping, and brain magnetic resonance imaging (MRI).

Results: Patients exhibited significantly higher tHcy and lower folate levels than controls; hyper-tHcy was significantly associated with the variables group (patients vs. controls), MTHFR genotype, and their interaction terms. BA was observed in 30.1% of patients and was significantly associated with hyper-tHcy (β = 0.45, p = 0.003) and polytherapy (β = 0.31, p < 0.001).

Discussion: Our investigation suggests that hyper-tHcy plays a role in the development of BA in patients with epilepsy. Although the real origin of this phenomenon is not yet fully elucidated, experimental data support the hypothesis of a link of the neuronal Hcy-mediated damage with oxidative stress and excitotoxicity.

Atrophy in various areas of the brain has been observed in 20–50% of patients with both partial and generalized epilepsies (Liu et al., 2005; Betting et al., 2006; Labate et al., 2006). The causes of this finding have not yet been fully elucidated and a number of possible risk factors have been explored, including, among others, duration of epilepsy, polytherapy, seizure frequency, age, and initial brain insults (Liu et al., 2003, Liu et al., 2005; Betting et al., 2006; Labate et al., 2006).

Homocysteine (Hcy), a sulfur-amino acid, is an intermediate product of methionine metabolism and is normally at plasma total (t) concentrations <15 μmol/L. Higher values are considered abnormal and are seen in approximately 5% of the general population. Higher percentages, from 10–50%, are observed in individuals addicted to chronic alcohol assumption or those with a number of systemic diseases, including hypertension, diabetes, and renal insufficiency (Herrmann et al., 2007). A large body of literature has suggested that hyper-tHcy is a risk factor for cardiovascular and neurodegenerative diseases (Herrmann et al., 2007). A possible causative role of hyper-tHcy in the development of brain atrophy (BA) has been also evidenced in elderly individuals who are at risk for Alzheimer’s disease (den Heijer et al., 2003).

Ten percent to 20% of patients with epilepsy exhibit elevated plasma levels of t-Hcy (Caccamo et al., 2004; Belcastro et al., 2007). This finding derives primarily from a complex interplay between the chronic intake of enzyme-inducing antiepileptic drugs (AEDs) and genetically determined mutations of methylene-tetrahydrofolate-reductase (MTHFR) (Caccamo et al., 2004; Belcastro et al., 2007). AEDs, in fact, deplete the organism of folate and other B vitamins (necessary cofactors in the metabolic pathways of Hcy), and MTHFR mutations cause a reduction in its enzymatic activity, which plays a key role in the transformation of Hcy to methionine (Herrmann et al., 2007). The present investigation has been carried out to explore the possibility that BA in patients with epilepsy is associated with elevated plasma levels of tHcy.

Patients and Methods

  1. Top of page
  2. Summary
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

Patients

Patients were enrolled on the basis of the following criteria: (1) age of 18–55 years; older patients were excluded to minimize the effect of age on both tHcy levels and BA; (2) firm diagnosis of epilepsy according to the clinical and electrocardiography (EEG) criteria of the International League Against Epilepsy (ILAE); (3) duration of epilepsy of at least 3 years; (4) absence of any other brain abnormalities apart from areas of atrophy; (5) no concomitant diseases known to be associated with hyper-tHcy, including cardiovascular diseases, diabetes mellitus, hypothyroidism, chronic renal failure, and hypertension; (6) no abnormal blood chemistry parameters; (7) no chronic use of alcohol, tobacco products, or vitamins; (8) nonvegetarian or particular diet requirements; and (9) no intake of other drugs known to affect plasma tHcy levels, apart from AEDs. A group of healthy volunteers with similar characteristics were investigated as controls.

Blood determinations

Fasting blood samples were collected in the morning at the time of magnetic resonance imaging (MRI). Plasma vitamin and tHcy concentrations were determined as previously described (Caccamo et al., 2004; Belcastro et al., 2007). C677T polymorphism analysis was made by using double-gradient density-acrylamide gel electrophoresis following a standard protocol (Caccamo et al., 2004).

Magnetic resonance imaging

All patients underwent brain MRI, using a 1.5 T scanner (Siemens, Munich, Germany). Each scan was visually examined independently by two expert radiologists, blinded to each other’s readings and to clinical, biochemical, and genetic data.

Statistical analysis

Continuous variables (biochemical measures and age) were analyzed using a Student’s t-test and categorical factors (distribution of sex and MTHFR genotypes) by a chi-square test. The effect of MTHFR genotypes on tHcy and folate plasma levels were evaluated with the factorial analysis of variance (ANOVA) followed by a Tukey’s post hoc test.

Within the entire group, correlation between plasma tHcy and folate and between tHcy and vitamin B12 levels was carried out by calculation of Pearson’s product-moment coefficients. The effect of tHcy plasma levels and AED therapy on the presence of BA was investigated by multiple regression analysis in a model also including MTHFR genotypes and age as independent predictors. Most of the statistics were well powered referring to a type I error of 0.05. Our ability to reject the null hypothesis was ≥0.8. All the analyses were carried out using Statistica V. 6 (Statsoft, Tulsa, OK, U.S.A.). The study protocol was approved by the local ethic committees. Signed informed consent was obtained from each participant, after reading of the protocol and discussion of the study procedure, risks, and benefits.

Results

  1. Top of page
  2. Summary
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

Demographic, clinical, biochemical, and genetic features of patients and controls are summarized in Table 1. Patients and controls did not differ significantly with respect to age, sex, MTHFR genotypes distribution, and vitamin B12 concentrations, whereas a significant difference was found for plasma tHcy and folate (Table 1). Within the entire sample, tHcy and folate plasma levels were significantly correlated (r = −0.7; p < 0.01). A significant effect of the variables group (patients vs. controls), MTHFR genotypes, and their interaction terms was found on tHcy levels. Moreover, patients who carried the TT677 MTHFR genotype exhibited higher plasma tHcy levels and lower folate than those with different MTHFR genotypes and controls (Table 2).

Table 1.   Demographic, clinical, biochemical, and genetic data of patients and controls
 Patients (n = 58)Controls (n = 60)p-value
  1. VPA, valproic acid; CBZ, carbamazepine; LTG, lamotrigine; MTHFR, methylene-tetrahydrofolate-reductase; PB, phenobarbital; LEV, levetiracetam; TPM, topiramate; na, not applicable.

Demographic data
 Male/female (n)33/2532/280.7
 Age (years)43.5 ± 13.143.8 ± 12.70.5
Clinical data
 Seizure types
  Primary generalized (n)18nana
  Temporal focal (n)29nana
  Extratemporal focal (n)11nana
  Therapy
  CBZ (n)12nana
  PB (n)11nana
  PB + CBZ (n)6nana
  PB + CBZ + VPA (n)2nana
  PB + VPA + LEV (n)2nana
  VPA + PB + LTG (n)3nana
  PHT + PB + TPM (n)3nana
  CBZ + TPM (n)7nana
  CBZ + PB + LEV (n)4nana
  VPA (n)5nana
  CBZ + PB + LTG (n)3nana
Biochemical data
 Hcy (μmol/L)19.5 ± 4.913.5 ± 9.1<0.001
 Folate (nmol/L)3.5 ± 1.34.5 ± 1.6<0.001
 Vitamin B12 (pg/ml)397 ± 191414 ± 1750.4
Genetic data
 CC677 MTHFR genotype (n)14180.5
 CT677 MTHFR genotype (n)30260.4
 TT677 MTHFR genotype (n)16140.6
Table 2.   Plasma tHcy of patients and controls grouped according to the MTHFR genotypes
 Patients (n = 58)Controls (n = 60)
nHcy (μmol/L)nHcy (μmol/L)
  1. Values are indicated as mean ± SD.

  2. ap < 0.001 in comparison with CC677 and TT677 MTHFR, CC677 MTHFR controls, and CT677 MTHFR controls.

  3. bp < 0.001 in comparison with other groups of patients and controls.

  4. MTHFR, methylene-tetrahydrofolate-reductase.

CC677 MTHFR genotype1810.5 ± 2.3148.7 ± 2.1
CT677 MTHFR genotype2617.0 ± 3.3a3015.3 ± 3.8
TT677 MTHFR genotype1433.8 ± 3.6b1618.9 ± 3.4

Seventeen patients (30.1%) showed BA. Of them, 5 (29.4%) had ventricular abnormalities (uni- or bilateral increased volume of the lateral ventricles), 2 (11.8%) focal gyral abnormalities, 3 (17.6%) hippocampal atrophy, and 7 (41.2%) had a diffuse cortical atrophy. Correlation coefficients of agreement (k) between the two radiologists were greater than 0.6.

BA was not associated with seizure types, age, or MTHFR genotypes (data not shown). A significant association was observed with both tHcy plasma levels (β = 0.45; p = 0.003) and polytherapy (β = 0.31 p = 0.0001). Because of the small sample size, other potential covariates, such as seizure frequency, EEG abnormalities, and EEG abnormality localization were not included in the regression model.

Discussion

  1. Top of page
  2. Summary
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

The preliminary results of the present investigation show that BA in patients with epilepsy is associated with elevated plasma tHcy levels and support data deriving from another study performed on elderly healthy individuals (den Heijer et al., 2003). Over the past two decades, a progressively increasing number of articles have appeared, strongly suggesting that hyper-tHcy is a risk factor for cerebrovascular and neurodegenerative diseases, including Parkinson’s disease and Alzheimer’s disease (Kuhn et al., 2001; Mattson & Shea, 2003; Zhang et al., 2005; Herrmann et al., 2007). The mechanism through which hyper-tHcy displays its detrimental effects has not yet been elucidated. Experimental data have demonstrated that Hcy at high levels affects mitochondria function via oxidative damage with consequent perturbation of the redox status (Mattson & Shea, 2003; Zou & Banerjee, 2005; Mattson, 2006).

In addition, Hcy potentiates the in vitro action of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a specific inhibitor of complex I (Duan et al., 2002), with consequent increase in highly reactive oxygen species (ROS) and hence decreased ATP production, altered calcium handling, and membrane lipo-peroxidation (Testa et al., 2005). Despite the body of experimental data and of clinical studies showing significant associations between hyper-tHcy and various diseases, the real pathogenetic role of hyper-tHcy in these diseases has not yet been clarified and the debate on whether it is a biochemical marker rather than a causative agent is still open (Herrmann et al., 2007).

The results of the present study are preliminary and should be taken cautiously. If confirmed, they might have important practical implications. Hyper-tHcy is easily modifiable through folate supplementation (Belcastro et al., 2007). Patients with epilepsy would, therefore, need to be routinely screened for plasma tHcy concentrations. An increased number of patients and analysis of additional variables would undoubtedly confer to the study a stronger scientific value, particularly duration of the disease and exposition to AEDs, types of AEDs, frequency of seizures, type and location of EEG abnormalities, and volumetric determinations of particular brain areas, especially amygdala and hippocampus.

Acknowledgment

  1. Top of page
  2. Summary
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

Disclosure: The authors declare no conflicts of interest.

References

  1. Top of page
  2. Summary
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References