Plasma homocysteine and aminothiol levels in idiopathic epilepsy patients receiving antiepileptic drugs

Introduction

Homocysteine is a sulfur containing amino acid formed during methionine metabolism. Plasma homocysteine level is an important risk factor for many diseases such as arterial and venous thrombosis, myocardial infarction and chronic renal insufficiency. Accordingly, hyperhomocysteinemia has been associated with cardiovascular diseases and is regarded as an independent risk factor for atherosclerosis [1]. Patients with long-term antiepileptic drug treatment often have hyperhomocysteinemia and low levels of serum folate, vitamin B12 and vitamin B6, all of which are associated with homocysteine metabolism [2], [3]. On the other hand, some antiepileptic drugs, i.e. carbamazepine, phenytoin, phenobarbital and primidone, have inducing effects on hepatic enzymes [4], [5], and may stimulate hepatic cytochrome P450 and glucuronyl transferase [6]. However, many of the lately developed antiepileptics are less likely to influence the activity of hepatic enzymes when compared with older drugs. The risk of hyperhomocysteinemia can be regarded negligible or even absent with these new drugs [7].

In the present study, our aim was to investigate the effects of valproic acid and two new generation antiepileptic drugs (lamotrigine and levetiracetam) on plasma homocysteine and aminothiols. We have used the commercial homocysteine method to measure plasma aminothiols namely cysteine, cysteinylglycine and glutathione.

Materials and methods

Validation studies

Stock solutions of cysteine, cysteinylglycine and glutathione (10 mmol/L) were prepared and used in validation studies. To demonstrate the linearity of the method, to draw calibration graphs and to determine the analytical measurement range serial dilutions (50, 100, 200 μmol/L for cysteine; 1, 5, 10, 50 μmol/L for glutathione and cysteinylglycine) were made and applied to the homocysteine sample preparation procedure. The area under the curve (AUC) of the peak retention times were calculated from the chromatograms. Cysteine, cysteinylglycine and glutathione concentrations were calculated from the corresponding concentration-AUC plots. A representative chromatogram and the concentration-AUC plots for cysteine, cysteinylglycine and glutathione is shown in Figure 1. To determine the limit of detection (LOD) values, pretreatment was carried out in accordance with the homocysteine sample preparation procedure. Twenty repetitive measurements were performed. LOD values were determined as 3.19 μmol/L for cysteine and 0.43 μmol/L for homocysteine. For glutathione and cysteinylglycine LOD values could not be calculated because their retention times were very close to each other and the peaks were not completely separated in all runs. For reproducibility studies, working standards (cysteine: 50, 100, 200 μmol/L, cysteinylglycine: 5, 10, 50 μmol/L, glutathione: 1, 5, 10 μmol/L) were used. Appropriate samples were prepared from these standards according to the homocysteine sample preparation procedure. Each concentration was studied 12 times (3 times on the first day, 3 times on the second day and 6 times on the third day). Percentage CV values for intra and inter day measurements were calculated. They were less than 10% except for two concentrations (Table 1). For the recovery study, a plasma pool was prepared from the leftover blood specimens. Recovery values were calculated as 97.7% for homocysteine and 97.9% for cysteine. Recovery values for glutathione and cysteinylglycine were not calculated because as mentioned above their retention times were very close and the peaks were not completely separated in all runs.

Figure 1:

A representative chromatogram and calibration plots for cysteine, cysteinylglycine and glutathione.

Table 1:

Intra day and inter day CV values for cysteine, cysteinylglycine and glutathione.

	Concentration (μmol/L)	CV (%)	CV (%)
		Intra day	Inter day
Cysteine	50	9.64	10.20
	100	6.68	3.10
	200	6.55	9.64
Cysteinylglycine	5	5.08	5.23
	10	6.15	3.02
	50	7.70	4.10
Glutathione	1	8.05	8.02
	5	6.89	2.52
	10	5.22	10.60

Results

Our controls were 38 healthy subjects aged 15–65 years without any health problems and the patient group comprised 47 idiopathic epilepsy who were under valproic acid, levetiracetam or lamotrigine monotherapy. No significant gender difference was present. Also there was no significant age difference between control and patient groups, and there was no significant difference in duration of drug use between different patient groups. We have used commercial immunoassay kits for serum folic acid and vitamin B12 determinations. Plasma aminothiols were assayed by the homocysteine method using HPLC with fluorescence detection where cysteine, cysteinylglycine and glutathione were assayed from the same chromatogram obtained for homocysteine. However, as mentioned in validation studies, cysteinylglycine and glutathione peaks could not be separated completely in all chromatograms. Therefore, in future studies the chromatography conditions used in measuring aminothiols needs to be improved by changing chromatography conditions such as flow-rate and pH.

Table 2 shows vitamin B12, folic acid and aminothiol levels of healthy controls and patients with idiopathic epilepsy receiving different antiepileptic drugs. There was no significant difference in vitamin B12 and folic acid levels of antiepileptic drug users compared to controls. Also, no significant difference was found for homocysteine, cysteine and cysteinylglycine levels between patients and controls. However, although there was no significant difference in glutathione levels of patients using levetiracetam compared to the controls, glutathione levels of lamotrigine and valproic acid users were significantly increased. Table 3 shows comparison of plasma aminothiol levels of controls and all idiopathic epilepsy patients. No significant difference was found in homocysteine and cysteinylglycine levels but cysteine and glutathione levels of antiepileptic drug users were significantly different than controls.

Discussion

In early studies on plasma homocysteine levels of epilepsy patients receiving phenytoin [8], [9], [10] fluctuations in homocysteine levels were reported. Further studies after phenytoin have been performed on patients receiving carbamazepine and valproic acid in which almost all patients were observed to have elevated homocysteine levels [3], [11], [12], [13], [14], [15], [16]. Later, effects of newer antiepileptics were studied. Gidal et al. [17] examined homocysteine, vitamin B12 and folic acid in patients who received valproic acid and lamotrigine monotherapy before and after treatment. Chronic treatment with lamotrigine did not effect blood concentrations of homocysteine, folic acid and vitamin B12. On the other hand, valproic acid caused increased vitamin B12 and decreased homocysteine concentrations whereas folic acid was not changed. Belcastro et al. [18] examined 259 epileptic patients and 231 healthy controls and reported that plasma homocysteine levels were significantly higher and folate levels were lower in patients. Patients who were treated with oxcarbazepine, topiramate, carbamazepine, and phenobarbital exhibited mean plasma homocysteine levels above the physiologic range. Conversely, homocysteine concentrations were observed to be unchanged in lamotrigine and levetiracetam groups [18].

Antiepileptic drugs may interfere with the absorption and metabolism of folate [19], [20]. It is unclear how antiepileptics cause changes in vitamin levels. One mechanism is by decreasing the activity of the enzyme required for re-methylation of methionine [8], [21], [22]. Other possible mechanisms involved are activation of the cytochrome P450 system, reducing absorption of vitamins by increasing intestinal pH, and interacting with the metabolism of vitamins [23], [24], [25]. While carbamazepine, phenytoin and phenobarbital induce cytochrome P450 system, recent reports on newly developed antiepileptics such as lamotrigine, topiramate, levetiracetam and oxcarbazepine suggest that they do not induce enzymes. Drugs that do not induce hepatic enzymes also have no effect on serum folic acid levels [17].

No significant difference between vitamin B12, folic acid and homocysteine levels of controls and patients under valproic acid, lamotrigine and levetiracetam monotherapy was observed in our study. However, valproic acid and lamotrigine users had higher glutathione levels than controls. Glutathione is a tripeptide synthesized from glutamate, cysteine and glycine. It protects cells against free radical related oxidative damage and is present at high levels in many tissues especially in the liver [26]. Glutathione is also involved in the transport of amino acids via gamma-glutamyl cycle. Therefore, it is thought that it serves as the storage and transport form of cysteine.

There is limited information on the relationship between homocysteine and aminothiols in epilepsy patients treated with different antiepileptic drugs. Apeland et al. [5] studied patients with hyperhomocysteinemia receiving antiepileptics who were also given pyridoxine, riboflavin, folic acid as supplements for 1 month. They observed that all redox products were decreased. Homocysteine will transform into cysteine and glutathione by trans-sulfuration pathway. This is not only a compensatory mechanism that prevents accumulation of homocysteine, but also provides cysteine and glutathione to the body. Thus the trans-sulfuration pathway assumes a protective role against oxidative stress while reducing homocysteine levels. As suggested by Vitvitsky et al. [27], the significant link between the trans-sulfuration pathway and the redox buffering capacity warrants investigations into potential neuroprotective benefits of nutritional modulation of redox homeostasis.

We admit that the chromatography conditions we have used in measuring aminothiols needs to be improved by changes such as flow-rate and pH. In any case, antiepileptic drugs resulted in increased glutathione compared to the control values. This may suggest impaired redox homeostasis causing increased glutathione synthesis in response to oxidative stress induced by these drugs. Since glutathione synthesis occurs in the cytoplasm with the aid of two enzymes, namely gamma-glutamylcysteine synthetase and glutathione synthetase, future studies determining the activities of these enzymes will be useful to clarify the underlying mechanism of the changes observed.