gms | German Medical Science

24. Jahrestagung der Deutschen Gesellschaft für Arterioskleroseforschung

Deutsche Gesellschaft für Arterioskleroseforschung

18.03. - 20.03.2010, Blaubeuren

Convulsive properties of D,L homocysteine and D,L homocysteine thiolactone in adult rats

Meeting Contribution

  • corresponding author O. Stanojlovic - Institute of Medical Physiology "Richard Burian", School of Medicine, University of Belgrade, Belgrade, Serbia
  • A. Rašic-Markovic - Institute of Medical Physiology "Richard Burian", School of Medicine, University of Belgrade, Belgrade, Serbia
  • D. Hrncic - Institute of Medical Physiology "Richard Burian", School of Medicine, University of Belgrade, Belgrade, Serbia
  • D. Djuric - Institute of Medical Physiology "Richard Burian", School of Medicine, University of Belgrade, Belgrade, Serbia

Deutsche Gesellschaft für Arterioskleroseforschung e.V.. 24. Jahrestagung der Deutschen Gesellschaft für Arterioskleroseforschung. Blaubeuren, 18.-20.03.2010. Düsseldorf: German Medical Science GMS Publishing House; 2011. Doc10dgaf09

DOI: 10.3205/10dgaf09, URN: urn:nbn:de:0183-10dgaf092

Published: March 23, 2011

© 2011 Stanojlovic et al.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en). You are free: to Share – to copy, distribute and transmit the work, provided the original author and source are credited.


Abstract

Introduction: Hyperhomocysteinemia is related to epileptogenesis and suboptimal control of seizures in the patients with epilepsy. Available data suggest that homocysteine can be harmful to human cells because of its metabolic conversion to homocysteine thiolactone, a reactive thioester.

The aim of the study was to investigate the convulsive properties of equimolar concentrations of D,L homocysteine and D,L homocysteine thiolactone in adult rats.

Methods: Adult male Wistar rats were divided into following groups: 1. saline-treated (C, n=10), 2. D, L homocysteine 8 mM/kg, i.p. (H; n=8), and 3. D,L homocysteine-thiolactone (Ht; n=8). Seizure behavior was assessed by the incidence, latency, number and intensity of seizure episodes. Seizure severity was described by a descriptive scale with grades 0–4.

Results: There were no behavioural signs of seizure activity in C group. The incidence of convulsions was lower in group H comparing to the Ht group but statistical significance was not attained. Latency to the first seizure onset was significantly higher in H comparing to the Ht group (p<0.01). Median number of seizure episodes and severity of seizure episodes per rat were significantly lower in H comparing to Ht group.

Conclusion: Our findings suggest that D,L homocysteine thiolactone exerts stronger convulsive effect comparing to the equimolar concentration of D,L homocysteine.


Introduction

Epilepsy is a major health problem that affects 1–2% of the population worldwide. Each type of epilepsy shares the common feature of persistently increased neuronal excitability that manifests sporadically as seizure generation [1], [2]. One of the principles for seizures arise is disruption of balance between excitation and inhibition. Among numerous candidates for excitatory neurotransmitters, homocysteine and its derivates represent favorite candidates [3] and similar to glutamine, exert a direct excitatory effect on N-methyl-D-aspartate and metabotropic glutamate receptors. Previous studies suggest that increased homocysteine levels may be related to epileptogenesis and/or suboptimal control of seizures in the patients with epilepsy [4], [5], on the other hand, severe hyperhomocysteinemia exhibit a wide range of clinical manifestations in patients including neurological abnormalities and atherosclerotic vascular diseases [6].

The aim of the study was to investigate the convulsive properties of equimolar concentrations of D,L homocysteine and D,L homocysteine thiolactone in adult rats.


Methods

All experimental procedures were carried out in accordance with The European Council Directive (86/609/EEC), and were approved by the Animal Care Committee of the University of Belgrade (298/5-2).

Two-month old Wistar male rats (170–200 g), obtained from the Military Medical Academy Breeding Laboratories, Belgrade (Serbia) were used. They were kept under controlled environmental conditions and housed individually with free access to standard laboratory animal chow and tap water. The acclimatization period lasted for 7 days.

The animals were divided into the following groups: 1. Controls, saline-injected (C, n=10); 2. D, L homocysteine 8.0 mmol/kg (H, n=8); 3. D, L homocysteine thiolactone 8.0 mmol/kg (Ht, n=8). Each rat was used only once. All the substances were applied intraperitoneally (i.p.). D,L homocysteine and D,L homocysteine thiolactone (Sigma-Aldrich Chemical Co., U.S.A.) were freshly prepared in saline and after adjusting the pH to 7.4 administered in a volume of 1.0 ml/100 g body weight.

Convulsive behaviour was assessed by the latency to the seizure onset, incidence of seizures (the number of convulsing animals out of total number of rats), the number of seizure episodes per rat and the seizure severity. Seizure severity was described by a descriptive scale with the following grades: 1 – head nodding, lower jaw twitching, 2 – myoclonic body jerks, bilateral forelimb clonus, 3 – generalized tonic-clonic convulsions, 4 – status epilepticus or frequent repeated episodes of clonic convulsions for an extended period of time (over 5 min).

Significance of the differences in the incidence of seizures and seizure grade distribution were evaluated by Fisher’s exact probability test. Since the normal distribution of the data on seizure latency, number and intensity of seizure episodes has not been estimated by Kolmogorov-Smirnov test, the non-parametric analyses (Kruskal-Wallis ANOVA and Mann Whitney U-test) were used to determine the statistical significance of the differences between the groups (*p<0.05, **p<0.01). The results were expressed as medians with 25th and 75th percentiles.


Results

No signs of seizure behavior were observed in C group. The seizure incidence was higher in Ht (100.0%) compared to the H (66.67%) group, but there was no statistical significance between the groups (Figure 1 [Fig. 1]).

Median latency to the first seizure episode significantly decreased in Ht [28 (21–39) min] (p<0.05) as compared to the H [76.5 (42–90) min] group (Figure 2A [Fig. 2]). Median number of seizure episodes per rat was significantly higher in Ht [4 (1–5)] (p<0.05) compared to the H [1.5 (0–4)] group (Figure 2B [Fig. 2]). Median seizure episode severity was significantly higher in Ht [2 (2–3)] (p>0.05) compared to the H [2 (1–2)] group (Figure 2C [Fig. 2]).


Discussion

Homocysteine is a sulphur-containing amino acid normally present in human plasma and its concentration ranges from 1 to 15µmol/L. Systemic administration of homocysteine or homocysteine thiolactone can trigger seizures in animals [7], [8] and patients with homocystinuria suffer from epileptic seizures [9]. Available data suggests that homocysteine can be harmful to human cells because of its metabolic conversion to a reactive thioester homocysteine thiolactone. Normal human plasma levels of homocysteine thiolactone vary from 0 to 34.8 nmol/L and account for 0.002–0.3% total plasma homocysteine [10].

Our results presented here demonstrate that D,L homocysteine thiolactone possess stronger convulsive potential demonstrated thought decreased latency and increased number and intensity of seizures, compared to the D,L homocysteine.

Homocysteine-thiolactone injected intravenously is extremely neurotoxic and it induces seizures and death in experimental animals. Its action could be ascribed to the increased glutamate release, blockade of GABA synthesis, or inhibition of glutamate decarboxylase [11]. It has been suggested that toxicity of homocysteine-thiolactone may be due to its metabolism to homocysteic acid, a potent neurotransmitter [10].


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