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The comparison of nail and serum trace elements in patients with epilepsy and healthy subjects
Progress in Neuro-Psychopharmacology & Biological Psychiatry 28 (2004) 99 – 104 www.elsevier.com/locate/pnpbp
The comparison of nail and serum trace elements in patients with epilepsy and healthy subjects ¨ zerolb, Mukaddes Gu¨lecßb, Bu¨nyamin Isßıkc, Nevin ˙Ilhand, Atilla ˙Ilhana,*, Elif O ¨ mer Akyolb Necip ˙Ilhan d, O a
Department of Neurology, I˙no¨nu¨ University Medical School, 44069, Malatya, Turkey b Department of Biochemistry, I˙no¨nu¨ University Medical School, Malatya, Turkey c Department of Family Medicine, Fatih University Medical School, Ankara, Turkey d Department of Biochemistry, FI˙rat University Medical School, Elazıg˘, Turkey Accepted 17 September 2003
Abstract The objective of this prospective study was to determine the levels of manganese (Mn), copper (Cu), and zinc (Zn) levels in both nail and serum from patients with epilepsy. For this purpose, levels of these elements were measured in 31 patients with epilepsy and 19 healthy subjects. Element analyses were carried out by atomic absorption spectrophotometer (AAS). Increased Mn levels were detected in nail of patients with epilepsy compared to healthy controls ( P < .008). The main nail Zn and Cu levels were found to be unchanged in epileptic patients compared to control subjects. There were no significant differences in serum Mn and Zn levels between epileptic patients and control subjects. However, there was a statistically significant increase in serum Cu levels in patients with epilepsy in comparison with control group ( P < .009). Our results demonstrate that some trace element levels may vary in epileptic patients, and because of the more stable status, the analysis of these element levels in some tissues such as nail might be superior to serum analysis. D 2003 Elsevier Inc. All rights reserved. Keywords: Epilepsy; Nail; Serum; Copper; Zinc; Manganese
1. Introduction It is widely accepted that trace elements play an important role in various metabolic processes. Zinc (Zn), copper (Cu), and manganese (Mn) are known to be necessary for the activity of some enzymes. In recent years, it has been suggested that epileptic patients might show some abnormalities in serum trace element concentrations (Ilhan et al., 1999; Davidson and Ward, 1988; Ghose and Taylor, 1983; Palm and Hallmans, 1982; Werther et al., 1986; Kurekci et Abbreviations: AMPA, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate; ATP, adenosine triphosphate; GABA, gamma-aminobutiric acid; MMT, methylcyclopentadienyl manganese tricarbonyl; MnTBAP, Mn tetrakis (4-benzoic acid) porphyrin; MPP+, 1-methyl-4-phenylpyridinium; NMDA, N-methyl-D-aspartate; 6-OHDA, 6-hydroxydopamine; ROS, reactive oxygen species; S.E.M., standard error of mean; SOD, superoxide dismutase. * Corresponding author. Tel.: +90-422-341-06-60/4903; fax: +90-422341-07-29. E-mail address: [email protected] (A. I˙lhan). 0278-5846/$ – see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2003.09.025
al., 1995). Seizure activity in epileptic patients and experimental models of epilepsy influences the metabolism of essential trace elements, e.g., zinc, iron, copper, and manganese, in the brain and peripheral tissues (Papavasiliou and Miller, 1983; Carl et al., 1989; Carl et al., 1990). Thus, it is important to clarify whether the concentration of essential elements in the brain is altered by enhancement of susceptibility to seizures or epileptic neuronal activity. However, the relationship between movement of essential trace elements and seizure activity is poorly understood. Although many authors have studied on the relationship between some trace elements and epilepsy, there are conflicting reports in the literature on this issue. The low Mn concentration is attributed by some investigators to the seizure activity associated with the epilepsy, whereas others propose that the low Mn concentration may be secondary to genetic mechanisms underlying the epilepsy. Chronic seizures caused a decrease in whole blood Mn levels but did not affect brain Mn concentrations. In a study (Carl et al., 1986), comparison of hospitalized epileptic
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patients with matched normals showed that the mean whole blood Mn concentration of the epileptic population was significantly lower than those of the normal population. The whole blood Mn concentration in the epileptics did not correlate either with seizure frequency or with anticonvulsant therapy. It was observed, however, that the patients whose epilepsy was a result of trauma had significantly higher blood Mn concentrations than patients whose history was negative for trauma (Carl et al., 1986). Some authors reported that epileptic patients have higher serum Zn concentrations than controls (Davidson and Ward, 1988; Ghose and Taylor, 1983; Werther et al., 1986; Kurekci et al., 1995; Kuzuya et al., 1993); while some others have observed normal (Higashi et al., 1982; Schott and Delves, 1978) and even low zinc concentrations (Palm and Hallmans, 1982) in epileptic patients under classical treatment. The authors have previously suggested that hair trace element analysis might be a diagnostic tool for epileptic patients (Ilhan et al., 1999). To illuminate if there is an additional role of nail trace element concentrations for the diagnosis of epilepsy, we had planned a clinical prospective study. Therefore, the aim of the present study is to enlighten whether there is a difference between serum and nail Cu, Zn, and Mn concentrations in patients with epilepsy and healthy subjects.
All of the materials (glass and plastic) used were thoroughly cleaned with a hot solution of nitric acid (20% v/v) for 48 h and rinsed three times with demineralized water and dried. Nail specimens were taken in the same day with blood sampling. In order to wipe out contaminating material such as dirtiness, nail specimens were put in an acetone/hexane (3/5, v/v) mixture for 24 h. Then they were washed several times with demineralized water and dried in an oven. After weighing the nail specimens, they were digested in a solution comprising concentrated nitric acid/perchloric acid mixture (1/5, v/v) by heating mildly until all perchloric acid fumes had disappeared and diluted with deionized water. All the element analyses were determined by atomic absorption spectrophotometer (AAS; Shimadzu AA-6701F) equipped
2. Methods 2.1. Subjects We studied 31 patients (16 females, 15 males), aged from 14 to 37 years, suffering from various types of epilepsy (11 focal epilepsy, 20 generalized epilepsy) and 19 healthy subjects (10 females, 9 males), aged from 16 to 36 years. The clinical diagnosis was made according to the criteria of the International League Against Epilepsy (Commission of the Classification and Terminology of the International League Against Epilepsy, 1981), and seizure type was classified based on the seizure description and electroencephalographic records. Epileptic patients with other diseases were excluded from the study. Routine laboratory tests were performed at the same time with blood and nail sampling. Informed consent was obtained from all the patients and healthy subjects. 2.2. Sample preparation and analytical methods Blood samples were taken in the morning (09:30 am) from the patients and controls approximately after 12 h fasting (before daily breakfast) between Monday and Friday. Ten milliliters of blood were taken from the antecubital veins of patients and controls without using anticoagulant and they were centrifuged at 3000 g for 15 min at room temperature. Serum samples were stored at 70 jC for the measurement of Cu, Zn, and Mn.
Fig. 1. Nail manganese (Mn), zinc (Zn), and copper (Cu) concentrations in epileptic patients and control groups. * P < .008 compared to control group. Bars represent means and error bars represent S.E.M.
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with a graphite furnace with deuterium background correction using a standard additional technique of Kirgbright (1980). Appropriate dilutions for different element analysis were also made before application of the samples to the AAS when needed. Each measurement was performed three times and averages were taken. Results were expressed in ppm for nail and in Ag/dl for serum. Serum Mn levels was expressed as Ag/l.
LSD. Bivariate comparisons were examined using Pearson rank correlation coefficients (r) and values were corrected for ties. Two-tailed significance values were used. Results were expressed as mean F S.E.M. P values < .05 were regarded as statistically significant.
2.3. Statistical analysis
The mean F S.E.M. values of Cu, Zn, and Mn concentrations in the nail and serum are shown in Figs. 1 and 2 for epilepsy patients and healthy controls. The concentration of Mn in nail from the patients was significantly higher than those of healthy control subjects ( P < .008) (Fig. 1). The main nail Zn and Cu levels were found to be unchanged in epileptic patients compared to control subjects. There was no significant differences in serum Mn and Zn levels between epileptic patients and control subjects. Likewise, the mean trace element levels in epileptics showed no significant difference according to gender and the type of seizure. On the other hand, there was statistically significant increase in serum Cu levels in patients with epilepsy in comparison with control group ( P < .009) (Fig. 2). In both study groups, the routine laboratory tests were normal.
Data were analyzed by using the SPSS for Windows computing program. One-way ANOVA tests were performed. Post Hoc multiple comparisons were done with
3. Results
4. Discussion 4.1. The relationships between trace elements and epilepsy In this study, we compared nail and serum Cu, Zn, and Mn levels of epilepsy patients and healthy controls in order to investigate the trace element status of the body. Trace elements in the hair and nail accumulate at concentrations higher than in blood and can reflect long-term variations in the corresponding blood levels (Hopps, 1977). Trace element levels are very low in serum but relatively high in hair and nail. It is important to emphasize that the serum is not suitable to determine the levels of some trace elements as serum element levels are widely variable in a day, even in an hour depending on food intake. Moreover, nail is a tissue that is easily sampled and transported and can be preserved indefinitely under appropriate conditions without deteriorating. Slow metabolic turnover rate of nail is an important source of knowledge about accumulation of elements over long periods of time (Akyol et al., 1997). For that reason, nail has been recognized as a valuable material for assessing trace elements. 4.2. The possible involvement of Mn and Cu in epilepsy
Fig. 2. Serum manganese (Mn), zinc (Zn), and copper (Cu) concentrations in epileptic patients and control groups. * P < .009 compared to control group. Bars represent means and error bars represent S.E.M.
The major finding of the present study is the increased nail Mn and serum Cu levels in patients with epilepsy compared to healthy controls. These results may indicate that Mn and Cu have significant impacts on the process of epilepsy. In the literature, low serum Mn levels were
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frequently reported in epileptic patients. High serum Mn levels were also reported in a few patients with seizure as case reports. Komaki et al. (1999) described a child having tremor and seizures because of Mn intoxication due to chronic total parenteral nutrition including Mn. They found that the blood Mn level was high and observed that the symptoms and MRI abnormalities disappeared after withdrawal of Mn administration. 4.3. The importance of Mn in the pathophysiology of epilepsy Methylcyclopentadienyl manganese tricarbonyl (MMT) is an organic Mn-containing compound, which is used as an additive in unleaded gasoline. One neurotoxic effect of MMT in mice is seizure activity. Fishman et al. (1987) have observed that MMT (organic manganese) or a closely related metabolite, not elemental Mn itself, is responsible for the seizure activity observed. They suggested that the seizure activity may be the result of an inhibitory effect of MMT at the GABA-A receptor linked chloride channel. Based on the growing body of evidence implicating reactive oxygen species (ROS) in both acute and chronic neurological disorders, several low molecular weight molecules that mimic antioxidant properties of endogenous enzymes have been developed and characterized, with an emphasis on superoxide dismutase (SOD) mimetics. On the other hand, Mn compose the prosthetic group of the major antioxidant enzyme SOD (Mn-SOD) localized in the cell mitochondria. Three major classes of these compounds are macrocyclic Mn complexes (Liu et al., 1994), salen – Mn complexes (Doctrow et al., 1997), and Mn porphyrins (Faulkner et al., 1994). The metalloporphyrin Mn tetrakis (4-benzoic acid) porphyrin (MnTBAP) possesses both SOD and catalase activity and has been shown to protect against peroxynitrite- and nitric oxide-induced suppression of mitochondrial respiration in J774 cells (Szabo et al., 1996), kainate- and paraquat-produced cell death in cortical neuron cultures (Patel et al., 1996), and hydrogen peroxide-mediated injury in endothelial cells (Faulkner et al., 1994). Evidence indicates, however, that metalloporphyrins such as MnTBAP do not cross the blood-brain barrier, which would hinder their use in treating neurological disorders. The macrocyclic Mn complexes have SOD activity, but not other ROS scavenging properties, and have shown efficacy in several biological models for disease (Riley and Weiss, 1994). The salen – manganese complexes might be used to prevent kainic acid-induced neuropathology (Rong et al., 1999). On the other hand, one of the superoxide dismutase/catalase mimetics, EUK143 (an analog of EUK-8), was found to reduced oxidative stress and neurotoxicity produced by 1-methyl-4-phenylpyridinium (MPP + ) and 6-hydroxydopamine (6-OHDA) in primary dopaminergic neuron cultures (Pong et al., 2000). These results suggest a potential role for synthetic SOD/
catalase mimetics as therapeutic agents against various neurological disorders. 4.4. Therapeutical approach to the epilepsy with trace elements It has been reported that supplementation, although developmentally encompassing and highly effective in elevating tissue Mn levels, had no effect on seizure latency or severity (Critchfield et al., 1993). Similarly, brain monoamine concentrations and glutamine synthetase activities were resistant to Mn supplementation. Our data show that Mn concentrations are elevated in nail of epileptic patients. This increased Mn concentration may be related to the increased antioxidant demand of these patients. We did not measure Mn-SOD activity in the present study; therefore, it will be very interesting to evaluate both Mn concentration and Mn-SOD activity together in epileptic patients in future studies. 4.5. The importance of Cu in the pathophysiology of epilepsy In the current study, there was statistically significant increase in serum Cu levels in patients with epilepsy in comparison with control group. Our findings were also consistent with previous studies investigating the possible involvement of Cu in seizure disorders. Copper and iron may participate in the Fenton reaction for the production of free radicals, but the roles of other trace elements in Fenton chemistry remain to be investigated. Horning et al. (2000) demonstrated that copper is a rapidly acting neurotoxin at physiological concentrations (10 –100 AM). Although little is known about the biochemical basis underlying Cu-mediated neurotoxicity, high concentrations of Cu have been shown to interfere with neuronal L-type calcium channels, which disrupts calcium homeostasis (Schulte et al., 1995). Because it is known that Cu ions have high affinities for certain amino acids (e.g., histidine), Cu can potentially bind to proteins and alter their structure and function. Furthermore, in contrast to Zn, Cu can enhance neurotoxicity indirectly by generating oxyradicals. Copper ions target thiol groups that are capable of reducing Cu + 2 to Cu + 1, which then can be reoxidized back to the Cu + 2 form in the presence of molecular oxygen. Subsequently, the molecular oxygen is converted to superoxide radical, which ultimately can lead to lipoperoxidation (Agarwal et al., 1989). Berg and Shi (1996) have suggested that the production of ROS underlies the role of Zn and Cu in some neurodegenerative conditions. 4.6. The importance of Zn in the pathophysiology of epilepsy We did not find any significant difference between serum and nail Zn levels of epileptic patients and controls at baseline
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evaluation. The discordance between Zn levels of serum at these studies might be resulted from the variable status of serum trace elements in a day. Therefore, we suggested that nail specimens would be a valuable material for such element analyses in epileptic patients to avoid false results. Although not statistically significant, we have found decreased Zn level in serum from patients with epilepsy ( P < .083). Alteration of Zn homeostasis in the brain may be associated with the etiology and manifestation of epileptic seizures (Sterman et al., 1988). Zinc plays an essential role in metalloenzyme function, control of gene transcription, and in neurotransmission and/or neuromodulation in the central nervous system (Frederickson and Moncrieff, 1994). In the brain, Zn is primarily localized in synaptic vesicles of glutamergic nerve terminals (Martinez-Guijarro et al., 1991; Perez-Clausell and Danscher, 1985) and is released during synaptic activity (Charton et al., 1985). Zinc inhibits Nmethyl-D-aspartate (NMDA) (Peters et al., 1987) and gamma-aminobutiric acid (GABA) (Christine and Choi, 1990) receptor-mediated currents and potentiates alpha-amino-3hydroxy-5-methyl-4-isoxazolepropionate (AMPA) (Westbrook and Mayer, 1987), glycine, and adenosine triphosphate (ATP) receptor-mediated currents (Smart et al., 1994). Also, Zn plays an important role in synthesizing GABA in neurons. The enhancement of Zn released from the brain may cause the decrease of GABA levels since Zn is associated with the activity of glutamic acid decarboxylase, which is necessary for GABA synthesis (Ebadi et al., 1984). Impairment of inhibitory synaptic transmission, mediated by GABA, may lead to an increase in susceptibility to seizures (Kamphuis et al., 1986). Zinc-containing terminals are prevalent in seizure-prone regions, such as the hippocampus where convulsant doses of kainic acid induce massive release and translocation of Zn from presynaptic boutons to postsynaptic neurons. The subcellular origin of this divalent cation appears to be glutamergic vesicles where histologically reactive Zn is colocalized and coreleased in response to potassium, electrical stimulation, seizure activity, and epilepsy. A relative deficiency of Zn ions in the presynaptic terminals of young rats could be responsible for increased seizure susceptibility since Zn ions may act as an anticonvulsant by limiting membrane depolarization by NMDA receptor activation (Fukahori et al., 1988). Consistent with that interpretation is the fact that young rats showed high susceptibility to kainate-induced seizure but revealed less neuronal death (Suh et al., 1996). Audiogenic seizure susceptibility in mice was increased by intracerebral injection of Zn (Chung and Johnson, 1983). On the other hand, audiogenic seizure activity was decreased by subcutaneous injection of Zn (Howell et al., 1986). Susceptibility to kindled seizures was decreased by dietary Zn loading, while this susceptibility in cats was increased by Zn deprivation (Sterman et al., 1986). Previous administration of Zn ions substantially reduces the frequency of noiseinduced tonic and clonic seizures and deaths in mice (Morton et al., 1990). These studies suggest that an optimal
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level of vesicular Zn ions protects the brain against seizure induction, while an excess amounts of Zn ions results in neuronal death (Suh et al., 1996). Thus, maintenance of Zn homeostasis in the brain may be important for prevention of seizure development, and a proper Zn supply to the brain may have a seizure-preventive effect. 4.7. What do the present findings mean? Our results demonstrate that some trace element levels may change in epileptic patients, and thus this changes may effect the neurological stability and neuronal conductivity. Although this study does not demonstrate a causal relation between some trace elements and seizure disorder and cannot be used as the basis for altering current approaches to therapy, it provides insight into the potentially important role of previously unrecognized factors in the pathophysiology. Studies are required to investigate the combined pathophysiological mechanisms of Mn and Cu pathogenicity.
5. Conclusion Our results demonstrate that some trace element levels may vary in epileptic patients suggesting a potential role of trace metals in the etiopathogenesis of epilepsy. Since variations in serum trace element status were reported in epileptic patients and because we found some differences in serum and nail trace element concentrations between patients with epilepsy and healthy controls, we conclude that nail trace element levels may be more reliable to demonstrate the trace element status in epileptic patients. These data need to be confirmed through the analysis of greater samples and it remains to be established, which are the mechanisms involved in the epileptogenesis. References Agarwal, K., Sharma, A., Talukder, G., 1989. Effects of copper on mammalian cell components. Chem. Biol. Interact. 69, 1 – 16. Akyol, O., Ersan, F., Akcay, F., Altuntas, I., Senol, M., Sasmaz, S., Yasar, A., 1997. Hair, nail, serum, and urine copper levels in users of copper intrauterine devices and interactions between copper and some other trace elements. Trace Elem. Electrolytes 14, 124 – 129. Berg, J.M., Shi, Y., 1996. The galvanization of biology: a growing appreciation for the roles of zinc. Science 271, 1081 – 1085. Carl, G.F., Keen, C.L., Gallagher, B.B., Clegg, M.S., Littleton, W.H., Flannery, D.B., Hurley, L.S., 1986. Association of low blood manganese concentrations with epilepsy. Neurology 36, 1584 – 1587. Carl, G.F., Critchfield, L.W., Thompson, J.L., McGinnis, L.S., Wheeler, G.A., Gallagher, B.B., Holmes, G.L., Hurley, L.S., Keen, C., 1989. Effect of kainate-induced seizures on tissue trace element concentrations in the rat. Neuroscience 33, 223 – 227. Carl, G., Critchfield, J.W., Thompson, J.L., Holmes, G.L., Gallagher, B.B., Keen, C.L., 1990. Genetically epilepsyprone rats are characterized by altered tissue trace element concentrations. Epilepsia 31, 247 – 252. Charton, G., Rovira, C., Ben-Ari, Y., Leviel, V., 1985. Spontaneous and evoked release of endogenous Zn in the hippocampal mossy fiber zone of the rat in situ. Exp. Brain Res. 58, 202 – 205.
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The comparison of nail and serum trace elements in patients with epilepsy and healthy subjects ¨ zerolb, Mukaddes Gu¨lecßb, Bu¨nyamin Isßıkc, Nevin ˙Ilhand, Atilla ˙Ilhana,*, Elif O ¨ mer Akyolb Necip ˙Ilhan d, O a
Department of Neurology, I˙no¨nu¨ University Medical School, 44069, Malatya, Turkey b Department of Biochemistry, I˙no¨nu¨ University Medical School, Malatya, Turkey c Department of Family Medicine, Fatih University Medical School, Ankara, Turkey d Department of Biochemistry, FI˙rat University Medical School, Elazıg˘, Turkey Accepted 17 September 2003
Abstract The objective of this prospective study was to determine the levels of manganese (Mn), copper (Cu), and zinc (Zn) levels in both nail and serum from patients with epilepsy. For this purpose, levels of these elements were measured in 31 patients with epilepsy and 19 healthy subjects. Element analyses were carried out by atomic absorption spectrophotometer (AAS). Increased Mn levels were detected in nail of patients with epilepsy compared to healthy controls ( P < .008). The main nail Zn and Cu levels were found to be unchanged in epileptic patients compared to control subjects. There were no significant differences in serum Mn and Zn levels between epileptic patients and control subjects. However, there was a statistically significant increase in serum Cu levels in patients with epilepsy in comparison with control group ( P < .009). Our results demonstrate that some trace element levels may vary in epileptic patients, and because of the more stable status, the analysis of these element levels in some tissues such as nail might be superior to serum analysis. D 2003 Elsevier Inc. All rights reserved. Keywords: Epilepsy; Nail; Serum; Copper; Zinc; Manganese
1. Introduction It is widely accepted that trace elements play an important role in various metabolic processes. Zinc (Zn), copper (Cu), and manganese (Mn) are known to be necessary for the activity of some enzymes. In recent years, it has been suggested that epileptic patients might show some abnormalities in serum trace element concentrations (Ilhan et al., 1999; Davidson and Ward, 1988; Ghose and Taylor, 1983; Palm and Hallmans, 1982; Werther et al., 1986; Kurekci et Abbreviations: AMPA, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate; ATP, adenosine triphosphate; GABA, gamma-aminobutiric acid; MMT, methylcyclopentadienyl manganese tricarbonyl; MnTBAP, Mn tetrakis (4-benzoic acid) porphyrin; MPP+, 1-methyl-4-phenylpyridinium; NMDA, N-methyl-D-aspartate; 6-OHDA, 6-hydroxydopamine; ROS, reactive oxygen species; S.E.M., standard error of mean; SOD, superoxide dismutase. * Corresponding author. Tel.: +90-422-341-06-60/4903; fax: +90-422341-07-29. E-mail address: [email protected] (A. I˙lhan). 0278-5846/$ – see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2003.09.025
al., 1995). Seizure activity in epileptic patients and experimental models of epilepsy influences the metabolism of essential trace elements, e.g., zinc, iron, copper, and manganese, in the brain and peripheral tissues (Papavasiliou and Miller, 1983; Carl et al., 1989; Carl et al., 1990). Thus, it is important to clarify whether the concentration of essential elements in the brain is altered by enhancement of susceptibility to seizures or epileptic neuronal activity. However, the relationship between movement of essential trace elements and seizure activity is poorly understood. Although many authors have studied on the relationship between some trace elements and epilepsy, there are conflicting reports in the literature on this issue. The low Mn concentration is attributed by some investigators to the seizure activity associated with the epilepsy, whereas others propose that the low Mn concentration may be secondary to genetic mechanisms underlying the epilepsy. Chronic seizures caused a decrease in whole blood Mn levels but did not affect brain Mn concentrations. In a study (Carl et al., 1986), comparison of hospitalized epileptic
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patients with matched normals showed that the mean whole blood Mn concentration of the epileptic population was significantly lower than those of the normal population. The whole blood Mn concentration in the epileptics did not correlate either with seizure frequency or with anticonvulsant therapy. It was observed, however, that the patients whose epilepsy was a result of trauma had significantly higher blood Mn concentrations than patients whose history was negative for trauma (Carl et al., 1986). Some authors reported that epileptic patients have higher serum Zn concentrations than controls (Davidson and Ward, 1988; Ghose and Taylor, 1983; Werther et al., 1986; Kurekci et al., 1995; Kuzuya et al., 1993); while some others have observed normal (Higashi et al., 1982; Schott and Delves, 1978) and even low zinc concentrations (Palm and Hallmans, 1982) in epileptic patients under classical treatment. The authors have previously suggested that hair trace element analysis might be a diagnostic tool for epileptic patients (Ilhan et al., 1999). To illuminate if there is an additional role of nail trace element concentrations for the diagnosis of epilepsy, we had planned a clinical prospective study. Therefore, the aim of the present study is to enlighten whether there is a difference between serum and nail Cu, Zn, and Mn concentrations in patients with epilepsy and healthy subjects.
All of the materials (glass and plastic) used were thoroughly cleaned with a hot solution of nitric acid (20% v/v) for 48 h and rinsed three times with demineralized water and dried. Nail specimens were taken in the same day with blood sampling. In order to wipe out contaminating material such as dirtiness, nail specimens were put in an acetone/hexane (3/5, v/v) mixture for 24 h. Then they were washed several times with demineralized water and dried in an oven. After weighing the nail specimens, they were digested in a solution comprising concentrated nitric acid/perchloric acid mixture (1/5, v/v) by heating mildly until all perchloric acid fumes had disappeared and diluted with deionized water. All the element analyses were determined by atomic absorption spectrophotometer (AAS; Shimadzu AA-6701F) equipped
2. Methods 2.1. Subjects We studied 31 patients (16 females, 15 males), aged from 14 to 37 years, suffering from various types of epilepsy (11 focal epilepsy, 20 generalized epilepsy) and 19 healthy subjects (10 females, 9 males), aged from 16 to 36 years. The clinical diagnosis was made according to the criteria of the International League Against Epilepsy (Commission of the Classification and Terminology of the International League Against Epilepsy, 1981), and seizure type was classified based on the seizure description and electroencephalographic records. Epileptic patients with other diseases were excluded from the study. Routine laboratory tests were performed at the same time with blood and nail sampling. Informed consent was obtained from all the patients and healthy subjects. 2.2. Sample preparation and analytical methods Blood samples were taken in the morning (09:30 am) from the patients and controls approximately after 12 h fasting (before daily breakfast) between Monday and Friday. Ten milliliters of blood were taken from the antecubital veins of patients and controls without using anticoagulant and they were centrifuged at 3000 g for 15 min at room temperature. Serum samples were stored at 70 jC for the measurement of Cu, Zn, and Mn.
Fig. 1. Nail manganese (Mn), zinc (Zn), and copper (Cu) concentrations in epileptic patients and control groups. * P < .008 compared to control group. Bars represent means and error bars represent S.E.M.
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with a graphite furnace with deuterium background correction using a standard additional technique of Kirgbright (1980). Appropriate dilutions for different element analysis were also made before application of the samples to the AAS when needed. Each measurement was performed three times and averages were taken. Results were expressed in ppm for nail and in Ag/dl for serum. Serum Mn levels was expressed as Ag/l.
LSD. Bivariate comparisons were examined using Pearson rank correlation coefficients (r) and values were corrected for ties. Two-tailed significance values were used. Results were expressed as mean F S.E.M. P values < .05 were regarded as statistically significant.
2.3. Statistical analysis
The mean F S.E.M. values of Cu, Zn, and Mn concentrations in the nail and serum are shown in Figs. 1 and 2 for epilepsy patients and healthy controls. The concentration of Mn in nail from the patients was significantly higher than those of healthy control subjects ( P < .008) (Fig. 1). The main nail Zn and Cu levels were found to be unchanged in epileptic patients compared to control subjects. There was no significant differences in serum Mn and Zn levels between epileptic patients and control subjects. Likewise, the mean trace element levels in epileptics showed no significant difference according to gender and the type of seizure. On the other hand, there was statistically significant increase in serum Cu levels in patients with epilepsy in comparison with control group ( P < .009) (Fig. 2). In both study groups, the routine laboratory tests were normal.
Data were analyzed by using the SPSS for Windows computing program. One-way ANOVA tests were performed. Post Hoc multiple comparisons were done with
3. Results
4. Discussion 4.1. The relationships between trace elements and epilepsy In this study, we compared nail and serum Cu, Zn, and Mn levels of epilepsy patients and healthy controls in order to investigate the trace element status of the body. Trace elements in the hair and nail accumulate at concentrations higher than in blood and can reflect long-term variations in the corresponding blood levels (Hopps, 1977). Trace element levels are very low in serum but relatively high in hair and nail. It is important to emphasize that the serum is not suitable to determine the levels of some trace elements as serum element levels are widely variable in a day, even in an hour depending on food intake. Moreover, nail is a tissue that is easily sampled and transported and can be preserved indefinitely under appropriate conditions without deteriorating. Slow metabolic turnover rate of nail is an important source of knowledge about accumulation of elements over long periods of time (Akyol et al., 1997). For that reason, nail has been recognized as a valuable material for assessing trace elements. 4.2. The possible involvement of Mn and Cu in epilepsy
Fig. 2. Serum manganese (Mn), zinc (Zn), and copper (Cu) concentrations in epileptic patients and control groups. * P < .009 compared to control group. Bars represent means and error bars represent S.E.M.
The major finding of the present study is the increased nail Mn and serum Cu levels in patients with epilepsy compared to healthy controls. These results may indicate that Mn and Cu have significant impacts on the process of epilepsy. In the literature, low serum Mn levels were
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frequently reported in epileptic patients. High serum Mn levels were also reported in a few patients with seizure as case reports. Komaki et al. (1999) described a child having tremor and seizures because of Mn intoxication due to chronic total parenteral nutrition including Mn. They found that the blood Mn level was high and observed that the symptoms and MRI abnormalities disappeared after withdrawal of Mn administration. 4.3. The importance of Mn in the pathophysiology of epilepsy Methylcyclopentadienyl manganese tricarbonyl (MMT) is an organic Mn-containing compound, which is used as an additive in unleaded gasoline. One neurotoxic effect of MMT in mice is seizure activity. Fishman et al. (1987) have observed that MMT (organic manganese) or a closely related metabolite, not elemental Mn itself, is responsible for the seizure activity observed. They suggested that the seizure activity may be the result of an inhibitory effect of MMT at the GABA-A receptor linked chloride channel. Based on the growing body of evidence implicating reactive oxygen species (ROS) in both acute and chronic neurological disorders, several low molecular weight molecules that mimic antioxidant properties of endogenous enzymes have been developed and characterized, with an emphasis on superoxide dismutase (SOD) mimetics. On the other hand, Mn compose the prosthetic group of the major antioxidant enzyme SOD (Mn-SOD) localized in the cell mitochondria. Three major classes of these compounds are macrocyclic Mn complexes (Liu et al., 1994), salen – Mn complexes (Doctrow et al., 1997), and Mn porphyrins (Faulkner et al., 1994). The metalloporphyrin Mn tetrakis (4-benzoic acid) porphyrin (MnTBAP) possesses both SOD and catalase activity and has been shown to protect against peroxynitrite- and nitric oxide-induced suppression of mitochondrial respiration in J774 cells (Szabo et al., 1996), kainate- and paraquat-produced cell death in cortical neuron cultures (Patel et al., 1996), and hydrogen peroxide-mediated injury in endothelial cells (Faulkner et al., 1994). Evidence indicates, however, that metalloporphyrins such as MnTBAP do not cross the blood-brain barrier, which would hinder their use in treating neurological disorders. The macrocyclic Mn complexes have SOD activity, but not other ROS scavenging properties, and have shown efficacy in several biological models for disease (Riley and Weiss, 1994). The salen – manganese complexes might be used to prevent kainic acid-induced neuropathology (Rong et al., 1999). On the other hand, one of the superoxide dismutase/catalase mimetics, EUK143 (an analog of EUK-8), was found to reduced oxidative stress and neurotoxicity produced by 1-methyl-4-phenylpyridinium (MPP + ) and 6-hydroxydopamine (6-OHDA) in primary dopaminergic neuron cultures (Pong et al., 2000). These results suggest a potential role for synthetic SOD/
catalase mimetics as therapeutic agents against various neurological disorders. 4.4. Therapeutical approach to the epilepsy with trace elements It has been reported that supplementation, although developmentally encompassing and highly effective in elevating tissue Mn levels, had no effect on seizure latency or severity (Critchfield et al., 1993). Similarly, brain monoamine concentrations and glutamine synthetase activities were resistant to Mn supplementation. Our data show that Mn concentrations are elevated in nail of epileptic patients. This increased Mn concentration may be related to the increased antioxidant demand of these patients. We did not measure Mn-SOD activity in the present study; therefore, it will be very interesting to evaluate both Mn concentration and Mn-SOD activity together in epileptic patients in future studies. 4.5. The importance of Cu in the pathophysiology of epilepsy In the current study, there was statistically significant increase in serum Cu levels in patients with epilepsy in comparison with control group. Our findings were also consistent with previous studies investigating the possible involvement of Cu in seizure disorders. Copper and iron may participate in the Fenton reaction for the production of free radicals, but the roles of other trace elements in Fenton chemistry remain to be investigated. Horning et al. (2000) demonstrated that copper is a rapidly acting neurotoxin at physiological concentrations (10 –100 AM). Although little is known about the biochemical basis underlying Cu-mediated neurotoxicity, high concentrations of Cu have been shown to interfere with neuronal L-type calcium channels, which disrupts calcium homeostasis (Schulte et al., 1995). Because it is known that Cu ions have high affinities for certain amino acids (e.g., histidine), Cu can potentially bind to proteins and alter their structure and function. Furthermore, in contrast to Zn, Cu can enhance neurotoxicity indirectly by generating oxyradicals. Copper ions target thiol groups that are capable of reducing Cu + 2 to Cu + 1, which then can be reoxidized back to the Cu + 2 form in the presence of molecular oxygen. Subsequently, the molecular oxygen is converted to superoxide radical, which ultimately can lead to lipoperoxidation (Agarwal et al., 1989). Berg and Shi (1996) have suggested that the production of ROS underlies the role of Zn and Cu in some neurodegenerative conditions. 4.6. The importance of Zn in the pathophysiology of epilepsy We did not find any significant difference between serum and nail Zn levels of epileptic patients and controls at baseline
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evaluation. The discordance between Zn levels of serum at these studies might be resulted from the variable status of serum trace elements in a day. Therefore, we suggested that nail specimens would be a valuable material for such element analyses in epileptic patients to avoid false results. Although not statistically significant, we have found decreased Zn level in serum from patients with epilepsy ( P < .083). Alteration of Zn homeostasis in the brain may be associated with the etiology and manifestation of epileptic seizures (Sterman et al., 1988). Zinc plays an essential role in metalloenzyme function, control of gene transcription, and in neurotransmission and/or neuromodulation in the central nervous system (Frederickson and Moncrieff, 1994). In the brain, Zn is primarily localized in synaptic vesicles of glutamergic nerve terminals (Martinez-Guijarro et al., 1991; Perez-Clausell and Danscher, 1985) and is released during synaptic activity (Charton et al., 1985). Zinc inhibits Nmethyl-D-aspartate (NMDA) (Peters et al., 1987) and gamma-aminobutiric acid (GABA) (Christine and Choi, 1990) receptor-mediated currents and potentiates alpha-amino-3hydroxy-5-methyl-4-isoxazolepropionate (AMPA) (Westbrook and Mayer, 1987), glycine, and adenosine triphosphate (ATP) receptor-mediated currents (Smart et al., 1994). Also, Zn plays an important role in synthesizing GABA in neurons. The enhancement of Zn released from the brain may cause the decrease of GABA levels since Zn is associated with the activity of glutamic acid decarboxylase, which is necessary for GABA synthesis (Ebadi et al., 1984). Impairment of inhibitory synaptic transmission, mediated by GABA, may lead to an increase in susceptibility to seizures (Kamphuis et al., 1986). Zinc-containing terminals are prevalent in seizure-prone regions, such as the hippocampus where convulsant doses of kainic acid induce massive release and translocation of Zn from presynaptic boutons to postsynaptic neurons. The subcellular origin of this divalent cation appears to be glutamergic vesicles where histologically reactive Zn is colocalized and coreleased in response to potassium, electrical stimulation, seizure activity, and epilepsy. A relative deficiency of Zn ions in the presynaptic terminals of young rats could be responsible for increased seizure susceptibility since Zn ions may act as an anticonvulsant by limiting membrane depolarization by NMDA receptor activation (Fukahori et al., 1988). Consistent with that interpretation is the fact that young rats showed high susceptibility to kainate-induced seizure but revealed less neuronal death (Suh et al., 1996). Audiogenic seizure susceptibility in mice was increased by intracerebral injection of Zn (Chung and Johnson, 1983). On the other hand, audiogenic seizure activity was decreased by subcutaneous injection of Zn (Howell et al., 1986). Susceptibility to kindled seizures was decreased by dietary Zn loading, while this susceptibility in cats was increased by Zn deprivation (Sterman et al., 1986). Previous administration of Zn ions substantially reduces the frequency of noiseinduced tonic and clonic seizures and deaths in mice (Morton et al., 1990). These studies suggest that an optimal
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level of vesicular Zn ions protects the brain against seizure induction, while an excess amounts of Zn ions results in neuronal death (Suh et al., 1996). Thus, maintenance of Zn homeostasis in the brain may be important for prevention of seizure development, and a proper Zn supply to the brain may have a seizure-preventive effect. 4.7. What do the present findings mean? Our results demonstrate that some trace element levels may change in epileptic patients, and thus this changes may effect the neurological stability and neuronal conductivity. Although this study does not demonstrate a causal relation between some trace elements and seizure disorder and cannot be used as the basis for altering current approaches to therapy, it provides insight into the potentially important role of previously unrecognized factors in the pathophysiology. Studies are required to investigate the combined pathophysiological mechanisms of Mn and Cu pathogenicity.
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