Evaluation of Acetylcholinesterase and Acetylcholine Levels in Children with Idiopathic Epilepsy

Objective of this work was to assess whether acetylcholinesterase and acetylcholine, levels that can be used as biomarkers for drug-resistant epilepsy in children with idiopathic epilepsy. Acetylcholinesterase and acetylcholine levels were measured in three groups of children, 30 children with drug resistant epilepsy,30 with seizures free and30 age and sex matched healthy children. Significant lower acetylcholinesterase was found in drug resistant epilepsy compared to seizure free epilepsy and healthy controls. Higher acetylcholine levels was found in seizure free epilepsy compared to drug resistant epilepsy and healthy controls. Stepwise linear regression analysis showed that low ACHE, high ACH, high severity score are significant independent factors associated with idiopathic epilepsy. Moreover, Receiver Operating Characteristic (ROC) analysis showed that severity score at cutoff of Chalfont score>60 had the highest sensitivity 86.7% and specificity 80% followed by serum ACHE at cutoff <3.212(ng/ml) with sensitivity 70% and specificity 100% and then serum ACH at cutoff >18.410(ng/ml)with sensitivity 70% and specificity 83.3% as predictors for idiopathic epilepsy. Increased circulating level of ACHand decreased ACHE may predict idiopathic epilepsy suggesting their role in the childhood idiopathic epilepsy’spathogenesis.

Epilepsy affected the development of learning and cognitive functions via a number of numerous factors: etiology 1 age of onset, seizure type 2,3 duration and severity, interictal epileptic form discharges, 4 drug treatment. Epilepsy is a devastating neurological and systemic disorder characterized by recurrent seizures 5 . Despite the rapid progression in clinical and pre-clinical epilepsy research, the pathogenesis of epilepsy still remains elusive Acetylcholinesterase (AChE) has a significant role in the pathogenesis of neurodegenerative diseases by inducing aggregation of pathological proteins, oxidative stress, apoptosis, and inflammatory response. Diminished AChE concentrations cause irregularly augmented concentrations of ACH in cholinergic synapses, producing unnecessary nicotinic receptors and muscarinic stimulation 6 . Researches associated to ACh on the vagus nerve for epilepsy were imperative to elucidate inflammations in epilepsy. The present work will explore the predictive role of AChEand ACH for idiopathic epilepsy and how it is related to clinical features of the patients.

Subjects
The study comprised three groups of children, 30 children with drug resistant epilepsy, 30 with seizures free and 30 age and sex matched healthy children.

Methods assessment of acetyl cholinesterase activity (ache)
Acetyl cholinesterase Activity was assessed by a double-antibody sandwich enzymelinked immunosorbent assay ELISA kit from Shanghai Biovision Co., Ltd, Jufengyuan Road, Baoshan District, Shanghai.

assessment of acetylcholine activity
The technique was designated for reversed-phase HPLC separation of acetylcholine of their homologues in serum, united with post columnfluorometric quantification and enzymatic derivatization. The separation happens on a polymeric resin derivatized with hydrophobic moiety and the mobile phase comprises Na 2 HPO 4 . 3-(p-hydroxyphenyl) sodium dodecylsulphate; propionate and post column enzyme reactor comprises immobilized choline oxidase, peroxidase and acetyl cholinesterase. Method is well suited for non-attended automatic operation and free of encountered interferences with electrochemical detection.

ethical approval
This research was approved by the Ethical Committee of Al-Azhar University (No: 00782) and followed the World Medical Association's Declaration of Helsinki. Furthermore, each participant in the study signed a written consent after a full description of the study.

Statistical analysis
Statistical analysis was performed using SPSS version 21 for windows. Data were expressed as mean ± standard deviation and compared to t-test to compare between two groups. ANOVA and post hoc tests for comparing between more than 2 groups. Data were expressed as percentages and frequencies, and were analyzed with the two-tailed chi square test.

ReSultS
Chalfont severity score was significantly increases in drug resistance epilepsy cases than seizure free and controls (Table 1). Significant lower levels of AChE was found in drug resistant epilepsy compared to seizure free epilepsy and healthy controls (p < 0.05). Lower ACH levels was found in seizure free epilepsy compared to drug resistant epilepsy and healthy controls (p < 0.05) ( Table 2). Stepwise linear regression analysis showed that ACHE, ACH, severity score are significant independent factors associated with idiopathic epilepsy (Table 3). Moreover, Receiver Operating Characteristic (ROC analysis showed that severity score at cutoff of Chlfont score>60 had the highest sensitivity 86.7% and specificity 80% followed by serum ACHE at cutoff < 3.212 (ng/ ml) with sensitivity 70% and specificity 100% and then serum ACH at cutoff > 18.410 (ng/ml) with sensitivity 70% and specificity 83.3% as predictors for idiopathic epilepsy.

diScuSSion
Proinflammatory mediators could amendexcitability of the neurons and affect neurotransmission causing reduction in the seizures threshold and increase neuronal damage 7,8 .Cytokines have been involved as inhibitors and mediators of various forms of neurodegeneration 9,10,11 . Proinflammatory cytokine in the innate immune response modulates fundamental processes in the brain 10,12 .  The influence of neuromodulators implicated in the impulses transmission on the pathogenesis is of immense significanceas epileptic seizures happen with the disturbance of the inhibitory-excitatory balance in the brain. Consequently, mutations/disorders of the elements at the ion channel level and receptor might causestimulus transmission abnormality and epileptic discharges. Diminished levels of AChE cause abnormally elevated levels of ACH in cholinergicsynapses, leading to exaggerated stimulation of nicotinic and muscarinic receptors [13][14][15] . Neuro transmitters mainly ACH has been involved in the epilepsy's pathogenes is as proved via their amendment in pre-clinical model of epileptic seizure 16 . Amongst the various participating influences underlying the seizures generation's mechanism, the role of neurotransmitters has been involved in the same. Neurotransmitters are endogenous constituents that transfer signals across the synapse and controlexcitatory/ inhibitory neuronal functions through fastening to their particular receptors. Usually it is stored in axon terminals, synaptic vesicles and secreted into the synapse following anappropriate signal. Liberated neuro transmitters carry out the connecting functions over the synaptic cleft and fastening to particular receptors. Gut microbiota could modulate brain behavior and function and is highly documented as an imperative factor in mediating the risk of epilepsy and the impacts of seizure interventions 17,18 concluSion In conclusion, investigating the relations of ACH and AChE that are the major actors in epilepsy has become an imperative aim to elucidate the underlying pathology in neurological disorders.

Conflicts of Interest
None.