Moushira M. Zaki¹, Rehab S.I. Moustafa³ , Mones M. Abu Shady³ , Ahmed Helal El Sayed⁴ and Eman R. Youness^2*

¹Biological Anthropology Department, National Research Centre, Cairo, Egypt.

²Medical Biochemistry Department, Medical Research Institute, National Research Centre, Cairo, Egypt.

³Child Health Department, Medical Research and Clinical Studies Institute, National Research Centre, Cairo, Egypt.

⁴Pediatric Department, Faculty of Medicine for Boys, Al-Azhar University, Cairo, Egypt.

Corresponding Author E-mail: hoctober2000@yahoo.com

DOI : https://dx.doi.org/10.13005/bpj/2653

Abstract

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. Methods: 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. Results: 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. Conclusion: Increased circulating level of ACHand decreased ACHE may predict idiopathic epilepsy suggesting their role in the childhood idiopathic epilepsy’spathogenesis

Keywords

Acetyl cholinesterase; acetylcholine; idiopathic epilepsy

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Copy the following to cite this article: Zaki M. M, Moustafa R. S. I, Shady M. M. A, Sayed A. H. E, Youness E. R. Evaluation of Acetylcholinesterase and Acetylcholine Levels in Children with Idiopathic Epilepsy. Biomed Pharmacol J 2023;16(2).

Copy the following to cite this URL: Zaki M. M, Moustafa R. S. I, Shady M. M. A, Sayed A. H. E, Youness E. R. Evaluation of Acetylcholinesterase and Acetylcholine Levels in Children with Idiopathic Epilepsy. Biomed Pharmacol J 2023;16(2). Available from: https://bit.ly/42TjLNe

Introduction

Epilepsy affected the development of learning and cognitive functionsvia a number of numerous factors: etiology ¹ age of onset, seizure type^2,3 duration and severity, interictal epileptic form discharges,⁴drug treatment. Epilepsy is a devastating neurological and systemic disorder characterized by recurrent seizures⁵. 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 and Methods 

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 sandwichenzyme-linked immunosorbent assay ELISA kit from Shanghai Biovision Co., Ltd, Jufengyuan Road, Baoshan District, Shanghai.

Assessment of acetylcholine Activity

Thetechniquewasdesignated 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 Na₂HPO₄. 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[MF6]  (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.

Table 1: Electroencephalographic findings and clinical data in children with drug resistant epilepsy & seizure free children.

	drug resistant epilepsy (n=30) Mean ± SD	seizure free (n=30) Mean ± SD	Independent T test/ chi square test
			t/x2	p-value
Age (years)	9.013±1.426	9.277±0.871	-0.863	0.392
Gender (N, %) Male Female	20 (66.7%) 10 (33.3%)	22 (73.3%) 8 (26.7%)	0.317	0.573
Age of onset of seizures (years)	3.233±1.670	3.783±2.104	1.122	0.267
Duration of disease (years)	5.687±1.523	6.667±3.384	1.446	0.153
Chalfont severity score	91.733±20.120	48.867±16.950	8.925	<0.0001
Type of epilepsy (N, %) Focal Generalized Focal with secondary generalization	22 (73.3%) 6 (20%) 2 (6.7%)	13 (43.3%) 13 (43.3%) 4 (12.3%)	5.560	0.062

*p < 0.05

Table 2: AChE and ACH serum levels in epilepticchildren and healthy children.

	Seizure free Children (n=30) Mean ± SD	Children with drug resistant epilepsy (n=30) Mean ± SD	Healthy children (n=30) Mean ± SD	ANOVA test
				F	p-value
(ng/ml)	3.646±6.601	1.969±1.129	4.550±2.870	71.656	<0.0001**
ACH (ng/ml)	29.705±18.042	12.818±5.253	10.065±7.811	23.755	<0.0001**
Post hoc analysis
	Seizure free vs drug resistantepilepsy	Seizure freevshealthy children	drug resistant epilepsy vs healthy children
AChE(ng/ml)	<0.0001*	0.202	<0.01**
ACH (ng/ml)	<0.0001*	0.375	<0.01**

**p < 0.01

Table 3: Liner stepwise regression analysis for prediction of drug resistantepilepsy in idiopathic epileptic children.

Coefficients		Unstandardized Coefficients		Standardized Coefficients	t	P-value	95.0% Confidence Interval for B
Model
		B	Std. Error	Beta			Lower Bound	Upper Bound
1	(Constant)	3.449	0.115		30.108	<0.0001*	3.22	3.678
	score	-0.013	0.002	-0.761	-8.925	<0.0001*	-0.017	-0.01
2	(Constant)	3.421	0.102		33.625	<0.0001*	3.217	3.625
	score	-0.008	0.002	-0.467	-4.492	<0.0001*	-0.012	-0.005
	ACHE	-0.03	0.007	-0.427	-4.105	<0.0001*	-0.045	-0.015
3	(Constant)	3.393	0.097		35.093	<0.0001*	3.199	3.586
	score	-0.007	0.002	-0.412	-4.112	<0.0001*	-0.011	-0.004
	ACHE	-0.059	0.013	-0.846	-4.723	<0.0001*	-0.084	-0.034
	ACH	0.014	0.005	0.433	2.799	0.007*	0.004	0.024

*p < 0.05

Table 4: Predictive values, specificity and Sensitivity for prediction of drug resistant epilepsy in babies with idiopathic epilepsy

variables	AUC	Cutoff point	Sensitivity	Specificity	95% Confidence Interval
					Lower Bound	Upper Bound
Chalfont score	0.943	>60	86.7%	80%	0.892	0.994
Serum ACHE (ng/ml)	0.995	<3.212	70%	100%	0.984	1.000
Serum ACH (ng/ml)	0.786	>18.410	70%	83.3%	0.659	0.913

AUC: area under curve;

Figure 1: ROC (Receiver Operating Characteristic) curve for predictors of drug resistant epilepsy in children with idiopathic epilepsy Click here to view Figure

Discussion

Proinflammatory mediators couldamendexcitability of the neurons and affect neurotransmission causingreductionin 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. Pro-inflammatory 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 immensesignificanceas 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 mightcausestimulus transmission abnormality and epileptic discharges. Diminished levels of AChEcause abnormally elevatedlevels of ACH in cholinergicsynapses, leading toexaggerated stimulation of nicotinic and muscarinic receptors^13–15.Neurotransmitters mainly ACH has been involved in the epilepsy’s pathogenesis as proved via their amendment in pre-clinical model of epileptic seizure¹⁶.Amongst the variousparticipating 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 followinganappropriate signal. Liberatedneurotransmitterscarry out the connecting functions over the synaptic cleft and fastening to particular receptors. Gut microbiotacould modulate brain behavior and function and is highlydocumented 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

There are no conflict of interest.

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