Manuscript accepted on :19-05-2023
Published online on: 26-06-2023
Plagiarism Check: Yes
Reviewed by: Dr. Sunil Jagtap and Dr. Reya Raaj
Second Review by: Dr. Hind Shakir
Final Approval by: Dr. Patorn Promchai
Moushira M. Zaki1, Rehab S.I. Moustafa3 , Mones M. Abu Shady3 , Ahmed Helal El Sayed4 and Eman R. Youness2*
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
Download this article as: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 type2,3 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) |
seizure free (n=30) |
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) |
Children with drug resistant epilepsy (n=30) |
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 |
Discussion
Proinflammatory mediators couldamendexcitability of the neurons and affect neurotransmission causingreductionin the seizures threshold and increase neuronal damage7,8.Cytokines have been involved as inhibitors and mediators of various forms of neurodegeneration9,10,11. 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 receptors13–15.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 interventions17,18
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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