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).
Manuscript received on :28-03-2023
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

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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

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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).

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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)
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 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.

References

  1. Beghi E, Cornaggia CM, Elia M. Proposal for a multicenter study on epilepsy and learning disorders in children. Epilepsia. 2001;42(1):10-12.
    CrossRef
  2. Bourgeois BFD, Prensky AL, Palkes HS, Talent BK, Busch SG. Intelligence in epilepsy: a prospective study in children. Ann Neurol Off J Am Neurol Assoc Child Neurol Soc. 1983;14(4):438-444.
    CrossRef
  3. Holmes GL. Pathogenesis of learning disabilities in epilepsy. Epilepsia. 2001;42:13-15.
    CrossRef
  4. Binnie CD. Cognitive performance, subtle seizures, and the EEG. Epilepsia. 2001;42:16-18.
    CrossRef
  5. Devinsky O, Vezzani A, O’Brien TJ, et al. Epilepsy Nat Rev Dis Primers 4: 18024. 2018.
    CrossRef
  6. Aboul Ezz HS, Khadrawy YA, Noor NA. The neuroprotective effect of curcumin and Nigella sativa oil against oxidative stress in the pilocarpine model of epilepsy: a comparison with valproate. Neurochem Res. 2011;36(11):2195-2204.
    CrossRef
  7. Vezzani A, Fujinami RS, White HS, et al. Infections, inflammation and epilepsy. Acta Neuropathol. 2016;131(2):211-234.
    CrossRef
  8. Suleymanova EM. Behavioral comorbidities of epilepsy and neuroinflammation: Evidence from experimental and clinical studies. Epilepsy Behav. 2021;117:107869. doi:https://doi.org/10.1016/ j.yebeh.2021.107869
    CrossRef
  9. Allan SM, Rothwell NJ. Cytokines and acute neurodegeneration. Nat Rev Neurosci. 2001;2(10):734-744.
    CrossRef
  10. Montgomery SL, Bowers WJ. Tumor necrosis factor-alpha and the roles it plays in homeostatic and degenerative processes within the central nervous system. J neuroimmune Pharmacol. 2012;7(1):42-59.
    CrossRef
  11. Rothwell NJ. The role of cytokines in neurodegeneration. In: Cytokines in the Nervous System. Springer; 1996:145-162.
    CrossRef
  12. Zalpoor H, Akbari A, Samei A, et al. The roles of Eph receptors, neuropilin-1, P2X7, and CD147 in COVID-19-associated neurodegenerative diseases: inflammasome and JaK inhibitors as potential promising therapies. Cell Mol Biol Lett. 2022;27(1):1-21.
    CrossRef
  13. Chen ET, Thornton JT, Mulchi Jr C. Early forming a hummingbird-like hovering neural network circuitry pattern with reentrant spatiotemporal energy-sensory orientation privileged to avoid “Epilepsy” based on a biomimetic acetylcholinesterase memcapacitor prosthesis. Sensors & Transducers. 2015;191(8):84-99.
  14. Mishra A, Goel RK. Psychoneurochemical investigations to reveal neurobiology of memory deficit in epilepsy. Neurochem Res. 2013;38(12):2503-2515.
    CrossRef
  15. Arend J, Kegler A, Caprara ALF, et al. Depressive, inflammatory, and metabolic factors associated with cognitive impairment in patients with epilepsy. Epilepsy Behav. 2018;86:49-57.
    CrossRef
  16. Akyuz E, Polat AK, Eroglu E, Kullu I, Angelopoulou E, Paudel YN. Revisiting the role of neurotransmitters in epilepsy: An updated review. Life Sci. 2021;265:118826.
    CrossRef
  17. Li X, Wang Q, Wu D, et al. The effect of a novel anticonvulsant chemical Q808 on gut microbiota and hippocampus neurotransmitters in pentylenetetrazole-induced seizures in rats. BMC Neurosci. 2022;23(1):7. doi:10.1186/s12868-022-00690-3
    CrossRef
  18. Strandwitz P. Neurotransmitter modulation by the gut microbiota. Brain Res. 2018;1693:128-133.
    CrossRef

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