Can serum orexin levels be used as a marker in childhood epilepsy?

Abstract

Objective

Epilepsy is one of the most common neurological diseases in the pediatric population. Orexins are excitatory peptides and associated with energy homeostasis, eating and drinking behaviors, sleep regulation, sleep-wake periods, analgesia, and cognitive activities such as attention, learning, and memory. The aim of this study was to reveal the relationship between plasma orexin levels and seizures in pediatric epilepsy patients with seizures, epilepsy patients in remission, and healthy control group with similar demographic characteristics.

Material and methods

The study was conducted between December 2021 and December 2022 in the Department of Pediatric Neurology of a tertiary hospital. 30 epilepsy patients who had a seizure in the last 24 h, 30 epilepsy patients in remission, and 17 healthy controls were included in the study. The Human Orexin‐A Enzyme‐Linked Immunosorbent Assay Kit was used to measure serum orexin‐A level. In the ERG and CG, blood samples were obtained during routine controls.

Results

No statistically significant difference was found between serum orexin levels of acute seizure group (ASG), epilepsy in remission group (ERG), and control group (CG). In the ASG, no significant difference was found when orexin levels were compared according to the time of blood sampling after seizure (0–8 h, 8–16 h, 16–24 h). In patients with seizures, the orexin levels of patients with focal seizures were compared with those of patients with generalized seizures. There was no statistically significant difference between the groups. Patients with seizures were evaluated according to electroencephalography (EEG) and cranial magnetic resonance imaging (MRI) findings, and no statistically significant difference was found between orexin levels between the groups.

Conclusion

Our study is the first to evaluate orexin levels in children with epilepsy. Further research is needed to evaluate the role of orexins in the pathophysiology of epilepsy. With the clear relationship between orexin and epilepsy, antiorexinergic drugs can be used in the treatment of epilepsy.

1. Introduction

Orexins (hypocretins) are excitatory peptides and are associated with energy homeostasis, eating and drinking behaviors, sleep regulation, sleep-wake periods, analgesia and cognitive activities such as attention, learning and memory [[1], [2], [3]]. They are secreted as preproorexin from the posterolateral region of the hypothalamus, and then transformed into peptides of 33 amino acids Orexin A (hypocretin-1) and 28 amino acids Orexin B (hypocretin-2) [4]. Orexin receptors (OX1R, OX2R) are expressed in cortical regions, hippocampus and thalamic, hypothalamic and brainstem nuclei. Orexin A shows equal affinity for OX1R and OX2R receptors, while Orexin B shows higher affinity for the OX2R receptor [5,6]. OX1R preferentially influences arousal (defined as onset of wakefulness), drug- and food-related reward, and OX2R preferentially influences wakefulness [3]. Orexinergic activity is highest in wakefulness, moderate in slow wave sleep (SWS) and completely absent in rapid eye movement (REM) sleep. The absence of orexinergic activity leads to the onset of REM sleep and widespread cortical desynchronization. This may reduce the risk of abnormal neuronal activity developing into interictal epileptiform discharges (IEDs) and seizures. In addition, REM sleep deprivation increases Orexin A levels in cerebrospinal fluid [7].

Orexins have been previously associated with both epilepsy and sleep disorders in human and in vivo studies. In particular, orexin receptor antagonists have shown promise in modulating seizure activity by targeting the pathways influenced by orexin signaling [8]. A study executed in rats found that orexin increased the seizure activity triggered by penicillin G [9]. Another study showed that inactivation of hippocampal orexin receptors reduced pentylenetetrazole-induced seizures in rats [10]. Similarly, orexin receptor antagonism was shown to improve sleep and reduce seizures in another study in Kcna1-null rats [11]. These findings highlight the importance of understanding the complex role of orexin in epilepsy pathophysiology, which this study aims to contribute to.

Different results were obtained in adult studies of the relationship between orexins and epilepsy. In a study conducted by Samzadeh et al., it was observed that levels of cerebrospinal fluid orexin A decreased following recurrent generalized tonic-clonic seizures. The study highlighted the potential significance of this finding in relation to post-seizure somnolence [12]. In a distinct study, the investigation encompassed the assessment of orexin A levels within cerebrospinal fluid (CSF) across three distinct cohorts: 21 healthy control subjects, 20 individuals with psychogenic non-epileptic seizures, and 39 patients diagnosed with primary generalized seizures. The results indicated significantly elevated CSF Orexin A levels in patients with primary generalized seizures [13].

To the best of our knowledge, there is no study investigating the relationship between epilepsy and orexin in the pediatric population. The aim of this study was to determine plasma orexin levels in epilepsy patients with seizures, epilepsy patients in remission and healthy controls with similar demographic characteristics and to determine the relationship between orexin and epilepsy and/or epileptic seizures.

2. Material and methods

This study was conducted between December 2021 and December 2022 at the Child Neurology Clinic. The individuals included in the study were categorized into three groups: the Acute Seizure Group (ASG), comprising 30 individuals diagnosed with epilepsy who had experienced a seizure within the last 24 h; the Epilepsy in Remission (ERG), consisting of 30 individuals diagnosed with epilepsy who had been seizure-free for the past 3 months; and the Control Group (CG) with 17 healthy children.

In the ASG, blood samples were obtained in the 24 h after the last seizure, and orexin levels were compared according to the time of blood sampling after seizure (0–8 h, 8–16 h, 16–24 h). In the ERG and CG, blood samples were obtained during routine controls.

The Human Orexin‐A Enzyme‐Linked Immunosorbent Assay Kit (Cat. No. E1296Hu from BT LAB) was used to measure orexin‐A. This assay is based on the quantitative sandwich enzyme immunoassay (ELISA) technique. In this assay, appropriate amounts of serum samples and anti-OX-A antibodies followed by streptavidin-HRP were added to the sample wells. After incubation and washing steps substrate solutions were added to each well. Finally, after incubation and adding stop solution, the resulting substrate is used to generate the color, and the optical density is measured at a wavelength of 450 nm. Measurements were carried out using ELISA plate reader Bio-Tek Synergy HT (Biotek Instruments Inc., Winooski, VT, USA). Intra-assay CV and inter-assay CV were <8 % and <10 %, respectively and assay range was between 5 pg/L and 2000 pg/L with sensitivity 2.53 pg/L.

The sample size for the research was determined through the utilization of the G-Power software program. With an assumed effect size of 0.45, a power of 80 %, and a significance level of 0.05, the calculated total sample size amounted to a minimum of 51 participants, ensuring at least 17 individuals in each group. This calculation ensured the adequacy of group distribution, including the control group. The control group was selected to match the demographic characteristics (age and gender) of the epilepsy groups, ensuring comparability across groups.

Although diet, sleep patterns, and concurrent medication use are known to influence orexin levels, these factors were not controlled for in the study design. This limitation was due to the retrospective nature of data collection and the difficulty in standardizing these variables across pediatric patients. Future studies should aim to account for these confounding factors to provide more comprehensive insights.

The data collected during the study were evaluated using the SPSS 20.0 software package (Statistical Package for the Social Sciences 20.0). For variables that did not exhibit a normal distribution in pairwise comparisons between groups, the Mann Whitney U Test was used while examining differences.

When investigating intergroup differences, a significance level of 0.05 was utilized. In cases where p < 0.05, significant differences between groups were indicated, while if p > 0.05, it was noted that there were no significant differences between groups. Chi-Square test was used to examine the dependency between variables. 0.05 was used as the significance level and it was stated that there was a significant dependence between the groups in case of p < 0.05 and there was no significant dependence between the groups in case of p > 0.05.

3. Results

The groups and their demographic data were presented in Table 1 (Table 1).

Table 1.

Groups and demographic characteristics.

	ASG	ERG	CG
Age (median ± SD) (years)	8,7 ± 4,5	10 ± 3,9	9,1 ± 4,4
Gender
Girl	11 (36,7 %)	10 (33,3 %)	7 (41,2 %)
Boy	19 (63,3 %)	20 (66,7 %)	10 (58,8 %)
Age (median ± SD) (years)	8,7 ± 4,5	10 ± 3,9	9,1 ± 4,4
Seizure Frequencies (seizures/month)
Single seizure	6 (20 %)	0 (0 %)	–
Rare seizures (2–4 seizures/month)	11 (36,7 %)	30 (100 %)	–
Frequent seizures (5–15 seizures/month)	5 (16,7 %)	0 (0 %)	–
Daily seizures (≥16 seizures/month)	8 (26,7 %)	0 (0 %)	–
Medication Usage
Not using medication	7 (23,3 %)	0 (0 %)	–
Using medication	23 (76,7 %)	30 (100 %)	–

∗ASG: Acute seuzire group, ERG: Epilepsy in remission group, CG: Control group.

No statistically significant difference was found when serum orexin levels of ASG, ERG and control groups were compared (p > 0.05) (Table 2).

Table 2.

Comparison of serum orexin levels between groups.

Plasma Orexin Levels (pg/L)	Mean	Median	Min	Max	SD	p
ASG (n = 30)	41,60	200	114	9624	2078,58	0.716
ERG (n = 30)	37,18	197,5	140	900	198,60
CG (n = 17)	37,62	211	144	360	62,46

∗ASG: Acute seuzire group, ERG: Epilepsy in remission group, CG: Control group.

∗∗Kruskal Wallis test was applied.

When comparing the post-seizure serum orexin levels in the ASG based on the time of blood collection (0–8, 8–16, 16–24 h), no statistically significant differences was found (p > 0.05) (Table 3).

Table 3.

Comparison of serum orexin levels in the ASG.

		n	Mean	Median	Min	Max	SD	Rank Mean	p	Binary Comparison
Plasma Orexin Levels (pg/L)	1/First 8 h	17	1494,11	216,00	114	9624	2658,67	16,21	0.155	1–2 1-3 2–3
	2/8–16 h	4	174,50	174,50	125	224	40,41	7,75
	3/16–24 h	9	569,44	234,00	160	1773	679,91	17,61

∗ASG: Acute seuzire group.

∗∗Kruskal Wallis test was applied.

The orexin levels of patients with focal seizures in the ASG and the ERG were compared with the orexin levels of patients with generalized seizures. No statistically significant difference was found between the groups (p > 0.05) (Table 4).

Table 4.

Comparison of serum orexin levels of patients with focal and generalized seizures.

			n	Mean	Median	Min	Max	SD	Rank Mean	U	p
Plasma Orexin Levels (pg/L)	ASG	Focal seizures	11	347,72	205,00	126	1760	470,83	146	80	0.307
		Generalized seizures	19	1442,00	226,00	114	9624	2524,72	319
		Total	30	1040,76	220,00	114	9624	2078,58
	ERG	Focal seizures	10	292,30	199,50	181	855	207,70	173	82	0.448
		Generalized seizures	20	293,10	196,00	140	900	199,41	292
		Total	30	292,83	197,50	140	900	198,60

∗ASG: Acute seuzire group, ERG: Epilepsy in remission group.

∗∗Mann Whitney U Test was applied.

ASG and ERG patients were divided into 3 groups according to electroencephalography (EEG) findings: normal EEG, focal abnormality in EEG and generalized abnormality in EEG. No statistically significant difference was found when the orexin levels of the groups were compared with each other (p > 0.05) (Table 5).

Table 5.

Comparison of serum orexin levels in ASG and ERG patients according to EEG findings.

			n	Mean	Median	Min	Max	SD	Rank Mean	p	Binary Comparison
Plasma Orexin Levels (pg/L)	ASG	1/Normal EEG	10	387,1	230	114	1773	493,88	15,30	0.749	1–2 1-3 2–3
		2/Focal abnormality	16	1567,6	220	126	9624	2728,27	16,34
		3/Generalized abnormality	4	567,5	202,5	191	855	469,51	12,63
	ERG	1/Normal EEG	2	523	523	191	855	469,51	21,00	0.647	1–2 1-3 2–3
		2/Focal abnormality	16	277,81	193,50	156	900	187,13	15,38
		3/Generalized abnormality	12	274,4	233,5	140	697	160,81	14,75

∗ASG: Acute seuzire group, ERG: Epilepsy in remission group group.

∗∗Kruskal Wallis test was applied.

ASG and ERG patients were divided into two groups as patients with pathologic EEG and patients with normal EEG. The orexin levels of the groups were compared and no statistically significant difference was found (p > 0.05) (Table 6).

Table 6.

Comparison of serum orexin levels in patients with pathologic EEG and normal EEG in ASG and ERG.

			n	Mean	Median	Min	Max	SD	Rank Mean	U	p
Plasma Orexin Levels (pg/L)	ASG	EEG Normal	10	387,1	230,00	114	1773	493,88	153	98	0.948
		Pathologic EEG	20	1367,6	210,50	125	9624	2478,22	312
		Total	30	387,1	230,00	114	1773	493,88
	ERG	EEG Normal	2	523	523,00	191	855	469,51	42	17	0.414
		PathologicEEG	28	276,39	197,50	140	900	173,18	423
		Total	30	292,83	197,50	140	900	198,60

∗ASG: Acute seuzire group, ERG: Epilepsy in remission group.

∗∗Mann Whitney U Test was applied.

ASG and ERG patients were divided into two groups as normal MRI and abnormal MRI according to cranial magnetic resonance imaging (MRI) results. The orexin levels of the groups were compared and no statistically significant difference was found (p > 0.05) (Table 7).

Table 7.

Comparison of serum orexin levels according to cranial MRI findings in ASG and ERG.

			n	Mean	Median	Min	Max	SD	Mean Rank	U	p
Plasma Orexin Levels (pg/L)	ASG	Normal MRI	15	991,66	209	125	9624	2431,15	162,00	42	0.265
		Abnormal MRI	8	1144,37	272	174	5884	1985,83	114,00
	ERG	Normal MRI	15	278,73	201	160	855	184,88	167,50	47	0.238
		Abnormal MRI	9	382,55	308	156	900	254,80	132,50

∗ASG: Acute seuzire group, ERG: Epilepsy in remission group.

∗∗Mann Whitney U Test was applied.

We also wanted to compare the orexin A levels of patients with sleep seizures and patients with awake seizures. However, the total number of patients with sleep seizures (8 patients) was not suitable for a statistically significant comparison.

4. Discussion

This is the first study evaluating serum orexin levels in pediatric epilepsy patients. In our study, no statistically significant difference was found when serum orexin levels of ASG, ERG, and control groups were compared. The findings of this study suggest that serum orexin levels alone may not serve as effective biomarkers for pediatric epilepsy. This highlights the need for further exploration of alternative biomarkers or a combination of biomarkers to better understand and diagnose epilepsy. Future research should also investigate whether orexin levels could be integrated into a broader framework of clinical and biochemical indicators to improve epilepsy management.

Epilepsy is a multifactorial disorder involving diverse neurochemical, genetic, and environmental factors. It is possible that orexin plays a more nuanced or indirect role in seizure regulation, rather than serving as a straightforward indicator. This complexity could account for the lack of significant differences observed in this study. In addition to the direct impact of orexin on seizure activity, alternative mechanisms may explain its role in epilepsy. Orexin is known to influence arousal states and sleep-wake cycles, which are closely related to seizure susceptibility. Disruptions in these cycles could alter the excitability of neuronal networks, potentially modulating seizure thresholds. Furthermore, orexin interacts with various neurotransmitter systems, including gamma-aminobutyric acid (GABA) and glutamate, which play critical roles in the pathophysiology of epilepsy. These interactions may provide indirect pathways through which orexin contributes to seizure dynamics.

The relationship between orexin levels and seizure activity is not definitively established in adult studies comparing seizure activity and serum orexin levels. Kacinski et al. evaluated orexin levels in a total of 49 sleep disorder patients, including 14 children with parasomnia, 25 children with epilepsy, and 10 children with seizures during sleep. Examination was performed with the use of polysomnography and the videoelectroencephalography Grass device. Blood samples were taken before the registration of sleep, after 2.5 h of sleep, or 0.5 h after the occurrence of clinical seizures. Serum orexin A levels were found to be significantly lower in epileptic children without seizures compared to parasomnic patients but higher in patients with seizures during polysomnographic examination [14]. Since there was no healthy control group in Kacinski et al.'s study, it is not possible to make an exact correlation between epilepsy and serum orexin levels based on their findings.

In a study that compared basal orexin levels between 40 adults with drug-resistant focal epilepsy and 37 healthy adult controls, orexin levels were found to be significantly lower in epilepsy patients. Similarly, in the same study, although not statistically significant, pre-seizure basal orexin levels were found to be higher when compared to post-seizure orexin levels [15]. In another study conducted by Çikrikler and colleagues involving a total of 80 adults, including 39 with focal epileptic seizures, 20 with psychogenic non-epileptic seizures (PNES), and 21 healthy controls, post-seizure orexin levels were found to be significantly higher in epileptic patients compared to both the PNES group and the healthy control group [13].

This study contrasts with findings in adult epilepsy, where significant relationships between orexin levels and seizure activity have been observed. These discrepancies may reflect age-related differences in orexin function and its role in epilepsy pathophysiology. Pediatric patients may exhibit distinct orexin dynamics compared to adults, influenced by developmental stages, hormonal factors, or neurochemical maturation. Investigating these age-related differences could provide valuable insights into the mechanisms underlying epilepsy and the role of orexin.

Experimental studies have suggested that orexin may trigger seizures, highlighting its potential involvement in epilepsy pathophysiology. Building on these findings, orexin receptor antagonists have shown promise in reducing seizure susceptibility in animal models. These antagonists, which block orexin signaling, have been explored as potential therapeutic agents for epilepsy, particularly in the context of their role in sleep disorders and seizure modulation [8]. Although this study does not provide direct evidence for the clinical application of these agents, it underscores the need for further research to evaluate their potential as part of a broader strategy for epilepsy treatment.

In our study, orexin levels of patients with ASG and ERG focal seizures were compared with orexin levels of patients with generalized seizures. No statistically significant difference was found between the groups. There are no studies comparing orexin levels in focal and generalized seizures in children and adults. However, in a study conducted in 69 adult patients, CSF orexin levels of 20 epilepsy patients with generalized seizures and 24 epilepsy patients in remission were evaluated and CSF orexin levels of patients with seizures were found to be lower than those of epilepsy patients without seizures and the control group [12]. In other studies evaluating epilepsy and orexin levels in adults, epilepsy patients with focal seizures were included in the study and, as mentioned above, no clear relationship between serum orexin levels and seizures could be established.

There is no study comparing EEG findings and orexin levels in epileptic patients in children or adults. In a study carried out with 20 male rats, it was found that orexins increased spike numbers and spike amplitude sizes during penicillin G-induced convulsions [9] In another study conducted by Sagedhnia et al. in rats, it was shown that 4-aminopyridine-induced seizures and EEG disturbances were improved by orexin A antagonists and it was suggested that orexin A antagonists could be used in seizure treatment [16]. In our study, in order to evaluate the relationship between EEG findings and orexin levels, ASG and ERG patients were divided into 3 groups according to their electroencephalography (EEG) findings: normal EEG, focal abnormalities in EEG and generalized abnormalities in EEG. When comparing the orexin levels among these groups, no statistically significant differences were observed. Similarly, ASG and ERG patients were further divided into those with pathological EEG findings and those with normal EEG findings. Upon comparing the orexin levels between these subgroups, no statistically significant differences were found.

In this study, ASG and ERG patients were categorized into two groups based on cranial magnetic resonance imaging (MRI) results: normal MRI and abnormal MRI. The orexin levels of these groups were compared, and no statistically significant differences was found. Although there are no studies comparing cranial MRI findings and orexin levels in pediatric patients, in a study conducted in adults, no statistically significant difference was found when the orexin levels of patients with temporal or extratemporal lesions on brain MRI were compared. In the same study, post-seizure nocturnal orexin levels were found to be higher in patients with ictal electroencephalography onset in the left hemisphere or lesions in the left temporal lobe. However, the exact reason for this was not fully explained, although it was suggested that it might be related to the left hemisphere's greater epileptogenicity [15].

The orexin levels demonstrated a wide range of variability across the study groups (ASG, ERG, and CG). This variability might be attributed to differences in individual patient characteristics, such as seizure type, seizure frequency, and timing of blood sample collection. While the Human Orexin-A Enzyme-Linked Immunosorbent Assay Kit used in this study ensures reliable measurements with an intra-assay CV of <8 % and an inter-assay CV of <10 %, the observed variability in orexin levels may have impacted the study findings, potentially obscuring minor group differences. This variability highlights the need for future studies to standardize factors such as blood sample collection timing and control for patient-specific variables to better delineate the relationship between orexin levels and epilepsy.

The first limitation of our study is the relatively small total number of participants and the lack of diversity, particularly in the control group. The small sample size may have contributed to the non-significant findings by reducing statistical power and increasing the likelihood of Type II errors. While the sample size was calculated to meet the minimal requirements for statistical validity, a larger and more diverse cohort would allow for more robust comparisons and potentially reveal subtle group differences.

Other limitations of our study include the lack of standardization for post-seizure orexin level assessment times and the failure to evaluate whether basal orexin levels vary by age. Additionally, the study did not analyze confounding variables such as sleep disturbances, comorbidities, or environmental factors, which are known to influence orexin levels.

Furthermore, a single time-point measurement for orexin was used, which may not fully capture fluctuations over time. This limitation restricts the interpretation of findings, as orexin levels are known to vary in response to sleep-wake cycles, metabolic states, and other factors. Future studies should consider repeated measurements at multiple time points to better understand the dynamics of orexin levels in epilepsy.

Nonetheless, this study holds significance as it is the first to assess orexin levels in pediatric epilepsy patients, and to examine the correlation between orexin levels, EEG, and cranial MRI findings. Although no statistically significant differences in orexin levels were observed between the study groups, the findings contribute to the growing body of knowledge on the complex role of orexin in epilepsy.

The variability in orexin levels and the multifactorial nature of epilepsy suggest that orexin levels alone may not serve as a direct biomarker for epilepsy. However, the interplay between orexin and seizure dynamics warrants further investigation. Future studies with larger and more diverse cohorts, standardized measurement protocols, and analyses of confounding variables could provide a clearer understanding of the role of orexin in epilepsy pathophysiology.

While our findings do not support the immediate consideration of anti-orexinergic drugs for epilepsy treatment, these agents could be explored in the future as part of a broader therapeutic strategy, once their role in seizure modulation is better understood.

CRediT authorship contribution statement

Mutluay Arslan: Writing – review & editing, Writing – original draft, Investigation. Canan Üstün: Writing – original draft, Supervision, Investigation. Ayşe Nur Coşkun: Investigation, Conceptualization. Hasan Çelik: Data curation. Hacı Nadir Yalçın: Data curation. Eda Karaismailoğlu: Formal analysis, Data curation. Erdim Sertoğlu: Validation, Investigation. Bülent Ünay: Writing – review & editing.

Data availability statement

The datasets generated and/or analyzed during the current study are not publicly available due to ethical restrictions but are available from the corresponding author on reasonable request.

Ethics statement

Ethics Committee approval was obtained on December 29, 2021 under reference number 2021/105 for the study. All patients and children in the control group, along with their parents, were informed about the study and provided informed consent with the Informed Voluntary Consent Form (ICF).

Funding

This study was not supported by any funding agent.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.