Search strategy

A comprehensive systematic search was conducted across three primary databases: PubMed (National Library of Medicine), Embase, and Web of Science in June 2025. Two independent reviewers applied the search term (“ghrelin” OR “nesfatin-1”) AND (“epilepsy” OR “seizure*” OR “epileptic syndrome*”) consistently across all platforms, with sources indexed through June 2025. The only filter applied was a restriction to English-language publications.

Quality assessment

The methodological quality of the studies included was evaluated using the Newcastle-Ottawa Scale for three domains: (1) Selection, (2) Comparability and (3) Outcome. Each study was awarded a maximum of nine stars, with scores of ≥ 7 considered high quality, 5-6 moderate quality, and ≤ 4 low quality. Two independent reviewers performed the assessments, and study inclusion required a minimum score of 5 points.

Selection of studies and evaluation

Studies underwent systematic evaluation at three levels: title, abstract, and full-text review according to predefined criteria. Inclusion criteria encompassed original research papers and case reports involving human subjects (both adult and pediatric) with a confirmed epilepsy diagnosis and reported ghrelin or nesfatin-1 measurements. Given the limited literature explicitly examining these peptides in epilepsy contexts, broad inclusion parameters were maintained.

Exclusion criteria encompassed commentaries, letters to editors, and review articles, studies with unavailable full texts, research involving non-epilepsy patients, animal or cell culture studies, research with limited or no relevance to ghrelin or nesfatin-1, pharmacological interventions other than ASMs, and non-English publications (Figure I).

Two independent reviewers screened titles, abstracts, and full-text articles for adherence to the inclusion criteria. Subsequently, two reviewers (O.Y. and M.Z.) conducted a supplementary manual search, reference and citation tracking to finalize the selection of papers. Any discrepancies or disagreements were resolved through collaborative discussion and consensus.

General findings

The summaries of studies evaluating ghrelin and nesfatin-1 in epilepsy patients are presented in Tables 1 and 2, respectively. A total of 449 records were identified through database searches. Three additional articles were identified through reference screening during the manuscript preparation process. After removing duplicates, 216 studies remained for title and abstract screening. Of these, 199 articles underwent full-text review, and 32 studies met all inclusion criteria and were included in the systematic review. The study selection process is detailed in the PRISMA flowchart (Figure I).

Exclusions were based on the following criteria:

The studies included comprised 27 on ghrelin and five on nesfatin-1, published between 2005 and 2025. Study designs included prospective case-control, cross-sectional, and cohort studies, with sample sizes ranging from 6 to 114 participants. All studies were hospital-based. Most studies compared epilepsy patients to healthy controls (HC), while some included additional sub- groups (e.g., generalized epilepsy, focal epilepsy, resistant or mild epilepsy). The main characteristics of the studies included are summarized in Table 1 (ghrelin) and Table 2 (nesfatin-1).

Ghrelin in epilepsy

This systematic review identified 27 studies investigating ghrelin in epilepsy patients conducted between 2005 and 2025. The majority focused on pediatric po-pulations (70.4%, n = 19), with eight studies (29.6%) involving adults. ELISA was the predominant measurement method (66.6%, n = 18), with blood samples collected after overnight fasting as the standard protocol.

Ghrelin levels showed heterogeneous patterns in epilepsy patients: 37.1% (10/27) of the studies reported decreases, 29.6% (8/27) reported increases, and 33.3% (9/27) reported no change – Aydin et al. [21], noted that although ghrelin levels in serum and saliva increased after treatment in newly diagnosed patients, they remained lower than in HC [21]. Notably, the correlation between saliva and serum ghrelin concentrations highlights the potential of saliva and urine as non-invasive alternatives for hormone measurement [22].

Ghrelin levels varied by epilepsy type [22-25]. Patients with generalized epilepsy (GE) demonstrated higher AG and DAG levels compared to HCs [23]. On the other hand, focal epilepsy (FE) patients showed higher plasma ghrelin levels than both GE patients [22, 24] and HC [26]. Another study identified lower total ghrelin and reduced ghrelin-to-DAG ratios in children with GE compared to other epilepsy types [27].

ASM effects on ghrelin levels showed variation across studies. Research on valproic acid (VPA) yielded conflicting results regarding weight gain mechanisms [28, 29], with some studies linking elevated ghrelin to increased appetite. In contrast, others found reduced levels in VPA-treated patients who developed obesity [22, 23, 30-33]. Age- related differences emerged, with varying ghrelin responses between prepubertal and pubertal children [24, 34]. Other ASMs, including levetiracetam, oxcarbazepine (OXC)and topiramate, showed minimal effects on ghre-lin levels [35-39].

Three studies examined ghrelin during ketogenic diet (KD) treatment. Two reported reduced ghrelin and DAG levels in children with drug-resistant epilepsy [40, 41], while one found no significant changes after 12 months in mixed pediatric and adult populations receiving the diet [42]. Notably, lower serum ghrelin levels were observed in drug-resistant epilepsy patients, suggesting ghrelin’s potential involvement in the pathophysiology of drug-resistant epilepsy [25, 37].

Ghrelin levels demonstrated dynamic changes following seizures, with decreases observed immediately after the seizure, followed by increases at 48 hours [43]. Higher ghrelin levels were associated with positive ASM responses, suggesting potential as biomarkers for treatment efficacy prediction [23, 44-46]. Lower ghrelin levels in drug-resistant epilepsy patients indicated possible involvement in treatment-resistant pathophysiology [25, 37].

Finally, ghrelin’s relationship with comorbidities in epilepsy has been explored. For example, OXC treatment has been associated with increased linear growth in children, whereas VPA reduced ghrelin levels without significantly affecting growth parameters [34].

Nesfatin-1 in epilepsy

Five studies investigated nesfatin-1 in epilepsy patients between 2009 and 2021, primarily focusing on adult po-pulations using prospective designs. All studies measured serum nesfatin-1 via ELISA using fasting samples, with one study additionally examining saliva samples [21].

Four studies (80%) reported elevated serum nesfatin-1 levels in epilepsy patients compared to HCs, parti-cularly in untreated patients or those with active seizures [21, 25, 43, 47]. Aydin et al. [21] demonstrated that while nesfatin-1 levels decreased following ASM treatment, they remained high compared to HC in both serum and saliva samples.

Yu et al. [47], observed higher nesfatin-1 levels in patients with frequent seizures compared to those with fewer seizures. In drug-resistant temporal lobe epilepsy (TLE), patients showed lower nesfatin-1 levels than non-drug- resistant patients, though this difference was not statistically significant [25].

Chen et al. [48], identified serum nesfatin-1 as a potential prognostic marker, with higher levels associated with poorer one-year survival outcomes in status epilepticus patients.

Additionally, nesfatin-1 demonstrated potential as a differential biomarker, with epileptic patients showing consistently elevated levels compared to patients with functional seizures (previously termed psychogenic non-epileptic seizures), who had lower levels. However, this finding was limited to male participants only [43]. The same authors also demonstrated that salivary glands produce nesfatin-1 at levels significantly lower than those in brain tissue, providing insight into its peripheral sources [21].

Ghrelin in epilepsy

The observed heterogeneity in ghrelin levels – 37.1% of studies reporting decreases, 29.6% increases, and 33.3% no change – reflects its complex interplay with epilepsy subtypes, ASM effects, and metabolic factors. Notably, increased ghrelin levels were particularly evident in patients with FE or SeLFE compared to those with GE or HC [24, 26]. This pattern aligns with emerging evidence of subtype-specific neuroendocrine dysregulation in epi-lepsy networks.

The relationship between ASMs and ghrelin levels remains one of the most clinically relevant yet inconsistent findings in our review. While some medications like levetiracetam and topiramate showed minimal effects on ghrelin levels, others, particularly VPA and carbamazepine, demonstrated more pronounced but variable impacts [50]. The observation that patients with better ASM responses tend to have higher ghrelin levels suggests a potential role for ghrelin as a biomarker for treatment efficacy. However, this requires validation in larger, controlled studies.

The relationship between VPA and ghrelin-mediated weight gain warrants particular attention due to its clinical significance. As an orexigenic hormone, ghrelin stimulates appetite via neuropeptide Y and agouti-related peptide pathways, theoretically contributing to VPA- associated weight gain [14]. However, our review reveals conflicting evidence: some studies link elevated ghrelin levels during early VPA therapy with increased appetite and weight gain [29], while others associate VPA-induced obesity with paradoxically reduced ghrelin levels [30, 31]. These discrepancies may stem from patient age, treatment duration, or metabolic factors.

The three studies examining ghrelin during KD treatment provide intriguing insights into metabolic adaptation in epilepsy. Standardized procedures are clearly needed in future KD research, as this discrepancy may reflect variations in study length, patient populations, or the degree of ketosis achieved.

Additionally, ghrelin levels were shown to decrease shortly after seizures but rebounded within 48 hours, indicating a possible role in postictal recovery [43, 51]. The strong correlation between saliva and serum ghrelin levels underscores the potential for non-invasive diagnostic tools [22].

Nesfatin-1 in epilepsy

With 80% of the studies reporting increased levels in epilepsy patients compared to HC, nesfatin-1 shows more consistent patterns than ghrelin, despite being represented by just five studies (Table 2). This consistency, combined with its correlation with seizure frequency and potential prognostic value, positions nesfatin-1 as an up-and-coming biomarker candidate.

The correlation between nesfatin-1 levels and seizure frequency suggests a direct relationship with epileptic activity, potentially reflecting the neuropeptide’s role in neuronal excitability or stress response pathways [52]. However, decreases were noted in drug-resistant TLE and following prolonged ASM use, indicating dynamic regulation in response to drug therapy and disease progression [25, 47].

The preliminary evidence linking elevated nesfatin-1 levels to poor survival outcomes in status epilepticus, while requiring validation, indicates that this neuropeptide may serve as a marker of disease severity and prognosis [48]. Furthermore, identifying salivary nesfatin-1 production at levels lower than those in brain tissue opens avenues for the development of non-invasive biomarkers [21].

Perhaps most clinically significant is the emerging evidence that nesfatin-1 levels differ between epileptic seizures and functional seizures. This finding, though currently limited to male patients, suggests potential utility in differential diagnosis – a persistent challenge in epilepsy care where misdiagnosis rates remain substantial [43].

Comparative analysis

There are significant distinctions between ghrelin and nesfatin-1 in terms of their possible clinical uses. While ghrelin exhibits inconsistent and frequently conflicting patterns, which may reflect its complex role in energy homeo-stasis and multiple regulatory pathways, nesfatin-1 demonstrates a more consistent elevation in epilepsy patients. This disparity may indicate their unique physiolo-gical functions: ghrelin’s principal role in hunger and energy equilibrium may be more influenced by confounding variables such as pharmacological effects, body composition, and feeding conditions, whereas nesfatin-1’s function in stress response and neuronal regulation may more accurately represent epileptic pathophysiology.

Both neuropeptides show potential for non-invasive assessment, with demonstrated correlations between serum and saliva concentrations. This capability may enhance point-of-care testing and continuous monitoring, addressing current limitations in epilepsy biomarker development.

Clinical implications and practical applications

The findings of this systematic review have several immediate and potential future clinical applications:

Immediate clinical applications can be: (1) Monitoring VPA-associated weight gain: ghrelin measurement could help identify patients at risk for significant weight gain early in VPA treatment, allowing for proactive dietary counseling and potentially dose adjustment or medi-cation switching [53-55]. (2) Non-invasive moni-toring: the demonstrated correlation between saliva/urine and serum levels of both peptides offers practical advantages for pediatric patients and repeated monitoring, avoiding frequent blood draws [21, 22]. (3) Differential diagnosis: nesfatin-1 shows promise in distinguishing epileptic seizures from functional seizures, which remains a significant clinical challenge affecting 20-30% of patients in epilepsy monitoring units [43].

Potential future applications can be: (1) Treatment response prediction: elevated ghrelin levels in responders to ASMs suggest potential use in personalizing medication selection and predicting treatment success before clinical response is evident [23, 44]. (2) Drug resistance identification: lower ghrelin and nesfatin-1 levels in drug- resistant epilepsy may help identify candidates for alternative therapies (surgery, neurostimulation, KD) earlier in the disease course [25, 37]. (3) Prognostic biomarker: nesfatin-1 levels correlating with seizure frequency and survival in status epilepticus suggest utility in risk stratification and monitoring [47, 48]. (4) Therapeutic target: understanding the mechanisms by which these peptides influence seizure threshold could lead to novel neuropeptide-based therapies, expanding beyond ACTH (currently the only neuropeptide treatment for epilepsy) [4, 14].

Although these applications are promising, several barriers to clinical implementation persist, including the lack of standardized measurement protocols, the absence of established reference ranges, cost considerations, and the need for validation in diverse populations. These challenges underscore the need for translational research that bridges laboratory findings to bedside applications [56, 57].

Limitations

Several limitations must be acknowledged when interpreting these findings. The methodological quality assessment revealed that some studies lack control groups, primarily due to ethical constraints in epilepsy research, limiting the strength of comparative conclusions [27, 40, 42, 58]. The predominance of cross-sectional studies limits causal inference; heterogeneity in study populations, epilepsy types, measurement methodologies, ASM regimens, and the geographical concentration of studies may affect the generalizability of findings. Additionally, many studies had relatively small sample sizes and short follow-up periods, which is particularly concerning for understanding long-term relationships between neuropeptide levels and epilepsy outcomes.

Currently, measurement of ghrelin and nesfatin-1 in clinical epilepsy care remains research-based rather than routine practice. While our review identifies potential applications, translation to clinical utility requires several unmet prerequisites.

Biomarker development progresses through three phases: discovery, validation, and clinical utility [55, 56], with current evidence indicating that these neuropeptides are currently in the early stages. The substantial gap between biomarker discovery and clinical implementation is well-recognized [59], with key requirements for clinical adoption including: (1) standardized, cost-effective assays; (2) clinically meaningful cutoff values validated across diverse populations [60, 61]; (3) prospective demonstration that biomarker-guided decisions improve patient outcomes [61]; and (4) cost-effectiveness evidence [62].

Epilepsy biomarker development faces unique challenges, including clinical heterogeneity and complex pathophysiology. Therefore, while the theoretical foundation is established through the studies reviewed here, practical clinical application awaits further translational research that bridges the critical gaps between laboratory findings and bedside implementation [6, 63].

We also acknowledge that meta-analysis and meta- regression methods, along with forest plot visualization, could have strengthened our findings. However, the significant heterogeneity in study designs, measurement methods (ELISA vs. RIA), patient populations (pediatric vs. adult, various epilepsy types), ASM regimens, timing of measurements, and outcome definitions precluded meaningful quantitative synthesis. The included studies used different cutoff values, units of measurement, and control group definitions, making statistical pooling unreliable and potentially misleading. Future studies with standardized protocols would enable robust meta-analytic approaches.

Future research

This review reveals some areas of research priority. First, large-scale, multicenter, prospective cohort studies with standardized measurement protocols are needed to validate the potential of biomarkers and establish reference ranges. Second, studies investigating which seizures influence the regulation of ghrelin and nesfatin-1 could provide insights into potential therapeutic targets. Third, intervention studies examining whether neuropeptide modulation affects seizure control or epilepsy-related comorbidities could establish therapeutic potential. Fourth, validation of non-invasive measurement techniques could facilitate clinical implementation.

Consensus rules for neuropeptide measurement in epilepsy research would benefit the field by establishing standardized procedures for sample collection, processing, and analysis, thereby enhancing comparability across subsequent studies and facilitating significant meta- analyses.