Nesfatin-1 and Ghrelin/GOAT as Novel Biomarkers in Adolescent Headache with Temporomandibular Disorders

Nesfatin-1 and Ghrelin/GOAT as Novel Biomarkers in Adolescent Headache with Temporomandibular Disorders | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Nesfatin-1 and Ghrelin/GOAT as Novel Biomarkers in Adolescent Headache with Temporomandibular Disorders Anna Sojka, Barbara Dorocka-Bobkowska, Yasmin Bartosik, Ozgun Yetkin, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7076570/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Nesfatin-1 and Ghrelin are recently discovered peptide hormones with roles beyond energy homeostasis. This study investigated Nesfatin-1 and Ghrelin O-acetyltransferase (Ghrelin/GOAT) levels in adolescents with headaches and analyzed their correlation with temporomandibular disorders (TMD) symptoms. Methods: We measured Nesfatin-1 and Ghrelin/GOAT levels in the serum and saliva of 44 adolescents with headaches (aged 10-17 years) and 20 controls without headaches or TMD symptoms. TMD was evaluated using Diagnostic Criteria for TMD (DC/TMD) axis I and II. Participants underwent comprehensive clinical examination and biochemical analysis. Results: Significant differences were found in Nesfatin-1 and Ghrelin/GOAT levels between patients and controls (p<0.001). Nesfatin-1 levels were consistently elevated in all headache patients' blood and saliva regardless of headache type or TMD status. Ghrelin/GOAT showed a distinctive tissue-specific pattern: serum concentrations were higher (p<0.001), while salivary levels were lower (p<0.001) compared to controls. Gender-specific differences were observed in TMD manifestations: muscle pain with palpation was more common in females (74% vs 38%, p<0.016), while psychological distress was more prevalent in males (90% vs 48%, p<0.003). No significant differences in neuroendocrine markers were found between migraine and tension-type headache groups. Conclusions: Altered Nesfatin-1 and Ghrelin/GOAT levels may serve as important biomarkers for adolescent headache, regardless of TMD status. The tissue-specific regulation of Ghrelin/GOAT and gender-specific clinical manifestations highlight the complex interplay between neuroendocrine factors and pain perception. These findings provide new insights into potential diagnostic markers and therapeutic targets for adolescent headache and TMD management. Key Words: Nesfatin-1 and Ghrelin/GOAT, headache, temporomandibular disorders, adolescents 1. INTRODUCTION Headache is one of the most prevalent complaints in pediatric neurological clinics ( 1 ). Globally, many studies have estimated the prevalence of headaches and migraines in children and adolescents to be between 54.4% and 58.4%, often leading to school absences and decreased academic performance ( 2 , 3 ). Temporomandibular disorders (TMD) and associated pain in the masticatory muscles, temporomandibular joint (TMJ), and surrounding tissues are the most common conditions of chronic orofacial pain or discomfort. They are the main cause of pain of non-dental origin in the oro-facial region ( 4 ). TMD frequently overlaps with headaches, especially with tension-type headaches (TTH), though this relationship is often overlooked in clinical practice. The International Classification of Headache Disorders, 3rd edition (beta version), (ICHD-3 beta), mentions that when the diagnosis of TMD is uncertain, the headache should be coded as ‘TTH or one of its subtypes’ (presumably with pericranial muscle tenderness) ( 5 ). Frequent headaches like migraine and TTH can have a significant impact on disability, as well as the quality of life, prompting the need for early recognition and treatment ( 6 ). Therefore, early identification, establishing a treatment plan, and implementing lifestyle changes can alter disease progression and improve the child's quality of life ( 7 ). The availability of reliable biomarkers for headache diagnosis would solve such diagnostic ambiguity. Some previous reports have described biomarkers for migraine in peripheral blood and cerebrospinal fluid ( 8 , 9 ). Nesfatin-1, discovered in 2006, is a satiety molecule involved in stress response and various physiological processes ( 10 ). It is present in stress-sensitive areas of the brain and has been implicated in glucose homeostasis, lipid metabolism, and anxiety behaviors ( 11 ). Similarly, Ghrelin, identified in 1999, functions as a neuropeptide in the central nervous system, affecting eating behavior, energy use, and memory retention ( 12 ). And ghrelin O-acyltransferase (GOAT) is a specific enzyme that modifies ghrelin with a medium-chain fatty acid and plays an important role in regulating ghrelin’s activities ( 13 ). The study aims were to investigate levels of Nesfatin-1 and the Ghrelin/GOAT, which can be analyzed in the future in clinical practice in young patients with headaches. The research questions are: Is there a correlation between the levels of Nesfatin-1, Ghrelin/GOAT, the occurrence of TMD symptoms and the difference between the TMD subgroups in patients with headaches? Can we investigate how ghrelin and nesfatin-1 levels could be used as a diagnostic procedure for headaches caused by different causes? Is there any gender distribution of TMD, parafunctional habits, and associated symptoms in adolescents with headaches? 2. MATERIALS AND METHODS 2.1. Study Population: This prospective study was conducted at the Department of Developmental Neurology, Poznan University of Medical Sciences, from November 2017 to December 2020. Among 700 patients initially screened, 44 adolescents met the inclusion criteria of confirmed migraine or TTH according to the ICHD-3 beta criteria ( 5 ). The diagnosis was established through a comprehensive assessment protocol, including parental questionnaires designed by the investigators, detailed medical history, and clinical examination by a certified child neurologist (M.Z. and B.S.). The control group comprised 20 age-matched adolescents without headache or TMD symptoms. The study employed stringent exclusion criteria for both groups. Primary exclusion criteria included: history of epilepsy or other central nervous system disorders; current or previous use of psychiatric medications; presence of severe medical conditions; comorbid psychotic disorders; current malignancy; body mass index (BMI) exceeding 27; and presence of chronic inflammatory, gastrointestinal, or nutritional disorders that could potentially affect Nesfatin-1 and Ghrelin levels. Additional exclusion criteria for the control group included any history of headaches or TMD symptoms. Subjects with BMI > 27 were excluded from the study as body mass significantly affects the secretion and circulating levels of both Nesfatin-1 and Ghrelin, which could confound the interpretation of results ( 14 , 15 ). The study was conducted according to the Declaration of Helsinki and approved by the Bioethical Committee of Poznan University of Medical Sciences (approval number 550/17) ( 16 ). Written informed consent was obtained from all participants and their legal guardians following a comprehensive explanation of the study protocol. Participant anonymity was maintained throughout the study, and participation involved no financial or other obligations. The study procedures were designed to minimize any potential burden on the participants. 2.2. Clinical Examination of TMD The examinations were made by dentists (Y.B. and A.S.M.) according to the protocol followed by the Diagnostic Criteria for TMD (DC/TMD) Axis II guidelines ( 17 , 18 ). A comprehensive clinical assessment included medical and dental history, specifically concerning parafunctional habits such as bruxism. The intraoral examination evaluated signs of bruxism, including dental wear facets, fractures, masseter hypertrophy, and soft tissue changes (linea alba, tongue scalloping). Two major diagnostic categories were assessed: myalgia and disc displacement with reduction (DDwR). Myalgia was evaluated through palpation of temporalis and masseter muscles and pain during jaw opening, while DDwR was identified by joint sounds (clicking or popping). Following DC/TMD protocols, pain provocation during examination was confirmed as familiar pain matching the patient's primary complaint. Functional measurements included maximum mouth opening (measured from maxillary to mandibular incisal edges at midline), with opening less than 40 mm considered restricted ( 19 ). Mandibular deviation was defined as displacement of 2 mm or more to either side during opening movement. The assessment also included distress evaluation using the patient health questionnaire-4 by DC/TMD Axis II - Patient Health Questionnaires (PHQ) PHQ-4, PHQ-9, generalized anxiety disorders (GAD) GAD-7 and oral behavior checklist (OBC) ( 18 ). 2.3. Biochemical Analysis: Fasting venous blood (5 mL) and saliva (2 mL) samples were collected between 08:00–09:00. Blood samples were centrifuged (3000 × g, 5 minutes), and the serum was stored at -20°C for later analysis. Saliva samples underwent double centrifugation (3046.4g, 2 × 10 minutes), with supernatant collection between cycles, and were stored at -20°C in 0.5 mL aliquots. Thyroid Stimulating Hormone (TSH), fasting plasma glucose, and total cholesterol were analyzed using a Cobas 8000 platform (Roche Diagnostics GmbH, Germany). Nesfatin-1 and Ghrelin/GOAT levels were duplicated using commercial enzyme-linked immunosorbent assay (ELISA) kits (Human NEFA/Nesfatin-1 and Human MBOAT4/GOAT). All analyses were performed blinded using reagents from the same manufacturer lot. Both saliva and blood were tested in two trials. 2.4. Statistical Analysis: Descriptive statistics included means, standard deviations, and medians for continuous variables. The Shapiro-Wilk test was used to assess the normality of distribution. Due to non-normal distribution, between-group comparisons were conducted using non-parametric tests: Mann-Whitney U test for two-group comparisons and Kruskal-Wallis test for multiple-group analyses. Categorical variables were presented as counts (N) and percentages, with differences between proportions analyzed using chi-square tests. Statistical significance was set at p < 0.05. All analyses were performed using Statistica v13.0 (Dell Inc., Round Rock, TX, USA). 3. RESULTS The study cohort comprised 44 adolescents with headaches (23 females [52.3%] and 21 males [47.7%] aged 10-17 years). All participants had experienced the last episodes of headaches within the previous three months, and this was the main reason for their admission to the Department. Based on ICHD-3 beta diagnostic criteria, 14 participants (31.8%) were diagnosed with migraine, while 30 (68.1%) met the criteria for TTH (5). According to DC/TMD Axis I criteria (17), the distribution of TMD was as follows: 18 patients (40.9%) with myalgia, five patients (11.3%) with DDwR, and 21 patients (48.0%) without TMD symptoms. The control group comprised 20 healthy adolescents (8 females [40%] and 12 males [60%], aged 12-17 years) without headache or TMD symptoms. 3.1. Characteristics of the Patients Table 1 presents the demographic and biochemical characteristics of patients with headaches compared to controls, stratified by sex. No statistically significant differences were observed between female and female controls or between male patients and male controls regarding anthropometric measurements (BMI, weight, height) or basic biochemical parameters (TSH, total cholesterol, glucose). The mean BMI values were comparable between groups (20.78 kg/m² in female patients vs. 19.53 kg/m² in female controls; 19.05 kg/m² in male patients vs. 19.90 kg/m² in male controls). However, significant differences were found in neuroendocrine markers. Nesfatin-1 levels in blood were markedly elevated in both female (39.12±18.28 ng/mL vs. 9.14±0.63 ng/mL, p<0.001) and male patients (43.97±14.47 ng/mL vs. 7.52±2.50 ng/mL, p<0.001) compared to their respective controls. Similarly, salivary Nesfatin-1 levels were significantly higher in females (10.57±9.62 ng/mL vs. 2.16±0.68 ng/mL, p<0.005) and male patients (11.16±9.19 ng/mL vs. 1.85±0.82 ng/mL, p<0.001). A distinctive pattern was observed with Ghrelin/GOAT: blood levels were significantly elevated in both female (6.35±2.75 ng/mL vs. 1.47±0.22 ng/mL, p<0.001) and male patients (6.21±2.46 ng/mL vs. 1.46±0.45 ng/mL, p<0.001), while salivary levels were markedly lower in both female (0.29±0.24 ng/mL vs. 1.65±0.58 ng/mL, p<0.001) and male patients (0.24±0.25 ng/mL vs. 1.63±0.78 ng/mL, p<0.001) compared to controls. Characteristic Female (N=23) mean±SD (median) Female Control mean±SD (median) p-value Male (N=21) mean±SD (median) Male Control mean±SD (median) p-value BMI [kg/m2] 21,05±2,41 (20,78) 20,65±4,10 (19,53) 0,603 20,23±2,53 (19,05) 19,74±2,63 (19,90) 0,708 Weight [kg] 51,91±9,25 (52,00) 45,25±16,74 (47,00) 0,416 48,67±13,67 (50,00) 48,63±17,42 (43,75) 0,721 Height [cm] 156,43±10,41 (155,00) 144,88±15,99 (153,50) 0,099 153,57±14,04 (155,00) 154,08±19,98 (149,50) 0,895 TSH level[µU/ml] 3,47±1,73 (3,41) 3,08±1,01 (2,60) 0,527 3,23±1,38 (2,83) 3,37±1,16 (3,34) 0,369 Total cholesterol[mg/dl] 161,70±25,66 (158,00) 158,50±29,49 (153,50) 0,635 162,81±34,33 (167,00) 161,50±24,25 (161,50) 0,808 Glucose level [mg/dl] 88,74±6,05 (88,00) 90,63±4,96 (88,50) 0,286 89,71±7,46 (93,00) 93,92±5,65 (94,50) 0,106 Nesfatin-1 (blood)[ng/mL] 39,12±18,28 (39,48) 9,14±0,63 (9,04) <0,001 43,97±14,47 (45,00) 7,52±2,50 (8,14) <0,001 Nesfatin-1 (saliva)[ng/mL] 10,57±9,62 (6,61) 2,16±0,68 (2,32) <0,005 11,16±9,19 (8,22) 1,85±0,82 (1,76) <0,001 Ghrelin/GOAT (blood)[ng/mL] 6,35±2,75 (5,86) 1,47±0,22 (1,43) <0,001 6,21±2,46 (6,64) 1,46±0,45 (1,39) <0,001 Ghrelin/GOAT (saliva)[ng/mL] 0,29±0,24 (0,21) 1,65±0,58 (1,41) <0,001 0,24±0,25 (0,18) 1,63±0,78 (1,37) <0,001 Table 1. Characteristics of the investigated headache patients and controls BMI (Body Mass Index), GOAT (Ghrelin O-acetyltransferase), TSH (Thyroid-Stimulating Hormone) 3.2. Prevalence of symptoms of TMD and parafunction according to a questionnaire between females and males with headache Gender-specific analysis revealed significant differences in TMD manifestations. Muscle pain upon palpation was significantly more prevalent in females than males (74% n=17/23 vs 38% n=8/21, p<0.016). Conversely, males exhibited a higher prevalence of TMD-free cases than females (66% n=14/21 vs 30% n=7/23, p<0.016). Psychological distress was significantly more prevalent in males compared to females (90% n=19/21 vs 48% n=11/23, p<0.003) (Table 2). Myalgia diagnosis, according to DC/TMD criteria, also being more frequent in females (52% n=12/23 vs. 28% n=6/23), but no significant difference (p<0.105). While both groups showed comparable rates of parafunctional habits, males demonstrated a higher frequency of both bite (52% n=11/21, 43% n=10/23, p<0.550) and non-bite (90% n=19/21, 82% n=19/23, p<0.447) parafunctions. Physical symptoms were similarly distributed between females and males (70% n=16/23 and 71% n=15/21, respectively). No significant differences were observed in other assessed parameters ( Table 2 )." Table 2. Gender Distribution of TMD, Parafunctional Habits, and Associated Symptoms in Adolescents with Headache Signs and symptoms of TMD Female (N=23) Male (N=21) p-value Pain symptoms in the temple area 16 (70%) 18 (86%) 0,203 Muscle pain with palpation 17 (74%) 8 (38%) 0,016 Bite parafunctions 10 (43%) 11 (52%) 0,550 Non-bite parafunctions Signs of bruxism 19 (82%) 10 (43%) 19 (90%) 10 (48%) 0,447 0,739 Physical symptoms N(%) Distress (Depression&Anxiety) N(%) Diagnosis of TMD – Myalgia Diagnosis of TMD – DDwR No TMD 16 (70%) 11 (48%) 12 (52%) 3 (13%) 7 (30%) 15 (71%) 19 (90%) 6 (28%) 2 (9%) 14 (66%) 0,942 0,003 0,105 0,673 0,016 P<0,05, TMD (Temporomandibular Disorders), DDwR (Disc displacement with reduction) 3.3. Neuroendocrine Markers, Physical Symptoms, and Parafunctional Habits in Adolescents with Headache: Comparison Among TMD Diagnostic Subgroups Comparison between TMD subgroups revealed significant differences in physical symptoms. Patients with myalgia showed a significantly higher prevalence of physical symptoms compared to the no-TMD group (89% vs 47%, p<0.006). Similarly, the DDwR group demonstrated significantly more physical symptoms than the no-TMD group (100% vs 47%, p<0.035). The Kruskal-Wallis test showed no significant differences in Nesfatin-1 (blood and saliva), Ghrelin/GOAT (blood and saliva), or TSH levels among Myalgia, DDwR, and no-TMD groups (Table 3). Table 3. Comparison of Neuroendocrine Markers and Clinical Manifestations Across TMD Diagnostic Subgroups in Headache Patients TMD Nesfatin-1 (ng/ml) Blood mean±SD (median) Nesfatin-1 (ng/ml) Saliva mean±SD (median) Ghrelin/ GOAT (ng/ml) Blood mean±SD (median) Ghrelin/ GOAT (ng/ml) Saliva mean±SD (median) Physical symptoms of TMD N(%) mean±SD (median) Distress N(%) Parafunctional bite or nonbite N(%) TSH (uU/ml) N(%) Myalgia(M) N=18 40,89±17,23 (40,99) 11,59±9,08 (10,30) 6,45±2,85 (6,34) 0,18±0,06 (0,16) 16(89%)* 12(67%)* 18(100%)* 3,66±1,88 (3,33) Disc displacement with reduction (DDwR) N=5 44,79±16,32 (50,00) 11,22±11,29 (8,22) 7,19±3,58 (6,19) 0,25±0,03 (0,25) 5(100%)** 5(100%)** 5(100%)** 3,74±1,34 (4,18) No TMD N=21 41,10±16,76 (45,00) 10,13±9,48 (4,48) 5,92±2,13 (5,61) 0,33±0,33 (0,21) 10(47%)*** 12(57%)*** 18(85%)*** 2,99±1,27 (2,56) p-value 0,628 0,823 0,764 0,067 *vs*** 0,006 *vs**0,486 **vs*** 0,035 *vs*** 0,522 *vs**0,149 **vs***0,077 *vs*** 0,119 *vs**0,393 **vs***0,392 0,289 p<0,05, GOAT (Ghrelin O-acetyltransferase), TTH (Tension-Type Headache), TMD (Temporomandibular Disorders), TSH (Thyroid-Stimulating Hormone), Distress (Depression and Anxiety) 3.4. Comparison of Neuroendocrine Markers and Clinical Parameters Between Migraine and Tension-Type Headache Groups No statistically significant differences between migraine and TTH groups were observed in the measured parameters. The Mann-Whitney test revealed similar Nesfatin-1 and Ghrelin/GOAT levels in both blood and saliva samples between groups. Additionally, no significant differences were found in TMD diagnoses (Myalgia and DDwR), parafunctional habits, psychological distress, or physical symptoms between migraine and TTH patients (Table 4). Table 4. Neuroendocrine Markers and Clinical Characteristics in Migraine versus Tension-Type Headache Patients Headache Nesfatin-1 (ng/ml) Blood mean±SD (median) Nesfatin-1 (ng/ml) Saliva mean±SD (median) Ghrelin/ GOAT (ng/ml) Blood mean± SD (median) Ghrelin/ GOAT (ng/ml) Saliva mean±SD (median) TSH (uU/ml) Myalgia TMD N(%) DDwR TMD N(%) Parafuntions bite or nonbite N(%) Distress (N(%) Physical Symptoms N(%) Migraine (N=14) 42,94±17,04 (42,94) 10,98±8,23 (8,62) 6,26±2,65 (5,91) 0,25±0,26 (0,16) 3,57±1,95 (2,89) 8(57%) 1(7%) 14(100%) 8(57%) 23(76%) TTH (N=30) 40,73±16,58 (41,70) 10,79±9,91 (4,52) 6,30±2,60 (6,09) 0,27±0,23 (0,21) 3,25±1,36 (3,20) 10(33%) 4(13%) 18(93%) 20(66%) 8(57%) p-value 0,998 0,457 0,990 0,296 0,990 0,131 0,555 0,399 0,564 0,224 p<0.05, Ghrelin/GOAT (Ghrelin O-acetyltransferase), TTH (Tension-Type Headache), TMD (Temporomandibular Disorders), TSH (Thyroid-Stimulating Hormone), DDwR (Disc displacement with reduction), Distress (Depression and Anxiety) 3.5. Comparison of Neuroendocrine Markers Between Headache Subgroups and Controls Nesfatin-1 and Ghrelin/GOAT levels revealed consistent patterns across all headache subgroups compared to controls (Table 5). Nesfatin-1 levels in blood were significantly elevated in migraine patients (42.94±17.04 ng/ml), TTH patients (40.73±16.58 ng/ml), those with TMD symptoms (41.74±16.75 ng/ml), and those without TMD symptoms (41.10±16.76 ng/ml) compared to controls (all p<0.001). Similarly, salivary Nesfatin-1 levels were markedly higher in all patient subgroups (ranging from 10.13±9.48 to 11.51±9.32 ng/ml) than in controls (1.97±0.77 ng/ml) (all p<0.001). Ghrelin/GOAT demonstrated a tissue-specific pattern: blood levels were significantly higher in all headache groups (ranging from 5.92±2.13 to 6.61±2.95 ng/ml) compared to controls (1.46±0.37 ng/ml) (all p<0.001), while salivary levels showed the opposite trend, being significantly lower in all patient groups (ranging from 0.19±0.06 to 0.33±0.33 ng/ml) compared to controls (1.64±0.69 ng/ml) (all p<0.001). Table 5. Comparison of Nesfatin-1 and Ghrelin/GOAT Levels Between Headache Subgroups, TMD Categories, and Controls. Migraine (N=14) Control (N=20) p-value TTH (N=30) Control (N=20) p-value TMD (N=23) Control (N=20) p-value No TMD (N=21) Control (N=20) p-value Nesfatin-1 (ng/ml) Blood mean±SD (median) 42,94± 17,04 (42,94) 8,16±2,10 (8,64) <0,001 40,73±16,58 (41,70) 8,16±2,10 (8,64) <0,001 41,74±16,75 (42,33) 8,16±2,10 (8,64) <0,001 41,10±16,76 (45,00) 8,16±2,10 (8,64) <0,001 Nesfatin-1 (ng/ml) Saliva mean±SD (median) 10,98± 8,23 (8,62) 1,97±0,77 (2,01) <0,001 10,79±9,91 (4,52) 1,97±0,77 (2,01) <0,001 11,51±9,32 (8,92) 1,97±0,77 (2,01) <0,001 10,13±9,48 (4,48) 1,97±0,77 (2,01) <0,001 Ghrelin/ GOAT (ng/ml) Blood mean±SD (median) 6,26±2,65 (5,91) 1,46±0,37 (1,41) <0,001 6,30±2,60 (6,09) 1,46±0,37 (1,41) <0,001 6,61±2,95 (6,19) 1,46±0,37 (1,41) <0,001 5,92±2,13 (5,61) 1,46±0,37 (1,41) <0,001 Ghrelin/ GOAT (ng/ml) Saliva mean±SD (median) 0,25±0,26 (0,16) 1,64±0,69 (1,38) <0,001 0,27±0,23 (0,21) 1,64±0,69 (1,38) <0,001 0,19±0,06 (0,19) 1,64±0,69 (1,38) <0,001 0,33±0,33 (0,21) 1,64±0,69 (1,38) <0,001 p<0,05, Ghrelin/GOAT (Ghrelin O-acetyltransferase), TTH (Tension-Type Headache), TMD (Temporomandibular Disorders) 4. DISCUSSION The current study provides new perspectives into the relationship between neuroendocrine markers and headaches with TMD in adolescents. The most notable finding was the consistent and significant increase of Nesfatin-1 levels in both blood and saliva among all headache groups, regardles of headache type or TMD status. This pattern and the unique tissue-specific changes in Ghrelin/GOAT levels suggest that these peptides may serve as potential biomarkers for headache disorders in adolescents. The elevated Nesfatin-1 levels reported in our headache patients correspond with recent information regarding this peptide's function beyond energy regulation. Nesfatin-1 has been associated with multiple stress-related disorders, as evidenced by Hofmann et al., who identified positive associations between Nesfatin-1 levels and scores of anxiety, depression, and perceived stress in individuals of normal weight ( 20 ). Xiao et al. showed a favorable correlation between plasma Nesfatin-1 levels and the degree of depression ( 21 ). Considering the recognized correlation between psychological discomfort and pain problems, our findings further substantiate that Nesfatin-1 may function as a neurobiological mediator connecting stress and pain perception in adolescents experiencing headaches. The tissue-specific distribution of Ghrelin/GOAT—augmented in blood yet diminished in saliva—represents a particularly intriguing finding. This varied expression indicates differential control of this peptide in distinct bodily fluids, possibly signifying localized inflammatory or stress responses. Previous research has shown elevated plasma Ghrelin levels in children with anxiety disorders ( 22 ), but our study is the first to demonstrate this distinct pattern in adolescents with headaches. The mechanism behind the inverse association between blood and salivary Ghrelin/GOAT levels requires additional examination and may yield insights into the pathophysiology of headache disorders. Our data revealed prominent gender-specific differences, with muscular discomfort upon palpation occurring much more frequently in females (74%) compared to males (38%, p < 0.016). In contrast, males demonstrated elevated levels of psychological distress (90% vs 48%, p < 0.003) and were more frequently asymptomatic for TMD (66% vs 30%, p < 0.016). These gender disparities align with previous research demonstrating higher TMD prevalence in females ( 23 , 24 ), possibly due to hormonal influences, different pain thresholds, and gender-specific stress responses. The paradoxical observation of increased psychological distress in boys, despite lower incidences of TMD symptoms, indicates intricate gender-specific correlations between psychological factors and pain expression in adolescents. The correlation between TMD and headache was apparent in our population, with 52% of headache patients exhibiting TMD symptoms. This association validates the findings of Nilsen et al. about the strong correlation between TMD pain and headache in teenagers ( 25 ). Liljestrom et al. also reported connections between TMD and headaches in teenagers, urging the diagnosis of TMD when headaches are accompanied by ear pain, difficulty in mouth opening, and tiredness ( 26 ). Our data support these suggestions, indicating that a thorough evaluation of TMD should be included in the standard assessment for adolescents presenting with headaches. The lack of significant differences in Nesfatin-1 and Ghrelin/GOAT levels between patients with migraine and those with TTH is particularly noteworthy. This finding suggests that changes in these neuroendocrine markers may represent overarching pain mechanisms instead of headache-specific pathways. Similarly, the lack of significant differences in these markers among different TMD subgroups (myalgia, DDwR, no TMD) reinforces their potential role as general biomarkers of pain and/or stress, rather than as indicators of specific diseases. Parafunctional habits were significantly widespread in our study group, with both bite and non-bite parafunctions observed in over 80% of cases. This prevalence is more than the reported ranges in literature (5.9–49.6%) ( 27 , 28 ) and highlights the possible contribution of these activities to the onset and persistence of TMD and headache symptoms. The elevated incidence of physical symptoms in individuals with myalgia (89%) and disc displacement with a decrease (100%) relative to those without TMD (47%) underscores the clinical importance of these conditions and their effect on adolescents' quality of life ( 28 ). Several limitations of our study should be acknowledged. The limited sample size, especially within some subgroups, constrains the generalizability of our results. The cross-sectional approach inhibits our ability to establish causality or ascertain whether the identified neuroendocrine abnormalities are primary factors or indirect effects of headache and TMD. Moreover, although we accounted for various potential confounding factors (BMI, medication usage, and other medical disorders), we cannot dismiss the impact of unmeasured variables on our findings. Future research should concentrate on longitudinal studies to determine the temporal link between neuroendocrine indicators and headache/TMD symptoms, along with the possible predictive usefulness of these markers for treatment efficacy. Investigating the processes governing the tissue-specific patterns of Ghrelin/GOAT may yield significant insights into the pathophysiology of headache diseases. Ultimately, interventional studies focusing on these neuroendocrine pathways may provide novel therapeutic strategies for adolescents suffering from headaches and TMD. 5. CONCLUSION The current study provides novel insights into the relationship between neuroendocrine markers and headaches with TMD in adolescents. In the present study, the plasma Nesfatin-1 and Ghrelin/GOAT levels were measured in the group of adolescents with headaches. To our knowledge, no similar publications were found to analyze these hormone levels in adolescents with headaches. The most striking finding was the consistently elevated Nesfatin-1 levels in headache patients' blood and saliva, regardless of TMD status (p < 0.001). Our study revealed a distinct pattern in Ghrelin/GOAT levels: elevated in blood but decreased in saliva (p < 0.001), suggesting tissue-specific regulation of these neuroendocrine markers. In conclusion, our findings reveal substantial changes in Nesfatin-1 and Ghrelin/GOAT levels in adolescents experiencing headaches, irrespective of headache classification or TMD status. The persistent increase of these markers across various clinical manifestations indicates their potential usefulness as biomarkers in adolescent headache problems. The gender-specific disparities in TMD symptoms and psychological suffering underscore the necessity of treating sex as a biological variable in pain studies. These findings enhance our comprehension of the intricate interactions among neuroendocrine variables, psychological distress, and pain perception in adolescents experiencing headaches and TMD. Declarations Author Contribution CRediT authorship contribution statement: Conceptualization: Anna Sojka, Marcin Zarowski, Barbara Steinborn, Barbara Dorocka-Bobkowska; Data curation, formal analysis, funding acquisition, investigation, methodology, project administration, resources, software: Anna Sojka, Yasmin Bartosik; Supervision: Marcin Zarowski, Barbara Steinborn, Barbara Dorocka-Bobkowska; Validation, visualization, writing: Anna Sojka, Ozgun Yetkin. All authors reviewed the results and approved the final version of the manuscript. Acknowledgement Conflicts of Interest/Funding: Authors disclose any financial and personal relationships with others or organizations that could inappropriately influence the work. The study was conducted according to the Declaration of Helsinki and approved by the Bioethical Committee of Poznan University of Medical Sciences (approval number 550/17) (16). Written informed consent was obtained from all participants and their legal guardians following a comprehensive explanation of the study protocol. Participant anonymity was maintained throughout the study, and participation involved no financial or other obligations. The study procedures were designed to minimize any potential burden on the participants. This publication was prepared without any external source of funding. Open access funding is provided by Poznan University of Medical Sciences within the Polish Consortium. References Dao JM, Qubty W (2018) Headache Diagnosis in Children and Adolescents. Curr Pain Headache Rep 22(3):17 Wöber-Bingöl C (2013) Epidemiology of migraine and headache in children and adolescents. Curr Pain Headache Rep 17(6):341 Abu-Arafeh I, Razak S, Sivaraman B, Graham C (2010) Prevalence of headache and migraine in children and adolescents: a systematic review of population-based studies. Dev Med Child Neurol 52(12):1088–1097 Gauer RL, Semidey MJ (2015) Diagnosis and treatment of temporomandibular disorders. Am Fam Physician 91(6):378–386 The International Classification of Headache Disorders, 3rd edition (beta version). Cephalalgia (2013) ;33(9):629–808 Hershey AD, Powers SW, Vockell AL, LeCates SL, Segers A, Kabbouche MA (2004) Development of a patient-based grading scale for PedMIDAS. Cephalalgia 24(10):844–849 Hershey AD (2010) Current approaches to the diagnosis and management of paediatric migraine. Lancet Neurol 9(2):190–204 van Dongen RM, Zielman R, Noga M, Dekkers OM, Hankemeier T, van den Maagdenberg AM et al (2017) Migraine biomarkers in cerebrospinal fluid: A systematic review and meta-analysis. Cephalalgia 37(1):49–63 Loder E, Rizzoli P (2006) Biomarkers in migraine: their promise, problems, and practical applications. Headache 46(7):1046–1058 Oh IS, Shimizu H, Satoh T, Okada S, Adachi S, Inoue K et al (2006) Identification of nesfatin-1 as a satiety molecule in the hypothalamus. Nature 443(7112):709–712 Aydin S, Dag E, Ozkan Y, Erman F, Dagli AF, Kilic N et al (2009) Nesfatin-1 and ghrelin levels in serum and saliva of epileptic patients: hormonal changes can have a major effect on seizure disorders. Mol Cell Biochem 328(1–2):49–56 Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K (1999) Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 402(6762):656–660 Kojima M, Hamamoto A, Sato T (2016) Ghrelin O -acyltransferase (GOAT), a specific enzyme that modifies ghrelin with a medium-chain fatty acid. J Biochem 160(4):189–194 Gutierrez-Castro K, Kornhauser-Araujo C, Gomez A, Luevano-Contreras C, Fafutis-Morris M, Gugliucci A et al (2020) Serum nesfatin-1 levels are higher in obese vs lean adolescents and associate with cardiometabolic dyslipidemia. J Clin Mol Med. ;4 Shiiya T, Nakazato M, Mizuta M, Date Y, Mondal MS, Tanaka M et al (2002) Plasma ghrelin levels in lean and obese humans and the effect of glucose on ghrelin secretion. J Clin Endocrinol Metab 87(1):240–244 World Medical Association Declaration (2013) of Helsinki: ethical principles for medical research involving human subjects. JAMA 310(20):2191–2194 Schiffman E, Ohrbach R, Truelove E, Look J, Anderson G, Goulet JP et al (2014) Diagnostic Criteria for Temporomandibular Disorders (DC/TMD) for Clinical and Research Applications: recommendations of the International RDC/TMD Consortium Network* and Orofacial Pain Special Interest Group†. J Oral Facial Pain Headache 28(1):6–27 Ohrbach R, Knibbe W (2018) Diagnostic criteria for temporomandibular disorders (DC/TMD) scoring manual for self-report instruments. University at Buffalo, NY, US and ACTA, Amsterdam, The Netherlands Zawawi KH, Al-Badawi EA, Lobo SL, Melis M, Mehta NR (2003) An index for the measurement of normal maximum mouth opening. J Can Dent Assoc 69(11):737–741 Hofmann T, Ahnis A, Elbelt U, Rose M, Klapp BF, Stengel A (2015) NUCB2/nesfatin-1 Is Associated with Elevated Levels of Anxiety in Anorexia Nervosa. PLoS ONE 10(7):e0132058 Xiao MM, Li JB, Jiang LL, Shao H, Wang BL (2018) Plasma nesfatin-1 level is associated with severity of depression in Chinese depressive patients. BMC Psychiatry 18(1):88 Ozmen S, Şeker A, Demirci E (2019) Ghrelin and leptin levels in children with anxiety disorders. J Pediatr Endocrinol Metab 32(10):1043–1047 Pallegama RW, Ranasinghe AW, Weerasinghe VS, Sitheeque MA (2005) Anxiety and personality traits in patients with muscle related temporomandibular disorders. J Oral Rehabil 32(10):701–707 AKHTER R, MORITA M, ESAKI M, NAKAMURA K, KANEHIRA T (2011) Development of temporomandibular disorder symptoms: a 3-year cohort study of university students. J Rehabil 38(6):395–403 Nilsson IM, List T, Drangsholt M (2013) Headache and co-morbid pains associated with TMD pain in adolescents. J Dent Res 92(9):802–807 Liljeström M-R, Le Bell Y, Anttila P, Aromaa M, Jämsä T, Metsähonkala L et al (2005) Headache Children with Temporomandibular Disorders have Several Types of Pain and other Symptoms. Cephalalgia 25(11):1054–1060 Machado E, Dal-Fabbro C, Cunali PA, Kaizer OB (2014) Prevalence of sleep bruxism in children: a systematic review. Dent Press J Orthod 19(6):54–61 Manfredini D, Restrepo C, Diaz-Serrano K, Winocur E, Lobbezoo F (2013) Prevalence of sleep bruxism in children: a systematic review of the literature. J Oral Rehabil 40(8):631–642 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7076570","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":493062025,"identity":"c9131537-075f-4975-8dfc-a58c5a7a4d56","order_by":0,"name":"Anna Sojka","email":"","orcid":"","institution":"Poznan University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Anna","middleName":"","lastName":"Sojka","suffix":""},{"id":493062026,"identity":"1608d220-f285-4524-8853-61456d11e77e","order_by":1,"name":"Barbara Dorocka-Bobkowska","email":"","orcid":"","institution":"Poznan University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Barbara","middleName":"","lastName":"Dorocka-Bobkowska","suffix":""},{"id":493062027,"identity":"92a6ee07-8881-49d9-8c4c-b9a88d1bb12a","order_by":2,"name":"Yasmin Bartosik","email":"","orcid":"","institution":"Poznan University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yasmin","middleName":"","lastName":"Bartosik","suffix":""},{"id":493062028,"identity":"58849a4e-b913-47aa-9b16-905d5cc0db6e","order_by":3,"name":"Ozgun Yetkin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6UlEQVRIiWNgGAWjYDADCWYGxgcMDAdI0AHUwmxAohYGBjYJorTw8x+/Jl3YZlMn2c57rJqn5o4cPwPzw0c38GiRbDhTJj2zLU1Cmpkv7TbPsWfGkg1sxsY5eLQYHOxJk+ZtOywhx8xjdpuH7XDihgM8bNJ4tRzmAWn5D9ZSzPOPGC3H2I8BtRwAOozHjBloHWEtkj08zNY855IlZzbzGEvO7TtsLNlMwC/AEHt4m6fMjl/i/BnDD2++HZbjZ29++BifFgYGHgMGRjYIk4kHRDLjVQ4C7A8YGP5AmIw/CKoeBaNgFIyCkQgAUUdDy6dkPCgAAAAASUVORK5CYII=","orcid":"","institution":"Poznan University of Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Ozgun","middleName":"","lastName":"Yetkin","suffix":""},{"id":493062029,"identity":"a35aa19d-5ca6-40c6-a20e-7d9e55ac39da","order_by":4,"name":"Barbara Steinborn","email":"","orcid":"","institution":"Poznan University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Barbara","middleName":"","lastName":"Steinborn","suffix":""},{"id":493062030,"identity":"0a60625a-2bf1-4d25-a182-df65f4db9034","order_by":5,"name":"Marcin Zarowski","email":"","orcid":"","institution":"Poznan University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Marcin","middleName":"","lastName":"Zarowski","suffix":""}],"badges":[],"createdAt":"2025-07-08 15:53:27","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-7076570/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7076570/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89208539,"identity":"4cd34d0d-790a-4b38-8e42-1fe75767813e","added_by":"auto","created_at":"2025-08-16 20:31:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1086871,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7076570/v1/cef85107-ea07-4eff-9a6b-d01e8eb23b25.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Nesfatin-1 and Ghrelin/GOAT as Novel Biomarkers in Adolescent Headache with Temporomandibular Disorders","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eHeadache is one of the most prevalent complaints in pediatric neurological clinics (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Globally, many studies have estimated the prevalence of headaches and migraines in children and adolescents to be between 54.4% and 58.4%, often leading to school absences and decreased academic performance (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Temporomandibular disorders (TMD) and associated pain in the masticatory muscles, temporomandibular joint (TMJ), and surrounding tissues are the most common conditions of chronic orofacial pain or discomfort. They are the main cause of pain of non-dental origin in the oro-facial region (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). TMD frequently overlaps with headaches, especially with tension-type headaches (TTH), though this relationship is often overlooked in clinical practice. The International Classification of Headache Disorders, 3rd edition (beta version), (ICHD-3 beta), mentions that when the diagnosis of TMD is uncertain, the headache should be coded as \u0026lsquo;TTH or one of its subtypes\u0026rsquo; (presumably with pericranial muscle tenderness) (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Frequent headaches like migraine and TTH can have a significant impact on disability, as well as the quality of life, prompting the need for early recognition and treatment (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Therefore, early identification, establishing a treatment plan, and implementing lifestyle changes can alter disease progression and improve the child's quality of life (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). The availability of reliable biomarkers for headache diagnosis would solve such diagnostic ambiguity. Some previous reports have described biomarkers for migraine in peripheral blood and cerebrospinal fluid (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eNesfatin-1, discovered in 2006, is a satiety molecule involved in stress response and various physiological processes (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). It is present in stress-sensitive areas of the brain and has been implicated in glucose homeostasis, lipid metabolism, and anxiety behaviors (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Similarly, Ghrelin, identified in 1999, functions as a neuropeptide in the central nervous system, affecting eating behavior, energy use, and memory retention (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). And ghrelin O-acyltransferase (GOAT) is a specific enzyme that modifies ghrelin with a medium-chain fatty acid and plays an important role in regulating ghrelin\u0026rsquo;s activities (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe study aims were to investigate levels of Nesfatin-1 and the Ghrelin/GOAT, which can be analyzed in the future in clinical practice in young patients with headaches. The research questions are:\u003c/p\u003e\u003cp\u003eIs there a correlation between the levels of Nesfatin-1, Ghrelin/GOAT, the occurrence of TMD symptoms and the difference between the TMD subgroups in patients with headaches?\u003c/p\u003e\u003cp\u003eCan we investigate how ghrelin and nesfatin-1 levels could be used as a diagnostic procedure for headaches caused by different causes?\u003c/p\u003e\u003cp\u003eIs there any gender distribution of TMD, parafunctional habits, and associated symptoms in adolescents with headaches?\u003c/p\u003e"},{"header":"2. MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Study Population:\u003c/h2\u003e\u003cp\u003eThis prospective study was conducted at the Department of Developmental Neurology, Poznan University of Medical Sciences, from November 2017 to December 2020. Among 700 patients initially screened, 44 adolescents met the inclusion criteria of confirmed migraine or TTH according to the ICHD-3 beta criteria (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). The diagnosis was established through a comprehensive assessment protocol, including parental questionnaires designed by the investigators, detailed medical history, and clinical examination by a certified child neurologist (M.Z. and B.S.). The control group comprised 20 age-matched adolescents without headache or TMD symptoms.\u003c/p\u003e\u003cp\u003eThe study employed stringent exclusion criteria for both groups. Primary exclusion criteria included: history of epilepsy or other central nervous system disorders; current or previous use of psychiatric medications; presence of severe medical conditions; comorbid psychotic disorders; current malignancy; body mass index (BMI) exceeding 27; and presence of chronic inflammatory, gastrointestinal, or nutritional disorders that could potentially affect Nesfatin-1 and Ghrelin levels. Additional exclusion criteria for the control group included any history of headaches or TMD symptoms. Subjects with BMI\u0026thinsp;\u0026gt;\u0026thinsp;27 were excluded from the study as body mass significantly affects the secretion and circulating levels of both Nesfatin-1 and Ghrelin, which could confound the interpretation of results (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe study was conducted according to the Declaration of Helsinki and approved by the Bioethical Committee of Poznan University of Medical Sciences (approval number 550/17) (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Written informed consent was obtained from all participants and their legal guardians following a comprehensive explanation of the study protocol. Participant anonymity was maintained throughout the study, and participation involved no financial or other obligations. The study procedures were designed to minimize any potential burden on the participants.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Clinical Examination of TMD\u003c/h2\u003e\u003cp\u003eThe examinations were made by dentists (Y.B. and A.S.M.) according to the protocol followed by the Diagnostic Criteria for TMD (DC/TMD) Axis II guidelines (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). A comprehensive clinical assessment included medical and dental history, specifically concerning parafunctional habits such as bruxism. The intraoral examination evaluated signs of bruxism, including dental wear facets, fractures, masseter hypertrophy, and soft tissue changes (linea alba, tongue scalloping). Two major diagnostic categories were assessed: myalgia and disc displacement with reduction (DDwR). Myalgia was evaluated through palpation of temporalis and masseter muscles and pain during jaw opening, while DDwR was identified by joint sounds (clicking or popping). Following DC/TMD protocols, pain provocation during examination was confirmed as familiar pain matching the patient's primary complaint. Functional measurements included maximum mouth opening (measured from maxillary to mandibular incisal edges at midline), with opening less than 40 mm considered restricted (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Mandibular deviation was defined as displacement of 2 mm or more to either side during opening movement. The assessment also included distress evaluation using the patient health questionnaire-4 by DC/TMD Axis II - Patient Health Questionnaires (PHQ) PHQ-4, PHQ-9, generalized anxiety disorders (GAD) GAD-7 and oral behavior checklist (OBC) (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Biochemical Analysis:\u003c/h2\u003e\u003cp\u003eFasting venous blood (5 mL) and saliva (2 mL) samples were collected between 08:00\u0026ndash;09:00. Blood samples were centrifuged (3000 \u0026times; g, 5 minutes), and the serum was stored at -20\u0026deg;C for later analysis. Saliva samples underwent double centrifugation (3046.4g, 2 \u0026times; 10 minutes), with supernatant collection between cycles, and were stored at -20\u0026deg;C in 0.5 mL aliquots. Thyroid Stimulating Hormone (TSH), fasting plasma glucose, and total cholesterol were analyzed using a Cobas 8000 platform (Roche Diagnostics GmbH, Germany). Nesfatin-1 and Ghrelin/GOAT levels were duplicated using commercial \u003cem\u003eenzyme-linked immunosorbent\u003c/em\u003e assay (ELISA) kits (Human NEFA/Nesfatin-1 and Human MBOAT4/GOAT). All analyses were performed blinded using reagents from the same manufacturer lot. Both saliva and blood were tested in two trials.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Statistical Analysis:\u003c/h2\u003e\u003cp\u003eDescriptive statistics included means, standard deviations, and medians for continuous variables. The Shapiro-Wilk test was used to assess the normality of distribution. Due to non-normal distribution, between-group comparisons were conducted using non-parametric tests: Mann-Whitney U test for two-group comparisons and Kruskal-Wallis test for multiple-group analyses. Categorical variables were presented as counts (N) and percentages, with differences between proportions analyzed using chi-square tests. Statistical significance was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. All analyses were performed using Statistica v13.0 (Dell Inc., Round Rock, TX, USA).\u003c/p\u003e\u003c/div\u003e"},{"header":"3. RESULTS","content":"\u003cp\u003eThe study cohort comprised 44 adolescents with headaches (23 females [52.3%] and 21 males [47.7%] aged 10-17 years). All participants had experienced the last episodes of headaches within the previous three months, and this was the main reason for their admission to the Department. Based on ICHD-3 beta diagnostic criteria, 14 participants (31.8%) were diagnosed with migraine, while 30 (68.1%) met the criteria for TTH (5). According to DC/TMD Axis I criteria (17), the distribution of TMD was as follows: 18 patients (40.9%) with myalgia, five patients (11.3%) with DDwR, and 21 patients (48.0%) without TMD symptoms. The control group comprised 20 healthy adolescents (8 females [40%] and 12 males [60%], aged 12-17 years) without headache or TMD symptoms.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.1. Characteristics of the Patients\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTable 1 presents the demographic and biochemical characteristics of patients with headaches compared to controls, stratified by sex. No statistically significant differences were observed between female and female controls or between male patients and male controls regarding anthropometric measurements (BMI, weight, height) or basic biochemical parameters (TSH, total cholesterol, glucose). The mean BMI values were comparable between groups (20.78 kg/m\u0026sup2; in female patients vs. 19.53 kg/m\u0026sup2; in female controls; 19.05 kg/m\u0026sup2; in male patients vs. 19.90 kg/m\u0026sup2; in male controls). However, significant differences were found in neuroendocrine markers. Nesfatin-1 levels in blood were markedly elevated in both female (39.12\u0026plusmn;18.28 ng/mL vs. 9.14\u0026plusmn;0.63 ng/mL, p\u0026lt;0.001) and male patients (43.97\u0026plusmn;14.47 ng/mL vs. 7.52\u0026plusmn;2.50 ng/mL, p\u0026lt;0.001) compared to their respective controls. Similarly, salivary Nesfatin-1 levels were significantly higher in females (10.57\u0026plusmn;9.62 ng/mL vs. 2.16\u0026plusmn;0.68 ng/mL, p\u0026lt;0.005) and male patients (11.16\u0026plusmn;9.19 ng/mL vs. 1.85\u0026plusmn;0.82 ng/mL, p\u0026lt;0.001). A distinctive pattern was observed with Ghrelin/GOAT: blood levels were significantly elevated in both female (6.35\u0026plusmn;2.75 ng/mL vs. 1.47\u0026plusmn;0.22 ng/mL, p\u0026lt;0.001) and male patients (6.21\u0026plusmn;2.46 ng/mL vs. 1.46\u0026plusmn;0.45 ng/mL, p\u0026lt;0.001), while salivary levels were markedly lower in both female (0.29\u0026plusmn;0.24 ng/mL vs. 1.65\u0026plusmn;0.58 ng/mL, p\u0026lt;0.001) and male patients (0.24\u0026plusmn;0.25 ng/mL vs. 1.63\u0026plusmn;0.78 ng/mL, p\u0026lt;0.001) compared to controls.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"630\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCharacteristic\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFemale (N=23)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003emean\u0026plusmn;SD\u003c/p\u003e\n \u003cp\u003e(median)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFemale Control\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003emean\u0026plusmn;SD\u003c/p\u003e\n \u003cp\u003e(median)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMale (N=21)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003emean\u0026plusmn;SD\u003c/p\u003e\n \u003cp\u003e(median)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMale Control\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003emean\u0026plusmn;SD\u003c/p\u003e\n \u003cp\u003e(median)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003eBMI [kg/m2]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e21,05\u0026plusmn;2,41\u003c/p\u003e\n \u003cp\u003e(20,78)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e20,65\u0026plusmn;4,10\u003c/p\u003e\n \u003cp\u003e(19,53)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e0,603\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e20,23\u0026plusmn;2,53\u003c/p\u003e\n \u003cp\u003e(19,05)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e19,74\u0026plusmn;2,63\u003c/p\u003e\n \u003cp\u003e(19,90)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e0,708\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003eWeight [kg]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e51,91\u0026plusmn;9,25\u003c/p\u003e\n \u003cp\u003e(52,00)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e45,25\u0026plusmn;16,74\u003c/p\u003e\n \u003cp\u003e(47,00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e0,416\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e48,67\u0026plusmn;13,67\u003c/p\u003e\n \u003cp\u003e(50,00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e48,63\u0026plusmn;17,42\u003c/p\u003e\n \u003cp\u003e(43,75)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e0,721\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003eHeight [cm]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e156,43\u0026plusmn;10,41\u003c/p\u003e\n \u003cp\u003e(155,00)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e144,88\u0026plusmn;15,99\u003c/p\u003e\n \u003cp\u003e(153,50)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e0,099\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e153,57\u0026plusmn;14,04\u003c/p\u003e\n \u003cp\u003e(155,00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e154,08\u0026plusmn;19,98\u003c/p\u003e\n \u003cp\u003e(149,50)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e0,895\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003eTSH level[\u0026micro;U/ml]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e3,47\u0026plusmn;1,73\u003c/p\u003e\n \u003cp\u003e(3,41)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e3,08\u0026plusmn;1,01\u003c/p\u003e\n \u003cp\u003e(2,60)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e0,527\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e3,23\u0026plusmn;1,38\u003c/p\u003e\n \u003cp\u003e(2,83)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e3,37\u0026plusmn;1,16\u003c/p\u003e\n \u003cp\u003e(3,34)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e0,369\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003eTotal cholesterol[mg/dl]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e161,70\u0026plusmn;25,66\u003c/p\u003e\n \u003cp\u003e(158,00)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e158,50\u0026plusmn;29,49\u003c/p\u003e\n \u003cp\u003e(153,50)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e0,635\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e162,81\u0026plusmn;34,33\u003c/p\u003e\n \u003cp\u003e(167,00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e161,50\u0026plusmn;24,25\u003c/p\u003e\n \u003cp\u003e(161,50)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e0,808\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003eGlucose level [mg/dl]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e88,74\u0026plusmn;6,05\u003c/p\u003e\n \u003cp\u003e(88,00)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e90,63\u0026plusmn;4,96\u003c/p\u003e\n \u003cp\u003e(88,50)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e0,286\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e89,71\u0026plusmn;7,46\u003c/p\u003e\n \u003cp\u003e(93,00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e93,92\u0026plusmn;5,65\u003c/p\u003e\n \u003cp\u003e(94,50)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e0,106\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003eNesfatin-1 (blood)[ng/mL]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e39,12\u0026plusmn;18,28\u003c/p\u003e\n \u003cp\u003e(39,48)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e9,14\u0026plusmn;0,63\u003c/p\u003e\n \u003cp\u003e(9,04)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;0,001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e43,97\u0026plusmn;14,47\u003c/p\u003e\n \u003cp\u003e(45,00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e7,52\u0026plusmn;2,50\u003c/p\u003e\n \u003cp\u003e(8,14)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;0,001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003eNesfatin-1 (saliva)[ng/mL]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e10,57\u0026plusmn;9,62\u003c/p\u003e\n \u003cp\u003e(6,61)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e2,16\u0026plusmn;0,68\u003c/p\u003e\n \u003cp\u003e(2,32)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;0,005\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e11,16\u0026plusmn;9,19\u003c/p\u003e\n \u003cp\u003e(8,22)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e1,85\u0026plusmn;0,82\u003c/p\u003e\n \u003cp\u003e(1,76)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;0,001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003eGhrelin/GOAT (blood)[ng/mL]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e6,35\u0026plusmn;2,75\u003c/p\u003e\n \u003cp\u003e(5,86)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e1,47\u0026plusmn;0,22\u003c/p\u003e\n \u003cp\u003e(1,43)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;0,001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e6,21\u0026plusmn;2,46\u003c/p\u003e\n \u003cp\u003e(6,64)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e1,46\u0026plusmn;0,45\u003c/p\u003e\n \u003cp\u003e(1,39)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;0,001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003eGhrelin/GOAT (saliva)[ng/mL]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e0,29\u0026plusmn;0,24\u003c/p\u003e\n \u003cp\u003e(0,21)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e1,65\u0026plusmn;0,58\u003c/p\u003e\n \u003cp\u003e(1,41)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;0,001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0,24\u0026plusmn;0,25\u003c/p\u003e\n \u003cp\u003e(0,18)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e1,63\u0026plusmn;0,78\u003c/p\u003e\n \u003cp\u003e(1,37)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;0,001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e Characteristics of the investigated headache patients and controls\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;BMI (Body Mass Index), GOAT (Ghrelin O-acetyltransferase), TSH (Thyroid-Stimulating Hormone)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2. Prevalence of symptoms of TMD and parafunction according to a questionnaire between females and males with headache\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGender-specific analysis revealed significant differences in TMD manifestations. Muscle pain upon palpation was significantly more prevalent in females than males (74% n=17/23 vs 38% n=8/21, p\u0026lt;0.016). Conversely, males exhibited a higher prevalence of TMD-free cases than females (66% n=14/21 vs 30% n=7/23, p\u0026lt;0.016). Psychological distress was significantly more prevalent in males compared to females (90% n=19/21 vs 48% n=11/23, p\u0026lt;0.003) (Table 2).\u003c/p\u003e\n\u003cp\u003eMyalgia diagnosis, according to DC/TMD criteria, also being more frequent in females (52% n=12/23 vs. 28% n=6/23), but no significant difference (p\u0026lt;0.105). \u0026nbsp; While both groups showed comparable rates of parafunctional habits, males demonstrated a higher frequency of both bite (52% n=11/21, 43% n=10/23, p\u0026lt;0.550) and non-bite (90% n=19/21, 82% n=19/23, p\u0026lt;0.447) parafunctions. \u0026nbsp;Physical symptoms were similarly distributed between females and males (70% n=16/23 and 71% n=15/21, respectively). No significant differences were observed in other assessed parameters (\u003cem\u003eTable 2\u003c/em\u003e).\u0026quot;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u003c/strong\u003e Gender Distribution of TMD, Parafunctional Habits, and Associated Symptoms in Adolescents with Headache\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 187px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSigns and symptoms of TMD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 128px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFemale\u003c/strong\u003e (N=23)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 141px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMale\u0026nbsp;\u003c/strong\u003e(N=21)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 148px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; p-value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 187px;\"\u003e\n \u003cp\u003ePain symptoms in the temple area\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 128px;\"\u003e\n \u003cp\u003e16\u0026nbsp;(70%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 141px;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; 18 (86%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 148px;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;0,203\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 187px;\"\u003e\n \u003cp\u003eMuscle pain with palpation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 128px;\"\u003e\n \u003cp\u003e17 (74%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 141px;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;8 (38%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 148px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;0,016\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 187px;\"\u003e\n \u003cp\u003eBite parafunctions\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 128px;\"\u003e\n \u003cp\u003e10 (43%)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 141px;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;11 (52%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 148px;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;0,550\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 196px;\"\u003e\n \u003cp\u003eNon-bite parafunctions\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eSigns of bruxism\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 119px;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; 19 (82%)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; 10 (43%) \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 141px;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;19 (90%)\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;10 (48%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 148px;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;0,447\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;0,739\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 187px;\"\u003e\n \u003cp\u003ePhysical symptoms N(%)\u003c/p\u003e\n \u003cp\u003eDistress\u0026nbsp;(Depression\u0026amp;Anxiety) N(%)\u003c/p\u003e\n \u003cp\u003eDiagnosis of TMD \u0026ndash; Myalgia\u003c/p\u003e\n \u003cp\u003eDiagnosis of TMD \u0026ndash; DDwR\u003c/p\u003e\n \u003cp\u003eNo TMD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 128px;\"\u003e\n \u003cp\u003e16 (70%)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;11 (48%)\u003c/p\u003e\n \u003cp\u003e12 (52%)\u003c/p\u003e\n \u003cp\u003e3 (13%)\u003c/p\u003e\n \u003cp\u003e7 (30%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 141px;\"\u003e\n \u003cp\u003e15 (71%)\u003c/p\u003e\n \u003cp\u003e19 (90%)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;6 (28%)\u003c/p\u003e\n \u003cp\u003e2 (9%)\u003c/p\u003e\n \u003cp\u003e14 (66%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 148px;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;0,942\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;0,003\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;0,105\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;0,673\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;0,016\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eP\u0026lt;0,05, TMD (Temporomandibular Disorders), DDwR (Disc displacement with reduction)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eNeuroendocrine Markers, Physical Symptoms, and Parafunctional Habits in Adolescents with Headache: Comparison Among TMD Diagnostic Subgroups\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eComparison between TMD subgroups revealed significant differences in physical symptoms. Patients with myalgia showed a significantly higher prevalence of physical symptoms compared to the no-TMD group (89% vs 47%, p\u0026lt;0.006). Similarly, the DDwR group demonstrated significantly more physical symptoms than the no-TMD group (100% vs 47%, p\u0026lt;0.035). The Kruskal-Wallis test showed no significant differences in Nesfatin-1 (blood and saliva), Ghrelin/GOAT (blood and saliva), or TSH levels among Myalgia, DDwR, and no-TMD groups (Table 3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3.\u003c/strong\u003e Comparison of Neuroendocrine Markers and Clinical Manifestations Across TMD Diagnostic Subgroups in Headache Patients\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"661\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTMD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNesfatin-1\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e(ng/ml)\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eBlood\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003emean\u0026plusmn;SD\u003c/p\u003e\n \u003cp\u003e(median)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNesfatin-1\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e(ng/ml)\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eSaliva\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003emean\u0026plusmn;SD\u003c/p\u003e\n \u003cp\u003e(median)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGhrelin/\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eGOAT\u003c/strong\u003e (ng/ml)\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eBlood\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003emean\u0026plusmn;SD\u003c/p\u003e\n \u003cp\u003e(median)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGhrelin/\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eGOAT\u003c/strong\u003e (ng/ml)\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eSaliva\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003emean\u0026plusmn;SD\u003c/p\u003e\n \u003cp\u003e(median)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePhysical symptoms of TMD\u003c/strong\u003e N(%)\u003c/p\u003e\n \u003cp\u003emean\u0026plusmn;SD\u003c/p\u003e\n \u003cp\u003e(median)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDistress\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eN(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eParafunctional bite or nonbite\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eN(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTSH\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e(uU/ml)\u003c/p\u003e\n \u003cp\u003eN(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eMyalgia(M)\u003c/p\u003e\n \u003cp\u003eN=18\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e40,89\u0026plusmn;17,23\u003c/p\u003e\n \u003cp\u003e(40,99)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003e11,59\u0026plusmn;9,08\u003c/p\u003e\n \u003cp\u003e(10,30)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 61px;\"\u003e\n \u003cp\u003e6,45\u0026plusmn;2,85\u003c/p\u003e\n \u003cp\u003e(6,34)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0,18\u0026plusmn;0,06\u003c/p\u003e\n \u003cp\u003e(0,16)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e16(89%)*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e12(67%)*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e18(100%)*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e3,66\u0026plusmn;1,88\u003c/p\u003e\n \u003cp\u003e(3,33)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eDisc displacement with reduction\u003c/p\u003e\n \u003cp\u003e(DDwR) N=5\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e44,79\u0026plusmn;16,32\u003c/p\u003e\n \u003cp\u003e(50,00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003e11,22\u0026plusmn;11,29\u003c/p\u003e\n \u003cp\u003e(8,22)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 61px;\"\u003e\n \u003cp\u003e7,19\u0026plusmn;3,58\u003c/p\u003e\n \u003cp\u003e(6,19)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0,25\u0026plusmn;0,03\u003c/p\u003e\n \u003cp\u003e(0,25)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e5(100%)**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e5(100%)**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e5(100%)**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e3,74\u0026plusmn;1,34\u003c/p\u003e\n \u003cp\u003e(4,18)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eNo TMD N=21\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e41,10\u0026plusmn;16,76\u003c/p\u003e\n \u003cp\u003e(45,00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003e10,13\u0026plusmn;9,48\u003c/p\u003e\n \u003cp\u003e(4,48)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 61px;\"\u003e\n \u003cp\u003e5,92\u0026plusmn;2,13\u003c/p\u003e\n \u003cp\u003e(5,61)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0,33\u0026plusmn;0,33\u003c/p\u003e\n \u003cp\u003e(0,21)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e10(47%)***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e12(57%)***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e18(85%)***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e2,99\u0026plusmn;1,27\u003c/p\u003e\n \u003cp\u003e(2,56)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e0,628\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003e0,823\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 61px;\"\u003e\n \u003cp\u003e0,764\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0,067\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e*vs*** \u003cstrong\u003e\u003cem\u003e0,006\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e*vs**0,486\u003c/p\u003e\n \u003cp\u003e**vs***\u003cstrong\u003e\u003cem\u003e0,035\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e*vs*** 0,522\u003c/p\u003e\n \u003cp\u003e*vs**0,149\u003c/p\u003e\n \u003cp\u003e**vs***0,077\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e*vs*** 0,119\u003c/p\u003e\n \u003cp\u003e*vs**0,393\u003c/p\u003e\n \u003cp\u003e**vs***0,392\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e0,289\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003ep\u0026lt;0,05, GOAT (Ghrelin O-acetyltransferase), TTH (Tension-Type Headache), TMD (Temporomandibular Disorders), TSH (Thyroid-Stimulating Hormone), Distress (Depression and Anxiety)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4. Comparison of Neuroendocrine Markers and Clinical Parameters Between Migraine and Tension-Type Headache Groups\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo statistically significant differences between migraine and TTH groups were observed in the measured parameters. The Mann-Whitney test revealed similar Nesfatin-1 and Ghrelin/GOAT levels in both blood and saliva samples between groups. Additionally, no significant differences were found in TMD diagnoses (Myalgia and DDwR), parafunctional habits, psychological distress, or physical symptoms between migraine and TTH patients (Table 4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4.\u003c/strong\u003e Neuroendocrine Markers and Clinical Characteristics in Migraine versus Tension-Type Headache Patients\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"668\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHeadache\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNesfatin-1\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e(ng/ml)\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eBlood\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003emean\u0026plusmn;SD\u003c/p\u003e\n \u003cp\u003e(median)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNesfatin-1\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e(ng/ml)\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eSaliva\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003emean\u0026plusmn;SD\u003c/p\u003e\n \u003cp\u003e(median)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGhrelin/\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eGOAT\u003c/strong\u003e (ng/ml)\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eBlood\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003emean\u0026plusmn;\u003c/p\u003e\n \u003cp\u003eSD\u003c/p\u003e\n \u003cp\u003e(median)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGhrelin/\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eGOAT\u003c/strong\u003e (ng/ml)\u003c/p\u003e\n \u003cp\u003eSaliva\u003c/p\u003e\n \u003cp\u003emean\u0026plusmn;SD\u003c/p\u003e\n \u003cp\u003e(median)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTSH\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e(uU/ml)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMyalgia\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eTMD\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eN(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDDwR\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eTMD\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eN(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eParafuntions\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ebite or nonbite\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eN(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDistress\u003c/strong\u003e (N(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePhysical\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eSymptoms\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eN(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003eMigraine\u003c/p\u003e\n \u003cp\u003e(N=14)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e42,94\u0026plusmn;17,04\u003c/p\u003e\n \u003cp\u003e(42,94)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e10,98\u0026plusmn;8,23\u003c/p\u003e\n \u003cp\u003e(8,62)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e6,26\u0026plusmn;2,65\u003c/p\u003e\n \u003cp\u003e(5,91)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e0,25\u0026plusmn;0,26\u003c/p\u003e\n \u003cp\u003e(0,16)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e3,57\u0026plusmn;1,95\u003c/p\u003e\n \u003cp\u003e(2,89)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e8(57%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e1(7%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e14(100%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e8(57%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e23(76%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003eTTH\u003c/p\u003e\n \u003cp\u003e(N=30)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e40,73\u0026plusmn;16,58\u003c/p\u003e\n \u003cp\u003e(41,70)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e10,79\u0026plusmn;9,91\u003c/p\u003e\n \u003cp\u003e(4,52)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e6,30\u0026plusmn;2,60\u003c/p\u003e\n \u003cp\u003e(6,09)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e0,27\u0026plusmn;0,23\u003c/p\u003e\n \u003cp\u003e(0,21)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e3,25\u0026plusmn;1,36\u003c/p\u003e\n \u003cp\u003e(3,20)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e10(33%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e4(13%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e18(93%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e20(66%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e8(57%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e0,998\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e0,457\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e0,990\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e0,296\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e0,990\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0,131\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e0,555\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e0,399\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0,564\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e0,224\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003ep\u0026lt;0.05, Ghrelin/GOAT (Ghrelin O-acetyltransferase), TTH (Tension-Type Headache), TMD (Temporomandibular Disorders), TSH (Thyroid-Stimulating Hormone), DDwR (Disc displacement with reduction), \u0026nbsp;Distress (Depression and Anxiety)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5. Comparison of Neuroendocrine Markers Between Headache Subgroups and Controls\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNesfatin-1 and Ghrelin/GOAT levels revealed consistent patterns across all headache subgroups compared to controls (Table 5). Nesfatin-1 levels in blood were significantly elevated in migraine patients (42.94\u0026plusmn;17.04 ng/ml), TTH patients (40.73\u0026plusmn;16.58 ng/ml), those with TMD symptoms (41.74\u0026plusmn;16.75 ng/ml), and those without TMD symptoms (41.10\u0026plusmn;16.76 ng/ml) compared to controls (all p\u0026lt;0.001). Similarly, salivary Nesfatin-1 levels were markedly higher in all patient subgroups (ranging from 10.13\u0026plusmn;9.48 to 11.51\u0026plusmn;9.32 ng/ml) than in controls (1.97\u0026plusmn;0.77 ng/ml) (all p\u0026lt;0.001). Ghrelin/GOAT demonstrated a tissue-specific pattern: blood levels were significantly higher in all headache groups (ranging from 5.92\u0026plusmn;2.13 to 6.61\u0026plusmn;2.95 ng/ml) compared to controls (1.46\u0026plusmn;0.37 ng/ml) (all p\u0026lt;0.001), while salivary levels showed the opposite trend, being significantly lower in all patient groups (ranging from 0.19\u0026plusmn;0.06 to 0.33\u0026plusmn;0.33 ng/ml) compared to controls (1.64\u0026plusmn;0.69 ng/ml) (all p\u0026lt;0.001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 5.\u003c/strong\u003e Comparison of Nesfatin-1 and Ghrelin/GOAT Levels Between Headache Subgroups, TMD Categories, and Controls.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"736\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMigraine\u0026nbsp;\u003c/strong\u003e(N=14)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl\u0026nbsp;\u003c/strong\u003e(N=20)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 44px;\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e\u0026nbsp; \u003cstrong\u003eTTH\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;(N=30)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl\u0026nbsp;\u003c/strong\u003e(N=20)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 44px;\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e\u0026nbsp; \u003cstrong\u003eTMD\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; (N=23)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl\u003c/strong\u003e (N=20)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 44px;\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp; No TMD\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; (N=21)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003eControl (N=20)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 44px;\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNesfatin-1\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e(ng/ml)\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eBlood\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003emean\u0026plusmn;SD\u003c/p\u003e\n \u003cp\u003e(median)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59px;\"\u003e\n \u003cp\u003e42,94\u0026plusmn;\u003c/p\u003e\n \u003cp\u003e17,04\u003c/p\u003e\n \u003cp\u003e(42,94)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e8,16\u0026plusmn;2,10\u003c/p\u003e\n \u003cp\u003e(8,64)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 44px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u0026lt;0,001\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e40,73\u0026plusmn;16,58\u003c/p\u003e\n \u003cp\u003e(41,70)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e8,16\u0026plusmn;2,10\u003c/p\u003e\n \u003cp\u003e(8,64)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 44px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u0026lt;0,001\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e41,74\u0026plusmn;16,75\u003c/p\u003e\n \u003cp\u003e(42,33)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e8,16\u0026plusmn;2,10\u003c/p\u003e\n \u003cp\u003e(8,64)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 44px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u0026lt;0,001\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e41,10\u0026plusmn;16,76\u003c/p\u003e\n \u003cp\u003e(45,00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e8,16\u0026plusmn;2,10\u003c/p\u003e\n \u003cp\u003e(8,64)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 44px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;0,001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNesfatin-1\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e(ng/ml)\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eSaliva\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003emean\u0026plusmn;SD\u003c/p\u003e\n \u003cp\u003e(median)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59px;\"\u003e\n \u003cp\u003e10,98\u0026plusmn;\u003c/p\u003e\n \u003cp\u003e8,23\u003c/p\u003e\n \u003cp\u003e(8,62)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e1,97\u0026plusmn;0,77\u003c/p\u003e\n \u003cp\u003e(2,01)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 44px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u0026lt;0,001\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e10,79\u0026plusmn;9,91\u003c/p\u003e\n \u003cp\u003e(4,52)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e1,97\u0026plusmn;0,77\u003c/p\u003e\n \u003cp\u003e(2,01)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 44px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u0026lt;0,001\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e11,51\u0026plusmn;9,32\u003c/p\u003e\n \u003cp\u003e(8,92)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e1,97\u0026plusmn;0,77\u003c/p\u003e\n \u003cp\u003e(2,01)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 44px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u0026lt;0,001\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e10,13\u0026plusmn;9,48\u003c/p\u003e\n \u003cp\u003e(4,48)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e1,97\u0026plusmn;0,77\u003c/p\u003e\n \u003cp\u003e(2,01)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 44px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;0,001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGhrelin/\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eGOAT\u003c/strong\u003e (ng/ml)\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eBlood\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003emean\u0026plusmn;SD\u003c/p\u003e\n \u003cp\u003e(median)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59px;\"\u003e\n \u003cp\u003e6,26\u0026plusmn;2,65\u003c/p\u003e\n \u003cp\u003e(5,91)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e1,46\u0026plusmn;0,37\u003c/p\u003e\n \u003cp\u003e(1,41)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 44px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u0026lt;0,001\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e6,30\u0026plusmn;2,60\u003c/p\u003e\n \u003cp\u003e(6,09)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e1,46\u0026plusmn;0,37\u003c/p\u003e\n \u003cp\u003e(1,41)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 44px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u0026lt;0,001\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e6,61\u0026plusmn;2,95\u003c/p\u003e\n \u003cp\u003e(6,19)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e1,46\u0026plusmn;0,37\u003c/p\u003e\n \u003cp\u003e(1,41)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 44px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u0026lt;0,001\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e5,92\u0026plusmn;2,13\u003c/p\u003e\n \u003cp\u003e(5,61)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e1,46\u0026plusmn;0,37\u003c/p\u003e\n \u003cp\u003e(1,41)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 44px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;0,001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 65px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGhrelin/\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eGOAT\u003c/strong\u003e (ng/ml)\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eSaliva\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003emean\u0026plusmn;SD\u003c/p\u003e\n \u003cp\u003e(median)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59px;\"\u003e\n \u003cp\u003e0,25\u0026plusmn;0,26\u003c/p\u003e\n \u003cp\u003e(0,16)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e1,64\u0026plusmn;0,69\u003c/p\u003e\n \u003cp\u003e(1,38)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 44px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u0026lt;0,001\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e0,27\u0026plusmn;0,23\u003c/p\u003e\n \u003cp\u003e(0,21)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e1,64\u0026plusmn;0,69\u003c/p\u003e\n \u003cp\u003e(1,38)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 44px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u0026lt;0,001\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e0,19\u0026plusmn;0,06\u003c/p\u003e\n \u003cp\u003e(0,19)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e1,64\u0026plusmn;0,69\u003c/p\u003e\n \u003cp\u003e(1,38)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 44px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u0026lt;0,001\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e0,33\u0026plusmn;0,33\u003c/p\u003e\n \u003cp\u003e(0,21)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e1,64\u0026plusmn;0,69\u003c/p\u003e\n \u003cp\u003e(1,38)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 44px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;0,001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003ep\u0026lt;0,05, Ghrelin/GOAT (Ghrelin O-acetyltransferase), TTH (Tension-Type Headache), TMD (Temporomandibular Disorders)\u003c/p\u003e"},{"header":"4. DISCUSSION","content":"\u003cp\u003eThe current study provides new perspectives into the relationship between neuroendocrine markers and headaches with TMD in adolescents. The most notable finding was the consistent and significant increase of Nesfatin-1 levels in both blood and saliva among all headache groups, regardles of headache type or TMD status. This pattern and the unique tissue-specific changes in Ghrelin/GOAT levels suggest that these peptides may serve as potential biomarkers for headache disorders in adolescents.\u003c/p\u003e\u003cp\u003eThe elevated Nesfatin-1 levels reported in our headache patients correspond with recent information regarding this peptide's function beyond energy regulation. Nesfatin-1 has been associated with multiple stress-related disorders, as evidenced by Hofmann et al., who identified positive associations between Nesfatin-1 levels and scores of anxiety, depression, and perceived stress in individuals of normal weight (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Xiao et al. showed a favorable correlation between plasma Nesfatin-1 levels and the degree of depression (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Considering the recognized correlation between psychological discomfort and pain problems, our findings further substantiate that Nesfatin-1 may function as a neurobiological mediator connecting stress and pain perception in adolescents experiencing headaches.\u003c/p\u003e\u003cp\u003eThe tissue-specific distribution of Ghrelin/GOAT\u0026mdash;augmented in blood yet diminished in saliva\u0026mdash;represents a particularly intriguing finding. This varied expression indicates differential control of this peptide in distinct bodily fluids, possibly signifying localized inflammatory or stress responses. Previous research has shown elevated plasma Ghrelin levels in children with anxiety disorders (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e), but our study is the first to demonstrate this distinct pattern in adolescents with headaches. The mechanism behind the inverse association between blood and salivary Ghrelin/GOAT levels requires additional examination and may yield insights into the pathophysiology of headache disorders.\u003c/p\u003e\u003cp\u003eOur data revealed prominent gender-specific differences, with muscular discomfort upon palpation occurring much more frequently in females (74%) compared to males (38%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.016). In contrast, males demonstrated elevated levels of psychological distress (90% vs 48%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.003) and were more frequently asymptomatic for TMD (66% vs 30%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.016). These gender disparities align with previous research demonstrating higher TMD prevalence in females (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e), possibly due to hormonal influences, different pain thresholds, and gender-specific stress responses. The paradoxical observation of increased psychological distress in boys, despite lower incidences of TMD symptoms, indicates intricate gender-specific correlations between psychological factors and pain expression in adolescents.\u003c/p\u003e\u003cp\u003eThe correlation between TMD and headache was apparent in our population, with 52% of headache patients exhibiting TMD symptoms. This association validates the findings of Nilsen et al. about the strong correlation between TMD pain and headache in teenagers (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Liljestrom et al. also reported connections between TMD and headaches in teenagers, urging the diagnosis of TMD when headaches are accompanied by ear pain, difficulty in mouth opening, and tiredness (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Our data support these suggestions, indicating that a thorough evaluation of TMD should be included in the standard assessment for adolescents presenting with headaches.\u003c/p\u003e\u003cp\u003eThe lack of significant differences in Nesfatin-1 and Ghrelin/GOAT levels between patients with migraine and those with TTH is particularly noteworthy. This finding suggests that changes in these neuroendocrine markers may represent overarching pain mechanisms instead of headache-specific pathways. Similarly, the lack of significant differences in these markers among different TMD subgroups (myalgia, DDwR, no TMD) reinforces their potential role as general biomarkers of pain and/or stress, rather than as indicators of specific diseases.\u003c/p\u003e\u003cp\u003eParafunctional habits were significantly widespread in our study group, with both bite and non-bite parafunctions observed in over 80% of cases. This prevalence is more than the reported ranges in literature (5.9\u0026ndash;49.6%) (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e) and highlights the possible contribution of these activities to the onset and persistence of TMD and headache symptoms. The elevated incidence of physical symptoms in individuals with myalgia (89%) and disc displacement with a decrease (100%) relative to those without TMD (47%) underscores the clinical importance of these conditions and their effect on adolescents' quality of life (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSeveral limitations of our study should be acknowledged. The limited sample size, especially within some subgroups, constrains the generalizability of our results. The cross-sectional approach inhibits our ability to establish causality or ascertain whether the identified neuroendocrine abnormalities are primary factors or indirect effects of headache and TMD. Moreover, although we accounted for various potential confounding factors (BMI, medication usage, and other medical disorders), we cannot dismiss the impact of unmeasured variables on our findings.\u003c/p\u003e\u003cp\u003eFuture research should concentrate on longitudinal studies to determine the temporal link between neuroendocrine indicators and headache/TMD symptoms, along with the possible predictive usefulness of these markers for treatment efficacy. Investigating the processes governing the tissue-specific patterns of Ghrelin/GOAT may yield significant insights into the pathophysiology of headache diseases. Ultimately, interventional studies focusing on these neuroendocrine pathways may provide novel therapeutic strategies for adolescents suffering from headaches and TMD.\u003c/p\u003e"},{"header":"5. CONCLUSION","content":"\u003cp\u003eThe current study provides novel insights into the relationship between neuroendocrine markers and headaches with TMD in adolescents. In the present study, the plasma Nesfatin-1 and Ghrelin/GOAT levels were measured in the group of adolescents with headaches. To our knowledge, no similar publications were found to analyze these hormone levels in adolescents with headaches.\u003c/p\u003e\u003cp\u003eThe most striking finding was the consistently elevated Nesfatin-1 levels in headache patients' blood and saliva, regardless of TMD status (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Our study revealed a distinct pattern in Ghrelin/GOAT levels: elevated in blood but decreased in saliva (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), suggesting tissue-specific regulation of these neuroendocrine markers.\u003c/p\u003e\u003cp\u003eIn conclusion, our findings reveal substantial changes in Nesfatin-1 and Ghrelin/GOAT levels in adolescents experiencing headaches, irrespective of headache classification or TMD status. The persistent increase of these markers across various clinical manifestations indicates their potential usefulness as biomarkers in adolescent headache problems. The gender-specific disparities in TMD symptoms and psychological suffering underscore the necessity of treating sex as a biological variable in pain studies. These findings enhance our comprehension of the intricate interactions among neuroendocrine variables, psychological distress, and pain perception in adolescents experiencing headaches and TMD.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eCRediT authorship contribution statement: Conceptualization: Anna Sojka, Marcin Zarowski, Barbara Steinborn, Barbara Dorocka-Bobkowska; Data curation, formal analysis, funding acquisition, investigation, methodology, project administration, resources, software: Anna Sojka, Yasmin Bartosik; Supervision: Marcin Zarowski, Barbara Steinborn, Barbara Dorocka-Bobkowska; Validation, visualization, writing: Anna Sojka, Ozgun Yetkin. All authors reviewed the results and approved the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eConflicts of Interest/Funding: Authors disclose any financial and personal relationships with others or organizations that could inappropriately influence the work. The study was conducted according to the Declaration of Helsinki and approved by the Bioethical Committee of Poznan University of Medical Sciences (approval number 550/17) (16). Written informed consent was obtained from all participants and their legal guardians following a comprehensive explanation of the study protocol. Participant anonymity was maintained throughout the study, and participation involved no financial or other obligations. The study procedures were designed to minimize any potential burden on the participants. This publication was prepared without any external source of funding. Open access funding is provided by Poznan University of Medical Sciences within the Polish Consortium.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDao JM, Qubty W (2018) Headache Diagnosis in Children and Adolescents. Curr Pain Headache Rep 22(3):17\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eW\u0026ouml;ber-Bing\u0026ouml;l C (2013) Epidemiology of migraine and headache in children and adolescents. Curr Pain Headache Rep 17(6):341\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAbu-Arafeh I, Razak S, Sivaraman B, Graham C (2010) Prevalence of headache and migraine in children and adolescents: a systematic review of population-based studies. Dev Med Child Neurol 52(12):1088\u0026ndash;1097\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGauer RL, Semidey MJ (2015) Diagnosis and treatment of temporomandibular disorders. Am Fam Physician 91(6):378\u0026ndash;386\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eThe International Classification of Headache Disorders, 3rd edition (beta version). Cephalalgia (2013) ;33(9):629\u0026ndash;808\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHershey AD, Powers SW, Vockell AL, LeCates SL, Segers A, Kabbouche MA (2004) Development of a patient-based grading scale for PedMIDAS. Cephalalgia 24(10):844\u0026ndash;849\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHershey AD (2010) Current approaches to the diagnosis and management of paediatric migraine. Lancet Neurol 9(2):190\u0026ndash;204\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003evan Dongen RM, Zielman R, Noga M, Dekkers OM, Hankemeier T, van den Maagdenberg AM et al (2017) Migraine biomarkers in cerebrospinal fluid: A systematic review and meta-analysis. Cephalalgia 37(1):49\u0026ndash;63\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLoder E, Rizzoli P (2006) Biomarkers in migraine: their promise, problems, and practical applications. Headache 46(7):1046\u0026ndash;1058\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOh IS, Shimizu H, Satoh T, Okada S, Adachi S, Inoue K et al (2006) Identification of nesfatin-1 as a satiety molecule in the hypothalamus. Nature 443(7112):709\u0026ndash;712\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAydin S, Dag E, Ozkan Y, Erman F, Dagli AF, Kilic N et al (2009) Nesfatin-1 and ghrelin levels in serum and saliva of epileptic patients: hormonal changes can have a major effect on seizure disorders. Mol Cell Biochem 328(1\u0026ndash;2):49\u0026ndash;56\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K (1999) Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 402(6762):656\u0026ndash;660\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKojima M, Hamamoto A, Sato T (2016) Ghrelin O -acyltransferase (GOAT), a specific enzyme that modifies ghrelin with a medium-chain fatty acid. J Biochem 160(4):189\u0026ndash;194\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGutierrez-Castro K, Kornhauser-Araujo C, Gomez A, Luevano-Contreras C, Fafutis-Morris M, Gugliucci A et al (2020) Serum nesfatin-1 levels are higher in obese vs lean adolescents and associate with cardiometabolic dyslipidemia. J Clin Mol Med. ;4\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShiiya T, Nakazato M, Mizuta M, Date Y, Mondal MS, Tanaka M et al (2002) Plasma ghrelin levels in lean and obese humans and the effect of glucose on ghrelin secretion. J Clin Endocrinol Metab 87(1):240\u0026ndash;244\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWorld Medical Association Declaration (2013) of Helsinki: ethical principles for medical research involving human subjects. JAMA 310(20):2191\u0026ndash;2194\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSchiffman E, Ohrbach R, Truelove E, Look J, Anderson G, Goulet JP et al (2014) Diagnostic Criteria for Temporomandibular Disorders (DC/TMD) for Clinical and Research Applications: recommendations of the International RDC/TMD Consortium Network* and Orofacial Pain Special Interest Group\u0026dagger;. J Oral Facial Pain Headache 28(1):6\u0026ndash;27\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOhrbach R, Knibbe W (2018) Diagnostic criteria for temporomandibular disorders (DC/TMD) scoring manual for self-report instruments. University at Buffalo, NY, US and ACTA, Amsterdam, The Netherlands\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZawawi KH, Al-Badawi EA, Lobo SL, Melis M, Mehta NR (2003) An index for the measurement of normal maximum mouth opening. J Can Dent Assoc 69(11):737\u0026ndash;741\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHofmann T, Ahnis A, Elbelt U, Rose M, Klapp BF, Stengel A (2015) NUCB2/nesfatin-1 Is Associated with Elevated Levels of Anxiety in Anorexia Nervosa. PLoS ONE 10(7):e0132058\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXiao MM, Li JB, Jiang LL, Shao H, Wang BL (2018) Plasma nesfatin-1 level is associated with severity of depression in Chinese depressive patients. BMC Psychiatry 18(1):88\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOzmen S, Şeker A, Demirci E (2019) Ghrelin and leptin levels in children with anxiety disorders. J Pediatr Endocrinol Metab 32(10):1043\u0026ndash;1047\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePallegama RW, Ranasinghe AW, Weerasinghe VS, Sitheeque MA (2005) Anxiety and personality traits in patients with muscle related temporomandibular disorders. J Oral Rehabil 32(10):701\u0026ndash;707\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAKHTER R, MORITA M, ESAKI M, NAKAMURA K, KANEHIRA T (2011) Development of temporomandibular disorder symptoms: a 3-year cohort study of university students. J Rehabil 38(6):395\u0026ndash;403\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNilsson IM, List T, Drangsholt M (2013) Headache and co-morbid pains associated with TMD pain in adolescents. J Dent Res 92(9):802\u0026ndash;807\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiljestr\u0026ouml;m M-R, Le Bell Y, Anttila P, Aromaa M, J\u0026auml;ms\u0026auml; T, Mets\u0026auml;honkala L et al (2005) Headache Children with Temporomandibular Disorders have Several Types of Pain and other Symptoms. Cephalalgia 25(11):1054\u0026ndash;1060\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMachado E, Dal-Fabbro C, Cunali PA, Kaizer OB (2014) Prevalence of sleep bruxism in children: a systematic review. Dent Press J Orthod 19(6):54\u0026ndash;61\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eManfredini D, Restrepo C, Diaz-Serrano K, Winocur E, Lobbezoo F (2013) Prevalence of sleep bruxism in children: a systematic review of the literature. J Oral Rehabil 40(8):631\u0026ndash;642\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7076570/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7076570/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground: Nesfatin-1 and Ghrelin are recently discovered peptide hormones with roles beyond energy homeostasis. This study investigated Nesfatin-1 and Ghrelin O-acetyltransferase (Ghrelin/GOAT) levels in adolescents with headaches and analyzed their correlation with temporomandibular disorders (TMD) symptoms.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMethods: We measured Nesfatin-1 and Ghrelin/GOAT levels in the serum and saliva of 44 adolescents with headaches (aged 10-17 years) and 20 controls without headaches or TMD symptoms. TMD was evaluated using Diagnostic Criteria for TMD (DC/TMD) axis I and II. Participants underwent comprehensive clinical examination and biochemical analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eResults: Significant differences were found in Nesfatin-1 and Ghrelin/GOAT levels between patients and controls (p\u0026lt;0.001). Nesfatin-1 levels were consistently elevated in all headache patients' blood and saliva regardless of headache type or TMD status. Ghrelin/GOAT showed a distinctive tissue-specific pattern: serum concentrations were higher (p\u0026lt;0.001), while salivary levels were lower (p\u0026lt;0.001) compared to controls. Gender-specific differences were observed in TMD manifestations: muscle pain with palpation was more common in females (74% vs 38%, p\u0026lt;0.016), while psychological distress was more prevalent in males (90% vs 48%, p\u0026lt;0.003). No significant differences in neuroendocrine markers were found between migraine and tension-type headache groups.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConclusions: Altered Nesfatin-1 and Ghrelin/GOAT levels may serve as important biomarkers for adolescent headache, regardless of TMD status. The tissue-specific regulation of Ghrelin/GOAT and gender-specific clinical manifestations highlight the complex interplay between neuroendocrine factors and pain perception. These findings provide new insights into potential diagnostic markers and therapeutic targets for adolescent headache and TMD management. Key Words: Nesfatin-1 and Ghrelin/GOAT, headache, temporomandibular disorders, adolescents \u0026nbsp;\u003c/p\u003e","manuscriptTitle":"Nesfatin-1 and Ghrelin/GOAT as Novel Biomarkers in Adolescent Headache with Temporomandibular Disorders","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-01 10:22:40","doi":"10.21203/rs.3.rs-7076570/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cfd170ff-0246-41fd-8dd3-5274867438b7","owner":[],"postedDate":"August 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-16T20:23:27+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-01 10:22:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7076570","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7076570","identity":"rs-7076570","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}