{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,3]],"date-time":"2026-08-03T03:11:51Z","timestamp":1785726711840,"version":"3.56.0"},"reference-count":51,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2026,2,4]],"date-time":"2026-02-04T00:00:00Z","timestamp":1770163200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Istanbul Atlas University Research Fund","award":["2023\/03"],"award-info":[{"award-number":["2023\/03"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["JCM"],"abstract":"<jats:p>Background: Botulinum toxin type A (BoNT-A) is an established preventive therapy for chronic migraine (CM), yet the accompanying neurochemical changes remain incompletely characterized. Objective: To evaluate the effects of BoNT-A on plasma substance P (SP), \u03b3-aminobutyric acid (GABA), glutamate, glutamine, and 5-hydroxytryptamine (5-HT), and on urinary 5-HT, and to explore relationships with clinical outcomes. Methods: In this prospective study, plasma neurotransmitters were analyzed in CM patients (n = 31) at baseline and one month after BoNT-A (155 U; PREEMPT protocol) and in healthy controls (n = 30). Plasma SP was measured using enzyme-linked immunosorbent assay (ELISA); plasma GABA, glutamate, and glutamine were quantified via liquid chromatography\u2013tandem mass spectrometry (LC\u2013MS\/MS) with isotopically labeled internal standards; plasma and urinary 5-HT were determined by high-performance liquid chromatography (HPLC). Clinical outcomes included monthly headache frequency, Visual Analog Scale (VAS), and Migraine Disability Assessment (MIDAS). Statistical analyses applied appropriate parametric or non-parametric tests with p &lt; 0.05 considered significant. Results: One month post-BoNT-A, headache frequency, MIDAS, and VAS were significantly reduced (all p &lt; 0.001). SP levels were significantly higher after BoNT-A than at baseline and versus controls. Plasma 5-HT increased post-BoNT-A, while urinary 5-HT decreased. Plasma GABA was elevated in patients versus controls without statistical significance. Glutamine was significantly higher before treatment, whereas the Glu\/Gln ratio increased after BoNT-A. Correlations revealed that higher GABA was associated with lower VAS and attack frequency post-treatment. Conclusions: BoNT-A provided short-term clinical improvement with distinct neurochemical changes, including increased plasma SP and 5-HT, decreased urinary 5-HT, reduced glutamine, and a higher Glu\/Gln ratio. These biomarkers, particularly Glu\/Gln, may serve as indicators of cortical excitability and therapeutic response in CM.<\/jats:p>","DOI":"10.3390\/jcm15031208","type":"journal-article","created":{"date-parts":[[2026,2,4]],"date-time":"2026-02-04T09:02:01Z","timestamp":1770195721000},"page":"1208","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Neurochemical Changes Following Botulinum Toxin Type A in Chronic Migraine: An LC\u2013MS\/MS and HPLC Evaluation of Plasma and Urinary Biomarkers"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-8893-2926","authenticated-orcid":false,"given":"Seyma","family":"Dumur","sequence":"first","affiliation":[{"name":"Department of Medical Biochemistry, Faculty of Medicine, Istanbul Atlas University, 34203 Istanbul, Turkey"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Demet","family":"Aygun","sequence":"additional","affiliation":[{"name":"Department of Neurology, Faculty of Medicine, Istanbul Atlas University, 34203 Istanbul, Turkey"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Era","family":"Gorica","sequence":"additional","affiliation":[{"name":"Center for Translational and Experimental Cardiology (CTEC), Department of Cardiology, University Hospital Z\u00fcrich, University of Z\u00fcrich, Wagistrasse 12, 8952 Schlieren, Switzerland"},{"name":"Department of Cardiac Surgery, University Hospital Zurich, R\u00e4mistrasse 100, 8091 Zurich, Switzerland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2362-7845","authenticated-orcid":false,"given":"Hafize","family":"Boyaci","sequence":"additional","affiliation":[{"name":"Fikret Biyal Central Biochemistry Laboratory, Istanbul University-Cerrahpasa, 34303 Istanbul, Turkey"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1052-5911","authenticated-orcid":false,"given":"Bagnu","family":"Dundar","sequence":"additional","affiliation":[{"name":"Department of Medical Biochemistry, Faculty of Medicine, Istanbul Atlas University, 34203 Istanbul, Turkey"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dildar","family":"Konukoglu","sequence":"additional","affiliation":[{"name":"Department of Medical Biochemistry, Cerrahpasa Faculty of Medicine, Istanbul University-Cerrahpasa, 34098 Istanbul, Turkey"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1347-8498","authenticated-orcid":false,"given":"Hafize","family":"Uzun","sequence":"additional","affiliation":[{"name":"Department of Medical Biochemistry, Faculty of Medicine, Istanbul Atlas University, 34203 Istanbul, Turkey"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,2,4]]},"reference":[{"key":"ref_1","unstructured":"GBD 2019 Diseases and Injuries Collaborators (2020). Global burden of 369 diseases and injuries in 204 countries and territories, 1990\u20132019: A systematic analysis for the Global Burden of Disease Study 2019. Lancet, 396, 1562."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"403","DOI":"10.1016\/bs.pbr.2020.06.008","article-title":"The genetics of migraine and the path to precision medicine","volume":"255","author":"Cader","year":"2020","journal-title":"Prog. Brain Res."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1866","DOI":"10.1056\/NEJMra1915327","article-title":"Migraine","volume":"383","author":"Ashina","year":"2020","journal-title":"N. Engl. J. Med."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Silvestro, M., Iannone, L.F., Orologio, I., Tessitore, A., Tedeschi, G., Geppetti, P., and Russo, A. (2023). Migraine Treatment: Towards New Pharmacological Targets. Int. J. Mol. Sci., 24.","DOI":"10.20944\/preprints202307.0175.v1"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"3284446","DOI":"10.1155\/2022\/3284446","article-title":"Botox (OnabotulinumtoxinA) for Treatment of Migraine Symptoms: A Systematic Review","volume":"2022","author":"Shaterian","year":"2022","journal-title":"Pain Res. Manag."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"11","DOI":"10.4103\/sjmms.sjmms_95_22","article-title":"Advances in CGRP Monoclonal Antibodies as Migraine Therapy: A Narrative Review","volume":"11","author":"Aditya","year":"2023","journal-title":"Saudi J. Med. Med. Sci."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"S9","DOI":"10.1046\/j.1526-4610.43.7s.3.x","article-title":"Evidence for antinociceptive activity of botulinum toxin type A in pain management","volume":"43","author":"Aoki","year":"2003","journal-title":"Headache"},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Valente, M., Lettieri, C., Russo, V., Janes, F., and Gigli, G.L. (2021). Clinical and Neurophysiological Effects of Botulinum Neurotoxin Type A in Chronic Migraine. Toxins, 13.","DOI":"10.3390\/toxins13060392"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"2319","DOI":"10.2147\/JPR.S113614","article-title":"Onabotulinum toxin A in the treatment of chronic migraine: Patient selection and special considerations","volume":"10","author":"Barbanti","year":"2017","journal-title":"J. Pain. Res."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"102476","DOI":"10.1016\/j.coph.2024.102476","article-title":"Native botulinum toxin type A vs. redesigned botulinum toxins in pain: What did we learn so far?","volume":"78","author":"Matak","year":"2024","journal-title":"Curr. Opin. Pharmacol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"163","DOI":"10.1186\/s10194-023-01698-8","article-title":"What to do with non-responders to CGRP(r) monoclonal antibodies: Switch to another or move to gepants?","volume":"24","author":"Vuralli","year":"2023","journal-title":"J. Headache Pain"},{"key":"ref_12","unstructured":"Graefe, S.B., Rahimi, N., and Mohiuddin, S.S. (2024, December 29). Biochemistry, Substance P, StatPearls, Available online: https:\/\/www.ncbi.nlm.nih.gov\/books\/NBK554583\/."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"227","DOI":"10.1007\/s00441-018-2922-y","article-title":"Substance P and pain chronicity","volume":"375","year":"2019","journal-title":"Cell Tissue Res."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Viudez-Mart\u00ednez, A., Torregrosa, A.B., Navarrete, F., and Garc\u00eda-Guti\u00e9rrez, M.S. (2024). Understanding the Biological Relationship between Migraine and Depression. Biomolecules, 14.","DOI":"10.3390\/biom14020163"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"2749","DOI":"10.1097\/j.pain.0000000000003289","article-title":"Region-specific changes in brain glutamate and gamma-aminobutyric acid across the migraine attack in children and adolescents","volume":"165","author":"Cho","year":"2024","journal-title":"Pain"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"100","DOI":"10.1177\/026988110001400201","article-title":"Regulation of serotonin release by GABA and excitatory amino acids","volume":"14","author":"Tao","year":"2000","journal-title":"J. Psychopharmacol."},{"key":"ref_17","first-page":"92","article-title":"Cerebrospinal fluid neurotransmitter amino acids in migraine","volume":"13","author":"Zukerman","year":"1993","journal-title":"Cephalalgia"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"102","DOI":"10.1016\/S0022-510X(98)00023-9","article-title":"Plasma levels of neuroexcitatory amino acids in patients with migraine or tension headache","volume":"156","author":"Alam","year":"1998","journal-title":"J. Neurol. Sci."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"735","DOI":"10.1177\/34.6.2422252","article-title":"Inter- and intracellular relationship of substance P-containing neurons with serotonin and GABA in the dorsal raphe nucleus: Combination of autoradiographic and immunocytochemical techniques","volume":"34","author":"Onteniente","year":"1986","journal-title":"J. Histochem. Cytochem."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"124039","DOI":"10.1016\/j.jchromb.2024.124039","article-title":"Liquid chromatography coupled to tandem mass spectrometry (LC-MS\/MS) methods for the therapeutic drug monitoring of cytotoxic anticancer drugs: An update","volume":"1236","author":"Briki","year":"2024","journal-title":"J. Chromatogr. B Analyt. Technol. Biomed. Life Sci."},{"key":"ref_21","unstructured":"(2018). Headache Classification Committee of the International Headache Society (IHS) The International Classification of Headache Disorders, 3rd edition. Cephalalgia, 38, 1\u2013211."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"126","DOI":"10.1186\/s10194-021-01339-y","article-title":"Minimal important change and responsiveness of the Migraine Disability Assessment Score (MIDAS) questionnaire","volume":"22","author":"Carvalho","year":"2021","journal-title":"J. Headache Pain"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"786","DOI":"10.1111\/j.1526-4610.2004.04146.x","article-title":"Validity and reliability of the Turkish Migraine Disability Assessment (MIDAS) questionnaire","volume":"44","author":"Siva","year":"2004","journal-title":"Headache"},{"key":"ref_24","first-page":"e088","article-title":"Validation of Digital Visual Analog Scale Pain Scoring With a Traditional Paper-based Visual Analog Scale in Adults","volume":"2","author":"Delgado","year":"2018","journal-title":"J. Am. Acad. Orthop. Surg. Glob. Res. Rev."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"493","DOI":"10.1093\/milmed\/usaa391","article-title":"Botulinum Toxin Type A for the Treatment of Post-traumatic Headache: A Randomized, Placebo-Controlled, Cross-over Study","volume":"186","author":"Zirovich","year":"2021","journal-title":"Mil. Med."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1779","DOI":"10.1007\/s10072-016-2665-z","article-title":"Effects of onabotulinumtoxinA treatment on efficacy, depression, anxiety, and disability in Turkish patients with chronic migraine","volume":"37","author":"Demiryurek","year":"2016","journal-title":"Neurol. Sci."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"69","DOI":"10.1016\/j.toxicon.2020.02.017","article-title":"Chronic migraine and Botulinum Toxin Type A: Where do paths cross?","volume":"178","author":"Martinelli","year":"2020","journal-title":"Toxicon"},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Heikkil\u00e4, H.M., Hielm-Bj\u00f6rkman, A.K., Innes, J.F., and Laitinen-Vapaavuori, O.M. (2017). The effect of intra-articular botulinum toxin A on substance P, prostaglandin E2, and tumor necrosis factor alpha in the canine osteoarthritic joint. BMC Vet. Res., 13.","DOI":"10.1186\/s12917-017-0990-y"},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Becker, W.J. (2020). Botulinum Toxin in the Treatment of Headache. Toxins, 12.","DOI":"10.3390\/toxins12120803"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"39","DOI":"10.1016\/j.pneurobio.2014.06.001","article-title":"Botulinum toxin A, brain and pain","volume":"119\u2013120","author":"Matak","year":"2014","journal-title":"Prog. Neurobiol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"619","DOI":"10.3390\/toxins14090619","article-title":"Botulinum Toxin-A Current Place in the Treatment of Chronic Migraine and Other Primary Headaches","volume":"14","author":"Domitrz","year":"2022","journal-title":"Toxins"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"245","DOI":"10.1016\/S0041-0101(99)00153-1","article-title":"Sensitivity of embryonic rat dorsal root ganglia neurons to Clostridium botulinum neurotoxins","volume":"38","author":"Welch","year":"2000","journal-title":"Toxicon"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"106","DOI":"10.1016\/S0021-5155(99)00197-5","article-title":"Presynaptic effects of botulinum toxin type A on the neuronally evoked response of albino and pigmented rabbit iris sphincter and dilator muscles","volume":"44","author":"Ishikawa","year":"2000","journal-title":"Jpn. J. Ophthalmol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"366","DOI":"10.1111\/j.1464-410X.2007.07312.x","article-title":"Botulinum toxin type A inhibits sensory neuropeptide release in rat bladder models of acute injury and chronic inflammation","volume":"101","author":"Lucioni","year":"2008","journal-title":"BJU Int."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"316","DOI":"10.1016\/j.pain.2010.02.024","article-title":"Peptide-mediated transdermal delivery of botulinum neurotoxin type A reduces neurogenic inflammation in the skin","volume":"149","author":"Carmichael","year":"2010","journal-title":"Pain"},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Filipovi\u0107, B., Matak, I., Bach-Rojecky, L., and Lackovi\u0107, Z. (2012). Central action of peripherally applied botulinum toxin type A on pain and dural protein extravasation in rat model of trigeminal neuropathy. PLoS ONE, 7.","DOI":"10.1371\/journal.pone.0029803"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"137","DOI":"10.1016\/j.neuroscience.2017.06.040","article-title":"Involvement of substance P in the antinociceptive effect of botulinum toxin type A: Evidence from knockout mice","volume":"358","author":"Matak","year":"2017","journal-title":"Neuroscience"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"319","DOI":"10.1038\/ncpuro1124","article-title":"Drug Insight: Biological effects of botulinum toxin A in the lower urinary tract","volume":"5","author":"Chancellor","year":"2008","journal-title":"Nat. Clin. Pract. Urol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"1203","DOI":"10.1093\/brain\/awz052","article-title":"Acute withdrawal and botulinum toxin A in chronic migraine with medication overuse: A double-blind randomized controlled trial","volume":"142","author":"Pijpers","year":"2019","journal-title":"Brain"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"97","DOI":"10.1016\/j.nicl.2018.01.016","article-title":"High brain serotonin levels in migraine between attacks: A 5-HT4 receptor binding PET study","volume":"18","author":"Deen","year":"2018","journal-title":"Neuroimage Clin."},{"key":"ref_41","doi-asserted-by":"crossref","unstructured":"Matak, I., B\u00f6lcskei, K., Bach-Rojecky, L., and Helyes, Z. (2019). Mechanisms of Botulinum Toxin Type A Action on Pain. Toxins, 11.","DOI":"10.3390\/toxins11080459"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"142","DOI":"10.1111\/j.1600-0854.2006.00520.x","article-title":"Traffic of botulinum toxins A and E in excitatory and inhibitory neurons","volume":"8","author":"Verderio","year":"2007","journal-title":"Traffic"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"331","DOI":"10.1016\/j.neuropharm.2013.02.011","article-title":"Involvement of \u03bc-opioid receptors in antinociceptive action of botulinum toxin type A","volume":"70","author":"Matak","year":"2013","journal-title":"Neuropharmacology"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"665","DOI":"10.1007\/s00702-013-1150-6","article-title":"Association of antinociceptive action of botulinum toxin type A with GABA-A receptor","volume":"121","year":"2014","journal-title":"J. Neural Transm."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"21338","DOI":"10.1016\/S0021-9258(19)36614-1","article-title":"Tetanus toxin and botulinum toxins type A and B inhibit glutamate, gamma-aminobutyric acid, aspartate, and met-enkephalin release from synaptosomes. Clues to the locus of action","volume":"267","author":"McMahon","year":"1992","journal-title":"J. Biol. Chem."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"2248","DOI":"10.1097\/j.pain.0000000000000634","article-title":"Combined glutamate and glutamine levels in pain-processing brain regions are associated with individual pain sensitivity","volume":"157","author":"Zunhammer","year":"2016","journal-title":"Pain"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"100","DOI":"10.1016\/j.pnpbp.2014.10.011","article-title":"Blockade of spinal glutamate recycling produces paradoxical antinociception in rats with orofacial inflammatory pain","volume":"57","author":"Yang","year":"2015","journal-title":"Prog. Neuropsychopharmacol. Biol. Psychiatry"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"1859","DOI":"10.1093\/brain\/awx130","article-title":"Cortical glutamate in migraine","volume":"140","author":"Zielman","year":"2017","journal-title":"Brain"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"55","DOI":"10.1186\/s10194-018-0885-8","article-title":"Increased thalamic glutamate\/glutamine levels in migraineurs","volume":"19","author":"Bathel","year":"2018","journal-title":"J. Headache Pain"},{"key":"ref_50","doi-asserted-by":"crossref","unstructured":"Noseda, R., Kainz, V., Borsook, D., and Burstein, R. (2014). Neurochemical pathways that converge on thalamic trigeminovascular neurons: Potential substrate for modulation of migraine by sleep, food intake, stress and anxiety. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0103929"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"117112","DOI":"10.1016\/j.neuroimage.2020.117112","article-title":"Regional balance between glutamate+glutamine and GABA+ in the resting human brain","volume":"220","author":"Steel","year":"2020","journal-title":"Neuroimage"}],"container-title":["Journal of Clinical Medicine"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2077-0383\/15\/3\/1208\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,2,4]],"date-time":"2026-02-04T09:16:31Z","timestamp":1770196591000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2077-0383\/15\/3\/1208"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,2,4]]},"references-count":51,"journal-issue":{"issue":"3","published-online":{"date-parts":[[2026,2]]}},"alternative-id":["jcm15031208"],"URL":"https:\/\/doi.org\/10.3390\/jcm15031208","relation":{},"ISSN":["2077-0383"],"issn-type":[{"value":"2077-0383","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,2,4]]}}}