{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T18:24:06Z","timestamp":1785435846974,"version":"3.56.0"},"reference-count":38,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2026,6,8]],"date-time":"2026-06-08T00:00:00Z","timestamp":1780876800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Ongoing Research Funding Program","award":["ORF-2026-1293-2"],"award-info":[{"award-number":["ORF-2026-1293-2"]}]},{"DOI":"10.13039\/501100002383","name":"King Saud University","doi-asserted-by":"crossref","id":[{"id":"10.13039\/501100002383","id-type":"DOI","asserted-by":"crossref"}]},{"id":[{"id":"https:\/\/ror.org\/02f81g417","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Molecules"],"abstract":"<jats:p>Background: Phenylketonuria (PKU) is a rare genetic disorder caused by mutations in the phenylalanine hydroxylase (PAH) gene, resulting in a deficiency of the enzyme responsible for metabolizing phenylalanine (Phe) and its accumulation. PKU can be identified through newborn screening (NBS) or genetic sequencing; however, both approaches have limitations, including high false-discovery rates and variants of uncertain significance (VUS). This study aims to identify a PKU metabolomic profile using unique biomarkers to enhance early diagnosis and improve treatment outcomes. Methods: Dried blood spot (DBS) samples from 65 patients diagnosed with PKU and matched healthy controls were collected through the NBS program. An untargeted metabolomics analysis was conducted using liquid chromatography-high-resolution mass spectrometry (LC-HRMS) to profile metabolites and investigate altered metabolic pathways in patients with PKU. Results: A total of 418 significantly dysregulated metabolites were identified in PKU patients. Among them, 90 metabolites were identified as endogenous human metabolites. The most significantly affected pathways were those related to the metabolism of aromatic amino acids and polysaccharides. Moreover, lipid metabolic pathways were dysregulated, including those involved in fatty acid and phospholipid biosynthesis. In addition to phenylalanine (AUC = 0.994), 1,11-Undecanedicarboxylic acid (UDCA) (AUC = 0.969) was significantly elevated in patients with PKU, suggesting it is a promising potential biomarker for PKU. Conclusions: Untargeted metabolomics revealed distinct metabolic alterations in patients with PKU, providing insights into disease pathophysiology. The identification of UDCA as a consistently elevated metabolite supports its potential utility as a supplementary biomarker for PKU diagnosis and monitoring. Further validation in larger cohorts, using a targeted metabolomics approach, is warranted.<\/jats:p>","DOI":"10.3390\/molecules31122000","type":"journal-article","created":{"date-parts":[[2026,6,8]],"date-time":"2026-06-08T05:43:46Z","timestamp":1780897426000},"page":"2000","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Pushing the Boundaries of Biomarker Discovery in Phenylketonuria: Metabolomic Profiling Reveals Novel Biomarkers and Their Associations with Phenylalanine"],"prefix":"10.3390","volume":"31","author":[{"given":"Reem","family":"AlMalki","sequence":"first","affiliation":[{"name":"Metabolomics Section, Precision Medicine Laboratory Department, Genomics Medicine Center of Excellence, King Faisal Specialist Hospital and Research Center, Riyadh 11211, Saudi Arabia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9222-464X","authenticated-orcid":false,"given":"Shereen M.","family":"Aleidi","sequence":"additional","affiliation":[{"name":"College of Pharmacy, University of Sharjah, Sharjah 27272, United Arab Emirates"},{"name":"Department of Biopharmaceutics and Clinical Pharmacy, School of Pharmacy, The University of Jordan, Amman 11942, Jordan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2244-6842","authenticated-orcid":false,"given":"Maha","family":"Al Mogren","sequence":"additional","affiliation":[{"name":"Metabolomics Section, Precision Medicine Laboratory Department, Genomics Medicine Center of Excellence, King Faisal Specialist Hospital and Research Center, Riyadh 11211, Saudi Arabia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shaima","family":"Almohsen","sequence":"additional","affiliation":[{"name":"Metabolomics Section, Precision Medicine Laboratory Department, Genomics Medicine Center of Excellence, King Faisal Specialist Hospital and Research Center, Riyadh 11211, Saudi Arabia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9595-8649","authenticated-orcid":false,"given":"Khalid M.","family":"Sumaily","sequence":"additional","affiliation":[{"name":"Clinical Biochemistry Unit, Pathology Department, College of Medicine, King Saud University, Riyadh 11461, Saudi Arabia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4222-0952","authenticated-orcid":false,"given":"Ahmed","family":"Alfares","sequence":"additional","affiliation":[{"name":"Metabolomics Section, Precision Medicine Laboratory Department, Genomics Medicine Center of Excellence, King Faisal Specialist Hospital and Research Center, Riyadh 11211, Saudi Arabia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9527-9424","authenticated-orcid":false,"given":"Anas M. Abdel","family":"Rahman","sequence":"additional","affiliation":[{"name":"Metabolomics Section, Precision Medicine Laboratory Department, Genomics Medicine Center of Excellence, King Faisal Specialist Hospital and Research Center, Riyadh 11211, Saudi Arabia"},{"name":"Department of Neuroscience and Renal, Bristol Medical School, Dorothy Hodgkin Building, Whitson Street, Bristol BS1 3NY, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,6,8]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Eichwald, T., da Silva, L.B., Staats Pires, A.C., Niero, L., Schnorrenberger, E., Filho, C.C., Esp\u00edndola, G., Huang, W.L., Guillemin, G.J., and Abdenur, J.E. (2023). Tetrahydrobiopterin: Beyond Its Traditional Role as a Cofactor. Antioxidants, 12.","DOI":"10.3390\/antiox12051037"},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Telegina, T.A., Vechtomova, Y.L., Borzova, V.A., and Buglak, A.A. (2023). Tetrahydrobiopterin as a Trigger for Vitiligo: Phototransformation during UV Irradiation. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms241713586"},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Walter, J.H., Lachmann, R.H., and Burgard, P. (2012). Hyperphenylalaninaemia. Inborn Metabolic Diseases: Diagnosis and Treatment, Springer.","DOI":"10.1007\/978-3-642-15720-2_17"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"36","DOI":"10.1038\/s41572-021-00267-0","article-title":"Phenylketonuria","volume":"7","author":"Blau","year":"2021","journal-title":"Nat. Rev. Dis. Primers"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"S86","DOI":"10.1016\/j.ymgme.2009.10.016","article-title":"Pathogenesis of cognitive dysfunction in phenylketonuria: Review of hypotheses","volume":"99","author":"Hoeksma","year":"2010","journal-title":"Mol. Genet. Metab."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Lo Bianco, M., Leonardi, R., Migliore, A., Moliteo, E., Sciacca, M., Rinella, S., Pappalardo, M.G., La Spina, L., Messina, M., and Iacobacci, R. (2025). Sapropterin Dihydrochloride Responsiveness in Phenylketonuria: A Case Series Exploring Gaps in Comprehensive Patient Monitoring. Nutrients, 17.","DOI":"10.3390\/nu17172892"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"624","DOI":"10.1002\/jimd.12732","article-title":"The clinical relevance of novel biomarkers as outcome parameter in adults with phenylketonuria","volume":"47","author":"Engelke","year":"2024","journal-title":"J. Inherit. Metab. Dis."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"291","DOI":"10.1002\/ped4.12343","article-title":"Newborn screening for genetic disorders: Current status and prospects for the future","volume":"6","author":"Ding","year":"2022","journal-title":"Pediatr. Investig."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"22","DOI":"10.1186\/s40246-022-00398-9","article-title":"Genetic etiology and clinical challenges of phenylketonuria","volume":"16","author":"Elhawary","year":"2022","journal-title":"Hum. Genom."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"716","DOI":"10.1097\/GIM.0b013e3181b3a61e","article-title":"Impact of false-positive newborn metabolic screening results on early health care utilization","volume":"11","author":"Lipstein","year":"2009","journal-title":"Genet. Med."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Zhu, Z., Gu, J., Genchev, G.Z., Cai, X., Wang, Y., Guo, J., Tian, G., and Lu, H. (2020). Improving the diagnosis of phenylketonuria by using a machine learning\u2013based screening model of neonatal MRM data. Front. Mol. Biosci., 7.","DOI":"10.3389\/fmolb.2020.00115"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"107678","DOI":"10.1016\/j.ymgme.2023.107678","article-title":"VUS: Variant of uncertain significance or very unclear situation?","volume":"140","author":"Kemp","year":"2023","journal-title":"Mol. Genet. Metab."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"6556","DOI":"10.1038\/s41598-022-10415-5","article-title":"Clinical diagnosis of metabolic disorders using untargeted metabolomic profiling and disease-specific networks learned from profiling data","volume":"12","author":"Thistlethwaite","year":"2022","journal-title":"Sci. Rep."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"136","DOI":"10.1016\/j.jchromb.2019.04.009","article-title":"Untargeted LC\/MS-based metabolic phenotyping (metabonomics\/metabolomics): The state of the art","volume":"1117","author":"Gika","year":"2019","journal-title":"J. Chromatogr. B"},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"van der Weerd, J.C., van Wegberg, A.M.J., Boer, T.S., Engelke, U.F.H., Coene, K.L.M., Wevers, R.A., Bakker, S.J.L., de Blaauw, P., Groen, J., and van Spronsen, F.J. (2024). Impact of Phenylketonuria on the Serum Metabolome and Plasma Lipidome: A Study in Early-Treated Patients. Metabolites, 14.","DOI":"10.3390\/metabo14090479"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"3675","DOI":"10.1007\/s00415-023-11703-4","article-title":"Characterisation and differential diagnosis of neurological complications in adults with phenylketonuria: Literature review and expert opinion","volume":"270","author":"Merkel","year":"2023","journal-title":"J. Neurol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"407","DOI":"10.1007\/s10545-017-0109-4","article-title":"Structural elucidation of novel biomarkers of known metabolic disorders based on multistage fragmentation mass spectra","volume":"41","author":"Coene","year":"2018","journal-title":"J. Inherit. Metab. Dis."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"6595","DOI":"10.1039\/C9AN01642B","article-title":"Metabolomics for improved treatment monitoring of phenylketonuria: Urinary biomarkers for non-invasive assessment of dietary adherence and nutritional deficiencies","volume":"144","author":"Wild","year":"2019","journal-title":"Analyst"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"107312","DOI":"10.1016\/j.envint.2022.107312","article-title":"The exposome as the science of social-to-biological transitions","volume":"165","author":"Vineis","year":"2022","journal-title":"Environ. Int."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"341","DOI":"10.1002\/iub.1150","article-title":"Phenylalanine hydroxylase: Function, structure, and regulation","volume":"65","author":"Flydal","year":"2013","journal-title":"IUBMB Life"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1417","DOI":"10.1016\/S0140-6736(10)60961-0","article-title":"Phenylketonuria","volume":"376","author":"Blau","year":"2010","journal-title":"Lancet"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"208","DOI":"10.2174\/1574886318666230331112800","article-title":"Current Landscape on Development of Phenylalanine and Toxicity of its Metabolites\u2014A Review","volume":"19","author":"Bose","year":"2024","journal-title":"Curr. Drug Saf."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"242","DOI":"10.1006\/abio.2000.4542","article-title":"Measurement of phenyllactate, phenylacetate, and phenylpyruvate by negative ion chemical ionization-gas chromatography\/mass spectrometry in brain of mouse genetic models of phenylketonuria and non-phenylketonuria hyperphenylalaninemia","volume":"280","author":"Sarkissian","year":"2000","journal-title":"Anal. Biochem."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Pinto, A., Ilgaz, F., Evans, S., van Dam, E., Rocha, J.C., Karabulut, E., Hickson, M., Daly, A., and MacDonald, A. (2023). Phenylalanine Tolerance over Time in Phenylketonuria: A Systematic Review and Meta-Analysis. Nutrients, 15.","DOI":"10.3390\/nu15163506"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"549","DOI":"10.1002\/1097-4547(20000901)61:5<549::AID-JNR10>3.0.CO;2-0","article-title":"Is there a relationship between 3-hydroxy-3-methylglutaryl coenzyme a reductase activity and forebrain pathology in the PKU mouse?","volume":"61","author":"Shefer","year":"2000","journal-title":"J. Neurosci. Res."},{"key":"ref_26","first-page":"100360","article-title":"Myelin lipid metabolism and its role in myelination and myelin maintenance","volume":"4","author":"Hu","year":"2023","journal-title":"Innovation"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"784","DOI":"10.1016\/j.ijbiomac.2022.09.062","article-title":"Dysmyelination and glycolipid interference caused by phenylalanine in phenylketonuria","volume":"221","author":"Rondelli","year":"2022","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"390","DOI":"10.14336\/AD.2015.0827","article-title":"Phenylketonuria Pathophysiology: On the Role of Metabolic Alterations","volume":"6","author":"Schuck","year":"2015","journal-title":"Aging Dis."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"118522","DOI":"10.1016\/j.biopha.2025.118522","article-title":"Phenylketonuria: A guide through the complex maze of its neurological pathophysiology providing a new perspective on treatment strategies","volume":"191","author":"Nulmans","year":"2025","journal-title":"Biomed. Pharmacother."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"317","DOI":"10.1007\/s10571-009-9455-6","article-title":"Experimental evidence that phenylalanine provokes oxidative stress in hippocampus and cerebral cortex of developing rats","volume":"30","author":"Fernandes","year":"2010","journal-title":"Cell. Mol. Neurobiol."},{"key":"ref_31","first-page":"69","article-title":"A Multiplatform Metabolomics Approach to Characterize Plasma Levels of Phenylalanine and Tyrosine in Phenylketonuria","volume":"32","author":"Blasco","year":"2017","journal-title":"JIMD Rep."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"811","DOI":"10.1093\/ajcn\/nqab400","article-title":"Plasma metabolomic profile changes in females with phenylketonuria following a camp intervention","volume":"115","author":"Schoen","year":"2022","journal-title":"Am. J. Clin. Nutr."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"D622","DOI":"10.1093\/nar\/gkab1062","article-title":"HMDB 5.0: The human metabolome database for 2022","volume":"50","author":"Wishart","year":"2022","journal-title":"Nucleic Acids Res."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"679","DOI":"10.1007\/s12098-021-03759-9","article-title":"Inborn errors of metabolism\u2014Approach to diagnosis and management in neonates","volume":"88","author":"Balakrishnan","year":"2021","journal-title":"Indian J. Pediatr."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"479","DOI":"10.1007\/s10545-010-9104-8","article-title":"The enzymology of mitochondrial fatty acid beta-oxidation and its application to follow-up analysis of positive neonatal screening results","volume":"33","author":"Wanders","year":"2010","journal-title":"J. Inherit. Metab. Dis."},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Alotaibi, A.Z., AlMalki, R.H., Mogren, M.A., Sebaa, R., Alanazi, M., Jacob, M., Alodaib, A., Alfares, A., and Rahman, A.M.A. (2024). Exploratory Untargeted Metabolomics of Dried Blood Spot Samples from Newborns with Maple Syrup Urine Disease. Int. J. Mol. Sci., 25.","DOI":"10.3390\/ijms25115720"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"W388","DOI":"10.1093\/nar\/gkab382","article-title":"MetaboAnalyst 5.0: Narrowing the gap between raw spectra and functional insights","volume":"49","author":"Pang","year":"2021","journal-title":"Nucleic Acids Res."},{"key":"ref_38","first-page":"92","article-title":"Multivariate analysis in metabolomics","volume":"1","author":"Worley","year":"2013","journal-title":"Curr. Metabolomics"}],"container-title":["Molecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1420-3049\/31\/12\/2000\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,6,10]],"date-time":"2026-06-10T04:15:14Z","timestamp":1781064914000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1420-3049\/31\/12\/2000"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,6,8]]},"references-count":38,"journal-issue":{"issue":"12","published-online":{"date-parts":[[2026,6]]}},"alternative-id":["molecules31122000"],"URL":"https:\/\/doi.org\/10.3390\/molecules31122000","relation":{},"ISSN":["1420-3049"],"issn-type":[{"value":"1420-3049","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,6,8]]}}}