{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T10:17:26Z","timestamp":1788862646639,"version":"build-2803163510"},"reference-count":36,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2025,7,2]],"date-time":"2025-07-02T00:00:00Z","timestamp":1751414400000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0"},{"start":{"date-parts":[[2025,7,2]],"date-time":"2025-07-02T00:00:00Z","timestamp":1751414400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0"}],"funder":[{"DOI":"10.13039\/100009619","name":"Japan Agency for Medical Research and Development","doi-asserted-by":"publisher","award":["im0210625h0001"],"award-info":[{"award-number":["im0210625h0001"]}],"id":[{"id":"10.13039\/100009619","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100006120","name":"Ministry of the Environment, Government of Japan","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100006120","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Sci Rep"],"DOI":"10.1038\/s41598-025-07926-2","type":"journal-article","created":{"date-parts":[[2025,7,1]],"date-time":"2025-07-01T20:24:16Z","timestamp":1751401456000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Development of a novel liquid chromatography-tandem mass spectrometry based enzymatic assay of 5,10-methylenetetrahydrofolate reductase"],"prefix":"10.1038","volume":"15","author":[{"given":"Kohei","family":"Sunoki","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Miyuki","family":"Watanabe","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shiho","family":"Aoki","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eriko","family":"Jimbo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hiroko","family":"Shimbo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tomohide","family":"Goto","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hitoshi","family":"Osaka","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,7,2]]},"reference":[{"key":"7926_CR1","unstructured":"Rosenblatt, D. S. & Fenton, W. A. Inherited disorders of folate and cobalamin transport and metabolism in The Metabolic and Molecular Bases of Inherited Disease (ed Scriver, C. R.) 3897\u20133907 (McGraw-Hill, (2001)."},{"key":"7926_CR2","doi-asserted-by":"publisher","first-page":"168","DOI":"10.1016\/j.jchromb.2018.06.008","volume":"1092","author":"J Nandania","year":"2018","unstructured":"Nandania, J., Kokkonen, M., Euro, L. & Velagapudi, V. Simultaneous measurement of folate cycle intermediates in different biological matrices using liquid chromatography-tandem mass spectrometry. J. Chromatogr. B Analyt Technol. Biomed. Life Sci. 1092, 168\u2013178. https:\/\/doi.org\/10.1016\/j.jchromb.2018.06.008 (2018).","journal-title":"J. Chromatogr. B Analyt Technol. Biomed. Life Sci."},{"key":"7926_CR3","doi-asserted-by":"publisher","first-page":"298","DOI":"10.1038\/nature13236","volume":"510","author":"J Fan","year":"2014","unstructured":"Fan, J. et al. Quantitative flux analysis reveals folate-dependent NADPH production. Nature 510, 298\u2013302. https:\/\/doi.org\/10.1038\/nature13236 (2014).","journal-title":"Nature"},{"key":"7926_CR4","doi-asserted-by":"crossref","unstructured":"Surtees, R., Leonard, J. & Austin, S. Association of demyelination with deficiency of cerebrospinal-fluid S-adenosylmethionine in inborn errors of methyl-transfer pathway. Lancet 338, 1550\u20131554. 10.1016\/0140\u20136736(91)92373-a (1991).","DOI":"10.1016\/0140-6736(91)92373-A"},{"key":"7926_CR5","doi-asserted-by":"publisher","first-page":"142","DOI":"10.1111\/cga.12232","volume":"57","author":"M Hiraoka","year":"2017","unstructured":"Hiraoka, M. & Kagawa, Y. Genetic polymorphisms and folate status. Congenit Anom. 57, 142\u2013149. https:\/\/doi.org\/10.1111\/cga.12232 (2017).","journal-title":"Congenit Anom."},{"key":"7926_CR6","unstructured":"Thomas, M. A. & Rosenblatt, D. S. Severe methylenetetrahydrofolate reductase deficiency in MTHFR Polymorphisms and Disease (ed Rozen, R.) 41\u201353 (Landes Bioscience, (2005)."},{"key":"7926_CR7","doi-asserted-by":"publisher","first-page":"21","DOI":"10.1007\/s10545-016-9991-4","volume":"40","author":"M Huemer","year":"2017","unstructured":"Huemer, M. et al. Guidelines for diagnosis and management of the cobalamin-related remethylation disorders cblc, cbld, cble, cblf, cblg, CblJ and MTHFR deficiency. J. Inherit. Metab. Dis. 40, 21\u201348. https:\/\/doi.org\/10.1007\/s10545-016-9991-4 (2017).","journal-title":"J. Inherit. Metab. Dis."},{"key":"7926_CR8","doi-asserted-by":"publisher","first-page":"758","DOI":"10.1016\/j.braindev.2011.05.014","volume":"33","author":"AN Prasad","year":"2011","unstructured":"Prasad, A. N., Rupar, C. A. & Prasad, C. Methylenetetrahydrofolate reductase (MTHFR) deficiency and infantile epilepsy. Brain Dev. 33, 758\u2013769. https:\/\/doi.org\/10.1016\/j.braindev.2011.05.014 (2011).","journal-title":"Brain Dev."},{"key":"7926_CR9","doi-asserted-by":"publisher","unstructured":"Gales, A. et al. Adolescence\/adult onset MTHFR deficiency May manifest as isolated and treatable distinct neuro-psychiatric syndromes. Orphanet J. Rare Dis. 13 https:\/\/doi.org\/10.1186\/s13023-018-0767-9 (2018).","DOI":"10.1186\/s13023-018-0767-9"},{"key":"7926_CR10","doi-asserted-by":"publisher","first-page":"521","DOI":"10.1016\/j.braindev.2010.08.013","volume":"33","author":"M Tsuji","year":"2011","unstructured":"Tsuji, M. et al. 5,10-Methylenetetrahydrofolate reductase deficiency with progressive polyneuropathy in an infant. Brain Dev. 33, 521\u2013524. https:\/\/doi.org\/10.1016\/j.braindev.2010.08.013 (2011).","journal-title":"Brain Dev."},{"key":"7926_CR11","doi-asserted-by":"publisher","unstructured":"Iida, S. et al. Rapidly progressive psychotic symptoms triggered by infection in a patient with methylenetetrahydrofolate reductase deficiency: a case report. BMC Neurol. 17 https:\/\/doi.org\/10.1186\/s12883-017-0827-0 (2017).","DOI":"10.1186\/s12883-017-0827-0"},{"key":"7926_CR12","doi-asserted-by":"publisher","first-page":"e58","DOI":"10.1111\/j.1528-1167.2011.03064.x","volume":"52","author":"M Arai","year":"2011","unstructured":"Arai, M. & Osaka, H. Acute leukoencephalopathy possibly induced by phenytoin intoxication in an adult patient with methylenetetrahydrofolate reductase deficiency. Epilepsia 52, e58\u201361. https:\/\/doi.org\/10.1111\/j.1528-1167.2011.03064.x (2011).","journal-title":"Epilepsia"},{"key":"7926_CR13","doi-asserted-by":"publisher","first-page":"777","DOI":"10.1002\/jimd.12323","volume":"44","author":"C Marelli","year":"2021","unstructured":"Marelli, C. et al. Clinical and molecular characterization of adult patients with late-onset MTHFR deficiency. J. Inherit. Metab. Dis. 44, 777\u2013786. https:\/\/doi.org\/10.1002\/jimd.12323 (2021).","journal-title":"J. Inherit. Metab. Dis."},{"key":"7926_CR14","doi-asserted-by":"publisher","first-page":"200","DOI":"10.5692\/clinicalneurol.54.200","volume":"54","author":"A Tamura","year":"2014","unstructured":"Tamura, A. et al. Posterior-predominant leukoencephalopathy which was caused by methylenetetrahydrofolate reductase deficiency and successfully treated with folic acid. Rinsho Shinkeigaku. 54, 200\u2013206. https:\/\/doi.org\/10.5692\/clinicalneurol.54.200 (2014).","journal-title":"Rinsho Shinkeigaku"},{"key":"7926_CR15","doi-asserted-by":"publisher","first-page":"188","DOI":"10.1001\/jamaneurol.2013.4915","volume":"71","author":"EF Diekman","year":"2014","unstructured":"Diekman, E. F., de Koning, T. J., Verhoeven-Duif, N. M., Rovers, M. M. & van Hasselt, P. M. Survival and psychomotor development with early betaine treatment in patients with severe methylenetetrahydrofolate reductase deficiency. JAMA Neurol. 71, 188\u2013194. https:\/\/doi.org\/10.1001\/jamaneurol.2013.4915 (2014).","journal-title":"JAMA Neurol."},{"key":"7926_CR16","doi-asserted-by":"publisher","first-page":"427","DOI":"10.1002\/humu.22970","volume":"37","author":"DS Froese","year":"2016","unstructured":"Froese, D. S. et al. Mutation update and review of severe methylenetetrahydrofolate reductase deficiency. Hum. Mutat. 37, 427\u2013438. https:\/\/doi.org\/10.1002\/humu.22970 (2016).","journal-title":"Hum. Mutat."},{"key":"7926_CR17","doi-asserted-by":"publisher","first-page":"1283","DOI":"10.1016\/j.ajhg.2021.05.009","volume":"108","author":"J Weile","year":"2021","unstructured":"Weile, J. et al. Shifting landscapes of human MTHFR missense-variant effects. Am. J. Hum. Genet. 108, 1283\u20131300. https:\/\/doi.org\/10.1016\/j.ajhg.2021.05.009 (2021).","journal-title":"Am. J. Hum. Genet."},{"key":"7926_CR18","doi-asserted-by":"publisher","first-page":"1137","DOI":"10.1203\/00006450-197711000-00004","volume":"11","author":"DS Rosenblatt","year":"1977","unstructured":"Rosenblatt, D. S. & Erbe, R. W. Methylenetetrahydrofolate reductase in cultured human cells. I. Growtha and metabolic studies. Pediatr. Res. 11, 1137\u20131141. https:\/\/doi.org\/10.1203\/00006450-197711000-00004 (1977).","journal-title":"Pediatr. Res."},{"key":"7926_CR19","doi-asserted-by":"publisher","first-page":"835","DOI":"10.1093\/clinchem\/48.6.835","volume":"48","author":"T Suormala","year":"2002","unstructured":"Suormala, T., Gamse, G. & Fowler, B. 5,10-Methylenetetrahydrofolate reductase (MTHFR) assay in the forward direction: residual activity in MTHFR deficiency. Clin. Chem. 48, 835\u2013843. https:\/\/doi.org\/10.1093\/clinchem\/48.6.835 (2002).","journal-title":"Clin. Chem."},{"key":"7926_CR20","doi-asserted-by":"publisher","unstructured":"Matthews, R. G. & Baugh, C. M. Interactions of pig liver methylenetetrahydrofolate reductase with methylenetetrahydropteroylpolyglutamate substrates and with dihydropteroylpolyglutamate inhibitors. Biochemistry. 19, 2040\u2013; (2045). https:\/\/doi.org\/10.1021\/bi00551a005 (1980).","DOI":"10.1021\/bi00551a005"},{"key":"7926_CR21","doi-asserted-by":"publisher","first-page":"167","DOI":"10.3390\/ijms23010167","volume":"23","author":"C Savojardo","year":"2021","unstructured":"Savojardo, C., Babbi, G., Baldazzi, D., Martelli, P. L. & Casadio, R. A glance into MTHFR deficiency at a molecular level. Int. J. Mol. Sci. 23, 167. https:\/\/doi.org\/10.3390\/ijms23010167 (2021).","journal-title":"Int. J. Mol. Sci."},{"key":"7926_CR22","doi-asserted-by":"publisher","first-page":"2261","DOI":"10.1038\/s41467-018-04735-2","volume":"9","author":"DS Froese","year":"2018","unstructured":"Froese, D. S. et al. Structural basis for the regulation of human 5,10-methylenetetrahydrofolate reductase by phosphorylation and S-adenosylmethionine Inhibition. Nat. Commun. 9, 2261. https:\/\/doi.org\/10.1038\/s41467-018-04735-2 (2018).","journal-title":"Nat. Commun."},{"key":"7926_CR23","doi-asserted-by":"publisher","first-page":"1725","DOI":"10.1373\/clinchem.2008.104711","volume":"54","author":"XK Zhang","year":"2008","unstructured":"Zhang, X. K. et al. Multiplex enzyme assay screening of dried blood spots for lysosomal storage disorders by using tandem mass spectrometry. Clin. Chem. 54, 1725\u20131728. https:\/\/doi.org\/10.1373\/clinchem.2008.104711 (2008).","journal-title":"Clin. Chem."},{"key":"7926_CR24","doi-asserted-by":"publisher","first-page":"761","DOI":"10.1111\/cge.13936","volume":"99","author":"R Delarosa-Rodriguez","year":"2021","unstructured":"Delarosa-Rodriguez, R. et al. Accuracy diagnosis improvement of Fabry disease from dried blood spots: enzyme activity, lyso-Gb3 accumulation and GLA gene sequencing. Clin. Genet. 99, 761\u2013771. https:\/\/doi.org\/10.1111\/cge.13936 (2021).","journal-title":"Clin. Genet."},{"key":"7926_CR25","doi-asserted-by":"publisher","first-page":"7349","DOI":"10.1021\/acs.analchem.8b00650","volume":"90","author":"M Schittmayer","year":"2018","unstructured":"Schittmayer, M., Birner-Gruenberger, R. & Zamboni, N. Quantification of cellular folate species by LC-MS after stabilization by derivatization. Anal. Chem. 90, 7349\u20137356. https:\/\/doi.org\/10.1021\/acs.analchem.8b00650 (2018).","journal-title":"Anal. Chem."},{"key":"7926_CR26","doi-asserted-by":"publisher","first-page":"253","DOI":"10.1006\/abio.2001.5421","volume":"299","author":"L Huang","year":"2001","unstructured":"Huang, L., Zhang, J., Hayakawa, T. & Tsuge, H. Assays of methylenetetrahydrofolate reductase and methionine synthase activities by monitoring 5-methyltetrahydrofolate and tetrahydrofolate using high-performance liquid chromatography with fluorescence detection. Anal. Biochem. 299, 253\u2013259. https:\/\/doi.org\/10.1006\/abio.2001.5421 (2001).","journal-title":"Anal. Biochem."},{"key":"7926_CR27","doi-asserted-by":"publisher","first-page":"466","DOI":"10.1046\/j.1365-2141.2002.03876.x","volume":"162","author":"C Tonetti","year":"2003","unstructured":"Tonetti, C. et al. Relations between molecular and biological abnormalities in 11 families from siblings affected with methylenetetrahydrofolate reductase deficiency. Eur. J. Pediatr. 162, 466\u2013475. https:\/\/doi.org\/10.1046\/j.1365-2141.2002.03876.x (2003).","journal-title":"Eur. J. Pediatr."},{"key":"7926_CR28","doi-asserted-by":"publisher","first-page":"611","DOI":"10.1002\/humu.22779","volume":"36","author":"P Burda","year":"2015","unstructured":"Burda, P. et al. Insights into severe 5,10-methylenetetrahydrofolate reductase deficiency: molecular genetic and enzymatic characterization of 76 patients. Hum Mutat. 36, 611\u2013621. https:\/\/doi.org\/10.1002\/humu.22779 (2015).","journal-title":"Hum Mutat"},{"key":"7926_CR29","doi-asserted-by":"publisher","first-page":"427","DOI":"10.1038\/d41586-017-07291-9","volume":"551","author":"E Dolgin","year":"2017","unstructured":"Dolgin, E. The most popular genes in the human genome. Nature 551, 427\u2013431. https:\/\/doi.org\/10.1038\/d41586-017-07291-9 (2017).","journal-title":"Nature"},{"key":"7926_CR30","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1016\/j.ejmg.2014.10.004","volume":"58","author":"SC Liew","year":"2015","unstructured":"Liew, S. C. & Gupta, E. D. Methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism: epidemiology, metabolism and the associated diseases. Eur. J. Med. Genet. 58, 1\u201310. https:\/\/doi.org\/10.1016\/j.ejmg.2014.10.004 (2015).","journal-title":"Eur. J. Med. Genet."},{"key":"7926_CR31","doi-asserted-by":"publisher","first-page":"901","DOI":"10.1007\/s10897-016-9956-7","volume":"25","author":"BL Levin","year":"2016","unstructured":"Levin, B. L. & Varga, E. M. T. H. F. R. Addressing genetic counseling dilemmas using Evidence-Based literature. J. Genet. Couns. 25, 901\u2013911. https:\/\/doi.org\/10.1007\/s10897-016-9956-7 (2016).","journal-title":"J. Genet. Couns."},{"key":"7926_CR32","doi-asserted-by":"publisher","first-page":"1044","DOI":"10.1086\/301825","volume":"62","author":"NM van der Put","year":"1998","unstructured":"van der Put, N. M. et al. A second common mutation in the methylenetetrahydrofolate reductase gene: an additional risk factor for neural-tube defects? Am. J. Hum. Genet. 62, 1044\u20131051. https:\/\/doi.org\/10.1086\/301825 (1998).","journal-title":"Am. J. Hum. Genet."},{"key":"7926_CR33","doi-asserted-by":"publisher","first-page":"661","DOI":"10.1093\/clinchem\/47.4.661","volume":"47","author":"NQ Hanson","year":"2001","unstructured":"Hanson, N. Q., Aras, O., Yang, F. & Tsai, M. Y. C677T and A1298C polymorphisms of the methylenetetrahydrofolate reductase gene: incidence and effect of combined genotypes on plasma fasting and post-methionine load homocysteine in vascular disease. Clin. Chem. 47, 661\u2013666. https:\/\/doi.org\/10.1093\/clinchem\/47.4.661 (2001).","journal-title":"Clin. Chem."},{"key":"7926_CR34","doi-asserted-by":"publisher","first-page":"401","DOI":"10.1007\/s10545-007-0445-x","volume":"30","author":"T Rummel","year":"2007","unstructured":"Rummel, T. et al. Intermediate hyperhomocysteinaemia and compound heterozygosity for the common variant c.677C\u2009>\u2009T and a MTHFR gene mutation. J. Inherit. Metab. Dis. 30, 401. https:\/\/doi.org\/10.1007\/s10545-007-0445-x (2007).","journal-title":"J. Inherit. Metab. Dis."},{"key":"7926_CR35","unstructured":"Committee for Medicinal Products for Human Use (CHMP). European Mediciens Agency. Guideline on bioanalytical method validation. (2011). https:\/\/www.ema.europa.eu\/en\/homepage"},{"key":"7926_CR36","unstructured":"U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER). Center for Veterinary Medicine (CVM). Bioanalytical Method Validation Guidance for Industry. (2018). https:\/\/www.fda.gov\/regulatory-information\/search-fda-guidance-documents\/bioanalytical-method-validation-guidance-industry"}],"updated-by":[{"DOI":"10.1038\/s41598-026-70774-1","type":"correction","label":"Correction","source":"publisher","updated":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T00:00:00Z","timestamp":1788825600000}}],"container-title":["Scientific Reports"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.nature.com\/articles\/s41598-025-07926-2.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s41598-025-07926-2","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s41598-025-07926-2.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T09:00:12Z","timestamp":1788858012000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.nature.com\/articles\/s41598-025-07926-2"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,7,2]]},"references-count":36,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2025,12]]}},"alternative-id":["7926"],"URL":"https:\/\/doi.org\/10.1038\/s41598-025-07926-2","relation":{},"ISSN":["2045-2322"],"issn-type":[{"value":"2045-2322","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,7,2]]},"assertion":[{"value":"18 February 2025","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"18 June 2025","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"2 July 2025","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"8 September 2026","order":5,"name":"change_date","label":"Change Date","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"Correction","order":6,"name":"change_type","label":"Change Type","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"A Correction to this paper has been published:","order":7,"name":"change_details","label":"Change Details","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"https:\/\/doi.org\/10.1038\/s41598-026-70774-1","URL":"https:\/\/doi.org\/10.1038\/s41598-026-70774-1","order":8,"name":"change_details","label":"Change Details","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"The authors declare no competing interests.","order":1,"name":"Ethics","label":"Competing interests","group":{"name":"EthicsHeading","label":"Declarations"}}],"article-number":"22674"}}