{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,3]],"date-time":"2026-08-03T08:47:37Z","timestamp":1785746857435,"version":"3.56.0"},"reference-count":29,"publisher":"MDPI AG","issue":"15","license":[{"start":{"date-parts":[[2026,8,3]],"date-time":"2026-08-03T00:00:00Z","timestamp":1785715200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biology"],"abstract":"<jats:p>Alkaptonuria (AKU) is a rare metabolic disorder caused by homogentisic acid accumulation and characterised by ochronosis, oxidative stress, chronic inflammation, and progressive connective tissue damage. This study aimed to define the circulating proteomic alterations associated with AKU and assess their relationship with nitisinone treatment. Plasma samples from 11 patients with AKU and 6 age- and sex-matched healthy controls were analysed by liquid chromatography coupled to tandem mass spectrometry using label-free quantification. Differentially abundant proteins were identified using thresholds of |log2FC| \u2265 1 and Benjamini\u2013Hochberg false discovery rate \u2264 0.01, followed by functional enrichment and treatment-stratified analyses. Twenty-two proteins were differentially abundant between AKU patients and controls. Complement components (C1R, C1S, C9, C4BPA, CPN2), fibronectin, clusterin, PGLYRP2, and haemoglobin subunits showed increased abundance, whereas most immunoglobulin chains, kallikrein, apolipoprotein A2, and alpha-1-antitrypsin showed decreased abundance. Functional enrichment highlighted complement activation, B-cell-mediated and humoral immune responses, immunoglobulin-related functions, platelet activation, and erythrocyte gas-exchange pathways. Correlation analysis linked several proteins, particularly CPN2, APOA2, C1R and C1S, to core biochemical parameters of disease activity. Treatment-stratified analysis identified fourteen proteins that remained significantly altered in both treated and untreated patients, forming a treatment-resistant core of the signature, while several complement-, coagulation-, and lipid-related proteins were significant only in one treatment subgroup. These findings define an AKU plasma proteomic signature dominated by complement activation and humoral immune alterations, together with extracellular matrix, erythrocyte-, and coagulation-associated changes. The persistence of most alterations across treatment groups suggests that residual systemic proteomic dysregulation remains despite nitisinone treatment.<\/jats:p>","DOI":"10.3390\/biology15151271","type":"journal-article","created":{"date-parts":[[2026,8,3]],"date-time":"2026-08-03T08:04:59Z","timestamp":1785744299000},"page":"1271","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Plasma Proteomic Signatures in Alkaptonuria"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0009-0000-6969-1493","authenticated-orcid":false,"given":"Rebecca","family":"Finetti","sequence":"first","affiliation":[{"name":"Department of Biotechnology, Chemistry and Pharmacy, University of Siena, 53100 Siena, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9281-034X","authenticated-orcid":false,"given":"Anna","family":"Visibelli","sequence":"additional","affiliation":[{"name":"Department of Biotechnology, Chemistry and Pharmacy, University of Siena, 53100 Siena, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-0009-4011","authenticated-orcid":false,"given":"Bianca","family":"Roncaglia","sequence":"additional","affiliation":[{"name":"Department of Biotechnology, Chemistry and Pharmacy, University of Siena, 53100 Siena, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alfonso","family":"Trezza","sequence":"additional","affiliation":[{"name":"Department of Biotechnology, Chemistry and Pharmacy, University of Siena, 53100 Siena, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4924-5423","authenticated-orcid":false,"given":"Luana","family":"Peruzzi","sequence":"additional","affiliation":[{"name":"Department of Biotechnology, Chemistry and Pharmacy, University of Siena, 53100 Siena, Italy"},{"name":"UOC Medicina Molecolare e Genetica, Azienda Ospedaliera Senese, 53100 Siena, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Barbara","family":"Marzocchi","sequence":"additional","affiliation":[{"name":"Department of Biotechnology, Chemistry and Pharmacy, University of Siena, 53100 Siena, Italy"},{"name":"UOC Laboratorio Patologia Clinica, Ospedale S. Maria alle Scotte, AOU Senese, 53100 Siena, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0263-7107","authenticated-orcid":false,"given":"Ottavia","family":"Spiga","sequence":"additional","affiliation":[{"name":"Department of Biotechnology, Chemistry and Pharmacy, University of Siena, 53100 Siena, Italy"},{"name":"Centro della Scienza e della Tecnica, Polo Universitario Grossetano, Via Ginori 41, 58100 Grosseto, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-1836-0353","authenticated-orcid":false,"given":"Mattia","family":"Cicogni","sequence":"additional","affiliation":[{"name":"Fondazione Toscana Life Sciences, Strada del Petriccio e Belriguardo, 53100 Siena, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2923-8157","authenticated-orcid":false,"given":"Laura","family":"Salvini","sequence":"additional","affiliation":[{"name":"Fondazione Toscana Life Sciences, Strada del Petriccio e Belriguardo, 53100 Siena, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Laura","family":"Tinti","sequence":"additional","affiliation":[{"name":"Fondazione Toscana Life Sciences, Strada del Petriccio e Belriguardo, 53100 Siena, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8329-1565","authenticated-orcid":false,"given":"Vittoria","family":"Cicaloni","sequence":"additional","affiliation":[{"name":"Fondazione Toscana Life Sciences, Strada del Petriccio e Belriguardo, 53100 Siena, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sara","family":"Cheleschi","sequence":"additional","affiliation":[{"name":"Fondazione Toscana Life Sciences, Strada del Petriccio e Belriguardo, 53100 Siena, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marco","family":"Rossi","sequence":"additional","affiliation":[{"name":"Fondazione Toscana Life Sciences, Strada del Petriccio e Belriguardo, 53100 Siena, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Cristina","family":"Tinti","sequence":"additional","affiliation":[{"name":"Fondazione Toscana Life Sciences, Strada del Petriccio e Belriguardo, 53100 Siena, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6976-9086","authenticated-orcid":false,"given":"Annalisa","family":"Santucci","sequence":"additional","affiliation":[{"name":"Department of Biotechnology, Chemistry and Pharmacy, University of Siena, 53100 Siena, Italy"},{"name":"Industry 4.0 Competence Center ARTES 4.0 Viale Rinaldo Piaggio, 56025 Pontedera, Italy"},{"name":"MetabERN, Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Via Aldo Moro, 53100 Siena, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,3]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"16","DOI":"10.1038\/s41572-024-00498-x","article-title":"Alkaptonuria","volume":"10","author":"Bernardini","year":"2024","journal-title":"Nat. Rev. Dis. Primers"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"70","DOI":"10.1016\/j.freeradbiomed.2015.02.021","article-title":"Oxidative stress and mechanisms of ochronosis in alkaptonuria","volume":"88","author":"Braconi","year":"2015","journal-title":"Free Radic. Biol. Med."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1053","DOI":"10.1111\/joa.14190","article-title":"An anatomical investigation of alkaptonuria: Novel insights into ochronosis of cartilage and bone","volume":"246","author":"Hughes","year":"2024","journal-title":"Am. J. Anat."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Milella, M.S., Geminiani, M., Trezza, A., Visibelli, A., Braconi, D., and Santucci, A. (2024). Alkaptonuria: From Molecular Insights to a Dedicated Digital Platform. Cells, 13.","DOI":"10.3390\/cells13121072"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1078","DOI":"10.1016\/j.joca.2018.05.017","article-title":"Inflammatory and oxidative stress biomarkers in alkaptonuria: Data from the DevelopAKUre project","volume":"26","author":"Braconi","year":"2018","journal-title":"Osteoarthr. Cartil."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"6011","DOI":"10.1002\/jcp.30284","article-title":"Homogentisic acid induces cytoskeleton and extracellular matrix alteration in alkaptonuric cartilage","volume":"236","author":"Galderisi","year":"2021","journal-title":"J. Cell. Physiol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"922","DOI":"10.1002\/jcb.22780","article-title":"Proteomic and redox-proteomic evaluation of homogentisic acid and ascorbic acid effects on human articular chondrocytes","volume":"111","author":"Braconi","year":"2010","journal-title":"J. Cell. Biochem."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1682","DOI":"10.1016\/j.bbadis.2012.07.011","article-title":"Alkaptonuria is a novel human secondary amyloidogenic disease","volume":"1822","author":"Millucci","year":"2012","journal-title":"Biochim. Biophys. Acta (BBA)-Mol. Basis Dis."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"135","DOI":"10.1016\/j.bbagen.2016.11.026","article-title":"Homogentisic acid induces aggregation and fibrillation of amyloidogenic proteins","volume":"1861","author":"Braconi","year":"2017","journal-title":"Biochim. Biophys. Acta (BBA)-Gen. Subj."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"797","DOI":"10.1007\/s10545-015-9842-8","article-title":"Amyloidosis in alkaptonuria","volume":"38","author":"Millucci","year":"2015","journal-title":"J. Inherit. Metab. Dis."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"48","DOI":"10.1016\/j.gene.2016.02.044","article-title":"Serum amyloid A1: Structure, function and gene polymorphism","volume":"583","author":"Sun","year":"2016","journal-title":"Gene"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Trezza, A., Roncaglia, B., Visibelli, A., Barletta, R., Peruzzi, L., Marzocchi, B., Braconi, D., Spiga, O., and Santucci, A. (2025). Integrated Clinomics and Molecular Dynamics Simulation Approaches Reveal the SAA1.1 Allele as a Biomarker in Alkaptonuria Disease Severity. Biomolecules, 15.","DOI":"10.3390\/biom15020194"},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Mastroeni, P., Trezza, A., Geminiani, M., Frusciante, L., Visibelli, A., and Santucci, A. (2024). HGA Triggers SAA Aggregation and Accelerates Fibril Formation in the C20\/A4 Alkaptonuria Cell Model. Cells, 13.","DOI":"10.3390\/cells13171501"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"762","DOI":"10.1016\/S2213-8587(20)30228-X","article-title":"Efficacy and safety of once-daily nitisinone for patients with alkaptonuria (SONIA 2): An international, multicentre, open-label, randomised controlled trial","volume":"8","author":"Ranganath","year":"2020","journal-title":"Lancet Diabetes Endocrinol."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Braconi, D., Geminiani, M., Psarelli, E.E., Giustarini, D., Marzocchi, B., Rossi, R., Bernardini, G., Spiga, O., Gallagher, J.A., and Sang, K.-H.L.Q. (2022). Effects of Nitisinone on Oxidative and Inflammatory Markers in Alkaptonuria: Results from SONIA1 and SONIA2 Studies. Cells, 11.","DOI":"10.3390\/cells11223668"},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Spiga, O., Cicaloni, V., Bernini, A., Zatkova, A., and Santucci, A. (2017). ApreciseKUre: An approach of Precision Medicine in a Rare Disease. BMC Med. Inform. Decis. Mak., 17.","DOI":"10.1186\/s12911-017-0438-0"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"e47","DOI":"10.1093\/nar\/gkv007","article-title":"limma powers differential expression analyses for RNA-sequencing and microarray studies","volume":"43","author":"Ritchie","year":"2015","journal-title":"Nucleic Acids Res."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"100722","DOI":"10.1016\/j.xinn.2024.100722","article-title":"Thirteen years of clusterProfiler","volume":"5","author":"Yu","year":"2024","journal-title":"The Innovation"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"25","DOI":"10.1038\/75556","article-title":"Gene ontology: Tool for the unification of biology","volume":"25","author":"Ashburner","year":"2000","journal-title":"Nat. Genet."},{"key":"ref_20","first-page":"D498","article-title":"The reactome pathway knowledgebase","volume":"48","author":"Jassal","year":"2020","journal-title":"Nucleic Acids Res."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1842","DOI":"10.1038\/s44321-025-00253-z","article-title":"Ontology-guided clustering enables proteomic analysis of rare pediatric disorders","volume":"17","author":"Itang","year":"2025","journal-title":"EMBO Mol. Med."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"e2200106","DOI":"10.1002\/prca.202200106","article-title":"Optimised plasma sample preparation and LC-MS analysis to support large-scale proteomic analysis of clinical trial specimens: Application to the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) trial","volume":"17","author":"Januszewski","year":"2023","journal-title":"Proteom.\u2013Clin. Appl."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"4982","DOI":"10.1021\/pr100646w","article-title":"Depletion of Abundant Plasma Proteins and Limitations of Plasma Proteomics","volume":"9","author":"Tu","year":"2010","journal-title":"J. Proteome Res."},{"key":"ref_24","first-page":"77","article-title":"Exploring the translational potential of clusterin as a biomarker of early osteoarthritis","volume":"32","author":"Kalvaityte","year":"2021","journal-title":"J. Orthop. Transl."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Kang, Y.-H., Varghese, P.M., Al Aiyan, A., Pondman, K., Kishore, U., and Sim, R.B. (2024). Complement-Coagulation Cross-talk: Factor H-mediated regulation of the Complement Classical Pathway activation by fibrin clots. Front. Immunol., 15.","DOI":"10.3389\/fimmu.2024.1368852"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1163","DOI":"10.1007\/s10545-011-9377-6","article-title":"Redox-proteomics of the effects of homogentisic acid in an in vitro human serum model of alkaptonuric ochronosis","volume":"34","author":"Braconi","year":"2011","journal-title":"J. Inherit. Metab. Dis."},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Michalik, S., Nteak, S.K., Drouin, N., Salazar, M.G., Hammer, E., Dhople, V.M., Holtfreter, S., Weiss, S., van den Toorn, H.W.P., and Br\u00f6ker, B.M. (2026). Immunoglobulin sub-class levels define inter-donor plasma variability: A longitudinal dual-lab study. Mol. Syst. Biol.","DOI":"10.1101\/2025.11.04.686385"},{"key":"ref_28","first-page":"S46","article-title":"Therapeutic blockade of interleukin-6 by tocilizumab in the management of AA amyloidosis and chronic inflammatory disorders: A case series and review of the literature","volume":"33","author":"Lane","year":"2015","journal-title":"Clin. Exp. Rheumatol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"271","DOI":"10.1016\/j.biocel.2016.08.016","article-title":"Comparative proteomics in alkaptonuria provides insights into inflammation and oxidative stress","volume":"81","author":"Braconi","year":"2016","journal-title":"Int. J. Biochem. Cell Biol."}],"container-title":["Biology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2079-7737\/15\/15\/1271\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,3]],"date-time":"2026-08-03T08:22:35Z","timestamp":1785745355000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2079-7737\/15\/15\/1271"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,3]]},"references-count":29,"journal-issue":{"issue":"15","published-online":{"date-parts":[[2026,8]]}},"alternative-id":["biology15151271"],"URL":"https:\/\/doi.org\/10.3390\/biology15151271","relation":{},"ISSN":["2079-7737"],"issn-type":[{"value":"2079-7737","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,3]]}}}