{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T18:07:43Z","timestamp":1785434863028,"version":"3.56.0"},"posted":{"date-parts":[[2026,2,12]]},"group-title":"Biochemistry","reference-count":41,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,2,12]],"date-time":"2026-02-12T00:00:00Z","timestamp":1770854400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["F32GM133047"],"award-info":[{"award-number":["F32GM133047"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["5R01NS124679-04"],"award-info":[{"award-number":["5R01NS124679-04"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["R01NS117538"],"award-info":[{"award-number":["R01NS117538"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100001659","name":"Deutsche Forschungsgemeinschaft","doi-asserted-by":"crossref","award":["4313887"],"award-info":[{"award-number":["4313887"]}],"id":[{"id":"10.13039\/501100001659","id-type":"DOI","asserted-by":"crossref"}]},{"id":[{"id":"https:\/\/ror.org\/006w34k90","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,2,12]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Management of patients with mitochondrial respiratory chain diseases is challenging, in part because of our incomplete understanding of pathogenesis and a lack of biomarkers. Unknown metabolites account for &gt;90% of detected features in modern metabolomics experiments and hold immense untapped promise for new basic and biomedical research. We recently used mass spectrometry-based metabolomics to identify and validate 19 circulating blood-based biomarkers for patients with the mitochondrial DNA (mtDNA) m.3243A&gt;G pathogenic variant, which is the most frequent cause of the mitochondrial disorder MELAS (\n                  <jats:underline>m<\/jats:underline>\n                  itochondrial\n                  <jats:underline>e<\/jats:underline>\n                  ncephalomyopathy, lactic\n                  <jats:underline>a<\/jats:underline>\n                  cidosis, and\n                  <jats:underline>s<\/jats:underline>\n                  troke-like episodes). However, the most significantly changing biomarker corresponded to an \u201cunknown\u201d metabolite. Here, we combine cheminformatics with analytical chemistry and identify that feature as 4,5-dihydroxyhexanoic acid (4,5-DHHA), a metabolite previously associated with inherited defects of gamma-aminobutyric acid (GABA) catabolism, but with no prior links to mitochondrial respiratory chain disorders. We validate this finding in an independent MELAS cohort and further show that 4,5-DHHA levels correlate with disease severity and are elevated in patients with other forms of mitochondrial disease and sepsis. Furthermore, brain 4,5-DHHA levels were elevated in two genetic mouse models of mitochondrial disease.\n                  <jats:italic>In vitro<\/jats:italic>\n                  and tissue culture experiments indicate that 4,5-DHHA is generated when the GABA catabolite succinic semialdehyde reacts with an intermediate of the pyruvate dehydrogenase reaction and is sensitive to mitochondrial complex I function. Our work identifies 4,5-DHHA as a robust plasma and urine marker of mitochondrial dysfunction in humans and reveals new connections between the respiratory chain and GABA metabolism.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Significance Statement<\/jats:title>\n                  <jats:p>\n                    Inborn errors of the mitochondrial respiratory chain cause severe, progressive diseases, yet effective treatments and biomarkers remain limited. Modern metabolomics detects thousands of molecules in biofluids, but the vast majority are unidentified. In this study, we investigate the most significantly altered blood metabolite in patients with the most common mitochondrial disease \u2013 MELAS (\n                    <jats:underline>m<\/jats:underline>\n                    itochondrial\n                    <jats:underline>e<\/jats:underline>\n                    ncephalomyopathy, lactic\n                    <jats:underline>a<\/jats:underline>\n                    cidosis, and\n                    <jats:underline>s<\/jats:underline>\n                    troke-like episodes) \u2013 and identify it as an 4,5-dihydroxyhexanoic acid (4,5-DHHA). We show that 4,5-DHHA is reproducibly elevated and correlates with severity. Levels are increased across multiple mitochondrial disorders as well as in sepsis and rise when respiratory chain function is impaired. These findings establish 4,5-DHHA as a promising biomarker of mitochondrial dysfunction and reveal a link to dysregulated GABA metabolism.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.02.10.705117","type":"posted-content","created":{"date-parts":[[2026,2,12]],"date-time":"2026-02-12T16:21:13Z","timestamp":1770913273000},"source":"Crossref","is-referenced-by-count":1,"title":["4,5-dihydroxyhexanoic acid is a robust circulating and urine marker of mitochondrial disease and its severity"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5023-0029","authenticated-orcid":false,"given":"Owen S.","family":"Skinner","sequence":"first","affiliation":[{"name":"Howard Hughes Medical Institute and the Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts, USA"},{"name":"Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA"},{"name":"Broad Institute, Cambridge, Massachusetts, USA"},{"name":"Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maria","family":"Miranda","sequence":"additional","affiliation":[{"name":"Howard Hughes Medical Institute and the Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts, USA"},{"name":"Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA"},{"name":"Broad Institute, Cambridge, Massachusetts, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fangcong","family":"Dong","sequence":"additional","affiliation":[{"name":"Howard Hughes Medical Institute and the Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts, USA"},{"name":"Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA"},{"name":"Broad Institute, Cambridge, Massachusetts, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tessa","family":"Struhl","sequence":"additional","affiliation":[{"name":"Howard Hughes Medical Institute and the Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts, USA"},{"name":"Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA"},{"name":"Broad Institute, Cambridge, Massachusetts, USA"},{"name":"MITRE Corporation, Stafford, Virginia, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Melissa A.","family":"Walker","sequence":"additional","affiliation":[{"name":"Howard Hughes Medical Institute and the Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts, USA"},{"name":"Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA"},{"name":"Broad Institute, Cambridge, Massachusetts, USA"},{"name":"Department of Neurology, Massachusetts General Hospital, Boston, Massachusetts, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Grigorij","family":"Schleifer","sequence":"additional","affiliation":[{"name":"Anesthesia Center for Critical Care Research of the Department of Anesthesia, Critical Care, and Pain Medicine, Harvard Medical School and Massachusetts General Hospital, Boston, Massachusetts, USA"},{"name":"Staff Unit for Medical & Scientific Tech Development & Coordination, University Hospital Bonn, Bonn, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Matthew T.","family":"Henke","sequence":"additional","affiliation":[{"name":"Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School and Blavatnik Institute, Boston, Massachusetts, United States"},{"name":"Department of Pharmaceutical Sciences, University of Illinois Chicago, Chicago, Illinois, 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