4,5-dihydroxyhexanoic acid is a robust circulating and urine marker of mitochondrial disease and its severity

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 >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>G pathogenic variant, which is the most frequent cause of the mitochondrial disorder MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes). However, the most significantly changing biomarker corresponded to an "unknown" 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. <i>In vitro</i> 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.