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

Background:

Mitochondrial diseases are genetic disorders that cause mitochondria to be dysfunctional. They are characterized by defects in the respiratory chain, resulting in a range of different symptoms with a widely variable age of onset. While individual mitochondrial diseases are rare, this group of genetic disorders affects 1 in 5,000 people (Wen et al., 2025). Currently, mitochondrial diseases cannot be cured, with clinical care focusing on managing symptoms. To uncover potential therapeutic targets and monitor disease progression, the authors of this preprint focus on identifying biomarkers for respiratory chain diseases.

In 2021, the group published a biomarker discovery study centering on patients with the most common mutation causing mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes, abbreviated MELAS. Using metabolomics and proteomics, the researchers identified classic markers of mitochondrial disease and novel metabolite biomarker families, resulting in 19 circulating biomarkers for MELAS, largely related to NADH-reductive stress (Sharma et al., 2021). However, the most significantly changed metabolite in their dataset remained unidentified, or “dark.” In this preprint, Skinner et al. (2026) identified this biomarker as 4,5-dihydroxyhexanoic acid (4,5-DHHA) and discovered that 4,5-DHHA correlates with disease severity across mitochondrial disease patient studies and mouse models.

What I liked/why this new work is important:

Combining mass spectrometry, patient studies, mouse models, and in vitro biochemistry, the authors identified 4,5-DHHA as a potential monitoring biomarker for respiratory chain diseases. In addition to providing a potential avenue for tracking disease progression, the identification of biomarkers can provide pathophysiological insights and new therapeutic strategies. I really appreciate how the authors mined existing datasets to support and increase the breadth of their findings.

Key findings:

Identification of an unknown metabolite biomarker

The authors mined their previously published untargeted metabolomics data collected using plasma samples from patients with MELAS (m.3243A>G pathogenic variant) and healthy controls (Sharma et al., 2021). The scientists divided the patients into two groups: patients who had experienced the stroke-like episodes that hallmark the disease were defined as the MELAS group, and patients who have the m.3242A>G mutation but had not experienced stroke-like episodes were defined as the Carrier group. They identified an unknown metabolite that showed the most significant difference between MELAS patients and controls, referring to it as U147 based on its mass-to-charge ratio. U147, detected in both plasma and urine, was increased in the MELAS group and to a lesser degree in the Carrier group. Using small molecule databases and literature searching, the authors created a list of candidate molecules consistent with the mass-to-charge ratio of U147. After comparison against standards and validation with gas chromatography- and liquid chromatography-mass spectrometry, they concluded that U147 was 4,5-DHHA.

4,5-DHHA as a biomarker for mitochondrial disease

The authors correlated 4,5-DHHA levels from their previously published patient cohort with three severity metrics to determine if 4,5-DHHA could be an effective monitoring biomarker for MELAS. They found strong correlations, with 4,5-DHHA outperforming lactate, a classical biomarker for mitochondrial disease, in two out of the three metrics. Further, by mining previously published datasets, the research team discovered that 4,5-DHHA was elevated in patients with mitochondrial myopathies, Leigh Syndrome French Canadian Variant, and septic shock, demonstrating that 4,5-DHHA could be an effective biomarker for various mitochondrial diseases and diseases characterized by respiratory chain dysfunction. They confirmed these results in mouse models of mitochondrial disease, showing that 4,5-DHHA was elevated in liver and brain in the Ndufs4^-/- model of Leigh Syndrome and in the brain of the previously published mouse model of thymidine kinase 2 deficiency.

4,5-DHHA biochemistry

The authors then sought to determine the mechanism of 4,5-DHHA synthesis using in vitro biochemistry. They propose a model in which succinic semialdehyde can be converted to 5-keto-4-hydroxyhexanoic acid (KHHA) by the pyruvate dehydrogenase complex, and then KHHA is converted to 4,5-DHHA by an unknown dehydrogenase. In cells, KHHA and 4,5-DHHA generation increased when complex I was inhibited, which matches the increase in 4,5-DHHA observed in patients and mice with respiratory chain dysfunction (as described above).

Future directions & questions for the authors:

1. I particularly appreciate the work captured in Fig. 4 to determine the biochemical origin of 4,5-DHHA. Do you have plans to pursue this mechanism? In particular, how would you try to identify the potential unknown dehydrogenase that converts KHHA to 4,5-DHHA?
2. While your results support 4,5-DHHA as a monitoring biomarker for respiratory chain diseases, you note that you were unable to correlate 4,5-DHHA levels and specific symptoms, both in the patient cohorts and mouse models. Is this a direction you hope to pursue in the future?
3. What are the most important limitations that need to be addressed before 4,5-DHHA is implemented as a monitoring biomarker for mitochondrial diseases in the clinic?

References:

Sharma, R., Reinstadler, B., Engelstad, K., Skinner, O. S., Stackowitz, E., Haller, R. G., Clish, C. B., Pierce, K., Walker, M. A., Fryer, R., Oglesbee, D., Mao, X., Shungu, D. C., Khatri, A., Hirano, M., Vivo, D. C. D., & Mootha, V. K. (2021). Circulating markers of NADH-reductive stress correlate with mitochondrial disease severity. The Journal of Clinical Investigation, 131(2). https://doi.org/10.1172/JCI136055.

Skinner, O. S., Miranda, M., Dong, F., Struhl, T., Walker, M. A., Schleifer, G., Henke, M. T., Clardy, J., Hirano, M., De Vivo, D. C., Schon, E. A., Engelstad, K., Siegmund, S. E., Laprise, C., Des Rosiers, C., Mootha, V. K., & Sharma, R. (2026) 4,5-dihydroxyhexanoic acid is a robust circulating and urine marker of mitochondrial disease and its severity. BioRxiv: The Preprint Server for Biology, 2026.02.10.705117. https://doi.org/10.64898/2026.02.10.705117.

Wen, H., Deng, H., Li, B., Chen, J., Zhu, J., Zhang, X., Yoshida, S., & Zhou, Y. (2025). Mitochondrial diseases: From molecular mechanisms to therapeutic advances. Signal Transduction and Targeted Therapy, 10(1), 9. https://doi.org/10.1038/s41392-024-02044-3.

Tags: biochemistry, biomarker, mass spectrometry, metabolite, mitochondrial disease, respiratory chain disease

(No Ratings Yet)