Abstract

The regulation of mitochondrial function by endogenous fatty acid metabolites is critical for cellular energy homeostasis and the control of reactive oxygen species (ROS) production. This study investigates the effects of two $\omega$-oxidation products of palmitic acid, **$\omega$-hydroxypalmitic acid (HPA)** and **$\alpha,\omega$-hexadecanedioic acid (HDA)**, on the bioenergetic processes of isolated rat liver mitochondria. Our primary objective was to characterize their roles as potential modulators of **free respiration** (State 4) and **hydrogen peroxide ($\text{H}_2\text{O}_2$) generation**. Using high-resolution respirometry and fluorometric assays, we found that HPA acts as a potent uncoupler, significantly activating free respiration and decreasing the mitochondrial membrane potential ($\Delta\Psi_m$), similar to classical protonophores. In contrast, HDA stimulated respiration without a significant drop in $\Delta\Psi_m$, suggesting a distinct mechanism, possibly involving the activation of a proton leak pathway or an uncoupling protein. Crucially, both HPA and HDA demonstrated a significant inhibitory effect on $\text{H}_2\text{O}_2$ generation, a key source of oxidative stress. These findings highlight HPA and HDA as important endogenous regulators that can fine-tune mitochondrial efficiency, suggesting a protective role for these metabolites in mitigating oxidative damage by promoting mild uncoupling and reducing ROS output.