Functional partitioning of lipoic acid decouples cellular abundance from mitochondrial utilization.

α-Lipoic acid (LA) is widely included in "mitochondrial cocktails" recommended to patients with primary mitochondrial disorders, yet its mechanism of action remains unclear. Here, we define the intracellular availability and functional utilization of LA in mammalian cells. We show that under typical culture conditions, free LA is near-completely absent in cells. Rather, any LA generated through mitochondrial fatty acid synthesis (mtFAS) remains in a protein-bound pool, as disruption of the mtFAS pathway does not alter free LA levels despite strong loss of protein lipoylation. Conversely, supplementation with exogenous LA markedly increases free intracellular LA in both control and mtFAS-deficient cells, but is incapable of restoring protein lipoylation, mitochondrial respiration, or cell proliferation in the absence of mtFAS. Instead, the cellular effects of LA supplementation resemble those of the antioxidant N-acetylcysteine. These findings clarify the mechanism of action of a widely used mitochondrial supplement and identify a fundamental disconnect between cellular LA abundance and mitochondrial utilization, challenging the rationale for using LA supplementation to restore mitochondrial function.