Absolute quantification of tricarboxylic acid (TCA) cycle intermediates in mouse ocular tissues reveals distinct tissue- and sex-specific mitochondrial metabolism

The mitochondrial tricarboxylic acid (TCA) cycle is central to cellular energy production and redox homeostasis in the eye, which must sustain exceptionally high metabolic activity to support vision. Retinal neurons, the retinal pigment epithelium (RPE), cornea, and lens perform distinct physiological functions and therefore exhibit divergent metabolic demands; however, the absolute concentrations of key TCA cycle intermediates and their variation across ocular tissues, between sexes, and over the course of the day remain poorly defined. Targeted gas chromatography–mass spectrometry was employed to quantify absolute concentrations of TCA cycle metabolites in mouse ocular tissues collected at 10 AM and 2 PM to assess time-of-day–dependent metabolic variation. Ratios of selected metabolites were subsequently calculated to gain insight into tissue-specific TCA cycle dynamics. Among all ocular tissues, the retina exhibited the highest concentrations of TCA cycle intermediates, particularly succinate, citrate, and malate, consistent with its high energy demands. The RPE/choroid displayed a balanced distribution of intermediates and the highest α-ketoglutarate (α-KG)/isocitrate ratio, indicative of robust mitochondrial oxidation and reductive carboxylation capacity. Corneal tissue was characterized by prominent malate enrichment, especially in females, suggesting a metabolic adaptation that supports redox regulation and antioxidant defense. In contrast, the lens exhibited uniformly low levels of most TCA intermediates, with the exception of succinate, consistent with minimal mitochondrial activity under physiologically hypoxic conditions. Notably, both the cornea and lens demonstrated significant sex-dependent and time-of-day–dependent differences in TCA cycle metabolite abundance. These findings reveal pronounced tissue-specific differences in mitochondrial metabolism across the eye, reflecting the unique functional and biochemical demands of each tissue. These metabolic signatures may underlie their susceptibility to mitochondrial dysfunction in various ocular diseases.