A multi-tiered workflow for examining organic acid profiles delineates tissue-specific changes in fatty acyl partitioning during aging.

Fatty acids (FAs), as the predominant organic acids, form a major component of the metabolome. We present a multi-tiered method that comprehensively captures FA diversity-including chain lengths (C2-C34), unsaturation, isomers, and endogenous forms-within a single biological specimen. This workflow quantifies the broadest range of free FAs reported to date. Integrated with two complementary tiers profiling the total FA pool from alkaline hydrolysis and esterified acyl compositions across lipid classes, our multi-tiered workflow enables the investigation of differential fatty acyl partitioning. Applying this platform to quantify >540 unique lipids (free and esterified forms) and polar carboxylic acids, we investigated FA remodeling in the brain, retina (eyeball), and skeletal muscles of young and aged mice. We found that aged glycolytic tissues preferentially partition odd-chain and diunsaturated FAs (with lower β-oxidizability) into triacylglycerols. Additionally, aging shifts the FA18:1 partitioning into diacylglycerols over anionic phospholipids, which may mitigate pro-aging lipid signatures in the skeletal muscle.