Isomer-Resolved Mass Spectrometry Imaging Reveals Regional and Age-Dependent Glycolipid Metabolism in Rat Brain

Glycolipids are essential for myelin integrity, but their extensive isomeric complexity, where isomers like galactosylceramide (GalCer) and glucosylceramide (GlcCer) have unique functions, has hindered our ability to map their metabolism in the brain. Existing mass spectrometry methods fail to resolve this complexity. We fundamentally overcome this barrier with a powerful new isomer-resolved mass spectrometry imaging workflow. By coupling ion mobility and mass spectrometry imaging with targeted enzymatic pretreatment, our method provides direct visualization of distinct de novo metabolic pathways for GalCer and GlcCer in situ. When applied to rat brains across the lifespan (2, 6, and 12 months), this technology revealed a previously unrecognized age-related decline that selectively affected the GalCer pathway, while the isomeric GlcCer pathway remained comparatively stable. We confirmed these metabolic alterations colocalize with myelin-associated proteins specifically in oligodendrocyte-rich regions, providing a functional link to altered oligodendrocyte-related lipid metabolism. This integrated approach provides a new paradigm for dissecting isomer-specific glycolipid metabolism, offering insight into lipid alterations in brain aging and opening new avenues for biomarker discovery in neurodegeneration.