Endocrine Abstracts

Ageing is associated with decline in mitochondrial function whereby dysregulation in carnitine metabolism is highly correlated to poor ageing phenotypes. L-Carnitine transports activated long-chain fatty-acids (FAs) across mitochondrial membranes for β-oxidation and energy production. It is hypothesised that accumulation of long-chain FAs within cells is related to disordered transportation and reflects lower cellular energy upon ageing. Our global aim is to determine the differences in composition and distribution of L-carnitine and acyl-L-carnitines (ACs) within aged mouse brains. Mass spectrometry imaging (MSI) allows concomitant spatial measurement of multiple molecules with direct histopathological correlation. Here we developed a universal MSI protocol to image L-carnitine (C0) and ACs of various chain lengths; acetyl-(C2), butyryl-(C4), hexanoyl-(C6), decanoyl-(C10), lauroyl-(C12), myristoyl-(C14), palmitoyl-(C16) and stearoyl-(C18). Matrix assisted laser desorption ionisation (MALDI)-MSI was performed on Synapt-G2Si-QToF and Bruker-12T-SolariX-Fourier-transform–ion-cyclotron-resonance (FT-ICR)-MS. Brains (male, C57BL6J, aged 3m) were cryosectioned (12 μm) and imaged (100 μm resolution). ACs (C0-C18) standards were used for method optimisation and validation. Carnitines readily ionised as [M+H] ⁺ ions. 2,5-Dihydroxybenzoic acid (DHB) was selected as MALDI matrix achieving x5 intense signals than α-cyano-4-hydroxycinnamic acid. Ionisation efficiency changed with chain length as follows; C16>C12>C10>C6>C4>C2>C0, reflected in different limits of detection (LODs). Solvent compositions (90%-50% aqueous methanol) were compared for DHB application and 50% selected improving LODs two-fold for long-chain ACs; C16(<0.1ug/mL) > C12(~0.1ug/mL) >C10(~0.2ug/mL) > C6(~0.2ug/mL) > C4(~1ug/mL) > C2(~5ug/mL) >C0(~100ug/mL). Using optimised parameters ACs were successfully imaged in control mouse brain tissue. Carnitine was more abundant in grey-matter, in contrast long-chain ACs (C14-18) were in white-matter. Identity was confirmed at high-mass resolution using FT-ICR-MS, which also offered improved LODs. MALDI-MSI represents a useful approach for imaging ACs (C0-C18) in brain tissue. Future work will investigate effects of age on composition and distribution of these ACs.