Endocrine Abstracts

Autotaxin (ATX) is a secreted enzyme that generates the lipid signalling molecule LPAs from LPCs. The ATX/lPA axis is strongly linked to fibrotic diseases and therapeutic inhibitors are in development. Assessing the balance of LPC/lPA in diseased target tissues is critical to inform pharmacokinetics/pharmacodynamics (PK-PD) and predict efficacy of ATX inhibitors in vivo. Mass spectrometry imaging (MSI) allows concomitant measurement of multiple molecules with histopathological correlation. We developed a universal MSI protocol to image LPA/lPC pairs simultaneously in fibrotic liver tissue as PK/PD markers. 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. Carbon tetrachloride treated (12 weeks; olive oil control) rat livers (n = 3/group) were cryosectioned (10μm) and imaged (150μm). LPA/lPC pairs (16:0, 18:0, 18:1), 17:0-LPA and 19:0-LPC were used for method optimisation and validation. LPCs ionised more readily as [M+H]⁺ and [M+Na]⁺ in equal abundance, whereas LPAs were observed as [M+2Na-H]⁺, limit-of-detections (LODs) off-tissue (LPC: 0.1μg/mL; LPA: 1ug/mL). Following matrix screening, 2,5-dihydroxybenzoic acid (DHB) performed best in 50% MeOH. Addition of 20-40 mM sodium acetate facilitated sodiated ion formation for both LPAs and LPCs. Automated spray parameters were optimised; low flow 0.05mL/min helped to achieve a smooth layer of DHB+CH₃COONa on tissue surface. Using optimised parameters, LPAs and LPCs were successfully imaged in control and fibrotic rat livers recording different abundances (LPA: 18:0≥18:1>16:0>18:2≥20:4; LPC: 16:0>18:0>18:1≥20:4≥18:2). Lipid identification was confirmed following assessment of specific fragmentation patterns (LPC: loss of -59Da and -205Da from head group; LPA: loss of H₂O -18Da) on and off-tissue vs reference standards. More sensitive and specific detection was achieved using the FT instrument; mass accuracy 1ppm with 3mDa mass resolution. MALDI-FT-ICR-MS represents a useful approach for imaging LPAs/lPCs simultaneously. Future work will investigate ATX inhibitors in preclinical efficacy models.