Isomer-resolved characterization of acylcarnitines reveals alterations in type 2 diabetes

Acylcarnitines (CARs) are metabolites of fatty acids that play crucial roles in various cellular energy metabolism pathways. The structural diversity of CAR species arises from several modifications localized on the fatty acyl chain and there is currently a lack of reports characterizing these detailed structures. High-performance liquid chromatography (HPLC)-electrospray mass spectrometry (ESI-MS) is the common tool for CARs analysis.

In this study, we improved the MS detection signals of CARs by adding NH₄HCO₃ as buffer in the mobile phase of LC system. We demonstrated that electron activated dissociation (EAD) on the ZenoTOF 7600 system is capable of localizing the hydroxyl group and methyl branching position in CARs. The benzophenone Paternò-Büchi (PB) reaction was used for derivatizing the carbon-carbon double bond (CC). The capability of profiling CARs with detailed structural information was demonstrated by analyzing complex lipid extracts from mouse plasma. Our results also provided visualization of isomers composition, including branched chain isomers of CAR 4:0 and CAR 5:0 and CC location isomers of unsaturated CARs. Notably, we observed significant changes in the relative compositions of branched-chain isomers of CAR 5:0 and CC location isomers of several unsaturated CARs in mouse plasma samples from type 2 diabetes (T2D) compared to normal controls, suggesting their potential as diagnostic indicators for T2D.

In this work, we enhanced the limit of detection for acylcarnitine species by incorporating ammonium bicarbonate into the LC system. The CC positions in the acyl chain of CARs were identified using Paternò-Büchi (PB) derivatization coupled with tandem mass spectrometry. Modifications such as methyl branching and hydroxyl groups along the acyl chain were localized through Electron-Activated Dissociation (EAD) on the Zeno-TOF 7600 system.