Jenny M Nilsson: Advances in achiral and chiral analytical methods enhance in vivo lipidomics discoveries

Abstract

Lipidomics aims to characterize the diversity and structural complexity of lipids in biological systems. However, no single analysis can capture the entire lipidome because lipids differ in abundance, polarity and ionization behavior. Analytical methods must therefore balance broad lipid-class coverage with sufficient structural resolution. Method development remains essential, since poor chromatographic performance or unresolved lipid isomers may limit detection, compromise relative quantitation and obscure biologically relevant changes.

The papers included in this thesis focus on analytical method development and applications of LC-MS-based lipidomics. In the first paper, ammonium bicarbonate was evaluated as a mobile-phase buffer to reduce the pronounced peak tailing of phosphatidic acids and other lipids containing accessible phosphate groups in reversed-phase analysis. Combined with an HST-CSH C18 column, ammonium bicarbonate improved peak shape and detectability which enabled analysis of several phosphorylated lipids that were previously poorly detected. This achiral method broadened the lipidome coverage obtainable by reversed-phase analysis. In the second paper, a chiral LC-MS method was developed to separate triacylglycerol regioisomers and enantiomers. Coupling of amylose tris(3-chloro-4-methylphenylcarbamate) columns increased chromatographic efficiency and resolution despite the low enantiomeric selectivity. The method also distinguished triacylglycerols differing in double-bond position and cis/trans configuration and provided reproducible determination of enantiomeric distributions in liver samples.

The developed methods were subsequently applied to biological studies. In the third paper, complementary achiral and chiral lipidomics revealed a possible time-dependent hepatic response to doxorubicin in rats. Short exposure time caused broad lipid depletion, whereas the later response involved selective remodeling of neutral lipids, polyunsaturated phospholipids, cardiolipins and triacylglycerol stereoisomers. In paper four, the enantio- and regioisomeric analysis showed that the triacylglycerols composition differed between tumor and non-tumor tissue in a mouse model of hepatocellular carcinoma. Doxorubicin produced the clearest tumor-associated stereochemical changes, while combined atorvastatin and doxorubicin treatment altered TG stereochemistry in both tumor and non-tumor tissue.

In conclusion, this thesis describes analytical developments that improves lipid detectability and coverage and reveal structural information not captured by conventional lipid profiling. The applications demonstrated that improved analytical resolution can strengthen the interpretation of treatment-, time- and tissue-dependent hepatic lipid remodeling.