Tissue-specific lipid extraction protocols intended for tailored mass spectrometry analysis and precision medicine

Dana Hicks

Lipidomics provides biological insights into the pathological mechanisms associated with cardiometabolic disorders and non-alcoholic fatty liver disease, with an important emphasis on the biological tissues predominantly affected, including liver, heart and adipose tissue. Robust tissue homogenisation and lipid extraction workflows are crucial to metabolic phenotyping studies and the accurate interpretation of lipid profiles. Currently, numerous methods are used to extract lipids from biological tissues, however a comprehensive analysis of the efficiency and reproducibility of these methods to extract multiple lipid classes simultaneously across different tissue types has not been performed. Here, comprehensive and reproducible tissue lipid extraction workflows are presented. Homogenisation was optimised using a bead-based approach, with murine adipose, liver and heart tissue. Extraction of the polar and neutral lipids from these tissues was assessed using methyl tert-butyl ether (MTBE), dichloromethane, isopropanol and butanol and methanol (BUME), for a comprehensive comparison of mono- and biphasic approaches. Multiple tests were performed to investigate the influence differing tissue types and the properties of solvent mixtures have on the lipid classes and individual species extracted. Targeted analysis of tissue extracts using ultra-high performance liquid chromatography (UHPLC-MS/MS), provided optimal sensitivity, specificity, and relative quantification of 20 lipid subclasses. The data generated from technical replicates, across each tissue type, were used to determine the most suitable overall approach with respect to coverage and reproducibility of the lipidome. Principal component analysis (PCA) and coefficient of variation (CV) were used to assess the reproducibility of lipid extraction workflows from the technical replicates, whereas relative quantification was used to assess interspecies lipid recovery. In summary, 702 reproducible lipid features were recovered from liver, 310 from heart and 889 from adipose tissue. Significantly, the optimal lipid extraction method was distinctive to each tissue type, becoming a notable contribution to precision lipidomic research.

Tissue-specific lipid extraction protocols intended for tailored mass spectrometry analysis and precision medicine

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