Evaluation of Tissue-Specific Extraction Protocols for Comprehensive Lipid Profiling

Background

Robust tissue pre-treatment and lipid extraction workflows are crucial to metabolic phenotyping studies and accurate interpretation of lipid profiles. Numerous methods for lipid extraction from tissues have been developed, but the choice of technique influences analysis. This study provides a comprehensive evaluation of six liquid-liquid extraction methods (three biphasic and three monophasic) used for lipidomic tissue analysis by liquid chromatography-mass spectrometry. Extraction methods were assessed for their suitability for comprehensive lipid profiling across diverse tissue types: adipose, liver, and heart. These techniques were compared using lyophilised and fresh frozen samples.

Results

The study revealed significant differences in the coverage and reliability of lipid species extracted using each technique, dependent on the tissue type. The optimal extraction method for adipose tissue was butanol:methanol (BUME) (3:1) which achieved the highest lipid coverage, yield and reproducibility (886 lipids with a coefficient of variation (CV) < 30%); methyl tert-butyl ether (MTBE) with ammonium acetate was most effective for liver tissue (707 lipids CV < 30%) and BUME (1:1) for heart tissue (311 lipids CV < 30%). These findings reveal that the most effective lipid extraction methods are highly tissue-specific, underscoring the critical need for bespoke protocols tailored to each tissue type. The optimised tissue-specific methods were validated using an intervention study in C57BL/6 mice to investigate diet-induced metabolic changes. The results demonstrated distinct discriminating lipid profiles unique to each tissue type, with 374 lipid species from 13 subclasses significantly different between high-fat diet (HFD) and normal diet (ND adipose tissue, while 485 lipid species from 17 subclasses were significantly different between HFD and ND liver tissue.

Significance and novelty

This study presents a new approach to studying lipid profiles derived from diverse tissues that substantially improve comprehensive lipid species’ detection sensitivity and reliability. Our systematic evaluation provides evidence that tailored tissue-specific extraction protocols are highly valuable in comprehensive lipidomics studies, offering robust tools for reliably identifying lipid changes and facilitates a deeper understanding of tissue-specific metabolic processes in diverse research and clinical applications.