Lipid Analysis in Live Caenorhabditis elegans Using Solution-State NMR Spectroscopy

Unsaturated fatty acids (UFAs) play key roles in essential cellular functions such as membrane dynamics, metabolism, and animal development. Disruptions in UFA metabolism are linked to metabolic, cardiovascular, and neurodegenerative disorders. Cellular UFAs composition and quantification are normally determined using methods such as gas chromatography and/or mass spectrometry, which require extraction procedures and prevent analysis of live specimens. Here, we describe a protocol that employs uniform ¹³C isotope labeling and high-resolution 2D solution-state nuclear magnetic resonance (NMR) spectroscopy to analyze lipid composition and fatty acid unsaturation directly in the model organism Caenorhabditis elegans. The approach enables in vivo assessment of lipid storage compositions with sufficient resolution and sensitivity to distinguish wild-type animals from those with altered fatty acid desaturation. Complementary analysis of total lipid extracts provides information regarding lipid molecules that are not detected in vivo, such as phospholipid molecules organized in biological membranes. Overall, this non-destructive NMR-based method offers a powerful tool for investigating lipid metabolism in C. elegans and other small model systems that can be isotopically enriched. Key features • Solution-state NMR spectroscopy is not destructive and can be used on live cells and multicellular organisms. • ¹³C isotopic enrichment is required for high-resolution NMR analysis of lipids in live C. elegans. • Lipid signals from live worms arise from the mobile lipid phase in lipid droplets. • NMR provides readouts of lipid compositions in live animals at a highly sensitive rate, enabling precise interpretation of the whole cell lipid metabolism.