Quantifying lipids in human cells using Raman spectroscopy

Omics methods should deliver information on cellular composition as well as their content and localization in cells. This is particularly important for lipids, both saturated and unsaturated fatty acids, which regulate key metabolic, signaling, and toxic processes. Furthermore, differences in local concentration can determine cell fate: activating endoplasmic reticulum stress, exacerbating lipotoxicity, or promoting metabolic adaptation. By using deuterated fatty acids as Raman probes (Rp), we can determine their local concentrations within cells, taking into account, for example, accumulation in lipid droplets, ER or membranes. Monitoring the incubation time and distribution of Rp allows for the assessment of the rate of lipid uptake and accumulation in the cell. Therefore, RS spectroscopy methods can be used to study the kinetics of lipid transport and metabolism, key in fluxomics. We quantitatively calibrate the C–D band intensities using deuterated lipid standards and compare the RS results with LC-MS and MF data to verify the quantitative accuracy of the Raman method. We demonstrate that despite using µM concentrations of dPA and dOA acids in the medium, local concentrations inside cells (especially in lipid droplets) can reach mM levels which demonstrates the importance of a local, not just total, lipid detection method.