An FT-IR Spectroscopy investigation on different methods of lipid extraction from HepG2 cells

The Fourier transform infrared (FTIR) spectroscopy is a non-invasive technique that is largely used for investigating different samples of biomedical interest. In particular, it is one of the most common and up-to-date techniques for studying lipidomics. Most cell membranes are made up of proteins and lipids (proteins 55%, lipids 42%, and carbohydrates 3%). Lipids are a primary class of biological molecules that play numerous vital roles in various processes inducing apoptosis, differentiation, chemotaxis, and other responses.

In the present work, we adopted FTIR spectroscopy for monitoring lipid extraction efficiency of different methods that have been used for extracting lipids from hepatocarcinoma cells (HepG2). The traditional Bligh and Dyer method, a gold standard for total lipid determination based on a mixture of chloroform/methanol (1:2, v/v) followed by the addition of water to create a biphasic system, has been compared with three methods involving different extraction solvents: Bume modified method, a new rapid and simple chloroform-free method based on butanol/methanol mixture (BUME); a simple and robust two steps method with the addition of KOH for the detection of sphingosine-1-phosphate (S1P) and related sphingolipids [4] and finally a method based on a mixture of isopropanol:water: ethyl acetate (30:10:60, v/v/v) for analysis of broad categories of sphingolipids.

Infrared spectra have been obtained in the 4000-600 cm^-1 wavenumber range. The spectra acquired from samples obtained with the cited methods showed the contributions of different functional groups. A qualitative comparison among them indicated that all the spectra exhibited similar lipid species profiles. The ratio values estimated using the absorbance of selected bands related to different cell lipid constituents have been evaluated for a quantitative comparison of the efficiency of the different extraction methods.