Determination of Fatty Acids in Phospholipids of Cheek Cells - Fast and Simple with the Help of a Selective Transmethylation and Chromatoprobe

Helmut Geppert, Central Analytical Laboratory, Brandenburg University of Technology at Cottbus, Germany

Many epidemiological studies have reported that n-3 and n-6 long-chain polyunsaturated fatty acids (LC-PUFAs) play important roles in human health and common diseases. Current studies use different biological compartments for fatty acids, such as whole blood, red blood cells, plasma, and adipose tissue. However, these approaches are invasive and not well accepted in population studies, particularly in infants and young children. For this reason, several studies have proposed the cheek cell sampling as an alternative matrix for evaluating fatty acids status. However the small sample size is a big challenge and the separation of phospholipids from the neutral lipids with thin-layer chromatography (TLC) or solid phase extraction (SPE) is very time consuming /1/.
With reference to the publication of Ichihara /2/, we have developed a new method for the selective transmethylation of the phospholipids in cheek cells without prior separation of the other lipids: the aqueous cheek cell suspension is treated directly with methanolic KOH and hexane. Glycerophospholipids will be rapidly transmethylated in the methanolic phase and the fatty acid methyl esters are simultaneously extracted into the organic phase. Triglycerides (TGs) and cholesterol esters remain unchanged and will be also extracted into the hexane phase. Free fatty acids remain as potassium salts in the methanolic phase.
For the detection of the fatty acid methyl esters in the matrix-containing hexane phase with GC-FID is that of Amirav et al. /3/ developed ChromatoProbe particularly well suited. Up to 20 μl organic phase can be easily injected and the non-volatile matrix (e.g. TGs) remains in the microvial.

/1/ Garza Puentes et al. (2015). Evaluation of less invasive methods to assess fatty acids from phospholipid fraction: cheek cell and capillary blood sampling. Int. J. Food Sci. Nutr. 66: 936–942
/2/ Ichihara et al. (2012): Simple procedure for fatty acid analysis of glycerophospholipids in Escherichia coli and Saccharomyces cerevisiae. J. Biosci. Bioeng. 114, 472e475
/3/ Amirav et al. (1997): A direct sample introduction device for mass spectrometry studies and GC-MS analysis". Europ. Mass. Spectrom. 3, 105-111