A time efficient adaptation of GC-FID method for the analysis of PBMC lipid composition

Peripheral Blood Mononuclear Cells (PBMC) comprise of lymphocytes and monocytes which are involved in the pathogenesis of chronic inflammatory diseases. A method for estimation of PBMC lipid composition was modified from an existing method for plasma to support our fish oil intervention study in mild-moderate asthmatics. This modification involved an hour long direct transesterification reaction using methanol and acetyl chloride followed by a modified GC-FID method. The identification of PBMC fatty acid composition was achieved by relative comparison with a 37 Fatty Acid component standard. The method was found to be reproducible and accurate with a within batch and between batch Coefficient of variation (CV) of <10%. The modified method was adapted for PBMC total lipid estimation and was considered suitable to detect changes in PBMC composition following fish oil supplementation. This method is quick, scalable, and cost effective for large scale population and intervention studies. Keywords: Fatty acids, PBMC, fish oil, Gas chromatography Introduction Fish oils comprise of two major omega 3 polyunsaturated fatty acids (PUFAs) namely Eicosapentaenoic Acid (EPA, 20:5) and Docosahexaenoic Acid (DHA, 22:6), these PUFAs are important structural components of cell membranes. In fish oil supplementation studies, an incorporation of EPA and DHA in total cell lipids and phospholipids, which occurs mostly at the expense of omega 6 PUFAs (primarily Arachidonic Acid, AA, 20:4), is expected. The extraction of fatty acids from cells and tissues is generally based on the original methods of Folch (1957) or Bligh & Dyer (1959), which have been widely applied and used on different types of cells (Damsgaard et al. 2008; Garcia-Larsen 2011; Grindel et al. 2013). Similarly, there are standard methods for derivatisation of lipids (acid-catalysed) (Lepage and Roy 1986; Morrison and Smith 1964, Matsumot et al. 2013) in a quantitative manner (Blau and Halket 1993; Christie 2003). Masood et al. (2005) proposed a method for measurement of fatty acids in plasma samples which is principally derived from Lepage & Roy (1986) with advantages of speed, reproducibility, lower toxicity and cost. While previous investigations have concentrated upon the use of plasma samples for the analysis of fatty acid composition of cells, the focus of this paper is the use of PBMC.