Characterization of Phospholipid Molecular Species by Means of HPLC-Tandem Mass Spectrometry

Abstract

Phospholipids are the main constituents of the permeability barrier of cells and subcellular organelles. The phospholipid bilayer is the environment in which isolated proteins or dynamic nanoassemblies of sterols, sphingolipids, and proteins, called lipid rafts, act their vital functions, such as energy transduction, signal transduction, solute transport, DNA replication, protein targeting and trafficking, cell-cell recognition, secretion and many others (Lingwood et al., 2010). Christie et al. (2011) reported a detailed description of the general structure of phospholipids. According to Sud et al., 2007, phoshopholipids represent the category of lipids with the highest variety of structures (7775), followed in descending order, by polyketides (6713), fatty acyls (3942), sphingolipids (3936), glycerolipids (3044), sterol lipids (2196) and others. The positive effects of dietary phospholipids (PL) on hepatic lipid metabolism, atherosclerosis, obesity-related disorders and cardiovascular disease is a consistent experimental evidence (Shirouchi et al., 2007). The daily intake of PL can vary from 2 to 8 g per day and represents 1-10% of total daily fat intake (Cohn et al., 2008). The main natural phospholipid is phosphatidylcholine (lecithin) which is completely absorbed in humans. Natural or synthetic PLs are extracted from eggs and soybean and used as drug delivery systems since decades (Papahadjopoulos, 1978). During brain development, one of the most efficient forms of supplying 3-fatty acids such as DHA (docosahexaenoic acid) are phospholipids (Bourre & Dumont, 2002). In animal studies, it was reported that krill oil (rich in 3-containing PLs) had stronger effects with respect to fish oil (rich in 3-containing triacylglycerols) in increasing the DHA level in rat brain (Di Marzo et al., 2010). Although 3 are predominantly linked in the position Sn-1,3 of triacylglycerols (TG) in seal blubber oil, they are esterified in the Sn-2 position of the TGs and PLs of eggs obtained after feeding laying hens with enriched diets. Moreover, more 3 fatty acids are incorporated in phosphatidylethanolamine (PE) and phosphatidylcholine (PC) than in TGs (Pacetti et al., 2005). Apoptotic events and age-related diseases are strictly related to oxidized phospholipids. Much research remains to be done for the PL characterization by Liquid chromatography (LC) – tandem mass spectrometry (MS) aimed to understand their biological and 656 Tandem Mass Spectrometry – Applications and Principles physiopathological activity (Domingues et al., 2008). In the food industry, phospholipids are considered among the best emulsifying agents. Commercial preparations derived from soybean and corn oils (commercial lecithins) are used extensively in manufactured foods such as bakery items, frostings, non-dairy creamers, confectionery products and ice creams. Staling and off-flavors are often related to the deterioration of the functional lipids in foods (Weihrauch et al., 1983). PLs extracts from eggs and/or fish products can play a pivotal role as an innovative food ingredient (Dumay et al., 2009; Commission Decision, 2000). Recently, a lipid extract rich of 3-containing PLs from krill (Euphausia superba) has been authorised by the EC as novel food/food ingredient (Commission Decision, 2009). Species, geographical origin, and production method of fish (i.e. wild/farmed) are the information which must be labelled in fishery and aquaculture products according to EU labelling regulations (Commission Regulation (EC) No 2065/2001). TGs are considered as markers for wild and farmed fish since their profile reflects the diet lipids (Standal et al., 2010), whereas the phospholipid profile is less affected by the diet and can be related to other variables, such as the species and stock (Joensen et al., 2000).