Materials

2.2.1 Phospholipids under investigation

Epikuron 145 (referred to as soya PL in text), B.N. 1-5-9005, Lucas Mayer, Hamburg, Germany.

Epikuron 200 (referred to as soya PC in text), B.N. 1-4-9018, Lucas Mayer, Hamburg, Germany.

Ovothin 180 (referred to as egg PL in text), B.N. 1-4-9240, Lucas Mayer, Hamburg, Germany.

Ovothin 200 (referred to as egg PC in text), B.N. 1-4-9248, Lucas Mayer, Hamburg, Germany.

2.2.2 Materials used in TLC identification of lipids

Acetone, AnalaR, B.N. K20840006 416, BDH Chemicals Ltd., Poole, UK.

Ammonia hydroxide (conc.), AnalaR, B.N. 7198610M, BDH Chemicals Ltd., Poole, UK.

a-napthol, research grade, B.N. 31075362, Fisons scientific equipment, UK. Chloroform, AnalaR, B.N. K22453741 550, BDH Chemicals Ltd., Poole, UK. Deionised water, pH approximately 5, from Elgastat, UHQ PS, Elguard. Iodine, GPR, B.N. 215379413, BDH Chemicals Ltd., Poole, UK.

Methanol, AnalaR, B.N. K20930670 424, BDH Chemicals Ltd., Poole, UK. Ninhydrin spray, B.N. A86271, BDH Chemicals Ltd., Poole, UK.

Phosphomoly^dic acid 20% w/v in ethanol, B.N. 9/30211022, Aldrich Chemical Co., USA.

Potassium permanganate, B.N. 1579800, BDH Chemicals Ltd., Poole, UK.

Silica gel 60 F2 5 4 Aluminium backed TLC plates, B.N. 540020711, BDH Chemicals

Ltd., Poole, UK.

Silver nitrate solution, B.N. X, BDH Chemicals Ltd., Poole, UK.

2.2.3 M aterials used in ^ P-NM R analysis

Chloroform- d, B.N. 00423MF, Aldrich Chemical Co., USA. Triethylphosphate, B.N. 123H2623, Sigma Chemical Co., USA.

2.2.4 M aterials for zeta potential

Lipofundin® 10% MCT, Braun, Germany. 2.3 M ethods

The phospholipid composition of commercially available egg yolk PL, egg yolk PC, soya bean PL and soya bean PC was assessed quantitatively and qualitatively. Two techniques were employed to determine the phospholipid compositions: ^‘P-NMR and TLC.

2.3.1 ^ P-N M R m ethodology

In the context o f phospholipid analysis, ^^P-NMR is usually employed to establish the structural organisation o f phospholipids. Specifically, it can be used to determine whether the phospholipids are arranged in either a micelle or a bilayer organisation. However, in this context, ^'P-NMR was employed to quantitatively determine the phospholipids present in the four lipid samples. It provided both qualitative and quantitative information on the lipids containing a phosphorus headgroup (Henderson et

Chapter Two- Composition o f unsaturated phospholipids

a l , 1974). The main disadvantage with this teehnique was that no data on non­ phosphorus containing components was generated, e.g. glycolipids.

The lipid sample (100 mg) was dissolved in 3 ml o f deuterated chloroform; methanol (2:1 v/v) containing approximately 5 mg o f an internal standard triethylphosphate. The ^*P-NMR parameters as described by Sotirhos et al. (1986) were employed. The ^'P-NM R was performed at 25 °C on a Briiker 250 MHZ N M R (Karlsruhe, Germany) at

161.7 MHZ. A pulse angle o f 45 ° and a ten second pulse were utilised. Two hundred accumulations were obtained before Fourier transformation o f the free induction decay. A ^*P-NMR spectrum o f a commercially available egg PC sample is provided in Figure

2.2.

II

Peak I II III Internal standard

5 -2 5 °C 1.60 1.40 0.66 0 Area under peak 2.2 1.0 113.8 9.7

Figure 2.2 P-NMR spectrum o f egg PC sample

The phospholipids are identified by their chemical shifts at 25 °C (6-25 °C) relative to the internal standard triethylphosphate. In the above example the three peaks at 0.66 (Peak III), 1.40 (Peak II) and 1.60 (Peak I) correspond to phosphatidylcholine, lyso- phosphatidyleholine and sphingomyelin respectively. The quantity o f each PL is directly proportional to the area under each peak. Therefore, the mol% o f each lipid can be calculated by comparing the integrated areas under each peaks. The mol% for each phosphorus containing lipid in this egg PC sample is shown in Table 2.6.

2.3.2 Thin L ayer C hrom atography (TLC)

TLC was used to qualitatively assess the lipid composition o f the four phospholipid samples. A variety o f solvent systems can be used to separate the various lipid

components (Fried, 1991; Hammond, 1993). However, for these investigations, two solvent systems were found to be particularly suitable:

1) Non-polar solvent system. (CMH) Chloroform: Methanol: Water (65:25:4 v/v)

2) Polar alkali solvent system. (CMAH) Chloroform: Methanol: Ammonia hydroxide: Water (46:14:1.6:1 v/v)

Both these solvent systems were made up fresh when required. Each solvent system was poured into a glass tank to a depth o f approximately 1.0 cm. Evaporation of the solvent from the tank was reduced by placing a glass lid on top of the tank. To reduce drifting of the lipid spots, filter paper was placed in the tank to uniformly distribute the solvent vapour within the tanks. Commercially available silica gel 60 with binder on aluminium backing (Merck, Germany) was used as the stationary phase. Approximately 0.2 g of lipid was dissolved in 1 ml of chloroform. Approximately 1.2 pi (equivalent to 0.2 mg of lipid) of this solution was carefully spotted from a glass capillary pipette 2 cm above the bottom edge of the plate. After the spot had dried, the plates were put in an equilibrated glass tank and the lid was immediately placed over the tank. Care was taken to position the plate horizontally and to ensure the edges of the plate were not damaged. After the solvent front had progressed 10 cm from the original position of the lipid spot, the plate was removed from the solvent system. The plate was subsequently dried in a vacuum oven at 50 °C for 10 minutes before subjecting the plate to the reagent.

2.3.2.1 TLC separation of PC samples

To separate the different lipids of the PC samples, the one dimensional TLC with the CMH solvent system, described in section 2.3.2, was found to be satisfactory.

2.3.2.2 TLC separation of PL samples

To separate the individual lipids from the more complex phospholipid mixtures two dimensional TLC was employed. This two dimensional technique enabled greater lipid separation than was possible with one dimensional TLC. The separation was carried out by applying approximately 0.4 mg of lipid in chloroform onto the bottom right hand comer of the plate- 2 cm from the bottom edge and 2 cm in from the right hand edge of the plate. The lipid spot was separated on the plate in the non-polar (CMH) solvent. After the solvent front had travelled 10 cm, the plate was removed from the non-polar solvent and dried in a pre-heated vacuum oven at 50 °C for approximately 10 minutes. The dried plate was then turned 90 ° clockwise and placed in the polar solvent (CMAH).

Chapter Two- Composition o f unsaturated phospholipids

The solvent front was allowed to travel 10 cm up the plate before removal from the polar solvent. The plates were dried in a vacuum oven at 50 °C prior to spraying with appropriate reagents.

2.3.2.3 Spray/vapour reagents used in TLC identification

A variety of reagents (Table 2.1) were used to locate and in some cases identify the individual lipid components (Kates, 1972; Dawson et al., 1986). The phospholipids were also identified based on their Rf values. The Rf values o f the lipids were calculated by measuring the distance travelled by each spot and dividing it by the distance of the solvent front. The Rf values were compared with literature values (Gunstone et al., 1994b). The presence of saccharides after separation was detected after dipping the plate in a silver nitrate solution and spraying with an alkaline spray.

Name o f spray/vapour Detects Appearance

Phosphomolybdic acid (5%) in ethanol Phospholipids Blue-black spots on yellow background Sulphuric acid-glacial acetic acid (1:1

vol/vol)

Sterols and sterol esters Red spots on white background Potassium permanganate crystals in

closed tank

Any lipid Black spots on a colourless background

Ninhydrin spray PE Violet spots on white background

Iodine crystals in closed tank Unsaturated fatty acids Brown spots on an off-white background a napthol 0.5% in methanol-water (1:1) Sterol glycosides Violet spots

Aqueous sulphuric acid (50%) All carbon containing material

Dark brown spots on a colourless background

1) Plate dipped in silver nitrate solution. (Produced by diluting 0.1 ml saturated

AgNo] to 20 ml in acetone / water) 2) After drying, spray with 0.5 M NaOH

in aq. ethanol.

Saccharides Silver spots

All plates were placed in a fume cupboard prior to spraying with reagents aerosolised with a hand held chlorofiurocarbon containing propellant canister.

2.3.3 Zeta potential measurements

Each lipid sample was dispersed and diluted in deionised water before measuring the zeta potential of the individual lipid sample using a Malvern Zetasizer IV (Malvern Instruments, UK). As a comparison, Lipofundin® 10% MCT, a commercially available medium chain triglyceride stabilised with soya PL, was also measured in deionised water. Each sample was measured three times. The average and standard deviation of each sample were calculated.

2.3.4 Gas chromatography (GC) to determine fatty acid profile of phospholipids The fatty acid profiles of each of the four phospholipids were determined by Lucas Meyer, the manufacturer of the PLs. The technique employed to analyse the fatty acids was capillary gas chromatography after hydrolysis and estérification of the fatty acids. The derivatised fatty acids were separated by GC and detected with flame ionisation (Lucas Meyer, 1995).

2.3.5 Purification of egg PC and soya PC

Approximately 20 g of egg PC and soya PC were refined further by Lipid Products (Nutfield, Redhill, UK) using a general elution protocol developed by Rouser et al. (1967). The purpose of this purification was to reduce the level of non-PC components. These purified PCs were used in some particle size experiments, described in section 3.4.4.5.I. The two purified PCs were analysed by TLC separation using the CMH solvent.