ANALYSIS OF PHOSPHOLIPID


MOLECULAR SPECIES

Phospholipids occur in nature as a combination of two fatty chains. Since these fatty chains can vary in length and degree of unsaturation, each natural phospholipid contains numerous molecular species. These species differ greatly in their chemical and biological properties and their identification and quantification are of great interest.
If separations of intact molecular species of phospholipids were proposed, they are not very efficient since no more than 5 or 6 compounds were obtained for phosphatidylcholine from rat liver (Christie WW et al., J Chromatogr 1985, 325, 473). More recently, the use of monolithic C18 silica column allowed the separation of ten phosphatidylcholine molecular species using a UV detection (Merlin JF et al., Anal Chim Acta 2006, 565, 163). The main advantage of this experimental design was that, under isocratic conditions, the separation is simpler and faster that those obtained with conventional columns.
For the separation of phosphatidylserine molecular species, a baseline separation of the five main species was observed in a bovine brain preparation (HPLC with a polystyrene/divinylbenzene column), but species identification was possible only with electrospray mass spectrometric detection (Larsen A et al. J Chromatogr. 2002, 774, 115). The same detection technique combined with liquid chromatography has been applied successfully to the determination of the sn-position of fatty acids in all erythrocyte phospholipids (Beermann C et al., Lipids 2005, 40, 211).

The best practical and sensitive strategy for resolution of phospholipid molecular species is the conversion of each subclass into an appropriate neutral derivative either absorbing UV or fluorescent. This transformation includes most frequently the removal of the polar head group by enzymatic hydrolysis.

The following application outlines the strategy we have adopted for the resolution of molecular species of diradylglycerols obtained from any glycerophospholipid. The method is also efficient to analyze molecular species of sphingomyelin.

ANALYTICAL STEPS

1- Isolation of individual phospholipids

Individual phospholipids are prepared by TLC with the previously reported procedure based on boric acid impregnated silica gel plates and an alkaline developing solvent. They are then eluted from silica gel as described.


2- Preparation of diradylglycerols (or ceramides)

2.1- Reagents

50 mM phosphate buffer pH 7.2 containing 2 mM calcium chloride, diethyl ether containing BHT as antioxidant (3 mg/100 ml).
Phospholipase C from Clostridium perfringens (welchii) (Type I from Sigma) recommended for the analyzis of phosphatidylcholine and sphingomyelin.
Phospholipase C from Bacillus cereus (Type V from Sigma) recommended for the other phospholipids.

2.2- Procedure

- Chloroform solutions of purified phospholipids are filtered on special 0.2 µm syringe microfilters (membrane of PTFE or Nylon compatible with strong solvents) to remove silica gel particles.
- Phospholipid solutions (containing up to 0.5 mg lipids) are evaporated in a screw-capped glass tube under nitrogen and 1 ml of phosphate buffer is added.
- Lipids are dispersed by a brief sonication (a turbid suspension is obtained), about 60U of phospholipase C are added (in phosphate buffer if provided in dry powder).
- 1.5 ml of diethyl ether are added and the mixture is vortexed for 1 h (2 or 3 h for phosphatidylserine or posphatidylinositol) in the dark.
- Centrifuge the mixture, collect the ether phase and wash two times the aqueous phase with diethyl ether, evaporate all the ether phases containing diradylglycerols.


3- Derivatization of diradylglycerols

Diradylglycerols obtained after pospholipase action must be processed imediatly.

3.1- When only small amounts of phospholipids (less than 100 µg) are available, fluorescent derivatives are more convenient if the analytical equipment is available. The recommended procedure using naproxen as a fluorescent tag is described elsewhere for the analysis of diacylglycerol molecular species. Their HPLC separation is also described.

3.2- When a higher amount of phospholipids is available (0.1-1 mg), UV absorbing derivatives are convenient for accurate analysis.

3.2.1- Materials and reagents

Dinitrobenzoyl chloride (DNBC) kept in a dry box, 4-dimethylaminopyridine (DMAP), pyridine (kept dry with 5 A molecular sieve), hexane, toluene,
0.1% sodium carbonate in water,
silica gel plates.

3.2.2- Derivatization procedure

Weigh directly in the tube containing dry DAG : 30 mg DNBC and 10 mg DMAP.
Keep about 20 min under vacuum (less than 1 mm Hg) and add 1 ml of dry pyridine.
Warm in a water bath at 60°C for 5 min.
After cooling, add 2 ml sodium carbonate solution and vortex.
Add 2 ml hexane, vortex 1 min and centrifuge. Collect the hexane phase and wash 2 times the lower phase with 2 ml hexane. Evaporate the hexane phases and dissolve the residue with 0.1 ml hexane.
Separate the diradylglycerols by TLC with toluene as developing solvent.
Locate the spots of derivatized DAG under UV light after primuline spray.
The Rf of the derivatives are: alkenylacyl, 0.56; alkylacyl, 0.39; a,3-diacyl, 0.3; 1,2-diacyl, 0.23; monoacyl, 0.1.
Spots are scraped and the molecular species eluted from the silica gel by 2 washings with 2 ml of hexane/diethyl ether (1/1, v/v). After the solvent evaporation under nitrogen, the lipids are dissolved in a small volume of acetonitrile.

3.2.3- Separation of molecular species by HPLC

The molecular species are separated using a reversed-phase column (Lichrospher RP18, 5 µm, 25 cm long from Merck) and acetonitrile/2-propanol (95/5, v/v) as eluent. We found that complex mixtures (i.e. from phosphatidylethanolamine) are better separated when the column is cooled at 13°C (plastic jacket with running water, Alltech). The detection is made with a UV spectrophotometer at 230 nm. The response is directly related to the molar amount of each molecular species.
The identification of the separated components is made as explained for the fluorescent derivatives of DAG.

OTHER TECHNOLOGIES

Gas chromatography

Capillary gas chromatography was used with success for the quantitative determination of molecular species of diacylglycerols either free or prepared from phosphatidycholine (Tserng KY et al., Anal Biochem 2003, 323, 84). Not all molecular species were separated, but all the major molecular species were readily separable and the nonpolar methylsiloxane column was used for more than 3 years with daily analyses of biological samples. 

Mass spectrometry analysis

Mass spectrometry allows the analyst to determine with a minimum of preparation the nature and amount of phospholipid molecular species. An example of analysis of phospholipid species using a narrow-bore normal-phase HPLC method coupled on-line with an electrospray ionisation ion-trap mass spectrometer is given by Uran S et al. (J Chromatogr B 2001, 758, 265). The combination of lyso-fragment mass, molecular ion and chromatographic retention time enables to identify each species for all the phospholipids present in human blood. The method is characterized by a low limit of detection (0.1-5 ng of injected substance). A similar technology was efficiently used to analyzed more than 90 phospholipid constituents in rat peritoneal surface (Gao F et al., Biochim Biophys Acta 2006, 1761, 667). The whole analysis was effected on about 50 pmol (40 ng) of injected phospholipids.
Tandem mass spectrometry (MS/MS) appears to be the most reliable and sensitive technology to determine the large array of phospholipid molecular species in animal tissues. Thus, the distribution of about 25 molecular species of the  four main phospholipids have been described in ten different rat tissues (Hicks AM et al., Biochim Biophys Acta 2006, 1761, 1022). Each tissue was found to possess unique species profiles that may be used to identify unknown tissues, especially when phosphatidylserine is considered.
The use of electrospray ionization coupled with mass spectrometry (ESI-MS) made possible the identification of about 450 phospholipid species in cell lipid extracts (review in Milne S et al., Methods 2006, 39, 92). Precise analyses of mammalian cell phospholipids using tandem electrospray ionization mass spectrometry, in conjunction with stable isotope labelling, have been obtained (Hunt AN et al., Methods 2006, 39, 104). 
 
The use of HPLC coupled on-line with a mass spectrometer was shown to be a very powerful tool for the analysis of intact molecular species (Malavolta M et al., J Chromatogr B 2004, 810, 173). More than 140 species from all phospholipids could be identified and quantified in blood mononuclear cells. A normal-phase liquid chromatography/quadrupole lilnear ion trap mass spectrometry method was applied with success for the separation of the main phospholipid species in human blood (Wang C et al., Anal Chim Acta 2004, 525, 1). A similar approach using a coupling between HPLC and tandem mass spectrometry allowed the separation and quantitation of all phospholipid species present in hen eggs (Pacetti D et al., J Chromatogr A 2005, 1097, 66).

A quantitative analysis of lysophosphatidic acid in plasma was proposed (Yoon HR et al. J Chromatogr B 2003, 788, 85). Using electrospray negative ionization tandem mass spectrometry, it was possible to separate all the species from the plasma matrix.

A novel methodology for the analysis of the molecular species of phosphatidylethanolamine and its lyso derivatives has been presented (Han X et al., J Lipid Res 2005, 46, 1548). Lipid extracts are directly treated with fluorenylmethoxylcarbonyl chloride (Fmoc) which derivatized PE and lysoPE species to their corresponding carbamates. The reaction solution is than directly analyzed with an electrospray ionization mass spectrometer. The detection limit is said to be in the attomole range per injection. That procedure may be probably extended to other phospholipids by using other derivatization reagents.

Synthesis of phospholipids

As standards, phospholipid species are generally synthesized by a previous generation of selected lysophospholipids from selected phospholipids with various lipases (Paltauf F et al., Prog Lipid Res 1994, 33, 239). The lysolipids are then reacylated enzymatically or chemically with the fatty acid of interest.
A non-enzymatic method for the synthesis of phospholipids was described using a simple and rapid acylating reaction by various acyl-CoAs in the presence of imidazole in water at room temperature (Testet E et al., J Lipid Res 2002, 13, 1150). The only limitation was the major production of N-acyl phosphatidylethanolamine when compared to that of phosphatidylethanolamine. These products need to be purified before further use.

A review by Van der Meeren P et al. of several HPLC separation techniques for phospholipid molecular species may be found in the book "Food analysis by HPLC" (Nollet LML Ed., Marcel Dekker inc, 1992).