Methodology
The total volume of the analytical column was 200 ml and the rotational speed was 500 rpm. The chromatographic system was composed
with a Gilson 307 solvent-delivery pump and a Pharmacia Frac 300
fraction collector.
The biphasic solvent system used was heptane-ethyl acetate-acetonitrile
(1:0.55: 1, v/v/v). These HPLC quality solvents were degassed before use.
Phosphatidylcholine was separated from other phospholipids at a flow
rate of 2 ml/min, using the system described as normal ascending and
reversed descending elution modes respectively. Fractions were collected
every 4 min and cooled in the dark before analysis.
High performance thin-layer chromatography
Lipidic content of fractions collected by centrifugal partition chromatography was analysed by high performance thin-layer chromatography
(HPTLC) using an automated multiple development apparatus AMD
(Camag, Muttenz, Switzerland). Phosphatidylcholine was identified on
precoated plates (Silica gel 60 F 254, Merck, Nogent-sur-Marne, France).
Standards and samples application was performed by the spray-on technique using a Linomat IV (Camag, Muttenz, Switzerland). Eluant system
consisted ofchloroform-methanol-water (65:25:4, v/v/v). Revelation was
done with primulin, which showed compounds sharing double bonds as
fluorescent spots under UV at 366 nanometres. This revelation was completed with crossing-tests: molybdene blue revealed group containing
phosphorus and Dragendorffs reagent was also used for choline.
Hydrolysis of phosphatidylcholine
The following method was adapted from Robertson & Lands (1962).
Each collected fraction of PC was dried under nitrogen, before being
suspended in 1 ml diethylether. 150 pl of freshly defreezed phospholipase were added and the mixture was shaken at a constant temperature of 30°C during 50 minutes. The preparation was then dried under
nitrogen and suspended in 1 ml chloroform-methanol (2:1, v/v). TLC
of hydrolyzecl PC allowed the identification of lyso-phosphatidylcholine (L-PC) and free fatty acids (FFAs) of n-2 position according to their
retention factor (Rf).
The hydrolysis was performed with phospholipase A2 (PLA2) from
Sigma (Saint-Quentin Fallavier, France). This enzyme (EC 3.1.1.4) from
bee Apis mellifera venom was chosen to hydrolyse partially PC in the
n-2 position. 5 mg of PLA2 (1222 U/mg prot.) were suspended in 4 ml
of 3 mM CaCl
2
. The preparation was shaken for a few minutes, aliquoted
and then stored at -80°C.
Analysis of molecular species by gas chromatography
L-PC and FFA spots of each collected fraction were scraped and submitted to methylation with BF 3 in 14% methanolic solution at 100°C
for 10 min for FFA and 20 min for L-PC according to the method of
Morrisson & Smith (1964). Fatty acid methyl esters were collected in
hexane, dried under nitrogen ancl suspended in pentane.
17
The total volume of the analytical column was 200 ml and the rotational speed was 500 rpm. The chromatographic system was composed
with a Gilson 307 solvent-delivery pump and a Pharmacia Frac 300
fraction collector.
The biphasic solvent system used was heptane-ethyl acetate-acetonitrile
(1:0.55: 1, v/v/v). These HPLC quality solvents were degassed before use.
Phosphatidylcholine was separated from other phospholipids at a flow
rate of 2 ml/min, using the system described as normal ascending and
reversed descending elution modes respectively. Fractions were collected
every 4 min and cooled in the dark before analysis.
High performance thin-layer chromatography
Lipidic content of fractions collected by centrifugal partition chromatography was analysed by high performance thin-layer chromatography
(HPTLC) using an automated multiple development apparatus AMD
(Camag, Muttenz, Switzerland). Phosphatidylcholine was identified on
precoated plates (Silica gel 60 F 254, Merck, Nogent-sur-Marne, France).
Standards and samples application was performed by the spray-on technique using a Linomat IV (Camag, Muttenz, Switzerland). Eluant system
consisted ofchloroform-methanol-water (65:25:4, v/v/v). Revelation was
done with primulin, which showed compounds sharing double bonds as
fluorescent spots under UV at 366 nanometres. This revelation was completed with crossing-tests: molybdene blue revealed group containing
phosphorus and Dragendorffs reagent was also used for choline.
Hydrolysis of phosphatidylcholine
The following method was adapted from Robertson & Lands (1962).
Each collected fraction of PC was dried under nitrogen, before being
suspended in 1 ml diethylether. 150 pl of freshly defreezed phospholipase were added and the mixture was shaken at a constant temperature of 30°C during 50 minutes. The preparation was then dried under
nitrogen and suspended in 1 ml chloroform-methanol (2:1, v/v). TLC
of hydrolyzecl PC allowed the identification of lyso-phosphatidylcholine (L-PC) and free fatty acids (FFAs) of n-2 position according to their
retention factor (Rf).
The hydrolysis was performed with phospholipase A2 (PLA2) from
Sigma (Saint-Quentin Fallavier, France). This enzyme (EC 3.1.1.4) from
bee Apis mellifera venom was chosen to hydrolyse partially PC in the
n-2 position. 5 mg of PLA2 (1222 U/mg prot.) were suspended in 4 ml
of 3 mM CaCl
2
. The preparation was shaken for a few minutes, aliquoted
and then stored at -80°C.
Analysis of molecular species by gas chromatography
L-PC and FFA spots of each collected fraction were scraped and submitted to methylation with BF 3 in 14% methanolic solution at 100°C
for 10 min for FFA and 20 min for L-PC according to the method of
Morrisson & Smith (1964). Fatty acid methyl esters were collected in
hexane, dried under nitrogen ancl suspended in pentane.
17