GLYCOGLYCEROLIPID
MOLECULAR SPECIES
If TLC
chromatography on silver nitrate-impregnated plates was formerly the method of
choice for separation of molecular species, reversed-phase HPLC is now adopted
by all glycolipid specialists.
Several elution systems were reported and, with ODS columns, mixtures of
methanol, acetonitrile and water are commonly used (review in: Heinz E,
Advances in lipid methodology. 3, Christie WW Ed, The oily Press 1996). An
example of HPLC separation of MGDG molecular species was reported using a
RP18 column and an isocratic elution with a mixture of methanol/water (9/1, v/v)
containing 5% water (Schmidt H et al., Biochem J 1993, 289, 777). Some
other examples of MGDG, DGDG and SQDG separation into molecular species by
reversed-phase HPLC may be found in the work of Kesselmeier J et al. (Anal
Biochem 1985, 144, 319).
As several difficulties arise in doing HPLC separations of intact molecules,
better results and more accurate data are obtained in releasing the diacylglycerol moiety and analyzing
the species after derivatization as for phospholipids .
The proposed method is
based on a sequence of periodate oxidation and hydrazine treatment resulting in
a cleavage of the glycosidic bonds in monoglycosyl-, diglycosyl- and
sulfoquinovosyl-diglycerides(Heinze FJ et al., Anal Biochem 1984, 139, 126).
Procedure
- the purified glycosyl diglycerides are dissolved in methanol (1 ml), mixed
with 1 ml of 0.2 M HIO4 in methanol and kept for 90 min at room
temperature in the dark.
- 4 ml of chloroform and 1.5 ml of 0.45% NaCl solution are added. After
vortexing and centrifugation, the lower phase is washed with 1.5 ml
ethylene glycol solution (5 % in water, w/v) and once again with the NaCl
solution. The mixture is evaporated under a stream nitrogen with the help of
addition of ethanol.
- the dry residue is dissolved in 0.5 ml of a solution of 1,1-dimethylhydrazine
(1 %, w/v, in isopropanol/chloroform/acetic acid/water, 3.5/3/1.5/1, v/v) and
kept in the dark for 4 h in the case of DGDG and one night in the case of MGDG
and SQDG. 3 ml of hexane are added, and the mixture is washed twice with 2 ml of
50 mM KH2PO4. After removal of the aqueous phase, the solvent mixture is
evaporated under a stream of nitrogen.
- The liberated diglycerides contained in the dry residue are derivatized with dinitrobenzoyl
chloride for UV detection and naproxen
for fluorescence detection. Then, the molecular species are separated by HPLC as
for the study of phospholipid molecular species.
The yield of diacylglycerols released from glycoglycerolipids is not more than
50% but they are representative of the intact lipids (similar fatty acid
patterns).
An alternative procedure for the determination of triacylglycerol molecular
species is using mass spectrometry (Murphy
RC et al., Anal Biochem 2007, 366, 59).
