Nomenclature and
structure
Triacylglycerols contain three fatty chains which vary in
length and degree of unsaturation, thus, forming several molecular species. The species
differ greatly in their physicochemical and biological properties and there is a great
interest in their identification and quantification.
General strategy of analysis
A- Determination of the amount of triacylglycerols
This quantification can be made by several approaches, we
propose below some of the possibilities.
1- An evaluation
of the total amount of triacylglycerols in an oil sample can be made by the determination
of glycerol as previously described after a saponification
step. Commercial enzymatic kits based on this principle are commercially available for the
ready quantification of triacylglycerols in plasma after generation of glycerol by lipase
hydrolysis. These kits cannot be used directly in the presence of organic
solvents but development of a simple and efficient extraction procedure using
solvents and detergents has been described (Rodriguez-Sureda
V et al., Anal Biochem 2005, 343, 277).
2- The
quantification of triacylglycerols is easily made in the µg range after separation by TLC in combination with GLC analysis of fatty acids.
The scraped spots are directly transmethylated with BF3/methanol after addition
of a known amount of an odd fatty acid (C17:0) as internal standard and a solvent able to
dissolve triacylglycerol without interfering with the reaction (Toluene, xylene). This
method gives the fatty acid profile and the total amount of triacylglycerols
(1.07 x mass of fatty acids) in the spot.
3- The
determination of the amount of triacylglycerols can be made readily by HPLC either
directly on a tissue extract or after TLC separation:
4- Determination of milk fat in mixed fats by
analysis of butyric acid (Molkentin J et al. Chromatographia 1998, 48,
758).
Since butyric acid (C4) occurs only in milk fat from cows and other mammals, but
not in animal adipose or vegetal fats, gas chromatographic analysis of C4 in the
mixed fat and the pure milk enables the content of milk fat to be calculated. If
a sample of the pure milk fat is not available, a typical mean C4 content may be
used instead (3.42 g / 100 g fat for European milk fats). Methyl valerate (C5)
was taken as internal standard, the ratio Q =
C4/C5 being considered.
Preparation of samples
An amount of fat (about 100 mg weighed to the nearest 0.1 mg) was dissolved
in 5 ml of methyl valerate in heptane (0.4 mg/ml). 1 ml of this solution was
mixed with 20 ml
Na methylate solution (2M in methanol) in a sample vial, shaken vigorously for 3
min and centrifuged for 1 min. After addition of 100 mg NaH2SO4,
2 H2O
the vial was recapped, mixed again for 2 min and centrifuged for 1 min. The
clear supernatant was used for GC analysis. A temperature program from 45 to
75°C was used to separate C4 and C5.
An alternative method to measure milk lipids was proposed using UV
spectrophotometry (Forcato DO et al., J Dairy Sci 2005, 88, 478). This
simple microtechnique is based upon the measurement of the absorbance of small
samples (about 50 ml of milk) in ethanol at 208 nm after cold precipitation of
proteins. This method is fast and efficient in estimating fat content in milk
samples without prior lipid extraction.
.
B- Study of the structure of triacylglycerol molecules
This can be made at three
levels according to the required information:
1. The first
level is the
investigation of the variety of fatty acids acylating the positions 1 and 3 (position
b)
and those acylating the position 2 (position a). This investigation needs an enzymatic
or chemical reaction.
2. The second level is an analysis of the various fatty acids acylating each carbon of the glycerol backbone. This exploration needs specific chemical reactions.
3. The third level is a HPLC separation of the intact triacylglycerol molecular species allowing their direct identification and quantification. In contrast to the two previous ways of research, no transformation or destruction of the molecules are needed.
Miscellaneous
Interesterification of
a mixture of triacylglycerols (fats and oils)
This chemical reaction catalysed by alkali produces a random
distribution of ester groups within the triacylglycerol molecules.
