Simple regiodistribution analysis of triacylglycerols limited to the distribution of fatty acids to the 2-position and to the 1,3-positions can be made by the analysis of hydrolysis products obtained either by :
enzymatic reaction or
A comprehensive review of all aspects of the stereospecific analysis of fatty acid distribution was released by Buchgraber M (Eur J Lipid Sci Technol 2004, 106, 621).
The methodology used the properties of a lipase (pancreatic lipase) which is specific for the primary ester bond of glycerides. The enzyme hydrolyses more rapidly saturated than polyunsaturated fatty acids and short than long chains. However, with triacylglycerols commonly found in animal or vegetal tissues, little fatty acid specificity is evident. Hydrolysis produces fatty acids and mainly 2-monoacylglycerol. Only that form needs to be isolated and transesterified for GLC analysis, since any 1- or 3-monoacylglycerol (migrating in the same TLC spot) are products of acyl migration. Furthermore, released fatty acids may not be similar to the mean composition of the positions 1 (a) and 3 (a'). This mean composition of each fatty acid is calculated from its proportion in the intact triacylglycerol and in position 2.
To determine quantitatively the fatty acid at the sn-2 position (b), an extensive triglyceride hydrolysis must be processed.
TLC plates (silicagel G) and tank
water bath (40°C)
- Incubation medium: 1 M Tris buffer pH 8 containing 2.2% CaCl2 (w/v) and 0.05% deoxycholate (w/v) - 10 mg/ml pancreatic lipase (w/v) in water
- 4 M HCl
- diethylether, ethanol, chloroform, chloroform/acetone (96/4, v/v)
- 2.3% boric acid in ethanol, primuline solution, BF3/methanol reagent
Evaporate a small volume of solution containing up to 5 mg triacylglycerol in a glass tube.
Add 1.35 ml of the incubation medium and warm at 40°C for 2 min
Add 0.1 ml of pancreatic lipase and vortex for 3 min (do some trials with unknown samples).
Stop the enzymatic reaction by addition of 1 ml ethanol and 1.5 ml 4 M HCl
Wash the aqueous phase two times with 5 ml diethylether, collect the ether phase in a glass tube and wash with 2 ml water.
The ether phase is evaporated and dissolved in 50-100 µl chloroform.
The products are separated by TLC on boric acid impregnated silicagel plates with chloroform/acetone as solvent system.
After spraying primuline, the spot corresponding to 2-monoacylglycerols is scraped off and directly transmethylated with BF3/methanol reagent.
A : origin, B : 1-MG, C : 2-MG, D : fatty acids
E : 1,2-DG, F : 1,3-DG, G : TG
Analyze the methylated fatty acids by GLC (position 2 or b) and, with the composition of an aliquot of the initial triacylglycerol, calculate the position a of each fatty acid by means of the relationship: position a = [(3 x triacylglycerol)-(position b)] / 2.
A critical study of analytical techniques for the determination of the partial positional distribution of fatty acids in triacylglycerols has shown that the method previously described is the most reliable and shows good correlation with fat melting point and hardness (Segura J. et al., Grasas Aceites 2015, 66, e076).
Extensive enzymatic hydrolysis
About 50 mg of fat are hydrolyzed
after addition of 40 mg pancreatic lipase in 4 ml of a Tris solution (1M, pH
8.0). Tubes are shaken and 0.4 ml of calcium chloride solution (22%, w/v) and
2 ml of sodium chloride solution (0.1%, w/v) are added. The tubes are incubated
at 40°C for 15 min under agitation. After cooling, 2 ml HCl (6M) and 1 ml diethyl
ether are added. Tubes are shaken for 1 min and centrifuged. The upper ether
layer is collected and evaporated for further analysis of fatty acids or monoglycerides.
A new approach of the asymmetrical stereochemical distribution of fatty acids in triacylglycerols has been described calculating the asymmetric a coefficient from the sn-2 fatty acid, and triacylglycerol composition of the oil (Martinez-Force E et al., Anal Biochem 2004, 334, 175). This coefficient reflects the relative content of fatty acids at the sn-1 and sn-3 positions.
These enzymatic methods
have been applied to various vegetable and animal oils with mainly C14-20 fatty
acids. Unfortunately, they
are not suitable for triacylglycerols
containing short chain and polyunsaturated fatty acids, because of lower lipase
hydrolysis rates. Thus, an alternative method has been developed using immobilized
lipase B from Candida antarctica (Watanabe Y. et al., J Oleo Sci
2015, 64, 1193). The method was applied with success to fish oil, milk
fat, and palm oil. Its repeatability and reproducibility have ben collaboratively
studied by 12 laboratories.
As difficulties are due to the
occurrence of unavoidable acyl migration, an analytical procedure based on ethyl
magnesium bromide deacylation was proposed (Turon F et al. Lipids 2002, 37,
This deacylation procedure was shown to lead to representative 2-monoacylglycerols (2-MAG), allowing the composition of the native triacylglycerols in the 2-position to be determined directly. The fatty acid composition in the 1,3-positions can then be estimated from the composition of the 2-MAG and TAG according to the formula 3 x TAG - (2 x 2-MAG).
All details on the experimental procedure (similar to that proposed for the regiospecific analysis of TAG) may be found in the original paper (Turon F et al 2002).