ANALYSIS OF TRIACYLGLYCEROLS
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.
Interesterification of a mixture of triacylglycerols (fats and oils)
This chemical reaction catalyzed by alkali produces a random distribution of ester groups within the triacylglycerol molecules.
As fatty acids are always present, even in small amounts, in the triacylglycerol pool of cell extracts, it can be necessary to eliminate these fatty acids to improve the triacylglycerol composition or to analyze the free fatty acid fraction. A simple procedure has been described for microalgal lipids but can be applied to any lipid extract (Paik MJ et al., J Chromatogr A 2009, 1216, 5917).
Procedure : Solid sodium carbonate (60 mg) pre-wetted with 0.1M KOH (100ml) was added with an aliquot of a solution of triacylglycerols in toluene containing free fatty acids. The triacylglycerol fraction was then extracted with 2-ml volumes of dichloromethane/hexane (1/4, vol/vol), (three times) by vortex-mixing and each extract was decanted by brief centrifugation. After solvent evaporation, the residue was tansmethylated for GLC analysis. The free fatty acids adsorbed to the solid sodium carbonate were recovered by acidification (to pH <2) of the particles with 6M HCl and then extracting with three successive 2 ml volumes of dichloromethane/hexane mixture, with vigorous vortex-mixing before each extraction. The combined extracts were dried over anhydrous Na2SO4 and were subjected to derivatization for further analysis. The pre-wetted sodium carbonate showed strong affinity toward the polar lipids, including phosphatidic acid, phosphatidylglycerol, lysophosphatidic acid, and monogalactosyl diglyceride.
Enzymatic and fluorometric determination of triacylglycerols in cow milk
Briefly, the access to the milk triacylglycerols is mediated by moderate heat treatment, detergents and the addition of phospholipase C. Triacylglycerol molecules are hydrolysed by lipoprotein lipase and lipase to free fatty acids and glycerol. The glycerol unit is subsequently oxidised by glycerol dehydrogenase to dihydroxy acetone, and the cofactor NAD+ is concomitantly reduced to NADH + H+. NADH + H+ equivalently reduces the non-fluorescent resazurin to resorufin mediated by the enzyme diaphorase. The developed resorufin is terminally estimated by its fluorescense compared with a standard curve (Larsen T et al., Food Chem 2011, 125, 1110).