The Lipid Library

STEREOSPECIFIC ANALYSIS OF TRIACYL-sn-GLYCEROLS

Abstract: The distribution of fatty acids on the glycerol backbone of a triacyl-sn-glycerol molecule often conveys important information about a fat. However, the first analysis procedures to have been developed were tedious and involved enzymatic hydrolysis. Two alternative approaches are described below using simple chemical degradative and derivatization steps followed by chiral chromatography.

For some years, it has been recognised that the compositions of the fatty acids on the different positions of the glycerol moiety of triacylglycerols can be very different. Position 2 is of course distinctive in that it is a secondary hydroxyl group, but it is not always recognised that the two primary positions are different stereochemically since there is a centre of asymmetry. Triacylglycerols therefore exist in enantiomeric forms. The positions are defined by a 'stereospecific numbering' (sn) system as sn-1, sn-2 and sn-3, and in natural triacyl-sn-glycerols, each can have a distinctive fatty acid composition. Our web page on triacylglycerols contains a more comprehensive account.

Pancreatic lipase hydrolysis is a relatively simple method for the determination of the composition of position 2, i.e. for “regiospecific” analysis. This can also be accomplished by some modern mass spectrometry methods. However, the compositions of positions sn-1 and sn-3 could until recently only be obtained by complex "stereospecific analysis" procedures with many steps involving degradation, synthesis, enzymatic hydrolysis and chromatographic separation of the products [1]. These methods have been used to determine the structures of many natural triacyl-sn-glycerols, and some surprisingly asymmetric fats have been found, with first prize going to milk fat in which all the short-chain fatty acids (butyric and hexanoic acids) are concentrated in position sn-3 with none in positions sn-1 and 2.

The Approach via Chiral Derivatizing Agents

Most lipid analysts, who are accustomed to organic solvents and homogeneous media, have an aversion to carrying out reactions such as enzymatic hydrolyses for which aqueous media are required. On the other hand, two alternative approaches to stereospecific analysis have been described that use simple chemical degradative and derivatization steps and the methodology of chiral chromatography described in our web page on this topic. It perhaps will demonstrate that this kind of methodology will have applications not too far from the main-stream of lipid analysis and research much sooner than many of my readers might have considered probable. Applications of this methodology to triacylglycerols have recently been reviewed [2].

The first of these methods was developed in my laboratory [3,4]. The first step is similar to that in other stereospecific analysis procedures, i.e. partial hydrolysis of the triacylglycerols giving among other products a mixture of sn-1,2-, 2,3- and 1,3-diacylglycerols. The reagent used for this hydrolysis step is ethyl magnesium bromide (a Grignard reagent), which is preferred over pancreatic lipase hydrolysis say because it exerts no known specificity for particular fatty acids. It appears to cause less acyl migration than occurs with other chemical reagents, although this aspect still requires improvement and there is always room for a better idea.

The second step involves reacting the product with a chiral derivatizing agent, (S)-(+)-1-(1-naphthyl)ethyl isocyanate, with purification of the resulting diacyl-sn-glycerol urethane derivatives by chromatography on solid-phase extraction columns containing an octadecylsilyl phase.

The third and key step involves resolution of the diacylglycerol urethanes by high-performance liquid chromatography (HPLC) merely on a column of silica gel. A simple isocratic mobile phase is employed, and the derivatives absorb strongly in the UV spectrum so detection is straightforward. 1,3-Diacyl-sn-glycerol urethanes elute early and this fraction is easily recovered. Because the derivatizing agent is chiral and a single enantiomer, the 1,2- and 2,3-diacyl-sn-glycerol urethanes are now diastereomers (see my web page on chiral chromatography for a definition), so they are separable in a non-chiral environment. In practice, the 1,2-diacyl-sn-glycerol derivatives elute ahead of the 2,3-diastereomers and the two distinct fractions can be collected.

Some separation of molecular species occurs also within each diastereomeric fraction, and while this might be considered an advantage in some circumstances, it is here something of a nuisance as it restricts the range of fatty acid components in the triacylglycerols that can be investigated. However, most of the common fats with C₁₆ and C₁₈ fatty acids are in the practical range.

The last step then involves methylating each of the fractions for analysis by GC with the highest precision possible. Then, the results for the compositions of each position are simply a matter of calculation. For example, as we know the fatty acid composition of the intact triacylglycerols and we have determined that of the 1,2-diacyl-sn-glycerol derivatives, it is simple arithmetic to calculate the composition of position sn-3. Similarly, the composition of position sn-1 can be calculated by difference once that of the 2,3-diacyl-sn-glycerols is known, and position sn-2 can be calculated from the results for the 1,3-diacylglycerols (or with greater accuracy by an independent analysis with pancreatic lipase). Thus, the compositions of all three positions are determined without resort to enzymes, using standard chromatography columns and derivatizing agents that are readily available.

This method has since been adapted to be used in conjunction with HPLC and mass spectrometry with electrospray ionization [5]. In addition, a variation upon the method in which monoacyl-sn-glycerol rather than diacylglycerol urethane derivatives are employed has been described and looks very promising [6].

The Approach via Chiral Chromatography Phases

Different but comparable approaches have been adopted by Professor Takagi and colleagues in Japan [7-9]. They converted either mono- or diacyl-sn-glycerols prepared from triacylglycerols to the 3,5-dinitrophenyl urethane (DNPU) derivatives for resolution by HPLC on columns containing a stationary phase with chiral moieties bonded chemically to a base of silica gel. The 3,5-dinitrophenyl moieties of the urethanes contribute to charge-transfer interactions with functional groups having pi electrons on the stationary phase and thus aid the resolution. 1 and 3-Monoacyl-sn-glycerols were prepared from triacylglycerols by partial hydrolysis with ethyl magnesium bromide; they were converted to the dinitrophenylurethane derivatives and then resolved on a chiral column. The distributions of fatty acids in each of positions sn-1, -2 and -3 could be calculated from the data.

By lowering the column temperature and slowing down the flow-rate, the method could even be applied to such complex triacyl-sn-glycerols as fish oils. This procedure will certainly be widely used in the future, as the special chiral columns and the reagent are now readily available. As with the procedure developed in my lab, there is no need to use lipases.

The only caveat I have with methods that utilize monoacylglycerol derivatives is with the extent of isomerization that may occur during their preparation from triacylglycerols with the aid of a Grignard reagent. This is a significant and well-documented but manageable problem when preparing diacylglycerols, and I would expect it to be worse when preparing monoacylglycerols. I am not aware of any publication in which this aspect has been tackled adequately.

Conclusions

This kind of methodology is unlikely to be used routinely with the common commercial fats, other than possibly as a means of detecting adulteration. It will be of greater value with specialist fats and is likely to be of great importance to biochemists and nutritionists. As this area is developing very rapidly at the moment, the above comments will certainly not be the last word on the subject.

References

1. Christie, W.W. The positional distributions of fatty acids in triglycerides. In: Analysis of Oils and Fats (ed. R.J. Hamilton & J.B. Rossell), Elsevier Applied Science, London, pp. 313-339 (1986).
2. Kuksis, A. and Itabashi, Y. Regio- and stereospecific analysis of glycerolipids. Methods, 36, 172-185 (2005).
3. Laakso, P. and Christie, W.W. Chromatographic resolution of chiral diacylglycerol derivatives: potential in the stereospecific analysis of triacyl-sn-glycerols. Lipids, 25, 349-353 (1990).
4. Christie, W.W., Nikolova-Damyanova, B., Laakso, P. and Herslof, B. Stereospecific analysis of triacyl-sn-glycerols via resolution of diastereomeric diacylglycerol derivatives by high-performance liquid chromatography on silica. J. Am. Oil Chem. Soc., 68, 695-701 (1991).
5. Agren, J.J. and Kuksis, A. Analysis of diastereomeric DAG naphthylethylurethanes by normal-phase HPLC with on-line electrospray MS. Lipids, 37, 613-619 (2002).
6. Petrosino, T., Riccieri, R., Blasi, F., Brutti, M., D'arco, G., Bosi, A., Maurelli, S., Cossignani, L., Simonetti, M.S. and Damiani, P. Original normal-phase high-performance liquid chromatographic separation of monoacylglycerol classes from extra virgin olive oil triacylglycerols for their stereospecific analysis. J. AOAC Int., 90, 1647-1654 (2007).
7. Takagi, T. and Ando, Y. Stereospecific analysis of acyl group distribution in triacylglycerols by HPLC with chiral column. J. Japan Oil Chem. Soc. (Yukagaku), 39, 622-628 (1990).
8. Takagi, T. and Ando, Y. Stereospecific analysis of triacyl-sn-glycerols by chiral HPLC. Lipids, 26, 542-547(1991).
9. Takagi, T. and Ando, Y. Stereospecific analysis of triacylglycerols by chiral-phase HPLC. Direct derivatization of partially hydrolyzed products. J. Japan Oil Chem. Soc. (Yukagaku), 40, 288-292 (1991).

This article has been updated appreciably from an earlier paper by the author that first appeared in Lipid Technology (Christie, W.W. Lipid Technology, 4, 72-74 (1992)).

Updated: 4/4/2008 Credits/disclaimer	Scottish Crop Research Institute (and MRS Lipid Analysis Unit), Invergowrie, Dundee (DD2 5DA), Scotland	Right click on the icon to open in a new window