TRIACYLGLYCEROLS

PART 2. BIOCHEMISTRY, METABOLISM AND ANALYSIS

1. Biosynthesis of Triacylglycerols

All eukaryotic organisms and even a few prokaryotes have the ability to synthesise triacylglycerols, and the process has been studied intensively in plants and animals especially. Many cell types and organs have the ability to synthesise triacylglycerols, but in animals the liver and intestines are most active, although most of the body stores of this lipid are in adipose tissue (see our web page on triacylglycerol composition). This lipid serves as a store of energy and a reserve of essential fatty acids, but it may also be produced as a protective measure to remove any excess of biologically active and potentially harmful lipids such as diacylglycerols and coenzyme A esters. Within all cell types, even those of the brain, triacylglycerols are stored as 'lipid droplets' (also termed ‘fat globules’, ‘oil bodies’, ‘lipid particles’, ‘adiposomes’, etc) enclosed by a monolayer of phospholipids and hydrophobic proteins, such as the adipose differentiation-related protein and perilipins in adipose tissue or oleosins in seeds.

Two main biosynthetic pathways are known, the sn-glycerol-3-phosphate pathway, which predominates in liver and adipose tissue, and a monoacylglycerol pathway in the intestines. In maturing plant seeds and some animal tissues, a third pathway has been recognized in which a diacylglycerol transferase is involved. The most important of these is the sn-glycerol-3-phosphate or Kennedy pathway illustrated below, first described by Professor Eugene Kennedy and colleagues in the 1950s, by means of which more than 90% of liver triacylglycerols are produced.

Kennedy pathway of triacylgycerol biosynthesis

In the sn-glycerol-3-phosphate or α-glycerophosphate pathway, the main source of the glycerol backbone is sn-glycerol-3-phosphate produced by the catabolism of glucose or to a lesser extent by the action of the enzyme glycerol kinase on free glycerol. Subsequent reactions occur in the endoplasmic reticulum. First, the precursor sn-glycerol-3-phosphate is esterified by a fatty acid coenzyme A ester in a reaction catalysed by a glycerol-3-phosphate acyltransferase at position sn-1 to form lysophosphatidic acid, and this is in turn acylated by an acylglycerophosphate acyltransferase in position sn-2 to form phosphatidic acid. The phosphate group is removed by the enzyme phosphatidic phosphohydrolase, and the resultant 1,2-diacyl-sn-glycerol is acylated by a diacylglycerol acyltransferase to form the triacyl-sn-glycerol. As the glycerol-3-phosphate acyltransferase has the lowest specific activity of these enzymes, this step may be the rate-limiting one.

Among other potential routes to the various intermediates, lysophosphatidic acid and phosphatidic acid can be synthesised in mitochondria, but must then be transported to the endoplasmic reticulum before they enter the pathway for triacylglycerol production. 1,2-Diacyl-sn-glycerols are produced by the action of phospholipase C on phospholipids. In addition, dihydroxyacetone-phosphate in peroxisomes or endoplasmic reticulum can be acylated by a specific acyltransferase to form 1-acyl dihydroxyacetone-phosphate, which is reduced by dihydroxyacetone-phosphate oxido-reductase to lysophosphatidic acid (part of the biosynthetic route to plasmalogens), which can then enter the pathway to triacylglycerols.

Alternative route to lysophosphosphatidic acid

In the glycerol-3-phosphate pathway, the starting material is of defined stereochemistry and each of the enzymes catalysing the various steps in the process is distinctive and can have preferences for particular fatty acids (as their coenzyme A esters) and for particular fatty acid combinations in the partially acylated intermediates. It should not be surprising, therefore, that natural triacylglycerols exist in enantiomeric forms with each position of the sn-glycerol moiety esterified by different fatty acids.

In the enterocytes of intestines after a meal, up to 75% of the triacylglycerols are formed via a monoacylglycerol pathway. 2-Monoacyl-sn-glycerols and free fatty acids released from dietary triacylglycerols by the action of pancreatic lipase within the intestines are taken up by the enterocytes. There, the monoacylglycerols are sequentially acylated via a triacylglycerol synthase complex with formation of sn-1,2-diacylglycerols mainly as the first intermediate in the process, though sn-2,3-diacylglycerols are also produced, and both are further acylated to form triacylglycerols.

Monoacylglycerol pathway of triacylglycerol biosynthesis

In the third biosynthetic pathway, which is less well known, triacylglycerols are synthesised by an acyl-CoA independent transacylation between two racemic diacylglycerols. The reaction was first detected in intestinal microvillus cells and is catalysed by a diacylglycerol transacylase. Both diacylglycerol enantiomers participate in the reaction with equal facility to transfer a fatty acyl group with formation of triacylglycerols and a 2-monoacyl-sn-glycerol. A similar reaction has been observed in seed oils.

Triacylglycerol formation via diacylglycerol transacylases

It has been suggested that the enzyme may function in remodelling triacylglycerols post synthesis, especially in oil seeds, and it is possible that it may be involved in similar processes in the liver, where extensive hydrolysis/re-esterification is known to occur.

In prokaryotes, the glycerol-3-phosphate pathway of triacylglycerol biosynthesis only occurs, but in yeast both glycerol-3-phosphate and dihydroxyacetone-phosphate can be the primary precursors and synthesis takes place in lipid droplets and the endoplasmic reticulum. In plants, many questions remain concerning the nature and compartmentalization of the process, as many different isoenzymes have been characterized in different organelles for each of the acylation steps. The terminal acylation step is catalysed by microsomal diacylglycerol acyltransferases with broad substrate specificities. However, little is known of how the membrane and storage lipids acquire their very different fatty acid compositions and positional distributions.

2. Triacylglycerol Function and Metabolism

Fat comprises about 40% of the energy intake in the human diet in Western countries, and a high proportion of this is triacylglycerols. The process of fat digestion is begun in the stomach by gastric or lingual lipases, the extent of which depending on species. However, this is relatively unimportant in comparison to the reaction with pancreatic lipase, which occurs in the duodenum. Entry of triacylglycerol degradation products into the duodenum stimulates synthesis of the hormone cholecystokinin and causes the gall bladder to release bile acids, which are strong detergents and act to emulsify the hydrophobic triacylglycerols so increasing the available surface area. In turn, cholecystokinin stimulates the release of the hydrolytic enzyme pancreatic lipase. The process of hydrolysis is regiospecific and results in the release of the fatty acids from the 1(3) positions of the triacylglycerols and formation of 2-monoacyl-sn-glycerols. Isomerization of the latter to 1(3)-monoacyl-sn-glycerols occurs to some extent that can be degraded completely to glycerol and free acids. The requirements and fatty acid specificity of pancreatic lipase are discussed in the next section.

Pancreatic lipase action

The free fatty acids and 2-monoacyl-sn-glycerols are rapidly taken up by the intestinal cells, via specific carrier molecules, and are esterified into triacylglycerols, as described above. There is evidence that the stereospecific structure of dietary triglycerides has an effect on the uptake of particular fatty acids and may influence the further lipid metabolism in humans. In particular, incorporation of palmitic acid into the position sn-2 of milk fat may be of benefit to the human infant (as a source of energy for growth and development), although it increases the atherogenic potential for adults.

Within the intestines, triacylglycerols are incorporated into lipoprotein complexes termed chylomicrons. These consist of a core of triacylglycerols together with some cholesterol esters, which is stabilized and rendered compatible with an aqueous environment by a surface film consisting of phospholipids, free cholesterol and one molecule of a truncated form of apoprotein B (48 kDa). These particles are secreted into the lymph and thence into the plasma for transport to the peripheral tissues for storage or structural purposes. Adipose tissue in particular secretes appreciable amounts of the enzyme lipoprotein lipase into the surrounding blood vessels, where it rapidly hydrolyses the triacylglycerols at the cell surface, releasing free fatty acids, most of which are absorbed into the adjacent adipocytes and re-utilized for triacylglycerol synthesis within the cell.

The chylomicrons remnants eventually reach the liver, where the remaining lipids are hydrolysed and absorbed. The fatty acids within the liver can be utilized for a variety of purpose, but a proportion is re-converted into triacylglycerols, which are exported into the plasma in the form of very-low-density lipoproteins (VLDL), consisting again of a triacylglycerol and cholesterol ester core, surrounded by phospholipids and free cholesterol, together with one molecule of full-length apoprotein B (100 kDa), apoprotein C and sometimes apoprotein E. These particles in turn are transported to the peripheral tissues, where they are hydrolysed and the free acids absorbed. Eventually, the remnants are returned to the liver. These processes are discussed in greater detail in our webpages dealing with lipoproteins.

Scottish thistle Within animal cells, a proportion of the fatty acids taken up from the circulation is converted to triacylglycerols as described briefly at the start of the previous section and incorporated into lipid droplets with their surrounding protective layer of phospholipids and hydrophobic proteins, such as perilipin. This process is especially important in adipose tissue, which is the major store of lipids in animals. When fatty acids are required by other tissues for energy or other purposes, they are released from the triacylglycerols by two enzymes mainly, termed 'the hormone-sensitive lipase' and the 'adipose triacylglycerol lipase'. Hormone-sensitive lipase is stimulated by the action of the hormones insulin and noradrenalin by a mechanism that involves phosphorylation of the enzyme by cAMP-protein kinase, thereby increasing its activity and causing it to translocate from the cytosol to the lipid droplet. Perilipin acts as a barrier in non-stimulated cells, but on stimulation, it is phosphorylated by the cAMP-protein kinase also; this changes its shape and reduces its hydrophobicity, and in the process activates lipolysis probably by exposing the triacylglycerols to the action of lipases. In addition to it activity towards triacylglycerols, hormone-sensitive lipase will hydrolyse diacylglycerols and cholesterol esters equally rapidly.

Less is known of the properties of the adipose triacylglycerol lipase, but it structurally related to the plant acyl-hydrolases. It is specific for triacylglycerols with little or no activity towards diacylglycerols. Free acids released by the combined action of these enzymes are exported into the plasma for transport to other tissues in the form of albumin complexes. Defects in these processes can have severe implications for the pathogenesis of diabetes and obesity in humans.

On the basis of profiling of the surface proteins and phospholipids, it has been argued that lipid droplets in cells other than adipose tissue should be considered as complex, metabolically active organelles that also function in the supply of fatty acids for various purposes, including membrane trafficking and possibly in the recycling of both simple and complex lipids. For example, within the liver, triacylglycerols are stored as lipid droplets in the cytoplasm adjacent to the endoplasmic reticulum, where a triacylglycerol hydrolase can effect lipolysis to di-and monoacylglycerols, which are more soluble in the membrane, which they are able to cross. They are then available for re-synthesis into triacylglycerols by luminally oriented acyltransferases before assembly into nascent lipoprotein complexes.

One specialized form of adipose tissue, brown fat, is highly vascularized and rich in mitochondria, which oxidize fat so rapidly that heat is generated. This appears to be especially important in young animals and in those recovering from hibernation. In addition, subcutaneous depots serve as insulation against cold in many terrestrial animals, as is obvious in the pig, which is surrounded by a layer of fat, and it is especially true for marine mammals. In the latter and in fish, the lipid depots are less dense than water and so aid buoyancy with the result that less energy is expended in swimming. More surprisingly, perhaps, triacylglycerols together with the structurally related glyceryl ether diesters and wax esters are the main components of the sonar lens used in echo-location by dolphins and some whales. The triacylglycerols are distinctive in that they contain two molecules of 3-methylbutyric (isovaleric) acid with one long-chain fatty acid. It appears that the relative concentrations of the various lipids in an organ in the head of the animals (termed the ‘melon’) vary in such a way that they are able to focus sound waves.

In plants, fatty acids are stored as triacylglycerols in lipid droplets in seeds, and their catabolism by β-oxidation within the peroxisomes to acetyl-CoA and thence to succinate via the glyoxylate cycle provides germinating seeds with energy, in addition to structural elements, before the seedlings develop the capacity to photosynthesize. Upon germination, the process begins with lipolysis at the surface of oil bodies, where the oleosins, which are specific structural proteins of plant oil bodies, are believed to serve to assist the docking of the lipase.

3. Regiospecific Analysis of Triacylglycerols by Hydrolysis with Pancreatic Lipase

The composition of position sn-2 of triacylglycerols can be determined by incubating them with the enzyme pancreatic lipase in an appropriate buffer. The fatty acids are hydrolysed from the primary positions leaving a 2-monoacyl-sn-glycerol, which can be isolated for determination of its fatty acid composition (see the last figure in the previous section).

Pig pancreatin, a powder obtained by dehydrating and defatting pig pancreas with acetone and diethyl ether, is the most widely used source of the enzyme; it is stable for long periods of time and is available from suppliers of biochemicals.

thistle All straight-chain saturated fatty acids in the normal chain-length range and most mono-, di- and trienoic acids are apparently hydrolysed from the primary positions of triacylglycerols at about the same rate, although the ester bonds of polyunsaturated fatty acids such as docosahexaenoic (e.g. in fish oils), trans-3-hexadecenoic (e.g. from some plant sources), γ-linolenic acid and phytanic acid to glycerol are hydrolysed more slowly, possibly as a result of steric hindrance caused by the proximity of substituent groups to the ester bonds. In addition, the enzyme hydrolyses triacylglycerol molecules that contain short-chain fatty acids more rapidly than molecules containing only longer-chain fatty acids. For example, with a triacylglycerol such as 1-butyro-2,3-dipalmitin, both the fatty acids on the primary positions were found to be hydrolysed at about the same rate but faster than from tripalmitin. Fortunately, when the enzyme is used with triacylglycerols containing a more normal range of fatty acids, little specificity is observed and the 2-monoacylglycerols produced can be considered in practice to be representative of those in the native triacylglycerols.

Calcium ions are essential and bile salts helpful for the reaction, and it is necessary that the triacylglycerols be well dispersed by vigorous shaking, as they must be in a micellar form for hydrolysis to occur. For this reason, methyl oleate or hexane have sometimes been added to relatively saturated fats, with high melting-points, as carriers; the latter may not always give reliable results, and pre-incubation at 42°C for 5 min has been recommended instead. Isooctane and cyclohexane have been recommended as carriers with microbial lipases. In structural studies, the concentrations of the various cations, bile salts and the enzyme, the pH of the buffer and the temperature are adjusted to their optima so that an appreciable degree of hydrolysis (50-60% is sufficient) occurs in a short time (1-2 min). Undesirable side reactions such as acyl migration are thereby minimized.

A semi-micro method developed by Luddy et al. (J. Am. Oil Chem. Soc., 41, 693-696 (1964)) is recommended as the best practical procedure for the purpose. In essence, a buffer solution (pH 8) containing bile salts and calcium chloride is added to the triacylglycerols at 40°C. After a brief equilibration period, the enzyme preparation is added and the mixture is shaken very vigorously for 1-2 minutes, when about 50% hydrolysis should have occurred. The reaction is then stopped by addition of acid, the lipid products are extracted, and the monoacylglycerols are isolated by thin-layer chromatography for methylation and gas chromatographic analysis.

As hydrolysis may not be completely random and as there may be some contamination from lipids endogenous to the enzyme preparation, or from fatty acids liberated from position sn-2 following acyl migration, the free fatty acids released may be somewhat different from the composition originally present in the primary positions of the triacylglycerols. The mean composition of each fatty acid in positions sn-1 and sn-3 can, however, be calculated from its concentration in the intact triacylglycerol and in position sn-2 by means of the relationship -

[position 1 & 3] = (3 x [triacylglycerol] - [position 2])/2

Values obtained are of course subject to the cumulative errors of the analyses.

The mould Rhizopus arrhizus secretes an extracellular lipase that also has an absolute specificity for the primary bonds of glycerolipids and so resembles pancreatic lipase in many respects. It differs in that it does not have an absolute requirement for calcium ions and is not activated by bile salts. Although it offers no appreciable advantages over pancreatic lipase in most instances, there are a few applications where it has been preferred. In addition, some purely chemical methods have been published.

4. Stereospecific Analysis of Triacyl-sn-glycerols - Methods involving Stereospecific Enzymes

No lipolytic enzyme has yet been isolated that is capable of distinguishing between position 1 and 3 of a triacyl-sn-glycerol. Nonetheless, a number of ingenious stereospecific analysis procedures have been developed for determining the compositions of positions sn-1, sn-2 and sn-3. Several methods have been described over the years, but nowadays there are two main approaches and one making use of specific lipases is described here.

Brockerhoff (J. Lipid Res., 6, 10-15 (1965)) devised the first stereospecific analysis procedure of general applicability. His method has been much modified by subsequent research workers but in essence has stood the test of time. The basic procedure is outlined below. α,β-Diacylglycerols (an equimolar mixture of the 1,2- and 2,3-sn-isomers) are first prepared by partial hydrolysis for conversion synthetically to phospholipid derivatives, which are in turn hydrolysed by the phospholipase A of snake venom, an enzyme which reacts only with the "natural" 1,2-diacyl-sn-glycerophosphatide. The products are a lysophosphatide that contains the fatty acids originally present in position sn-1, free fatty acids released from position sn-2 and the unchanged "unnatural" 2,3-diacyl-sn-phosphatide. After isolation of each product and transesterification, the fatty acid compositions (in mol per cent) are determined by gas chromatography. In addition, the composition of position sn-2 is determined independently by means of pancreatic lipolysis as a check.

Brockerhoff procedure for stereospecific analysis

Only the fatty acid composition of position sn-3 is not determined directly by this method, but the amount of each fatty acid in this position can be calculated from the analysis of the original triacylglycerol and those of positions sn-1 and sn-2, or from the analysis of the 2,3-diacyl-sn-phosphatide, i.e. for each component

[position 3] = 3 x [triacylglycerol] - [position sn-1] - [position sn-2]

or [position 3] = 2 x [2,3-diacyl-sn-phosphatide] - [position sn-2]

It is worthwhile carrying out both calculations as a check on accuracy.

The key to success with the procedure lies in the preparation of the intermediate α,β-diacylglycerols, which must be generated in a random manner so that the fatty acid compositions of the various positions are identical to those in the original triacylglycerols. There must be no selectivity for specific fatty acids or fatty acid combinations, and the least possible acyl migration during their formation. Hydrolysis with pancreatic lipase was used initially but a Grignard reagent, specifically ethyl magnesium bromide, is now preferred as it has no known fatty acid specificities and causes less acyl migration than other methods. In practice, such acyl migration as does occur causes some contamination (6-10%) of the 1,3-diacylglycerols but much less of the α,β-diacylglycerols in which the primary positions are virtually unchanged. I must state that I am not convinced that the problem has been fully solved. The α,β-diacylglycerols required for the procedure must not come in contact with polar solvents or be heated, and they must be isolated immediately by means of TLC on layers of silica gel G impregnated with boric acid for conversion without delay to phosphatides, which can in turn be purified by TLC or other methods.

In the original Brockerhoff procedure, phosphatidylphenols were prepared from the diacylglycerols, but it was later shown that it is relatively easy to prepare phosphatidylcholines, the chromatographic properties of which are better understood. They can also be hydrolysed with some stereo-selectivity with phospholipase C, offering an additional analytical option.

Full experimental details of a recommended procedure, which incorporates features developed in a number of laboratories, have been published (Christie, 1986; see reading list below). Although the procedure is rather complex and involves a number of steps, it is capable of reasonable precision in the hands of skilled workers. Analyses should not be accepted unless the results for major components in positions sn-2 and sn-3, determined by both available methods, agree within 4% (absolute).

The main drawback to this approach to the stereospecific analysis of triacyl-sn-glycerols is that the fatty acid composition of position sn-3 is not determined directly, so that the proportionate errors in the results for minor fatty acids in this position can be considerable. Small negative values are sometimes obtained and small positive values may arise in calculations for a component that is not present in the position at all. This could no doubt be overcome by reacting the unchanged 2,3-diacyl-sn-phosphatide with the lipase of Rhizopus arrhizus to release the fatty acids from position sn-3 for direct analysis.

5. Stereospecific Analysis of Triacyl-sn-glycerols - Methods involving Chiral Chromatography

In the last few years, alternative approaches to stereospecific analysis have been described that use simple chemical degradative and derivatization steps and the methodology of chiral chromatography. One such method was developed in our laboratory. The principle is dependent on the fact that diastereomeric compounds can be resolved by adsorption chromatography on silica gel, because they have different physical and chemical properties. Thus a chiral derivatizing agent (R)-X will react with a racemic substance (R,S)-Y -

The degree of separation of the two diastereomers in a chromatographic system will depend on the chiral structures of X and Y and the manner of their interactions with the mobile and stationary phases. In addition, it is noteworthy that the order of elution of the diastereomeric derivatives is reversed if the other enantiomer of the reagent, i.e. (S)-X, can be employed. (S)-(+)-1-(1-Naphthyl)ethyl urethane derivatives of diacyl-sn-glycerols are preferred for the purpose. With the HPLC column of silica gel used in this work, it appears that the presence of the hydrogen atom on the nitrogen between the chiral centres is essential to the separation process; it is presumed to be a primary site for hydrogen bonding to silanols on the adsorbent surface.

Chiral urethane detivative of diacylglycerols The first step in the stereospecific analysis procedure is identical to that in most other methods, i.e. partial hydrolysis of the triglycerides with ethyl magnesium bromide to a mixture of sn-1,2-, 2,3- and 1,3-diacylglycerols, amongst other products. Next, the products are reacted with a chiral derivatizing agent, (S)-(+)-1-(1-naphthyl)ethyl isocyanate and the diacyl-sn-glycerol urethane derivatives are isolated by chromatography on solid-phase extraction columns containing an octadecylsilyl phase; by-products of low molecular-weight and excess derivatizing reagents are eluted first and discarded, before the required diacylglycerol urethane derivatives are recovered.

The most important step involves resolution of the diacylglycerol urethanes by HPLC on a column of silica gel. A simple isocratic mobile phase is employed, and since the derivatives absorbed strongly in the UV spectrum, detection is straightforward. 1,3-Diacylglycerol urethanes elute early and this fraction is easily recovered; however, as they are more susceptible to acyl migration than the other products of interest, they are not used further. As the derivatizing agent is chiral and a single enantiomer, the 1,2- and 2,3-diacyl-sn-glycerol urethanes formed from it are now diastereomers, so they are separable in a non-chiral environment.

Stereospecific analysis via chiral chromatography

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 also occurs 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. Fortunately, most of the common fats with C₁₆ and C₁₈ fatty acids are in the practical range, and it may be possible with further development to extend this eventually.

The final step involves methylation of each of the fractions for analysis by GC with the highest precision possible, when the results for the positional distributions are simply a matter for calculation. For example, as the fatty acid composition of the intact triacylglycerols is known, and that of the 1,2-diacyl-sn-glycerol derivatives has been determined, it is simple arithmetic to calculate the composition of position sn-3. Similarly, the composition of position sn-1 can be calculated once that of the 2,3-diacyl-sn-glycerols is known. That of position sn-2 is calculated by difference (or by an independent hydrolysis with pancreatic lipase). Thus, the composition of all three positions is determined, without resort to enzymes, by using standard chromatography columns and derivatizing agents that are available from commercial sources.

diacylglycerols - 3,5-dinitrophenyl urethane derivatives Professor Takagi and colleagues in Japan adopted a related but distinct procedure. They converted mono- and 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 (e.g. Sumipax^TM OA-4100). 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 resolution. In their preferred technique, 1-, 2- and 3-monoacyl-sn-glycerols (rather than diacylglycerols) are prepared from triacylglycerols by partial hydrolysis with ethyl magnesium bromide. The 1(3)-forms are separated from the 2-isomers by TLC on silica gel impregnated with boric acid, before being converted to the DNPU derivatives. Then, the 1- and 3-monoacyl-sn-glycerol derivatives are resolved on a chiral HPLC column. After methylation and GC analysis, the distributions of fatty acids in each of positions sn-1, sn-2 and sn-3 can be calculated from the data. By means of lowering the column temperature and slowing down the flow-rate, the method can even be applied to such complex triacyl-sn-glycerols as fish oils.

6. Molecular Species Composition of Triacylglycerols

It is well known that natural triacylglycerols exist in the form of a large number of distinct molecular species. Three basic methods of analysis are available – silver ion chromatography, reversed-phase chromatography and high-temperature gas chromatography. In silver ion chromatography, separation is in essence according to the degree of unsaturation of the triacylglycerol molecules, while in reversed-phase chromatography separation is both by the combined degree of unsaturation and chain-lengths of the fatty acid moieties. Originally, thin-layer techniques only were available, but nowadays most analysts resort to high-performance liquid chromatography. While some excellent separations of triacylglycerols by gas chromatography have been published, the technology is close to its absolute limits, and quantification presents difficulties that are not always recognized. Separation can be by chain-length only or can include degree of unsaturation with appropriate column types. These techniques are discussed in the section of this website - Selected topics in the analysis of lipids – or you can consult my recent book cited below. Nowadays, mass spectrometry is being used increasingly for the purpose with atmospheric-pressure chemical ionization of electrospray ionization being favoured. These techniques can be used in conjunction with liquid chromatography or on the intact lipid, and give information both on molecular species compositions and on regiospecific distributions of fatty acids

The Lipid Library