An Introduction to Lipids and Gas Chromatography

Chapter 2 Sections A to D. Lipid Structures

A.  Definitions

There is no satisfactory universally accepted definition of a "lipid", although most chemists and biochemists who work with these fascinating natural products have a firm intuitive understanding of the term. Most general textbooks describe lipids rather loosely as a group of compounds, which have in common a ready solubility in organic solvents such as chloroform, ethers and alcohols - a definition which encompasses steroids, carotenoids, terpenes and bile acids (generic terms in their own right!) in addition to fatty acids and glycerolipids, for example. The author has recently criticised this concept of a lipid as unnecessarily broad [168], and has proposed a definition which hearkens back to the origins of the term, i.e.

"Lipids are fatty acids and their derivatives, and substances related biosynthetically or functionally to these compounds."

The fatty acids are compounds synthesised in nature via condensation of malonyl-coenzyme A units by a fatty acid synthetase complex. By such definitions, cholesterol (but not steroid hormones) can be considered a lipid, as are phospholipids and glycolipids. Gangliosides, which are acidic glycolipids, are soluble in water and would not be treated as a lipid if some of the looser definitions were accepted. In the subsequent text of this book, the strict definition of lipid given above was followed in selecting the subject matter. (Further discussion of this definition is available on this website here..)

Some further terms which have proved their worth, especially in discussing the chromatographic separation of lipids [163,168], are -

  • Simple lipids- those which on saponification yield at most two types of primary hydrolysis product (fatty acids, glycerol, etc.) per mole, e.g. triacylglycerols and cholesterol esters.

  • Complex lipids- those which on saponification yield three or more primary products per mole, e.g. phospholipids and glycolipids.

The phospholipids and glycolipids can themselves be further subdivided into glycero- and sphingolipids (see below).

If a thorough analysis of a lipid sample from an organism is intended, it is necessary to separate it into simpler classes, according to the nature of the constituent parts of the molecules, and these components in turn may then have to be identified and quantified. The principal purpose of this book is to describe the analysis of lipids by means of gas chromatography, a technique which requires that the lipids be volatilised. The fatty acids and most of the simple lipids can sometimes be analysed directly by this method, but the complex lipids must be hydrolysed to simpler less-polar moieties, for example to the diacylglycerol and ceramide backbones common to glycerophospholipids and sphingolipids, respectively, prior to separation. It is therefore the aliphatic part of the molecule which is of primary importance for gas chromatography, and the following text will concentrate on this aspect.

There are a number of books available that deal with lipids and their structures, and the author has found those cited to be of particular value [319,367]. Literally thousands of papers have appeared over the last 25 years detailing the structures and compositions of lipids from particular tissues and species, as determined by modern chromatographic methods, but there appears to have been very little effort to collate and critically compare these data in any systematic way, or to relate the compositions of lipids to their functions. Among other consequences of this, there remain anomalies and gaps in our knowledge. Comprehensive accounts of the lipids of the tissues of ruminant animals [162], tissue and membrane phospholipid compositions [395,970] and triacylglycerol compositions [125,553,686,824] have appeared, however, and there are miscellaneous reviews of the compositions of specific lipid classes or tissues in the literature. The author recently attempted to summarise the essential features of lipid composition in a succinct manner [168] and there is information elsewhere on this on this web site for for tissues from animals and plants & microorganisms. This cannot be repeated here, and a brief summary only of lipid structure and composition follows.

The nomenclature proposed by IUPAC-IUB commissions [415,416] has been followed throughout (see also our web page on Nomenclature).

 

B.  The Fatty Acids

The fatty acids of plant, animal and microbial origin generally contain even numbers of carbon atoms in straight chains, with a carboxyl group at one extremity and with double bonds of the cis configuration in specific positions in relation to this. In animal tissues, the common fatty acids vary in chain length from 14 to 22, but on occasion can span the range from 2 to 36 or even more. Individual groups of microorganisms can contain fatty acids with 80 or more carbon atoms, but higher plants usually exhibit a more limited chain-length distribution. Fatty acids from animal tissues may have one to six double bonds, those from algae may have up to five, while those of the higher plants rarely have more than three; microbial fatty acids only occasionally have more than one. Hydroxy fatty acids are synthesised in some animal tissues, but fatty acids with other functional groups, when present, have usually been taken up from the food chain. Plant and microbial fatty acids, on the other hand, can contain a wide variety of functional groups including trans-double bonds, acetylenic bonds, epoxyl, hydroxyl, keto and ether groups, and cyclopropene, cyclopropane and cyclopentene rings. Fatty acid structure and physical properties have been reviewed by Gunstone [318]. Further information on fatty acid structures, composition and functions is available on this web site here..

 

1.  Saturated fatty acids

The most abundant saturated fatty acids in animal and plant tissues are straight-chain compounds with 14, 16 and 18 carbon atoms, but all the possible odd- and even-numbered homologues with 2 to 36 carbon atoms have been found in nature in esterified form. They are named systematically from the saturated hydrocarbon with the same number of carbon atoms, the final -e being changed to -oic. Thus, the fatty acid with 16 carbon atoms and the structural formula -

CH3(CH2)14COOH

  - is systematically named hexadecanoic acid, although it is more usual to see the trivial name palmitic acid in the literature. It may also be termed a "C16" fatty acid or with greater precision as "16:0", the number before the colon specifying the number of carbon atoms, and that after the colon, the number of double bonds. A list of the common saturated fatty acids together with their trivial names and shorthand designations is given in Table 2.1. A comprehensive list of the trivial names of fatty acids has been published elsewhere [773].

 

Table 2.1. Saturated fatty acids of general formula
CH3(CH2)nCOOH
 Systematic name Shorthand
designation
Trivial name
ethanoic 2:0 acetic
butanoic 4:0 butyric
hexanoic 6:0 caproic
octanoic 8:0 caprylic
nonanoic 9:0 pelargonic
decanoic 10:0 capric
undecanoic 11:0  
dodecanoic 12:0 lauric
tridecanoic 13:0  
tetradecanoic 14:0 myristic
pentadecanoic 15:0  
hexadecanoic 16:0 palmitic
heptadecanoic 17:0 margaric
octadecanoic 18:0 stearic
nonadecanoic 19:0  
eicosanoic 20:0 arachidic
heneicosanoic 21:0  
docosanoic 22:0 behenic
tetracosanoic 24:0 lignoceric
 

 

Acetic acid is not often found in association with fatty acids of higher molecular weight in esterified form in lipid molecules, but it does occur esterified to glycerol in some seed oils and in ruminant milk fats. In certain vegetable oils, it has been detected in linkage to the hydroxyl group of a hydroxy fatty acid, which is in turn esterified to glycerol.

Lipid-bound C4 to C12 fatty acids are in essence only found in milk fats in animal tissues, while the medium-chain compounds occur in seed oils, such as coconut oil. Palmitic acid is one of the most abundant fatty acids in nature and is found in the lipids of all organisms. Stearic acid is also relatively common.

Odd-chain fatty acids are synthesised by many microorganisms, and are produced, but to a very limited extent, in animal tissues when the fatty acid synthetase accepts propionyl-coenzyme A as the primer molecule.

The higher saturated fatty acids are solid at room temperature and are comparatively inert chemically. (Further information on saturated fatty acids is available at our web site here..).

 

2.  Monoenoic fatty acids

Straight-chain even-numbered fatty acids with 10 to more than 30 carbon atoms and containing one cis-double bond have been characterised from natural sources. The double bond can be in a variety of different positions, and this is specified in the systematic nomenclature in relation to the carboxyl group. Thus, the most abundant monoenoic fatty acid in tissues is probably cis-9-octadecenoic acid, also termed oleic acid, and it has the structure –

CH3(CH2)7CH=CH(CH2)7COOH

In the shorthand nomenclature, it is designated 18:1. The position of the double bond can be denoted in the form (n-x), where n is the chain length of the fatty acid and x is the number of carbon atoms from the double bond in the terminal region of the molecule, i.e. oleic acid is 18:1(n-9). Although this contradicts the convention that the position of functional groups should be related to the carboxyl carbon, it is of great convenience to lipid biochemists. Animal and plant lipids frequently contain families of monoenoic fatty acids with similar terminal structures, but with different chain lengths, that may arise from a common precursor either by chain elongation or by beta-oxidation (Fig. 2.1(a)). The (n-x) nomenclature helps to point out such relationships. Some obvious examples can be seen in Table 2.2.

Biosynthetic relationships between unsaturated fatty acids

Figure 2.1.  Biosynthetic relationships between unsaturated fatty acids. (a) Elongation and retroconversion of oleic acid; (b) elongation and desaturation of linoleic acid; (c) biosynthesis of prostaglandin E2 from arachidonic acid; (d) elongation and desaturation of α-linolenic acid; (e) elongation and desaturation of oleic acid.

 

Table 2.2. Monoenoic fatty acids of general formula
CH3(CH2)mCH=CH(CH2)nCOOH

Systematic name

Trivial name

Shorthand designation

cis-9-tetradecenoic myristoleic 14:1(n-5)
cis-9-hexadecenoic palmitoleic 16:1(n-7)
trans-3-hexadecenoic - -
cis-6-octadecenoic petroselinic 18:1(n-12)
cis-9-octadecenoic oleic 18:1(n-9)
cis-11-octadecenoic cis-vaccenic 18:1(n-7)
trans-9-octadecenoic elaidic -
trans-11-octadecenoic vaccenic  -
cis-9-eicosenoic gadoleic 20:1(n-11)
cis-11-eicosenoic gondoic 20:1(n-9)
cis-13-docosenoic erucic 22:1(n-9)
cis-15-tetracosenoic nervonic 24:1(n-9)
 

 

Various positional isomers exist in nature and cis-6-octadecenoic acid (petroselinic acid) is found in seed oils of the Umbelliferae, for example, while cis-11-octadecenoic acid is the major unsaturated fatty acid in many bacterial species. Many different isomers may indeed exist in a lipid sample from a single natural source.

Monoenoic fatty acids with double bonds of the trans-configuration are also found on occasion in nature. For example, trans-3-hexadecenoic acid is always present as a substantial component of plant chloroplast lipids. trans-11-Octadecenoic acid (vaccenic) is formed as a by-product of biohydrogenation in the rumen, and thence finds its way into the tissues of ruminant animals, and via meat and dairy products into human tissues. In addition, trans-isomers are formed during industrial hydrogenation of fats and oils, as in margarine manufacture.

cis-Monoenoic fatty acids with 18 carbons or less melt below room temperature (trans-isomers have somewhat higher melting points). Because of the presence of the double bond, they are more susceptible to oxidation than are the saturated fatty acids. (Further information on mono-unsaturated fatty acids is available at our web site here..).

 

3.  Polyunsaturated fatty acids

Polyunsaturated fatty acids (often abbreviated to PUFA) of animal origin can be subdivided into families according to their derivation from specific biosynthetic precursors. In each instance, the families contain from two up to a maximum of six cis-double bonds, separated by single methylene groups (methylene-interrupted unsaturation), and they have the same terminal structure. A list of some of the more important of these acids is contained in Table 2.3.

Table 2.3. Polyunsaturated fatty acids of general formula
CH3(CH2)m(CH=CHCH2)x(CH2)nCOOH
Systematic name Trivial name Shorthand
designation
9,12-octadecadienoic* linoleic 18:2(n-6)
6,9,12-octadecatrienoic γ-linolenic 18:3(n-6)
8,11,14-eicosatrienoic homo- γ -linolenic 20:3(n-6)
5,8,11,14-eicosatetraenoic arachidonic 20:4(n-6)
4,7,10,13,16-eicosapentaenoic - 20:5(n-6)
9,12,15-octadecatrienoic α-linolenic 18:3(n-3)
5,8,11,14,17-eicosapentaenoic EPA 20:5(n-3)
7,10,13,16,19-docosapentaenoic - 22:5(n-3)
4,7,10,13,16,19-docosahexaenoic DHA 22:6(n-3)
5,8,11-eicosatrienoic Mead’s acid 20:3(n-9)
* The double bond configuration in each instance is cis.

 

Linoleic acid (cis-9,cis-12-octadecadienoic acid) is the most widespread fatty acid of this type, and it is found in most animal and plant tissues. It is designated 18:2(n-6), using the same shorthand nomenclature as before (methylene-interrupted cis-double bonds are assumed). It is an essential fatty acid in animal diets, as it cannot be synthesised in animal tissues yet is required for normal growth, reproduction and healthy development. The enzymes in animals are only able to insert new double bonds between an existing double bond and the carboxyl group. Linoleic acid therefore serves as the precursor of a family of fatty acids that is formed by desaturation and chain elongation, in which the terminal (n-6) structure is retained (Fig. 2.1(b)). Of these, arachidonic acid (20:4(n-6)) is particularly important as an essential component of the membrane phospholipids and as a precursor of the prostaglandins (Fig. 2.1(c)). These compounds have profound pharmacological effects and are the subject of intensive study. cis-6,cis-9,cis-12-Octadecatrienoic acid (18:3(n-6)), an important intermediate in the biosynthesis of arachidonic acid and a constituent of certain seed oils, has been the object of considerable research in its own right.

The enzymes in plant tissues are capable of inserting a double bond in the terminal region of an existing unsaturated fatty acid, and linolenic acid (cis-9,cis-12,cis-15-octadecatrienoic acid or 18:3(n-3)) is the end point of biosynthesis in most higher plants. When it is absorbed into animal tissues through the diet, it forms the precursor of a further family of polyunsaturated fatty acids with an (n-3) terminal structure (Fig. 2.1(d)). These fatty acids are also essential dietary components, especially in fish, although the requirement in mammals is probably appreciably less than that for the (n-6) series. Nonetheless, 20:5(n-3) and 22:6(n-3) fatty acids appear to have special functions in the phospholipids of nervous tissue and in the eye, and both are precursors of specific prostanoids.

Many other similar families of fatty acids exist in nature, and that derived from oleic acid (Fig. 2.1(e)) tends to assume greater importance in animals suffering from essential fatty acid deficiency.

Polyunsaturated fatty acids with more than one methylene group between the double bonds, such as cis-5,cis-11- and cis-5,cis-13-eicosadienoic acids occur in marine invertebrates and some other organisms, but are rarely found in animals.

Some plant species synthesise fatty acids with one or more double bonds of the trans-configuration (e.g. trans-9,trans-12-octadecenoic acid), with conjugated double bond systems (e.g. cis-9,trans-11,trans-13-octadecatrienoic or α-eleostearic acid), or with acetylenic bonds (e.g. octadec-cis-9-en-12-ynoic or crepenynic acid). The natural occurrence of such fatty acids has been reviewed [71,856].

In general, polyunsaturated fatty acids have low melting points, and they are susceptible to oxidative deterioration or autoxidation (see Section E.2). (Further information on polyunsaturated fatty acids is available at our web site here..).

 

4.  Branched-chain and cyclopropane fatty acids

Branched-chain fatty acids occur widely in nature, but tend to be present as minor components except in bacteria, where they appear to replace unsaturated fatty acids functionally. Usually, the branch consists of a single methyl group, either on the penultimate (iso) or antepenultimate (anteiso) carbon atoms (Fig. 2.2). In the biosynthesis of these fatty acids, the primer molecules for chain elongation by the fatty acid synthetase are 2-methylpropanoic and 2-methylbutanoic acids, respectively. Methyl branches can be found in other positions of the chain (on even-numbered carbon atoms), if methylmalonyl-coenzyme A rather than malonyl-coenzyme A is used in for chain extension; this can occur in bacteria and in animal tissues, especially those of ruminant animals, where polymethyl-branched fatty acids even can be synthesised [275].

Formulae of some branched-chain and cyclic fatty acids

Figure 2.2.  The structures of some branched-chain and cyclic fatty acids.

 

The commonest polymethyl-branched fatty acid is probably phytanic or 3,7,11,15-tetramethylhexadecanoic acid, which is a metabolite of phytol, and can be found in trace amounts in many animal tissues. It becomes a major component of the plasma lipids in Refsum's syndrome, a rare condition in which there is a deficiency in the enzymatic fatty acid alpha-oxidation system. Lough [562] has reviewed the occurrence and biochemistry of this and other isoprenoid fatty acids. Similar fatty acids are present in the lipids of the preen gland of birds and in those of tubercle bacilli.

The Mycobacteria and certain related species contain a highly distinctive range of very-long-chain α-branched β-hydroxy fatty acids, known as the mycolic acids, i.e. of the form -

RCH(OH)CH(R')COOH

Different species synthesise mycolic acids with quite characteristic structures and Mycobacteria, for example, produce C60 to C90 acids with C20 to C24 α-branches; the Nocardiae synthesise C38 to C60 fatty acids with C10 to C16 branches. They may also contain additional carbonyl groups, methyl branches, cyclopropane rings and isolated double bonds [367]. (Further information on branched-chain fatty acids is available at our web site here..).

Fatty acids with a cyclopropane ring in the aliphatic chain, such as lactobacillic or 11,12-methylene-octadecanoic acid, are found in the lipids of several gram-negative and a few gram-positive bacterial families of the order Eubacteriales.

 

5.  Oxygenated and cyclic fatty acids

In animal tissues, 2-hydroxy fatty acids are frequent components of the sphingolipids and they are also present in skin and wool wax. 4- and 5-Hydroxy fatty acids, which form lactones on hydrolysis, and keto acids are found in cow's milk. As part of the "arachidonic acid cascade", a large number of hydroperoxy, hydroxy and epoxy fatty acids (eicosanoids) are formed enzymatically as intermediates in the biosynthesis of prostanoids [713], e.g.

Arachidonic acid metabolism

This is a particularly active area of research at present, and novel structures and new pharmacological activities continue to be revealed. (Further information on eicosanoids fatty acids is available at our web site here..).

A large number of hydroxy fatty acids occur in seed oils [71,856], and the best known of these is probably ricinoleic or 12-hydroxy-cis-9-octadecenoic acid, which is the principal constituent of castor oil. Polyhydroxy fatty acids are present in plant cutins, while aleuritic or 9,10,16-trihydroxyhexadecanoic acid is one of the main components of shellac. Vernolic or 12,13-epoxy-cis-9-octadecenoic acid is one of a number of epoxy fatty acids to have been detected in seed oils.

Fatty acids containing a furanoid ring have been found in the reproductive tissues of fish, especially during starvation, in the simple lipid components, but their function is not known. Their primary origin is in plants and algae, and they are known to be components of at least one seed oil and of rubber latex. (Further information on oxygenated fatty acids is available at our web site here..).

Cyclopropane fatty acids were mentioned in the previous section. They sometime accompany cyclopropene fatty acids in seed oils of the Malvaceae and Bombacaceae among others. For example, sterculic acid is present in very small amounts in cotton seed oil, and if it is not removed during refining it can have a pharmacological effect on the consumer by inhibiting the desaturase enzyme systems. Fatty acids containing a cyclopentene ring are found in seed oils of the Flacourtiaceae, which are used in the treatment of leprosy, although there is no evidence that the acids themselves any have therapeutic value. A fatty acid with a cyclohexane ring has been found in rumen bacteria, and also occurs in the tissues of ruminants. (Further information on cyclic fatty acids is available at our website here..).

 

C.  Simple Lipids

1.  Triacylglycerols and related compounds

Triacylglycerols (commonly termed "triglycerides") consist of a glycerol moiety, each hydroxyl group of which is esterified to a fatty acid. In nature, these compounds are synthesised by enzyme systems, which determine that a centre of asymmetry is created about carbon-2 of the glycerol backbone, and they exist in different enantiomeric forms, i.e. with different fatty acids in each position. A "stereospecific numbering" system has been recommended to describe these forms [415,416]. In a Fischer projection of a natural L-glycerol derivative (Fig. 2.3), the secondary hydroxyl group is shown to the left of C-2; the carbon atom above this then becomes C-1 and that below becomes C-3. The prefix "sn" is placed before the stem name of the compound. If the prefix is omitted, then either the stereochemistry is unknown or the compound is racemic. Smith [857] has reviewed glyceride chirality.

The structures of some of the more important lipid classes

Figure 2.3. The structures of some of the more important lipid classes.

 

Nearly all the commercially important fats and oils of animal and plant origin consist almost exclusively of this simple lipid class. The fatty acid composition can vary enormously. In seed oils, the C18 unsaturated fatty acids tend to predominate. In animal fats, especially those of adipose tissue origin, the fatty acid composition reflects that of the diet to some extent, but C16 and C18 fatty acids are the most abundant components. Fish triacylglycerols and those of marine mammals differ from others in that they contain a high proportion of C20 and C22 polyunsaturated fatty acids. The compositions of natural oils and fats have been reviewed recently [686].

A full analysis of a triacylglycerol requires that not only the total fatty acid composition be determined but also the distribution of fatty acids in each position. In addition, the proportions of the individual molecular species must be known and gas chromatography, among other techniques, has been used in analyses of this kind.

Some seed and fungal lipids have been found with triacylglycerol components that contain hydroxy fatty acids, the hydroxyl group of which is esterified to an additional fatty acid. These lipids are known as estolides.

Diacylglycerols (less accurately termed "diglycerides") and monoacylglycerols ("monoglycerides") contain two moles and one mole of fatty acids per mole of glycerol, respectively, and are rarely present at greater than trace levels in fresh animal and plant tissues. Collectively, they are sometimes known as "partial glycerides". 1,2-Diacyl-sn-glycerols are important as intermediates in the biosynthesis of triacylglycerols and other lipids. In addition, it has become evident that they are important intracellular messengers, generated on hydrolysis of phosphatidylinositol and related compounds by specific enzymes of the phospholipase C type, and that they are involved in the regulation of vital processes in mammalian cells [388]. 2-Monoacyl-sn-glycerols are formed as intermediates or end products of the enzymatic hydrolysis of triacylglycerols.

Acyl migration occurs rapidly with such compounds, especially on heating, in alcoholic solvents or when protonated reagents are present, so special procedures are required for their isolation or analysis if the stereochemistry is to be retained. (Further information on triacylglycerols is available at our web site here..).

 

2.  Alkyldiacylglycerols and neutral plasmalogens

Many glycerolipids, including simple lipids, phospholipids and glycolipids, and especially those of animal or microbial origin, contain aliphatic residues linked by an ether or a vinyl ether bond to glycerol. Their occurrence, chemistry and biochemistry have been comprehensively reviewed [586]. Alkyldiacylglycerols are lipids in which a long-chain alkyl moiety is joined via an ether linkage to position 1 of L-glycerol, positions 2 and 3 being esterified with conventional fatty acids. The alkyl groups tend to be saturated or monoenoic of chain-length 16, 18 or 20. On hydrolysis, fatty acids (2 moles) and a glycerol ether (1 mole) are the products. The trivial names chimyl, batyl and selachyl alcohol are used for 1-hexadecyl-, 1-octadecyl- and 1-octadec-9-enylglycerol respectively. Alkyl ethers are found in small amounts only in most animal tissues, but they can be the major lipid class in the lipids of some marine animals.

The ether linkage in glycerol ethers is stable to both acidic and basic hydrolysis, although the ester bonds are readily hydrolysed as in all glycerolipids.

Neutral plasmalogens are related compounds in which position 1 of L-glycerol is linked by a vinyl ether bond (the double bond is of the cis-configuration) to an alkyl moiety. They have been detected in small amounts only in a few animal tissues. Although the vinyl ether linkage is stable to basic hydrolysis conditions, it is disrupted by acid (and by mercury salts) with the formation of a long-chain aldehyde, i.e.

Hydrolysis of plasmalogens

The principal aldehydes usually are saturated or monoenoic compounds, 16 or 18 carbon atoms in chain length. Their chemical and physical properties have been reviewed [576,578]. (Further information on ether lipids is available at our web site here..).

 

3.  Cholesterol and cholesterol esters

Cholesterol is by far the most common member of a group of steroids with a tetracyclic ring system; it has a double bond in one of the rings and one free hydroxyl group (Fig. 2.3). It is found both in the free state, where it has a vital role in maintaining membrane fluidity, and in esterified form, i.e. as cholesterol esters. The latter are hydrolysed or transesterified much more slowly than most other O-acyl lipids. (The correct generic term is indeed cholesterol rather than cholesteryl esters, but the individual components are designated cholesteryl palmitate, etc.).

Plant tissues contain related sterols, such as β-sitosterol, ergosterol and stigmasterol, but trace amounts only of cholesterol, and these may also be present in esterified form. Steroid hormones and bile acids are structurally related compounds, which differ in function from the lipids as defined above, and they are not considered further in this book. (Further information on sterols is available at our website here..).

 

4.  Wax esters and other simple lipids

Wax esters in their most abundant form consist of fatty acids esterified to long-chain alcohols with similar aliphatic chains to the acids. They are found in animal, plant and microbial tissues and have a variety of functions, such as acting as energy stores, waterproofing and even echolocation. The fatty acids may be straight-chain saturated or monoenoic with up to 30 carbons, but branched-chain and α- and ω-hydroxy acids are present on occasion; similar features are found in the alcohol moieties.

Waxes in general can contain a wide range of different compounds, including aliphatic diols, free alcohols, hydrocarbons (especially squalene), aldehydes, ketones, hydroxy-ketones, β-diketones and sesquiterpenes. The composition and biochemistry of waxes in nature, and methods for their analysis, have been reviewed in a comprehensive monograph [491]. (Further information on waxes is available at our website here..).

 

D.  Complex Lipids

1.  Glycerophospholipids

The structures of a typical glycerophospholipid, i.e. phosphatidylcholine, is shown in Figure 2.3. 1,2-Diacyl-sn-glycerol-3-phosphorylcholine (commonly termed "lecithin") is usually the most abundant lipid in the membranes of animal tissues, and is often a major lipid component of plant membranes, and sometimes of microorganisms. Together with the other choline-containing phospholipid, sphingomyelin, it comprises much of the lipid in the external monolayer of the plasma membrane of animal cells. It shares with other glycerophospholipids a 1,2-diacyl-sn-glycerol backbone, and this part of the molecule can be generated by hydrolysis with phospholipase C and converted to a nonpolar derivative for analysis by GLC or other techniques. (Only in the Archaebacteria do the complex lipids have the opposite 2,3-dialkyl-sn-glycerol structure [216]). The polar head group of all phospholipids prevents direct analysis by means of GLC. While this has disadvantages for certain biochemical applications, analysis via the diacylglycerol derivatives does have the merit that all phospholipids are treated in the same way, regardless of the structure of the parent compound.

In the phospholipids of animals and microorganisms, analogues containing vinyl ether and ether bonds are much more abundant than in the simple lipids. In this instance, it has been suggested that they should be termed "plasmenylcholine" and "plasmanylcholine", respectively. Phospholipid classes isolated by chromatographic means tend to be a mixture of the diacyl, alkylacyl and alkenylacyl forms. To indicate that this is so, they are sometimes termed the "diradyl" form of the appropriate phospholipid. One ether-containing phospholipid, in particular, which is presently being studied intensively because it can exert profound biological effects at minute concentrations, is 1-alkyl-2-acetyl-sn-glycero-phosphorylcholine or "platelet-activating factor" (often abbreviated to PAF). The chemistry and biochemistry of this compound have been reviewed recently [353,866].

Lysophosphatidylcholine, which contains only one fatty acid moiety in each molecule, generally in position sn-1, is sometimes present in tissues also but as a minor component; it is more soluble in water than most other lipids and can be lost during extraction, unless precautions are taken.

Phosphatidic acid or 1,2-diacyl-sn-glycerol-3-phosphate is found naturally in trace amounts only in tissues, but it is important metabolically as a precursor of most other glycerolipids. It is strongly acidic and is usually isolated as a mixed salt. As it is somewhat water soluble, it may be necessary to take special precautions during the extraction of tissues to ensure quantitative recovery.

Phosphatidylglycerol or 1,2-diacyl-sn-glycerol-3-phosphoryl-11-sn-glycerol tends to be a trace constituent of tissues, although it does appear to have important functions in lung surfactant and in plant chloroplasts. Diphosphatidylglycerol (or cardiolipin) is related structurally to phosphatidylglycerol, and is an abundant constituent of mitochondrial lipids, especially in heart muscle; its occurrence and properties have been reviewed [410]. These lipids also are acidic.

Phosphatidylethanolamine (once trivially termed "cephalin") is frequently the second most abundant phospholipid class in animal and plant tissues, and can be the major lipid class in microorganisms. It often contains a relatively high proportion of the aliphatic moieties as the ether forms. The amine group can be methylated enzymically, as part of a vital cellular process, to yield as intermediates first phosphatidyl-N-monomethylethanolamine and then phosphatidyl-N,N-dimethylethanolamine; the eventual product is phosphatidylcholine [943]. N-Acyl-phosphatidylethanolamine is a minor component of some plant tissues, and is also found in animal tissues under certain conditions. Lysophosphatidylethanolamine contains only one mole of fatty acid per mole of lipid.

Phosphatidylserine is a weakly acidic lipid, so is generally isolated from tissues in salt form. It is present in most tissues of animals and plants and is also found in microorganisms. Its biochemistry has been reviewed [81]. N-Acylphosphatidylserine has been detected in certain animal tissues.

Phosphatidylinositol, containing the optically inactive form of inositol - myoinositol, is a common constituent of animal, plant and microbial lipids. Often in animal tissues, it is accompanied by small amounts of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate (polyphosphoinositides). These compounds have a rapid rate of metabolism in animal cells, and with their diacylglycerol metabolites have a major role in regulating vital processes. The topic has been reviewed [388].

Each phospholipid class in a tissue has a distinctive fatty acid composition, probably related to its function but in a way that is still only partly understood. In a tissue such as liver, for example, most of the glycerophospholipids contain substantial proportions of the longer-chain polyunsaturated components. Typically, phosphatidylcholine contains 50% of saturated fatty acids, while arachidonic acid constitutes 20% of the total. Phosphatidylethanolamine has a similar proportion of saturated fatty acids, but somewhat less linoleic acid and correspondingly more of the C20 and C22 polyunsaturated fatty acids. Characteristically, high proportions of stearic and arachidonic acids are present in the phosphatidylinositol, while the composition of the phosphatidylserine is similar except that the 22:6(n-3) fatty acid substitutes for part of the arachidonic acid. Cardiolipin or diphosphatidylglycerol differs markedly from all the other glycerophospholipids in that the single fatty acid, linoleic acid, can comprise nearly 60% of the total. Similar general compositional trends are seen, although the absolute values may differ, in comparing the same lipids in other tissues and, with dietary influences superimposed, in comparing the corresponding lipids of other species.

As with the triacylglycerols, the fatty acids have distinctive positional distributions in phospholipids, with saturated components generally being concentrated in position sn-1 and unsaturated in position sn-2; they also exist in specific characteristic combinations in molecular species. Links to more information on each phospholipid class can be obtained in the section of the web site "A Lipid Primer".

 

2.  Glyceroglycolipids

Plant tissues especially tend to contain appreciable amounts of lipids in which 1,2-diacyl-sn-glycerols are joined by a glycosidic linkage at position sn-3 to a carbohydrate moiety. Their structures and compositions have been reviewed [366,367]. The main components are the mono- and digalactosyldiacylglycerols, but related lipids have been found containing up to four galactose units, or in which one or more of these is replaced by glucose moieties. In addition, a 6-O-acyl-monogalactosyldiacylglycerol is occasionally a component of plant tissues. A further unique plant glycolipid is sulfoquinovosyldiacylglycerol or the "plant sulfolipid", and contains a sulfonic acid residue linked by a carbon-sulfur bond to the carbohydrate moiety of a monoglycosyldiacylglycerol; it is found exclusively in the chloroplasts. Usually these lipids contain a high proportion of an 18:3(n-3) fatty acid, sometimes accompanied by 16:3(n-3).

Monogalactosyldiacylglycerols are known to be present in small amounts in brain and nervous tissue in some animal species, and a range of complex glyceroglycolipids have been isolated and characterized from intestinal tract and lung tissue. Such compounds would be destroyed by certain of the methods used in the isolation of glycosphingolipids, with which they frequently co-chromatograph, and may be more widespread than is generally thought. A complex sulfolipid, termed "seminolipid", of which the main component is 1-O-hexadecyl-2-O-hexadecanoyl-3-O-(3'-sulfo-β-D-galactopyranosyl-sn-glycerol, is the principal glycolipid in testis and sperm. The glycoglycerolipids of animal origin have also been reviewed [850].

Glycolipids, unlike phospholipids, are soluble in acetone and this property can be used in isolating them by chromatographic means. Links to more information on each glyceroglycolipid class can be obtained in the section of the web site "A Lipid Primer".

 

3.  Sphingolipids

Long-chain bases (sphingoids or sphingoid bases) are the characteristic structural unit of the sphingolipids, the chemistry and biochemistry of which have been thoroughly reviewed [460,974,981]. The bases are long-chain (12 to 22 carbon atoms) aliphatic amines, containing two or three hydroxyl groups, and often a distinctive trans-double bond in position 4 (see Fig. 2.3). The commonest or most abundant is sphingosine ((2S,3R,4E)-2-amino-4-octadecen-1,3-diol). More than 80 long-chain bases have been found in animals, plants and microorganisms, and many of these may occur in a single tissue, but always as part of a complex lipid as opposed to in the free form. The aliphatic chains can be saturated, monounsaturated and diunsaturated, with double bonds of either the cis or trans configuration, and they can also have methyl substituents. In addition, saturated and monoenoic straight- and branched-chain trihydroxy bases are found. The commonest long-chain base of plant origin, for example, is phytosphingosine ((2S,3S,4R)-2-amino-octadecanetriol). For shorthand purposes, a nomenclature similar to that for fatty acids can be used; the chain length and number of double bonds are denoted in the same manner with the prefixes "d" and "t" to designate di- and tri-hydroxy bases respectively. Thus, sphingosine is d18:1 and phytosphingosine is t18:0.

Ceramides contain fatty acids linked to the amine group of a long-chain base by an amide bond. Generally, they are present at low levels only in tissues, but are important as intermediates in the biosynthesis of the complex sphingolipids. The acyl groups of ceramides are long-chain (up to C26, but occasionally longer) odd- and even-numbered saturated or monoenoic fatty acids and related 2-D-hydroxy fatty acids. Polyunsaturated fatty acids are rarely present at greater than trace levels. Ceramides are the basic aliphatic building blocks of the sphingolipids, and they can sometimes be generated from sphingolipids by analogous methods to those used for the diacylglycerol moiety of phospholipids. It is this part of the molecule, together with the long-chain base and fatty acid constituents, which are most relevant to a text on gas chromatography.

Sphingomyelin consists of a ceramide unit linked at position 1 to phosphorylcholine, and it is found as a major component of the complex lipids of all animal tissues, but is not present in plants or microorganisms. It resembles phosphatidylcholine in many of its physical properties, and can apparently substitute in part for this in membranes. Sphingosine is usually the most abundant long-chain base constituent, together with sphinganine and C20 homologues.

The most widespread glycosphingolipids are the monoglycosylceramides (or cerebrosides), and they consist of the basic ceramide unit linked at position 1 by a glycosidic bond to glucose or galactose (Fig. 2.3). They were first found in brain lipids, where the principal form is a monogalactosylceramide, but they are now known to be ubiquitous constituents of animal tissues. In addition, they are found in plants (monoglucosylceramides only), where the main long-chain base is phytosphingosine.

Di-, tri- and tetraglycosylceramides (oligoglycosylceramides) are usually present also in animal tissues. The most common diglycosylceramide is lactosylceramide, and it can be accompanied by related compounds containing further galactose or galactosamine residues, for example. Tri- and tetraglycosylceramides with a terminal galactosamine moiety are sometimes termed "globosides", while glycolipids containing fucose are known as "fucolipids". Oligoglycosylceramides with more than 20 carbohydrate residues have been isolated from animal tissues, those from intestinal cells having been studied with particular intensity. They appear to form part of the immune response system. Although certain of these lipids have been found on occasion to have distinctive long-chain base and fatty acid compositions, the complex glycosyl moiety is considered to be of primary importance for their immunological function and therefore has received most attention from investigators.

Sulfate esters of galactosylceramide and lactosylceramide (often referred to as "sulfatides" or "lipid sulfates"), with the sulfate group linked to position 3 of the galactosyl moiety, are major components of brain lipids and are also found in trace amounts in other tissues; their chemistry and biochemistry have been reviewed [252].

Complex plant sphingolipids, the phytoglycosphingolipids, which contain glucosamine, glucuronic acid and mannose linked to the ceramide via phosphorylinositol, were isolated and characterised from seeds initially, but related compounds are also known to be present in other plant tissues and in fungi.

Gangliosides are highly complex oligoglycosylceramides, which contain one or more sialic acid groups (N-acyl, especially acetyl, derivatives of neuraminic acid, abbreviated to "NANA"), in addition to glucose, galactose and galactosamine. They were first found in the ganglion cells of the central nervous system, hence the name, but are now known to be present in most animal tissues. The nature of the long-chain base and fatty acid components of each ganglioside can vary markedly between tissues and species and is related in some way to its function. Links to more information on each sphingolipid class can be obtained in the section of the web site "A Lipid Primer".

 

Abbreviations

The following abbreviations are employed at various points in the text of these chapters:

amu, atomic mass units; BDMS, tert-butyldimethylsilyl; BHT, 2,6-di-tert-butyl-p-cresol; CI, chemical ionisation; DNP, dinitrophenyl; ECL, equivalent chain length; ECN, equivalent carbon number; EI, electron-impact ionisation; FCL, fractional chain length; GC, gas chromatography; GLC, gas-liquid chromatography; HPLC, high-performance liquid chromatography; IR, infrared; MS, mass spectrometry; NMR, nuclear magnetic resonance; PAF, platelet-activating factor; ODS, octadecylsilyl; TLC, thin-layer chromatography; TMS, trimethylsilyl; UV, ultraviolet.

 

This document is part of the book Gas Chromatography and Lipids by William W. Christie and published in 1989 by P.J. Barnes & Associates (The Oily Press Ltd), who retain the copyright.

Go to Part 2 of this Chapter

 

Updated June 27, 2011