Search

Lipid science is now an important and respected field of endeavour involving many individual sciences though this was not always true for organic chemists. The reasons for this change are discussed. Some current topics of interest are described including dietary recommendations, transgenic genetic modification of oilseeds, developments in analytical methods, and conjugated linoleic acid. Finally some significant features of the production and consumption of these materials are outlined.

•In considering oilseed crops with modified fatty acid composition three question have to be addressed: why is it nesessary to have oils and fats with composition different from that supplied by nature? how can such changes be accomplished? and what modified material is already available or likely to be so in the near future?•Our increased understanding of the nutritional necessary to provide healthy food and of the physical properties required for good quality spreads or frying oils allow us to write specifications for ideal oils in terms of their fatty acid composition. However, this may not be sufficient. We may need to define triacylglyderol composition and, at the same time, to manage the minor components which also have a marked influence on shelf life and on dietary properties.•Our desire to modify fatty acid composition is at least one century old and over that period of time a range of methods-some technological and some biological-have been developed. This lecture is confined to the consideration of producing modified seed oils by conventional seed breeding or by transgenic modification.•Efforts have been directed particularly to changing the levels of saturated acids, oleic acid, and linolenic acid-sometimes raising these and somtimes lowering them. Thses changes have been applied to the major oilseeds-soybean, rapeseed/sanola, and sunflower - and also to linseed. However, because of inadequqte control of triacylglycerol composition, and the minor components, the new oils have not always displayed the desired properties.

The fatty acid compositions of the seeds from four Cephalotaxus species or varieties (plum yews; Cephalotaxaceae) and two Podocarpus species (podocarps; Podocarpaceae) have been established. These compositions were compared with those previously published for some Taxaceae species (Taxus and Torreya). Cephalotaxaceae, Podocarpaceae, and Taxaceae belong to the Taxares suborder. Δ5-Olefinic acids are present in the seed lipids from all species analyzed. In Cephalotaxus, Podocarpus, and Torreya, the prominent Δ5-olefinic acid that occurs is the trienoic acid 5,11,14–20:3 (sciadonic) acid, comprising from 6.7 to 26.4% of total fatty acids. In these species, the Δ5,11 structure is largely favored over the Δ5,9 structure: the 5,9–18:2 (taxoleic) and 5,9,12–18:3 (pinolenic) acids are at the limit of detection, in contrast to Taxus and most Pinaceae species, where these two Δ5-olefinic acids generally predominate. 14-Methylhexadecanoic acid, an habitual though minor component of Pinaceae and Ginkgo biloba seed lipids, could not be detected in Cephalotaxus species studied here and was tentatively identified in trace amounts only in one Podocarpus species. In addition to sciadonic acid, Cephalotaxus and Podocarpus seeds are characterized by unusually high amounts of 11,14–20:2 acid, in the range of 3.1–12.0%. This contrasts with most of the 170 species of conifers analyzed so far (from the families Pinaceae, Cupressaceae, Taxodiaceae, Taxaceae, and Sciadopityaceae, which belong to the Pinares suborder), where this acid is generally ≤2%. A close resemblance between Torreya grandis and three of the Cephalotaxus species analyzed might be indicative of some phyletic relationship between the families Cephalotaxaceae and Taxaceae. 13C nuclear magnetic resonance spectroscopy of the seed oils from C. drupaceae and P. andinus has shown that Δ5-olefinic acids are apparently excluded from the internal position of triacylglycerols, which is a characteristic common to all Coniferales species analyzed so far, and consequently of great antiquity.

The fatty acid compositions of the seeds from three Taxus (yew) species and one Torreya species belonging to the Taxaceae family [Taxus cuspidata (Japanese yew), T. chinensis (Chinese yew), T. baccata (English yew), and Torreya grandis (Chinese nutmeg yew)] have been established. These compositions were compared with those previously published for T. canadensis (Canadian yew) and Torreya nucifera. In Taxus species, as well as in Torreya species, Δ5-olefinic acids are present in the seed lipids from all species analyzed. In Taxus, 5,9-18:2 (taxoleic) acid is the prominent Δ5-olefinic acid. It represents between 9.5 and 16.2% of total fatty acids. Other Δ5-olefinic acids that occur in low amounts are 5,9,12-18:3 (

The conjugated linoleic acid methyl cis-9,trans-11-octadecadienoate has been prepared on a large scale from methyl ricinoleate. Methyl ricinoleate was purified from castor esters by a partition method. It was converted to the mesylate, which was reacted with a base (1,8-diazabicyclo[5,4,0]-undec-7-ene) to give a product that contained 66% of the desired ester. Two urea crystallizations produced a product containing 83% methyl cis-9,trans-11-octadecadienoate, the identity of which was confirmed by gas chromatography linked to mass spectrometry and by Fourier transform infrared spectroscopy. The remaining impurities were methyl cis-9,cis-11- and cis-9-,trans-12-octadecadienoate.

The13C nuclear magnetic resonance spectra ofVernonia galamensis seed oil and of epoxidized palm super olein, soybean oil and linseed oil have been recorded and interpreted. The chemical shifts of the major signals are assigned and semi-quantitative results are derived. The spectroscopic procedure provides a useful method of analyzing oils that contain epoxy acids. The epoxide function differs from a double bond in its influence on the chemical shifts of nearby carbon atoms.

The high-resolution13C nuclear magnetic resonance spectra of twelve hydrogenated fats have been examined. Each spectrum contains 50–100 signals and reveals much about the nature of the acyl chains of both double-bond position and configuration. The signals for the ω1, ω2 and ω3 carbon atoms give information on thecis andtrans isomers of the Δ15, Δ14, Δ13 and Δ12 18:1 esters, respectively. Allylic signals distinguish betweencis andtrans esters, and the proportion of totalcis to totaltrans isomers can be obtained from these. Olefinic signals are the most informative, and most of these have been assigned. This leads to a semi-quantitative estimate of the various 18:1 isomers present. Assignments are based mainly on information already in the literature, but some were confirmed after urea fractionation of the acids from a hydrogenated oil in whichcis andtrans monoene acids were separately concentrated.

Based on the interpretation of the high-resolution13C nuclear magnetic resonance spectra of 15 spreading fats, baking fats, vegetable creams and an infant formula, it is possible to decide whether the sample contains butterfat, lauric oils, partially hydrogenated fat, linoleic acid or linolenic acid. The data have been interpreted in a semiquantitative manner, and this provides an insight into the nature of the material present in each sample.

Nine seed oils from three different conifer families, already examined by gas chromatography and known to contain diene, triene, and tetraene C18 and C20Δ5 acids, have been reexamined by high-resolution13C nuclear magnetic resonance spectroscopy. The Δ5 acids are apparent only in the α-chains. This location is independent of chainlength, double-bond number, and the species considered and is probably a general factor of conifer seed oils. The spectra confirm the presence of oleic, linoleic, α-linolenic, and of Δ5 acids and give quantitative information about (total) n-6, n-3, and Δ5 acids that is in accord with that obtained by gas chromatography.

When acting as an antioxidant, ethoxyquin is converted mainly to a 1,8′-dimer and to a quinone-imine, with the latter itself acting as a powerful antioxidant. The 8-methyl derivative of ethoxyquin was prepared in the expectation that this would not form a dimer but more quinone-imine and, therefore, be a more powerful antioxidant. On oxidation of 8-methylethoxyquin, the dimer was absent and the quinone-imine was formed in high yield. Despite this, 8-methylethoxyquin was a less efficient antioxidant than ethoxyquin in fish oil and meal.

The polymorphism of 25 glycerol trioctadecenoates with double bonds ranging from Δ4-Δ17 was investigated by differential scanning calorimetry. Triglycerides withcis bonds in odd positions Δ7-Δ13 exhibited three intermediate melting (β′-) forms, but those withcis bonds in even positions, exceptcis Δ4, lacked β′-forms. Among thetrans compounds, only Δ11, 13, and 14 showed β′-forms. Thecis andtrans Δ5 triglycerides were unusual, because they readily assumed low melting (α-) forms that were not easily converted to high melting (β-) forms. β-Form mp of compounds in each series (cis ortrans) alternated depending upon double bond position; an even position correlated with high mp. Heats of fusion (ΔHf) for β-forms, likewise, fluctuated with double bond position but nonuniformly;trans Δ6 had the highest ΔHf (43 cal/g),cis Δ12 the lowest (21 cal/g).

The ability of a series of 18 carbon acetylenic fatty acids to fulfill the unsaturated fatty acid requirements of Escherichia coli and Saccharomyces cerevisiae was investigated. Despite their high melting points (>40°C), several isomers of the acetylenic fatty acids were as efficient or more efficient in supporting growth than the analogous fatty acid having a cis -double bond. The efficiencies of the different positional isomers in supporting cell proliferation varied from essentially 0 cells per fmol for the 2–5 and 13–17 isomers to high values when the acetylenic bond was near the center of the chain: e.g. 45 E. coli and 5.5 S. cerevisiae cells/fmol for the 10 isomer. A striking ineffectiveness of the 9 isomer was observed with E. coli . The 7, 8 and 10 isomers were at least 10-fold more efficient than any of the other positional isomers in supporting the growth of E. coli . In contrast, the 9 isomer was among the most effective acetylenic fatty acids tested with the yeast mutant. Chromatographic analysis of the extracted lipids indicated that each of the acetylenic isomers tested (except Δ2 and Δ3) could be esterified by the prokaryotic and eukaryotic microorganisms. The content of unsaturated plus cyclopropane acids observed when growth ceased in E. coli cultures supplemented with growth-limiting concentrations of the acetylenic fatty acids ranged from approx. 15 mol% for the 8 isomer to approx. 35 mol% for the 14 and 17 isomers. The 8–11 isomers were observed to be esterified predominantly at the two position in phosphatidylethanolamine of E. coli and in phosphatidylcholine of S. cerevisiae .

A series of positional isomers of trans-octadecenoic acid were tested for their ability to support growth of microbial mutants that could not synthesize unsaturated fatty acids. The bacterial strain used (a variant of Escherichia coli 30E) grew with supplements of the trans isomers only at high temperatures (38 degrees) and with acids containing the trans-ethylenic bond between carbon atoms 8 through 13. The yeast mutant (Saccharomyces cerevisiae KD46) grew only with the 9-trans-octadecenoate giving cell yields about one-fifth those obtained with oleate. Although the trans isomers had little effect on the growth of the bacteria in the presence of oleate, they inhibited the growth of yeast with oleate. Inhibition was strongest for the 4, 6, 7, 11, and 12 isomers, almost negligible for the 8 isomer and of differing intermediate degrees for the others. The inhibitory effects had no correlation with the melting points of the acids and appeared to reflect selective action(s) on the metabolism of the cell. When the net yield of the yeast cultures with oleate was lowered by the effect of added trans acids, there was a marked accumulation of triglycerides and nonesterified acids in the cells. The marked increase in triglyceride content while phospholipid per cell remained relatively constant suggest that trans acids, in addition to forming inadequate membrane lipids, may also interfere with a basic control point in lipid metabolism.

Positional isomers ofcis-methyleneoctadecanoic acid differed greatly in their efficiency for growth of an unsaturated fatty acid auxotroph ofEscherichia coli upon glucose as a carbon source. The 8, 9, and 11 isomers were more efficient in producing cells (60–70 cells/fmole) than the others (0–7 cells/fmole), although all isomers were found esterified to a similar extent into cellular lipid. WithSaccharomyces cerevisiae mutants, all isomers between 6 and 12 supported some growth of the eukaryotic cells, and the 7 and 9 isomers were slightly more efficient than the 8-isomer. WhenE. coli were grown with glycerol, all isomers from 5 to 14 supported growth, and those with the substituent near the center of the acyl chain had the greatest efficiency (70 cells/fmole). With the glycerol medium, the pattern of efficiencies for the variouscis-methylene acyl chains resembled the broad selectivity reported earlier for thecis-ethylenic isomers in glucose medium, which agreed closely with predictions based upon the physical property of their phospholipid derivatives. Thus, metabolism of glycerol appeared to allow the cyclopropane acyl chains to support cell functions to the limits expected for bulk phase chain-chain fluidity considerations. This broad specificity was also obtained when cells were grown on glucose with cyclic AMP added to the culture. Therefore, the selective inadequacies of the 5, 6, 7, 10, 12 and 13 isomers in supporting cell growth on glucose may occur through an interaction modified by cAMP and dependent upon reduced cellular levels of cyclic AMP. The highly selective pattern of efficiency of thecis-methylene acids forE. coli growth on glucose resembles that with the acetylenic acids, but was shifted one carbon atom toward the methyl terminus. This observed selectivity pattern seems due to interactions of the individual acyl chains with cellular protein(s) rather than to chain-chain interactions in a bulk phase. The ability of certain positional isomers to support cell function equally well in both nutrient conditions suggests that the role of those acyl chain isomers may be independent of metabolite flux or cyclic nucleotide contents of the cell, whereas the actions of other isomeric fatty acids seem closely related to the metabolic status of the cell. A highly selective role for different fatty acids in modulating cellular function seems possible on the basis of the current evidence.

The distribution of oleic, linoleic, linolenic, petroselinic, hexadec-9 and 11-enoic, sterculic, four conjugated octadecatrienoic acids, isolinolenic, and octadeca-6,9,12,15-tetraenoic acid in vegetable triglycerides has been studied by hydrolysis with pancreatic lipase. The results, discussed in terms of a selectivity factor, indicate that these unsaturated acids do not compete equally for the secondary hydroxyl group.

The component glycerides of linseed, wild rose, candlenut, soya and stillingia oils have been estimated by chromatography on thin layers of silica impregnated with silver nitrate. The separated glycerides are identified, qualitatively and quantitatively, by gas-liquid chromatography (GLC) of their methyl esters in presence of added methyl heptadecanoate as an internal standard. The results agree with those obtained by lipolysis or calculated directly from the component acids on the basis of the theory of positional distribution.

Coenzyme A thiol esters of the complete series of positional isomers of cis-methylene octadecanoic acid were synthesized. Their activities as substrates for acyl-CoA: phospholipid acyltransferases of rat liver microsomes were determined with positional isomers of acylglycerophosphoryl choline and acylglycerophosphoryl ethanolamine as acceptors. The results showed that the acyltransferases discriminated between the acyl-CoA isomers in quite different ways. Certain acyl-CoA esters were transferred at much different rates with different acylglycerophospholipid acceptors, suggesting that different enzymes were acting with the different acceptors. Comparison of the rates of cis-cyclopropane acid derivatives and cis-octadecenoic acid derivatives showed that the presence of π bonds in the acyl residue is not an important factor for the enzyme or enzymes transferring cis-acyl groups to the 1 position of acylglycerophospholipids. On the other hand, the presence of a π bond can cause higher or lower transfer rates to the 2-hydroxyl depending upon the location of the cis-functional group along the acyl chain.

The hydrogenation of a range of double-bond positional and configurational octadecenoic acid isomers (cis (delta 2 and delta 4 to delta 13) and trans (delta 2 and delta 5 to delta 13] to stearic acid by a rumen Fusocillus sp. were examined. The cis and trans delta 5 to delta 13 isomers were all hydrogenated to some extent by late-log-phase cultures added to suspensions of individual isomers and incubated for a further 3 h. Of the cis-isomers, delta 5 to delta 11 (79-73% conversion to stearic acid) were the preferred substrates. delta 12-cis- (30%) and delta 13-cis-isomers (5%) were poorly hydrogenated. Of the trans-isomers, delta 8, delta 9 and delta 10 were 45% converted to stearic acid, the other isomers were poorly hydrogenated. These results are in agreement with less extensive studies using sheep rumen micro-organisms. When cultures were grown from small inocula in media containing individual isomers more extensive hydrogenation was found than with late-log-phase cultures. At 24 h, cis delta 2, delta 4 and delta 5 gave the highest conversions to stearic acid (90%) followed by the cis delta 6 to delta 12 and trans delta 8 to delta 10 isomers (approximately 75%), although at 6 and 12 h delta 9-trans gave higher yields of stearic acid than delta 9-cis, probably because the growth of the cis cultures showed a longer log-phase.

An unsaturated fatty acid auxotroph of Escherichia coli was grown with a series of cis-octadecenoate isomers in which the location of the double bond varied from positions 3 to 17. Each of these fatty acid isomers was incorporated into the cellular lipids, but cyclopropane derivatives were formed to at least a 3-fold greater extent from the cis-9 and cis-11 isomers than from any other positional isomers. The extent of cyclopropane acid formation was observed to be highly dependent on the rate of shaking of the culture. A culture shaking at 340 rev./min converted 8.7% of its oleate to the cyclopropane derivative at stationary phase, whereas a parellel culture shaken at 110 rev./min converted 66% of the oleate to a cyclopropane acid. The inability to observe selectivity or form derivatives from isomers other than the is-9 and cis-11 isomers seems to be due to enzyme specificity rather than a secondary affect of the abnormal unconverted fatty acids on the cell, because the cis-9 isomer is converted to its cyclopropane derivative even in cells grown with abnormal unreactive positional isomers. The preferred substrates for cyclopropanecarboxylic acid formation contained a cis ethylenic bond at either the 9 position or the -7 position. In combination with results of previous studies the specificity reported here supports a concept that two different enzymes may participate in cyclopropane ring synthesis. One enzyme activity may recognize its substrate by the distance from the π-bond to the carboxyl group and the other by the distance to the methyl group.

The specificity of the acyl-CoA:phospholipid acyltransferases has been studied using the 16 positional isomers of cis-octadecenoic acid. The results showed that the acyl-transferases acting at both the 1- and 2-positions of acyl-glycero-3-phosphorylcholine (acyl-GPC) discriminated between the acyl-CoA isomers in quite different ways. 1. 1. Acyl-CoA:1-acyl-GPC acyltransferase activity showed a distinct preference for the 9-, and 12-isomers. Of these three, the 9-octadecenoate (oleate) was the preferred substrate having a rate of 98 nmoles/min per mg. 2. 2. Acyl-CoA:2-acyl-GPC acyltransferase reacted more rapidly with the 8-,10-,12-,13- and 15-isomers, and of these the 12-octadecenoate had the fastest rate (121 nmoles/min per mg). 3. 3. As the enzymes were allowed to age at 4°, the activity was lost at slightly different rates for each isomer. The enzyme(s) esterifying the 1-position seemed to loose activity fairly uniformly with all isomers so that a similar pattern of reactivities was observed over a period of several days. The enzyme(s) esterifying the 2-position, however, differed in that after 2 days, the rate for the 9-isomer had dropped below that for the 12-isomer. This result suggests that different enzymes may exist for different acyl-CoA isomers. 4. 4. High concentrations of sucrose (0.8 M) tended to stabilize the activities with the 9- and 12-isomers, but did not change the fact that the activity for the 9-isomer was lost more rapidly. Albumin, contrary to our expectations, increased the rate of loss of activity. 5. 5. The enzyme activities were purified 10- to 15-fold above that of the crude tissue homogenate by treating the microsomal particles with sodium deoxycholate and albumin. 6. 6. Acyltransferase rates for the esterification of the naturally occurring 7-,9-,11-, and 13-octadecenoates to position 1 and 2 of diacyl-GPC indicated a preferred position for each acid which is in accord with that reported for the distribution of these monoenoic acids in phospholipids isolated from rat liver.

SUMMARY Although acetylenic acids are not normally found in mammalian tissues, octadecynoyl-CoA esters can serve as substrates for rat liver microsomal acyl-CoA: phospholipid acyltransferases. The relatively high rate of transfer to the 2-hydroxyl of I-acylglycerol-3-phosphorylcholine by the 5-, 9-, and I2-positional cis, tram and yne isomers of acids supports the concept that n-bonds at these positions facilitate esterification irrespective of configuration. The preferred positional isomers for transfer to the I-hydroxyl, however, are different for the cis and yne derivatives, indicating a sensitivity of the enzyme(s) to configuration. In the latter transfer, an alternating selectivity occurred with the acetylenic isomers between 8 to 13 that was opposite to the pattern for the cis-octadecenoyl transfers. 1NTRODUCTION Although unsaturated fatty acids may be generally regarded as desirable constituents of cellular lipids, the physicochemical properties that make them desirable are not clearly demonstrated. Acyl-CoA : phospholipid acyltransferases’ have relatively high selectivities’ for transfer to both the I- and 2-position of phosphoglycerides and these selectivities show a good correlation with the naturally-occurring distributions of acids between these positions3-‘. Since the two positions often contain saturated and unsaturated esters, respectively, the transferase activity forming the primary ester may select for some property of saturated acids and that forming the secondary ester for some properties of unsaturated acids. In our studies of the criteria by which an acyl chain may be selected for incorporation into phospholipids, we have examined positional isomers of cis-5,6 and trans-ethyleneic acids (ref. 7) as well as cis-cyclopropane derivatives*. A significant property of naturally-occurring saturated acids seems to be the absence of cis-9 or cis-I I configurations which are not favored by the transferase activity to the r-position. Unsaturated derivatives, that are normally esterified to the z-position, seem to have n-bonds at particular locations along the hydrocarbon chain with less regard