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Deuterium labeled monoacid triglycerides were synthesized and their mass spectra were measured. The spectra provided further support for proposed (1) structures of principal ions, knowledge about the formation of [M-18]+, the interexchange of hydrogen atoms between 2 and the 5,6 or 7 positions and the explusion of part of the alkyl chain.

The adaptation of a wide range of isolation methods to the separation of epoxy components from seed oils has been accomplished. The application of these methods to samples ranging from a few micrograms to 50 g. or more has been considered, and recommendations as to the most suitable methods for specific sample sizes are made. Most of the procedures described are equally suitable for the isolation of hydroxy and other oxy-acids and esters. Some of them, in conjunction with gas-liquid chromatography, provide methods for total analysis of oils containing oxy-acids, which are more accurate and convenient than current analytical methods.

Homologous series of synthetic long-chain mono- esters and diesters of 1,2-ethanediol were analyzed by mass spectrometry, and the patterns of fragmentation were studied. Under electron impact saturated ethanediol monoesters yielded prominent ions characteristic of the shortchain diol, such as the rearranged ion formed by 2,3-cleavage (m/e 104) and the ion caused by 3,4-cleavage (m/e 117). Fragments characteristic of the constituent long-chain moieties were the acylium ions (RCO)+, (RCO - 1)+, and the ions (RC(OH)e)+. The mass spectra of ethanediol diesters exhibited very intense peaks due to the ions formed by loss of the acyloxy group, (M - RCOO)+, or one carboxylic acid, (M - RCOOH)+. These ions are distinctive for diol diesters. Precise mass measurements by high resolution mass spectrometry verified the composition of the ion fragments. Spectral studies of some monoesters and diesters of 1,3- propanediol, 1,4-butanediol, 2,3-butanediol, and also of some monounsaturated homologues, demonstrated that mass spec- trometry is very suitable for the identification, distinction, and analysis of diol lipids.

Le probleme de la separation des acides gras est expose en detail. Les methodes physiques couramment appliquees a une macro- ou micro-echelle sont discutees et l'on donne des exemples tires de la litterature ou de methodes recemment utilisees dans les recherches. Celles-ci comprennent la cristallisation, la distillation fractionnee, la distribution par contre-courant, la chromatographie en phase gazeuse et la spectrographie de masse.

1. 1. Ethyl cis -9, trans -12-linoleate does not exhibit essential fatty acid activity as judged by effects upon dermal symptoms, water consumption, or growth. Both double bonds in the linoleate molecule must be cis if it is to possess essential fatty acid potency. 2. 2. Ethyl cis -9, trans -12-linoleate is unable to “spark” the utilization of linolenate as judged by dermal symptoms and water intake. However, the cis , trans isomer of linoleate plus linolenate gave greater growth than either supplement alone. 3. 3. All groups receiving linolenate showed high pentaenoic acid and hexaenoic acid contents, and diminished tetraenoic acid contents. The data suggest that in the group receiving linoleate plus linolenate, hexaenoic acid contents are less and pentaenoic acid contents are higher than in the group receiving linolenate alone. 4. 4. Trans double bonds derived from cis -9, trans -12-linoleate are retained by the animal and are deposited in the tissue lipides.

IN rrs MOST broad definition, chromatography is a method of separation based upon differences in concentration of molecules in or on the mobile and non-mobile phases of a flowing system. The mobile phase may be gaseous or liquid, the non-mobile phase may be liquid or solid. The number of possible experimental arrangements for effecting chromatographic separ~ tions is infinite, and the technique in its present state of development ia largely empirical. However, the method since its first use by Tsw~rT has found an ever increasing range of application both for separations and for quantitative analysis of difficultly separable substances of a wide variety. The theory and practice of chromatography has been summarized in several major publications. STa~X~ 1 and ZECH~EISTF~ and CHOL~OKY s have summarized the developmental literature on chromatography. In a subsequent book, ZECHMEISTER 3 has summarized the advances made in chromatography from 1938 to 1947. CAssrvY~ has recently treated the subject from the more theoretical aspect, and for a discussion of the principles involved in chromatography and a treatment of the effects of numerous variables upon adsorption and separation, the reader is referred to this latter volume. Although chromatography is a routine tool in separations and analyses in some fields, it has not generally reached that stage of development in its application ~ lipid separations. At prc~ent it is largely a research tool rather than a routine method in fat and oil chemistry. The attempts t o use this method in fat chemistry have been summarized in several reviews s-#. Recent developments indicate an expanded usefulness of the method, and it now appears that micro methods based upon various types of chromatography ~ l l soon place very effective tools in the hands of the lipid chemist. The chromatographic techniques thus far developed for use in lipid separations fall into five general classes. Elution analysis (classical chromatography), frontal analysis, displacement analysis, partition chromatography and paper partition chromatography will be discussed each in turn. The author has restricted himself in the present treatment to fats, oils, f a t ty acids and other lipids of biological importance, with the exception of the steroids and carotenoids.

The effect of isomeric cis -octadecenoic acids on growth of monkey kidney (MK 2 ) cells was variable. 1. 1. When 10 μ/ml of the 5-, 6-, 7-, 8-, 9-, 10-, 11-, l3-, 14-,16–l8:1 isomers were added to medium, a stimulation in growth of MK 2 cells occurred. 2. 2. Isomeric octadecenoic acids 3–18:1 or 17–18:1 (10 μg/ml) were inhibitory for MK 2 cell growth. 3. 3. Inhibition of cell growth was observed within 24 h when the medium contained 20 to 40 μg/ml of 3–18:1, and after 72 h almost all cells had lysed. A slight stimulation in growth of cells did occur with 3–18: 1 when the concentration was 5 μg/ml or less. 4. 4. The addition of as little as 2.5 μ/ml of 9–18:1 to medium containing the inhibitory level of 3–18:1 (20 μ/ml) increased the cell population to 50 % of the control population. 5. 5. When a combination of 20 μ/ml each of 9–18:1 and 3–18:1 was added to the medium an increase in cell growth was observed but not a complete reversal of the inhibition. 6. 6. Cells which had been maintained in a basal medium up to 96 h before the isomer was added responded similarly to cells which had the isomer added at 24 h. 7. 7. MK 2 cell growth was inhibited by 3–18: 1 and stimulated by 8–18: 1. This was opposite to the response observed for growth of a bacterium, Leptospira interrogans serotype patoc .

Several ions in the mass spectra of wax esters were related to the molecular structures. Assigned structures of ions were confirmed by deuterium labeling. A simple, direct method for quantititive analyses of mixtures was developed. The method involved a comparison of sets of three ions, RCO2H(+), RCO2H2 (+) and [R'-1](+) from all compounds in the mixture. The method was found applicable for mixtures of unsaturated wax esters after reduction with tetradeuterio hydrazine.

A method for the analysis of fatty esters employing separation by thin-layer chromatography and quantitative determinationvia their hydroxamic acids has been developed. Esters of different types are separated on silica gel plates, the spots or zones are scraped from the plates, the esters are extracted from the silica gel, and the iron hydroxamic acid complexes are formed. The latter are then measured by colorimetry. The method is suitable for analysis of mixtures of ordinary, epoxy, monohydroxy, and dihydroxy fatty esters as well as for mixtures of mono-, di-, and triglycerides. Used in conjunction with gas chromatography this method permits the fatty acid composition of seed oils containing oxygenated fatty acids to be measured in about 3 hours. The amount required for the total analysis is 1 to 10 mg. per determination, depending upon composition.

A method combining thermal fragmentation and mass spectrometry for the determination of the position of double bonds in an unsaturated ester is presented. The thermal fragmentation of methyl esters of deuterated long chain carboxylic acids yields a homologous series of olefins plus a homologous series of unsaturated esters. The positions of the deuterium atoms in the original ester are revealed by the deuterium content of its fragments as determined by mass spectrometry. Therefore, the positions of double bonds of a polyunsaturated acid can be determined by pyrolysis after saturation by deuterium. The structures of the unsaturated fragments are ascertained by mass spectrometric method, and the formation of the ion [M – 32] in the mass spectrometric fragmentation of unsaturated methyl esters is studied by means of deuterium labeling.

1. 1. The two possible pathways which lead from linoleic acid to arachidonic acid were simultaneously studied in liver microsomes from fat-deficient rats and from fat-fed rats. 2. 2. The overall yields of the conversion of linoleic acid to eicosa-8,11,14-trienoic and arachidonic acids were calculated and gave a ratio of 16 to 1 for the pathway via γ-linolenic acid over the pathway via eicosa-11,14-dienoic acid in the fat-fed rat, and a ratio of 6 to 1 in the fat-deficient rat. 3. 3. The side reactions to the alternate pathways were also investigated. A relatively high rate of chain elongation was found for eicosa-8,11-dienoic acid which minimizes the importance of the pathway via this acid. 4. 4. The inhibition and enhancement of the different reactions in the two pathways were tested under the same conditions using either mixtures of fatty acids extracted from rat livers or single fatty acids of the linoleic acid family. 5. 5. The interactions of the different acids in chain elongation and desaturation reactions are of small magnitude. However, the desaturation of eicosa-8,11,14-trienoic acid is strongly enhanced by linoleic and γ-linolenic acids which may account for its non-accumulation in tissue lipids.

Gas-liquid chromatography, several versions of paper chromatography, and alkaline isomerization have been applied to one preparation ofChlorella fatty acid methyl esters. GLC outranks other methods in rapidity, reproducibility, and resolving power. The presence of nonvolatile components in the sample can lead to erroneous results. Among PC methods which might arise from autoxidation and polymerization are detected. Esters or acids with 14 or fewer C atoms should be analyzed as nonvolatile derivatives. AI, as used here, introduces a systematic error in the determination of linoleic and linolenic acids. The quantitative results of the methods are in agreement for the major components, which all belong to the C10 and C18 series. A number of minor components have been revealed by GLC and/or PC. They are tentatively identified as

Coenzyme A thiol esters of the complete series of positional isomers of methylene-interrupted cis,cis-octadecadienoic acid were synthesized. Their rates of reaction, catalyzed by acyl-CoA:phospholipid acyltransferases in liver microsomal preparations from four species, ranged from 0 to 87 mµmoles per min per mg of protein. In general, acyl transfer was relatively rapid with compounds containing the cis-ethylenic bonds near the methyl end of the fatty acid chain. The commonly found 9,12-isomer, linoleate, was not rapidly esterified at position 1 of 2-acylglycero-3-phosphorylcholine, although six other positional isomers reacted rapidly at the primary hydroxyl. In contrast to the 9,12-isomer, the 8,11- and 10,13-isomers were esterified rapidly to position 1 of lecithin and slowly to position 2. The alternating activities of the 8,11-, 9,12-, and 10,13-isomers and their inverse effects at positions 1 and 2 indicate that the structure near carbon atoms 8, 9, and 10 is critical in the metabolism of polyunsaturated acids.

The metabolism of 9,15-octadecadienoate, 12,15-octadecadienoate and 7,13-eicosadienoate was investigated in the fatdeficient rat. The liver lecithins of those animals receiving 9,15- and 12,15-octadecadienoate, as well as the animal remaining on the fat-deficient control, were isolated. The fatty acid distributional pattern was determined by the use of lecithinase A. All three of the experimental dienoic acids were incorporated into tissue lipids. None of the acids, however, was converted to longer chain polyunsaturated fatty acids. The 9,15-octadecadienoate was esterified almost exclusively to the beta-position of lecithin, whereas 12,15-octadecadienoate was about equally distributed between the alpha- and beta-positions.

The complete series of methylene-interrupted cis,cis -octadecadienoic acids (analogues of linoleic acid), from the 2,5- to 14,17-isomer, has been synthesized. The 4,7- to 10,13-isomers were prepared by coupling a 1-bromoalk-2-yne with the di-Grignard complex of an (ˑ)-acetylenic acid and semi-hydrogenation of the resulting diynoic acid to a cis,cis -diene with Lindlar's catalyst. The 2,5- and 3,6-isomers and the 11,14- to 14,17-isomers were synthesized by first preparing the corresponding diacetylenic alcohols which were then oxidized to the acids and semi-hydrogenated. Purity of the intermediates and final compounds was established by gas-liquid and thin-layer chromatography. The compounds were characterized by melting points, critical solution temperatures, infrared spectroscopy, mass spectrometry, and nuclear magnetic resonance spectroscopy. Hydrogenation followed by gas chromatography confirmed the chain length and indicated absence of branched chain material. The positions of the double bonds were confirmed by ozonolysis. The 2,5-isomer has distinguishing features in its mass spectrum but only subtle distinctions can be detected in the spectra of the rest. The NMR spectra of the 2,5- to 5,8- and 11,14- to 14,17-isomers, however, all have features which uniquely identify them, though the 6,9- to 10,13-isomers have spectra which are virtually identical.

T HE USUAL definition of atherosclerosis suggests that the disease is a fatty degeneration of the arterial wall. It is becoming increasingly apparent that the disease accompanies abnormalities in the metabolism of various lipids, and the number of lipids involved in the process seems to be growing as investigators delve into the matter. At this stage of our knowledge, it cannot be stated dogmatically that abnormalities of lipid metabolism are the cau.�e of the disease. Such may be the case, however, for imbalances induced in lipid metabolism have led to lesions resembling atherosclerosis in many respects. The number of lipid substances in nature is legion, and most of these are not well characterized. The variety of structures is great, and their single common property is that they occur together because of similar characteristics of solubility. The state of affairs in lipid chemistry is rather parallel to that of protein chemistry. We know the structures of many of the simple units which together make up the complex lipids, and we are continually finding new units previously unsuspected within the lipid family. The compositions of the more complex lipids are known only empirically, and the detailed structures have yet to be worked out for many compounds with molecular weights of less than I ,OUU. The structures of proteolipids and lipoproteins are not known, and for the present we must be content with knowing the approximate composition of these substances. Even the determination of composition of

Rats were fed various levels of either ethyl linoleate, ethyl arachidonate, or ethyl linolenate. Weight gain, fat-deficiency status, and fatty acid composition of the liver lipids were determined. Dietary linoleate, fed in excess of 1% of calories, maintained good growth and cured fat deficiency. Increasing amounts of dietary linoleate were stored in the liver lipids and converted into fatty acids of the linoleate family—20:4 and 22:4. The concentration of 20:3 was decreased. Dietary arachidonate cured fat deficiency three times more effectively than linoleate. Increasing amounts of dietary arachidonate were stored in liver lipids and converted to 22:5ω6. The level of 20:3 was lowered three times more effectively than when linoleate was fed. No fatty acids of the linolenate family were synthesized from linoleate or arachidonate. Dietary linolenate did not support weight gain as efficiently as did linoleate or arachidonate. Fat-deficiency symptoms could not be cured completely. Increasing amounts of dietary linolenate increased the levels of fatty acids of the linolenate family; linolenic acid was stored, and 20:5, 22:5ω3, and 22:6 were synthesized from linolenate. The level of 20:3 was lowered in the same fashion as when linoleate or arachidonate was fed. Thr level of 20:4 was decreased with increasing amounts of dietary linolenate.

The mechanisms of mass spectrometric fragmentation and their applications to the elucidation of structure of lipids related to fatty acids are reviewed. The mechanism of fragmentation of saturated fatty acids and the mass spectrometric determination of the position of the double bond in unsaturated esters are emphasized. The use of pyrolysis-mass spectrometry for locating double bonds is introduced. Patterns of fragmentation of wax esters and glycerides are also given.

Fifteen approximately random, mixed, triacylglycerols, each of which contained 12∶0, 14∶0, 9, 12–18∶2, 16∶0 and only one positional isomer ofcis 18∶1 (Δ2 through Δ16), were synthesized. These mixtures were used as substrates for the lipase from the microorganismGeotrichum candidum to define the specificity of the enzyme for unsaturated fatty acids. Comparatively small quantities of the 18∶1 isomers, other than 9–18∶1, were hydrolyzed. Relatively large amounts of 18∶2 were released from all substrates. There was no preference between 9–18∶1 and 18∶2. The positional isomers other than 9–18∶1 accumulated in the di- and mono-acylglycerols.

SUMMARY Chromatographic procedures for the detection and evaluation of oxygenated fatty acids are described. Emphasis has been placed on epoxy acids, but these methods promise to be of great value in studies of all classes of oxygenated acids. Paper chromatography of fatty acids and their esters has been developed for the examination of mixtures containing oxygenated derivatives. The method of adsorption chromatography on thin layers of silicic acid has been shown to be a powerful tool in studies of epoxy acids and hydroxy acids. Gas-liquid chromatography (GLC) of epoxy esters has been studied using both polar and nonpolar columns and has great utility in the detection and analysis of these compounds in mixtures. These methods have been applied to the examination of the epoxy components of six seed oils. Thin-layer and GLC proved particularly useful in this study and together demonstrated the presence of at least three distinct epoxy acids in each of these oils. Some conclusions as to the probable structures of these epoxy components are presented on the basis of their chromatographic characteristics in relation to known substances.

One of the practical and increasing prob lems in biochemical and nutritional work involving tissue lipid analysis arises from the multiplicity of variables encountered. Alkaline isomerization and associated tech niques allow the quantitative measurement of some 6 or 7 fatty acid fractions, both in the diet and in each of the tissues. Gas chromatography methods may well in crease this number three- or fourfold. Bio chemically many of these fatty acids are related to each other and to other dietary components by mechanisms that are still to be established; but not all of the meas urable fractions are positively and linearly related with each other. Holman ('60) has used the negative correlation between triene and tetraene fractions and their sharply curvilinear relationships to dietary linoleate as a basis for detecting a defi ciency of essential fatty acids (EFA) in experimental animals, and for estimating their minimum requirement.

Displacement chromatography has been applied to model mixtures of some steroid compounds. From results gained, it appears that this type of chromatographic development can be used to advantage in separation of quantities of steroids as little as 50 mg. It also appears that carrier displacement chromatography may be applicable for the separation of much smaller quantities of steroids.

a) Displacement chromatography using a charcoal-isopropanol-methyl behenate system has been successfully applied to the isolation of docosahexaenoic acid and ethyl docosahexaenoate from cod liver oil concentrates. b) Methyl eicosapentaenoate was isolated from cod liver oil methyl esters by combined elution chromatography on silicic acid and displacement chromatography. c) Using silicic acid as adsorbent and petroleum ether-chloroform as solvent, ethyl docosapentaenoate was isolated from cod liver oil ethyl esters. d) With a spinning band fractionation column it was found that fractional distillation of methyl esters of cod liver oil at 0.5 mm. Hg. resulted in increasing conjugation in the distillate as the fractionation proceeded. Fractional distillation is to be avoided as a final step in purification of polyunsaturated esters. e) The end absorption of conjugated pentaenoic acids is so large at the characteristic maximum for hexaene (3,750 A) that simultaneous equations are required for the calculation of hexaenoic and pentaenoic acids. f) Fifteen minutes of treatment with 21% KOH-ethylene glycol at 180° is not optimum for isomerization of ethyl docosapentaenoate from cod liver oil. The optimum isomerization times for hexaenoic acids of various sources are 4 to 6 minutes, considerably different from the 15 minutes established for arachidonate.

Mass spectra of several triglycerides of specific structure or with specific deuterium labeling have been measured with a low resolution mass spectrometer. With saturated triglycerides the abundances of ions characteristic of the component acids, [M-RCO2]+, increase with increasing chain length, and [M-RCO2CH2]+ decrease with increasing chain length. Unsaturation in the acyl moiety causes the abundant formation of [RCO-1]+. Structures have been suggested for a number of the main peaks obtained from saturated triglycerides, and high resolution spectra of one triglyceride agree with the postulated structures. The peaks, [RCO+74]+, [RCO+115]+ and [RCO+128+14n]+, represent structures which contain the glyceryl portion of the triglyceride, since in case of the replacement of its hydrogens with deuteriums, these peaks are shifted accordingly. Evidence which indicates the possibility of determining the location of unsaturation by the interruption of homology of the [RCO+128+14n]+ series, brought about by the addition of deuterium to the unsaturated linkages, is introduced. Further evidence is also presented, which indicates that the [M-RCO2CH2]+ ions arise from the positions 1 and 3 and, in agreement with earlier studies from other laboratories, it is thus possible to identify the acyl groups attached to the 1 and 2 positions of the glyceryl moiety.

The fatty acid Composition of lipids from erythrocytes and depot fat of rats whose diet was supplemented with various levels of linoleate, arachidonate, or linolenate has been investigated. Dietary linoleate and linolenate are incorporated into the depot fat. The ratio of eicosatrienoic acid to eicosatetraenoic acid, derived from the analysis of depot fat, decreases with increasing amounts of all three dietary essential fatty acids (EFA) in the same manner as observed in organ lipids. The composition of the unsaturated fatty acids of erythrocyte lipids is altered readily by increasing amounts of dietary EFA. The concentration of 20: 3 is lowered extensively by all three EFA. The synthesis of arachidonate is inhibited by dietary linolenate and 20:5 and 22:5ω3 (double bond between third and fourth carbow from ω end) can be detected only in animals given linolenate.

An orujo oil (sulfur olive oil) has been shown to contain several elasses of oxygenated fatty acids amounting to more than 10% of the total oil. Epoxy acids amount to 3.6% of the fatty acids of this oil and monohydroxy acids constitute a turther 6%. The major epoxy acid has been proven to betrans-9¬10-epoxystearic acid.

SummaryRigid exclusion of essential fatty acids from the diet of young swine leads to a deficiency disease not characterized by consistent or early skin lesions. The severity of the dietary regime imposed caused high mortality, but of the 8 pigs that survived beyond the normal weanling age, skin lesions were observed in one and aortic lesions in 5, at or before 98 days on experiment. The data suggest that essential fatty acids are required by swine and that deficiency of essential fatty acids may lead to impairment of the arterial wall.