Determination of the glyceride structure of fats: Distribution of individual saturated and unsaturated acids1

A method has been devised which gives the distribution of saturated and unsaturated fatty acids. It involves fractionation of the triglycerides into groups on the basis of total unsaturation by employing chromatography on a silicic acid-silver nitrate column. The glyceride composition of each fraction is then determined by gas-liquid chromatography (GLC) of the oxidized glycerides. Using this method, the glyceride composition of lard and cocoa butter was determined to give quantitative amt of 24 and 18 glycerides, respectively. Duplicate analyses agreed to within ±0.5%. The fatty acid composition calculated from the glyceride composition agreed to within ±1.5% with that of the original fat. This approach provides a new basis for the evaluation of the glyceride tyes in natural fats and for the first time permits the quantitative determination of all the chemically different glycerides of myristic, palmitic, stearic, oleic, linoleic and linolenic acids in a fat. Presented at AOCS Meeting in Minneapolis, 1963. Issued at NRC 7947. National Research Council Postdoctorate Fellow. Prairie Regional Laboratory, Saskatoon, Sask.     

Fatty acids,fatty alcohols,and wax esters fromLimnanthes douglassi (Meadowfoam) seed oil

Limanathes douglasii seed oil glycerides contain fatty acids which predominantly (97%) have 20 or more carbon atoms. Fatty acids were prepared by saponification; fatty alcohols, by sodium reduction of the glycerides; and liquid wax esters, byp-toluenesulfonic acid-catalyzed reaction of the fatty acids with the fatty alcohols. Solid waxes were prepared by hydrogenation of the glyceride oil and of the wax esters. Chemical and physical constants were determined forLimnanthes douglasii seed oil and its derivatives. The liquid wax esters had properties very similar to those of jojoba (Simmondsia chinensis) seed oil. The solid hydrogenated wax ester was identical in physical appearance and melting point to hydrogenated jojoba seed oil. A laboratory of the Northern Utilization Research and Development Division, Agricultural Research Service, USDA.     

Structure of unsaturated vegetable oil glycerides: Direct calculation from fatty acid composition

Composition and structure of unsaturated glycerides of vegetable oils can be calculated directly from the fatty acid composition of the oil. Fatty acid distribution on the 2 position as normally determined by lipase hydrolysis is calculated from the composition of the whole oil by applying the following three rules in their respective order: Saturated fatty acids and those with chain length greater than 18 carbons are first distributed equally and randomly on the 1 and 3 position of the glycerol moiety; oleic and linolenic acids are treated equally, or as a unit, and distributed equally and randomly on all three glyceride positions with any excess from the 1 and 3 position being added to the 2 position; and all remaining positions are filled by linoleic acid. Remarkably good agreement between the calculated and experimentally determined fatty acid distributions is shown for soybean, linseed, safflower and many other vegetable oils whose compositions are reported in the literature. An association between oleic and linolenic acid within the glyceride structure of some vegetable oils is evident. Presented in part at the T. P. Hilditch Symposium Annual Meeting AOCS, Houston, Texas, 1965. No. Utiliz. Res. Dev. Div., ARS, USDA.     

DC-Trennung von Ricinusölfettsäure-Partialglyceriden

Thin-Layer Chromatographic Separation of Partial Glycerides of Castor Oil Fatty Acids Partial glycerides of castor oil fatty acids and hydrogenated castor oil fatty acids were prepared by esterification or glycerolysis and fractionated, together with commercial products, by TLC (especially by two-dimensional technique) on silicagel 60 precoated plates. By comparison of the two-dimensional chromatograms of the partial esters of castor oil fatty acids with synthetic standards, such as partial glycerides of ricinoleic, di- and tri-ricinoleic acids, estolides of castor oil fatty acids esterified to partial glycerides, and partial esters of castor oil fatty acids with 1,3-propanediol, the substances that could be identified were partial glycerides of ricinoleic, diricinoleic and triricinoleic and tetraricinoleic acids as well as partial glycerides, which contained, in addition to ricinoleic, diricinoleic and triricinoleic acids, fatty acids without hydroxyl groups as terminal estolide chain. The TLC enables an insight into the complex character of the glyceride composition of partial glycerides of castor oil fatty acids.     

The structural analysis of wheat flour glycerolipids

The compositions of the fatty acids in the 1, 2 and 3 positions of the principal glycerolipids and their various stereoisomers were determined. Fatty acids in the 1 and 3 positions of triglycerides were similar in composition and less unsaturated than those in the 2 position. Fatty acids in the 1,2-, 1,3- and 2,3-diglycerides were distributed in a pattern which indicated isomerization ofsn-1,2-diacylglycerol. Lysophosphatidyl choline (the principal monoacyl lipid) consisted of about 80% 1-acyl and 20% 2-acyl isomers. The fatty acid compositions indicated that most of the 2-lysophosphatidyl choline was formed by isomerization of 1-lysophosphatidyl choline. Most of the digly cerides and lysophosphatidyl choline were synthesized in the ripening wheat grain. However a small proportion of these partial glycerides and all of the other minor partial glycerides (monoglycerides, digalactosyl monoglycerides) appeared to be the result of limited lipolysis of the corresponding diacyl lipids in the wheat or in the freshly-milled flour. Fatty acids in the 2 position of all the fully acylated glycerides were very similar in composition, but there were considerable differences in the 1 position fatty acids.     

Chromatography of polar triglycerides on silicic Acid Columns

Triglycerides containing polar fatty acids are resolved by silicic acid chromatography into molecular species containing increased amounts of the polar acids. Natural fats like isano, oiticica, castor, or kamala seed oils with both nonpolar fatty acids and polar hydroxy or keto acids have been resolved into component glycerides according to the degree of polarity. Silicic acid chromatography offers a means of obtaining specific glycerides from fats for detailed studies on glyceride composition and structure. Presented at the AOCS Meeting, Cincinnati, October 1965. No. Utiliz. Res. Dev. Div., ARS, USDA.     

Fatty acids,fatty alcohols,wax esters,and methyl esters fromCrambe abyssinica andLunaria annua seed oils

Crambe abyssinica andLunaria annua, members of the Cruciferae family, have seed oil glycerides containing ca. 55–65% of C₂₂ and C₂₄ unsaturated fatty acids. Fatty acids were prepared by saponification; fatty alcohols, by sodium reduction of glycerides; liquid wax esters, byp-toluenesulfonic acid-catalyzed reaction of fatty acids with fatty alcohols; and methyl esters, by reaction of fatty acids with diazomethane. Solid hydrogenated glyceride oils and wax esters were compared with several commercial waxes. Chemical and physical constants were determined for the seed oils and their derivatives. Position of unsaturation in theCrambe fatty acids was determined by gas chromatographic analysis of the permanganate-periodate degradation products. The major dicarboxylic acid was brassylic (C₁₃), proving the docosenoic acid to be erucic. Presented in part at the AOCS meeting in New Orleans, La., 1962. A laboratory of the No. Utiliz. Res. & Dev. Div., ARS, U.S.D.A.     

Composition of the fat extracted from the seeds of theobroma bicolor

The fat fromTheobroma bicolor was analyzed for glyceride content by thin layer chromatography (TLC), and for fatty acid composition and triglyceride (carbon number) composition by gas liquid chromatography (GLC). The fat was then separated into glycerides of different degrees of unsaturation by means of silver nitrate TLC. Then, the bands were examined by GLC before and after conversion to methyl esters. From the results obtained, the distribution of the fatty acids on the individual glycerides was calculated. The fat consisted of 96.5% triglyceride with only 2.5% diglyceride and 1.7% free fatty acid. The major fatty acids present were 42.3% C18:0, 45,2% C18:1, and 6.0% C16:1. Most of the triglycerides were of carbon number 52 (18.0%) and 54 (77.6%). The major triglycerides were 38.6% 1-stearyl-2,3 diolein (SOO), 25.4% 2-oleyl-1,3 distearin (SOS) and 13.8% 1-palmito-2-oleyl-stearin (POS). Only 44.3% of the fat consisted of monounsaturated triglycerides.     

Fatty acid composition of fats of differing melting points extracted from ram subcutaneous tissue

The observation that the subcutaneous fat of pasture-fed Southdown rams consists of two distinct regions is reported. Fatty acid composition of fat from the outer and inner regions of subcutaneous tissue taken from the rib region of eight Southdown rams fed pasture were determined. Relative to the harder inner regions (mean melting point 43.1°C), the softer outer regions (mean melting point 31.8°C) were shown to contain more 9∶0-, 15∶0-, 17∶0-, 17∶1-, 18∶1-cis and total 18∶1 fatty acids; less 14∶0-, 16∶0-, 18∶0- and 18∶1-trans fatty acids; and a greater variety and a greater concentration of branched-chain components. Proportions of medium chain-length fatty acids other than 9∶0, did not differ between the layers. The fatty acid contents of serial samples taken at 1-mm intervals through these tissues were determined. Changes in concentrations of components among samples were gradual through the tissues. There was no clear connective tissue sheet, as has been reported for pigs. The inner region of the tissues contains apparently nonrandom fluctuating changes in fatty acid composition.     

Stereospecific analysis of some cruciferae species

Results of stereospecific analysis of nine Cruciferae seed oils are presented. The major fatty acids in the oils investigated are nonrandomly distributed, not only between inner and outer positions, but also between sn-1 and sn-3 positions. When the positional data were plotted versus erucic acid content, a regular and characteristic distribution pattern for each fatty acid was obtained. Category I acids (saturated acids and acids with more than 18 carbon atoms) prefer either of the outer positions. Erucic acid, however, has a clear preference for the sn-3 position. When in the outer positions, Category II acids (unsaturated acids with 18 carbon atoms) as a rule prefer the sn-1 to the sn-3 position. This tendency increases with decreasing unsaturation.     

Nachweis der Glyceride veresterter Olivenöl-Fettsäuren

Detection of the Glycerides of Esterified Olive Oil Fatty Acids Natural olive oil and ester oil with the same fatty acid composition contain approximately the same amount of dipalmito-olein (ca. 4%) and palmito-diolein (ca. 20%). The positional isomers of these glycerides which contain palmitic acid in the 2-position, occur in very small amount in olive oil but are present in considerably larger amount in ester oil. The same is also true for the corresponding stearic acid containing glycerides. The dipalmito-olein and palmito-diolein containing fractions were isolated by silver nitrate chromatography. The characteristic ratios of the positional isomers (POP/PPO and POO/OPO) in both the oils are determined indirectly by enzymatic hydrolysis of the fatty acids in 1,3-position and analysing the 2-monoglyceride fatty acids gas chromatographically.     

On the Mechanism of the Thermal Polymerization of Linseed Oil

The monomeric and polymeric glycerides present in thermally polymerized linseed oils can be separated quantitatively by molecular distillation. Analysis of the fatty acids of monomeric and polymeric glycerides indicate the occurence of intra- and intermolecular condensations respectively. Whereas in the former reaction, leading to the formation of bicyclic, dimeric fatty acid groups within the orginal glycerides, no increase in the molecular weight of the oil takes place, in the latter one, characterized by an increase in the molecular weight, fatty acids of different glyceride molecules are involved. It is shown, however, that the overall increase in molecular weight is due to the dimeric glycerides which are formed by interesterification reactions between glyceride monomers containing dimeric fatty acid groups, the latter resulting from intramolecular condensation.     

Glyceride distribution in adipose and liver glycerides of animals

The glyceride distribution in depot fats from a series of animals was determined by pancreatic lipase hydrolysis, isolation of hydrolytic products by thin-layer chromatography (TLC), and fatty acid analysis by gas-liquid chromatography (GLC). Distribution of the principal types of glycerides (S₃, S₂U, SU₂ U₃) in the internal and external adipose tissue fats from the same pig was nonrandom. The percentages of palmitic acid at the 2-position in these adipose fats were comparable. However, liver glycerides from this same animal differed strikingly from adipose glycerides, having, for example, only ca. 15% of its palmitic acid in the 2-position compared with > 80% for adipose fats. The liver glycerides of lamb, rabbit, and dog also differed considerably from adipose glycerides in glyceride distribution and in percentages of individual fatty acids in 2-position. The composition of adipose glycerides from Lamb, beef, deer, rabbit, chicken, and dog in terms of the four principal glyceride types approached closely the values calculated for random. When positional isomers were considered, however, only the adipose glycerides of the dog conformed to random distribution. Presented at the AOCS meeting, Chicago, October 11–14, 1964. E. Utiliz. Res. and Devel. Div., ARS, USDA.     

Partial fractionation of fatty acid triglycerides on a silicic acid column

A silicic acid column chromatographic method for the fractionation of glyceride types, employing an all-glass apparatus which permits gradient changes in solvent mixtures under pressure is described. Data on seven natural fats are discussed on the basis of the iodine values of individual fractions. The general trend in elution with the increasing concentration of ethyl ether in n-hexane is governed by a) chain length and b) unsatuation of the constitutent fatty acids. Glycerides containing short-chain acids are more strongly adsorbed than those containing long-chain acids and glycerides containing unsaturated fatty acids are more strongly adsorbed in direct relation to unsaturation than those containing saturated acids of the same chain-length.     

Fatty acid specificity of glyceride synthesis by homogenates of bovine mammary tissue

Fatty acid esterification by cell free preparations of bovine mammary tissue was investigated to determine if the type of long chain fatty acid supplied might influence the rate of triglyceride synthesis by that tissue. Homogenates of lactating bovine mammary tissue esterified¹⁴C-fatty acids into glycerides at rates dependent upon chain length and degree of unsaturation. Palmitic, stearic, oleic and linoleic acids were esterified at rates consistent with their concentration in milk fat. A comparison of free fatty acid concentrations of mammary tissue with levels saturating esterification suggested that supply of fatty acids does not limit glyceride synthesis. Certain combinations of fatty acids were facilitory, competitive or inhibitory to esterification. Stearic acid complimented esterification of palmitic and oleic acids. Unlabeledtrans-11-octadecenoic acid did not compete with¹⁴C-palmitate as efficiently in the esterification process as did unlabeledcis-9-octadecenoic acid, indicating that the mammary gland may preferentially esterify thecis-isomer of C-18∶1. Linoleic acid inhibited esterification of palmitic, stearic and oleic acids. Michigan Agricultural Experiment Station Journal Article No. 5100.     

The separation of glycerides by liquid-liquid column partition chromatography

A liquid-liquid partition chromatography method was developed to separate triglycerides. The solvent was a two phase mixture of acetone, heptane, and water supported on silane treated celite. A study was made of the best means for equilibrating the solvents and support, packing the column, and introducing the sample. The effect of various operating variables such as flow rate, sample size, column length, and solvent compositions was studied using trilaurin and trimyristin as model glycerides. Under the best conditions achieved, it was calculated that glycerides differing by two carbon atoms or one double bond would not separate completely, but glycerides differing by two double bonds or four carbon atoms would be separated. Cocoa butter, a relatively simple triglyceride, was fractionated, and the fatty acid composition of each fraction was determined by gas chromatography. The glyceride composition was calculated and compared with theoretical compositions. The results indicate that useful glyceride separation can be obtained with this system. Probably even more useful separations could be obtained if a more sensitive device were used to detect the triglycerides in the effluent. This would allow the use of solvent compositions which give larger retention volumes and more plate efficiency. Journal Paper No. J-4517 of the Iowa Agricultural and Home Economics Experiment Station, Ames, Iowa, Project No. 1517. Presented at the AOCS meeting in Toronto, Canada, 1962. Based on a thesis presented by B. C. Black in partial fulfillment of the requirements for a master’s degree.     

Neutral lipids from the temporal gland of the African elephant,Loxodonta africana

Lipids from inactive and active temporal glands of the African elephant,Loxodonta africana, were isolated and fractionated. The inactive gland had a much higher total lipid content per gram of fresh tissue than the active gland. Lipids from the inactive gland consisted predominantly of neutral lipids while the active gland contained large quantities of phospholipids. Neutral lipids from the active gland contained much more hydrocarbons, cholesterol, and alkoxy glycerides than neutral lipids from an inactive gland. The alkoxy glyceride fraction did not contain any alkenyl glycerides. The hydrocarbons consisted of a mixture containing predominantly straight chain even numbered saturated and unsaturated hydrocarbons from C₁₈–C₃₀. Fatty acids from various fractions were investigated by gas liquid chromatography. Those from the active gland were characterized by a higher percentage of unsaturated acids. The change, from inactive to the active state involves mainly a reduction in palmitic and an increase in oleic acid content.     

Application of thin-layer chromatography to the quantitative estimation of tissue triglycerides II. Influence of methyl parathion on the composition of liver triglycerides in the rat

Methyl parathion fed at 10 ppm in a high protein low fat diet inhibited 46.9% of the total liver carboxylesterase activity. The total fatty acid composition of liver triglycerides was not significantly altered. However, the methyl parathion-fed rats showed a higher percentage of saturated acids in the 2-position of the glyceride molecule. Triglyceride analysis employing the multiple TLC-GLC technique (5) also showed a slightly higher percentage of saturated glycerides and those containing 1 and 2 double bonds than those in the control group. Triglyceride patterns in both groups were in general agreement with those calculated from the fatty acid distribution as suggested by Vander Wal (11). Presented at the AOCS meeting in Houston, Texas, April, 1965.     

The component triglycerides of palm-kernel oil

Triglycerides of palm-kernel oil were fractionated by gas liquid chromatography into 13 groups based on their carbon number of 30 to 54. These groups represent 99.9% of the total glyceride content. The proportion of triglyceride types in each group was calculated from the component fatty acids. These groups are defined by the nature of their constituent fatty acids but the position of the acids on glycerol is unknown. These 87 types, 24 of which are given in detail, were found. The two major components are trilaurin (19.8%) and dilauromyristin (14.1%). Only 18 types, occurring in an amount greater than 1%, together represent 80% of the total glycerides. Comparison of the glyceride content with that of coconut oil revealed many similarities between the two oils.     

The effect of dietary copper on the structure and physical properties of adipose tissue triglycerides in pigs

The inner back fat of control pigs had a higher melting point and stearic acid-oleic acid ratio than did the outer back fat of the control animals or the inner and outer back fats of pigs given the control diet supplemented with 250 ppm copper. By a combination of argentation thin layer chromatography and pancreatic lipase hydrolysis, it was shown that in the inner back fat of the control animals the proportions of two of the more saturated glyceride species were significantly greater and the proportions of two of the more unsaturated species were less than in the outer back fat of the control animals and in the inner and outer back fats of the copper-fed animals. These findings probably account for the observed differences in melting point. Stereospecific analyses of the glycerides demonstrated that the increased content of stearic acid in the inner back fat of the control pigs was distributed between the 1 and 3 positions.