Gas chromatographic resolution of butteroil and synthetic triglycerides beyond their carbon numbers

Preparative gas chromatography was used to isolate the C₂₆ to C₃₈ triglycerides of butteroil in groups of uniform molecular weight. Determination of fatty acids allowed a preliminary assignment of triglyceride structure and an estimation of the relative proportions of the various glyceride types. A basis for a further gas chromatographic resolution of saturated triglycerides of identical molecular weight but differing in composition and positional distribution of fatty acids was recognized in the easy separation of various derivatives of 1- and 2-monoglycerides. The study indicated that it might be practical to separate positional isomers of those triglycerides which contain fatty acids of both short and long chain lengths. Other promising routes to increased glyceride resolution were suggested by experiments using chromatographic conditions approaching gas-solid systems.     

Pancreatic lipase hydrolysis of triglycerides by a semimicro technique

Procedures are described for rapid lipase hydrolysis of triglycerides, isolation of the hydrolytic products by TLC and their conversion to methyl esters and fatty acid analysis by GLC. The techniques are applicable to a few mg of triglycerides or fats. Examples of data obtained with purified triglycerides indicate that the specific action of pancreatic lipase for the 1,3 ester groups is nearly absolute and the technique may be used as a criterion of purity of di- and tri-acid triglycerides. Ca. 83% of the palmitic but only 10~12% of stearic and C₁₈ unsaturated acids of commercial lard occur in 2-position. Presented in part at the AOCS Meeting, Minneapolis, 1963. A laboratory of E. Utiliz. Res. & Dev. Div., ARS, USDA.     

Gas-liquid chromatography of triglycerides from erucic acid oils and fish oils

By critically selecting optimum operating conditions, quantitative gas-liquid chromatography of triglycerides has been extended to molecules containing substantial amounts of C₂₀, C₂₂, and C₂₄ fatty acids. The triglycerides of four erucic acid oils (water cress, rapessed, nasturtium, andLunaria annua) and two fully hydrogenated fish oils (menhaden and tuna) have been quantitatively analyzed by this technique. The average fatty acid chain length calculated from the triglyceride composition of each oil agreed closely with that determined by GLC of its respective methyl esters. Several conclusions about the triglyceride composition of the fats analyzed are discussed. Winner, AOCS Bond Award. Presented at the AOCS Meeting in Cincinnati, October 1965.     

Structure of bovine milk fat triglycerides: II. Long chain lengths

The long chain triglycerides of bovine milk fat were isolated by thin layer chromatography, and their chemical structure determined by combined thin layer and gas liquid chromatography, and a stereospecific analysis of a molecular distillate of butteroil of comparable composition. The milk fat fraction (39% of total) contained C₈–C₂₀ fatty acids which were distributed among the glycerides of 40–56 acyl carbon atoms in a manner not unlike that found for the same acids in the short chain triglycerides. Although individual triglycerides were not identified, the specific distribution of the fatty acids could best be accounted for by assuming a common pool of long chain 1,2-diglyceride precursors from which the bulk of both short and long chain triglycerides are synthesized by a stereospecific introduction of C₄–C₁₈ fatty acids in position 3 of sn-glycerol. This hypothesis is compatible with the results of stereospecific analyses of the short and long chain fractions and of the total butteroil. It is supported by the nonrandom distributions demonstrated for the molecular weights of the milk fat triglycerides of different degrees of saturation. Presented in part at the AOCS meeting, Philadelphia, October, 1966.     

Triglyceride structure of milk fats

The enantiomeric nature of the triglycerides of bovine milk fat was reinvestigated by determining the stereospecific distribution of fatty acids in rearranged butterfat, following partial hydrolysis with pancreatic lipase, and in certain molecular distillates of native butterfat, following Grignard degradation. The results with rearranged butterfat confirmed the validity of pancreatic lipase hydrolysis as a means of generating representative diglycerides from milk fat triglycerides. The Grignard degradation and lipolysis gave identical distributions for fatty acids when included as part of the assay system in the stereospecific analysis. Characteristically, butyric acid and the other short chain acids occupied the 3 position in the native butterfat, while in the rearranged oil they were distributed more or less randomly. Gas chromatographic analysis of the short chain glycerides on polyester columns allowed an effective resolution of butyryl, caproyl and caprylyl glycerides of identical numbers of total acyl carbons and double bonds. The method was especially well suited for resolution of the 2,3-diglycerides, which were recovered either as the more polar fraction from thin layer chromatography of the X-1,2-diacylglycerols, or by acetolysis of the residual phenolphosphatides resulting from phospholipase A digestion. It was shown that butyric acid in the 3 position was preferentially paired with myristic, palmitic and oleic acid in the 2 position, and palmitic and oleic acid in the 1 position, which was also characteristic of the other short chain acids. One of eight papers presented in the symposium “Milk Lipids,” AOCS Meeting, Ottawa, September 1972.     

Evaluation of <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> Lipase for the Determination of Regiodistribution in Triacylglycerols with Medium Chain Fatty Acids

The nutritional profile and rheological behaviors of lipids is both due to fatty acid composition and regiodistribution on external and internal positions of triacylglycerol. Actual methods for regiodistribution analysis having some restrictions, there is still a need for investigating a safe, simple and environmentally friendly method for the sn-2 position analysis that could especially be used for the analysis of fats containing medium and short chain fatty acids. The objective of this study was to evaluate the 1,3-selectivity and typoselectivity of Rhizopus oryzae lipase in the presence of short/medium chain fatty acids in partial hydrolysis conditions used for regiodistribution analysis. Structured triacylglycerols containing eight-carbon-chain length fatty acids in the sn -2 position were chemically synthesized using DCC/DMAP coupling agent and purification steps by flash-chromatography. The final product showed very high purity and was used as the substrate for 1,3-selectivity evaluation. Typoselectivity was assessed by investigating partial hydrolysis of equimolar blends of homogeneous TAG. This study confirmed the 1,3-selectivity of Rhizopus oryzae lipase in the hydrolysis conditions used, and revealed that this lipase was less influenced by fatty acids chain length than pancreatic lipase. Considering this, Rhizopus oryzae lipase appeared to be a good candidate for regiodistribution analysis of fats containing medium and short chain fatty acids.     

Modification of goat milk fat triglycerides by immobilized lipase

The lipid fraction of goat milk was subjected to transesterification using a commercially available immobilized lipase to decrease the amount of short- and medium-chain fatty acids (C₄–C₁₄) by enrichment of the reaction mixture with long-chain (C_18:1 and C_18:2) fatty acids. Aliquots were taken during, transesterification at different reaction times and analyzed for triglycerides and their fatty acid components. The gas chromatographic analyses of triglycerides (previously isolated by thinlayer chromatography) showed that at 6 h reaction under the experimental conditions led to the greatest reduction of the low molecular weight triglycerides (C₂₂–C₃₈) and concomitantly to the greatest increase in the higher molecular weight triglycerides (C₄₈–C₅₄). These changes correlated with the variations observed in the fatty acids of the triglyceride fraction.     

Monoenoic fatty acids in human brain lipids: Isomer identification and distribution

The carbon chain length distribution and the double bond positional isomer composition of the monoenoic fatty acids of the lipids of total human brain tissue have been determined using gas chromatography and gas chromatography/mass spectrometry of the fatty acid methyl and picolinyl esters. The even chain length monoenoic C₁₆ to C₂₈ fatty acids contain predominantly two positional isomer series, the n−7 and n−9cis homologues, whose relative proportion varies significantly with chain length. The odd chain length long-chain fatty acids consist of n−8 and n−10 isomers, whereas the odd chain length very long-chain (more than 22 carbon) fatty acids are n−7 and n−9 isomers.     

Specific distribution of fatty acids in the milk fat triglycerides of goat and sheep

The triglycerides of the fat globules of sheep and goat milk were isolated and separated into short and long chain lengths by silicic acid column chromatography. The short chain lengths comprised major triglycerides with 34–44 acyl carbon atoms and accounted for nearly 50% of the total milk fat. The long chain lengths contained major triglycerides with 40–54 acyl carbons. Stereospecific analyses of the short chain triglyceride fraction showed that of the 20–23 moles per cent of C₄−C₈ fatty acids present, at least 95% were specifically attached to the glycerol molecule in the position corresponding to carbon 3 ofsn-glycerol. The distribution of the other fatty acids (C₁₀ or greater) did not show such marked specificity for either the 1 or the 2 position. Although individual triglycerides were not identified, the specific placement of the fatty acids could best the accounted for by assuming a common pool of long chain 1,2-diglycerides which served as precursors of the bulk of both short and long chain triglycerides during milk fat synthesis. Presented in part at the AOCS Meeting, New York, October 1968.     

Composition of wax esters,triglycerides and diacyl glyceryl ethers in the jaw and blubber fats of the Amazon River dolphin (Inia geoffrensis)

The lower jaw fat of the Amazon River dolphinInia geoffrensis contains 52.8% wax ester, 44.7% triglyceride and 2.5% diacyl glyceryl ether, while its dorsal blubber fat is >98% triglyceride. Examination of the intact lipids, the derived fatty acids and the derived fatty alcohols by gas chromatography reveals that the blubber triglycerides show characteristics of freshwater fish fats, but the jaw fat lipids have several distinctive features. Jaw fat wax esters, triglycerides and diacyl glyceryl ethers are all rich in C₁₀, C₁₂ and C₁₄ fatty acids and contain no polyunsaturated acids. The fatty alcohols in the wax esters are over 90% saturated. The major carbon numbers in the jaw fat triglycerides (C₃₈–C₄₆) are considerably lower than those of the blubber triglycerides (C₄₈–C₅₄). The possible adaptation of the jaw lipids for use in the underwater echolocation process of this dolphin is discussed.     

The triglycerides of sable fish (anaplopoma fimbria). II. fatty acid distribution in triglyceride fractions as determined with pancreatic lipase

Sable fish muscle lipids were fractionated on a silicic acid column with mixtures of chloroform and methanol as eluting solvents. Three main peaks containing only triglycerides were isolated; 11 additional peaks contained phosphorous. Each of the 3 triglyceride peaks was separately fractionated into 300 fractions on silica gel columns impregnated with silver nitrate. Mixtures of petroleum ether and ethyl ether were the eluting solvents. About 25 distinct fractions were isolated from each column. The fractions were characterized for fatty acid content by gas chromatography of the methyl esters. The results showed that the fractionation did not depend upon the presence of single fatty acids but upon total unsaturation. Fatty acid distribution within each fraction was determined with the use of hog pancreatic lipase, followed by thin-layer chromatography and gas chromatography. Presented at the AOCS Meeting in Houston, Texas, 1965.     

Fatty acid composition of oil in snow crab (Chionoecetes opilio) by gas chromatography/mass spectrometry

The hepatopancreatic fatty acid extract of the snow crab contains a high percentage (26%) of odd-carbon-numbered fatty acids and a substantial quantity (29%) of methyl-branched fatty acids, as indicated by gas chromatography/mass spectrometry (GC/MS) and gas liquid chromatography (GLC). A wide distribution in chain length of the fatty acids (C₁₀ to C₂₆) and in positional isomers of the linear monoenes are also indicated by GC/MS.     

Studies in Acidolysis

Acidolysis of a natural triglyceride oil with free fatty acids of different chain length has been studied to find out the degree of interchange of fatty acids, the mode of distribution of the free acids incorporated in the triglyceride molecules and the glyceride composition of the oil after acidolysis. The results as determined by gas-liquid chromatography and pancreatic lipase hydrolysis indicate that the degree of interchange of fatty acids depends on the chain length of the free acids, and the incorporation of the free acids in the glyceride molecules occurs in a purely random manner. Glyceride compositions of the oils are also randomly rearranged after acidolysis reaction.     

Fatty Acid Specificity of Candida cylindracea Lipase

Candida cylindracea lipase (SIGMA) was tested against triglycerides (TG) and wax esters (WE) of marine origin as substrates. Under the same conditions, wax esters were hydrolysed at a lower rate than the triglycerides. The C₁₄ to C₁₈ saturated and monounsaturated fatty acids were preferentially hydrolysed whereas the longer chain monoenes (20:1 and 22:1) and particularly the polyunsaturated fatty acids (18:4,20:5 and 22:6) were resistant to the hydrolysis in triglycerides as well as in wax esters. No specificity was demonstrated for the fatty alcohols in the wax esters.     

Acid lipase of the castor bean

The acid lipase of the castor bean is present in the dormant seed. It is extracted from the fat pad obtained by centrifuging a macerate of the seed in pH 7.0 buffer containing cysteine and ethylene diaminetetraacetic acid. The pH optimum of the enzyme is 4.2; it is rather heat-stable, and is inhibited by mercurials and sulfhydryl reagents. Maximum hydrolysis of saturated triglycerides occurs with fatty acids of chain length C₄ to C₈; unsaturated C₁₈ triglycerides are hydrolyzed at a slightly lower rate. This lipase is a three-component system consisting of the apoenzyme, a lipid cofactor (a cyclic tetramer of ricinoleic acid), and a protein activator (a small, heat-stable glycoprotein which appears to be related to some of the castor allergens). Maximum lipolysis requires all three components. Lipase activity is associated with the spherosomes, the subcellular site of oil storage in the endosperm. Presented at the AOCS-AACC Joint Meeting, Washington, D.C., April 1968. So. Utiliz. Res. Dev. Div., ARS, USDA.     

Distinctive medium chain wax esters,triglycerides, and diacyl glyceryl ethers in the head fats of the pacific beaked whale,Berardius bairdi

Lipids were extracted from the mandibular fat body (jaw), the fatty forehead (melon), and the dorsal blubber of a Pacific beaked whale (Berardius bairdi) and separated into lipid classes by preparative thin layer chromatography. The head fats were mixtures of wax esters and triglycerides with a very small amount of diacyl glyceryl ether. The blubber fat contained 97% was ester and 3% triglyceride. Gas liquid chromatography (GLC) of the intact lipid classes indicated an unusually low C₂₆–C₃₀ range for most of the jaw and melon wax esters compared to the more normal C₃₂–C₄₀ molecules found in the blubber. Distinctive lower molecular weight C₂₄–C₄₀ triglycerides occurred in the head fats vs. the usual C₄₄–C₅₈ range in the blubber. Most diacyl glyceryl ethers were in the C₃₅–C₄₆ range, below the molecular weight of hexadecyldipalmitoyl glyceryl ether (C₄₈). GLC of the derived fatty acid methyl esters showed that the lower molecular weight neutral lipids in the head fats were due to high levels of iso-10∶0, n−10∶0, iso-11∶0, iso-12∶0, n−12∶0, and iso-13∶0 acids. The wax ester fatty alcohols and the alkoxy chains of the glyceryl ethers were mostly the C₁₄–C₂₀ chain lengths commonly observed in marine organisms. The distinctive medium chain neutral lipids in the jaw and melon fats of this whale may be related to the postulated acoustical role of these tissues in echolocation.     

Determination of triglyceride structure via silver nitrate-TLC

A procedure for the quantitative analysis of component triglycerides by thin-layer chromatography (TLC) with silver nitrate-silicic acid coated plates in combination with lipase hydrolysis is described. The method is demonstrated on synthetic triglyceride mixtures and applied to several natural fats. The results indicate that the fatty acids of natural fats are not distributed among the triglycerides in accordance with any simple mathematical formula and methods based on mathematical distribution patterns cannot be generally applied. Supported in part by a grant from the Special Dairy Industry Board of the National Dairy Council and U.S. Public Health Service Grant No. HE 05735.     

Distribution of individual fatty acids in the crystallization fractions of lard

Distribution of the individual fatty acids in the triglycerides of lard was determined by fractional crystallization, partial enzymatic hydrolysis with steapsin, and fatty acid analyses by GLC. It was found that none of the individual fatty acids corresponded to a random distribution in the crystallization fractions, but that the distribution of the total saturated and total unsaturated acids was very nearly random. The short chain fatty acids, C₁₄ and C₁₆, both saturated and unsaturated, were found to be more predominant in the 2-position than in the 1- and 3-positions of the lard triglycerides. All of the C₁₈ fatty acids were found to be more predominant in the 1- and 3-positions.     

Fatty alcohols

“Fatty” or higher alcohols are mostly C₁₁ to C₂₀ monohydric compounds. In probably no other homologous aliphatic series is the current balance between natural and synthetic products so vividly evident. Natural sources, such as plant or animal esters (waxes), can be made to yield straight chain (normal) alcohols with a terminal (primary) hydroxyl, along with varying degrees of unsaturation. In the past, socalled fatty alcohols were prepared commercially by three general processes from fatty acids or methyl esters, occasionally triglycerides. Fatty acids add hydrogen in the carboxyl group to form fatty alcohols when treated with hydrogen under high pressure and suitable metal catalysts. By a similar reaction, fatty alcohols are prepared by the hydrogenation of glycerides or methyl esters. Fatty alcohols are also prepared by the sodium reduction of esters of fatty acids in a lower molecular weight alcohol. The sodium reduction method was ordinarily too expensive; it was displaced early by the other methods; finally most unsaturated alcohols made by this route were largely replaced. Methyl ester reduction continues to provide perhaps 20% of the saturated fatty alcohols, and selective hydrogenation with the use of special catalysts such as copper or cadmium oxides was developed for the production of oleyl alcohol. Synthetic or petroleum technology for long chain alcohols include the Ziegler process, useful for straight chain, even-numbered saturated products. A second is the carbonylation and reduction of olefins affording medium or highly branched chain alcohols. Paraffin oxidation affords mixed primary alcohols. Fatty alcohols undergo the usual reactions of alcohols. They may be reacted with ethylene oxide to yield a series of polymeric polyoxyethylene alcohols or with acetylene under pressure to yield vinyl ethers or with vinyl acetate to give vinyl ethers.     

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.