Suitability ofGeotrichum candidum lipase for the stereospecific analysis of some triglycerides

A procedure is described for determining the stereospecific structure of triacid triglycerides containing oleic acid. The method utilizes the unique specificity of the lipase system fromGeotricum candidum for hydrolyzing fatty acids which containcis-9-unsaturation.The procedure involves a pancreatic lipase hydrolysis of 10-20 mg of substrate to determine the fatty acids in the beta-position and the incubation of another 50 mg of triglyceride withG. candidum lipase to obtain diglyceride for further treatment. The alpha,alpha- and alpha,beta-diglycerides are collected separately, converted to phenyl phosphatides and treated with phospholipase A. The analysis of the monoglycerides, produced with pancreatic lipase and the analysis of the beta-lysophosphatides allows the determination of the ratios of the original 2 position oleic acidsn-triglycerides while the analysis of the alpha-lysophosphatides aids in the determination of the originalsn-triglycerides which contained oleic acid in the 3 position. The 1 position oleic acidsn-triglycerides are calculated by difference.Racemic and enantiomeric triglycerides containing palmitic, stearic and oleic acids were synthesized and used to establish the limits of the new method. The good agreement between the actual and observed values for a mixture of isomers indicates that the procedure will be useful in the analysis of triacid triglycerides which contain oleic acid. The application of the procedure to triacid triglycerides which contain other unsaturated fatty acids is discussed.     

Intramolecular fatty acid distribution in milk fat triglycerides of monkeys

Pancreatic lipase hydrolysis was used to determine the distribution of fatty acids in the milk triglycerides of four species of monkeys and of human milk. The patterns of the major fatty acids were generally similar in all species examined, but there were some differences in the relative concentrations of individual fatty acids esterified at either the 2 or 1,3 positions. Caprylic, stearic, oleic, and linoleic acids were found predominantly at the 1,3 positions; in contrast, lauric, myristic, palmitic, and palmitoleic were concentrated at the 2 position. Monkey milk fats had greater proportions of these acids at the respective positions than did bovine milk fat. Also, the monkey fats were relatively uniform both in total unsaturated fatty acids (41–48%) and in the proportion of these esterified at the 2 position (19–26%). In general, both the fatty acid composition and the specific distribution of fatty acids in the monkey milk fats more closely resembled the patterns in human milk fat than did those in ruminant milk fats.     

Positional distribution of fatty acids in the triglycerides of bovine milk fat with elevated levels of linoleic acid

A stereospecific distribution of fatty acids in bovine milk fat containing 15.5% linoleic acid has been compared with the distribution in bovine milk fat containing a normal level (1.8%) of linoleic acid. The positional distribution was obtained by the separate analysis of milk fat triglycerides of high, medium, and low molecular weight. The order of preference for linoleic acid in the high molecular weight triglycerides was position 3>position 2 >position 1. There was an accompanying altered distribution of myristic acid and palmitic acid in favor of position 1 at the expense of position 3. However, the proportions of myristic acid and palmitic acid in position 2, relative to positions 1 and 3 were identifical in the high molecular weight fractions of the two milk fats. The distribution of linoleic acid in the medium molecular weight triglycrides of linoleic-rich milk fat was position 1=position 2>position 3. This resulted in a change in the distribution of 18 carbon monounsaturated fatty acids in favor of position 2 at the expense of position 1, but the distribution of myristic acid and palmitic acid was virtually unaltered. The distribution of linoleic acid in the low molecular weight triglycerides was position 2>position 1>position 3. The amounts of myristic acid and palmitic acid in position 2 and of palmitic acid in position 1 decreased in the low molecular weight triglycerides of the milk fat containing elevated levels of linoleic acid. Changes in the distribution of fatty acids which were observed in the separate analysis of the high, medium, and low molecular weight triglycerides were not apparent when comparing the distribution in the total milk fats. For example, the distribution of myristic acid and palmitic acid appeared to be unchanged, while the distribution of 18 carbon monounsaturated fatty acids was slightly altered in favor of positions 2 and 3. Moreover, linoleic acid showed an almost equal preference for the three positions of the glycerol moiety in milk fat containing elevated levels of this fatty acid with some concentration at position 2.     

Effect of dietary fat supplementation on the composition and positional distribution of fatty acids in ruminant and porcine glycerides

Dietary fats which were protected from ruminal metabolism were fed to ruminants, and the constituent fatty acids subsequently appeared in the glycerides of tissues and secretory products. These dietary fat induced alterations in tissue lipid composition were particularly apparent when the fat source was enriched with linoleic acid. Similarly, when pigs were fed linoleic-enriched fats, the linoleic acid was incorporated into the adipose tissue triglycerides. Stereospecific analyses were carried out on triglycerides from various tissues and secretory products obtained from animals fed control or linoleate-enriched diets. The analysis of adipose tissue triglycerides showed that linoleate and oleate were preferentially esterified to positions 2 and 3 (cattle and sheep), and positions 1 and 3 (pigs). Of the other major adipose tissue fatty acids, palmitate was preferentially esterified at position 1 (ruminants) and position 2 (pigs), and stearate was preferentially esterified at positions 1 and 3 (ruminants), and position 1 (pigs). Stereospecific analysis of high mol wt milk triglycerides showed that linoleate was either evenly distributed on all three positions (goats), or predominantly on position 3 (cows). Furthermore, the incorporation of this linoleate did not markedly alter the positional specificity of the other major milk triglyceride fatty acids. Of these fatty acids, the short and medium chain length acids (butyratelaurate) were mainly on position 3, myristate and palmitate on positions 1 and 2, and stearate and oleate evenly distributed. Thoracic duct lymph triglycerides from sheep tended to show preferential incorporation of linoleate at position 3, palmitate at position 2, and stearate at position 1 and 3; oleate, on the other hand, tended to be evenly distributed on all three positions of the lymph triglyceride. The stereospecific arrangement of fatty acids in sheep liver triglycerides was similar to that of lymph triglycerides, and this may reflect the uptake of intact or partially hydrolysed chylomicron and/or very low density lipoprotein triglycerides by the liver. There were also some analogies in the stereospecific arrangement of fatty acids on ruminant lymph and milk triglycerides and this may reflect an incomplete hydrolysis of chylomicron and/or very low density lipoprotein triglycerides prior to uptake by the mammary gland. An unusual feature of lymph from sheep fed linoleate was the presence of phospholipids which contained large amounts of linoleate in ca. equal proportion at both positions 1 and 2 of the phospholipid molecule.     

Some properties of an exocellular lipase fromRhizopus arrhizus

Rhizopus arrhizus, a mold of the mucor family, excretes an active lipase when cultured properly. This lipase has a mol wt of 43,000 and a high carbohydrate content, Upon storage at 4C in aqueous solution, lipase I is slowly converted by proteolysis to a more cationic form, lipase II, which has a lower mol wt (32,000) and no carbohydrate.Rhizopus lipase shows the same positional specificity on long chain triglycerides as pancreatic lipase; it has no preferential side chain specificity against oleic vs. palmitic acid. Like pancreatic lipase,Rhizopus lipase acts on micelles of short chain triglycerides and is inhibited by high concentrations of bile acids; however, in the presence of deoxycholate,Rhizopus lipase does not require added Ca⁺⁺ for full activity.     

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.     

Selective hydrolysis of polyunsaturated fatty acid-containing oil withGeotrichum candidum lipase

Polyunsaturated fatty acids (PUFA), especially docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), can be concentrated in glycerides by hydrolyzing tuna oil withGeotrichum candidum lipase, the main components in the resulting oil being triglycerides. The reaction mechanism of this selective hydrolysis was investigated. Although the lipase acted well on the esters of oleic, linoleic, and α-linolenic acids, it did not affect the esters of γ-linolenic acid, arachidonic acid, EPA, and DHA as much. The action of PUFA-glycerides was mono-> di- > triglycerides. Furthermore, the condensation of PUFA-partial glycerides and PUFA occurred even in the presence of a large amount of water, and the partial glycerides converted to the triglycerides by transacylation. These results suggested that the PUFA-rich triglycerides were accumulated in the glyceride fraction by the following mechanism: The PUFA-partial glycerides generated by the hydrolysis were converted to PUFA-triglycerides by condensation and transacylation reactions. As the PUFA-triglycerides formed were the poor substrates of lipase, they were accumulated in the reaction mixture.     

Digestion of butyrate glycerides by pancreatic lipase

The racemic triglycerides, glyceryl-1-palmitate-2,3-dibutyrate (PBB), glyceryl-1-butyrate-2,3-dipalmitate (PPB), glyceryl-2-butyrate-1,3-dipalmitate (PBP), and the diglyceride, racemic glyceryl-1-palmitate-3-butyrate (P-B) were synthesized and digested with pancreatic lipase. Each triglyceride was mixed with equimolar amounts of triolein (OOO) prior to incubation. The following order of digestion rates was observed: PBB>PPB>PBP>P-B. There was no evidence for short-chain fatty acid specificity; however the triglycerides containing butyric acid were hydrolyzed more rapidly than OOO. Based upon the fatty acid composition of partial glycerides, digestion of butyrate glycerides was not a simple phenomenon. For example, in the digestion of PBB, butyric acid accumulated faster than palmitic acid in the diglycerides, and monobutyrin was found to accumulate when the diglyceride, P-B, was digested. As evidenced by the fatty acid composition of the monoglycerides, positional specificity of pancreatic lipase was always maintained. Scientific contribution No. 245, Agricultural Experiment Station, University of Connecticut, Storrs. Presented in part at the AOCS Meeting, Philadelphia, October 1967.     

Studien zur Glyceridstruktur von Fetten I: Vergleichende Untersuchungen an Modellsubstanzen über den Einsatz von Pankreaslipase und Methylmagnesiumbromid zur strukturspezifischen Triglyceridspaltung

Studies on the Glyceride Structure of Fats I: Comparative Studies with Model Substances on the Use of Pancreas Lipase and Methyl Magnesium Bromide for the Stereospecific Cleavage of Triglycerides Using model substances the optimum conditions of reaction are determined for the stereospecific cleavage of triglycerides by pancreas lipase and methyl magnesium bromide. Studies with pancreas lipase showed that this enzyme having a strict positional specificity attacks the primary ester bonds (1- and 3-positions), however, unsaturated and short chain fatty acids are cleaved more easily than the saturated fatty acids. Isomerization of the partial glycerides has not been detected. Studies with methyl magnesium bromide revealed that the Grignard reagent does not have any positional specificity in the cleavage of the fatty acids. In the recommended procedure 1,2- and 1,3-diglycerides, 1- and 2-monoglycerides as well as tert. alcohols are formed. A ratio of 2:1 between 1,2- and 1,3-diglycerides correspond to the secondary positions of the triglyceride molecule. Even in this case isomerization of the partial glycerides has not been detected.     

Stereospecific analysis of triglycerides

Stereospecific analysis determines how the fatty acids of triglycerides are distributed over the three different positions of the glycerol. The special problem is the differentiation of position I-1 and L-3 of glycerol. In the presently known methods, triglycerides are first degraded to mixtures of diglycerides, either by the action of a lipase or by degradation with a Grignard reagent. The isomeric diglycerides are then resolved with the help of a stereospecific enzyme, either a diglyceride kinase or (after conversion of the diglycerides to phospholipids) a phospholipase. It is then possible to analyze or calculate the fatty acid composition for each position on the glycerol. The key to a successful stereospecific analysis is the preparation of a representative diglyceride mixture by a truly random degradation of the triglyceride. The Grignard degradation is the most reliable method, but it is not always applicable, and it is accompanied by some isomerization of glycerides. There is room for improvement in the method. Analyse of natural fats have shown most of them to be asymmetric, i.e., the composition of fatty acids in position 1 differs markedly from that of position 3. Several rules of fatty acid distribution have become apparent. Presented at the 62 nd Annual AOCS Meeting, Houston, May 1971.     

Die Verdauung von Speisefetten mit Pankreaslipasen und ihre Beeinflussung durch das Fettsäuremuster

The Influence of Fatty Acids in Triglycerides on the Digestion of Dietary Fats by Pancreatic Lipase The digestion of dietary fats by pancreatic lipase was studied in in-vitro-experiments. We tested the following fats: coconut, butterfat, cocoabutter, lard and oil of corn germ. The breakdown of triglycerides was followed up by monitoring the free fatty acids and glycerol. Additionally we analyzed the fatty acid distribution by gas-liquid chromatography of triglycerides, 1,2-diglycerides and 2-monoglycerides. Fatty acids with a chain length from C₁₀C₂₀ were determined by gas chromatography. Short chain fatty acids were not regarded separately. As pancreatic lipase has a positional specificity for the 1- and 3-position of a triglyceride there is information on the distribution of fatty acids in fats and of their digestion by such experiments. For the resorption of the fatty acids it may be of a certain importance in which position it is esterified in the fat when it is hydrolysed in gut. The hydrolysis of fats used in these experiments was quite different. This can be explained by the fatty acid distribution, the chain length and by a varying rate of emulsification of fats in an aqueous phase.     

Triglyceride composition ofSapindus mukorossi seed oil

The fatty acid composition ofSapindus mukorossi seed oil was determined by spectrophotometry, urea complexation, and gas liquid chromatography (GLC). The percentages of individual acids were found to be: palmitic, 4.0; stearic, 0.2; arachidic, 4.4; oleic 62.8; linoleic, 4.6; linolenic, 1.6; and eicosenoic, 22.4. Triglyceride composition was calculated from the fatty acid compositions of the native oil and of the monoglycerides produced from it by pancreatic lipase hydrolysis. The oil is composed of 0.1, 2.1, 22.0, and 75.8% trisaturated, monounsaturated disaturated, diunsaturated monosaturated, and triunsaturated glycerides, respectively. The special characteristic of theSapindus mukorossi seed oil is its content of 26.3 and 26.7% triolein and eicoseno-di-oleins, respectively.     

Triglyceride composition of native and rearranged butter and coconut oils

Triglyceride gas chromatography was used for quantitative fractionation by carbon number of native and rearranged butter and coconut oils. Significant differences in the triglyceride type distributions between the corresponding native and chemically modified fats were found. Increased proportions of both short and long chain triglycerides occurred in the rearranged butterfat. In reconstituted coconut oil there was a shift towards the shorter chain length triglycerides. The natural and the rearranged triglyceride distributions of the two oils were shown to differ from the random distributions calculated for the corresponding fatty acid complements. The butterfat triglyceride compositions showed greater deviations from random than did the coconut oils. The modified oils approached random distributions more closely than did the native ones. In both cases the deviations from truly random populations appeared to be in the direction anticipated on the basis of chemical reactivity.     

Triglyceride composition of tobacco seed oil

Fatty acid and triglyceride compositions of Indian tobacco seed oil (Nicotiana rustica) have been determined by combination of the techniques of systematic crystallization at low temperatures, pancreatic lipase hydrolysis, and gas liquid chromatography of methyl esters. The percentages of individual fatty acids were found to be linoleic 71.2, oleic 15.7, palmitic 8.4, and stearic 3.8. The special characteristic of the tobacco seed oil is its content of 37.4, 22.8, 4.9, 19.7, 9.3, and 3.3% of trilinolein, dilinoleoolein, dioleolinolein, dilinoleosaturated, linoleooleosaturated, and linoleodisaturated glycerides, respectively. In the present investigation, preference of linoleic acid over oleic acid for the 2-position of glycerol moiety was not observed.     

Triglyceride composition ofmadhuca butyraceae seed fat

Fatty acid and triglyceride compositions of phulwara butter (Madhuca butyraceae seed fat) have been determined by combination of the techniques of systematic crystallization at low temperatures, pancreatic lipase hydrolysis, and gas liquid chromatography of methyl esters. The percentages of individual fatty acids were found to be palmitic, 55.6; stearic, 5.2; oleic, 35.9; and linoleic, 3.3. The special characteristic of the phulwara butter is its content of POP, 52.5%; PLP, 4.9%; POSt, 8.6%; POO, 14.4% and PPP, 7.7% (P, palmitic; St. stearic; O, oleic; and L, linoleic). 2-Monoglycerides obtained by lipolysis of this fat and its least soluble fraction contained 13,0% and 29.3% saturated acids, respectively. Phulwara butter may be a potential source of palmitic acid for the pharmaceutical industry.     

Triacylglycerol structure of human colostrum and mature milk

Because triacylglycerol (TAG) structure influences the metabolic fate of its component fatty acids, we have examined human colostrum and mature milk TAG with particular attention to the location of the very long chain polyunsaturated fatty acid on the glycerol backbone. The analysis was based on the formation of various diacylglycerol species from human milk TAG upon chemical (Grignard degradation) or enzymatic degradation. The structure of the TAG was subsequently deduced from data obtained by gas chromatographic analysis of the fatty acid methyl esters in the diacylglycerol subfractions. The highly specific TAG structure observed was identical in mature milk and colostrum. The three major fatty acids (oleic, palmitic and linoleic acids) each showed a specific preference for a particular position within milk TAG: oleic acid for thesn-1 position, palmitic acid for thesn-2 position and linoleic acid for thesn-3 position. Linoleic and α-linolenic acids exhibited the same pattern of distribution and they were both found primarily in thesn-3 (50%) andsn-1 (30%) positions. Their longer chain analogs, arachidonic and docosahexaenoic acids, were located in thesn-2 andsn-3 positions. These results show that polyunsaturated fatty acids are distributed within the TAG molecule of human milk in a highly specific fashion, and that in the first month of lactation the maturation of the mammary gland does not affect the milk TAG structure.     

Lipid composition of morris hepatoma 5123c,and of livers and blood plasma from host and normal rats

The lipid composition of Morris hepatoma 5123c was analyzed together with that of liver and blood plasma from both normal and tumor-bearing rats. The results showed that the liver of tumor-bearing rats contained higher amounts of glycerides, cholesteryl esters, free fatty acids and phospholipids than the liver of normal rats. In the blood plasma of tumor-bearing rats, there was an increase of free cholesterol and triglycerides; this latter difference, however, was not statistically significant. Acyl chain changes in the liver of tumor-bearing rats consisted of an increase of palmitic and oleic acids and a decrease of stearic and arachidonic acids in phosphatidylinositol. Morris hepatoma 5123c contained a lower amount of triglycerides than the livers (both host and normal) and showed a significant decrease of total phospholipids when compared to the host liver. The major acyl chain changes found in Morris hepatoma 5123c compared with both normal and host rat livers were: a) a higher percentage of arachidonic acid together with a lower proportion of palmitic acid in cholesteryl esters; b) an increase of stearic and arachidonic acids and a decrease of palmitic acid in triglycerides; and c) a higher level of palmitic and oleic acids associated with a lower percentage of stearic and C₂₂ polyunsaturated acids in phosphatidylcholine.     

The triglyceride composition ofMyrica carolinensis fruit coat fat (bayberry tallow)

The triglycerides ofMyrica carolinensis fruit coat fat (bayberry tallow) contain only three fatty acids: myristic (21.5 mole %), palmitic (77.5%), and stearic (1.0%). The component triglycerides of this simple fat were determined by gas-liquid chromatography and pancreatic lipase hydrolysis. Triglycerides with carbon numbers 42, 44, 46, 48, and 50 were found. Lipase hydrolysis showed a preferential but not exclusive esterification of myristic acid at the 2-position. The triglyceride composition calculated from the combined experimental results did not conform to a random or 1,3-random-2-random distribution pattern. Regional differences in fatty acid and triglyceride composition within the fruit coat were also observed. Presented at the AOCS meeting in Houston, Texas, 1965.     

Synthesis and analysis of optically active triglycerides

Physical-chemical methods for steric analysis of asymmetrical triglycerides and 1,3-diglycerides have been investigated. Aliphatic triglycerides exhibit optical rotation at the D line and in the ultraviolet to 313 mμ when they contain greatly different fatty acid substituents, such as isobutyric, sorbic or others contrasted with plamitic and similars. However, optical rotation is not demonstrable with triglyceride antipodes which have only palmitic, oleic and similar component acids. Since their optical rotation is not apparent, they are called cryptoactive glycerides. Presence or absence of the piezoelectric effect permits diagnosis of antipode or racemate with crystalline tri- and diglycerides. Mixed melting point diagrams of one antipode and/or of the racemate with the triglyceride of unknown configuration can be used to identify the latter. X-ray diffraction patterns of antipode and racemic glycerides are different, and configuration assignment is possible by making comparisons. The optical rotation, α _d = +0.19°, of dolphin oil was found to be due to S(+)-methylethylacetic acid which occurs as a component acid in the triglycerides. The optical activity ofeuphorbiaceae triglycerides is discussed and some pertinent model compounds have been prepared. 2-Oleo-palmitostearin from fresh cocoa beans was found to be the racemic compound. Application of the Cahn-Ingold-Prelog R,S nomenclature to glycerides is outlined. Numerous glycerides have been synthesized in stereospecific form and their physical data are given. Some comments are made on stereospecific analysis of triglycerides without separating them.     

Chromatographic separation of lactone precursors and tentative identification of the γ-lactones of 4-hydroxy octanoic and 4-hydroxy nonanoic acids in butterfat

A combination of silicic acid column and thin layer chromatography which separates γ- and δ-hydroxy acid containing glycerides from butterfat is described. This procedure facilitates the isolation of the γ- and δ-hydroxy acids (lactone precursors) while avoiding lactonization. The method indicated that the hydroxy acids are entirely esterified to the glyceride in fresh butterfat. Hydrolysis, using pancreatic lipase, showed that these polar acids are located on the α-positions of the glycerides. The γ-lactones of 4-hydroxy octanoic and 4-hydroxy nonanoic acids were tentatively identified by gas liquid chromatography. These occurred in quantities of 0.25–0.5 ppm and approximately 0.2 ppm respectively in the butterfat samples investigated. Authorized for publication on June 22, 1966, as Paper No. 3156 in the journal series of the Pennsylvania Agricultural Experiment Station.