R. A. Barford

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.

Direct conversion of lipid components to their fatty acid methyl esters

SummaryMethyl esters were prepared from cholesteryl esters, phospholipids, and glycerides in substantially quantitative yields by methanolysis with large excess of sodium or potassium methoxide in absolute methanol.A silicic acid chromatographic adsorption column technique was described, which was effective in separating methyl esters from unsaponifiables such as sterols, pigments, etc., and free acids.Conditions for complete methanolysis of glyceride fats and oils requiring only 5 min. of reflux time were described.Quantitative conversion of fatty acids to methyl esters was accomplished by direct esterification with absolute methanol containing 4% HCL or H2SO4 and by methylation with diazomethane.

A rapid and quantitative procedure for the preparation of methyl esters of butteroil and other fats

A simple and convenient method for the quantitative preparation of methyl esters of fatty acids from glyceride fats and oils is described. The procedure, using potassium methylate as catalyst and a heating interval of 2 min at 65C in a closed vial, is applicable to fats containing both low and high molecular weight fatty acids such as butteroil. The methyl esters of samples ranging from a few mg to 30 mg are isolated by CS2 extraction and a TLC technique. A similar procedure using sulfuric acid in methanol as catalyst is described for the conversion of free fatty acids to methyl esters. For the routine analysis by GLC of fats and oils such as lard, tallow, soybean, cottonseed oil or butteroil, no isolation of the methyl ester is required. A CS2 extraction carried out in the reaction vial allows the GLC analysis immediately after the reaction period (2 min).

Fatty acids of cows' milk. A. Techniques employed in supplementing gas-liquid chromatography for identification of fatty acids

Milk fat methyl esters were subjected to distillation and silicic acid column chromatography to provide fractions of less complexity for gas-liquid chromatographic analysis. It was still necessary however to employ supplemental techniques for identification. Chromatograms were obtained with polyester columns of different polarity on all the fractions and necessary reference samples. While many of the components were identified in the usual way by plots of relative retention time versus number of carbon atoms, iodine values for total unsaturation and ultraviolet spectrophotometry for conjugated and nonconjugated polyunsaturated acids were essential for positive identification of some components. Similarly, examination by infrared spectrophotometry confirmed the presence or absence of conjugated diene ascis-trans, trans-trans or both. Isolatedtrans or terminal double bonds were also determined in this way. Gas-liquid chromatograms of some fractions showed incompletely resolved peaks attributable to the presence of methyl esters of odd-carbon atom, branched-chain, and unsaturated acids. Hydrogenation and rechromatographing provided more positive determination of the structure of these components. Further confirmation of identity of some peaks on the chromatogram was achieved by collection of the appropriate fractions and examination of the collected material. At least 60 fatty acids were identified, including several not previously reported, such as odd-numbered carbon chain length monoethenoid acids from C15 to C23.

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.

Alcoholysis ofVernonia anthelmintica seed oil and isolation of methyl epoxyoleate

The methyl esters ofVernonia anthelmintica seed oil were prepared without oxirane destruction by methanolysis using potassium methylate as catalyst. A single crystallization of the methyl esters from petroleum ether at −60C was found to be a convenient means of preparing large samples of methyl epoxyoleate of 93.6% purity in 92% of theory yield as based on oxirane titration. Further purification was achieved by recrystallization from petroleum ether at −40C to give a product at 97% purity in 54% of theory yield. Gas-liquid chromatography and thin layer chromatography along with associated techniques were employed to obtain the fatty acid composition of the crystallization fractions. Fifteen fatty acid methyl esters, including the methyl 12,13-epoxyoleate were identified.

Fatty acids of lard. A. Identification by gas-liquid chromatography

AbstratA fresh commercially rendered lard was separated into its component fatty acids by subjecting the methyl esters to gas-liquid chromatography on nonpolar and polar columns. The lard methyl esters were first chromatographed on a silicone column. This column achieved separation of the component esters principally on the basis of chain length, and fractions which were represented by a single peak or series of peaks were collected. The collected fractions were then rechromatographed on an ethylene glycol succinate polyester column to separate and identify the saturated and the unsaturated esters. Qualitative evidence was obtained for the presence of 29 fatty acids ranging in chain length from 10 to 20 carbon atoms. Included were the esters of the following: Satrated acids (10,12,14,15,16,17,18,19,20, and 22 C-atoms), monounsaturated acids (14,16,17,18,19, 20, and 22 C-atoms), and polyunsturated acids (18∶2,20∶2,22∶2,18∶3,20∶3,22∶3,20∶4,22∶4,20∶5, and 22∶5).3. Peaks for several additional trace components were also observed.

The hydrolysis of long chain trisaturated triglycerides by pancreatic lipase

groups), 5.8 and 4.8 r (glycerol protons). Peak ratios were as expected for a saturated monoaeetotriglyceride. (I) showed specific optical rotations in a Cary recording spectropolarimeter (hexane, 25 ~ , c = 4) of --0.5 ~ at the sodium D line, --0.8 ~ at 450 mr*, and --1.9 ~ at 350 m~. Hydrogenation of the material did not alter the rotation. On transesterifieation of the aceto-triglyeeride in the spectropolarimetrie cell with 5% tiC1 (anhydrous) in methanol:hexane (4:1), the specific rotation at 350 m~ decreased from +2.3 ~ at 15 rain to +1.8 ~ at 45 rain, +0.6 ~ at 120 rain, and 0.0 ~ at 195 rain. At the end of this time, TLC showed the presence of only methyl esters and glycerol. The disappearance of optieal activity with the elimination of the chiral center in the glycerol, in conjunction with the other data presented, precludes the presence of any other chiral centers previously reported in triglyeerides optically active because of asymmetry in a fatty acid moiety. Comparison with synthetic products of known configuration shows that the natural material is essentially all (S)-a-aceto-triglyceride. This work will be described later in greater detail.

A satisfactory glc column for the determination of epoxyoleic acid in seed oils

A number of stationary phases were evaluated for their suitability for the analysis of methyl epoxyoleate. Ethylene glycol succinate polyester, Carbowax 20M and Apiezon “L≓ all showed evidence of decomposition or alteration of the compound and also a loss of area on the chromatogram. A silicone rubber (SE-30) column did not exhibit this tendency and was found satisfactory for the determination of methyl epoxyoleate.A limitation of the use of an SE-30 column in the analysis of mixed esters is its inability to separate effectively fatty acid esters of the same chain length. However, by chromatographing the sample on a polyester column as well as on an SE-30 column, all of the fatty acid components of the oil can be determined quantitatively.

Observations on the effect of cholesterol and fat supplementation on the composition of rabbit liver and plasma lipides

Abstract 1. 1. A study has been made of liver and plasma lipides from rabbits fed commercial chow, chow supplemented with beef tallow and cholesterol, and chow plus corn oil and cholesterol. 2. 2. Animals fed the supplemented diets showed substantial increases in liver and plasma total lipides, particularly in cholesteryl esters; those receiving tallow and cholesterol supplements showed the greatest increases. 3. 3. Monoenoic acid was the principal fatty acid of the liver and plasma cholesteryl esters from rabbits on augmented diets. This was particularly true in animals receiving tallow and cholesterol supplementation. 4. 4. The fatty acid composition of the liver phospholipides was relatively uniform, whereas that of the plasma showed large increases in each fatty acid in the supplemented groups. The amounts of dienoic and tetraenoic acids were greatest in the plasma phospholipides of the tallow-cholesterol supplemented animals.