Stereospecific analysis of triacylglycerols rich in long-chain polyunsaturated fatty acids

Six oils of marine, algal, and microbial origin were analyzed for stereospecific distribution of component fatty acids. The general procedure involved preparation ofsn-1,2-(2,3)-diacylglycerols by partial deacylation with ethylmagnesium bromide or pancreatic lipase, separation of X-1,3- andsn-1,2(2,3)-diacylglycerols by borate thin-layer chromatography, resolution of thesn-1,2- andsn-2,3-enantiomers by chiral phase high-performance liquid chromatography following preparation of dinitrophenylurethane derivatives, and determination of the fatty acid composition by gas chromatography. Unexpected complications arose during a stereospecific analysis of triacylglycerols containing over 33% of either 20∶4 or 22∶6 fatty acids. Thesn-1,2(2,3)-diacylglycerols made up of two long-chain polyunsaturated acids migrated with the X-1,3-diacylglycerols and required separate chiral phase resolution. Furthermore, the enzymatic method yieldedsn-1,2(2,3)-diacylglycerols, overrepresenting the polyenoic species due to their relative resistance to lipolysis, but prolonged digestion yielded correct composition for the 2-monoacylglycerols. The final positional distribution of the fatty acids was established by pooling and normalizing the data from subfractions obtained by norman- and chiral-phase separation of diacylglycerols. The molecular species of X-1,3-,sn-1,2- andsn-2,3-diacylglycerol dinitrophenylurethanes were identified by chiral-phase liquid chromatography/mass spectrometry with electrospray ionization, which demonstrated a preferential association of the paired long-chain acids with thesn-1,2- andsn-2,3-diacylglycerol isomers.     

Identification of natural diacylglycerols as the 3,5-dinitrophenylurethanes by chiral phase liquid chromatography with mass spectrometry

This study reports a facile identification of the molecular species of enantiomeric diacylglycerols by combining chiral phase high-performance liquid chromatography with positive chemical ionization mass spectrometry. For this purpose the 3,5-dinitrophenylurethane (DNPU) derivatives ofsn-1,2(2,3)-diacylglycerols are separated on an (R+)-naphthylethylamine polymer column (25 cm × 0.46 cm ID) using an isocratic solvent system made up of hexane/dichloroethane/acetonitrile (85∶10∶5, by vol) or isooctane/tert-butyl methyl ether/acetonitrile/isopropanol (80∶10∶5∶5, by vol). About 1% of the column effluent (1 mL/min) was admitted to a quadrupole mass spectrometer (Hewlett-Packard, Palo Alto, CA)via direct liquid inlet interface, and positive chemical ionization spectra were recorded over the range of 200–900 mass units. The DNPU derivatives of diacylglycerols yield characteristic [M-DNPU]⁺ and [RCO+74]⁺ ions for each diacylglycerol species from which the molecular weight and exact pairing of fatty acids can be unequivocally obtained. The characteristic ions appear to be generated in nearly correct mass proportions as indicated by preliminary quantitative comparisons. The abbreviations 14∶0, 16∶1, 18∶2, etc. represent normal chain fatty acids of 14, 16, 18, etc. acyl carbons and 0, 1, 2, etc. double bonds, respectively; 16∶0–18∶1, etc. represent diacylglycerols containing 16∶0 and 18∶1 fatty acids of unspecified positional distribution;sn indicates stereospecific numbering of glycerol carbons;sn-1,2-diacylglycerols andsn-2,3-diacylglycerols are enantiomeric diacylglycerols of unspecified fatty acid composition;rac-1,2-diacylglycerols are racemic diacylglycerols representing equal amounts ofsn-1,2-andsn-2,3-enantiomers;sn-1,2(2,3)-diacylglycerols are a mixture ofsn-1,2-andsn-2,3-diacylglycerols of unspecified proportion of enantiomers and unspecified fatty acid compisition and positional distribution; X-1,3-diacylglycerols are diacylglycerols of unspecified fatty acid composition and reverse isomer content.     

Studies of triacyglycerol structure of very low density lipoproteins of normolipemic subjects and patients with type III and type IV hyperlipoproteinemia

The triacylglycerols of very low density lipoproteins (VLDL-TG) were analyzed in samples from normal subjects and patients with Frederickson’s Type III and Type IV hyperlipoproteinemia. VLDL were obtained by conventional ultracentrifugation, and the triacylglycerols were isolated by thin-layer chromatography (TLC). Representative sn-1,2(2,3)- and sn-1,3-diacylglycerols were generated by Grignard degradation of the triacylglycerols, and were resolved by TLC on borate-treated silica gel. The molecular association of the fatty acids in the diacylglycerol moieties was determined by gasliquid chromatography with mass spectrometry (GC/MS) of the tertiary-butyldimethylsilyl ethers. The positional distribution of the fatty acids was established by the Brockerhoff stereospecific analysis. The results showed a marked asymmetry in the distribution of the fatty acids in all samples, with the saturated acids predominantly in the sn-1-position and the unsaturated fatty acids distributed about equally between the sn-2- and sn-3-positions. In all instances, the molecular species composition of the sn-1,2-, sn-2,3- and sn-1,3-diacylglycerols was found to be similar to that calculated for 1-random 2-random 3-random distribution of triacylglycerols. There were marked differences in the quantitative composition of the molecular species of the VLDL-TG between normal subjects and patients, but these discrepancies were attributed to differences in the fatty acid composition of the samples.     

Milk fat structure of a patient with type 1 hyperlipidemia

Structural analyses were performed on milk fat samples obtained 3–10 days postpartum from a lactating patient with primary Type 1 hyperlipidemia. The milk triacylglycerols contained 3–7% C₁₀, 14–21% C₁₂, 20–30% C₁₄, 22–26% C₁₆ and 20–30% C₁₈ (largely oleic) acids. Gas liquid chromatographic (GLC) analyses of the X-1,3- and X-1,2-diacylglycerols on polar siloxane columns showed a markedly non-random association of acyl chains. Stereospecific analyses indicated that the short chain length fatty acids were confined essentially to the sn-3-position of the triacylglycerol molecule. Furthermore, these acids were largely absent from the phosphatidylcholines and the endogenous sn-1,2-diacylglycerols of the milk fat. It is concluded that the short chain fatty acids are incorporated into the milk triacylglycerols during the final stage of biosynthesis via the phosphatidic acid pathway, and that the overall fatty acid distribution is consistent with the 1-random 2-random 3-random hypothesis.     

Evidence for acyl transfer reactions between neutral glycerolipids in dogfish (Squalus acanthias) serum

Radioactively labeled triacylglycerols, 1,3-dioleoyl-2-[1-¹⁴C]-palmitoylglycerol and 1,3-[9,10-³H]-dioleoyl-2-palmitoylglycerol, were incubated with dogfish (Squalus acanthias) serum for periods of up to 10.0 hr. Changes in the positional distributions of carbon-14 and tritium within the triacylglycerols in 5.0 hr and 10.0 hr indicate that intermolecular and intramolecular shifts occur among the fatty acids. In addition, a maximum of 8.3% of the carbon-14 and 5.9% of the tritium was incorporated into 1-alkyl-2,3-diacylglycerols; essentially all of this incorporated radioactivity was associated with the acyl chains in the 1.5 and 5.0 hr incubations. In the 10.0 hr incubations, however, 25% of the tritium incorporated into the 1-alkyl-2,3-diacylglycerols was associated with the 0-alkyl chains. Radioactivity was not incorporated significantly into free fatty acids in the 1.5 and 5.0 hr incubations. These results indicate that acyl transfer reactions take place among molecular species of triacylglycerols, as well as between triacylglycerols and 1-alkyl-2,3-diacylglycerols. In the latter case, the conversions appear to be operative in the virtual absence of net biosynthesis.     

Comparative studies of triacylglycerol structure of very low density lipoproteins and chylomicrons of normolipemic subjects and patients with type II hyperlipoproteinemia

The triacylglycerols of very low density lipoproteins (VLDL) and of chylomicrons were analyzed in the fasting and postabsorptive states from normolipemic subjects and patients with Frederickson's Type II hyperlipoproteinemia, who subsisted on free choice diets, standard diets excluding lard, or were given a breakfast enriched in lard. The VLDL and chylomicrons were obtained by conventional ultracentrifugation, and the triacylglycerols were isolated by thin-layer chromatography (TLC). Representative sn-1,2, 2n-2-3- and sn-1,3-diacylglycerols were generated by partial Grignard degradation of the triacylglycerols and a stereospecific hydrolysis by phospholipase C of the mixed sn-1,2(2,3)-diacyl phosphatidylcholines prepared as intermediates. Representative sn-2-acylglycerols were obtained by hydrolysis with pancreatic lipase. Positional distribution of the fatty acids was established by subtracting in turn the fatty acid composition of the sn-2-position from the fatty acid composition of the sn-1,2- and sn-2,3-diacylglycerols. The molecular association of the fatty acids in the diacylglycerol moieties was determined by gas-liquid chromatography with mass spectrometry (GC/MS) of the tertiary-butyldimethylsilyl (t-BDMS) ethers. The molecular association of the fatty acids in the triacylglycerols was determined by 1-random 2-random 3-random calculation following experimental validation of the distribution. The results confirm a marked asymmetry in the positional distribution of the fatty acids in all triacylglycerol samples, with the palmitic acid predominantly in the sn-1-position, the unsaturated acids about equally divided between the sn-2-and sn-3-positions, and the stearic acid divided about equally between the sn-1- and sn-3-positions. The overall structure of the VLDL and chylomicron triacylglycerols from patients and control subjects was characterized by a noncorrelative distribution of fatty acids under all dietary conditions.     

Determination of molecular species of oil triacylglycerols by reversed-phase and chiral-phase high-performance liquid chromatography

A method is described for the determination of molecular species of oil triacylglycerols. The method is based on the analytical separation of the enantiomericsn-1,2-andsn-2,3-diacylglycerols, derived from triacylglycerols, by high-performance liquid-chromatography (HPLC) on a chiral column containing N-(R)-1-(α-naphthyl)ethylaminocarbonyl-(S)-valinecarbonyl-(S)-valine as stationary phase. Model triacyl-glycerol molecules comprising three known fatty acids were isolated from peanut oil and cottonseed oil by a combination of argentation-TLC and reversed-phase HPLC and submitted to partial chemical deacylation. The derivedsn-1,2(2,3)-diacylglycerols were analyzed and fractionated as 3,5-dinitrophenyl urethane derivatives by reversed-phase HPLC according to chainlength and unsaturation. From thesn-1,2(2,3)-diacylglycerol composition and the diacylglycerolsn-1,2-andsn-2,3-enantiomer composition, the individual molecular species of four peanut oil triacylglycerols and one cottonseed oil triacylglycerol were identified and quantitated. The method can be applied to triacylglycerols of any other oil or fat.     

Determination of positional distribution of short-chain fatty acids in bovine milk fat on chiral columns

The positional distribution of acetic and butyric acids in bovine milk fat triacylglycerols was determined by chiral-phase high-performance liquid chromatography (HPLC) of the derived diacylglycerols. Enriched fractions of acetic and butyric acid-containing triacylglycerols were isolated by normal-phase thin-layer chromatography (TLC) from a molecular distillate of butter oil, and they were fully hydrogenated. Mixedsn-1,2(2,3)- andX-1,3-diacylglycerols of short- and long-chainlength, which were generated by partial Grignard degradation of the hydrogenated triacylglycerols, were isolated by borate-TLC. The enantiomericsn-1,2-andsn-2,3-diacylglycerols and theX-1,3-diacylglycerols as their 3,5-dinitrophenylurethanes were resolved by HPLC on chiral columns. Both acetic and butyric acids were exclusively associated with thesn- 2,3- andX-1,3-diacylglycerols of short and long chainlength. These results establish the presence of acetic and butyric acids in thesn-3-position of bovine milk fat triacylglycerols. Other short-and medium-chainlength acids were found in progressively increasing proportions also in thesn-1- andsn-2-positions.     

Regioselective analysis of triacylglycerols by lipase hydrolysis

A modified procedure for the regiospecific analysis of triacylglycerols (TAG) with a 1,3-specific lipase is described. After partial lipase hydrolysis of the triacylglycerol, the released free fatty acids (FFA) and 1,2(2,3)-diacylglycerols (DAG) were isolated by thin-layer chromatography (TLC) and converted to fatty acid methyl esters (FAME). The FAME were analyzed by gas-liquid chromatography (GLC). The 1,3-specific lipases used in this study included supported preparations from strains ofMucor miehei andRhizopus oryzae. The method also was applied to the regiospecific analyses of tung nut and Chinese melon seed oil triacyglycerols, both of which contain high proportions of α-elaeostearic acid. The TAG composition of the oils was substantiated in parallel analysis of the oils by highperformance liquid chromatography with chemical ionization mass spectrometric detection of intact TAG.     

Preparation of 1,3-diacylglycerols and 1-alkyl-3-acylglycerols in the presence of quaternary ammonium salt

A series of symmetrical and racemic 1,3-diacylglycerols and 1-alkyl-3-acylglycerols were prepared simply, and in high yields, by the reaction of carboxylic acids with glycidyl esters or glycidyl ethers in the presence of quaternary ammonium salt. The effect of reaction conditions on the yield, the ratio of 1,3- and 1,2-diacylglycerols, and the stability of 1,3-diglycerides are also discussed.     

Rapid separations of diacyl- and dialkylglycerol enantiomers by high performance liquid chromatography on a chiral stationary phase

Rapid and practical separations of 1,2-and 2,3-diacyl-and dialkyl-sn-glycerol enantimers as their 3,5-dinitrophenylurethane derivatives were carried out by normal-phase high performance liquid chromatography on a chiral stationary phase, N-(R)-1-(α-naphthyl)ethylaminocarbonyl-(S)-valine chemically bonded to γ-aminopropyl silanized silica. Complete separations of the racemates into enantiomers were achieved for both of the diacyl-and dialkylglycerols within 10 min using a stainless steel column (25 cm long) packed with the 5-μ particles, an isocratic elution with a mixture of hexane/ethylene dichloride/ethanol as a mobile phase and a UV detector. Thesn-1,2-enantiomers were eluted ahead of the correspondingsn-2,3-enantiomers. Satisfactory separation of thesn-1,3-diacylglycerols from the corresponding enantiomers and the separation of the homologues differing in acyl and alkyl groups were also observed. The formations of hydrogen bonding and charge transfer complex between the urethane derivatives and the stationary phase may contribute to the enantiomer separations.     

Reversed-Phase high-performance liquid chromatographic analysis of triacylglycerol autoxidation products with ultraviolet and evaporative light-scattering detectors

The oxidation products of triacylgycerol standards (trilinolenin, trilinolein and triolein) and a natural mixture of rapeseed oil triacylglycerols were analyzed with a reversed-phase high-performance liquid chromatographic method. The oxidation products were detected with ultraviolet (UV) and evaporative light-scattering detectors (ELSD). The chromatographic profiles obtained with these two detectors were similar for all samples except triolein. ELSD is a mass detector and can detect the oxidation products of triolein and other compounds that do not have conjugated dienes in their structure. The two detectors can be used in series. The sensitivity of ELSD approached that of the UV detector used. ELSD seems to be a good universal detector type for monitoring autoxidation products of edible oils.     

Stereochemical course of diacylglycerol formation in rat intestine

The biosynthesis of diacylglycerols from 2-monoacylglycerols and free fatty acids was examined in evert sacs of rat intestinal mucosa. By means of alternate labeling of the monoacylglycerols and fatty acids, and conventional stereo-specific analysis, it was shown that the main products of synthesis were thesn-1,2-diacylglycerols (53–63%), butsn-2,3-diacylglycerols (37–47%) were also formed in significant amounts. The total yield and proportions of the isomeric diacylglycerols recovered appeared to vary with the nature of the monoacylglycerol and the complexity of the free fatty acid mixture supplied.     

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.     

Lipid methodology — chromatography and beyond. Part II. GC/MS,LC/MS and specific enzymic hydrolysis of glycerolipids

The combination of specific enzymic degradation with GC/MS or LC/MS identification and quantitation of enantiomeric diacylglycerols and reverse isomers has greatly improved the methods of structural analysis of triacylglycerols, so that in many instances complete characterization of both major and minor species is possible. The techniques described for the analysis of triacylglycerols and sn-1,2- and sn-2,3-diacylglycerols are also applicable to the X-1,3-diacylglycerols and X-1-monoacylglycerols following coversion to triacylglycerols by acylation with appropriate fatty acids. For many applications, however, a combination of specific enzymic hydrolysis with a GLC analysis of the products on polar capillary columns may be adequate for the identification and quantitation of the major molecular species of both triacylglycerols and diacylglycerols.     

Enzymatic esterification of glycerol I. Lipase-catalyzed synthesis of regioisomerically pure 1,3-sn-diacylglycerols

Regioisomerically pure 1,3-sn-diacylglycerols are conveniently prepared in high yields (>80%) and in large quantities by enzymatic esterification of glycerol in the presence of various 1,3-selective lipases(Chromobacterium viscosum, Rhizopus delemar, Rhizomucor miehei) and a variety of different acyl donors like free fatty acids, fatty acid alkyl esters and vinyl esters. All reactions are carried out in aprotic organic solvents of low water content, namelyn-hexane, diethyl ether or tBuOMe. The creation of an artificial interphase between the solvent-immiscible hydrophilic glycerol and the hydrophobic reaction media by the adsorption of glycerol onto a solid support prior to use was essential for the success of these transformations. The effects of reaction conditions and the regioselectivities of the lipases on the product yields are described in detail.     

High-Resolution NMR Spectroscopy: An Alternative Fast Tool for Qualitative and Quantitative Analysis of Diacylglycerol (DAG) Oil

Multinuclear (¹H, ¹³C, ³¹P) and multidimensional NMR spectroscopy was employed for the analysis of diacylglycerol (DAG) oil and the quantification of its acylglycerols and acyl chains composition. A number of gradient selected two dimensional NMR techniques (TOCSY, HSQC-DEPT, HSQC-TOCSY, and HMBC) facilitated the assignment of the complex one dimensional ¹H- and ¹³C-NMR spectra. In several cases, the aforementioned 2D-NMR techniques offered solid proof of earlier assignments based on chemical shift changes induced by the presence and relative positions of double bonds within the acyl chains. Integration of the appropriate signals in the NMR spectra of the three nuclei allowed the determination of DAG oil composition, which was found to be within the limits accepted for this oil, namely 1-monoacylglycerols 0.40–0.60%; 2-monoacylglycerols 0.40–0.50%; 1,3-diacylglycerols 57–62%; 1,2-diacylglycerols 28–32%; triacylglycerols 9–11%; saturated fatty acids 3–5%; oleic acid 37–45%; linoleic acid 49–53%; and linolenic acid 5–6.5%; tocopherols 0.24–0.27%. The compositional results obtained from the NMR spectra of the three nuclei were compared and discussed in terms of repeatability and ease of performance.     

Crystallization and Polymorphism of 1,3-Acyl-Palmitoyl-<Emphasis Type="Italic">rac</Emphasis>-Glycerols

Crystallization and melting behavior, small-angle X-ray scattering, X-ray powder diffraction and infra-red absorbance were measured for nine 1,3-acyl-palmitoyl-rac-glycerols (1,3-acetoyl-, -butyroyl-, -hexanoyl-, -octanoyl-, -decanoyl-, -lauroyl, -myristoyl- and -oleoyl-palmitoyl-rac-glycerol and 1,3-dipalmitoyl-glycerol). All but one of the prepared 1,3-diacylglycerols (1,3-DAG) were β-stable with 1,3-acetoyl-palmitoyl-rac-glycerol being the exception (β′-stable). Small-angle X-ray scattering indicates that molecules in β-tending diacid 1,3-DAG adopt a herringbone-type configuration similar to monoacid 1,3-DAG. In this configuration acyl chains of the same length associate and regular chain-end matching between terminal methyl groups delineate lamellae. In contrast, molecules in crystalline 1,3-acetoyl-palmitoyl-rac-glycerol are oriented similar to those of 1(3)-monoacylglycerol. Interestingly, DSC curves indicate five of the nine diacid compounds have meta-stable forms—suggesting these forms are quite common for diacid 1,3-DAG. Meta-stable forms are observed in the melting curve when the difference in length between acyl chains is large (1,3-acetoyl-, -butyroyl- and -hexanoyl-palmitoyl-rac-glycerol), and in the crystallization curve when the difference is moderate (1,3-decanoyl- and -lauroyl-palmitoyl-rac-glycerol).     

The purification and specificity of a lipase fromVernonia anthelmintica seed

Acetone powders prepared fromVernonia anthelmintica seed catalyzed the release of 6.4 to 9.6 μ-moles of free fatty acids per milligram of protein when blended with olive oil and phosphate buffer and shaken for 20 min at 43 C. A 20 fold purification was achieved by differential centrifugation of an ammonium hydroxide extract of the acetone powder. Results from Sephadex G-200 chromatography and polyacrylamide gel electrophoresis suggested that the lipase activity was associated with a molecule of molecular weight greater than 200,000. Free fatty acids, 1,2- and 1,3-diglycerides, monoglycerides and glycerol were found in the digestion products. With most substrates the 1,2-to 1,3-diglyceride ratio was approximately 2∶1 and monoglycerides tended to accumulate. Analysis of the digestion products from synthetic triglycerides of known structure indicated that both primary and secondary ester positions of the triglyceride molecule were hydrolyzed and that considerable isomerization of 1,2-diglyceride to 1,3-diglyceride occurred. The monoglyceride was consistently lower than the 1,2-diglyceride and in the majority of cases also lower than the 1,3-diglyceride in the fatty acid originally present in the 2 position of the triglyceride. No fatty acid preference was observed. Scientific contribution No. 316. Presented in part at the AOCS Meeting, Philadelphia, October 1966.     

Distribution of the major fatty acids of human milk betweensn-2 andsn-1,3 positions of triacylglycerols

Human milk triacylgycerols (TAG) were analyzed by tandem mass spectrometry. The SIMPLEX method and a simple linear model were used to interpret the distribution of fatty acids between thesn-2 andsn-1,3 positions in 24 major molecular weight groups of TAG. The number of regio-isomeric pairs of TAG varied between 3 and 18 in each of these groups. Hexadecanoic (16∶0), tetradecanoic (14∶0) and dodecanoic acids (12∶0) typically occupied thesn-2 position in TAG containing less than 54 acyl carbons, whereas long-chain C18 and C20 acids were predominantly located at the primary positions. The positions of the three fatty acids within a TAG molecule were shown to depend on the fatty acid combination. The maximum of 12∶0 in thesn-2 position appeared at acyl carbon number (ACN) 48, the maxima of 14∶0 were at ACN 44 and ACN 50, and for 16∶0 at ACN 46 and 52.