The regiospecific position of 18∶1 cis and trans monoenoic fatty acids in milk fat triacylglycerols

TAG of butterfat were fractionated according to the type and degree of unsaturation into six fractions by silver-ion HPLC. The fractions containing TAG with either cis-or trans-monoenoic FA were collected and fractionated further by reversed-phase HPLC to obtain fractions containing cis TAG of ACN:DB (acyl carbon number:double bonds) 48∶1, 50∶1, and 52∶1 as well as trans 48∶1, 50∶1, and 52∶1. The FA compositions of these fractions were elucidated by GC. The MW distribution of each fraction was determined by ammonia negative-ion CI-MS. Each of the [M-H]⁻ parent ions was fractionated further by collision-induced dissociation with argon, which gave information on the location of cis-and trans-FA between the primary and secondary positions of TAG. The results suggest that the sn-positions of the monoenoic cis-and trans-FA depend on the two other FA present in the molecule. With 14∶0 FA in the TAG molecule, the 18∶1 FA in the sn-2 position are mostly present as cis-isomers. When there is no 14∶0 in the TAG molecule, the trans-18∶1 isomers seem to be more common in the sn-2 position. Also when other long-chain FA are present, the trans-isomers are more likely to be located in the secondary (sn-2) position.     

Stereospecific distribution of fatty acids in triacylglycerols of olive oils

The positional distribution of fatty acids (FA) in triacylglycerols (TAG) of 47 virgin olive oils from diverse cultivars grown in distinct areas of North‐Eastern Italy was studied. Few data were previously available on oils from these geographical areas. The effects of climatic and geographical conditions on the stereospecific distribution of TAG in olive oil were confirmed. Moreover, the results of the stereospecific analysis were used to evaluate the preferential esterification position of each FA on the basis of the degree of unsaturation and the chain length. The data of the stereospecific analysis of olive oil TAG can contribute to the determination of the selectivity of olive fruit acyltransferases for distinct FA.     

Determination of the positional distribution of fatty acids in butterfat triacylglycerols

Triacylglycerol (TAG) standards were separated by analytical high-performance liquid chromatography (HPLC) with laser light-scattering detection (LLSD). A high sensitivity for TAGs was observed with LLSD whereas poor sensitivity was observed with ultraviolet detection. The HPLC-LLSD analytical separation of butterfat TAGs showed that the TAGs were eluted according to increasing carbon number. Preparative HPLC-LLSD was used to characterize butterfat TAGs that contained hypercholesterolemic fatty acids (laurate, myristate, palmitate) with carbon chainlengths of 12 or greater. These TAG fractions accounted for 29.2% of the total butterfat TAGs. Analysis of the positional distribution of fatty acids of selected butterfat TAGs containing hypercholesterolemic fatty acids showed the presence of positional isomers in each of these fractions. These butterfat TAGs also showed the predominant presence of hypercholesterolemic fatty acids at thesn-2 position. The characterization of the positional distribution of hypercholesterolemic fatty acids in butterfat TAGs is the first step for the determination of the metabolic role of the positional distribution in the hypercholesterolemic effects of butter.     

The composition of new zealand milk fat triacylglycerols by reversed-phase high-performance liquid chromatography

A reversed-phase high-performance liquid chromatography (HPLC) method was developed for the purpose of analyzing milk fat triacylglycerols by evaporative light scattering detection. With a binary solvent system, comprising dichloromethane and acetonitrile, the method allowed a separation of 61 distinct peaks, on the basis of chainlength and number of double bonds. Triacylglycerols of differing partition numbers were clearly resolved, and the resolution between peaks of the same partition number was high. An argentation thin-layer chromatography method, separating triacylglycerols on the basis of chainlength and degree of unsaturation, provided nine band extracts that were analyzed by HPLC. By using existing fatty acid methyl ester data of these bands, an identity for each HPLC peak has been proposed.     

Separation of milk fat triacylglycerols by argentation thin-layer chromatography

Argentation thin-layer chromatography was investigated as a method of obtaining detailed compositional information about milk fat. A modified argentation thin-layer chromatography procedure, developed to optimize the separation of the complex mixture of total milk fat triacylglycerols, provided nine different groups of triacylglycerols, based on both the degree of unsaturation and the total length of fatty acid groups. Fatty acid methyl ester (FAME) analysis was performed to determine the composition of each band. Separation on the basis of chainlength was most pronounced among the fully saturated triacylglycerol groups, as evidenced by the high level of C_4:0 and C_6:0 in bands 7 and 8, respectively. For the cis-monoenoic triacylglycerols, the separation of C_4:0 and C_6:0 was less distinct. The cis,cis dienes and other dienoic, trienoic, or tetraenoic species were principally observed in two bands of retention factor <0.08 on the chromatography plate. Minimal cross-contamination of bands was observed, with the exception of the lowest of the trisaturate bands, band 7, in which trans-monoenes were found to be present. Three samples from different points of the New Zealand dairy season were separated by argentation thin-layer chromatography, and their FAME distributions were measured. In addition to differences in the masses of band extracts from these samples, levels of C_10:0 and C_12:0 in all bands, and levels of monounsaturates in the dienoic and trienoic bands, were found to differ. These changes were generally consistent with a pattern of decreasing fat hardness over the November to March period of a typical dairy season.     

Molecular weight distribution and regioisomeric structure of triacylglycerols in some common human milk substitutes

Triacylglycerol (TAG) molecular weight distribution and regioisomeric structure of selected molecular weight species in human milk and in 32 human milk substitutes was determined. Negative ion chemical ionization mass spectrometry was used to determine the molecular weight distribution and collisionally induced dissociation tandem mass spectrometry applied to identify the sn-2 and sn-1/3 positions of fatty acids in TAG. The main molecular weight species of human milk TAG in decreasing order of abundance were 52∶2, 52∶3, 52∶1, 54∶3, 50∶2, 50∶1, 54∶4, 48∶1, 54∶2, 48∶2, 46∶1, 52∶4, and 50∶3 (acyl carbon number/number of double bonds), constituting 83 mol% of total TAG molecular species. In human milk substitutes, the proportion of the corresponding molecular weight species varied from 33 to 87 mol%. The main TAG regioisomers within the molecular weight species 52∶2, 52∶3, and 50∶1 in human milk were 18∶1-16∶0-18∶1 (83 mol%), 18∶1-16∶0-18∶2 (83 mol%), and 18∶1-16∶0-16∶0 (80 mol%), respectively. In human milk substitutes, the corresponding proportions varied in a wide range of 0–82 mol%, 0–100 mol%, and 0–73 mol%, respectively. Although TAG structures in some human milk substitutes closely resembled those in human milk, the great variation among samples leads to the conclusion that it is still possible to improve the TAG composition in human milk substitutes by applying novel methods to synthesize structured TAG.     

Oxidative interactions of cholesterol with triacylglycerols

Triacylglycerols (TGs) accelerated the decomposition of cholesterol at 130°C. Addition of stearic and linoleic acids also accelerated cholesterol decomposition and produced characteristic cholesterol oxide profiles, qualitatively different from those produced in the presence of TGs. Milk fat accelerated cholesterol decomposition at 130°C and produced a cholesterol oxide pattern similar to that arising from the addition of pure TG. Not only did TGs affect cholesterol oxidation, but cholesterol influenced the decomposition of TGs. Addition of cholesterol accelerated the destruction of TGs at the beginning of heating while protecting them later. A similar pattern,i.e., acceleration followed by protection, could also be seen when triacylglycerols were heated in the presence of other triacylglycerols. The results of this work demonstrate that the stability of lipid components in complex mixtures is influenced by interactions among these components and/or their decomposition products. Such interactions do not merely shift,i.e., accelerate or delay, the oxidation rate, they may also modify the shape of the oxidation curve itself.     

High-performance liquid chromatography of human milk triacylglycerols and gas chromatography of component fatty acids

Human milk triacylglycerols were separated by high-performance liquid chromatography. A 5-μ Supelcosil LC-18 column (Supelco, Inc., Bellefonte, PA) was used with acetone/acetonitrile (64∶36, vol/vol) as mobile phase. Triacylglycerols were tentatively identified based on theoretical carbon number and relative retention time. Despite changes resulting from dietary fat variation, the major component triacylglycerols were those composed of palmitic, oleic and linoleic acids. Triacylglycerols with palmitic, stearic and oleic acids were present as minor components. Fatty acids were quantified by gas chromatography relative to an internal standard. Ratios of n−6/n−3 fatty acids were found to be high than previously reported. Based on a paper presented at the Symposium on Milk Lipids held at the AOCS Annual Meeting, Baltimore, MD, April 1990.     

Quantitative analysis of synthetic mixtures of triacylglycerols with fatty acids from caprylic to stearic

Separation and quantitation of triacylglycerols tricaprylin to tristearin was achieved by high-performance liquid chromatography with refractive index detection and an eluent composed of propionitrile and butyronitrile (80:20, vol/vol). Peak identification was based on the logarithms of retention times, and quantitation was achieved by way of theoretical relative response factors. The validity of the response factor calculations was tested by analysis of primary standard mixtures of saturated triacylglycerols and of triacylglycerols obtained by interesterification of binary mixtures of monoacid triacylglycerols.     

Identification of triacylglycerols in the enzymatic transesterification of rapeseed and butter oil

A blend of rapeseed and butter oil was transesterified using immobilized Thermomyces lanuginosus lipase (Lipozyme® TL IM) as catalyst. The reaction was followed by reversed-phase HPLC and the triacylglycerol peaks were tentatively identified from their elution times by calculating equivalent carbon numbers. Further identification was made using HPLC-electrospray tandem mass spectrometry. A few of the triacylglycerols detected were typical combinations of fatty acids originating from rapeseed oil, such as α-linolenic acid, and short-chain fatty acids from butter oil. Due to the regioselectivity of the lipase, the transesterification reaction involved mainly fatty acids in the sn-1 and sn-3 positions. However, significant changes in the fatty acid composition in the sn-2 position were detected after 6 h.     

Triacylglycerols of winter butterfat containing configurational isomers of monoenoic fatty acyl residues. I. Disaturated monoenoic triacylglycerols

The triacylglycerols of winter butterfat were fractionated according to the type and degree of unsaturation into six fractions by silver ion high-performance liquid chromatography (Ag-HPLC). The acyl carbon number distribution of the triacylglycerols in each fraction was elucidated by reversed-phase HPLC and mass spectrometry (MS). The MS analysis of each fraction gave deprotonated triacylglycerol [M - H]⁻ ions which were produced by chemical ionization with ammonia. The daughter spectrum of each of the [M - H]⁻ ions provided information on its fatty acid constituents. Successful fractionation of triacylglycerols differing in the configuration of one fatty acyl residue by Ag-HPLC was important because geometrical isomers could not be distinguished by the MS system used. In addition to the fatty acid compositions, reversed-phase HPLC analysis demonstrated the purity of the collected fractions: molecules having acis-trans difference were separated nearly to the baseline. Triacylglycerols differing in the configuration of one fatty acyl residue were not equally distributed in relation to their acyl carbon numbers. This indicates that during the biosynthesis of triacylglycerols,cis- andtrans-fatty acids are processed differently. Although the fatty acid compositions of the corresponding molecular weight species of disaturatedtrans- and disaturatedcis-monoenoic triacylglycerols were similar, there may be differences in the amounts of different fatty acid combinations or in the distribution of fatty acids between the primary and secondary glycerol positions. In addition to the main components, it was possible to analyze minor triacylglycerols, such as molecules containing one odd-chain fatty acid, by the MS system used.     

Triacylglycerols of winter butterfat containing configurational isomers of monoenoic fatty acyl residues. II. Saturated dimonoenoic triacylglycerols

Triacylglycerols of Finnish winter butterfat containing one saturated and two monoenoic fatty acyl residues were studied. With silver ion high-performance liquid chromatography (HPLC), molecules were separated according to the difference in the configuration of one fatty acyl moiety. The distribution of the saturatedcis,trans-dimonoenoic and saturatedcis,cis-dimonoenoic triacylglycerols according to their acyl carbon numbers was compared by means of reversed-phase HPLC and tandem mass spectrometry. Furthermore, two examples of the fatty acid composition of a specified molecular weight species were shown. The fatty acid compositions of corresponding saturatedcis,trans-dimonoenoic and saturatedcis,cis-dimonoenoic triacylglycerols were similar; however, there may be differences in the proportions of different fatty acid combinations or in the distribution of fatty acids between primary and secondary glycerol positions.     

Prediction of the goat milk fatty acids by near infrared reflectance spectroscopy

Near infrared reflectance spectroscopy (NIRS) was used to predict the goat milk fatty acid (FA) profile. The ability of cow milk broad‐based calibration equations to predict the goat milk FA profile was assessed. Three hundred twenty‐eight samples in the calibration set and 108 in the validation set were analyzed. We showed that the bias and unexplained error were significant for most of the FA despite an adequate standardized Mahalanobis distance (index to establish the boundaries of a population of samples) which allowed us to test the ability of bovine models to predict goat milk FA profiles. To better predict the goat milk FA composition, a specific goat model was investigated. The cross‐validation coefficient of determination (R²CV; proportion of variance explained by the model in cross‐validation) and residual predictive deviation (RPD; index which allows to standardize the standard error of prediction (SEP)) were >0.90 and 3, for saturated, monounsaturated, and unsaturated FA, total trans FA, isomer cis9trans11 of CLA, cis9‐, trans10‐, and trans11‐C18:1, respectively. Monitoring of the equation performance of milk FA included the calculation of the bias and unexplained error. Practical applications: In this work, we evaluated the ability of NIRS to predict FA composition of goat milk and tested the ability of using broad‐based cow milk calibration equations to monitor an independent set of goat milk samples. We tested the hypothesis that the calibration equations obtained for FA from cow milk could be applied on goat milk. As the spectra of goat milk are similar to those of cow milk when they are measured by the standardized Mahalanobis distance (index to establish the boundaries of a population of samples), we can accept the hypothesis. It will be possible to use the existing calibration equations for predicting FA profile on spectra of milk of a different specie. However, the rejection of the hypothesis would put in doubt the use of Mahalanobis distance as the unique index for testing the performance of calibration equations on different milk populations for predicting FA.     

Regional distribution in the fatty acids of triacylglycerols and phospholipids within soybean seeds (Glycine max L.)

The fatty acid distribution of triacylglycerols (TAG) and major phospholipids (PL) within soybean seeds (Glycine max L.) was investigated in relation to their tocopherol contents. The lipids extracted from four cultivars were separated by thin‐layer chromatography into seven fractions. Tocopherols were predominantly detected in the axis, followed by cotyledons and seed coat. The major lipid components were TAG and PL, while hydrocarbons, steryl esters, free fatty acids and diacylglycerols (sn‐1,3 and sn‐1,2) were also present in minor proportions. With a few exceptions, the dominant PL components were phosphatidylcholine, followed by phosphatidylethanolamine or phosphatidylinositiol. Significant differences (p <0.05) in fatty acid distribution existed when different soybean cultivars were examined. However, the principal characteristics of the fatty acid distribution in the TAG were evident among four cultivars; unsaturated fatty acids were predominantly concentrated in the sn‐2 position, and saturated fatty acids primarily occupied the sn‐1 or sn‐3 position in the oils of the soybeans.     

Lactational changes in concentration and distribution of ganglioside molecular species in human breast milk from Chinese mothers

Gangliosides play a critical role in human brain development and function. Human breast milk (HBM) is an important dietary source of gangliosides for the growing infant. In this study, ganglioside concentrations were measured in the breast milk from a cross‐sectional sample of Chinese mothers over an 8‐month lactation period. The average total ganglioside concentration increased from 13.1 mg/l during the first month to 20.9 mg/l by 8 months of lactation. The average concentration during the typically solely breast‐feeding period of 1?6 months was 18.9 mg/l. This is the first study to report the relative distribution of the individual ganglioside molecular species through lactation for any population group. The ganglioside molecular species are made up of different fatty acid moieties that influence the physical properties of these gangliosides, and hence affect their function. The GM₃ molecular species containing long‐chain acyl fatty acids had the most prominent changes, increasing in both concentration and relative distribution. The equivalent long‐chain acyl fatty acid GD₃ molecular species typically decreased in concentration and relative distribution. The lactational trends for both concentration and relative distribution for the very long‐chain acyl fatty acid molecular species were more varied. The major GM₃ and GD₃ molecular species during lactation were d40:1 and d42:1, respectively. An understanding of ganglioside molecular species distribution in HBM is essential for accurate application of mass spectrometry methods for ganglioside quantification.     

Stereospecific analysis and mass spectrometry of triacylglycerols fromarabidopsis thaliana (L.) heynh. columbia seed

Arabidopsis thaliana continues to be an excellent model organism for studying plant molecular genetics and biochemistry. In particular, the generation and analysis of mutant lines has facilitated the study of fatty acid biosynthesis, lipid bioassembly and the regulation of these processes. In view of its importance in understanding the pathways specific to seed storage lipid biosynthesis, we report here, for the first time, stereospecific and mass spectral analyses of the triacylglycerols present inA. thaliana (L.) Heynh. Columbia wild-type seed. The use of NH ₄ ⁺ -chemical-ionization mass spectrometry/mass spectrometry is described as a powerful technique in analyzing even trace amounts of individual triacylglycerol species.     

Method for characterization of triacylglycerols and fat-soluble vitamins in edible oils and fats by supercritical fluid chromatography

This study demonstrates the usefulness of capillary supercritical fluid chromatography (SFC) for the characterization of triacylglycerols of edible oils and fats. Triacylglycerols were separated according to the acyl carbon number and the degree of unsaturation on a 25% cyanopropyl/25% phenyl/50% methylpolysiloxane stationary phase. Valuable information concerning the triacylglycerol composition of berry oils was obtained, despite the overlapping of certain triacylglycerol peaks. Simultaneous analysis of fat-soluble vitamins and triacylglycerols is not practical by capillary SFC with flame-ionization detection because of the low concentration of naturally-occurring fat-soluble vitamins in edible oils. Therefore, higher loading of the sample, which led to overloading of triacylglycerols, was required to get reasonable peaks for fat-soluble vitamins. The method was applied to the characterization of triacylglycerols and tocopherols in sea buckthorn pulp and seed oil, and cloudberry seed oil without any sample purification prior to SFC. In addition, the stationary phase proved useful for separating the more complex mixtures of triacylglycerols found in milk fat and in fish oil.     

Quantitation of acyl migration during lipase-catalyzed acidolysis, and of the regioisomers of structured triacylglycerols formed

Various MLM-type (M, medium-chain fatty acids; L, long-chain fatty acids) structured triacylglycerols were produced in pilot- or small-scale packed-bed reactors by lipasecatalyzed acidolysis. The incorporation and acyl migration of octanoic acid were measured by gas chromatography and Grignard degradation, and ranged from 39.0 to 48.7% and 0.6 to 9.3%, respectively. Quantitation of triacylglycerol molecular species was performed by ammonia negative ion chemical ionization (NICI) mass spectrometry (MS). The proportion of ACN (acyl carbon number) 34 species that contained one C₁₈ fatty acid and two C_8∶0′ in samples analyzed, varied from 12.5 to 23.2%. The selected regioisomers MLM and MML within the ACN 34 species group were quantified by NICI tandem MS (MS/MS) and were in the range of 97.1 to 98.4% and 1.6 to 2.9%, respectively. There was no correlation between the level of acyl migration during lipase-catalyzed esterification and the level of regioisomers of the selected MLM-type triacylglycerols in the structured lipid samples.     

Distribution of Δ5-olefinic acids in the triacylglycerols fromPinus koraiensis andPinus pinaster seed oils

Purified triacylglycerols (TAG) fromPinus koraiensis andP. pinaster seed oils, which are interesting and commercially available sources of Δ5-olefinic acids (i.e.,cis-5,cis-9,cis-12 18:3 andcis-5,cis-11,cis-14 20:3 acids) were fractionated by reversed-phase high-performance liquid chromatography, and each fraction was examined by capillary gas-liquid chromatography for its fatty acid composition. A structure could be assigned to more than 92% of TAG from both oils. In both instances, ca. 48% of the TAG were shown to contain at least one δ5-olefinic acid. In the great majority of TAG, our data showed that there is only one molecule of δ5-olefinic acid per molecule of TAG. This is compatible with theoretical calculations based on the proportion of total δ5-olefinic in the oils. Thecis-5,cis-9,cis-12 18:3 acid (14.2 and 8.6% of total fatty acids in the seed oils ofP. koraiensis andP. pinaster, respectively) and thecis-5,cis-11,cis-14 20:3 acid (1.1 and 8.1% of total fatty acids in the seed oils ofP. koraiensis andP. pinaster, respectively) are preferentially associated with two molecules of linoleic acid, and to a lesser extent, to one molecule of linoleic acid and one molecule of oleic acid, or two oleic acid molecules. However, several other combinations occur, each in low amounts. The distribution of δ5-olefinic acids in TAG is evidently not random. Combining these results with the known preferential esterification of δ5-olefinic acids to the 1,3-positions of TAG would suggest that most of these acids are present at only one of these positions at a time.