The digestion of dietary triacylglycerols

Dietary triacylglycerols (TAGs) are the major lipid components in the human diet and they are carriers of energy as well as important fatty acids. Many factors affect the digestion and absorption of TAGs. Evidence is accumulating that, in addition to the overall fatty acid profile, the TAG structure and the species composition are of importance when considering the nutritional effects of a dietary fat. There is good evidence that in addition to its short-term effects in the intestine on absorption of fatty acids the TAG structure also has long-term effects resulting from differences in the profile of absorbed fatty acids. Observations on the different atherogenic potential of dietary fats have given us a clear indication of the importance of the TAG structure for absorption of saturated fatty acids. In this context, one may focus on the effects of the structure of dietary fats as such, or one may speculate additionally on the possibilities of modifying the structure of fats to affect their absorption and the distribution of the fatty acids in the body after digestion and uptake. In this review we will summarize diverse aspects of TAG digestion and absorption, as well as the influences of the fatty acid composition and the intramolecular structure of dietary TAGs on their digestion and absorption.     

Chemistry and liquid chromatography methods for the analyses of primary oxidation products of triacylglycerols

Abstract

Triacylglycerols (TAGs) are one of the major components of the cells in higher biological systems, which can act as an energy reservoir in the living cells. The unsaturated fatty acid moiety is the key site of oxidation and formation of oxidation compounds. The TAG free radical generates several primary oxidation compounds. These include hydroperoxides, hydroxides, epidioxides, hydroperoxy epidioxides, hydroxyl epidioxides, and epoxides. The presence of these oxidized TAGs in the cell increases the chances of several detrimental processes. For this purpose, several liquid chromatography (LC) methods were reported in their analyses. This review is therefore focused on the chemistry, oxidation, extraction, and the LC methods reported in the analyses of oxidized TAGs. The studies on thin-layer chromatography were mostly focused on the total oxidized TAGs separation and employ hexane as major solvent. High-performance LC (HPLC) methods were discussed in details along with their merits and demerits. It was found that most of the HPLC methods employed isocratic elution with methanol and acetonitrile as major solvents with an ultraviolet detector. The coupling of HPLC with mass spectrometry (MS) highly increases the efficiency of analysis as well as enables reliable structural elucidation. The use of MS was found to be helpful in studying the oxidation chemistry of TAGs and needs to be extended to the complex biological systems.     

Effects of palm oil and transesterified palm oil on chylomicron and VLDL triacylglycerol structures and postprandial lipid response

The effects of positional distribution of triacylglycerol (TAG) fatty acids to TAG structures in chylomicrons and VLDL, and to postprandial lipemia, were studied in 10 healthy premenopausal women using a 6-h oral fat load test and a randomized, double-blind cross-over design. Molecular level information of TAG regioisomerism was obtained with a tandem mass spectrometric method. The positional distribution of fatty acids in chylomicron TAGs was similar to the respective dietary fat; 79% of the analyzed regioisomers in palm oil and 84% of the analyzed regioisomers in transesterified oil were found in chylomicron TAGs 3 h after the oral fat loads. VLDL TAGs were equal after the two fat loads in all but one regioisomer. Similarities in the fatty acid compositions of chylomicron TAGs suggest that palmitic acid was absorbed equally from both test fats. The proportion of palmitoleic acid in the chylomicrons was increased. Fat with palmitic acid predominantly in the sn-1 and sn-3 positions caused a larger incremental area of total TAGs in plasma and reduced plasma insulin values at the beginning of the postprandial response (0-90 min) compared with fat with palmitic acid randomly distributed. The relationship between TAG molecular structures in dietary fats and in lipoproteins provides new means for understanding the effects of fatty acid positional distribution on human lipid metabolism.     

Triacylglyceride measurement in small quantities of homogenised insect tissue: comparisons and caveats

Triacylglycerides (TAGs) are the most important stored energy reserve in eukaryotes and are regularly measured in insects. Quantitative analysis of TAGs is complicated by their diversity of structure, and there are concerns with the quantitative accuracy of commonly used analytical methods. We used thin layer chromatography coupled to a flame ionisation detector (TLC-FID), an accurate method that is not sensitive to saturation or chain length of fatty acids, to quantify TAG content in small amounts of insect tissue, and used it to validate three high-throughput lipid assays (gravimetric, vanillin, and enzymatic). The performance of gravimetric assays depended on the solvent used. Folch reagent (chloroform: methanol 2:1 v/v) was a good index of TAG content, but overestimated lipid content due to the extraction of structural lipid and non-lipid components. Diethyl ether produced reasonable quantitative measurements but lacked precision and could not produce a repeatable rank-order of samples. The vanillin assay was accurate both as a quantitative method and as an index, preferably with a standard of mixed fatty acid composition. The enzymatic assay did not accurately or precisely quantify TAGs under our assay conditions. We conclude that the vanillin assay is suitable as a high-throughput method for quantifying TAG providing fatty acid composition does not change among treatment groups. However, if samples contain significant quantities of di- or mono-acylglycerides, or the fatty acid composition differs across treatment groups, TLC-FID is recommended.     

Simplifying Hansen Solubility Parameters for Complex Edible Fats and Oils

Hansen solubility parameters (HSPs), often used to predict the miscibility between two compounds, are an alternative tool in evaluating the ability of the solvent to interact via dispersion, dipole-dipole, and hydrogen bonding interactions. The aim of this paper is to find a simple way to calculate HSPs for complex mixtures of triglycerides (TAGs). HSPs were calculated using two approaches: the first assumes that the contributions to the dispersion, dipole-dipole, and hydrogen bonding interactions may be subdivided into larger functional moieties (i.e., fatty acids and fatty acid methyl esters) that are additive, while the second approach assumes that vegetable oils are comprised of mixtures of simple TAGs in the same mass fractions as the fatty acids. The HSPs obtained using the two approaches are compared to reference values determined using the “Hansen Solubility Parameters in Practice” software (HSPiP) considering the complex TAG profile for each vegetable oil.HSPs for vegetable oils, obtained with the HSPiP software, did not correspond well to the HSPs obtained from the group contribution approach, when using fatty acids, fatty acids + glycerol or fatty acid methyl esters. In contrast, the HSPs calculated for vegetable oils, assuming that all TAGs are simple and in the same mass fractions as the fatty acids, provide similar values to the HSPs obtained from the HSPiP software. Therefore, it is possible to calculate the HSPs for complex oils by simply knowing the fatty acid composition. Knowledge of HSPs may be used to rationalize the ability of certain low molecular weight molecules to develop organogels in vegetable oils as well as the crystallization of triglycerides.     

The determination of the asymmetrical stereochemical distribution of fatty acids in triacylglycerols

The fatty acid distribution in triacylglycerols (TAGs) is a factor that contributes to the intrinsic properties of oils from different species variants. Many hypotheses have been proposed to explain the specific distribution of fatty acids in the different naturally occurring oils. Currently, the 1,3-random-2-random theory is more or less accepted, but it has been widely shown that most vegetable oils do not behave randomly in the sn-1 and sn-3 stereochemical positions. For this reason, complex methodologies have been developed to analyze the fatty acid composition of the three stereochemical positions in TAGs. In this article, we propose that by calculating the asymmetric alpha coefficient, the stereochemical asymmetry of fatty acids in TAG molecular species can be defined. This coefficient reflects the relative content of fatty acids at the sn-1 and sn-3 positions and may overcome the problems found mainly with complex sn-1, sn-2, and sn-3 stereochemical analysis of fatty acids in TAG. The alpha coefficient is calculated from the fatty acid, sn-2 fatty acid, and TAG composition of the oil. Indeed, through this coefficient, it has been possible to show that, despite having the same overall content, the stearic acid distribution in the sn-1 and sn-3 positions is not random in some oils.     

The metabolism of structured triacylglycerols

The triacylglycerol (TAG) structure in addition to the overall fatty acid profile is of importance when considering the nutritional effect of a dietary fat. This review aims at summarizing our current knowledge of the digestion, absorption, uptake, and transport of structured TAGs, with particular emphasis on the following aspects: gastric emptying, specificity of pancreatic lipase, lymphatic transport and clearance of chylomicrons, effects of lipid structure on tissue lipid compositions and the fecal loss of fats. So an overview will be provided for how the structure and fatty acid composition of TAGs affect their absorption and the distribution of the fatty acids in the body following digestion and absorption.     

Triacylglyceride composition and fatty acyl saturation profile of a psychrophilic and psychrotolerant fungal species grown at different temperatures

Pseudogymnoascus destructans is a psychrophilic fungus that infects cutaneous tissues in cave dwelling bats, and it is the causal agent for white nose syndrome (WNS) in North American (NA) bat populations. Geomyces pannorum is a related psychrotolerant keratinolytic species that is rarely a pathogen of mammals. In this study, we grew P. destructans and G. pannorum in static liquid cultures at favourable and suboptimal temperatures to: 1) determine if triacylglyceride profiles are species-specific, and 2) determine if there are differences in fatty acyl (FA) saturation levels with respect to temperature. Total lipids isolated from both fungal spp. were separated by thin-layer chromatography and determined to be primarily sterols (∼15 %), free fatty acids (FFAs) (∼45 %), and triacylglycerides (TAGs) (∼50 %), with minor amounts of mono-/diacylglycerides and sterol esters. TAG compositions were profiled by matrix-assisted laser desorption–ionization time-of-flight mass spectrometry (MALDI–TOF). Total fatty acid methyl esters (FAMEs) and acyl lipid unsaturation levels were determined by gas chromatography–mass spectrometry (GC–MS). Pseudogymnoascus destructans produced higher proportions of unsaturated 18C fatty acids and TAGs than G. pannorum. Pseudogymnoascus destructans and G. pannorum produced up to a two-fold increase in 18:3 fatty acids at 5 °C than at higher temperatures. TAG proportion for P. destructans at upper and lower temperature growth limits was greater than 50 % of total dried mycelia mass. These results indicate fungal spp. alter acyl lipid unsaturation as a strategy to adapt to cold temperatures. Differences between their glycerolipid profiles also provide evidence for a different metabolic strategy to support psychrophilic growth, which may influence P. destructans' pathogenicity to bats.     

拟南芥TAG1基因对脂类合成调控作用的研究进展

三酰甘油（TAG）是真核生物中能量贮存的最主要形式。植物中贮存的三酰甘油是食用油类和工业用油的主要来源。TAG1基因的表达产物甘油二酯酰基转移酶（DGAT）能够调控三酰甘油的合成。as11是TAG1基因突变获得的脂类代谢相关突变体。该文概述了拟南芥（Arabidopsis thaliana）突变体as11的生物学特征及TAG1基因对脂类合成调控的最新进展。     

拟南芥TAG1 基因对脂类合成调控作用的研究进展

三酰甘油(TAG)是真核生物中能量贮存的最主要形式。植物中贮存的三酰甘油是食用油类和工业用油的主要来源。TAG1基因的表达产物甘油二酯酰基转移酶(DGAT)能够调控三酰甘油的合成。as11是TAG1基因突变获得的脂类代谢相关突变体。该文概述了拟南芥(Arabidopsis thaliana)突变体as11的生物学特征及TAG1基因对脂类合成调控的最新进展。     

Changes in Triacylglycerol Composition in Maturing Sea Buckthorn (Hippophaë rhamnoides) Seeds

Sea buckthorn (Hippophaë rhamnoides L.) seeds on the 29th, 53rd, 80th, and 107th day after pollination were used for determining, by lipase hydrolysis, the qualitative and quantitative composition of the triacylglycerol (TAG) positional types, groups, and positional species, as well as the factor of selectivity of incorporation of unsaturated fatty acids, octadecenoic, linoleic, and linolenic, into the sn-2-position of TAGs. Until the 80th day after pollination, there was a predominant formation of triunsaturated TAGs, which included linolenic and linoleic acid residues. After the 80th day, the absolute content of these major components of total TAGs markedly decreased, and an increase in total TAG content was mainly accounted for by the rise in the level of those TAG species, which included saturated fatty acids, palmitic and stearic (monosaturated–diunsaturated and disaturated–monounsaturated), as well as in the level of sn-2-octadecenoyl species belonging to the triunsaturated and palmito–diunsaturated types of TAGs. At each maturation stage, the quantitative dynamics of separate TAG species was determined by the content of fatty acid species available for TAG formation and the factor of selectivity of these species. The decrease in the content of a certain group of triunsaturated TAGs found here seems to be caused by their metabolization during seed maturation.     

Triacylglycerol catabolism in the prawn Macrobrachium borellii (Crustacea: Palaemoniade)

While invertebrates store neutral lipids as their major energy source, little is known about triacylglycerol (TAG) class composition and their differential catabolism in aquatic arthropods. This study focuses on the composition of the main energy source and its catabolism by lipase from the midgut gland (hepatopancreas) of the crustacean Macrobrachium borellii. Silver-ion thin-layer chromatography of prawn large TAG deposit (80% of total lipids) and its subsequent fatty acid analysis by gas chromatography allowed the identification of 4 major fractions. These are composed of fatty acids of decreasing unsaturation and carbon chain length, the predominant being 18:1n-9. Fraction I, the most unsaturated one, contained mainly 20:5n-3; fraction II 18:2n-6; fraction III 18:1n-9 while the most saturated fraction contained mostly 16:0. Hepatopancreas main lipase (Mr 72 kDa) cross-reacted with polyclonal antibodies against insect lipase, was not dependent on the presence of Ca²⁺ and had an optimum activity at 40 °C and pH 8.0. Kinetic analysis showed a Michaelis–Menten behavior. A substrate competition assay evidenced lipase specificity following the order: 18:1n-9-TAG > PUFA-enriched-TAG > 16:0-TAG different from that in vertebrates. These data indicate there is a reasonable correspondence between the fatty acid composition of TAG and the substrate specificity of lipase, which may be an important factor in determining which fatty acids are mobilized during lipolysis for oxidation in crustaceans.     

Characterisation of castor oil by on-line and off-line non-aqueous reverse-phase high-performance liquid chromatography-mass spectrometry (APCI and UV/MALDI)

A non-aqueous reverse-phase HPLC method, based on two columns in series, has been used to separate the major triacylglycerols (TAGs) from commercial castor oil and to perform either on-line negative ion atmospheric pressure chemical ionisation (APCI), or off-line positive ion matrix-assisted laser desorption ionisation (MALDI)/MS. The resulting Mass Spectra showed chloride-attached TAG molecules [M + CI]- in the case of negative-ion APCI, and sodium-attached TAG molecules [M + Na]+ in the case of positive-ion MALDI. For MALDI time-of-flight (TOF)/MS, a liquid binary matrix system consisting of sodium ferrocyanide and glycerol was applied, resulting in excellent TAG sensitivity, which was necessary for the determination of trace amounts of TAGs in castor oil. Both techniques allowed unambiguous molecular mass determination of the intact TAG molecules with no thermal degradation. Furthermore, seamless post source decay (PSD) fragment ion analysis by means of a curved field reflector TOF mass spectrometer allowed the determination of the fatty acid composition of each individual TAG. Castor oil contained eight different TAGs which were successfully determined by both APCI and MALDI techniques. In each TAG, at least two units of 12-hydroxy-9-octadecenoic acid (ricinoleic acid) were present. The following fatty acids were determined by seamless PSD fragment ion analysis and APCI/MALDI molecular mass determination as TAG substructures: ricinoleic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, dihydroxy stearic acid and eicosenoic acid. Triricinolein was the dominating TAG.     

Characterization of the triacylglycerol profile in marine diatoms by ultra performance liquid chromatography coupled with electrospray ionization–quadrupole time-of-flight mass spectrometry

Diatoms are considered to have great potential as new biofuel sources because they can effectively accumulate triacylglycerols (TAGs). Detailed structure information of TAG in diatoms is much needed not only for the assessment of biofuel quality such as fatty acid chain length and unsaturation degree but also for the tracing of biosynthetic precursors because the biosynthesis of TAG is typically completed by utilizing the diacylglycerol acyltransferase in the cytoplasm. In this report, a comprehensive characterization of TAGs in marine diatoms was performed using ultra performance liquid chromatography–electrospray ionization–quadrupole time-of-flight mass spectrometry. Many types of major TAGs were identified for the first time in these diatoms: 12 TAGs in Chaetoceros debilis, 9 TAGs in Phaeodactylum tricornutum Bohlin, 16 TAGs in Nitzschia closterium f. minutissima, 16 TAGs in Thalassiosira weissflogii, 13 TAGs in Thalassiosira sp., 16 TAGs in Stephanodiscus asteaea and 7 TAGs in Skeletonema costatum. Semi-quantification of TAGs in these diatoms was also carried out, and it was found that the contents of individual TAGs ranged from 0.5?±?0.1 to 217.9?±?8.1 nmol mg^?1 total lipids. In addition, the total lipid contents in diatoms ranged from 143.6?±?16.3 to 201.1?±?16.3 mg g^?1 dry microalgae and the total TAG contents ranged from 36.8?±?9.5 to 793.2?±?54.4 nmol mg^?1 total lipids. By comparative analysis of the compositions and concentrations of major TAGs in the seven algal strains, N. closterium f. minutissima with high abundance of TAGs containing the most monounsaturated fatty acids (mainly palmitoleic acid) was considered as one of the most promising diatom strains for microalgal biofuel production. Additionally, based on the information of sn-2 fatty acid obtained (mainly C16 in the sn-2 position), we propose the hypothesis that TAGs in diatoms are mainly derived from lipids in chloroplasts through the prokaryotic biosynthesis pathway, including monogalactosyldiacylglycerol and digalactosyldiacylglycerol.     

Identification and characterization of a type-2 diacylglycerol acyltransferase (DGAT2) from Rhodosporidium diobovatum

Triacylglycerols (TAGs), synthesized in the microsomal membranes of eukaryotes, serve as a primary storage form of carbon and energy in microorganisms. For this reason, TAGs produced by organisms have great potential to become biofuels and facilitate researchers to look for alternative renewable sources of energy. The present study describes the identification and functional characterization of a type-2 diacylglycerol acyltransferase from Rhodosporidium diobovatum, designated as RdDGAT, which catalyzed the final step of TAG synthesis. A full-length cDNA clone for RdDGAT was obtained, and its biological activity was proven by being expressed in a Saccharomyces cerevisiae quadruple mutant that was defective in TAG synthesis. Enzymatic assays were performed and finally the existence of TAGs in the transformed Saccharomyces cerevisiae quadruple mutant was determined using the method of thin-layer chromatography. Substrate preference experiments revealed that RdDGAT preferred unsaturated fatty acids over saturated ones. Through further analysis, we assume that the evolution and expression characteristics of the RdDGAT gene perhaps is the result of adaption to its oligotrophic and cold living environment.     

Separation and Identification of Fatty Acids

An improved method is described for separating saturated fatty acids by reversed-phase paper chromatography. The 2,4-dinitrophenylhydrazides prepared from saturated fatty acids from C₂ to C₂₂ are run upward on paper impregnated with tetralin, using 90% methanol—acetic acid—tetralin, 80% ethanol—acetic acid—tetralin, or 90% methanol—tetralin as the moving solvent. The simultaneous separations of all even-carbon acids from C₆ to C₂₂, all odd-carbon acids from C₇ to C₁₉, and also all odd- and even-carbon acids from C₇ to C₁₉ can be successfully performed by means of this paper chromatography. The method is useful for the detection of component saturated fatty acids in natural fats.     

Production of trans-free interesterified fat using indigenously immobilized lipase

Abstract

Enzymatic interesterification was carried out between high-oleic canola oil and fully hydrogenated soybean oil using indigenously immobilized Thermomyces lanuginosus lipas substrate concentration, moisture content of enzyme, and enzyme load. Interesterification resulted in a decrease in the concentration of tri-unsaturated and trisaturated TAG and an increase of mono- and di-saturated TAG as observed by reversed-phase HPLC. The alteration in TAG composition and the presence of new TAG species after interesterification was correlated with extended plasticity characterized by lower slip melting point with a significant change in functionality and consistency of the interesterified product. Thermal and structural properties of the blends before and after interesterification were assessed by differential scanning calorimetry (DSC), X-ray diffraction and polarized light microscopy. Trans-fat analysis indicated the absence of any trans fatty acid in the final interesterified product. The resultant interesterified products with varying slip melting points can be used in the formulation of healthier fat and oil products and address a critical industrial demand for trans free formulations for base-stocks of spreads, margarines, and confectionary fats.     

Quantitative Mapping of Triacylglycerol Chain Length and Saturation Using Broadband CARS Microscopy

Lipid droplets (LDs), present in many cell types, are highly dynamic organelles that store neutral lipids, primarily triacylglycerols (TAGs). With the discovery of new LD functions (e.g., in immune response, protein clearage, and occurrence with disease), new methods to study LD chemical composition in situ are necessary. We present an approach for in situ, quantitative TAG analysis using label-free, coherent Raman microscopy that allows deciphering LD TAG composition in different biochemically complex samples with submicrometer spatial resolution. Employing a set of standard TAGs, we generate a spectral training matrix capturing the variation caused in Raman-like spectra by TAG backbone, chain length, and number of double bonds per chain, as well as the presence of proteins or other diluting molecules. Comparing our fitting approach to gas chromatography measurements for mixtures of standard TAGs and food oils, we find the root mean-square error for the prediction of TAG chemistry to be 0.69 CH₂ and 0.15 #C=C. When progressing to more complex samples such as oil emulsions and LDs in various eukaryotic cells, we find good agreement with bulk gas chromatography measurements. For differentiated adipocytes, we find a significant increase in the number of double bonds in small LDs (below 2 μm in diameter) compared to large LDs (above 2 μm in diameter). Coupled with a relatively limited sample preparation requirement, this approach should enable rapid and accurate TAG LD analysis for a variety of cell biology and technological applications.     

Quantitative profiling and pattern analysis of triacylglycerol species in Arabidopsis seeds by electrospray ionization mass spectrometry

Plant triacylglycerols (TAGs), or vegetable oils, provide approximately 25% of dietary calories to humans and are becoming an increasingly important source of renewable bioenergy and industrial feedstocks. TAGs are assembled by multiple enzymes in the endoplasmic reticulum from building blocks that include an invariable glycerol backbone and variable fatty acyl chains. It remains a challenge to elucidate the mechanism of synthesis of hundreds of different TAG species in planta. One reason is the lack of an efficient analytical approach quantifying individual molecular species. Here we report a rapid and quantitative TAG profiling approach for Arabidopsis seeds based on electrospray ionization tandem mass spectrometry with direct infusion and multiple neutral loss scans. The levels of 93 TAG molecular species, identified by their acyl components, were determined in Arabidopsis seeds. Quantitative TAG pattern analyses revealed that the TAG assembly machinery preferentially produces TAGs with one elongated fatty acid. The importance of the selectivity in oil synthesis was consistent with an observation that an Arabidopsis mutant overexpressing a patatin‐like phospholipase had enhanced seed oil content with elongated fatty acids. This quantitative TAG profiling approach should facilitate investigations aimed at understanding the biochemical mechanisms of TAG metabolism in plants.