Sequential one-step extraction and analysis of triacylglycerols and fatty acids in plant tissues

A method for plant tissue digestion and triacylglycerol (TAG) extraction followed by transmethylation of TAGs to produce the fatty acid methyl esters (FAMEs) from small storage tissue samples is presented. The method allows the analysis of both TAGs and FAMEs from the same sample. Several reagent mixtures and different experimental conditions were tested on sliced sunflower seeds. The best results were obtained using a mixture that was 33.3% a solution of NaCl (0.17 M) in methanol and 66.6% heptane by volume. The TAGs in the heptane solution were transmethylated with a mixture containing methanol:toluene:dimethoxypropane:H(4)SO(2) (39:20:5:2, by vol). The method was also tested on other oil seed storage tissue (soybean) and fruit tissues from olive and acorn. In all cases, sunflower, soybean, olive, and acorn, the TAGs and FAMEs composition data obtained by this method were quite similar to data from a standard analysis method. In samples with high protein content, such as soybean and sunflower seeds, the TAG extraction was incomplete. The water content of fruit samples did not interfere with TAG extraction obtained by this method.     

Distribution of unusual fatty acids in the triacylglycerols of sea buckthorn mesocarp oil

The structure of unusual fatty acid (FA) components of triacylglycerols (TAGs) of mature sea buckthorn (Hippophae rhamnoides L.) mesocarp oil was determined by GLC and MS, and the positional-species composition (PSC) of these TAGs was estimated using the methods of TAG chemical deacylation, TLC, GLC, and computer calculation. It was shown that the unusual FAs comprised n-cis-Δ9-hexadecenoic, n-cis-Δ9,12-hexadecadienoic (palmitolinoleic), and n-cis-Δ11-octadecenoic (cis-vaccenic) acids. The hexadecenoic acid predominated in the oil, and in its distribution in TAGs, it was similar to the total FAs differing from them only in some prevalence in the triunsaturated TAGs and in the TAGs with a shorter acyl chain, as well as in the sn-2 position of TAGs. Palmitolinoleic (16:2) acid comprised only 5% of total FAs, and it was exclusively concentrated in the sn-2 position of TAGs. As regards its distribution between various positional types and forms of TAGs, the 16:2 acid was similar to oleate and total FAs. As compared to the total TAGs, the TAGs with 16:2 acid were characterized by a lower FA chain length as well as by a highest unsaturation. The TAGs with vaccenic acid (V-TAGs) considerably exceeded O-TAGs, i.e., the TAGs containing oleic acid, another 18:1 positional isomer, both in their content in the total TAGs and in their unsaturation. In the composition of positional types and fractions of various unsaturation, O-TAGs were similar to the total TAGs, while V-TAGs were characterized by a very unusual structure, viz., a very high triunsaturated TAG level and an extremely low concentration of 1,3-disaturated-2-monounsaturated TAGs. In addition, oleic acid, like most other unsaturated FAs, was incorporated predominantly in the sn-2 position of TAGs, while vaccenic acid, being also unsaturated, was nevertheless by 90% concentrated in the sn-1,3 positions of V-TAGs. Unusual FAs were related to each other in the mechanism of their biosynthesis. In fact, hexadecenoic acid biosynthesis produced by palmitic acid desaturation, was, on the one hand, further desaturated forming palmitolinoleic acid, and, on the other hand, converted to vaccenic acid via C₂ elongation.     

Triacylglycerol accumulation and profiling in the model diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum (Baccilariophyceae) during starvation

Although substantial economic barriers exist, marine diatoms such as Thalassiosira pseudonana and Phaeodactylum tricornutum hold promise as feedstock for biodiesel because of their ability to manufacture and store triacylglycerols (TAGs). The recent sequencing of these two marine diatom genomes by the United States Department of Energy Joint Genome Institute and the development of improved systems for genetic manipulation should allow a more systematic approach to understanding and maximizing TAG production. However, in order to best utilize these genomes and genetic tools, we must first gain a deeper understanding of the nutrient-mediated regulation of TAG anabolism. By determining both the yield and molecular species distribution of TAGs we will, in the future, be able to fully characterize the effects of genetic manipulation. Here, we lay the groundwork for understanding TAG production in T. pseudonana and P. tricornutum, as a function of nitrate and silicate depletion. Diatoms were starved of either nitrate or silicate, and TAGs were extracted with hexane from lyophilized samples taken at various time intervals following starvation. The timing of TAG production and the relative abundance of TAGs were estimated by fluorescence spectroscopy using Nile red and the total yield per biomass determined by gravimetric assay. TAGs were analyzed using thin layer chromatography, gas chromatography–mass spectrometry, and electrospray ionization mass spectrometry to identify the major TAG species produced during the growth curve. Under our conditions, the TAG yield from T. pseudonana is about 14–18% of total dry weight. The TAG yield from P. tricornutum is about 14% of total dry weight. Silicate-starved T. pseudonana accumulated an average of 24% more TAGs than those starved for nitrate; however, the chemotypes of the TAGs produced were generally similar regardless of the starvation condition employed.     

Thin-Layer Chromatography of Coordination Ag+-Complexes of Plant Diacylglycerols

Coordination complexes of unsaturated rac-1,2-diacylglycerols (DAGs) with silver ions were separated by adsorption and reversed-phase TLC (Ag-TLC and Ag-rpTLC, respectively). During the Ag-TLC, the silver ion complexes were shown to be formed by the DAG coordination centers of various structures and only on the adsorbent surface. Separation of the complexes proceeds according to the adsorption mechanism, and there is an inverse exponential functional relationship between the DAG mobility and their double bond number. Meanwhile, during the Ag-rpTLC, the Ag⁺-complexes are formed only with double bonds, only in solution, and at a 1 : 1 ratio. The complexes are fractionated by partitioning between two liquid phases, and the relationship between the mobility and unsaturation of these complexes is directly proportional. Nevertheless, despite all the differences between the two TLC methods, the polarity of DAGs with a bent configuration of their acyl chains greatly exceeds that of DAGs with the same unsaturation but with the acyl-chain conformation close to extended: it is two to three times greater in Ag-TLC and 30–40% greater in Ag-rpTLC. In addition, the relationship between the mobility and unsaturation of DAG complexes exhibits quantitative rather than qualitative differences in both versions of argentation TLC. Thus, under all conditions of argentation liquid chromatography, the mobility of complexes and, therefore, their polarity are determined not only by their composition (unsaturation), but also by the spatial structure (conformation) of their molecules.     

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.     

Methods for the analysis of triacylglycerols

This article discusses the methods most commonly employed in the analysis of the triacylglycerols (TAGs) in natural fats and considers the main advantages and disadvantages of each and the techniques for optimising analytical conditions. Complete analysis of the composition of a natural fat calls for a method of extracting and purifying the triglyceride fraction, normally by preparatory thin-layer and column chromatography. Determination of the individual components of triglyceride mixtures still entails certain difficulties, namely, the separation and identification of the TAGs in natural fats. High-performance liquid chromatography (HPLC) offers significant advantages over gas and thin-layer chromatography. Many workers have developed non-aqueous, reversed-phase HPLC systems capable of successfully resolving complex mixtures of TAGs, and combining reversed-phase (RP) HPLC and argentation chromatography may improve the results. Identification of the TAGs separated by HPLC becomes an extremely complex task if many different fatty acids are involved and if the sn-stereoscopic positions on the glycerol are to be determined. Enzymatic analysis and chiral-phase chromatography are capable of localising fatty acids on the TAG molecule. In closing, some of the most interesting biomedical applications of TAG analysis are summarised.     

Dynamics of Fatty‐Acid Composition of Neutral Acylglycerols in Maturing Euonymus Fruits

The dynamics of the fatty‐acid (FA) composition of neutral acylglycerols (NAGs) composed of 1,2,3‐triacyl‐sn‐glycerols (TAGs) and 3‐acetyl‐1,2‐diacyl‐sn‐glycerols (acDAGs) was determined in the fruit seeds and arils of three Euonymus L. species at three stages of their maturity. The NAG composition comprised 29 FAs, linoleic, oleic, palmitic, and α‐linolenic acids being predominant. Noticeable amounts of other FAs, such as lauric, myristic, hexadec‐9‐enoic, stearic, (Z)‐vaccenic, and arachidic acid, etc., could also be present. In the course of maturation, the qualitative composition of major FAs remained nearly unchanged, while the unsaturation index of FAs in seeds and in TAGs, as well as, but to a lesser extent, in arils and in acDAGs, respectively, always decreased. This decline was brought about by a sharp fall of the α‐linolenate level, a decrease of the linoleate content, and a corresponding rise in the oleate content. It is suggested that, in both seeds and arils, both classes of NAGs were formed at the expense of the same FA pool; the quantitative composition of this pool was characteristic of a given fruit part and strongly changed during maturation. The accumulation of TAGs in E. europaeus fruits was accompanied by a conversion of hexadec‐9‐enoic acid into (Z)‐vaccenic acid via the C₂‐elongation reaction.     

Novel chromatographic resolution of chiral diacylglycerols and analysis of the stereoselective hydrolysis of triacylglycerols by lipases

In the present study, we propose a general and accessible method for the resolution of enantiomeric 1,2-sn- and 2,3-sn-diacylglycerols based on derivatization by isocyanates, which can be easily used routinely by biochemists to evaluate the stereopreferences of lipases in a time course of triacylglycerol (TAG) hydrolysis. Diacylglycerol (DAG) enantiomers were transformed into carbamates using achiral and commercially available reagents. Excellent separation and resolution factors were obtained for diacylglycerols present in lipolysis reaction mixtures. This analytical method was then applied to investigate the stereoselectivity of three model lipases (porcine pancreatic lipase, PPL; lipase from Rhizomucor miehei, MML; and recombinant dog gastric lipase, rDGL) in the time course of hydrolysis of prochiral triolein as a substrate. From the measurements of the diglyceride enantiomeric excess it was confirmed that PPL was not stereospecific (position sn-1 vs sn-3 of triolein), whereas MML and rDGL preferentially hydrolyzed the ester bond at position sn-1 and sn-3, respectively. The enantiomeric excess of DAGs was not constant with time, decreasing with the course of hydrolysis. This was due to the fact that DAGs can be products of the stereospecific hydrolysis of TAGs and substrates for stereospecific hydrolysis into monoacylglycerols.     

Lipid metabolism in the moss Dicranum scoparium: effect of light conditions and heavy metals on the accumulation of acetylenic triacylglycerols

Lipid metabolism in the moss Dicranum scoparium Hedw. was studied using radiolabelling from [1-¹⁴C]acetate. The effect of two environmental parameters, light and heavy metals, on such metabolism was examined. Radiolabelling was approximately linear for 48 h after which the radioactivity in the total and polar lipid fractions remained constant in the light, whereas it declined in the dark. Pulse-chase experiments confirmed that lipid labelling was influenced by light exposure. Light exposure altered the pattern of polar lipid labelling, especially of those lipids associated with chloroplast membranes. Within the neutral lipids, diacylglycerols and triacylglycerols (TAGs) were the major classes labelled. D. scoparium contained up to 45% of total acyl moieties as 9,12,15-octadecatrien-6-ynoic (18:3A) acid and this was found in a TAG subfraction which could be separated by TLC. Although TAGs always represented 65–75% of total neutral lipid labelling, the proportion of TAGs containing 18:3A was increased with time and by light but reduced by exposure to environmentally relevant levels of Cu²⁺ and Pb²⁺. The data suggest that 18:3A is produced in D. scoparium by the action of a bi-functional Δ6-desaturase on α -linolenate. Furthermore, important environmental factors (such as light and heavy metals) significantly change the metabolism of TAGs containing 18:3A.     

Changes in the distribution of South Korean forest vegetation simulated using thermal gradient indices

To predict changes in South Korean vegetation distribution, the Warmth Index (WI) and the Minimum Temperature of the Coldest Month Index (MTCI) were used. Historical climate data of the past 30 years, from 1971 to 2000, was obtained from the Korea Meteorological Administration. The Fifth-Generation National Center for Atmospheric Research (NCAR) /Penn State Mesoscale Model (MM5) was used as a source for future climatic data under the A1B scenario from the Special Report on Emission Scenario (SRES) of the Intergovernmental Panel on Climate Change (IPCC). To simulate future vegetation distribution due to climate change, the optimal habitat ranges of Korean tree species were delimited by the thermal gradient indices, such as WI and MTCI. To categorize the Thermal Analogy Groups (TAGs) for the tree species, the WI and MTCI were orthogonally plotted on a two-dimensional grid map. The TAGs were then designated by the analogue composition of tree species belonging to the optimal WI and MTCI ranges. As a result of the clustering process, 22 TAGs were generated to explain the forest vegetation distribution in Korea. The primary change in distribution for these TAGs will likely be in the shrinkage of areas for the TAGs related to Pinus densiflora and P. koraiensis, and in the expansion of the other TAG areas, mainly occupied by evergreen broad-leaved trees, such as Camellia japonica, Cyclobalanopsis glauca, and Schima superba. Using the TAGs to explain the effects of climate change on vegetation distribution on a more regional scale resulted in greater detail than previously used global or continental scale vegetation models.     

Thin layer chromatography of coordinated Ag+-complexes of plant diacylglycerols

Coordination complexes of unsaturated rac-1,2-diacylglycerols (DAGs) with silver ions were separated by adsorption and reversed-phase TLC (Ag-TLC and Ag-rpTLC, respectively). During the Ag-TLC, the silver ion complexes were shown to be formed by the DAG coordination centers of various structures and only on the adsorbent surface. Separation of the complexes proceeds according to the adsorption mechanism, and there is an inverse exponential functional relationship between the DAG mobility and their double bond number. Meanwhile, during the Ag-rpTLC, the Ag(+)-complexes are formed only with double bonds, only in solution, and at a 1:1 ratio. The complexes are fractionated by partitioning between two liquid phases, and the relationship between the mobility and unsaturation of these complexes is directly proportional. Nevertheless, despite all the differences between the two TLC methods, the polarity of DAGs with a bent configuration of their acyl chains greatly exceeds that of DAGs with the same unsaturation but with the acyl-chain conformation close to extended: it is two to three times greater in Ag-TLC and 30-40% greater in Ag-rpTLC. In addition, the relationship between the mobility and unsaturation of DAG complexes exhibits quantitative rather than qualitative differences in both versions of argentation TLC. Thus, under all conditions of argentation liquid chromatography, the mobility of complexes and, therefore, their polarity are determined not only by their composition (unsaturation), but also by the spatial structure (conformation) of their molecules.     

Phosphorylation of the lipid droplet localized glycerol‑3‑phosphate acyltransferase Gpt2 prevents a futile triacylglycerol cycle in yeast

The proteome of lipid droplets, storage compartments of triacylglycerols (TAGs), comprises TAG synthesizing and TAG degrading enzymes. Thus, to prevent a futile cycle the activity of enzymes catalyzing key steps in TAG turnover has to be strictly coordinated. The first and committed reaction of TAG synthesis is catalyzed by a glycerol‑3‑phosphate acyltransferase (GPAT). Here we demonstrate that in the model organism yeast the lipid droplet associated GPAT Gpt2 requires phosphorylation at a conserved motif to prevent a futile TAG cycle. Phosphorylation deficiency at the conserved motif increases the enzyme activity of Gpt2 and consequently enhances TAG synthesis. In proliferating cells the phosphorylation deficient GPAT-form contributes to TAG metabolism similar to control. However, during lipolysis the increased activity of phosphorylation deficient Gpt2 causes a constant TAG level by using TAG-released fatty acids as substrate for TAG synthesis. These data strongly indicate that phosphorylation of Gpt2 at a conserved motif plays a critical role in coordinating the synthesis and degradation of TAGs.     

Metabolic engineering of oilseed crops to produce high levels of novel acetyl glyceride oils with reduced viscosity,freezing point and calorific value

Seed oils have proved recalcitrant to modification for the production of industrially useful lipids. Here, we demonstrate the successful metabolic engineering and subsequent field production of an oilseed crop with the highest accumulation of unusual oil achieved so far in transgenic plants. Previously, expression of the Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) gene in wild‐type Arabidopsis seeds resulted in the accumulation of 45 mol% of unusual 3‐acetyl‐1,2‐diacyl‐sn‐glycerols (acetyl‐TAGs) in the seed oil (Durrett et al., 2010 PNAS 107:9464). Expression of EaDAcT in dgat1 mutants compromised in their ability to synthesize regular triacylglycerols increased acetyl‐TAGs to 65 mol%. Camelina and soybean transformed with the EaDAcT gene accumulate acetyl‐triacylglycerols (acetyl‐TAGs) at up to 70 mol% of seed oil. A similar strategy of coexpression of EaDAcT together with RNAi suppression of DGAT1 increased acetyl‐TAG levels to up to 85 mol% in field‐grown transgenic Camelina. Additionally, total moles of triacylglycerol (TAG) per seed increased 20%. Analysis of the acetyl‐TAG fraction revealed a twofold reduction in very long chain fatty acids (VLCFA), consistent with their displacement from the sn‐3 position by acetate. Seed germination remained high, and seedlings were able to metabolize the stored acetyl‐TAGs as rapidly as regular triacylglycerols. Viscosity, freezing point and caloric content of the Camelina acetyl‐TAG oils were reduced, enabling use of this oil in several nonfood and food applications.     

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.     

Enhancement of extraplastidic oil synthesis in Chlamydomonas reinhardtii using a type‐2 diacylglycerol acyltransferase with a phosphorus starvation–inducible promoter

When cultivated under stress conditions, many plants and algae accumulate oil. The unicellular green microalga Chlamydomonas reinhardtii accumulates neutral lipids (triacylglycerols; TAGs) during nutrient stress conditions. Temporal changes in TAG levels in nitrogen (N)‐ and phosphorus (P)‐starved cells were examined to compare the effects of nutrient depletion on TAG accumulation in C. reinhardtii. TAG accumulation and fatty acid composition were substantially changed depending on the cultivation stage before nutrient starvation. Profiles of TAG accumulation also differed between N and P starvation. Logarithmic‐growth‐phase cells diluted into fresh medium showed substantial TAG accumulation with both N and P deprivation. N deprivation induced formation of oil droplets concomitant with the breakdown of thylakoid membranes. In contrast, P deprivation substantially induced accumulation of oil droplets in the cytosol and maintaining thylakoid membranes. As a consequence, P limitation accumulated more TAG both per cell and per culture medium under these conditions. To enhance oil accumulation under P deprivation, we constructed a P deprivation‐dependent overexpressor of a Chlamydomonas type‐2 diacylglycerol acyl‐CoA acyltransferase (DGTT4) using a sulphoquinovosyldiacylglycerol 2 (SQD2) promoter, which was up‐regulated during P starvation. The transformant strongly enhanced TAG accumulation with a slight increase in 18 : 1 content, which is a preferred substrate of DGTT4. These results demonstrated enhanced TAG accumulation using a P starvation–inducible promoter.     

Fasting triacylglycerol status, but not polyunsaturated/saturated fatty acid ratio, influences the postprandial response to a series of oral fat tolerance tests

Elevated postprandial lipemia is emerging as a risk factor for obesity-related chronic diseases, such as type 2 diabetes and cardiovascular disease, and is associated with alterations in several metabolic biomarkers of disease. Our goal was to examine the effects of specific polyunsaturated/saturated fatty acid (P/S) ratios on postprandial triacylglycerol (TAG) concentrations and metabolic biomarkers in men with different fasting TAG concentrations through a series of oral fat tolerance tests (OFTT) consisting solely of emulsified lipid. Otherwise healthy men with high (>1.69 mmol/L) fasting TAG (HTAG, n=8) and low fasting TAG (LTAG, n=8) underwent three OFTTs with specific P/S ratios of 0.2, 1.0 and 2.0, respectively, and a total lipid load of 1 g/kg subject body mass. All subjects received each treatment separated by at least 1 week. Postprandial plasma TAG fatty acid composition reflected fatty acids present in the OFTT. All other metabolic responses were independent of the P/S ratio ingested. An accelerated increase in postprandial TAGs was observed in HTAG compared to LTAG. Interleukin (IL)-6 and soluble intercellular adhesion molecule (sICAM)-1 were significantly elevated in HTAG at baseline (P<.05). IL-6 increased significantly following each OFTT (P<.05) in both groups. Postprandial glucose and CRP were significantly exaggerated (P<.05) in HTAG. Overall, HTAG subjects had an accelerated postprandial TAG response and increased concentrations of several inflammatory markers following an OFTT, in the absence of an insulin response. However, P/S ratio had no influence on postprandial lipid and inflammatory parameters.     

An ultraviolet spectrophotometric assay for the screening of sn-2-specific lipases using 1,3-O-dioleoyl-2-O-α-eleostearoyl-sn-glycerol as substrate

In the present study, we propose a continuous assay for the screening of sn-2 lipases by using triacylglycerols (TAGs) from Aleurites fordii seed (tung oil) and a synthetic TAG containing the α-eleostearic acid at the sn-2 position and the oleic acid (OA) at the sn-1 and sn-3 positions [1,3-O-dioleoyl-2-O-α-eleostearoyl-sn-glycerol (sn-OEO)]. Each TAG was coated into a microplate well, and the lipase activity was measured by optical density increase at 272 nm due to transition of α-eleostearic acid from the adsorbed to the soluble state. The sn-1,3-regioselective lipases human pancreatic lipase (HPL), LIP2 lipase from Yarrowia lipolytica (YLLIP2), and a known sn-2 lipase, Candida antarctica lipase A (CALA) were used to validate this method. TLC analysis of lipolysis products showed that the lipases tested were able to hydrolyze the sn-OEO and the tung oil TAGs, but only CALA hydrolyzed the sn-2 position. The ratio of initial velocities on sn-OEO and tung oil TAGs was used to estimate the sn-2 preference of lipases. CALA was the enzyme with the highest ratio (0.22 ± 0.015), whereas HPL and YLLIP2 showed much lower ratios (0.072 ± 0.026 and 0.038 ± 0.016, respectively). This continuous sn-2 lipase assay is compatible with a high sample throughput and thus can be applied to the screening of sn-2 lipases.     

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.     

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.