Quantitative Analysis of TAG in Oils Using Lithium Cationization and Direct‐Infusion ESI Tandem Mass Spectrometry

This study was undertaken to determine whether triple‐stage mass spectrometry (MS³) could be employed to obtain quantitative and regioisomeric data from complex oil samples without the need for a chromatographic step in the analysis protocol. Lithium‐7 trifluoroacetate and electrospray ionization were used to form lithium adducts of the triacylglycerols (TAG) in a fish oil sample. The first‐generation precursor ion was the lithium‐TAG adduct, the second‐generation precursor ion was formed by loss of a neutral acid side chain in the first fragmentation. The ions used for analysis were formed in the second fragmentation by loss of the lactones of the acid side chains remaining after the first fragmentation. This analysis scheme provided quantitative and regioisomeric data without interference from TAG in the sample other than TAG with the same acyl carbon number, one more double bond, and two acyl side chains in common with the analyte. Even in this case a majority of the interferences could be estimated and compensated. Analysis of synthetic samples containing the fish oil matrix indicated that both absolute and relative quantitative data could be obtained with average errors of approximately 5 %. The method proved well suited to routine analyses of complex oil samples.     

Comprehensive and quantitative profiling of lipid species in human milk,cow milk and a phospholipid‐enriched milk formula by GC and MS/MSALL

Here we present a workflow for in‐depth analysis of milk lipids that combines gas chromatography (GC) for fatty acid (FA) profiling and a shotgun lipidomics routine termed MS/MS^ALL for structural characterization of molecular lipid species. To evaluate the performance of the workflow we performed a comparative lipid analysis of human milk, cow milk, and Lacprodan^® PL‐20, a phospholipid‐enriched milk protein concentrate for infant formula. The GC analysis showed that human milk and Lacprodan have a similar FA profile with higher levels of unsaturated FAs as compared to cow milk. In‐depth lipidomic analysis by MS/MS^ALL revealed that each type of milk sample comprised distinct composition of molecular lipid species. Lipid class composition showed that the human and cow milk contain a higher proportion of triacylglycerols (TAGs) as compared to Lacprodan. Notably, the MS/MS^ALL analysis demonstrated that the similar FA profile of human milk and Lacprodan determined by GC analysis is attributed to the composition of individual TAG species in human milk and glycerophospholipid species in Lacprodan. Moreover, the analysis of TAG molecules in Lacprodan and cow milk showed a high proportion of short‐chain FAs that could not be monitored by GC analysis. The results presented here show that complementary GC and MS/MS^ALL analysis is a powerful approach for characterization of molecular lipid species in milk and milk products. Practical applications : Milk lipid analysis is routinely performed using gas chromatography. This method reports the total fatty acid composition of all milk lipids, but provides no structural or quantitative information about individual lipid molecules in milk or milk products. Here we present a workflow that integrates gas chromatography for fatty acid profiling and a shotgun lipidomics routine termed MS/MS^ALL for structural analysis and quantification of molecular lipid species. We demonstrate the efficacy of this complementary workflow by a comparative analysis of molecular lipid species in human milk, cow milk, and a milk‐based supplement used for infant formula. A workflow for milk lipid analysis based on combined gas chromatography and high‐resolution shotgun lipidomics. In‐depth structural characterization and quantification of molecular lipid species in milk and milk products.     

Quantification of the Molecular Species of TAG and DAG in Lesquerella (Physaria fendleri) Oil by HPLC and MS

Ten diacylglycerols (DAG) and 74 triacylglycerols (TAG) in the seed oil of Physaria fendleri were recently identified by high‐performance liquid chromatography (HPLC) and mass spectrometry (MS). These acylglycerols (AG) were quantified by HPLC with evaporative light scattering detector and electrospray ionization mass spectrometry of the lithium adducts of the AG in the HPLC fractions of lesquerella oil. The MS¹ ion signal intensities of molecular ions [M + Li]⁺ in HPLC fractions of an HPLC peak were used to estimate the ratios of AG in the HPLC peak. The ratios of TAG with the same mass in HPLC fractions were estimated by the ratios of the sums of MS² ion signal intensities from the neutral loss of the three fatty acids [M + Li ? FA]⁺. The ratio of DAG with the same mass were estimated by the ratio of the sums of two MS² ion signal intensities [M + Li ? FA]⁺ and [FA + Li]⁺ from the two different FA of a DAG. We have estimated the contents of ten molecular species of DAG and 74 molecular species of TAG in P. fendleri oil using this new method. The content of ten DAG combined was about 1 % and 74 TAG was about 98 %. The contents of DAG in decreasing order were: LsLs (0.25 %), LsLn (0.25 %), LsO (0.24 %), and LsL (0.11 %); and the contents of TAG in decreasing order were: LsLsO (31.3 %), LsLsLn (24.9 %), LsLsL (15.8 %), LsL‐OH20:2 (4.3 %), LsO‐OH20:2 (2.8 %), and LsLn‐OH20:2 (2.5 %).     

<Superscript>1</Superscript>H- and <Superscript>13</Superscript>C-NMR Characterization of the Molecular Components of the Lipid Fraction of Pecorino Sardo Cheese

In this work the molecular fatty components of Pecorino Sardo Protected Designation of Origin (PS PDO) cheese were characterized through an exhaustive investigation of the ¹H- and ¹³C-NMR spectra of the extracted lipids. Several fatty acids (FA), such as long chain saturated, oleic, linoleic, linolenic, butyric, capric, caprylic, caproic, trans vaccenic, conjugated linoleic acid (cis9, trans11–18:2), and caproleic (9–10:1) were unambiguously detected. The positional isomery of some acyl groups in the glycerol backbone of triacylglycerols (TAG) was assessed. Furthermore, the NMR signals belonging to sn-1,2/2,3, sn-1,3 diacylglycerols (DAG), and free fatty acids (FFA) were analysed as a measure of lipolytic processes on cheese. Lastly, ¹H-NMR resonances of saturated aldehydes and hydroperoxides were detected, their very low intensity indicating that the lipid oxidation process can be considered to be of minor relevance in Pecorino Sardo cheese.     

Regiospecific determination of short-chain triacylglycerols in butterfat by normal-phase HPLC with on-line electrospray-tandem mass spectrometry

This study uses normal-phase HPLC with on-line positive ion electrospray mass spectrometry (ESI-MS) to obtain quantitative compositional data on both synthetic and butterfat short-chain TAG. The product ion tandem MS of standards averaged 11.1 times lower in abundance of the ion formed by cleavage of FA from the sn-2-position for the pairs of regioisomers in the TAG classes: L/L/S-L/S/L and L/S/S-S/L/S, where L denotes long and S short acyl chain (C₂−C₆). The molar correction factors, determined for 42 regioisomeric pairs of short-chain TAG of 20 randomized mixture of standards, differed by 1.4–80% as the ratios varied between 0.217 and 5.847. Butterfat TAG were resolved into four fractions on short flash chromatography grade silica gel columns. Pairs of regioisomers in the TAG classes L/S/S-S/L/S with predominance of L/S/S isomers and the sole regioisomers in the TAG classes L/L(M)/S were identified by tandem MS, where M denotes either 8∶0 or 10∶0 acyl chain. The total proportion of L/L(M)/S isomers was estimated at 34.7 and that of L/S/S-S/L/S at 1.0 mol%, including a small proportion of S/S/S. In contrast to previous work, the present data indicate the presence of a small proportion of butyric and caproic acids in the sn-1-position. The overall distribution of the FA in the short-chain TAG of butterfat, calculated from direct MS measurements, was consistent with the results of indirect determinations based on stereospecific analyses of total butterfat.     

Determination of positional distribution of butyryl groups in milkfat triacylglycerols,triacylglycerol mixtures,and isolated positional isomers of triacylglycerols by gas chromatography and1H nuclear magnetic resonance spectroscopy

Positional isomers (1-butyryl-2X-3Y-rac-glycerol and 2-butyryl-1X-3Y-rac-glycerol;X,Y=long-chain acyls) of saturated triacylglycerols (TAG) with 34 and 40 acyl carbons were shown to separate in two chromatographic peaks on immobilized phenyl(65%) methylsilicone column by gas-liquid chromatography, and on reversed-phase ODS-1 column by high-performance liquid chromatography. The analysis of 500-MHz¹H nuclear magnetic resonance (NMR) spectra showed distinct differences between 2-butyryl-1X-3Y-rac-glycerol and 1-butyryl-2X-3Y-rac-glycerol isomers in the resonance signals of methylene and methine protons of glycerol backbone, and carbon-2 methylene of acyl groups, and methyl protons of butyryl group. The¹H NMR spectra of three interesterified mixtures of three monoacid TAG containing saturated butyrate and caproate TAG and unsaturated butyrate TAG showed that triplets of methyl protons of butyryl groups atsn-1(3)- andsn-2-positions in saturated and unsaturated TAG had similar chemical shifts and that the chemical shift of caproyl methyl protons was different from those of butyryl methyl protons. The positional distribution of butyryl groups in isolated positional isomers of butyrate TAG, interesterified TAG mixtures, and natural and interesterified butteroil can be determined by integration of these signals.     

13C NMR spectra of TAG: An easy way to distinguish milks from different animal species

In order to differentiate milks from different species, we carried out a comparative analysis of TAG from cow, buffalo, goat, and sheep milk fat based on ¹³C NMR experiments. NMR spectroscopy, although less sensitive than other techniques, does not require an extensive chemical manipulation of samples and can easily highlight the differences in the content of short-chain acyl groups in the four milk species. The resonances were assigned and quantified, and by using only three NMR parameters in data clustering with fuzzy logic analysis, we were able to distinguish goats' milk from sheep's milk, and both of these milks from cows' and buffaloes' milks. This appears to be an important result, considering the ease and rapidity with which milk identification can be obtained. From ¹³C NMR spectra of TAG, the positional distribution of FA chains on the glycerol backbone can also be easily evaluated. In particular, analysis of the positional distribution of monounsaturated FA revealed that it may be species-specific, and we are currently analyzing larger data sets in order to evaluate the use of this parameter as a suitable approach to address the issue of milk authenticity.     

Comprehensive Quantitation Using Two Stable Isotopically Labeled Species and Direct Detection of <Emphasis Type="Italic">N</Emphasis>-Acyl Moiety of Sphingomyelin

Sphingomyelin (ceramide‐phosphocholine, CerPCho) is a common sphingolipid in mammalian cells and is composed of phosphorylcholine and ceramide as polar and hydrophobic components, respectively. In this study, a qualitative liquid chromatography‐electrospray ionization tandem mass spectrometry (LC–ESI–MS/MS/MS) analysis is proposed in which CerPCho structures were assigned based on product ion spectra corresponding to sphingosylphosphorylcholine and N‐acyl moieties. From MS/MS/MS analysis of CerPCho, we observed product ion spectra of the N‐acyl fatty acids as [RCO₂]^? ions as well as sphingosylphosphorylcholine. A calibration curve for CerPCho was constructed using two stable isotopically labeled CerPCho species and then used to quantify the CerPCho species in HeLa cells as a proof‐of‐principle study. The present study proposes an accurate method for quantifying and assigning structures to each CerPCho species in crude biologic samples by LC–ESI–MS/MS/MS analysis.     

The Updated Bottom Up Solution Applied to Atmospheric Pressure Photoionization and Electrospray Ionization Mass Spectrometry

The Updated Bottom Up Solution (UBUS) was recently applied to atmospheric pressure chemical ionization (APCI) mass spectrometry (MS) of triacylglycerols (TAG). This report demonstrates that the UBUS applies equally well to atmospheric pressure photoionization (APPI) MS and to electrospray ionization (ESI) MS. Critical Ratio 1 (CR1), the [MH]⁺/Σ[DAG]⁺ or [MNH₄]⁺/Σ[DAG]⁺ ratio, does not exhibit the same strongly sigmoidal shape as it does by APCI‐MS. CR1 varies more widely for APPI‐MS than by APCI‐MS, having a maximum value of 11.8, indicating a much greater effect of unsaturation on ion ratios in APPI‐MS than APCI‐MS. Critical Ratio 2, the [AA]⁺/[AB]⁺ ratio for Type II TAG or [AC]⁺/([AB]⁺+[BC]⁺) ratio for Type III TAG, allows quantification of regioisomers of TAG, and shows good agreement for APPI‐MS to regioisomer quantification determined by APCI‐MS. Critical Ratio 3, the [BC]⁺/[AB]⁺ ratio for Type III TAG, reveals new trends relating the degree of unsaturation by APPI‐MS, and shows that structural assignments made by ESI‐MS are in good agreement to APCI‐MS data. In addition to providing valuable structural information, the Critical Ratios also constitute a reduced data set that allows APPI‐MS or ESI‐MS mass spectra to be reconstructed when processed through the UBUS. Quantification by APPI‐MS of vitamin D in the gelcaps gave values of 42.90 ± 0.83 μg, or 1716 ± 33 international units, in good agreement with APCI‐MS.     

Rapid MS method for analysis of cocoa butter TAG

Ammonia negative ion CI-MS was applied to analyze the M.W. distribution and regioisomeric structure of TAG in cocoa butter and in cocoa butter equivalents. The M.W. distribution results obtained for a reference cocoa butter were consistent with corresponding results obtained in an intercomparison study by chromatographic methods. Minor but statistically significant differences were observed when proportions of the three major M.W. species (52∶1, 54∶1, and 50∶1; acyl carbon number/number of double bonds) in a mixture of nine cocoa butters and in mixtures containing 10 or 20% (w/w) of specific cocoa butter equivalents were compared. Tandem MS was used to determine the regioisomeric structure of the three major TAG M.W. species in cocoa butter and in cocoa butter equivalents. The regioisomeric structure in cocoa butter and in all the equivalents analyzed were nearly identical, oleic acid being located primarily in the sn-2 position. These results cannot be exploited in detecting added foreign fats in this case. However, the present study shows that useful TAG composition data, which may be used to detect foreign fats in cocoa butter by applying chemometric data evaluation, can be obtained by MS in a significantly shorter time compared to chromatographic methods.     

Acyl Migration Kinetics of 2-Monoacylglycerols from Soybean Oil via <Superscript>1</Superscript>H NMR

The acyl migration kinetics of neat 2-monoacylglycerol (2-MAG) to form 1-MAG was determined using ¹H NMR spectroscopy to monitor the β-proton integration ratios of the two species over time. 2-MAG was synthesized by the Novozym 435-catalyzed alcoholysis of soybean oil and isolated by solvent extraction or molecular distillation at a mole fraction (X _2-MAG) of 0.94 relative to total MAG. The kinetics parameters of the neat 2-MAG acyl migration were investigated over the temperature range of 23–80 °C. The 2-MAG mol fraction remained unchanged at 23 °C over the course of 168 h and reached an equilibrium of X _2-MAG = 0.09 at only 80 °C. Modeling of the kinetics data revealed a 2-MAG half life (t _1/2) of 3,500 and 22.8 h at 23 and 80 °C, respectively, with an activation energy of 79.0 ± 6.5 kJ mol⁻¹. The use of ¹H NMR spectroscopy proved an expedient method for monitoring the acyl migration in 2-MAG compared to other reported methods (e.g. GC, HPLC, and ¹³C-NMR spectroscopy), requiring no sample manipulation and minimizing the deleterious effects of high temperatures and solvent exposure. Product names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product to the exclusion of others that may be suitable.     

Analysis of the polyethylene glycol glucosides and FA esters thereof by atmospheric-pressure ionization MS

Polyethylene glycol (PEG) glucosides (PEGG) and the PEGG esters of lauric acid were analyzed by atmospheric-pressure ionization MS (API-MS) with electrospray ionization. Straightforward mass characterization of the complex mixtures could be achieved without prior chromatographic separation. The constituents were identified on the basis of quasi-molecular ions. Individual components could be observed as protonated molecular ions [M+H]⁺ and/or as their NH₄ ⁺, Na⁺, or K⁺ adducts in positive ion mode. The mass spectrometric investigation showed that mixtures of PEGG consisted of monoglucoside, diglucoside, polyglucoside, and free PEG. The esterification product is a mixture of two types of nonionic surfactants: PEG-laurates and PEGG-laurates. The reasons for distortion of the quasi-molecular ion intensities and the stabilization of adduct ions were discussed. The rapid and highly sensitive API-MS analysis technique proposed here is well suited for direct characterization of complex mixtures and suitable for development as a routine analytical method.     

Adapted MS/MSALL Shotgun Lipidomics Approach for Analysis of Cardiolipin Molecular Species

Cardiolipin (Ptd₂Gro) is a complex, doubly charged phospholipid located in the inner mitochondrial membrane where it plays an essential role in regulating bioenergetics. Abnormalities in Ptd₂Gro content or composition have been associated with mitochondrial dysfunction in a variety of disease states. Here, we report the development of an adapted high‐resolution data‐independent acquisition (DIA) MS/MS^ALL shotgun lipidomic method to enhance the accuracy and reproducibility of Ptd₂Gro molecular species quantitation from biological samples. Utilizing the doubly charged molecular ions and the isotopic pattern with negative mode electrospray ionization mass spectrometry (ESI‐MS) using an adapted MS/MS^ALL approach, we profiled more than 150 individual Ptd₂Gro species, including monolysocardiolipin (MLPtd₂Gro). The method described in this study demonstrated high reproducibility, sensitivity, and throughput with a wide dynamic range. This high‐resolution MS/MS^ALL shotgun lipidomics approach could be extended to screening aberrations of Ptd₂Gro metabolism involved in mitochondrial dysfunction in various pathological conditions and diseases.     

Recoveries of rat lymph FA after administration of specific structured <Superscript>13</Superscript>C-TAG

The potential of the specific structured TAG MLM [where M-caprylic acid (8∶0) and L=linoleic acid (18∶2n−6)] is the simultaneous delivery of energy and EFA. Compared with long-chain TAG (LLL), they may be more rapidly hydrolyzed and absorbed. This study examined the lymphatic recoveries of intragastrically administered L^*L^*, M^*M^*M^*, ML^*M, and ML^*L^* (where ^*=¹³C-labeled FA) in rats. Lymph lipids were separated into lipid classes and analyzed by GC combustion isotope ratio MS. The recoveries of lymph TAG 18∶2n-6 8 h after administration of L^*L^*L^*, ML^*M, and ML^*L^* were 38.6, 48.4, and 49.1%, respectively, whereas after 24 h the recoveries were approximately 50% in all experimental groups. The exogenous contribution to lymph TAG 18∶2n-6 was approximately 80 and 60% at maximum absorption of the specific structured TAG and L^*L^*L^*, respectively, 3–6 h after administration. The tendency toward more rapid recovery of exogenous long-chain FA following administration of specific structured TAG compared with long-chain TAG was probably due to fast hydrolysis. The lymphatic recovery of 8∶0 was 2.2% 24 h after administration of M^*M^*M^*. This minor lymphatic recovery of exogenous 8∶0 was probably due to low stimulation of chylomicron formation. These results demonstrate tendencies toward faster lymphatic recovery of long-chain FA after administration of specific structured TAG compared with long-chain TAG.     

Characterization of sacha inchi (<Emphasis Type="Italic">Plukenetia volubilis</Emphasis> L.) oil by FTIR spectroscopy and <Superscript>1</Superscript>H NMR. Comparison with linseed oil

Three oil samples obtained from sacha inchi (Plukenetia volubilis L.) seeds were studied by means of FTIR and ¹H NMR. Frequency data of the most significant bands of the IR spectrum of this oil are given. These data show that sacha inchi oil has a high degree of unsaturation. The same fact is deduced from the ratio between the absorbance of the bands due to the stretching vibrations of the cis olefinic CH double bonds at 3010.5 cm⁻¹ and to the methylene symmetrical stretching vibrations at 2855.1 cm⁻¹. The proportions of monounsaturated, polyunsaturated, and saturated acyl groups were predicted from the frequency of some IR bands, and these were in satisfactory agreement with the values obtained through FAME generation and their quantification by GC. Likewise, simple observation of the ¹H NMR spectra provided a great deal of information about sacha inchi oil, with regard not only to the relative proportions of the different acyl groups but also to their nature. Thus, the presence of γ-linolenic acyl groups was discounted. Furthermore, the area of some ¹H NMR signals was used to determine the proportion of saturated and mono-, di-, and triunsaturated acyl groups, which also were in satisfactory agreement with the values obtained by classical methods. IR and ¹H NMR determinations take very little time in comparison with classical methods and do not require chemical manipulation or transformation of the sample. A comparison was also made between the compositions of sacha inchi and linseed oil. Both oils are important sources of the healthful n−3 linolenic acyl groups, and sacha inchi also contains high proportions of the n−6 linoleic acyl groups.     

<Superscript>13</Superscript>C-NMR Regioisomeric Analysis of EPA and DHA in Fish Oil Derived Triacylglycerol Concentrates

The regio-isomeric distribution of the omega-3 polyunsaturated fatty acids (PUFA) cis-5,8,11,14,17-eicosapentaenoic acid (EPA) and cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) in the triacylglycerols (TAG) of anchovy/sardine fish oil was determined by ¹³C nuclear magnetic resonance (NMR) analysis under quantitative conditions. From the measurements of sn-1,3 and sn-2 carbonyl peak areas it was established that EPA was mainly located in the sn-1,3 positions, whereas DHA primarily occupied the sn-2 position. Reconstituted TAG prepared by Candida antarctica lipase-B (CALB) glycerolysis of the ethyl ester (EE) or the free fatty acid (FFA) forms of anchovy/sardine fish oil, displayed a different pattern: EPA was equally distributed, while DHA was preferentially attached to the sn-1,3 positions. TAG concentrates of varying EPA and DHA molar fractions were prepared by CALB-catalyzed glycerolysis of the corresponding EE and FFA. ¹³C-NMR analysis of the purified products revealed a lack of CALB regioselectivity for EPA and a slight sn-1,3 regioselectivity for DHA. Since this pattern was observed in all cases of this study, it was concluded that the lipase regioselectivity during TAG synthesis is independent of both the acyl donor type (carboxylic acid or ester) and the fatty acid content of the oil substrate.     

Analysis of Triacylglycerols and Free Fatty Acids in Algae Using Ultra-Performance Liquid Chromatography Mass Spectrometry

To assess the suitability of microalgal strains for biodiesel production the lipid content and composition, especially individual triacylglycerols (TAG) and free fatty acids (FFA) must be determined. In this study, the compositions and concentrations of TAG and FFA were analysed in four halophytic algal species, Dunaliella salina, D. tertiolecta, D. bardawil, and D. granulata. These species were selected as part of a larger screen to identify species suitable for biofuel feedstocks. An accelerated solvent extraction instrument was used for lipids and fatty acid extraction using a dichloromethane–hexane solvent system. Ultra-performance liquid chromatography coupled with mass spectrometry (MS) detection was optimized and applied to the quantitative analysis of TAG and FFA in the different algal extracts. Individual TAG were characterized structurally using direct electrospray ionization (ESI) MS and MS/MS techniques. Cationic adducts (NH₄ ⁺) of TAG were detected and quantified in the positive ESI MS and MS/MS modes, while the negative ESI mode was used for FFA analysis. Over 20 TAG were identified and quantified in the four Dunaliella strains. Analysis of FFA compositions demonstrated that the most abundant FFA in these four algal species were palmitic, linolenic, linoleic, and oleic acids.     

Chemometric classification of olive cultivars based on compositional data of oils

European Union regulations provide important guidelines for maintaining the Protected Designation of Origin (PDO) of olive oil and other foods. This includes characterization of foods based on variety (cultivar) and geographical origin, as this may be used as a criterion for determining authenticity and quality. Therefore, analytical method standards need to be established to ensure these criteria. This study describes how cultivar differences can be established between Italian oils, obtained from single varieties, based on acid, sterol, and TAG differences determined by chemometrics. TAG and FA composition provided the best basis for differentiation of olive oils among cultivars. The results were compared with those obtained using ¹³C NMR analysis, and a similar differentiation between oils of different cultivars was achieved. ¹³C NMR provides useful information on the acyl composition and on the positional distribution of the glycerol moiety and can be used for classification of cultivars based on oil composition. Furthermore, the advantages of this technique come from the rapidity with which information can be obtained and from the very simple preparation procedure required for analysis.     

Oleate acutely stimulates the secretion of triacylglycerol by cultured rat hepatocytes by accelerating the emptying of the secretory compartment

The acute effects of addition of oleate on the rate of triacylglycerol (TAG) secretion by cultured rat hepatocytes were studied by monitoring the use of endogenous (¹⁴C-prelabeled) acyl moieties and exogenous (³H-labeled) oleate for the synthesis of secreted TAG simultaneously. Inclusion of exogenous oleate in the medium stimulated the secretion of the endogenous ¹⁴C-labeled acyl moieties by 55–100%. To find out whether the stimulation was due to increased endogenous TAG mobilization or an increased rate of processing of TAG within the endoplasmic reticulum (ER) secretory machinery, use was made of the inhibition of apolipoprotein B (apoB) synthesis (but not degradation) by Ca²⁺ mobilization from the ER. Inhibition of apoB synthesis stopped entry of acyl moieties (from endogenous and exogenous sources) into the secretory pathway. However, even when entry of acyl moieties into the secretory pathway was totally inhibited, exogenous oleate was still able to stimulate (twofold) the secretion [¹⁴C]TAG, indicating that oleate stimulates the emptying of prelabeled TAG from the secretory compartment at a point distal to apoB synthesis and nascent particle formation. These data indicate that exogenous oleate, besides providing additional acyl moieties for incorporation into secreted TAG, stimulates the secretion of endogenous TAG in a manner (i) that is independent of effects on apoB synthesis and/or degradation and (ii) that involves the enhanced processing of TAG resident within the ER secretory pathway.     

Nontargeted Lipidomic Characterization of Porcine Organs Using Hydrophilic Interaction Liquid Chromatography and Off-Line Two-Dimensional Liquid Chromatography–Electrospray Ionization Mass Spectrometry

Lipids form a significant part of animal organs and they are responsible for important biological functions, such as semi‐permeability and fluidity of membranes, signaling activity, anti‐inflammatory processes, etc. We have performed a comprehensive nontargeted lipidomic characterization of porcine brain, heart, kidney, liver, lung, spinal cord, spleen, and stomach using hydrophilic interaction liquid chromatography (HILIC) coupled to electrospray ionization mass spectrometry (ESI/MS) to describe the representation of individual lipid classes in these organs. Detailed information on identified lipid species inside classes are obtained based on relative abundances of deprotonated molecules [M?H]^? in the negative‐ion ESI mass spectra, which provides important knowledge on phosphatidylethanolamines and their different forms of fatty acyl linkage (ethers and plasmalogens), phosphatidylinositols, and hexosylceramides containing nonhydroxy‐ and hydroxy‐fatty acyls. The detailed analysis of identified lipid classes using reversed‐phase liquid chromatography in the second dimension was performed for porcine brain to determine more than 160 individual lipid species containing attached fatty acyls of different acyl chain length, double‐bond number, and positions on the glycerol skeleton. The fatty acid composition of porcine organs is determined by gas chromatography with flame ionization detection after the transesterification with sodium methoxide.