Position Of Double Bond Research Articles

Elucidation of phosphatidylcholine isomers using two dimensional liquid chromatography coupled in-line with ozonolysis mass spectrometry

In this study, two dimensional (2D) liquid chromatography (LC) was coupled in-line with ozonolysis-mass spectrometry (O3-MS) for the elucidation of phosphatidylcholine (PC) structures in phospholipid extracts. In O3-MS, PC molecules carried by mobile phase pass through a length of gas-permeable tubing surrounded by ozone vapor resulting in oxidative cleavage of double bonds. The characteristic aldehyde products are then directly analyzed by eletrospray ionization (ESI)-MS, allowing assignment of the double bond positions within the PC fatty acyl chains. The on-line 2D-LC method employs hydrophilic interaction chromatography as the first dimension to separate phospholipid classes and reversed phase chromatography in the second dimension to further separate PC molecular species. Thus, by combing the separation power of 2D-LC with in-line O3-MS (2D-LC/O3-MS), PC species in complex mixtures can be identified including the detailed structure of their two fatty acid chains. When the 2D-LC/O3-MS method was applied to a rat liver phospholipid extract, 19 PC molecular species were identified with fatty acid isomers unambiguously assigned.

Identification and significance of unsaturated archaeal tetraether lipids in marine sediments

Studies of archaeal glycerol dibiphytanyl glycerol tetraethers (GDGTs) in the environment and cultures have exclusively focused on compounds with fully saturated alkyl moieties. Here we report a number of novel unsaturated GDGTs (uns-GDGTs) whose alkyl chains contain up to six double bonds and zero to two cyclopentyl moieties. The identification of these lipids was achieved via comparison of lipid distribution before and after hydrogenation, characteristic retention time patterns, and diagnostic ions using liquid chromatography/tandem mass spectrometry (LC/MS/MS), and ether cleavage products using gas chromatography/mass spectrometry (GC/MS). Isomerism resulting from different unsaturation patterns in the alkyl moieties was observed and specific positions of double bonds in the biphytene and biphytadiene moieties were tentatively assigned. Uns-GDGTs were detected in sediment and microbial mat samples as both core lipids (CLs) and intact polar lipids (IPLs) associated with mono- or diglycosyl or phosphatidylglycerol headgroups. However, these lipids were overlooked in past investigations because conventional methods for archaeal lipid analysis are unsuitable for uns-GDGTs. Samples from distinct marine environments (Black Sea, Cariaco Basin, Discovery Basin, Eastern Mediterranean Sea, upwelling area off NW Africa, and seep sites off Crimea and Pakistan) were screened for uns-GDGTs using a new LC/MS protocol. The results show that uns-GDGTs contribute significantly to the archaeal lipid pool in anoxic methane-rich environments (Black Sea, Cariaco Basin, and both seep sites) but they were barely detected in the oxic or hypersaline settings. The characteristic distribution of uns-GDGTs implies that they are attractive targets for future studies aiming at the chemotaxonomy of uncultivated archaea and regulation of uns-GDGT biosynthesis.

Thermodynamic Parameters of the Interactions between Lapachol and Isolapachol Sodium Salts and Chitosan Flakes

The adsorption process of lapachol and isolapachol sodium salts (LSS and ISS, respectively) on chitosan flakes was investigated. The aim of this work is to analyze the interaction between chitosan flakes and LSS and ISS, bioactive quinones, looking for the enhancement of their stability and for their controlled release. The effects of contact time, quinone concentration, temperature, and ionic strength on the adsorption of LSS and ISS onto chitosan flakes were studied using a batch adsorption technique. The kinetics of adsorption follows the pseudo-second-order model. Isotherm data were analyzed using the Langmuir and Freundlich models. The adsorption process was found to be endothermic for LSS–chitosan and exothermic for ISS–chitosan. The results have shown the importance of the double bond position in the side chain of the quinone. The stability of both quinones was improved after adsorption on chitosan. An increase in ionic strength decreases the amount adsorbed for both quinones around 50 % for LSS an...

1,3-Dipolar cycloaddition of BC2N nanotubes: A DFT study

It seems the isolated CC units in the zigzag BC2N nanotubes could be considered as preactivated sites for exohedral functionalization. Therefore, we theoretically investigate the [2+3] cycloaddition reactions of 1,3 dipoles (1,3 DCs) such as nitriloxide (HCN+O−), azide (NN+NH−) and azomethine ylides (H2CNH+CH2-) with BC2N nanotubes in terms of geometry, energies, and electronic structures. Negative values of 1,3 DC reaction energies indicate the exothermic character of adsorption process of BC2N nanotubes. Our results show the ZZ-3 (n, 0) BC2N nanotubes in which borons and nitrogens are located in trans positions of CC double bonds lead to the most energetically favorable configuration for 1,3 DC products. Further, Er values for different (n, 0) BC2N nanotubes are in the series of (3,0)<(4,0)<(5,0), indicating BC2N-NTs with smaller diameter (higher curvature) might be more favorable because of both energetic and structural considerations. Er values for different 1,3 dipoles are in the series of (H2CNH+CH2-)<(HCN+O−)< (NN+NH−), in agreement with the distortion/interaction model for the cycloaddition reactions. Variation of HOMO–LUMO gaps (Eg) for 1,3 DC products shows a decreasing trend with increasing of tube diameter while Eg is almost independent of the type of 1,3 dipoles.

Pinpointing Double Bonds in Lipids by Paternò‐Büchi Reactions and Mass Spectrometry

AbstractThe positions of double bonds in lipids play critical roles in their biochemical and biophysical properties. In this study, by coupling Paternò–Büchi (P‐B) reaction with tandem mass spectrometry, we developed a novel method that can achieve confident, fast, and sensitive determination of double bond locations within various types of lipids. The P‐B reaction is facilitated by UV irradiation of a nanoelectrospray plume entraining lipids and acetone. Tandem mass spectrometry of the on‐line reaction products via collision activation leads to the rupture of oxetane rings and the formation of diagnostic ions specific to the double bond location.

Pinpointing Double Bonds in Lipids by Paternò‐Büchi Reactions and Mass Spectrometry

The positions of double bonds in lipids play critical roles in their biochemical and biophysical properties. In this study, by coupling Paternò-Büchi (P-B) reaction with tandem mass spectrometry, we developed a novel method that can achieve confident, fast, and sensitive determination of double bond locations within various types of lipids. The P-B reaction is facilitated by UV irradiation of a nanoelectrospray plume entraining lipids and acetone. Tandem mass spectrometry of the on-line reaction products via collision activation leads to the rupture of oxetane rings and the formation of diagnostic ions specific to the double bond location.

Ligand determinants of fatty acid activation of the pronociceptive ion channel TRPA1

Background and purpose. Arachidonic acid (AA) and its derivatives are important modulators of cellular signalling. The transient receptor potential cation channel subfamily A, member 1 (TRPA1) is a cation channel with important functions in mediating cellular responses to noxious stimuli and inflammation. There is limited information about the interactions between AA itself and TRPA1, so we investigated the effects of AA and key ethanolamide and amino acid/neurotransmitter derivatives of AA on hTRPA1.Experimental approach. HEK 293 cells expressing hTRPA1 were studied by measuring changes in intracellular calcium ([Ca]i) with a fluorescent dye and by standard whole cell patch clamp recordings.Key results. AA (30 μM) increased fluorescence in hTRPA1 expressing cells by 370% (notional EC50 13 μM). The covalent TRPA1 agonist cinnamaldehyde (300 μM) increased fluorescence by 430% (EC50, 11 μM). Anandamide (230%) and N-arachidonoyl tyrosine (170%) substantially activated hTRPA1 at 30 μM, however, N-arachidonoyl conjugates of glycine and taurine were less effective while N-acyl conjugates of 5-HT did not affect hTRPA1. Changing the acyl chain length or the number and position of double bonds reduced fatty acid efficacy at hTRPA1. Mutant hTRPA1 (Cys621, Cys641 and Cys665 changed to Ser) could be activated by AA (100 μM, 40% of wild type) but not by cinnamaldehyde (300 μM).Conclusions and implications. AA is a more potent activator of TRPA1 than its ethanolamide or amino acid/neurotransmitter derivatives and acts via a mechanism distinct from that of cinnamaldehyde, further underscoring the likelyhood of multiple pharmacologically exploitable sites on hTRPA1.

Characterization of Cyanobacterial Hydrocarbon Composition and Distribution of Biosynthetic Pathways

Cyanobacteria possess the unique capacity to naturally produce hydrocarbons from fatty acids. Hydrocarbon compositions of thirty-two strains of cyanobacteria were characterized to reveal novel structural features and insights into hydrocarbon biosynthesis in cyanobacteria. This investigation revealed new double bond (2- and 3-heptadecene) and methyl group positions (3-, 4- and 5-methylheptadecane) for a variety of strains. Additionally, results from this study and literature reports indicate that hydrocarbon production is a universal phenomenon in cyanobacteria. All cyanobacteria possess the capacity to produce hydrocarbons from fatty acids yet not all accomplish this through the same metabolic pathway. One pathway comprises a two-step conversion of fatty acids first to fatty aldehydes and then alkanes that involves a fatty acyl ACP reductase (FAAR) and aldehyde deformylating oxygenase (ADO). The second involves a polyketide synthase (PKS) pathway that first elongates the acyl chain followed by decarboxylation to produce a terminal alkene (olefin synthase, OLS). Sixty-one strains possessing the FAAR/ADO pathway and twelve strains possessing the OLS pathway were newly identified through bioinformatic analyses. Strains possessing the OLS pathway formed a cohesive phylogenetic clade with the exception of three Moorea strains and Leptolyngbya sp. PCC 6406 which may have acquired the OLS pathway via horizontal gene transfer. Hydrocarbon pathways were identified in one-hundred-forty-two strains of cyanobacteria over a broad phylogenetic range and there were no instances where both the FAAR/ADO and the OLS pathways were found together in the same genome, suggesting an unknown selective pressure maintains one or the other pathway, but not both.

Types of Oilseed and Adipose Tissue Influence the Composition and Relationships of Polyunsaturated Fatty Acid Biohydrogenation Products in Steers Fed a Grass Hay Diet

The current study evaluated the composition and relationships of polyunsaturated fatty acid biohydrogenation products (PUFA-BHP) from the perirenal (PRF) and subcutaneous fat (SCF) of yearling steers fed a 70% grass hay diet with concentrates containing either sunflower-seed (SS) or flaxseed (FS). Analysis of variance indicated several groups or families of structurally related FA, and individual FA within these were affected by a number of novel oilseed by fat depot interactions (P<0.05). Feeding diets containing SS increased the proportions of non-conjugated 18:2 BHP (i.e., atypical dienes, AD) and conjugated linoleic acids (CLA) with the first double bond from carbon 7 to 9, trans-18:1 isomers with double bonds from carbon 6 to 12, and these PUFA-BHP had greater proportions in SCF compared to PRF (P<0.05). Enrichment of conjugated linolenic acids, AD and CLA isomers with the first double bond in position 11 or 12, and t-18:1 isomers with double bonds from carbon 13 to 16 were achieved by feeding diets containing FS, with PRF having greater proportions than SCF (P<0.05). Principal component analysis visually confirmed interaction effects on these groups/families of FA, and further confirmed or suggested a number of relationships between PUFA-BHP. Feeding SS or FS in a grass hay diet and exploiting adipose tissue differences, therefore, present unique opportunities to differentially enrich a number of PUFA-BHP which seem to have positive health potential in humans (i.e., t11-18:1, c9,t11-18:2 and c9,t11,c15-18:3).

What Happens for Sterol Dynamics When Cholesterol is Enzymatically Oxidized?

Cholesterol is an essential component of the mammalian cell membrane. Thermal fluctuations of cholesterol from its equilibrium position, along the bilayer normal, have important consequences in its cellular trafficking. In this work, we have investigated the effect of replacing part of membrane cholesterol with one of its oxidative products, cholestenone (4-cholesten-3-one, a ketosterone that differs from cholesterol only in the hydroxyl headgroup and the position of double bond), on the transbilayer movement (flip-flop). In the same spirit, we have studied desorption of these sterols from a membrane towards the aqueous phase. The reason why we focus on cholestenone is that it is one of the most common products when cholesterol is being oxidized, thus serving as an example of cholesterol oxidation. The results from our atomistic molecular dynamics simulations show that replacing the hydroxyl group of cholesterol by the keto group found in cholestenone has a significant effect on inter-leaflet translocation of the sterol. To discuss the cause and consequences of the observed effects, we used umbrella (free energy) simulations to calculate the potential of mean force for flip-flop as well as for desorption of these two sterols from the membrane into the water phase. The results brought out that even seemingly tiny changes in sterol structure have considerable implications for sterol dynamics. As cholesterol oxidation is often used to deplete cholesterol in experimental conditions, our results highlight that it is essential to understand the membrane behavior of the introduced lipid species, in this case cholestenone.

Effects of Lipid Composition on Biological Membrane Electrostatics

A complete understanding of how biological membranes function can be obtained only by considering how their various components influence membrane properties and their interactions with biomolecules. In membrane charge transport processes, such as ion permeation, or the movements of membrane-active peptides, a better understanding of lipid-dependent membrane electrostatics is needed. We have varied lipid components that influence membrane chemistry, structure, hydration and packing in ways that will alter the membrane's electrostatic (dipole) potential. We have compared simulations with different electrolyte composition to study the electrostatic influences of ions. We have simulated zwitterionic and 1:1 mixtures with charged lipids, to analyse how the head group controls the dipole potential. We varied the glycerol backbone of the lipids, studying ether lipids that have no carbonyl groups, leading to changes in dipole potential by up to 250 mV. We discuss an effect of interfacial water on the dipole potential and explain the origin of the change. We have simulated a 1:1 mixture of mono- and di-myristoylphosphatidylcholine to isolate the contribution from the single carbonyl group. Lipid chain length effects have been studied using saturated and unsaturated bilayers of different thickness, spanning approximately 7 A. Simulations of monounsaturated and polyunsaturated bilayers were compared to evaluate the roles of double bond position and extent of unsaturation. Differences in dipole potential for all systems are in a good agreement with experiment. Important contributions from the different lipid components along with methods to extract membrane electrostatic properties will be discussed.

Structural characterization of glycerophospholipids by combinations of ozone- and collision-induced dissociation mass spectrometry: the next step towards “top-down” lipidomics

The complete structural elucidation of complex lipids, including glycerophospholipids, using only mass spectrometry represents a major challenge to contemporary analytical technologies. Here, we demonstrate that product ions arising from the collision-induced dissociation (CID) of the [M + Na](+) adduct ions of phospholipids can be isolated and subjected to subsequent gas-phase ozonolysis - known as ozone-induced dissociation (OzID) - in a linear ion-trap mass spectrometer. The resulting CID/OzID experiment yields abundant product ions that are characteristic of the acyl substitution on the glycerol backbone (i.e., sn-position). This approach is shown to differentiate sn-positional isomers, such as the regioisomeric phosphatidylcholine pair of PC 16:0/18:1 and PC 18:1/16:0. Importantly, CID/OzID provides a sensitive diagnostic for the existence of an isomeric mixture in a given sample. This is of very high value for the analysis of tissue extracts since CID/OzID analyses can reveal changes in the relative abundance of isomeric constituents even within different tissues from the same animal. Finally, we demonstrate the ability to assign carbon-carbon double bond positions to individual acyl chains at specific backbone positions by adding subsequent CID and/or OzID steps to the workflow and that this can be achieved in a single step using a hybrid triple quadrupole-linear ion trap mass spectrometer. This unique approach represents the most complete and specific structural analysis of lipids by mass spectrometry demonstrated to date and is a significant step towards comprehensive top-down lipidomics.

Synthesis of long-chain fatty acid derivatives as a novel anti-Alzheimer’s agent

In order to develop new drugs for Alzheimer’s disease, we prepared 17 fatty acid derivatives with different chain lengths and different numbers and positions of double bonds by using Wittig reaction and stereospecific hydrogenation of triple bonds as key reactions. Among them, (4Z,15Z)-octadecadienoic acid (10) and (23Z,34Z)-heptatriacontadienoic acid (16) showed the most potent neurite outgrowth activities on Aβ(25–35)-treated rat cortical neurons, which activities were comparable to that of a positive control, NGF. Both fatty acids 10 and 16 possess two (Z)-double bonds at the n-3 and n-14 positions, which might be important for the neurite outgrowth activity.

Effect of Monoglyceride Structure and Cholesterol Content on Water Permeability of the Droplet Bilayer

The process of water permeation across lipid membranes has significant implications for cellular physiology and homeostasis, and its study may lead to a greater understanding of the relationship between the structure of lipid bilayer and the role that lipid structure plays in water permeation. In this study, we formed a droplet interface bilayer (DIB) by contacting two aqueous droplets together in an immiscible solvent (squalane) containing bilayer-forming surfactant (monoglycerides). Using the DIB model, we present our results on osmotic water permeabilities and activation energy for water permeation of an associated series of unsaturated monoglycerides as the principal component of droplet bilayers, each having the same chain length but differing in the position and number of double bonds, in the absence and presence of a varying concentration of cholesterol. Our findings suggest that the tailgroup structure in a series of monoglyceride bilayers is seen to affect the permeability and activation energy for the water permeation process. Moreover, we have also established the insertion of cholesterol into the droplet bilayer, and have detected its presence via its effect on water permeability. The effect of cholesterol differs depending on the type of monoglyceride. We demonstrate that the DIB can be employed as a convenient model membrane to rapidly explore subtle structural effects on bilayer water permeability.

Stereochemistry of C18 Monounsaturated Cork Suberin Acids Determined by Spectroscopic Techniques Including 1 H-NMR Multiplet Analysis of Olefinic Protons

Suberin is a biopolyester responsible for the protection of secondary plant tissues, and yet its molecular structure remains unknown. The C18:1 ω-hydroxyacid and the C18:1 α,ω-diacid are major monomers in the suberin structure, but the configuration of the double bond remains to be elucidated. To unequivocally define the configuration of the C18:1 suberin acids. Pure C18:1 ω-hydroxyacid and C18:1 α,ω-diacid, isolated from cork suberin, and two structurally very close C18:1 model compounds of known stereochemistry, methyl oleate and methyl elaidate, were analysed by NMR spectroscopy, Fourier transform infrared (FTIR) and Raman spectroscopy, and GC-MS. The GC-MS analysis showed that both acids were present in cork suberin as only one geometric isomer. The analysis of dimethyloxazoline (DMOX) and picolinyl derivatives proved the double bond position to be at C-9. The FTIR spectra were concordant with a cis-configuration for both suberin acids, but their unambiguous stereochemical assignment came from the NMR analysis: (i) the chemical shifts of the allylic (13) C carbons were shielded comparatively to the trans model compound, and (ii) the complex multiplets of the olefinic protons could be simulated only with (3) JHH and long-range (4) JHH coupling constants typical of a cis geometry. The two C18:1 suberin acids in cork are (Z)-18-hydroxyoctadec-9-enoic acid and (Z)-octadec-9-enedoic acid.

Locating double bonds in lipids – New approaches to the use of ozonolysis

AbstractSince the location of double bonds within a lipid molecule is critical to its biological function, methods that reveal details of this structural arrangement are of importance. Ozone can react with double bonds resulting in ozonolysis products that have masses characteristic of the exact location of these double bonds within fatty acid residues. This paper describes the use of the ozonolysis reaction for the assignment of double bond positions, with a focus on the recent developments in coupling ozonolysis reactions in‐situ and in‐line with mass spectrometry, even for use with complex lipid samples.

A comprehensive evaluation of the cetane numbers of fatty acid methyl esters

The cetane number (CN), being related to ignition and combustion behavior, is a prime indicator of the quality of diesel fuels, including those derived from renewable resources such as biodiesel. While many effects of compound structure are known or have been postulated, experimental data have not always been available for the various ester components of biodiesel and a comprehensive evaluation including some structural features not yet experimentally investigated such as double bond configuration and position is lacking. In this work, existing CN data of fatty esters are complemented by studying C18 esters with differing double bond positions and double bond configurations. For the first time, CNs, determined as derived cetane number (DCNs), of neat trans fatty acid methyl esters, methyl elaidate (methyl 9(E)-octadecenoate) and methyl linolelaidate (methyl 9(E),12(E)-octadecenoate), were determined as were the CNs of the C18:1 positional isomers methyl petroselinate (methyl 6(Z)-octadecenoate) and methyl asclepate (methyl 11(Z)-octadecenoate). The CNs of the positional and geometric isomers of methyl oleate are close to the CN of methyl oleate. These data are compared to other previously determined CN data. Furthermore, the applicability of CN data is evaluated using an extensive collection of CN determinations for methyl oleate as “standard” fatty acid (methyl) ester. The average CN value for methyl oleate is in the range of 56–58 with a CN of 57 suggested for calculating purposes. Similarly, uncertainty ranges could be established for CNs of other fatty materials. The data are also useful for predicting CNs of compounds for which CNs are not readily available or easily determinable.

Unprecedented separation of long chain alkenones from gas chromatography with a poly(trifluoropropylmethylsiloxane) stationary phase

Long chain alkenones (LCAs) have played a central role in reconstructing paleo-sea surface temperature throughout much of the world oceans. The standard method for LCA separation and quantification utilizes gas chromatography (GC) with a non-polar column and flame ionization detection (FID). The method has remained nearly unchanged since the debut of alkenone paleothermometry over three decades ago. The conventional stationary phase used [poly(dimethylsiloxane)] is adequate for calculation of the U37K′ index for most marine samples, but performs poorly in the separation of methyl and ethyl C38 LCAs and fails to separate certain lacustrine LCAs, including structural isomers featuring differing double bond and carbonyl positions. Here, we demonstrate major improvements in the selectivity and resolution of numerous LCAs using a mid-polarity, dipole selective poly(trifluoropropylmethylsiloxane) GC stationary phase with FID. Various columns featuring this phase achieved baseline resolution of all C38 LCAs in a marine sediment and Emiliania huxleyi culture samples, improving peak resolution of C38 LCAs by>350%. Such dipole selective stationary phases also reveal novel tri-unsaturated isomeric LCAs in lacustrine samples, for which we provide the first report and preliminary characterization. We conduct a thorough evaluation of LCA retention behavior on poly(trifluoropropylmethylsiloxane) and poly(dimethylsiloxane) columns and report an optimal range of operating conditions for both phases.

Hexadecenoic Fatty Acid Isomers: A Chemical Biology Approach for Human Plasma Biomarker Development

Hexadecenoic fatty acids are monounsaturated lipid components, which are interesting targets of plasma lipidomic studies and biomarker development. The main positional isomers, palmitoleic (9-cis-16:1) and sapienic acids (6-cis-16:1), have an endogenous origin from palmitic acid, the former being recognized as a component of adipose tissue with signaling activity, whereas the latter is mainly reported as a component of sebum. The trans 16:1 isomers are attributed so far to dietary sources of industrial and dairy fats, whereas the endogenous formation due to the free radical-mediated isomerization can represent an emerging, yet unexplored, pathway connected to cellular stress. Herein, we report a chemical biology approach for the development of hexadecenoic fatty acids as plasma biomarkers, with the first synthesis of 6-trans-16:1 and the efficient analytical setup with unambiguous assignment of 16:1 double bond position and geometry, which was applied to human commercial LDL and plasma cholesteryl esters. Sapienic acid was identified together with its geometrical trans isomer for the first time. The quantitation of hexadecenoic fatty acid isomers evidenced their different levels in the two lipid classes and LDL fractions, making us foresee interesting applications to the metabolic evaluation of fatty acid pathways. These findings open new perspectives for plasma lipidomics involving monounsaturated fatty acids, highlighting future developments for their evaluation in different health conditions including free radical stress.

Identification and Quantitation of Fatty Acid Double Bond Positional Isomers: A Shotgun Lipidomics Approach Using Charge-Switch Derivatization

The specific locations of double bonds in mammalian lipids have profound effects on biological membrane structure, dynamics and lipid second messenger production. Herein, we describe a shotgun lipidomics approach that exploits charge-switch derivatization with N-(4-aminomethylphenyl) pyridinium (AMPP) and tandem mass spectrometry for identification and quantification of fatty acid double bond positional isomers. Through charge-switch derivatization of fatty acids followed by positive-ion mode ionization and fragmentation analysis, a marked increase in analytic sensitivity (low fmol/μL) and the identification of double bond positional isomers can be obtained. Specifically, the locations of proximal double bonds in AMPP-derivatized fatty acids are identified by diagnostic fragment ions resulting from the markedly reduced 1,4-hydrogen elimination from the proximal olefinic carbons. Additional fragmentation patterns resulting from allylic cleavages further substantiated the double bond position assignments. Moreover, quantification of fatty acid double bond positional isomers is achieved by the linear relationship of the normalized intensities of characteristic fragment ions vs the isomeric compositions of discrete fatty acid positional isomers. The application of this approach for the analysis of fatty acids in human serum demonstrated the existence of two double bond isomers of linolenic acid (i.e., Δ(6,9,12) 18:3, γ-linolenic acid (GLA), and Δ(9,12,15) 18:3, α-linolenic acid (ALA)). Remarkably, the isomeric ratio of GLA vs ALA esterified in neutral lipids was 3-fold higher than the ratio of their nonesterified moieties. Through this developed method, previously underestimated or unidentified alterations in fatty acid structural isomers can be determined facilitating the identification of novel biomarkers and maladaptive alterations in lipid metabolism during disease.