Nonpolar Lipid Classes Research Articles

Ultrahigh-Performance Supercritical Fluid Chromatography-Mass Spectrometry in the Clinical Lipidomic Analysis.

Ultrahigh-performance supercritical fluid chromatography-mass spectrometry (UHPSFC/MS) method is optimized for the high-throughput quantitation of lipids in human serum and plasma with an emphasis on robustness and accurate quantitation. Bridged ethylene hybrid (BEH) silica column (100×3mm; 1.7μm) is used for the separation of 17 nonpolar and polar lipid classes in 4.4min using the positive ion electrospray ionization mode. The lipid class separation approach in UHPSFC/MS results in the coelution of all lipid species within one lipid class in one chromatographic peak, including two exogenous internal standards (IS) per lipid class, which provides the optimal conditions for robust quantitation. The method was validated according to European Medicines Agency and Food and Drug Administration recommendations. UHPSFC/MS combined with LipidQuant software allows a semiautomated process to determine lipid concentrations with a total run time of only 8min including column equilibration, which enables the analysis of 160 samples per day.

A lipidomics platform to analyze the fatty acid compositions of non-polar and polar lipid molecular species from plant tissues: Examples from developing seeds and seedlings of pennycress (Thlaspi arvense).

The lipidome comprises the total content of molecular species of each lipid class, and is measured using the analytical techniques of lipidomics. Many liquid chromatography-mass spectrometry (LC-MS) methods have previously been described to characterize the lipidome. However, many lipidomic approaches may not fully uncover the subtleties of lipid molecular species, such as the full fatty acid (FA) composition of certain lipid classes. Here, we describe a stepwise targeted lipidomics approach to characterize the polar and non-polar lipid classes using complementary LC-MS methods. Our "polar" method measures 260 molecular species across 12 polar lipid classes, and is performed using hydrophilic interaction chromatography (HILIC) on a NH2 column to separate lipid classes by their headgroup. Our "non-polar" method measures 254 molecular species across three non-polar lipid classes, separating molecular species on their FA characteristics by reverse phase (RP) chromatography on a C30 column. Five different extraction methods were compared, with an MTBE-based extraction chosen for the final lipidomics workflow. A state-of-the-art strategy to determine and relatively quantify the FA composition of triacylglycerols is also described. This lipidomics workflow was applied to developing, mature, and germinated pennycress seeds/seedlings and found unexpected changes among several lipid molecular species. During development, diacylglycerols predominantly contained long chain length FAs, which contrasted with the very long chain FAs of triacylglycerols in mature seeds. Potential metabolic explanations are discussed. The lack of very long chain fatty acids in diacylglycerols of germinating seeds may indicate very long chain FAs, such as erucic acid, are preferentially channeled into beta-oxidation for energy production.

Benchmarking One-Phase Lipid Extractions for Plasma Lipidomics.

A key element of successful lipidomics analysis is a sufficient extraction of lipid molecules typically by two-phase systems such as chloroform-based Bligh and Dyer (B&D). However, numerous metabolomics and lipidomics studies today apply easy to use one-phase extractions. In this work, quantitative flow injection analysis high-resolution mass spectrometry was applied to benchmark the lipid recovery of popular one-phase extraction methods for human plasma samples. The following organic solvents were investigated: methanol (MeOH), ethanol (EtOH), 2-propanol (IPA), 1-butanol (BuOH), acetonitrile (ACN) and the solvent mixtures BuOH/MeOH (3:1) and MeOH/ACN (1:1). The recovery of polar lysophospholipids was sufficient for all tested solvents. However, nonpolar lipid classes such as triglycerides (TG) and cholesteryl esters (CE) revealed extraction efficiencies less than 5% due to precipitation in polar solvents EtOH, MeOH, MeOH/ACN, and ACN. Sample pellets also contained a substantial amount of phospholipids, for example, more than 75% of total phosphatidylcholine and sphingomyelin for ACN. The loss of lipids by precipitation was directly related to the polarity of solvents and lipid classes. Although, lipid recovery increased with the volume of organic solvent, recovery in polar MeOH remains incomplete also for less polar lipid classes such as ceramides. Addition of stable isotope-labeled internal standards prior to lipid extraction could compensate for insufficient lipid recovery for polar lipid classes including lysolipids and phospholipids but not for nonpolar CE and TG. In summary, application of one-phase extractions should be limited to polar lipid classes unless sufficient recovery/solubility of nonpolar lipids has been demonstrated. The presented data reveal that appropriate lipid extraction efficiency is fundamental to achieve accurate lipid quantification.

Lipidomic profiling of Pleurotus ostreatus by LC/MS Q-TOF analysis

In this paper, for the first time a lipidomic analysis on Pleurotus ostreatus species was performed by liquid chromatography quadrupole time-of-flight mass spectrometry (LC/MS Q-TOF). Twenty-seven lipid classes, including polar and non-polar lipid classes, were detected. Free fatty acids (FA) were the predominant fraction (>57%), followed by fatty acid ester of hydroxyl fatty acid and ceramide. C18 chain length and two double bonds were the main structural characteristics for FA. Phosphatydilcholine, phosphatydiletanolamine, and glycerophosphates showed high percentages of polyunsaturated fatty acids. Unconventional fatty acids, such as odd and oxygenated chains, were detected. The highest odd/even ratio was found in hexosylceramides and sphingomyelin, while oxygenated chains were mainly represented in ceramides. As a preliminary approach, the results of lipid molecular species, subjected to principal component analysis and discriminant analysis, were able to differentiate P. ostreatus samples on the base of grown substrate. The results of the comprehensive analysis of P. ostreatus lipids are useful to evaluate the lipid nutritional value and could facilitate exploitation of P. ostreatus consumption.

Retention dependences support highly confident identification of lipid species in human plasma by reversed-phase UHPLC/MS.

Reversed-phase ultrahigh-performance liquid chromatography-mass spectrometry (RP-UHPLC/MS) method was developed with the aim to unambiguously identify a large number of lipid species from multiple lipid classes in human plasma. The optimized RP-UHPLC/MS method employed the C18 column with sub-2-μm particles with the total run time of 25 min. The chromatographic resolution was investigated with 42 standards from 18 lipid classes. The UHPLC system was coupled to high-resolution quadrupole-time-of-flight (QTOF) mass analyzer using electrospray ionization (ESI) measuring full-scan and tandem mass spectra (MS/MS) in positive- and negative-ion modes with high mass accuracy. Our identification approach was based on m/z values measured with mass accuracy within 5 ppm tolerance in the full-scan mode, characteristic fragment ions in MS/MS, and regularity in chromatographic retention dependences for individual lipid species, which provides the highest level of confidence for reported identifications of lipid species including regioisomeric and other isobaric forms. The graphs of dependences of retention times on the carbon number or on the number of double bond(s) in fatty acyl chains were constructed to support the identification of lipid species in homologous lipid series. Our list of identified lipid species is also compared with previous publications investigating human blood samples by various MS-based approaches. In total, we have reported more than 500 lipid species representing 26 polar and nonpolar lipid classes detected in NIST Standard reference material 1950 human plasma.

Accurate Lipid Quantification of Tissue Homogenates Requires Suitable Sample Concentration, Solvent Composition, and Homogenization Procedure-A Case Study in Murine Liver.

Lipidomics aim to quantify lipid species in all kinds of samples, including tissues. To subject a fixed amount of sample to various workflows, tissue homogenates were frequently prepared at defined concentrations in water or by addition of organic solvents. Here, we investigated this first step of tissue lipidomics by quantitative flow injection analysis coupled to Fourier-Transform mass spectrometry (FTMS). The influence of sample concentration, solvent composition, and homogenization procedure on the recovery of lipids was studied in murine liver. Liver homogenates were prepared either by grinding tissue in liquid nitrogen or by bead-based homogenization. Ground samples were dissolved at different concentrations in water, methanol, and water/methanol = 1/1 (v/v). Here, lipid recovery depends on solvent composition and sample concentration. The recovery of nonpolar lipid classes, including triglycerides and cholesteryl ester, was decreased in methanolic homogenates. In contrast, due to superior dispersion of precipitates, bead-based homogenization resulted in efficient lipid recovery independent of the solvent composition. However, lipid distribution within samples, i.e., lipid content of supernatant and pellet following centrifugation, was altered substantially by solvent composition. In conclusion, accurate lipid quantification of tissue homogenates requires evaluation of solvent composition, sample concentration, as well as the homogenization method to guarantee efficient lipid recovery. Due to a potential loss of lipids, removal of precipitates by centrifugation prior to lipid extraction should be avoided.

Profile of fatty acid lipid fractions of omega-3 fatty acid-enriched table eggs.

The omega-3 fatty acid (FA) enrichment of yolk is a key means one of the main objectives to improve the nutraceutical properties of eggs. We evaluated the effect of the dietary inclusion of extruded linseed fed to laying hens on the fatty acid composition of the polar and non-polar lipid classes of the eggs. Two groups of 36 Lohmann White Leghorn layers (65weeks old) were each fed one of two different diets for a period of 12weeks. The two diets consisted of a conventional cereal-based diet concentrate (C) and a diet concentrate containing 5% linseed (L). The inclusion of linseed in the diet increased the content of α-linolenic (C18:3n-3), eicosapentaenoic (C20:5n-3) and docosahexaenoic (C22:6n-3) acids in neutral lipids, while a concomitant decrease in arachidonic acid (C20:4n-6) was observed. As regards the polar fraction, the fatty acid composition was slightly affected by the dietary treatments except for C18:0 (+1.14 fold), C18:2n-6 (+1.23 fold), C18:3n-3 (+2.8 fold) and C22:6n-3 (+1.41 fold). Principal component analysis demonstrated that very long-chain FAs were more representative of polar lipids, except for C20:5n-3, while neutral lipids were characterized by dietary n-3 FA (C18:3n-3).

Coral Lipidomes and Their Changes during Coral Bleaching

Numerous species of coral polyps form the structural basis of tropical coral reefs. Coral polyp tissues are rich in lipids. Currently, information on the composition of fatty acids and classes of coral lipid is reviewed. However, each lipid class represents a complex spectrum of molecular species of lipids, which is defined as the lipidome of a biological system. Scientific publications on human and higher terrestrial animal lipidomes exceed those on the lipidome of marine organisms by two orders of magnitude, and the data on coral lipidomes are very scattered. The existence of symbiotic coral species is completely dependent on the presence of intracellular microalgae (zooxanthellae), the loss of which is called coral bleaching and leads to the death of the entire coral reef. The bleaching causes significant changes in the lipid profile of corals. This paper summarizes information on the composition of common lipids, fatty acids, and molecules of polar and nonpolar lipid classes of octocoral and hexacoral polyps and their symbionts. We discuss general mechanisms of coral bleaching and show the importance of lipid indicators in the study of this process. The transition from classical integral indicators to the lipidomic analysis opens up new possibilities in the study of biochemistry and ecology of corals.

Validation of lipidomic analysis of human plasma and serum by supercritical fluid chromatography-mass spectrometry and hydrophilic interaction liquid chromatography-mass spectrometry.

Ultrahigh-performance supercritical fluid chromatography-mass spectrometry (UHPSFC/MS) has a great potential for the high-throughput lipidomic quantitation of biological samples; therefore, the full optimization and method validation of UHPSFC/MS is compared here with ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC/MS) in hydrophilic interaction liquid chromatography (HILIC) mode as the second powerful technique for the lipid class separation. First, the performance of six common extraction protocols is investigated, where the Folch procedure yields the best results with regard to recovery rate, matrix effect, and precision. Then, the full optimization and analytical validation for eight lipid classes using UHPSFC/MS and HILIC-UHPLC/MS methods are performed for the same sample set and applied for the lipidomic characterization of pooled samples of human plasma, human serum, and NIST SRM 1950 human plasma. The choice of appropriate internal standards (IS) for individual lipid classes has a key importance for reliable quantitative workflows illustrated by the selectivity while validation and the calculation of the quantitation error using multiple internal standards per lipid class. Validation results confirm the applicability of both methods, but UHPSFC/MS provides some distinct advantages, such as the successful separation of both non-polar and polar lipid classes unlike to HILIC-UHPLC/MS, shorter total run times (8 vs. 10.5min), and slightly higher robustness. Various types of correlations between methods (UHPSFC/MS and HILIC-UHPLC/MS), biological material (plasma and serum), IS (laboratory and commercially mixtures), and literature data on the standard reference material show the intra- and inter-laboratory comparison in the quantitation of lipid species from eight lipid classes, the concentration differences in serum and plasma as well as the applicability of non-commercially available internal standard mixtures for lipid quantitation.

Mass spectrometric investigation of cardiolipins and their oxidation products after two-dimensional heart-cut liquid chromatography

The anionic phospholipid class of cardiolipins (CL) is increasingly attracting scientific attention in the recent years. CL can be found as a functional component of mitochondrial membranes in almost all living organisms. Changes in the CL composition are favored by oxidative stress. Based on this finding, the investigation of CL and their oxidation products in relation to various disease patterns, including neurodegenerative ones, is moving into the focus of current research. The analysis of this diverse lipid class is still challenging and requires sensitive and selective methods. In this work, we demonstrate an online two-dimensional liquid chromatography (2D-LC) approach by means of a heart-cut setup. In the first dimension, a fast hydrophilic interaction liquid chromatography (HILIC) method was developed for the separation of CL and their oxidation products from other phospholipid classes, but more important from nonpolar lipid classes, such as triacylglycerol and cholesterol. Those classes can negatively affect the electrospray ionization and also the chromatography. For the heart-cut approach, the CL fraction was selectively transferred to a loop using a six-port valve followed by the transfer to a reversed phase (RP) column in second dimension. On the RP column, the transferred CL fraction including the oxidation products were separated according to the hydrophobicity of acyl chain moieties. Matrix effects were significantly reduced compared to the one-dimensional LC-MS method. In addition, the total separation time had not to be prolonged by shifting the equilibration step of the RP column parallel to the separation in first dimension. The heart-cut LC-LC approach was applied to artificially oxidized lipid extracts of bovine heart and yeast by means of Fenton reaction. In summary, 42 species have been identified by high resolution mass spectrometry and database matching. 31 species thereof have been further characterized by MS/MS experiments.

Analysis of artificially oxidized cardiolipins and monolyso-cardiolipins via liquid chromatography/high-resolution mass spectrometry and Kendrick mass defect plots after hydrophilic interaction liquid chromatography based sample preparation.

Cardiolipins (CL) are a special lipid class which plays a main role in energy metabolism in mitochondria and is involved in apoptosis. In contrast to other glycerophospholipids, they contain four fatty acyl residues which results in a high structural diversity. Oxidation, for example by reactive oxygen species, or lyso forms such as monolyso-CL (MLCL), increases this diversity. Mass spectrometric analysis and computational identification of CL, MLCL and their oxidation products is therefore a challenging task. In order to distinguish CL, MLCL and their oxidation products, a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed. A hydrophilic interaction liquid chromatography (HILIC)-based solid-phase extraction (SPE) clean-up approach was developed for CL enrichment. Graphical analysis of CL, MLCL and their oxidation products was carried out by a three-dimensional Kendrick mass defect (3D-KMD) plot module, as well as a refined lipid search module of the open-source metabolomics data mining software MZmine 2. The HILIC-based SPE clean-up enabled complete separation of polar and nonpolar lipid classes. A yeast (Saccharomyces cerevisiae) lipid extract, which was artificially oxidized by means of the Fenton reaction, was analyzed by the developed LC/MS/MS method. CL species with differences in chain length and degree of unsaturation have been separated by high-performance liquid chromatography (HPLC). In total 66 CL, MLCL and oxidized species have been identified utilizing 3D-KMD plots in combination with database matching using MZmine 2. For further characterization of annotated species, MS/MS experiments have been utilized. 3D-KMD plots capturing chromatographic and high-resolution mass spectrometry data have been successfully used for graphical identification of CL, MLCL as well as their oxidized species. Therefore, we chose multiple KMD bases such as hydrogen and oxygen to visualize the degree of unsaturation and oxidation capturing chromatographic data by means of a color-coded paint scale as the third dimension. In combination with database matching, the analysis of low concentrated lipid species in complex samples has been significantly improved.

Analytical separations for lipids in complex, nonpolar lipidomes using differential mobility spectrometry

Secretions from meibomian glands located within the eyelid (commonly known as meibum) are rich in nonpolar lipid classes incorporating very-long (22-30 carbons) and ultra-long (>30 carbons) acyl chains. The complex nature of the meibum lipidome and its preponderance of neutral, nonpolar lipid classes presents an analytical challenge, with typically poor chromatographic resolution, even between different lipid classes. To address this challenge, we have deployed differential mobility spectrometry (DMS)-MS to interrogate the human meibum lipidome and demonstrate near-baseline resolution of the two major nonpolar classes contained therein, namely wax esters and cholesteryl esters. Within these two lipid classes, we describe ion mobility behavior that is associated with the length of their acyl chains and location of unsaturation. This capability was exploited to profile the molecular speciation within each class and thus extend meibum lipidome coverage. Intriguingly, structure-mobility relationships in these nonpolar lipids show similar trends and inflections to those previously reported for other physicochemical properties of lipids (e.g., melting point and phase-transition temperatures). Taken together, these data demonstrate that differential ion mobility provides a powerful orthoganol separation technology for the analysis of neutral lipids in complex matrices, such as meibum, and may further provide a means to predict physicochemical properties of lipids that could assist in inferring their biological function(s).

UHPSFC/ESI-MS Analysis of Lipids.

This new analytical approach for high-throughput and comprehensive lipidomic analysis of biological samples using ultrahigh-performance supercritical fluid chromatography (UHPSFC) with electrospray ionization-mass spectrometry (ESI-MS) is based on lipid class separation using 1.7μm particle bridged ethylene hybrid silica columns and a gradient of methanol-water-ammonium acetate mixture as a modifier. The method enables a fast separation of 30 nonpolar and polar lipid classes within 6-min analysis time covering six main lipid categories including fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterols, and prenols. Individual lipid species within lipid classes are identified based on positive- and negative-ion full scan and tandem mass spectra measured with high mass accuracy and high resolving power. The method is used for the quantitative analysis of lipid species in biological tissues using internal standards for each lipid class. This high-throughput, comprehensive, and accurate UHPSFC/ESI-MS method is suitable for the lipidomic analysis of large sample sets in clinical research.

Retention behavior of lipids in reversed-phase ultrahigh-performance liquid chromatography–electrospray ionization mass spectrometry

Reversed-phase ultrahigh-performance liquid chromatography (RP-UHPLC) method using two 15cm sub–2μm particles octadecylsilica gel columns is developed with the goal to separate and unambiguously identify a large number of lipid species in biological samples. The identification is performed by the coupling with high-resolution tandem mass spectrometry (MS/MS) using quadrupole – time-of-flight (QTOF) instrument. Electrospray ionization (ESI) full scan and tandem mass spectra are measured in both polarity modes with the mass accuracy better than 5ppm, which provides a high confidence of lipid identification. Over 400 lipid species covering 14 polar and nonpolar lipid classes from 5 lipid categories are identified in total lipid extracts of human plasma, human urine and porcine brain. The general dependences of relative retention times on relative carbon number or relative double bond number are constructed and fit with the second degree polynomial regression. The regular retention patterns in homologous lipid series provide additional identification point for UHPLC/MS lipidomic analysis, which increases the confidence of lipid identification. The reprocessing of previously published data by our and other groups measured in the RP mode and ultrahigh-performance supercritical fluid chromatography on the silica column shows more generic applicability of the polynomial regression for the description of retention behavior and the prediction of retention times. The novelty of this work is the characterization of general trends in the retention behavior of lipids within logical series with constant fatty acyl length or double bond number, which may be used as an additional criterion to increase the confidence of lipid identification.

High-Throughput and Comprehensive Lipidomic Analysis Using Ultrahigh-Performance Supercritical Fluid Chromatography-Mass Spectrometry.

New analytical approach for high-throughput and comprehensive lipidomic analysis of biological samples using ultrahigh-performance supercritical fluid chromatography (UHPSFC) with electrospray ionization-mass spectrometry (ESI-MS) is presented in this work as an alternative approach to established shotgun MS or high-performance liquid chromatography-MS. The lipid class separation is performed by UHPSFC method based on 1.7 μm particle-bridged ethylene hybrid silica column with a gradient of methanol-water-ammonium acetate mixture as a modifier. All parameters of UHPSFC conditions are carefully optimized and their influence on the chromatographic behavior of lipids is discussed. The final UHPSFC/ESI-MS method enables a fast separation of 30 nonpolar and polar lipid classes within 6 min analysis covering 6 main lipid categories including fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterols, and prenols. Individual lipid species within lipid classes are identified based on positive and negative-ion full-scan and tandem mass spectra measured with high mass accuracy and high resolving power. Developed UHPSFC/ESI-MS method is applied for the analysis of porcine brain extract as a complex lipidomic sample, where 24 lipid classes containing 436 lipid species are identified. The method is validated for the quantitative analysis of lipid species in biological tissues using internal standards for each lipid class. This high-throughput, comprehensive and accurate UHPSFC/ESI-MS method is suitable for the lipidomic analysis of large sample sets in the clinical research.

Determination of nonpolar and polar lipid classes in human plasma, erythrocytes and plasma lipoprotein fractions using ultrahigh-performance liquid chromatography-mass spectrometry

A novel normal-phase (NP) ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC/MS) method is developed for a separation and quantitation of nonpolar lipid classes occurring in human plasma, erythrocytes and plasma lipoprotein fractions. The baseline class separation of cholesteryl esters (CE), cholesterol, triacylglycerols (TG), regioisomers of 1,2- and 1,3-diacylglycerols (DG) and 1-monoacylglycerols (1-MG) is achieved using an optimized hexane - 2-propanol–acetonitrile mobile phase within 18min for all nonpolar lipid classes or only 9min excluding monoacylglycerols not detected in studied samples. The determination of individual nonpolar lipid classes is performed by the response factor approach and the use of dioleoyl ethylene glycol as a single internal standard. Polar lipid classes, such as phosphatidylglycerols (PG), phosphatidylethanolamines (PE), phosphatidylcholines (PC), sphingomyelins (SM) and lysophosphatidylcholines (LPC), are separated by hydrophilic interaction liquid chromatography (HILIC) using 5mmol/L aqueous ammonium acetate–methanol–acetonitrile gradient within 13minutes. The quantitation of polar lipid classes is done by a similar approach as for nonpolar lipid classes, but a different internal standard (sphingosyl PE d17:1/12:0) is used. The complementary information on fatty acyl profiles after the transesterification of the total lipid extract is obtained by gas chromatography with flame ionization detection (GC/FID). The applicability of developed methodology for fast and comprehensive characterization of blood lipidome is illustrated on samples of human plasma, erythrocytes, high-density lipoprotein (HDL), low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) fractions.

Single run HPLC separation coupled to evaporative light scattering detection unravels wheat flour endogenous lipid redistribution during bread dough making

Wheat flour lipids present a complex mixture of components with varying polarities and have been classified as either starch or non-starch lipids, the latter being further subdivided in free and bound lipids depending on the extraction method and solvents used. A novel separation and detection method for wheat lipids in flour and dough that separates both non-polar and polar lipid classes in a single run HPLC analysis using a monolithic silica column and a quaternary gradient and detects them with evaporative light scattering is presented. The method was applied to study in-depth the changes in lipid distribution during bread dough development, thereby monitoring the fate of the different lipid classes. Full dough development was not necessary to decrease the extractability of both non-polar and polar free lipids and increase their levels in the bound lipid extract. The data for the first time also showed which specific lipid classes were redistributed and transferred from starch to gluten, as confirmed by analyzing starch fractions. In addition, not all redistributed lipids interacted in a similar way with the gluten proteins. Major changes occurred during the first 30 s of mixing after which slight changes were observed for triacylglycerols, free fatty acids, lysophosphatidylcholine, and N-acylphosphatidylethanolamine.

양식산 및 천연산 고등어근육의 일반성분과 지질성분 비교

Proximate compositions and fatty acid profiles of cultured and wild mackerel (Scomber japonicus) muscles were compared. Protein content ranged from approximately 16% to 18% and was higher in wild fish than in cultured ones. Lipid content was between two to four times higher in large and small cultured fish (20.1-20.5%) compared with same sized wild fish. The prominent non-polar lipid (NL) class in fish muscles was triglyceride, and additionally, free sterol was among the prominent NL classes in wild fish muscles. Prominent phospholipid (PL) classes in cultured and wild fish muscles were phosphatidylethanolamine and phosphatidylcholine, with the former being higher in cultured fish and the latter higher in wild fish. Prominent fatty acids of total lipid were 16:0, 18:1n-9, 22:6n-3 (docosahexaenoic acid, DHA), 20:5n-3 (eicosapentaenoic acid, EPA), 16:1n-7, 18:0 and 14:0, while 18:2n-6 was among the prominent fatty acids in cultured fish. The n-3 polyunsaturated fatty acids (PUFA, DHA+EPA) content (in mg/100 g of muscle tissue) was higher in cultured fish (2,711 mg in large fish and 2,572 mg in small fish) than in wild fish (2,431 mg in large fish and 1,398 mg in small fish). In conclusion, we have been able to demonstrate that cultured mackerel could also be a good sources of n-3 PUFA, such as DHA and EPA.

A Simple Mycobacterial Monomycolated Glycerol Lipid Has Potent Immunostimulatory Activity

It is a long held belief that the strong immunostimulatory activity of the Mycobacterium bovis bacillus Calmette-Guérin vaccine and Freund's complete adjuvant is due to specific mycobacterial cell envelope components, such as lipids and polysaccharides. Implicated mycobacterial lipids include, among others, the so-called cord factor or trehalose dimycolate, but limited information is available regarding the precise molecular nature of the stimulatory components responsible for the interaction with human APCs. In this regard, the majority of research aimed at identifying and characterizing individual immunostimulatory mycobacterial lipids has been performed in the murine system using bone marrow-derived dendritic cells. In this study, it is documented that potent immunostimulatory activity lies within the bacillus Calmette-Guérin nonpolar lipid class. This activity can be narrowed down to a remarkably simple monomycolyl glycerol (MMG) with the ability to stimulate human dendritic cells as assessed by enhanced expression of activation markers and the release of proinflammatory cytokines. A synthetic analog of MMG based on 32 carbons (C(32)) was found to exhibit comparable levels of immunostimulatory activities. Immunization of mice with the tuberculosis vaccine candidate, Ag85B-ESAT-6, in MMG or the synthetic analog using cationic liposomes as the delivery vehicle was found to give rise to a prominent Th1 response characterized by significant levels of IFN-gamma. Together, this development opens up the possibility of producing a novel class of chemically defined lipid adjuvants to enhance the activity of new vaccine formulations, directed against infectious agents including tuberculosis.

Quantitative evaluation of sebum lipid components with nuclear magnetic resonance

A NMR spectroscopic method is described that enables the quantitation of specific lipid classes and components, independent of fatty acid composition. We demonstrate this method for measuring cholesterol, squalene, and pools of sterol esters, wax esters (WEs), and triglyceride (TG) components in sebum and meibum. When 600 MHz NMR equipment is used in conjunction with highly sensitive cryogenically cooled probes, this method has adequate sensitivity, and for some applications, advantages over commonly used HPLC-evaporative light-scattering detection and mass spectrometry-based approaches. This method is shown to be useful for preclinical and clinical monitoring of the efficacy of sebum-reducing agents in animals and humans. In Syrian hamsters, 3% topical flutamide and 20 mg/kg oral isotretinoin reduced sterol esters by 18.7% and 30.0%, respectively, and reduced WEs by 32.9% and 31.8%, respectively, as measured in a punch biopsy of the ear. In a 72 patient clinical methodology study, the assay delivered reproducible and noninvasive measurements of WEs, cholesteryl esters, TGs, and squalene from Sebutape skin blots. The quantitative results of sebum analysis obtained by the NMR method correlate well with those obtained with HPLC-based approaches. This approach may be broadly applicable to cases in which fatty acid-independent quantification of lipid classes is desired.