Distinctive Lipid Characteristics of Colorectal Cancer Revealed through Non-targeted Lipidomics Analysis of Tongue Coating.

The metabolites and microbiota in tongue coating display distinct characteristics in certain digestive disorders, yet their relationship with colorectal cancer (CRC) remains unexplored. Here, we employed liquid chromatography coupled with tandem mass spectrometry to analyze the lipid composition of tongue coating using a nontargeted approach in 30 individuals with colorectal adenomas (CRA), 32 with CRC, and 30 healthy controls (HC). We identified 21 tongue coating lipids that effectively distinguished CRC from HC (AUC = 0.89), and 9 lipids that differentiated CRC from CRA (AUC = 0.9). Furthermore, we observed significant alterations in the tongue coating lipid composition in the CRC group compared to HC/CRA groups. As the adenoma-cancer sequence progressed, there was an increase in long-chain unsaturated triglycerides (TG) levels and a decrease in phosphatidylethanolamine plasmalogen (PE-P) levels. Furthermore, we noted a positive correlation between N-acyl ornithine (NAOrn), sphingomyelin (SM), and ceramide phosphoethanolamine (PE-Cer), potentially produced by members of the Bacteroidetes phylum. The levels of inflammatory lipid metabolite 12-HETE showed a decreasing trend with colorectal tumor progression, indicating the potential involvement of tongue coating microbiota and tumor immune regulation in early CRC development. Our findings highlight the potential utility of tongue coating lipid analysis as a noninvasive tool for CRC diagnosis.

Deep profiling of plasmalogens by coupling the Paternò-Büchi derivatization with tandem mass spectrometry.

Plasmalogens are a special class of glycerophospholipids characterized by a vinyl ether bond (-C = C-O-) at the sn-1 position of the glycerol backbone. Altered plasmalogen profiles have been observed in neurodegenerative diseases and cancers. Profiling of plasmalogens requires specifying the vinyl ether bond and differentiating them from various types of isobars and isomers. Herein, by coupling C = C derivatization via offline Paternò-Büchi reaction with liquid chromatography-tandem mass spectrometry, we have developed a sensitive workflow for analysis of plasmalogens from biological samples. Using bovine heart lipid extract as a model system, we profiled more than 100 distinct structures of plasmenylethanolamines (PE-Ps) and plasmenylcholines (PC-Ps) at the C = C location level, far exceeding previous reports. Analysis of human glioma and normal brain tissue samples revealed elevated n-10 C = C isomers of PE-Ps in the glioma tissue samples. These findings suggest that the developed workflow holds potential in aiding the study of altered metabolism of plasmalogens in clinical samples.

Phosphatidylcholine-Plasmalogen-Oleic Acid Has Protective Effects against Arachidonic Acid-Induced Cytotoxicity.

Plasmalogens are a group of glycerophospholipids containing a vinyl-ether bond at the sn-1 position in the glycerol backbone. Cellular membrane plasmalogens are considered to have important roles in homeostasis as endogenous antioxidants, differentiation, and intracellular signal transduction pathways including neural transmission. Therefore, reduced levels of plasmalogens have been suggested to be associated with neurodegenerative diseases such as Alzheimer's disease. Interestingly, although arachidonic acid is considered to be involved in learning and memory, it could be liberated and excessively activate neuronal activity to the excitotoxic levels seen in Alzheimer's disease patients. Here, we examined the protective effects of several kinds of plasmalogens against cellular toxicity caused by arachidonic acid in human neuroblastoma SH-SY5Y cells. As a result, only phosphatidylcholine-plasmalogen-oleic acid (PC-PLS-18) showed protective effects against arachidonic acid-induced cytotoxicity based on the results of lactate dehydrogenase release and ATP depletion assays, as well as cellular morphological changes in SH-SY5Y cells. These results indicate that PC-PLS-18 protects against arachidonic acid-induced cytotoxicity, possibly via improving the stability of the cellular membrane in SH-SY5Y cells.

Compositional Study of Phospholipids from the Dried Big Head and Opossum Shrimp, Mussel, and Sea Cucumber Using 31P NMR Spectroscopy: Content and Fatty Acid Composition of Plasmalogen.

Herein, we present a qualitative and quantitative analysis of the compositions of plasmalogens and phospholipids (PLs) in dried big head shrimp (Solenocera melantho), opossum shrimp (Neomysis awatschensis), mussel (Mytilus galloprovincialis), and sea cucumber (Apostichopus japonicus). We also analyze the fatty acid composition of the extracted lipids, phosphatidyl choline (PtdCho), and plasmalogen choline (PlsCho) from each sample. In big head shrimp, opossum shrimp, and mussel, phosphatidyl choline (PtdCho) was the most abundant PL at 1677.9, 1603, and 1661.6 mg/100 g of dried sample, respectively, whereas the most abundant PL in sea cucumber was PlsCho (206.9 mg/100 g of dried sample). In all four samples, plasmalogen ethanolamine (PlsEtn) was higher than phosphatidyl ethanolamine (PtdEtn). The content (mg/100 g of dried sample) of PlsCho was highest in mussel (379.0), and it was higher in big head shrimp (262.3) and opossum shrimp (245.6) than sea cucumber (206.9). The contents (mg/100 g of dried sample) of PlsEtn were in the order of mussel (675.4) > big head shrimp (629.5) > opossum shrimp (217.9) > sea cucumber (51.5). For analyzing the fatty acids at the sn-2 position of PlsCho, the consecutive treatment with phospholipase A1, solid phase extraction, thin-layer chromatography (TLC), and GC-FID were applied. The most abundant fatty acid was eicosapentaenoic acid (EPA, C20:5, n-3) in big head shrimp and sea cucumber, palmitoleic acid (C16:1, n-7) in opossum shrimp, and docosadienoic acid (C22:2, n-6) in mussel.

Life without air.

An early exposure to lipid biochemistry in the laboratory of Konrad Bloch resulted in a fascination with the biosynthesis, structures, and functions of bacterial lipids. The discovery of plasmalogens (1-alk-1'-enyl, 2-acyl phospholipids) in anaerobic Gram-positive bacteria led to studies on the physical chemistry of these lipids and the cellular regulation of membrane lipid polymorphism in bacteria. Later studies in several laboratories showed that the formation of the alk-1-enyl ether bond involves an aerobic process in animal cells and thus is fundamentally different from that in anaerobic organisms. Our work provides evidence for an anaerobic process in which plasmalogens are formed from their corresponding diacyl lipids. Studies on the roles of phospholipases in Listeria monocytogenes revealed distinctions between its phospholipases and those previously discovered in other bacteria and showed how the Listeria enzymes are uniquely fitted to the intracellular lifestyle of this significant human pathogen.

Synthesis of a plasmenylethanolamine.

A concise synthesis of a plasmenylethanolamine (PlsEtn-[16:0/18:1 n-9]), known as antioxidative phospholipids commonly found in cell membranes, has been achieved from an optically active known diol through 8 steps. The key transformations for the synthesis of PlsEtn-[16:0/18:1 n-9] are (1) regio- and Z-selective vinyl ether formation via the alkylation of a lithioalkoxy allyl intermediate with an alkyl iodide, and (2) a one-pot phosphite esterification-oxidation sequence to construct the ethanolamine phosphonate moiety in the presence of the vinyl ether functionality. The piperidine salt of synthetic PlsEtn-[16:0/18:1 n-9] was desalinated through reversed-phase high-performance liquid chromatography purification.

Unequivocal Mapping of Molecular Ether Lipid Species by LC-MS/MS in Plasmalogen-Deficient Mice.

Deficient ether lipid biosynthesis in rhizomelic chondrodysplasia punctata and other disorders is associated with a wide range of severe symptoms including small stature with proximal shortening of the limbs, contractures, facial dysmorphism, congenital cataracts, ichthyosis, spasticity, microcephaly, and mental disability. Mouse models are available but show less severe symptoms. In both humans and mice, it has remained elusive which of the symptoms can be attributed to lack of plasmanyl or plasmenyl ether lipids. The latter compounds, better known as plasmalogens, harbor a vinyl ether double bond conferring special chemical and physical properties. Discrimination between plasmanyl and plasmenyl ether lipids is a major analytical challenge, especially in complex lipid extracts with many isobaric species. Consequently, these lipids are often neglected also in recent lipidomic studies. Here, we present a comprehensive LC-MS/MS based approach that allows unequivocal distinction of these two lipid subclasses based on their chromatographic properties. The method was validated using a novel plasmalogen-deficient mouse model, which lacks plasmanylethanolamine desaturase and therefore cannot form plasmenyl ether lipids. We demonstrate that plasmanylethanolamine desaturase deficiency causes an accumulation of plasmanyl species, a too little studied but biologically important substance class.

Recovery of brain DHA-containing phosphatidylserine and ethanolamine plasmalogen after dietary DHA-enriched phosphatidylcholine and phosphatidylserine in SAMP8 mice fed with high-fat diet.

BACKGROUND: Glycerophospholipids were the main components of cerebral cortex lipids, and there was a close association between lipid homeostasis and human health. It has been reported that dietary DHA-enriched phosphatidylcholine (DHA-PC) and phosphatidylserine (DHA-PS) could improve brain function. However, it was unclear that whether supplementation of DHA-PC and DHA-PS could change lipid profiles in the brain of dementia animals. METHODS: SAMP8 mice was fed with different diet patterns for 2 months, including high-fat diet and low-fat diet. After intervention with DHA-PC and DHA-PS for another 2 months, the lipid profile in cerebral cortex was determined by lipidomics in dementia mice. RESULTS: High-fat diet could significantly decrease the levels of DHA-containing PS/pPE, DPA-containing PS, and AA-containing PE, which might exhibit the potential of lipid biomarkers for the prevention and diagnosis of AD. Notably, DHA-PC and DHA-PS remarkably recovered the lipid homeostasis in dementia mice. These might provide a potential novel therapy strategy and direction of dietary intervention for patients with cognitive decline. CONCLUSIONS: DHA-PC and DHA-PS could recover the content of brain DHA-containing PS and pPE in SAMP8 mice fed with high-fat diet.

[New functional food ingredient - lipid module, source of astaxantine and plasmalogenes].

The aim of this work was to study the impact of modification of fatty acids composition of laboratory animals' diet in the presence of plasmalogenes (PG), astaxanthin (ASTA) and their combination on animals' adaptation potential in stress conditions. MATERIAL AND METHODS: The fatty acids composition of diet was altered with the use of lipid module, containing 88.7% of high oleic sunflower oil, 6.3% of coconut oil and 5% of micralagae Schizochytrium sp. oil. The experiment was conducted with the use of 80 male Wistar rats with initial body weight 125±5 g. Selected animals (n=50) were divided into 5 groups according to their activity in tests «Open field¼ (OF) and «Elevated plus-maze¼ (EPM). The animals of control group 1st were not exposed to physiological tests - this was an intact control group. The animals of control groups 1st and 2nd were treated with standard half-synthetic diets for 30 days [381 kcal/100 g of dry food, 20.1% of casein on calories, 10% of fat (the mixture of lard and sunflower oil in 1:1 ratio)]. In the diet of 3d group animals the sunflower oil (50% of fat in the diet) was substituted with lipid module, enriched with ASTA (4.0±0.3 mg/day/kg b.w.); in the diet of group 4 animals - with lipid module, enriched with PG (79.0±2.0 mg/day/kg b.w.); in the diet of group 5 animals - with lipid module, enriched with ASTA and PG (the same doses). The evaluation of anxiety level and total exploration activity was conducted in «elevated plus maze¼ test. On the 31st day of experiment, animals were exposed to forced swim test for evaluation of their physical endurance and capacity in the conditions of high stress. RESULTS AND DISCUSSION: The introduction into the animal diet of lipid module, enriched with PG and/or ASTA had pronounced hypolipidemic effect, lowered total serum cholesterol against a background of decrease in low-density lypoproteins (LDL) level. The concentration of ω-3 polyunsaturated fatty acid (PUFA) - docosahexaenoic acid in liver cells of animals treated with lipid module enriched with PG and/or ASTA increased more the 3 times, while ω-6 linoleic acid decreased twice. The consumption of enriched with ASTA lipid module inhibited the increase in blood corticosterone level after stress (exhausting physical exercise) and lowered it to the control animals values, thus showing adaptogenic effect. The animals treated with lipid module enriched with ASTA (group 3) spent significantly less time in open arms of the maze in comparison with the first test, what may show the increase in anxiety level. The introduction of PG into lipid module neutralized this effect. The forced swim test with load showed no increase in working ability and endurance of animals of all tested groups. CONCLUSION: The further study of adaptogenic action of PG in combination with ASTA in the composition of lipid module compared to the similar effect of traditional phospholipids is of special interest.

A novel assay for the introduction of the vinyl ether double bond into plasmalogens using pyrene-labeled substrates.

Plasmanylethanolamine desaturase (PEDS) (EC 1.14.99.19) introduces the 1-prime double bond into plasmalogens, one of the most abundant phospholipids in the human body. This labile membrane enzyme has not been purified and its coding sequence is unknown. Previous assays for this enzyme used radiolabeled substrates followed by multistep processing. We describe here a straight-forward method for the quantification of PEDS in enzyme incubation mixtures using pyrene-labeled substrates and reversed-phase HPLC with fluorescence detection. After stopping the reaction with hydrochloric acid in acetonitrile, the mixture was directly injected into the HPLC system without the need of lipid extraction. The substrate, 1-O-pyrenedecyl-2-acyl-sn-glycero-3-phosphoethanolamine, and the lyso-substrate, 1-O-pyrenedecyl-sn-glycero-3-phosphoethanolamine, were prepared from RAW-12 cells deficient in PEDS activity and were compared for their performance in the assay. Plasmalogen levels in mouse tissues and in cultured cells did not correlate with PEDS levels, indicating that the desaturase might not be the rate limiting step for plasmalogen biosynthesis. Among selected mouse organs, the highest activities were found in kidney and in spleen. Incubation of intact cultivated mammalian cells with 1-O-pyrenedecyl-sn-glycerol, extraction of lipids, and treatment with hydrochloric or acetic acid in acetonitrile allowed sensitive monitoring of PEDS activity in intact cells.

Inorganic mercury and cadmium induce rigidity in eukaryotic lipid extracts while mercury also ruptures red blood cells.

Hg and Cd are non-essential toxic heavy metals that bioaccumulate in the tissues of living systems but less is known about their interactions with Eukaryotic lipid bilayers. Microscopy experiments showed that Hg and Cd changed the cell morphology of rabbit erythrocytes while Hg also induced cell rupture. As membranes are one of the first available targets, our study aimed to better understand metal-lipid interactions that could lead to toxic effects. Fluorescence spectroscopy (Laurdan Generalized Polarization) and dynamic light scattering were used to analyze metal-induced changes in membrane fluidity and the size of liposomes composed of Brain (Porcine), Liver (Bovine), Heart (Bovine) and Yeast (S. cerevisiae) lipid extracts. Under physiological chloride and pH levels, Hg irreversibly cleaves plasmalogens resulting in an increase in membrane rigidity. These lipids are enriched in Brain, Heart and Erythrocyte membranes and are important in signalling and the protection against oxidative stress. Interestingly, Hg had a heavily reduced effect on the plasmalogen-free Yeast extract membrane. In contrast, Cd induced rigidity by targeting negatively charged phosphatidic acid, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol and cardiolipin in these extracts. Metal-induced liposome aggregation depended on the proportion of negatively charged lipids/plasmalogen and even the order of metal addition. Our results show that data from model systems correlate with trends observed in complex biological extracts and red blood cells and serve as a predictive tool for analyzing metal-lipid interactions. The determination of the specific lipid targets for Hg and Cd provides new insights how these metals exert toxic effects on cell membranes.

The biophysical properties of ethanolamine plasmalogens revealed by atomistic molecular dynamics simulations.

Given the importance of plasmalogens in cellular membranes and neurodegenerative diseases, a better understanding of how plasmalogens affect the lipid membrane properties is needed. Here we carried out molecular dynamics simulations to study a lipid membrane comprised of ethanolamine plasmalogens (PE-plasmalogens). We compared the results to the PE-diacyl counterpart and palmitoyl-oleyl-phosphatidylcholine (POPC) bilayers. Results show that PE-plasmalogens form more compressed, thicker, and rigid lipid bilayers in comparison with the PE-diacyl and POPC membranes. The results also point out that the vinyl-ether linkage increases the ordering of sn-1 chain substantially and the ordering of the sn-2 chain to a minor extent. Further, the vinyl-ether linkage changes the orientation of the lipid head group, but it does not cause changes in the head group and glycerol backbone tilt angles with respect to the bilayer normal. The vinyl-ether linkage also packs the proximal regions of the sn-1 and sn-2 chains more closely together which also decreases the distance between the rest of the sn-1 and sn-2 chains.

Chemical Derivatization and Ultrahigh Resolution and Accurate Mass Spectrometry Strategies for "Shotgun" Lipidome Analysis.

Lipids play critical structural and functional roles in the regulation of cellular homeostasis, and it is increasingly recognized that the disruption of lipid metabolism or signaling or both is associated with the onset and progression of certain metabolically linked diseases. As a result, the field of lipidomics has emerged to comprehensively identify and structurally characterize the diverse range of lipid species within a sample of interest and to quantitatively monitor their abundances under different physiological or pathological conditions. Mass spectrometry (MS) has become a critical enabling platform technology for lipidomic researchers. However, the presence of isobaric (i.e., same nominal mass) and isomeric (i.e., same exact mass) lipids within complex lipid extracts means that MS-based identification and quantification of individual lipid species remains a significant analytical challenge. Ultrahigh resolution and accurate mass spectrometry (UHRAMS) offers a convenient solution to the isobaric mass overlap problem, while a range of chromatographic separation, differential extraction, intrasource separation and selective ionization methods, or tandem mass spectrometry (MS/MS) strategies may be used to address some types of isomeric mass lipid overlaps. Alternatively, chemical derivatization strategies represent a more recent approach for the separation of lipids within complex mixtures, including for isomeric lipids. In this Account, we highlight the key components of a lipidomics workflow developed in our laboratory, whereby certain lipid classes or subclasses, namely, aminophospholipids and O-alk-1'-enyl (i.e., plasmalogen) ether-containing lipids, are shifted in mass following sequential functional group selective chemical derivatization reactions prior to "shotgun" nano-ESI-UHRAMS analysis, "targeted" MS/MS, and automated database searching. This combined derivatization and UHRAMS approach resolves both isobaric mass lipids and certain categories of isomeric mass lipids within crude lipid extracts, with no requirement for extensive sample handling prior to analysis, with additional potential for enhanced ionization efficiencies, improved molecular level structural characterization, and multiplexed relative quantification. When integrated with a monophasic method for the simultaneous global extraction of both highly polar and nonpolar lipids, this workflow has been shown to enable the sum composition level identification and relative quantification of 500-600 individual lipid species across four lipid categories and from 36 lipid classes and subclasses, in only 1-2 min data acquisition time and with minimal sample consumption. Thus, while some analytical challenges remain to be addressed, shotgun lipidomics workflows encompassing chemical derivatization strategies have particular promise for the analysis of samples with limited availability that require rapid and unbiased assessment of global lipid metabolism.

Catalytic Z-selective olefin cross-metathesis for natural product synthesis.

Alkenes are found in many biologically active molecules, and there are a large number of chemical transformations in which alkenes act as the reactants or products (or both) of the reaction. Many alkenes exist as either the E or the higher-energy Z stereoisomer. Catalytic procedures for the stereoselective formation of alkenes are valuable, yet methods enabling the synthesis of 1,2-disubstituted Z alkenes are scarce. Here we report catalytic Z-selective cross-metathesis reactions of terminal enol ethers, which have not been reported previously, and of allylic amides, used until now only in E-selective processes. The corresponding disubstituted alkenes are formed in up to >98% Z selectivity and 97% yield. These transformations, promoted by catalysts that contain the highly abundant and inexpensive metal molybdenum, are amenable to gram-scale operations. Use of reduced pressure is introduced as a simple and effective strategy for achieving high stereoselectivity. The utility of this method is demonstrated by its use in syntheses of an anti-oxidant plasmalogen phospholipid, found in electrically active tissues and implicated in Alzheimer's disease, and the potent immunostimulant KRN7000.

Membrane lipid profile of in vitro-produced embryos is affected by vitrification but not by long-term dietary supplementation of polyunsaturated fatty acids for oocyte donor beef heifers.

Dietary rumen-protected polyunsaturated fatty acids (PUFAs) rich in linoleic acid (LA) may affect embryo yield, and LA can modulate the molecular mechanisms of lipid uptake in bovine blastocysts produced in vitro. In embryos, membrane lipids, such as phosphatidylcholines (PCs) and sphingomyelins (SMs), affect cryopreservation success. The aim of the present study was to evaluate embryonic developmental rates after the IVF of oocytes retrieved from Nellore heifers fed for approximately 90 days with rumen-protected PUFAs rich in LA. In addition, we evaluated embryo cryotolerance and the membrane structure lipid composition using matrix-assisted laser desorption ionisation mass spectrometry of fresh and vitrified embryos. Embryo development to the blastocyst stage (mean 43.2%) and embryo survival after vitrification and warming (mean 79.3%) were unaffected by diet. The relative abundance of one lipid species (PC ether (PCe; 38:2, which means that this lipid has 38 carbon atoms and 2 double bonds in the fatty acyl residues) was increased after PUFAs supplementation. However, 10 ions were affected by cryopreservation; ions consistent with PC 32:0, PC 34:1, SM 24:1, PC 40:6 or PC 42:9, PC plasmalogen (PCp) 44:10 or PC 42:7, triacylglycerol (TAG) 54:9 and a not assigned ion (m/z 833.2) were lower in blastocysts that survived to the cryopreservation process compared with fresh blastocysts, whereas the abundance of the ions PC 36:3 or PC 34:0, PCe 38:2 or PC 36:6 and PC 36:5 or PCe 38:1 were increased after cryopreservation. Thus, the results demonstrate that the mass spectrometry profiles of PC, SM and TAG species differ significantly in bovine blastocysts upon cryopreservation. Because the lipid ion abundances of fresh and vitrified-warmed embryos were distinct, they can be used as potential markers of post-cryopreservation embryonic survival.

Human native, enzymatically modified and oxidized low density lipoproteins show different lipidomic pattern.

In the present paper we have performed comparative lipidomic analysis of two prototypic atherogenic LDL modifications, oxidized LDL and enzymatically modified LDL. Oxidization of LDL was carried out with different chemical modifications starting from the same native LDL preparations: (i) by copper oxidation leading to terminally oxidized LDL (oxLDL), (ii) by moderate oxidization with HOCl (HOCl LDL), (iii) by long term storage of LDL at 4°C to produce minimally modified LDL (mmLDL), or (iv) by 15-lipoxygenase, produced by a transfected fibroblast cell line (LipoxLDL). The enzymatic modification of LDL was performed by treatment of native LDL with trypsin and cholesteryl esterase (eLDL). Free cholesterol (FC) and cholesteryl esters (CE) represent the predominant lipid classes in all LDL preparations. In contrast to native LDL, which contains about two-thirds of total cholesterol as CE, enzymatic modification of LDL decreased the proportion of CE to about one-third. Free cholesterol and CE in oxLDL are reduced by their conversion to oxysterols. Oxidization of LDL preferentially influences the content of polyunsaturated phosphatidylcholine (PC) and polyunsaturated plasmalogen species, by reducing the total PC fraction in oxLDL. Concomitantly, a strong rise of the lysophosphatidylcholine (LPC) fraction can be found in oxLDL as compared to native LDL. This effect is less pronounced in eLDL. The mild oxidation of LDL with hypochlorite and/or lipoxygenase does not alter the content of the analyzed lipid classes and species in a significant manner. The lipidomic characterization of modified LDLs contributes to the better understanding their diverse cellular effects.

Metabolism and functions of lipids in myelin.

Rapid conduction of nerve impulses requires coating of axons by myelin sheaths, which are lipid-rich and multilamellar membrane stacks. The lipid composition of myelin varies significantly from other biological membranes. Studies in mutant mice targeting various lipid biosynthesis pathways have shown that myelinating glia have a remarkable capacity to compensate the lack of individual lipids. However, compensation fails when it comes to maintaining long-term stability of myelin. Here, we summarize how lipids function in myelin biogenesis, axon-glia communication and in supporting long-term maintenance of myelin. We postulate that change in myelin lipid composition might be relevant for our understanding of aging and demyelinating diseases. This article is part of a Special Issue titled Brain Lipids.

Human Milk Plasmalogens Are Highly Enriched in Long-Chain PUFAs.

BACKGROUND: Human milk contains unique glycerophospholipids, including ethanolamine-containing plasmalogens (Pls-PEs) in the milk fat globule membrane, which have been implicated in infant brain development. Brain Pls-PEs accumulate postnatally and are enriched in long-chain polyunsaturated fatty acids (LC-PUFAs), particularly docosahexaenoic acid (DHA). Fatty acid (FA) composition of Pls-PEs in milk is poorly understood because of the analytical challenges in separating Pls-PEs from other phospholipids in the predominating presence of triacylglycerols. The variability of Pls-PE FAs and the potential role of maternal diet remain unknown. OBJECTIVES: Our primary objectives were to establish improved methodology for extracting Pls-PEs from human milk, enabling FA analysis, and to compare FA composition between Pls-PEs and 2 major milk phospholipids, phosphatidylcholine and phosphatidylethanolamine. Our secondary objective was to explore associations between maternal DHA intake and DHA in milk phospholipids and variability in phospholipid-DHA within a woman. METHODS: Mature milk was collected from 25 women, with 4 providing 3 milk samples on 3 separate days. Lipids were extracted, and phospholipids were removed by solid phase extraction. Pls-PEs were separated by using normal-phase HPLC, recovered and analyzed for FAs by GLC. Diet was assessed by using a validated food-frequency questionnaire. RESULTS: Pls-PE concentration in human milk was significantly higher in LC-PUFAs than phosphatidylethanolamine and phosphatidylcholine, including arachidonic acid (AA) and DHA. The mean ± SD concentration of AAs in Pls-PEs was ∼2.5-fold higher than in phosphatidylethanolamine (10.5 ± 1.71 and 3.82 ± 0.92 g/100 g, respectively). DHA in Pls-PEs varied across women (0.95-6.51 g/100 g), likely independent of maternal DHA intake. Pls-PE DHA also varied within a woman across days (CV ranged from 9.8% to 28%). CONCLUSIONS: Human milk provides the infant with LC-PUFAs from multiple lipid pools, including a source from Pls-PEs. The biological determinants of Pls-PE FAs and physiological relevance to the breastfed infant remain to be elucidated.

Hydrolysis of plasmalogen by phospholipase A1 from Streptomyces albidoflavus for early detection of dementia and arteriosclerosis.

OBJECTIVES: To obtain an ethanolamine plasmalogen (PlsEtn)-hydrolyzing enzyme and to develop an assay that would help determine PlsEtn concentrations in human serum as an indicator of Alzheimer-type dementia and of arteriosclerosis. RESULTS: Phospholipase A1s, SaPLA1 and SvPLA1 from, respectively, Streptomyces albidoflavus NA297 and S. avermitilis JCM5070-but not phospholipase B from Streptomyces sp. NA684, PLA2-Nagase from S. avermitilis, PLA2IIL from S. violaceoruber nor LIPOMOD 699L (porcine phospholipase)-hydrolyzed choline plasmalogen (PlsCho) and PlsEtn (PlsCho preferred over PlsEtn). Using a combination of SaPLA1, lysoplasmalogen-specific phospholipase D (LyPls-PLD), with amine oxidase, an end-point assay was developed for measuring serum PlsEtn concentration. The standard curve, generated using various amounts of PlsEtn in this assay, was linear between 0 and 0.2 mM. PlsEtn concentrations in forty-seven serum samples, determined independently by this enzyme-based assay and (125)I-HPLC method, exhibited a linear relationship, indicating that the assay is suitable for fast and accurate measurement of serum PlsEtn concentration. CONCLUSIONS: An assay, developed using SaPLA1, LyPls-PLD, and AOX, selectively measured PlsEtn levels in blood samples. This assay could be a useful diagnostic tool for early stage detection of diseases such as Alzheimer-type dementia and arteriosclerosis.

Serum choline plasmalogens, particularly those with oleic acid in sn-2, are associated with proatherogenic state.

Serum plasmalogens (Pls) (1-O-alk-1'-enyl-2-acyl glycerophospholipids) are of particular interest for studies on metabolic disorders associated with oxidative stress and chronic inflammation. Serum levels of Pls are known to correlate positively with HDL-cholesterol (HDL-C); however, few studies have examined serum Pls molecular species in association with pathophysiological conditions and their clinical significance. To clarify these, we determined serum levels of individual ether glycerophospholipids in Japanese asymptomatic cohorts (n = 428; 362 male and 66 female subjects) by LC/MS/MS, and examined their correlations with clinical parameters. We found that the proportion of choline Pls (PlsCho) among total serum phospholipids was significantly lower in the male group over 40 years old and was associated with multiple risk parameters more strongly than HDL-C. The abundance of serum PlsCho with oleic acid (18:1) in sn-2 exhibited the strongest positive correlation with serum concentrations of adiponectin and HDL-C, while being inversely associated with waist circumference and the serum levels of TG and small dense LDL-cholesterol. The characterization of serum ether glycerophospholipids verified the specificity of PlsCho, particularly the ones with 18:1 in sn-2, as a sensitive biomarker for the atherogenic state.