Improved Butanol–Methanol (BUME) Method by Replacing Acetic Acid for Lipid Extraction of Biological Samples

Extraction of lipids from biological samples is a critical step in lipidomics, especially for shotgun lipidomics where lipid extracts are directly infused into a mass spectrometer. The butanol–methanol (BUME) extraction method was originally developed to extract lipids from plasma samples with 1 % acetic acid. Considering some lipids are sensitive to acidic environments, we modified this protocol by replacing acetic acid with lithium chloride solution and extended the modified extraction to tissue samples. Although no significant reduction of plasmalogen levels in the acidic BUME extracts of rat heart samples was found, the modified method was established to extract various tissue samples, including rat liver, heart, and plasma. Essentially identical profiles of the majority of lipid classes were obtained from the extracts of the modified BUME and traditional Bligh–Dyer methods. However, it was found that neither the original, nor the modified BUME method was suitable for 4‐hydroxyalkenal species measurement in biological samples.     

Disseminated Neoplasia in the Soft-Shell Clam Mya arenaria: Membrane Lipid Composition and Functional Parameters of Circulating Cells

In a previous study we compared lipid composition and functional parameters of circulating cells from Cerastoderma edule affected or not by disseminated neoplasia (neoplastic cells vs hemocytes) (Le Grand et al. Chem Phys Lipids 167:9–20 2013 ). Neoplastic cells presented morpho‐functional modifications concomitant to striking membrane lipid alterations: the proportion of particular plasmalogen molecular species was drastically decreased. We wanted to test whether this pattern was representative of bivalve neoplastic cells. For the purpose, a similar study was conducted on another bivalve species affected by disseminated neoplasia, the soft‐shell clam (Mya arenaria). Although total reactive oxygen species production was unaffected, M. arenaria neoplastic cells presented some functional alterations: phagocytosis activity was reduced by 33 %. However, lipid compositions were not drastically altered. Particularly, sterol and plasmalogen levels did not differ between both cell types (about 43 % of membrane lipids and 35 % of phospholipids, respectively in hemocytes and neoplastic cells). This could be related to the fact that disseminated neoplasia was not related to hemolymph cell proliferation in M. arenaria (0.9 ± 0.2 10⁶cell mL^?1, considering both healthy and neoplastic clams, n = 6). Nevertheless this study highlighted minor but specific alterations of membrane lipid composition in M. arenaria neoplastic cells. The only phospholipid subclass in which the fatty acid profile strongly differed between both cell types was serine plasmalogen (PlsSer), with neoplastic cells presenting lower specific enrichment of 20:1n‐11 in PlsSer. Such specific alteration of membrane lipid composition strengthened the assumption of an implication of key plasmalogen molecular species in this leukemia‐like disease in bivalves.     

Over-expression of the Anti-apoptotic Protein Bcl-2 Affects Membrane Lipid Composition in HL-60 Cells

We studied modifications induced at the membrane lipid level by over-expression of the anti-apoptotic protein Bcl-2. When total cell phospholipids were analyzed, the transformation led to a moderate decrease in poly-unsaturated fatty acids, compensated by an increase in mono-unsaturated species. At the mitochondrial membrane level, the changes were more important and occurred in saturated and dimethyl acetal fatty acids, which became more abundant, while unsaturated fatty acid content diminished. This indicates a decline in oxidation-sensitive fatty acids (unsaturated species) together with a gain in oxidation-insensitive saturated fatty acids and in plasmalogen (as detected by dimethyl acetal fatty acids) considered as oxygen species scavengers. Theses changes, combined with the protective role of Bcl-2 against oxidation due to its effect on the redox potential, should protect cells from apoptosis starting in mitochondria. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Lipidomic Analysis of Clostridium cadaveris and Clostridium fallax

The lipidomes of Clostridium fallax and Clostridium cadaveris were studied using thin-layer chromatography (TLC) and normal phase liquid chromatography/mass spectrometry (NPLC/MS). Both species contain diradylglycerol (DRG), monohexosyldiradylglycerol (MHDRG), monohexosyl monoacylglycerol (MHMAG), phosphatidylglycerol (PtdGro), and phosphatidylethanolamine (PtdEtn). DRG, MHDRG, PtdEtn, and PtdGro are present in both diacyl and alk-1-enyl acyl (plasmalogen) forms. Both species contain cardiolipin (Ptd₂Gro), which is present in tetraacyl, monoalkenyl-triacyl, and dialkenyl-diacyl forms. Both species contain small amounts of phosphatidylcholine (PtdCho). The presence of octadecadienoic (18:2) acyl chains in some PtdCho species indicates that they arise from the medium because no 18:2 is seen in the other lipids and clostridia generally lack the capacity to synthesize polyunsaturated fatty acids. The major lipidomic differences between these two species are that C. fallax contains a glycerolacetal of plasmenylethanolamine while C. cadaveris contains an ethanolamine-phosphate-modified diacylglycerol. The significance of these lipid compositions is discussed.     

Identification of Plasmalogen Cardiolipins from Pectinatus by Liquid Chromatography–High Resolution Electrospray Ionization Tandem Mass Spectrometry

High resolution electrospray ionization tandem mass spectrometry (HR-ESI–MS/MS) was used to analyze cardiolipins (Ptd₂Gro) including their plasmalogen forms from three species of the anaerobic beer-spoilage bacterial genus Pectinatus. Cardiolipins including their plasmalogens were analyzed by HR-ESI–MS/MS on Orbitrap and almost 100 cardiolipins (i.e. tetra-acyl—Ptd₂Gro, plasmenyl-tri-acyl—PlsPtd₂Gro, and di-plasmenyl-di-acyl—Pls₂Ptd₂Gro) of three classes were identified. The structures of the molecular species that consist of various regioisomers and structurally similar compounds were revealed in detail. The high resolution mass spectrometry allowed the unambiguous structural assignment of Ptd₂Gro, PlsPtd₂Gro, and Pls₂Ptd₂Gro in the three species of Pectinatus, which contain predominantly odd numbered fatty acids.     

Separation and Determination of Functional Complex Lipids from Chicken Skin

Complex lipids including sphingolipid and plasmalogens were expected to be used as functional supplement, although their physiological activities have not been fully demonstrated. Although these complex lipids exist voluminously in brain and nervous tissues, hardly any animal resources of these lipids have been used since the outbreak of bovine spongiform encephalopathy. Thus, the chemical composition and concentration method of complex lipids from the skin of mature laying hens, a huge amount of which is wasted every year, has been investigated. Total lipid yield (32 g/100 g) prepared from chicken skin contained 2% complex lipids. Total lipids predominantly consisted of triacylglycerol (TAG), with phosphatidylcholine, sphingomyelin (SM) and phosphatidylethanolamine (PE) generally predominant as complex lipids. PE was primarily plasmalogens (62 mol%), of which arachidonic acid (47.6 mol%) and docosahexaenoic acid (11.2 mol%) were the predominant fatty acids. The component sphingoid base of sphingomyelin was almost totally 4-trans sphingenine (sphingosine). The complex lipids were able to be separated from an ethanol extract of minced skin in good yield by solvent fractionation with a hexane/ethanol system. Moreover, highly purified SM (>95 wt%) was prepared by a combination of solvent fractionation and alkaline/acidic hydrolysis from the ethanol extract. Thus, it was shown that culled chicken skin could be a potential resource of the antioxidant phospholipid plasmalogens and human-type sphingolipid.     

Calcium-Independent Phospholipase A<Subscript>2</Subscript> in Rabbit Ventricular Myocytes

We have previously reported that the majority of phospholipase A(2) (PLA(2)) activity in rabbit ventricular myocytes is membrane-associated, calcium-independent (iPLA(2)), selective for arachidonylated plasmalogen phospholipids and inhibited by the iPLA(2)-selective inhibitor bromoenol lactone (BEL). Here, we identified the presence of iPLA(2) in rabbit ventricular myocytes, determined the full length sequences for rabbit iPLA(2)beta and iPLA(2)gamma and compared their homology to the human isoforms. Rabbit iPLA(2)beta encoded a protein with a predicated molecular mass of 74 kDa that is 91% identical to the human iPLA(2)beta short isoform. Full length iPLA(2)gamma protein has a predicated molecular mass of 88 kDa and is 88% identical to the human isoform. Immunoblot analysis of iPLA(2)beta and gamma in membrane and cytosolic fractions from rabbit and human cardiac myocytes demonstrated a similar pattern of distribution with both isoforms present in the membrane fraction, but no detectable protein in the cytosol. Membrane-associated iPLA(2) activity was inhibited preferentially by the R enantiomer of bromoenol lactone [(R)-BEL], indicating that the majority of activity is due to iPLA(2)gamma.