Decreased sphingomyelin (t34:1) is a candidate predictor for lung squamous cell carcinoma recurrence after radical surgery: a case-control study.

BACKGROUND: To reduce disease recurrence after radical surgery for lung squamous cell carcinomas (SQCCs), accurate prediction of recurrent high-risk patients is required for efficient patient selection for adjuvant chemotherapy. Because treatment modalities for recurrent lung SQCCs are scarce compared to lung adenocarcinomas (ADCs), accurately selecting lung SQCC patients for adjuvant chemotherapy after radical surgery is highly important. Predicting lung cancer recurrence with high objectivity is difficult with conventional histopathological prognostic factors; therefore, identification of a novel predictor is expected to be highly beneficial. Lipid metabolism alterations in cancers are known to contribute to cancer progression. Previously, we found that increased sphingomyelin (SM)(d35:1) in lung ADCs is a candidate for an objective recurrence predictor. However, no lipid predictors for lung SQCC recurrence have been identified to date. This study aims to identify candidate lipid predictors for lung SQCC recurrence after radical surgery. METHODS: Recurrent (n = 5) and non-recurrent (n = 6) cases of lung SQCC patients who underwent radical surgery were assigned to recurrent and non-recurrent groups, respectively. Extracted lipids from frozen tissue samples of primary lung SQCC were analyzed by liquid chromatography-tandem mass spectrometry. Candidate lipid predictors were screened by comparing the relative expression levels between the recurrent and non-recurrent groups. To compare lipidomic characteristics associated with recurrent SQCCs and ADCs, a meta-analysis combining SQCC (n = 11) and ADC (n = 20) cohorts was conducted. RESULTS: Among 1745 screened lipid species, five species were decreased (≤ 0.5 fold change; P < 0.05) and one was increased (≥ 2 fold change; P < 0.05) in the recurrent group. Among the six candidates, the top three final candidates (selected by AUC assessment) were all decreased SM(t34:1) species, showing strong performance in recurrence prediction that is equivalent to that of histopathological prognostic factors. Meta-analysis indicated that decreases in a limited number of SM species were observed in the SQCC cohort as a lipidomic characteristic associated with recurrence, in contrast, significant increases in a broad range of lipids (including SM species) were observed in the ADC cohort. CONCLUSION: We identified decreased SM(t34:1) as a novel candidate predictor for lung SQCC recurrence. Lung SQCCs and ADCs have opposite lipidomic characteristics concerning for recurrence risk. TRIAL REGISTRATION: This retrospective study was registered at the UMIN Clinical Trial Registry ( UMIN000039202 ) on January 21, 2020.

UHPLC-Q-Orbitrap HRMS-based quantitative lipidomics reveals the chemical changes of phospholipids during thermal processing methods of Tan sheep meat.

Thermal processing affects the lipid compositions of meat products. The study determined the effects of boiled, steamed and roasted processing methods on the lipidomics profiles of Tan sheep meat with a validated UPLC-Q-Orbitrap HRMS combined lipid screening strategy method. Combined with sphingolipid metabolism, the boiled approach was the suitable choice for atherosclerosis patients for more losses of sphingomyelin than ceramide in meat. The similarly less losses of phosphatidylcholine and lysophosphatidylcholine showed in glycerophospholipid metabolism implied that steamed Tan sheep meat was more suitable for the populations of elderly and infants. Furthermore, a total of 90 lipids with significant difference (VIP > 1) in 6 lipid subclasses (sphingomyelin, ceramide, lysophosphatidylcholine, phosphatidylcholine, phosphatidylethanolamines, triacylglycerol,) were quantified among raw and three types of thermal processed Tan sheep meat, further providing useful information for identification of meat products with different thermal processing methods (LOD with 0.14-0.31 µg kg-1, LOQ with 0.39-0.90 µg kg-1).

Simultaneous quantification of the lipids phosphatidylcholine, 3-sn-phosphatidylethanolamine, sphingomyelin, and L-α-lysophosphatidylcholine extracted from the tissues of the invasive golden apple snail (Pomacea canaliculata) using UHPLC-ESI-MS/MS.

Lipids such as phosphatidylcholine (PC), 3-sn-phosphatidylethanolamine (PE), sphingomyelin (SM) and L-α-lysophosphatidylcholine (LPC) are the major components of biological membranes and play important roles in physiological functions. Here, PC, PE, SM, and LPC were extracted from golden apple snails (GAS, Pomacea canaliculata) and GAS flesh (GASF) using an ethanol/hexane sequential scheme and quantified simultaneously using ultra-high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS) to evaluate whether the GAS could be the source of the four lipids. Our results suggest that ethanol extracts contained the most crude lipids, and the yield of dry (evaporated) lipids were 3.45 g per 100 g fresh GASF and 1.82 g per 100 g of fresh GAS. Quantification of the lipids using UHPLC-ESI-MS/MS suggested that GAS contained PE, PC, SM and LPC, with SM being the most abundant lipid (after purification: 1.71 and 1.42 mg g-1 dry weight from 100 g of GASF and GAS, respectively). The method we used is cost-effective, and the recovery rates of ethanol and hexane ranged from 80-91% and 87-91% respectively. Overall, GAS and GASF are potential raw materials for lipids such as SM and PC extraction using the ethanol/hexane method. Comparatively, lipids extraction from the GAS is more effective and timesaving. Our finding would provide a way to utilize GAS and potentially control its invasion.

Obese Mice with Dyslipidemia Exhibit Meibomian Gland Hypertrophy and Alterations in Meibum Composition and Aqueous Tear Production.

BACKGROUND: Dyslipidemia may be linked to meibomian gland dysfunction (MGD) and altered meibum lipid composition. The purpose was to determine if plasma and meibum cholesteryl esters (CE), triglycerides (TG), ceramides (Cer) and sphingomyelins (SM) change in a mouse model of diet-induced obesity where mice develop dyslipidemia. METHODS: Male C57/BL6 mice (8/group, age = 6 wks) were fed a normal (ND; 15% kcal fat) or an obesogenic high-fat diet (HFD; 42% kcal fat) for 10 wks. Tear production was measured and meibography was performed. Body and epididymal adipose tissue (eAT) weights were determined. Nano-ESI-MS/MS and LC-ESI-MS/MS were used to detect CE, TG, Cer and SM species. Data were analyzed by principal component analysis, Pearson's correlation and unpaired t-tests adjusted for multiple comparisons; significance set at p ≤ 0.05. RESULTS: Compared to ND mice, HFD mice gained more weight and showed heavier eAT and dyslipidemia with higher levels of plasma CE, TG, Cer and SM. HFD mice had hypertrophic meibomian glands, increased levels of lipid species acylated by saturated fatty acids in plasma and meibum and excessive tear production. CONCLUSIONS: The majority of meibum lipid species with saturated fatty acids increased with HFD feeding with evidence of meibomian gland hypertrophy and excessive tearing. The dyslipidemia is associated with altered meibum composition, a key feature of MGD.

A Targeted Mass Spectrometric Analysis Reveals the Presence of a Reduced but Dynamic Sphingolipid Metabolic Pathway in an Ancient Protozoan, Giardia lamblia.

Giardia lamblia, a single-celled eukaryote, colonizes and thrives in the small intestine of humans. Because of its compact and reduced genome, Giardia has adapted a "minimalistic" life style, as it becomes dependent on available resources of the small intestine. Because Giardia expresses fewer sphingolipid (SL) genes-and glycosphingolipids are critical for encystation-we investigated the SL metabolic cycle in this parasite. A tandem mass spectrometry (MS/MS) analysis reveals that major SLs in Giardia include sphingomyelins, sphingoid bases, ceramides, and glycosylceramides. Many of these lipids are obtained by Giardia from the growth medium, remodeled at their fatty acyl chains and end up in the spent medium. For instance, ceramide-1-phosphate, a proinflammatory molecule that is not present in the culture medium, is generated from sphingosine (abundant in the culture medium) possibly by remodeling reactions. It is then subsequently released into the spent medium. Thus, the secretion of ceramide-1-phospate and other SL derivatives by Giardia could be associated with inflammatory bowel disease observed in acute giardiasis. Additionally, we found that the levels of SLs increase in encysting Giardia and are differentially regulated throughout the encystation cycle. We propose that SL metabolism is important for this parasite and, could serve as potential targets for developing novel anti-giardial agents.

Deep Profiling of Immunosuppressive Glycosphingolipids and Sphingomyelins in Wild Cordyceps.

Deep profiling of glycosphingolipids and sphingomyelins in wild Cordyceps was carried out by using offline chromatographic enrichment followed by ultrahigh performance liquid chromatography-ultrahigh definition-quadrupole time-of-flight mass spectrometry (UHPLC-UHD-Q-TOF-MS). A total of 119 glycosphingolipids (72 new ones) and 87 sphingomyelins (43 new ones) were identified from wild Cordyceps on the basis of the accurate mass and MS/MS fragmentations, isotope patterns, sphingolipid (SPL) database matching, confirmation by SPL standards, and the reversed-phase liquid chromatographic retention rule. This study is the most comprehensive report on the identification of glycosphingolipids and sphingomyelins from fungus. A subsequent lipopolysaccharide-induced mouse splenic lymphocyte proliferation assay showed that the Cordyceps glycosphingolipid fraction exhibits higher immunosuppressive activity compared to that of Cordyceps sphingomyelins. Our findings provided insight into the chemical diversity of sphingolipids in Cordyceps and chemical evidence for the therapeutic application of wild Cordyceps.

Endometriosis foci differentiation by rapid lipid profiling using tissue spray ionization and high resolution mass spectrometry.

Obtaining fast screening information on molecular composition of a tissue sample is of great importance for a disease biomarkers search and for online surgery control. In this study, high resolution mass spectrometry analysis of eutopic and ectopic endometrium tissues (90 samples) is done using direct tissue spray mass spectrometry in both positive and negative ion modes. The most abundant peaks in the both ion modes are those corresponding to lipids. Species of three lipid classes are observed, phosphatidylcholines (PC), sphingomyelins (SM) and phosphoethanolamines (PE). Direct tissue analysis gives mainly information on PC and SM lipids (29 species) in positive ion mode and PC, SM and PE lipids (50 species) in negative ion mode which gives complementary data for endometriosis foci differentiation. The biggest differences were found for phospholipids with polyunsaturated acyls and alkils. Although, tissue spray shows itself as appropriate tool for tissue investigation, caution should be paid to the interpretation of mass spectra because of their higher complexity with more possible adducts formation and multiple interferences must be taken into account. The present work extends the application of direct tissue analysis for the rapid differentiation between endometriotic tissues of different foci.

Method to Measure Sphingomyelin Synthase Activity Changes in Response to CD95L.

Sphingomyelin synthases 1 and 2 convert the anti-oncometabolite ceramide to sphingomyelin, the most abundant sphingolipid in plasma membrane. CD95L-induced ceramide increase is associated with the caspase-dependent inhibition of sphingomyelin synthesis, which enhances the mitochondrial route to apoptosis. Knocking down sphingomyelin synthase 1 or inhibiting sphingomyelin synthesis facilitates ceramide accumulation, cytochrome c release from mitochondria, and caspase-9 activation in cancer cell upon CD95L treatment. Here, we describe a method to monitor in situ sphingomyelin synthase activity changes triggered by CD95L.

Characterisation of plasmalemmal shedding of vesicles induced by the cholesterol/sphingomyelin binding protein, ostreolysin A-mCherry.

Ostreolysin A (OlyA) is a 15-kDa protein that binds selectively to cholesterol/sphingomyelin membrane nanodomains. This binding induces the production of extracellular vesicles (EVs) that comprise both microvesicles with diameters between 100nm and 1µm, and larger vesicles of around 10-µm diameter in Madin-Darby canine kidney cells. In this study, we show that vesiculation of these cells by the fluorescent fusion protein OlyA-mCherry is not affected by temperature, is not mediated via intracellular Ca2+ signalling, and does not compromise cell viability and ultrastructure. Seventy-one proteins that are mostly of cytosolic and nuclear origin were detected in these shed EVs using mass spectroscopy. In the cells and EVs, 218 and 84 lipid species were identified, respectively, and the EVs were significantly enriched in lysophosphatidylcholines and cholesterol. Our collected data suggest that OlyA-mCherry binding to cholesterol/sphingomyelin membrane nanodomains induces specific lipid sorting into discrete patches, which promotes plasmalemmal blebbing and EV shedding from the cells. We hypothesize that these effects are accounted for by changes of local membrane curvature upon the OlyA-mCherry-plasmalemma interaction. We suggest that the shed EVs are a potentially interesting model for biophysical and biochemical studies of cell membranes, and larger vesicles could represent tools for non-invasive sampling of cytosolic proteins from cells and thus metabolic fingerprinting.

Effects of n-6 and n-3 polyunsaturated acid-rich soybean phosphatidylcholine on membrane lipid profile and cryotolerance of human sperm.

OBJECTIVE: To study the effects of n-6 and n-3 polyunsaturated acid-rich soybean phosphatidylcholine (soy-PC) on sperm cryotolerance with regard to sperm membrane lipid profile, membrane surface integrity, and routine semen parameters. DESIGN: Experimental study. SETTING: University-affiliated tertiary hospital. PATIENT(S): A total of 20 normospermic fertile men. INTERVENTION(S): Semen samples examined for differences in semen parameters, sperm membrane lipid profile, and plasma membrane surface both before and after cryopreservation using basic freezing medium with N-tris(hydroxymethyl)-methyl-2-aminoethane sulfonic acid (TES) and tris-(hydroxymethyl)-aminomethane (TRIS) supplemented with purified soy-PC (TEST-PC) or egg yolk (TEST-Y), both alone or in association (TEST-Y-PC). MAIN OUTCOME MEASURE(S): Conventional semen parameters and membrane lipid profile by matrix-assisted laser/desorption ionization mass spectrometry (MALDI-MS). RESULT(S): Postthaw sperm cell motility, vitality, and morphology parameters were similar for soy-PC (TEST-PC) and egg yolk (TEST-Y) cryoprotectants. However, sperm exposed to TEST-Y-PC presented better kinetic parameters, which were similar to the original quality of the fresh semen. Human sperm MALDI-MS lipid profiles revealed that the relative abundance of glycerophospholipids of m/z 760.44 [PC (34:1)+H]+, 781.55 [SM (20:0) +Na]+, 784.55 [PC (36:3) +H]+, 806.64 [PC (38:6) +H]+, 807.64 [SM (22:1) +Na]+, and 809.64 [SM (22:0) +Na]+ increased in soy-PC samples (TEST-PC). Nonetheless, only one lipid (m/z 781.55, [SM (20:0) +Na]+) statistically significantly changed when sperm was cryopreserved in TEST-Y-PC. CONCLUSION(S): Sphingomyelin was defined as a prospective biomarker of soy-PC treatment, and it could be related to the positive cryoprotective effects of soy-PC in human sperm, opening new perspectives to design of a more efficient synthetic cryoprotectant medium containing purified egg yolk biomolecules combined with soy-PC.

Phase diagram of a polyunsaturated lipid mixture: Brain sphingomyelin/1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine/cholesterol.

Phospholipids having a polyunsaturated acyl chain are abundant in biological membranes, but their behavior in lipid mixtures is difficult to study. Here we elucidate the nature of such mixtures with this report of the first ternary phase diagram containing the polyunsaturated lipid SDPC in mixtures of BSM/SDPC/Chol (brain sphingomyelin/1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine/cholesterol). These mixtures show coexisting macroscopic liquid-disordered (Ld) and liquid-ordered (Lo) phase separation, with phase boundaries determined by FRET and by fluorescence microscopy imaging of giant unilamellar vesicles (GUVs). Surprisingly, SDPC mixes with BSM/Chol similarly to how DOPC and POPC mix with BSM/Chol. Notably, intermediate states are produced within the Ld+Lo liquid-liquid immiscibility region upon addition of fourth component POPC. These mixtures of BSM/SDPC/POPC/Chol exhibit nanoscopic Ld+Lo domains over a very large volume of composition space, possibly because Ld/Lo line tension is not high.

Unique thermal behavior of sphingomyelin species with nonhydroxy and 2-hydroxy very-long-chain (C28-C32) PUFAs.

In rat germ cells and spermatozoa, sphingomyelin (SM) contains molecular species with nonhydroxy (n) and 2-hydroxy (h) very-long-chain polyunsaturated fatty acids (V), the most abundant being SMs with (n- and h-) 28:4n-6, 30:5n-6, and 32:5n-6 as acyl chains. The aim of this study was to gain information about their thermotropic behavior and interactions with other lipids. After isolation from rat testis, multilamellar and giant unilamellar vesicles from these SMs were examined using fluorescent probes. Only n-32:5 SM and h-32:5 SM displayed a gel-liquid transition temperature (Tt ∼ 21-22°C), the rest remaining in the liquid state in the 5°C-45°C range. The degree of order was larger in bilayers of any of the h-V SMs than in those of their chain-matched n-V SMs. Both, but n-V SM relatively more than h-V SM, decreased the Tt of dimyristoylphosphatidylcholine as their proportion increased in binary phosphatidylcholine:SM liposomes. In contrast to the established ability of 16:0 SM to form lateral cholesterol/SM-rich ordered domains in ternary dioleoylphosphatidylcholine:cholesterol:SM bilayers, neither n-V SM nor h-V SM showed a tendency to do so. Thus, these SMs are in the fluid state and are not involved in this type of domains in spermatozoa at physiological temperatures. However, this state could be altered at the very low temperatures at which these gametes are usually preserved.

Tracing and separating plasma components causing matrix effects in hydrophilic interaction chromatography-electrospray ionization mass spectrometry.

Matrix effects on electrospray ionization were investigated for plasma samples analysed by hydrophilic interaction chromatography (HILIC) in gradient elution mode, and HILIC columns of different chemistries were tested for separation of plasma components and model analytes. By combining mass spectral data with post-column infusion traces, the following components of protein-precipitated plasma were identified and found to have significant effect on ionization: urea, creatinine, phosphocholine, lysophosphocholine, sphingomyelin, sodium ion, chloride ion, choline and proline betaine. The observed effect on ionization was both matrix-component and analyte dependent. The separation of identified plasma components and model analytes on eight columns was compared, using pair-wise linear correlation analysis and principal component analysis (PCA). Large changes in selectivity could be obtained by change of column, while smaller changes were seen when the mobile phase buffer was changed from ammonium formate pH 3.0 to ammonium acetate pH 4.5. While results from PCA and linear correlation analysis were largely in accord, linear correlation analysis was judged to be more straight-forward in terms of conduction and interpretation.

Orally administered sphingomyelin in bovine milk is incorporated into skin sphingolipids and is involved in the water-holding capacity of hairless mice.

BACKGROUND: We previously reported that dietary sphingomyelin (SM) concentrate from bovine milk improves epidermal functions. SM is a known precursor of ceramide (Cer) in the stratum corneum (SC). Neither the uptake nor distribution of orally administered SM nor its effects on epidermal functions have been demonstrated. OBJECTIVE: We evaluated the effects of dietary SM on epidermal functions, and the distribution and fate of its radiolabeled metabolites in mice orally administered [4,5-(3)H-sphinganyl] sphingomyelin ((3)H-SM). METHODS: Bovine milk SM (98% purity) was administered orally to 13-week-old hairless mice at 142 mg/kg per day for eight weeks. Their SC hydration, transepidermal water loss (TEWL), and SC Cer content were measured. (3)H-SM was then administered orally to 10-week-old hairless mice. Its distribution and metabolites in the skin were evaluated with whole-body autoradiography, liquid scintillation counting, and thin-layer chromatography. RESULTS: SC hydration in the SM-administered mice was higher than that in control mice, whereas their TEWL and Cer contents did not differ. Radioactivity was distributed extensively in the bodies of the experimental mice and decreased gradually with time. In contrast, the radioactivity in the SC remained constant after its administration, and radiolabeled SM and Cer were detected in the skin. This suggests that dietary SM is transferred to the skin and then converted to Cer in the SC. CONCLUSIONS: Orally administered SM is incorporated into skin SM and converted to SC Cer, which is involved in the water-holding capacity of the SC.

Sticholysin II: a pore-forming toxin as a probe to recognize sphingomyelin in artificial and cellular membranes.

Sphingomyelin is a major component of membrane rafts, and also is a precursor of many bioactive molecules. The sphingomyelin plays important biological roles and alterations of its metabolism are the basis of some genetic disorders such as the Niemann Pick disease. A complete understanding of its biological role is frustrated by the lack of efficient tools for its recognition in the cell. Sticholysin II (StnII) is a 20 kDa protein from the sea-anemone Stichodactyla helianthus which shows a cytotoxic activity by forming oligomeric aqueous pores in the cell plasma membrane. A recent NMR analysis indicates that the sticholysin II binds specifically to sphingomyelin by two domains that recognize respectively the hydrophilic (i.e. phosphorylcholine) and the hydrophobic (i.e. ceramide) moieties of the molecule. Aim of our research has been to verify the possible employ of an antibody against the StnII to investigate the localization and the dynamics of sphingomyelin in cell membranes. For this purpose, we developed a monoclonal antibody (named A10) against the toxin and we tested its ability to bind StnII after binding to sphingomyelin. A10 antibody is able to recognize the sticholysin II both in its native form and after SDS treatment, being the protein still suitable for many analytic techniques such as ELISA, western blotting and immunofluorescence. The high affinity of the toxin for the sphingomyelin in cell membranes has been demonstrated by microscopic immuno-localization and western blot analysis; both methods confirmed that sphingomyelin is the molecular acceptor for StnII also in cell membranes. Finally, we studied the specificity of the toxin for sphingomyelin by a cell membrane-double labelling method, using cholera toxin, specific for the ganglioside GM1, and sticholysin II. The results obtained show that there is no cross-reactivity between the two toxins, confirming that sticholysin II is able to discriminate among membrane domains with sphingomyelin with respect to those enriched with gangliosides.

An efficient hydrophilic interaction liquid chromatography separation of 7 phospholipid classes based on a diol column.

A hydrophilic interaction liquid chromatography (HILIC) - ion trap mass spectrometry method was developed for separation of a wide range of phospholipids. A diol column which is often used with normal phase chromatography was adapted to separate different phospholipid classes in HILIC mode using a mobile phase system consisting of acetonitrile, water, ammonium formate and formic acid. An efficient between-class separation of seven phospholipid classes including phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinostol, phosphatidylcholine, phosphatidylserine, sphingomyelin and lysophosphatidylcholine was successfully achieved within 14 min using a gradient elution which starts with 90% of organic solvent and ends with 70% of organic solvent. 53 mM formic acid (in both organic phase and aqueous phase) and 60mM ammonium formate (only in aqueous phase) were used as mobile phase modifier. The relatively high amount of ammonium formate was essential to obtain well-shaped peaks of each phospholipid class, especially phosphatidylserines; actually, no negative effect due to ammonium formate was observed for electrospray-mass spectrometry detection in real-life samples. Good chromatographic separation between different lipid classes was obtained (Rs, from 0.73 to 4.97) and well-shaped peaks (tailing factor, from 0.98 to 1.20) were obtained. The developed method was fully validated and the satisfactory performance characteristics such as linearity (R(2), 0.990-0.999), retention time stability (RSD<1%), within day repeatability (RSD, 5-13%), between day variation (RSD, 7-14%) and recoveries (99.6-115.5%) indicated the gradient HILIC method was appropriate for profiling of plasma phospholipids. The method was successfully applied to separate phospholipids extracts from human plasma, mouse plasma and rat plasma.

Unique sphingomyelin patches are targets of a beta-cell-specific antibody.

To devise successful imaging and therapeutic strategies, the identification of ß-cell surface markers is one of the challenges in diabetes research that has to be resolved. We previously showed that IC2, a rat monoclonal IgM antibody, can be used for ex vivo determination of ß-cell mass by imaging. Further progress toward the development of an antibody-based imaging agent was hampered by the lack of knowledge regarding the nature and composition of the IC2 antigen. Here, we show a series of systematic experiments involving classical lipid extraction and chromatography techniques combined with immunochemistry, which led to the identification of sphingomyelin as the target antigen assembled in the form of patches on the ß-cell surface. Our findings were verified by modulating SM by enzymatic cleavage, downregulation, upregulation, and perturbation of membrane SM and observation of corresponding changes in IC2 binding. Cholesterol participates in stabilization of these patches, as its removal results in loss of IC2 binding. We believe that these findings have implications for identifying future ligands for the proposed antigen for imaging purposes as well as for potential therapy, as sphingomyelin has been shown to play a role in the apoptotic cascade in pancreatic ß cells.

A new high-performance liquid chromatographic method with evaporative light scattering detector for the analysis of phospholipids. Application to Iberian pig subcutaneous fat.

A new method for the analysis of phospholipids by normal-phase HPLC is described using a silica column. Addition of ammonia and triethylamine to a gradient based on chloroform/methanol/water promoted a good and rapid separation of phospholipid classes (20 min run). The use of an evaporative light scattering detector permitted an accurate analysis of a mixture of phospholipids. Calibration curves were linear within different range for each phospholipid class. The LOD and LOQ obtained were below 0.03 and 0.05 mg kg⁻¹ for all cases, respectively. Besides, a new method for the separation of phospholipids from total lipids before HPLC analysis by a solid-phase extraction (SPE) with Si cartridges has been developed. This methodology gave a good recovery ranging from 97 to 117%. The method was validated with a standard mixture of phospholipids. This method has been applied to characterize the phospholipid fraction of subcutaneous fat from Iberian pig. Cardiolipin, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylcholine, and sphingomyelin have been described for first time in these samples. The fatty acid composition of the different phospholipid classes and their HPLC electrospray ionization mass spectrometry have been used for characterizing the molecular species present in each one.

Biochemical studies on sphingolipid of Artemia franciscana (I) isolation and characterization of sphingomyelin.

Sphingomyelin was isolated from cysts of the brine shrimp Artemia franciscana using QAE-Sephadex A25, Florisil and Iatrobeads column chromatographies. The chemical structure was identified using thin-layer chromatography, gas-liquid chromatography, infrared spectroscopy and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The ceramide moiety of sphingomyelin consisted of stearic, arachidic, and behenic acids as fatty acids, and hexadeca-4- and heptadeca-4-sphingenines as sphingoids. By comparative analysis, the ceramide component of Artemia sphingomyelin appears unique in invertebrates and vertebrates. Biological functions of sphingomyelin have largely been investigated using mammalian-derived sphingomyelin. In mammals, a wide variety of molecular species of sphingomyelins have been reported, especially derived from nerve tissue, while the lower animal Artemia contains this unusual sphingomyelin perhaps because of having a much simpler nervous system. The purified unusual sphingomyelin derived from Artemia franciscana might be a very useful tool in elucidating the functions and mechanisms of action of this mediator.

Simultaneous preparation of purified plasmalogens and sphingomyelin in human erythrocytes with phospholipase A1 from Aspergillus orizae.

A method for the simultaneous purification of plasmalogens and sphingomyelin (SM) in human erythrocytes is described. Treatment of total lipids with n-hexane/acetone (1:1 v/v) resulted in selective precipitation of SM. Both the supernatant and the precipitate fractions were incubated with a phospholipase A(1) (PLA1) from Aspergillus orizae for 3.5 h. The PLA1-treated lipids were extracted with n-hexane/isopropanol, the hexane layer was obtained using a Na(2)SO(4) solution, and the hexane layer was further washed with water. At this step, the relative concentration of the plasmalogens was 92% of the total phospholipids in the supernatant fraction, and that of SM was 97.7% in the precipitate fraction. Each fraction was applied to high performance liquid chromatography (HPLC) for further purification. The plasmalogen and SM obtained were almost free of the other lipids. The purity of the plasmalogens and SM was monitored by HPLC, which can separate intact plasmalogens from their diacyl analogs.