Sphingosine 1-phosphate (S1P) is a lipid mediator formed by the metabolism of sphingomyelin. In vertebrates, S1P is secreted into the extracellular environment and signals via G protein-coupled S1P ...receptors to regulate cell-cell and cell-matrix adhesion, and thereby influence cell migration, differentiation and survival. The expression and localization of S1P receptors is dynamically regulated and controls vascular development, vessel stability and immune cell trafficking. In addition, crucial events during embryogenesis, such as angiogenesis, cardiogenesis, limb development and neurogenesis, are regulated by S1P signalling. Here, and in the accompanying poster, we provide an overview of S1P signalling in development and in disease.
► An efficient HILIC separation of 7 phospholipid classes. ► The relatively high amount of ammonium formate is beneficial to separation of PLs. ► The possible mechanism of HILIC separation of ...phospholipids on diol-bonded column. ► IPA (isopropanol) method for extracting phospholipids from plasma.
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 60
mM 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.
Abstract
Background
Recent studies suggest that associations of ceramides (Cer) and sphingomyelins (SM) with health outcomes differ according to the fatty acid acylated to the sphingoid backbone. The ...purpose of this study was to assess associations of Cer and SM species with mortality.
Methods
The study population included participants from the Cardiovascular Health Study (CHS), a community-based cohort of adults aged ≥65 years who were followed from 1992–2015 (n = 4612). Associations of plasma Cer and SM species carrying long-chain (i.e., 16:0) and very-long-chain (i.e., 20:0, 22:0, 24:0) saturated fatty acids with mortality were assessed using Cox proportional hazards models.
Results
During a median follow-up of 10.2 years, 4099 deaths occurred. High concentrations of Cer and SM carrying fatty acid 16:0 were each associated with an increased risk of mortality. Conversely, high concentrations of several ceramide and sphingomyelin species carrying longer fatty acids were each associated with a decreased risk of mortality. The hazard ratios for total mortality per 2-fold difference in each Cer and SM species were: 1.89 (95% CI), 1.65–2.17 for Cer-16, 0.79 (95% CI, 0.70–0.88) for Cer-22, 0.74 (95% CI, 0.65–0.84) for Cer-24, 2.51 (95% CI, 2.01–3.14) for SM-16, 0.68 (95% CI, 0.58–0.79) for SM-20, 0.57 (95% CI, 0.49–0.67) for SM-22, and 0.66 (0.57–0.75) for SM-24. We found no association of Cer-20 with risk of death.
Conclusions
Associations of Cer and SM with the risk of death differ according to the length of their acylated saturated fatty acid. Future studies are needed to explore mechanisms underlying these relationships.
Display omitted
•Golden apple snail (GAS) contains abundant of lipids that can be extracted easily.•Ethanol/hexane method is cost-effective for GAS lipids extraction.•Lipids extracted from GAS can be ...purified effectively using acetone-hexane mixture.•Sphingomyelin is the most compound extracted from GAS using ethanol/hexane.
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.
Alterations of sphingolipids and their metabolizing enzymes play a role in various diseases. However, peripheral biomarkers for such changes are limited. Particularly, in the increasingly reported ...involvement of neutral sphingomyelinase (NSM) with four described isoforms in tissues or cells, a peripheral marker is lacking. We here describe the detection of an NSM activity in human serum and plasma samples which hydrolyses fluorescently labeled sphingomyelin to ceramide in a time- and volume-dependent manner. Reaction rates were linear up to 10 days, and serum volumes above 2 vol-% were inhibitory. Biochemical properties were different from acid sphingomyelinase (ASM) with respect to detergent specificity (sodium deoxycholate), pH profile (pH 7-9), and cation dependence: Serum NSM activity was inhibited by EDTA ≥ 1 µM and restored in EDTA-anticoagulated plasma with the addition of ≥ 100 µM Co
. It was independent of Mg
, the typical cofactor of cellular NSM species, and even inhibited by Mg
≥ 20 mM. Serum NSM activity was not correlated with ASM activity and was independent of sex and age in 24 healthy adults. Since human peripheral NSM activity is very low and activities in rodents are even lower or undetectable, future research should aim to increase the reaction rate and determine the source of this enzymatic activity. The established activity could serve as a future biomarker or therapeutic target in diseases affected by sphingolipid derangements.
•Phospholipids and milk fat globules were compared between five mammalian milk.•Human milk contains a higher amount of sphingomyelin than other mammalian milk.•The phospholipids acyl chain ...unsaturation are highest in human milk.•Milk fat globules mean size and distribution showed obvious differences across species.•A similar TAG core-membrane lipid structure was observed in all mammalian milk.
We compared phospholipids (PLs) content, their molecular species, and milk fat globules size and microstructure in the milk of five mammalian species, including human, cow, goat, yak, and donkey. The absolute quantification of major PLs was determined using 31P NMR and their fatty acid composition with GC. The molecular species of PLs were analysed using LC-MS where a total of 9 PL species, including one sphingomyelin (SM), six glycerophospholipid (GPL), and two lysoglycerophospholipids (lyso-GPLs), were identified. PLs profile shows an obvious difference among the species, with human milk showing higher SM content and more unsaturated fatty acyls than other mammalian milk. The mammalian milk show a similar core-membrane lipid structure but obvious different size distribution. These data provide a basis for better construction of infant formulas to provide PLs requirements and a similar milk fat globule structure for infants.
Sphingomyelin (SM) has been proposed to form cholesterol-dependent raft domains and sphingolipid domains in the plasma membrane (PM). How SM contributes to the formation and function of these domains ...remains unknown, primarily because of the scarcity of suitable fluorescent SM analogs. We developed new fluorescent SM analogs by conjugating a hydrophilic fluorophore to the SM choline headgroup without eliminating its positive charge, via a hydrophilic nonaethylene glycol linker. The new analogs behaved similarly to the native SM in terms of their partitioning behaviors in artificial liquid order-disorder phase-separated membranes and detergent-resistant PM preparations. Single fluorescent molecule tracking in the live-cell PM revealed that they indirectly interact with each other in cholesterol- and sphingosine backbone-dependent manners, and that, for ∼10-50 ms, they undergo transient colocalization-codiffusion with a glycosylphosphatidylinositol (GPI)-anchored protein, CD59 (in monomers, transient-dimer rafts, and clusters), in CD59-oligomer size-, cholesterol-, and GPI anchoring-dependent manners. These results suggest that SM continually and rapidly exchanges between CD59-associated raft domains and the bulk PM.
The definition of new reliable markers for neonatal maturity evaluation is crucial in canine clinical practice. Concerns about the safety of amniotic sampling in pregnant dogs have prevented its ...collection for diagnostic purposes. Moreover, amniotic fluid had been considered waste material until the latest studies reported amniocentesis as a reliable and safe procedure, even in the canine species. In our study, amniotic fluid (n = 63) collected at birth from ten dogs undergoing elective Caesarean sections at term was analysed to discover new potential indices of canine neonatal maturity. Based on gestational age, mothers and puppies were divided into two groups: the early group (≤65 days from luteinizing hormone (LH) surge, n = 5) and the late group (>65 days from LH surge, n = 5). Amniotic parameters of the lightest and heaviest puppy in individual/each litter, with a birth weight difference of at least 20% among littermates, were also compared. In particular, the content of lecithin, sphingomyelin, surfactant protein A (SP-A), cortisol, and pentraxin 3 (PTX3) in amniotic fluid, which is considered predictive of foetal development in humans, were investigated. Maternal serum SP-A and cortisol were also measured simultaneously.
All amniotic parameters were detectable in canine amniotic fluid. Interestingly, the concentrations of different amniotic parameters correlated with each other. Lecithin was positively correlated with sphingomyelin (p < 0.0001), maternal SP-A (p < 0.0005), and the ratio of amniotic and maternal cortisol (p < 0.004). Amniotic SP-A was inversely correlated to maternal SP-A (p < 0.05), lecithin (p < 0.005), and lecithin-sphingomyelin ratio (p < 0.05). A positive correlation was also recorded between amniotic and maternal cortisol (p < 0.008). Considering that all puppies were born alive and mature, these data could provide a potential range of expected amniotic values in full-term new-born dogs. Furthermore, since gestational age was positively correlated with both maternal and amniotic cortisol (p < 0.0001) and amniotic PTX3 (p < 0.05), amniotic fluid seems to be an attractive, innovative, and minimally invasive matrix with potential diagnostic and prognostic utility for the investigation of canine maturity.
•Lecithin, sphingomyelin, SP-A, cortisol, PTX3 were all detectable in canine amniotic fluid.•Gestational age was correlated to amniotic cortisol and PTX3.•Canine amniotic fluid composition may be predictive of newborn pups maturity.
Sphingomyelins (SMs) are an essential class of lipids widely existing in different organisms. The sphingoid base and N-acyl chain are two building blocks which can undergo different types of ...modifications during lipogenesis, including desaturation, hydroxylation, and methyl branching. Current lipidomic analysis methods cannot provide detailed information on these structural motifs. Herein, we developed a tandem mass spectrometric method by harnessing radical-directed dissociation (RDD) from collision-induced dissociation (CID) of the bicarbonate anion adduct of SM (M + HCO3−). A major RDD channel produced a high-abundance fragment carrying the intact N-acyl chain, termed as “N-acyl fragment”, allowing the assignment of the sphingoid base/N-acyl composition and relative quantitation of compositional isomers of SM at high sensitivity. RDD also produced intrachain fragments in lower abundances, which helped localization of methyl branching and hydroxylation in SM. The acetone Paternò–Büchi (PB) reaction was found to be capable of derivatizing the Δ4 carbon–carbon double bond (CC) in sphingosine (SPH) base and producing CC diagnostic ions upon CID, albeit at much lower efficiencies than those of the isolated CC in alkyl chains. A liquid chromatography–mass spectrometry workflow was developed by incorporating MS2 CID of SM via M + HCO3− and PB-MS2 CID. The capability of profiling SM with detailed structural information was demonstrated by analyzing complex lipid extracts from porcine brain and Caenorhabditis elegans. These results provided visualization of the sphingoid base/N-acyl compositional isomers of SM lipids and revealed large structural diversity from each sample. These included identification of the sphingadiene base d18:1(Δ4,14), CC location isomers in N-acyls, C-2 hydroxylation of N-acyls, and iso-methyl branched SPH base.
Transferrin (Tf) endocytosis and recycling are essential for iron uptake and the regulation of cell proliferation. Tf and Tf receptor (TfR) complexes are internalized via clathrin-coated pits ...composed of a variety of proteins and lipids and pass through early endosomes to recycling endosomes. We investigated the role of sphingomyelin (SM) synthases (SMS1 and SMS2) in clathrin-dependent trafficking of Tf and cell proliferation. We employed SM-deficient lymphoma cells that lacked SMSs and that failed to proliferate in response to Tf. Transfection of SMS1, but not SMS2, enabled these cells to incorporate SM into the plasma membrane, restoring Tf-mediated proliferation. SM-deficient cells showed a significant reduction in clathrin-dependent Tf uptake compared with the parental SM-producing cells. Both SMS1 gene transfection and exogenous short-chain SM treatment increased clathrin-dependent Tf uptake in SM-deficient cells, with the Tf being subsequently sorted to Rab11-positive recycling endosomes. We observed trafficking of the internalized Tf to late/endolysosomal compartments, and this was not dependent on the clathrin pathway in SM-deficient cells. Thus, SMS1-mediated SM synthesis directs Tf-TfR to undergo clathrin-dependent endocytosis and recycling, promoting the proliferation of lymphoma cells.
Background: Sphingomyelin synthase (SMS) catalyzes the synthesis of sphingomyelin.
Results: Sphingomyelin-deficient cells failed to proliferate in response to transferrin. Transfection of SMS1 enabled these cells to generate sphingomyelin, promoting clathrin-dependent uptake of transferrin and its dependent proliferation.
Conclusion: SMS1 is indispensable for transferrin internalization and cell proliferation.
Significance: Our findings provide new insights into the role of SMS1 in transferrin biology.
PDF
