Sphingomyelin Content Research Articles

Molecular Simulations of Mixed Lipid Bilayers with Sphingomyelin, Glycerophospholipids, and Cholesterol.

The highly diversified composition of lipid bilayers across living cells is crucial for many biological processes. Lipid bilayers mainly consist of phosphatidylcholines (PC), phosphatidylethanolamines (PE), sphingomyelin (SM), and cholesterol, with eukaryotic membranes containing high percentage of sphingomyelin and cholesterol. In this study, we have modeled bilayers with different concentration of PC, PE, and SM to understand the changes in bilayer properties with varied SM concentrations. In addition, membrane models with 33% cholesterol have been simulated to understand the influence of cholesterol. To quantitatively access the structure and dynamics of membranes, deuterium order parameters (SCD), mass density profiles, lipid relaxation times, clustering analysis, and radial distribution functions are calculated. The SCDs compare favorably with past NMR experiments and increase with an increase in SM content. The surface area calculations showed that on addition of 50% palmitoyl-SM (PSM) surface area decreases (60.0 ± 0.6 Å2) from that of pure POPC (64.7 Å2), which is further lowered in the presence of cholesterol (44.4 ± 0.2 Å2). The lipid axial relaxation time decreases with increase in concentration of glycerophospholipids. The accuracy of these lipid membranes allows for future studies with more complex lipid mixtures containing SM to represent the diversity of lipids in natural membranes.

Lipidomics and RNA-Seq Study of Lipid Regulation in Aphis gossypii parasitized by Lysiphlebia japonica

The cotton–melon aphid, Aphis gossypii Glover, is a major insect pest worldwide. Lysiphlebia japonica (Ashmead) is an obligate parasitic wasp of A. gossypii, and has the ability to regulate lipid metabolism of the cotton-melon aphid. Lipids are known to play critical roles in energy homeostasis, membrane structure, and signaling. However, the parasitoid genes that regulate fat metabolism and lipid composition in aphids are not known. 34 glycerolipids and 248 glycerophospholipids were identified in this study. We have shown that a 3-day parasitism of aphids can induce significant changes in the content and acyl chain composition of triacylglycerols (TAGs) and subspecies composition of glycerophospholipids content and acyl chains. It also upregulate the expression of several genes involved in triacylglycerol synthesis and glycerophospholipid metabolism. Pathway analysis showed that a higher expression of genes involved in the tricarboxylic acid cycle and glycolysis pathways may contribute to TAGs synthesis in parasitized aphids. Interestingly, the higher expression of genes in the sphingomyelin pathway and reduced sphingomyelin content may be related to the reproductive ability of A. gossypii. We provide a comprehensive resource describing the molecular signature of parasitized A. gossypii particularly the changes associated with the lipid metabolism and discuss the biological and ecological significance of this change.

Membrane permeation of arginine-rich cell-penetrating peptides independent of transmembrane potential as a function of lipid composition and membrane fluidity

Cell-penetrating peptides (CPPs) are prominent delivery vehicles to confer cellular entry of (bio-) macromolecules. Internalization efficiency and uptake mechanism depend, next to the type of CPP and cargo, also on cell type. Direct penetration of the plasma membrane is the preferred route of entry as this circumvents endolysosomal sequestration. However, the molecular parameters underlying this import mechanism are still poorly defined. Here, we make use of the frequently used HeLa and HEK cell lines to address the role of lipid composition and membrane potential. In HeLa cells, at low concentrations, the CPP nona-arginine (R9) enters cells by endocytosis. Direct membrane penetration occurs only at high peptide concentrations through a mechanism involving activation of sphingomyelinase which converts sphingomyelin into ceramide. In HEK cells, by comparison, R9 enters the cytoplasm through direct membrane permeation already at low concentrations. This direct permeation is strongly reduced at room temperature and upon cholesterol depletion, indicating a complex dependence on membrane fluidity and microdomain organisation. Lipidomic analyses show that in comparison to HeLa cells HEK cells have an endogenously low sphingomyelin content. Interestingly, direct permeation in HEK cells and also in HeLa cells treated with exogenous sphingomyelinase is independent of membrane potential. Membrane potential is only required for induction of sphingomyelinase-dependent uptake which is then associated with a strong hyperpolarization of membrane potential as shown by whole-cell patch clamp recordings. Next to providing new insights into the interplay of membrane composition and direct permeation, these results also refute the long-standing paradigm that transmembrane potential is a driving force for CPP uptake.

Cytochrome P450 1A2 N‐terminal Site Directed Mutagenesis: Effects on Lipid Microdomain Localization

The protein components of the cytochrome P450 monooxygenase system consist of cytochromes P450 (CYPs), NADPH‐dependent cytochrome P450 reductase (CPR), and cytochrome b5. They form functional complexes that catalyze the oxidation of exogenous and endogenous molecules, rendering them more water soluble. In eukaryotes, these membrane bound proteins are found in many tissues and are located primarily in the endoplasmic reticulum. The lipid milieu into which the P450 system proteins reside is heterogenous, consisting of ordered and disordered domains. Ordered domains have a higher content of saturated fatty acids, cholesterol, and sphingomyelin than disordered domains, whereas disordered domains have a higher content of phospholipids with more unsaturated fatty acid chains. When a bilayer membrane is treated with non‐ionic detergent such as Brij 98, the disordered domains readily solubilize whereas ordered regions remain intact. Our lab has shown that various P450 system proteins reside in different lipid microdomains. For example, despite their high level of amino acid sequence similarity, CYP1A2 and CYP1A1 reside in ordered and disordered regions, respectively. Furthermore, our lab has shown that the N‐terminal amino acid sequences of CYP1A2 and CYP1A1 contribute to their specific localization in the ER membrane. The goal of this study is to use site directed mutagenesis to identify which N‐terminal amino acids may be responsible in the lipid raft localization of CYP1A2 and how this effects CYP1A2 enzymatic activity. CYP1A2 N‐terminal amino acids were mutated to their CYP1A1 counterparts and expressed in HEK293‐T cells, and their microdomain localization determined. Although substitution of the NH2‐terminal amino acids of CYP1A2 with that of CYP1A1 leads to changes in their microdomain localization, the results show that Serine4Aspartic Acid and Proline5Phenylalanine mutations alone do not alter microdomain localization of the CYP1A proteins.Support or Funding InformationThis work was supported, in whole or in part, by National Institutes of Health, NIEHS, United StatesPublic Health Service Grants ES004344, and ES013648.

Using precursor ion scan of 184 with liquid chromatography-electrospray ionization-tandem mass spectrometry for concentration normalization in cellular lipidomic studies

Cellular lipidomic studies have been favored approaches in many biomedical research areas. To provide fair comparisons of the studied cells, it is essential to perform normalization of the determined concentration before lipidomic analysis. This study proposed a cellular lipidomic normalization method by measuring the phosphatidylcholine (PC) and sphingomyelin (SM) contents in cell extracts. To provide efficient analysis of PC and SM in cell extracts, flow injection analysis-electrospray ionization-tandem mass spectrometry (FIA-ESI-MS/MS) with a precursor ion scan (PIS) of m/z 184 was used, and the parameters affecting the performance of the method were optimized. Good linearity could be observed between the cell extract dilution factor and the reciprocal of the total ion chromatogram (TIC) area in the PIS of m/z 184 within the dilution range of 1- to 16-fold (R2 = 0.998). The calibration curve could be used for concentration adjustment of the unknown concentration of a cell extract. The intraday and intermediate precisions were below 10%. The accuracy ranged from 93.0% to 105.6%. The performance of the new normalization method was evaluated using different numbers of HCT-116 cells. Sphingosine, ceramide (d18:1/18:0), SM (d18:1/18:0) and PC (16:1/18:0) were selected as the representative test lipid species, and the results showed that the peak areas of each lipid species obtained from different cell numbers were within a 20% variation after normalization. Finally, the PIS of 184 normalization method was applied to study ischemia-induced neuron injury using oxygen and glucose deprivation (OGD) on primary neuronal cultured cells. Our results showed that the PIS of 184 normalization method is an efficient and effective approach for concentration normalization in cellular lipidomic studies.

The human plasma-metabolome: Reference values in 800 French healthy volunteers; impact of cholesterol, gender and age.

Metabolomic approaches are increasingly used to identify new disease biomarkers, yet normal values of many plasma metabolites remain poorly defined. The aim of this study was to define the “normal” metabolome in healthy volunteers. We included 800 French volunteers aged between 18 and 86, equally distributed according to sex, free of any medication and considered healthy on the basis of their medical history, clinical examination and standard laboratory tests. We quantified 185 plasma metabolites, including amino acids, biogenic amines, acylcarnitines, phosphatidylcholines, sphingomyelins and hexose, using tandem mass spectrometry with the Biocrates AbsoluteIDQ p180 kit. Principal components analysis was applied to identify the main factors responsible for metabolome variability and orthogonal projection to latent structures analysis was employed to confirm the observed patterns and identify pattern-related metabolites. We established a plasma metabolite reference dataset for 144/185 metabolites. Total blood cholesterol, gender and age were identified as the principal factors explaining metabolome variability. High total blood cholesterol levels were associated with higher plasma sphingomyelins and phosphatidylcholines concentrations. Compared to women, men had higher concentrations of creatinine, branched-chain amino acids and lysophosphatidylcholines, and lower concentrations of sphingomyelins and phosphatidylcholines. Elderly healthy subjects had higher sphingomyelins and phosphatidylcholines plasma levels than young subjects. We established reference human metabolome values in a large and well-defined population of French healthy volunteers. This study provides an essential baseline for defining the “normal” metabolome and its main sources of variation.

Novel Interconnections in Lipid Metabolism Revealed by Overexpression of Sphingomyelin Synthase-1

This study investigates the consequences of elevating sphingomyelin synthase 1 (SMS1) activity, which generates the main mammalian sphingolipid, sphingomyelin. HepG2 cells stably transfected with SMS1 (HepG2-SMS1) exhibit elevated enzyme activity in vitro and increased sphingomyelin content (mainly C22:0- and C24:0-sphingomyelin) but lower hexosylceramide (Hex-Cer) levels. HepG2-SMS1 cells have fewer triacylglycerols than controls but similar diacylglycerol acyltransferase activity, triacylglycerol secretion, and mitochondrial function. Treatment with 1 mm palmitate increases de novo ceramide synthesis in both cell lines to a similar degree, causing accumulation of C16:0-ceramide (and some C18:0-, C20:0-, and C22:0-ceramides) as well as C16:0- and C18:0-Hex-Cers. In these experiments, the palmitic acid is delivered as a complex with delipidated BSA (2:1, mol/mol) and does not induce significant lipotoxicity. Based on precursor labeling, the flux through SM synthase also increases, which is exacerbated in HepG2-SMS1 cells. In contrast, palmitate-induced lipid droplet formation is significantly reduced in HepG2-SMS1 cells. [14C]Choline and [3H]palmitate tracking shows that SMS1 overexpression apparently affects the partitioning of palmitate-enriched diacylglycerol between the phosphatidylcholine and triacylglycerol pathways, to the benefit of the former. Furthermore, triacylglycerols from HepG2-SMS1 cells are enriched in polyunsaturated fatty acids, which is indicative of active remodeling. Together, these results delineate novel metabolic interactions between glycerolipids and sphingolipids.

Prepartal overfeeding alters the lipidomic profiles in the liver and the adipose tissue of transition dairy cows

Physiological adaptations in the energy metabolism of dairy cows during the periparturient period are partly mediated by insulin resistance (IR), which may subsequently induce metabolic disorders postpartum. The molecular mechanisms underlying IR in dairy cows are largely unknown. This study aimed to find a novel insight into the molecular mechanisms underlying IR in dairy cows during the periparturient period by analyzing the effects of prepartal overfeeding on the lipidomic profiles in the liver and adipose tissue (AT). Sixteen cows were allocated to controlled-energy and high-energy feeding groups. Lipidomic profiling was conducted on liver and adipose tissue samples collected at 8 days prior to the predicted parturition, and 1 day (only AT) and 9 days after the actual parturition. Five ceramides (Cers) were identified to be significantly increased by prepartal overfeeding in AT in the analysis of the variance between groups within time points. Principal component-linear discriminant analysis showed that lipidomic profiles between the feeding groups were mainly characterized by phosphatidylcholines (PC), phosphatidylethanolamines (PE), lysophophosphatidylcholines (LysoPC), and lysophosphatidylethanolamines (LysoPE) in the liver, and by Cer, PE, and phosphatidylinositols (PI) in AT. Lipid class levels indicated that prepartal overfeeding elevated the concentration of PE, PI, LysoPC, LysoPE, and sphingomyelin in the liver, and increased the concentration of Cer in AT during the periparturient period. Prepartal overfeeding significantly altered the concentrations of various sphingolipids, phospholipids, and lysophospholipids in the liver and AT of dairy cows during the periparturient period.

Sphingomyelin Content in Breast Milk and Infant Formula: A Nutrient That May Affect Neurodevelopment

Sphingomyelin Content in Breast Milk and Infant Formula: A Nutrient That May Affect Neurodevelopment

The Role of Sphingomyelin (SM) in Tissue Factor (TF) Encryption: ATP-Induced Activation of Acid Sphingomyelinase in Macrophages Decrypts Tissue Factor By Hydrolysis of SM in the Outer Leaflet

While tissue factor (TF)-mediated blood coagulation is essential for maintaining hemostasis, the aberrant activation of TF-mediated coagulation is a major determinant of thrombotic occlusions, the precipitating event in acute myocardial infarction, unstable angina, and ischemic stroke. Typically, TF on cell surfaces exists in inactive coagulant status (cryptic TF). Cell injury leads conversion of cryptic TF to coagulant active/prothrombotic TF. Molecular differences between cryptic and procoagulant TF and the mechanisms that are responsible for the conversion from one to the other form are poorly understood and often controversial. A majority of the evidence in the literature suggest that level of anionic phospholipids, such as phosphatidylserine (PS), in the outer leaflet of the plasma membrane plays a critical role in regulating TF procoagulant activity at the cell surface. However, other pathways, such as the thioredoxin system or thiol-disulfide exchange pathways involving protein-disulfide isomerase (PDI), were also shown to contribute to TF activation by inducing structural changes in TF. It is unknown at present whether TF on cell surfaces of naïve cells exists primarily in the cryptic state because of the limited availability of anionic phospholipids at the outer leaflet or phospholipids present in the outer leaflet play an active role in maintaining TF in the cryptic state. In the outer leaflet of mammalian plasma membrane, sphingomyelin (SM) constitutes up to 50% of the total phospholipids present on the cell surface. It is possible that a high SM content in the outer leaflet may be responsible for maintaining TF in its cryptic state at the cell surface in naïve cells, and the hydrolysis of SM on the outer leaflet mediated by factors released in cell injury contributes to TF activation. The present study was carried out to investigate this possibility.First, we tested the potential effect of SM on TF activity in a reconstituted system in which full-length TF was reconstituted into phospholipid vesicles composed of varying molar concentrations of SM with the remainder of the vesicle consisting of phosphatidylcholine (PC). SM, at 35 mol % or higher concentration in the proteoliposome, inhibited TF coagulant activity significantly as measured in factor X activation assay. Ceramide, having a similar sphingosine backbone as of SM, had no inhibitory effect on TF-FVIIa activation of FX. Measurement of FVIIa-TF amidolytic activity showed that SM does not inhibit the amidolytic activity of FVIIa-TF, indicating that SM neither affects FVIIa binding to TF nor TF-FVIIa cleavage of the small substrate peptide. SM also inhibited significantly TF activity of TF reconstituted in PC/PS (94%:6% mol/mol) vesicles. Next, human monocyte-derived macrophages (MDMs) were treated with varying concentrations of bacterial sphingomyelinase (b-SMase) to hydrolyze SM in the outer leaflet. b-SMase treatment increased cell surface TF activity in a dose-dependent manner. SMase treatment also enhanced the release of TF-bearing microparticles (MPs). SMase treatment had no significant effect on cell surface prothrombinase activity or annexin V binding to MDMs, indicating that b-SMase treatment did not increase PS availability at the cell surface under our experimental conditions. Similar to that observed in bone marrow-derived mouse macrophages, ATP (200 µM) stimulation of MDMs increased cell surface TF activity by about 3-fold and triggered the release of TF+ MPs. Immunofluorescence confocal microscopy revealed that ATP stimulation induced in the translocation of acid(a)-SMase from intracellular compartments to the outer leaflet of the plasma membrane. Treatment of MDMs with sphingomyelinase inhibitors, desipramine and imipramine (1 and 5 µM), or silencing a-SMase with siRNA markedly reduced the ATP-induced increased TF activity at the cell surface and TF+ MPs release. Finally, ATP stimulation was shown to increase the hydrolysis of SM in the outer leaflet of MDMs markedly. a-SMase inhibitors or silencing of a-SMase attenuated the ATP-induced SM hydrolysis. In summary, our data indicate that SM plays a critical role in maintaining TF in the cryptic state in resting cells. Activation/translocation of a-SMase to the outer leaflet following the activation of ATP receptor P2X7 leads to hydrolysis of SM and thus relieves the inhibitory effect of SM on TF, leading to TF decryption and the release of TF+ MPs. DisclosuresNo relevant conflicts of interest to declare.

Changes in the Sphingolipid Content in Tissues and Behavioral Modifications of Rats Subjected to Neurogenic Stress: Role of Sphingomyelinases

We studied changes in the level of ceramide and sphingomyelin (SM) in the neocortex and peripheral tissues of rats after chronic (7-day-long) neurogenic stress (sessions of nociceptive stimulation in a chamber with an electrified floor) and after such stress induced against the background of course (14 days) injections of the tricyclic antidepressant imipramine. The latter agent, as is known, is a functional inhibitor of acid sphingomyelinase (aSMase). Introductions of imipramine considerably prevented manifestations of a state similar to depression (depression-like state, DLS). Suppression of motor/research activity was weakened, manifestations of emotional phenomena (acts of defecation and urination) decreased, and the latency of leaving the center of the open field became shorter. Imipramine injected into stressed animals decreased the ceramide/SM ratio and the level of ceramide in the neocortex, liver, and blood serum; the concentration of SM in the blood serum increased somewhat, as compared with the corresponding indices in rats subjected to isolated action of stress. Such changes in the levels of sphingolipids were also manifested after direct action of imipramine on isolated slices of the neocortex of stressed rats. The obtained results are indicative of the important role of the level of activity of aSMase in alterations of the ceramide/SM ratio in the neocortex of rats and in changes in the corresponding modifications of their behavior. Levels of sphingolipids in the blood serum can be the important index of the efficacy of the action of antidepressants under conditions of chronic stress and after the development of the DLS.

Choline absorption and evaluation of bioavailability markers when supplementing choline to lactating dairy cows.

The metabolites of choline have a central role in many mammalian biological processes, and choline supplementation to the periparturient dairy cow improves hepatic lipid metabolism. However, variability in responses to choline supplementation has highlighted a lack of understanding of choline absorption in the lactating dairy cow. Our objective was to determine net choline absorption by measuring net portal fluxes of choline and choline metabolites in cows receiving either dietary supplements of rumen-protected choline (RPC) or abomasal delivery of choline (ADC). We also evaluated markers for choline bioavailability by examining relationships between net portal absorption of choline and choline metabolites in plasma and milk. Five late-lactation Holstein cows were used in a 5×5 Latin square design, with 5-d treatment periods and a 2-d interval between periods. Treatments were (1) control (0g/d of choline), (2) 12.5g/d of choline fed as RPC, (3) 25g/d of choline fed as RPC, (4) 12.5g/d of choline provided as ADC, and (5) 25g/d of choline provided as ADC. At the end of each 5-d period, milk was sampled and 9 blood samples were collected simultaneously from an artery and portal vein at 30-min intervals. Plasma, milk, and feed ingredient concentrations of acetylcholine, betaine, free choline, glycerophosphocholine, lysophosphatidylcholine, phosphatidylcholine, phosphocholine, and sphingomyelin were quantified by hydrophilic interaction liquid chromatography-tandem mass spectrometry. With an increasing dose of ADC, the net portal flux of free choline increased and regression analysis indicated 61% net absorption of the infused dose. Among the choline metabolites, only concentrations of betaine, free choline, and phosphocholine increased in both arterial plasma (3.9, 1.9, and 0.4 times, respectively) and milk (2.5, 1.4, and 1.0 times, respectively) with 25g/d of ADC relative to the control. For RPC, the net portal flux of free choline was low relative to ADC (13%), which was similar to the relative difference observed in the concentrations and yields of milk free choline and betaine (averaged 21%). When evaluating markers for choline bioavailability, betaine was the leading candidate. Betaine in plasma and milk (alone or in combination with phosphocholine) was strongly associated with net free choline portal flux (coefficient of determination ranging from 0.64 to 0.79). In summary, free choline supply to the lactating dairy cow increases only specific choline metabolites in plasma and milk, which can be potential markers for choline bioavailability.

Effects of Chronic Neurogenic Stress on Behavior of Rats and Contents of Sphingolipids in Their Brain and Peripheral Tissues

We studied the contents of ceramide and sphingomyelin (SM) in the hippocampus, neocortex, and peripheral tissues of rats and the behavior characteristics of these animals in the open field at different times after the induction of chronic neurogenic stress. The experiments were carried out on young Wistar rats weighing 180–220 g, with a mean level of behavioral mobility. Within the poststress period, animals demonstrated significant suppression of manifestations of motor and orientational/research activities, and intensification of autonomic manifestations of fear and anxiety (with a delay of leaving the field center). One day after termination of stressing, the content of ceramide in the hippocampus increased against the background of a drop in the SM level, but this did not occur in the neocortex, liver, and blood serum. The contents of phosphatidylcholine and phosphatidylethanolamine in the brains of animals exposed to stress did not change. Eight days after stressing, the level of ceramide in the brain structures and blood serum was significantly higher than in the control. These data suggest that the action of chronic neurogenic stress leads to activation of metabolism of sphingolipids and intensification of production of ceramide in the brain and blood serum. Accumulation of ceramide in the serum within a remote period after termination of the action of stress can serve as a marker of the stable development of a state of emotional tension accompanied by typical signs of depression-like behavior.

Sphingomyelin content of erythrocyte membranes is in linear association with membranes' loading with cholesterol in coronary artery disease patients

Sphingomyelin content of erythrocyte membranes is in linear association with membranes' loading with cholesterol in coronary artery disease patients

Lipids of the liver microsomal fraction in the ground squirrel Spermophilus undulatus during hibernation

Winter sleep of the ground squirrel Spermophilus undulatus was accompanied by a 20% decrease in phospholipid content (µg phospholipid per 1 mg protein) in microsomal fractions of the liver as compared with summer-active squirrels. The phosphatidylcholine level (mol %) in hibernating squirrels was lower than in summer-active squirrels, and the content of sphingomyelin (mol %) during the torpor bout was higher than in winter- and summer-active squirrels. The cholesterol, fatty acid, monoglyceride, and diglyceride levels in the microsomal fraction of the liver were elevated during hibernation. Pronounced seasonal changes in the lipid/protein ratio implicate the lipids of the liver microsomal fraction in adaptation of the ground squirrel to hibernation.

Pharmacological Activation of Peroxisome Proliferator-Activated Receptor {Delta} Increases Sphingomyelin Synthase Activity in THP-1 Macrophage-Derived Foam Cell.

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors, which mediate glucose and lipid homeostasis by regulating the expression of a large number of transcription factors. Sphingomyelin synthase (SMS) is a key enzyme in the synthesis of sphingomyelin (SM), and its expression and activity have been reported to be associated with atherosclerosis (AS). Although there have been many functional PPAR and SMS studies on atherosclerosis in recent years, few have investigated the correlation between the activation of PPARδ and the activity of SMS. In his study, macrophage-induced foam cells were utilized to model important pathological changes that occur in AS. The influence of PPARδ agonism by GW501516 on SMS and its product molecule SM were measured. Results indicated that the activation of PPARδ was correlated in a positive manner with the activity of SMS2, and the content of SM was dose dependently increased by GW501516. Together, this study represents the first to suggest that PPARδ activation may be a potential risk of AS through enhancing activity of SMS2.

Comparative lipidomics and proteomics analysis of platelet lipid rafts using different detergents

Lipid rafts play a pivotal role in physiological functions of platelets. Their isolation using nonionic mild detergents is considered as the gold standard method, but there is no consensual detergent for lipid raft studies. We aimed to investigate which detergent is the most suitable for lipid raft isolation from platelet membrane, based on lipidomics and proteomics analysis.Platelets were obtained from healthy donors. Twelve sucrose fractions were extracted by three different detergents, namely Brij 35, Lubrol WX, and Triton X100, at 0.05% and 1%. After lipidomics analysis and determination of fractions enriched in cholesterol (Ch) and sphingomyelin (SM), proteomics analysis was performed.Lipid rafts were mainly observed in 1–4 fractions, and non-rafts were distributed on 5–12 fractions. Considering the concentration of Ch and SM, Lubrol WX 1% and Triton X100 1% were more suitable detergents as they were able to isolate lipid raft fractions that were more enriched than non-raft fractions. By proteomics analysis, overall, 822 proteins were identified in platelet membrane. Lipid raft fractions isolated with Lubrol WX 0.05% and Triton X100 1% contained mainly plasma membrane proteins. However, only Lubrol WX 0.05 and 1% and Triton X100 1% were able to extract non-denaturing proteins with more than 10 transmembrane domains.Our results suggest that Triton X100 1% is the most suitable detergent for global lipid and protein studies on platelet plasma membrane. However, the detergent should be adapted if investigation of an association between specific proteins and lipid rafts is planned.

Evolution of phospholipid contents during the production of quark cheese from buttermilk

We report the evolution of phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylcholine (PC), phosphatidylserine (PS), and sphingomyelin (SM) contents during the production of quark cheese from buttermilk by successive ultrafiltration concentration, enrichment with cream, concurrent homogenization and pasteurization, fermentative coagulation, and separation of quark from whey by further ultrafiltration. Buttermilk is richer than milk itself in phospholipids that afford desirable functional and technological properties, and is widely used in dairy products. To investigate how phospholipid content is affected by end-product production processes such as ultrafiltration, homogenization, pasteurization or coagulation, we measured the phospholipids at several stages of each of 5 industrial-scale quark cheese production runs. In each run, 10,000L of buttermilk was concentrated to half volume by ultrafiltration, enriched with cream, homogenized, pasteurized, inoculated with lactic acid bacteria, incubated to coagulation, and once more concentrated to half volume by ultrafiltration. Phospholipid contents were determined by HPLC with evaporative light scattering detection in the starting buttermilk, concentrated buttermilk, ultrafiltrate, cream-enriched concentrated buttermilk (both before and after concurrent homogenization and pasteurization), coagulate, and quark, and also in the rinsings obtained when the ultrafiltration equipment was washed following initial concentration. The average phospholipid content of buttermilk was approximately 5 times that of milk, and the phospholipid content of buttermilk fat 26 to 29 times that of milk fat. Although phospholipids did not cross ultrafiltration membranes, significant losses occurred during ultrafiltration (due to retention on the membranes) and during the homogenization and pasteurization process. During coagulation, however, phospholipid content rose, presumably as a consequence of the proliferation of the inoculated lactic acid bacteria. In spite of these changes in total phospholipid content, the relative proportions of the phospholipids studied remain fairly stable throughout quark production (PE>PC>SM>PS>PI) and similar to those found in the milk of the region, except that SM content was lower than in milk.

LONG–TERM FOOD RESTRICTION PREVENTS THE AGE-RELATED CHANGES OF THE CONTENT OF BIOLOGYCALLY ACTIVE SPHINGO- AND GLYCEROLIPIDS СONTENS IN THE RAT TISSUES.

Age peculiarities of the calorie-restricted diet effects on the contents biologically active sphingo- and glycerolipids were studied in the heart, liver and brain of 3- and 24-month-old rats. Rats were either kept on the ad libitum diet or on a calorie restricted diet (70-80% reduction in total calories) without reduction in essential nutrients. It has been determined that calorie restricted diet decreased the ceramide, sphingomyelin, cardiolipin and phosphatidic acid levels in the all investigated tissues of the rats. At the same time, calorie restriction diet prevented the age-induced ceramide and phosphatidic acid accumulation, ceramide/sphingomyelin ratio elevation, and sphingomyelin and cardiolipin content decrease in the tissues of 24-month-old rats. In addition, tissue specificity of calorierestricted diet effects has been determined. The Elevated levels of cardiolipin and phosphatidic acid were determined in the heart and liver of 24 months-old rats under calorie-restricted diet, in comparison to control animals, whereas in the brain the caloric restriction diet had the opposite effects. These results suggest that calorie-restricted diet may prevent the development of age-associated pathologies due to the modulation of biologically active lipid turnover in the old tissues.

Phospholipid analysis in sera of horses with allergic dermatitis and in matched healthy controls.

BackgroundLipids have become an important target for searching new biomarkers typical of different autoimmune and allergic diseases. The most common allergic dermatitis of the horse is related to stings of insects and is known as insect bite hypersensitivity (IBH) or summer eczema, referring to its recurrence during the summer months. This intense pruritus has certain similarities with atopic dermatitis of humans. The treatment of IBH is difficult and therefore new strategies for therapy are needed. Autoserum therapy based on the use of serum phospholipids has recently been introduced for horses. So far, serum lipids relating to these allergic disorders have been poorly determined. The main aim of this study was to analyse phospholipid profiles in the sera of horses with allergic dermatitis and in their healthy controls and to further assess whether these lipid profiles change according to the clinical status after therapy.MethodsSera were collected from 10 horses with allergic dermatitis and from 10 matched healthy controls both before and 4 weeks after the therapy of the affected horses. Eczema horses were treated with an autogenous preparation made from a horse’s own serum and used for oral medication. Samples were analysed for their phospholipid content by liquid chromatography coupled to a triple-quadrupole mass spectrometer (LC-MS). Data of phospholipid concentrations between the groups and over the time were analysed by using the Friedman test. Correlations between the change of concentrations and the clinical status were assessed by Spearman’s rank correlation test.ResultsThe major phospholipid classes detected were phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylinositol (PI) and phosphatidylethanolamine (PE). Eczema horses had significantly lower total concentrations of PC (p < 0.0001) and SM (p = 0.0115) than their healthy controls. After a 4-week therapy, no significant differences were found between the groups. Changes in SM concentrations correlated significantly with alterations in clinical signs (p = 0.0047).ConclusionsHorses with allergic dermatitis have an altered phospholipid profile in their sera as compared with healthy horses and these profiles seem to change according to their clinical status. Sphingomyelin seems to have an active role in the course of equine insect bite hypersensitivity.