Differences in phosphatidylcholine profiles and identification of characteristic phosphatidylcholine molecules in meat animal species and meat cut locations.

Phosphatidylcholine (PC) is an essential component of the plasma membrane. Its profile varies with species and tissues. However, the PC profiles in meat have not been explored in depth. This study aimed to investigate the differences in PC profiles between various meat animal species and meat cut sites, along with the identification of characteristic PC molecules. The results demonstrated that the PC profiles of chicken meat differed from those of other species. Significant differences were also observed between the PC profiles of pork meat and the meat obtained from other species. The amount of PCs containing ether bonds was high in pork meat. PCs containing an odd number of carbon atoms were characteristic of beef and lamb meats. Furthermore, PC profiles differed based on the muscle location in chicken and pork. These results suggest that the PC profiles of skeletal muscles are indicators of animal species and muscle location.

Serum lipids profiling perturbances in patients with ischemic heart disease and ischemic cardiomyopathy.

BACKGROUND: Ischemic heart disease (IHD) is a common cardiovascular disorder associated with inadequate blood supply to the myocardium. Chronic coronary ischemia leads to ischemic cardiomyopathy (ICM). Despite their rising prevalence and morbidity, few studies have discussed the lipids alterations in these patients. METHODS: In this cross-sectional study, we analyzed serum lipids profile in IHD and ICM patients using a lipidomics approach. Consecutive consenting patients admitted to the hospital for IHD and ICM were enrolled. Serum samples were obtained after overnight fasting. Non-targeted metabolomics was applied to demonstrate lipids metabolic profile in control, IHD and ICM patients. RESULTS: A total of 63 and 62 lipids were detected in negative and positive ion mode respectively. Among them, 16:0 Lyso PI, 18:1 Lyso PI in negative ion mode, and 19:0 Lyso PC, 12:0 SM d18:1/12:0, 15:0 Lyso PC, 17:0 PC, 18:1-18:0 PC in positive ion mode were significantly altered both in IHD and ICM as compared to control. 13:0 Lyso PI, 18:0 Lyso PI, 16:0 PE, 14:0 PC DMPC, 16:0 ceramide, 18:0 ceramide in negative ion mode, and 17:0 PE, 19:0 PC, 14:0 Lyso PC, 20:0 Lyso PC, 18:0 PC DSPC, 18:0-22:6 PC in positive ion mode were significantly altered only in ICM as compared to IHD and control. CONCLUSION: Using non-targeted lipidomics profiling, we have successfully identified a group of circulating lipids that were significantly altered in IHD and ICM. The lipids metabolic signatures shed light on potential new biomarkers and therapeutics for preventing and treating ICM.

Plasma metabolomic profiling of amino acids and polar lipids in Iranian obese adults.

BACKGROUND: Obesity, widely recognized as a serious health concern, is characterized by profoundly altered metabolism. However, the intermediate metabolites involved in this change remain largely unknown. OBJECTIVE: We conducted targeted metabolomics profiling to identify moieties associated with adult obesity. METHODS: In this case-control study of Iranian adults, 200 obese patients were compared with 100 controls based on 104 metabolites profiled by a targeted metabolomic approach using liquid chromatography coupled to triple quadrupole mass spectrometry (LC-MS/MS). The analysis comprised acylcarnitines, diacyl-phosphatidylcholines (PCaa), acyl-alkyl-phosphatidylcholines (PCae), sphingomyelins (SM), lyso-phospholipids (LPC) and amino acids. We performed multivariable linear regression to identify metabolites associated with obesity, adjusting for age, sex, total energy intake, total physical activity, smoking, and alcohol consumption. The Bonferroni correction was used to adjust for multiple testing. RESULTS: A pattern of 19 metabolites was significantly associated with obesity. Branched chain amino acids, alanine, glutamic acid, proline, tyrosine LPCa C16:1, PCaa C32:1, PCaa C32:2 and PCaa C38:3 were positively, while serine, asparagine, LPCa C18:1, LPCa C18:2, LPCe C18:0, PCae C34:3, PCae C38:4 and PCae C40:6 were negatively associated with obesity (all p < 0.00048). CONCLUSIONS: A metabolomic profile containing 9 amino acids and 10 polar lipids may serve as a potential biomarker of adult obesity. Further studies are warranted to replicate these findings as well as investigate potential changes in this profile after weight reduction.

Trimethylation Enhancement Using (13)C-Diazomethane ((13)C-TrEnDi): Increased Sensitivity and Selectivity of Phosphatidylethanolamine, Phosphatidylcholine, and Phosphatidylserine Lipids Derived from Complex Biological Samples.

Significant sensitivity enhancements in the tandem mass spectrometry-based analysis of complex mixtures of several phospholipid classes has been achieved via (13)C-TrEnDi. (13)C-TrEnDi-modified phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylcholine (PC) lipids extracted from HeLa cells demonstrated greater sensitivity via precursor ion scans (PISs) than their unmodified counterparts. Sphingomyelin (SM) species exhibited neither an increased nor decreased sensitivity following modification. The use of isotopically labeled diazomethane enabled the distinction of modified PE and modified PC species that would yield isobaric species with unlabeled diazomethane. (13)C-TrEnDi created a PE-exclusive PIS of m/z 202.1, two PS-exclusive PISs of m/z 148.1 and m/z 261.1, and a PIS of m/z 199.1 for PC species (observed at odd m/z values) and SM species (observed at even m/z values). The standardized average area increase after TrEnDi modification was 10.72-fold for PE species, 2.36-fold for PC, and 1.05-fold for SM species. The sensitivity increase of PS species was not quantifiable, as there were no unmodified PS species identified prior to derivatization. (13)C-TrEnDi allowed for the identification of 4 PE and 7 PS species as well as the identification and quantitation of an additional 4 PE and 4 PS species that were below the limit of detection (LoD) prior to modification. (13)C-TrEnDi also pushed 24 PE and 6 PC lipids over the limit of quantitation (LoQ) that prior to modification were above the LoD only.

Improved specificity of serum phosphatidylcholine detection based on side-chain losses during negative electrospray ionization tandem mass spectrometry.

Many current tandem mass spectrometry (MS) methods for measuring phosphatidylcholines (PtdChos) rely only on precursor ion scanning of the common 184 m/z phosphocholine fragment with positive electrospray ionization (+ESI), and thus measure pools of PtdChos rather than specific isoforms. In this paper, we developed and compared an isotope dilution, tandem MS method capable of quantifying PtdChos based on specific fatty acid side-chains to the traditional 184 m/z method. The method is based on the detection of PtdCho ammonium formate (AmF) adduct as parent ions and fatty acid fragment daughter ions under negative electrospray ionization (-ESI). Accuracy, imprecision, and recovery were below 15 %, with acceptable linearity (R 2 > 0.99) up to 5 µg/mL. We used the method to analyze the distributions of PtdChos with common side-chain combinations among 60 subjects and showed that it was possible for two individuals to have the same PtdCho pool concentration based on detection of the 184 m/z fragment, but up to a fourfold difference in the levels of specific isoforms comprising the pool based on our method. We then compared the results of both methods across 572 patients with mild cognitive impairment (MCI), Alzheimer's disease (AD), or no impairment (NI), which showed that statistically significant associations between specific PtdCho isoforms and AD were masked with the 184 m/z method. Our findings demonstrate the importance of isoform specificity for quantifying PtdChos, and suggest caution when interpreting analytical data based on pools of biomarkers.

Lipidomic analysis of local inflammation models shows a specific systemic acute phase response to lipopolysaccharides.

Toll-like receptors (TLR) are crucial for recognizing bacterial, viral or fungal pathogens and to orchestrate the appropriate immune response. The widely expressed TLR2 and TLR4 differentially recognize various pathogens to initiate partly overlapping immune cascades. To better understand the physiological consequences of both immune responses, we performed comparative lipidomic analyses of local paw inflammation in mice induced by the TLR2 and TLR4 agonists, zymosan and lipopolysaccharide (LPS), respectively, which are commonly used in models for inflammation and inflammatory pain. Doses for both agonists were chosen to cause mechanical hypersensitivity with identical strength and duration. Lipidomic analysis showed 5 h after LPS or zymosan injection in both models an increase of ether-phosphatidylcholines (PC O) and their corresponding lyso species with additional lipids being increased only in response to LPS. However, zymosan induced stronger immune cell recruitment and edema formation as compared to LPS. Importantly, only in LPS-induced inflammation the lipid profile in the contralateral paw was altered. Fittingly, the plasma level of various cytokines and chemokines, including IL-1ß and IL-6, were significantly increased only in LPS-treated mice. Accordingly LPS induced distinct changes in the lipid profiles of ipsilateral and contralateral paws. Here, oxydized fatty acids, phosphatidylcholines and phosphatidylethanolamines were uniquely upregulated on the contralateral side. Thus, both models cause increased levels of PC O and lyso-PC O lipids at the site of inflammation pointing at a common role in inflammation. Also, LPS initiates systemic changes, which can be detected by changes in the lipid profiles.

Evaluation of air oxidized PAPC: A multi laboratory study by LC-MS/MS.

Oxidized LDL (oxLDL) has been shown to play a crucial role in the onset and development of cardiovascular disorders. The study of oxLDL, as an initiator of inflammatory cascades, led to the discovery of a variety of oxidized phospholipids (oxPLs) responsible for pro-inflammatory actions. Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) is frequently used by the scientific community as a representative oxPL mixture to study the biological effects of oxidized lipids, due to the high abundance of PAPC in human tissues and the biological activities of oxidized arachidonic acids derivatives. Most studies focusing on oxPAPC effects rely on in-house prepared mixtures of oxidized species obtained by exposing PAPC to air oxidation. Here, we described a multi-laboratory evaluation of the compounds in oxPAPC by LC-MS/MS, focusing on the identification and relative quantification of the lipid peroxidation products (LPPs) formed. PAPC was air-oxidized in four laboratories using the same protocol for 0, 48, and 72 h. It was possible to identify 55 different LPPs with unique elemental composition and characterize different structural isomeric species within these. The study showed good intra-sample reproducibility and similar qualitative patterns of oxidation, as the most abundant LPPs were essentially the same between the four laboratories. However, there were substantial differences in the extent of oxidation, i.e. the amount of LPPs relative to unmodified PAPC, at specific time points. This shows the importance of characterizing air-oxidized PAPC preparations before using them for testing biological effects of oxidized lipids, and may explain some variability of effects reported in the literature.

Analysis of fragmented oxidized phosphatidylcholines in human plasma using mass spectrometry: Comparison with immune assays.

Circulating oxidized phospholipids are increasingly recognized as biomarkers of atherosclerosis. Clinical association studies have been mainly performed using an immune assay based on monoclonal antibody E06, which recognizes a variety of molecular species of oxidized phosphatidylcholine (OxPC) in lipoproteins, cell membranes or covalently bound to plasma proteins. Accumulating evidence shows that individual molecular species of OxPC demonstrate different biological activities and have different half-life times. Therefore, it is likely that certain molecular species can be associated with pathology more strongly than others. This hypothesis can only be tested using LC-MS/MS allowing quantification of individual molecular species of OxPCs. In order to ensure that laborious LC-MS/MS methods do not simply replicate the results of a technically simpler E06-OxPCs assay, we have performed relative quantification of 8 truncated molecular species of OxPCs in plasma of 132 probands and compared the data with the results of the E06-OxPCs and OxLDL assays. We have found a strong correlation between individual molecular species of OxPCs but only a weak correlation of LC-MS/MS-OxPCs data with the E06-OxPCs assay and no correlation with the OxLDL assay. Furthermore, in contrast to the results of E06-OxPCs or OxLDL assays, 7 out of 8 OxPC species were associated with hypertension. The data suggest that the results of the LC-MS/MS-OxPCs assay do not replicate the results of two ELISA-based lipid oxidation tests and therefore may produce additional diagnostic information. These findings necessitate development of simplified mass spectrometric procedures for high-throughput and affordable analysis of selected molecular species of OxPCs.

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.

Molecular species of phosphatidylcholine in familial lecithin-cholesterol acyltransferase deficiency: effect of enzyme supplementation.

We studied the molecular species composition of plasma phosphatidylcholine (PC) in three patients with familial deficiency of lecithin-cholesterol acyltransferase in order to determine whether certain species are increased in the absence of this enzyme activity. Compared to normal plasma, the deficient plasma contained significantly higher percentages of 16:0-18:2 and 18:0-18:2 species and lower percentages of 16:0-20:4 and 18:0-20:4 species. The bulk of the total plasma PC as well as the abnormal composition of molecular species were found in the very-low-density and low-density lipoprotein (VLDL + LDL) fractions. When the deficient plasma was incubated with partially purified enzyme from normal human plasma, there was a significant reduction in the amounts of most major species, mainly in the VLDL + LDL fraction. When the selectivity factors were calculated by dividing the percentage contribution of each species for cholesterol esterification by its percentage concentration, the highest selectivity factors were found for 16:0-18:2, 18:1-18:1 and 18:1-18:2. The order of selectivity of the enzyme for various species was very similar to that obtained earlier using normal HDL as substrate. These results show that lecithin-cholesterol acyltransferase has significant effects on the molecular species composition of plasma PC and the deficiency of the enzyme results in accumulation of certain PC species normally used by the enzyme, as well as in abnormal distribution of these species among the lipoproteins.

Effects of intravenously infused fish oil on platelet fatty acid phospholipid composition and on platelet function in postoperative trauma.

BACKGROUND: The aim of this study was to assess the short-term effect of IV infusion of fish oil emulsion on the fatty acid profiles of platelet phosphatidylcholine and phosphatidylethanolamine and on platelet function in postoperative patients. METHODS: Over a 7-day period, 10 patients received a 20% soybean fat emulsion with an added 10% marine fish oil emulsion, whereas 9 controls received only 20% soybean fat emulsion. RESULTS: By comparison with controls, in patients receiving fish oil, (1) a large increase in eicosapentaenoic acid (20:5n-3) was observed in both platelet phosphatidylcholine (1.55% +/- 0.17% vs 0.38% +/- 0.06% by weight, p < .01) and phosphatidylethanolamine 2.21% +/- 0.18% vs 0.66% +/- 0.08% by weight, p < .01); (2) eicosapentaenoic acid (20:5n-3)/arachidonic acid (20:4n-6) ratios doubled in both platelet phosphatidylcholine (p < .01) and phosphatidylethanolamine (p < .05); (3) with collagen as aggregating factor, maximal reaction speed decreased (p < .02) and latency increased (p < .002); and (4) no toxic effect, in particular no increase of postoperative bleeding and no perturbation of hepatic and renal function, was observed during the fish oil infusion. CONCLUSIONS: A short-term IV infusion of fish oil clearly modifies the platelet composition and changes some parameters of platelet function.

Identification of character impact odorants of different soybean lecithins.

The potent odorants of standardized, enzymatically hydrolyzed, and deoiled soybean lecithins were characterized systematically by combined gas chromatography/mass spectrometry and olfactometry. Sixty-one odorants were identified; 53 of these odor-active compounds have not previously been reported as odorants of soybean lecithin flavor. By aroma extract dilution analysis and modified combined hedonic and response measurement the following odorants showed the highest flavor dilution factors and CHARM values: (E,E)-2, 4-decadienal (deep-fried), (E)-beta-damascenone (apple-like), 2, 3-diethyl-5-methylpyrazine (roasty, earthy), (E)-2-nonenal (cardboard-like), trans-4,5-epoxy-(E)-2-decenal (metallic), 1-nonen-3-one (mushroom-like), 2-ethyl-3,5-dimethylpyrazine (roasty, earthy), and 1-octen-3-one (mushroom-like). Enzymatic hydrolysis intensified especially the roasty sensation of 2, 3-diethyl-5-methylpyrazine, whereas deoiling effected a general significant decrease in olfactory perception on the nitrogen-containing compounds. In addition, sensory profiles of nasal and retronasal lecithin odor were performed.

Alk-1-enylacyl, alkylacyl, and diacyl subclasses of native ethanolamine and choline glycerophospholipids can be quantified directly by phosphorus-31 NMR in solution.

We show that phosphorus-31 nuclear magnetic resonance spectroscopy can be used to distinguish and to quantify the alk-1-enylacyl, alkylacyl, and diacyl glycerophosphoethanolamine (GPE) subclasses, and the respective glycerophosphocholine (GPC) subclasses, in their native form without prior degradation or derivatization, provided the phospholipids are observed in the nonaggregated state. Monomeric phospholipid distribution is ascertained by recording the spectra, after removal of metal ions, on CDCl3/CD3OD/D2O (50:50:15, by vol) solutions. The utility of this approach is exemplified for the ethanolamine glycerophospholipids (EPL) from bovine brain and the choline glycerophospholipids (CPL) from bovine heart. Sharp and well-resolved resonances are obtained for alkylacylGPE (+0.395 ppm; re 1% H3PO4), alkenylacylGPE (+0.353 ppm), and diacylGPE (+0.315 ppm), and for alkylacylGPC (-0.383 ppm), alkenyl-acylGPC (-0.436 ppm) and diacylGPC (-0.451 ppm). Integrated peak areas are shown to closely correlate with dose. Accurate quantitation of EPL and CPL subclasses at submicromolar levels can further be facilitated by use of synthetic dialkylGPE (+0.602 ppm) and dialkylGPC (-0.196 ppm) as internal standards. The method is simple, rapid, sensitive and reproducible, and permits the complete resolution and direct quantitation of all ethanolamine and choline glycerophospholipid subclasses quite independent of fatty chain length and degree of unsaturation.

Rapid separation of the major phospholipid classes on a single aminopropyl cartridge.

A rapid method for the separation of the individual phospholipid classes phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI) by a single solid-phase extraction was developed. PC, PE, PS and PI were sequentially eluted from aminopropyl bonded silica with acetonitrile/n-propanol (2∶1, vol/vol), methanol, isopropanol/methanolic HCl (4∶1, vol/vol) and methanol/methanolic HCl (9∶1, vol/vol). Standard recoveries were over 95% for PC and PE and over 85% for PS and PI with undistorted fatty acid composition. The separation of complex lipid mixtures on aminopropyl minicolumns can be refined to the level of individual phospholipid classes.

Fatty acids of serine, ethanolamine, and choline plasmalogens in some marine bivalves.

The FA composition of glycerophospholipid (GPL) classes and subclasses was investigated in whole animals of three marine bivalve mollusks: the Japanese oyster Crassostrea gigas, the blue mussel Mytilus edulis, and the Manila clam Ruditapes philippinarum. Individual organs (gills, mantle, foot, siphon, and muscle) of the Manila clam also were examined. The PS plasmalogen (PSplsm), PE plasmalogen (PEplsm), and PC plasmalogen (PCplsm) subclasses were isolated by HPLC, and their individual FA compositions were examined using GC. Plasmalogen forms of PS and PE, when compared to their respective diacyl forms, were found to be specifically enriched with non-methylene-interrupted (NMI) FA (7,15-22:2, 7,13-22:2, and their precursors) and 20:1 n-11 FA. Such a clear specific association was not found for PCplsm. Interestingly, this trend was most apparent in PSplsm, and the above FA were found to be, respectively, the predominant PUFA and monounsaturated FA in the PSplsm isolated from the three species. This specificity was maintained in all the analyzed organs of the Manila clam but varied in proportions: The highest level of plasmalogens, NMI FA, and 20:1 n-11 was measured in gills and the lowest was in muscle. These results represent the first comprehensive report on a FA composition of the PSplsm subclass isolated from mollusks. The fact that NMI FA and 20:1 n-11, which are thought to be biosynthesized FA, were mainly associated with aminophospholipid plasmalogens (PE and PS) is likely to have a functional significance in bivalve membranes.

[Clinical pharmacology of hepatoprotectors]. / Klinichna farmakolohiia hepatoprotektoriv.

Data from published literature are summarized together with findings secured in the author's investigations on clinical pharmacology of hepatoprotectors. Classification of hepatoprotectors is submitted, their effects on the functional condition and metabolism in hepatocytes are discussed. Details are given of clinical and pharmacological properties of hepabene, legalon, essentiale, sirepara, wobenzime, phebichol, antral, thiotriazone.

Characterization and analysis of the subclasses and molecular species of choline phosphoglycerides from porcine heart by successive chemical hydrolyses and reverse phase high-performance liquid chromatography.

Choline phosphoglycerides (CPG) represent the major fraction of heart phospholipids. Since depletion of membrane phospholipids and accumulation of lyso-compounds, particularly lysophosphatidylcholines, have been implicated in arrhythmogenesis, it was of great interest to study the composition of this major phospholipid fraction of the heart at a molecular level in an established animal model. The data presented here describe the first report on the detailed chemical examination of CPG and resolution, characterization and quantitative analysis of the molecular species of this phospholipid fraction from porcine heart by high performance liquid chromatography (HPLC). This fraction constitutes 37.5 +/- 0.7% (n = 21) of the total phospholipids and upon successive mild acid and alkaline hydrolyses revealed the presence of essentially three subclasses: diacyl-, alkenylacyl-, and alkylacyl glycerophosphorylcholines, in a relative abundance of 57.7 +/- 2.2% (n = 8), 37.3 +/- 1.3% (n = 8) and 4.6 +/- 0.2% (n = 8), respectively. The fourth subclass, dialkyl CPG was found only in minute amounts (0.43 +/- 0.05%, n = 8) and the presence of dialkenyl and alkenylalkyl analogues could not be detected. Alternatively, by converting the CPG fraction to benzoate derivatives after phospholipase C digestion, it was possible to isolate and quantitate subclass composition by TLC/spectroscopy or both subclass compositions and molecular species analysis by HPLC directly by a UV detector online with the column. By these techniques, subclass composition was found to be very similar to that obtained by the chemical hydrolysis technique. By HPLC, up to 25 species can be identified and quantitated in each subclass, their identity being confirmed by gas-liquid chromatography, after their isolation from the column. The analyses showed that up to 74% of the diacyl moiety consisted of 16:0-18:2 (34%), 16:0-18:1 (27%), and 18:0-18:2 (13%) species, while the major species of the alkenylacyl moiety were 16:0-18:2 (44%) 16:0-18:1 (13%), 16:0-20:4 (12%) and 18:1-18:2 (9%) making up more than 75% of the total mass of this subclass. The major molecular species of the alkylacyl moiety was 16:0-18:2, constituting up to 47% of this fraction, while others constituted about 10% (16:0-18:1), 9% (18:1-18:2), 8% (16:0-20:4) and 6% (18:0-18:2), making up 80% of the total mass. The ether chain composition of akylacyl CPG whether determined after isolation of this fraction by the chemical hydrolysis technique or by HPLC was indistinguishable.(ABSTRACT TRUNCATED AT 400 WORDS)

Molecular species of phosphatidylcholine and phosphatidylethanolamine in subcellular membranes from rat liver.

Four subfractions of phosphatidycholine and phosphyatidylethanolamine according to the degree of unsaturation of their fatty acids have been separated from lipid extracts of microsomes, and inner and outer mitochondrial membranes. The predominant species found in the three membranes contained one saturated and one unsaturated fatty acid. In microsomes completely saturated species of both phosphatidylcholine and phosphatideylethanolamine were practically nonexistent. In outer mitochondrial membranes species with two unsaturated fatty acids were absent. In the inner mitochondrial membranes, however, disaturated species and those with two unsaturated fatty acids were found.