Liquid chromatography-tandem mass spectrometry for the determination of sphingomyelin species from calf brain, ox liver, egg yolk, and krill oil

In this study, molecular species of sphingomyelin (SM) in egg yolk, calf brain, ox liver, and krill oil were investigated. Classes of phospholipids (PLs) were purified, identified, and quantified by normal phase semipreparative high-performance liquid chromatography (HPLC) combined with evaporative light scattering detectors (ELSD). For SM molecular species identification, pure SM collected through a flow splitter was loaded to HPLC-electrospray ionization-tandem mass spectrometry (LC-ESI-MS(2)), with 100% methanol containing 5 mM ammonium formate as mobile phase. In addition to classes of PLs, the used approach allowed the determination of profiles of SM species in egg yolk, ox liver, and calf brain, whereas krill oil turned out not to contain any SM. It also allowed the separation and identification of SM subclasses, as well as tentative identification of species with the same molecular mass, including isomers. The results showed that egg yolk contained the highest proportion of (d18:1-16:0)SM (94.1%). The major SM molecular species in ox liver were (d18:1-16:0)SM (25.5%), (d18:1-23:0)SM (19.7%), (d18:1-24:0)SM (13.2%), and (d18:1-22:0)SM (12.5%). Calf brain SM was rich in species such as (d18:1-18:0)SM (40.7%), (d18:1-24:1)SM (17.1%), and (d18:1-20:0)SM (10.8%).     

南美白对虾空气油炸过程中脂质组学快检技术研究

为实时监测南美白对虾在空气油炸过程中脂质组学轮廓变化,本研究采用iKnife-REIMS联用技术、主成分分析(PCA)和正交偏最小二乘判别分析(OPLS-DA)探究了不同空气油炸温度(140、170、200℃,10 min)对南美白对虾肌肉组织脂质组成的影响。结果表明,经结构鉴定和相对含量测定,南美白对虾样品中共检出10种脂肪酸与31种磷脂分子,其中,亚油酸(m/z 279,21.88%)、EPA(m/z 301,16.59%)与DHA(m/z 327,15.14%)为主要脂肪酸离子;[PE 36:1-H]^-(m/z 744,20.16%)与[PE 38:5-H]^-/[PC O-36:5-H]^-(m/z 764,15.92%)为主要磷脂离子。随着油炸温度的升高,油炸南美白对虾中饱和脂肪酸与甘油磷脂酸(PA)分子的相对含量呈上升趋势,而不饱和脂肪酸、磷脂酰乙醇胺(PE)、磷脂酰胆碱(PC)与磷脂酰肌醇(PI)分子的相对含量不断减少。通过共享与特有化合物结构分析图(SUS-plot)确定了6个潜在标记物(m/z 277、m/z 770、m/z 810、m/z 818、m/z 844及m/z 836),可用于空气油炸样品的实时鉴别。经方法学验证,该iKnife-REIMS联用实时检测方法的灵敏度和精密度均可满足空气油炸过程中南美白对虾脂肪酸和磷脂的脂质组学轮廓分析测试要求。本研究结果为食品加工过程中脂质组学变化研究提供了新的检测技术手段。     

Lipid profiling by electrospray ionization tandem mass spectrometry and the identification of lipid phosphorylation by kinases in potato stolons

There is limited information about the involvement of lipids and esterified fatty acids in signaling pathways during plant development. The purpose of this study was to evaluate the lipid composition and molecular species of potato (Solanum tuberosum L., cv. Spunta) stolons and to identify phosphorylated lipids in the first two developmental stages of tuber formation. Lipid profiling was determined using ESI-MS/MS, a useful method for the determination of the biosynthesis and catabolism of lipids based on their fatty acid composition. The most prevalent compound identified in this study was phosphatidic acid (PA); digalactosyldiacylglycerol (DGDG) was the second most abundant compound. A 34:2 species was identified in PA, phosphatidylcholine (PC), phosphatidylinositol (PI), and phosphatidylethanolamine (PE). The identification of lipid phosphorylation by kinases was revealed by the presence of the phosphorylated lipids. PA was metabolized to diacylglycerol pyrophosphate (DGPP) by phosphatidic acid kinase (PAK). This work establishes a correlation between lipid fatty acid composition and lipid metabolism enzymes at the beginning of tuber formation and is the first report of PAK activity in the early events of potato tuber formation.     

Inorganic mercury interactions with lipid components of biological membranes:31P-NMR study of Hg(II) binding to headgroups of micellar phospholipids

Phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylcholine (PC) in micellar phase in water have been studied by high resolution phosphorus-31 nuclear magnetic resonance (³¹P-NMR), in order to follow inorganic mercury Hg(II) binding to the lipid headgroups. Decrease of the NMR peak area is observed upon HgCl₂ addition, with greater effect on PE and PS compared to PC. This is interpreted by Hg(II) binding to several phospholipid headgroups, linking different micelles together and forming by extension a large “insoluble” phospholipid-mercury network that is undetectable by high-resolution³¹P-NMR. The extent of phospholipid aggregation depends on the mercury-to-lipid molar ratio, and apparent Hg(II) affinities to phospholipid headgroups are in the order PE>PS>>PC. When HgCl₂ is added to mixed micelles prepared with two lipids (PE/PC or PS/PC), co-precipitation is observed for both components in similar proportions.     

Characterization of phospholipid molecular species in the edible parts of bony fish and shellfish

The phospholipid molecular species of freshwater (pangasius, Nile perch, trout), marine fish fillets (horse mackerel, European hake, common sole, European anchovy, European pilchard, Atlantic mackerel) and the edible muscle foot of bivalves (clam, mussel, oyster) commonly available in the Italian market during spring and summer were characterized by means of normal-phase high performance liquid chromatography coupled online with positive electrospray ionization ion-trap tandem mass spectrometry. From principal component analysis (PCA), it was observed that the total fatty acid profile was not suitable to differentiate among the shellfish genera. The fatty acid molecular combinations of phosphatidylcholine, the main phospholipid class, as well as phosphatidylinositol and phosphatidylethanolamine allowed for the differentiation of shellfish from the bony fishes. Phosphatidylserine and phosphatidylethanolamine plasmalogen profile allowed for the discrimination of each bony fish or shellfish genus since PS and pPE classes included a large number of fatty acid combinations that were specific for a fish genus or group.     

Changes produced in the antioxidative activity of phospholipids as a consequence of their oxidation

The antioxidative activities of native and oxidized soybean phosphatidylcholine (PC), phosphatidylthanolamine (PE), and phosphatidylinositol (PI) in the protection of soybean oil heated in the dark under air at 60 degrees C were studied in an attempt to clarify the consequences that phospholipid oxidation has on antioxidative activities. The three native phospholipids protected the oil when assayed at 200 ppm, and phospholipid oxidation decreased the antioxidative activity of both PC and PI. However, slightly oxidized PE was more antioxidative than native PE, most likely as a consequence of the formation by amino-carbonyl reactions of pyrrolized phospholipids, which were determined and for which antioxidative properties are known. Nevertheless, further increases in PE oxidation produced a decrease in its antioxidative activity. These results suggest that two opposite reactions are competing in the antioxidative activity of amino phospholipids upon oxidation: fatty acid chain oxidation, which decreases phospholipid antioxidative activity, and amino-carbonyl reactions, which produce derivatives with antioxidant properties. This last property may be useful to increase the antioxidative activity of commercial lecithins containing amino phospholipids.     

Quantitative relationship between trimethylamine oxide aldolase activity and formaldehyde accumulation in white muscle from gadiform fish during frozen storage

The accumulation of formaldehyde and the resulting deterioration of seafood products during frozen storage are primarily caused by the enzymatic activity of trimethylamine oxide aldolase (TMAOase). A screening of muscle samples from 24 species showed TMAOase activity in only the nine gadiform species that were analyzed. Enzyme activities in the major white muscle of gadiform fish showed large variations between species as well as between individuals. A frozen storage experiment showed a similarly large variation in the rate of formaldehyde accumulation, which could be accounted for by the endogenous white muscle in situ TMAOase activity. This TMAOase activity also correlated with the rate of insolubilization of otherwise high ionic strength soluble protein. A simple model describing the accumulation of free formaldehyde during frozen storage of gadiform fish is proposed. The model is based on a storage time-dependent decay of substrate-saturated white muscle TMAOase activity.     

Bioconcentration and elimination of sulfamethazine and its main metabolite in sturgeon (Acipenser schrenkii)

A steady-state bioconcentration and elimination of sulfamethazine (SM(2)) in the sturgeon (A. schrenkii) was conducted in flow-through aqueous conditions. Two treated groups of fish were exposed to concentrations of 1.00 and 0.10 mg/L of SM(2), respectively. SM(2) and its main metabolite, N(4)-acetyl-SM(2), were determined in both fish muscle and water during the 8-day uptake period and the subsequent 6-day elimination period. Rapid uptakes of the drug were observed in both treated groups. Muscle tissue residues plateaued after approximately 3 days. The bioconcentration factor in muscle (BCF(m)) in the low-concentration drug solution was 1.19 and that in the high-concentration-treated level was 0.61. The calculated biodegradation index was 3.72%. The elimination half-times (t(1/2)) of the two treatment levels were 19.44 and 23.52 h, respectively. The result indicates that SM(2) will neither bioconcentrate in individual aquatic organisms nor biomagnify in the food chain, although the BCF(m) was relatively higher under the low-concentration exposure.     

采用UHPLC-QTOF-MS技术筛选亚麻籽油脂质分子标志物

为了探究亚麻籽油中脂质的真实属性和发掘其有价值的脂质分子,该研究基于超高效液相色谱-四级杆飞行时间质谱联用技术（Ultra High Performance Liquid Chromatography-Quadrupole Time-of-Flight Spectroscopy, UHPLC-QTOF-MS）表征了亚麻籽油的脂质轮廓。结果表明：在正离子模式下检测到15个脂质分子小类共668种脂质分子;在负离子模式下检测到31个脂质分子小类共404种脂质分子;共有7个脂质分子小类脂质在正、负离子下均被检测到;甜菜碱脂、甘油糖脂、神经节苷脂、鞘磷脂、神经酰胺、羟基脂肪酸的脂肪酸酯、酰基肉碱和植物固醇在亚麻籽油首次在该研究中被发现,它们都具有特殊的生物学功能。多元变量统计分析结果表明每两个品种亚麻籽油间相对定量值差异显著的脂质分子均超过200种（P<0.05）,并筛选出6种甘油磷酸肌醇、1种甘油磷酸胆碱、1种甘油磷酸乙醇胺和1种三酰基甘油作为亚麻籽A油的标志性脂质分子;6种三酰基甘油和1种半单酰基甘油磷酸酯作为亚麻籽B油的标志性脂质分子;1种神经节苷脂和1种硫代异鼠李糖甘油二酯作为亚麻籽C油的标志性脂质分子。总之,该研究在亚麻籽油中鉴定出39个脂质分子小类共1 072种脂质分子,其中有22个脂质分子小类共415种脂质分子首次在亚麻籽油中被检测到,此外不同品种亚麻籽油在脂质分子层面存在显著差异（P<0.05）,这些脂质分子作为标志物,可用于植物油的品质判别、营养评价,真伪鉴别和安全性评价等,同时该研究也为其他植物油的分析提供方法参考。     

A rapid-test for screening biochar effects on seed germination

We developed a rapid-test to screen for effects of biochar on seed germination and soils. Crop seeds were placed in containers and covered with 15 g of soil with 1% biochar by weight. Two agricultural soils from South Carolina USA were used. Eighteen biochars were produced from six primary feedstocks [pine chips (PC), poultry litter (PL), swine solids (SS), switchgrass (SG); and two blends of PC and PL, 50% PC/50% PL (55), and 80% PC/20% PL (82)]. Each feedstock was pyrolyzed at 350, 500 and 700°C. There were few biochar effects on seed germination. Shoot dry weight was increased for carrot, cucumber, lettuce, oat, and tomato; primarily with biochars containing PL. Soil pH, electrical conductivity and extractable phosphorus primarily increased with PL, SS, 55, and 82 treatments for both soil types and across species. This method can be an early indicator of biochar effects on seed germination and soil health.     

Comparative evaluation of the emulsifying properties of phosphatidylcholine after enzymatic acyl modification

The ability of enzymatically synthesized structured phosphatidylcholine (PC) containing caprylic acid to form and stabilize oil-in-water emulsions prepared with different triglycerides [medium chain triglycerides (MCT), soybean oil, and enzymatically synthesized structured lipids] was examined and compared with natural soybean PC and deoiled lecithin. Emulsions were prepared with varying oil and emulsifier concentrations. The particle size distribution, creaming stability, and viscosity were measured for the evaluation of the emulsifying properties. With an increase in the oil concentration, there was an increase in particle size, viscosity, and creaming layer. With an increase in the phospholipid (PL) concentration, there was usually a decrease in particle size and an increase in viscosity, where the emulsion stability was increased. General emulsions prepared with structured lipids resulted in smaller particle sizes as compared to MCT and soybean oil. Deoiled lecithin was able to increase the viscosity more significantly and give smaller particle sizes as compared to the other emulsifiers, thus producing more stable emulsions. However, in certain cases, structured PC was superior to deoiled lecithin and soybean PC. This observation was made for emulsions prepared with soybean oil or structured lipid at an oil/water ratio of 10:90. At an oil/water ratio of 30:70, the deoiled lecithin performed better as compared to the other PLs with all oil types. However, structured PC produced more stable emulsions as compared to natural soybean PC in MCT and soybean oil.     

Stereospecific analysis of triacylglycerol and phospholipid fractions of five wild freshwater fish from Poyang Lake

The fatty acids (FA) compositions and positional distributions in triacylglycerols (TAG) and phospholipids (PL) of five wild freshwater fish (Squaliobarbus curriculus, Erythroculter ilishaeformis, Pseudobagrus fulvidraco, Bostrichthys sinensis, and Siniperca kneri Garman) from Poyang Lake (the largest freshwater lake of China) were studied. For TAG, S. kneri German had the highest content (13.59%) of n - 3 polyunsaturated fatty acids (PUFA) and E. ilishaeformis had the lowest ratio of (n - 6)/(n - 3) (0.65). PL had a high content of PUFA, which declined in the order of phosphatidylethanolamine (PE) > phosphatidylcholine (PC) > TAG. 9c11t-18:2 accounted for 6.38-50.77% of total conjugated linoleic acids (CLA). The highest level of odd-branched chain fatty acids (OBCFA) was 26.7% in B. sinensis. The study revealed that the distribution of FA among the sn positions was not random: monounsaturated fatty acids (MUFA) and PUFA preferred positions 1 and 3 and saturated fatty acids (SFA) position 2 of TAG, while SFA and MUFA predominated over sn-1-PL and PUFA over sn-2-PL.     

Collagen content in farmed Atlantic salmon (Salmo salar L.)

To clarify fish flesh quality problems and softening of fish muscle tissue during chilled storage, the collagen content, types I and V, and its changes in solubility during storage on ice in muscle of farmed Atlantic salmon (Salmo salar L.) were analyzed. The contents of acid-soluble, pepsin-soluble, and insoluble collagen in white muscle were determined in fresh fish muscle and after 3 days of storage in ice. The total collagen content was 2.9 g kg(-)(1) fresh weight. During storage on ice, a progressive change in solubility of muscle collagen was found. For acid- and pepsin-soluble collagen fractions, significantly higher and lower values, respectively, were detected. The presence and quantification of types I and V collagen in the different collagen fractions was determined also, but no significant difference in solubility during storage was found. The result suggested that collagen fibers of Atlantic salmon have a high solubility in acid solutions and contain few cross-links.     

Growth-inhibiting effects of Coptis japonica root-derived isoquinoline alkaloids on human intestinal bacteria.

The growth-inhibiting activity of Coptis japonica (Makino) root-derived materials toward eight human intestinal bacteria was examined using an impregnated paper disk method and compared to that of four commercially available isoquinoline alkaloids [berberine sulfate (BS), berberine iodide (BI), palmatine chloride (PC), and palmatine sulfate(PS)], as well as that of Thea sinensis leaf-derived epigallocatechin gallate (EGCG). The biologically active constituents of the Coptis extract were characterized as the isoquinoline alkaloids berberine chloride (BC), palmatine iodide (PI), and coptisine chloride (CC) by spectral analysis. The growth responses varied with both chemical and bacterial strain used. In a test using 500 microg/disk, BC and PI produced a clear inhibitory effect against Bifidobacterium longum, Bifidobacterium bifidum, Clostridium perfringens, and Clostridium paraputrificum, whereas weak or no inhibition was observed in Bifidobacterium adolescentis, Lactobacillus acidophilus, Lactobacillus casei, and Escherichia coli. At 1000 microg/ disk, CC revealed weak or no growth inhibition toward all test bacteria, whereas EGCG exhibited weak growth inhibition against only C. perfringens and C. paraputrificum. Among various isoquinoline alkaloids, BC exhibited more potent inhibitory activity toward C. perfringens than BI and BS, whereas the inhibitory effect was more pronounced in PI compared to PC and PS. The Coptis root-derived materials did not promote growth of B. longum and C. perfringens.     

Mass spectrometric identification of molecular species of phosphatidylcholine and lysophosphatidylcholine extracted from shark liver

The profile and structural characterization of molecular species of phosphatidylcholine (PC) and lysophosphatidylcholine (LysoPC) from shark liver using liquid chromatographic/electrospray ionization mass spectrometry (LC-ESI/MS) and tandem mass spectrometry (MS/MS) are described for the first time in this paper. The presence of (i) a relatively high content of ether PC species, such as 1-O-alkyl- and 1-alk-1'-enyl-2-polyunsaturated PC species (about 20%), and (ii) a high percentage of docosahexaenoic acid (DHA)-containing LysoPC (about 27%) is the characteristic of this marine material. 1-Hexadecanoyl-2-docosahexaenoyl-PC (16:0/22:6; about 24%) and 1-docosahexaenoyl-2-hydroxyl-LysoPC (22:6; about 27%) are the two most abundant species in shark liver. The other polyunsaturated PC species including ether PC are tentatively identified as 1-heptadecanoyl-2-docosahexaenoyl-PC (17:0/22:6), 1-octadecyl-2-docosahexaenoyl-PC (alkyl-18:0/22:6), 1-hexadecyl-2-docosahexaenoyl-PC (alkyl-16:0/22:6), 1-octadecenyl-2-eicosapentaenoyl-PC (alkyl-18:1/20:5), 1-octadecenyl-2-eicosatetraenoyl-PC (alkyl-18:1/20:4), 1-hexadecyl-2-docosapentaenoyl-PC (alkyl-16:0/22:5), 1-(1 Z-hexadecenyl)-2-docosahexaenoyl-PC (alkenyl-16:0/22:6), and 1-octadecenoyl-2-docosahexaenoyl-PC (18:1/22:6). These results establish that high contents of ether DHA-PC and DHA-LysoPC species can be obtained from shark liver.     

Stability and mineralization of organic phosphorus incorporated into model humic polymers

The stability of the phosphate ester linkage in phosphoserine (PS) and phosphoethanolamine (PE) was evaluated after incorporation of these compounds into model humic polymers. Humic polymers prepared by oxidation of a mixture of substituted phenols in the presence of either PS or PE resulted in model humic materials containing from 0.25 to 0.94% P, values within the range found for organic P in natural soil humic materials. The organic P contained in model humic polymers was resistant to hydrolysis with 1 n HC1 and 1 n NaOH and resistance of the P ester to hydrolysis with 6 n HCl was increased through incorporation into model humic polymers. Organic P in model humic polymers was also stabilized towards hydrolysis with acid and alkaline phosphomonoesterases. Less than 11% of the organic P in polymers containing PS and PE was hydrolyzed by acid or alkaline phosphatase. The incorporation of PE into a model humic polymer markedly reduced the amount of P mineralized during incubation in soil when compared to P mineralized in soils treated with PE. For all environmental conditions imposed during soil incubations (i.e. pH, aeration, temperature), only 20% of the P in model humic polymer containing PE was released during a 16-week period. In contrast, > 60% of the P in either PS and PE added individually to soils or PS and PE intimately mixed with preformed model humic polymer and then added to soils was released during the initial 7 days of soil incubation. The results suggest that a portion of the unidentified organic P in soils may arise from the incorporation of organic compounds containing both amine and phosphate ester functional groups into humic materials and that the organic P thus formed is resistant to both chemical and enzymatic hydrolysis.     

Ascorbyl palmitate, gamma-tocopherol, and EDTA affect lipid oxidation in fish oil enriched salad dressing differently

The aim of the study was to investigate the ability of gamma-tocopherol, ethylenediaminetetraacetate (EDTA), and ascorbyl palmitate to protect fish oil enriched salad dressing against oxidation during a 6 week storage period at room temperature. The lipid-soluble gamma-tocopherol (220 and 880 microg g-1 of fish oil) reduced lipid oxidation during storage by partly retarding the formation of lipid hydroperoxides (PV) and by decreasing the concentrations of individual volatile oxidation products by 34-39 and 42-66%, respectively. EDTA (10 and 50 microg g-1 of dressing) was the most efficient single antioxidant, and overall peroxide values and volatiles were reduced by approximately 70 and 77-86%, respectively. Conversely, prooxidant effects were observed with a high concentration of ascorbyl palmitate (300 microg g-1 of fish oil), whereas a low concentration was slightly antioxidative (50 microg/g of fish oil). Finally, a combination of all three antioxidants completely inhibited oxidation during storage, indicating that the prooxidant effects of ascorbyl palmitate were reverted or overshadowed by EDTA and gamma-tocopherol.     

Proteome analysis elucidating post-mortem changes in cod (Gadus morhua) muscle proteins

Proteome analysis was successfully applied to study the alterations in fish muscle proteins during ice storage. The processes occurring during post-mortem metabolism are known to lead to characteristic changes in the texture and taste of fish muscle. Endogenous proteases are anticipated to play the major role in these processes, although the exact mechanisms during fish meat tenderization have yet to be depicted. Protein changes in cod (Gadus morhua) muscle were followed during 8 days of storage. Within the partial proteome (pI 3.5-8.0, MW 13-35 kDa) significant changes were found in 11 protein spots. In nine protein spots the intensity increased, and for eight of these the increases were significant (p < 0.05) within the first 2 h post-mortem. In contrast, two protein spots decreasing in intensity showed significant (p < 0.03) changes after 8 days, thereby indicating that in the fish muscle different biochemical processes are involved in the protein changes observed post-mortem.     

Plant sterols and antioxidant parameters in enriched beverages: storage stability

Plant sterols (PS) stability, antioxidant parameters, and color were studied during 6 months of storage at 4, 24, and 37 °C in three PS-enriched functional beverages. Beverages were skimmed milk with fruit juice and PS (MFJPS), fruit juice and PS (FJPS), and skimmed milk with PS (MPS). No loss in total PS content occurred during storage observing the same values at any given storage time point. Total carotenoids decreased 36% with storage at two months and then remained stable. Total polyphenols showed fluctuations throughout the storage, remaining stable at 6 months and reaching initial values. The antioxidant capacity (TEAC method) increased 18% at 6 months, and there was an increase in color over time and temperature, probably due to Maillard reaction compound formation. The increase in total antioxidant capacity might have helped PS maintenance throughout storage, these beverages being a good PS source even after 6 months of storage.     

Preliminary study on puffer fish proteome-species identification of puffer fish by two-dimensional electrophoresis

The aims of this work were to determine the differential characterization of the urea soluble protein components of puffer fish species and to establish a preliminary proteomic database using an immobilized pH gradient two-dimensional electrophoresis (2DE) technique. The puffer fish muscle proteins resolved into 171-260 spots in the 2DE gels, with a pI range of 3-10 and molecular mass range of 7.4-205.0 kDa, following Comassie blue staining. Puffer fish muscle proteins fell in the region with pI values of 3.5-7.0, and molecular masses of 7.4-45.0 kDa were well-resolved and were good for species comparison. The more acidic proteins of lower molecular masses showed species specific characteristics. Therefore, the species of puffer fish can be differentiated from the comparison of the characteristic 2DE protein patterns.