TLC and <Superscript>31</Superscript>P-NMR Analysis of Low Polarity Phospholipids

High-performance TLC and ³¹P-NMR were assessed as methods of observing the presence of numerous low polarity phospholipids: bis-phosphatidic acid (BPA), semi-lyso bis-phosphatidic acid (SLBPA), N-acyl phosphatidylethanolamine (NAPE), N-(1,1-dimethyl-3-oxo-butyl)-phosphatidylethanolamine (diacetone adduct of PE, DOBPE), N-acetyl PE, phosphatidylmethanol (PM), phosphatidylethanol (PEt), phosphatidyl-n-propanol (PP), phosphatidyl-n-butanol (PB). Both techniques are non-discriminative and do not require the prior isolation of individual lipids. It appears that 2D TLC is superior to ³¹P NMR in the analysis of low polarity phospholipids. All phosphatidylalcohols were well separated by 2D TLC. However, some compounds which can present difficulty in separation by 2D-TLC (e.g., SLBPA and NAPE; or DOBPE and N-acetyl PE) were easily distinguished using ³¹P NMR so the methods are complimentary. A disadvantage of 2D TLC is that Rf values can vary with different brands and batches of TLC plates. The chemical shifts of ³¹P NMR were less variable, and so a library of standards may not be necessary for peak identification. Another advantage of ³¹P NMR is the ease of quantification of phospholipids. The applicability of the methods was tested on natural extracts of fish brain and cabbage stem.     

Chemical Nature and Diversity of Phosphorus in New Zealand Pasture Soils Using <Superscript>31</Superscript>P Nuclear Magnetic Resonance Spectroscopy and Sequential Fractionation

Information on the phosphorus (P) forms of pasture soils is central to agricultural and environmental sustainability. We used a combination of ³¹P nuclear magnetic resonance (NMR) spectroscopy of NaOH–EDTA extracts and sequential fractionation to investigate P forms, with an emphasis on organic P in relation to environmental and biophysical variables, in 24 diverse pasture soils taken from around New Zealand (organic C 19–102 g kg⁻¹, total P 116–2746 mg kg⁻¹, pH 5.2–7.0). Soils were separated by cluster analysis of soil physicochemical properties and sequentially extracted P pools into those either derived from volcanic-ash materials or not. This separation was also evident for organic P species in NaOH–EDTA extracts, which removed about 75% of total soil organic P. The major organic P compound class was monoesters (24–60% of extracted P), made up of 14 to 91% myo-inositol hexakisphosphate. The next largest organic P class was diesters (0–4% of P extracted), which were enriched in volcanic-ash soils (monoester to diester ratio = 14) compared to non volcanic-ash soils (ratio = 30). Correlation analysis indicated that mean annual temperature had a significant negative and positive effect on monoester and diester concentrations, respectively. This was attributed to better physical protection of monoesters (especially inositol phosphates) from mineralization, and increased diesters from microbial activity and biological turnover. The anomalous behaviour of volcanic-ash soils was attributed to slow mineralization and the sampling of soils at different times of year without full knowledge of the large and highly dynamic microbial biomass P pool which is predominantly diesters.     

Detection and Quantification of Free Glycerol in Virgin Olive Oil by <Superscript>31</Superscript>P-NMR Spectroscopy

³¹P-Nuclear magnetic resonance (NMR) spectroscopy was employed to detect and quantify free glycerol in virgin olive oils originating from various regions of Greece. This analytical method was based on the derivatization of the hydroxyl groups of glycerol with the tagging reagent 2-chloro-4,4,5,5-tetramethyldioxaphospholane, and identification of the phosphitylated compound on the basis of ³¹P chemical shifts. Quantification of glycerol in olive oils was accomplished by integration of the appropriate signals in the ³¹P NMR spectrum and the use of the phosphitylated cyclohexanol as internal standard. A linear correlation was observed between the glycerol content and 1,3-diacylglycerols and free acidity indicating that glycerol is the final product of the partial hydrolysis of triacylglycerols.     

Investigation of the Structure-Directing Effect of Surfactants on the Distribution of Silicate Species Using <Superscript>29</Superscript>Si-NMR Spectroscopy

Silicate anions were studied using ²⁹Si NMR to investigate the structure-directing effect of a surfactant (sodium dodecylsulfate-SDS) on the distribution of silicate species present in alkaline silicate solutions. The distribution of the anions in the presence of polyelectrolytes was also investigated by ²⁹Si NMR through the use of different bases including tetramethyl ammonium hydroxide (TMAOH) and sodium hydroxide (NaOH). Both SDS and the polyelectrolytic solutions were shown to stabilize low molecular weight silicate species and thus inhibit polymerization.

A 31P-NMR study of peroxo species formed during oxidation of cyclohexene with hydrogen peroxide in tri-n-butylphosphate catalyzed by heteropolyacids

In the oxidation of cyclohexene with H₂O₂in monophasic tri-n-butylphosphate (TBP) solution catalyzed by Keggin-type 12-heteropolyacids, i.e., H₃PMo_12-xW _x O₄₀(x=0–12), several peroxo species were observed by ³¹P-NMR spectroscopy in lower field than the original heteropolyacids. Their composition varied regularly with that of the starting catalyst. The P-containing peroxo species formed was deduced as [PM₄O₈(O₂)₈]^3-(M = Mo, W). The peroxo species formed more easily with a decrease in the W content, x of H₃PMo_12-xW_xO₄₀. It was further indicated from the reactivity with cyclohexene and the comparison with catalytic performance that W-rich peroxo species were catalytically more active than Mo-rich peroxo species for the oxidation of cyclohexene in this reaction system.     

Quantification of soybean oil ethanolysis with <Superscript>1</Superscript>H NMR

In this study, proton NMR spectroscopy (200 MHz) was used for quantifying the content of ethyl esters in known mixtures of soybean oil and ethyl soyate (biodiesel). For this purpose, the peak areas of ester ethoxy and glycerol methylenic peaks in the region of 4.05–4.40 ppm were measured and a calibration plot of the respective peak areas vs. the known composition of the oil/ethyl ester mixtures was used. The transesterification values determined in this way were compared with viscosity and total glycerol determinations and a good correlation was obtained. Therefore, for routine analysis, the conversion (in %) of oil to ethyl esters was determined. The methodology presented in this work proved to be quicker and simpler than others reported in the literature, such as GC and/or HPLC.     

Visualization of Bile Homeostasis Using <Superscript>1</Superscript>H-NMR Spectroscopy as a Route for Assessing Liver Cancer

Changes in bile synthesis by the liver or alterations in the enterohepatic circulation due to a variety of etiological conditions may represent a novel source of liver disease-specific biomarkers. Bile from patients with liver diseases exhibited significant changes in the levels of glycine- and taurine-conjugated bile acids, phospholipids, cholesterol and urea relative to non-liver disease controls. Cholangiocarcinoma and non-malignant liver diseases (NMLD) showed the most significant alterations. Further, hepatocellular carcinoma (HCC) could be differentiated from NMLD (p = 0.02), as well as non-liver disease controls (p = 0.02) based on the amounts of bile acids, phospholipids and/or cholesterol. HCC also differed with cholangiocarcinoma although not significantly. Urea increases somewhat in non-malignant liver disease relative to non-liver disease controls, while the bile acids, phospholipids and cholesterol all decrease significantly. The ratio between some major bile metabolites also distinguished NMLD (p = 0.004–0.01) from non-liver disease controls. This snapshot view of bile homeostasis, is obtainable from a simple nuclear magnetic resonance (NMR) approach and demonstrates the enormous opportunity to assess liver status, explore biomarkers for high risk diseases such as cancers and improve the understanding of normal and abnormal cellular functions.     

有机磷酸31P-NMR谱图研究

报道了目前国内外常用水处理药剂中的四类有机磷化合物的＾３１Ｐ－ＮＭＲ谱。其中包括小分子有机磷酸、大分子有机磷酸、有机亚磷酸和有机次磷酸。     

The use of <Superscript>1</Superscript>H NMR for yield determination in the regioselective epoxidation of squalene

¹H NMR can be used to determine the epoxide yield rapidly in the oxidation of squalene. Moreover, unequivocal distinction can be made between internal and terminal epoxide bonds. To underline the power of this technique, different stoichiometric and catalytic epoxidation procedures were carried out using squalene as substrate. They were characterized in terms of substrate conversion and regioselectivity of the epoxide fraction.     

Acyl Migration Kinetics of 2-Monoacylglycerols from Soybean Oil via <Superscript>1</Superscript>H NMR

The acyl migration kinetics of neat 2-monoacylglycerol (2-MAG) to form 1-MAG was determined using ¹H NMR spectroscopy to monitor the β-proton integration ratios of the two species over time. 2-MAG was synthesized by the Novozym 435-catalyzed alcoholysis of soybean oil and isolated by solvent extraction or molecular distillation at a mole fraction (X _2-MAG) of 0.94 relative to total MAG. The kinetics parameters of the neat 2-MAG acyl migration were investigated over the temperature range of 23–80 °C. The 2-MAG mol fraction remained unchanged at 23 °C over the course of 168 h and reached an equilibrium of X _2-MAG = 0.09 at only 80 °C. Modeling of the kinetics data revealed a 2-MAG half life (t _1/2) of 3,500 and 22.8 h at 23 and 80 °C, respectively, with an activation energy of 79.0 ± 6.5 kJ mol⁻¹. The use of ¹H NMR spectroscopy proved an expedient method for monitoring the acyl migration in 2-MAG compared to other reported methods (e.g. GC, HPLC, and ¹³C-NMR spectroscopy), requiring no sample manipulation and minimizing the deleterious effects of high temperatures and solvent exposure. Product names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product to the exclusion of others that may be suitable.     

Bile Acids Conjugation in Human Bile Is Not Random: New Insights from <Superscript>1</Superscript>H-NMR Spectroscopy at 800 MHz

Bile acids constitute a group of structurally closely related molecules and represent the most abundant constituents of human bile. Investigations of bile acids have garnered increased interest owing to their recently discovered additional biological functions including their role as signaling molecules that govern glucose, fat and energy metabolism. Recent NMR methodological developments have enabled single-step analysis of several highly abundant and common glycine- and taurine- conjugated bile acids, such as glycocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, and taurochenodeoxycholic acid. Investigation of these conjugated bile acids in human bile employing high field (800 MHz) ¹H-NMR spectroscopy reveals that the ratios between two glycine-conjugated bile acids and their taurine counterparts correlate positively (R ² = 0.83–0.97; p = 0.001 × 10⁻²–0.006 × 10⁻⁷) as do the ratios between a glycine-conjugated bile acid and its taurine counterpart (R ² = 0.92–0.95; p = 0.004 × 10⁻³–0.002 × 10⁻¹⁰). Using such correlations, concentration of individual bile acids in each sample could be predicted in good agreement with the experimentally determined values. These insights into the pattern of bile acid conjugation in human bile between glycine and taurine promise useful clues to the mechanism of bile acids’ biosynthesis, conjugation and enterohepatic circulation, and may improve our understanding of the role of individual conjugated bile acids in health and disease. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization of sacha inchi (<Emphasis Type="Italic">Plukenetia volubilis</Emphasis> L.) oil by FTIR spectroscopy and <Superscript>1</Superscript>H NMR. Comparison with linseed oil

Three oil samples obtained from sacha inchi (Plukenetia volubilis L.) seeds were studied by means of FTIR and ¹H NMR. Frequency data of the most significant bands of the IR spectrum of this oil are given. These data show that sacha inchi oil has a high degree of unsaturation. The same fact is deduced from the ratio between the absorbance of the bands due to the stretching vibrations of the cis olefinic CH double bonds at 3010.5 cm⁻¹ and to the methylene symmetrical stretching vibrations at 2855.1 cm⁻¹. The proportions of monounsaturated, polyunsaturated, and saturated acyl groups were predicted from the frequency of some IR bands, and these were in satisfactory agreement with the values obtained through FAME generation and their quantification by GC. Likewise, simple observation of the ¹H NMR spectra provided a great deal of information about sacha inchi oil, with regard not only to the relative proportions of the different acyl groups but also to their nature. Thus, the presence of γ-linolenic acyl groups was discounted. Furthermore, the area of some ¹H NMR signals was used to determine the proportion of saturated and mono-, di-, and triunsaturated acyl groups, which also were in satisfactory agreement with the values obtained by classical methods. IR and ¹H NMR determinations take very little time in comparison with classical methods and do not require chemical manipulation or transformation of the sample. A comparison was also made between the compositions of sacha inchi and linseed oil. Both oils are important sources of the healthful n−3 linolenic acyl groups, and sacha inchi also contains high proportions of the n−6 linoleic acyl groups.     

<Superscript>1</Superscript>H NMR characterization of milk lipids: A comparison between cow and buffalo milk

Characterization of the lipid fraction of raw cow and buffalo milk samples, collected in different breeding areas in Apulia, a region of southern Italy, were performed by means of ¹H NMR. The aim of this work was to establish whether FA composition data obtained by ¹H NMR can be used in the differentiation of buffalo and cow milk samples according to species. A complete assignment of the signals present in the spectrum was attempted by COSY, heteronuclear coherence spectra. Quantification of FA was carried out by inserting the integrals of particular peaks in suitable calculations. Multivariate statistical analysis, conducted on the results of the quantification, permitted buffalo and cow milks to be distinguished.     

Roles of adsorbed OH and adsorbed H in the oxidation of hydrogen and the reduction of UO<Stack><Subscript>2</Subscript><Superscript>2+</Superscript></Stack> ions at Pt electrodes under non-conventional conditions

The roles of adsorbed hydroxyl radicals, OH, at a high temperature and adsorbed hydrogen atoms, H, in an acidic solution were investigated in the electrochemical reactions on Pt electrode by using potentiodynamic polarisation experiment, cyclic voltammetry and constant-potential electrolysis combined with UV/VIS analysis. From the analysis of the polarisation curves obtained from Pt electrode in a 0.185 M H₃BO₃ solution at 473 K, it was found that the reducing capability of dissolved hydrogen is significantly enhanced due to the increases of the mass transfer and the electron transfer rates. Especially, it is suggested that the stable Pt-OH_ad plays a significant role in the passivation reaction in the potential range from 0.60 to 0.75 V_SHE. From the analyses of the experimental results for the electrochemical reduction of UO₂²⁺ ions on Pt surface in a 1.0 M HClO₄ solution, it is recognised that the reduction reaction of UO₂²⁺ to U⁴⁺ ions is strongly dependent on the hydrogen atoms adsorbed on Pt electrode (indirect reduction of UO₂²⁺) as well as on the electrons transferred from Pt electrode (direct reduction of UO₂²⁺). In addition, the reduction mechanism of UO₂²⁺ ions involved in Pt-H_ad is also proposed.     

Role of the functional group position in producing viscoelasticity in micellar solutions: A <Superscript>1</Superscript>H NMR study

¹H NMR spectra of cetyltrimethylammonium bromide (CTAB) micellar solutions in the absence and presence of sodium anthranilate (NaAn) have been recorded. Studies were performed at a fixed CTAB concentration in the presence of different NaAn concentrations. The specificity of the functional group attached to the salt in producing changes (i.e., viscoelasticity) in CTAB micellar solutions was explored. It was concluded that the nature of the salt counterion together with its intercalation/adsorption in the head group region is the principal cause of viscoelasticity.     

Rapid Identification and Relative Quantification of the Phospholipid Composition in Commercial Lecithins by <Superscript>31</Superscript>P NMR

³¹P NMR analysis of samples prepared in a sodium cholate detergent system was used as a method for the identification and quantification of enzymatic hydrolysis products of lecithin. To precisely characterize all of the hydrolysis products from commercial lecithin, a series of enzymatic reactions of each phospholipid with phospholipase PLA1 were conducted and monitored by ³¹P NMR at different times. Twenty-six phosphorus-containing hydrolysis products from six classes of phospholipids (PC, PI, PS, PE, PG, PA) were found and determined by ³¹P NMR measurement. The impact of pH on the chemical shift values for these hydrolysis products was observed and reported. To the best of our knowledge, this is the first report of ³¹P-NMR chemical shift values for entire lyso-phospholipids hydrolyzed from 6 classes of phospholipids. Rapid and routine analysis of phospholipid composition in commercial lecithins by ³¹P NMR was achieved without the need of phospholipid standards.     

Fractionation of Soybean Globulins Using Ca<Superscript>2+</Superscript> and Mg<Superscript>2+</Superscript>: A Comparative Analysis

The substitution of CaCl₂ by MgCl₂ was undertaken in Deak’s two-step process of separating the soybean 11S and 7S globulins, aiming at higher purities and lower phytic acid (PA) contents of recovered protein fractions. The effects of pH and the addition of NaCl were also evaluated. Compared with CaCl₂, MgCl₂ reduced the PA content of the 11S-rich fraction by 63–71% but increased that of the 7S-rich fraction by 14–28%, depending on pH. Correspondingly, more Ca²⁺ was recovered in the 11S-rich fraction, while more Mg²⁺ co-precipitated with the 7S-rich fraction. NaCl increased the purity of the 11S-rich fraction and reduced its PA content, but the purity of the 7S-rich fraction was reduced by using 50–100 mM NaCl. Lowering pHs from 6.4 and 4.8 to 5.6 and 4.0 in the two precipitation steps increased the yield of both fractions. The optimized fractionating procedure was as follows: the 11S-rich fraction was precipitated at pH 5.8 by using 5 mM MgCl_2, 10 mM NaHSO₃ and 20 mM NaCl, followed by the precipitation of the 7S-rich fraction at pH 4.5. The new method provided both fractions with satisfactory protein yields (22% for 11S and 16% for 7S), purities (88% for 11S and 80% for 7S) and PA contents (0.356% for 11S and 0.882% for 7S).     

Discrimination of Cod Liver Oil According to Wild/Farmed and Geographical Origins by GC and <Superscript>13</Superscript>C NMR

The objective of this study was to test the possibility of using lipid profiles obtained by gas chromatography (GC) and ¹³C nuclear magnetic resonance (NMR) in authentication of cod liver oils according to wild/farmed and geographical origin. GC and ¹³C-NMR data of cod liver oil from wild and farmed fish from different locations in Norway and Scotland were obtained, and analyzed by principal component analysis (PCA) and linear discriminant analysis (LDA) to test if it was possible to differentiate oil from wild and cultured cod (Gadus morhua L.), and to further elucidate differences between fish from the different farms/catch area. Cod liver oils of wild and farmed origin were clearly separated in the PCA score plot both from GC and NMR data. From NMR data it was also possible to observe groupings based on geographical origin (farm/catch area) of the different samples. Using LDA with cross validation the wild/farmed classification rates were 97% for GC data and 100% for NMR data. In the classification of cod liver oils according to geographical origin (38 samples from six different farms/catch area), the correct classification rate was 63% for GC data and 95% for NMR data.     

Nitrogen Leaching from <Superscript>15</Superscript>N Labelled Cow Urine and Dung Applied to Grassland on a Sandy Soil

Nitrogen (N) leaching under grazed pastures can be very high directly under urine spots. The amount of N which is returned by one excretion of urine or dung can locally exceed 1000 kg ha⁻¹ a⁻¹ which is far more than the uptake by surrounding plants during one grazing period. We therefore quantified the contribution of N deriving from urine and dung to the total N leaching under urine and dung patches. Dung N and urine N was separately sampled from a cow feed with ¹⁵N labelled grassilage, and were amended on lysimeters in October 2000 and October 2001. Lysimeters (350 mm diameter and 800 mm length) were filled with sand, and an intact grass sod from a pasture, 4 lysimeter each were amended with the ¹⁵N labelled dung and urine; 4 lysimeters without an application of dung or urine served as control. During 11 months after dung and urine amendment the amount of leachate was monitored and leachate was analysed for nitrate, ammonium and total N. ¹⁵N in these fractions was measured. Dung and urine applications of 1052 and 1030 kg N ha⁻¹ in autumn increased N leaching. Leaching loss of nitrate and dissolved organic N deriving from dung was only 37 kg N ha⁻¹ in both years, whereas under urine patches 447 kg nitrate-N ha⁻¹, 108 kg N ha⁻¹ ammonia-N and 53 kg ha⁻¹ dissolved organic N leached on average of both experimental years. N not deriving from dung and urine exceeded the leached N under the control by about 36 and 136 kg ha⁻¹ on average of both years, suggesting the contribution of different priming processes.