Determination of Glycerol in Wines Using <Superscript>31</Superscript>P-NMR Spectroscopy

³¹P-NMR spectroscopy was employed to detect and quantify glycerol in red wines from various regions of Greece. This novel analytical method was based on the derivatization of the hydroxyl groups of glycerol with 2-chloro-4,4,5,5-tetramethyl dioxaphospholane, and identification of the phosphitylated compound on the basis of ³¹P chemical shifts. Quantification of glycerol in wines was accomplished by integration of appropriate signals in the ³¹P-NMR spectrum and the use of the phosphitylated cyclohexanol as the internal standard. The method was reproducible (CV (%) = 2.35) and accurate (CV (%) = 1.34). Its applicability to glycerol quantification in wines was tested against a weighted amount of a glycerol-model compound by linear regression analysis (R = 0.999; intercept = 0.074 ± 0.078; slope = 0.998 ± 0.003; p = 0.000). Furthermore, the NMR method was compared to the AOAC official method (HPLC) using the Bland and Altman statistical analysis. The distribution of the data points in the bias plot showed that 100% of the measurements of glycerol in 16 wine samples from various regions of Greece were within the limits of agreement of the two methods.     

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.     

Quantitative Analysis of Dairy Phospholipids by 31P NMR

³¹P NMR analysis of samples prepared in a sodium cholate detergent system was assessed as a method for the quantitative measurement of dairy phospholipids. Major phospholipid (PL) classes measured included: phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), sphingomyelin (SM) and dihydrosphingomyelin (DHSM). The ³¹P NMR method was validated by comparison with a quantitative two-dimensional thin-layer chromatography (2D-TLC) technique. The 2D-TLC system was more sensitive, able to detect some minor compounds not observed by ³¹P NMR. However, ³¹P NMR is more suited to routine analysis, with sample analysis taking 36 min. The method was also more versatile and sample analysis was possible on high phospholipid containing materials without prior lipid extraction (e.g. buttermilk protein concentrate, beta serum liquid).     

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.     

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.     

Quantitative characterization of phospholipids in milk fat <Emphasis Type="Italic">via</Emphasis><Superscript>31</Superscript>P NMR using a monophasic solvent mixture

The phospholipids (PL) occurring in both ewe and cow milk fat globule membrane were identified and quantitatively determined using ³¹P NMR spectroscopy with inverse gated decoupled sequences, which allowed a rigorous quantitative analysis. A strict relation between amount and distribution of PL and type of feeding was found. The method was calibrated over a mixture of PL standards. A recently introduced solvent constituted by a monophasic dimethylformamide/triethylamine/guanidinium hydrochloride solvent mixture was used. Compared to the traditional chloroform/methanol/water-EDTA solvent, the new solvent mixture shows very similar accuracy and precision from a quantitative point of view. The monophasic solvent overcomes the partition problems related to a biphasic system, and slightly enlarges the range of ³¹P NMR chemical shifts, thus improving the resolution. In addition, the new solvent apparently displays a lower chemical shift dependence on the various PL concentrations. The limit of the method is mainly determined by the formation of adducts between triethylamine and some PL, namely, PE, monomethylphosphati-dylethanolamine, phosphatidylethanolamine plasmalogens, and some lyso-PL. However, the new ³¹P NMR signals arising from these adducts could be easily quantified in the determination of PE.     

Brain levels of <Emphasis Type="Italic">N</Emphasis>-acylethanolamine phospholipids in mice during pentylenetetrazol-induced seizure

The N-acylethanolamine phospholipids (NAPE) are precursors for N-acylethanolamines (NAE), including anandamide (20∶4-NAE), which is a ligand for the cannabinoid receptors. Previously, NAPE were believed to be found only in injured tissue, e.g., after neurodegenerative insults. Neuronal injury may occur in response to seizure activity. Therefore, we investigated the effect of pentylenetetrazol (PTZ)-induced seizures in PTZ-kindled mice on the level of NAPE in the brain. Male NMRI mice were kindled with PTZ injections 3 times/wk, thereby developing clonic seizures in response to PTZ. Mice were killed within 30 min after the clonic seizure on the test day (12th injection) and the brains were collected. Eight species of NAPE were analyzed as the glycerophospho-N-acylethanolamines by high-performance liquid chromatography-coupled electrospray ionization mass spectrometry. No effect of the PTZ kindling on the NAPE levels in murine brains was observed. Total NAPE in control mice cortex (n=4) was 16.4±3.0 μmol/g wet weight of which 20∶4-NAPE accounted for 3.6 mol%, and the major species was 16∶0-NAPE, accounting for 52.1 mol%. Determination of the activity of NAPE-hydrolyzing phospholipase D and of N-acyltransferase in brain membrane preparations from adult and 3-d-old mice revealed an enzyme pattern in the adult mice that was favorable for NAE accumulation as opposed to NAPE accumulation. Thus, there was no difference in NAPE levels; at present, however, this does not exclude that NAE may accumulate during seizure.     

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.     

Synthesis, X-ray structure and Hg, Se CP MAS NMR studies on the first tris(imidazolidine-2-selone) mercury complex: {Chloro-tris[N-methyl-2(3H)-imidazolidine-2- selone]mercury(II)}chloride

A novel cationic Hg(II) complex has been synthesized with N-methyl-imidazolidine-2-selone ligand. The tris(N-Methyl-imidazolidine-2-selone) mercury(II) complex, [(MeImSe)₃HgCl]⁺Cl⁻ (1), has been characterized by single crystal X-ray analysis and CP MAS ¹⁹⁹Hg and ⁷⁷Se NMR.     

Characterization of diacylglycerol oil mayonnaise emulsified using phospholipase A<Subscript>2</Subscript>-treated egg yolk

Mayonnaise samples were prepared using DAG oil as the oil phase; the properties and stability were compared with mayonnaise samples prepared from TAG oil. Normal (nontreated) egg yolk and phospholipase A₂ (PLA2)-treated egg yolk were used as emulsifiers. The DAG oil mayonnaise prepared with nontreated egg yolk (DAG-M) was unstable, with cracks forming within 4 wk at 40°C. This type of fracture was not observed for TAG oil mayonnaise prepared with nontreated egg yolk (TAG-M). ³¹P NMR and quantitative analyses of phospholipids suggested that the phospholipids of egg yolk lipoprotein were dissolved in DAG-M oil droplets, which might result in the coalescence and fracture of the DAG-M. Phospholipids were not dissolved in TAG-M oil droplets. No crack formation was observed for DAG oil mayonnaise prepared with PLA2-treated egg yolk (DAG-PLM) after storage for more than 4 wk at 40°C. ³¹P NMR spectroscopy and quantitative analyses of phospholipids indicated that the dissolution of phospholipid molecules into the oil droplets was almost prevented in DAG-PLM. The stability of DAG-PLM is improved probably because the PLA2 treatment changes the molecular polarity of egg yolk phospholipids and prevents them from dissolution into the DAG oil droplets from oil/water interface.     

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.     

Novel Cyclopropane Fatty Acids from the Phospholipids of the Caribbean Sponge <Emphasis Type="Italic">Pseudospongosorites suberitoides</Emphasis>

The cyclopropane fatty acids 17-methyl-trans-4,5-methyleneoctadecanoic acid, 18-methyl-trans-4,5-methylenenonadecanoic acid, and 17-methyl-trans-4,5-methylenenonadecanoic acid were characterized for the first time in nature in the phospholipids (mainly PE, PG and PS) of the hermit-crab sponge Pseudospongosorites suberitoides. Pyrrolidine derivatization was the key in identifying the position of the cyclopropyl and methyl groups in the acyl chains and ¹H NMR was used to determine the trans stereochemistry of the cyclopropane ring. The phospholipids from the sponge also contained an interesting series of iso-anteiso Δ^5,9 fatty acids with chain-lengths between 17 and 21 carbons, with the fatty acids (5Z,9Z)-18-methyl-5,9-nonadecadienoic acid and the (5Z,9Z)-17-methyl-5,9-nonadecadienoic acid being described for the first time in sponges. The anteiso α-methoxylated fatty acid 2-methoxy-12-methyltetradecanoic acid was also identified for the first time in nature in the phospholipids of this interesting marine sponge. The novel cyclopropyl fatty acids could have originated from the phospholipids of a cyanobacterium living in symbiosis with the sponge.     

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.     

On the Quantification of Lignin Hydroxyl Groups With 31P and 13C NMR Spectroscopy

Factors affecting the accuracy of the analysis of lignin hydroxyl and carboxyl groups with ³¹P NMR have been further elucidated. Two modifications of ³¹P NMR analysis of lignin, namely the protocols using 1,3,2-dioxaphospholane (PR-I) and 2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane (PR-II) as phosphorylation reagents with different internal standards, were studied. The previous ³¹P NMR standard protocol with PR-II underestimated OH groups by about 30%, whereas the ³¹P NMR standard protocol with PR-I tended to produce overestimated data. It has been shown that cholesterol is not an appropriate internal standard, resulting in underestimated values for OH groups due to incomplete baseline resolution. The best internal standard has been found to be endo-N-hydroxy-5-norbornene-2,3-dicarboximide. Strong care should be taken related to the stability of the internal standards to avoid inflated results due to IS degradation. Under modified optimized conditions, both methods show a good correlation with the ¹³C NMR protocol in the quantification of hydroxyl groups as average, with the variability between the methods in the range of 5–15%. However, the ³¹P NMR protocols report COOH content that is twice as low as that of ¹³C NMR data. Finally, the best approach for the use of the ³¹P and ¹³C NMR methods in lignin analysis is discussed.     

Preparation of phospholipids highly enriched with n-3 polyunsaturated fatty acids by lipase

The immobilized 1,3-regiospecific Rhizomucor miehei lipase (Lipozyme™) was employed to catalyze the transesterification reaction (acidolysis) of 1,2-diacyl-sn-glycero-3-phosphatidylcholine with n-3 polyunsaturated fatty acids under nonaqueous solvent-free conditions. With a concentrate of 55% eicosapentaenoic acid (EPA) and 30% docosahexaenoic acid (DHA) and pure phosphatidylcholine from egg yolk, phospholipids of 32% EPA and 16% DHA content were obtained, presumably as a mixture of phosphatidylcholine and lysophosphatidylcholine. ³¹P nuclear magnetic resonance (NMR) analysis turned out to be a valuable technique to study the details of the reactions involved. It revealed that when 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine was transesterified with 98% pure EPA, a substantial amount of hydrolysis side reaction took place (39%), leading to a product mixture of 39% phosphatidylcholine, 44% lysophosphatidylcholine, and 17% sn-glycerol-3-phosphatidylcholine. The lysophosphatidylcholine constituent comprised 70% EPA, whereas the phosphatidylcholine component contained 58% EPA. The ³¹P NMR technique provided valid information about the mechanism of the reaction. It became evident that a high dosage of lipase containing 5% water afforded optimal conditions for the optimal extent of EPA incorporation into the phospholipids, under which the extent of hydrolysis side reaction remained relatively high.     

Synthesis,characterization and antibacterial activity of quaternized <Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">O</Emphasis>-(2-carboxyethyl) chitosan

N,O-(2-carboxyethyl)chitosan (N,O-2-CEC) was prepared from chitosan with 3-chloropropionic acid as modifying agent and NaOH as catalyst. Different quaternary ammonium groups were introduced into N,O-2-CEC by the reaction between N,O-2-CEC and different 2,3-epoxypropyl trialkyl ammonium chlorides in the presence of 25% NaOH aqueous solution, and obtained different quaternized N,O-2-carboxyethyl chitosans (QCECs). Structures of QCECs were characterized by FT-IR, ¹HNMR and gel permeation chromatography (GPC). Antimicrobial activity of QCECs was evaluated against a gram-negative bacterium Escherichia coli and a gram-positive bacterium Staphylococcus aureus. Compared with N,O-2-CEC and quaternized chitosans, the QCECs had much stronger antimicrobial activity, which increased with increasing chain length of the alkyl in the quaternary ammonium groups. The presence of benzyl in quaternary ammonium groups could endow QCECs with much better antimicrobial activity.     

Highly selective and sensitive Th<Superscript>4+</Superscript>-PVC-based membrane sensor based on 2-(diphenylphosphorothioyl)-<Emphasis Type="Italic">N</Emphasis>′,<Emphasis Type="Italic">N</Emphasis>′-diphenylacetamide

A Th⁴⁺ ion-selective membrane sensor was fabricated from poly (vinyl chloride) (PVC) matrix membrane containing 2-(diphenylphosphorothioyl)-N′,N′-diphenyl acetamide (DPTD) as a neutral carrier, potassium tetrakis (p-chlorophenyl) borate (KTpClPB) as anionic excluder and o-nitrophenyloctyl ether (NPOE) as a plasticizing solvent mediator. The effects of the membrane composition, pH and additive anionic influence on the response properties were investigated. The sensor, comprising 30% PVC, 63% solvent mediator, 4% ionophore and 3% anionic additive demonstrates the best potentiometric response characteristics. It displays Nernstian behavior (15.2 ± 0.5 mV per decade) over the concentration range 1.0 × 10⁻²–1.0 × 10⁻⁶ M. The detection limit of the electrode is 6.3 × 10⁻⁷ M (∼140 ng/ml). The response time of the electrode is 30 s .The sensor can be used in the pH range 3.0–9.0 for about 6 weeks. The membrane sensor was used as an indicator electrode in the potentiometric titration of Th⁴⁺ ions with EDTA. It was successfully applied to the determination of thorium ions in binary mixture.     

Differentiation of Fish Oils According to Species by <Superscript>13</Superscript>C-NMR Regiospecific Analyses of Triacyglycerols

The aim of this study was to use ¹³C-nuclear magnetic resonance (NMR) regiospecific analyses of triacylglycerols to distinguish fish oils from different fish species for authentication purposes. ¹³C-NMR data of muscle lipids from Atlantic salmon (Salmo salar L.), mackerel (Scomber scombrus) and herring (Clupea harengus) were obtained, and the distribution of omega-3 polyunsaturated fatty acids between the sn-1,3 and sn-2 glycerol chains calculated from the carbonyl region. The results show that there were significant differences in the sn-2 position specificity of the fatty acids 22:6n-3, 20:5n-3 and 18:4n-3 among the species investigated. The most pronounced difference was that herring had a higher proportion of its 22:6n-3 in the sn-2 position compared to the two other species. Principal component analysis of data points in the carbonyl-region showed that there were also differences in the level and regiospecific distribution of monounsaturated/saturated fatty acids, which made it possible to distinguish oils of these three species solely from the carbonyl region of ¹³C-NMR spectra.     

Meningioma phospholipid profiles measured by31P nuclear magnetic resonance spectroscopy

Fourteen cases of intracranial meningioma were characterized after chloroform/methanol extraction by³¹P nuclear magnetic resonance (NMR) spectroscopy at 202.4 MHz. Each phospholipid class detected in the extracts was identified and quantitated in terms of its molar percentage relative to the total phospholipids measured. The following phospholipids were assayed by³¹P NMR: phosphatidylglycerol, phosphatidic acid, diphosphatidylglycerol, ethanolamine plasmalogen, phosphatidylethanolamine (PE), lysophosphatidylinositol, phosphatidylserine, sphingomyelin, lysophosphatidylcholine (LPC), phosphatidylinositol (PI), sphingosylphosphorylcholine and phosphatidylcholine. In addition, two unidentified phospholipids were detected with resonances at 0.13 and −0.78 ppm, respectively. Three distinct types of spectra were obtained on the extracts and grouped accordingly for comparison purposes. Type 1 tumors showed unusual³¹P NMR profiles with low levels of PE and PI and elevated levels of LPC; type 2 tumors were characterized by low levels of the ethanolamine phospholipids and near equivalent levels of PI and LPC. The spectra of type 1 and type 2 tumors were characteristic of degenerative cells that lacked membrane permeability associated with loss of ethanolamine plasmalogen in the presence of significant phospholipid turnover. Meningiomas belonging to the third spectral type showed characteristics similar to those of normal tissues with normal levels of PE and ethanolamine plasmalogen, as well as very low levels of LPC relative to PI. Type 3 tumors lacked the characteristic signs of degeneration noted in type 1 and type 2 tumors. The data corroborate and augmentin vivo spectroscopic findings reported earlier and demonstrate the value of³¹P NMR spectroscopic phospholipid analysis on lipid extracts for the characterization of meningiomas.