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.     

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.     

A31P nuclear magnetic resonance investigation of acyl group transfer from phosphatidylcholine to yield lysophosphatidylcholine in human plasma

³¹P nuclear magnetic resonance (NMR) spectroscopy was used to measure the rate of acyl transfer from phosphatidylcholine (lecithin, PC) in whole plasma and in high density lipoprotein (HDL). Spectral deconvolution was used to resolve overlapping resonances in the³¹P NMR spectra of the phospholipids. Mean values of the acyl group transfer rates from PC in plasma and HDL were 36 μmol L⁻¹h⁻¹ and 19 μmol L⁻¹h⁻¹, respectively. The reciprocal nature of the decrease in the spectral peak intensities of PC, compared to the increase in the intensities of the lysolecithin (lysoPC) peaks, suggested a substrate/product relationship consistent with the action of lecithin:cholesterol acyltransferase (LCAT), the enzyme responsible for the esterification of free cholesterol in plasma. LCAT involvement was confirmed by measuring the cholesterol esterification rate based on the¹³C NMR spectra obtained on lipid extracts from plasma that had been incubated at 37°C. Within experimental error, the rate of lysoPC formation in plasma was shown to be equal to that of cholesteryl ester formation.     

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.     

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).     

Vitamin E and the susceptibility of erythrocytes and reconstituted liposomes to oxidative stress in aged diabetics

A remarkable increase in the permeability of erythrocyte ghosts and liposomal membranes composed of erythrocyte lipids from aged diabetics was revealed by measuring [¹⁴C]glucose leakage. There were no significant differences in the contents of free cholesterol or phospholipids, or in the cholesterol/phospholipid ratio between diabetic and normal erythrocyte membranes, but significantly higher amounts of unsaturated fatty acids, arachidonic acid and docosahexaenoic acid were observed in the erythrocyte membranes of diabetics. Reconstituted liposomes prepared from aged diabetic erythrocyte lipids were highly susceptible to superoxide-induced oxidative stress. Vitamin E was highly effective in suppressing the peroxidative lysis of liposomes composed of diabetic erythrocyte lipids. The effect of superoxide dismutase (SOD) on the inhibition of peroxidation of unsaturated lipids within liposomal membranes was less than that of vitamin E.     

Quantitative Analysis of Sunflower Lecithin Adulteration with Soy Species by NMR Spectroscopy and PLS Regression

NMR spectroscopy was used to distinguish pure sunflower lecithin from that blended with soy species, and to quantify the degree of such adulteration. Sample preparation included liquid extraction of lecithin blends and measurement of polar and non‐polar fractions using ³¹P and ¹H NMR spectrometry. Several phospholipid species, linolenic acid and stachyose were found to be characteristic for sunflower lecithin authentication. For quantitative analysis, partial least squares regression (PLS) was utilized for modeling NMR data of authentic lecithin samples and in‐house prepared blends (n = 80). The models based on phospholipid, fatty acid and saccharide distributions were validated using independent test sets. PLS based on saccharide composition is able to estimate lecithin falsification regarding its vegetable origin with the sensitivity and root mean square error of validation below 1 % w/w and 3.5 % w/w, respectively. Prediction error was improved by modeling the whole lecithin profile (phospholipids, fatty acids, and saccharides). Repeatability and precision expressed in coefficients of variations was estimated to be below 8 %. The developed approach allowed the evaluation of the composition of lecithin blends of unknown soy and sunflower content on the basis of multiple chemical components.     

Phospholipid identification and quantification of membrane vesicle subfractions by 31P−1H two-dimensional nuclear magnetic resonance

An approach to the direct quantification of phospholipids from two-dimensional ³¹P−¹H nuclear magnetic resonance (NMR) spectroscopy with isotropic proton mixing has been developed as a general method for phospholipid analysis of minor membrane vesicle subfractions. Membrane vesicles were subfractionated by sedimentation to density equilibrium in a sucrose gradient, and a modified Folch method was employed to extract their phospholipids. The coefficient for the NMR detection efficiency of each phospholipid and the relative mole percentage of the phospholipids present in the membrane vesicles were calculated. We demonstrate low detection limits such that relative concentrations of phospholipids in membrane subfractions may be determined even in the submicromolar range.     

17O NMR determination of proton sites in solid heteropoly acid H3PW12O40.31P,29Si and17O NMR,FT-IR and XRD study of H3PW12O40 and H4SiW12O40 supported on carbon

¹⁷O MAS NMR spectra for solid heteropoly acid (HPA) H₃PW₁₂O₄₀ are reported. Comparison of solid-state and solution¹⁷O resonances shows that in the solid dehydrated H₃PW₁₂O₄₀ terminal W=O oxygen atoms are the predominant protonation sites. H₃PW₁₂O₄₀ and H₄SiW₁₂O₄₀ supported on chemically activated carbon have been studied by means of NMR, FT-IR and XRD. The carbon-supported HPA's retain their Keggin structure and form finely dispersed HPA species. No HPA crystal phase is developed even at an HPA loading as high as 45 wt%.³¹P,²⁹Si and¹⁷O MAS NMR spectra for bulk and carbon-supported HPA's indicate interaction of the HPA Keggin units with the carbon surface, causing large line broadening in the NMR spectra.     

NMR study of the lipid profile of mullet raw roe and bottarga

Samples of mullet (Mugil cephalus) roes from two different fishing areas were studied by means of ¹H, ¹³C and ³¹P NMR and GC techniques. The lipid fraction of unprocessed roes and that of the corresponding salted and dried commercial products “bottarga” were analyzed and the data compared. Roes of mullets from different origin showed different composition regarding mainly triacylglycerols, monounsaturated and n‐3 polyunsaturated fatty acids. The NMR spectra of processed roes, compared to those of the corresponding raw materials, showed a release of free fatty acids and the formation of other minor molecular components, such as free fatty alcohols, lysophospholipids, and diacylglycerols, thus indicating that hydrolytic processes were the main consequence of manufacturing. An increase of cholesterol ester concentration after processing was also detected. Through the analysis of the ³¹P NMR spectra, it was possible to follow the fate of the phospholipids and the formation of lysophospholipids induced by the salting and drying procedures.     

Slow exchange of erythrocyte and plasma phospholipids

³²P-Orthophosphate was injected intravenously into a pig, and the specific activities of serum phospholipids and phosphatidylcholine, serum and urine inorganic phosphorus, and erythrocyte phospholipids were followed for the next eight days. The specific activities of serum phospholipids, phosphatidylcholine, and inorganic phosphorus declined monoexponentially and were not significantly different from each other after the first day. These data imply that there are no large poorly labeled pools of inorganic phosphorus or phospholipids supplying serum phospholipid. Phospholipid metabolism is sufficiently rapid to obscure any immediate precursor product relationship. The specific activity of erthrocyte phospholipid rose slowly to a maximum on ca. the fourth day. The data were consistent with a precursor product relationship between serum phospholipids and part of the erythrocyte phospholipids.     

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.     

Erythrocyte membrane lipid alterations in undernourished cerebral palsied children during high intakes of a soy oil-based enteral formula

Five undernourished children with severe cerebral palsy (CP) were tube-fed sufficient volumes of Isocal^TM to allow rapid weight gain. Isocal^TM provided, on average, 88% of their daily energy intake for at least 25 days. The purpose of our study was to correct the undernutrition and to analyze the major erythrocyte phospholipids before and after feeding periods for possible feeding and disease-related differences. The fatty acid profiles of erythrocyte membranes from CP children were compared with those from 12 healthy children and with the fatty acid composition of the formula. There were no clinical or biochemical indications of essential fatty acid deficiency. The feeding of a soy oil-based formula increased the proportions of 18∶2n−6 in the phospholipids. The increases occurred predominantly in phosphatidylcholine followed by phosphatidylethanolamine. Despite such large dietary intakes of soy oil, no changes were observed in the phospholipid concentrations of 20∶4n−6, 18∶3n−3, 20∶5n−3, or in the C₂₂n−6 and C₂₂n−3 fatty acids. These findings are consistent with an inhibition of the Δ6 desaturase by high dietary linoleate.     

Characterization of fluorine-, aluminum-, silicon-, and phosphorus-containing complexes in wet-process phosphoric acid using nuclear magnetic resonance spectroscopy

Wet-process phosphoric acid is one product of the reaction between phosphate rock and sulfuric acid. The limiting step in this process occurs when the acid is filtered from the reaction slurry, which also contains calcium sulfate (gypsum). The acid-soluble impurities present in the phosphate rock (e.g., fluorine, silicon, and aluminum) form complexes in wet-process acid which can alter the optimum size and habit of gypsum crystals, thereby reducing the filtration rates. Fluorine-containing complexes are strongly suspected of being potent modifiers of the crystal habit of gypsum. However, the identities of the complexes responsible for the habit modification have not been established. The identities of the complexes formed in phosphoric acid (28% P₂O₅) containing additions of fluorine (HF and H₂SiF₆) and aluminum [Al(NO₃)₃ · 9H₂O or AlF₃ · 9H₂O] were established in this study by using fluorine-19 (¹⁹F) and phosphorus-31 (³¹P) nuclear magnetic resonance (NMR) spectroscopies. Peaks due to aluminum fluoride, fluorosilicate, and fluoroaluminum phosphate complexes were observed in the NMR spectra recorded from these solutions. In addition, the¹⁹F and³¹P NMR spectra of wet-process acids were recorded. These spectra contained peaks assigned to the hexafluorosilicate ion (major species), along with aluminum fluoride and fluoroaluminum phosphate complexes (minor species).     

Alterations in heart and kidney membrane phospholipids in hypertension as observed by 31P nuclear magnetic resonance

Abnormalities of phospholipids in hypertension have previously been described in human erythrocyte, platelet, and plasma lipoproteins. Since the heart and kidney are adversely affected by hypertension, we investigated possible alterations in their membrane phospholipids, which could play a role in the derangement of intracellular ion balance widely observed in hypertension. The phospholipid compositions of heart and kidney from spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats were determined by using ³¹P nuclear magnetic resonance (NMR) spectroscopy. Absolute contents of all phospholipids in hypertensive hearts and kidneys were significantly higher than in normotensive hearts and kidneys. Expressed as a fraction of total phospholipid, cardiolipin (CL) and phosphatidylethanolamine plasmalogen (PEp) were significantly increased in SHR hearts compared to WKY hearts (CL and PEp were 7.95±0.22% and 13.16±0.35% in SHR vs. 7.01±0.20% and 11.19±0.42% in WKY rats, P<-0.05), but phosphatidylethanolamine (PE) and phosphatidylcholine (PC) were significantly decreased in SHR (PE and PC were 22.46±0.37% and 44.81±0.43% in SHR vs. 24.02±0.44% and 46.01±0.50% in WKY rats, p≤0.05). In the phospholipids extracted from rat kidneys, the percentage of PE was significantly higher for SHR than for WKY rats (20.37±0.60% vs. 18.43±0.37%, P≤0.05), while PEp and phosphatidylserine (PS) were significantly lower for SHR (PEp and PS were 10.22±0.36% and 8.42 ±0.28% in SHRs vs. 11.29±0.36% and 9.71±0.40% in WKY rats, P≤0.05). The above alterations in phospholipid composition might contribute to the higher oxygen consumption in the hypertensive heart and abnormal intracellular ion concentrations and ion transport in the heart and the kidney in hypertension.     

Copper(II)-catalyzed lipid peroxidation in liposomes and erythrocyte membranes

Cu⁺⁺ was uniquely capable of catalyzing the peroxidation of rat erythrocyte membrane lipid in the presence of 10 mM H₂O₂, whereas several other transition metal ions were without significant effect. In contrast, peroxidation of soybean phospholipid liposomes could be catalyzed with decreasing efficiency by Co⁺⁺, Cu⁺⁺, Pb⁺⁺, or Cr⁺⁺⁺ also in the presence of H₂O₂. The effect of imidazole on Cu⁺⁺-catalyzed lipid peroxidation was stimulatory in liposomes and inhibitory in membrane preparations, whereas EDTA, histidine, citrate and alanine inhibited peroxidation in both systems. EDTA could stop the peroxidation after initiation, but catalase could not, indicating that Cu⁺⁺ alone was necessary for the propagation of the chain reaction. Competitive inhibition studies with various scavengers of hydroxyl radicals or singlet oxygen and the absence of significant reaction enhancement by D₂O indicated that neither of these reactive oxygen species was a major mediator in the Cu⁺⁺-H₂O₂ oxidative system. A copper-oxygen complex may be directly involved in the initiation of peroxidation. Normal erythrocyte membranes and phospholipid liposomes also differ in their sensitivities toward external oxidative stress. In the absence of H₂O₂, Cu⁺⁺ (0.2 mM) was capable of catalyzing lipid peroxidation in liposomes, aged erythrocyte membranes and membranes from vitamin-E-deficient rats; however, freshly prepared membranes from control rats and liposomes containing α-tocopherol required H₂O₂ greater than 2 mM for the catalytic effect of Cu⁺⁺ to be observed.     

Synthesis and NMR studies of the polymer membranes based on poly(4-vinylbenzylboronic acid) and phosphoric acid

Proton conduction in novel anhydrous membranes based on host polymer, poly(4-vinylbenzylboronic acid), (P4VBBA) and phosphoric acid, (H₃PO₄) as proton solvent was studied. The materials were prepared by the insertion of the proton solvent into P4VBBA at different stoichiometric ratios to get P4VBBA·xH₃PO₄ composite electrolytes. Homopolymer and the composite materials were characterized by FT-IR, ¹¹B MAS NMR and ³¹P MAS NMR. ¹¹B MAS NMR results suggested that acid doping favors or leads to a four-coordinated boron arrangement. ³¹P MAS NMR results illustrated the immobilization of phosphoric acid to the polymer through condensation with boron functional groups (B-O-P and/or B-O-P-O-B). Thermogravimetric analysis (TGA) showed that the condensation of composite materials starts approximately at 140 °C. An exponential weight loss above this temperature was attributed to intermolecular condensation of acidic units forming cross-linked polymer. The insertion of phosphoric acid into the matrix softened the materials shifting T_g to lower temperatures. The temperature dependence of the proton conductivity was modeled with Arrhenius relation. P4VBBA·2H₃PO₄ has a maximum proton conductivity of 0.0013 S/cm at RT and 0.005 S/cm at 80 °C.     

Formation of pervaporation membranes from polyphosphazenes having hydrophilic and hydrophobic pendant groups: Synthesis and characterization

A series of new polyphosphazene polymers were synthesized using three different pendant groups with the goal of probing structure–function relationships between pendant group substitution and polymer swelling/water flux through thin dense films. Formation of polymers with relative degrees of hydrophilicity was probed by varying the stoichiometry of the pendant groups attached to the phosphazene backbone: p‐methoxyphenol, 2‐(2‐methoxyethoxy)ethanol, and o‐allylphenol. The polymers in this study were characterized using NMR, thermal methods, and dilute solution light‐scattering techniques. These techniques revealed that the polymers were amorphous high polymers (M_w = 10⁵–10⁷) with varying ratios of pendant groups as determined by integration of the ¹H‐ and ³¹P‐NMR spectra. Thin dense film membranes were solution‐cast with azo‐bis(cyclohexane)carbonitrile included in the matrix and crosslinked using thermal initiation. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 80: 422–431, 2001     

Identification of carrot inositol phospholipids by fats atom bombardment mass spectrometry

Inositol phospholipids from carrot cell membranes grown in suspension cultured were purified by thinlayer chromatography (TLC) or column chromatography and tentatively identified by co-migration on TLC with animal inositol phospholipid standards. For more rigorous chemical characterization, carrot inositol phospholipids were then analyzed by negative ion fast atom bombardment mass spectrometry (FABMS). One phosphatidylinositol (PI), two lysophosphatidylinositols (LPI), and one phosphatidylinositol monophosphate (PIP) were identified in the carrot samples by the observation of ions [M-H]⁻ and numerous fragment ions in the negative FAB mass spectra. MS/MS analysis were carried out to obtain further structural information of these phospholipids using a double-focusing mass spectrometer in which the magnetic sector (B) and the electrostatic analyzer (E) were scanned at a constant ratio (B/E). These B/E linked scans provided fragment ions of selected precursor ions while eliminating matrix and other contaminating ions. No molecular ions were detected for lysophosphatidylinositol monophosphate (LPIP) or phosphatidylinositol bisphosphate (PIP₂), but fragment ions corresponding to these structures were observed. The primary fatty acids present in the carrot inositol phospholipids were linoleic (18∶2) and palmitic (16∶0) acids, whereas animal lipids contained arachidonic (20∶4), stearic (18∶0), linoleic, and palmitic acids. The only phosphatidylinositol found in carrot cells was palmitoyl linoleoyl PI.     

Solid state 19F NMR study of crystal transformation in PVDF and its nanocomposites

The polymorphism of poly(vinylidene fluoride) (PVDF) and its nanocomposites was studied by means of solid state nuclear magnetic resonance spectroscopy. ¹³C cross polarization magic angle spinning (¹³C CP MAS) NMR spectra were recorded using simultaneous high‐power decoupling on both the proton and fluorine channels. Both ¹H → ¹³C and ¹⁹F → ¹³C CP experiments were conducted, giving identical results apart from intensity variations due to the CP efficiency. Two main resonances for the CF₂ and the CH₂ groups were observed for both neat PVDF (PVDF‐C0) and the nanocomposite containing 2 wt% clay (PVDF‐C2) samples. ¹⁹F CP MAS spectra were obtained from long proton spin‐lock experiments with a shorter contact time. The results showed two strong resonances at ?84 and ?98 ppm with equal intensities, representing the α‐form crystalline structure of PVDF. It was shown that the clay induces the crystallization of PVDF in β‐form. Our earlier investigations using thermal analysis and X‐ray scattering methods also showed crystal transformation of PVDF in its clay nanocomposites. POLYM. ENG. SCI. 46:1684–1690, 2006. © 2006 Society of Plastics Engineers