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.     

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.     

Effect of Oils Extracted from Plant Seeds on the Growth and Lipolytic Activity of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> Yeast

This study was aimed at evaluating the capability of Yarrowia lipolytica W29 for the synthesis of lipolytic enzymes in a medium containing plant oils from non‐conventional sources with some components displaying bioactivity. Oils from almond, hazelnut, and coriander seeds were obtained by using n‐hexane (Soxhlet method) and a chloroform/methanol mixture of solvents (Folch method), and their effect on the growth and lipolytic activity of Y. lipolytica was compared. A comparison of these two extraction methods showed that the extraction with n‐hexane was less effective regarding the oil extraction yields than the extraction conducted according to Folch's procedure. The lipolytic activity of the studied yeast was higher in the culture media containing oils extracted with the Soxhlet method than the Folch method but it was lower compared to olive oil medium. Among all oils tested, almond oil extracted with n‐hexane was the best inducer of extracellular lipases synthesized by Y. lipolytica. Its lipolytic activity achieved the maximum value of 2.33 U/mL after 48 h of culture. After 24 h of culture, it was close to the value obtained for the medium containing olive oil. Almond oil was a source of oleic and linoleic acids, which may determine differences in the lipolytic activity. The linoleic acid content in almond oil was higher than that found in other oils. When n‐hexane was used for extraction, the resultant oils were characterized by lower contents of polyphenols and poorer antioxidative activity.     

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.     

<Superscript>1</Superscript>H- and <Superscript>13</Superscript>C-NMR Characterization of the Molecular Components of the Lipid Fraction of Pecorino Sardo Cheese

In this work the molecular fatty components of Pecorino Sardo Protected Designation of Origin (PS PDO) cheese were characterized through an exhaustive investigation of the ¹H- and ¹³C-NMR spectra of the extracted lipids. Several fatty acids (FA), such as long chain saturated, oleic, linoleic, linolenic, butyric, capric, caprylic, caproic, trans vaccenic, conjugated linoleic acid (cis9, trans11–18:2), and caproleic (9–10:1) were unambiguously detected. The positional isomery of some acyl groups in the glycerol backbone of triacylglycerols (TAG) was assessed. Furthermore, the NMR signals belonging to sn-1,2/2,3, sn-1,3 diacylglycerols (DAG), and free fatty acids (FFA) were analysed as a measure of lipolytic processes on cheese. Lastly, ¹H-NMR resonances of saturated aldehydes and hydroperoxides were detected, their very low intensity indicating that the lipid oxidation process can be considered to be of minor relevance in Pecorino Sardo cheese.     

Optimization of an Oil Extraction Process for Algae from the Treatment of Manure Effluent

Increasing interest in the coupling of biological wastewater treatment processes with the generation of value-added products (such as oil containing ω-3 fatty acids (FA)) has stimulated efforts in adapting extraction methods for treatment byproducts. This study’s objective was to compare a high temperature/pressure extraction method (accelerated solvent extraction) (ASE) and a manual extraction method (modified Folch extraction) with regard to their ability to extract total oil from three algae samples from the treatment of dairy manure effluent. The efficiency of total oil and FA extraction with three solvents (chloroform/methanol, isopropanol/hexane, and hexane) was also evaluated using the ASE method. Results showed that the ASE method yielded higher values for total oil content compared to the Folch method but similar values for FA content and composition after four extraction cycles with chloroform/methanol. However, the ASE method yielded much higher amounts of FA in the first cycle (85–95% of total extracted) compared to the Folch method (44–55% of total extracted in the first cycle). As expected, the extraction efficiency of the ASE method for FA was dependent on the extraction solvent. FA content values using ASE with chloroform/methanol > isopropanol/hexane > hexane. FA content values using the Folch method or ASE with chloroform/methanol were not significantly influenced by sample particle size within the size range of 0.1–1 mm.     

Synthesis,characterization and evaluation of phosphorylated resins in the removal of Pb<Superscript>2+</Superscript> from aqueous solutions

Phosphinic-derivative poly(styrene-co-divinylbenzene)-based on PS–DVB copolymers with different porosity degrees have been prepared by aromatic electrophilic substitution reaction using PCl₃/AlCl₃ followed by base-promoted hydrolysis. The phosphorylation reaction was analyzed by infra-red spectroscopy (FTIR), scanning electron microscopy (SEM), and thermogravimetry (TG/DTG). In addition, the phosphorous content of the phosphorylated copolymers was determined by spectrophotometry using the method based on sodium molybdate reactant so that the extension of that modification could be assessed. The performance of the phosphorylated resins in the extraction of Pb²⁺ from aqueous solutions in a batch system was also evaluated. The Pb²⁺ content was determined by atomic absorption spectrometry (AAS). These materials presented excellent extraction capacity under the contact time of 30 min and pH 6.     

Quantitative extraction of hamster liver lipid and cholesterol with supercritical carbon dioxide

A quantitative method for liver lipid and cholesterol extraction with supercritical CO₂ and ethanol entrainer (SCE) is reported and compared with the Folch (chloroform/methanol) procedure. Mean values for lipid and cholesterol in hamster livers (n=48) were similar between the SCE and Folch methods. Correlation coefficients between the two methods were 0.9866 for total lipid and 0.9546 for cholesterol. Similar mean values and high correlations between the two methods validate the SCE procedure as a precise alternative method for quantitative liver lipid extractions. The SCE method also reduces the use of hazardous solvents.     

Structural and Activity Investigation into Al<Superscript>3+</Superscript>, La<Superscript>3+</Superscript> and Ce<Superscript>3+</Superscript> Addition to the Phosphomolybdate Heteropolyanion for Isobutane Selective Oxidation

Abstract  

Twelve phosphomolybdate compounds were synthesized via cationic exchange and were of the form: M _x H_3–3x [PMo₁₂O₄₀] (M = Al, La or Ce; 0 ≤ x ≤ 1). These compounds were analyzed by XRD and adsorption isotherm. Aluminum addition causes a primitive cubic phase, while lanthanum and cerium yield body-centered structures. La and Ce addition reduces surface area of phosphomolybdate structure. Temperature-programmed experiments for the selective oxidation of isobutane yielded methacrolein, 3-methyl-2-oxetanone (lactone), acetic acid (not with aluminous compounds), propene (only with aluminous compounds), carbon dioxide and water. The preference for propene rather than acetic acid formation with Al³⁺ may be due to the smaller cation size, or primitive cubic structure. These products form via two distinct reaction processes, labeled categories 1 and 2. Category 1 formation is associated with isobutane forming products on the surface, but reaction rate determined by bulk migration of charged particles. Category 2 formation is concerned with isobutane penetrating deep within the bulk of the substrate and forming products which subsequently desorb in a series of bell-shaped humps. Methacrolein forms via both category 1 and 2, whilst all other products form via category 2 exclusively. Kinetic analysis showed apparent activation barriers for category 1 methacrolein formation range from 67 ± 2 kJ mol⁻¹ to >350 kJ mol⁻¹, and occur in groups with small, medium and large activation barriers. The addition of +3 metal cations to the phosphomolybdate anion increase thermal stability, significantly decreasing deactivation; IR spectroscopy shows that the Keggin structure remains intact during temperature-programmed experiments with the Al, La and Ce salts.     

Application of a Nickel-Bispidine Complex as Pre-Catalyst for C(<Emphasis Type="Italic">sp</Emphasis><Superscript>2</Superscript>)–C(<Emphasis Type="Italic">sp</Emphasis><Superscript>3</Superscript>) Bond Formations

Abstract  

In the present study, the nickel-catalyzed cross coupling of aryl halides with benzyl zinc bromides or dialkyl zinc reagents to create C(sp ²)–C(sp ³) bonds has been explored. As pre-catalyst the well-defined and easy-accessible (bispidine)Ni(NO₃)₂ complex has been applied. After investigation of different reaction parameters a broad scope of C(sp ²)–C(sp ³) bond formations were feasible under mild reaction conditions.     

Purification of Extracted Fatty Acids from the Microalgae <Emphasis Type="Italic">Spirulina</Emphasis>

The fatty acid (FA) analysis of microalgae Spirulina was studied by applying accelerated solvent extraction (ASE) and followed by purification using solid-phase extraction (SPE). The objective was to develop a sensitive and reliable purification procedure to remove pigment in lipids co-extracted from Spirulina. Four extraction solvents were used for the ASE lipids extraction. The extraction efficiency was ranked in the following order: chloroform:methanol > dichloromethane:methanol > ethanol > hexane. The major composition of fatty acids were examined. Hexane and chloroform:methanol were compared for the purification step. The amounts of sorbent (Silica gel H), sample, and the volume of eluent were optimized during SPE procedure. This purification step can successfully remove the pigments from extracted lipids. For 0.1 g algae sample, chloroform:methanol (2:1, v/v) was the optimal extraction solvent, 0.3 g silica gel was the optimal amount of sorbent, with 7 mL for the volume of eluent. For hexane as the extraction solvent, 0.5 g algae sample, 0.3 g silica gel was the optimal amount of sorbent, 5 mL was the optimal volume of eluent. The calibration curve was produced comprised from five samples that contained FAME concentrations which was ranged from 0.1 to 10 mg/L (R ² > 0.99). The recoveries of fatty acids were 67.97–134.37%, 74.20–99.13% and 98.34–115.42%, with standard deviations (SD) of three replicate detections ranged from 1.09 to 8.41%.     

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.     

A rapid method for extraction of total lipids from whey protein concentrates and separation of lipid classes with solid phase extraction

A modified procedure for extraction of total lipids from whey protein concentrates was developed such that stable emulsion with extracting solvents was avoided and the solvent system remained monophasic. Nonlipid contaminants from the extract were removed using gel filtration instead of traditional aqueous washing to prevent any loss of polar lipids. The extraction of total lipids by the modified procedure was complete and comparable with a reference procedure. Traditional thin-layer chromatography is tedious and more qualitative than quantitative for lipid class separation. Total lipids were further separated into free fatty acids, phospholipids, cholesterol ester, triacylglycerol, cholesterol, diacylglycerol, and monoacylglycerol, using modified solid phase extraction procedure. Columns with 2 g amino propyl packing allowed separation of up to 80 mg of total lipids into lipid classes gravimetrically. The values for anhydrous milk fat for all lipid classes agreed with those in the literature. Separation of total lipids into lipid classes with solid phase extraction is easy, quantitative, and can also be performed on a preparative scale.     

11α hydroxylation of 16α, 17‐epoxyprogesterone in biphasic ionic liquid/water system by Aspergillus ochraceus

BACKGROUND: The improved efficiency of steroid biotransformation using the biphasic system is generally attributed to the positive effect on the solubility of substrate in aqueous media. A promising alternative for the application of organic solvents in biphasic systems is the use of ionic liquids (ILs). This study aims to investigate the applicability of the biphasic ILs/water system for 11α hydroxylation of 16α, 17‐epoxyprogesterone (HEP) by Aspergillus ochraceus. RESULTS: Of the seven ILs tested, [C₃mim][PF₆] exhibited the best biocompatibility, with markedly improved biotransformation efficiency. In the [C₃mim][PF₆]‐based biphasic system, substrate conversion reached 90% under the condition in which buffer pH, volume ratio of buffer to ILs, cell concentration, and substrate concentration were 4.8, 10/1, 165 g L^?1 and 20 g L^?1, respectively. This is more efficient than that of the monophasic aqueous system. The effects of the cations and anions of these ILs on the 11α hydroxylation of 16α, 17‐epoxyprogesterone (HEP) by A. ochraceus is also discussed. CONCLUSION: The above results showed that IL/water biphasic system improved the efficiency of 11α hydroxylation of 16α, 17‐epoxyprogesterone (HEP) by A. ochraceus, thus suggesting the potential industrial application of ILs‐based biphasic systems for steroid biotransformation. © 2012 Society of Chemical Industry     

Synthesis and photophysical behavior of a water-soluble fluorescein-bearing polymer for Fe<Superscript>3+</Superscript> ion sensing

An acrylic monomer bearing xanthene group, acryloylfluorescein (Ac-Flu) was synthesized from fluorescein and acryloyl chloride in the presence of triethylamine in dry dichloromethane (CH₂Cl₂) at room temperature. The synthesized Ac-Flu was identified by IR, MS and ¹HNMR spectra. Copolymer of Ac-Flu and acrylamide (AM) was synthesized with thermal initiator and it was characterized by the method of IR, UV–Vis and DSC. The photophysical behaviors of Ac-Flu and its copolymer were explored by recording the fluorescence spectra in solution, solid state and film in detail. In addition, the ability of the copolymer to detect different metal cations (Mn²⁺, Fe³⁺, Fe²⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺) in aqueous solution was investigated. The results showed that poly(Ac-Flu-co-AM) had a good linear response between the logarithm of concentration of Fe³⁺ (lg[Fe³⁺]) against the relative fluorescence intensity for Fe³⁺ concentration. The results suggest that this copolymer may offer potential as a reusable polymer sensor for Fe³⁺ ion in aqueous solution.     

Enantioselective separation of R,S-phenylsuccinic acid by biphasic recognition chiral extraction

A new process has been developed to separate phenylsuccinic acid (H₂A) enantiomers, based on the oppositely preferential recognition of hydrophobic and hydrophilic chiral selectors in organic and aqueous phases, respectively, which is named as biphasic recognition chiral extraction (BRCE). BRCE system is established by adding hydrophobic l-iso-butyl tartrate in organic phase and hydrophilic β-cyclodextrin (β-CD) derivative in aqueous phase, which preferentially recognize S-H₂A and R-H₂A, respectively. The studies performed involve two enantioselective extractions in a biphasic system, where H₂A enantiomers form four complexes with β-CD derivative in aqueous phase and l-iso-butyl tartrate in organic phase, respectively. Here it is shown that the efficiency of the extraction depends, often strongly, on a number of process variables, including the types of organic solvents and β-CD derivatives, iso-butyl tartrate configurations, the concentrations of the extractants and H₂A enantiomers, pH and temperature. Phase-equilibria in BRCE systems is governed by the complex chemical equilibria in both the organic and aqueous phases. By changing the monophasic recognition chiral extraction (MRCE) system into BRCE system, the enantioselectivity increases from 1.501 to 2.862. The maximum enantioselectivity for H₂A enantiomers is obtained at pH≤2.5 and the ratio of 2:1 of [l-(+)-iso-butyl tartrate] to [HP-β-CD]. The experimental results show that BRCE is of much stronger chiral separation ability than MRCE, which is due to utilization of the separation abilities of both tartrate and β-CD derivative. It may be very helpful to optimize the extraction systems and realize the large-scale production of pure enantiomers.     

Generation of phospholipid artefacts during extraction of developing soybean seeds with methanolic solvents

The major phospholipids of soybean cotyledons during development were phosphatidylcholine (45–55%), phosphatidylethanolamine (24–28%), and phosphatidylinositol (15–18%) when the tissue was steam-killed prior to extraction of the lipids. The only other phospholipids of any significance (4–6%) was identified as phosphatidylglycerol. Phosphatidic acid was a minor constituent (<1%), and neither N-acyl phosphatidylethanolamine norbis-phosphatidic acid were detected in appreciable (>0.1% of the total lipid phosphorus) quantities. When fresh cotyledons were rapidly homogenized in mixtures of chloroform and methanol or in methanol alone, phosphatidylmethanol was formed in variable amounts (0–20% of the total phospholipid), and when cotyledons were soaked in methanol prior to homogenizing, phosphatidylmethanol became the major phospholipid, accounting for up to 75% of the total lipid phosphorus. Phosphatidylmethanol was formed by the phospholipase D-catalyzed transphosphatidylation of phosphatidylcholine and phosphatidylethanolamine during extraction.     

Role of Mo<Superscript>6+</Superscript> during nickel electrodeposition from sulfate solutions

The effect of Mo⁶⁺ on the current efficiency, deposit quality, surface morphology, crystallographic orientations and polarisation behaviour of the cathode during electrodeposition of nickel from sulfate solutions was investigated. Mo⁶⁺ did not have a significant effect on current efficiency over the concentration range 2–100 mg dm⁻³. However; a decrease in current efficiency by a magnitude of more than 20% was seen at 500 mg dm⁻³. The quality of the nickel deposit with reference to the visual appearance and contamination level varied with varying concentration of Mo⁶⁺; this was also reflected in the morphology and crystallographic orientations of the deposits. Addition of Mo⁶⁺ to the electrolyte introduced two new crystal planes i.e., (220) and (311). Depolarisation of the cathode was noted at lower concentrations of Mo⁶⁺ (2–40 mg dm⁻³) whereas polarisation of the cathode was observed at Mo⁶⁺ concentration >40 mg dm⁻³ .The effect of Mo⁶⁺ on parameters such as Tafel slope (b), transfer coefficient (α) and exchange current density (i ₀) were also determined.     

Comparison of methylene chloride and chloroform for the extraction of fats from food products

Ten food products with a wide range of total fat, fatty acid and sterol content were obtained from a supermarket in the Washington, DC area. These food products were extraced by a new method, using methylene chloride/methanol as the extraction solvent. The results were compared to the Folch et al. procedure, in which chloroform/methanol is the extraction solvent. Total fat was determined, and fatty acid methyl esters and sterol butyrates were prepared and measured by gas liquid chromatography. An analysis of variance indicated that methylene chloride is as effective as chloroform, a suspected carcinogen, in the Folch et al. extraction of total fat, fatty acids and sterols from foods. Presented in part at the 64th Federation of American Societies for Experimental Biology Annual Meeting, Anaheim, CA, April 1980.     

Utilization of dairy beta stream to produce phospholipids products by salt precipitation and solvent fractionation

Dairy phospholipids (PLs) have unique functional and nutraceutical properties over PLs obtained from other sources that typically lack phosphatidylserine (PS) and sphingomyelin (SM). In this study, an underutilized dairy by-product, beta stream, was used for the production of enriched dairy PLs products and membrane proteins by solvent extractions. After calcium salt precipitation of the membrane components, various green (i.e., safer or bioderived) solvent systems were evaluated for their capabilities for PLs extraction from the PLs-protein-salt precipitate in comparison to the standard Folch extraction (chloroform: methanol, 2:1, v/v). Among the five solvent systems (Folch alternatives) evaluated, the combination of hexane and isopropanol (H:IP, 3:2, v/v) was identified as the best for total lipid and PLs extraction, resulting in a recovery of 74.4% and 65.7% (w/w), respectively. EDTA chelating of the calcium ions was applied to produce a salt-free membrane protein product. A treatment with 0.2% EDTA was able to significantly reduce the calcium content to 0.20 mg/g protein compared to 58.81 mg/g in the initial calcium-precipitated MFGM. EDTA chelating of cations also facilitated the extraction of lipids during H:IP (3:2) treatment, which increased the total recovery of total lipid and PLs to 90.7% and 82.5% (w/w), respectively. Overall, this study demonstrates a great potential for the utilization of beta stream and other low-solids content aqueous dairy by-products for PLs recovery and further fractionation. Value-added dairy products (PLs and membrane proteins) can be obtained using green solvents identified in this study, and the process is industrially scalable.